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Cysteine-bound persulfides (Cys-SSH) in proteins are sulfur carrier intermediates in the synthesis of essential cofactors such as iron-sulfur clusters, molybdenum cofactor, vitamin (thiamine), and thionucleosides (thiolated tRNA). Protein-bound persulfides are also used for signaling purposes as a carrier of the "H 2 S" signal. Several methods have been developed to detect and quantify cysteine-bound persulfides in protein and monitor their exchange. The main challenge in developing these techniques is to discriminate persulfidated cysteine from cysteine and other cysteine modifications. It is also critical to develop ratiometric methods to quantify the level of persulfidation in the protein of interest. We describe here a ratiometric method to label and quantify protein-bound persulfides relying on alkylation and gel-shift assays. This method is based on the derivation of cysteine and persulfides with "heavy" alkylating agents, followed by specific cleavage of the sulfur-sulfur bond of the alkylated persulfide by a reducing agent and separation of the alkylated species by electrophoresis. A persulfide is thus revealed by the appearance of a species lacking one alkylation unit under reducing conditions. We call this alkylation-reduction band-shift (ARBS) assay. Moreover, the quantification of the bands corresponding to the persulfidated and non-persulfidated species in the same lane provides a ratiometric quantification allowing determination of the level of persulfidation of individual cysteine. Other cysteine modifications such as disulfides, sulfenic, sulfinic, sulfonic acids, nitrosothiols, and sulfenamides preclude alkylation. Thus, they may appear as false positives, but they are ruled out by the analysis under nonreducing conditions since these species do not behave as persulfides under these conditions., (© 2021. Springer Science+Business Media, LLC, part of Springer Nature.)

Background: The incidence of inflammatory bowel diseases (IBDs) tended to increase for several decades. Diet is suspected to be a major determinant of the occurrence of these diseases. This prospective study aimed to assess the associations among occurrence of IBD, dietary patterns, and ultra-processed food in the French NutriNet-Santé cohort., Methods: Participants of the NutriNet-Santé cohort who completed at least three 24-hour dietary records were included. Incident IBD cases were identified from 3 questionnaires and confirmed by phone or email interview. Major dietary patterns (DPs) were computed using a principal component analysis (PCA) based on 29 food groups' consumption, whereas proportions of ultra-processed foods (UPFs) were obtained using the NOVA classification. Multivariable Poisson models were performed to evaluate associations among DP quintiles, UPF proportion (UPFp) in the diet, and incident IBD., Results: A total of 105,832 participants were included, contributing 238,924 person-years in a mean follow-up of 2.3 ± 2.2 years. Among them, 75 participants reported an incident IBD. Three major DPs were retained: "healthy," "traditional," and "western." No significant association was found for DPs and UPFp after adjustments for covariates., Conclusions: In this study, neither DPs nor UPF proportion in the diet were significantly associated with the risk of incident IBD after adjustments for covariates. Further studies are needed to investigate the long-term association between diet and IBD., (© 2020 Crohn’s & Colitis Foundation. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Purpose: Dietary intakes are reflected in plasma by the presence of hundreds of exogenous metabolites and variations in endogenous metabolites. The exploration of diet-related plasma metabolic profiles could help to better understand the impact of overall diet on health. Our aim was to identify metabolomic signatures reflecting overall diet in women from the French general population., Methods: This cross-sectional study included 160 women in the SU.VI.MAX cohort with detailed dietary data (≥ 10 24-h dietary records) selected according to their level of adherence to the French dietary recommendations, represented by the validated score mPNNS-GS; 80 women from the 10th decile of the score were matched with 80 women from the 1st decile. Plasma metabolomic profiles were acquired using untargeted UPLC-QToF mass spectrometry analysis. The associations between metabolomic profiles and the mPNNG-GS, its components and Principal Component Analyses-derived dietary patterns were investigated using multivariable conditional logistic regression models and partial correlations., Results: Adherence to the dietary recommendations was positively associated with 3-indolepropionic acid and pipecolic acid (also positively associated with fruit and vegetable intake and a healthy diet)-2 metabolites linked to microbiota and inversely associated with lysophosphatidylcholine (LysoPC(17:1)), acylcarnitine C9:1 (also inversely associated with a healthy diet), acylcarnitine C11:1 and 2-deoxy-D-glucose. Increased plasma levels of piperine and Dihydro4mercapto-3(2H) furanone were observed in women who consumed a Western diet and a healthy diet, respectively. Ethyl-β-D-glucopyranoside was positively associated with alcohol intake. Plasma levels of LysoPC(17:1), cholic acid, phenylalanine-phenylalanine and phenylalanine and carnitine C9:1 decreased with the consumption of vegetable added fat, sweetened food, milk and dairy products and fruit and vegetable intakes, respectively., Conclusion: This study highlighted several metabolites from both host and microbial metabolism reflecting the long-term impact of the overall diet., Trial Registration: SU.VI.MAX, clinicaltrials.gov NCT00272428. Registered 3 January 2006, https://clinicaltrials.gov/show/NCT00272428.

Background: Excessive sugar intake is now recognized as a key risk factor for obesity, type 2 diabetes, and cardiovascular diseases. In contrast, evidence on the sugar-cancer link is less consistent. Experimental data suggest that sugars could play a role in cancer etiology through obesity but also through inflammatory and oxidative mechanisms and insulin resistance, even in the absence of weight gain., Objective: The objective was to study the associations between total and added sugar intake and cancer risk (overall, breast, and prostate), taking into account sugar types and sources., Methods: In total, 101,279 participants aged >18 y (median age, 40.8 y) from the French NutriNet-Santé prospective cohort study (2009-2019) were included (median follow-up time, 5.9 y). Sugar intake was assessed using repeated and validated 24-h dietary records, designed to register participants' usual consumption for >3500 food and beverage items. Associations between sugar intake and cancer risk were assessed by Cox proportional hazard models adjusted for known risk factors (sociodemographic, anthropometric, lifestyle, medical history, and nutritional factors)., Results: Total sugar intake was associated with higher overall cancer risk (n = 2503 cases; HR for quartile 4 compared with quartile 1: 1.17; 95% CI: 1.00, 1.37; Ptrend = 0.02). Breast cancer risks were increased (n = 783 cases; HRQ4vs.Q1 = 1.51; 95% CI: 1.14, 2.00; Ptrend = 0.0007). Results remained significant when weight gain during follow-up was adjusted for. In addition, significant associations with cancer risk were also observed for added sugars, free sugars, sucrose, sugars from milk-based desserts, dairy products, and sugary drinks (Ptrend ≤ 0.01)., Conclusions: These results suggest that sugars may represent a modifiable risk factor for cancer prevention (breast in particular), contributing to the current debate on the implementation of sugar taxation, marketing regulation, and other sugar-related policies. This trial was registered at clinicaltrials.gov as NCT03335644., (Copyright © The Author(s) on behalf of the American Society for Nutrition 2020.)

Background: Organic food (OF) consumption has substantially increased in high income countries, mostly driven by environmental concerns and health beliefs. Lower exposure to synthetic pesticides has been systematically documented among consumers of organic products compared to non-consumers. While experimental studies suggest that pesticides currently used in food production may be associated with type 2 diabetes (T2D), no well-conducted prospective studies have investigated the potential association between consumption of organic products and the risk of T2D, controlling for potential confounding factors. The objective of this prospective study was to estimate the association between OF consumption and the risk of T2D., Methods: A total of 33,256 participants (76% women, mean (SD) age: 53 years (14)) of the French NutriNet-Santé prospective cohort study who completed the organic food frequency questionnaire were included (2014-2019). The proportion of OF in the diet (as weight without drinking water) was computed. The associations between the proportion of OF in the diet (as 5% increment and as quintiles) and the risk of T2D were estimated using multivariable Hazard Ratio (HR) and 95% confidence interval (95% CI) derived from proportional hazards models adjusted for confounders (sociodemographic, anthropometric, lifestyle, medical and nutritional factors)., Results: During follow-up (mean = 4.05 y, SD = 1.03 y, 134,990 person-years), 293 incident cases of T2D were identified. After adjustment for confounders including lifestyle (physical activity, smoking status, alcohol consumption) and nutritional quality of the diet assessed by the adherence to the French food-based dietary guidelines, OF consumption was associated with a lower risk of T2D. Participants with the highest quintile of OF consumption, compared with those with the lowest quintile, had 35% lower risk of T2D (95% CI = 0.43-0.97). Each increment of 5% in the proportion of OF in the diet was associated with 3% lower risk of T2D (HR 0.97, 95% CI = 0.95-0.99)., Conclusions: In this large prospective cohort study, OF consumption was inversely associated with the risk of T2D. Further experimental and prospective studies should be conducted to confirm these observations., Clinical Trial Registry: The study was registered at ClinicalTrials.gov ( NCT03335644 ).

Objective: There is sparse evidence for the association of suitable food substitutions for red and processed meat on the risk of type 2 diabetes. We modeled the association between replacing red and processed meat with other protein sources and the risk of type 2 diabetes and estimated its population impact., Research Design and Methods: The European Prospective Investigation into Cancer (EPIC)-InterAct case cohort included 11,741 individuals with type 2 diabetes and a subcohort of 15,450 participants in eight countries. We modeled the replacement of self-reported red and processed meat with poultry, fish, eggs, legumes, cheese, cereals, yogurt, milk, and nuts. Country-specific hazard ratios (HRs) for incident type 2 diabetes were estimated by Prentice-weighted Cox regression and pooled using random-effects meta-analysis., Results: There was a lower hazard for type 2 diabetes for the modeled replacement of red and processed meat (50 g/day) with cheese (HR 0.90, 95% CI 0.83-0.97) (30 g/day), yogurt (0.90, 0.86-0.95) (70 g/day), nuts (0.90, 0.84-0.96) (10 g/day), or cereals (0.92, 0.88-0.96) (30 g/day) but not for replacements with poultry, fish, eggs, legumes, or milk. If a causal association is assumed, replacing red and processed meat with cheese, yogurt, or nuts could prevent 8.8%, 8.3%, or 7.5%, respectively, of new cases of type 2 diabetes., Conclusions: Replacement of red and processed meat with cheese, yogurt, nuts, or cereals was associated with a lower rate of type 2 diabetes. Substituting red and processed meat by other protein sources may contribute to the prevention of incident type 2 diabetes in European populations., (© 2020 by the American Diabetes Association.)

Background: Ultra-processed food (UPF) consumption has increased drastically worldwide and already represents 50%-60% of total daily energy intake in several high-income countries. In the meantime, the prevalence of overweight and obesity has risen continuously during the last century. The objective of this study was to investigate the associations between UPF consumption and the risk of overweight and obesity, as well as change in body mass index (BMI), in a large French cohort., Methods and Findings: A total of 110,260 adult participants (≥18 years old, mean baseline age = 43.1 [SD 14.6] years; 78.2% women) from the French prospective population-based NutriNet-Santé cohort (2009-2019) were included. Dietary intakes were collected at baseline using repeated and validated 24-hour dietary records linked to a food composition database that included >3,500 different food items, each categorized according to their degree of processing by the NOVA classification. Associations between the proportion of UPF in the diet and BMI change during follow-up were assessed using linear mixed models. Associations with risk of overweight and obesity were assessed using Cox proportional hazard models. After adjusting for age, sex, educational level, marital status, physical activity, smoking status, alcohol intake, number of 24-hour dietary records, and energy intake, we observed a positive association between UPF intake and gain in BMI (β Time × UPF = 0.02 for an absolute increment of 10 in the percentage of UPF in the diet, P < 0.001). UPF intake was associated with a higher risk of overweight (n = 7,063 overweight participants; hazard ratio (HR) for an absolute increase of 10% of UPFs in the diet = 1.11, 95% CI: 1.08-1.14; P < 0.001) and obesity (n = 3,066 incident obese participants; HR10% = 1.09 (1.05-1.13); P < 0.001). These results remained statistically significant after adjustment for the nutritional quality of the diet and energy intake. Study limitations include possible selection bias, potential residual confounding due to the observational design, and a possible item misclassification according to the level of processing. Nonetheless, robustness was tested and verified using a large panel of sensitivity analyses., Conclusions: In this large observational prospective study, higher consumption of UPF was associated with gain in BMI and higher risks of overweight and obesity. Public health authorities in several countries recently started to recommend privileging unprocessed/minimally processed foods and limiting UPF consumption., Trial Registration: ClinicalTrials.gov NCT03335644 (https://clinicaltrials.gov/ct2/show/NCT03335644)., Competing Interests: The authors have declared that no competing interests exist.

Background: Mounting evidence, yet with varying levels of proof, suggests that dietary fibers (DFs) may exert a protective role against various chronic diseases, but this might depend on the DF type and source., Objectives: Our objectives were to assess the associations between the intake of DFs of different types [total (TDF), soluble (SF), insoluble (IF)] and from different sources (fruits, vegetables, whole grains, legumes, potatoes and tubers) and the risk of cardiovascular diseases (CVDs), cancer, type 2 diabetes (T2D), and mortality in the large-scale NutriNet-Santé prospective cohort (2009-2019)., Methods: Overall, 107,377 participants were included. Usual DF intake was estimated from validated repeated 24-h dietary records over the first 2 y following inclusion in the cohort. Associations between sex-specific quintiles of DF intake and the risk of chronic diseases and mortality were assessed using multiadjusted Cox proportional hazards models., Results: T2D risk was inversely associated with TDFs [HR for quintile 5 compared with quintile 1: 0.59 (95% CI: 0.42, 0.82), P-trend <0.001], SFs [HR: 0.77 (0.56, 1.08); P-trend = 0.02], and IFs [HR: 0.69 (0.50, 0.96); P-trend = 0.004]. SFs were associated with a decreased risk of CVD [HR: 0.80 (0.66, 0.98); P-trend = 0.01] and colorectal cancer [HR: 0.41 (0.21, 0.79); P-trend = 0.01]. IFs were inversely associated with mortality from cancer or CVDs [HR: 0.65 (0.45, 0.94); P-trend = 0.02]. TDF intake was associated with a decreased risk of breast cancer [HR:: 0.79 (0.54, 1.13); P-trend = 0.04]. DF intake from fruit was associated with the risk of several chronic diseases., Conclusions: Our results suggest that DF intake, especially SFs and DFs from fruits, was inversely associated with the risk of several chronic diseases and with mortality. Further studies are needed, involving different types and sources of fiber. Meanwhile, more emphasis should be put on DFs in public health nutrition policies, as DF intake remains below the recommended levels in many countries. This trial was registered at clinicaltrials.gov as NCT03335644., (Copyright © The Author(s) on behalf of the American Society for Nutrition 2020.)

Chlorite dismutase is a unique heme enzyme that catalyzes the conversion of chlorite to chloride and molecular oxygen. The enzyme is highly specific for chlorite but has been known to bind several anionic and neutral ligands to the heme iron. In a pH study, the enzyme changed color from red to green in acetate buffer pH 5.0. The cause of this color change was uncovered using UV-visible and EPR spectroscopy. Chlorite dismutase in the presence of acetate showed a change of the UV-visible spectrum: a redshift and hyperchromicity of the Soret band from 391 to 404 nm and a blueshift of the charge transfer band CT1 from 647 to 626 nm. Equilibrium binding titrations with acetate resulted in a dissociation constant of circa 20 mM at pH 5.0 and 5.8. EPR spectroscopy showed that the acetate bound form of the enzyme remained high spin S = 5/2, however with an apparent change of the rhombicity and line broadening of the spectrum. Mutagenesis of the proximal arginine Arg183 to alanine resulted in the loss of the ability to bind acetate. Acetate was discovered as a novel ligand to chlorite dismutase, with evidence of direct binding to the heme iron. The green color is caused by a blueshift of the CT1 band that is characteristic of the high spin ferric state of the enzyme. Any weak field ligand that binds directly to the heme center may show the red to green color change, as was indeed the case for fluoride.

Background: More than 330 food additives (e.g. artificial sweeteners, emulsifiers, dyes) are authorized in Europe, with a great variability of use across food products., Objective: The objective of this study was to investigate the distribution and co-occurrence of food additives in a large-scale database of foods and beverages available on the French market., Design: The open access crowdsourced Open Food Facts database (https://world.openfoodfacts.org/) was used to retrieve the composition of food and beverage products commonly marketed on the French market (n = 126,556), based on the ingredients list. Clustering of food additive variables was used in order to determine groups of additives frequently co-occurring in food products. The clusters were confirmed by network analysis, using the eLasso method., Results: Fifty-three-point eight percent of food products contained at least 1 food additive and 11.3% at least 5. Food categories most likely to contain food additives (in more than 85% of food items) were artificially sweetened beverages, ice creams, industrial sandwiches, biscuits and cakes. The most frequently used food additives were citric acid, lecithins and modified starches (>10,000 products each). Some food additives with suspected health effects also pertained to the top 50: sodium nitrite, potassium nitrate, carrageenan, monosodium glutamate, sulfite ammonia caramel, acesulfame K, sucralose, (di/tri/poly) phosphates, mono- and diglycerides of fatty acids, potassium sorbate, cochineal, potassium metabisulphite, sodium alginate, and bixin (>800 food products each). We identified 6 clusters of food additives frequently co-occurring in food products., Conclusions: Food additives are widespread in industrial French products and some clusters of additives frequently co-occurring in food products were identified. These results pave the way to future etiological studies merging composition data to food consumption data to investigate their association with chronic disease risk, in particular potential 'cocktail effects'.

Importance: Ultraprocessed foods (UPF) are widespread in Western diets. Their consumption has been associated in recent prospective studies with increased risks of all-cause mortality and chronic diseases such as cancer, cardiovascular diseases, hypertension, and dyslipidemia; however, data regarding diabetes are lacking., Objective: To assess the associations between consumption of UPF and risk of type 2 diabetes (T2D)., Design, Setting, and Participants: In this population-based prospective cohort study, 104 707 participants aged 18 years or older from the French NutriNet-Santé cohort (2009-2019) were included. Dietary intake data were collected using repeated 24-hour dietary records (5.7 per participant on average), designed to register participants' usual consumption for more than 3500 different food items. These were categorized according to their degree of processing by the NOVA classification system., Main Outcomes and Measures: Associations between UPF consumption and risk of T2D were assessed using cause-specific multivariable Cox proportional hazard models adjusted for known risk factors (sociodemographic, anthropometric, lifestyle, medical history, and nutritional factors)., Results: A total of 104 707 participants (21 800 [20.8%] men and 82 907 [79.2%] women) were included. Mean (SD) baseline age of participants was 42.7 (14.5) years. Absolute T2D rates in the lowest and highest UPF consumers were 113 and 166 per 100 000 person-years, respectively. Consumption of UPF was associated with a higher risk of T2D (multi-adjusted hazard ratio [HR] for an absolute increment of 10 in the percentage of UPF in the diet, 1.15; 95% CI, 1.06-1.25; median follow-up, 6.0 years; 582 252 person-years; 821 incident cases). These results remained statistically significant after adjustment for several markers of the nutritional quality of the diet, for other metabolic comorbidities (HR, 1.13; 95% CI, 1.03-1.23), and for weight change (HR, 1.13; 95% CI, 1.01-1.27). The absolute amount of UPF consumption (grams per day) was consistently associated with T2D risk, even when adjusting for unprocessed or minimally processed food intake (HR for a 100 g/d increase, 1.05; 95% CI, 1.02-1.08)., Conclusions and Relevance: In this large observational prospective study, a higher proportion of UPF in the diet was associated with a higher risk of T2D. Even though these results need to be confirmed in other populations and settings, they provide evidence to support efforts by public health authorities to recommend limiting UPF consumption., Trial Registration: ClinicalTrials.gov Identifier: NCT03335644.

Background: Diet has been recognized as a modifiable risk factor for breast cancer. Highlighting predictive diet-related biomarkers would be of great public health relevance to identify at-risk subjects. The aim of this exploratory study was to select diet-related metabolites discriminating women at higher risk of breast cancer using untargeted metabolomics., Methods: Baseline plasma samples of 200 incident breast cancer cases and matched controls, from a nested case-control study within the Supplémentation en Vitamines et Minéraux Antioxydants (SU.VI.MAX) cohort, were analyzed by untargeted LC-MS. Diet-related metabolites were identified by partial correlation with dietary exposures, and best predictors of breast cancer risk were then selected by Elastic Net penalized regression. The selection stability was assessed using bootstrap resampling., Results: 595 ions were selected as candidate diet-related metabolites. Fourteen of them were selected by Elastic Net regression as breast cancer risk discriminant ions. A lower level of piperine (a compound from pepper) and higher levels of acetyltributylcitrate (an alternative plasticizer to phthalates), pregnene-triol sulfate (a steroid sulfate), and 2-amino-4-cyano butanoic acid (a metabolite linked to microbiota metabolism) were observed in plasma from women who subsequently developed breast cancer. This metabolomic signature was related to several dietary exposures such as a "Western" dietary pattern and higher alcohol and coffee intakes., Conclusions: Our study suggested a diet-related plasma metabolic signature involving exogenous, steroid metabolites, and microbiota-related compounds associated with long-term breast cancer risk that should be confirmed in large-scale independent studies., Impact: These results could help to identify healthy women at higher risk of breast cancer and improve the understanding of nutrition and health relationship., (©2019 American Association for Cancer Research.)

Background: Breast cancer is a major cause of death in occidental women. The role of metabolism in breast cancer etiology remains unclear. Metabolomics may help to elucidate novel biological pathways and identify new biomarkers to predict breast cancer long before symptoms appear. The aim of this study was to investigate whether untargeted metabolomic signatures from blood draws of healthy women could contribute to better understand and predict the long-term risk of developing breast cancer., Methods: A nested case-control study was conducted within the SU.VI.MAX prospective cohort (13 years of follow-up) to analyze baseline plasma samples of 211 incident breast cancer cases and 211 matched controls by LC/MS. Multivariable conditional logistic regression models were computed., Results: A total of 3,565 ions were detected and 1,221 were retained for statistical analysis. A total of 73 ions were associated with breast cancer risk ( P < 0.01; FDR ≤ 0.2). Notably, we observed that a lower plasma level of O-succinyl-homoserine (OR = 0.70, 95%CI = [0.55-0.89]) and higher plasma levels of valine/norvaline [1.45 (1.15-1.83)], glutamine/isoglutamine [1.33 (1.07-1.66)], 5-aminovaleric acid [1.46 (1.14-1.87)], phenylalanine [1.43 (1.14-1.78)], tryptophan [1.40 (1.10-1.79)], γ-glutamyl-threonine [1.39 (1.09-1.77)], ATBC [1.41 (1.10-1.79)], and pregnene-triol sulfate [1.38 (1.08-1.77)] were associated with an increased risk of developing breast cancer during follow-up. Conclusion: Several prediagnostic plasmatic metabolites were associated with long-term breast cancer risk and suggested a role of microbiota metabolism and environmental exposure., Impact: After confirmation in other independent cohort studies, these results could help to identify healthy women at higher risk of developing breast cancer in the subsequent decade and to propose a better understanding of the complex mechanisms involved in its etiology., (©2019 American Association for Cancer Research.)

Purpose: Lipid intakes such as saturated (SFA), monounsaturated (MUFA) and polyunsaturated (PUFA) fatty acids have been widely studied regarding cardiovascular health, but their relevance to cancer is unclear. Inconsistent epidemiological results may be explained by varied mechanisms involving PUFAs and redox balance, inflammatory status and cell signalling, along with interactions with other dietary components such as antioxidants, dietary fibre and more generally fruits and vegetable intakes. Therefore, this study aimed to investigate the associations between lipid intakes and cancer risk, and their potential modulation by vitamin C, vitamin E, dietary fibre and fruit and vegetable intakes., Methods: This prospective study included 44,039 participants aged ≥ 45 years from the NutriNet-Santé cohort (2009-2017). Dietary data were collected using repeated 24 h-dietary records. Multivariable Cox models were performed to characterize associations., Results: SFA intake was associated with increased overall [n = 1722 cases, HR Q5vsQ1  = 1.44 (1.10-1.87), p-trend = 0.008] and breast [n = 545 cases, HR Q5vsQ1  = 1.98 (1.24-3.17), p-trend = 0.01] cancer risks. n-6 PUFA [HR Q5vsQ1  = 0.56 (0.32-0.97), p-trend = 0.01] and MUFA (HR Q5vsQ1  = 0.41 [0.18-0.0.95), p-trend = 0.009] intakes were associated with a decreased risk of digestive cancers (n = 190 cases). Associations between n-6 PUFA, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) intakes and digestive cancer risk were modulated by dietary fibre, vitamin C and fruit and vegetable intakes., Conclusion: These findings suggested that SFA intake could increase overall and breast cancer risks while some unsaturated fatty acids could decrease digestive cancer risk. However, in line with mechanistic hypotheses, our results suggest that intakes of fruits and vegetables and their constituents (antioxidants, fibre) may interact with PUFAs to modulate these associations.

Background: Ultra-processed food (UPF) consumption has increased over the last decades in Westernized countries. Our objective was to investigate for the first time the association between the proportion of UPF (%UPF) in the diet and incident depressive symptoms in the NutriNet-Santé cohort., Methods: The sample included 20,380 women and 6350 men (aged 18-86 years) without depressive symptoms at the first Center for Epidemiologic Studies Depression Scale (CES-D) measurement, using validated cut-offs (CES-D score ≥ 17 for men and ≥ 23 for women). The proportion of UPF in the diet was computed for each subject using the NOVA classification applied to dietary intakes collected by repeated 24-h records (mean = 8; SD = 2.3). The association between UPF and depressive symptoms was evaluated using multivariable Cox proportional hazards models., Results: Over a mean follow-up of 5.4 years, 2221 incident cases of depressive symptoms were identified. After accounting for a wide range of potential confounders, an increased risk of depressive symptoms was observed with an increased %UPF in the diet. In the main model adjusted for sociodemographic characteristics, body mass index, and lifestyle factors, the estimated hazard ratio for a 10% increase in UPF was 1.21 (95% confidence interval = 1.15-1.27). Considering %UPF in food groups, the association was significant only for beverages and sauces or added fats., Conclusion: Overall, UPF consumption was positively associated with the risk of incident depressive symptoms, suggesting that accounting for this non-nutritional aspect of the diet could be important for mental health promotion.

Importance: Growing evidence indicates that higher intake of ultraprocessed foods is associated with higher incidence of noncommunicable diseases. However, to date, the association between ultraprocessed foods consumption and mortality risk has never been investigated., Objective: To assess the association between ultraprocessed foods consumption and all-cause mortality risk., Design, Setting, and Participants: This observational prospective cohort study selected adults, 45 years or older, from the French NutriNet-Santé Study, an ongoing cohort study that launched on May 11, 2009, and performed a follow-up through December 15, 2017 (a median of 7.1 years). Participants were selected if they completed at least 1 set of 3 web-based 24-hour dietary records during their first 2 years of follow-up. Self-reported data were collected at baseline, including sociodemographic, lifestyle, physical activity, weight and height, and anthropometrics., Exposures: The ultraprocessed foods group (from the NOVA food classification system), characterized as ready-to-eat or -heat formulations made mostly from ingredients usually combined with additives. Proportion (in weight) of ultraprocessed foods in the diet was computed for each participant., Main Outcomes and Measures: The association between proportion of ultraprocessed foods and overall mortality was the main outcome. Mean dietary intakes from all of the 24-hour dietary records available during the first 2 years of follow-up were calculated and considered as the baseline usual food-and-drink intakes. Mortality was assessed using CépiDC, the French national registry of specific mortality causes. Hazard ratios (HRs) and 95% CIs were determined for all-cause mortality, using multivariable Cox proportional hazards regression models, with age as the underlying time metric., Results: A total of 44 551 participants were included, of whom 32 549 (73.1%) were women, with a mean (SD) age at baseline of 56.7 (7.5) years. Ultraprocessed foods accounted for a mean (SD) proportion of 14.4% (7.6%) of the weight of total food consumed, corresponding to a mean (SD) proportion of 29.1% (10.9%) of total energy intake. Ultraprocessed foods consumption was associated with younger age (45-64 years, mean [SE] proportion of food in weight, 14.50% [0.04%]; P < .001), lower income (<€1200/mo, 15.58% [0.11%]; P < .001), lower educational level (no diploma or primary school, 15.50% [0.16%]; P < .001), living alone (15.02% [0.07%]; P < .001), higher body mass index (calculated as weight in kilograms divided by height in meters squared; ≥30, 15.98% [0.11%]; P < .001), and lower physical activity level (15.56% [0.08%]; P < .001). A total of 602 deaths (1.4%) occurred during follow-up. After adjustment for a range of confounding factors, an increase in the proportion of ultraprocessed foods consumed was associated with a higher risk of all-cause mortality (HR per 10% increment, 1.14; 95% CI, 1.04-1.27; P = .008)., Conclusions and Relevance: An increase in ultraprocessed foods consumption appears to be associated with an overall higher mortality risk among this adult population; further prospective studies are needed to confirm these findings and to disentangle the various mechanisms by which ultraprocessed foods may affect health.

Nutrition is often used by cancer survivors as a lever to take charge of their own health. However, some dietary behaviors are not currently recommended for patients without medical supervision. Our study aimed at evaluating weight-loss restrictive diets and fasting practices among cancer survivors of the NutriNet-Santé cohort, as well as related socio-demographic and lifestyle factors. In October 2016, 2,741 cancer survivors had completed a specific questionnaire about their practices. Fasting and non-fasting patients (respectively dieting and non-dieting) were compared using logistic regression models. Analyses were weighted according to the age, gender, and cancer location distribution of French cancer cases. 13.8% had already practiced weight-loss restrictive diet as their diagnosis. They were more likely to be women, professionally active, overweight/obese, to use dietary supplements and to have breast cancer (all p < 0.05). 6.0% had already fasted, 3.5% as their diagnosis. They were more likely to be younger, with higher educational level, higher incomes, professionally active, to have a healthy weight, and to use dietary supplements (all p < 0.05). Fasting was associated with the opinion that such practice could improve cancer prognosis (p < 0.0001). Patients who received nutritional information from health care professionals were less likely to practice fasting or weight-loss restrictive diet (0.42[0.27-0.66], p < 0.0001 and 0.49[0.38-0.64], p < 0.0001 respectively). Our study provided original results suggesting that weight-loss restrictive diets are widely practiced by cancer survivors. Fasting was less common in our study though non negligible. Sources of nutritional information received as cancer diagnosis seemed to be a key determinant of these practices., (© 2018 UICC.)

Circadian disruption has been classified as probably carcinogenic to humans by the World Health Organization's International Agency for Research on Cancer. The circadian clock is subject to environmental factors, particularly light exposure and food intake rhythms. However, the association between nutritional circadian behaviours and cancer is not well understood. We investigated the longitudinal associations between number of eating episodes, night-time fasting duration, time of first and last eating episodes, as well as nutritional quality of last eating episode, respectively, with breast and prostate cancer risks, the two main cancer locations in women and men respectively. This prospective study included 41,389 day-working adults in the French NutriNet-Santé cohort (2009-2016) who completed at least three 24 h dietary records during the first 2 years of follow-up. Multivariable Cox models were computed. 1,732 first primary incident cancer cases were diagnosed during the follow-up, among which 428 breast and 179 prostate cancers. After adjustment for covariates including sleep duration, late eaters (last eating episode after 9:30 pm) had an increased risk of breast (Hazard ratio [HR] = 1.48 [1.02-2.17], p = 0.03) and prostate (HR = 2.20 [1.28-3.78], p = 0.004) cancers. However, no association was observed between cancer risk and number of eating episodes, night-time fasting duration, time of first eating episode or macronutrient composition of the last eating episode. This large cohort study suggests that circadian perturbations resulting from late time of last food intake may be involved in carcinogenesis at different locations. Beyond nutritional quality of food intake, nutritional circadian regulation should be further investigated in the context of cancer prevention., (© 2018 UICC.)

Diverse plant products (e.g. fruits, vegetables, legumes) are associated with decreased cancer risk at several locations while red and processed meat were found to increase cancer risk. A pro plant-based dietary score reflecting the relative contribution of consumed plant vs animal products was developed, and was associated with lower overall mortality, type 2 diabetes and cardiovascular disease risk, among omnivorous adults. For the first time, we investigated the prospective associations between this pro plant-based dietary score and cancer risk. This study included 42,544 men and women of the French NutriNet-Santé prospective cohort (2009-2016) aged ≥45 years who completed at least three 24-hr-dietary records during the first year of follow-up. The risk of developing cancer was compared across sex-specific tertiles of pro plant-based dietary score by multivariable Cox models. In total, 1,591 first primary incident cancer cases were diagnosed during follow-up, among which 487 breast, 243 prostate, 198 digestive and 68 lung cancers. A higher pro plant-based dietary score was associated with decreased risks of overall (HR t3vs.t1 =0.85; 95% CI 0.76, 0.97; P trend =0.02), digestive (HR t3vs.t1 =0.68; 95% CI 0.47; 0.99; P trend  = 0.04) and lung (HR t3vs.t1 =0.47; 95% CI 0.25, 0.90; P trend =0.02) cancer, though no substantial associations were found for breast or prostate cancers. This large cohort study supports a beneficial role of higher intakes of plant-based products along with lower intakes of animal products, within a balanced omnivorous diet, regarding primary cancer prevention. These results are consistent with mechanistic evidence from experimental studies., (© 2018 UICC.)

Background: Helping consumers make healthier food choices is a key issue for the prevention of cancer and other diseases. In many countries, political authorities are considering the implementation of a simplified labelling system to reflect the nutritional quality of food products. The Nutri-Score, a five-colour nutrition label, is derived from the Nutrient Profiling System of the British Food Standards Agency (modified version) (FSAm-NPS). How the consumption of foods with high/low FSAm-NPS relates to cancer risk has been studied in national/regional cohorts but has not been characterized in diverse European populations., Methods and Findings: This prospective analysis included 471,495 adults from the European Prospective Investigation into Cancer and Nutrition (EPIC, 1992-2014, median follow-up: 15.3 y), among whom there were 49,794 incident cancer cases (main locations: breast, n = 12,063; prostate, n = 6,745; colon-rectum, n = 5,806). Usual food intakes were assessed with standardized country-specific diet assessment methods. The FSAm-NPS was calculated for each food/beverage using their 100-g content in energy, sugar, saturated fatty acid, sodium, fibres, proteins, and fruits/vegetables/legumes/nuts. The FSAm-NPS scores of all food items usually consumed by a participant were averaged to obtain the individual FSAm-NPS Dietary Index (DI) scores. Multi-adjusted Cox proportional hazards models were computed. A higher FSAm-NPS DI score, reflecting a lower nutritional quality of the food consumed, was associated with a higher risk of total cancer (HRQ5 versus Q1 = 1.07; 95% CI 1.03-1.10, P-trend < 0.001). Absolute cancer rates in those with high and low (quintiles 5 and 1) FSAm-NPS DI scores were 81.4 and 69.5 cases/10,000 person-years, respectively. Higher FSAm-NPS DI scores were specifically associated with higher risks of cancers of the colon-rectum, upper aerodigestive tract and stomach, lung for men, and liver and postmenopausal breast for women (all P < 0.05). The main study limitation is that it was based on an observational cohort using self-reported dietary data obtained through a single baseline food frequency questionnaire; thus, exposure misclassification and residual confounding cannot be ruled out., Conclusions: In this large multinational European cohort, the consumption of food products with a higher FSAm-NPS score (lower nutritional quality) was associated with a higher risk of cancer. This supports the relevance of the FSAm-NPS as underlying nutrient profiling system for front-of-pack nutrition labels, as well as for other public health nutritional measures., Competing Interests: The authors have declared that no competing interests exist.

Several national and international authorities have proposed nutritional and lifestyle recommendations with the aim of improving health of the general population. Scores of adherence to these recommendations can be calculated at the individual level. Here, we investigated the associations between four nutritional scores and overall, breast, prostate, and colorectal cancer risk in a large prospective population-based cohort: the cancer-specific World Cancer Research Fund/American Institute for Cancer Research (WCRF/AICR) score, the Alternate Healthy Eating Index 2010 (AHEI-2010), a score based on adherence to the Mediterranean diet (MEDI-LITE), and the French National Nutrition Health Program-Guideline Score (PNNS-GS). This study included 41,543 participants aged ≥40 years from the NutriNet-Santé cohort (2009-2017). A total of 1,489 overall incident cancers were diagnosed. A one-point increment of the WCRF/AICR score was significantly associated with decreased overall [12%; 95% confidence interval (CI), 8%-16%; P < 0.0001], breast (14%; 95% CI, 6%-21%; P = 0.001), and prostate (12%; 95% CI, 0%-22%; P = 0.05) cancer risks. Hazard ratio for colorectal cancer risk was 0.86 (95% CI, 0.72-1.03; P = 0.09). The PNNS-GS score was associated with reduced colorectal cancer risk ( P = 0.04) and AHEI-2010 was associated with reduced overall cancer risk ( P = 0.03). The WCRF/AICR score performed best. Compared with other tested scores, it included a stronger penalty for alcohol, which is a major risk factor for several cancer sites. Better adherence to nutritional recommendations, especially those designed for cancer prevention, could substantially contribute to decreased cancer incidence. Significance: This large prospective population-based cohort study suggests that following dietary recommendations such as the ones proposed by the World Cancer Research Fund/American Institute for Cancer Research could significantly contribute to cancer prevention. Cancer Res; 78(15); 4427-35. ©2018 AACR ., (©2018 American Association for Cancer Research.)

The study of the structure, function, folding and conformational transitions of cytochrome c is of great interest because this protein plays an important role in biological electron transport and apoptosis. The different native and non-native conformations have been studied extensively under equilibrium conditions at different pH values, however, kinetic studies are rare because they require technically challenging rapid mixing and spectroscopic monitoring techniques. Here we present the refolding kinetics of acid denatured cytochrome c using the pH jump technique from pH 2 to pH 4.7 in combination with a new ultrafast continuous flow mixing device that allows time resolved measurements to the microsecond time scale. Our results show that the initial refolding of denatured oxidized cytochrome c occurs very rapidly with a time constant τ = 10 μs, and is followed by discrete refolding steps with time constants of 56 and 208 μs. Electron paramagnetic resonance analysis of the different intermediates, obtained by microsecond freeze hyper quenching showed that the first two intermediates are predominantly high spin, and the third intermediate is the low spin species with complete His/Met coordination. The initial rapid phase is characterized by the formation of high spin species distinct from the completely unfolded state. We interpret this as the formation of a five coordinate species with His18 as the axial ligand or six coordinate with water and His18 as the axial ligands., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Unhealthy behaviours are linked to a higher risk of eye diseases, but their combined effect on visual function is unknown. We aimed to examine the individual and combined associations of diet, physical activity, smoking and alcohol consumption with visual impairment among French adults. 38 903 participants aged 18-73 years from the CONSTANCES nationwide cohort (2012-2016) with visual acuity measured and who completed, lifestyle, medical and food frequency questionnaires were included. Visual impairment was defined as a presenting visual acuity <20/40 in the better eye. After full multivariate adjustment, the odds for visual impairment increased with decreasing diet quality (p for trend = 0.04), decreasing physical activity (p for trend = 0.02) and increasing smoking pack-years (p for trend = 0.03), whereas no statistically significant association with alcohol consumption was found. Combination of several unhealthy behaviours was associated with increasing odds for visual impairment (p for trend = 0.0002), with a fully-adjusted odds ratio of 1.81 (95% CI 1.18 to 2.79) for participants reporting 2 unhealthy behaviours and 2.92 (95% CI 1.60 to 5.32) for those reporting 3 unhealthy behaviours. An unhealthy lifestyle including low/intermediate diet quality, low physical activity and heavy smoking was associated with visual impairment in this large population-based study.

Background: Combination of metabolomics and epidemiological approaches opens new perspectives for ground-breaking discoveries. The aim of the present study was to investigate for the first time whether plasma untargeted metabolomic profiles, established from a simple blood draw from healthy women, could contribute to predict the risk of developing breast cancer within the following decade and to better understand the aetiology of this complex disease., Methods: A prospective nested case-control study was set up in the Supplémentation en Vitamines et Minéraux Antioxydants (SU.VI.MAX) cohort, including 206 breast cancer cases diagnosed during a 13-year follow-up and 396 matched controls. Untargeted nuclear magnetic resonance (NMR) metabolomic profiles were established from baseline plasma samples. Multivariable conditional logistic regression models were computed for each individual NMR variable and for combinations of variables derived by principal component analysis., Results: Several metabolomic variables from 1D NMR spectroscopy were associated with breast cancer risk. Women characterized by higher fasting plasma levels of valine, lysine, arginine, glutamine, creatine, creatinine and glucose, and lower plasma levels of lipoproteins, lipids, glycoproteins, acetone, glycerol-derived compounds and unsaturated lipids had a higher risk of developing breast cancer. P-values ranged from 0.00007 [odds ratio (OR)T3vsT1=0.37 (0.23-0.61) for glycerol-derived compounds] to 0.04 [ORT3vsT1=1.61 (1.02-2.55) for glutamine]., Conclusion: This study highlighted associations between baseline NMR plasma metabolomic signatures and long-term breast cancer risk. These results provide interesting insights to better understand complex mechanisms involved in breast carcinogenesis and evoke plasma metabolic disorders favourable for carcinogenesis initiation. This study may contribute to develop screening strategies for the identification of at-risk women for breast cancer well before symptoms appear.

Purpose: While many cancer patients are affected by weight loss, others tend to gain weight, which may impact prognosis and risk of recurrence and of second cancer. The aim of this prospective study was to investigate weight variation between before and after cancer diagnosis and socio-demographic, economic, lifestyle and clinical factors associated with moderate-to-severe weight gain., Methods: 1051 incident cases of first primary cancer were diagnosed in the NutriNet-Santé cohort between 2009 and 2015. Weight was prospectively collected every 6 months since subjects' inclusion (i.e. an average of 2y before diagnosis). Mean weights before and after cancer diagnosis were compared with paired Student's t-test. Factors associated with moderate-to-severe weight gain (≥5% of initial weight) were investigated by age and sex-adjusted logistic regression., Results: Weight loss was observed in men (-3.54±4.39kg in those who lost weight, p=0.0002) and in colorectal cancer patients (-3.94±4.40kg, p=0.001). Weight gain was observed in breast and skin cancers (2.83±3.21kg, p=0.04, and 2.96±2.75kg, p=0.04 respectively). Women (OR=1.75[1.06-2.87],p=0.03), younger patients (2.44[1.51-3.70],p<0.0001), those with lower income (OR=1.30[1.01-1.72],p-trend=0.007), lower education (OR=1.32[1.03-2.70],p-trend=0.03), excess weight before diagnosis (OR=1.64[1.12-2.42],p=0.01), lower physical activity (OR=1.28[1.01-1.64],p=0.04) and those who stopped smoking (OR=4.31[1.99-9.35],p=0.005]) were more likely to gain weight. In breast cancer patients, induced menopause was associated with weight gain (OR=4.12[1.76-9.67]), but no association was detected for tumor characteristics or treatments., Conclusion: This large prospective cohort provided original results on weight variation between before and after cancer diagnosis, highlighting different weight trajectories. Socio-demographic and economic factors appeared to influence the risk of weight gain, illustrating social inequalities in health., Competing Interests: CONFLICTS OF INTEREST The authors declare that they have no conflicts of interest.

Specialized infrared spectroscopic techniques have been developed that allow studying the secondary structure of membrane proteins and the influence of crucial parameters like lipid content and detergent. Here, we focus on an ATR-FTIR spectroscopic study of Af-Amt1 and the influence of LDAO/glycerol on its structural integrity. Our results clearly indicate that infrared spectroscopy can be used to identify the adapted sample conditions.

Experimental results suggested that iron-induced lipid peroxidation may explain the direct associations observed between red/processed meat intakes and colorectal and breast cancer risk. However, epidemiological evidence is lacking. Thus, we investigated the association between dietary iron intake and breast cancer risk, and its potential modulation by an antioxidant supplementation and lipid intake. This prospective study included 4646 women from the SU.VI.MAX trial (daily low-dose antioxidants vs. placebo). 188 incident breast cancers were diagnosed (median follow-up=12.6y). Dietary iron intake was assessed using repeated 24h dietary records. Multivariable Cox proportional hazards models were computed. Dietary iron intake was associated with an increased breast cancer risk (HRT3vs.T1=1.67 (1.02-2.71), P-trend=0.04). This association was observed in the placebo group (HRT3vs.T1=2.80 (1.42-5.54), P-trend=0.003), but not in the antioxidant-supplemented group (P-trend=0.7, P-interaction=0.1). Besides, in the placebo group, the increased breast cancer risk associated with dietary iron intake was more specifically observed in women with higher lipid intake (P-trend=0.046). These findings suggest that dietary iron intake may be associated with an increased breast cancer risk, especially in women who did not received antioxidants during the trial and who consumed more lipids. This supports the experimental results suggesting that breast cancer risk may be increased by iron-induced lipid peroxidation.

People have been exposed to a lot of information regarding vitamin D, with evidence suggesting that vitamin D may be involved in numerous health conditions, subsequently creating concerns about vitamin D insufficiency. As a result, what do people really know or believe about this topic? In this cross-sectional study, we assessed vitamin D-related knowledge and beliefs in 59,273 French adults (NutriNet-Santé cohort) using a specific questionnaire. Answers to this questionnaire were weighted according to the French sociodemographic distribution and compared across individual characteristics, using χ²-tests. Physicians and media were identified as key information providers. Participants did not always accurately cite vitamin D sources (e.g., 72% only for sun exposure, fatty fish: 61%) or established health effects (e.g., bone health: 62%-78%). Conversely, they mentioned incorrect sources and health effects for which there is no consensus yet (e.g., skin cancer). These findings were modulated by age/generational and socioeconomic factors. A strong inconsistency was also observed between participants' true vitamin D status (plasma 25-hydroxyvitamin D concentration) and their opinion about it. This study, the first in Europe with such a large sample, stresses the need for simple and up-to-date supports of communication for the public and healthcare professionals regarding sources and health effects of vitamin D., Competing Interests: The authors declare no conflict of interest. The founding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.

Physical activity (PA) but also reduced sedentary behavior may be associated with better prognosis and lower risk of recurrence in cancer patients. Our aim was to quantify the variations in PA and time spent sedentary between before and after diagnosis, relying on prospective data in French adults. We also investigated sociodemographic and lifestyle factors associated with these variations.Subjects (n = 942) were incident cancer cases diagnosed in the NutriNet-Santé cohort between 2009 and 2015. PA and sedentary behavior were prospectively collected with the 7-day short version of the IPAQ questionnaire every year since subjects' inclusion (i.e., an average of 2 year before diagnosis). All PA and sitting time points before and after diagnosis was compared by mixed model. Factors associated with decrease in PA and increase in sitting time were investigated using logistic regressions.Overall and vigorous PA decreased after diagnosis (P = 0.006, -32.8 ± 36.8 MET-hour/week on average, in those who decreased their overall PA and P = 0.005, -21.1 ± 36.8 MET-hour/week for vigorous PA, respectively), especially in prostate (-39.5 ± 36.3 MET-hour/week) and skin (-35.9 ± 38 MET-hour/week) cancers, in men (-40.8 ± 46.3MET-hour/week), and in those professionally inactive (-34.2 ± 37.1 MET-hour/week) (all P < 0.05). Patients with higher PA level before diagnosis were more likely to decrease their PA (odds ratio [OR]: 4.67 [3.21-6.81], P < 0.0001). Overweight patients more likely to decrease moderate PA (OR: 1.45 [1.11-1.89], P = 0.006) and walking (OR: 1.30 [1.10-1.70], P = 0.04). Sitting time increased (P = 0.02, +2.44 ± 2.43 hour/day on average, in those who increased their sitting time), especially in women (+2.48 ± 2.48 hour/day), older patients (+2.48 ± 2.57 hour/day), and those professionally inactive (2.41 ± 2.40 hour/day) (all P < 0.05). Patients less sedentary before diagnosis were more likely to increase their sitting time (OR: 3.29 [2.45-4.42], P < 0.0001).This large prospective study suggests that cancer diagnosis is a key period for change in PA and sedentary behavior. It provides insights to target the subgroups of patients who are at higher risk of decreasing PA and increasing sedentary behavior after cancer diagnosis., Competing Interests: The authors have no conflict of interest to disclose.

Far infrared spectra of complex molecular structures like lipid membranes or proteins show large and broad continuum modes that include contributions of the internal hydrogen bonding of the assembled structures. Here we corroborate the pH triggered structural rearrangement in pH-sensitive liposomes with a clear shift of the far-infrared mode from 170 to 159 cm(-1). This spectral change was accompanied by the broadening of the hydrogen bonding signature by about 25 cm(-1) and correlates with the well-known hydrogen bonding dependent shifts of the ν(PO2(-))(as) vibration of the lipid headgroup in the mid infrared and with further shifts of functional group vibrations. Far infrared spectroscopy is thus a useful tool for the investigation of conformational changes in large molecular structures.

Proton transfer across membranes and membrane proteins is a central process in biological systems. Zn(2+) ions are capable of binding to acidic residues, often found within such specific pathways, thereby leading to a blockage. Here we probed Zn(2+) inhibition of the proton-pumping NADH:ubiquinone oxidoreductase from Escherichia coli by means of electrochemically induced FTIR difference spectroscopy. Numerous conformational changes were identified including those that arise from the reorganization of the membrane arm upon electron transfer in the peripheral arm of the protein. Signals at very high wavenumbers (1781 and 1756 cm(-1)) point to the perturbation of acidic residues in a highly hydrophobic environment upon Zn(2+) binding. In variant D563N(L), which lacks part of the proton pumping activity (residue located on the horizontal amphipathic helix), the spectral signature of Zn(2+) binding is changed. Our data support a role for this residue in proton translocation., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)