Methionine Restriction Impairs Degradation of a Protein that Aberrantly Engages the Endoplasmic Reticulum Translocon.

Proteins that persistently engage endoplasmic reticulum (ER) translocons are degraded by multiple translocon quality control (TQC) mechanisms. In Saccharomyces cerevisiae , the model translocon-associated protein Deg1 -Sec62 is subject to ER-associated degradation (ERAD) by the Hrd1 ubiquitin ligase and, to a lesser extent, proteolysis mediated by the Ste24 protease. In a recent screen, we identified nine methionine-biosynthetic genes as candidate TQC regulators. Here, we found methionine restriction impairs Hrd1-independent Deg1 -Sec62 degradation. Beyond revealing methionine as a novel regulator of TQC, our results urge caution when working with laboratory yeast strains with auxotrophic mutations, often presumed not to influence cellular processes under investigation.

Lipid biosynthesis perturbation impairs endoplasmic reticulum-associated degradation.

The relationship between lipid homeostasis and protein homeostasis (proteostasis) is complex and remains incompletely understood. We conducted a screen for genes required for efficient degradation of Deg1-Sec62, a model aberrant translocon-associated substrate of the endoplasmic reticulum (ER) ubiquitin ligase Hrd1, in Saccharomyces cerevisiae. This screen revealed that INO4 is required for efficient Deg1-Sec62 degradation. INO4 encodes one subunit of the Ino2/Ino4 heterodimeric transcription factor, which regulates expression of genes required for lipid biosynthesis. Deg1-Sec62 degradation was also impaired by mutation of genes encoding several enzymes mediating phospholipid and sterol biosynthesis. The degradation defect in ino4Δ yeast was rescued by supplementation with metabolites whose synthesis and uptake are mediated by Ino2/Ino4 targets. Stabilization of a panel of substrates of the Hrd1 and Doa10 ER ubiquitin ligases by INO4 deletion indicates ER protein quality control is generally sensitive to perturbed lipid homeostasis. Loss of INO4 sensitized yeast to proteotoxic stress, suggesting a broad requirement for lipid homeostasis in maintaining proteostasis. A better understanding of the dynamic relationship between lipid homeostasis and proteostasis may lead to improved understanding and treatment of several human diseases associated with altered lipid biosynthesis.

Genome Sequence of Arthrobacter Phage NathanVaag.

We report the genome sequence of bacteriophage NathanVaag, an actinobacteriophage isolated from soil in El Paso, Texas, that infects Arthrobacter sp. strain ATCC 21022. The 49,645-bp genome contains 73 predicted protein-coding genes. Based on gene content similarity to phages in the Actinobacteriophage Database, NathanVaag is assigned to phage cluster AO1.

Molecular and cellular evolution of the primate dorsolateral prefrontal cortex.

The granular dorsolateral prefrontal cortex (dlPFC) is an evolutionary specialization of primates that is centrally involved in cognition. We assessed more than 600,000 single-nucleus transcriptomes from adult human, chimpanzee, macaque, and marmoset dlPFC. Although most cell subtypes defined transcriptomically are conserved, we detected several that exist only in a subset of species as well as substantial species-specific molecular differences across homologous neuronal, glial, and non-neural subtypes. The latter are exemplified by human-specific switching between expression of the neuropeptide somatostatin and tyrosine hydroxylase, the rate-limiting enzyme in dopamine production in certain interneurons. The above molecular differences are also illustrated by expression of the neuropsychiatric risk gene FOXP2, which is human-specific in microglia and primate-specific in layer 4 granular neurons. We generated a comprehensive survey of the dlPFC cellular repertoire and its shared and divergent features in anthropoid primates.

Relationships Between Resilience, Mental Well-Being, and COVID-19 Worries in Collegiate Student-Athletes.

The onset of the COVID-19 pandemic was associated with robust declines in well-being for collegiate student-athletes. Worries about COVID-19 have frequently been associated with worsening well-being; therefore, it is important to examine protective factors against well-being decrements. Resilience, one's ability to respond to stress and adversity, may be one such factor. Despite this possible influence, resilience has not yet been studied in student-athletes in this context as the pandemic has progressed. Therefore, the purpose of this study was to examine the moderating role of resilience on the relationship between COVID-19 worries and well-being. In this cross-sectional design, National Collegiate Athletic Association Division III athletes (N = 91) at one university completed surveys on COVID-19 worries, resilience, and well-being between February and March 2021. All competitions had been postponed until the Spring 2021 semester. The findings revealed a negative correlation between COVID-19 worries and well-being (r = -0.21, p = 0.05) and a positive correlation between resilience and well-being (r = 0.44, p < 0.001). Additionally, multiple regression and simple slopes analyses showed that individuals with higher resilience endorsed greater scores of well-being, even when COVID-19 worries increased (ß = 0.38, p = 0.02). In conclusion, our results suggest that resilience had a moderating effect on the relationship between COVID-19 worries and well-being.

Brain Endothelial Cells: Metabolic Flux and Energy Metabolism.

The neurovascular unit (NVU) consists of multiple cell types including brain endothelial cells, pericytes, astrocytes, and neurons that function collectively to maintain homeostasis within the CNS microenvironment. As the principal barrier-forming component of the NVU, the endothelial cells perform an array of complex functions that require substantial energy resources. The principal metabolic pathways for producing ATP are glycolysis and mitochondrial oxidative phosphorylation. While previous studies have demonstrated that glycolysis is a primary pathway for most endothelial cells, details about the energy producing pathways of brain endothelial cells are not fully characterized. The contributions of glycolysis and mitochondrial respiration to energy metabolism are quantifiable using metabolic flux analysis that measures cellular oxygen consumption and acidification (proton production) in a closed microtiter plate format. ATP production rates are then calculated. The bioenergetics of the human brain microvascular endothelial cell line, hCMEC/D3, indicate that these cells exhibit relatively elevated rates of glycolytic flux and glycolytic ATP production, thus confirming their glycolytic nature even in the presence of abundant oxygen. Furthermore, energy producing pathways involving mitochondrial respiration are relatively low, although contributing significantly to total ATP production. Interestingly, the bioenergetics of the hCMEC/D3 cells are relatively similar to those of human primary brain microvascular endothelial cells (hBVECs). These findings allow a quantitative understanding of the bioenergetics of brain endothelial cells in a cultured and proliferative state and also provide a platform for comparative studies of disease states and conditions involving exposures to drugs or metabolic disruptors.

The calmodulin redox sensor controls myogenesis.

Muscle aging is accompanied by blunted muscle regeneration in response to injury and disuse. Oxidative stress likely underlies this diminished response, but muscle redox sensors that act in regeneration have not yet been characterized. Calmodulin contains multiple redox sensitive methionines whose oxidation alters the regulation of numerous cellular targets. We have used the CRISPR-Cas9 system to introduce a single amino acid substitution M109Q that mimics oxidation of methionine to methionine sulfoxide in one or both alleles of the CALM1 gene, one of three genes encoding the muscle regulatory protein calmodulin, in C2C12 mouse myoblasts. When signaled to undergo myogenesis, mutated myoblasts failed to differentiate into myotubes. Although early myogenic regulatory factors were present, cells with the CALM1 M109Q mutation in one or both alleles were unable to withdraw from the cell cycle and failed to express late myogenic factors. We have shown that a single oxidative modification to a redox-sensitive muscle regulatory protein can halt myogenesis, suggesting a molecular target for mitigating the impact of oxidative stress in age-related muscle degeneration.

Inequalities in education and national income are associated with poorer diet: Pooled analysis of individual participant data across 12 European countries.

BACKGROUND: Malnutrition linked to noncommunicable diseases presents major health problems across Europe. The World Health Organisation encourages countries to conduct national dietary surveys to obtain data to inform public health policies designed to prevent noncommunicable diseases. METHODS: Data on 27334 participants aged 19-64y were harmonised and pooled across national dietary survey datasets from 12 countries across the WHO European Region. Weighted mean nutrient intakes were age-standardised using the Eurostat 2013 European Standard Population. Associations between country-level Gross Domestic Product (GDP) and key nutrients and nutrient densities were investigated using linear regression. The potential mitigating influence of participant-level educational status was explored. FINDINGS: Higher GDP was positively associated with total sugar intake (5·0% energy for each 10% increase in GDP, 95% CI 0·6, 9·3). Scandinavian countries had the highest vitamin D intakes. Participants with higher educational status had better nutritional intakes, particularly within lower GDP countries. A 10% higher GDP was associated with lower total fat intakes (-0·2% energy, 95% CI -0·3, -0·1) and higher daily total folate intakes (14µg, 95% CI 12, 16) in higher educated individuals. INTERPRETATION: Lower income countries and lower education groups had poorer diet, particularly for micronutrients. We demonstrate for the first time that higher educational status appeared to have a mitigating effect on poorer diet in lower income countries. It illustrates the feasibility and value of harmonising national dietary survey data to inform European policy regarding access to healthy diets, particularly in disadvantaged groups. It specifically highlights the need for strong policies supporting nutritional intakes, prioritising lower education groups and lower income countries.

Traditional methods v. new technologies - dilemmas for dietary assessment in large-scale nutrition surveys and studies: a report following an international panel discussion at the 9th International Conference on Diet and Activity Methods (ICDAM9), Brisbane, 3 September 2015.

The aim of the present paper is to summarise current and future applications of dietary assessment technologies in nutrition surveys in developed countries. It includes the discussion of key points and highlights of subsequent developments from a panel discussion to address strengths and weaknesses of traditional dietary assessment methods (food records, FFQ, 24 h recalls, diet history with interviewer-assisted data collection) v. new technology-based dietary assessment methods (web-based and mobile device applications). The panel discussion 'Traditional methods v. new technologies: dilemmas for dietary assessment in population surveys', was held at the 9th International Conference on Diet and Activity Methods (ICDAM9), Brisbane, September 2015. Despite respondent and researcher burden, traditional methods have been most commonly used in nutrition surveys. However, dietary assessment technologies offer potential advantages including faster data processing and better data quality. This is a fast-moving field and there is evidence of increasing demand for the use of new technologies amongst the general public and researchers. There is a need for research and investment to support efforts being made to facilitate the inclusion of new technologies for rapid, accurate and representative data.

Measures of low food variety and poor dietary quality in a cross-sectional study of London school children.

BACKGROUND/OBJECTIVES: The use of simple screening tools to measure nutritional adequacy in a public health context in developed countries are currently lacking. We explore the relationship between food variety and nutrient intake of London school children using a simple tool with potential use for screening for inadequate diets. SUBJECTS/METHODS: A cross-sectional survey was carried out in 2010. The survey included 2579 children aged 7-10 years in 52 primary schools in East London in the United Kingdom. The analysis included 2392 children (93% of the original sample). Food variety was assessed as the total number of listed foods recorded over 24 h using the validated Child and Diet Assessment Tool (CADET) comprising 115 listed foods divided into 16 food categories. Dietary quality was determined by the proportion of children meeting recommended intakes of individual micronutrients, namely, calcium, iron, zinc, folate, vitamin A and vitamin C. RESULTS: The mean number of CADET-listed foods consumed daily by children was 17.1 (95% CI: 16.8, 17.5). Children who consumed fewer than 11 foods on the collection day had particularly low nutrient intakes. Children consuming three different vegetables and two different fruits on average consumed 19-20 listed foods. It was estimated between 4 and 20% of children did not meet the recommended levels for individual micronutrients during the period of data collection. CONCLUSIONS: A simple method using food counts to assess daily food variety may help public health nutritionists identify groups of children at risk of inadequate diets.

An exploration of socio-economic and food characteristics of high trans fatty acid consumers in the Dutch and UK national surveys after voluntary product reformulation.

Trans fatty acids (TFA) increase the risk of mortality and chronic diseases. TFA intakes have fallen since reformulation, but may still be high in certain, vulnerable, groups. This paper investigates socio-economic and food consumption characteristics of high TFA consumers after voluntary reformulation in the Netherlands and UK. Post-reformulation data of adults aged 19-64 was analysed in two national surveys: the Dutch National Food Consumption Survey (DNFCS) collected 2007-2010 using 2*24hr recalls (N = 1933) and the UK National Diet and Nutrition Survey (NDNS) years 3&4 collected 2010/11 and 2011/12 using 4-day food diaries (N = 848). The socio-economic and food consumption characteristics of the top 10% and remaining 90% TFA consumers were compared. Means of continuous data were compared using t-tests and categorical data means using chi-squared tests. Multivariate logistic regression models indicated which socio-demographic variables were associated with high TFA consumption. In the Dutch analyses, women and those born outside the Netherlands were more likely to be top 10% TFA consumers than men and Dutch-born. In the UK unadjusted analyses there was no significant trend in socio-economic characteristics between high and lower TFA consumers, but there were regional differences in the multivariate logistic regression analyses. In the Netherlands, high TFA consumers were more likely to be consumers of cakes, buns & pastries; cream; and fried potato than the remaining 90%. Whereas in the UK, high TFA consumers were more likely to be consumers of lamb; cheese; and dairy desserts and lower crisps and savoury snack consumers. Some socio-demographic differences between high and lower TFA consumers were evident post-reformulation. High TFA consumers in the Dutch 2007-10 survey appeared more likely to obtain TFA from artificial sources than those in the UK survey. Further analyses using more up-to-date food composition databases may be needed.

Interventions to reduce consumption of sugar-sweetened beverages or increase water intake: evidence from a systematic review and meta-analysis.

A systematic review and meta-analyses were conducted to evaluate the effects of interventions to reduce sugar-sweetened beverages (SSB) or increase water intakes and to examine the impact of behaviour change techniques (BCTs) in consumption patterns. Randomized and nonrandomized controlled trials published after January 1990 and until December 2016 reporting daily changes in intakes of SSB or water in volumetric measurements (mL d-1 ) were included. References were retrieved through searches of electronic databases and quality appraisal followed Cochrane principles. We calculated mean differences (MD) and synthesized data with random-effects models. Forty studies with 16 505 participants were meta-analysed. Interventions significantly decreased consumption of SSB in children by 76 mL d-1 (95% confidence interval [CI] -105 to -46; 23 studies, P < 0.01), and in adolescents (-66 mL d-1 , 95% CI -130 to -2; 5 studies, P = 0.04) but not in adults (-13 mL d-1 , 95% CI -44 to 18; 12 studies, P = 0.16). Pooled estimates of water intakes were only possible for interventions in children, and results were indicative of increases in water intake (MD +67 mL d-1 , 95% CI 6 to 128; 7 studies, P = 0.04). For children, there was evidence to suggest that modelling/demonstrating the behaviour helped to reduce SSB intake and that interventions within the home environment had greater effects than school-based interventions. In conclusion, public health interventions - mainly via nutritional education/counselling - are moderately successful at reducing intakes of SSB and increasing water intakes in children. However, on average, only small reductions in SSBs have been achieved by interventions targeting adolescents and adults. Complementary measures may be needed to achieve greater improvements in both dietary behaviours across all age groups.

An evaluation of diabetes targeted apps for Android smartphone in relation to behaviour change techniques.

BACKGROUND: Mobile applications (apps) could support diabetes management through dietary, weight and blood glucose self-monitoring, as well as by promoting behaviour change. The present study aimed to evaluate diabetes apps for content, functions and behaviour change techniques (BCTs). METHODS: Diabetes self-management apps for Android smartphones were searched for on the Google Play Store. Ten apps each from the following search terms were included; 'diabetes', 'diabetes type 1', 'diabetes type 2', 'gestational diabetes'. Apps were evaluated by being scored according to their number of functions and BCTs, price, and user rating. RESULTS: The mean (SD) number of functions was 8.9 (5.9) out of a possible maximum of 27. Furthermore, the mean (SD) number of BCTs was 4.4 (2.6) out of a possible maximum of 26. Apps with optimum BCT had significantly more functions [13.8; 95% confidence interval (CI) = 11.9-15.9] than apps that did not (4.7; 95% CI = 3.2-6.2; P < 0.01) and significantly more BCTs (5.8; 95% CI = 4.8-7.0) than apps without (3.1; 95% CI = 2.2-4.1; P < 0.01). Additionally, apps with optimum BCT also cost more than other apps. In the adjusted models, highly rated apps had an average of 4.8 (95% CI = 0.9-8.7; P = 0.02) more functions than lower rated apps. CONCLUSIONS: 'Diabetes apps' include few functions or BCTs compared to the maximum score possible. Apps with optimum BCTs could indicate higher quality. App developers should consider including both specific functions and BCTs in 'diabetes apps' to make them more helpful. More research is needed to understand the components of an effective app for people with diabetes.

Strengthening the Reporting of Observational Studies in Epidemiology - nutritional epidemiology (STROBE-nut): An extension of the STROBE statement.

Concerns have been raised about the quality of reporting in nutritional epidemiology. Research reporting guidelines such as the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) statement can improve quality of reporting in observational studies. Herein, we propose recommendations for reporting nutritional epidemiology and dietary assessment research by extending the STROBE statement into Strengthening the Reporting of Observational Studies in Epidemiology - Nutritional Epidemiology (STROBE-nut). Recommendations for the reporting of nutritional epidemiology and dietary assessment research were developed following a systematic and consultative process, co-ordinated by a multidisciplinary group of 21 experts. Consensus on reporting guidelines was reached through a three-round Delphi consultation process with 53 external experts. In total, 24 recommendations for nutritional epidemiology were added to the STROBE checklist. When used appropriately, reporting guidelines for nutritional epidemiology can contribute to improve reporting of observational studies with a focus on diet and health.

Mechanical parameters and flight phase characteristics in aquatic plyometric jumping.

Plyometric jumping is a commonly prescribed method of training focused on the development of reactive strength and high-velocity concentric power. Literature suggests that aquatic plyometric training may be a low-impact, effective supplement to land-based training. The purpose of the present study was to quantify acute, biomechanical characteristics of the take-off and flight phase for plyometric movements performed in the water. Kinetic force platform data from 12 young, male adults were collected for counter-movement jumps performed on land and in water at two different immersion depths. The specificity of jumps between environmental conditions was assessed using kinetic measures, temporal characteristics, and an assessment of the statistical relationship between take-off velocity and time in the air. Greater peak mechanical power was observed for jumps performed in the water, and was influenced by immersion depth. Additionally, the data suggest that, in the water, the statistical relationship between take-off velocity and time in air is quadratic. Results highlight the potential application of aquatic plyometric training as a cross-training tool for improving mechanical power and suggest that water immersion depth and fluid drag play key roles in the specificity of the take-off phase for jumping movements performed in the water.

Dietary fibre intake and risk of ischaemic and haemorrhagic stroke in the UK Women's Cohort Study.

BACKGROUND: Stroke risk is modifiable through many risk factors, one being healthy dietary habits. Fibre intake was associated with a reduced stroke risk in recent meta-analyses; however, data were contributed by relatively few studies, and few examined different stroke types. METHODS: A total of 27,373 disease-free women were followed up for 14.4 years. Diet was assessed with a 217-item food frequency questionnaire and stroke cases were identified using English Hospital Episode Statistics and mortality records. Survival analysis was applied to assess the risk of total, ischaemic or haemorrhagic stroke in relation to fibre intake. RESULTS: A total of 135 haemorrhagic and 184 ischaemic stroke cases were identified in addition to 138 cases where the stroke type was unknown or not recorded. Greater intake of total fibre, higher fibre density and greater soluble fibre, insoluble fibre and fibre from cereals were associated with a significantly lower risk for total stroke. For total stroke, the hazard ratio per 6 g/day total fibre intake was 0.89 (95% confidence intervals: 0.81-0.99). Different findings were observed for haemorrhagic and ischaemic stroke in healthy-weight or overweight women. Total fibre, insoluble fibre and cereal fibre were inversely associated with haemorrhagic stroke risk in overweight/obese participants, and in healthy-weight women greater cereal fibre was associated with a lower ischaemic stroke risk. In non-hypertensive women, higher fibre density was associated with lower ischaemic stroke risk. CONCLUSIONS: Greater total fibre and fibre from cereals are associated with a lower stroke risk, and associations were more consistent with ischaemic stroke. The different observations by stroke type, body mass index group or hypertensive status indicates potentially different mechanisms.

Measuring diet in primary school children aged 8-11 years: validation of the Child and Diet Evaluation Tool (CADET) with an emphasis on fruit and vegetable intake.

BACKGROUND/OBJECTIVES: The Child And Diet Evaluation Tool (CADET) is a 24-h food diary that measures the nutrition intake of children aged 3-7 years, with a focus on fruit and vegetable consumption. Until now CADET has not been used to measure nutrient intake of children aged 8-11 years. To ensure that newly assigned portion sizes for this older age group were valid, participants were asked to complete the CADET diary (the school and home food diary) concurrently with a 1-day weighed record. SUBJECTS/METHODS: A total of 67 children with a mean age of 9.3 years (s.d.: ± 1.4, 51% girls) participated in the study. Total fruit and vegetable intake in grams and other nutrients were extracted to compare the mean intakes from the CADET diary and Weighed record using t-tests and Pearson's r correlations. Bland-Altman analysis was also conducted to assess the agreement between the two methods. RESULTS: Correlations comparing the CADET diary to the weighed record were high for fruit, vegetables and combined fruit and vegetables (r=0.7). The results from the Bland-Altman plots revealed a mean difference of 54 g (95% confidence interval: -88, 152) for combined fruit and vegetables intake. CADET is the only tool recommended by the National Obesity Observatory that has been validated in a U.K. population and provides nutrient level data on children's diets. CONCLUSIONS: The results from this study conclude that CADET can provide high-quality nutrient data suitable for evaluating intervention studies now for children aged 3-11 years with a focus on fruit and vegetable intake.

Understanding the impacts of care farms on health and well-being of disadvantaged populations: a protocol of the Evaluating Community Orders (ECO) pilot study.

INTRODUCTION: Care farms, where all or part of the farm is used for therapeutic purposes, show much potential for improving the health and well-being of a range of disadvantaged groups. Studies to date have been qualitative or observational, with limited empirical evidence of the effectiveness of care farms in improving health and well-being. Understanding the underlying mechanisms that lead to improvements for different disadvantaged groups is a further gap in the evidence. Participants in this study are offenders serving community orders. Their low socioeconomic status and poor health outcomes relative to the general population exemplifies disadvantage. METHODS AND ANALYSIS: This paper describes the protocol of a study to understand the impacts of care farms and to pilot the design and tools for a study to assess cost-effectiveness of care farms in improving the quality of life of offenders. As a pilot study, no power calculation has been conducted. However, 150 offenders serving community sentences on care farms and 150 on other probation locations (eg, litter picking, painting) will be recruited over a 1-year period. Changes in quality of life, measured by Clinical Outcome in Routine Evaluation-Outcome Measure, health and reconvictions of offenders at care farms compared to other probation locations will be analysed to inform the sample size calculation for the follow on study. The feasibility of recruitment, retention, collecting cost data and modelling cost-effectiveness will also be assessed. The study will use qualitative methods to explore the experiences of offenders attending care farms and perceptions of probation and care farm staff on the processes and impacts of the intervention. ETHICS AND DISSEMINATION: Findings will be published and inform development of a natural experiment and will be disseminated to probation services, care farms and academics. University of Leeds Ethical Review Board approved: SoMREC/13/014. National Offender Management Service (NOMS) approved: 2013-257.

Dietary patterns derived with multiple methods from food diaries and breast cancer risk in the UK Dietary Cohort Consortium.

BACKGROUND/OBJECTIVES: In spite of several studies relating dietary patterns to breast cancer risk, evidence so far remains inconsistent. This study aimed to investigate associations of dietary patterns derived with three different methods with breast cancer risk. SUBJECTS/METHODS: The Mediterranean Diet Score (MDS), principal components analyses (PCA) and reduced rank regression (RRR) were used to derive dietary patterns in a case-control study of 610 breast cancer cases and 1891 matched controls within four UK cohort studies. Dietary intakes were collected prospectively using 4- to 7-day food diaries and resulting food consumption data were grouped into 42 food groups. Conditional logistic regression models were used to estimate odds ratios (ORs) for associations between pattern scores and breast cancer risk adjusting for relevant covariates. A separate model was fitted for post-menopausal women only. RESULTS: The MDS was not associated with breast cancer risk (OR comparing first tertile with third 1.20 (95% CI 0.92; 1.56)), nor the first PCA-derived dietary pattern, explaining 2.7% of variation of diet and characterized by cheese, crisps and savoury snacks, legumes, nuts and seeds (OR 1.18 (95% CI 0.91; 1.53)). The first RRR-derived pattern, a 'high-alcohol' pattern, was associated with a higher risk of breast cancer (OR 1.27; 95% CI 1.00; 1.62), which was most pronounced in post-menopausal women (OR 1.46 (95% CI 1.08; 1.98)). CONCLUSIONS: A 'high-alcohol' dietary pattern derived with RRR was associated with an increased breast cancer risk; no evidence of associations of other dietary patterns with breast cancer risk was observed in this study.