Identification of novel F2-isoprostane metabolites by specific UDP-glucuronosyltransferases.

UDP-glucuronosyltransferases (UGTs) catalyze the conjugation of glucuronic acid with endogenous and exogenous lipophilic small molecules to facilitate their inactivation and excretion from the body. This represents approximately 35 % of all phase II metabolic transformations. Fatty acids and their oxidized eicosanoid derivatives can be metabolized by UGTs. F2-isoprostanes (F2-IsoPs) are eicosanoids formed from the free radical oxidation of arachidonic acid. These molecules are potent vasoconstrictors and are widely used as biomarkers of endogenous oxidative damage. An increasing body of evidence demonstrates the efficacy of measuring the ß-oxidation metabolites of F2-IsoPs rather than the unmetabolized F2-IsoPs to quantify oxidative damage in certain settings. Yet, the metabolism of F2-IsoPs is incompletely understood. This study sought to identify and characterize novel phase II metabolites of 15-F2t-IsoP and 5-epi-5-F2t-IsoP, two abundantly produced F2-IsoPs, in human liver microsomes (HLM). Utilizing liquid chromatography-mass spectrometry, we demonstrated that glucuronide conjugates are the major metabolites of these F2-IsoPs in HLM. Further, we showed that these molecules are metabolized by specific UGT isoforms. 15-F2t-IsoP is metabolized by UGT1A3, 1A9, and 2B7, while 5-epi-5-F2t-IsoP is metabolized by UGT1A7, 1A9, and 2B7. We identified, for the first time, the formation of intact glucuronide F2-IsoPs in human urine and showed that F2-IsoP glucuronidation is reduced in people supplemented with eicosapentaenoic and docosahexaenoic acids for 12 weeks. These studies demonstrate that endogenous F2-IsoP levels can be modified by factors other than redox mechanisms.

Preoperative Treatment of Locally Advanced Rectal Cancer.

BACKGROUND: Pelvic radiation plus sensitizing chemotherapy with a fluoropyrimidine (chemoradiotherapy) before surgery is standard care for locally advanced rectal cancer in North America. Whether neoadjuvant chemotherapy with fluorouracil, leucovorin, and oxaliplatin (FOLFOX) can be used in lieu of chemoradiotherapy is uncertain. METHODS: We conducted a multicenter, unblinded, noninferiority, randomized trial of neoadjuvant FOLFOX (with chemoradiotherapy given only if the primary tumor decreased in size by <20% or if FOLFOX was discontinued because of side effects) as compared with chemoradiotherapy. Adults with rectal cancer that had been clinically staged as T2 node-positive, T3 node-negative, or T3 node-positive who were candidates for sphincter-sparing surgery were eligible to participate. The primary end point was disease-free survival. Noninferiority would be claimed if the upper limit of the two-sided 90.2% confidence interval of the hazard ratio for disease recurrence or death did not exceed 1.29. Secondary end points included overall survival, local recurrence (in a time-to-event analysis), complete pathological resection, complete response, and toxic effects. RESULTS: From June 2012 through December 2018, a total of 1194 patients underwent randomization and 1128 started treatment; among those who started treatment, 585 were in the FOLFOX group and 543 in the chemoradiotherapy group. At a median follow-up of 58 months, FOLFOX was noninferior to chemoradiotherapy for disease-free survival (hazard ratio for disease recurrence or death, 0.92; 90.2% confidence interval [CI], 0.74 to 1.14; P = 0.005 for noninferiority). Five-year disease-free survival was 80.8% (95% CI, 77.9 to 83.7) in the FOLFOX group and 78.6% (95% CI, 75.4 to 81.8) in the chemoradiotherapy group. The groups were similar with respect to overall survival (hazard ratio for death, 1.04; 95% CI, 0.74 to 1.44) and local recurrence (hazard ratio, 1.18; 95% CI, 0.44 to 3.16). In the FOLFOX group, 53 patients (9.1%) received preoperative chemoradiotherapy and 8 (1.4%) received postoperative chemoradiotherapy. CONCLUSIONS: In patients with locally advanced rectal cancer who were eligible for sphincter-sparing surgery, preoperative FOLFOX was noninferior to preoperative chemoradiotherapy with respect to disease-free survival. (Funded by the National Cancer Institute; PROSPECT ClinicalTrials.gov number, NCT01515787.).

Nucleus of the solitary tract A2 neurons control feeding behaviors via projections to the paraventricular hypothalamus.

Hindbrain NTS neurons are highly attuned to internal physiological and external environmental factors that contribute to the control of food intake but the relevant neural phenotypes and pathways remain elusive. Here, we investigated the role of NTS A2 neurons and their projections in the control of feeding behaviors. In male TH Cre rats, we first confirmed selective targeting of NTS A2 neurons and showed that chemogenetic stimulation of these neurons significantly suppressed dark cycle food intake, deprivation re-feed and high fat diet intake. Despite reducing intake, activation of NTS A2 neurons had no effect on food approach, anxiety-like behaviors, locomotor activity, blood glucose levels nor did it induce nausea/malaise, thus revealing a selective role for these neurons in the consummatory aspect of food intake control. Pathway-specific mapping and manipulation of NTS A2 neurons showed that these effects were mediated by NTS A2 neurons projecting to the paraventricular nucleus of the hypothalamus (PVH) because chemogenetic activation of these projections, but not projections to bed nucleus of the stria terminalis (BNST), reduced food intake. Cell-type specific analyses demonstrated that activation of NTS A2 neurons recruited both PVH oxytocin (OT)- and corticotropin-releasing factor (CRF)-expressing neurons, and plasma analyses showed increased plasma corticosterone following NTS A2 stimulation. While we also showed that chemogenetic inhibition of NTS A2 neurons attenuated the intake inhibitory effects of CCK, the specificity of transgene expression was low. Together, these findings showed that NTS A2 neurons are sufficient to control the consummatory aspects of feeding, regardless of energy status or food palatability and identified their projections to PVH, but not BNST, in food intake control.

Ca:Mg ratio, medium-chain fatty acids, and the gut microbiome.

BACKGROUND & AIMS: Ketogenic medium-chain fatty acids (MCFAs) with profound health benefits are commonly found in dairy products, palm kernel oil and coconut oil. We hypothesize that magnesium (Mg) supplementation leads to enhanced gut microbial production of MCFAs and, in turn, increased circulating MCFAs levels. METHODS: We tested this hypothesis in the Personalized Prevention of Colorectal Cancer Trial (PPCCT) (NCT01105169), a double-blind 2 × 2 factorial randomized controlled trial enrolling 240 participants. Six 24-h dietary recalls were performed for all participants at the baseline and during the intervention period. Based on the baseline 24-h dietary recalls, the Mg treatment used a personalized dose of Mg supplementation that would reduce the calcium (Ca): Mg intake ratio to around 2.3. We measured plasma MCFAs, sugars, ketone bodies and tricarboxylic acid cycle (TCA cycle) metabolites using the Metabolon's global Precision Metabolomics™ LC-MS platform. Whole-genome shotgun metagenomics (WGS) sequencing was performed to assess microbiota in stool samples, rectal swabs, and rectal biopsies. RESULTS: Personalized Mg treatment (mean dose 205.58 mg/day with a range from 77.25 to 389.55 mg/day) significantly increased the plasma levels of C7:0, C8:0, and combined C7:0 and C8:0 by 18.45%, 25.28%, and 24.20%, respectively, compared to 14.15%, 10.12%, and 12.62% decreases in the placebo arm. The effects remain significant after adjusting for age, sex, race and baseline level (P = 0.0126, P = 0.0162, and P = 0.0031, respectively) and FDR correction at 0.05 (q = 0.0324 for both C7:0 and C8:0). Mg treatment significantly reduced the plasma level of sucrose compared to the placebo arm (P = 0.0036 for multivariable-adjusted and P = 0.0216 for additional FDR correction model) whereas alterations in daily intakes of sucrose, fructose, glucose, maltose and C8:0 from baseline to the end of trial did not differ between two arms. Mediation analysis showed that combined C7:0 and C8:0 partially mediated the effects of Mg treatment on total and individual ketone bodies (P for indirect effect = 0.0045, 0.0043, and 0.03, respectively). The changes in plasma levels of C7:0 and C8:0 were significantly and positively correlated with the alterations in stool microbiome α diversity (r = 0.51, p = 0.0023 and r = 0.34, p = 0.0497, respectively) as well as in stool abundance for the signatures of MCFAs-related microbiota with acyl-ACP thioesterase gene producing C7:0 (r = 0.46, p = 0.0067) and C8:0 (r = 0.49, p = 0.003), respectively, following Mg treatment. CONCLUSIONS: Optimizing Ca:Mg intake ratios to around 2.3 through 12-week personalized Mg supplementation leads to increased circulating levels of MCFAs (i.e. C7:0 and C8:0), which is attributed to enhanced production from gut microbial fermentation and, maybe, sucrose consumption.

The Fish Oil to Reduce Tobacco Use iN Expectant mothers (FORTUNE) feasibility trial.

BACKGROUND: Three small clinical trials have suggested that supplementation with n-3 long-chain polyunsaturated fatty acids (eicosapentaenoic acid and docosahexaenoic acid) found in fish oils may reduce nicotine cravings and at higher doses reduce cigarette consumption. Pregnant women who smoke have fewer pharmacologic options to aid them with smoking cessation. Although n-3 long-chain polyunsaturated fatty acid supplementation has been studied in pregnancy, few studies have evaluated doses of ≥4 g per day, and no previous studies have selectively enrolled pregnant women who smoke. High-dose n-3 long-chain polyunsaturated fatty acids may aid cessation but could be poorly tolerated in pregnant women who smoke because of gastrointestinal side effects. OBJECTIVE: We conducted a feasibility trial to determine the tolerability of high-dose n-3 long-chain polyunsaturated fatty acid supplementation in pregnant women who smoked. We hypothesized that n-3 long-chain polyunsaturated fatty acid doses of 4.2 g a day would be well-tolerated relative to an olive oil placebo. We assessed red blood cell phospholipid membrane concentrations at baseline and end of therapy (4 weeks) and piloted outcomes for a future efficacy trial of n-3 long-chain polyunsaturated fatty acid supplementation for smoking cessation in pregnancy. STUDY DESIGN: We recruited 28 pregnant women between the gestational ages of 6 and 36 weeks who reported daily cigarette smoking and were motivated to quit to participate in a double-blind placebo-controlled randomized feasibility trial of 4.2 g per day of n-3 long-chain polyunsaturated fatty acid supplementation. Participants reported cigarettes per day, completed the Fagerström Test for Cigarette Dependence, and provided blood, urine, and exhaled CO samples. We used repeated-measures analysis of variance to pilot analyses of changes in cigarettes per day and Fagerström Test for Cigarette Dependence scores. RESULTS: At baseline, red blood cell membrane eicosapentaenoic acid concentrations were negatively correlated with cigarettes per day (r=-0.44; P=.04). By 4 weeks, circulating n-3 long-chain polyunsaturated fatty acid levels increased by 18% in the n-3 long-chain polyunsaturated fatty acid supplementation arm vs a decrease of 3% in the placebo arm. Occurrence of gastrointestinal side effects such as burping, heartburn, diarrhea, abdominal pain, or nausea did not differ statistically between study arms. At 4 weeks, participants allocated to the n-3 long-chain polyunsaturated fatty acids arm reported a median of 3 cigarettes per day (interquartile range, 1-8) vs 7 cigarettes per day (interquartile range, 1-14) in the placebo arm, which was not statistically significant (P=.99). Participants allocated to the n-3 long-chain polyunsaturated fatty acids arm had a decrease of 1 (interquartile range, 0-1) on the Fagerström Test for Cigarette Dependence score vs 0 (interquartile range, 0-0) for placebo (P=.46). CONCLUSION: High-dose n-3 long-chain polyunsaturated fatty acids may be tolerated in pregnant women who smoke; however, there was a high level of participant dropout, with more participants allocated to the fish oil arm becoming lost to follow-up. These results will inform the design of a future large-scale randomized controlled trial to test the impact of fish oil supplements on smoking cessation in pregnancy.

N-3 Long Chain Fatty Acids Supplementation, Fatty Acids Desaturase Activity, and Colorectal Cancer Risk: A Randomized Controlled Trial.

INTRODUCTION: n-3 long-chain polyunsaturated fatty acids (LCPUFA) have anti-inflammatory effects and may reduce colorectal cancer risk. The purpose of this study was to evaluate the effects of n-3 LCPUFA supplementation on markers of rectal cell proliferation and apoptosis and examine how genetic variation in desaturase enzymes might modify this effect. METHODS: We conducted a randomized, double-blind, control six-month trial of 2.5 grams of n-3 LCPUFA per day compared to olive oil. Study participants had a history of colorectal adenomas. Randomization was stratified based on the gene variant rs174535 in the fatty acid desaturase 1 enzyme (FADS1). Our primary outcome was change in markers of rectal epithelial proliferation and apoptosis. RESULTS: A total of 141 subjects were randomized. We found no difference in apoptosis markers between participants randomized to n-3 LCPUFA compared to olive oil (P = 0.41). N-3 LCPUFA supplementation increased cell proliferation in the lower colonic crypt compared to olive oil (P = 0.03) however baseline indexes of proliferation were different between the groups at randomization. We found no evidence that genotype modified the effect. CONCLUSIONS: Our study did not show evidence of a proliferative or pro-apoptotic effect on n-3 LCPUFA supplementation on rectal mucosa regardless of the FADS genotype.ClinicalTrials.gov Identifier: NCT01661764Supplemental data for this article is available online at https://dx.doi.org/10.1080/01635581.2021.1955286.

Investigating N-3 Fatty Acids to prevent Neonatal Tobacco-related outcomeS (INFANTS): study protocol for a double-blind, randomized, placebo-controlled parallel clinical trial of n-3 polyunsaturated fatty acids in pregnant smokers.

BACKGROUND: Tobacco use during pregnancy is the most important modifiable risk factor associated with adverse pregnancy outcomes, increasing the risk of preterm birth, intrauterine growth restriction and sudden infant death syndrome. Fewer than half of pregnant smokers can quit on their own. Identifying safe and effective therapies to prevent tobacco-related adverse pregnancy outcomes and/or increase smoking cessation in pregnant women would have a substantial public health impact. Cigarette smoking is associated with a relative deficiency in circulating n-3 long-chain polyunsaturated fatty acid (n-3 LCPUFA) levels. A recent analysis found that smokers taking n-3 LCPUFAs during pregnancy had a reduction in preterm labor risk when compared to non-smokers. Studies have shown that supplemental n-3 LCPUFAs may also reduce nicotine cravings and daily cigarette use. Thus, smokers may benefit from supplemental n-3 LCPUFAs by lowering the risk of preterm labor and/or increased smoking cessation. To address important remaining knowledge gaps, we propose the Investigating N-3 Fatty Acids to prevent Neonatal Tobacco related outcomeS (INFANTS). METHODS: The INFANTS study is a multicenter, randomized, double-blind, placebo-controlled study that will randomize 400 pregnant smokers to either supplemental n-3 LCPUFAs or placebo. Participants will be enrolled between 12 and 24 weeks' gestation and followed until 6 weeks after delivery. We will recruit from clinical centers throughout Middle Tennessee. We will assess smoking behavior after 12 weeks of supplementation using self-report and validated biomarkers of tobacco exposure. We will measure response to supplementation using biological markers of n-3 LCPUFA status. Our primary endpoint will be preterm labor as reflected by gestational age at delivery. Our secondary endpoint will be change from baseline in cigarettes per day at 12 weeks. DISCUSSION: This study tests the hypothesis that smoking-induced n-3 LCPUFA deficiencies contribute to tobacco-related adverse pregnancy outcomes and that supplementation of n-3 LCPUFAs in pregnant smokers may prevent these complications. If our study demonstrates that supplemental n-3 LCPUFAs are effective at reducing the risk of tobacco-related adverse neonatal outcomes and/or reducing tobacco use during pregnancy, our results could have an immediate and major impact on pregnancy care and neonatal outcomes. TRIAL REGISTRATION: ClinicalTrials.gov NCT04417595. Registered on April 21, 2020.

Pharmacogenetic study in gastric cancer patients treated with adjuvant fluorouracil/leucovorin or epirubicin/cisplatin/fluorouracil before and after chemoradiation on CALGB 80101 (Alliance).

There is a lack of pharmacogenetic predictors of outcome in gastric cancer patients. The aim of this study was to assess previously identified candidate genes associated with 5-fluorouracil (5-FU), cisplatin, or epirubicin toxicity or response in a cohort of resected gastric cancer patients treated on CALGB (Alliance) 80101. Gastric or gastroesophageal cancer patients randomized to adjuvant 5-FU/leucovorin or epirubicin/cisplatin/5-FU before and after 5-FU chemoradiation were genotyped for single nucleotide polymorphisms (SNPs) in GSTP1 (rs1695), ERCC1 (rs11615 and rs3212986), XRCC1 (rs25487), UGT2B7 (rs7439366) and the 28 base-pair tandem repeats in TYMS (rs34743033). Logistic regression and log rank tests were used to assess the association between each SNP and incidence of grade 3/4 neutropenia and leukopenia, overall (OS) and progression-free survival (PFS), respectively. Toxicity endpoint analyses were adjusted for the treatment arm, while OS and PFS were also adjusted for performance status, sex, age, lymph node involvement, and primary tumor site and size. Of 281 subjects with successful genotyping results and available clinical (toxicity and efficacy) data, 166 self-reported non-Hispanic White patients were included in the final analysis. There was a lack of evidence of an association among any SNPs tested with grade 3/4 neutropenia and leukopenia or OS and PFS. Age, lymph node involvement, and primary tumor size were significantly associated with OS and PFS. This study failed to confirm results of previous gastric cancer pharmacogenetic studies.

The changing incidence of childhood-onset type 1 diabetes in Wales: Effect of gender and season at diagnosis and birth.

AIMS: Determinants of the changing incidence of childhood-onset type 1 diabetes remain uncertain. We determined the recent time-trend of type 1 diabetes incidence in Wales and explored the role of vitamin D by evaluating the influence of season both at diagnosis and at birth. METHODS: Data from all Welsh paediatric units 1990-2019, and from primary care to determine ascertainment. RESULTS: Log-linear modelling indicated a non-linear secular trend in incidence with peak and subsequent decline. The peak occurred around June 2010: 31∙3 cases/year/100,000 children aged < 15y. It occurred earlier in children younger at diagnosis and earlier in boys. There were more cases in males aged <2y and >12y but more in females aged 9-10 y. More were diagnosed in winter. Also, children born in winter had less risk of future diabetes. CONCLUSIONS: The risk of developing type 1 diabetes before age 15y in Wales is no longer increasing. The data on season are consistent with a preventative role for vitamin D both during pregnancy and later childhood. Metereological Office data shows increasing hours of sunlight since 1980 likely to increase vitamin D levels with less diabetes. Additional dietary supplementation with vitamin D might further reduce the incidence of type 1 diabetes.

Evaluation of determinants for age disparities in the survival improvement of colon cancer: results from a cohort of more than 486,000 patients in the United States.

Over the past two decades, elderly colon cancer patients experienced less improvement in survival than their younger counterparts, yet the contributing factors remain unknown. We aimed to evaluate factors that may contribute to the age disparity of survival improvement among patients with colon cancer. Using data from the National Cancer Database, we identified patients diagnosed with colon cancer between 2004 and 2012 with follow-up data up to 2017. The hazard ratios (HR) and 95% confidence intervals (CI) for 5-year OS associated with study variables were estimated using multivariable Cox regression. Among 486,284 patients included in this study, elderly patients (aged ≥75) had a lower adherence to National Comprehensive Cancer Network (NCCN) treatment guidelines (% of non-adherence: 45.3%) than younger patients (aged <50, 19.3%; P<0.001). After adjusting for demographics, access to care and clinical characteristics, compared with patients diagnosed between 2004 and 2006, younger and older patients diagnosed between 2010 and 2012 experienced 16% (HR: 0.84, 95% CI: 0.81-0.88) and 6% (HR: 0.94, 95% CI: 0.93-0.95) reductions in mortality (P for interaction=1.42×10-5), respectively. After an additional adjustment for guideline adherence status, no significant difference in the improvement of survival was noted (P for interaction=0.17). The association patterns were similar regardless of tumor stage, race, and high comorbidity scores (all P for interaction>0.05). Several patient-related factors were identified in association with noncompliance to NCCN guidelines, including comorbidity status. However, over 60% of noncompliance elderly patients had a Charlson comorbidity score of 0. The observed age disparity in survival improvement among colon cancer patients was primarily explained by a slower improvement in adherence to NCCN treatment guidelines in elderly than younger patients. Many older adults were not receiving recommended therapies despite minimal comorbidities. Our findings call for measures to increase adherence to treatment guidelines among elderly patients to improve survival.

Mendelian Randomization Analysis of n-6 Polyunsaturated Fatty Acid Levels and Pancreatic Cancer Risk.

BACKGROUND: Whether circulating polyunsaturated fatty acid (PUFA) levels are associated with pancreatic cancer risk is uncertain. Mendelian randomization (MR) represents a study design using genetic instruments to better characterize the relationship between exposure and outcome. METHODS: We utilized data from genome-wide association studies within the Pancreatic Cancer Cohort Consortium and Pancreatic Cancer Case-Control Consortium, involving approximately 9,269 cases and 12,530 controls of European descent, to evaluate associations between pancreatic cancer risk and genetically predicted plasma n-6 PUFA levels. Conventional MR analyses were performed using individual-level and summary-level data. RESULTS: Using genetic instruments, we did not find evidence of associations between genetically predicted plasma n-6 PUFA levels and pancreatic cancer risk [estimates per one SD increase in each PUFA-specific weighted genetic score using summary statistics: linoleic acid odds ratio (OR) = 1.00, 95% confidence interval (CI) = 0.98-1.02; arachidonic acid OR = 1.00, 95% CI = 0.99-1.01; and dihomo-gamma-linolenic acid OR = 0.95, 95% CI = 0.87-1.02]. The OR estimates remained virtually unchanged after adjustment for covariates, using individual-level data or summary statistics, or stratification by age and sex. CONCLUSIONS: Our results suggest that variations of genetically determined plasma n-6 PUFA levels are not associated with pancreatic cancer risk. IMPACT: These results suggest that modifying n-6 PUFA levels through food sources or supplementation may not influence risk of pancreatic cancer.

Progressive Metabolic Dysfunction and Nutritional Variability Precedes Necrotizing Enterocolitis.

Necrotizing Enterocolitis (NEC) is associated with prematurity, enteral feedings, and enteral dysbiosis. Accordingly, we hypothesized that along with nutritional variability, metabolic dysfunction would be associated with NEC onset. Methods: We queried a multicenter longitudinal database that included 995 preterm infants (<32 weeks gestation) and included 73 cases of NEC. Dried blood spot samples were obtained on day of life 1, 7, 28, and 42. Metabolite data from each time point included 72 amino acid (AA) and acylcarnitine (AC) measures. Nutrition data were averaged at each of the same time points. Odds ratios and 95% confidence intervals were calculated using samples obtained prior to NEC diagnosis and adjusted for potential confounding variables. Nutritional and metabolic data were plotted longitudinally to determine relationship to NEC onset. Results: Day 1 analyte levels of alanine, phenylalanine, free carnitine, C16, arginine, C14:1/C16, and citrulline/phenylalanine were associated with the subsequent development of NEC. Over time, differences in individual analyte levels associated with NEC onset shifted from predominantly AAs at birth to predominantly ACs by day 42. Subjects who developed NEC received significantly lower weight-adjusted total calories (p < 0.001) overall, a trend that emerged by day of life 7 (p = 0.020), and persisted until day of life 28 (p < 0.001) and 42 (p < 0.001). Conclusion: Premature infants demonstrate metabolic differences at birth. Metabolite abnormalities progress in parallel to significant differences in nutritional delivery signifying metabolic dysfunction in premature newborns prior to NEC onset. These observations provide new insights to potential contributing pathophysiology of NEC and opportunity for clinical care-based prevention.

Ca:Mg Ratio, APOE Cytosine Modifications, and Cognitive Function: Results from a Randomized Trial.

BACKGROUND: Deterioration of ionized calcium (Ca2+) handling in neurons could lead to neurodegenerative disease. Magnesium (Mg) antagonizes Ca during many physiologic activities, including energy metabolism and catalyzation of demethylation from 5-methylcytosine(5-mC) to 5-hydroxymethylcytosine(5-hmC). OBJECTIVE: To test the hypothesis that actively reducing the Ca:Mg intake ratio in the diet through Mg supplementation improves cognitive function, and to test whether this effect is partially mediated by modified cytosines in Apolipoprotein E (APOE). METHODS: This study is nested within the Personalized Prevention of Colorectal Cancer Trial (PPCCT), a double-blind 2×2 factorial randomized controlled trial, which enrolled 250 participants from Vanderbilt University Medical Center. Target doses for both Mg and placebo arms were personalized. RESULTS: Among those aged > 65 years old who consumed a high Ca:Mg ratio diet, we found that reducing the Ca:Mg ratio to around 2.3 by personalized Mg supplementation significantly improved cognitive function by 9.1% (p = 0.03). We also found that reducing the Ca:Mg ratio significantly reduced 5-mC at the cg13496662 and cg06750524 sites only among those aged > 65 years old (p values = 0.02 and 0.03, respectively). Furthermore, the beneficial effect of reducing the Ca:Mg ratio on cognitive function in those aged over 65 years was partially mediated by reductions in 5-mC levels (i.e., cg13496662 and cg06750524) in APOE (p for indirect effect = 0.05). CONCLUSION: Our findings suggest that, among those age 65 and over with a high dietary Ca:Mg ratio, optimal Mg status may improve cognitive function partially through modifications in APOE methylation. These findings, if confirmed, have significant implications for the prevention of cognitive aging and Alzheimer's disease.Clinical Trial Registry number and website: #100106 https://clinicaltrials.gov/ct2/show/NCT03265483.

Dorsal vagal complex and hypothalamic glia differentially respond to leptin and energy balance dysregulation.

Previous studies identify a role for hypothalamic glia in energy balance regulation; however, a narrow hypothalamic focus provides an incomplete understanding of how glia throughout the brain respond to and regulate energy homeostasis. We examined the responses of glia in the dorsal vagal complex (DVC) to the adipokine leptin and high fat diet-induced obesity. DVC astrocytes functionally express the leptin receptor; in vivo pharmacological studies suggest that DVC astrocytes partly mediate the anorectic effects of leptin in lean but not diet-induced obese rats. Ex vivo calcium imaging indicated that these changes were related to a lower proportion of leptin-responsive cells in the DVC of obese versus lean animals. Finally, we investigated DVC microglia and astroglia responses to leptin and energy balance dysregulation in vivo: obesity decreased DVC astrogliosis, whereas the absence of leptin signaling in Zucker rats was associated with extensive astrogliosis in the DVC and decreased hypothalamic micro- and astrogliosis. These data uncover a novel functional heterogeneity of astrocytes in different brain nuclei of relevance to leptin signaling and energy balance regulation.

New Author Guidelines for Displaying Data and Reporting Data Analysis and Statistical Methods in Experimental Biology.

The American Society for Pharmacology and Experimental Therapeutics has revised the Instructions to Authors for Drug Metabolism and Disposition, Journal of Pharmacology and Experimental Therapeutics, and Molecular Pharmacology These revisions relate to data analysis (including statistical analysis) and reporting but do not tell investigators how to design and perform their experiments. Their overall focus is on greater granularity in the description of what has been done and found. Key recommendations include the need to differentiate between preplanned, hypothesis-testing, and exploratory experiments or studies; explanations of whether key elements of study design, such as sample size and choice of specific statistical tests, had been specified before any data were obtained or adapted thereafter; and explanations of whether any outliers (data points or entire experiments) were eliminated and when the rules for doing so had been defined. Variability should be described by S.D. or interquartile range, and precision should be described by confidence intervals; S.E. should not be used. P values should be used sparingly; in most cases, reporting differences or ratios (effect sizes) with their confidence intervals will be preferred. Depiction of data in figures should provide as much granularity as possible, e.g., by replacing bar graphs with scatter plots wherever feasible and violin or box-and-whisker plots when not. This editorial explains the revisions and the underlying scientific rationale. We believe that these revised guidelines will lead to a less biased and more transparent reporting of research findings.

Central leptin signaling transmits positive valence.

Hunger resulting from food deprivation is associated with negative affect. This is supported by recent evidence showing that hunger-sensitive neurons drive feeding through a negative valence teaching signal. However, the complementary hypothesis that hormonal signals of energy surfeit counteract this negative valence, or even transmit positive valence, has received less attention. The adipose-derived hormone leptin signals in proportion to fat mass, is an indicator of energy surplus, and reduces food intake. Here, we showed that centrally-delivered leptin reduced food intake and conditioned a place preference in food-restricted as well as ad libitum fed rats. In contrast, leptin did not reduce food intake nor condition a place preference in obese rats, likely due to leptin resistance. Despite a well-known role for hindbrain leptin receptor signaling in energy balance control, hindbrain leptin delivery did not condition a place preference in food-restricted rats, suggesting that leptin acting in midbrain or forebrain sites mediates place preference conditioning. Supporting the hypothesis that leptin signaling induces a positive affective state, leptin also decreased the threshold for ventral tegmental area brain stimulation reward. Together, these data suggest that leptin signaling is intrinsically preferred, and support the view that signals of energy surfeit are associated with positive affect. Harnessing the positive valence of signals such as leptin may attenuate the negative affect associated with hunger, providing a compelling new approach for weight loss maintenance.

Randomized Trial of Standard Adjuvant Chemotherapy Regimens Versus Capecitabine in Older Women With Early Breast Cancer: 10-Year Update of the CALGB 49907 Trial.

PURPOSE: Older women with breast cancer remain under-represented in clinical trials. The Cancer and Leukemia Group B 49907 trial focused on women age 65 years and older. We previously reported the primary analysis after a median follow-up of 2.4 years. Standard adjuvant chemotherapy showed significant improvements in recurrence-free survival (RFS) and overall survival compared with capecitabine. We now update results at a median follow-up of 11.4 years. PATIENTS AND METHODS: Patients age 65 years or older with early breast cancer were randomly assigned to either standard adjuvant chemotherapy (physician's choice of either cyclophosphamide, methotrexate, and fluorouracil or cyclophosphamide and doxorubicin) or capecitabine. An adaptive Bayesian design was used to determine sample size and test noninferiority of capecitabine. The primary end point was RFS. RESULTS: The design stopped accrual with 633 patients at its first sample size assessment. RFS remains significantly longer for patients treated with standard chemotherapy. At 10 years, in patients treated with standard chemotherapy versus capecitabine, the RFS rates were 56% and 50%, respectively (hazard ratio [HR], 0.80; P = .03); breast cancer-specific survival rates were 88% and 82%, respectively (HR, 0.62; P = .03); and overall survival rates were 62% and 56%, respectively (HR, 0.84; P = .16). With longer follow-up, standard chemotherapy remains superior to capecitabine among hormone receptor-negative patients (HR, 0.66; P = .02), but not among hormone receptor-positive patients (HR, 0.89; P = .43). Overall, 43.9% of patients have died (13.1% from breast cancer, 16.4% from causes other than breast cancer, and 14.1% from unknown causes). Second nonbreast cancers occurred in 14.1% of patients. CONCLUSION: With longer follow-up, RFS remains superior for standard adjuvant chemotherapy versus capecitabine, especially in patients with hormone receptor-negative disease. Competing risks in this older population dilute overall survival benefits.

Effects of fish oil supplementation on eicosanoid production in patients at higher risk for colorectal cancer.

Fish oil supplementation may represent a potential chemopreventive agent for reducing colorectal cancer risk. The mechanism of action of fish oil is unknown but presumed to be related to eicosanoid modification. The purpose of this study was to evaluate the effects of fish oil supplementation on the levels of urinary and rectal eicosanoids. We conducted a randomized, double-blind, controlled trial of 2.5 g of fish oil per day compared with olive oil supplementation over a 6-month period. Study participants had a history of colorectal adenomas. Randomization was stratified based on the gene variant rs174535 in the fatty acid desaturase 1 enzyme (FADS1), which affects tissue levels of arachidonic acid. A total of 141 participants were randomized. Urinary prostaglandin E2 metabolite (PGE-M) was measured at baseline, 3, and 6 months and rectal prostaglandin E2 (PGE2) at baseline and 6 months. Repeated-measures linear regression was used to determine the effect of the intervention on each outcome measure. Overall, fish oil supplementation was found to reduce urinary PGE-M production compared with olive oil (P=0.03). Fish oil did not reduce rectal PGE2 overall; however, it did significantly reduce PGE2 in the subgroup of participants not using aspirin or NSAIDs (P=0.04). FADS1 genotype did not seem to modify effects of fish oil on PGE2 production. We conclude that fish oil supplementation has a modest but beneficial effect on eicosanoids associated with colorectal carcinogenesis, particularly in those not taking aspirin or NSAIDs.

Assessing bioactivity-exposure profiles of fruit and vegetable extracts in the BioMAP profiling system.

The ToxCast program has generated in vitro screening data on over a thousand chemicals to assess potential disruption of important biological processes and assist in hazard identification and chemical testing prioritization. Few results have been reported for complex mixtures. To extend these ToxCast efforts to mixtures, we tested extracts from 30 organically grown fruits and vegetables in concentration-response in the BioMAP® assays. BioMAP systems use human primary cells primed with endogenous pathway activators to identify phenotypic perturbations related to proliferation, inflammation, immunomodulation, and tissue remodeling. Clustering of bioactivity profiles revealed separation of these produce extracts and ToxCast chemicals. Produce extracts elicited 87 assay endpoint responses per item compared to 20 per item for ToxCast chemicals. On a molar basis, the produce extracts were 10 to 50-fold less potent and when constrained to the maximum testing concentration of the ToxCast chemicals, the produce extracts did not show activity in as many assay endpoints. Using intake adjusted measures of dose, the bioactivity potential was higher for produce extracts than for agrichemicals, as expected based on the comparatively small amounts of agrichemical residues present on conventionally grown produce. The evaluation of BioMAP readouts and the dose responses for produce extracts showed qualitative and quantitative differences from results with single chemicals, highlighting challenges in the interpretation of bioactivity data and dose-response from complex mixtures.