bluuebunny/crossref_metadata_2025 huggingface dataset:
Identifying vulnerable steps in the coenzyme A biosynthesis pathway of M. tuberculosis
Authors: Mizrahi Valerie , Evans Joanna , Trujillo Carolina , Eoh Hyungjin , Ehrt Sabine , Schnappinger Dirk , Boshoff Helena , Rhee Kyu , Barry Clifton E.
Published: November 2023

Enzymes in the pantothenate and coenzyme A (CoA) biosynthesis pathways have attracted considerable interest as potential targets for the development of drugs against a number of human pathogens, including M. tuberculosis. While potent inhibitors have been developed against M. tuberculosis pantothenate synthase and pantothenate kinase, these have failed to translate into compounds with significant whole‐cell activity. These findings illustrate the challenges inherent in target‐led approaches to tuberculosis drug discovery, which is confounded by issues of permeability, metabolism and efflux, by a lack of understanding of target vulnerability.

To identify vulnerable steps in the pantothenate and CoA biosynthesis pathway in M. tuberculosis, a set of anhydrotetracycline (ATc)‐regulated mutants of M. tuberculosis was constructed using a transcriptional silencing system. The mutants were used to assess the impact of depletion of specific targets in the pantothenate and CoA pathway on growth and survival of the organism. Mutants with inducer‐dependent growth phenotypes were obtained for panB, panC, coaBC, coaD and coaE whereas those in panB, panE and panK were refractory to ATc‐regulated growth. While transcriptional silencing of panB, panC or coaE was bacteriostatic, coaBC silencing was apparently bactericidal in vitro, as determined by CFU enumeration. CoaBC was likewise shown to be required for growth and persistence of M. tuberculosis in vivo based on enumeration of bacillary loads in the lungs of mice in which coaBC was silenced immediately post‐infection, during acute infection, or during chronic infection. However, by taking advantage of the fact that M. tuberculosis is capable of CoaBC bypass via CoA salvage, coaBC silencing was shown to result in a transient, ‘non‐growing but metabolically active’ state from which non‐culturable bacilli can be rescued by uptake and assimilation of pantethine. These findings highlight the limitations of colony‐forming ability as a proxy for viability in M. tuberculosis. In further studies, the impact of silencing of these genes is being investigated by analysing changes in the metabolomic profile of M. tuberculosis with the aim of identifying similarities and differences in the metabolic response to target depletion at different steps of the pathway.

Support or Funding Information

This work was supported by grants from the TB Drug Accelerator program of the Bill and Melinda Gates Foundation (to C.E.B. III, D.S., K.R. and V.M.), the South African Medical Research Council, the National Research Foundation of South Africa and an International Research Scholar's grant from the HHMI (to V.M.) and, in part, by the Intramural Research Program of NIAID (H.B. and C.E.B. III).
Alkaloid Biosynthesis In Plants
Authors: O'Connor Sarah
Published: November 2023

Plants are– arguably– the world's best chemists. All plants synthesize thousands of complicated molecules that they use to protect themselves from predators, attract pollinators and communicate with other plants. Thousands of years ago, humans realized that many of these plant‐derived molecules also have a powerful impact on human health and well‐being. Advances in genomic and transcriptomic sequencing have rapidly advanced our understanding of the complex metabolic pathways that produce these high‐value chemicals. Here we show how transcriptomic and genomic data can be used to elucidate the alkaloid pathways of Madagascar Periwinkle, a medicinal plant that produces compounds that are used to treat a variety of cancers.

Support or Funding Information

BBSRC

ERC
Molecular Controls In Plant Hormone Signaling
Authors: Jez Joseph
Published: November 2023

Plants respond to developmental cues and abiotic and biotic stresses by controlling both the level and activity of phytoormones. Recent structural biology studies provide new insights on control mechanism before and after binding of auxin and jasmonates to their respective receptors. Pre‐receptor action by acyl acid amido synthetases of the GH3 family modulate hormone levels by amino acid conjugation. The first crystal structures of benzoate‐specific Arabidopsis thaliana AtGH3.12/PBS3 and jasmonic acid (JA)‐specific AtGH3.11/JAR1, combined with biochemical analysis, define features for the conjugation of amino acids to diverse acyl acid substrates and highlight the importance of conformational changes in the C‐terminal domain for catalysis. These results demonstrate how a highly adaptable three‐dimensional scaffold is used for the evolution of promiscuous activity across an enzyme family for modulation of plant signaling molecules. A second molecular control mechanism in auxin responses involves protein‐protein interactions mediated by the Phox/Bem1p (PB1) domains of Auxin Response Factors (ARF) and Aux/IAA co‐repressors to regulate transcriptional events modulated by the phytohormone auxin. These structures reveal a two‐pronged interaction and evolutionarily conservation of this core recognition feature, along with co‐varying interface sequences allows for versatile PB1‐mediated interactions in auxin signaling. Combined structural, functional, and sequence analysis provides new molecular insights on plant hormone response systems.
Determination of CYP1A2 Activity in the US Population by Use of Caffeine Metabolite Ratios in Spot Urine Samples: NHANES 2009–2010
Authors: Rybak Michael E. , Sternberg Maya R. , Pao Ching‐I , Pfeiffer Christine M.
Published: November 2023

The cytochrome P450 enzyme CYP1A2 is a majorphase I enzyme in the liver, accounting for nearly 15 % of P450 content. It isintegral in the metabolism of numerous drugs, the elimination of environmentaltoxins, and the activation of procarcinogens such as dietary aryl‐andheterocyclic amines, mycotoxins, and tobacco‐specific nitrosamines. Genetic, lifestyle, dietary and environmental factors can affect CYP1A2 activity. CYP1A2is also the most important enzyme in caffeine metabolism, catalyzing thedemethylation of caffeine and its subsequent metabolites. A metabolite ratio (MR) of these compounds in the urine, resulting from either a test dose or regular dietary intake of caffeine, can be used to determine CYP1A2 activity. In this study we described CYP1A2 activity in the US population ≥6 y by selected demographic, lifestyle and physiologic variables. We used spot urine concentrations of 5‐acetylamino‐6‐amino‐ 3‐methyluracil(AAMU), 1‐methyluric acid (1U), 1‐methylxanthine (1X), and 1,7‐ dimethyluricacid (17U) from the cross‐sectional NHANES 2009–2010 to calculate CYP1A2 activity by use of the MR [(AAMU+1U+1X)/17U]. We excluded individuals taking prescription medications known to interfere with caffeine metabolism and individuals with evidence of insufficient caffeine exposure to reliably calculate the CYP1A2 MR (n = 2197). We used Spearman correlation to examine the relation of CYP1A2 MR with continuous variables, and performed bivariate comparisons of unadjusted geometric mean (GM) CYP1A2 MRs for categorical variables. CYP1A2 activity was higher in males vs. females (Wald F P <0.0001) and highest in non‐Hispanic blacks, followed by non‐Hispanic whites, and lowest in Hispanics (Wald F P = 0.0002). An inverse relation was observed for CYP1A2 MR with age (Spearman ρ = −0.32, P <0.001). CYP1A2 activity was lower in individuals with evidence of inflammation (serum CRP ≥5 mg/L vs. <5 mg/L; Wald F P <0.0001) and in diabetics(yes vs. no based on a physician's diagnosis; Wald F P = 0.0221). GM CYP1A2 MR was lower in non‐smokers vs. smokers(i.e. serum cotinine ≤10 ng/mL vs. >10 ng/mL) (Wald F P <0.0001), and no association was observed with alcohol consumption (limited to individuals ≥20 y). Our associations of CYP1A2 activity with age, sex, race‐ethnicity, smoking status, and inflammation were generally consistent with observations in other studies. We believe our study is the first report of using urine caffeine MRs in spot urine samples to estimate CYP1A2 activity in a representative sample of the US population.
Altered fatty acid metabolism remodels the hypothalamic metabolome to stimulate feeding behavior in BCO2 knockout mice
Authors: Guo Xin , Wu Lei , Soh Traces , Chowanadisai Winyoo , Clarke Stephen , Lucas Edralin , Smith Brenda , Wang Weiqun , Medeiros Denis , Lin Dingbo
Published: November 2023

Objective

β,β‐carotene‐9′,10′‐oxygenase2 (BCO2) is a carotenoid cleavage enzyme localized to the inner membrane of mitochondria and is broadly expressed in many tissues, such as liver and hypothalamus. We recently found that systemic depletion of BCO2 impaired lipid metabolism in a mouse model of diet‐induced obesity. The aim of this study is to explore the mechanism by which BCO2‐regulated lipid metabolism impacts hypothalamic metabolome and subsequent stimulation of feeding behavior.

Methods

After weaning, male BCO2 knockout (KO) and wild type (WT, 129S6) mice were fed a standard chow for 3 weeks. Food intake, body weight, and fasting blood glucose level were monitored on a weekly basis. Gluconeogenesis was examined via a pyruvate tolerance test. At the end of study, mice were sacrificed after a 3 hour fast. Blood and hypothalamic tissues were collected. Plasma biomarkers of lipid metabolism were tested, including cholesterol, lipoproteins, triglycerides (TG), and nonesterified fatty acids (NEFA). Fresh hypothalamic mitochondria were isolated and subjected to mitochondrial respiratory activity assays. Metabolomics profiling assay on hypothalamic tissues was conducted. Western blot was employed to determine altered lipid metabolism and energy homeostasis.

Results

Average daily food intake was significantly increased (14 %, p<0.01) in the KO mice compared to the WT controls. The KO mice also exhibited a higher (p<0.05) fasting NEFA and blood glucose compared to the WT mice. However, the body weight of the KO mice was reduced (p<0.05) compared to age and gender‐matched WT mice. The KO mice developed pyruvate intolerance. Enhanced expression of hypothalamic acetyl‐CoA carboxylases, peroxisome proliferator‐activated receptor α, and forkhead box O1, and suppression of AMP‐activated protein kinase confirmed impaired lipid metabolism and disturbed energy homeostasis in KO mice. Hypothalamic metabolomics results showed decreases in oleate and acyl‐carnitine contents, suppression of long chain fatty acid transport into mitochondria, and inhibition of bile acid biosynthesis in KO mice. The mitochondrial respiratory capacity of complex II was significantly decreased in the hypothalamus of KO mice.

Conclusions

BCO2 may exert a role in regulation of hypothalamic mitochondrial metabolism. Ablation of BCO2 results in decreased fatty acid oxidation, impaired lipid sensing, and potentiated gluconeogenesis
Dietary Glycine Alters One‐Carbon Metabolic kinetics in vivo
Authors: Huang Yu‐Hsuan , Chiang En‐Pei Isabel
Published: November 2023

Folate‐dependent one‐carbon metabolism is required for nucleotide biosynthesis and for the remethylation of homocysteine to methionine. Glycine‐N methyltransferase (GNMT) is a major hepatic enzyme that converts S‐adenosylmethionine to S‐adenosylhomocysteine while generating sarcosine from glycine, hence it can regulate mediating methyl group availability in mammalian cells. The metabolic role of GNMT in folate‐mediated one‐carbon metabolism and how glycine supply may affect one carbon metabolic kinetics are not fully understood. In the present study, we investigated how GNMT expression may affect cellular one carbon flow under various glycine conditions. Hepatocyte‐derived cell‐lines with and without GNMT expression were cultured in minimum essential medium (MEM) with and without high glycine supply. Methylene‐tetrahydrofolate (mTHF) utilization and endogenous formate biosynthesis were carefully investigated using stable isotopic tracers and GC‐MS. We discovered that high glycine inhibits the M+1 specie enrichments in deoxythymidine (dT+1) from 2,3,3 D3‐serine, suggesting that high glycine may inhibit endogenous formate production in these in vitro models. In a parallel experiment that exogenous formate was used as the 1C source, enrichments in dT+1 significantly increased from exogenous formate, supporting our hypothesis that high glycine inhibits endogenous format production for thymidine synthesis. On the other hand, high glycine promotes M+1 specie enrichments in methionine from 2,3,3 D3‐serine, indicating that high glycine supply may favor the utilization of methyleneTHF derived one carbon for homocysteine remethylation. High glycine supply can promote folate dependent methionine synthesis in our cell models. Furthermore, our preliminary in vivo experiments demonstrated that high dietary glycine can promote folate dependent methionine synthesis, consistent with our in vitro findings. These results showed that high dietary glycine promotes methionine remethylation in vitro and in vivo.

Support or Funding Information

supported by MOST 104‐2320‐B005‐010‐MY3; MOST104‐2911‐I005‐301; and Ministry of Education, Taiwan, R.O.C. under the ATU plan
Vitamin B12 Deficiency Defined Using Total Cobalamin, Holo‐Transcobalamin II, and an Algorithm of Both Total Cobalamin and Holo‐Transcobalamin II in Vegetarians, Vegans, and Omnivores
Authors: Arnold Taylor , Knurick Jessica , Johnston Carol
Published: November 2023

Vitamin B12 (cobalamin) is a water‐soluble vitamin essential for purine and pyrimidine synthesis, DNA methylation, and the myelination of nerve cells. As vitamin B12 is only found in animal products, vegetarians and vegans are at an inherently higher risk of developing a deficiency than the omnivorous population. The standard measures used to evaluate vitamin B12 nutriture in the clinical setting include total cobalamin, as well as homocysteine and methylmalonic acid (MMA). Each of these markers have flaws, however, as total cobalamin includes biologically inactive cobalamin, homocysteine can be influenced by folate status, and MMA is not present in significant amounts when vitamin B12 nutriture is adequate or excessive. Because of this, holo‐transcobalamin II (TCII), which is the carrier of biologically active vitamin B12, is becoming increasingly popular in research settings to accurately detect cobalamin status. Consequently, there continues to be a discrepancy between the standard analysis methods used in the clinical and research settings. In this study, we sought to compare deficiency prevalence in omnivores, lacto‐ovo vegetarians, and vegans using three different definitions of vitamin B12 deficiency: serum B12 < 148 pmol/L, serum TCII < 35 pmol/L, and serum B12 < 243 pmol/L + TCII < 35 pmol/L. To accomplish this, we analyzed blood samples of 82 subjects between the ages of 19 and 50 years old (27 omnivores, 27 lacto‐ovo vegetarians, and 28 vegans). Chi squared tests revealed significant differences in deficiency detection across the three deficiency definitions (p=0.046). In particular, there was a significant difference in the frequency of deficiency detected using serum B12 (4.9%) and serum TCII (17.1%) (p=0.025) in the total study population. The prevalence of deficiency detected when using serum TCII, rather than serum total cobalamin, appeared to increase more in lacto‐ovo vegetarians (from 7.4% to 25.9%) and vegans (from 3.6% to 17.9%) than in omnivores (from 2.7% to 7.4%), although this difference was not significant. Changing the clinical standard to detect vitamin B12 deficiency may improve deficiency detection, particularly at early onset. In addition, using TCII to detect deficiency cases may improve detection of marginal cases, which can allow practitioners to treat individuals who are deficient before symptoms progress. Future research is needed to examine the feasibility of using TCII in a clinical setting.

Support or Funding Information

Graduate and Professional Student Association, Arizona State University
Food Insecurity is Associated with Depression and Stress Among a Cohort of Pregnant Kenyan Women of Mixed HIV Status
Authors: Tsai Irene , Krumdieck Natalie R. , Collins Shalean , Widen Elizabeth M. , Wekesa Pauline , Onono Maricianah , Young Sera L.
Published: November 2023

Food insecurity (FI) is well established to be harmful among children, adolescents, and adults, but little is known about either its prevalence or covariates during pregnancy. Given the importance of maternal health for the lifelong well‐being of children, a greater understanding of FI may provide insight into potentially modifiable determinants of maternal and child health. Therefore, we examined (1) the population prevalence of food insecurity among all pregnant women attending 7 urban and rural antenatal clinics in Nyanza province, Kenya, and (2) assessed the predictors of FI at baseline among women enrolled in Pith Moromo, an ongoing observational pregnancy cohort study in this region.

Cohort participants (n=363) were enrolled at <30 weeks gestational age, and were purposively sampled to achieve equal proportions of HIV‐infected and –uninfected women across low, moderate, and severe tertiles of food insecurity. FI was measured by clinic‐based study staff using an individually focused food insecurity access scale (IFIAS, Natamba et al., 2014). Staff assessed depression using the Center for Epidemiologic Studies Depression Scale (CES‐D), and assessed stress using the Perceived Stress Scale (PSS). An asset score was created by assessing patterns of asset ownership, and weighted using principal component analysis. Linear regression was used to evaluate predictors of FI at enrollment into the cohort, including maternal age, gestational age, HIV status, marital status, perceived stress, and urban/peri‐urban/rural locality. In the population‐based sample (n=1,188), the mean score for FI was 12.9 (SD 7.4), 39.44%, 34.54%, and 26.01% of women were in the low, moderate, and severe FI groups. Among participants in the cohort, the mean score for FI was 13.6 (SD 7.1). In multivariate linear models among cohort participants, CES‐D score was positively associated with FI (β 0.11, 95% confidence interval 0.00 to 0.21, p<0.05), and greater perceived stress was also associated with higher FI (β 0.19 score, 95% confidence interval 0.00 to 0.39, p<0.05). Greater asset index scores were associated with lower FI (β −1.43, 95% confidence interval −2.02 to −0.85, p<0.001). Maternal HIV and marital status were not significantly associated with FI.

In sum, FI is experienced by many pregnant women in this region. In our cohort, we observed significant positive correlations between FI and psychosocial indicators, including probable maternal depression and perceived stress. Understanding the relationships between mental health and food insecurity may help inform the development of psychosocial health interventions to improve mental health and/or food security among pregnant women.

Support or Funding Information

Support: SLY NIH K01 MH098902
ZnT2‐Mediated Zn Transport Is Required for Vesicle Acidification And Milk Secretion during Lactation
Authors: Lee Sooyeon , Rivera Olivia C , Kelleher Shannon L
Published: November 2023

Successful lactation requires optimal regulation of mammary gland differentiation, milk secretion and milk composition. Recently, we determined that the loss of the vesicular zinc (Zn) transporter ZnT2 impairs mammary gland differentiation, reduces milk secretion and leads to suboptimal milk composition in lactating mice. In this study, we aimed to determine how the loss of ZnT2 compromises milk secretion during lactation. We hypothesized that ZnT2‐mediated Zn transport regulates vesicle acidification, which is critical for vesicle trafficking and secretion. First, we studied the role of ZnT2 in vesicle trafficking in mammary epithelial cells (MECs) in vitro. We found that while ZnT2‐expressing cells successfully trafficked beta‐casein‐containing vesicles toward the membrane in response to the lactogenic hormone prolactin, beta‐casein‐containing vesicles were retained in the perinuclear region in ZnT2‐attenuated MECs, suggesting that ZnT2 is important for vesicle trafficking and secretion. Vesicle trafficking and secretion in neuronal and pancreatic cells requires vesicle acidification through the vacuolar ATPase (V‐ATPase) proton pump, which generates a vesicular pH gradient that permits trafficking and exocytosis of secretory components. To test the hypothesis that ZnT2 regulates vesicle acidification, we first determined if ZnT2 and V‐ATPase interact, and determined the localization of V‐ATPase using immunofluorescence in mouse mammary glands. We found that the cytoplasmic subunit V1 domain was localized to the apical membrane of MECs, indicating active vesicular secretion. In contrast, the V1 domain remained cytoplasmic in the MECs of ZnT2‐null mice. We next measured vesicular pH using the fluorescent ratiometric probe Lysosensor DND‐160 in isolated MECs. We found that ZnT2‐attenuated MECs failed to acidify vesicles (pH 6.0) while acidification was augmented in ZnT2‐overexpressing cells (pH 4.9) compared to mock‐transfected cells (pH 5.5). Importantly, we found that ZnT2 is directly associated with both the cytoplasmic V1 and the membrane‐bound V0 domains of V‐ATPase, suggesting a role for ZnT2 in V‐ATPase complex assembly. Taken together, these results establish that ZnT2 is critical for V‐ATPase complex assembly, which is coupled to proton gradient formation to acidify vesicles for milk secretion.

Support or Funding Information

Intramural funds to SLK
Acidic Human Milk Oligosaccharides Vary Across Populations and Over Lactation
Authors: Newburg David S , Chen Ceng , Cline Allison , Morrow Ardythe L
Published: November 2023

Background

Human milk oligosaccharides (HMOS) inhibit pathogens, promote colonization through a prebiotic effect, and inhibit inflammation of the intestinal mucosa. The content of HMOS can vary qualitatively and quantitatively among individuals and over the course of lactation.

Objective

To compare the acidic HMOS composition of milk from mothers in Cincinnati, OH, Shanghai, China, and Mexico City, Mexico.

Methods

A total of 120 mothers per site were included, and 760 milk samples were analyzed by LC‐MS as part of the Global Exploration of Human Milk (GEHM) study. Eligibility required healthy, singleton, term delivery and > 75% of feedings from breast milk for at least 3 months. Milk samples were collected between 9AM–1PM at 2, 4, 13 and 26 wks postpartum using Medela Symphony breast pumps, and represented the contents of one breast that was not used for feeding for two hours prior to pumping.

Results

Total oligosaccharides decreased over lactation, from 5.8 (week 2) to 2.8 g/L (week 26, p<0.001). However, the most rapid decline in HMOS occurred in the acidic oligosaccharide fraction, from 2.4 (week 2) to 0.4 g/L (week 26, p<0.001). Milk from Shanghai was significantly lower than the other two sites in total HMOS (at 1 month: 4.1 vs 5.1 g/L, p<0.001). The most abundant individual acidic HMOS across the populations were 3′‐sialyllactose (3′SL), 6′‐sialyllactose (6′SL), and disialyllacto‐N‐tetraose (DSLNT). Comparing populations, 3′SL was higher (p<0.001), 6′SL was lower (p<0.001), and DSLNT was lower (p<0.001) in Mexican than Cincinnati or Shanghai mothers. The concentrations of 6′SL and DSLNT, but not of 3′SL, declined over the first 26 weeks of lactation.

Conclusions

Concentrations of acidic HMOS differ among populations, and change over the course of lactation. The biological ramifications of these differences warrant investigation.

Support or Funding Information

Supported in part by HD013021, HD059140, and Mead‐Johnson
Term Infant Formula Supplemented with Human Milk Oligosaccharides (2′Fucosyllactose and Lacto‐N‐neotetraose) Shifts Stool Microbiota and Metabolic Signatures Closer to that of Breastfed Infants
Authors: Steenhout Philippe , Sperisen Peter , Martin François‐Pierre , Sprenger Norbert , Wernimont Susan , Pecquet Sophie , Berger Bernard
Published: November 2023

Human milk oligosaccharides (HMOs) may provide benefits to formula‐fed infants in part by influencing the establishment of the intestinal microbiota. We evaluated effects of infant formula with 2 HMOs (2′Fucosyllactose [2′FL] and Lacto‐N‐neotetraose [LNnT]) on the early intestinal microbiota in infants (ClinicalTrials.gov NCT01715246).

Healthy term infants ages 0–14 days were randomly assigned to intact protein cow's milk‐based infant formula (Control, n 87) or the same formula with 1.0 g/L 2′FL and 0.5 g/L LNnT (Test, n 88); a reference group of breastfed infants was also included (n 38). Stool samples were collected at age 3 months for assessment of microbiota using 16S rRNA gene sequencing and metagenomics; metabolic signature was assessed using proton NMR‐based metabolite profiling.

The global average microbial composition profile, established for infants with available stool samples who had adhered to the study protocol, showed a similar pattern for Control (n 65) and Test (n 58) at the genus level, although Test was closer to breastfed (n 34) than Control. Calculation of microbial alpha diversity and comparison of the global microbiota composition using random permutations of the redundancy analysis (RDA) confirmed that Test was different from Control at the genus level (p<0.001) and closer to the breastfed group. Statistical analyses identified several taxa differentially present in Control and Test: Bifidobacterium (p=0.01), Escherichia (p=0.008), unclassified Coprobacillaceae (p=0.01), unclassified Peptostreptococcaceae (p=0.026), Dorea (p=0.033), and Megamonas (p=0.035). Correction for False Discovery Rate confirmed the first three observations. Main discriminants between Test and Control by random forest analysis were Bifidobacterium, Escherichia and Peptostreptococcaceae. Clinically relevant pathogens were rare; however, Clostridium difficile toxin A/B was detected in 14% of Test and 26% of Control (OR 0.47, CI 0.17–1.27, p=0.15). Multivariate analysis identified phenylalanine, isoleucine, tyrosine, fecal organic acids and fucosylated compounds as influential metabolites that discriminated between the Test, Control and breastfed groups. Globally, the values observed in Test were more similar to those observed in the breastfed group compared with Control, a finding that may indicate reduced protein fermentation.

Together these findings indicate that the addition of 2′FL and LNnT to a starter infant formula shifts the stool microbiota and metabolic signature towards those observed in breastfed infants, both in several aspects of composition and function. Further studies are warranted to evaluate whether such a shift in gut ecology towards the breastfed standard leads to health benefits.

Support or Funding Information

The study was funded by Nestec S.A.
Metabolic Phenotype and Increased Adiposity in a Mouse Model of Chronic Gestational Alcohol Exposure
Authors: Amos‐Kroohs Robyn M , Nelson David W , Yen Chi‐Liang Eric , Smith Susan M
Published: November 2023

Fetal Alcohol Spectrum Disorder (FASD) is the largest preventable cause of neurodevelopmental disability and recent studies suggest that prenatal alcohol exposure (PAE) also increases obesity risk at adolescence. Children with FASD exhibit hyperphagia and have inappropriate dietary intake which may contribute to increased obesity risk. We developed a mouse model of gestational binge alcohol exposure (PAE) to delineate the contributions of metabolic dysregulation, eating behavior and diet response in offspring to increased obesity risk. Pregnant C57Bl/6J females were gavaged with 3 g/kg alcohol (EtOH) daily from gestational day (GD) 12.5 to 17.5 in two half doses, two hours apart. Isocaloric maltodextrin (MD) and medium chain triglycerides (MCT) were included as nutritional controls. A sham (H2O) treatment was used to control for gavage stress. At 17 weeks, sex matched pairs from each litter were investigated for the following endpoints: obesity via dual‐energy X‐ray absorptiometry (DXA), carbohydrate metabolism using glucose tolerance testing (GTT), blood pressure and heart rate using arterial probes as well as indirect calorimetry in metabolic cages. Although lighter during lactation, EtOH pups gained significantly more weight post‐weaning than MD and MCT groups (p<0.05). EtOH offspring have increased fat mass compared with other groups at 17 weeks (p<0.05) by DXA. Male EtOH, MD, and MCT offspring exhibited increased basal glucose levels and significantly increased tolerance during GTT in comparison with H2O offspring (p<0.05) despite similar weights between groups. EtOH offspring have increased systolic blood pressure (p<0.05) and heart rate (p<0.05). In metabolic chambers, all offspring were challenged with high sugar (70%) and high fat (60%) diets. Metabolic parameters such as food intake, VO2, and respiratory exchange ratio were similar between groups, although slight differences were found in feeding behavior patterns and food intake during diet challenges between EtOH and all control groups. After a month on high fat diet, male EtOH offspring have exacerbated weight and fat mass gain (p<0.05). After a month on high fat diet, female EtOH offspring exhibit significantly increased basal glucose levels (p>0.05) and increased glucose tolerance during GTT. Taken together, these data suggest that PAE may be associated with a metabolic syndrome. Up to 1 in 3 women report drinking during pregnancy, so PAE may be a previously unrecognized factor for increased obesity risk in the context of an obesogenic environment.

Support or Funding Information

AA22999. AA11085, 2T32DK007665
Effects of DHA & Choline Supplementation on Developmental Outcomes of the Typically Developing and Fetal Alcohol Exposed Rat Pup
Authors: Canipe L Grant , Jackson Thomas W , Cheatham Carol L
Published: November 2023

Objective

Fetal Alcohol Exposure (FAE) causes a range of neurodevelopmentally abnormal phenotypes. Effects on hippocampus and frontal lobe, areas that preferentially accrete docosahexaenoic acid (DHA), are seen consistently, possibly due to changes to the epigenome during gestation. DHA and choline play an integral role in neural development and subsequent cognitive function, but their effects are generally studied in isolation. Recent work in our lab has shown that DHA and choline act together in support of infant and toddler cognition. Thus, a nutritional intervention using a combination of DHA and choline may have promise for the amelioration of FAE. The primary goal of this study was to examine the effects of DHA and choline supplementation on development in typically‐developing and alcohol‐exposed pups. The secondary goal was to evaluate the feasibility of DHA and choline as a prenatal supplement for rat dams by designing a supplemented liquid‐ethanol diet.

Methods

Rat dams were randomly assigned to four treatment groups, with EtOH and/or DHA & choline (S) (EtOH, EtOH‐S, pair‐fed, and pair‐fed‐S), and a fifth group added as ad libitum control. The experimental diet was administered from gestational day 5 to 20. Diet was isocaloric (~100 cal/day/rat) with 35% of daily calories supplied by either EtOH or maltose dextrin. Dams were supplemented daily with DHA (10.8 mg) and choline (155 mg). Food intake was measured for the EtOH‐fed dams; weight‐matched dams were fed this amount (pair‐fed). On postnatal day 2 through 21, markers for development were assessed on bar‐holding and negative geotaxis in pups (n=65). Days to first success (latency) on each test were used as a measure of development.

Results

We successfully developed a palatable DHA‐ and choline‐supplemented liquid diet. PROC GLM (SAS Ver 9.4) was used to test group differences. FAE resulted in significantly longer latency to success on bar‐holding in EtOH compared to pair‐fed and control animals (p<0.001). There were no significant differences in latencies on either task between the EtOH‐S group and pair‐fed‐S. The EtOH‐S group had significantly shorter latency on both tasks than did the EtOH group (all p<0.001). Finally, there were no significant differences between the EtOH‐S group and the ad libitum control (p<0.10 with the trend in favor of EtOH‐S), and both performed significantly better than the EtOH group (p<0.01).

Conclusion

The mitigating effect of DHA and choline supplementation on FAE shows promise and warrants further investigation. The addition of an ad libitum supplemented group is needed to strengthen our conclusion. However, the data suggest a positive developmental impact of DHA & choline supplementation on the typically‐developing and alcohol‐exposed fetus.

Support or Funding Information

NIAAA K01‐AA021134
Intake of Genistein and Daidzein Ameliorates Adiposity and Metabolic Syndrome in High Fat Fed C57BL/6J Mice
Authors: Luo Ting , Miranda Omar , Adamson Allysa , Shay Neil F
Published: November 2023

Introduction

Genistein and daidzein are isoflavones abundant in soybean. These isoflavones have been shown to have a weak but significant estrogenic effect and other studies have shown isoflavones may impact cancer and metabolic disease. Our prior research (Luo et al. 2014) indicated metabolic improvements in male mice fed a high‐fat (HF) diet supplemented with Novasoy, a dietary supplement containing a mixture of soy isoflavones. With two related, but slightly different chemical structures, it is plausible that dietary genistein and daidzein influence metabolism differently.

Objective

The objective of the present study was to determine if diet supplementation with genistein (G) or daidzein (D) would have a differential impact on body weight, body composition, and other physiologic and metabolic parameters in high‐fat fed male C57BL/6J mice.

Methods

C57BL/6J mice (n=8 per group) were provided either low‐fat diet (LF, 10% kcal fat), high‐fat diet (HF, 45% kcal fat), or HF plus ~0.2% of either G or D for 10 weeks. Dietary levels of isoflavones are equivalent to intake of dietary supplements in humans. Body weight was recorded weekly and food intake bi‐weekly.

Results

Dietary supplementation with G or D led to decreased body weight compared to HF‐fed mice. Furthermore, body weight gain in G‐fed mice was profoundly impacted and lower in value vs. all groups including LF‐fed mice, weight gains were as follows: LF: 7.0 ± 0.6 g, HF: 18.6 ± 1.0 g, G: 2.9 ± 0.7 g, D: 11.9 ± 1.4 g. In glucose tolerance testing, both G and D intake were associated with a trend of decreased area under the curve (AUC), consistent with improved glucose sensitivity vs. HF‐fed mice. Serum insulin levels were consistent with this finding, with concentrations in G‐fed mice significantly lower than HF‐fed and equivalent to LF‐fed mice (P < 0.01). Serum resistin concentrations showed a trend of lower levels vs. HF‐fed mice in G‐ and D‐fed mice (P < 0.01). Serum MCP‐1 levels were measured by ELISA and G‐fed mice had trended to lower MCP‐1 levels compared to HF‐ and D‐fed mice (P < 0.10). Liver tissue sections were evaluated for fat content and relative fat content was LF: 9.0 ± 1.7 %, HF: 30.8 ± 3.8 %, G: 15 ± 3.7 %, D: 30 ± 6.3 %. Hepatic gene expression levels were measured by RT‐PCR, and G‐fed mice had higher levels of several genes related to lipid metabolism including stearoyl‐CoA desaturase‐1 and sterol regulatory element‐binding protein 1a.

Conclusion

The profound impact of genistein intake on weight gain was unanticipated and needs to be further explored to determine if it may be of use as a dietary supplement to help promote weight loss or to help improve glucose sensitivity.

Support or Funding Information

Oregon Agricultural Experiment Station
Adherence to a Mediterranean Diet, and Effects on Cardiometabolic Health in Older Australians; Results from the Mediterranean Diet for Cognition and Cardiovascular Health in the Elderly (MedLey) Trial
Authors: Davis Courtney Rose , Bryan Janet , Wilson Carlene , Hodgson Jonathan , Woodman Richard , Murphy Karen
Published: November 2023

The MedLey study aimed to assess the impact of adherence to the Mediterranean diet (MedDiet) for six months on cardiometabolic health indicators in a population of older Australian adults. We recruited 166 healthy male and female (57% female) adults aged 65 years or older. Volunteers were randomly allocated (stratified on gender, age and BMI) into either a control group (habitual diet) (n=81) or the MedDiet group (n=85). We measured adherence using a 15‐point MedDiet adherence score (higher scores reflect higher adherence) based on dietary intake data collected using 3‐day weighed food records completed at baseline, two and four months. Cardiometabolic measures included fasting plasma lipids, high‐sensitivity C‐reactive protein (hs‐CRP), F2‐isoprostanes, glucose, insulin and blood pressure (not presented here). These were measured at baseline and after three and six months. We used a simple linear mixed effects model to compare groups over time.

Seventy volunteers in the MedDiet group and 67 in the HabDiet group completed the study and were included in the analysis. Adherence scores at baseline were 7.1 and 7.4 for the MedDiet and HabDiet groups, respectively (P for between group difference =0.424). Adherence in the MedDiet group significantly increased by 2.1 (SEM 0.37) points at two months and 2.6 (SEM 0.37) points at four months relative to the control (P <0.001). There was an overall significant effect of diet over the 6 months on triglycerides and plasma F2‐isoprostanes (P<0.05 for between group interaction for both). In the MedDiet group, compared to the HabDiet group, after adjustment for baseline values, mean (SEM) triglyceride concentrations were significantly lower at three (−0.149 (0.04) mmol/L, P<0.001) and six months (−0.094 (0.04) mmol/L, P<0.05), and F2‐isoprostane concentrations were significantly lower at three (−103 (25.9) pmol/L, P<0.001) and 6 months (−65.4 (26.4), pmol/L, P=0.013). There were no significant differences between groups for total, HDL or LDL cholesterol, hs‐CRP, glucose or insulin.

Increased adherence to the MedDiet resulted in a significantly lower F2‐isoprostanes over six months, indicating a positive effect on oxidative damage to lipids in vivo. Triglyceride concentrations were also lower with the MedDiet, but the MedDiet had little effect on glycaemic control, hs‐CRP or circulating cholesterol concentrations.

Support or Funding Information

This project was supported by a grant from the National Health Medical Research Council of Australia. Sponsor Prof Linda Tapsell, American Society Nutrition, Affiliation, ltapsell@uow.edu.au.
Effects of omega‐3 supplementation during pregnancy and youth on neurodevelopment and cognition in childhood: a systematic review and meta‐analysis
Authors: Shulkin Masha L , Pimpin Laura , Bellinger David , Kranz Sarah , Duggan Christopher , Fawzi Wafaie , Mozaffarian Dariush
Published: November 2023

BACKGROUND

Long‐chain omega‐3 fatty acids are thought to be crucial for optimal neurodevelopment in early life.

OBJECTIVES

To investigate the effect of omega‐3 supplementation during pregnancy and infancy on child cognitive and developmental outcomes.

METHODS

We searched PubMed, Cochrane Library, EMBASE, and PsychInfo through May 2015 without language or publication year restrictions for randomized controlled trials (RCTs) of omega‐3 supplementation (>3 months) i.e. docosahexanoic acid (DHA) and eicosapentaenoic acid (EPA), and quantitative measure of neurodevelopment or cognition. Full‐text inclusion decisions and data extractions were performed independently and in duplicate. Our primary outcome was the standardized mean difference in Bayley Scales of Infant Development (BSID) score between intervention groups in RCTs. Other outcomes included the Weschler Intelligence Scale for Children, Weschler Preschool and Primary Scale of Intelligence, Kaufman Brief Intelligence Test, Kaufman Assessment Battery for Children, Peabody Picture Vocabulary Test, and other standardized measures.

RESULTS

Among 571 abstracts, we identified 15 trials with 20 intervention arms involving 2,525 children. Trials used DHA + EPA (N=6 arms), DHA only (N=2), DHA + arachidonic acid (AA) (N=10), or DHA + EPA + AA (N=2); either prenatally (mean 20 weeks gestation; N=4 arms) or within the first few days of birth (N=16). Mean supplementation duration was 7.3 months; and age at outcome assessment, 16 months. In pooled analyses, both maternal and infant supplementation similarly improved neurodevelopment: standardized mean difference (SMD) in BSID= 0.21 (95% CI: 0.01, 0.41) and 0.24 (0.00, 0.48) respectively (Figure 1). Among BSID subscales, DHA and/or EPA raised the psychomotor developmental index (N=8 arms; SMD 0.40; 95% CI: 0.10, 0.70), while DHA + AA raised the mental developmental index (N=15 arms; SMD 0.17; 95% CI: 0.00, 0.35). Pooled findings for other outcomes will be presented.

CONCLUSION

Omega‐3 supplementation during either pregnancy or infancy improves child neurodevelopment. These findings indicate the importance of sufficient polyunsaturated fatty acid intake by pregnant women and young children.

Support or Funding Information

Bill & Melinda Gates Foundation
Feeding a Maternal Diet Enriched in Docosahexaenoic Acid to Lactating Dams Improves the Ex Vivo Tolerance Response to Egg Protein in Suckled Pups
Authors: Richard Caroline , Lewis Erin D , Goruk Suzan , Field Catherine J
Published: November 2023

An important T cell mediated process that occurs early in life is the development of oral tolerance (OT). Food allergies are believed to be the result of a failure to develop OT to a dietary antigen. The objective of this study was to determine the effect of feeding a maternal diet supplemented with docosahexaenoic acid (DHA) while containing adequate amounts of arachidonic acid (AA) during the suckling period on the development of the immune system and OT in offspring.

Sprague‐Dawley dams were randomized to one of the two nutritionally adequate experimental diets 24–48h prior to parturition: control diet (N=12, 0.4% AA and 0% DHA of total fatty acids) or high DHA diet (N=8, 0.4% AA and 0.9% DHA of total fatty acids). Diets were fed to dams ad libitum throughout the suckling period (21 days) and matched for macronutrient, micronutrient and fatty acid content differing only in the composition of omega‐3. At 14‐days pups from each dam were randomly assigned to a mucosal OT challenge for 5 days: placebo (sucrose) or ovalbumin (OVA). At 3 weeks, pups were euthanized and immune cell phenotypes and cytokine production by mitogen‐ (Concanavalin A (ConA), lipopolysaccharide (LPS)) or OVA‐stimulated splenocytes were measured by direct immunofluorescence assay and ELISA, respectively.

Feeding dams a high DHA diet increased the proportion of DHA and AA found in breast milk vs. the control diet (both P<0.01). Regardless of the OT challenge, feeding a high DHA diet led to a higher production of interferon‐γ by splenocytes stimulated with ConA and a higher production of interleukin (IL)‐6, IL‐10 and tumor necrosis factor‐α after LPS stimulation (all P<0.05). OVA‐stimulated splenocytes from suckled pups fed the high DHA diet produced significantly more IL‐10 and less transforming growth factor‐β compared to the control diet (both P<0.02), suggestive of better OT. The OT challenge with OVA resulted in a lower production of IL‐6 by LPS‐stimulated splenocytes and led to a higher proportion of cytotoxic T cells expressing CD28 and total cells expressing CD27 (all P<0.02) compared to non‐tolerized pups.

Our results suggest that feeding a maternal diet enriched in DHA during the suckling period improves the ability of splenocytes to respond ex vivo to mitogens and the tolerance response to a dietary antigen. Irrespective of the suckling diet, pups who had been orally tolerized to OVA had a lower response to a bacterial antigen despite the presence of more activated T cells and memory immune cells.

Support or Funding Information

Supported from the Natural Sciences and Engineering Research Council of Canada. CR holds a scholarship from Canadian Institutes for Health Research and Fonds de recherche en santé du Québec.
Transgenic Up‐Regulation Of Claudin‐6 Decreases Diesel Particulate Matter (DPM)‐Induced Pulmonary Inflammation
Authors: Bodine Jared , Gassman Jason , Milner Dallin C. , Lewis Adam , Dunaway Todd , Egbert Kaleb , Christiansen Connor , Christiansen Andrew , Monson Troy , Broberg Dallin , Arroyo Juan , Reynolds Paul R
Published: November 2023

Cldn6 is a tetraspanin transmembrane protein found in tight junctional complexes and is implicated in maintaining lung epithelial barriers. In the present study, we tested the hypothesis that abundant Cldn‐6 influences inflammation in mice exposed to acute environmental diesel particulate matter (DPM). Mice were subjected to ten exposures of nebulized DPM over a period of 20 days via a nose only inhalation system (Sireq industries). Using real time RT‐PCR, we discovered that the Cldn6 gene was up‐regulated in control mice exposed to DPM but DPM significantly decreased Cldn‐6 expression in lung‐specific transgenic mice that up‐regulate Cldn‐6 (Cldn‐6 TG). DPM caused increased cell diapedesis into bronchoalveolar lavage fluid (BALF) obtained from control mice, however, Cldn‐6 TG mice had less total cells in BALF following DPM exposure. Because Cldn‐6 TG mice diminished cell diapedesis, other inflammatory intermediates were screened in order to characterize the impact of increased Cldn‐6 on inflammation. In summary, MAPKs, NF‐kB and cytokines including TNF‐alpha and IL‐1beta were all differentially regulated in Cldn6 TG mice and controls following DPM exposure. These results demonstrate that epithelial barriers organized by Cldn‐6 mediate, at least in part, diesel‐induced inflammation. Further work may show that Cldn‐6 is a key target in understanding pulmonary epithelial gateways exacerbated by environmental pollution.

Support or Funding Information

This work was supported by a grant from the Flight Attendant's Medical Research Institute (FAMRI, PRR) and a BYU Mentoring Environment Grant (PRR).