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Ethanolamine phosphate phospholyase (ETNPPL) is an enzyme that irreversibly degrades phospho-ethanolamine (p-ETN), an intermediate in the Kennedy pathway of phosphatidylethanolamine (PE) biosynthesis. PE is the second most abundant phospholipid in mammalian membranes. Disturbance of hepatic phospholipid homeostasis has been linked to the development of metabolic dysfunction-associated steatotic liver disease (MASLD). We generated whole-body Etnppl knockout mice to investigate the impact of genetic deletion of Etnppl on hepatic lipid metabolism. Primary hepatocytes isolated from Etnppl -/- mice showed increased conversion of [3H]ethanolamine to [ 3 H]p-ETN and [ 3 H]PE compared to Etnppl +/+ mice. Male and female Etnppl +/+ and Etnppl -/- mice were fed either a chow or a western-type diet (WTD). Irrespective of diet, Etnppl -/- mice had elevated fasting levels of total plasma cholesterol, triglyceride (TG) and apolipoprotein B100 (VLDL particles). Interestingly, hepatic TG secretion was unchanged between groups. Although hepatic lipids (phosphatidylcholine (PC), PE, TG, and cholesterol) were not different between mice, RNA sequencing analysis showed downregulation in genes related to cholesterol biosynthesis in Etnppl -/- mice. Furthermore, hepatic low-density lipoprotein receptor-related protein1 (LRP1) protein level was lower in female Etnppl -/- mice, which may indicate reduced uptake of remnant VLDL particles from circulation. Hepatic PE levels were only increased in WTD-fed female Etnppl -/- mice, not chow diet-fed mice. However, hepatic lipid accumulation and metabolic dysfunction-associated steatohepatitis (MASH) development were unchanged between Etnppl +/+ and Etnppl -/- mice. To conclude, ETNPPL has a role in regulating plasma lipoprotein metabolism independent of hepatic TG levels., (© 2024 The Author(s). The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

Background: Phosphatidylcholine (PC) derived from eggs has been shown to beneficially modulate T cell response and intestinal permeability under the context of a high-fat diet., Objectives: The objective of this study was to determine whether there is a differential effect of plant and animal-derived sources of PC on immune function., Methods: Four-week-old male Wistar rats were randomly assigned to consume 1 of 4 diets (n = 10/group) for 12 wk, all containing 1.5 g of total choline/kg of diet but differing in choline forms: 1-Control Low-Fat [CLF, 20% fat, 100% free choline (FC)]; 2-Control High-Fat (CHF, 50% fat, 100% FC); 3-High-Fat Egg-derived PC (EPC, 50% fat, 100% Egg-PC); 4-High-Fat Soy-derived PC (SPC, 50% fat, 100% Soy-PC). Immune cell functions and phenotypes were measured in splenocytes by ex vivo cytokine production after mitogen stimulation and flow cytometry, respectively., Results: The SPC diet increased splenocyte IL-2 production after PMA+I stimulation compared with the CHF diet. However, the SPC group had a lower proportion of splenocytes expressing the IL-2 receptor (CD25+, P < 0.05). After PMA+I stimulation, feeding EPC normalized splenocyte production of IL-10 relative to the CLF diet, whereas SPC did not (P < 0.05). In mesenteric lymph node lymphocytes, the SPC diet group produced more IL-2 and TNF-α after PMA+I stimulation than the CHF diet, whereas the EPC diet group did not., Conclusions: Our results suggest that both egg- and soy-derived PC may attenuate high-fat diet-induced T cell dysfunction. However, egg-PC enhances, to a greater extent, IL-10, a cytokine involved in promoting the resolution phase of inflammation, whereas soy-PC appears to elicit a greater effect on gut-associated immune responses., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: Obesity is associated with increased intestinal permeability and a diminished immune response. Phosphatidylcholine (PC), a form of choline found in eggs, has been shown to beneficially modulate T-cell response in the context of obesity when provided as the sole form of choline in the diet., Objective: This study aimed to determine the impact of varying doses of PC as part of a high-fat diet (HFD) on immune cell function and intestinal permeability., Methods: Male Wistar rats 4 wk of age were randomly assigned to consume 1 of 6 diets for 12 wk containing the same amount of total choline but differing in the forms of choline: 1-control low-fat (CLF, 20% fat, 100% free choline [FC]); 2-control high-fat (CHF, 50% fat, 100% FC); 3-100% PC (100PC, 50% fat, 100% egg-PC); 4-75% PC (75PC, 50% fat, 75% egg-PC+25% FC); 5-50% PC (50PC, 50% fat, 50% egg-PC+50% FC); and 6-25% PC (25PC; 50% fat, 25% egg-PC+75% FC). Intestinal permeability was measured by fluorescein isothiocyanate-dextran. Immune function was assessed by ex vivo cytokine production of splenocytes and cells isolated from the mesenteric lymph node (MLN) after stimulation with different mitogens., Results: Feeding the CHF diet increased intestinal permeability compared with the CLF diet, and doses of PC 50% or greater returned permeability to levels similar to that of the CLF diet. Feeding the CHF diet lowered splenocyte production of interleukin (IL)-1β, IL-2, IL-10, and tumor necrosis factor-alpha, and MLN production of IL-2 compared with the CLF group. The 50PC diet most consistently significantly improved cytokine levels (IL-2, IL-10, tumor necrosis factor-alpha) compared with the CHF diet., Conclusions: Our results show that a dose of 50% of total choline derived from egg-PC can ameliorate HFD-induced intestinal permeability and immune cell dysfunction., (Copyright © 2023 American Society for Nutrition. Published by Elsevier Inc. All rights reserved.)

Elevated circulating dipeptidyl peptidase-4 (DPP4) is a biomarker for liver disease, but its involvement in gluconeogenesis and metabolic associated fatty liver disease progression remains unclear. Here, we identified that DPP4 in hepatocytes but not TEK receptor tyrosine kinase-positive endothelial cells regulates the local bioactivity of incretin hormones and gluconeogenesis. However, the complete absence of DPP4 (Dpp4-/-) in aged mice with metabolic syndrome accelerates liver fibrosis without altering dyslipidemia and steatosis. Analysis of transcripts from the livers of Dpp4-/- mice displayed enrichment for inflammasome, p53, and senescence programs compared with littermate controls. High-fat, high-cholesterol feeding decreased Dpp4 expression in F4/80+ cells, with only minor changes in immune signaling. Moreover, in a lean mouse model of severe nonalcoholic fatty liver disease, phosphatidylethanolamine N-methyltransferase mice, we observed a 4-fold increase in circulating DPP4, in contrast with previous findings connecting DPP4 release and obesity. Last, we evaluated DPP4 levels in patients with hepatitis C infection with dysglycemia (Homeostatic Model Assessment of Insulin Resistance > 2) who underwent direct antiviral treatment (with/without ribavirin). DPP4 protein levels decreased with viral clearance; DPP4 activity levels were reduced at long-term follow-up in ribavirin-treated patients; but metabolic factors did not improve. These data suggest elevations in DPP4 during hepatitis C infection are not primarily regulated by metabolic disturbances.

Background: In humans, the development of gut-associated lymphoid tissue (GALT) occurs in the first years of life and can be influenced by diet., Objectives: The objective of this study was to determine the effect of dietary choline on the development of gut-associated lymphoid tissue (GALT)., Methods: Three feeding trials were conducted in female Sprague-Dawley rats. Beginning 3 d before parturition (studies 1 and 3) or at day 10 of gestation (study 2), control dams consumed a 100% free choline (FC) diet until the end of the lactation period. In studies 1 and 3, test dams consumed a high-glycerophosphocholine (HGPC) diet [75% glycerophosphocholine (GPC), 12.5% phosphatidylcholine (PC), 12.5% FC] and a 100% PC diet, respectively (both 1 g of choline/kg diet). In study 2, test dams consumed a high-sphingomyelin (SM) and PC (SMPC) diet (34% SM, 37% PC, 17% GPC, 7% FC, 5% phosphocholine) or a 50% PC diet (50% PC, 25% FC, 25% GPC), both 1.7 g of choline/kg diet. Immune cell phenotypes and ex vivo cytokine production by mitogen-stimulated immune cells were measured., Results: Feeding of the HGPC diet lowered T-cell IL-2 (44%), IFN-γ (34%), and TNF-α (55%) production in mesenteric lymph nodes (MLNs) compared with control. Feeding both SMPC and 50% PC diets during the lactation and weaning periods increased IL-2 (54%) and TNF-α (46%) production after T-cell stimulation compared with control. There was a lower production of IL-2 (46%), IL-6 (66%), and TNF-α (45%), and a higher production of IL-10 (44%) in both SMPC and 50% PC groups following ovalbumin stimulation compared with control in MLNs. Feeding a diet containing 100% PC increased the production of IFN-γ by 52% after T-cell stimulation compared with control., Conclusion: Feeding a diet containing a mixture of choline forms with a high content of lipid-soluble forms during both the lactation and weaning periods enhances ex vivo immune responses from the GALT in female Sprague-Dawley offspring., (Copyright © 2022 American Society for Nutrition.)

Background: Allergic asthma (AA) and allergic rhinoconjunctivitis (ARC) are common comorbid environmentally triggered diseases. We hypothesized that severe AA/ARC reflects a maladaptive or unrestrained response to ubiquitous aeroallergens., Methods: We performed provocation studies wherein six separate cohorts of persons (total n = 217) with ARC, with or without AA, were challenged once or more with fixed concentrations of seasonal or perennial aeroallergens in an aeroallergen challenge chamber (ACC)., Results: Aeroallergen challenges elicited fully or partially restrained vs. unrestrained evoked symptom responsiveness, corresponding to the resilient and adaptive vs. maladaptive AA/ARC phenotypes, respectively. The maladaptive phenotype was evoked more commonly during challenge with a non-endemic versus endemic seasonal aeroallergen. In an AA cohort, symptom responses evoked after house dust mite (HDM) challenges vs. recorded in the natural environment were more accurate and precise predictors of asthma severity and control, lung function (FEV1), and mechanistic correlates of maladaptation. Correlates included elevated levels of peripheral blood CD4+ and CD8+ T-cells, eosinophils, and T-cell activation, as well as gene expression proxies for ineffectual epithelial injury/repair responses. Evoked symptom severity after HDM challenge appeared to be more closely related to levels of CD4+ and CD8+ T-cells than eosinophils, neutrophils, or HDM-specific IgE., Conclusions: Provocation studies support the concept that resilience, adaptation, and maladaptation to environmental disease triggers calibrate AA/ARC severity. Despite the ubiquity of aeroallergens, in response to these disease triggers in controlled settings (ie, ACC), most atopic persons manifest the resilient or adaptive phenotype. Thus, ARC/AA disease progression may reflect the failure to preserve the resilient or adaptive phenotype. The triangulation of CD8+ T-cell activation, airway epithelial injury/repair processes and maladaptation in mediating AA disease severity needs more investigation., (© 2021 The Authors. Allergy published by European Academy of Allergy and Clinical Immunology and John Wiley & Sons Ltd.)

Two hemibiotrophic pathogens, Colletotrichum acutatum (Ca) and C . gloeosporioides (Cg), cause anthracnose fruit rot and anthracnose crown rot in strawberry ( Fragaria × ananassa Duchesne), respectively. Both Ca and Cg can initially infect through a brief biotrophic phase, which is associated with the production of intracellular primary hyphae that can infect host cells without causing cell death and establishing hemibiotrophic infection (HBI) or quiescent (latent infections) in leaf tissues. The Ca and Cg HBI in nurseries and subsequent distribution of asymptomatic infected transplants to fruit production fields is the major source of anthracnose epidemics in North Carolina. In the absence of complete resistance, strawberry varieties with good fruit quality showing rate-reducing resistance have frequently been used as a source of resistance to Ca and Cg. However, the molecular mechanisms underlying the rate-reducing resistance or susceptibility to Ca and Cg are still unknown. We performed comparative transcriptome analyses to examine how rate-reducing resistant genotype NCS 10-147 and susceptible genotype 'Chandler' respond to Ca and Cg and identify molecular events between 0 and 48 h after the pathogen-inoculated and mock-inoculated leaf tissues. Although plant response to both Ca and Cg at the same timepoint was not similar, more genes in the resistant interaction were upregulated at 24 hpi with Ca compared with those at 48 hpi. In contrast, a few genes were upregulated in the resistant interaction at 48 hpi with Cg. Resistance response to both Ca and Cg was associated with upregulation of MLP-like protein 44, LRR receptor-like serine/threonine-protein kinase, and auxin signaling pathway, whereas susceptibility was linked to modulation of the phenylpropanoid pathway. Gene regulatory network inference analysis revealed candidate transcription factors (TFs) such as GATA5 and MYB-10, and their downstream targets were upregulated in resistant interactions. Our results provide valuable insights into transcriptional changes during resistant and susceptible interactions, which can further facilitate assessing candidate genes necessary for resistance to two hemibiotrophic Colletotrichum spp. in strawberry., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Adhikari, Aryal, Redpath, Van den Broeck, Ashrafi, Philbrick, Jacobs, Sozzani and Louws.)

Background: Oak and birch pollens are strongly cross-reactive. It is unknown how robust this cross-reactivity is in patients without natural exposure to pollen of both trees. We assessed the symptom response to birch pollen in subjects with skin-prick test (SPT) results positive to oak and birch but only naturally exposed to oak by using an allergen challenge chamber. Methods: The subjects with SPT results positive to oak and birch had their serum-specific immunoglobulin E (ssIgE) to oak and birch antigens measured. Residential historical data were obtained. The subjects were exposed to birch pollen (3500 ± 700 grains/m³) in two consecutive 3-hour challenges. Symptoms were recorded at baseline and 30-minute intervals. Results: Twenty-four subjects, 12 men; ages 20-58 years, completed the study. Sixteen subjects (66.7%) responded with high Total Symptom Scores (TSS) ≥10 of a maximum 21. Twelve subjects (50%) had ssIgE values ≥0.70 kU/L to oak, 10 of whom had ssIgE values ≥0.70 kU/L to birch. These 10 subjects had a significantly higher maximum TSS than the rest. Also, 15 subjects without a previous natural exposure to birch pollen responded with TSS equivalent to the 9 subjects with previous exposure. Conclusion: Virginia live oak ssIgE levels of patients allergic to oak and birch correlated with the symptom response to birch pollen exposure, even without previous natural exposure to birch. The subjects naive to birch pollen responded to birch pollen exposure with symptoms comparable with both those with previous sustained exposure and also those who resided in endemic areas, as reported by other researchers.

Obesity is associated with immune dysfunction including an impaired T-cell function characterized by a lower IL-2 (proliferation marker) production after stimulation. Phosphatidylcholine (PC), a form of choline mostly found in eggs, has been shown to beneficially modulate T-cell responses during the lactation period by increasing the production of IL-2. To determine the impact of egg-PC as part of a high-fat diet on immune function we randomly fed male Wistar rats one of three diets containing the same amount of total choline but differing in the form of choline: 1-Control low fat [CLF, 10% wt/wt fat, 100% free choline (FC)]; 2- Control high-fat (CHF, 25% wt/wt fat, 100% FC); 3- PC high-fat (PCHF, 25% wt/wt, 100% PC). After 9 weeks of feeding, rats were euthanized. Cell phenotypes and ex vivo cytokine production by splenocytes stimulated with phorbol 12-myristate 13-acetate plus ionomycin (PMA+I), lipopolysaccharide (LPS) and pokeweed (PWM) were measured by flow cytometry and ELISA, respectively. Rats fed the PCHF diet had a lower proportion of CD3+ cells when compared to both the CLF and the CHF. Following PMA+I stimulation, splenocytes from the CHF group produced less IL-2 and TNF-α compared to CLF and PCHF groups. No significant differences in cytokine production were found among groups after LPS and PWM stimulation. Our results show that feeding a high-fat diet impairs T-cell responses, as measured by ex vivo cytokine production, which can be attenuated by providing egg-PC., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Azarcoya-Barrera, Wollin, Veida-Silva, Makarowski, Goruk, Field, Jacobs and Richard.)

Background & Aims: Dysregulation of liver lipid metabolism is associated with the development and progression of nonalcoholic fatty liver disease, a spectrum of liver diseases including nonalcoholic steatohepatitis (NASH). In the liver, insulin controls lipid homeostasis by increasing triglyceride (TAG) synthesis, suppressing fatty acid oxidation, and enhancing TAG export via very low-density lipoproteins. Downstream of insulin signaling, the mechanistic target of rapamycin complex 1 (mTORC1), is a key regulator of lipid metabolism. Here, we define the role of hepatic mTORC1 activity in mouse models of NASH and investigate the mTORC1-dependent mechanisms responsible for protection against liver damage in NASH., Methods: Utilizing 2 rodent NASH-promoting diets, we demonstrate that hepatic mTORC1 activity was reduced in mice with NASH, whereas under conditions of insulin resistance and benign fatty liver, mTORC1 activity was elevated. To test the beneficial effects of hepatic mTORC1 activation in mouse models of NASH, we employed an acute, liver-specific knockout model of TSC1 (L-TSC-KO), a negative regulator of mTORC1., Results: L-TSC-KO mice are protected from and have improved markers of NASH including reduced steatosis, decreased circulating transaminases, and reduced expression of inflammation and fibrosis genes. Mechanistically, protection from hepatic inflammation and fibrosis by constitutive mTORC1 activity occurred via promotion of the phosphatidylcholine synthesizing enzyme, CCTα, and enhanced very low-density lipoprotein-triglyceride export. Additionally, activation of mTORC1 protected from hepatic steatosis via negative feedback of the mTORC2-AKT-FOXO-SREBP1c lipogenesis axis., Conclusions: Collectively, this study identifies a protective role for liver mTORC1 signaling in the initiation and progression of NASH in mice via dual control of lipid export and synthesis., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Folate and choline are interconnected metabolically. The MTHFD1 R653Q SNP is a risk factor for birth defects and there are concerns that choline deficiency may interact with this SNP and exacerbate health risks. 80-90% of women do not meet the Adequate Intake (AI) for choline. The objective of this study was to assess the effects of choline deficiency on maternal one-carbon metabolism and reproductive outcomes in the MTHFD1-synthetase deficient mouse ( Mthfd1S ), a model for MTHFD1 R653Q. Mthfd1S +/+ and Mthfd1S +/- females were fed control (CD) or choline-deficient diets (ChDD; 1/3 the amount of choline) before mating and during pregnancy. Embryos were evaluated for delays and defects at 10.5 days gestation. Choline metabolites were measured in the maternal liver, and total folate measured in maternal plasma and liver. ChDD significantly decreased choline, betaine, phosphocholine, and dimethylglycine in maternal liver ( p < 0.05, ANOVA), and altered phosphatidylcholine metabolism. Maternal and embryonic genotype, and diet-genotype interactions had significant effects on defect incidence. Mild choline deficiency and Mthfd1S +/- genotype alter maternal one-carbon metabolism and increase incidence of developmental defects. Further study is required to determine if low choline intakes contribute to developmental defects in humans, particularly in 653QQ women.

Allergen exposure chambers (AECs) can be used for controlled exposure to allergenic and non-allergenic airborne particles in an enclosed environment, in order to (i) characterize the pathological features of respiratory diseases and (ii) contribute to and accelerate the clinical development of pharmacological treatments and allergen immunotherapy for allergic disease of the respiratory tract (such as allergic rhinitis, allergic rhinoconjunctivitis, and allergic asthma). In the guidelines of the European Medicines Agency for the clinical development of products for allergen immunotherapy (AIT), the role of AECs in determining primary endpoints in dose-finding Phase II trials is emphasized. Although methodologically insulated from the variability of natural pollen exposure, chamber models remain confined to supporting secondary, rather than primary, endpoints in Phase III registration trials. The need for further validation in comparison with field exposure is clearly mandated. On this basis, the European Academy of Allergy and Clinical Immunology (EAACI) initiated a Task Force in 2015 charged to gain a better understanding of how AECs can generate knowledge about respiratory allergies and can contribute to the clinical development of treatments. Researchers working with AECs worldwide were asked to provide technical information in eight sections: (i) dimensions and structure of the AEC, (ii) AEC staff, (iii) airflow, air processing, and operating conditions, (iv) particle dispersal, (v) pollen/particle counting, (vi) safety and non-contamination measures, (vii) procedures for symptom assessments, (viii) tested allergens/substances and validation procedures. On this basis, a minimal set of technical requirements for AECs applied to the field of allergology is proposed., (© 2021 The Authors. Allergy published by European Academy of Allergy and Clinical Immunology and John Wiley & Sons Ltd.)

Mitochondrial energy production and function rely on optimal concentrations of the essential redox-active lipid, coenzyme Q (CoQ). CoQ deficiency results in mitochondrial dysfunction associated with increased mitochondrial oxidative stress and a range of pathologies. What drives CoQ deficiency in many of these pathologies is unknown, just as there currently is no effective therapeutic strategy to overcome CoQ deficiency in humans. To date, large-scale studies aimed at systematically interrogating endogenous systems that control CoQ biosynthesis and their potential utility to treat disease have not been carried out. Therefore, we developed a quantitative high-throughput method to determine CoQ concentrations in yeast cells. Applying this method to the Yeast Deletion Collection as a genome-wide screen, 30 genes not known previously to regulate cellular concentrations of CoQ were discovered. In combination with untargeted lipidomics and metabolomics, phosphatidylethanolamine N-methyltransferase (PEMT) deficiency was confirmed as a positive regulator of CoQ synthesis, the first identified to date. Mechanistically, PEMT deficiency alters mitochondrial concentrations of one-carbon metabolites, characterized by an increase in the S-adenosylmethionine to S-adenosylhomocysteine (SAM-to-SAH) ratio that reflects mitochondrial methylation capacity, drives CoQ synthesis, and is associated with a decrease in mitochondrial oxidative stress. The newly described regulatory pathway appears evolutionary conserved, as ablation of PEMT using antisense oligonucleotides increases mitochondrial CoQ in mouse-derived adipocytes that translates to improved glucose utilization by these cells, and protection of mice from high-fat diet-induced insulin resistance. Our studies reveal a previously unrecognized relationship between two spatially distinct lipid pathways with potential implications for the treatment of CoQ deficiencies, mitochondrial oxidative stress/dysfunction, and associated diseases., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Purpose: To determine the effect of feeding buttermilk-derived choline metabolites on the immune system development in Sprague-Dawley rat pups., Methods: Sprague-Dawley dams were randomized to one of the three diets containing 1.7 g/kg choline: 1-Control (100% free choline (FC)), 2-Buttermilk (BM, 37% phosphatidylcholine (PC), 34% sphingomyelin (SM), 17% glycerophosphocholine (GPC), 7% FC, 5% phosphocholine), and 3-Placebo (PB, 50% PC, 25% FC, 25% GPC) until the end of the lactation period. At weaning, pups continued on the same diet as their mom. Cell phenotypes and cytokine production by mitogen-stimulated splenocytes isolated from 3- and 10-week-old pups were measured., Results: At 3 weeks, BM-pups had a higher proportion of cytotoxic T cells (CTL; CD3 + CD8 +) while both BM- and PB-pups had an increased proportion of cells expressing CD28 + , CD86 + and CD27 + (all p > 0.05). Following ConA stimulation, splenocytes from BM- and PB-pups produced more TNF-α and IFN-γ and after LPS stimulation produced more IL-10 and TNF-α (all p > 0.05). Starting at week 6 of age, BM-pups had a higher body weight. At 10 weeks, both the BM- and PB-pups had a higher proportion of CTL expressing CD27 + . After ConA stimulation, splenocytes from BM- and PB-pups produced more IL-2, IFN-γ and IL-6 and more IL-10 after LPS stimulation (all p > 0.05)., Conclusion: The proportion of lipid soluble forms of choline in the diet during lactation and weaning periods influence the immune system development in rat offspring., (© 2021. Springer-Verlag GmbH Germany, part of Springer Nature.)

Background: Signifying the 2-compartments/1-disease paradigm, allergic rhinoconjunctivitis (ARC) and asthma (AA) are prevalent, comorbid conditions triggered by environmental factors (eg, house dust mites [HDMs]). However, despite the ubiquity of triggers, progression to severe ARC/AA is infrequent, suggesting either resilience or adaptation., Objective: We sought to determine whether ARC/AA severity relates to maladaptive responses to disease triggers., Methods: Adults with HDM-associated ARC were challenged repetitively with HDMs in an aeroallergen challenge chamber. Mechanistic traits associated with disease severity were identified., Results: HDM challenges evoked maladaptive (persistently higher ARC symptoms), adaptive (progressive symptom reduction), and resilient (resistance to symptom induction) phenotypes. Symptom severity in the natural environment was an imprecise correlate of the phenotypes. Nasal airway traits, defined by low inflammation-effectual epithelial integrity, moderate inflammation-effectual epithelial integrity, and higher inflammation-ineffectual epithelial integrity, were hallmarks of the resilient, adaptive, and maladaptive evoked phenotypes, respectively. Highlighting a crosstalk mechanism, peripheral blood inflammatory tone calibrated these traits: ineffectual epithelial integrity associated with CD8 + T cells, whereas airway inflammation associated with both CD8 + T cells and eosinophils. Hallmark peripheral blood maladaptive traits were increased natural killer and CD8 + T cells, lower CD4 + mucosal-associated invariant T cells, and deficiencies along the TLR-IRF-IFN antiviral pathway. Maladaptive traits tracking HDM-associated ARC also contributed to AA risk and severity models., Conclusions: Repetitive challenges with HDMs revealed that maladaptation to disease triggers may underpin ARC/AA disease severity. A combinatorial therapeutic approach may involve reversal of loss-of-beneficial-function traits (ineffectual epithelial integrity, TLR-IRF-IFN deficiencies), mitigation of gain-of-adverse-function traits (inflammation), and blocking of a detrimental crosstalk between the peripheral blood and airway compartments., (Published by Elsevier Inc.)

Choline is an essential nutrient required for various biological processes. Eggs, dairy, and meat are rich in phosphatidylcholine (PC), whereas cereal and legumes are rich in free choline. Excess dietary choline leads to increase plasma trimethylamine N-oxide (TMAO). Epidemiological studies suggest that plasma TMAO is a biomarker for atherosclerosis and it has been suggested that a lower intake of eggs and meat would reduce choline consumption and thus reduce atherosclerosis development. To investigate whether the form of dietary choline influences atherosclerosis development in Ldlr -/- , we randomly fed Ldlr -/- male mice (aged 8 - 10 wk) one of the three 40% (calories) high fat diets (with 0.5% w/w of cholesterol): Control (0.1% w/w free-choline, CON), choline-supplemented (0.4% free-choline, CS), or PC-supplemented (0.1% free-choline and 0.3% choline from PC, PCS). After 12-wk of dietary intervention, the animals were euthanized and tissues and blood collected. Aortic atherosclerotic plaque area, plasma choline, lipid metabolites, and spleen and peripheral blood cell phenotypes were quantified. Surprisingly, the PCS group had significantly lower atherosclerotic lesions while having 2-fold higher plasma TMAO levels compared with both CON and CS groups (P<0.05). In the fasting state, we found that PCS decreased plasma very low-density lipoprotein-cholesterol (VLDL-C) and apolipoprotein B48 (APOB48), and increased plasma high-density lipoprotein-cholesterol (HDL-C). However, very low-density lipoprotein (VLDL) secretion was not affected by dietary treatment. We observed lower levels of circulating pro-atherogenic chemokines in the PCS group. Our study suggests that increased dietary PC intake does not induce a pro-atherogenic phenotype., (Copyright © 2021. Published by Elsevier Inc.)

Dietary choline, which is converted to phosphatidylcholine (PC) in intestinal enterocytes, may benefit inflammatory bowel disease patients who typically have reduced intestinal choline and PC. The present study investigated the effect of dietary choline supplementation on colitis severity and intestinal mucosal homoeostasis using a Citrobacter rodentium-induced colitis model. C57BL/6J mice were fed three isoenergetic diets differing in choline level: choline-deficient (CD), choline-sufficient (CS) and choline-excess (CE) for 3 weeks prior to infection with C. rodentium. The effect of dietary choline levels on the gut microbiota was also characterised in the absence of infection using 16S rRNA gene amplicon sequencing. At 7 d following infection, the levels of C. rodentium in CD mice were significantly greater than that in CS or CE groups (P < 0·05). CD mice exhibited greater damage to the surface epithelium and goblet cell loss than the CS or CE mice, which was consistent with elevated pro-inflammatory cytokine and chemokine levels in the colon. In addition, CD group exhibited decreased concentrations of PC in the colon after C. rodentium infection, although the decrease was not observed in the absence of challenge. Select genera, including Allobaculum and Turicibacter, were enriched in response to dietary choline deficiency; however, there was minimal impact on the total bacterial abundance or the overall structure of the gut microbiota. Our results suggest that insufficient dietary choline intake aggravates the severity of colitis and demonstrates an essential role of choline in maintaining intestinal homoeostasis.

Background & Aims: Patients with ulcerative colitis have low concentrations of the major membrane lipid phosphatidylcholine (PC) in gastrointestinal mucus, suggesting that defects in colonic PC metabolism might be involved in the development of colitis. To determine the precise role that PC plays in colonic barrier function, we examined mice with intestinal epithelial cell (IEC)-specific deletion of the rate-limiting enzyme in the major pathway for PC synthesis: cytidine triphosphate:phosphocholine cytidylyltransferase-α (CTα IKO mice)., Methods: Colonic tissue of CTα IKO mice and control mice was analyzed by histology, immunofluorescence, electron microscopy, quantitative polymerase chain reaction, Western blot, and thin-layer chromatography. Histopathologic colitis scores were assigned by a pathologist blinded to the experimental groupings. Intestinal permeability was assessed by fluorescein isothiocyanate-dextran gavage and fecal microbial composition was analyzed by sequencing 16s ribosomal RNA amplicons. Subsets of CTα IKO mice and control mice were treated with dietary PC supplementation, antibiotics, or 4-phenylbutyrate., Results: Inducible loss of CTα in the intestinal epithelium reduced colonic PC concentrations and resulted in rapid and spontaneous colitis with 100% penetrance in adult mice. Colitis development in CTα IKO mice was traced to a severe and unresolving endoplasmic reticulum stress response in IECs with altered membrane phospholipid composition. This endoplasmic reticulum stress response was linked to the necroptotic death of IECs, leading to excessive loss of goblet cells, formation of a thin mucus barrier, increased intestinal permeability, and infiltration of the epithelium by microbes., Conclusions: Maintaining the PC content of IEC membranes protects against colitis development in mice, showing a crucial role for IEC phospholipid equilibrium in colonic homeostasis. SRA accession number: PRJNA562603., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

The liver is the central organ regulating cholesterol synthesis, storage, transport, and elimination. Mouse carboxylesterase 1d (Ces1d) and its human ortholog CES1 have been described to possess lipase activity and play roles in hepatic triacylglycerol metabolism and VLDL assembly. It has been proposed that Ces1d/CES1 might also catalyze cholesteryl ester (CE) hydrolysis in the liver and thus be responsible for the hydrolysis of HDL-derived CE; this could contribute to the final step in the reverse cholesterol transport (RCT) pathway, wherein cholesterol is secreted from the liver into bile and feces, either directly or after conversion to water-soluble bile salts. However, the proposed function of Ces1d/CES1 as a CE hydrolase is controversial. In this study, we interrogated the role hepatic Ces1d plays in cholesterol homeostasis using liver-specific Ces1d-deficient mice. We rationalized that if Ces1d is a major hepatic CE hydrolase, its absence would (1) reduce in vivo RCT flux and (2) provoke liver CE accumulation after a high-cholesterol diet challenge. We found that liver-specific Ces1d-deficient mice did not show any difference in the flux of in vivo HDL-to-feces RCT nor did it cause additional liver CE accumulation after high-fat, high-cholesterol Western-type diet feeding. These findings challenge the importance of Ces1d as a major hepatic CE hydrolase., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Prolonged, isocaloric, time-restricted feeding (TRF) protocols can promote weight loss, improve metabolic dysregulation, and mitigate non-alcoholic fatty liver disease (NAFLD). In addition, 3-day, severe caloric restriction can improve liver metabolism and glucose homeostasis prior to significant weight loss. Thus, we hypothesized that short-term, isocaloric TRF would improve NAFLD and characteristics of metabolic syndrome in diet-induced obese male mice. After 26 weeks of ad libitum access to western diet, mice either continued feeding ad libitum or were provided with access to the same quantity of western diet for 8 h daily, over the course of two weeks. Remarkably, this short-term TRF protocol modestly decreased liver tissue inflammation in the absence of changes in body weight or epidydimal fat mass. There were no changes in hepatic lipid accumulation or other characteristics of NAFLD. We observed no changes in liver lipid metabolism-related gene expression, despite increased plasma free fatty acids and decreased plasma triglycerides in the TRF group. However, liver Grp78 and Txnip expression were decreased with TRF suggesting hepatic endoplasmic reticulum (ER) stress and activation of inflammatory pathways may have been diminished. We conclude that two-week, isocaloric TRF can potentially decrease liver inflammation, without significant weight loss or reductions in hepatic steatosis, in obese mice with NAFLD.

Inhibition of eukaryotic elongation factor 1A1 (EEF1A1) with the marine compound didemnin B decreases lipotoxic HepG2 cell death in vitro and improves early stage non-alcoholic fatty liver disease (NAFLD) in young genetically obese mice. However, the effects of didemnin B on NAFLD in a model of long-term diet-induced obesity are not known. We investigated the effects of didemnin B on NAFLD severity and metabolic parameters in western diet-induced obese mice, and on the cell types that contribute to liver inflammation and fibrosis in vitro. Male 129S6 mice were fed either standard chow or western diet for 26 weeks, followed by intervention with didemnin B (50 μg/kg) or vehicle by intraperitoneal (i.p.) injection once every 3 days for 14 days. Didemnin B decreased liver and plasma triglycerides, improved oral glucose tolerance, and decreased NAFLD severity. Moreover, didemnin B moderately increased hepatic expression of genes involved in ER stress response (Perk, Chop), and fatty acid oxidation (Fgf21, Cpt1a). In vitro, didemnin B decreased THP-1 monocyte proliferation, disrupted THP-1 monocyte-macrophage differentiation, decreased THP-1 macrophage IL-1β secretion, and decreased hepatic stellate cell (HSteC) proliferation and collagen secretion under both basal and lipotoxic (high fatty acid) conditions. Thus, didemnin B improves hepatic steatosis, glucose tolerance, and blood lipids in obesity, in association with moderate, possibly hormetic, upregulation of pathways involved in cell stress response and energy balance in the liver. Furthermore, it decreases the activity of the cell types implicated in liver inflammation and fibrosis in vitro. These findings highlight the therapeutic potential of partial protein synthesis inhibition in the treatment of NAFLD., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: Buttermilk contains a mixture of choline forms; it is high in phosphatidylcholine (PC) and sphingomyelin (SM), which could have an impact on immune system development and function., Objectives: We aimed to determine the effect of feeding buttermilk-derived choline forms during pregnancy and lactation on maternal immune function., Methods: Sprague Dawley dams (n = 8 per diet) were randomly assigned midway through pregnancy (10 d of gestation) to 1 of 3 experimental diets, containing 1.7 g/kg choline: control [100% free choline (FC)]; buttermilk [37% PC, 34% SM, 17% glycerophosphocholine (GPC), 7% FC, 5% phosphocholine]; or placebo (50% PC, 25% FC, 25% GPC). Dams consumed the same diet until the end of the lactation period (21 d after parturition). Cell phenotypes and cytokine production by mitogen-stimulated splenocytes were measured and compared using 1-factor ANOVA test in order to asses the effect of diet on immune fuction of lactating dams (main outcome)., Results: After ConA stimulation, splenocytes from dams in the buttermilk group produced more IL-2 (30%), TNF-α (30%), and IFN-γ (42%) compared with both the placebo and control diets. Placebo-fed dams had a higher proportion of CD8+ cells expressing CD152+ (22%) in spleen, and splenocytes from dams that were fed the buttermilk and the placebo diets produced about 50% and 53% more IL-10 after LPS and OVA stimulation, respectively, compared with the control group., Conclusions: Feeding buttermilk-derived choline forms during pregnancy and lactation had a beneficial impact on the immune system of Sprague Dawley rat dams, especially on T-cell function., (Copyright © The Author(s) on behalf of the American Society for Nutrition 2020.)

Background: Choline, an essential nutrient, is required for cell membranes, lipoprotein secretion, and methyl-group metabolism. Recently, it has been proposed that excess dietary choline consumption is metabolized to trimethylamine (TMA) by the gut microbiota; TMA is then oxidized to trimethylamine N-oxide (TMAO) in the liver. Epidemiological studies have clearly shown a positive correlation between plasma TMAO concentrations and cardiovascular events. Furthermore, some studies have shown an association between excess dietary choline, plasma TMAO concentrations, and atherosclerotic lesion size in apoE knockout (Apoe-/-) mice., Objective: The aim of this study was to further investigate the relation between dietary choline and atherosclerosis in 2 atherogenic mouse models, the LDL receptor knockout (Ldlr-/-) and Apoe-/- mice., Methods: Six feeding trials were performed in Ldlr-/- (40% high-fat diet) and Apoe-/- (unpurified diet) male mice, aged 8-10 wk. Mice randomly received control diet (0.1% choline), or choline- (1% choline), betaine- (0.1% choline and 0.9% betaine), or TMAO- (0.1% choline and 0.12% or 0.2% TMAO) supplemented diet for ≤28 wk. After the dietary intervention, the animals were killed and tissues and blood collected. Aortic atherosclerotic plaque area, plasma lipids, and choline metabolites were quantified., Results: In Ldlr-/- mice, dietary supplementation for 8 wk with choline or TMAO increased plasma TMAO concentrations by 1.6- and 4-fold, respectively. After 16 wk, there was a 2-fold increase in plasma TMAO after dietary TMAO supplementation. In Apoe-/- mice, dietary supplementation with choline, betaine, or TMAO for 12 wk did not increase plasma TMAO concentrations. However, choline and TMAO supplementation for 28 wk significantly increased plasma TMAO concentrations by 1.8- and 1.5-fold, respectively. Contrary to predictions, atherosclerotic lesion size was not altered by any of the dietary interventions, irrespective of mouse model., Conclusions: In our study, high intakes of dietary choline or TMAO supplementation did not influence atherosclerosis development in Ldlr-/- or Apoe-/- male mice., (Copyright © American Society for Nutrition 2019.)

Anthracnose fruit rot and anthracnose crown rot (ACR) caused by two species complexes of the fungus referred to as Colletotrichum acutatum and Colletotrichum gloeosporioides , respectively, are major pathogens of strawberry in North Carolina. Anthracnose epidemics are common when susceptible cultivars and asymptomatic planting stocks carrying quiescent Colletotrichum infection or hemibiotrophic infection (HBI) are planted. The main objective of this study was to assess resistance to HBI and ACR in strawberry. Strawberry cultivars and breeding lines were spray inoculated with isolates of C. acutatum or C. gloeosporioides . Four epidemiological parameters providing estimates of rate-reducing resistance to HBI and ACR in strawberry cultivars and lines were evaluated in repeated experiments in controlled environments in a greenhouse. HBI severity, measured as the percentage of total leaf area covered by acervuli, was estimated visually and by image analysis. ACR severity was rated weekly for wilt symptoms, and relative area under disease progress curve scores were calculated for comparing strawberry cultivars and lines. Significant differences ( P ≤ 0.005) in HBI severity were found among strawberry genotypes; however, the correlations were not remarkable between Colletotrichum species ( r = 0.4251). Although significant variation in resistance was observed for ACR, this was also weakly correlated ( r = 0.2430) with resistance to C. gloeosporioides HBI. Overall, rate-reducing resistance to HBI and ACR in strawberry identified in this study could be utilized in breeding programs to develop durable resistance to anthracnose in North Carolina.

This article introduces the open-source Subject-Mediated Automatic Remote Testing Apparatus (SMARTA) for visual discrimination tasks, which aims to streamline and ease data collection, eliminate or reduce observer error, increase interobserver agreement, and automate data entry without the need for an internet connection. SMARTA is inexpensive and easy to build, and it can be modified to accommodate a variety of experimental designs. Here we describe the utility and functionality of SMARTA in a captive setting. We present the results from a case study of color vision in ruffed lemurs (Varecia spp.) at the Duke Lemur Center in Durham, North Carolina, in which we demonstrate SMARTA's utility for two-choice color discrimination tasks, as well as its ability to streamline and standardize data collection. We also include detailed instructions for constructing and implementing the fully integrated SMARTA touchscreen system.

Phosphatidylethanolamine (PE) N -methyltransferase (PEMT) accounts for ∼30% of hepatic phosphatidylcholine (PC) biosynthesis. Pemt -/- mice fed a high-fat diet are protected against diet-induced obesity (DIO) and insulin resistance (IR) but develop nonalcoholic fatty liver disease (NAFLD) associated with a decreased PC:PE ratio. We investigated whether the lack of hepatic PEMT or the lack of PEMT in other tissues (where it is expressed at low levels) is responsible for or contributes to the protection against DIO and IR in Pemt -/- mice. Furthermore, we investigated whether decreasing PEMT expression with antisense oligonucleotides (ASOs) would result in metabolic benefits in both lean and obese mice without negatively impacting liver health. We both restored hepatic PEMT in Pemt -/- mice via adeno-associated virus delivery and decreased hepatic PEMT with ASOs in wild-type and ob/ob mice. Weight gain, insulin sensitivity, and indices of liver function were determined. We report that the protection against DIO and IR and the development of NAFLD is dependent on hepatic PEMT activity. NAFLD, associated with a significant decrease in the hepatic PC:PE ratio, was exacerbated by PEMT deficiency in obese mice, suggesting that phospholipid insufficiency promotes NAFLD progression during obesity or overnutrition. Hepatic PEMT is critical for maintaining phospholipid balance, which is crucial for a healthy liver.-Wan, S., van der Veen, J. N., Bakala N'Goma, J.-C., Nelson, R. C., Vance, D. E., Jacobs, R. L. Hepatic PEMT activity mediates liver health, weight gain, and insulin resistance.

Background: A variety of antigens have been identified as causative of hypersensitivity pneumonitis (HP), which is characterized by inflammation to the lung parenchyma that is induced by exposure. Goose and duck down (GDD) bedding is often overlooked by physicians as a potential cause, yet the use of GDD has markedly increased in recent years, paralleling an increased frequency of reports of GDD-induced HP., Objective: To determine the frequency of GDD as the causative antigen in patients with HP who use bedding that contains GDD., Methods: Patients referred with a working diagnosis of HP underwent a detailed environmental history. Those who were using GDD were asked to remove it as an avoidance procedure. Signs, symptoms, spirometry, and inflammatory markers were followed up at weekly intervals for up to 1 month to determine the effect of remediation., Results: Eighty patients with HP were seen during an 8-year period. Thirty-two patients (40%) were using GDD bedding. Of these 32 patients, 12 (37.5% of those exposed and 15% of the total HP population experienced remission (or nonprogression) of disease by simply avoiding GDD bedding. Eleven (92%) of these 12 patients were female. In patients with GDD-induced HP, lung biopsy patterns were varied., Conclusion: Approximately one-third of patients with HP, who slept with GDD, had persistent improvement or remission with simple avoidance. The higher incidence of GDD-induced HP in females may be hormonal and/or sociocultural related. Lung biopsy findings were across the spectrum of histopathologic patterns. Avoidance-challenge techniques were effective in confirming diagnoses and causation and mitigating the need for additional remediation., (Copyright © 2019 American College of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

Background: GSP301 is an investigational fixed-dose combination nasal spray that contains the antihistamine, olopatadine hydrochloride (HCl), and the corticosteroid, mometasone furoate. Objective: To evaluate the efficacy and safety of GSP301 in patients with seasonal allergic rhinitis (SAR). Methods: In this double-blind, randomized, parallel-group study, patients (≥12 years of age) with SAR were equally randomized to intranasal GSP301 (olopatadine 665 μg and mometasone 25 μg), olopatadine HCl (665 μg), mometasone furoate (25 μg), or placebo for 14 days of twice-daily treatment. The primary end point was the mean change from baseline in the average A.M. and P.M. 12-hour reflective Total Nasal Symptom Score (rTNSS) analyzed by using mixed-effect model repeated measures ( p < 0.05 indicates statistical significance). Additional assessments included instantaneous TNSS (iTNSS), individual nasal symptoms, reflective Total Ocular Symptom Score (rTOSS) and instantaneous Total Ocular Symptom Score (iTOSS), onset of action, Physician-assessed Nasal Symptom Score (PNSS), quality of life, and adverse events (AE). Results: A total of 1180 patients were randomized. Over 14 days of treatment, GSP301 significantly improved average A.M. and P.M. rTNSS versus placebo (least squares mean difference -0.98 [95% confidence interval, -1.38 to -0.57]; p < 0.001) and versus olopatadine (p = 0.003), and approached statistical significance versus mometasone (p = 0.059). GSP301 also significantly improved average A.M. and P.M. iTNSS versus placebo and both monotherapies (p < 0.05, all). Further, GSP301 significantly improved individual nasal symptoms, overall ocular symptoms (rTOSS and iTOSS), and overall quality of life versus placebo (p < 0.01, all). Onset of action for GSP301 was observed within 15 minutes and was maintained at all subsequent time points assessed. Results for the PNSS also were significant for GSP301 versus placebo (p < 0.001). The percentages of patients with treatment-emergent AEs treated with GSP301, olopatadine, mometasone, and placebo were 12.9, 12.5, 7.1, and 9.4%, respectively. Conclusion: GSP301 was efficacious and well tolerated for the treatment of SAR symptoms compared with placebo, with a rapid onset of action of 15 minutes in patients ≥12 years of age.Clinical trial NCT02631551, www.clinicaltrials.gov.

Background: Controlled allergen challenge facilities (CACF), in disparate geographic regions with dissimilar engineering and base populations, have historically functioned as single, independent sites in clinical allergy trials. We aimed to demonstrate "between-unit reproducibility" to allow controlled challenge trials of participants using 2 CACFs., Objective: To compare and standardize 2 CACFs located in Kingston, Ontario, Canada, and San Antonio, Texas, by examining participant-reported symptom severity during qualifying and treatment visits and evaluating response to treatment, while using the same allergen., Methods: At 2 different CACFs, participants were enrolled in a double-blind, placebo-controlled, crossover intervention trial with cetirizine 10 mg. Different distribution devices delivered common short ragweed pollen via laminar air flow and maintained an airborne concentration of 3500 ± 700 grains/m 3 in both facilities. A 1-hour "sham" run with no pollen release preceded a priming exposure of 3 hours and was followed 3 days later by a qualifying/treatment 5-hour exposure. At least 14 days later, another priming exposure was followed by the crossover exposure and treatment., Results: Forty-eight and 43 subjects completed the study at Kingston and San Antonio, respectively. Demographics were similar. Fewer than 10% exhibited symptoms with sham exposure. No significant differences were found between the 2 facilities in maximal total rhinoconjunctivitis symptom score, total nasal symptom score, and total ocular symptom score, nor in areas under the curve. In both facilities, no significant effects of cetirizine 10 mg over placebo were detected., Conclusion: The results were equivalent, demonstrating that the 2 CACFs can be used together in dual-center clinical trials and show the possibility of multicenter trials involving multiple CACFs., (Copyright © 2019 American College of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

Phosphatidylethanolamine N-methyltransferase (PEMT) is an important enzyme in hepatic phosphatidylcholine (PC) biosynthesis. Pemt -/- mice fed a high-fat diet are protected from obesity and whole-body insulin resistance. However, Pemt -/- mice develop severe nonalcoholic steatohepatitis (NASH). Because NASH is often associated with hepatic insulin resistance, we investigated whether the increased insulin sensitivity in Pemt -/- mice was restricted to nonhepatic tissues or whether the liver was also insulin sensitive. Strikingly, the livers of Pemt -/- mice compared with those of Pemt +/+ mice were not insulin resistant, despite elevated levels of hepatic triacylglycerols and diacylglycerols, as well as increased hepatic inflammation and fibrosis. Endogenous glucose production was lower in Pemt -/- mice under both basal and hyperinsulinemic conditions. Experiments in primary hepatocytes and hepatoma cells revealed improved insulin signaling in the absence of PEMT, which was not due to changes in diacylglycerols, ceramides, or gangliosides. On the other hand, the phospholipid composition in hepatocytes seems critically important for insulin signaling such that lowering the PC:phosphatidylethanolamine (PE) ratio improves insulin signaling. Thus, treatments to reduce the PC:PE ratio in liver may protect against the development of hepatic insulin resistance.-Van der Veen, J. N., Lingrell, S., McCloskey, N., LeBlond, N. D., Galleguillos, D., Zhao, Y. Y., Curtis, J. M., Sipione, S., Fullerton, M. D., Vance, D. E., Jacobs, R. L. A role for phosphatidylcholine and phosphatidylethanolamine in hepatic insulin signaling.

Information on the inheritance of resistance to Colletotrichum gloeosporioides and C. acutatum hemibiotrophic infections (HBI) in strawberry leaf tissue and the genetic control of anthracnose crown rot (ACR) in crown tissue are relatively unknown. Six parental genotypes were crossed in a half-diallel mating design to generate 15 full-sib families. HBI and ACR experiments were conducted concurrently. Both seedlings and parental clones were inoculated with 1 × 10 6 conidia/ml of C. gloeosporioides or C. acutatum. Percent sporulating leaf area, wilt symptoms, and relative area under the disease progress curve were calculated to characterize resistance among genotypes and full-sib families. Low dominance/additive variance ratios for C. acutatum HBI (0.13) and C. gloeosporioides ACR (0.20) were observed, indicating additive genetic control of resistance to these traits. Heritability estimates were low for C. acutatum HBI (0.25) and C. gloeosporioides HBI (0.16) but moderate for C. gloeosporioides ACR (0.61). A high genetic correlation (r A = 0.98) between resistance to C. acutatum HBI and C. gloeosporioides HBI was observed, suggesting that resistance to these two Colletotrichum spp. may be controlled by common genes in strawberry leaf tissue. In contrast, negative genetic correlations between ACR and both HBI traits (r A = -0.85 and -0.61) suggest that resistance in crown tissue is inherited independently of resistance in leaf tissue in the populations tested. Overall, these findings provide valuable insight into the genetic basis of resistance, and the evaluation and deployment of resistance to HBIs and ACR in strawberry breeding programs.

Background: Group I Paks are serine/threonine kinases that function as major effectors of the small GTPases Rac1 and Cdc42, and they regulate cytoskeletal dynamics, cell polarity, and transcription. We previously demonstrated that Pak1 and Pak2 function redundantly to promote skeletal myoblast differentiation during postnatal development and regeneration in mice. However, the roles of Pak1 and Pak2 in adult muscle homeostasis are unknown. Choline kinase β (Chk β) is important for adult muscle homeostasis, as autosomal recessive mutations in CHKβ are associated with two human muscle diseases, megaconial congenital muscular dystrophy and proximal myopathy with focal depletion of mitochondria., Methods: We analyzed mice conditionally lacking Pak1 and Pak2 in the skeletal muscle lineage (double knockout (dKO) mice) over 1 year of age. Muscle integrity in dKO mice was assessed with histological stains, immunofluorescence, electron microscopy, and western blotting. Assays for mitochondrial respiratory complex function were performed, as was mass spectrometric quantification of products of choline kinase. Mice and cultured myoblasts deficient for choline kinase β (Chk β) were analyzed for Pak1/2 phosphorylation., Results: dKO mice developed an age-related myopathy. By 10 months of age, dKO mouse muscles displayed centrally-nucleated myofibers, fibrosis, and signs of degeneration. Disease severity occurred in a rostrocaudal gradient, hindlimbs more strongly affected than forelimbs. A distinctive feature of this myopathy was elongated and branched intermyofibrillar (megaconial) mitochondria, accompanied by focal mitochondrial depletion in the central region of the fiber. dKO muscles showed reduced mitochondrial respiratory complex I and II activity. These phenotypes resemble those of rmd mice, which lack Chkβ and are a model for human diseases associated with CHKβ deficiency. Pak1/2 and Chkβ activities were not interdependent in mouse skeletal muscle, suggesting a more complex relationship in regulation of mitochondria and muscle homeostasis., Conclusions: Conditional loss of Pak1 and Pak2 in mice resulted in an age-dependent myopathy with similarity to mice and humans with CHKβ deficiency. Protein kinases are major regulators of most biological processes but few have been implicated in muscle maintenance or disease. Pak1/Pak2 dKO mice offer new insights into these processes.

Dietary recommendations for cheese usually promote low (LOW)- over regular (REG)-fat versions due to the saturated fat. Conversely, epidemiological evidence shows that cheese consumption associates with reduced risk of type 2 diabetes. To investigate how cheese influences diabetes-related outcomes, a feeding trial comparing LOW and REG cheese was conducted in high-fat, lard-based diet (HFD)-fed insulin-resistant rats followed by evaluation of potential mechanisms. After 4 weeks of HFD, LOW or REG was added at 7 and 10 g/100 g diet, respectively, for another 8 weeks. Following either an oral glucose or insulin tolerance test to assess glucoregulation, rats were euthanized and serum was collected for metabolomic and lipid analyses. Hepatic tissue was used to measure glucoregulatory enzymes and lipid content. Both LOW and REG improved insulin sensitivity without effect on oral glucose tolerance, insulin secretion or body weight. Serum metabolomics identified 33 metabolites of interest, with 21 being phosphatidylcholines (PCs) or lysophosphatidylcholines (LPCs). HFD rats had significantly reduced LPC C16:1, C17:0, C18:1, C20:3 and C24:0, and these effects were normalized by LOW or REG cheese. Fourteen PC species were lowest in the HFD group and normalized by cheese feeding. Serum choline was elevated sevenfold in HFD- but not cheese-fed rats compared with rats fed low-fat diet. Liver triglyceride was elevated by LOW feeding. In conclusion, inclusion of both LOW and REG cheeses in the diet of insulin-resistant rats improves in vivo glucoregulation. This is associated with altered phospholipid metabolism, including cheese-mediated normalization of species that are decreased by high-fat feeding., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Phosphatidylethanolamine N -methyltransferase (PEMT) is a hepatic integral membrane protein localized to the endoplasmic reticulum (ER). PEMT catalyzes approximately 30% of hepatic phosphatidylcholine (PC) biosynthesis. Pemt -/- mice fed a high-fat diet (HFD) develop steatohepatitis. Interestingly, portions of the ER located close to the canaliculus are enriched in PEMT. Phospholipid balance and asymmetrical distribution by adenosine triphosphatase phospholipid transporting 8B1 (ATP8B1) on the canalicular membrane is required for membrane integrity and biliary processes. We hypothesized that PEMT is an important supplier of PC to the canaliculus and that PEMT activity is critical for the maintenance of canalicular membrane integrity and bile formation following HFD feeding when there is an increase in overall hepatic PC demand. Pemt +/+ and Pemt -/- mice were fed a chow diet, an HFD, or a choline-supplemented HFD. Plasma and hepatic indices of liver function and parameters of bile formation were determined. Pemt -/- mice developed cholestasis, i.e, elevated plasma bile acid (BA) concentrations and decreased biliary secretion rates of BAs and PC, during HFD feeding. The maximal BA secretory rate was reduced more than 70% in HFD-fed Pemt -/- mice. Hepatic ABCB11/bile salt export protein, responsible for BA secretion, was decreased in Pemt -/- mice and appeared to be retained intracellularly. Canalicular membranes of HFD-fed Pemt -/- mice contained fewer invaginations and displayed a smaller surface area than Pemt +/+ mice. Choline supplementation (CS) prevented and reversed the development of HFD-induced cholestasis. Conclusion: We propose that hepatic PC availability is critical for bile formation. Dietary CS might be a potential noninvasive therapy for a specific subset of patients with cholestasis.

Phosphatidylethanolamine N-methyltransferase (PEMT) converts phosphatidylethanolamine (PE) to phosphatidylcholine (PC), mainly in the liver. Pemt -/- mice are protected from high-fat diet (HFD)-induced obesity and insulin resistance, but develop severe non-alcoholic fatty liver disease (NAFLD) when fed a HFD, mostly due to impaired VLDL secretion. Oxidative stress is thought to be an essential factor in the progression from simple steatosis to steatohepatitis. Vitamin E is an antioxidant that has been clinically used to improve NAFLD pathology. Our aim was to determine whether supplementation of the diet with vitamin E could attenuate HFD-induced hepatic steatosis and its progression to NASH in Pemt -/- mice. Treatment with vitamin E (0.5 g/kg) for 3 weeks improved VLDL-TG secretion and normalized cholesterol metabolism, but failed to reduce hepatic TG content. Moreover, vitamin E treatment was able to reduce hepatic oxidative stress, inflammation and fibrosis. We also observed abnormal ceramide metabolism in Pemt -/- mice fed a HFD, with elevation of ceramides and other sphingolipids and higher expression of mRNAs for acid ceramidase (Asah1) and ceramide kinase (Cerk). Interestingly, vitamin E supplementation restored Asah1 and Cerk mRNA and sphingolipid levels. Together this study shows that vitamin E treatment efficiently prevented the progression from simple steatosis to steatohepatitis in mice lacking PEMT., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Rationale: Trade in rhinoceros horn is regulated or banned internationally in recognition of its impact on wild populations worldwide. Enforcement of the laws and regulations depends on successfully identifying when violations occur, which is complicated by the presence of alternative/imitation rhinoceros horn keratin (e.g., bovid horn keratin). In this study, we assess the potential for Direct Analysis in Real Time (DART) ionization paired with Time-Of-Flight Mass Spectrometry (DART-TOFMS) to classify different keratin types from four taxonomic groups: rhinoceros, bovid, domestic horse, and pangolin., Methods: The spectra of 156 keratin samples from all five rhinoceros species (horn keratin), eight genera of bovids (horn keratin), domestic horses (hoof keratin), and all extant species of pangolins (scale keratin) were collected. Fisher ratio analysis identified the most important ions that characterized each class and these ions were used for the training model, which consisted of 143 spectra. Kernel Discriminant Analysis (KDA) was used to classify the different groups., Results: The spectra collected for each taxonomic group are distinctive. The chemotypes demonstrate that the spectra of rhinoceros, bovids, and domestic horse are similar to each other, whereas the chemotypes of pangolins show a different chemical profile. The model built by KDA resolved each taxonomic group: 95% of samples were correctly assigned using leave-one-out cross validation. The 13 blind samples not used in model development were all correctly classified to taxonomic source., Conclusions: DART-TOFMS appears to be a reliable approach for taxonomic identification of keratin. This analysis can be carried out with a small sliver of keratin, with minimal sample preparation, inexpensively and quickly, making it a potential valuable tool for identification of rhinoceros horn and other keratin types., (Published 2018. This article is a US Government work and is in the public domain in the USA.)

The varied answers to the question "What is a species?" provoke more than lively debates in academic circles. They pose practical problems for law enforcement. Commercial wildlife trade threatens many primate species and is regulated through such laws and international agreements as the U.S. Endangered Species Act and the Convention on International Trade in Endangered Species of Wild Fauna and Flora. Enforcing legislation relies on the ability to identify when violations occur. Species-defining characters may not be preserved in wildlife trade items. For example, pelage patterns and behavioral characters (e.g., vocalizations) are absent from skulls. Accordingly, identifying victims of illegal trade can be difficult, which hinders enforcement. Moreover, identifying new species and "splitting" of currently recognized species can result in enforcement lags and regulatory loopholes. Although such negative consequences should not hinder scientific advancement, we suggest that they be considered by primate taxonomists and provide recommendations to prevent unintended conservation consequences., (Published 2018. This article is a U.S. Government work and is in the public domain in the USA.)

Cyclic fatty acid monomers (CFAM) generated through domestic or industrial heating of vegetable oils may alter liver enzymes and induce hepatomegaly and steatosis, but the underlying mechanisms are not clearly understood. This study aimed to assess the effects of CFAM on liver and plasma lipids and to determine whether these effects are modulated by dietary lipids. Thirty-six (36) male Wistar rats were fed either of the four isoenergetic diets consisting of canola oil or soybean oil with/without 500 mg/100 g CFAM of total fat for 28 days. Rats fed CFAM had higher liver total lipids ( p  =   0.03) and triacylglycerols (TAG) ( p  =   0.02), but less hepatic phosphatidylcholine ( p  =   0.02) compared to those fed the non-CFAM diets. CFAM did not alter liver phosphatidylethanolamine N -methyltransferase (PEMT) activity and CTP: phosphocholine cytidylyltransferase (CT-α) protein levels. Rats fed CFAM diets had higher levels of plasma total cholesterol (TC), VLDL + LDL cholesterol, higher ratio of TC to HDL cholesterol, and lower levels of HDL cholesterol compared with rats fed non-CFAM diets ( p  <   0.05). Plasma alanine transaminase (ALT) was decreased with CFAM, but plasma insulin, glucose, and TAG did not vary among the four diet groups ( p  <   0.05). Rats fed canola oil and CFAM had higher plasma levels of aspartate transaminase (AST) and AST/ALT ratio compared with the other three diet groups. These results indicate that CFAM may provoke an accumulation of TAG in the liver related to a decrease in phosphatidylcholine (PC) levels, but the effect of CFAM on PC concentrations may not occur through impairment of the two main PC biosynthesis pathways.

Background: Phosphatidylethanolamine N-methyltransferase (PEMT) converts phosphatidylethanolamine to phosphatidylcholine. Pemt-/-/low density lipoprotein receptor (Ldlr)-/- mice have significantly reduced plasma lipids and are protected against atherosclerosis. Recent studies have shown that choline can be metabolized by the gut flora into trimethylamine-N-oxide (TMAO), which is an emerging risk factor for atherosclerosis., Objective: The objective of this study was to determine whether ectopic hepatic PEMT expression or choline supplementation would promote atherosclerosis in Pemt-/-/Ldlr-/- mice., Methods: Male 8- to 10-wk-old Pemt+/+/Ldlr-/- (SKO) and Pemt-/-/Ldlr-/- (DKO) mice were injected with an adeno-associated virus (AAV) expressing green fluorescent protein (GFP) or human PEMT and fed a Western diet (40% of calories from fat, 0.5% cholesterol) for 8 wk. In a separate experiment, 8- to 10-wk-old SKO and half of the DKO male mice were fed a Western diet with normal (3 g/kg) choline for 12 wk. The remaining DKO mice [choline-supplemented (CS) DKO] were fed a CS Western diet (10 g choline/kg). Plasma lipid concentrations, choline metabolites, and aortic atherosclerosis were measured., Results: Plasma cholesterol, plasma TMAO, and aortic atherosclerosis were reduced by 60%, 40%, and 80%, respectively, in DKO mice compared with SKO mice. AAV-PEMT administration increased plasma cholesterol and TMAO by 30% and 40%, respectively, in DKO mice compared with AAV-GFP-treated DKO mice. Furthermore, AAV-PEMT-injected DKO mice developed atherosclerotic lesions similar to SKO mice. In the second study, there was no difference in atherosclerosis or plasma cholesterol between DKO and CS-DKO mice. However, plasma TMAO concentrations were increased 2.5-fold in CS-DKO mice compared with DKO mice., Conclusions: Reintroducing hepatic PEMT reversed the atheroprotective phenotype of DKO mice. Choline supplementation did not increase atherosclerosis or plasma cholesterol in DKO mice. Our data suggest that plasma TMAO does not induce atherosclerosis when plasma cholesterol is low. Furthermore, this is the first report to our knowledge that suggests that de novo choline synthesis alters TMAO status.

Purpose: The present study was designed to determine the effects of both choline form and availability on maternal immune function during lactation., Methods: Sprague-Dawley rats were randomized to one of the three diets 24-48 h before parturition and fed ad libitum until 21 days postnatal: 1 g/kg choline as free choline (C, n = 11), the current form, and amount of choline in commercial diets; 1 g/kg choline as phosphatidylcholine (PC1, n = 11); or 2.5 g/kg choline as PC (PC2.5, n = 8). Choline metabolites in offspring stomach contents were quantified. At 21 days, lymphocytes from mothers' mesenteric lymph nodes and spleens were isolated and phenotypes and ex vivo cytokine production after mitogen exposure were determined., Results: There was a higher proportion of choline and a lower proportion of lyso-PC in stomach contents (representing dam's milk) of C pups compared to PC1. In the mesenteric lymph nodes, feeding PC1 compared to C led to a higher IL-2 production after Concanavalin A (ConA) stimulation and a higher proportion of T cells (CD3+) and a lower proportion of B cells [immunoglobulin (Ig)κ, CD45RA+, and IgM+; P < 0.05]. Splenocytes from the PC1 group produced more IL-6 and TNF-α after lipopolysaccharides stimulation compared to C (P < 0.05). Splenocytes from the PC2.5 group produced more IL-2 and IL-6 after ConA stimulation compared to PC1 (P < 0.05)., Conclusions: Feeding choline as PC in the maternal diet improved the ability of immune cells to respond ex vivo to mitogens and increasing the amount of PC in the diet further improved T cell proliferation.

De novo phosphatidylcholine (PC) synthesis via CTP:phosphocholine cytidylyltransferase-α (CTα) is required for VLDL secretion. To determine the precise role of de novo PC synthesis in intestinal lipid metabolism, we deleted CTα exclusively in the intestinal epithelium of mice (CTα IKO mice). When fed a chow diet, CTα IKO mice showed normal fat absorption despite a ∼30% decrease in intestinal PC concentrations relative to control mice, suggesting that biliary PC can fully support chylomicron secretion under these conditions. However, when fed a high-fat diet, CTα IKO mice showed impaired passage of FAs and cholesterol from the intestinal lumen into enterocytes. Impaired intestinal lipid uptake in CTα IKO mice was associated with lower plasma triglyceride concentrations, higher plasma glucagon-like peptide 1 and peptide YY, and disruption of intestinal membrane lipid transporters after a high-fat meal relative to control mice. Unexpectedly, biliary bile acid and PC secretion was enhanced in CTα IKO mice due to a shift in expression of bile-acid transporters to the proximal intestine, indicative of accelerated enterohepatic cycling. These data show that intestinal de novo PC synthesis is required for dietary lipid absorption during high-fat feeding and that the reacylation of biliary lyso-PC cannot compensate for loss of CTα under these conditions., (Copyright © 2018 Kennelly et al.)

Food protein and lipid based nanoparticles have attracted recent interest as a means of delivering nutraceuticals. Nanoparticle encapsulation of nutraceuticals faces challenges to overcome for it to be readily applied in the food industry, such as low encapsulation efficiency for hydrophilic compounds and poor stability once in the gastrointestinal tract. This research introduces a new protein-lipid composite nanoparticle with a three-layered structure (a barley protein layer, α-tocopherol layer and phospholipid layer) and an inner aqueous compartment to load hydrophilic nutraceuticals. This delivery system showed efficient encapsulation of vitamin B12 (69%) and controlled release behavior in simulated gastrointestinal media. An in vitro cell evaluation demonstrated that nanoparticles could internalize into Caco-2 cells via energy-dependent endocytosis and significantly increase the uptake and transport efficiency of vitamin B12 in this model. In vivo, the developed vitamin B12 loaded nanoparticle showed increased serum vitamin B12 levels upon oral administration and reduced the methylmalonic acid level more efficiently than the free form in rats. A 14-day in vivo toxicity study showed no evidence of toxicity in rats implying the safety of the developed nanoparticles in long term use. Overall, the results of this study show the great potential of the developed nanoparticles in increasing the absorption of vitamin B12 upon oral administration.

Genetic analyses of parentage sometimes reveal that "socially monogamous" (pair-living) species do not reside in strict family groups. Circumstances such as adult turnovers and extra-pair copulations, among others, may result in non-nuclear families. These genetic relationships within groups have implications for interpreting social behaviors. Red-bellied lemurs (Eulemur rubriventer) live in groups generally comprising an adult male-female pair plus immatures, and early genetic analyses of parentage in a relatively small sample suggested they mate monogamously. However, previous research on this taxon has also identified scenarios in which non-nuclear families might result, such as adult turnovers. To assess the potential occurrence of non-nuclear families in this "socially monogamous" taxon, as well as the social conditions under which they might occur, we combined behavioral observations of wild red-bellied lemurs in Ranomafana National Park with genetic parentage analysis of immatures from 17 groups. We found that the majority of groups (75%) represented nuclear family groups. However, 25% of groups represented non-nuclear families at some point during the study. The social factors that resulted in non-nuclear families were varied and included at least one adult turnover, and potentially delayed female dispersals and extra-pair copulations. Our results suggest that red-bellied lemurs are generally reproductively monogamous, with only limited evidence that non-nuclear families result from non-monogamous reproduction. However, similar to other pair-living primates, red-bellied lemurs appear to exhibit flexibility in their social organization and mating strategies. Multiple lines of evidence should be considered when inferring parent-offspring relationships within pair-living groups., (© 2018 Wiley Periodicals, Inc.)