Relationship between a common variant in the fatty acid desaturase (FADS) cluster and eicosanoid generation in humans.

Dramatic shifts in the Western diet have led to a marked increase in the dietary intake of the n-6 polyunsaturated fatty acid (PUFA), linoleic acid (LA). Dietary LA can then be converted to arachidonic acid (ARA) utilizing three enzymatic steps. Two of these steps are encoded for by the fatty acid desaturase (FADS) cluster (chromosome 11, 11q12.2-q13) and certain genetic variants within the cluster are highly associated with ARA levels. However, no study to date has examined whether these variants further influence pro-inflammatory, cyclooxygenase and lipoxygenase eicosanoid products. This study examined the impact of a highly influential FADS SNP, rs174537 on leukotriene, HETE, prostaglandin, and thromboxane biosynthesis in stimulated whole blood. Thirty subjects were genotyped at rs174537 (GG, n = 11; GT, n = 13; TT, n = 6), a panel of fatty acids from whole serum was analyzed, and precursor-to-product PUFA ratios were calculated as a marker of the capacity of tissues (particularly the liver) to synthesize long chain PUFAs. Eicosanoids produced by stimulated human blood were measured by LC-MS/MS. We observed an association between rs174537 and the ratio of ARA/LA, leukotriene B4, and 5-HETE but no effect on levels of cyclooxygenase products. Our results suggest that variation at rs174537 not only impacts the synthesis of ARA but the overall capacity of whole blood to synthesize 5-lipoxygenase products; these genotype-related changes in eicosanoid levels could have important implications in a variety of inflammatory diseases.

Global analysis of condition-specific subcellular protein distribution and abundance.

Cellular control of protein activities by modulation of their abundance or compartmentalization is not easily measured on a large scale. We developed and applied a method to globally interrogate these processes that is widely useful for systems-level analyses of dynamic cellular responses in many cell types. The approach involves subcellular fractionation followed by comprehensive proteomic analysis of the fractions, which is enabled by a data-independent acquisition mass spectrometry approach that samples every available mass to charge channel systematically to maximize sensitivity. Next, various fraction-enrichment ratios are measured for all detected proteins across different environmental conditions and used to group proteins into clusters reflecting changes in compartmentalization and relative conditional abundance. Application of the approach to characterize the response of yeast proteins to fatty acid exposure revealed dynamics of peroxisomes and novel dynamics of MCC/eisosomes, specialized plasma membrane domains comprised of membrane compartment occupied by Can1 (MCC) and eisosome subdomains. It also led to the identification of Fat3, a fatty acid transport protein of the plasma membrane, previously annotated as Ykl187.

Environment-responsive transcription factors bind subtelomeric elements and regulate gene silencing.

Subtelomeric chromatin is subject to evolutionarily conserved complex epigenetic regulation and is implicated in numerous aspects of cellular function including formation of heterochromatin, regulation of stress response pathways and control of lifespan. Subtelomeric DNA is characterized by the presence of specific repeated segments that serve to propagate silencing or to protect chromosomal regions from spreading epigenetic control. In this study, analysis of genome-wide chromatin immunoprecipitation and expression data, suggests that several yeast transcription factors regulate subtelomeric silencing in response to various environmental stimuli through conditional association with proto-silencing regions called X elements. In this context, Oaf1p, Rox1p, Gzf1p and Phd1p control the propagation of silencing toward centromeres in response to stimuli affecting stress responses and metabolism, whereas others, including Adr1p, Yap5p and Msn4p, appear to influence boundaries of silencing, regulating telomere-proximal genes in Y' elements. The factors implicated here are known to control adjacent genes at intrachromosomal positions, suggesting their dual functionality. This study reveals a path for the coordination of subtelomeric silencing with cellular environment, and with activities of other cellular processes.

Low rate of SARS-CoV-2 incident infection identified by weekly screening PCR in a prospective year-long cohort study.

BACKGROUND: Asymptomatic and pre-symptomatic SARS-CoV-2 infections may contribute to ongoing community transmission, however, the benefit of routine screening of asymptomatic individuals in low-risk populations is unclear. METHODS: To identify SARS-CoV-2 infections 553 seronegative individuals were prospectively followed for 52 weeks. From 4/2020-7/2021, participants submitted weekly self-collected nasal swabs for rtPCR and completed symptom and exposure surveys. RESULTS: Incident SARS2-CoV-2 infections were identified in 9/553 (1.6%) participants. Comparisons of SARS2-CoV-2(+) to SARS2-CoV-2(-) participants revealed significantly more close contacts outside the household (median: 5 versus 3; p = 0.005). The incidence of infection was higher among unvaccinated/partially vaccinated than among fully vaccinated participants (9/7,679 versus 0/6,845 person-weeks; p = 0.004). At notification of positive test result, eight cases were symptomatic and one pre-symptomatic. CONCLUSIONS: These data suggest that weekly SARS2-CoV2 surveillance by rtPCR did not efficiently detect pre-symptomatic infections in unvaccinated participants.

Angiotensin II receptor I auto-antibodies following SARS-CoV-2 infection.

BACKGROUND: Coronavirus disease 2019 (COVID-19) is associated with endothelial activation and coagulopathy, which may be related to pre-existing or infection-induced pro-thrombotic autoantibodies such as those targeting angiotensin II type I receptor (AT1R-Ab). METHODS: We compared prevalence and levels of AT1R-Ab in COVID-19 cases with mild or severe disease to age and sex matched negative controls utilizing multivariate logistic and quantile regression adjusted for comorbidities including hypertension, diabetes, and heart disease. RESULTS: There were trends toward increased prevalence (50% vs. 33%, p = 0.1) and level of AT1R-Ab (median 9.8 vs. 6.1 U/mL, p = 0.06) in all cases versus controls. When considered by COVID-19 disease severity, there was a trend toward increased prevalence of AT1R-Ab (55% vs. 31%, p = 0.07), as well as significantly higher AT1R-Ab levels (median 10.7 vs. 5.9 U/mL, p = 0.03) amongst individuals with mild COVID-19 versus matched controls. In contrast, the prevalence (42% vs. 37%, p = 0.9) and level (both medians 6.7 U/mL, p = 0.9) of AT1R-Ab amongst those with severe COVID-19 did not differ from matched controls. CONCLUSIONS: These findings support an association between COVID-19 and AT1R-Ab, emphasizing that vascular pathology may be present in individuals with mild COVID-19 as well as those with severe disease.

Acyl-lipid desaturases and Vipp1 cooperate in cyanobacteria to produce novel omega-3 PUFA-containing glycolipids.

BACKGROUND: Dietary omega-3 (n-3), long chain (LC-, ≥ 20 carbons), polyunsaturated fatty acids (PUFAs) derived largely from marine animal sources protect against inflammatory processes and enhance brain development and function. With the depletion of natural stocks of marine animal sources and an increasing demand for n-3 LC-PUFAs, alternative, sustainable supplies are urgently needed. As a result, n-3 18-carbon and LC-PUFAs are being generated from plant or algal sources, either by engineering new biosynthetic pathways or by augmenting existing systems. RESULTS: We utilized an engineered plasmid encoding two cyanobacterial acyl-lipid desaturases (DesB and DesD, encoding Δ15 and Δ6 desaturases, respectively) and "vesicle-inducing protein in plastids" (Vipp1) to induce production of stearidonic acid (SDA, 18:4 n-3) at high levels in three strains of cyanobacteria (10, 17 and 27% of total lipids in Anabaena sp. PCC7120, Synechococcus sp. PCC7002, and Leptolyngbya sp. strain BL0902, respectively). Lipidomic analysis revealed that in addition to SDA, the rare anti-inflammatory n-3 LC-PUFA eicosatetraenoic acid (ETA, 20:4 n-3) was synthesized in these engineered strains, and ~ 99% of SDA and ETA was complexed to bioavailable monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) species. Importantly, novel molecular species containing alpha-linolenic acid (ALA), SDA and/or ETA in both acyl positions of MGDG and DGDG were observed in the engineered Leptolyngbya and Synechococcus strains, suggesting that these could provide a rich source of anti-inflammatory molecules. CONCLUSIONS: Overall, this technology utilizes solar energy, consumes carbon dioxide, and produces large amounts of nutritionally important n-3 PUFAs and LC-PUFAs. Importantly, it can generate previously undescribed, highly bioavailable, anti-inflammatory galactosyl lipids. This technology could therefore be transformative in protecting ocean fisheries and augmenting the nutritional quality of human and animal food products.

ESCRT-III is required for scissioning new peroxisomes from the endoplasmic reticulum.

Dynamic control of peroxisome proliferation is integral to the peroxisome's many functions. The endoplasmic reticulum (ER) serves as a source of preperoxisomal vesicles (PPVs) that mature into peroxisomes during de novo peroxisome biogenesis and support growth and division of existing peroxisomes. However, the mechanism of PPV formation and release from the ER remains poorly understood. In this study, we show that endosomal sorting complexes required for transport (ESCRT)-III are required to release PPVs budding from the ER into the cytosol. Absence of ESCRT-III proteins impedes de novo peroxisome formation and results in an aberrant peroxisome population in vivo. Using a cell-free PPV budding assay, we show that ESCRT-III proteins Vps20 and Snf7 are necessary to release PPVs from the ER. ESCRT-III is therefore a positive effector of membrane scission for vesicles budding both away from and toward the cytosol. These findings have important implications for the evolutionary timing of emergence of peroxisomes and the rest of the internal membrane architecture of the eukaryotic cell.

Allele-specific methylation in the FADS genomic region in DNA from human saliva, CD4+ cells, and total leukocytes.

Background: Genetic variants within the fatty acid desaturase (FADS) gene cluster (human Chr11) are important regulators of long-chain (LC) polyunsaturated fatty acid (PUFA) biosynthesis in the liver and consequently have been associated with circulating LC-PUFA levels. More recently, epigenetic modifications such as DNA methylation, particularly within the FADS cluster, have been shown to affect LC-PUFA levels. Our lab previously demonstrated strong associations of allele-specific methylation (ASM) between a single nucleotide polymorphism (SNP) rs174537 and CpG sites across the FADS region in human liver tissues. Given that epigenetic signatures are tissue-specific, we aimed to evaluate the methylation status and ASM associations between rs174537 and DNA methylation obtained from human saliva, CD4+ cells and total leukocytes derived from whole blood. The goals were to (1) determine if DNA methylation from these peripheral samples would display similar ASM trends as previously observed in human liver tissues and (2) evaluate the associations between DNA methylation and circulating LC-PUFAs. Results: DNA methylation at six CpG sites spanning FADS1 and FADS2 promoter regions and a putative FADS enhancer region were determined in two Caucasian cohorts of healthy volunteers: leukocytes in cohort 1 (n = 89, median age = 43, 35% male) and saliva and CD4+ cells in cohort 2 (n = 32, median age = 41, 41% male). Significant ASM between rs174537 and DNA methylation at three CpG sites located in the FADS2 promoter region (i.e., chr11:61594865, chr11:61594876, chr11:61594907) and one CpG site in the putative enhancer region (chr11:61587979) were observed with leukocytes. In CD4+ cells, significant ASM was observed at CpG sites chr11:61594876 and chr11:61584894. Genotype at rs174537 was significantly associated with DNA methylation from leukocytes. Similar trends were observed with CD4+ cells, but not with saliva. DNA methylation from leukocytes and CD4+ cells also significantly correlated with circulating omega-6 LC-PUFAs. Conclusions: We observed significant ASM between rs174537 and DNA methylation at key regulatory regions in the FADS region from leukocyte and CD4+ cells. DNA methylation from leukocytes also correlated with circulating omega-6 LC-PUFAs. These results support the use of peripheral whole blood samples, with leukocytes showing the most promise for future nutrigenomic studies evaluating epigenetic modifications affecting LC-PUFA biosynthesis in humans.

An indicator cell assay for blood-based diagnostics.

We have established proof of principle for the Indicator Cell Assay Platform™ (iCAP™), a broadly applicable tool for blood-based diagnostics that uses specifically-selected, standardized cells as biosensors, relying on their innate ability to integrate and respond to diverse signals present in patients' blood. To develop an assay, indicator cells are exposed in vitro to serum from case or control subjects and their global differential response patterns are used to train reliable, disease classifiers based on a small number of features. In a feasibility study, the iCAP detected pre-symptomatic disease in a murine model of amyotrophic lateral sclerosis (ALS) with 94% accuracy (p-Value = 3.81E-6) and correctly identified samples from a murine Huntington's disease model as non-carriers of ALS. Beyond the mouse model, in a preliminary human disease study, the iCAP detected early stage Alzheimer's disease with 72% cross-validated accuracy (p-Value = 3.10E-3). For both assays, iCAP features were enriched for disease-related genes, supporting the assay's relevance for disease research.

Alterations in levels and ratios of n-3 and n-6 polyunsaturated fatty acids in the temporal cortex and liver of vervet monkeys from birth to early adulthood.

Deficiencies in omega-3 (n-3) long chain polyunsaturated fatty acids (LC-PUFAs) and increases in the ratio of omega-6 (n-6) to n-3 LC-PUFAs in brain tissues and blood components have been associated with psychiatric and developmental disorders. Most studies have focused on n-3 LC-PUFA accumulation in the brain from birth until 2years of age, well before the symptomatic onset of such disorders. The current study addresses changes that occur in childhood and adolescence. Postmortem brain (cortical gray matter, inferior temporal lobe; n=50) and liver (n=60) from vervet monkeys fed a uniform diet from birth through young adulthood were collected from archived tissues. Lipids were extracted and fatty acid levels determined. There was a marked reduction in the ratio of n-6 LC-PUFAs, arachidonic acid (ARA) and adrenic acid (ADR), relative to the n-3 LC-PUFA, docosahexaenoic acid (DHA), in temporal cortex lipids from birth to puberty and then a more gradual decrease though adulthood. This decrease in ratio resulted from a 3-fold accumulation of DHA levels while concentrations of ARA remained constant. Early childhood through adolescence appears to be a critical period for DHA accretion in the cortex of vervet monkeys and may represent a vulnerable stage where lack of dietary n-3 LC-PUFAs impacts development in humans.

DNA methylation in an enhancer region of the FADS cluster is associated with FADS activity in human liver.

Levels of omega-6 (n-6) and omega-3 (n-3), long chain polyunsaturated fatty acids (LcPUFAs) such as arachidonic acid (AA; 20:4, n-6), eicosapentaenoic acid (EPA; 20:5, n-3) and docosahexaenoic acid (DHA; 22:6, n-3) impact a wide range of biological activities, including immune signaling, inflammation, and brain development and function. Two desaturase steps (Δ6, encoded by FADS2 and Δ5, encoded by FADS1) are rate limiting in the conversion of dietary essential 18 carbon PUFAs (18C-PUFAs) such as LA (18:2, n-6) to AA and α-linolenic acid (ALA, 18:3, n-3) to EPA and DHA. GWAS and candidate gene studies have consistently identified genetic variants within FADS1 and FADS2 as determinants of desaturase efficiencies and levels of LcPUFAs in circulating, cellular and breast milk lipids. Importantly, these same variants are documented determinants of important cardiovascular disease risk factors (total, LDL, and HDL cholesterol, triglycerides, CRP and proinflammatory eicosanoids). FADS1 and FADS2 lie head-to-head (5' to 5') in a cluster configuration on chromosome 11 (11q12.2). There is considerable linkage disequilibrium (LD) in this region, where multiple SNPs display association with LcPUFA levels. For instance, rs174537, located ∼ 15 kb downstream of FADS1, is associated with both FADS1 desaturase activity and with circulating AA levels (p-value for AA levels = 5.95 × 10(-46)) in humans. To determine if DNA methylation variation impacts FADS activities, we performed genome-wide allele-specific methylation (ASM) with rs174537 in 144 human liver samples. This approach identified highly significant ASM with CpG sites between FADS1 and FADS2 in a putative enhancer signature region, leading to the hypothesis that the phenotypic associations of rs174537 are likely due to methylation differences. In support of this hypothesis, methylation levels of the most significant probe were strongly associated with FADS1 and, to a lesser degree, FADS2 activities.

Multidimensional cost-benefit analysis to guide evidence-based environmental enrichment: providing bedding and foraging substrate to pen-housed monkeys.

Refinement of animal care and housing is an important shared goal-and challenge-of the team of research, veterinary, and animal care personnel charged with ensuring the wellbeing of laboratory animals. This study addresses 2 issues central to decision-making and implementation of environmental enhancement: methods for useful and comprehensive cost analysis and evaluation of engineering, husbandry, and facilities considerations. The study was undertaken to analyze the feasibility and cost of providing wood shavings as a floor cover for pen-housed monkeys. The beneficial effects of bedding for the welfare of laboratory-housed animals have long been validated. Our study illustrates a workable team-based procedure for comprehensive cost analysis of an important environmental enhancement and demonstrates that the animal welfare benefit is accompanied by decreased husbandry costs. An engineering solution to the potential challenge that wood shavings pose in terms of clogging water pipes was successful. Another successful outcome was the reduction in water (estimated at 192,000 gal annually) and chemicals used to clean housing areas. Emphasis on rigorous evaluation and objective measures of cost and benefit, as well as inclusion of the many factors and teams involved in animal research, holds strong potential for building a better foundation from which to contribute effective changes and improvements in laboratory animal welfare. Taken together, the findings of this study demonstrate that team-based, integrative, and scientific evaluation of environmental enhancement is an effective approach to guide selection of strategies with maximal potential for improving animal welfare.