Cardiac Molecular Analysis Reveals Aging-Associated Metabolic Alterations Promoting Glycosaminoglycans Accumulation via Hexosamine Biosynthetic Pathway.

Age is a prominent risk factor for cardiometabolic disease, often leading to heart structural and functional changes. However, precise molecular mechanisms underlying cardiac remodeling and dysfunction exclusively resulting from physiological aging remain elusive. Previous research demonstrated age-related functional alterations in baboons, analogous to humans. The goal of this study is to identify early cardiac molecular alterations preceding functional adaptations, shedding light on the regulation of age-associated changes. Unbiased transcriptomics of left ventricle samples are performed from female baboons aged 7.5-22.1 years (human equivalent ≈30-88 years). Weighted-gene correlation network and pathway enrichment analyses are performed, with histological validation. Modules of transcripts negatively correlated with age implicated declined metabolism-oxidative phosphorylation, tricarboxylic acid cycle, glycolysis, and fatty-acid ß-oxidation. Transcripts positively correlated with age suggested a metabolic shift toward glucose-dependent anabolic pathways, including hexosamine biosynthetic pathway (HBP). This shift is associated with increased glycosaminoglycan synthesis, modification, precursor synthesis via HBP, and extracellular matrix accumulation, verified histologically. Upregulated extracellular matrix-induced signaling coincided with glycosaminoglycan accumulation, followed by cardiac hypertrophy-related pathways. Overall, these findings revealed a transcriptional shift in metabolism favoring glycosaminoglycan accumulation through HBP before cardiac hypertrophy. Unveiling this metabolic shift provides potential targets for age-related cardiac diseases, offering novel insights into early age-related mechanisms.

Comparative lifespan and healthspan of nonhuman primate species common to biomedical research.

There is a critical need to generate age- and sex-specific survival curves to characterize chronological aging consistently across nonhuman primates (NHP) used in biomedical research. Accurate measures of chronological aging are essential for inferences into genetic, demographic, and physiological variables driving differences in NHP lifespan within and between species. Understanding NHP lifespans is relevant to public health because unraveling the demographic, molecular, and clinical bases of health across the life course in translationally relevant NHP species is fundamentally important to the study of human aging. Data from more than 110,000 captive individual NHP were contributed by 15 major research institutions to generate sex-specific Kaplan-Meier survival curves using uniform methods in 12 translational aging models: Callithrix jacchus (common marmoset), Chlorocebus aethiops sabaeus (vervet/African green), Macaca fascicularis (cynomolgus macaque), M. fuscata (Japanese macaque), M. mulatta (rhesus macaque), M. nemestrina (pigtail macaque), M. radiata (bonnet macaque), Pan troglodytes spp. (chimpanzee), Papio hamadryas spp. (baboon), Plecturocebus cupreus (coppery titi monkey), Saguinus oedipus (cotton-top tamarin), and Saimiri spp. (squirrel monkey). After employing strict inclusion criteria, primary analysis results are based on 12,269 NHP that survived to adulthood and died of natural/health-related causes. A secondary analysis was completed for 32,616 NHP that died of any cause. For the primary analyses, we report ages of 25th, 50th, 75th, and 85th percentiles of survival, maximum observed ages, rates of survivorship, and sex-based differences captured by quantile regression models and Kolmogorov-Smirnov tests. Our findings show a pattern of reduced male survival among catarrhines (African and Asian primates), especially macaques, but not platyrrhines (Central and South American primates). For many species, median lifespans were lower than previously reported. An important consideration is that these analyses may offer a better reflection of healthspan than lifespan. Captive NHP used in research are typically euthanized for humane welfare reasons before their natural end of life, often after diagnosis of their first major disease requiring long-term treatment with reduced quality of life (e.g., endometriosis, cancer, osteoarthritis). Supporting the idea that these data are capturing healthspan, for several species typical age at onset of chronic disease is similar to the median lifespan estimates. This data resource represents the most comprehensive characterization of sex-specific lifespan and age-at-death distributions for 12 biomedically relevant species, to date. The results clarify the relationships among NHP ages and will provide a valuable resource for the aging research community, improving human-NHP age equivalencies, informing investigators of the expected survival rates of NHP assigned to studies, providing a metric for comparisons in future studies, and contributing to our understanding of the factors that drive lifespan differences within and among species.

Perinatal maternal undernutrition in baboons modulates hepatic mitochondrial function but not metabolites in aging offspring.

We previously demonstrated in baboons that maternal undernutrition (MUN), achieved by 70 % of control nutrition, impairs fetal liver function, but long-term changes associated with aging in this model remain unexplored. Here, we assessed clinical phenotypes of liver function, mitochondrial bioenergetics, and protein abundance in adult male and female baboons exposed to MUN during pregnancy and lactation and their control counterparts. Plasma liver enzymes were assessed enzymatically. Liver glycogen, choline, and lipid concentrations were quantified by magnetic resonance spectroscopy. Mitochondrial respiration in primary hepatocytes under standard culture conditions and in response to metabolic (1 mM glucose) and oxidative (100 µM H2O2) stress were assessed with Seahorse XFe96. Hepatocyte mitochondrial membrane potential (MMP) and protein abundance were determined by tetramethylrhodamine ethyl ester staining and immunoblotting, respectively. Liver enzymes and metabolite concentrations were largely unaffected by MUN, except for higher aspartate aminotransferase levels in MUN offspring when male and female data were combined. Oxygen consumption rate, extracellular acidification rate, and MMP were significantly higher in male MUN offspring relative to control animals under standard culture. However, in females, cellular respiration was similar in control and MUN offspring. In response to low glucose challenge, only control male hepatocytes were resistant to low glucose-stimulated increase in basal and ATP-linked respiration. H2O2 did not affect hepatocyte mitochondrial respiration. Protein markers of mitochondrial respiratory chain subunits, biogenesis, dynamics, and antioxidant enzymes were unchanged. Male-specific increases in mitochondrial bioenergetics in MUN offspring may be associated with increased energy demand in these animals. The similarity in systemic liver parameters suggests that changes in hepatocyte bioenergetics capacity precede detectable circulatory hepatic defects in MUN offspring and that the mitochondria may be an orchestrator of liver programming outcome.

Differential mitochondrial bioenergetics and cellular resilience in astrocytes, hepatocytes, and fibroblasts from aging baboons.

Biological resilience, broadly defined as the ability to recover from an acute challenge and return to homeostasis, is of growing importance to the biology of aging. At the cellular level, there is variability across tissue types in resilience and these differences are likely to contribute to tissue aging rate disparities. However, there are challenges in addressing these cell-type differences at regional, tissue, and subject level. To address this question, we established primary cells from aged male and female baboons between 13.3 and 17.8 years spanning across different tissues, tissue regions, and cell types including (1) fibroblasts from skin and from the heart separated into the left ventricle (LV), right ventricle (RV), left atrium (LA), and right atrium (RA); (2) astrocytes from the prefrontal cortex and hippocampus; and (3) hepatocytes. Primary cells were characterized by their cell surface markers and their cellular respiration was assessed with Seahorse XFe96. Cellular resilience was assessed by modifying a live-cell imaging approach; we previously reported that monitors proliferation of dividing cells following response and recovery to oxidative (50 µM-H2O2), metabolic (1 mM-glucose), and proteostasis (0.1 µM-thapsigargin) stress. We noted significant differences even among similar cell types that are dependent on tissue source and the diversity in cellular response is stressor-specific. For example, astrocytes had a higher oxygen consumption rate and exhibited greater resilience to oxidative stress (OS) than both fibroblasts and hepatocytes. RV and RA fibroblasts were less resilient to OS compared with LV and LA, respectively. Skin fibroblasts were less impacted by proteostasis stress compared to astrocytes and cardiac fibroblasts. Future studies will test the functional relationship of these outcomes to the age and developmental status of donors as potential predictive markers.

Genetic characterization of a captive marmoset (Callithrix jacchus) colony using genotype-by-sequencing.

The marmoset is a fundamental nonhuman primate model for the study of aging, neurobiology, and many other topics. Genetic management of captive marmoset colonies is complicated by frequent chimerism in the blood and other tissues, a lack of tools to enable cost-effective, genome-wide interrogation of variation, and historic mergers and migrations of animals between colonies. We implemented genotype-by-sequencing (GBS) of hair follicle derived DNA (a minimally chimeric DNA source) of 82 marmosets housed at the Southwest National Primate Research Center (SNPRC). Our primary goals were the genetic characterization of our marmoset population for pedigree verification and colony management and to inform the scientific community of the functional genetic makeup of this valuable resource. We used the GBS data to reconstruct the genetic legacy of recent mergers between colonies, to identify genetically related animals whose relationships were previously unknown due to incomplete pedigree information, and to show that animals in the SNPRC colony appear to exhibit low levels of inbreeding. Of the >99,000 single-nucleotide variants (SNVs) that we characterized, >9800 are located within gene regions known to harbor pathogenic variants of clinical significance in humans. Overall, we show the combination of low-resolution (sparse) genotyping using hair follicle DNA is a powerful strategy for the genetic management of captive marmoset colonies and for identifying potential SNVs for the development of biomedical research models.

Genetic regulatory effects in response to a high-cholesterol, high-fat diet in baboons.

Steady-state expression quantitative trait loci (eQTLs) explain only a fraction of disease-associated loci identified through genome-wide association studies (GWASs), while eQTLs involved in gene-by-environment (GxE) interactions have rarely been characterized in humans due to experimental challenges. Using a baboon model, we found hundreds of eQTLs that emerge in adipose, liver, and muscle after prolonged exposure to high dietary fat and cholesterol. Diet-responsive eQTLs exhibit genomic localization and genic features that are distinct from steady-state eQTLs. Furthermore, the human orthologs associated with diet-responsive eQTLs are enriched for GWAS genes associated with human metabolic traits, suggesting that context-responsive eQTLs with more complex regulatory effects are likely to explain GWAS hits that do not seem to overlap with standard eQTLs. Our results highlight the complexity of genetic regulatory effects and the potential of eQTLs with disease-relevant GxE interactions in enhancing the understanding of GWAS signals for human complex disease using non-human primate models.

Differential mitochondrial bioenergetics and cellular resilience in astrocytes, hepatocytes, and fibroblasts from aging baboons.

Biological resilience, broadly defined as ability to recover from acute challenge and return to homeostasis, is of growing importance to the biology of aging. At the cellular level, there is variability across tissue types in resilience and these differences likely to contribute to tissue aging rate disparities. However, there are challenges in addressing these cell-type differences at regional, tissue and subject level. To address this question, we established primary cells from aged male and female baboons between 13.3-17.8 years spanning across different tissues, tissue regions, and cell types including: (1) fibroblasts from skin and from heart separated into left ventricle (LV), right ventricle (RV), left atrium (LA) and right atrium (RA), (2) astrocytes from the prefrontal cortex and hippocampus and (3) hepatocytes. Primary cells were characterized by their cell surface markers and their cellular respiration assessed with Seahorse XFe96. Cellular resilience was assessed by modifying a live-cell imaging approach we previously reported that monitors proliferation of dividing cells following response and recovery to oxidative (50µM-H2O2), metabolic (1mM-glucose) and proteostasis (0.1µM-thapsigargin) stress. We noted significant differences even among similar cell types that are dependent on tissue source and the diversity in cellular response is stressor specific. For example, astrocytes were more energetic and exhibited greater resilience to oxidative stress (OS) than both fibroblasts and hepatocytes. RV and RA fibroblasts were less resilient to OS compared with LV and LA respectively. Skin fibroblasts were less impacted by proteostasis stress compared to astrocytes and cardiac fibroblasts. Future studies will test the functional relationship of these outcomes to age and developmental status of donors as potential predictive markers.

Female baboon adrenal zona fasciculata and zona reticularis regulatory and functional proteins decrease across the life course.

Debate exists on life-course adrenocortical zonal function trajectories. Rapid, phasic blood steroid concentration changes, such as circadian rhythms and acute stress responses, complicate quantification. To avoid pitfalls and account for life-stage changes in adrenocortical activity indices, we quantified zonae fasciculata (ZF) and reticularis (ZR) across the life-course, by immunohistochemistry of key regulatory and functional proteins. In 28 female baboon adrenals (7.5-22.1 years), we quantified 12 key proteins involved in cell metabolism, division, proliferation, steroidogenesis (including steroid acute regulatory protein, StAR), oxidative stress, and glucocorticoid and mitochondrial function. Life-course abundance of ten ZF proteins decreased with age. Cell cycle inhibitor and oxidative stress markers increased. Seven of the 12 proteins changed in the same direction for ZR and ZF. Importantly, ZF StAR decreased, while ZR StAR was unchanged. Findings indicate ZF function decreased, and less markedly ZR function, with age. Causes and aging consequences of these changes remain to be determined.

Cardiac Molecular Analysis Reveals Aging-Associated Metabolic Alterations Promoting Glycosaminoglycans Accumulation Via Hexosamine Biosynthetic Pathway.

Age is a prominent risk factor for cardiometabolic disease, and often leads to heart structural and functional changes. However, precise molecular mechanisms underlying cardiac remodeling and dysfunction resulting from physiological aging per se remain elusive. Understanding these mechanisms requires biological models with optimal translation to humans. Previous research demonstrated that baboons undergo age-related reduction in ejection fraction and increased heart sphericity, mirroring changes observed in humans. The goal of this study was to identify early cardiac molecular alterations that precede functional adaptations, shedding light on the regulation of age-associated changes. We performed unbiased transcriptomics of left ventricle (LV) samples from female baboons aged 7.5-22.1 years (human equivalent ~30-88 years). Weighted-gene correlation network and pathway enrichment analyses were performed to identify potential age-associated mechanisms in LV, with histological validation. Myocardial modules of transcripts negatively associated with age were primarily enriched for cardiac metabolism, including oxidative phosphorylation, tricarboxylic acid cycle, glycolysis, and fatty-acid ß-oxidation. Transcripts positively correlated with age suggest upregulation of glucose uptake, pentose phosphate pathway, and hexosamine biosynthetic pathway (HBP), indicating a metabolic shift towards glucose-dependent anabolic pathways. Upregulation of HBP commonly results in increased glycosaminoglycan precursor synthesis. Transcripts involved in glycosaminoglycan synthesis, modification, and intermediate metabolism were also upregulated in older animals, while glycosaminoglycan degradation transcripts were downregulated with age. These alterations would promote glycosaminoglycan accumulation, which was verified histologically. Upregulation of extracellular matrix (ECM)-induced signaling pathways temporally coincided with glycosaminoglycan accumulation. We found a subsequent upregulation of cardiac hypertrophy-related pathways and an increase in cardiomyocyte width. Overall, our findings revealed a transcriptional shift in metabolism from catabolic to anabolic pathways that leads to ECM glycosaminoglycan accumulation through HBP prior to upregulation of transcripts of cardiac hypertrophy-related pathways. This study illuminates cellular mechanisms that precede development of cardiac hypertrophy, providing novel potential targets to remediate age-related cardiac diseases.

Characterizing Early Cardiac Metabolic Programming via 30% Maternal Nutrient Reduction during Fetal Development in a Non-Human Primate Model.

Intra-uterine growth restriction (IUGR) is a common cause of fetal/neonatal morbidity and mortality and is associated with increased offspring predisposition for cardiovascular disease (CVD) development. Mitochondria are essential organelles in maintaining cardiac function, and thus, fetal cardiac mitochondria could be responsive to the IUGR environment. In this study, we investigated whether in utero fetal cardiac mitochondrial programming can be detectable in an early stage of IUGR pregnancy. Using a well-established nonhuman IUGR primate model, we induced IUGR by reducing by 30% the maternal diet (MNR), both in males (MNR-M) and in female (MNR-F) fetuses. Fetal cardiac left ventricle (LV) tissue and blood were collected at 90 days of gestation (0.5 gestation, 0.5 G). Blood biochemical parameters were determined and heart LV mitochondrial biology assessed. MNR fetus biochemical blood parameters confirm an early fetal response to MNR. In addition, we show that in utero cardiac mitochondrial MNR adaptations are already detectable at this early stage, in a sex-divergent way. MNR induced alterations in the cardiac gene expression of oxidative phosphorylation (OXPHOS) subunits (mostly for complex-I, III, and ATP synthase), along with increased protein content for complex-I, -III, and -IV subunits only for MNR-M in comparison with male controls, highlight the fetal cardiac sex-divergent response to MNR. At this fetal stage, no major alterations were detected in mitochondrial DNA copy number nor markers for oxidative stress. This study shows that in 90-day nonhuman primate fetuses, a 30% decrease in maternal nutrition generated early in utero adaptations in fetal blood biochemical parameters and sex-specific alterations in cardiac left ventricle gene and protein expression profiles, affecting predominantly OXPHOS subunits. Since the OXPHOS system is determinant for energy production in mitochondria, our findings suggest that these early IUGR-induced mitochondrial adaptations play a role in offspring's mitochondrial dysfunction and can increase predisposition to CVD in a sex-specific way.

Integrated multi-omics analysis of brain aging in female nonhuman primates reveals altered signaling pathways relevant to age-related disorders.

The prefrontal cortex (PFC) has been implicated as a key brain region responsible for age-related cognitive decline. Little is known about aging-related molecular changes in PFC that may mediate these effects. To date, no studies have used untargeted discovery methods with integrated analyses to determine PFC molecular changes in healthy female primates. We quantified PFC changes associated with healthy aging in female baboons by integrating multiple omics data types (transcriptomics, proteomics, metabolomics) from samples across the adult age span. Our integrated omics approach using unbiased weighted gene co-expression network analysis to integrate data and treat age as a continuous variable, revealed highly interconnected known and novel pathways associated with PFC aging. We found Gamma-aminobutyric acid (GABA) tissue content associated with these signaling pathways, providing 1 potential biomarker to assess PFC changes with age. These highly coordinated pathway changes during aging may represent early steps for aging-related decline in PFC functions, such as learning and memory, and provide potential biomarkers to assess cognitive status in humans.

Multi-omics Analysis of Aging Liver Reveals Changes in Endoplasmic Stress and Degradation Pathways in Female Nonhuman Primates.

The liver is critical for functions that support metabolism, immunity, digestion, detoxification, and vitamin storage. Aging is associated with severity and poor prognosis of various liver diseases such as nonalcoholic fatty liver disease (NAFLD). Previous studies have used multi-omic approaches to study liver diseases or to examine the effects of aging on the liver. However, to date, no studies have used an integrated omics approach to investigate aging-associated molecular changes in the livers of healthy female nonhuman primates. The goal of this study was to identify molecular changes associated with healthy aging in the livers of female baboons ( Papio sp., n=35) by integrating multiple omics data types (transcriptomics, proteomics, metabolomics) from samples across the adult age span. To integrate omics data, we performed unbiased weighted gene co-expression network analysis (WGCNA), and the results revealed 3 modules containing 3,149 genes and 33 proteins were positively correlated with age, and 2 modules containing 37 genes and 216 proteins were negatively correlated with age. Pathway enrichment analysis showed that unfolded protein response (UPR) and endoplasmic reticulum (ER) stress were positively associated with age, whereas xenobiotic metabolism and melatonin and serotonin degradation pathways were negatively associated with age. The findings of our study suggest that UPR and a reduction in reactive oxygen species generated from serotonin degradation could protect the liver from oxidative stress during the aging process in healthy female baboons.

Genetic regulatory effects in response to a high cholesterol, high fat diet in baboons.

Steady-state expression quantitative trait loci (eQTLs) explain only a fraction of disease-associated loci identified through genome-wide association studies (GWAS), while eQTLs involved in gene-by-environment (GxE) interactions have rarely been characterized in humans due to experimental challenges. Using a baboon model, we found hundreds of eQTLs that emerge in adipose, liver, and muscle after prolonged exposure to high dietary fat and cholesterol. Diet-responsive eQTLs exhibit genomic localization and genic features that are distinct from steady-state eQTLs. Furthermore, the human orthologs associated with diet-responsive eQTLs are enriched for GWAS genes associated with human metabolic traits, suggesting that context-responsive eQTLs with more complex regulatory effects are likely to explain GWAS hits that do not seem to overlap with standard eQTLs. Our results highlight the complexity of genetic regulatory effects and the potential of eQTLs with disease-relevant GxE interactions in enhancing the understanding of GWAS signals for human complex disease using nonhuman primate models.

Maternal obesity alters the placental transcriptome in a fetal sex-dependent manner.

Infants born to obese mothers have an increased risk of developing obesity and metabolic diseases in childhood and adulthood. Although the molecular mechanisms linking maternal obesity during pregnancy to the development of metabolic diseases in offspring are poorly understood, evidence suggests that changes in the placental function may play a role. Using a mouse model of diet-induced obesity with fetal overgrowth, we performed RNA-seq analysis at embryonic day 18.5 to identify genes differentially expressed in the placentas of obese and normal-weight dams (controls). In male placentas, 511 genes were upregulated and 791 genes were downregulated in response to maternal obesity. In female placentas, 722 genes were downregulated and 474 genes were upregulated in response to maternal obesity. The top canonical pathway downregulated in maternal obesity in male placentas was oxidative phosphorylation. In contrast, sirtuin signaling, NF-kB signaling, phosphatidylinositol, and fatty acid degradation were upregulated. In female placentas, the top canonical pathways downregulated in maternal obesity were triacylglycerol biosynthesis, glycerophospholipid metabolism, and endocytosis. In contrast, bone morphogenetic protein, TNF, and MAPK signaling were upregulated in the female placentas of the obese group. In agreement with RNA-seq data, the expression of proteins associated with oxidative phosphorylation was downregulated in male but not female placentas of obese mice. Similarly, sex-specific changes in the protein expression of mitochondrial complexes were found in placentas collected from obese women delivering large-for-gestational-age (LGA) babies. In conclusion, maternal obesity with fetal overgrowth differentially regulates the placental transcriptome in male and female placentas, including genes involved in oxidative phosphorylation.

Development of a novel air-liquid interface airway tissue equivalent model for in vitro respiratory modeling studies.

The human airways are complex structures with important interactions between cells, extracellular matrix (ECM) proteins and the biomechanical microenvironment. A robust, well-differentiated in vitro culture system that accurately models these interactions would provide a useful tool for studying normal and pathological airway biology. Here, we report the development and characterization of a physiologically relevant air-liquid interface (ALI) 3D airway 'organ tissue equivalent' (OTE) model with three novel features: native pulmonary fibroblasts, solubilized lung ECM, and hydrogel substrate with tunable stiffness and porosity. We demonstrate the versatility of the OTE model by evaluating the impact of these features on human bronchial epithelial (HBE) cell phenotype. Variations of this model were analyzed during 28 days of ALI culture by evaluating epithelial confluence, trans-epithelial electrical resistance, and epithelial phenotype via multispectral immuno-histochemistry and next-generation sequencing. Cultures that included both solubilized lung ECM and native pulmonary fibroblasts within the hydrogel substrate formed well-differentiated ALI cultures that maintained a barrier function and expressed mature epithelial markers relating to goblet, club, and ciliated cells. Modulation of hydrogel stiffness did not negatively impact HBE differentiation and could be a valuable variable to alter epithelial phenotype. This study highlights the feasibility and versatility of a 3D airway OTE model to model the multiple components of the human airway 3D microenvironment.

Moderate maternal nutrient reduction in pregnancy alters fatty acid oxidation and RNA splicing in the nonhuman primate fetal liver.

Fetal liver tissue collected from a nonhuman primate (NHP) baboon model of maternal nutrient reduction (MNR) at four gestational time points (90, 120, 140, and 165 days gestation [dG], term in the baboon is ∼185 dG) was used to quantify MNR effects on the fetal liver transcriptome. 28 transcripts demonstrated different expression patterns between MNR and control livers during the second half of gestation, a developmental period when the fetus undergoes rapid weight gain and fat accumulation. Differentially expressed transcripts were enriched for fatty acid oxidation and RNA splicing-related pathways. Increased RNA splicing activity in MNR was reflected in greater abundances of transcript splice variant isoforms in the MNR group. It can be hypothesized that the increase in splice variants is deployed in an effort to adapt to the poor in utero environment and ensure near-normal development and energy metabolism. This study is the first to study developmental programming across four critical gestational stages during primate fetal liver development and reveals a potentially novel cellular response mechanism mediating fetal programming in response to MNR.

Potential miRNA biomarkers and therapeutic targets for early atherosclerotic lesions.

Identification of potential therapeutic targets and biomarkers indicative of burden of early atherosclerosis that occur prior to advancement to life-threatening unstable plaques is the key to eradication of CAD prevalence and incidences. We challenged 16 baboons with a high cholesterol, high fat diet for 2 years and evaluated early-stage atherosclerotic lesions (fatty streaks, FS, and fibrous plaques, FP) in formalin-fixed common iliac arteries (CIA). We used small RNA sequencing to identify expressed miRNAs in CIA and in baseline blood samples of the same animals. We found 412 expressed miRNAs in CIA and 356 in blood samples. Eight miRNAs (miR-7975, -486-5p, -451a, -191-5p, -148a-3p, -17-5p, -378c, and -144-3p) were differentially expressed between paired fatty streak lesion and no-lesion sites of the tissue, and 27 miRNAs (e.g., miR-92a-3p, -5001, -342-3p, miR-28-3p, -21-5p, -221-3p, 146a-5p, and -16-5p) in fibrous plaques. The expression of 14 blood miRNAs significantly correlated with extent of lesions and the number of plaques. We identified coordinately regulated miRNA-gene networks in which miR-17-5p and miR-146a-5p are central hubs and miR-5001 and miR-7975 are potentially novel miRNAs associated with early atherosclerosis. In summary, we have identified miRNAs expressed in lesions and in blood that correlate with lesion burden and are potential therapeutic targets and biomarkers. These findings are a first step in elucidating miRNA regulated molecular mechanisms that underlie early atherosclerosis in a baboon model, enabling translation of our findings to humans.

An Isobaric Labeling Approach to Enhance Detection and Quantification of Tissue-Derived Plasma Proteins as Potential Early Disease Biomarkers.

The proteomic analysis of plasma holds great promise to advance precision medicine and identify biomarkers of disease. However, it is likely that many potential biomarkers circulating in plasma originate from other tissues and are only present in low abundances in the plasma. Accurate detection and quantification of low abundance proteins by standard mass spectrometry approaches remain challenging. In addition, it is difficult to link low abundance plasma proteins back to their specific tissues or organs of origin with confidence. To address these challenges, we developed a mass spectrometry approach based on the use of tandem mass tags (TMT) and a tissue reference sample. By applying this approach to nonhuman primate plasma samples, we were able to identify and quantify 820 proteins by using a kidney tissue homogenate as reference. On average, 643 ± 16 proteins were identified per plasma sample. About 58% of proteins identified in replicate experiments were identified both times. A ratio of 50 µg kidney protein to 10 µg plasma protein, and the use of the TMT label with the highest molecular weight (131) for the kidney reference yielded the largest number of proteins in the analysis, and identified low abundance proteins in plasma that are prominently found in the kidney. Overall, this methodology promises efficient quantification of plasma proteins potentially released from specific tissues, thereby increasing the number of putative disease biomarkers for future study.

Cell-type deconvolution of bulk RNA-Seq from kidney using opensource bioinformatic tools.

Traditional bulk RNA-Seq pipelines do not assess cell-type composition within heterogeneous tissues. Therefore, it is difficult to determine whether conflicting findings among samples or datasets are the result of biological differences or technical differences due to variation in sample collections. This report provides a user-friendly, open source method to assess cell-type composition in bulk RNA-Seq datasets for heterogeneous tissues using published single cell (sc)RNA-Seq data as a reference. As an example, we apply the method to analysis of kidney cortex bulk RNA-Seq data from female (N=8) and male (N=9) baboons to assess whether observed transcriptome sex differences are biological or technical, i.e., variation due to ultrasound guided biopsy collections. We found cell-type composition was not statistically different in female versus male transcriptomes based on expression of 274 kidney cell-type specific transcripts, indicating differences in gene expression are not due to sampling differences. This method of cell-type composition analysis is recommended for providing rigor in analysis of bulk RNA-Seq datasets from complex tissues. It is clear that with reduced costs, more analyses will be done using scRNA-Seq; however, the approach described here is relevant for data mining and meta analyses of the thousands of bulk RNA-Seq data archived in the NCBI GEO public database.

Severe acute respiratory coronavirus virus 2 (SARS-CoV-2) infections occurring in healthcare workers after booster vaccination: A comparison of delta versus omicron variants.

In this study, we used genomic sequencing to identify variants of severe acute respiratory coronavirus virus 2 (SARS-CoV-2) in healthcare workers with coronavirus disease 2019 (COVID-19) after receiving a booster vaccination. We compared symptoms, comorbidities, exposure risks, and vaccine history between the variants. Postbooster COVID-19 cases increased as the SARS-CoV-2 omicron variant predominated.