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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 β-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., (© 2024 The Author(s). Advanced Science published by Wiley‐VCH GmbH.)

Studies have consistently shown that psychiatric genetic counseling (pGC) helps people with psychiatric conditions by increasing empowerment and self-efficacy, and addressing emotions like guilt. Yet, it is not routinely provided. Genetic counselors and trainees express low confidence in their ability to provide meaningful pGC, especially in the absence of adequate training. Therefore, to address this gap a "Psychiatric Genetic Counseling for Genetic Counselors" (PG4GC) workshop was developed and delivered to 13 groups of participants (primarily qualified genetic counselors and trainees) between 2015 and 2023 (10 workshops were delivered in-person, and three virtually). Participants completed quantitative questionnaires both before and after completing the workshop to assess their comfort, knowledge, behavior, and feeling of being equipped to provide pGC. In total, 232 individuals completed the pre-workshop questionnaire and 154 completed the post-workshop questionnaire. Participants felt more comfortable, knowledgeable, and equipped to provide pGC, and reported being more likely to address psychiatric concerns after the workshop, regardless of whether they were trainees or practicing professionals and whether they completed the workshop in-person or virtually. This study suggests that the PG4GC workshop is an effective educational tool in pGC training that may aid in broader implementation of the service., (© 2024 The Authors. American Journal of Medical Genetics Part B: Neuropsychiatric Genetics published by Wiley Periodicals LLC.)

The postmenopausal decrease in circulating estradiol (E2) levels has been shown to contribute to several adverse physiological and psychiatric effects. To elucidate the molecular effects of E2 on the brain, we examined differential gene expression and DNA methylation (DNAm) patterns in the nonhuman primate brain following ovariectomy (Ov) and subsequent subcutaneous bioidentical E2 chronic treatment. We identified several dysregulated molecular networks, including MAPK signaling and dopaminergic synapse response, that are associated with ovariectomy and shared across two different brain areas, the occipital cortex (OC) and prefrontal cortex (PFC). The finding that hypomethylation (p = 1.6 × 10 -51 ) and upregulation (p = 3.8 × 10 -3 ) of UBE2M across both brain regions provide strong evidence for molecular differences in the brain induced by E2 depletion. Additionally, differential expression (p = 1.9 × 10 -4 ; interaction p = 3.5 × 10 -2 ) of LTBR in the PFC provides further support for the role E2 plays in the brain, by demonstrating that the regulation of some genes that are altered by ovariectomy may also be modulated by Ov followed by hormone replacement therapy (HRT). These results present real opportunities to understand the specific biological mechanisms that are altered with depleted E2. Given E2's potential role in cognitive decline and neuroinflammation, our findings could lead to the discovery of novel therapeutics to slow cognitive decline. Together, this work represents a major step toward understanding molecular changes in the brain that are caused by ovariectomy and how E2 treatment may revert or protect against the negative neuro-related consequences caused by a depletion in estrogen as women approach menopause., (© 2024. The Author(s).)

Background & Aims: Deployment of law enforcement operational canines (OpK9s) risks injuries to the animals. This study's aim was to assess the current status of states' OpK9 (veterinary Emergency Medical Services [VEMS]) laws and care protocols within the United States., Methods: Cross-sectional standardized review of state laws/regulations and OpK9 VEMS treatment protocols was undertaken. For each state and for the District of Columbia (DC), the presence of OpK9 legislation and/or care protocols was ascertained. Information was obtained through governmental records and from stakeholders (eg, state EMS medical directors and state veterinary boards).The main endpoints were proportions of states with OpK9 laws and/or treatment protocols. Proportions are reported with 95% confidence intervals (CIs). Fisher's exact test ( P <.05) assessed whether presence of an OpK9 law in a given jurisdiction was associated with presence of an OpK9 care protocol, and whether there was geographic variation (based on United States Census Bureau regions) in presence of OpK9 laws or protocols., Results: Of 51 jurisdictions, 20 (39.2%) had OpK9 legislation and 23 (45.1%) had state-wide protocols for EMS treatment of OpK9s. There was no association ( P = .991) between presence of legislation and presence of protocols. There was no association ( P = .144) between presence of legislation and region: Northeast 66.7% (95% CI, 29.9-92.5%), Midwest 50.0% (95% CI, 21.1-78.9%), South 29.4% (95% CI, 10.3-56.0%), and West 23.1% (95% CI, 5.0-53.8%). There was significant ( P = .001) regional variation in presence of state-wide OpK9 treatment protocols: Northeast 100.0% (95% CI, 66.4-100.0%), Midwest 16.7% (95% CI, 2.1-48.4%), South 47.1% (95% CI, 23.0-72.2%), and West 30.8% (95% CI, 9.1-61.4%)., Conclusion: There is substantial disparity with regard to presence of OpK9 legal and/or clinical guidance. National collaborative guidelines development is advisable to optimize and standardize care of OpK9s. Additional attention should be paid to educational and training programs to best utilize the limited available training budgets.

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., Competing Interests: Declaration of Competing Interest The authors report no conflicts of interest., (Copyright © 2023 Elsevier Inc. All rights reserved.)

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.

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.

Maternal obesity (MO) during pregnancy is linked to increased and premature risk of age-related metabolic diseases in the offspring. However, the underlying molecular mechanisms still remain not fully understood. Using a well-established nonhuman primate model of MO, we analyzed tissue biopsies and plasma samples obtained from post-pubertal offspring (3-6.5 y) of MO mothers (n = 19) and from control animals born to mothers fed a standard diet (CON, n = 13). All offspring ate a healthy chow diet after weaning. Using untargeted gas chromatography-mass spectrometry metabolomics analysis, we quantified a total of 351 liver, 316 skeletal muscle, and 423 plasma metabolites. We identified 58 metabolites significantly altered in the liver and 46 in the skeletal muscle of MO offspring, with 8 metabolites shared between both tissues. Several metabolites were changed in opposite directions in males and females in both liver and skeletal muscle. Several tissue-specific and 4 shared metabolic pathways were identified from these dysregulated metabolites. Interestingly, none of the tissue-specific metabolic changes were reflected in plasma. Overall, our study describes characteristic metabolic perturbations in the liver and skeletal muscle in MO offspring, indicating that metabolic programming in utero persists postnatally, and revealing potential novel mechanisms that may contribute to age-related metabolic diseases later in life., (© 2022 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

Background: Reliable and effective label-free quantification (LFQ) analyses are dependent not only on the method of data acquisition in the mass spectrometer, but also on the downstream data processing, including software tools, query database, data normalization and imputation. In non-human primates (NHP), LFQ is challenging because the query databases for NHP are limited since the genomes of these species are not comprehensively annotated. This invariably results in limited discovery of proteins and associated Post Translational Modifications (PTMs) and a higher fraction of missing data points. While identification of fewer proteins and PTMs due to database limitations can negatively impact uncovering important and meaningful biological information, missing data also limits downstream analyses (e.g., multivariate analyses), decreases statistical power, biases statistical inference, and makes biological interpretation of the data more challenging. In this study we attempted to address both issues: first, we used the MetaMorphues proteomics search engine to counter the limits of NHP query databases and maximize the discovery of proteins and associated PTMs, and second, we evaluated different imputation methods for accurate data inference. We used a generic approach for missing data imputation analysis without distinguising the potential source of missing data (either non-assigned m/z or missing values across runs)., Results: Using the MetaMorpheus proteomics search engine we obtained quantitative data for 1622 proteins and 10,634 peptides including 58 different PTMs (biological, metal and artifacts) across a diverse age range of NHP brain frontal cortex. However, among the 1622 proteins identified, only 293 proteins were quantified across all samples with no missing values, emphasizing the importance of implementing an accurate and statiscaly valid imputation method to fill in missing data. In our imputation analysis we demonstrate that Single Imputation methods that borrow information from correlated proteins such as Generalized Ridge Regression (GRR), Random Forest (RF), local least squares (LLS), and a Bayesian Principal Component Analysis methods (BPCA), are able to estimate missing protein abundance values with great accuracy., Conclusions: Overall, this study offers a detailed comparative analysis of LFQ data generated in NHP and proposes strategies for improved LFQ in NHP proteomics data., (© 2022. The Author(s).)

Gas chromatography-coupled mass spectrometry (GC-MS) has been used in biomedical research to analyze volatile, non-polar, and polar metabolites in a wide array of sample types. Despite advances in technology, missing values are still common in metabolomics datasets and must be properly handled. We evaluated the performance of ten commonly used missing value imputation methods with metabolites analyzed on an HR GC-MS instrument. By introducing missing values into the complete (i.e., data without any missing values) National Institute of Standards and Technology (NIST) plasma dataset, we demonstrate that random forest (RF), glmnet ridge regression (GRR), and Bayesian principal component analysis (BPCA) shared the lowest root mean squared error (RMSE) in technical replicate data. Further examination of these three methods in data from baboon plasma and liver samples demonstrated they all maintained high accuracy. Overall, our analysis suggests that any of the three imputation methods can be applied effectively to untargeted metabolomics datasets with high accuracy. However, it is important to note that imputation will alter the correlation structure of the dataset and bias downstream regression coefficients and p -values.

Background: Improvements in antenatal medicine and surgical management for conditions associated with spina bifida such as hydrocephalus have extended the lifespan for individuals with spina bifida (SB) into adulthood. Decisions and education regarding reproductive care and pregnancies for patients with spina bifida are increasingly important. Pregnancy in these patients can be particularly challenging due to physical limitations, previous abdominal surgery for urinary or bowel management and presence of a ventriculoperitoneal shunt. To date, little research has examined the unique challenges that women with spina bifida face during pregnancy., Objective: The purpose of this descriptive study is to characterize the successful pregnancy histories of SB women and describe how pregnancy affected their mobility as well as bladder and bowel management., Study Design: We conducted semi-structured interviews with women followed in our adult multidisciplinary SB clinic who previously had successful pregnancies. Questions regarding perinatal issues, obstetrical complications, urinary tract infections (UTI) and neurological changes were asked. Baseline mobility, bladder and bowel management were compared with changes during and after pregnancy., Results: 121 women of childbearing age were followed per year by our adult multidisciplinary spina bifida clinic between 2009 and 2016. We identified 6 women who successfully carried 8 pregnancies to term. There were no miscarriages. Four women had ventriculoperitoneal (VP) shunts. No children were born with neural tube defects. Mean age at first pregnancy was 23.5 years. Average gestational age at delivery was 37 weeks. 50% of the women had a spontaneous vaginal delivery. Five of six women intended to get pregnant; only one patient consumed folic acid regularly prior to pregnancy. Two of six women had bladder augmentation surgery, one of whom had urologic changes during pregnancy that persisted after childbirth. The other patient had a concomitant bladder neck sling procedure and did not have urologic issues during pregnancy. 50% of the patients experienced bladder-bowel dysfunction during their pregnancy. While 67% patients had full baseline ambulatory function, 4 patients had decreased mobility and required additional assistance during pregnancy. All returned to their baseline functionality afterwards., Conclusion: Six of our patients had eight successful pregnancies, with no children born with neural tube defects. New changes to mobility, bladder and bowel management were experienced by over half of the women during their pregnancies. Future studies should focus on the role of multidisciplinary teams in reproductive health education and perinatal management of changes to activities of daily living during pregnancy in this population., Competing Interests: Conflicts of interest None., (Copyright © 2021 Journal of Pediatric Urology Company. Published by Elsevier Ltd. All rights reserved.)

Objectives: Systemic sclerosis (SSc) is a complex disease of unknown aetiology in which inflammation and fibrosis lead to multiple organ damage. There is currently no effective therapy that can halt the progression of fibrosis or reverse it, thus studies that provide novel insights into disease pathogenesis and identify novel potential therapeutic targets are critically needed., Methods: We used global gene expression and genome-wide DNA methylation analyses of dermal fibroblasts (dFBs) from a unique cohort of twins discordant for SSc to identify molecular features of this pathology. We validated the findings using in vitro, ex vivo and in vivo models., Results: Our results revealed distinct differentially expressed and methylated genes, including several transcription factors involved in stem cell differentiation and developmental programmes ( KLF4 , TBX5 , TFAP2A and homeobox genes) and the microRNAs miR-10a and miR-10b which target several of these deregulated genes. We show that KLF4 expression is reduced in SSc dFBs and its expression is repressed by TBX5 and TFAP2A . We also show that KLF4 is antifibrotic, and its conditional knockout in fibroblasts promotes a fibrotic phenotype., Conclusions: Our data support a role for epigenetic dysregulation in mediating SSc susceptibility in dFBs, illustrating the intricate interplay between CpG methylation, miRNAs and transcription factors in SSc pathogenesis, and highlighting the potential for future use of epigenetic modifiers as therapies., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2022. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: Triggering receptor expressed on myeloid cells (TREM)-1 is a key mediator of innate immunity previously associated with the severity of inflammatory disorders, and more recently, the inferior survival of lung and liver cancer patients. Here, we investigated the prognostic impact and immunological correlates of TREM1 expression in breast tumors., Methods: Breast tumor microarray and RNAseq expression profiles (n=4,364 tumors) were analyzed for associations between gene expression, tumor immune subtypes, distant metastasis-free survival (DMFS) and clinical response to neoadjuvant chemotherapy (NAC). Single-cell (sc)RNAseq was performed using the 10X Genomics platform. Statistical associations were assessed by logistic regression, Cox regression, Kaplan-Meier analysis, Spearman correlation, Student's t-test and Chi-square test., Results: In pre-treatment biopsies, TREM1 and known TREM-1 inducible cytokines (IL1B, IL8) were discovered by a statistical ranking procedure as top genes for which high expression was associated with reduced response to NAC, but only in the context of immunologically "hot" tumors otherwise associated with a high NAC response rate. In surgical specimens, TREM1 expression varied among tumor molecular subtypes, with highest expression in the more aggressive subtypes (Basal-like, HER2-E). High TREM1 significantly and reproducibly associated with inferior distant metastasis-free survival (DMFS), independent of conventional prognostic markers. Notably, the association between high TREM1 and inferior DMFS was most prominent in the subset of immunogenic tumors that exhibited the immunologically hot phenotype and otherwise associated with superior DMFS. Further observations from bulk and single-cell RNAseq analyses indicated that TREM1 expression was significantly enriched in polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) and M2-like macrophages, and correlated with downstream transcriptional targets of TREM-1 ( IL8 , IL-1B , IL6 , MCP- 1, SPP1, IL1RN, INHBA ) which have been previously associated with pro-tumorigenic and immunosuppressive functions., Conclusions: Together, these findings indicate that increased TREM1 expression is prognostic of inferior breast cancer outcomes and may contribute to myeloid-mediated breast cancer progression and immune suppression., Competing Interests: AS is an employee of SignaBlok, Inc. LM has an advisory role for Bristol Myers Squibb. AT has research funding (to the institution) from Sanofi, stock ownership of Gilead Sciences, Johnson and Johnson, Bristol Myers Squibb and Pfizer, and advisory roles for BeyondSpring and Lilly. The remaining 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 © 2021 Pullikuth, Routh, Zimmerman, Chifman, Chou, Soike, Jin, Su, Song, Black, Print, Bedognetti, Howard-McNatt, O’Neill, Thomas, Langefeld, Sigalov, Lu and Miller.)

Background: We performed expression quantitative trait locus (eQTL) analysis in single classical (CL) and non-classical (NCL) monocytes from patients with systemic lupus erythematosus (SLE) to quantify the impact of well-established genetic risk alleles on transcription at single-cell resolution., Methods: Single-cell gene expression was quantified using qPCR in purified monocyte subpopulations (CD14 ++ CD16 - CL and CD14 dim CD16 + NCL) from SLE patients. Novel analysis methods were used to control for the within-person correlations observed, and eQTLs were compared between cell types and risk alleles., Results: The SLE-risk alleles demonstrated significantly more eQTLs in NCLs as compared to CLs (p = 0.0004). There were 18 eQTLs exclusive to NCL cells, 5 eQTLs exclusive to CL cells, and only one shared eQTL, supporting large differences in the impact of the risk alleles between these monocyte subsets. The SPP1 and TNFAIP3 loci were associated with the greatest number of transcripts. Patterns of shared influence in which different SNPs impacted the same transcript also differed between monocyte subsets, with greater evidence for synergy in NCL cells. IRF1 expression demonstrated an on/off pattern, in which expression was zero in all of the monocytes studied from some individuals, and this pattern was associated with a number of SLE risk alleles. We observed corroborating evidence of this IRF1 expression pattern in public data sets., Conclusions: We document multiple SLE-risk allele eQTLs in single monocytes which differ greatly between CL and NCL subsets. These data support the importance of the SPP1 and TNFAIP3 risk variants and the IRF1 transcript in SLE patient monocyte function., (© 2021. The Author(s).)

Systemic lupus erythematosus (SLE) is a chronic, multisystem, autoimmune inflammatory disease with genomic and non-genomic contributions to risk. We hypothesize that epigenetic factors are a significant contributor to SLE risk and may be informative for identifying pathogenic mechanisms and therapeutic targets. To test this hypothesis while controlling for genetic background, we performed an epigenome-wide analysis of DNA methylation in genomic DNA from whole blood in three pairs of female monozygotic (MZ) twins of European ancestry, discordant for SLE. Results were replicated on the same array in four cell types from a set of four Danish female MZ twin pairs discordant for SLE. Genes implicated by the epigenetic analyses were then evaluated in 10 independent SLE gene expression datasets from the Gene Expression Omnibus (GEO). There were 59 differentially methylated loci between unaffected and affected MZ twins in whole blood, including 11 novel loci. All but two of these loci were hypomethylated in the SLE twins relative to the unaffected twins. The genes harboring these hypomethylated loci exhibited increased expression in multiple independent datasets of SLE patients. This pattern was largely consistent regardless of disease activity, cell type, or renal tissue type. The genes proximal to CpGs exhibiting differential methylation (DM) in the SLE-discordant MZ twins and exhibiting differential expression (DE) in independent SLE GEO cohorts (DM-DE genes) clustered into two pathways: the nucleic acid-sensing pathway and the type I interferon pathway. The DM-DE genes were also informatically queried for potential gene-drug interactions, yielding a list of 41 drugs including a known SLE therapy. The DM-DE genes delineate two important biologic pathways that are not only reflective of the heterogeneity of SLE but may also correlate with distinct IFN responses that depend on the source, type, and location of nucleic acid molecules and the activated receptors in individual patients. Cell- and tissue-specific analyses will be critical to the understanding of genetic factors dysregulating the nucleic acid-sensing and IFN pathways and whether these factors could be appropriate targets for therapeutic intervention.

Intracerebral hemorrhage (ICH) is a severe neurological disorder with no proven treatment. Inflammation after ICH contributes to clinical outcomes, but the relevant molecular mechanisms remain poorly understood. In studies of peripheral leukocyte counts and mRNA-sequencing (mRNA-seq), our group previously reported that monocytes and Interleukin-8 (IL-8) were important contributors to post-ICH inflammation. microRNA (miRNA) are powerful regulators of gene expression and promising therapeutic targets. We now report findings from an integrated analysis of miRNA-seq and mRNA-seq in peripheral blood mononuclear cells (PBMCs) from a swine ICH model. In 10 pigs, one PBMC sample was collected immediately prior to ICH induction and a second 6 h later; miRNA-seq and mRNA-seq were completed for each sample. An aggregate score calculation determined which miRNA regulated the differentially expressed mRNA. Networks of molecular interactions were generated for the combined miRNA/target mRNA. A total of 227 miRNA were identified, and 46 were differentially expressed after ICH (FDR < 0.05). The anti-inflammatory miR-181a was decreased post-ICH, and it was the most highly connected miRNA in the miRNA/mRNA bioinformatic network analysis. miR-181a has interconnected pathophysiology with IL-8 and monocytes; in prior studies, we found that IL-8 and monocytes contributed to post-ICH inflammation and ICH clinical outcome, respectively. miR-181a was a significant mediator of post-ICH inflammation and is promising for further study, including as a potential therapeutic target. This investigation also demonstrated feasible methodology for miRNA-seq/mRNA-seq analysis in swine that is innovative, and with unique challenges, compared with transcriptomics research in more established species., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC part of Springer Nature.)

Objectives: Gullah African Americans are descendants of formerly enslaved Africans living in the Sea Islands along the coast of the southeastern U.S., from North Carolina to Florida. Their relatively high numbers and geographic isolation were conducive to the development and preservation of a unique culture that retains deep African features. Although historical evidence supports a West-Central African ancestry for the Gullah, linguistic and cultural evidence of a connection to Sierra Leone has led to the suggestion of this country/region as their ancestral home. This study sought to elucidate the genetic structure and ancestry of the Gullah., Materials and Methods: We leveraged whole-genome genotype data from Gullah, African Americans from Jackson, Mississippi, African populations from Sierra Leone, and population reference panels from Africa and Europe to infer population structure, ancestry proportions, and global estimates of admixture., Results: Relative to non-Gullah African Americans from the Southeast US, the Gullah exhibited higher mean African ancestry, lower European admixture, a similarly small Native American contribution, and increased male-biased European admixture. A slightly tighter bottleneck in the Gullah 13 generations ago suggests a largely shared demographic history with non-Gullah African Americans. Despite a slightly higher relatedness to populations from Sierra Leone, our data demonstrate that the Gullah are genetically related to many West African populations., Discussion: This study confirms that subtle differences in African American population structure exist at finer regional levels. Such observations can help to inform medical genetics research in African Americans, and guide the interpretation of genetic data used by African Americans seeking to explore ancestral identities., (© 2021 The Authors. American Journal of Physical Anthropology published by Wiley Periodicals LLC.)

Background: Study design is a critical aspect of any experiment, and sample size calculations for statistical power that are consistent with that study design are central to robust and reproducible results. However, the existing power calculators for tests of differential expression in single-cell RNA-seq data focus on the total number of cells and not the number of independent experimental units, the true unit of interest for power. Thus, current methods grossly overestimate the power., Results: Hierarchicell is the first single-cell power calculator to explicitly simulate and account for the hierarchical correlation structure (i.e., within sample correlation) that exists in single-cell RNA-seq data. Hierarchicell, an R-package available on GitHub, estimates the within sample correlation structure from real data to simulate hierarchical single-cell RNA-seq data and estimate power for tests of differential expression. This multi-stage approach models gene dropout rates, intra-individual dispersion, inter-individual variation, variable or fixed number of cells per individual, and the correlation among cells within an individual. Without modeling the within sample correlation structure and without properly accounting for the correlation in downstream analysis, we demonstrate that estimates of power are falsely inflated. Hierarchicell can be used to estimate power for binary and continuous phenotypes based on user-specified number of independent experimental units (e.g., individuals) and cells within the experimental unit., Conclusions: Hierarchicell is a user-friendly R-package that provides accurate estimates of power for testing hypotheses of differential expression in single-cell RNA-seq data. This R-package represents an important addition to single-cell RNA analytic tools and will help researchers design experiments with appropriate and accurate power, increasing discovery and improving robustness and reproducibility.

Cells from the same individual share common genetic and environmental backgrounds and are not statistically independent; therefore, they are subsamples or pseudoreplicates. Thus, single-cell data have a hierarchical structure that many current single-cell methods do not address, leading to biased inference, highly inflated type 1 error rates, and reduced robustness and reproducibility. This includes methods that use a batch effect correction for individual as a means of accounting for within-sample correlation. Here, we document this dependence across a range of cell types and show that pseudo-bulk aggregation methods are conservative and underpowered relative to mixed models. To compute differential expression within a specific cell type across treatment groups, we propose applying generalized linear mixed models with a random effect for individual, to properly account for both zero inflation and the correlation structure among measures from cells within an individual. Finally, we provide power estimates across a range of experimental conditions to assist researchers in designing appropriately powered studies.

Background: Treatment failure in eosinophilic esophagitis (EoE) is common. We hypothesize that DNA methylation differs between patients by treatment response to topical steroids (oral viscous budesonide), thus offering the potential to inform targeting therapies., Objective: We sought to identify differentially methylated sites and affiliated genes in pre-treatment oesophageal cells between responders and non-responders and test for accelerated epigenetic ageing in oesophageal cells of EoE patients., Methods: DNA was extracted from prospectively collected and biobanked oesophageal biopsies from 36 Caucasian treatment naïve EoE patients at diagnosis. Methylation assays were completed using the Infinium HumanMethylation450 BeadChip. Normalized β values for each CpG site were tested (t test) for differential methylation. Further, 353 CpG probes were used to estimate epigenetic age for each patient and a linear regression model tested whether chronologic age and epigenetic age differed. Epigenetic age results were confirmed in an independent cohort of healthy controls., Results: Eighteen CpG sites were differentially methylated by treatment response (P < .00001). The mean epigenetic age and chronological age were 56.1 ± 11.1 and 36.7 ± 12.3 years, a mean age difference of 19.3 ± 5.2 years (P < .0001); accelerated ageing was not observed in the oesophageal cells of healthy controls., Conclusions and Clinical Relevance: EoE patients that respond versus do not respond to treatment have differences in their methylation profile, including enrichment of genes in pathways consistent with cellular injury and repair due to environmental stress and cell adhesion and barrier integrity. EoE also appears to accelerate cellular ageing. Whether treatment can arrest or reverse accelerated epigenetic ageing and the implications for long-term disease progression is important areas for future research., (© 2020 John Wiley & Sons Ltd.)

Systemic lupus erythematosus (SLE) is a multi-organ autoimmune disorder with a prominent genetic component. Individuals of African ancestry (AA) experience the disease more severely and with an increased co-morbidity burden compared to European ancestry (EA) populations. We hypothesize that the disparities in disease prevalence, activity, and response to standard medications between AA and EA populations is partially conferred by genomic influences on biological pathways. To address this, we applied a comprehensive approach to identify all genes predicted from SNP-associated risk loci detected with the Immunochip. By combining genes predicted via eQTL analysis, as well as those predicted from base-pair changes in intergenic enhancer sites, coding-region variants, and SNP-gene proximity, we were able to identify 1,731 potential ancestry-specific and trans-ancestry genetic drivers of SLE. Gene associations were linked to upstream and downstream regulators using connectivity mapping, and predicted biological pathways were mined for candidate drug targets. Examination of trans-ancestral pathways reflect the well-defined role for interferons in SLE and revealed pathways associated with tissue repair and remodeling. EA-dominant genetic drivers were more often associated with innate immune and myeloid cell function pathways, whereas AA-dominant pathways mirror clinical findings in AA subjects, suggesting disease progression is driven by aberrant B cell activity accompanied by ER stress and metabolic dysfunction. Finally, potential ancestry-specific and non-specific drug candidates were identified. The integration of all SLE SNP-predicted genes into functional pathways revealed critical molecular pathways representative of each population, underscoring the influence of ancestry on disease mechanism and also providing key insight for therapeutic selection., (Copyright © 2020 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Objective: Surgical treatment of herniated lumbar disc (HLD) remains rare in children. The purpose of this study was to evaluate for potential disease risk factors leading to surgery based on a large single-center experience., Methods: Data for all patients who had undergone surgical treatment for HLD between December 2008 and December 2016 at a single pediatric tertiary care referral center were collected and compared to data for a healthy control population obtained through a Youth Risk Behavior Surveillance System (YRBSS) survey in order to determine relevant disease risk factors. Univariate and multivariate logistic regression were used to determine the effect of potential risk factors., Results: Twenty-seven patients in the disease cohort and 5212 healthy controls from the general population were included in the risk factor analysis. The mean body mass index was significantly higher in the disease population (30.2 vs 24.0 kg/m2, p < 0.0001). Children who had undergone microdiscectomy were more likely to be obese (OR 7.4, 95% CI 3.46-15.8, p < 0.001). No association was found between lumbar microdiscectomy and sports participation (OR 1.0, 95% CI -0.002 to 0.005, p = 0.37)., Conclusions: Microdiscectomy remains a viable and safe option in the setting of failed conservative management for pediatric HLD. Childhood obesity is a risk factor for HLD and many other diseases, which increases its importance as a public health priority.

Background: Systemic sclerosis (SSc) is a rare autoimmune fibrosing disease with an incompletely understood genetic and non-genetic etiology. Defining its etiology is important to allow the development of effective predictive, preventative, and therapeutic strategies. We conducted this epigenomic study to investigate the contributions of DNA methylation to the etiology of SSc while minimizing confounding due to genetic heterogeneity., Methods: Genomic methylation in whole blood from 27 twin pairs discordant for SSc was assayed over 450 K CpG sites. In silico integration with reported differentially methylated cytosines, differentially expressed genes, and regulatory annotation was conducted to validate and interpret the results., Results: A total of 153 unique cytosines in limited cutaneous SSc (lcSSc) and 266 distinct sites in diffuse cutaneous SSc (dcSSc) showed suggestive differential methylation levels in affected twins. Integration with available data revealed 76 CpGs that were also differentially methylated in blood cells from lupus patients, suggesting their role as potential epigenetic blood biomarkers of autoimmunity. It also revealed 27 genes with concomitant differential expression in blood from SSc patients, including IFI44L and RSAD2. Regulatory annotation revealed that dcSSc-associated CpGs (but not lcSSc) are enriched at Encyclopedia of DNA Elements-, Roadmap-, and BLUEPRINT-derived regulatory regions, supporting their potential role in disease presentation. Notably, the predominant enrichment of regulatory regions in monocytes and macrophages is consistent with the role of these cells in fibrosis, suggesting that the observed cellular dysregulation might be, at least partly, due to altered epigenetic mechanisms of these cells in dcSSc., Conclusions: These data implicate epigenetic changes in the pathogenesis of SSc and suggest functional mechanisms in SSc etiology.

Background: Sensorineural hearing loss (SNHL) is a common form of hearing loss that can be inherited or triggered by environmental insults; auditory neuropathy spectrum disorder (ANSD) is a SNHL subtype with unique diagnostic criteria. The genetic factors associated with these impairments are vast and diverse, but causal genetic factors are rarely characterized., Methods: A family dyad, both cochlear implant recipients, presented with a hearing history of bilateral, progressive SNHL, and ANSD. Whole-exome sequencing was performed to identify coding sequence variants shared by both family members, and screened against genes relevant to hearing loss and variants known to be associated with SNHL and ANSD., Results: Both family members are successful cochlear implant users, demonstrating effective auditory nerve stimulation with their devices. Genetic analyses revealed a mutation (rs35725509) in the TMTC2 gene, which has been reported previously as a likely genetic cause of SNHL in another family of Northern European descent., Conclusion: This study represents the first confirmation of the rs35725509 variant in an independent family as a likely cause for the complex hearing loss phenotype (SNHL and ANSD) observed in this family dyad., (© 2018 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals, Inc.)

Introduction: Increasing attention has been focused on parental leave, but little is known about early leave and parental experiences for male and female attending physicians. Our goal was to describe and quantify the parental leave experiences of a nationally representative sample of emergency physicians (EP)., Methods: We conducted a web-based survey, distributed via emergency medicine professional organizations, discussion boards, and listservs, to address study objectives., Results: We analyzed data from 464 respondents; 56% were women. Most experienced childbirth while employed as an EP. Fifty-three percent of women and 60% of men reported working in a setting with a formal maternity leave policy; however, 36% of women and 18% of men reported dissatisfaction with these policies. Most reported that other group members cover maternity-related shift vacancies; a minority reported that pregnant partners work extra shifts prior to leave. Leave duration and compensation varied widely, ranging from no compensated leave (18%) to 12 or more weeks at 100% salary (7%). Supportive attitudes were reported during pregnancy (53%) and, to a lesser degree (43%), during leave. Policy improvement suggestions included the development of clear, formal policies; improving leave duration and compensation; adding paternity and adoption leave; providing support for physicians working extra to cover colleagues' leave; and addressing breastfeeding issues., Conclusion: In this national sample of EPs, maternity leave policies varied widely. The duration and compensation during leave also had significant variation. Participants suggested formalizing policies, increasing leave duration and compensation, adding paternity leave, and changing the coverage for vacancies to relieve burden on physician colleagues., Competing Interests: Conflicts of Interest: By the WestJEM article submission agreement, all authors are required to disclose all affiliations, funding sources and financial or management relationships that could be perceived as potential sources of bias. No author has professional or financial relationships with any companies that are relevant to this study. There are no conflicts of interest or sources of funding to declare.

Purpose: Research regarding the effect of trainees on emergency department (ED) operations has demonstrated mixed results. In this study, the authors evaluated the effect of trainees on ED length of stay (LOS), door to medical provider (DTMP) time, and door to disposition decision (DTDD) time while accounting for covariates known to influence ED efficiency and timeliness., Method: The authors used retrospective cohort data for ED visits to Maine Medical Center's mixed adult and pediatric ED for the calendar years 2005 through 2009. Each visit was coded indicating the type of provider conducting the visit (student-attending, resident-attending, midlevel provider, or attending group). Ordinary least squares regression analyses were performed to examine the relationships between provider groups and ED LOS, DTMP time, and DTDD time. Hierarchical regression models were constructed to control for the confounding effects of triage acuity, time of year, laboratory testing, radiographic testing, and patient characteristics., Results: The analysis of 246,142 visits found significant intergroup differences across provider groups for each outcome (P < .001). Multiple regression modeling revealed that treatment by trainees was a significant predictor of longer LOS (medical students and residents), shorter DTMP time (residents), and longer DTDD time (medical students and residents), after controlling for covariates., Conclusions: Laboratory and radiographic testing accounted for a much larger proportion of variation in outcomes than did trainees. The small increases in LOS and DTDD time are balanced by the decrease in DTMP time and the intangible benefits of educating trainees.

The behaviour of clamping the shell against the substratum may play an important role in the limpet adhesion mechanism because friction generated by this behaviour resists dislodgement by shear forces. This paper describes the development of an apparatus to analyse limpet clamping activity in relation to known forces, including simulated wave activity and predator attack. The results show that Cellana tramoserica clamps its shell in a closely regulated manner consistent with an active role in the limpet adhesion mechanism. Limpets clamped sharply for several seconds in response to single disturbances such as tapping the shell. In response to more continuous disturbance simulating a concerted predator attack, limpets clamped tightly for several minutes. In response to lifting forces applied to the shell, limpets clamped at a set proportion of the lifting force, even if the lift force was a highly dynamic wave profile. This behaviour has implications for numerical models that attempt to describe limpet adhesion because it shows that limpets cannot be represented by a simple mechanical analogue and that the clamping behaviour must be accounted for if useful predictions are to be drawn.

The LD50 values and survival times of three pomacentrid species and two blennies were measured after being subjected to the venom of one of their predators, the olive sea snake, Aipysurus laevis. The species differed significantly in the speed of their responses to the venom. At high venom doses, blennies had higher survival times than pomacentrids, and in the latter group Dascyllus survived longer than did species of Chromis. In part, this may be related to differences between blennies and pomacentrids in degree of cutaneous respiration. Relative survival time was influenced by venom dosage; ranking order of species' survival times was different at low doses than at high ones, and taxonomic correlations broke down.

Venom from the olive sea snake, Aipysurus laevis, was injected into three species of prey fish, Chromis nitida, Dascyllus aruanus and Istiblennius meleagris. Their behaviour and ventilatory patterns were observed for 48 hr. Six progressive stages of envenomation, involving impairment of the locomotory and ventilatory systems, were identified. There were significant interspecific differences in time taken to reach various stages. In nature, the action of the venom not only would facilitate subduing the prey, but recovering it if it escaped.