Comprehensive Multi-omics Analysis Reveals Mitochondrial Stress as a Central Biological Hub for Spaceflight Impact.

Spaceflight is known to impose changes on human physiology with unknown molecular etiologies. To reveal these causes, we used a multi-omics, systems biology analytical approach using biomedical profiles from fifty-nine astronauts and data from NASA's GeneLab derived from hundreds of samples flown in space to determine transcriptomic, proteomic, metabolomic, and epigenetic responses to spaceflight. Overall pathway analyses on the multi-omics datasets showed significant enrichment for mitochondrial processes, as well as innate immunity, chronic inflammation, cell cycle, circadian rhythm, and olfactory functions. Importantly, NASA's Twin Study provided a platform to confirm several of our principal findings. Evidence of altered mitochondrial function and DNA damage was also found in the urine and blood metabolic data compiled from the astronaut cohort and NASA Twin Study data, indicating mitochondrial stress as a consistent phenotype of spaceflight.

U1 snRNP determines mRNA length and regulates isoform expression.

U1 snRNP (U1), in addition to its splicing role, protects pre-mRNAs from drastic premature termination by cleavage and polyadenylation (PCPA) at cryptic polyadenylation signals (PASs) in introns. Here, a high-throughput sequencing strategy of differentially expressed transcripts (HIDE-seq) mapped PCPA sites genome wide in divergent organisms. Surprisingly, whereas U1 depletion terminated most nascent gene transcripts within ~1 kb, moderate functional U1 level decreases, insufficient to inhibit splicing, dose-dependently shifted PCPA downstream and elicited mRNA 3' UTR shortening and proximal 3' exon switching characteristic of activated immune and neuronal cells, stem cells, and cancer. Activated neurons' signature mRNA shortening could be recapitulated by U1 decrease and antagonized by U1 overexpression. Importantly, we show that rapid and transient transcriptional upregulation inherent to neuronal activation physiology creates U1 shortage relative to pre-mRNAs. Additional experiments suggest cotranscriptional PCPA counteracted by U1 association with nascent transcripts, a process we term telescripting, ensuring transcriptome integrity and regulating mRNA length.

MitoScape: A big-data, machine-learning platform for obtaining mitochondrial DNA from next-generation sequencing data.

The growing number of next-generation sequencing (NGS) data presents a unique opportunity to study the combined impact of mitochondrial and nuclear-encoded genetic variation in complex disease. Mitochondrial DNA variants and in particular, heteroplasmic variants, are critical for determining human disease severity. While there are approaches for obtaining mitochondrial DNA variants from NGS data, these software do not account for the unique characteristics of mitochondrial genetics and can be inaccurate even for homoplasmic variants. We introduce MitoScape, a novel, big-data, software for extracting mitochondrial DNA sequences from NGS. MitoScape adopts a novel departure from other algorithms by using machine learning to model the unique characteristics of mitochondrial genetics. We also employ a novel approach of using rho-zero (mitochondrial DNA-depleted) data to model nuclear-encoded mitochondrial sequences. We showed that MitoScape produces accurate heteroplasmy estimates using gold-standard mitochondrial DNA data. We provide a comprehensive comparison of the most common tools for obtaining mtDNA variants from NGS and showed that MitoScape had superior performance to compared tools in every statistically category we compared, including false positives and false negatives. By applying MitoScape to common disease examples, we illustrate how MitoScape facilitates important heteroplasmy-disease association discoveries by expanding upon a reported association between hypertrophic cardiomyopathy and mitochondrial haplogroup T in men (adjusted p-value = 0.003). The improved accuracy of mitochondrial DNA variants produced by MitoScape will be instrumental in diagnosing disease in the context of personalized medicine and clinical diagnostics.

Regulation of nuclear epigenome by mitochondrial DNA heteroplasmy.

Diseases associated with mitochondrial DNA (mtDNA) mutations are highly variable in phenotype, in large part because of differences in the percentage of normal and mutant mtDNAs (heteroplasmy) present within the cell. For example, increasing heteroplasmy levels of the mtDNA tRNALeu(UUR) nucleotide (nt) 3243A > G mutation result successively in diabetes, neuromuscular degenerative disease, and perinatal lethality. These phenotypes are associated with differences in mitochondrial function and nuclear DNA (nDNA) gene expression, which are recapitulated in cybrid cell lines with different percentages of m.3243G mutant mtDNAs. Using metabolic tracing, histone mass spectrometry, and NADH fluorescence lifetime imaging microscopy in these cells, we now show that increasing levels of this single mtDNA mutation cause profound changes in the nuclear epigenome. At high heteroplasmy, mitochondrially derived acetyl-CoA levels decrease causing decreased histone H4 acetylation, with glutamine-derived acetyl-CoA compensating when glucose-derived acetyl-CoA is limiting. In contrast, α-ketoglutarate levels increase at midlevel heteroplasmy and are inversely correlated with histone H3 methylation. Inhibition of mitochondrial protein synthesis induces acetylation and methylation changes, and restoration of mitochondrial function reverses these effects. mtDNA heteroplasmy also affects mitochondrial NAD+/NADH ratio, which correlates with nuclear histone acetylation, whereas nuclear NAD+/NADH ratio correlates with changes in nDNA and mtDNA transcription. Thus, mutations in the mtDNA cause distinct metabolic and epigenomic changes at different heteroplasmy levels, potentially explaining transcriptional and phenotypic variability of mitochondrial disease.

Specifications of the ACMG/AMP standards and guidelines for mitochondrial DNA variant interpretation.

Mitochondrial DNA (mtDNA) variant pathogenicity interpretation has special considerations given unique features of the mtDNA genome, including maternal inheritance, variant heteroplasmy, threshold effect, absence of splicing, and contextual effects of haplogroups. Currently, there are insufficient standardized criteria for mtDNA variant assessment, which leads to inconsistencies in clinical variant pathogenicity reporting. An international working group of mtDNA experts was assembled within the Mitochondrial Disease Sequence Data Resource Consortium and obtained Expert Panel status from ClinGen. This group reviewed the 2015 American College of Medical Genetics and Association of Molecular Pathology standards and guidelines that are widely used for clinical interpretation of DNA sequence variants and provided further specifications for additional and specific guidance related to mtDNA variant classification. These Expert Panel consensus specifications allow for consistent consideration of the unique aspects of the mtDNA genome that directly influence variant assessment, including addressing mtDNA genome composition and structure, haplogroups and phylogeny, maternal inheritance, heteroplasmy, and functional analyses unique to mtDNA, as well as specifications for utilization of mtDNA genomic databases and computational algorithms.

The association of mitochondrial DNA haplogroups with POAG in African Americans.

Mitochondrial dysfunction has been implicated in the pathogenesis of primary open-angle glaucoma (POAG). However, the potential significance of mitochondrial DNA (mtDNA) haplogroups to POAG has not been evaluated in the overaffected African American population. To investigate the association of mtDNA haplogroups with POAG and its phenotypic characteristics, genotyping data from 4081 African American subjects (1919 cases and 2162 controls) was analyzed using 1293 positions on mtDNA. The overall frequency of mtDNA haplogroups in the Primary Open-Angle African American Glaucoma Genetics (POAAGG) study cohort was 37% L3, 29% L2, 21% L1, 4% L0, and 10% non-African haplogroups (non-L). When all haplogroups (L0, L1, L2, and non-L) were compared against theL3 reference group, after adjusting by age and principal component of ancestry, the non-L3 haplogroups showed higher risk of POAG (OR-1.19, p = 0.02), with a particularly strong association among males (OR = 1.41, p = 0.003). More specifically the non-L group was associated with higher POAG risk than the L3 haplogroup (OR = 1.77, p = 0.007, Bonferroni adjusted p = 0.027) and to the L3e (n = 256, OR = 1.92, p = 0.007, Bonferroni adjusted p = 0.029). No significant association was found when genders were analyzed together or in female only analysis. There were no significant differences in various POAG endophenotypes across mtDNA haplogroups. This study expands our knowledge of mitochondrial genetics and mtDNA haplogroup associations in African American POAG. Further work is needed to better understand the functional role of mtDNA polymorphisms and their interactions with nuclear genes that affect POAG.

Mitochondrial DNA associations with East Asian metabolic syndrome.

Mitochondrial dysfunction has repeatedly been reported associated with type 2 diabetes mellitus (T2DM) and metabolic syndrome (MS), as have mitochondrial DNA (mtDNA) tRNA and duplication mutations and mtDNA haplogroup lineages. We identified 19 Taiwanese T2DM and MS pedigrees from Taiwan, with putative matrilineal transmission, one of which harbored the pathogenic mtDNA tRNALeu(UUR) nucleotide (nt) 3243A>G mutation on the N9a3 haplogroup background. We then recruited three independent Taiwanese cohorts, two from Taipei (N = 498, mean age 52 and N = 1002, mean age 44) and one from a non-urban environment (N = 501, mean age 57). All three cohorts were assessed for an array of metabolic parameters, their mtDNA haplogroups determined, and the haplogroups correlated with T2DM/MS phenotypes. Logistic regression analysis revealed that mtDNA haplogroups D5, F4, and N9a conferred T2DM protection, while haplogroups F4 and N9a were risk factors for hypertension (HTN), and F4 was a risk factor for obesity (OB). Additionally, the 5263C>T (ND2 A165V) variant commonly associated with F4 was associated with hypertension (HTN). Cybrids were prepared with macro-haplogroup N (defined by variants m.ND3 10398A (114T) and m.ATP6 8701A (59T)) haplogroups B4 and F1 mtDNAs and from macro-haplogroup M (variants m.ND3 10398G (114A) and m.ATP6 8701G (59A)) haplogroup M9 mtDNAs. Additionally, haplogroup B4 and F1 cybrids were prepared with and without the mtDNA variant in ND1 3394T>C (Y30H) reported to be associated with T2DM. Assay of mitochondria complex I in these cybrids revealed that macro-haplogroup N cybrids had lower activity than M cybrids, that haplogroup F cybrids had lower activity than B4 cybrids, and that the ND1 3394T>C (Y30H) variant reduced complex I on both the B4 and F1 background but with very different cumulative effects. These data support the hypothesis that functional mtDNA variants may contribute to the risk of developing T2DM and MS.

Individualized iterative phenotyping for genome-wide analysis of loss-of-function mutations.

Next-generation sequencing provides the opportunity to practice predictive medicine based on identified variants. Putative loss-of-function (pLOF) variants are common in genomes and understanding their contribution to disease is critical for predictive medicine. To this end, we characterized the consequences of pLOF variants in an exome cohort by iterative phenotyping. Exome data were generated on 951 participants from the ClinSeq cohort and filtered for pLOF variants in genes likely to cause a phenotype in heterozygotes. 103 of 951 exomes had such a pLOF variant and 79 participants were evaluated. Of those 79, 34 had findings or family histories that could be attributed to the variant (28 variants in 18 genes), 2 had indeterminate findings (2 variants in 2 genes), and 43 had no findings or a negative family history for the trait (34 variants in 28 genes). The presence of a phenotype was correlated with two mutation attributes: prior report of pathogenicity for the variant (p = 0.0001) and prior report of other mutations in the same exon (p = 0.0001). We conclude that 1/30 unselected individuals harbor a pLOF mutation associated with a phenotype either in themselves or their family. This is more common than has been assumed and has implications for the setting of prior probabilities of affection status for predictive medicine.

Integrative DNA, RNA, and protein evidence connects TREML4 to coronary artery calcification.

Coronary artery calcification (CAC) is a heritable and definitive morphologic marker of atherosclerosis that strongly predicts risk for future cardiovascular events. To search for genes involved in CAC, we used an integrative transcriptomic, genomic, and protein expression strategy by using next-generation DNA sequencing in the discovery phase with follow-up studies using traditional molecular biology and histopathology techniques. RNA sequencing of peripheral blood from a discovery set of CAC cases and controls was used to identify dysregulated genes, which were validated by ClinSeq and Framingham Heart Study data. Only a single gene, TREML4, was upregulated in CAC cases in both studies. Further examination showed that rs2803496 was a TREML4 cis-eQTL and that the minor allele at this locus conferred up to a 6.5-fold increased relative risk of CAC. We characterized human TREML4 and demonstrated by immunohistochemical techniques that it is localized in macrophages surrounding the necrotic core of coronary plaques complicated by calcification (but not in arteries with less advanced disease). Finally, we determined by von Kossa staining that TREML4 colocalizes with areas of microcalcification within coronary plaques. Overall, we present integrative RNA, DNA, and protein evidence implicating TREML4 in coronary artery calcification. Our findings connect multimodal genomics data with a commonly used clinical marker of cardiovascular disease.

Assessing the capability of massively parallel sequencing for opportunistic pharmacogenetic screening.

PURPOSE: The aim of the study was to assess exome data for preemptive pharmacogenetic screening for 203 clinically relevant pharmacogenetic variant positions from the Pharmacogenomics Knowledgebase and Clinical Pharmacogenetics Implementation Consortium and identify copy-number variants (CNVs) in CYP2D6. METHODS: We examined the coverage and genotype quality of 203 pharmacogenetic variant positions in 973 exomes compared with five genomes and with five genotyping chip data sets. Then, we determined the agreement of exome and chip genotypes by evaluating concordance in a three-way comparison of exome, genome, and chip-based genotyping at 1,929 variant positions in five individuals. Finally, we evaluated the utility of exomes for detecting CYP2D6 CNVs. RESULTS: For 5 individuals examined for 203 pharmacogenetic variants (5 × 203 = 1,015), 998/1,015 were identified by genome, 849/1,015 were identified by exome, and 295/1,015 by genotyping chip. Thirty-six pharmacogenetic star allele variants with moderate to strong Clinical Pharmacogenetics Implementation Consortium (CPIC) therapeutic recommendations were identified in 973 exomes. Exomes had high (98%) genotype concordance with chip-based genotyping. CYP2D6 CNVs were identified in 57/973 exomes. CONCLUSIONS: Exomes outperformed the current chip-based assay in detecting more important pharmacogenetic variant positions and CYP2D6 CNVs for preemptive pharmacogenetic screening. Tools should be developed to derive pharmacogenetic variants from exomes.Genet Med 19 3, 357-361.

Kupffer cells modulate hepatic fatty acid oxidation during infection with PR8 influenza.

In response to infection, patients with inborn errors of metabolism may develop a functional deterioration termed metabolic decompensation. The biochemical hallmarks of this disruption of metabolic homeostasis are disease specific and may include acidosis, hyperammonemia or hypoglycemia. In a model system previously published by our group, we noted that during influenza infection, mice displayed a depression in hepatic mitochondrial enzymes involved in nitrogen metabolism. Based on these findings, we hypothesized that this normal adaptation may extend to other metabolic pathways, and as such, may impact various inborn errors of metabolism. Since the liver is a critical organ in inborn errors of metabolism, we carried out untargeted metabolomic profiling of livers using mass spectrometry in C57Bl/6 mice infected with influenza to characterize metabolic adaptation. Pathway analysis of metabolomic data revealed reductions in CoA synthesis, and long chain fatty acyl CoA and carnitine species. These metabolic adaptations coincided with a depression in hepatic long chain ß-oxidation mRNA and protein. To our surprise, the metabolic changes observed occurred in conjunction with a hepatic innate immune response, as demonstrated by transcriptional profiling and flow cytometry. By employing an immunomodulation strategy to deplete Kupffer cells, we were able to improve the expression of multiple genes involved in ß-oxidation. Based on these findings, we are the first to suggest that the role of the liver as an immunologic organ is central in the pathophysiology of hepatic metabolic decompensation in inborn errors of metabolism due to respiratory viral infection.

U1 snRNP protects pre-mRNAs from premature cleavage and polyadenylation.

In eukaryotes, U1 small nuclear ribonucleoprotein (snRNP) forms spliceosomes in equal stoichiometry with U2, U4, U5 and U6 snRNPs; however, its abundance in human far exceeds that of the other snRNPs. Here we used antisense morpholino oligonucleotide to U1 snRNA to achieve functional U1 snRNP knockdown in HeLa cells, and identified accumulated unspliced pre-mRNAs by genomic tiling microarrays. In addition to inhibiting splicing, U1 snRNP knockdown caused premature cleavage and polyadenylation in numerous pre-mRNAs at cryptic polyadenylation signals, frequently in introns near (<5 kilobases) the start of the transcript. This did not occur when splicing was inhibited with U2 snRNA antisense morpholino oligonucleotide or the U2-snRNP-inactivating drug spliceostatin A unless U1 antisense morpholino oligonucleotide was also included. We further show that U1 snRNA-pre-mRNA base pairing was required to suppress premature cleavage and polyadenylation from nearby cryptic polyadenylation signals located in introns. These findings reveal a critical splicing-independent function for U1 snRNP in protecting the transcriptome, which we propose explains its overabundance.

Dysregulation of synaptogenesis genes antecedes motor neuron pathology in spinal muscular atrophy.

The motor neuron (MN) degenerative disease, spinal muscular atrophy (SMA) is caused by deficiency of SMN (survival motor neuron), a ubiquitous and indispensable protein essential for biogenesis of snRNPs, key components of pre-mRNA processing. However, SMA's hallmark MN pathology, including neuromuscular junction (NMJ) disruption and sensory-motor circuitry impairment, remains unexplained. Toward this end, we used deep RNA sequencing (RNA-seq) to determine if there are any transcriptome changes in MNs and surrounding spinal cord glial cells (white matter, WM) microdissected from SMN-deficient SMA mouse model at presymptomatic postnatal day 1 (P1), before detectable MN pathology (P4-P5). The RNA-seq results, previously unavailable for SMA at any stage, revealed cell-specific selective mRNA dysregulations (~300 of 11,000 expressed genes in each, MN and WM), many of which are known to impair neurons. Remarkably, these dysregulations include complete skipping of agrin's Z exons, critical for NMJ maintenance, strong up-regulation of synapse pruning-promoting complement factor C1q, and down-regulation of Etv1/ER81, a transcription factor required for establishing sensory-motor circuitry. We propose that dysregulation of such specific MN synaptogenesis genes, compounded by many additional transcriptome abnormalities in MNs and WM, link SMN deficiency to SMA's signature pathology.

Secondary variants in individuals undergoing exome sequencing: screening of 572 individuals identifies high-penetrance mutations in cancer-susceptibility genes.

Genome- and exome-sequencing costs are continuing to fall, and many individuals are undergoing these assessments as research participants and patients. The issue of secondary (so-called incidental) findings in exome analysis is controversial, and data are needed on methods of detection and their frequency. We piloted secondary variant detection by analyzing exomes for mutations in cancer-susceptibility syndromes in subjects ascertained for atherosclerosis phenotypes. We performed exome sequencing on 572 ClinSeq participants, and in 37 genes, we interpreted variants that cause high-penetrance cancer syndromes by using an algorithm that filtered results on the basis of mutation type, quality, and frequency and that filtered mutation-database entries on the basis of defined categories of causation. We identified 454 sequence variants that differed from the human reference. Exclusions were made on the basis of sequence quality (26 variants) and high frequency in the cohort (77 variants) or dbSNP (17 variants), leaving 334 variants of potential clinical importance. These were further filtered on the basis of curation of literature reports. Seven participants, four of whom were of Ashkenazi Jewish descent and three of whom did not meet family-history-based referral criteria, had deleterious BRCA1 or BRCA2 mutations. One participant had a deleterious SDHC mutation, which causes paragangliomas. Exome sequencing, coupled with multidisciplinary interpretation, detected clinically important mutations in cancer-susceptibility genes; four of such mutations were in individuals without a significant family history of disease. We conclude that secondary variants of high clinical importance will be detected at an appreciable frequency in exomes, and we suggest that priority be given to the development of more efficient modes of interpretation with trials in larger patient groups.

Identification of candidate genes involved in coronary artery calcification by transcriptome sequencing of cell lines.

BACKGROUND: Massively-parallel cDNA sequencing (RNA-Seq) is a new technique that holds great promise for cardiovascular genomics. Here, we used RNA-Seq to study the transcriptomes of matched coronary artery disease cases and controls in the ClinSeq® study, using cell lines as tissue surrogates. RESULTS: Lymphoblastoid cell lines (LCLs) from 16 cases and controls representing phenotypic extremes for coronary calcification were cultured and analyzed using RNA-Seq. All cell lines were then independently re-cultured and along with another set of 16 independent cases and controls, were profiled with Affymetrix microarrays to perform a technical validation of the RNA-Seq results. Statistically significant changes (p < 0.05) were detected in 186 transcripts, many of which are expressed at extremely low levels (5-10 copies/cell), which we confirmed through a separate spike-in control RNA-Seq experiment. Next, by fitting a linear model to exon-level RNA-Seq read counts, we detected signals of alternative splicing in 18 transcripts. Finally, we used the RNA-Seq data to identify differential expression (p < 0.0001) in eight previously unannotated regions that may represent novel transcripts. Overall, differentially expressed genes showed strong enrichment (p = 0.0002) for prior association with cardiovascular disease. At the network level, we found evidence for perturbation in pathways involving both cardiovascular system development and function as well as lipid metabolism. CONCLUSIONS: We present a pilot study for transcriptome involvement in coronary artery calcification and demonstrate how RNA-Seq analyses using LCLs as a tissue surrogate may yield fruitful results in a clinical sequencing project. In addition to canonical gene expression, we present candidate variants from alternative splicing and novel transcript detection, which have been unexplored in the context of this disease.

The genome sequence of Molossusnigricans (Chiroptera, Molossidae; Miller, 1902).

We present a genome assembly from an individual male Molossus nigricans (Chordata; Mammalia; Chiroptera; Molossidae). The genome sequence is 2.41 gigabases in span. The majority of the assembly is scaffolded into 24 chromosomal pseudomolecules, with the X sex chromosome assembled.

VWA3A-derived ependyma promoter drives increased therapeutic protein secretion into the CSF.

Recombinant adeno-associated viral vectors (rAAVs) are a promising strategy to treat neurodegenerative diseases because of their ability to infect non-dividing cells and confer long-term transgene expression. Despite an ever-growing library of capsid variants, widespread delivery of AAVs in the adult central nervous system remains a challenge. We have previously demonstrated successful distribution of secreted proteins by infection of the ependyma, a layer of post-mitotic epithelial cells lining the ventricles of the brain and central column of the spinal cord, and subsequent protein delivery via the cerebrospinal fluid (CSF). Here we define a functional ependyma promoter to enhance expression from this cell type. Using RNA sequencing on human autopsy samples, we identified disease- and age-independent ependyma gene signatures. Associated promoters were cloned and screened as libraries in mouse and rhesus macaque to reveal cross-species function of a human DNA-derived von Willebrand factor domain containing 3A (VWA3A) promoter. When tested in mice, our VWA3A promoter drove strong, ependyma-localized expression of eGFP and increased secreted ApoE protein levels in the CSF by 2-12× over the ubiquitous iCAG promoter.

Core mitochondrial genes are down-regulated during SARS-CoV-2 infection of rodent and human hosts.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral proteins bind to host mitochondrial proteins, likely inhibiting oxidative phosphorylation (OXPHOS) and stimulating glycolysis. We analyzed mitochondrial gene expression in nasopharyngeal and autopsy tissues from patients with coronavirus disease 2019 (COVID-19). In nasopharyngeal samples with declining viral titers, the virus blocked the transcription of a subset of nuclear DNA (nDNA)-encoded mitochondrial OXPHOS genes, induced the expression of microRNA 2392, activated HIF-1α to induce glycolysis, and activated host immune defenses including the integrated stress response. In autopsy tissues from patients with COVID-19, SARS-CoV-2 was no longer present, and mitochondrial gene transcription had recovered in the lungs. However, nDNA mitochondrial gene expression remained suppressed in autopsy tissue from the heart and, to a lesser extent, kidney, and liver, whereas mitochondrial DNA transcription was induced and host-immune defense pathways were activated. During early SARS-CoV-2 infection of hamsters with peak lung viral load, mitochondrial gene expression in the lung was minimally perturbed but was down-regulated in the cerebellum and up-regulated in the striatum even though no SARS-CoV-2 was detected in the brain. During the mid-phase SARS-CoV-2 infection of mice, mitochondrial gene expression was starting to recover in mouse lungs. These data suggest that when the viral titer first peaks, there is a systemic host response followed by viral suppression of mitochondrial gene transcription and induction of glycolysis leading to the deployment of antiviral immune defenses. Even when the virus was cleared and lung mitochondrial function had recovered, mitochondrial function in the heart, kidney, liver, and lymph nodes remained impaired, potentially leading to severe COVID-19 pathology.

Correlated changes between regulatory cis elements and condition-specific expression in paralogous gene families.

Gene duplication is integral to evolution, providing novel opportunities for organisms to diversify in function. One fundamental pathway of functional diversification among initially redundant gene copies, or paralogs, is via alterations in their expression patterns. Although the mechanisms underlying expression divergence are not completely understood, transcription factor binding sites and nucleosome occupancy are known to play a significant role in the process. Previous attempts to detect genomic variations mediating expression divergence in orthologs have had limited success for two primary reasons. First, it is inherently challenging to compare expressions among orthologs due to variable trans-acting effects and second, previous studies have quantified expression divergence in terms of an overall similarity of expression profiles across multiple samples, thereby obscuring condition-specific expression changes. Moreover, the inherently inter-correlated expressions among homologs present statistical challenges, not adequately addressed in many previous studies. Using rigorous statistical tests, here we characterize the relationship between cis element divergence and condition-specific expression divergence among paralogous genes in Saccharomyces cerevisiae. In particular, among all combinations of gene family and TFs analyzed, we found a significant correlation between TF binding and the condition-specific expression patterns in over 20% of the cases. In addition, incorporating nucleosome occupancy reveals several additional correlations. For instance, our results suggest that GAL4 binding plays a major role in the expression divergence of the genes in the sugar transporter family. Our work presents a novel means of investigating the cis regulatory changes potentially mediating expression divergence in paralogous gene families under specific conditions.

Transcriptome and metabolome after porcine hemodynamic-directed CPR compared with standard CPR and sham controls.

Objective: The effect of cardiac arrest (CA) on cerebral transcriptomics and metabolomics is unknown. We previously demonstrated hemodynamic-directed CPR (HD-CPR) improves survival with favorable neurologic outcomes versus standard CPR (Std-CPR). We hypothesized HD-CPR would preserve the cerebral transcriptome and metabolome compared to Std-CPR. Design: Randomized pre-clinical animal trial. Setting: Large animal resuscitation laboratory at an academic children's hospital. Subjects: Four-week-old female piglets (8-11 kg). Interventions: Pigs (1-month-old), three groups: 1) HD-CPR (compression depth to systolic BP 90 mmHg, vasopressors to coronary perfusion pressure 20 mmHg); 2) Std-CPR and 3) shams (no CPR). HD-CPR and Std-CPR underwent asphyxia, induced ventricular fibrillation, 10-20 min of CPR and post-resuscitation care. Primary outcomes at 24 h in cerebral cortex: 1) transcriptomic analysis (n = 4 per treatment arm, n = 8 sham) of 1727 genes using differential gene expression and 2) metabolomic analysis (n = 5 per group) of 27 metabolites using one-way ANOVA, post-hoc Tukey HSD. Measurements and main results: 65 genes were differentially expressed between HD-CPR and Std-CPR and 72 genes between Std-CPR and sham, but only five differed between HD-CPR and sham. Std-CPR increased the concentration of five AA compared to HD-CPR and sham, including the branched chain amino acids (BCAA), but zero metabolites differed between HD-CPR and sham. Conclusions: In cerebral cortex 24 h post CA, Std-CPR resulted in a different transcriptome and metabolome compared with either HD-CPR or sham. HD-CPR preserves the transcriptome and metabolome, and is neuroprotective. Global molecular analyses may be a novel method to assess efficacy of clinical interventions and identify therapeutic targets. Institutional protocol number: IAC 16-001023.