Degree of Cyclooxygenase-2 Inhibition Modulates Blood Pressure Response to Celecoxib and Naproxen.

Background: Non-steroidal anti-inflammatory drugs (NSAIDs) increase the risk of adverse cardiovascular events via suppression of cyclooxygenase (COX)-2-derived prostacyclin (PGI2) formation in heart, vasculature, and kidney. The Prospective Randomized Evaluation of Celecoxib Integrated Safety versus Ibuprofen Or Naproxen (PRECISION) trial and other large clinical studies compared the cardiovascular risk of traditional NSAIDs (i.e. naproxen), which inhibit both COX isozymes, with NSAIDs selective for COX-2 (i.e. celecoxib). However, whether pharmacologically equipotent doses were used - that is, whether a similar degree of COX-2 inhibition was achieved - was not considered. We compared drug target inhibition and blood pressure response to celecoxib at the dose used by most patients in PRECISION with the lowest recommended naproxen dose for osteoarthritis, which is lower than the dose used in PRECISION. Methods: Sixteen healthy participants (19-61 years) were treated with celecoxib (100 mg every 12h), naproxen (250 mg every 12h), or placebo administered twice daily for seven days in a double-blind, crossover design randomized by order. On Day 7 when drug levels had reached steady state, the degree of COX inhibition was assessed ex vivo and in vivo. Ambulatory blood pressure was measured throughout the final 12h dosing interval. Results: Both NSAIDs inhibited COX-2 activity relative to placebo, but naproxen inhibited COX-2 activity to a greater degree (62.9±21.7%) than celecoxib (35.7±25.2%; p<0.05). Similarly, naproxen treatment inhibited PGI2 formation in vivo (48.0±24.9%) to a greater degree than celecoxib (26.7±24.6%; p<0.05). Naproxen significantly increased blood pressure compared to celecoxib (differences in least-square means of mean arterial pressure: 2.5 mm Hg (95% CI: 1.5, 3.5); systolic blood pressure: 4.0 mm Hg (95% CI: 2.9, 5.1); diastolic blood pressure: 1.8 mm Hg (95% CI: 0.8, 2.8); p<0.05 for all). The difference in systolic blood pressure relative to placebo was associated with the degree of COX-2 inhibition (p<0.05). Conclusions: Celecoxib 200 mg/day inhibited COX-2 activity to a lesser degree than naproxen 500 mg/day, resulting in a less pronounced blood pressure increase. While the PRECISION trial concluded the non-inferiority of celecoxib regarding cardiovascular risk, this is based on a comparison of doses that are not equipotent.ClinicalTrials.gov identifier: NCT02502006 (https://clinicaltrials.gov/study/NCT02502006).

Three-dimensional chromatin reorganization regulates B cell development during ageing.

The contribution of three-dimensional genome organization to physiological ageing is not well known. Here we show that large-scale chromatin reorganization distinguishes young and old bone marrow progenitor (pro-) B cells. These changes result in increased interactions at the compartment level and reduced interactions within topologically associated domains (TADs). The gene encoding Ebf1, a key B cell regulator, switches from compartment A to B with age. Genetically reducing Ebf1 recapitulates some features of old pro-B cells. TADs that are most reduced with age contain genes important for B cell development, including the immunoglobulin heavy chain (Igh) locus. Weaker intra-TAD interactions at Igh correlate with altered variable (V), diversity (D) and joining (J) gene recombination. Our observations implicate three-dimensional chromatin reorganization as a major driver of pro-B cell phenotypes that impair B lymphopoiesis with age.

BEERS2: RNA-Seq simulation through high fidelity in silico modeling.

Simulation of RNA-seq reads is critical in the assessment, comparison, benchmarking and development of bioinformatics tools. Yet the field of RNA-seq simulators has progressed little in the last decade. To address this need we have developed BEERS2, which combines a flexible and highly configurable design with detailed simulation of the entire library preparation and sequencing pipeline. BEERS2 takes input transcripts (typically fully length messenger RNA transcripts with polyA tails) from either customizable input or from CAMPAREE simulated RNA samples. It produces realistic reads of these transcripts as FASTQ, SAM or BAM formats with the SAM or BAM formats containing the true alignment to the reference genome. It also produces true transcript-level quantification values. BEERS2 combines a flexible and highly configurable design with detailed simulation of the entire library preparation and sequencing pipeline and is designed to include the effects of polyA selection and RiboZero for ribosomal depletion, hexamer priming sequence biases, GC-content biases in polymerase chain reaction (PCR) amplification, barcode read errors and errors during PCR amplification. These characteristics combine to make BEERS2 the most complete simulation of RNA-seq to date. Finally, we demonstrate the use of BEERS2 by measuring the effect of several settings on the popular Salmon pseudoalignment algorithm.

Epigenetic signature of human immune aging in the GESTALT study.

Age-associated DNA methylation in blood cells convey information on health status. However, the mechanisms that drive these changes in circulating cells and their relationships to gene regulation are unknown. We identified age-associated DNA methylation sites in six purified blood-borne immune cell types (naive B, naive CD4+ and CD8+ T cells, granulocytes, monocytes, and NK cells) collected from healthy individuals interspersed over a wide age range. Of the thousands of age-associated sites, only 350 sites were differentially methylated in the same direction in all cell types and validated in an independent longitudinal cohort. Genes close to age-associated hypomethylated sites were enriched for collagen biosynthesis and complement cascade pathways, while genes close to hypermethylated sites mapped to neuronal pathways. In silico analyses showed that in most cell types, the age-associated hypo- and hypermethylated sites were enriched for ARNT (HIF1ß) and REST transcription factor (TF) motifs, respectively, which are both master regulators of hypoxia response. To conclude, despite spatial heterogeneity, there is a commonality in the putative regulatory role with respect to TF motifs and histone modifications at and around these sites. These features suggest that DNA methylation changes in healthy aging may be adaptive responses to fluctuations of oxygen availability.

Sexual dimorphism in the response to chronic circadian misalignment on a high-fat diet.

Longitudinal studies associate shiftwork with cardiometabolic disorders but do not establish causation or elucidate mechanisms of disease. We developed a mouse model based on shiftwork schedules to study circadian misalignment in both sexes. Behavioral and transcriptional rhythmicity were preserved in female mice despite exposure to misalignment. Females were protected from the cardiometabolic impact of circadian misalignment on a high-fat diet seen in males. The liver transcriptome and proteome revealed discordant pathway perturbations between the sexes. Tissue-level changes were accompanied by gut microbiome dysbiosis only in male mice, biasing toward increased potential for diabetogenic branched chain amino acid production. Antibiotic ablation of the gut microbiota diminished the impact of misalignment. In the United Kingdom Biobank, females showed stronger circadian rhythmicity in activity and a lower incidence of metabolic syndrome than males among job-matched shiftworkers. Thus, we show that female mice are more resilient than males to chronic circadian misalignment and that these differences are conserved in humans.

BEERS2: RNA-Seq simulation through high fidelity in silico modeling.

Simulation of RNA-seq reads is critical in the assessment, comparison, benchmarking, and development of bioinformatics tools. Yet the field of RNA-seq simulators has progressed little in the last decade. To address this need we have developed BEERS2, which combines a flexible and highly configurable design with detailed simulation of the entire library preparation and sequencing pipeline. BEERS2 takes input transcripts (typically fully-length mRNA transcripts with polyA tails) from either customizable input or from CAMPAREE simulated RNA samples. It produces realistic reads of these transcripts as FASTQ, SAM, or BAM formats with the SAM or BAM formats containing the true alignment to the reference genome. It also produces true transcript-level quantification values. BEERS2 combines a flexible and highly configurable design with detailed simulation of the entire library preparation and sequencing pipeline and is designed to include the effects of polyA selection and RiboZero for ribosomal depletion, hexamer priming sequence biases, GC-content biases in PCR amplification, barcode read errors, and errors during PCR amplification. These characteristics combine to make BEERS2 the most complete simulation of RNA-seq to date. Finally, we demonstrate the use of BEERS2 by measuring the effect of several settings on the popular Salmon pseudoalignment algorithm.

Aging exacerbates the brain inflammatory micro-environment contributing to α-synuclein pathology and functional deficits in a mouse model of DLB/PD.

BACKGROUND: Although ɑ-synuclein (ɑ-syn) spreading in age-related neurodegenerative diseases such as Parkinson's disease (PD) and Dementia with Lewy bodies (DLB) has been extensively investigated, the role of aging in the manifestation of disease remains unclear. METHODS: We explored the role of aging and inflammation in the pathogenesis of synucleinopathies in a mouse model of DLB/PD initiated by intrastriatal injection of ɑ-syn preformed fibrils (pff). RESULTS: We found that aged mice showed more extensive accumulation of ɑ-syn in selected brain regions and behavioral deficits that were associated with greater infiltration of T cells and microgliosis. Microglial inflammatory gene expression induced by ɑ-syn-pff injection in young mice had hallmarks of aged microglia, indicating that enhanced age-associated pathologies may result from inflammatory synergy between aging and the effects of ɑ-syn aggregation. Based on the transcriptomics analysis projected from Ingenuity Pathway Analysis, we found a network that included colony stimulating factor 2 (CSF2), LPS related genes, TNFɑ and poly rl:rC-RNA as common regulators. CONCLUSIONS: We propose that aging related inflammation (eg: CSF2) influences outcomes of pathological spreading of ɑ-syn and suggest that targeting neuro-immune responses might be important in developing treatments for DLB/PD.

Hidden neural networks for transmembrane protein topology prediction.

Hidden Markov Models (HMMs) are amongst the most successful methods for predicting protein features in biological sequence analysis. However, there are biological problems where the Markovian assumption is not sufficient since the sequence context can provide useful information for prediction purposes. Several extensions of HMMs have appeared in the literature in order to overcome their limitations. We apply here a hybrid method that combines HMMs and Neural Networks (NNs), termed Hidden Neural Networks (HNNs), for biological sequence analysis in a straightforward manner. In this framework, the traditional HMM probability parameters are replaced by NN outputs. As a case study, we focus on the topology prediction of for alpha-helical and beta-barrel membrane proteins. The HNNs show performance gains compared to standard HMMs and the respective predictors outperform the top-scoring methods in the field. The implementation of HNNs can be found in the package JUCHMME, downloadable from http://www.compgen.org/tools/juchmme, https://github.com/pbagos/juchmme. The updated PRED-TMBB2 and HMM-TM prediction servers can be accessed at www.compgen.org.

DNA methylation signatures reveal that distinct combinations of transcription factors specify human immune cell epigenetic identity.

Epigenetic reprogramming underlies specification of immune cell lineages, but patterns that uniquely define immune cell types and the mechanisms by which they are established remain unclear. Here, we identified lineage-specific DNA methylation signatures of six immune cell types from human peripheral blood and determined their relationship to other epigenetic and transcriptomic patterns. Sites of lineage-specific hypomethylation were associated with distinct combinations of transcription factors in each cell type. By contrast, sites of lineage-specific hypermethylation were restricted mostly to adaptive immune cells. PU.1 binding sites were associated with lineage-specific hypo- and hypermethylation in different cell types, suggesting that it regulates DNA methylation in a context-dependent manner. These observations indicate that innate and adaptive immune lineages are specified by distinct epigenetic mechanisms via combinatorial and context-dependent use of key transcription factors. The cell-specific epigenomics and transcriptional patterns identified serve as a foundation for future studies on immune dysregulation in diseases and aging.

CAMPAREE: a robust and configurable RNA expression simulator.

BACKGROUND: The accurate interpretation of RNA-Seq data presents a moving target as scientists continue to introduce new experimental techniques and analysis algorithms. Simulated datasets are an invaluable tool to accurately assess the performance of RNA-Seq analysis methods. However, existing RNA-Seq simulators focus on modeling the technical biases and artifacts of sequencing, rather than on simulating the original RNA samples. A first step in simulating RNA-Seq is to simulate RNA. RESULTS: To fill this need, we developed the Configurable And Modular Program Allowing RNA Expression Emulation (CAMPAREE), a simulator using empirical data to simulate diploid RNA samples at the level of individual molecules. We demonstrated CAMPAREE's use for generating idealized coverage plots from real data, and for adding the ability to generate allele-specific data to existing RNA-Seq simulators that do not natively support this feature. CONCLUSIONS: Separating input sample modeling from library preparation/sequencing offers added flexibility for both users and developers to mix-and-match different sample and sequencing simulators to suit their specific needs. Furthermore, the ability to maintain sample and sequencing simulators independently provides greater agility to incorporate new biological findings about transcriptomics and new developments in sequencing technologies. Additionally, by simulating at the level of individual molecules, CAMPAREE has the potential to model molecules transcribed from the same genes as a heterogeneous population of transcripts with different states of degradation and processing (splicing, editing, etc.). CAMPAREE was developed in Python, is open source, and freely available at https://github.com/itmat/CAMPAREE .

Sex-dependent compensatory mechanisms preserve blood pressure homeostasis in prostacyclin receptor-deficient mice.

Inhibitors of microsomal prostaglandin E synthase 1 (mPGES-1) are in the early phase of clinical development. Deletion of mPges-1 in mice confers analgesia, restrains atherogenesis, and fails to accelerate thrombogenesis, while suppressing prostaglandin E2 (PGE2), but increasing the biosynthesis of prostacyclin (PGI2). In low-density lipoprotein receptor-deficient (Ldlr-/-) mice, this last effect represents the dominant mechanism by which mPges-1 deletion restrains thrombogenesis, while suppression of PGE2 accounts for its antiatherogenic effect. However, the effect of mPges-1 depletion on blood pressure (BP) in this setting remains unknown. Here, we show that mPges-1 depletion significantly increased the BP response to salt loading in male Ldlr-/- mice, whereas, despite the direct vasodilator properties of PGI2, deletion of the I prostanoid receptor (Ipr) suppressed this response. Furthermore, combined deletion of the Ipr abrogated the exaggerated BP response in male mPges-1-/- mice. Interestingly, these unexpected BP phenotypes were not observed in female mice fed a high-salt diet (HSD). This is attributable to the protective effect of estrogen in Ldlr-/- mice and in Ipr-/- Ldlr-/- mice. Thus, estrogen compensates for a deficiency in PGI2 to maintain BP homeostasis in response to high salt in hyperlipidemic female mice. In male mice, by contrast, the augmented formation of atrial natriuretic peptide (ANP) plays a similar compensatory role, restraining hypertension and oxidant stress in the setting of Ipr depletion. Hence, men with hyperlipidemia on a HSD might be at risk of a hypertensive response to mPGES-1 inhibitors.

Comparative evaluation of full-length isoform quantification from RNA-Seq.

BACKGROUND: Full-length isoform quantification from RNA-Seq is a key goal in transcriptomics analyses and has been an area of active development since the beginning. The fundamental difficulty stems from the fact that RNA transcripts are long, while RNA-Seq reads are short. RESULTS: Here we use simulated benchmarking data that reflects many properties of real data, including polymorphisms, intron signal and non-uniform coverage, allowing for systematic comparative analyses of isoform quantification accuracy and its impact on differential expression analysis. Genome, transcriptome and pseudo alignment-based methods are included; and a simple approach is included as a baseline control. CONCLUSIONS: Salmon, kallisto, RSEM, and Cufflinks exhibit the highest accuracy on idealized data, while on more realistic data they do not perform dramatically better than the simple approach. We determine the structural parameters with the greatest impact on quantification accuracy to be length and sequence compression complexity and not so much the number of isoforms. The effect of incomplete annotation on performance is also investigated. Overall, the tested methods show sufficient divergence from the truth to suggest that full-length isoform quantification and isoform level DE should still be employed selectively.

RNA-seq analysis of galaninergic neurons from ventrolateral preoptic nucleus identifies expression changes between sleep and wake.

BACKGROUND: Previous studies show that galanin neurons in ventrolateral preoptic nucleus (VLPO-Gal) are essential for sleep regulation. Here, we explored the transcriptional regulation of the VLPO-Gal neurons in sleep by comparing their transcriptional responses between sleeping mice and those kept awake, sacrificed at the same diurnal time. RESULTS: RNA-sequencing (RNA-seq) analysis was performed on eGFP(+) galanin neurons isolated using laser captured microdissection (LCM) from VLPO. Expression of Gal was assessed in our LCM eGFP(+) neurons via real time qPCR and showed marked enrichment when compared to LCM eGFP(-) cells and to bulk VLPO samples. Gene set enrichment analysis utilizing data from a recent single-cell RNA-seq study of the preoptic area demonstrated that our VLPO-Gal samples were highly enriched with galanin-expressing inhibitory neurons, but not galanin-expressing excitatory neurons. A total of 263 genes were differentially expressed between sleep and wake in VLPO-Gal neurons. When comparing differentially expressed genes in VLPO-Gal neurons to differentially expressed genes in a wake-active neuronal region (the medial prefrontal cortex), evidence indicates that both systemic and cell-specific mechanisms contribute to the transcriptional regulation in VLPO-Gal neurons. In both wake-active and sleep-active neurons, ER stress pathways are activated by wake and cold-inducible RNA-binding proteins are activated by sleep. In contrast, expression of DNA repair genes is increased in VLPO-Gal during wakefulness, but increased in wake-active cells during sleep. CONCLUSION: Our study identified transcriptomic responses of the galanin neurons in the ventrolateral preoptic nucleus during sleep and sleep deprivation. Data indicate that VLPO contains mainly sleep-active inhibitory galaninergic neurons. The VLPO galanin neurons show responses to sleep and wake similar to wake-active regions, indicating these responses, such as ER stress and cold-inducible RNA-binding proteins, are systemic affecting all neuronal populations. Region-specific differences in sleep/wake responses were also identified, in particular DNA repair. Our study expands knowledge about the transcriptional response of a distinct group of neurons essential for sleep.

Age attenuates the transcriptional changes that occur with sleep in the medial prefrontal cortex.

Sleep abnormalities are common with aging. Studies show that sleep plays important roles in brain functions, and loss of sleep is associated with increased risks for neurological diseases. Here, we used RNA sequencing to explore effects of age on transcriptome changes between sleep and sleep deprivation (SD) in medial prefrontal cortex and found that transcriptional changes with sleep are attenuated in old. In particular, old mice showed a 30% reduction in the number of genes significantly altered between sleep/wake and, in general, had smaller magnitudes of changes in differentially expressed genes compared to young mice. Gene ontology analysis revealed differential age effects on certain pathways. Compared to young mice, many of the wake-active functions were similarly induced by SD in old mice, whereas many of the sleep-active pathways were attenuated in old mice. We found similar magnitude of changes in synaptic homeostasis genes (Fos, Arc, and Bdnf) induced by SD, suggesting intact synaptic upscaling on the transcript level during extended wakefulness with aging. However, sleep-activated processes, such as DNA repair, synaptogenesis, and axon guidance, were sensitive to the effect of aging. Old mice expressed elevated levels of immune response genes when compared to young mice, and enrichment analysis using cell-type-specific markers indicated upregulation of microglia and oligodendrocyte genes in old mice. Moreover, gene sets of the two cell types showed age-specific sleep/wake regulation. Ultimately, this study enhances understanding of the transcriptional changes with sleep and aging, providing potential molecular targets for future studies of age-related sleep abnormalities and neurological disorders.

Comparative evaluation of RNA-Seq library preparation methods for strand-specificity and low input.

Library preparation is a key step in sequencing. For RNA sequencing there are advantages to both strand specificity and working with minute starting material, yet until recently there was no kit available enabling both. The Illumina TruSeq stranded mRNA Sample Preparation kit (TruSeq) requires abundant starting material while the Takara Bio SMART-Seq v4 Ultra Low Input RNA kit (V4) sacrifices strand specificity. The SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian (Pico) by Takara Bio claims to overcome these limitations. Comparative evaluation of these kits is important for selecting the appropriate protocol. We compared the three kits in a realistic differential expression analysis. We prepared and sequenced samples from two experimental conditions of biological interest with each of the three kits. We report differences between the kits at the level of differential gene expression; for example, the Pico kit results in 55% fewer differentially expressed genes than TruSeq. Nevertheless, the agreement of the observed enriched pathways suggests that comparable functional results can be obtained. In summary we conclude that the Pico kit sufficiently reproduces the results of the other kits at the level of pathway analysis while providing a combination of options that is not available in the other kits.

JUCHMME: a Java Utility for Class Hidden Markov Models and Extensions for biological sequence analysis.

SUMMARY: JUCHMME is an open-source software package designed to fit arbitrary custom Hidden Markov Models (HMMs) with a discrete alphabet of symbols. We incorporate a large collection of standard algorithms for HMMs as well as a number of extensions and evaluate the software on various biological problems. Importantly, the JUCHMME toolkit includes several additional features that allow for easy building and evaluation of custom HMMs, which could be a useful resource for the research community. AVAILABILITY AND IMPLEMENTATION: http://www.compgen.org/tools/juchmme, https://github.com/pbagos/juchmme. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Distinct vascular genomic response of proton and gamma radiation-A pilot investigation.

The cardiovascular biology of proton radiotherapy is not well understood. We aimed to compare the genomic dose-response to proton and gamma radiation of the mouse aorta to assess whether their vascular effects may diverge. We performed comparative RNA sequencing of the aorta following (4 hrs) total-body proton and gamma irradiation (0.5-200 cGy whole body dose, 10 dose levels) of conscious mice. A trend analysis identified genes that showed a dose response. While fewer genes were dose-responsive to proton than gamma radiation (29 vs. 194 genes; q-value ≤ 0.1), the magnitude of the effect was greater. Highly responsive genes were enriched for radiation response pathways (DNA damage, apoptosis, cellular stress and inflammation; p-value ≤ 0.01). Gamma, but not proton radiation induced additionally genes in vasculature specific pathways. Genes responsive to both radiation types showed almost perfectly superimposable dose-response relationships. Despite the activation of canonical radiation response pathways by both radiation types, we detected marked differences in the genomic response of the murine aorta. Models of cardiovascular risk based on photon radiation may not accurately predict the risk associated with proton radiation.

Analysis of HETEs in human whole blood by chiral UHPLC-ECAPCI/HRMS.

The biosynthesis of eicosanoids occurs enzymatically via lipoxygenases, cyclooxygenases, and cytochrome P450, or through nonenzymatic free radical reactions. The enzymatic routes are highly enantiospecific. Chiral separation and high-sensitivity detection methods are required to differentiate and quantify enantioselective HETEs in complex biological fluids. We report here a targeted chiral lipidomics analysis of human blood using ultra-HPLC-electron capture (EC) atmospheric pressure chemical ionization/high-resolution MS. Monitoring the high-resolution ions formed by the fragmentation of pentafluorobenzyl derivatives of oxidized lipids during the dissociative EC, followed by in-trap fragmentation, increased sensitivity by an order of magnitude when compared with the unit resolution MS. The 12(S)-HETE, 12(S)-hydroxy-(5Z,8E,10E)-heptadecatrienoic acid [12(S)-HHT], and 15(S)-HETE were the major hydroxylated nonesterified chiral lipids in serum. Stimulation of whole blood with zymosan and lipopolysaccharide (LPS) resulted in stimulus- and time-dependent effects. An acute exposure to zymosan induced ∼80% of the chiral plasma lipids, including 12(S)-HHT, 5(S)-HETE, 15(R)-HETE, and 15(S)-HETE, while a maximum response to LPS was achieved after a long-term stimulation. The reported method allows for a rapid quantification with high sensitivity and specificity of enantiospecific responses to in vitro stimulation or coagulation of human blood.