Abstract Pulmonary hypertension (PH) is a vasculopathy that results in elevated mean pulmonary arterial pressures over 20mmHg. Despite significant advances in research, PH still has a high mortality rate, and there is currently no cure for the disease. As with all biomedical fields, PH researchers have embraced the power of next generation technologies such as microarrays and RNA sequencing. Most of these data can be found on public repositories, which is usually a requirement for publication. While these repositories are rich sources of data, they require intermediate to advanced bioinformatics skills to access, download, and make these data useful. Here we present P ulmonary H ypertension E ngine for L inked E xperiments ( PHELEX ), which represents a comprehensive catalogue of all RNA sequencing data related to PH that is currently available on the Gene Expression Omnibus (GEO), hosted by the US National Centre for Biotechnology Information (NCBI). We identified 2,278 bulk RNA sequencing samples from human, mouse and rat, and built a searchable tool based on the metadata that is associated with each sample. PHELEX is a functional tool that allows selected studies to be highlighted, and parsed through Confidence, an analysis tool we have created, which will model the data based on user-defined classifiers, perform differential gene expression and pathway analysis, and present these data using standard graphics, and text-file results. PHELEX also allows PH researchers to cross-cut between discrete studies, facilitating de novo understanding of these data. As a robust searchable repository of genomic data, we hope that PHELEX will accelerate PH innovation and discovery, by allowing researchers to mine existing genomic data and thus better understand the molecular signatures that underpin PH.

RATIONALE:Sepsis is a state of life-threatening organ dysfunction in the setting of infection. It is biologically heterogeneous, as evidenced by whole blood transcriptomic analyses that reveal distinct molecular subtypes based on gene expression. At the cellular level, sepsis is primarily mediated by neutrophils, a leukocyte population increasingly recognized as heterogeneous across several domains. We sought to further characterize neutrophil heterogeneity by identifying neutrophil subsets in critically ill patients with severe sepsis based on multidimensional mass cytometry analysis. METHODS:We generated time series mass cytometry (CyTOF) data from whole blood samples taken from 17 patients with sepsis admitted to an intensive care unit who were enrolled in a randomized controlled trial of high-dose vitamin C. We analyzed these data using unsupervised machine learning techniques to identify distinct neutrophil subtypes. We characterized the resulting subtypes and described their changes over time. RESULTS:We analyzed approximately 1.5 million cytometry events gated as neutrophils. We identified five clusters that reveal complex heterogeneity across multiple neutrophil markers of maturation and activation including olfactomedin-4 (OLFM4), CD177, glucose transporter 1 (GLUT1), CD16, and lipocalin-2. The two dominant clusters differed primarily in the abundance of OLFM4, a neutrophil granule protein. Between Day 1 and Day 7, there was an increased proportion of neutrophils in the dominant OLFM4-expressing cluster, a difference primarily driven by increased abundance of OLFM4 in the placebo group, but not the vitamin C group. CONCLUSIONS:Our findings point to complex multidimensional heterogeneity among neutrophils in sepsis, thereby extending the current concept of neutrophil heterogeneity with new data from critically ill patients with sepsis derived from mass cytometry. Variation in the temporal differences in cluster proportions between treatment arms may suggest opportunities for precision sepsis treatment.

AbstractBackgroundHypertension is a polygenic, complex disease that impacts men and women differently; whilst the incidence of high blood pressure (BP) is roughly equal over a lifetime, men typically are at higher risk of developing the disease earlier in life, before 50 years of age. There is adequate evidence that the brain is critical for the BP setpoint. The paraventricular nucleus (PVN) of the hypothalamus is an integrative structure that can influence not only neurohumoral responses to blood pressure changes, but also sympathetic drive. Here we manipulate the androgenic status of both male and female spontaneously hypertensive rats (SHRs) to determine how this changes gene expression within the PVN of these animals.MethodsSHR (8-weeks old) were either sham-operated or orchiectomized, whereas all females were oophorectomized, half of which received 10mg testosterone propionate subcutaneously. Mean arterial pressure (MAP) and testosterone (T) were measured by carotid cannulation and ELISA respectively. Sequencing was performed on hand-punched PVN sections and subjected to robust bioinformatic analysis.ResultsIn total, 6,571 differentially regulated genes (DRGs) are regulated in the PVN of male and female rats. High T (endogenous or replaced) correlates with higher MAP in both sexes. Orchidectomy-induced T depletion resulted in the significant regulation of 5,104 genes, involved in thousands of biological roles, including ones related to hormone and sex-hormone signalling. In the female SHR, testosterone replacement in oophorectomized animals induced the regulation of 1,727 genes, sharing many biological functions with those in the high T males. We validated key genes by qPCR to determine false discovery rate.ConclusionsT status in hypertensive rats correlates with MAP, and consistent changes in PVN transcriptome

BACKGROUND AND AIMS:Multiple germline gene variants promote familial and idiopathic pulmonary arterial hypertension (PAH); however, none are consistently identified in associated PAH with connective tissue disease (APAH-CTD). Moreover, the role of somatic variants in genes mediating clonal haematopoiesis of indeterminate potential (CHIP) in PAH is unknown. Here, somatic and germline DNMT3A variants and CHIP gene variants in PAH were evaluated. METHODS:Exome sequencing (ES) was compared between PAH Biobank participants (n = 1832 European ancestry/2572 total), vs. gnomAD controls (7509 European ancestry/141 456 total). Subsequently, targeted panel sequencing (TPS) of 22 CHIP genes, including DNMT3A, was performed in PAH (n = 1659) vs. controls (n = 3644). Somatic CHIP variants in the UK Biobank using ES (controls = 448 239; PAH = 2559) were also assessed. DNMT3A mRNA expression was measured in peripheral blood mononuclear cells (PBMCs) of patients with scleroderma APAH-CTD (n = 50), idiopathic PAH (n = 30), scleroderma without PAH (n = 19), and healthy controls (n = 41). Hemodynamic were evaluated in haematopoietic Dnmt3a-knockout mice. RESULTS:Predicted deleterious germline DNMT3A variants were increased in subjects of European ancestry (6/1832) vs. controls (6/7509) (relative risk [RR] = 4.1, P = .018). In the entire PAH Biobank cohort (n = 2572), DNMT3A germline and somatic variants were further enriched (PAH: 1.28% vs. controls: .43%, P = 1.65 × 10-10). Eight DNMT3A mutations (.39%) were likely germline (female/male: 7/1) and 25 (.82%) likely somatic (female/male: 21/4), including 13/33 APAH-CTD participants. TPS identified CHIP in 242 PAH subjects (48% DNMT3A). DNMT3A- and all-CHIP variants were associated with PAH after correcting for age, sex, and age-CHIP interactions (odds ratio [OR]: 25.44, P = 4.50 × 10-5; OR: 23.35, P = 2.87 × 10-8, respectively). In the UK Biobank, CHIP mutations were increased in PAH (PAH = 5.35% vs. Control = 3.45%, P ≤ .0001). DNMT3A was reduced in PAH-PBMCs (area under curve [AUC] = .82) (P < .0001). Haematopoietic Dnmt3a-knockout in mice caused inflammatory PAH, which was attenuated by IL-1β antibody therapy. CONCLUSIONS:Germline DNMT3A variants and somatic variants of DNMT3A and CHIP genes increase the risk of PAH, including APAH-CTD, promote inflammation, and constitute potential biomarkers and therapeutic targets.

Triphenyl phosphate (TPHP) is a high-production-volume flame retardant and plasticizer that is widely detected in the environment and in biomonitoring studies. TPHP exposure has been linked to endocrine disruption, metabolic disruption, genotoxicity, and neurodevelopmental effects in vitro and in vivo. The diverse toxicological outcomes across studies suggest disruption of fundamental regulatory processes, such as epigenetic control of gene expression. Here, we used an immortalized embryonic cell line derived from steelhead trout (STE-137) to investigate coordinated transcriptional and epigenetic responses to TPHP exposure. Cells were exposed to 0 or 80 μM TPHP for 24 h, followed by RNA sequencing (RNA-seq) or whole-genome bisulfite sequencing (WGBS). Differential expression analysis identified 1622 significant genes, with significant enrichment of DNA replication and repair, cell cycle regulation, and endocrine signaling pathways and prominent lipid metabolism genes. Weighted gene co-expression network analysis revealed modules highly correlated with exposure, including those enriched for protein and amino acid metabolism, ion transport, and genomic stability. WGBS methylome analysis detected 382 differentially methylated regions (DMRs), the majority hypermethylated and within gene bodies. Notable alterations included a DMR in the htr2cl1 gene, encoding a serotonin receptor, and the brca2 gene, a key DNA damage enzyme. Integration of RNA-seq and WGBS datasets identified nine genes with both expression and methylation changes, alongside altered gene expression of several key epigenetic regulators. Our results provide molecular evidence for early initiating events relevant to adverse outcome pathways and highlight the importance of epigenetic endpoints in developmental toxicity assessment.

RNA-seq quantifies the abundance of transcripts within a biological sample and performs differential analysis between different conditions to reveal regulated gene signatures. Three challenges exist: (1) different analytical packages can often report different expression patterns and false-discovery-rates and P-values; (2) the effective use of these analytical packages requires substantial knowledge of programming and bioinformatics; and (3) there are a lack of intuitive methods to prioritize target genes for further investigation. To address these challenges, we developed Confidence, a web-based application to perform simultaneous statistical analysis of RNA-seq count data. Confidence incorporates the Confidence Score (CS), ranging from 1 to 4 to aid in gene prioritization, where 1 represents low confidence and 4 represents high confidence. The Confidence web-based application was designed for rapid and intuitive analysis of standard experimental metadata and gene count inputs providing a web-based, ‘wide-net’ approach to RNA-seq analysis. Gene scoring allows for unbiased gene selection and identification of novel genes strongly associated with disease/treatment models across multiple species. Pathway analysis has been integrated so that highly confident genes can be placed into biological context. Confidence provides a new strategy for target prioritization in RNA-seq analysis and the generation of publication-quality figures, which we demonstrate here using a published database.

BACKGROUND: DRP1 (dynamin-related protein 1) mediates mitochondrial fission and permits rapid cell cycle progression in hyperproliferative cells by coordinating nuclear and mitochondrial division, a process called mitotic fission. However, DRP1 alone appears insufficient to complete fission, and the link between fission and cell cycle progression is unknown. We hypothesize that DNM2 (dynamin 2) interacts with DRP1 to complete mitochondrial fission and regulate cell cycle progression. We show that DNM2 is upregulated in pulmonary artery smooth muscle cells (PASMCs) in human and rodent pulmonary arterial hypertension (PAH), contributing to disease pathophysiology. METHODS: Mitochondrial morphology, protein colocalization, and fission were assessed using stimulated emission depletion microscopy, protein interactions by immunoprecipitation, and transcriptomics by RNA sequencing. DNM2 was quantified in PASMC and lungs from patients with PAH and rats with pulmonary hypertension (PH), induced by monocrotaline or sugen5416/hypoxia. siDNM2’s effects on cell proliferation, cell cycle progression, and apoptosis were assessed by flow cytometry. Single-cell RNA sequencing was performed on publicly available data sets. siDNM2 was nebulized to monocrotaline- and sugen5416/hypoxia-PH rats, and disease regression was quantified by cardiac catheterization and histology. RESULTS: DNM2 is increased in PAH PASMC. DNM2 interacts with DRP1 via its GTPase domain, permitting mitochondrial translocation and promoting fission. siDNM2 inhibits fission and cell proliferation and increases apoptosis. siDNM2 causes G1/G0 blockade by downregulating RGCC (regulator of cell cycle) with downstream effects on CDK (cyclin-dependent kinase) 4, cyclin D1, and p27 kip1 . Conversely, augmenting DNM2 in normal PASMC induces fission and accelerates proliferation. Upregulation of DNM2 in PAH is due to decreased miR-124-3p (microRNA-124-3p) and activation of STAT3 (signal transducer and activator of transcription 3). An miR-124-3p-STAT3-DNM2-DRP1-RGCC pathway accelerates mitotic fission and is upregulated in PASMC, airway epithelium, endothelial cells, fibroblasts, and macrophages in PAH. Nebulized siDNM2 regresses established PH in vivo in rats of both sexes. CONCLUSIONS: DNM2 is a mediator in the terminal steps of DRP1-dependent fission and constitutes a novel therapeutic target in PAH.

Supplementary Table S9: mRNAs affected by tomivosertib.

Supplementary Fig. S4: Proteomic profiling. Heatmap showing the Normalized Enrichment Score (NES) for the main independent pathways enriched in at least 2 of the patients in the cohort.

Supplementary Fig. S9: Imaging mass cytometry. (A) Heatmap of signaling markers expression within cell clusters and (B) Neighbourhood analysis showing cell-to-cell interactions over all patients before and after treatment with tomivosertib.

Supplementary Fig. S5: Translatomic profiling. Scatter plots demonstrating translation efficiency (TE) (top), Ribo-Seq (middle), RNA-Seq) (bottom) the fold change difference between ON MNKi and Pre-MNKi conditions for three different patients, patient 13 (A), patient 12 (B), and patient 16 (C). Only reads aligning with CDS were considered for this analysis. The x-axis represents the normalized number of reads corresponding to the experiment/condition of minimal expression. Red and blue dots denote significantly up or down-regulated mRNAs, respectively (Z-score > 2 and log2 FC > 0.5 for up and Z-score < -2 and log2 FC < -0.5 for down).

Supplementary Fig. S6: Translatomic profiling. UpSet plots display overlap of the translationally downregulated (A) and upregulated (B) mRNAs between each patient. The numbers above the bars represent the number of mRNAs that exclusively belonged to the conditions marked below the bars. The horizontal bar graph on each plot's left side shows the number of differentially translated mRNAs in each patient.

Supplementary Table S2. Representativeness of Study Participants.

Supplementary Table S3: Prior treatments. Summary of breast cancer surgery, radiation therapy, and systemic anti-cancer therapy.

Rationale Mitochondria in ductus arteriosus (DA) smooth muscle cells (DASMC) are oxygen sensors that trigger O2-induced vasoconstriction at birth; however, the molecular mechanisms of mitochondrial oxygen sensing are not fully understood. Many redox sensor proteins are conserved in the mammalian adult homeostatic oxygen sensing system, including NDUFS2 (NADH:Ubiquinone oxidoreductase core subunit S2), a component of mitochondrial Complex I that contributes to oxygen sensing in adult pulmonary arteries. Here we compared the role of NDUFS2 in DA oxygen sensing, to that of other Complex I subunits and putative O2-sensor subunits, including: NADH:Ubiquinone oxidoreductase core subunit S1 (NDUFS1), NADH:Ubiquionone oxidoreductase core subunit S7 (NDUFS7), Ubiquinol-cytochrome c reductase, Rieske iron-sulfur polypeptide 1 (UQCRFS1), and Cytochrome c oxidase subunit 4I2 (COX4I2). Methods Human DASMC were grown in hypoxia (2.5% O2, pO2=41mmHg). Oxygen responsiveness of DASMC was assessed, measuring O2-induced changes in intracellular calcium, [Ca2+]i, cell length, and mitochondrial reactive oxygen species (mROS) production. DASMC were treated for 48-hours with silencing RNA (siRNA) targeting NDUFS2, NDUFS1, NDUFS7, UQCRFS1, or COX4I2; knockdown was confirmed using qPCR and immunoblot. Mitochondrial metabolic consequences were assessed with micropolarimetry and Complex I, III, and IV activity assays. 3’RNA sequencing was used to explore the impact of gene knockdown on the DASMC molecular signature. Results The O2-induced increase in [Ca2+]i in siControl-treated cells (+18.6±2.3%) was reduced by siNDUFS2 (+5.5±1.5%, p<0.0001), but unchanged by other siRNAs. siNDUFS2 also uniquely depressed O2-induced DASMC shortening (from −18.4±1.1% to −8.9±0.77%, p<0.0001), and mROS generation (+24±4.9% untreated versus −6.6±5.4% siNDUFS2, p<0.0001). The mitochondrial antioxidant MitoTEMPO also inhibited mROS (+2.9±4.5%, p=0.001) and attenuated oxygen-induced cell shortening (8.43±0.91%, p=0.0003). Knockdown of NDUFS2 and other ETC subunits did not inhibit mitochondrial respiration or ETC activity. Transcriptomics revealed unique changes in mitochondrial pathways with siNDUFS2. Conclusions NDUFS2 regulates mROS and acts as a mitochondrial oxygen sensor in human DASMC. ### Competing Interest Statement The authors have declared no competing interest. Canadian Institutes of Health Research, 183762

Supplementary Table S8: Protein changes by patient.

Supplementary Table S6: Summary of all adverse events.

Supplementary Table S1: Patient characteristics. Age, ECOG performance status, HER2 status, steroid hormone receptor status, and biopsy site data are summarized.

Purpose: Preclinical data motivate clinical evaluation of inhibitors of MAPK-interacting kinases 1 and 2 (MNK1/2). We conducted a phase 1b clinical trial to study target engagement and safety of tomivosertib, a MNK1/2 inhibitor, alone and in combination with paclitaxel.Patients and Methods: Eligible patients had metastatic breast cancer resistant to standard-of-care treatments. Biopsies were obtained at baseline and during treatment with tomivosertib, and then tomivosertib was continued with the addition of paclitaxel until disease progression or toxicity. Serum drug levels were measured, and pharmacodynamic endpoints included IHC, proteomics, translatomics, and imaging mass cytometry.Results: Tomivosertib alone and in combination with paclitaxel was well tolerated. There was no pharmacokinetic interaction between the drugs. We observed a clear reduction in phosphorylation of eIF4E at S209, a major substrate of MNK1/2, and identified tomivosertib-induced perturbations in the proteome, translatome, and cellular populations of biopsied metastatic breast cancer tissue.Conclusions: We conclude that tomivosertib effectively inhibits MNK1/2 activity in metastatic breast cancer tissue and that it can safely be combined with paclitaxel in future phase II studies. We demonstrate feasibility of using proteomic profiles, translatomic profiles, and spatial distribution of immune cell infiltrates for clinical pharmacodynamic studies.