Selective deployment of transcription factor paralogs with submaximal strength facilitates gene regulation in the immune system.

In multicellular organisms, duplicated genes can diverge through tissue-specific gene expression patterns, as exemplified by highly regulated expression of RUNX transcription factor paralogs with apparent functional redundancy. Here we asked what cell-type-specific biologies might be supported by the selective expression of RUNX paralogs during Langerhans cell and inducible regulatory T cell differentiation. We uncovered functional nonequivalence between RUNX paralogs. Selective expression of native paralogs allowed integration of transcription factor activity with extrinsic signals, while non-native paralogs enforced differentiation even in the absence of exogenous inducers. DNA binding affinity was controlled by divergent amino acids within the otherwise highly conserved RUNT domain and evolutionary reconstruction suggested convergence of RUNT domain residues toward submaximal strength. Hence, the selective expression of gene duplicates in specialized cell types can synergize with the acquisition of functional differences to enable appropriate gene expression, lineage choice and differentiation in the mammalian immune system.

Control of inducible gene expression links cohesin to hematopoietic progenitor self-renewal and differentiation.

Cohesin is important for 3D genome organization. Nevertheless, even the complete removal of cohesin has surprisingly little impact on steady-state gene transcription and enhancer activity. Here we show that cohesin is required for the core transcriptional response of primary macrophages to microbial signals, and for inducible enhancer activity that underpins inflammatory gene expression. Consistent with a role for inflammatory signals in promoting myeloid differentiation of hematopoietic stem and progenitor cells (HPSCs), cohesin mutations in HSPCs led to reduced inflammatory gene expression and increased resistance to differentiation-inducing inflammatory stimuli. These findings uncover an unexpected dependence of inducible gene expression on cohesin, link cohesin with myeloid differentiation, and may help explain the prevalence of cohesin mutations in human acute myeloid leukemia.

Chromatin jets define the properties of cohesin-driven in vivo loop extrusion.

Complex genomes show intricate organization in three-dimensional (3D) nuclear space. Current models posit that cohesin extrudes loops to form self-interacting domains delimited by the DNA binding protein CTCF. Here, we describe and quantitatively characterize cohesin-propelled, jet-like chromatin contacts as landmarks of loop extrusion in quiescent mammalian lymphocytes. Experimental observations and polymer simulations indicate that narrow origins of loop extrusion favor jet formation. Unless constrained by CTCF, jets propagate symmetrically for 1-2 Mb, providing an estimate for the range of in vivo loop extrusion. Asymmetric CTCF binding deflects the angle of jet propagation as experimental evidence that cohesin-mediated loop extrusion can switch from bi- to unidirectional and is controlled independently in both directions. These data offer new insights into the physiological behavior of in vivo cohesin-mediated loop extrusion and further our understanding of the principles that underlie genome organization.

DNA synthesis is required for reprogramming mediated by stem cell fusion.

Embryonic stem cells (ESCs) can instruct the conversion of differentiated cells toward pluripotency following cell-to-cell fusion by a mechanism that is rapid but poorly understood. Here, we used centrifugal elutriation to enrich for mouse ESCs at sequential stages of the cell cycle and showed that ESCs in S/G2 phases have an enhanced capacity to dominantly reprogram lymphocytes and fibroblasts in heterokaryon and hybrid assays. Reprogramming success was associated with an ability to induce precocious nucleotide incorporation within the somatic partner nuclei in heterokaryons. BrdU pulse-labeling experiments revealed that virtually all successfully reprogrammed somatic nuclei, identified on the basis of Oct4 re-expression, had undergone DNA synthesis within 24 hr of fusion with ESCs. This was essential for successful reprogramming because drugs that inhibited DNA polymerase activity effectively blocked pluripotent conversion. These data indicate that nucleotide incorporation is an early and critical event in the epigenetic reprogramming of somatic cells in experimental ESC-heterokaryons.

Z-nucleic-acid sensing triggers ZBP1-dependent necroptosis and inflammation.

The biological function of Z-DNA and Z-RNA, nucleic acid structures with a left-handed double helix, is poorly understood1-3. Z-DNA-binding protein 1 (ZBP1; also known as DAI or DLM-1) is a nucleic acid sensor that contains two Zα domains that bind Z-DNA4,5 and Z-RNA6-8. ZBP1 mediates host defence against some viruses6,7,9-14 by sensing viral nucleic acids6,7,10. RIPK1 deficiency, or mutation of its RIP homotypic interaction motif (RHIM), triggers ZBP1-dependent necroptosis and inflammation in mice15,16. However, the mechanisms that induce ZBP1 activation in the absence of viral infection remain unknown. Here we show that Zα-dependent sensing of endogenous ligands induces ZBP1-mediated perinatal lethality in mice expressing RIPK1 with mutated RHIM (Ripk1mR/mR), skin inflammation in mice with epidermis-specific RIPK1 deficiency (RIPK1E-KO) and colitis in mice with intestinal epithelial-specific FADD deficiency (FADDIEC-KO). Consistently, functional Zα domains were required for ZBP1-induced necroptosis in fibroblasts that were treated with caspase inhibitors or express RIPK1 with mutated RHIM. Inhibition of nuclear export triggered the Zα-dependent activation of RIPK3 in the nucleus resulting in cell death, which suggests that ZBP1 may recognize nuclear Z-form nucleic acids. We found that ZBP1 constitutively bound cellular double-stranded RNA in a Zα-dependent manner. Complementary reads derived from endogenous retroelements were detected in epidermal RNA, which suggests that double-stranded RNA derived from these retroelements may act as a Zα-domain ligand that triggers the activation of ZBP1. Collectively, our results provide evidence that the sensing of endogenous Z-form nucleic acids by ZBP1 triggers RIPK3-dependent necroptosis and inflammation, which could underlie the development of chronic inflammatory conditions-particularly in individuals with mutations in RIPK1 and CASP817-20.

Publisher Correction: Z-nucleic-acid sensing triggers ZBP1-dependent necroptosis and inflammation.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Skeletal Muscle SIRT3 Deficiency Contributes to Pulmonary Vascular Remodeling in Pulmonary Hypertension Due to Heart Failure With Preserved Ejection Fraction.

BACKGROUND: Pulmonary hypertension (PH) is a major complication linked to adverse outcomes in heart failure with preserved ejection fraction (HFpEF), yet no specific therapies exist for PH associated with HFpEF (PH-HFpEF). We have recently reported on the role of skeletal muscle SIRT3 (sirtuin-3) in modulation of PH-HFpEF, suggesting a novel endocrine signaling pathway for skeletal muscle modulation of pulmonary vascular remodeling. In this study, we attempted to define the processes by which skeletal muscle SIRT3 defects affect pulmonary vascular health in PH-HFpEF. METHODS AND RESULTS: Skeletal muscle-specific Sirt3 knockout mice (Sirt3skm-/-) exhibited reduced pulmonary vascular density accompanied by pulmonary vascular proliferative remodeling and elevated pulmonary pressures. Using mass spectrometry-based comparative secretome analysis, we demonstrated elevated secretion of LOXL2 (lysyl oxidase homolog 2) in SIRT3-deficient skeletal muscle cells. Elevated circulation and protein expression levels of LOXL2 were also observed in plasma and skeletal muscle of Sirt3skm-/- mice, a rat model of PH-HFpEF, and humans with PH-HFpEF. In addition, expression levels of CNPY2 (canopy fibroblast growth factor signaling regulator 2), a known proliferative and angiogenic factor, were increased in pulmonary artery endothelial cells and pulmonary artery smooth muscle cells of Sirt3skm-/- mice and animal models of PH-HFpEF. CNPY2 levels were also higher in pulmonary artery smooth muscle cells of subjects with obesity compared with nonobese subjects. Moreover, treatment with recombinant LOXL2 protein promoted pulmonary artery endothelial cell migration/proliferation and pulmonary artery smooth muscle cell proliferation through regulation of CNPY2-p53 signaling. Last, skeletal muscle-specific Loxl2 deletion decreased pulmonary artery endothelial cell and pulmonary artery smooth muscle cell expression of CNPY2 and improved pulmonary pressures in mice with high-fat diet-induced PH-HFpEF. CONCLUSIONS: This study demonstrates a systemic pathogenic impact of skeletal muscle SIRT3 deficiency in remote pulmonary vascular remodeling and PH-HFpEF. This study suggests a new endocrine signaling axis that links skeletal muscle health and SIRT3 deficiency to remote CNPY2 regulation in the pulmonary vasculature through myokine LOXL2. Our data also identify skeletal muscle SIRT3, myokine LOXL2, and CNPY2 as potential targets for the treatment of PH-HFpEF.

Plasma Proteomics Identifies B2M as a Regulator of Pulmonary Hypertension in Heart Failure With Preserved Ejection Fraction.

BACKGROUND: Pulmonary hypertension (PH) represents an important phenotype in heart failure with preserved ejection fraction (HFpEF). However, management of PH-HFpEF is challenging because mechanisms involved in the regulation of PH-HFpEF remain unclear. METHODS: We used a mass spectrometry-based comparative plasma proteomics approach as a sensitive and comprehensive hypothesis-generating discovery technique to profile proteins in patients with PH-HFpEF and control subjects. We then validated and investigated the role of one of the identified proteins using in vitro cell cultures, in vivo animal models, and independent cohort of human samples. RESULTS: Plasma proteomics identified high protein abundance levels of B2M (ß2-microglobulin) in patients with PH-HFpEF. Interestingly, both circulating and skeletal muscle levels of B2M were increased in mice with skeletal muscle SIRT3 (sirtuin-3) deficiency or high-fat diet-induced PH-HFpEF. Plasma and muscle biopsies from a validation cohort of PH-HFpEF patients were found to have increased B2M levels, which positively correlated with disease severity, especially pulmonary capillary wedge pressure and right atrial pressure at rest. Not only did the administration of exogenous B2M promote migration/proliferation in pulmonary arterial vascular endothelial cells but it also increased PCNA (proliferating cell nuclear antigen) expression and cell proliferation in pulmonary arterial vascular smooth muscle cells. Finally, B2m deletion improved glucose intolerance, reduced pulmonary vascular remodeling, lowered PH, and attenuated RV hypertrophy in mice with high-fat diet-induced PH-HFpEF. CONCLUSIONS: Patients with PH-HFpEF display higher circulating and skeletal muscle expression levels of B2M, the magnitude of which correlates with disease severity. Our findings also reveal a previously unknown pathogenic role of B2M in the regulation of pulmonary vascular proliferative remodeling and PH-HFpEF. These data suggest that circulating and skeletal muscle B2M can be promising targets for the management of PH-HFpEF.

Effect of estrogen receptor α on cardiopulmonary adaptation to chronic developmental hypoxia in a rat model.

Humans living at high-altitude (HA) have adapted to this environment by increasing pulmonary vascular and alveolar growth. RNA sequencing data from a novel murine model that mimics this phenotypical response to HA suggested estrogen signaling via estrogen receptor alpha (ERα) may be involved in this adaptation. We hypothesized ERα was a key mediator in the cardiopulmonary adaptation to chronic hypoxia and sought to delineate the mechanistic role ERα contributes to this process by exposing novel loss-of-function ERα mutant (ERαMut) rats to simulated HA. ERα mutant or wild-type (wt) rats were exposed to normoxia or hypoxia starting at conception and continued postnatally until 6 wk of age. Both wt and ERαMut animals born and raised in hypoxia exhibited lower body mass and higher hematocrits, total alveolar volumes (Va), diffusion capacities of carbon monoxide (DLCO), pulmonary arteriole (PA) wall thickness, and Fulton indices than normoxia animals. Right ventricle adaptation was maintained in the setting of hypoxia. Although no major physiologic differences were seen between wt and ERαMut animals at either exposure, ERαMut animals exhibited smaller mean linear intercepts (MLI) and increased PA total and lumen areas. Hypoxia exposure or ERα loss-of-function did not affect lung mRNA abundance of vascular endothelial growth factor, angiopoietin 2, or apelin. Sexual dimorphisms were noted in PA wall thickness and PA lumen area in ERαMut rats. In summary, in room air-exposed rats and rats with peri- and postnatal hypoxia exposure, ERα loss-of-function was associated with decreased alveolar size (primarily driven by hypoxic animals) and increased PA remodeling.NEW & NOTEWORTHY By exposing novel loss-of-function estrogen receptor alpha (Erα) mutant rats to a novel model of human high-altitude exposure, we demonstrate that ERα has subtle but inconsistent effects on endpoints relevant to cardiopulmonary adaptation to chronic hypoxia. Given that we observed some histologic, sex, and genotype differences, further research into cell-specific effects of ERα during hypoxia-induced cardiopulmonary adaptation is warranted.

LUBAC is essential for embryogenesis by preventing cell death and enabling haematopoiesis.

The linear ubiquitin chain assembly complex (LUBAC) is required for optimal gene activation and prevention of cell death upon activation of immune receptors, including TNFR1 1 . Deficiency in the LUBAC components SHARPIN or HOIP in mice results in severe inflammation in adulthood or embryonic lethality, respectively, owing to deregulation of TNFR1-mediated cell death2-8. In humans, deficiency in the third LUBAC component HOIL-1 causes autoimmunity and inflammatory disease, similar to HOIP deficiency, whereas HOIL-1 deficiency in mice was reported to cause no overt phenotype9-11. Here we show, by creating HOIL-1-deficient mice, that HOIL-1 is as essential for LUBAC function as HOIP, albeit for different reasons: whereas HOIP is the catalytically active component of LUBAC, HOIL-1 is required for LUBAC assembly, stability and optimal retention in the TNFR1 signalling complex, thereby preventing aberrant cell death. Both HOIL-1 and HOIP prevent embryonic lethality at mid-gestation by interfering with aberrant TNFR1-mediated endothelial cell death, which only partially depends on RIPK1 kinase activity. Co-deletion of caspase-8 with RIPK3 or MLKL prevents cell death in Hoil-1-/- (also known as Rbck1-/-) embryos, yet only the combined loss of caspase-8 with MLKL results in viable HOIL-1-deficient mice. Notably, triple-knockout Ripk3-/-Casp8-/-Hoil-1-/- embryos die at late gestation owing to haematopoietic defects that are rescued by co-deletion of RIPK1 but not MLKL. Collectively, these results demonstrate that both HOIP and HOIL-1 are essential LUBAC components and are required for embryogenesis by preventing aberrant cell death. Furthermore, they reveal that when LUBAC and caspase-8 are absent, RIPK3 prevents RIPK1 from inducing embryonic lethality by causing defects in fetal haematopoiesis.

SOX17 Deficiency Mediates Pulmonary Hypertension: At the Crossroads of Sex, Metabolism, and Genetics.

Rationale: Genetic studies suggest that SOX17 (SRY-related HMG-box 17) deficiency increases pulmonary arterial hypertension (PAH) risk. Objectives: On the basis of pathological roles of estrogen and HIF2α (hypoxia-inducible factor 2α) signaling in pulmonary artery endothelial cells (PAECs), we hypothesized that SOX17 is a target of estrogen signaling that promotes mitochondrial function and attenuates PAH development via HIF2α inhibition. Methods: We used metabolic (Seahorse) and promoter luciferase assays in PAECs together with the chronic hypoxia murine model to test the hypothesis. Measurements and Main Results: Sox17 expression was reduced in PAH tissues (rodent models and from patients). Chronic hypoxic pulmonary hypertension was exacerbated by mice with conditional Tie2-Sox17 (Sox17EC-/-) deletion and attenuated by transgenic Tie2-Sox17 overexpression (Sox17Tg). On the basis of untargeted proteomics, metabolism was the top pathway altered by SOX17 deficiency in PAECs. Mechanistically, we found that HIF2α concentrations were increased in the lungs of Sox17EC-/- and reduced in those from Sox17Tg mice. Increased SOX17 promoted oxidative phosphorylation and mitochondrial function in PAECs, which were partly attenuated by HIF2α overexpression. Rat lungs in males displayed higher Sox17 expression versus females, suggesting repression by estrogen signaling. Supporting 16α-hydroxyestrone (16αOHE; a pathologic estrogen metabolite)-mediated repression of SOX17 promoter activity, Sox17Tg mice attenuated 16αOHE-mediated exacerbations of chronic hypoxic pulmonary hypertension. Finally, in adjusted analyses in patients with PAH, we report novel associations between a SOX17 risk variant, rs10103692, and reduced plasma citrate concentrations (n = 1,326). Conclusions: Cumulatively, SOX17 promotes mitochondrial bioenergetics and attenuates PAH, in part, via inhibition of HIF2α. 16αOHE mediates PAH development via downregulation of SOX17, linking sexual dimorphism and SOX17 genetics in PAH.

Illuminating Epigenetics and Inheritance in the Immune System with Bioluminescence.

The remarkable process of light emission by living organisms has fascinated mankind for thousands of years. A recent expansion in the repertoire of catalytic luciferase enzymes, coupled with the discovery of the genes and pathways that encode different luciferin substrates, means that bioluminescence imaging (BLI) is set to revolutionize longitudinal and dynamic studies of gene control within biomedicine, including the regulation of immune responses. In this review article, we summarize recent advances in bioluminescence-based imaging approaches that promise to enlighten our understanding of in vivo gene and epigenetic control within the immune system.

Remarkable variation in androecial morphology is closely associated with corolla traits in Western Hemisphere Justiciinae (Acanthaceae: Justicieae).

BACKGROUND AND AIMS: Few studies of angiosperms have focused on androecial evolution in conjunction with evolutionary shifts in corolla morphology and pollinator relationships. The Western Hemisphere clade of Justiciinae (Acanthaceae) presents the rare opportunity to examine remarkable diversity in staminal morphology. We took a phylogenetically informed approach to examine staminal diversity in this hypervariable group and asked whether differences in anther thecae separation is associated with phylogenetically informed patterns of variation in corolla morphology. We further discuss evidence for associations between anther diversity and pollinators in this lineage. METHODS: For the Dianthera/Sarotheca/Plagiacanthus (DSP) clade of Western Hemisphere Justiciinae, we characterized floral diversity based on a series of corolla measurements and using a model-based clustering approach. We then tested for correlations between anther thecae separation and corolla traits, and for shifts in trait evolution, including evidence for convergence. KEY RESULTS: There is evolutionary vagility in corolla and anther traits across the DSP clade with little signal of phylogenetic constraint. Floral morphology clusters into four distinct groups that are, in turn, strongly associated with anther thecae separation, a novel result in Acanthaceae and, to our knowledge, across flowering plants. These cluster groups are marked by floral traits that strongly point to associations with pollinating animals. Specifically, species that are known or likely to be hummingbird pollinated have stamens with parallel thecae, whereas those that are likely bee or fly pollinated have stamens with offset, divergent thecae. CONCLUSIONS: Our results suggest that anther thecae separation is likely under selection in concert with other corolla characters. Significant morphological shifts detected by our analyses corresponded to putative shifts from insect to hummingbird pollination. Results from this study support the hypothesis that floral structures function in an integrated manner and are likely subject to selection as a suite. Further, these changes can be hypothesized to represent adaptive evolution.

Cohesins functionally associate with CTCF on mammalian chromosome arms.

Cohesins mediate sister chromatid cohesion, which is essential for chromosome segregation and postreplicative DNA repair. In addition, cohesins appear to regulate gene expression and enhancer-promoter interactions. These noncanonical functions remained unexplained because knowledge of cohesin-binding sites and functional interactors in metazoans was lacking. We show that the distribution of cohesins on mammalian chromosome arms is not driven by transcriptional activity, in contrast to S. cerevisiae. Instead, mammalian cohesins occupy a subset of DNase I hypersensitive sites, many of which contain sequence motifs resembling the consensus for CTCF, a DNA-binding protein with enhancer blocking function and boundary-element activity. We find cohesins at most CTCF sites and show that CTCF is required for cohesin localization to these sites. Recruitment by CTCF suggests a rationale for noncanonical cohesin functions and, because CTCF binding is sensitive to DNA methylation, allows cohesin positioning to integrate DNA sequence and epigenetic state.

Necroptosis-based CRISPR knockout screen reveals Neuropilin-1 as a critical host factor for early stages of murine cytomegalovirus infection.

Herpesviruses are ubiquitous human pathogens that cause a wide range of health complications. Currently, there is an incomplete understanding of cellular factors that contribute to herpesvirus infection. Here, we report an antiviral necroptosis-based genetic screen to identify novel host cell factors required for infection with the ß-herpesvirus murine cytomegalovirus (MCMV). Our genome-wide CRISPR-based screen harnessed the capacity of herpesvirus mutants that trigger antiviral necroptotic cell death upon early viral gene expression. Vascular endothelial growth factor (VEGF) and semaphorin-binding receptor Neuropilin-1 (Nrp-1) emerge as crucial determinants of MCMV infection. We find that elimination of Nrp-1 impairs early viral gene expression and reduces infection rates in endothelial cells, fibroblasts, and macrophages. Furthermore, preincubation of virus with soluble Nrp-1 dramatically inhibits infection by reducing virus attachment. Thus, Nrp-1 is a key determinant of the initial phase of MCMV infection.

Initiation and maintenance of pluripotency gene expression in the absence of cohesin.

Cohesin is implicated in establishing and maintaining pluripotency. Whether this is because of essential cohesin functions in the cell cycle or in gene regulation is unknown. Here we tested cohesin's contribution to reprogramming in systems that reactivate the expression of pluripotency genes in the absence of proliferation (embryonic stem [ES] cell heterokaryons) or DNA replication (nuclear transfer). Contrary to expectations, cohesin depletion enhanced the ability of ES cells to initiate somatic cell reprogramming in heterokaryons. This was explained by increased c-Myc (Myc) expression in cohesin-depleted ES cells, which promoted DNA replication-dependent reprogramming of somatic fusion partners. In contrast, cohesin-depleted somatic cells were poorly reprogrammed in heterokaryons, due in part to defective DNA replication. Pluripotency gene induction was rescued by Myc, which restored DNA replication, and by nuclear transfer, where reprogramming does not require DNA replication. These results redefine cohesin's role in pluripotency and reveal a novel function for Myc in promoting the replication-dependent reprogramming of somatic nuclei.

Overexpression of Decay Accelerating Factor Mitigates Fibrotic Responses to Lung Injury.

CD55 or decay accelerating factor (DAF), a ubiquitously expressed glycosylphosphatidylinositol (GPI)-anchored protein, confers a protective threshold against complement dysregulation which is linked to the pathogenesis of idiopathic pulmonary fibrosis (IPF). Since lung fibrosis is associated with downregulation of DAF, we hypothesize that overexpression of DAF in fibrosed lungs will limit fibrotic injury by restraining complement dysregulation. Normal primary human alveolar type II epithelial cells (AECs) exposed to exogenous complement 3a or 5a, and primary AECs purified from IPF lungs demonstrated decreased membrane-bound DAF expression with concurrent increase in the endoplasmic reticulum (ER) stress protein, ATF6. Increased loss of extracellular cleaved DAF fragments was detected in normal human AECs exposed to complement 3a or 5a, and in lungs of IPF patients. C3a-induced ATF6 expression and DAF loss was inhibited using pertussis toxin (an enzymatic inactivator of G-protein coupled receptors), in murine AECs. Treatment with soluble DAF abrogated tunicamycin-induced C3a secretion and ER stress (ATF6 and BiP expression) and restored epithelial cadherin. Bleomycin-injured fibrotic mice subjected to lentiviral overexpression of DAF demonstrated diminished levels of local collagen deposition and complement activation. Further analyses showed diminished release of DAF fragments, as well as reduction in apoptosis (TUNEL and caspase 3/7 activity), and ER stress-related transcripts. Loss-of-function studies using Daf1 siRNA demonstrated worsened lung fibrosis detected by higher mRNA levels of Col1a1 and epithelial injury-related Muc1 and Snai1, with exacerbated local deposition of C5b-9. Our studies provide a rationale for rescuing fibrotic lungs via DAF induction that will restrain complement dysregulation and lung injury.

Skeletal muscle blood flow during exercise is reduced in a rat model of pulmonary hypertension.

Pulmonary arterial hypertension (PAH) is characterized by exercise intolerance. Muscle blood flow may be reduced during exercise in PAH; however, this has not been directly measured. Therefore, we investigated blood flow during exercise in a rat model of monocrotaline (MCT)-induced pulmonary hypertension (PH). Male Sprague-Dawley rats (∼200 g) were injected with 60 mg/kg MCT (MCT, n = 23) and vehicle control (saline; CON, n = 16). Maximal rate of oxygen consumption (V̇o2max) and voluntary running were measured before PH induction. Right ventricle (RV) morphology and function were assessed via echocardiography and invasive hemodynamic measures. Treadmill running at 50% V̇o2max was performed by a subgroup of rats (MCT, n = 8; CON, n = 7). Injection of fluorescent microspheres determined muscle blood flow via photo spectroscopy. MCT demonstrated a severe phenotype via RV hypertrophy (Fulton index, 0.61 vs. 0.31; P < 0.001), high RV systolic pressure (51.5 vs. 22.4 mmHg; P < 0.001), and lower V̇o2max (53.2 vs. 71.8 mL·min-1·kg-1; P < 0.0001) compared with CON. Two-way ANOVA revealed exercising skeletal muscle blood flow relative to power output was reduced in MCT compared with CON (P < 0.001), and plasma lactate was increased in MCT (10.8 vs. 4.5 mmol/L; P = 0.002). Significant relationships between skeletal blood flow and blood lactate during exercise were observed for individual muscles (r = -0.58 to -0.74; P < 0.05). No differences in capillarization were identified. Skeletal muscle blood flow is significantly reduced in experimental PH. Reduced blood flow during exercise may be, at least in part, consequent to reduced exercise intensity in PH. This adds further evidence of peripheral muscle dysfunction and exercise intolerance in PAH.

A RADseq phylogeny of Barleria (Acanthaceae) resolves fine-scale relationships.

Barleria is a genus of approximately 300 species of herbs, shrubs or, rarely, trees, that is broadly distributed across the Paleotropics. The genus is especially diverse in Tanzania, Angola, and Madagascar. A recent molecular study sampled 53 Barleria species and gathered data for five molecular markers (i.e., four chloroplast loci and the nuclear nrITS) to find support for the recognition of two subgenera previously circumscribed based on morphology, subg. Barleria and subg. Prionitis. That study further reconstructed four previously recognized sections (i.e., Fissimura, Prionitis, Somalia, Stellatohirta) as monophyletic, while three others (i.e., Barleria, Cavirostrata, Chrysothrix) were recovered as para- or polyphyletic. The present study aimed to reconstruct phylogenetic relationships within Barleria based on a broader sample of taxa and many more characters. We sampled 190 accessions representing 184 taxa, including varieties and subspecies. The dataset includes 167 of the ca. 300 species currently recognized or about 56% of total species diversity. We relied heavily on herbarium specimens to sample across the taxonomic breadth and geographic range of Barleria. Single nucleotide polymorphism data were generated using double-digest restriction-site associated DNA sequencing (ddRADseq). The maximum likelihood phylogeny corroborated the topology estimated from the chloroplast and nrITS data, but with greatly increased resolution and support for fine-scale relationships. A coalescent analysis failed to resolve distant evolutionary relationships across Barleria and between Barleria and outgroups, but recovered the same or similar topologies within each Barleria section. Importantly, the ddRADseq phylogeny recovered seven major lineages within subg. Barleria and resolved a polytomy that included B. cristata, the type species of the genus. The topology suggests at least four independent dispersal events to Madagascar followed by three subsequent radiations. Our results broadly inform our understanding of diversity and evolution in one of the largest genera of Acanthaceae, representing an important step towards a stable subgeneric classification for the genus.