Transporters involved in pH and K+ homeostasis affect pollen wall formation, male fertility, and embryo development.

Flowering plant genomes encode multiple cation/H+ exchangers (CHXs) whose functions are largely unknown. AtCHX17, AtCHX18, and AtCHX19 are membrane transporters that modulate K+ and pH homeostasis and are localized in the dynamic endomembrane system. Loss of function reduced seed set, but the particular phase(s) of reproduction affected was not determined. Pollen tube growth and ovule targeting of chx17chx18chx19 mutant pollen appeared normal, but reciprocal cross experiments indicate a largely male defect. Although triple mutant pollen tubes reach ovules of a wild-type pistil and a synergid cell degenerated, half of those ovules were unfertilized or showed fertilization of the egg or central cell, but not both female gametes. Fertility could be partially compromised by impaired pollen tube and/or sperm function as CHX19 and CHX18 are expressed in the pollen tube and sperm cell, respectively. When fertilization was successful in self-pollinated mutants, early embryo formation was retarded compared with embryos from wild-type ovules receiving mutant pollen. Thus CHX17 and CHX18 proteins may promote embryo development possibly through the endosperm where these genes are expressed. The reticulate pattern of the pollen wall was disorganized in triple mutants, indicating perturbation of wall formation during male gametophyte development. As pH and cation homeostasis mediated by AtCHX17 affect membrane trafficking and cargo delivery, these results suggest that male fertility, sperm function, and embryo development are dependent on proper cargo sorting and secretion that remodel cell walls, plasma membranes, and extracellular factors.

Clinical and Biological Insights from the University of California San Francisco Prospective and Longitudinal Cohort.

INTRODUCTION: Sarcoidosis is a systemic inflammatory disease characterized by non-necrotizing granulomas in involved organs, most commonly the lung. Description of patient characteristics in the Western United States is limited. Furthermore, blood-based measures that relate to clinical sarcoidosis phenotypes are lacking. We present an analysis of a prospective, longitudinal sarcoidosis cohort at a Northern Californian academic medical center. METHODS: We enrolled 126 sarcoidosis subjects and 64 healthy controls and recorded baseline demographic and clinical characteristics. We used regression models to identify factors independently associated with pulmonary physiology. We tested whether blood transcript levels at study entry could relate to longitudinal changes in pulmonary physiology. RESULTS: White, non-Hispanics composed ~70% of subjects. Hispanics and Blacks had a diagnostic biopsy at an age ~7 years younger than whites. Obstructive, but not restrictive, physiology characterized Scadding Stage IV patients. Subjects reporting use of immunosuppression had worse FEV1%p, FVC%p, and DLCO%p compared to subjects never treated, regardless of Scadding stage. We defined sarcoidosis disease activity by a drop in pulmonary function over 36 months and found that subjects meeting this definition had significant repression of blood gene transcripts related to T cell receptor signaling pathways, referred to as the "TCR factor." CONCLUSION: Obstructive pulmonary physiology defined Stage IV patients which were mostly white, non-Hispanics. Genes comprising the composite gene expression score, TCR factor, may represent a blood-derived measure of T-cell activity and an indirect measure of active sarcoidosis inflammation. Validation of this measure could translate into individualized treatment for sarcoidosis patients.

DIVE: a data intensive visualization engine.

SUMMARY: Modern scientific investigation is generating increasingly larger datasets, yet analyzing these data with current tools is challenging. DIVE is a software framework intended to facilitate big data analysis and reduce the time to scientific insight. Here, we present features of the framework and demonstrate DIVE's application to the Dynameomics project, looking specifically at two proteins. AVAILABILITY AND IMPLEMENTATION: Binaries and documentation are available at http://www.dynameomics.org/DIVE/DIVESetup.exe.

Longitudinal analysis of sarcoidosis blood transcriptomic signatures and disease outcomes.

Previously, we demonstrated concordance in differentially expressed genes in sarcoidosis blood and lung, implicating shared dysfunction of specific immune pathways. In the present study, we hypothesised that expression levels of candidate genes in sarcoidosis blood could predict and track with disease outcomes longitudinally. We applied Ingenuity Pathway Analysis to a cross-sectional derivation microarray dataset (n=38) to identify canonical pathways and candidate genes associated with sarcoidosis. In a separate longitudinal sarcoidosis cohort (n=103), we serially measured 48 candidate gene transcripts, and assessed their relation to disease chronicity and severity. In the cross-sectional derivation study, pathway analysis showed upregulation of genes related to interferon signalling and the role of pattern recognition receptors, and downregulation of T-cell receptor (TCR) signalling pathways in sarcoidosis. In the longitudinal cohort, factor analysis confirmed coregulation of genes marking these pathways and identified CXCL9 as an additional candidate pathway. CXCL9 and TCR factors discriminated between chronic versus nonprogressive disease, and CXCL9 predicted disease outcomes longitudinally. Interferon factor was similarly increased in both disease phenotypes. Factors associated with lung function decline included decreased TCR factor and increased CXCL9. These findings demonstrate blood transcriptomic signatures reflecting TCR signalling and CXCL9 predict sarcoidosis chronicity and correlate with disease severity longitudinally.

Brief exposure to secondhand smoke reversibly impairs endothelial vasodilatory function.

INTRODUCTION: We sought to determine the effects of brief exposures to low concentrations of tobacco secondhand smoke (SHS) on arterial flow-mediated dilation (FMD, a nitric oxide-dependent measure of vascular endothelial function), in a controlled animal model never before exposed to smoke. In humans, SHS exposure for 30 min impairs FMD. It is important to gain a better understanding of the acute effects of exposure to SHS at low concentrations and for brief periods of time. METHODS: We measured changes in FMD in rats exposed to a range of real-world levels of SHS for durations of 30 min, 10 min, 1 min, and 4 breaths (roughly 15 s). RESULTS: We observed a dose-response relationship between SHS particle concentration over 30 min and post-exposure impairment of FMD, which was linear through the range typically encountered in smoky restaurants and then saturated at higher concentrations. One min of exposure to SHS at moderate concentrations was sufficient to impair FMD. CONCLUSIONS: Brief SHS exposure at real-world levels reversibly impairs FMD. Even 1 min of SHS exposure can cause reduction of endothelial function.

Interferon-inducible chemokines reflect severity and progression in sarcoidosis.

BACKGROUND: Identification of serum proteins that track with disease course in sarcoidosis may have clinical and pathologic importance. We previously identified up-regulated transcripts for interferon-inducible chemokines CXCL9, and CXCL10, in blood of sarcoidosis patients compared to controls. The objective of this study was to determine whether proteins encoded by these transcripts were elevated in serum and identified patients with remitting vs. chronic progressive sarcoidosis longitudinally. METHODS: Serum levels of CXCL9, CXCL10, and proteins associated with inflammation and/or disease activity (sIL2R, ACE, ESR and CRP) were measured in a prospective cohort of sarcoidosis subjects and controls. Comparisons were made between groups and clinical course using pulmonary function measures and a severity score developed by Wasfi et al. RESULTS: In a cross-sectional analysis of 36 non-immunosuppressed sarcoidosis subjects, serum CXCL9, CXCL10, and sIL2R were significantly elevated compared to 46 controls (p < 0.0001). CXCL9 and CXCL10 were strongly inter-correlated (p = 0.0009). CXCL10 and CXCL9 were inversely correlated with FVC% predicted and DLCO% predicted, respectively. CXCL10 and CXCL9 significantly correlated with sarcoidosis severity score. sIL2R, ESR, CRP, and ACE serum levels did not correlate with pulmonary function measures or severity score. In the longitudinal analysis of 26 subjects, changes in serum CXCL10 level over time corresponded with progression versus remission of disease. CONCLUSIONS: Interferon-γ-inducible chemokines, CXCL9 and CXCL10, are elevated in sarcoidosis and inter-correlated with each other. Chemokine levels correlated with measures of disease severity. Serial measurements of CXCL10 corresponded to clinical course.

Renal improvement and remission in a patient with refractory ANCA-associated vasculitis treated with avacopan.

Antineutrophil cytoplasmic autoantibody (ANCA)-associated vasculitis is associated with end-organ damage resulting in significant morbidity and mortality. Most recently, avacopan, an orally administered selective antagonist of the C5a receptor, was approved by the US Food and Drug Administration as an adjunctive treatment of adult patients with severe, active ANCA-associated vasculitis (granulomatosis with polyangiitis and microscopic polyangiitis) in combination with standard therapy including glucocorticoids. This case study describes a 58-year-old Asian female with severe ANCA-associated vasculitis and acute renal failure who responded to adjunctive therapy with avacopan despite being refractory to rituximab and glucocorticoid therapy.

An analysis of connective tissue disease-associated interstitial lung disease at a US Tertiary Care Center: better survival in patients with systemic sclerosis.

OBJECTIVE: To compare survival of patients with connective tissue disease-associated interstitial lung disease (CTD-ILD) versus idiopathic pulmonary fibrosis (IPF) and patients with systemic sclerosis-associated ILD (SSc-ILD) versus other CTD-ILD followed at our center. METHODS: We used the Stanford ILD database, which contains prospectively collected information on patients with ILD evaluated at our tertiary care center from 2002 to 2009. Survival at last followup from time of ILD diagnosis was calculated using the Kaplan-Meier estimator. Prognostic factors for survival in the overall cohort (IPF and CTD-ILD) and in the CTD-ILD group were identified with univariate and multivariate Cox regression models. RESULTS: Of 427 patients with ILD, 148 (35%) had IPF and 76 (18%) had CTD-ILD at the baseline visit. The cumulative incidence of CTD was 4%. After a median followup of 4 years, 67 patients (36.4%) had died and 4 (2.2%) were lost to followup. Patients with IPF (n = 122) and CTD-ILD (n = 62) experienced similar survival rates (5-year survival about 50%). Patients with SSc-ILD (n = 24) experienced better survival than those with other CTD-ILD (n = 38), with 1-year, 3-year, and 5-year survival rates of 100%, 90%, and 77%, respectively, versus 78%, 42%, and 38% (p = 0.01). The presence of SSc in patients with CTD-ILD decreased the risk of death by > 80% even after correcting for age at ILD diagnosis, sex, and ethnicity (HR = 0.17, 95% CI 0.04-0.83). CONCLUSION: Survival in patients with SSc-ILD was better than in patients with other CTD-ILD, potentially related to routine screening for and early detection of ILD in patients with SSc at our center.

Donor myocardial infarction impairs the therapeutic potential of bone marrow cells by an interleukin-1-mediated inflammatory response.

Delivery of bone marrow cells (BMCs) to the heart has substantially improved cardiac function in most rodent models of myocardial infarction (MI), but clinical trials of BMC therapy have led to only modest improvements. Rodent models typically involve intramyocardial injection of BMCs from distinct donor individuals who are healthy. In contrast, autologous BMCs from individuals after MI are used for clinical trials. Using BMCs from donor mice after MI, we discovered that recent MI impaired BMC therapeutic efficacy. MI led to myocardial inflammation and an increased inflammatory state in the bone marrow, changing the BMC composition and reducing their efficacy. Injection of a general anti-inflammatory drug or a specific interleukin-1 inhibitor to donor mice after MI prevented this impairment. Our findings offer an explanation of why human trials have not matched the success of rodent experiments and suggest potential strategies to improve the success of clinical autologous BMC therapy.

Human potassium chloride cotransporter 1 (SLC12A4) promoter is regulated by AP-2 and contains a functional downstream promoter element.

Most K-Cl cotransport in the erythrocyte is attributed to potassium chloride cotransporter 1 (KCC1). K-Cl cotransport is elevated in sickle erythrocytes, and the KCC1 gene has been proposed as a modifier gene in sickle cell disease. To provide insight into our understanding of the regulation of the human KCC1 gene, we mapped the 5' end of the KCC1 cDNA, cloned the corresponding genomic DNA, and identified the KCC1 gene promoter. The core promoter lacks a TATA box and is composed of an initiator element (InR) and a downstream promoter element (DPE), a combination found primarily in Drosophila gene promoters and rarely observed in mammalian gene promoters. Mutational analyses demonstrated that both the InR and DPE sites were critical for full promoter activity. In vitro DNase I footprinting, electrophoretic mobility shift assays, and reporter gene assays identified functional AP-2 and Sp1 sites in this region. The KCC1 promoter was transactivated by forced expression of AP-2 in heterologous cells. Sequences encoding the InR, DPE, AP-2, and Sp1 sites were 100% conserved between human and murine KCC1 genes. In vivo studies using chromatin immunoprecipitation assays with antihistone H3 and antihistone H4 antibodies demonstrated hyperacetylation of this core promoter region.