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Mediators and Organ Crosstalk in Sterile and Infectious Inflammation View all 26 Articles
Valentin A. Pavlov
Northwell Health, United States
Susan Carpenter
University of California, Santa Cruz, United States
Petros Andrikopoulos
William Harvey Research Institute, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, United Kingdom
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Front. Immunol., 05 February 2018 | https://doi.org/10.3389/fimmu.2017.01977
Severity of Systemic Inflammatory Response Syndrome Affects the Blood Levels of Circulating Inflammatory-Relevant MicroRNAs
Stefano Caserta1*†, Manuela Mengozzi1, Florian Kern1,2, Sarah F. Newbury1, Pietro Ghezzi1 and Martin J. Llewelyn1,2
1Brighton and Sussex Medical School, University of Sussex, Falmer, United Kingdom
2Brighton and Sussex University Hospitals NHS Trust, Brighton, United Kingdom
The systemic inflammatory response syndrome (SIRS) is a potentially lethal response triggered by diverse forms of tissue injury and infection. When systemic inflammation is triggered by infection, the term sepsis is used. Understanding how inflammation is mediated and regulated is of enormous medical importance. We previously demonstrated that circulating inflammatory-relevant microRNAs (CIR-miRNAs) are candidate biomarkers for differentiating sepsis from SIRS. Here, we set out to determine how CIR-miRNA levels reflect SIRS severity and whether they derive from activated immune cells. Clinical disease severity scores and markers of red blood cell (RBC) damage or immune cell activation were correlated with CIR-miRNA levels in patients with SIRS and sepsis. The release of CIR-miRNAs modulated during SIRS was assessed in immune cell cultures. We show that severity of non-infective SIRS, but not sepsis is reflected in the levels of miR-378a-3p, miR-30a-5p, miR-30d-5p, and miR-192-5p. These CIR-miRNA levels positively correlate with levels of the redox biomarker, peroxiredoxin-1 (Prdx-1), which has previously been shown to be released by immune cells during inflammation. Furthermore, in vitro activated immune cells produce SIRS-associated miR-378a-3p, miR-30a-5p, miR-30d-5p, and miR-192-5p. Our study furthers the understanding of the origin, role, and trafficking of CIR-miRNAs as potential regulators of inflammation.
The systemic inflammatory response syndrome [SIRS (1)] can be triggered by diverse forms of injury including burns, ischemia, autoimmune diseases, injuries including surgery and infection. The severity of illness seen in SIRS varies widely but severe SIRS accompanied by multiple organ dysfunction syndrome (MODS), especially in the setting of sepsis (a condition which shares common clinical manifestations with SIRS), may be lethal (2). Even with optimal medical care, mortality rates in severe sepsis increase to around 50% (3). In this respect, clinical scores, such as the sequential organ failure assessment [SOFA (4)] and the acute physiology and chronic health evaluation II [APACHE II (5)], are useful to evaluate SIRS severity and patient mortality risk.
During SIRS, immune cell activation spreads to the whole body driving severe immunopathology (1, 6, 7). Damage- and/or pathogen-associated molecular pattern molecules (respectively, DAMPs and PAMPs) released after injury (or infection) initiate, via toll-like receptor (TLR) signals (6, 8), an activation cascade in immune and endothelial cells leading to inflammatory cytokine production [e.g., tumor necrosis factor (TNF) α, IL-1, IL-6, and IL-8], in a so-called "cytokine storm" (9, 10). Activated inflammatory cells migrate to affected organs and release secondary inflammatory mediators (1), including reactive oxygen species (ROS) and nitrogen species (11–15), resulting in oxidative stress (16, 17). Inflammatory cell migration to tissues and endothelial cell activation (18) lead to disseminated intravascular coagulation (19) aggravating organ damage. Increased levels of TNFα, IL-1, and IL-6 have been reported to occur more often in sepsis than SIRS (1, 20–22). Anti-inflammatory mediators (e.g., TGFβ and cytokine antagonists) are also present in plasma during SIRS, with soluble cytokine receptors often found at concentration higher than the respective cytokines (20, 23), hence compensatory anti-inflammatory responses [CAR (24)] may be activated during SIRS. Understanding the responses which occur in non-infective inflammation and sepsis and how they are regulated will aid development of diagnostics and therapeutics in these major inflammatory diseases.
MicroRNAs (miRNAs) are small (~23 nt) RNAs that function as post-transcriptional gene regulators (25, 26). By annealing to complementary sequences in the 3′ untranslated region (3′ UTR) of target mRNAs, they reduce expression of specific proteins by promoting target degradation or inhibition of translation (26). The role of miRNAs in SIRS remains unclear, although many inflammatory cytokines, mediators, and their regulators are miRNA targets (27). Approximately 100–200 miRNAs are found in human blood in health and disease (28–30). Previous studies (31, 32) suggest that exogenous miRNAs may be detected by TLRs (33, 34), particularly TLR7 (32), as potential DAMPs leading to inflammatory signals. We previously identified a pool of circulating inflammatory-relevant miRNA (CIR-miRNAs) that robustly distinguish sepsis from non-infective SIRS (35). We were the first to find that, in these conditions, CIR-miRNA levels inversely correlate with those of inflammatory cytokines (35), suggesting that they may be part of the CAR.
To extend these observations, we set out to determine how CIR-miRNAs reflect SIRS severity and whether they are likely to arise directly from immune activation. In this study, we investigated the correlation of plasma levels of CIR-miRNAs with disease severity and other inflammatory parameters. We found that SIRS severity significantly impacts on the levels of miR-378a-3p, miR-30a-5p, miR-30d-5p, and miR-192-5p. These CIR-miRNAs are unlikely to derive from red blood cell (RBC) damage consequent to hemolysis or coagulopathy. Instead their levels positively correlate with levels of the redox biomarker, Prdx-1, which is released by immune cells during inflammation (36, 37). Finally, we show that activated immune cells produce SIRS-associated miR-378a-3p, miR-30a-5p, miR-30d-5p, and miR-192-5p, in vitro. Our study has implications for furthering the understanding of the origin and role and trafficking of CIR-miRNAs in SIRS, sepsis, and potentially other inflammatory disease.
Patients and Healthy Donors
The patient population used in this study was previously described in detail (35, 38). Briefly, patients comprised unselected adult admissions to a mixed medical/surgical intensive/high-dependency care unit (ICU/HDU) at an English acute hospital (Brighton and Sussex University Hospitals NHS Trust). Patients were categorized as having non-infective SIRS (n = 44) or sepsis (n = 29), following standard criteria (38). SIRS severity was defined as: severe (SOFA ≥ 6) and non-severe (SOFA ≤ 3); patients with intermediate SOFA scores of 4–5 were excluded. Only patients with abdominal sepsis were included in this study. Blood samples were collected within <6 h from admission and time of sample collection did not affect levels of CIR-miRNAs (35). Healthy donors were recruited at Brighton and Sussex Medical School (BSMS, University of Sussex, Falmer, Brighton, UK). Refer to Materials and Methods in Supplementary Material for further details, including details of any medication targeting inflammation that patients were taking at the time of admission.
Hemoglobin, Prdx-1, and Cytokine Analysis in Plasma Samples
Red blood cell lysis can bias miRNA content in plasma (39, 40). The concentration of free hemoglobin (Hb) was independently measured in patient plasma by the Harboe spectrophotometric method (41, 42) and hemolytic samples (43–45) were excluded, as carried out previously (35). Prdx-1 plasma levels were independently measured using an ELISA kit for human Prdx-1 (#ABIN418422) from Antibodies Online (Aachen, Germany). Plasma cytokines (IL-6, IL-8) and anti-/inflammatory mediators [pro-calcitonin (PCT), C-reactive protein (CRP), and soluble CD25 (sCD25)] were measured on Luminex LX200 and ELISA (sCD25) as previously described (38).
Human Samples and Primary Cell Cultures
Human peripheral blood mononuclear cells (PBMCs) from healthy donors were freshly isolated by centrifugation over Ficoll–Hypaque density gradient as previously described (46). Cells were washed in sterile PBS (ThermoFisherScientific) and counted with 0.1% Trypan Blue cell viability exclusion dye (Sigma). Thereafter, 20 × 106 viable PBMCs were resuspended in 10 ml (2 × 106 cells/ml) of complete media: RPMI containing 100 IU/ml penicillin, 100 µg/ml streptomycin, 2 mM l-glutamine (all from ThermoFisherScientific) and 10% of exosome-depleted, heat-deactivated fetal calf serum (System Biosciences). PBMCs were stimulated with the bacterial superantigen (SAg), streptococcal pyrogenic exotoxin K/L [SPE-K/L (47)] as described before (46), in parallel to unstimulated control cultures (24-well plates, 1 ml/well). After 5 days, cells were harvested and assayed for viability with Trypan-blue (0.1%) (Figure S7 in Supplementary Material). Equal volumes of culture supernatants were then recovered, frozen at −80°C and total RNA was then extracted as described below.
RNA Extraction and MicroRNA Real-Time qPCR Array
Plasma and supernatant RNA was extracted using the miRCURY™ RNA isolation—biofluids kit (Exiqon, Denmark). After thawing on ice, an RNA spike-in template mixture was added to the samples. Eight milliliters of supernatant per sample was mixed with 2.4 ml of Lysis solution BF containing 16.67 µg/ml of MS2 bacteriophage RNA (UniSp6), prior to RNA purification, as specified by the manufacturer's instructions. Extracted total RNA was stored in a −80°C freezer. RNA was reverse transcribed (RT) and cDNA analyzed using the miRCURY LNA™ Universal RT miRNA PCR, Polyadenylation, and cDNA synthesis method. For plasma samples derived from patients (≥8 biological replicates), each microRNA was assayed by qPCR (miRNA Ready-to-Use PCR, Pick-&-Mix using ExiLENT SYBR® Green master mix) in two independent technical repeats, including negative controls (no-template from the RT reaction) using a LightCycler® 480 Real-Time PCR System (Roche). SAg stimulation experiments were performed as 10 independent biological replicates. Assays returning three crossing point (Cp) values less than the negative control and Cp < 37 were accepted. The stability values of candidate normalizers were assessed using the "NormFinder" software (48). Any qPCR data were normalized to the average Cp of internal normalizers [miR-320a and miR-486-5p (35)] in plasma samples and, in the case of culture supernatants, the Cp of normalizer spike-in (UniSp6); (delta Cp, dCp = normalizer Cp − assay Cp). Refer to the Materials and Methods in the Supplementary Material for further details.
Datasets were analyzed using the GraphPad Prism 6 and/or IBM SPSS Statistics 22 software. The D'Agostino and Pearson omnibus and Shapiro–Wilk tests were used to test normal data distribution; data were considered normally distributed only if they passed both tests. If not normally distributed, medians with interquartile ranges (IQRs, rather than means and SD) are shown and Mann–Whitney U Test (rather than t-tests) was used to calculate p values in 2-group comparisons. Correlations between SOFA and APACHE II scores and plasma levels of CIR-miRNAs and inflammatory cytokines/mediators were evaluated using the Spearman rho (ρ) and significances of the correlations determined as indicated. Generally, 0.35 ≤ ρ ≤ 1 or −1 ≤ ρ ≤ −0.35 were considered as moderate-to-strong correlation trends (49, 50). The confidence in the predictive value of each correlation was assessed by individual p values; in general, correlations trends were considered significant only if p ≤ 0.05 (51). In addition, for the qPCR miRNA array dataset, a Benjamini–Hochberg multiple comparison correction (52) was used to control for the number of false positives, using false discovery rates of 15% (Tables 1 and 2; Tables S1 and S2 in Supplementary Material) and 5% (Tables 3 and 4) which correspond to a ~1/6 and 1/20 chance of false positives, respectively. Such correction excludes that the significance of correlation of any parameter (such as disease severity, free Hb and Prdx-1) with any of the 43 miRNA tests run in parallel was not simply due to the chance of multiple testing. Unless stated differently in figure legends, levels of significance were assigned as: *p ≤ 0.05; **p ≤ 0.005; and ***p ≤ 0.0005. MiRNA and cytokine, Hb, and Prdx-1 analyses were conducted blind to the clinical data.
Table 1. Correlations of circulating inflammatory-relevant microRNAs (CIR-miRNAs) with severity of systemic inflammatory response syndrome (SIRS) as detected by sequential organ failure assessment (SOFA).
Table 2. Correlations of circulating inflammatory-relevant microRNAs (CIR-miRNAs) with severity of sepsis as detected by sequential organ failure assessment (SOFA).
Table 3. Correlations of circulating inflammatory-relevant microRNAs (CIR-miRNAs) with free hemoglobin levels in non-infective systemic inflammatory response syndrome (SIRS).
Table 4. Correlations of circulating inflammatory-relevant microRNAs (CIR-miRNAs) with peroxiredoxin-1 levels in non-infective systemic inflammatory response syndrome (SIRS).
Severity of Non-Infective SIRS Positively Correlates with the Levels of CIR-miRNAs
To determine whether CIR-miRNA levels are regulated by the severity of SIRS, Cp of single miRNAs were compared to the mean Cp of internal normalizers [as previously identified (35)] to give dCp values that increase with the abundancy of specific miRNAs. Severity of disease, according to the SOFA score, was then correlated with dCp in non-infective SIRS patients. Thirteen CIR-miRNAs showed generally positive trends correlating with SIRS severity (Table 1). Significantly, 8 miRNAs showed a positive correlation with the disease severity (Table 1, black bold), with miR-378a-3p, miR-30a-5p, miR-30d-5p, and miR-192-5p further passing the Benjamini–Hochberg correction for multiple comparisons (52) (Figure 1A). We further determined whether CIR-miRNA levels correlate with disease severity in sepsis patients (Figure 1B). None of the CIR-miRNAs correlating with disease severity in SIRS showed similar significant trends in sepsis (Figure 1B; Table 2). Although miR-22-3p, miR-375, miR-122-5p, miR-192-5p, and miR-378a-3p maintained modestly positive non-significant trends, multiple CIR-miRNAs showed generally inverse trends with sepsis severity (Table 2).
Figure 1. Circulating inflammatory-relevant microRNA levels correlate with the severity of non-infective systemic inflammatory response syndrome (SIRS). In miRNA qPCR arrays, within each patient's specimen, crossing point (Cp) of a single miRNA is compared to the mean Cp of 2 normalizers (miR-486-5p and miR-320a) to give delta Cp (dCp). dCp of non-infective SIRS (A) and sepsis (B) patients were analyzed in correlation analyses with disease severity, as determined by the sequential organ failure assessment (SOFA) score. (A) Non-parametric correlation of SOFA scores with the plasma levels of miR-378a-3p, miR-30a-5p, miR-30d-5p, and miR-192-5p in non-infective SIRS patients (n = 43 for all miRNAs). (B) Non-parametric correlation of SOFA scores with the plasma levels of miR-378a-3p (n = 26), miR-30a-5p (n = 24), miR-30d-5p (n = 27), and miR-192-5p (n = 27) in infective SIRS (sepsis) patients. Each symbol represents an individual patient. Correlation trends are shown with the linear regression model including Spearman rho (ρ) and the significances of the correlations (*p ≤ 0.05; **p ≤ 0.005; and ***p ≤ 0.0005 or ns, non-significant).
In comparison to pro-inflammatory cytokines (IL-8 and IL-6) and soluble mediators of inflammation and stress such as CRP, PCT, free hemoglobin (Hb), and Prdx-1, CIR-miRNAs showed more robust correlations with disease severity (compare Figures 1A and 2A). None of these inflammatory mediators showed a significant correlation with SIRS severity, although some non-significant trends were apparent (Figure 2A). While levels IL-8, IL-6, CRP, and PCT increased in sepsis compared to non-infective SIRS, only IL-6 positively correlated with sepsis severity (Figure 2B).
Figure 2. Correlations of plasma levels of inflammatory cytokines and stress mediators with the severity of non-infective systemic inflammatory response syndrome and sepsis. The levels of inflammatory cytokines (interleukin-IL-8 and IL-6) and stress mediators: C-reactive protein (CRP), pro-calcitonin (PCT), free hemoglobin (Hb), and peroxiredoxin-1 (Prdx-1) were measured by ELISA in the plasma of non-infective SIRS (A) and sepsis (B) patients. Thereafter, the correlation with the severity of disease, as determined by the sequential organ failure assessment (SOFA) score was investigated. (A) Non-parametric correlation of SOFA scores with the plasma levels of IL-8 (n = 43), IL-6 (n = 43), CRP (n = 43), PCT (n = 42), free Hb (n = 43), and Prdx-1 (n = 41) in non-infective SIRS patients. (B) Non-parametric correlation of SOFA scores with the plasma levels of IL-8 (n = 26), IL-6 (n = 27), CRP (n = 27), PCT (n = 27), free Hb (n = 27), and Prdx-1 (n = 24) in sepsis patients. Each triangle represents an individual patient. Correlation trends are shown with the linear regression model, including Spearman rho (ρ) and the significances of the correlations (* p ≤ 0.05; **p ≤ 0.005; and ***p ≤ 0.0005 or ns, non-significant).
Similar analyses were performed using the APACHE II in alternative to the SOFA severity score. In agreement with our previous analysis, CIR-miRNA levels positively and significantly correlated with the APACHE II score in non-infective SIRS (Figure S1A and Table S1 in Supplementary Material), but not in sepsis (Figure S1B and Table S2 in Supplementary Material). In addition, CIR-miRNAs in comparison to IL-6, CRP, PCT, and free Hb, generally showed more robust correlations with the APACHE II score, while IL-8 and Prdx-1 had significant positive correlation with the APACHE II score in non-infective SIRS (Figure S2A in Supplementary Material), but not in sepsis (Figure S2B in Supplementary Material).
CIR-miRNAs Significantly Discriminate Severe from Non-Severe SIRS
Consistent with a steady accumulation of CIR-miRNAs in more severe disease, dCp values were higher in severe rather than non-severe SIRS (Figure 3A) for the most significantly affected CIR-miRNAs. Hence, miR-378a-3p, miR-30a-5p, miR-30d-5p, and miR-192-5p distinguished non-severe from severe SIRS patients (Figure 3A). Also miR-122-5p, miR-101-3p, miR-21-5p, miR-148a-3p, and miR-532-5p (which all had non-significant positive trends to increase with SIRS severity, Table 1) significantly discriminated severe from non-severe SIRS (Figure S3 in Supplementary Material). By contrast, none of the inflammatory cytokines and stress mediators we measured in our cohort discriminated SIRS severity groups, including IL-8 (Figure 3B) and IL-6 (Figure S3B in Supplementary Material).
Figure 3. Circulating inflammatory-relevant microRNA biomarkers discriminate the severity of systemic inflammatory response syndrome (SIRS) better than inflammatory and stress mediators. In miRNA qPCR arrays, within each patient's specimen, crossing point (Cp) of individual miRNAs were normalized as in Figure 1 and analyzed in patients with non-severe (open circles) and severe (filled circles) non-infective SIRS. Each symbol represents an individual patient. (A) Dot plots show delta Cp values for miR-378a-3p, miR-30a-5p, miR-30d-5p, and miR-192-5p in non-severe (n = 21) and severe (n = 22) non-infective SIRS patients, together with the level of significance. (B) Dot plots show concentration of IL-8, pro-calcitonin (PCT), peroxiredoxin-1 (Prdx-1) and free hemoglobin (Hb) in non-severe (n = 21) and severe (n = 22, apart from Prdx-1 in which n = 20) non-infectious SIRS patients, together with the level of significance.
SIRS-Relevant CIR-miRNAs Are Unlikely to Derive from RBCs
We next investigated where the CIR-miRNAs regulated by SIRS severity may originate. We reasoned that if they derived from RBCs that contain multiple miRNAs (released in the blood after physiological hemolysis or coagulopathy), then CIR-miRNA levels would positively correlate with the amount of free Hb. Instead, around 95% of the analyzed CIR-miRNAs (40/43) showed inverse correlation trends with free Hb (Tables 3; Table S6 in Supplementary Material). Many CIR-miRNAs, including miR-378a-3p, miR-30a-5p, miR-30d-5p, and miR-192-5p (Figure 4A) and miR-101-3p, miR-21-5p, miR-22-3p, miR-423-5p, and miR-122-5p (Figure S4 in Supplementary Material) significantly inversely correlated with free Hb levels even after a stringent Benjamini–Hochberg correction (52), supporting that CIR-miRNAs relevant in severe SIRS do not derive from RBCs. By contrast, free Hb levels positively correlated with miRNAs abundant in RBCs (29), miR-486-5p and miR-451a (Figure 4B; Table 3), consistent with a RBC origin.
Figure 4. Circulating inflammatory-relevant microRNA levels inversely correlate with markers of red-blood cell lysis. In miRNA qPCR arrays, within each patient's specimen, crossing point (Cp) of individual miRNAs (open squares) were normalized as in Figure 1 and analyzed in correlation with levels of free Hb, which is derived from the lysis of red blood cells. Each square represents an individual patient. Correlation trends are shown with the linear regression model including Spearman rho (ρ) and the significances of the correlations (*p ≤ 0.05; **p ≤ 0.005; and ***p ≤ 0.0005 or ns, non-significant) for (A) miR-378a-3p, miR-30a-5p, miR-30d-5p, and miR-192-5p and (B) miR-486-5p in non-infective systemic inflammatory response syndrome patients (n = 43).
CIR-miRNAs Positively Correlate with Redox Biomarker Prdx-1
We sought to investigate whether SIRS-relevant CIR-miRNAs derive from immune cells (i.e., white blood cells). To do this, we used Prdx-1, a marker that we had previously found to be released by immune cells after inflammatory stimuli (36, 37). Consistently, in the entire SIRS cohort, Prdx-1 levels correlated with markers of immune activation (IL-8 and sCD25), rather than free Hb (Figure S5 in Supplementary Material), demonstrating that Prdx-1 did not increase due to RBC hemolysis. We then analyzed the correlation of Prdx-1 with CIR-miRNA dCp (Figure 5; Table 4). Around 40% (18/43) of CIR-miRNAs showed moderate-to-strong correlation trends with Prdx-1 levels, with a prevalence of positive relationships (Tables 4; Table S7 in Supplementary Material). Levels of 10 CIR-miRNAs, including miR-378a-3p, miR-30a-5p, and miR-192-5p (Figure 5A) and miR-101-3p, miR-21-5p, miR-22-3p, and miR-122-5p (Figure S6 in Supplementary Material) significantly positively correlated with those of Prdx-1, suggesting that the amounts of CIR-miRNAs modulated in non-infective SIRS mirror those of this inflammatory biomarker. By contrast, miRNAs abundant in RBCs, miR-486-5p (Figure 5B) and miR-451a (Table S7 in Supplementary Material), did not show positive correlation with Prdx-1.
Figure 5. Circulating inflammatory-relevant microRNA levels positively correlate with markers of immunological stress, peroxiredoxin-1 (Prdx1). In miRNA qPCR arrays, within each patient's specimen, crossing point (Cp) of individual microRNAs (miRNAs) (open rhombi) were normalized as in Figure 1 and analyzed in correlation with levels of plasma inflammatory stress marker, Prdx-1. Each symbol represents an individual patient. Correlation trends are shown with the linear regression model including Spearman rho (ρ) and the significances of the correlations (*p ≤ 0.05; **p ≤ 0.005; and ***p ≤ 0.0005 or ns, non-significant) for (A) miR-378a-3p, miR-30a-5p, miR-30d-5p, and miR-192-5p, and (B) miR-486-5p in non-infective systemic inflammatory response syndrome patients (n = 41).
Stimulated Immune Cells Produce CIR-miRNAs Affected by Severity of SIRS
To determine whether immune cells could produce miR-378a-3p, miR-30a-5p, miR-30d-5p, and miR-192-5p, PBMCs of 10 healthy donors were stimulated in vitro with bacterial SAg, known to drive massive inflammatory cell activation. After 5 days, viability of the cell cultures was determined using the Trypan-blue dye exclusion assay (Figure S7 in Supplementary Material). Relative to a normalizer spike-in, we found increased amounts of miR-378a-3p, miR-30a-5p, miR-30d-5p, and miR-192-5p in culture supernatants of stimulated PBMCs compared to unstimulated controls (Figure 6A). The increase of miRNAs in the supernatants varied across the miRNA species between 10- and 100-fold (Figure 6B), with the notable exception of miR-122-5p that was not significantly increased (Figures 6A,B). In cultures derived from four donors, levels of miR-10b-5p, which did not discriminate non-severe from severe SIRS (Figure S3 in Supplementary Material), did not increase upon activation (Figures 6A,B).
Figure 6. Levels of circulating inflammatory-relevant microRNAs affected by the severity of systemic inflammatory response syndrome (SIRS) are increased in supernatants of blood-derived immune cell cultures. PBMCs derived from 10 individuals were freshly purified from blood and equal cell numbers were then incubated in complete media in the presence of exosome-free bovine serum, strictly at the concentration of 2 × 106 cells/ml, in replicate wells. Half of the cultures were stimulated with the SPE-KL bacterial superantigen (SAg) from 5 days, in comparison to unstimulated controls (unstim). On day 5, cells were harvested and equal volumes of supernatants were recovered after two sequential spins prior to RNA extraction as detailed in the Section "Materials and Methods." The presence of microRNAs regulated (miR-378a-3p, miR-30a-5p, miR-30d-5p, miR-192-5p, and miR-122-5p) or unaffected (miR-10b-5p) by the severity of non-infective SIRS was assessed in Q-PCR array in multiple biological replicates. (A) Within each donor's specimen, crossing point (Cp) of a single miRNA is compared to the Cp of a spike-in RNA added to supernatants just prior to the RNA-purification to give delta Cp (dCp). dCp were then linearized to give the relative expression of individual miRNAs in supernatants from unstimulated (black bars; n = 10, 7, 5, 7, 10, 4, respectively, for miR-378a-3p, miR-30a-5p, miR-30d-5p, miR-192-5p, miR-122-5p, and miR-10b-5p) compared to SAg-activated cells (gray bars; n = 10, 10, 8, 9, 10, and 9, respectively, for miR-378a-3p, miR-30a-5p, miR-30d-5p, miR-192-5p, miR-122-5p, and miR-10b-5p). (B) Average miRNA fold-induction in 4–10 individuals were calculated as the average ratio of miRNA levels detected in supernatants from SAg-activated cells compared to unstimulated cultures.
We report here that, during non-infective SIRS, the blood levels of CIR-miRNAs increase in parallel with the severity of inflammation. Levels of CIR-miRNAs do not correlate with those of free Hb, indicating that they do not derive from RBCs during SIRS. Instead, CIR-miRNAs positively correlate with levels of the inflammatory mediator and redox enzyme, Prdx-1 which is released by immune cells in inflammation. Consistently miR-378a-3p, miR-30a-5p, miR-30d-5p, and miR-192-5p affected by severity of SIRS are produced by immune cells upon activation. As CIR-miRNAs are increasingly proposed as biomarkers for sepsis, cancer, and other disease (35, 53–55), their inflammatory cell origin should be considered in future research.
In our study, CIR-miRNAs distinguish non-severe from severe SIRS better than inflammatory cytokines and mediators. Inflammatory cytokines are thought to be released during SIRS as part of the cytokine storm, during MODS (1). As in our current study, plasma levels of cytokines have not previously been found to reliably increase in severe SIRS (1), perhaps due to low cytokine concentration and short half-life in samples (56, 57). By contrast, regulators of inflammatory mediators are abundant in the blood of SIRS patients (20), as we found to be the case for the CIR-miRNAs affected by SIRS severity (Figure 3; Figure S3 in Supplementary Material). In addition, it should be noted that blood levels of miR-378a-3p, miR-30a-5p, miR-30d-5p, and miR-192-5p were regulated by disease severity, irrespectively from whether patients were taking anti-inflammatory drugs (at the time of admission) (Figure S8 and Table S8 in Supplementary Material).
CIR-miRNAs may act as regulators of inflammation (35) by targeting the 3′UTR of mRNAs encoding pro-inflammatory cytokines/mediators (58–61). Many miRNAs are involved in inflammation/immune function (27), including miR-378 (62, 63), miR-30a/d (64, 65), miR-192 (64–68), and others found in this study (69). Interestingly, the more severe SIRS is, the more CIR-miRNAs are released, potentially counteracting systemic inflammation as part of the CAR (24). Significantly, direct or indirect targets of miR-378, miR-30a/d, and miR-192 include, among other genes, IL-1A (70), IL-1R (71), IL-17 (72), IL-17RA, and IL-17RE (73). In contrast, CIR-miRNA levels did not correlate with sepsis severity (compare Tables 1 and 2; Tables S1 and S2 in Supplementary Material), suggesting that inflammatory pathways leading to CIR-miRNA accumulation in blood are dysregulated in sepsis. Consequently, upon sepsis progression, the negative regulation exerted by CIR-miRNAs may be restrained, thus boosting immunopathology. Inflammatory cytokine (IL-1, IL-6, etc.) levels increased consistently much more in sepsis than in SIRS (35), therefore inflammatory mRNAs may act as a "sponge" to sequestrate miRNAs in sepsis, but not in other trauma including SIRS (74). Although miR-30a was recently associated with induction of myeloid derived suppressor cells in cancer (75), the question of whether CIR-miRNAs are anti-inflammatory and ameliorate sepsis outcome remains to be further investigated.
Alternatively, CIR-miRNAs may act as modulators of inflammation via indirect effects (i.e., by not targeting directly inflammatory cytokine expression). For instance, at least in mice, miR-378 is known to target phosphoinositide 3 kinase (PI3K) expression in liver cells, affecting liver metabolism with systemic effects which may be relevant during systemic inflammatory disease and MODS (76). Furthermore, PI3K pathway is also crucially regulated during immune cell proliferation, differentiation, and apoptosis (77), all of which may affect immune cell function during SIRS. Similarly, miR-30a has been shown to target the expression of Blimp-1 (78) (an important differentiation factor in immune cells) and various members of the calcineurin signaling pathway, including NFATc3 (79) at least in podocytes and cardiomyocytes, which might have implications for MODS and inflammation. Finally, miR-192 has been shown to suppress cell-proliferation pathways in myeloma (80) and other leukemic cells (81). However for all abovementioned cases, whether the same targets are also regulated in normal immune cells remains so far elusive.
Altogether, our results have implications for clinical practice and future therapeutic interventions. In particular in ICU/HDU, using miRNAs to distinguish non-infective SIRS from sepsis and severe from non-severe SIRS could help guide patient management, for example, triaging patients based on severity, informing decisions based on prognosis, and helping target therapy including the need for and choice of antibiotics. In future, miRNAs may become markers and/or targets for novel immunotherapy for acute inflammatory conditions, potentially providing a non-antibiotic alternative intervention.
The origin of circulating miRNAs is unclear. RBCs contain miRNAs that they release upon hemolysis (39, 40). To exclude artifacts from sample processing, we removed hemolytic samples and used plasma rather than serum, as the latter contains more Hb (and RBC miRNAs) due to coagulation damage (82, 83). Coagulopathy may drive pathophysiological levels of hemolysis and release of CIR-miRNAs, as seen for miR-486-5p and miR-451a [abundant in RBCs (29)]. Although free Hb levels did not significantly increase with SIRS severity, levels of multiple CIR-miRNAs inversely correlate with free Hb, indicating that these do not derive from RBCs in SIRS as in sepsis.
Our data suggest that CIR-miRNAs affected in SIRS may derive from inflammatory cells activated during disease. In particular, CIR-miRNAs may be released in the blood in association with inflammation-induced oxidative stress, as suggested by the positive correlation between Prdx-1 and multiple CIR-miRNAs. Prdx-1 is an anti-oxidant enzyme that mediates the elimination of H2O2 and can be secreted as a dimer by macrophages, upon TLR triggering (36, 37). Its expression is regulated mainly by the transcription factor Nrf2, activated by ROS and various other reactive, electrophilic species (84). Its induction may be an indicator of a protective response to oxidative stress (85, 86). Because Prdx-1 is associated with immune cells activation (37), we asked whether SIRS-relevant CIR-miRNAs were derived from circulating immune cells upon activation. We found that miRNAs affected by SIRS severity were indeed increased >10-fold in supernatants of PBMC cultures after activation with bacterial SAg that drove ~1.5-fold cell-expansion. Thus, sustained immune cell activation may explain the massive release of miRNAs regulated during severe SIRS. Macrophages/monocytes, T and NK cells could be possible candidates for the release of CIR-miRNAs.
Of note, activated PBMCs did not produce significant increase in the supernatant levels of miR-10b-5p, which was not affected by SIRS severity (Figure S3 in Supplementary Material). Unlike other miRNAs, the modest (<2-fold) increase of miR-122-5p upon SAg stimulation may be explained simply by cell proliferation, suggesting that PBMCs contribute little to miR-122-5p blood levels. As hepatocytes highly express miR-122 and increased blood levels of miR-122 were previously associated with liver pathology (87), epithelial cell release of miR-122 and potentially other CIR-miRNAs during SIRS should be further investigated in future.
In conclusion, our work opens up a number of questions about CIR-miRNAs for future investigation. Currently, the exact function of circulating miRNAs in inflammatory conditions remains unknown. CIR-miRNAs released by cells in the body after inflammatory damage may act as DAMPs that bind to TLR ligands (33), a property that is shared by Prdx-1 (86). For instance, small RNAs contained in exosomes can be phagocytosed in myeloid-origin, antigen-presenting like cells and trigger TLR7 signaling, in vitro (31). In this context, miRNAs may act as pro-inflammatory DAMPs to drive inflammatory cytokines (including IL-6) production downstream of intracellular TLRs (31). However, we previously found that pro-inflammatory cytokine levels inversely correlate with blood levels of CIR-miRNAs, suggesting that CIR-miRNAs may negatively regulate inflammation in vivo (35). In mice, regulatory T cells (Tregs) have been shown to dampen the activity of conventional T cells by miRNA transfer (88). Yet, it is unclear whether miRNAs from Tregs can exert suppression at distance. Furthermore, beyond targeting cytokine and inflammatory mediator expression, CIR-miRNAs may regulate inflammation also via indirect mechanisms, or a complex combination of direct and indirect mechanisms, by acting in concert on distinct targets, in multiple cell types, and in different organs. The dynamic contribution of immune cells to blood miRNAs has been frequently overlooked, despite inflammation clearly affecting them. Thus, while differences in miRNA transfer may exist in vivo compared to in vitro, future research is needed to clarify the role of CIR-miRNAs in immunopathological and homeostatic conditions. In particular, more research is needed to address which immune cells specifically produce CIR-miRNAs to be released in the blood, including when and how. Also the mode of trafficking and the cellular and molecular targets of CIR-miRNAs need to be identified yet. In future, such research may allow to harness CIR-miRNAs as immunomodulatory drugs useful for therapeutic purposes in inflammatory conditions as in other diseases.
Written informed consent or consultee approval to enroll was secured for all study participants (patients and healthy donors). This study was approved by the North Wales Research Ethics Committee (Central and East, reference 10/WNo03/19) and the BSMS Research Governance and Ethics Committee (reference: 13/182/LLE) and carried out in accordance with the approved guidelines. All data were anonymized.
SC and MM performed experiments and analyzed the data; SC, PG, SN, FK, and ML conceived and designed experiments; SC, SN, and ML wrote the first manuscript draft. All authors critically contributed to the final version of the manuscript.
The authors have no conflict of interest directly related to the current manuscript. However, SC, FK, SN, and ML are inventors in a patent submitted by the University of Sussex for the use of CIR-miRNAs as biomarkers of sepsis.
We thank staff and patients who participated in the clinical study. We thank Dr. Helen Stewart for reading the manuscript and other BSMS members at the University of Sussex for participating in useful discussions. We thank Dr. Antonio Sorrentino and Dr. Michael Thorsen (Exiqon), for assistance.
SC is supported by a Brighton and Sussex Medical School Internal Fellowship (University of Sussex). This work was funded by the University of Sussex Research Development Fund Round 3 (RDF 3-021). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
The Supplementary Material for this article can be found online at http://www.frontiersin.org/articles/10.3389/fimmu.2017.01977/full#supplementary-material.
miRNA, microRNA; CIR-miRNA, circulating inflammatory-relevant microRNA; SIRS, systemic inflammatory response syndrome; SOFA, sequential organ failure assessment; APACHE II, acute physiology and chronic health evaluation II; MODS, multiple organ dysfunction syndrome; CAR, compensatory anti-inflammatory responses; DAMPs, damage-associated molecular pattern molecules; PAMPs, pathogen-associated molecular pattern molecules; TLR, toll-like receptor; ROS, reactive oxygen species; TNF, tumor necrosis factor; TGF, tumor growth factor; CRP, C-reactive protein; sCD25, soluble CD25; Prdx-1, peroxiredoxin-1; Hb, hemoglobin; IL-, interleukin; PCT, pro-calcitonin; SAg, superantigen; SPE-K/L, streptococcal pyrogenic exotoxin K/L; PBMCs, peripheral blood mononuclear cells; RBCs, red blood cells; PBS, phosphate-buffered saline; FCS, fetal calf serum; RPMI, Roswell Park Memorial Institute medium; RT, reverse transcription/transcribed; IQR, interquartile ranges.
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Keywords: systemic inflammatory response syndrome, sepsis, microRNA, inflammation, immune cells, sequential organ failure assessment score, miR-378, miR-30
Citation: Caserta S, Mengozzi M, Kern F, Newbury SF, Ghezzi P and Llewelyn MJ (2018) Severity of Systemic Inflammatory Response Syndrome Affects the Blood Levels of Circulating Inflammatory-Relevant MicroRNAs. Front. Immunol. 8:1977. doi: 10.3389/fimmu.2017.01977
Received: 15 September 2017; Accepted: 20 December 2017;
Valentin A. Pavlov, Northwell Health, United States
Petros Andrikopoulos, William Harvey Research Institute (WHRI), United Kingdom
Susan Carpenter, University of California, Santa Cruz, United States
Copyright: © 2018 Caserta, Mengozzi, Kern, Newbury, Ghezzi and Llewelyn. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
*Correspondence: Stefano Caserta, S.Caserta@hull.ac.uk
†Present address: Stefano Caserta, School of Life Sciences, The University of Hull, Hull, United Kingdom
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<!doctype html>

    <title>Code coverage report for src/wizard.component.ts</title>
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    <link rel="stylesheet" href="../prettify.css" />
    <link rel="stylesheet" href="../base.css" />
    <meta name="viewport" content="width=device-width, initial-scale=1">

      all files / src/ wizard.component.ts

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import { Component, OnInit, Output, EventEmitter, ContentChildren, QueryList, AfterContentInit } from '@angular/core';
import { WizardStepComponent } from './wizard-step.component';

@Component({
  selector: 'form-wizard',
  template:
  `<div class="card">
    <div class="card-header">
      <ul class="nav nav-justified">
        <li class="nav-item" *ngFor="let step of steps" [ngClass]="{'active': step.isActive, 'enabled': !step.isDisabled, 'disabled': step.isDisabled}">
          <a (click)="goToStep(step)">{{step.title}}</a>
        </li>
      </ul>
    </div>
    <div class="card-block">
      <ng-content></ng-content>
    </div>
    <div class="card-footer" [hidden]="isCompleted">
        <button type="button" class="btn btn-secondary float-left" (click)="previous()" [hidden]="!hasPrevStep || !activeStep.showPrev">Previous</button>
        <button type="button" class="btn btn-secondary float-right" (click)="next()" [disabled]="!activeStep.isValid" [hidden]="!hasNextStep || !activeStep.showNext">Next</button>
        <button type="button" class="btn btn-secondary float-right" (click)="complete()" [disabled]="!activeStep.isValid" [hidden]="hasNextStep">Done</button>
    </div>
  </div>`
  ,
  styles: [
    '.card { height: 100%; }',
    '.card-header { background-color: #fff; padding: 0; font-size: 1.25rem; }',
    '.card-block { overflow-y: auto; }',
    '.card-footer { background-color: #fff; border-top: 0 none; }',
    '.nav-item { padding: 1rem 0rem; border-bottom: 0.5rem solid #ccc; }',
    '.active { font-weight: bold; color: black; border-bottom-color: #1976D2 !important; }',
    '.enabled { border-bottom-color: rgb(88, 162, 234); }',
    '.disabled { color: #ccc; }'
  ]
})
export class WizardComponent implements OnInit, AfterContentInit {
  @ContentChildren(WizardStepComponent) wizardSteps: QueryList<WizardStepComponent>;
  private _steps: Array<WizardStepComponent> = [];
  private _isCompleted: boolean = false;

  @Output() onStepChanged: EventEmitter<WizardStepComponent> = new EventEmitter<WizardStepComponent>();

  constructor() { }

  ngOnInit() {
  }

  ngAfterContentInit() {
    this.wizardSteps.forEach(step => this._steps.push(step));
    this._steps[0].isActive = true;
  }

  private get steps(): Array<WizardStepComponent> {
    return this._steps;
  }

  private get isCompleted(): boolean {
    return this._isCompleted;
  }

  private get activeStep(): WizardStepComponent {
    return this._steps.find(step => step.isActive);
  }

  private set activeStep(step: WizardStepComponent) {
    if (step !== this.activeStep && !step.isDisabled) {
      this.activeStep.isActive = false;
      step.isActive = true;
      this.onStepChanged.emit(step);
    }
  }

  private get activeStepIndex(): number {
    return this._steps.indexOf(this.activeStep);
  }

  private get hasNextStep(): boolean {
    return this.activeStepIndex < this._steps.length - 1;
  }

  private get hasPrevStep(): boolean {
    return this.activeStepIndex > 0;
  }

  goToStep(step: WizardStepComponent) {
    this.activeStep = step;
  }

  next() {
    if (this.hasNextStep) {
      let nextStep: WizardStepComponent = this._steps[this.activeStepIndex + 1];
      this.activeStep.onNext.emit();
      nextStep.isDisabled = false;
      this.activeStep = nextStep;
    }
  }

  previous() {
    if (this.hasPrevStep) {
      let prevStep: WizardStepComponent = this._steps[this.activeStepIndex - 1];
      this.activeStep.onPrev.emit();
      prevStep.isDisabled = false;
      this.activeStep = prevStep;
    }
  }

  complete() {
    this._isCompleted = true;
    this.activeStep.onComplete.emit();
  }

}

  Code coverage
  generated by istanbul at Mon Feb 13 2017 20:11:49 GMT-0600 (CST)

redpajama_set_name: RedPajamaGithub


On Sunday, Democratic presidential candidate Pete Buttigieg — the mayor of South, Bend, Indiana, who would become the first openly gay President if elected — criticized Vice President Mike Pence for his anti-gay rights policies.
As governor of Indiana, Pence, a conservative Christian, signed the Religious Freedom Restoration Act, which critics said allowed businesses to discriminate against LGBTQ people on religious grounds. Pence also has a track record of speaking out against policies that would protect the rights of LGBTQ people. And his wife, Karen Pence, accepted a part-time teaching job this year at a private Christian school that bars LGBTQ students and employees.
Buttigieg came out as gay in an essay for the South Bend Tribune while campaigning for re-election in 2015, when Pence was governor of his state. Buttigieg said Sunday that he had worried about how his community would respond.
Buttigieg, who was re-elected in 2015 by a wide margin, also discussed his sexual identity, his marriage and his faith on Sunday.
If elected, Buttigieg would become the first openly gay president and, at age 37, would also be the youngest.


text:
Dave Flynn
Described as a "Genre-Jumping Genius" (Tradreview.com) Dave Flynn is an award-winning composer/guitarist and founder of the unique Irish Memory Orchestra.
Amanda Feery
Amanda Feery is an Irish composer currently based in the U.S., where she is completing a PhD in Composition at Princeton University.
Hormoz Farhat
Hormoz Farhat was born in Iran.
Ciaran Farrell
Irish composer Ciarán Farrell was born in Dublin, Ireland and graduated from Trinity College Dublin in 1997.
Eibhlís Farrell
Eibhlis Farrell is from Rostrevor in Co. Down and studied composition with Raymond Warren, Charles Wuorinen and Robert Moevs.
David Fennessy
David Fennessy was born in Maynooth, Co Kildare. He began his musical studies at the DIT Conservatory of Music, studying guitar with John Feeley and composition with Eibhlis Farrell.
Howard Ferguson
Howard Ferguson was born in Belfast in 1908. At the Royal College of Music, London, he studied piano with R. O. Morris and conducting with Sir Malcom Sargent and later studied with Harold Samuel.
Sinéad Finegan
Sinéad Finegan completed an M. Phil in Music and Media Technology from Trinity College, Dublin in 2011.
Aloys Fleischmann
Aloys Fleischmann was born in Munich to Irish-based German parents.
Philip Flood
Philip Flood was born in Belfast and, after completing a degree in music at Queen's University, moved to London in 1986 where he studied with Robert Saxton and Simon Emmerson.
Paul Flynn
Paul Flynn is from Lucan, Co. Dublin. In 2000 he graduated with a BA in music from Trinity College Dublin, specialising in composition with Kevin O'Connell and Donnacha Dennehy.
Redmond Friel
Together with T. C.

redpajama_set_name: RedPajamaCommonCrawl


text:
Courrèges names Nicolas Di Felice new Artistic Director
Courrèges has announced the appointment of Nicolas Di Felice as the house's new Artistic Director, effective today.
"I've always dreamed of Courrèges, of its total and enveloping universe. It's a house that means a lot to me and in which I recognize myself. Its simplicity, its clarity, but also its optimism: I am honored to continue to keep alive these values with, I hope, just as much passion and enthusiasm as the founder," said Di Felice in a release.
Of Belgian origin, 37-year-old Di Felice studied at the noted fashion school of Brussels, La Cambre, before then working for Balenciaga, Christian Dior and Louis Vuitton.
The appointment comes four months after the house named Adrien Da Maia to be its new president.
"The nomination of Nicolas as artistic director marks a great day in the history of Courrèges. His vision for the brand, clear and modern, links perfectly with the timeless values of the house," said Da Maia.
Di Felice succeeds Yolanda Zobel, who was only named artistic director in 2018; she followed the design duo of Sébastien Meyer and Arnaud Vaillant, the founders of Indie label Coperni, who were dismissed after a three-year stint in 2018.
The father of French Space Age minimalism, Courrèges is noted for its signature futuristic style and obsession with white. Despite multiple recent travails, it remains an iconic French marque and one with real hidden potential, so Di Felice debut – in March 2021 – will be closely watched.
Founded in 1961 by André and Coqueline Courrèges, the house was acquired at the end of 2017 by Artemis, the key investment arm of the Pinault family. The Pinault's control Kering, the world's second-largest luxury group, which includes such stellar marques as Saint Laurent, Gucci, Alexandre McQueen, and Bottega Veneta.


There was a time, not so long ago, that the term "athleisure" could have gone the way of "normcore"—talked about ad nauseam but then forgotten, destined never to be used in the style lexicon again. But no—while the exalted wearing of all things basic seems to have slipped off the tongue of pop culture, athleisure is here to stay, affecting big brands and retailers in an officially permanent way. Numbers at the end of last year showed Americans spent $1 billion more dollars on apparel and shoes than the year before, and all credit went to active wear.
Koral Activewear tank and Onzie leggings, available at Carbon38.
"It's definitely here to stay," confirmed Elle Strauss, fashion director at Shopbop, an e-commerce giant that currently carries 34 active-wear brands. "Over the past year it's gone from practical workout wear to having more of a street vibe. It's less about performance and more about looking relaxed and cool." Strauss said she first noticed the trend creeping in five years ago and counts fitness brands collaborating with designers as part of the sea change (reference Mary Katrantzou's breathtakingly gorgeous creations for Adidas, plus Stella McCartney's longtime partnership with the brand).
A scad of brands and designers have beefed up their offerings with dedicated fitness categories, including Free People, Net-a-Porter's cheeky Net-a-Sporter, Trina Turk, and Cynthia Rowley, whose passion for surfing meant she started with wetsuits. "After performance and functionality is achieved, designing ready-to-wear and fitness is exactly the same," she told us.
The latest to throw her hat in the ring is Tory Burch, who announced her intentions last spring, saying "I find a lot of women wear what they wear to the gym all day long" (she already has a popular accessory range for Fit Bit). Tory Sport should be with us sometime this year, according to WWD, and will be available online on its own site (torysport.com, which currently is empty) and in a new New York City store. "It's creating another brand. It's being designed in-house and marries function and style. It will be yoga, running, some tennis, and golf," she has said. The Tory team didn't have anything else to add at this time.
Tory Burch's Fit Bit bracelet.
Consider: where Lululemon's initial explosion was met with some snide remarks about it being adopted by shoppers who wanted to appear as if they were gym-bound (while really only running daily errands), the athleisure movement is marked by people who really did log the calorie burn but simply chose to keep their workout togs on because they were so chic.
"As fitness becomes ever more entwined in day-to-day life, women's expectations have changed. It's not good enough for performance pieces to just perform. Today's woman is looking for items that she can blend into her overall wardrobe," Peterson said. And while today's hit-the-gym-hard woman might toss a sweat-soaked shirt post-spin or a run, she's keeping on her leggings and it shows—the category has grown far beyond the plain black crop.
Paparazzi shots mined for style inspiration are just as likely to show stars in workout clothes, like this one of Taylor Swift and Gigi Hadid walking together.
"Workout clothing in general is being mixed into the wardrobe, but it's most apparent in bottoms, and in tights in particular," Peterson said. "It used to be that women searched for a favorite pair of denim jeans. Now it's her favorite pair of leggings." Luxury-priced options from lines like Michi are crazy covetable, spurred by the celebrity supermodels spotted in them, and the idea of shelling out $175 for a pair suddenly doesn't seem quite as laughable (they're currently sold out on Nordstrom and Urban Outfitters' websites). Splits59 offers one of its most stylish pairs for $120.
Carbon38's Caroline Gogolak and Katie Johnson.
That early experience has translated easily into adulthood, with the site's stylish workout pieces replacing ballet school basics. "Ten times out of 10 I find myself reaching for leggings over jeans. They're sexier, more comfortable, and more flattering than denim," Johnson said. When launching their site, the duo worked hard to figure out just who was shopping, and it turned out, she was a lot like them.
"When we started the business, we made a pact to get to know our first 1,000 customers personally," she explained. That meant the women did everything, answering customer service calls, packing orders, and testing product. "The feedback loop was invaluable. The shopper wants performance and style. Every piece has to support her through a marathon and still look great with a blazer and heels." There's no way of knowing how many pieces are worn off the gym floor, but Johnson estimates 90 percent are worn beyond working out.
That figure might be higher than the airtime the majority of our workout duds get, but if things keep going the way they have been, it could become the norm before long.


From the moment that Google mentioned the words 'indexed apps' and 'ranking' in the same sentence in February, it was clear that 2015 was going to be the year of the app (once mobilegeddon had passed, of course).
And it's not just the traditional SEO industry that's benefitting from the new focus on apps and content: new app deep linking methods from Facebook, Twitter and other third-party tools are making it easier to integrate your app content with your other digital marketing efforts.
For the purposes of this guide we have focussed on app deep linking methods that have some sort of web element. All of these methods have their own markup and configuration, so it's worth investigating all of the ones you require before implementing them.
We have provided links throughout the post to more information on implementation, rather than trying to cover all the different methods in one post.
Google App Indexing (more information below): Get app content indexed in search results. Users can either view the content on the app if they have it installed, or install it if not.
Facebook App Links (more information below): Allow users to access content that was shared from your app, using an app alternative to web content, or app-only content.
Twitter App Cards: Create Twitter Cards to deep link to app content or allow users to install the app from a tweet.
Bing App Linking: Show your app and its content in Windows 8.1 Smart Search and Windows Phone search.
Deeplink.me: Build URLs for app content and link them from social media or external sites.
Branch Metrics: Allow users to share deep linked app content, access app content from ads, see app content before they install, see personalized welcome screens and 'text' themselves the app from a desktop device.
Google's App Indexing only works for Google mobile search. If the URL in the search results has a corresponding app version (and the user has the app installed on their device), Google will direct the user to the app page, instead of the web page.
If a deep-linked app is not installed on the user's device, then Google will prompt them to install the app.
Google will eventually crawl app-only content independently from the web version, so at some point you won't need to provide a corresponding page on the website to show up in Google's search results. But at the moment you do.
Use the App Indexing API (Android apps only) to send just the page title and description and prevent Google having to crawl the whole page with web annotations.
App indexing as a Google ranking factor?
There is also an additional ranking boost available if you use the App Indexing API (only available for Android apps). Google's Mariya Moeva has said this is because Google is able to collect additional engagement data (such as usage times and visited pages) to help determine how useful the app is to users.
Android apps can respond to regular HTTP requests and use them to deep link into apps. HTTP scheme is also a requirement for iOS apps, as Google's Mariya Moeva mentioned in a recent Google Webmaster Hangout about App Indexing. Using HTTP scheme, you don't need to markup your site — just set the app to respond to HTTP requests.
Make sure your content is the same on your app and your website, and only include direct content links in your deep linking (don't link to ads or any other page type). App content can be shorter than the web version as Google can recognize it's the same, but the more the app content tallies with the web content, the easier it is to get the content validated.
As with website content, make sure a user lands on the content they were searching for without having to go through a paywall, pop-ups or commercials.
Backend APIs need to be 'unlocked' in your Robots.txt file so Google can crawl the content in your app and check that the content is the same on the app page and web page.
Keep deep links that execute native ARM code out of your annotations, otherwise app deep linking might not work on all platforms.
Use a noindex.xml file to specify URIs that you want to exclude from app deep links, in the same way as you would noindex URLs with a robots noindex tag.
However, you can only exclude using an exact URI (example: uri="http://recipe-app.com/recipes/hidden_uri").
Or just a beginning phrase (example: uriPrefix="recipe-app://hidden_prefix").
These restrictions mean the noindex.xml file is not as powerful as robots.txt and is harder to use with complicated setups.
The website should have a unique URL for every language and there is one corresponding app deep link for each of these URLs.
The website has unique URLs for each language but there is only one corresponding deep link: the app uses the system language to display the content in the user's language.
While it's tempting to promote your app as hard as possible now that there are SEO benefits relating to app indexing, bear in mind that using app install interstitials that get in the way of your web content can also have a negative effect on the search performance of your site as a whole (which could outweigh the ranking benefits of using app indexing in the first place).
Sites that hide too much content with app interstitials are no longer be considered mobile-friendly, which presumably means those sites will no longer have the mobile friendly tag in search results or the mobile-friendly ranking boost.
The mobile-friendly testing tool and the Search Console Mobile Usability report were updated in September to indicate whether a site is hiding too much content with an app install interstitial on the transition from a search result, so it's worth double-checking that there are no issues on your site as soon as possible.
Test whether Google can see your app and view the API response (title, description, API URL, etc) using the Fetch as Google for Apps tool. You can either test an APK version that has been published on Google Play or upload an unpublished APK version.
Verify your Android app in Search Console and Google Play to compare and validate (not to discover) your app and its content. As of September 2015, you can compare how many people clicked on the search result links with the number who clicked on the search result 'install app' button. Verification and data for iOS is not yet available in Search Console.
If your site is included in Google News, add your apps in the Google News Publisher Center in the Mobile Apps section to help Google News direct users to your app content.
Use DeepCrawl's Custom Extraction feature to find and analyze just the URLs that have app deep linking markup applied.
When someone shares content from within an app that has Facebook App Links applied, anyone clicking on that link will be able to access that content through the app. This can be done using an app alternative to existing web content, or app-only content, and works either from 'web to app' or 'app to app'. The feature works with Android, iOS and Windows phones.
If the user clicking on the link does not have the app installed, they will be directed to the app store for their device or a web version of the same page if it's available.
For web content with an app alternative: add the required metadata to your page's <head> tag.
For app-only content: use Facebook's App Links Hosting API to host your App Links.
Set up App Links analytics using Bolt SDK on iOS or Android. Once this is working, you'll be able to view traffic and usage information in Facebook Insights.
Use DeepCrawl's Custom Extraction feature to find URLs that include Facebook App Links meta data and analyze how they behave.
We hope that you've found this post useful in learning more about app indexing.
You can read more about this topic in a Google Webmaster Hangout that covered app indexing.
Additionally, if you're interested in keeping up with Google's latest updates and best practice recommendations then why not loop yourself in to our emails?


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text:
Philonotula es un género de musgos hepáticas de la familia Bartramiaceae. Comprende 10 especies descritas y de estas, solo 8 aceptadas.

Taxonomía
El género fue descrito por Georg Ernst Ludwig Hampe y publicado en Videnskabelige Meddelelser fra Dansk Naturhistorisk Forening i Kjøbenhavn 1877–1878: 258. 1877.  La especie tipo es: Philonotula curta Hampe.

Especies aceptadas
A continuación se brinda un listado de las especies del género Philonotula aceptadas hasta diciembre de 2014, ordenadas alfabéticamente. Para cada una se indica el nombre binomial seguido del autor, abreviado según las convenciones y usos.
 Philonotula flexipes Müll. Hal.
 Philonotula guyabayana Schimp.
 Philonotula ligulatula (Müll. Hal.) Paris
 Philonotula reticulatum (Hook. f. & Wilson) Mitt.
 Philonotula runcinata (Müll. Hal. ex Ångstr.) Besch.
 Philonotula strictiuscula Müll. Hal.
 Philonotula tahitensis (Müll. Hal.) Besch.
 Philonotula vescoana Besch.

Referencias

Bartramiaceae

redpajama_set_name: RedPajamaWikipedia


In its judgement of 17 September 2015, the Court of Justice (9th Chamber) has further specified how to interpret Art. 5 (3) of Regulation (EC) No. 261/2004 (see my article of 08 December 2014 in this column).
In the case the Court of Justice has now decided upon, a passenger had requested compensation payments for a delayed flight from Ecuador to Amsterdam. According to the airline, the delay was due to an unexpected technical problem with the fuel pump and a hydro-mechanical unit which was not attributable to defective maintenance and which was not detected during regular maintenance.
The Court of Justice had to decide whether a technical problem which occurred unexpectedly falls within the definition of "extraordinary circumstances" within the meaning of Art. 5 (3) of Regulation No. 261/2004. With reference to recital 14 in that regulation, it pointed out that a problem may be classified as extraordinary only if it relates to an event which is not inherent in the normal exercise of the activity of the air carrier concerned and is beyond the actual control of that carrier on account of its nature or origin.
However, since the functioning of aircraft inevitably gives rise to technical problems, these cannot be classified regularly as "extraordinary". The event at hand might have been unforeseen, however it does not fall within the definition of "extraordinary circumstances" within the meaning of Art. 5 (3) of Regulation No. 261/2004.
Therefore, it is irrelevant whether the air carrier could have adopted reasonable measures to deal with the event.
Art. 5 (3) of Regulation No. 261/2004 shall be interpreted in that a technical problem which occurred unexpectedly cannot fall within the definition of "extraordinary circumstances" even if it is not attributable to defective maintenance and was not detected during regular maintenance.


text:
Georgia Football Player Devin Willock, Staff Member Chandler LeCroy Killed in Car Crash
by Tony Gray
Days after the University of Georgia football team won their second consecutive national championship, two members died in a car accident.
Recruiting team member Chandler LeCroy, 24, was the driver of an SUV with offensive lineman Devin Willock, 20, and two other UGA team members leaving a championship celebration Sunday morning.
Athens-Clarke County Police Department officers responded to a single vehicle crash on Barnett Shoals Road around 2:45 a.m., according to a report in The Atlanta Journal-Constitution. Responding officers determined the 2021 Ford Expedition driven by LeCroy was traveling south when the vehicle veered from the outside lane of the road, striking two power poles and several trees before coming to a stop.
Willock reportedly died at the scene as a result of his injuries. LeCroy, 24, was transported to a nearby hospital, where she died from her injuries.
Two other University of Georgia football team members in the vehicle were injured and reported to be in stable condition, according to a statement from the UGA Athletic Association.
"We are all heartbroken and devastated with the loss of Devin Willock and Chandler LeCroy," said head football coach Kirby Smart in a statement. "Devin was an outstanding young man in every way and was always smiling. He was a great teammate and a joy to coach.
"Chandler was a valuable member of our football staff and brought an incredible attitude and energy every single day."
"Devin and Chandler were two special people who meant so much to the University of Georgia, our football program and our athletic department," athetic director Josh Brooks said in a statement. "We ask that everyone keep their families in your prayers during this very difficult time."
The Journal-Constitution noted numerous reports identifying Warren McClendon and Tory Bowles as the surviving passengers of the crash. University of Georgia officials did not confirm their identities, though, the report added.
McClendon is a junior offensive tackle. He just declared for the NFL draft Saturday, the report continued.
Ms. Bowles was reportedly posting photos of recruiting prospects celebrating with the Bulldogs in their locker room Saturday.


The 3 Gunas (qualities / attitudes) in yoga consist of Sattvic, Rajasic and Tamasic.
Sattvic – qualities such as goodness, pure, positive, etc.
Rajasic – qualities such as stimulating, driven, dynamic, etc.
Tamasic – qualities such as dull, inactive, lethargy, violent, ignorant, etc.
I will just share a reflection of how I draw connections of these to some experiences at certain stages of my life.
We have moments where we are generally positive about work, school and life in general (Sattvic). I feel positive about my work as a Primary School teacher. I relish the impact I have on the children under my charge and the opportunity for meaningful interactions on the most minute of details. Since completing my teacher training over 10 years ago, positive experiences at work influenced my decision to undertake leadership roles as a subject head for co-curricular activities in the school I was with with. While work load might have increased significantly in some areas, the fact that I was in a better position to impact students' school experience beyond of academic platforms kept me going.
Later, I decided to take up appointment as a head of department of character education, which oversees the character education and non-academic programmes, leadership and holistic development of students within the school. With the increased responsibility, I had less personal time and it got to a stage where I began questioning my reasons for going to work daily. I saw less relevance in the meaning and nature of my daily activities at work which affected my time for quality interactions with my students. I felt my life had taken a really toxic turn (Tamasic) as it began affecting my sleep patterns, perceptions of life and generally positive demeanor.
As I arrived at a crossroad of my career, I made the decision to take a step back by relinquishing my appointment and to return to the ground as a Happy Ordinary PE teacher (HOPE). As part of my efforts to rejuvenate myself, I took a study break from work and embarked on a MSc in Exercise & Sport Studies programme. While upgrading myself in terms of professional knowledge, it served to renew my sense of purpose as an educator (Rajasic) at the same time.
The ups and downs I had experienced has brought about a better understanding that our individual lives consist of elements of each of these three qualities, with some more prominent than others at various point of our lives.