Intertidal macroalgae are exposed to many abiotic stress factors, and they must regularly react to changes in their environment. We used RNA-seq to describe how Porphyra umbilicalis (Rhodophyta) changes gene expression patterns to interact with different habitats. Tissue samples were taken from a typical habitat along the open-coast of the Northwest Atlantic, as well as from a rare, atypical habitat in an estuarine tidal rapid environment. Differential gene expression analyses suggest that pathogic bacteria and viruses may be a significant factor influencing the transcriptome in the human-impacted estuarine environment, but the atypical habitat does not necessarily induce more stress in Porphyra umbilicalis growing there. We found genes related to nitrogen transport are over-expressed in tissue from the open-coastal site compared to those from the estuarine site, where environmental N levels approach hypertrophic levels. Low N levels impede growth, but high levels are toxic to cells, and we use qPCR to show this species regulates expression of a putative high-affinity NH4+ transporter under low and high N conditions. Differences in expression of this transporter in these habitats appear to be inherited from parent to offspring and have general implications for adaptation to habitat in other species that are capable of asexual reproduction, as well as more specific implications for this species' use in aquaculture.
Abiotic stress is a nearly universal factor affecting photosynthetic eukaryotes. Some organisms deal with stressful environmental changes better than others, but few are capable of coping with variable environmental conditions better than intertidal algae. Porphyra umbilicalis Kützing is a species of marine red algae of the North Atlantic that is known for its tolerance of desiccation (Smith and Berry 1986), rapid osmotic changes (Reed et al. 1980), and subzero temperatures (Wang et al. 2011, Green and Neefus 2014). Some of this tolerance is due to cell structure and anatomy, but the physiological mechanisms by which they respond to abiotic stress are not well understood. As a candidate for potential use in integrated multitrophic aquaculture (IMTA) to remove NH4+ produced from fish effluent (Blouin et al. 2007, Kim et al. 2007, Day et al. 2009, Green and Neefus 2016), and as a potential model organism for Rhodophyta (Gantt et al. 2010, Blouin et al. 2011), a better understanding of the effect of environment on gene expression by which it copes with stress and N uptake is necessary.
Porphyra umbilicalis typically lives on rocky substrates in the high intertidal zone, but it can also be found in estuarine tidal rapid locations on bridge pilings such as in the mouth of the Great Bay Estuary system in Dover Point, NH, USA. (West et al. 2005, Eriksen et al. 2016). Estuarine rapids are rare in the Gulf of Maine, and experience high velocity currents and changes in salinity and temperature with tidal changes. Eriksen et al. (2016) identified six Porphyra umbilicalis genotypes among populations in the Gulf of Maine based on a limited set of polymorphic microsatellite loci but showed isolation by distance is a more important factor influencing population structure than selection for habitat. This suggests the populations acclimate to different stress conditions in different habitats through physiological adjustments.
Mathieson et al. (1981b, 1981a) compared the macroalgal species composition and environmental conditions at Dover Point (DP) and a fully marine location nearby at Fort Stark (FS), located on Jaffrey Point in New Castle, NH, USA. They describe a greater range in salinities (22–31 ppt DP; 26–32 ppt FS) and water temperature (1–22 °C DP; 1–20 °C FS) at DP. Over the last 30 years, the Great Bay Estuary System and DP has experienced increasingly high N levels, largely due to discharge from sewer treatment plants in surrounding towns, and non-point sources including run-off from increasing amounts of impervious pavement and development (PREP 2013, Trowbridge et al. 2014). Nitrogen levels from 2008 to 2010 measured at nearby Cedar Point by Nettleton (2012) were classified as hypertrophic and ranged from 5.03–57.98 μM total N. In comparison, NH4+ levels at FS collected from 1973 to 1974 by Norall and Mathieson (1974) ranged from approximately 0–0.08 μM NH4+, while NO3− concentrations ranged from 0.02 to 0.23 μM. Ammonium nitrogen levels in the vicinity of DP at that time ranged from 0.06 to 0.65 μM, and NO3− concentrations ranged from 0 to 0.02 μM (Norall and Mathieson 1974).
We used RNA-seq to identify differentially expressed (DE) genes within and among populations growing in these habitats at DP and FS, NH, USA. This sampling allows us to compare gene expression between two genotypes in a single habitat (FS), but also to compare gene expression among different habitats (DP and FS) using P. umbilicalis tissue with the same genotype but also with different genotypes. Our goal was to compare gene expression patterns among libraries to describe the extent to which environment and genotype affect gene expression, to determine if one environment elicits a greater stress response, or N metabolism response than the other, and if so, to begin to describe the physiological mechanisms by which Porphyra umbilicalis copes with stress. The results shed light on how this organism interacts with its environment in atypical habitats and has implications for N uptake in IMTA systems.
We collected tissue during high tide from two locations at Fort Stark, Jaffrey Point, New Hampshire (43° 03′ 28.3″ N 70° 42′ 41.7″ W) and from the U.S.-4/N.H.-16 bridge pilings at Dover Point, New Hampshire (43° 07′ 11.5″ N 70° 49′ 38.5″ W), mid-day in late July and early August 2012 (Fig. 1). At FS, whole thalli were removed from rocks within a 3 m radius at two locations while submersed or partially submersed. The thalli were patted dry, visible epiphytes and amphipods were carefully removed, and the tissue was flash frozen in liquid N in the field. Tissue from DP was collected during high tide from a single bridge piling where they were known to occur, inspected for preliminary species identification, cleaned of visible epiphytes, and then patted dry and frozen in liquid N in the field. We made no effort to eliminate microscopic epiphytes or endophytes, nor bacterial contaminants on or within the tissue that was demonstrated to be abundant in an earlier study by Miranda et al. (2013). A small amount of each thallus from each population was put aside while in the field in a 1.8-mL cryogenic tube for species confirmation and genotyping. Samples were returned to the lab and stored at − 80 °C until processing.
We extracted DNA from the small samples set aside for genotyping in a 96-well plate format using a modification of the method by Page (2010) described in Eriksen et al. (2016). Species identification was confirmed using restriction fragment length polymorphisms (RFLP) as described by Teasdale et al. (2002).
Tissue from each population was genotyped at three polymorphic microsatellite loci according to Eriksen et al. (2016). The thallus samples were then pooled into libraries based on genotype as genotype 3 (g3) or genotype 6 (g6) (Table 1); no individuals from DP were assayed as g3. The number of individuals collected was relatively low due to small population size. The pools of tissue were ground to a fine powder in a mortar and pestle with liquid N. We extracted the total nucleic acids from the tissue as described above and in Eriksen et al. (2016). Following extraction, the total nucleic acids were treated with RNase-free DNase (1500 Kunitz units, Qiagen, Germantown, MD, USA) per the manufacturer's instructions at the maximum volume. DNase was removed with equal volumes of 24:1 chloroform to isoamyl alcohol and centrifuged at 2440×g for 5 min at 4 °C. The supernatant was pipetted off, and 1/10 volumes of 3 M sodium acetate was added. The RNA was then precipitated with two volumes of cold 100% ethanol at − 20 °C for 1 h. The tubes were subsequently centrifuged at 12,000×g for 10 min to pellet the RNA, which was then washed with 500 μL cold 70% ethanol twice before being re-pelleted by centrifugation at 12,000×g for 2 min. The quality and quantity of RNA was assessed by evaluating concentration and A260/A280 and A260/A230 ratios on a NanoDrop 2000c spectrophotometer (ThermoFisher Scientific, Grand Island, NY, USA). Integrity of the RNA samples was measured on an Agilent 2100 Bioanalyzer at the Hubbard Center for Genomic Studies at the University of New Hampshire, Durham, USA; RNA integrity numbers (RIN) were high and ranged from 8.8 for the DP pool, to 9.2 for the FS g6 pool.
Library preparation by polyA cDNA isolation and sequencing on a HiSeq2000 platform was performed by Otogenetics Corporation (Norcross, GA, USA). Reads with adaptor contamination and low support bases were filtered out by the bioinformatics core at Otogenetics. The sequences were then mapped to the reference library Porphyra umbilicalis non-redundant EST contigs (assembly B, available at http://porphyra.rutgers.edu/ downloaded February, 2013; Chan et al. 2012a) using CLC Workbench; all sequences that did not map to the reference library were treated as contaminant sequence and eliminated from the dataset. Therefore, the results represent expression of Porphyra genes rather than expression of the meta-transcriptome to the extent that the EST database was culled of contaminant sequences (Chan et al. 2012a). Differential expression analysis was conducted in CLC Workbench, using RPKM (reads per kb per million reads) values to evaluate gene expression levels, and Kal's Z test to evaluate significance. Contigs with at least a twofold difference in expression and a significance level less than a Bonferroni-adjusted value of α were considered DE among the libraries.
The DE sequences were submitted to the KEGG Automated Annotation Server (KAAS) under default parameters for annotation. We used BLAST2GO to conduct BLASTX searches with a less stringent evalue cutoff of 1e−3 against the NCBI non-redundant database for sequence and pathway annotation (Conesa et al. 2005; Conesa and Götz 2008; Götz et al. 2008). Mapping and Interpro Scan analyses were conducted with BLAST2GO default values. Analysis of Gene Ontology (GO) annotations was done in WEGO (Ye et al. 2006). GO enrichment analysis was conducted in R v. 3.2.3 using the package topGO (Alexa and Rahnenfuhrer 2010). Heat maps for expression levels were created using the R package gplots (Warnes et al. 2016).
The expression levels of one DE contig (P_umbilicalis_esContig4469) that was annotated as an amt was evaluated by qPCR under low and high N conditions in tissue from multiple populations maintained in culture under common garden conditions. Tissue was sampled from the field at FS and DP and assayed as g3. Neutral spores were harvested from multiple individuals and grown to size in culture from spores according to Redmond et al. (2014). Cultures were maintained in 1–2-L Erlenmeyer flasks containing modified Von Stosch enriched (VSE) seawater (Ott 1966) with NH4Cl serving as the N source at 5 mM concentration. Salinity was measured weekly and adjusted to 30 ppt. Constant aeration was applied, ambient temperatures were maintained at 15 °C, and tissue was kept under a constant day neutral photoperiod (12:12 light to dark) at a 30-s average value of 30 μmol photons m−2 s−1.
Pieces of tissue between 0.2–0.3 g (fresh weight, patted dry) from three different thalli from each population were placed in 250-mL flasks containing either sterile seawater for low N conditions (< 5 μM NH4+), and an equal amount of tissue from the same three thalli was placed in Von Stosch enriched (VSE) seawater under high N conditions (5 mM NH4+). Different pieces of tissue from the same thallus were thus evaluated under both low and high N conditions. The samples were left to acclimate to the N conditions for 10–14 days under the culture conditions described above. Von Stosch enriched water was replaced in high N experimental treatments every 2 days to ensure constant high N conditions; however, the unenriched sterile seawater was not replaced to allow the N to become depleted.
Following the acclimation period, the tissue was removed from the culture medium, patted dry, and immediately frozen in liquid nitrogen. The RNA was extracted via the methods described above and assessed for quality using a NanoDrop 2000c spectrophotometer. We used the GoTaq® Probe 2-step RT-qPCR system (Promega, Madison, WI, USA) to prepare the cDNA.
The transcription elongation factor 1 (ElF1) is among the optimal reference genes tested for comparative gene expression analysis related to stress tolerance and development in Pyropia yezoensis (Kong et al. 2015). We identified contig P_umbilicalis_esisotig06418 as a putative ElF1, containing a putative zinc-binding domain with four conserved cysteine residues and an elongation transcription factor 1-like domain (pfam05129). For our reference gene, we designed primers to amplify a region of this contig (5′-CGT CGA CCC AGT TGC TAT AC-3′, and 5′-CTG CGA ACA CGA GCA GT-3′). Sequences acquired from tissue using these primers were a reciprocal best BLASTx hit with 97% identity and an evalue of 7e−35 to a transcription elongation factor from P. yezoensis (Uji et al. 2010).
The primers for the putative Porphyra umbilicalis amt target gene were designed to amplify a 128-bp region of P_umbilicalis_esContig4469. The forward primer (5′-GCG TAC TAC TTG TTC GGG TAT G-3′) was designed to span an exon-exon boundary, over an intron that was discovered in contig P_umbilicalis_esContig961 between base 210 and 360 (Eriksen 2014). Quantitative PCR was conducted in triplicate for each sample using a Roche LightCycler 2.0 and 20 μL reactions containing 1X GoTaq® Probe qPCR Master Mix (Promega), 100 nM reference gene primers (above), and 250 nM reference gene probe (5′-/56-FAM/CAG GGC GGT/ZEN/GAT GGT CAT CTC ATG/3IABkFQ/-3′; Integrated DNA Technologies), 900 nM of the target gene forward primer and reverse primer (5′-GCA AAC GTC CAC TGA AAG AAC-3′), 250 nM of the probe (5′-/5HEX/TCG GAC GAC/ZEN/ACG CAA GAG TTT CAT/3IABkFQ/-3′), and 16.5–39.5 ng prepared cDNA. Thermal cycling conditions included a one-time, 2 min hot start at 95 °C, then a cycle consisting of a denaturation step of 15 s at 95 °C, an annealing step at 55 °C for 10 s, and an extension step at 60 °C for 30 s which was repeated for 55 times, and followed by a single cooling step at 40 °C for 30 s.
Relative differential expression of the target amt gene was assessed using the calculations described by Pfaffl (2012) against expression of ElF1 as the reference gene. The normalizer sample was cDNA from total RNA extracted from cultured Schoodic Point tissue growing under standard culture conditions as described above. We also sampled tissue under high and low N conditions for cultured tissue from Schoodic Point (g1) and Quoddy Head (g4), ME as outgroup samples (Fig. 1).
Statistical analyses of expression were done in IBM SPSS v. 21. We used a two-way ANOVA to determine the effects of population and N level on relative expression. The full dataset was log transformed according to Zar (1999) in order to meet the homogeneity of variance and normality assumptions of the two-way ANOVA. Simple main effects with a Bonferroni adjustment were used to determine which populations had significant differences in gene expression.
There were 299 contigs with at least a twofold significant difference in expression when the two FS libraries were compared (FSg3 × FSg6) (Table 2). The KEGG Automated Annotation Server (KAAS) annotated relatively few of the contigs that were DE among the two genotypes from the same environment, and assigned them to 59 different pathways (Table 3). BLAST2GO found significant hits for more contigs (Table 4) and assigned GO annotations to about 92% of the DE contigs.
There were more contigs DE among the FS g3 (FSg3) and DP (DPg3) libraries than among the two FS libraries (Table 2). More of the DE contigs were annotated by KAAS (Table 3) and BLAST2GO (Table 4); contigs were assigned to nearly three times as many pathways. Compared to the FSg3 × FSg6 comparison, there was also a greater abundance of DE putative Porphyra contigs associated with biosynthesis of secondary metabolites, plant-microbe-interaction pathways such as "infection by Vibrio cholerae," and various plant antibiotic biosynthesis pathways (Table 5).
The greatest number of DE contigs was found in the comparison between the FS g6 (FSg6) and DPg3 libraries (Table 2). However, fewer of the DE contigs were annotated by KAAS (Table 3) than in the comparison between the FSg3 × DPg3 libraries. Similar to the comparison of FSg3 × DPg3, there was also an abundance of DE contigs associated with plant-microbe-interaction pathways (Table 5). BLAST2GO found significant hits for only about 60% of DE contigs (Table 4).
Differentially expressed candidate genes related to salinity and temperature stress.
Among contigs annotated by the BLAST2GO, 499 were identified as putatively coding for enzymes related to oxidation-reduction process (GO:0055114). Hierarchical clustering of expression patterns breaks these contigs into two groups (Fig. 2). In one group, contigs are highly upregulated in FSg6 and significantly downregulated in DPg3 libraries; in the other group, contigs are downregulated in both FS libraries and upregulated in DPg3. Genes involved in ROS control such as l-ascorbate peroxidase (APX) and catalase (CAT) were found in both groups, and expression levels had no clear correlation to habitat. Two contigs were identified as putative manganese superoxide dismutases (Mn-SOD), and both were over-expressed in the DPg3 library (Fig. 2). Fifteen contigs were annotated to GO:0009408, response to heat, but there is no clear upregulation of all heat shock proteins in any library (Fig. 3). Only one homolog of the kinases involved in the mitogen-activated protein kinase (MAPK) signaling cascade involved in stress signaling in green plants was found among the libraries, and there was no clear upregulation of these contigs among the libraries (Additional files 1 and 2). Calmodulins, Ca2+-dependent protein kinases, and other genes involved in calcium signaling in response to osmotic stress in green plants are not among the contigs found in these libraries.
There are 16 contigs found in all libraries that were annotated as GO:0006807, nitrogen compound metabolic process. Three contigs are annotated as NH4+ transporters (AMT), one is annotated as a nitrate transporter, and three are annotated as nitrate reductase (NR) (Fig. 4).
The transporters are consistently over-expressed in the FSg6 library and under-expressed in the DP library. Two contigs annotated as putative NH4+ transporters are over-expressed in FS tissue compared to DP tissue in comparisons of FSg3 × DPg3 and FSg6 × DPg3 libraries. The contig P_umbilicalis_esContig4469 annotated also as a putative ammonium transporter and was over-expressed in both FS libraries by over eightfold compared to the DPg3 library. Of the contigs annotated as putative nitrate reductases, two are over-expressed in FSg6, and the third is over-expressed in the DP library.
All populations experienced significantly higher relative expression of a putative NH4+ transporter P_umbilicalis_esContig4469 (hereafter NH4-4469) gene under low N conditions than high N conditions (Fig. 5). Individuals from FS experienced the greatest difference in relative expression under high and low N conditions (mean difference in relative expression = 0.65 ± 0.21 SD; F(1, 70) = 98.72, P < 0.001); individuals from DP also experienced a difference in relative expression to a lesser extent that was still significant (mean difference relative expression = 0.2 ± 0.21 SD; F(1,70) = 6.139, P = 0.003).
Under low N conditions, there was a significant difference among populations in relative expression of this gene (F(3, 70) = 16.307, P < 0.001, partial η2 = 0.411). FS tissue had significantly higher relative expression rates than DP and Quoddy Head (P < 0.001 for both). The difference in relative expression under low N conditions for tissue from FS and Schoodic Point was not significant at a Bonferroni-adjusted alpha = 0.017 (mean difference relative expression 0.24, SE = 0.08, P = 0.02).
Under high N conditions, relative expression of this putative NH4+ transporter was low in all populations. There were no significant differences in relative expression patterns among populations under high N conditions (F(3, 70) = 1.856, P = 0.145, partial η2 = 0.074).
We looked at differential expression patterns in Porphyra umbilicalis growing in a typical open-coastal habitat and a rare, atypical habitat in an estuarine tidal rapid habitat. Our goal was to understand how this species adjusts gene expression to acclimate to these different environments, and to understand N metabolism in this species that is a candidate for NH4+ removal in IMTA systems. We find evidence that environment has a greater effect on gene expression differences than genotype alone, though there is a cumulative effect of environment and genotype on gene expression. There is no evidence that one environment elicits a higher stress response than another, but the high N load at DP has a significant effect on N transporter activity that appears to be trans-generationally inherited.
The comparison of differential expression among libraries represents a breakdown of some of the factors affecting gene regulation. The comparison of expression patterns among FSg3 and FSg6 libraries represents differences due to genotype within the same environment, if we assume the micro-habitats of the two collection locations are roughly similar. Relative to the other comparisons among libraries, this comparison yielded the fewest DE contigs and the fewest annotations. The list of DE genes were enriched for innocuous terms related to cellular parts, and do not include terms that may be related to cellular stress such as oxidation-reduction process, phosphorylation, or protein ubiquitination (Table 3). Genotypic differences among these samples may thus be interpreted as a minor factor influencing expression levels compared to the effects of environment as illustrated in the other library comparisons (Table 2), and a factor that largely influences the expression of genes of unknown function, as only 21 and 41% of the DE contigs were annotated by KAAS and BLAST2GO, respectively (Tables 3 and 4).
The comparison of expression patterns among FSg3 and DPg3 libraries may represent differences due to environment only, because the tissue collected from DP was assayed as the same genotype as the FS material. These genotypes are only the same to the limited extent which the three polymorphic EST-SSR loci described in Eriksen et al. (2016) can identify genotypes, but these markers represented the most accurate way to identify genotypes available at the time of study. This comparison yielded many more DE contigs than the FSg3 × FSg6 comparison (Table 2), and more with known functions (Tables 3 and 4). There was a higher proportion of DE genes with GO terms associated with immune system processes and viral reproduction. The difference in expression levels of immune system processes and viral reproduction among comparisons of environment is also reflected in KAAS data, which annotated more contigs attributed to plant-microbe-interactions, such as viral infection, Vibrio cholerae infection, and plant-pathogen interactions (Table 5). This suggests that the heavily human-impacted estuarine environment harbors more pathogens that macroalgae populations must contend with when living there. Seaweed has been shown to act as a reservoir for various pathogenic Vibrio populations (Mahmud et al. 2007, Mahmud et al. 2008, Gonzalez et al. 2014, Martinez and Padilla 2016).
The comparison of patterns among FSg6 and DPg3 libraries represents the interactive effect of genotype and environment on expression. It contains the most DE contigs (Table 2), but fewer were of known function (Tables 3 and 4). It more closely resembles the FSg3 × DPg3 comparison in terms of the abundance of pathogen and infection-related contigs (Table 5). For some contigs (such as those associated with GO terms related to the establishment of localization, molecular transducer activity, and transporter activity), there appears to be a cumulative effect of environment and genotype on expression of the pathway.
The environment thus appears to be the largest factor influencing differential expression among the libraries, and environment-by-genotype interactions also appear to be significant. The effects of pathogens and microbes appear to have a significant effect on expression levels of putative Porphyra genes via upregulation of genes involved in infection pathways, plant-pathogen interaction pathways, and various antibiotic synthesis pathways in the DPg3 library.
Temperature and salinity conditions are two environmental factors that populations in these habitats may experience differently, possibly leading to a stress response. Populations under temperature and salinity stress should have elevated levels of expression of genes encoding antioxidant proteins (Dring 2005). Sampath-Wiley et al. (2008) found elevated levels of stress-related antioxidant enzymes such as glutathione reductase (GTR) and CAT in emersed Porphyra umbilicalis tissue found in a typical open-coastal habitat under high temperature stress. Using microarrays to evaluate expression patterns of Chondrus crispus, Collén et al. (2007) found that genes for antioxidant proteins such as APX, CAT, dehydroascorbate reductase, glutathione peroxidase, GTR, methionine sulfoxide reductase, manganese superoxide dismutase, and thioredoxin were downregulated in hypo-osmotic conditions compared to the non-stress control. A different study found upregulation of APX in Gracilaria changii exposed to hypo-osmotic conditions compared to the control exposed to 30 ppt salinity (Teo et al. 2009).
In the RNA-seq libraries analyzed in this study, there was no consistent pattern in expression of ROS contigs; some were upregulated in FS libraries, and others were upregulated in DP libraries (Fig. 2, Additional files 1 and 2). Contigs that are annotated as phosphorylation genes that are involved in stress signaling pathways in green plants, such as the mitogen-activated kinases (MAPK/MAPKK/MAPKKK, though only MAPK appears to exist in P. umbilicalis), also show no consistent pattern (Additional file 2). Heat shock proteins show no consistent pattern (Fig. 3). Based on these data, there is thus no clear evidence to suggest one or the other population is under more stress during submersed conditions in these habitats. Porphyra umbilicalis has non-rigid cell walls made of mannan and xylan (Lobban and Harrison 1997) and porphyran (Morrice et al. 1984) that allow the cell wall to expand and contract along with the plasma membrane under different osmotic conditions. The difference in salinity at the time of sampling was small and likely not outside of the range which P. umbilicalis is capable of coping with anatomical adjustments. Further work will be necessary to investigate specific hypotheses about the physiological response of this species to abiotic stress.
Nitrogen (N) is an essential and often a limiting nutrient for macroalgae; it plays an important role in construction of amino acids and pigments such as chlorophyll and phycobilins (Reed 1990). Low nutrient stress may exacerbate damage to the photosystems via other abiotic stress factors and lead to ROS production by slowing the electron transport chain, but high levels of N cause the intermediaries of N assimilation (NO2− and NH4+) to accumulate to toxic levels if the N assimilation process is not properly regulated within the cell (Taiz and Zeiger 2010; Raven 1980).
Nitrogen metabolism is thus tightly regulated by the cell, and the first step of regulation is intake. In barley, high-affinity NH4+ transport systems are active at low concentrations of NH4+, but they are downregulated when internal N concentrations reach sufficient levels (Glass et al. 2001). In the Porphyra umbilicalis EST database, Chan et al. (2012a) found seven contigs that code for putative NH4+ transporters (Chan et al. 2012b); thus, it seems reasonable to assume that transporters with different affinities for NH4+ may be present within the Porphyra genome as well.
Nitrogen uptake appears to be downregulated in DP tissue based on RNA-seq data (Fig. 4). Two contigs annotated as putative ammonium transporters (amts) and another annotated as a nitrate transporter were under-expressed in DP tissue. The contig P_umbilicalis_esContig4469 (NH4-4469) is identified by BLAST searches as a high-affinity transporter, and qPCR confirmed decreased expression of this gene under high N conditions, and increased expression under low N which is consistent with high-affinity transporters (Fig. 5).
The difference in expression patterns from the RNA-seq data of amt NH4-4469 (high expression in FS tissue and lower expression in DP tissue) is predicted based on environmental N levels; however, the difference persisted in tissue grown under similar N conditions in culture. Tissue grown from neutral spores from field collected tissue from FS and DP and maintained in culture under the same high NH4+ conditions (5 mM) maintained significant differences in expression patterns of this amt when later exposed to low N conditions (Fig. 5). Tissue grown from neutral spores from parental tissue collected at FS had significantly higher expression of this putative high-affinity amt under low N conditions than tissue grown from spores from DP. Similarly, tissue grown from FS spores had significantly higher expression levels of this amt gene than outgroup tissue grown from Quoddy Head spores, which is also exposed to atypically high environmental N levels (Garside and Garside 2004) similar to DP.
The finding suggests tissue from FS is genetically predisposed to thrive under lower N conditions by upregulating expression of a gene necessary to take in more NH4+ when it is scarce; or vice versa, that tissue from DP is genetically predisposed to thrive under potentially toxic loads of N. No sequence polymorphism among genotypes was found within a 465-bp region containing both coding and intron sequence of this gene using Sanger sequencing (Eriksen 2014), though upstream sequence data were not available. Heritability of expression patterns from parent to offspring encompasses the field of epigenetics, but the mechanism by which that information is passed through generations when the offspring are generated through sexual reproduction is complicated by erasure of genomic imprints during gametogenesis and embryogenesis (Gibson and Weir 2005); however, the asexual life cycle of the populations of Porphyra umbilicalis in this study theoretically could allow for simple transmission of environmentally induced epigenetic signals. Environmentally induced methylation changes are heritable in apomictic dandelions (Verhoeven et al. 2010) and has been shown to be an important mechanism of acclimation and adaptation in dandelion range expansion (Verhoeven and Preite 2014, Preite et al. 2015). Adaptation to environment by epigenetic signals may explain why previous studies using EST-SSR markers found no evidence of genetic structure in populations in open-coastal and estuarine environments in the Gulf of Maine (Eriksen et al. 2016). While the data are merely suggestive at this point, further exploration of polymorphism within the regulatory regions, or epigenetic signals in the regulatory region of this amt gene may be warranted.
Our results have implications for use of Porphyra umbilicalis in integrated multitrophic aquaculture (IMTA). The species has been suggested as a possible partner in IMTA systems (Blouin et al. 2007); however, the levels at which fish experience nitrogen toxicity in aquaculture systems is relatively low (Randall and Tsui 2002) compared to the NH4+ concentrations at which P. umbilicalis thrives in culture. Populations of P. umbilicalis representing different asexual lineages may have different genetically controlled abilities to take up NH4+ from fish effluent at lower environmental concentrations, depending on the expression of high-affinity NH4+ transporters such as NH4-4469. Researchers seeking to identify lineages with an increased ability to take up NH4+ from aquaculture systems might look for populations growing in low-nitrogen habitats such as open-coastal sites away from human influences.
Drs. W. Kelley Thomas, Leland Jahnke, Arthur Mathieson, and Chris Neefus provided invaluable input and advice. Greg Smith and Anna helped with collecting, and Dr. Andrew Page and Shay Polsky provided technical assistance with a ThermoFisher Scientific NanoDrop 2000c. Jobriah Anderson at the HCGS performed all genotyping and Sanger sequencing, as well as helped with the Agilent Bioanalyzer. Dr. Kazu Okamoto of the HCGS performed the differential expression analysis. We also thank Drs. Kirk Broders and Franz Lichtner for the technical assistance with the Roche Light Cycler 2.0.
This work was supported by the Leslie S. Hubbard Marine Program Endowment to Support Marine Research Development/Equipment. Partial funding was provided by the New Hampshire Agricultural Experiment Station. This is Scientific Contribution Number 2784. This work was supported by the USDA National Institute of Food and Agriculture Hatch Project 0222799.
The dataset supporting the conclusions of this article as well as Additional files are available in the figshare.com repository, https://doi.org/10.6084/m9.figshare.5467027 and https://figshare.com/s/2c5b2cf2155b4562f469.
RLE designed and implemented all experiments, analyzed the data, and wrote the manuscript. ASK contributed to the experimental design and provided funding and manuscript comments. Both authors read and approved the final manuscript.
The samples at Schoodic Point, Acadia National Park, were collected under permit number #ACAD-2012-SCI-0013 issued to Dr. Jessica Muhlin from April 16, 2012, to April 25, 2012.
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Sylvie Binda elected on the Pharmabiotic Research Institute board of directors
Sylvie Binda, vice-president research and development at Lallemand Health Solutions has been elected as a new member of the board of directors of Pharmabiotic Research Institute (PRI), as announced during the annual general assembly and member event, in Rotterdam, the Netherlands.
Lallemand Health Solutions is one of the founding members of the PRI, a non-profit organization created in 2010, aiming at clarifying the specific scientific and regulatory requirements of Microbiotic Medicinal Products in Europe. Over the years, the PRI has gathered an unmatched expertise around the development and registration of therapeutic and diagnostic products emerging from microbiome science. This expertise is shared with the members in a collaborative approach. In addition, the PRI is recognized as the expert of microbiome regulatory science by stakeholders and involved in discussions on the development of new standards in this field.
"It's an honor for me and a great mark of trust to have been elected by the Pharmabiotic Research Institute members to sit on the board of directors and I will do my utmost to uphold and contribute to the mission and the vision of the organization," confirms Mrs. Binda.
ABOUT PHARMABIOTIC RESEARCH INSTITUTE
PRI counts more than 100 member organizations, representing various expertise and interests in the microbiome development field, from early-stage academic researchers all the way to contract manufacturers and commercialization.
ABOUT LALLEMAND HEALTH SOLUTIONS
Lallemand Health Solutions produces probiotics for the dietary supplements, pharmaceutical, food, and nutricosmetic industries. With its manufacturing processes adhering to the highest quality standards and its ambitious research program, Lallemand Health Solutions provides probiotic strains that help millions of people improve their health and well-being.
Marilou Luneau, Content Manager
mluneau@lallemand.com

redpajama_set_name: RedPajamaCommonCrawl


We were finally able to add some new features to the client's billing backend (www.maiahost.com/clients).
Added new functionality to the "Hosting Account" page. Now you are able to view the primary domain name on that account as well as a list of the "additional hosting features" you might have added on. Those are: Dedicated IP Address (Yes, No) $24 per year; Additional Domain Hosting Slots (Num) $12 per year or $1 per month; Additional Storage Space (Num GBs) $12 per GB per year or $1 per gb per month; Additional Bandwidth/Traffic (Num GBs) $12 per 3GB per year or $1 per 3GBs per month.
NEW: My Domains page. On this page you will be able to see all domains registered with our services either as free (with yearly account payment you get one free domain registration/renewal) or paid ones. On this page you will be able to see the domains expiration dates and have option to renew each of them. The registration/renewal price is $12 per year.
If you wish to register new domain or add more features to your account you will have to contact our sales department via live chat or email (email is best) and we will provide you with payment links and etc.


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Interview: Cage The Elephant talks Tell Me I'm Pretty, Grammy nominations, and classic movies
Posted by Aesthetic Magazine ⋅ January 19, 2016 ⋅ 2 Comments
Filed Under Cage the Elephant, Dan Auerbach, GRAMMYs, The Black Keys
By: Lucy Sky –
Cage The Elephant.
Speaking humbly of their Grammy nomination, Matt Schultz is all about making music. While his style may have changed slightly in their most recent release, his attitude seemingly has not.
Check out our new interview below where we discuss working with Dan Auerbach of The Black Keys on Cage The Elephant's new album, Tell Me I'm Pretty, classic movies, and more!
Your vocals on the new album have transformed a bit from that rambunctious rock n' roll sound to quieter vocals and more acoustic guitar. Were there any specific reasons or motives behind that transition, other than provoking emotional responses from listeners?
What's been kind of funny, is I've always leaned a little more heavily into a folk sound, but the kind of artists that I listen to most of the time are more towards Bob Dylan, Neil Young, Paul Simon, guys like that for my own listening. Which is kind of funny because I think that's probably not so frequently portrayed in the music that we make until recently. I think the most [difficult] on this record was practicing restraint. I had some really great creative conversations with friends over the past couple years. One of the conversations [I] had, I was talking to a friend who was an actor, and [we] were talking about how in everyday life, a lot of people tend to downplay emotional response. It's much more reserved.
When you're given the license to express an emotion, a sentiment, or a feeling – a free license – sometimes people will give an honest portrayal of how they actually feel, which doesn't read as real, because in everyday life, we're constantly contradicting in the way we project those things. The idea is that we're always restraining ourselves and when you're given the opportunity to do whatever you want, sometimes you take advantage of that and you go overboard. It's not even overboard, you're just being honest, but it reads as overboard because we're constantly restraining ourselves. The idea was to have the same kind of intensity and have that tension there, but restrained. I don't know if it really worked, but that was the idea behind it.
You're known for your explosive, unruly, live performances. How do you think that will translate live, with the more emotional and personal tracks on Tell Me I'm Pretty?
I think this record doesn't necessarily ask you to move around, or be chaotic. It's more of being a thing, or something to stand in the sentiment of the song, rather than moving for the sake of entertainment. I don't think it's going to be any less animated, or anything like that. I'm hoping it'll be a little more impactful, because it's a solid thing I'll have to be during the performance, rather than just relying on my nervous energy to carry it through.
What specifically do you think Dan Auerbach brought to this album as the producer and do you think the fact that you had toured together in the past brought a special something to the recording process?
I think Dan brought a lot to the recording process and the further we get away from the whole experience of recording, the more I realize how impactful he was during it. He bases a lot on reaction and mostly first reaction, so he's really good at keeping you from holding yourself back, or getting in your own way – as far as overthinking things and that kind of stuff, self-sabotage. He also has a super strong grasp of tones and textures and how to make those things work together. Balance, how to create a classic sound that doesn't feel like your grandparents music, that doesn't feel archaic. Those are just a few of the things. He's really great too at toning things down, but in a way that makes the song more impactful, creating more space in a track.
You've been highly acclaimed even since your sophomore album hitting number 2 on the Billboard Top 200 chart in 2011 as one of the top rock albums of the year. It's said that Dan emphasizes first takes, how did that affect the writing and recording process?
I think it affected it dramatically. In the past, we've spent sometimes very little to hardly any time at all working on material before we went into the studio. Then what we would do is just build each song in the studio. Everything was under a microscope, to where we would structure a song, tear it apart, look at it, see how the pieces worked, [and] put them back together, until we found a balance of what was wanted. On this record, being the first record without Lincoln and with a new producer, we spent extensive time working on the songs prior to going into the studio. So I think that was one of the things that was a big difference on this record [as well].
How would you compare that process to that of Melophobia?
They're both good, I like them both. There's so many different ways to explore creativity, and I don't see any certain way as being the best, but I think each time you're just looking to discover new things that you can apply to the next process and hopefully get a little better. It's funny, I think sometimes we always perceive growth as movement forward and I think that it's very important to remember that sometimes you have to move backwards, sometimes you have to move to the side, things like that and pick up certain things and continue on.
We had really wanted to get back to a more classic sound. On Melophobia we spent a lot of time working on songwriting and structuring and also just on big production and on this record it was a much simpler approach and we wanted to get back to a more raw, more gritty sound, a more classic sound that couldn't necessarily be pinned down to a specific time period. When you're thinking about that kind of thing, especially when you're presenting it kind of in this I wouldn't say Rock N' Roll package, but definitely influenced by Rock N' Roll and I think it has a lot of that in it.
Cage The Elephant's new album, Tell Me I'm Pretty, featured Dan Auerbach of The Black Keys as producer.
What kind of influence, if any, did The Black Keys have on the album?
I don't know if The Black Keys per say had any influence on the record, I would almost even argue that Dan's a very strong songwriter, but I think his real strong suit might be his production.
His fingerprint is undoubtedly on the record and that was a very conscious decision on all of our parts together. There's not a lot of guys that are performing that kind of sound at the level that Dan is. I definitely wouldn't call that a Black Keys sound, there's a lot of bands that have done that sound in the past, I think Dan has just been one of the more successful ones.
Headlining the KROQ Halloween Costume Ball in LA, the release of "Mess Around" and pre-order of Tell Me I'm Pretty all happened the day before Halloween. Was that purposeful and how exciting, stressful or relieving was it to have that all happen in the same day?
I don't know if it was purposeful. I'm sure it was to someone in our team, our management or someone who planned it out, but I really didn't have much involvement in that. I generally stay stressed out, so it wasn't like it moved the meter much for me. I think relieving is the word. With this record, for some reason it just seems like each record we make, it becomes more difficult for me. Not necessarily the task, or what it demands, in honesty, with every record, I'm always striving to be a little more transparent – to learn something.
In the past, when I was younger, I put so much stock in persona and I feel like that really hindered the potency of whatever it was that I was writing. By listening to a couple songs in the past couple years, I realized when people were very honest, it sounded way more poetic than say writing to be perceived as poetic, or intellectual, or something like that. So with each record I've been trying to be more transparent and vaguely specific. On this record it got to the place where it was just a little too close, almost like each song was a journal entry, and if you have any deep seeded hatred of yourself like I do, I had a little trouble enjoying the record or even believing that others might enjoy it. After we released the record and saw the response, the nice thing is that I've been able to go back and vicariously enjoy it through other people.
The video for "Mess Around" featured footage from the 1902 film A trip to the Moon and other Georges Melies, what was the inspiration behind that?
I had seen a remastered version of A Trip to the Moon where Air did the soundtrack to it. When I first saw it, I thought maybe in recent times they had hand colored the film with digital technology. I at first was inspired and thought that would be a great way to do photographs of the band and started to research it. Then we started thinking about making a music video that way and when I started to look up creative groups that did that, I found out they didn't exist. Then when I dug deeper, I realized all that stuff was done in the early 1900's, late 1800's and they had these hand tinting companies that had like 200 artists on the floor. They did it like an assembly line, each artist would be assigned a color and then the frame would be passed around and they would color whatever object they were coloring, which is why some of the colors popped around in different places, which is really awesome. It was actually mistakes, but it's beautiful regardless. So I got fascinated, [but] realized we couldn't make a video like that. I was really disappointed when I found that out. But then, my wife is French, and I was talking to some of her friends and they told me that some George Melies films were public domain, so we reached out to the organization that takes care of his films and they were really excited that we were interested in using them. Then we made the video that way.
How did being nominated for a Grammy for Melophobia feel?
It felt great. Hopefully you don't make records to garner accolades or acknowledgments in that way, but it definitely was a confirmation of what we believed in the record and the direction that we were headed. We were really very flattered.
You said that the inspiration was a plenty for this album and that after finishing the album, there was still material in your minds. What can fans expect for the future of Cage The Elephant?
I don't know. Hopefully we'll stay true to the convictions we have about music and that approach, as far as expressing certain sentiments and ideas, that kind of thing, but continuously evolve according to whatever learnings we pick up along the way of the creative process.
You've toured with the Black Keys, Foo Fighters, Muse and Queens of the Stone Age. As musicians and personally, what did you take from those experiences?
I think the biggest thing that I've taken away from watching a lot of the people that I've looked up to and bands and artists that we love is work ethic. Most of the bands that we've toured with have been extremely hard workers. I feel like the creative world is kind of like fishing. It takes a lot of time, you're not always going to catch the big fish, but you're hoping that every time you cast a line, you can catch a fish. With those guys and girls and groups, I think it's just they're consistent in how hard they work and how present they are.
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2 thoughts on "Interview: Cage The Elephant talks Tell Me I'm Pretty, Grammy nominations, and classic movies"
This is fabulous. Thanks so much for sharing.
Posted by Track Queen | January 19, 2016, 3:52 pm
Pingback: Interview: Cage The Elephant talks Tell Me I'm Pretty, Grammy nominations, and classic movies | Lucy Sky - February 1, 2016


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Q: How to use superscript in geom_text? I am trying to use superscript in geom_text. But I am unable to do that. I am using the following code
library(caret)
library(tidyverse)

summ <- iris %>%
  group_by(Species) %>%
  summarise(R = cor(Sepal.Length, Petal.Length, use="pairwise.complete.obs"),
            RMSE = RMSE(Sepal.Length, Petal.Length)) %>%
  mutate_if(is.numeric, round, digits=2)

p <- ggplot(data=iris, aes(x = Sepal.Length, y = Petal.Length)) +
  geom_point(color="blue",alpha = 1/3) +
  facet_wrap(Species ~ ., scales="free") +
  geom_smooth(method=lm, fill="black", formula = y ~ x) +
  xlab("Sepal Length") +
  ylab("Petal Length") + theme_bw() +
  theme(panel.grid.major = element_blank(), panel.grid.minor = element_blank())

# Here we create our annotations data frame.
df.annotations <- data.frame()

# R
df.annotations <- rbind(df.annotations,
                        cbind(as.character(summ$Species),
                              paste("R", summ$R,
                                    sep = " = ")))

# RMSE
df.annotations <- rbind(df.annotations,
                        cbind(as.character(summ$Species),
                              paste("RMSE", summ$RMSE,
                                    sep = " = ")))

# This here is important, especially naming the first column
# Species
colnames(df.annotations) <- c("Species", "label")

vertical_adjustment = ifelse(grepl("\\bR\\b",df.annotations$label), 1.5, 3)

p + geom_text(data = df.annotations, aes(x=-Inf, y=+Inf, label=label),
              hjust = -0.1, vjust = vertical_adjustment, size=3.5)

My expected output is

How can I add the unit for RMSE after the value and superscript 2 after R i.e. R^2? You can also see that there is a slight shift in the alignment of RMSE with respect to R. How can I make them into the same line?

A: This could be achieved like so:

*

*Set parse=TRUE in geom_text

*Make use of ?plotmath to add superscripts to your labels

# Here we create our annotations data frame.
df.annotations <- data.frame(
  Species = rep(summ$Species, 2),
  label = c(
    paste0("~R^{2} == ", summ$R),
    paste0("~RMSE == ", summ$RMSE, "~m^{3} ~m^{-3}")
  )
)

vertical_adjustment = ifelse(grepl("\\bR\\b", df.annotations$label), 1.5, 3)

p + geom_text(data = df.annotations, aes(x=-Inf, y=+Inf, label=label),
              hjust = 0, vjust = vertical_adjustment, size=3.5, parse = TRUE)

A: Another approach is to use the ggtext package, which enables markdown formatting in ggplot text objects. I find this syntax much easier to use than R's plotmath, especially for things like italics and color:
df.annotations <- data.frame(
  Species = rep(summ$Species, 2),
  label = c(
    paste0("R2 = ", summ$R),
    paste0("RMSE = ", summ$RMSE, "m3 m-3")
  )
)

vertical_adjustment = ifelse(grepl("\\bR\\b", df.annotations$label), 1.5, 2.5)

p + geom_richtext(data = df.annotations, aes(x=-Inf, y=+Inf, label=label),
             hjust = 0, vjust = vertical_adjustment, size=3.5, label.size = 0)

redpajama_set_name: RedPajamaStackExchange


Welcome to THE PAIN STOP.
Here is some information if you have not visited before.
More information on each therapy can be found at the bottom of this page.
Appointments, especially the first one, are usually for an hour. For physical therapies, I work through clothes so please wear light, loose clothing eg t-shirt and shorts/leggins.
If you have not seen me before, please put 'new' after your name. When you leave your email address, you will receive a confirmation email with the time of your appointment and directions to Chelsea House Clinic. There is a car park behind the building.
If this appointment is for someone else, please put their name in the appointment slot. You can put your own in brackets after if you wish. If the appointment is for a baby the appointment will be for 30 min. Please put 'baby' after the name. It would be helpful if baby isn't fed in the hour before treatment thanks.
The system won't let you book in the 12 hours before the appointment time so if you're looking for a cancellation or on the day appointment, please phone Chelsea House Clinic on 01467 624888 and they will book you in if there is a free appointment. Please note that they will not book future appointments to avoid double booking, and will only have my diary on Mondays and Wednesdays when I am working.
If you are having any issues with finding an appointment or with the booking process, please don't hesitate to leave a message on my phone (07733 288035) and I'll call you back to help.
To see what data I hold on you and why, I have a written statement below in line with new Data Protection legislation. If you want to skip this, please scroll down for information on therapy options and booking.
As part of visiting me for therapy, I will need to have a record of your personal details, date of birth, address, telephone numbers, email and relevant medical information relating to your session. A unique reference number will also be allocated to you.
All data will be held in a locked filing cabinet or on a computer protected with a password.
No client files are left on surfaces for other clients/staff to read.
All notes will be kept secure for a period of 8 years for adults and will then be destroyed if you are no longer attending clinic. All children's notes will be kept until adult age ( 21 ), and then destroyed if no longer attending clinic.
Your practitioner is collecting data at the start of your first session. Some information maybe requested by email or text message to ensure the smooth running of your treatment. On occasion data from relevant medical notes / letters and scans may also form part of the data collected and held by your practitioner.
Data is collected to record, guide and supervise the your progress and be able to communicate effectively with the you for the best outcomes. It is also used to compare progress week to week and to highlight changes, red flags, yellow flags, action to be taken and a detailed dialogue of treatment provided.
Data also helps us to carry out relevant research from time to time. All clients can opt in or opt out of this. Again unique reference codes will be used to transfer data.
No sensitive/identifiable data is sent by email together in the same posting. Unique reference codes are used.
All computers/laptops and tablets are locked with pass codes and not left unattended. Only individuals with permission to read notes can access this data.
Clients have a right to see what information is being held about them and be given copies of this information if requested at no cost to them.
Clients have a right to request deletion of data, although this can be overridden in the interest of the client, especially regarding medical matters.
Practitioners take data protection and privacy seriously and promote this philosophy to all the industry in relation to protecting client data.
Bowen Technique, Emmett Technique plus other muscle relaxation techniques used to address pain. Bowen uses light pressure using fingers or thumbs, rolling gently over muscles, ligaments and tendons. Emmett uses very light finger pressure on precise places on muscles or little switches to provoke change.
Generally scars should be more than 6 weeks old unless in severe pain in which please phone me direct to discuss before booking please. It is believed that this work can help with pain resulting from adhesions.
Generally scars should be more than 6 weeks old unless in severe pain in which please phone me direct to discuss before booking please.


There is a growing need for specialized, intensive transitionally focused training for the numbers of young adults graduating from our traditional school systems that are diagnosed with autism spectrum, developmental and intellectual disabilities and are moderately to significantly impaired.
Austin's Place was started by parents of these special persons so that, simply put, their child would have a safe place to go to each day to continue to refine the life skills necessary to become contributing members in their local communities after graduation. It is the more intense behavioral and sensory needs of these specific types of learners that often limit these young adults from effectively participating into traditional adult vocational support programs. Austin's Place is designed to provide specialized and intensive programming for these young adults to address specific behavioral or sensory limitations, while still focusing on developing vocational skills and increasing community integration.
We have learned through educating these exceptional learners that is takes cooperation and collaboration. Therefore, families, instructors and therapists will work together to develop and implement quality and comprehensive programming to meet the global needs of these young adults.
Austin's Place is designed to serve these special persons and meet their individual needs in an innovative, strength-based and positive fashion. Young adults attending programming at Austin's Place will participate in daily routines and training opportunities to develop optimal levels of independence, while still accounting for their own capacities and limitations. Programming also incorporates the interests and strengths of consumers so that there is true motivation on the part of consumers to learn and progress. Instructional priorities are placed on developing independence in self care, community engagement as well as developing usable vocational skills in future settings.
Persons interested in exploring the options available for consumers at Austin's Place should contact 724-352-ASTN (2786).
Austin's Place is aptly named after Austin Wilczynski, Bonnie and Victor's son, for whom this new program is inspired by. Austin, diagnosed with multiple disabilities is a recent graduate of the Watson Institute.


Kick up the heat as Naren Young at Saxon + Parole dishes the secrets to their chili-infused cocktail.
Brisket chili is topped with chipotle cheddar biscuits and baked to perfection. A perfect autumn meal for halftime and to warm you to the bone.
Packed with vegetables and lean bison meat, a warm bowl of hearty chili will fuel you on a busy fall day. Make a double batch, you will want leftovers.
Black beans and cannellini combines with fresh cilantro, cumin, and jalapeño for a light soup that is a great first fall chili.
Creamy, sweet, savory, with the perfect tender crumb. Cornbread is truly an art that is worth mastering. Check out these tips and have it down to dunk in your chili all autumn long.
A drizzle across your Pad Thai or drenching French fries in a spicy firestorm, there's no denying Sriracha is on its way to being King of the Condiments.
A traditional Szechuan-style chicken poached with spicy chili oil sauce for ultra tender meat. Get the recipe.
A flavorful, simply-made bowl of rajma with fresh roti. Be ready for a creamy, not-too-spicy dish with a dollop of cream and a fried chili on top.
Marinated steak served with a cilantro and yogurt sauce that is also incorporated into a black rice salad with refreshing cucumbers.


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Insights to the Peak 10 Success
Thursday, Jun 28 2012 10:00AM
Speaker(s): David Jones, CEO, Peak 10
Location: Peak 10 Corporate Headquarters 8809 Lenox Pointe Drive, Suite G Charlotte, NC 28273
"David Jones, CEO, Peak 10, will personally share his journey to build one of our region's most admired companies"
Date: Thursday, June 28, 2012
Location: Peak 10 Corporate Headquarters,
8809 Lenox Pointe Drive, Suite G
Parking: Plenty of free parking
Who is David Jones and why should you listen to him? Because he is a Rock Solid, yet humble, CEO!
The Peak 10 tagline is "Rock Solid People. Rock Solid Solutions. Rock Solid Results." That sums up David and his company very well!
A startup in 2000. Surviving the collapse of the .Com bubble in 2001. Growing exponentially throughout the decade and The Great Recession. Now the leading independent data center and managed services provider in the U.S. Peak 10 foresaw the possibility of cloud computing. But it still took the exceptional leadership of entrepreneur and visionary David Jones to make that dream a reality. David Jones, the CEO and President of Peak 10, took a startup vision and transformed it into a company with the strongest balance sheet and income statement in the industry, yielding a 12.5 EBITDA multiple and a $410M deal when they sold to Welsh Carson in 2010.
David and his same executive team (with continued ownership in the company) boast a solid forecast for long-term growth and continued profitability, which will position Peak 10 well for another remarkable exit!
In addition to sharing personal insights, David will present an overview of Peak 10 to include:
Historical Path to Expansion and Growth
Valuation Drivers
Operational Discipline
Cloud and Outsourcing Trends
Leadership Lessons and Principles
Link to Bio for David Jones and his Leadership Team
http://www.peak10.com/company-profile/people
10:00 am – 12:00 pm Presentation by David Jones, CEO of Peak 10
12:00 pm - 1:00 pm Lunch and Networking
Program Cost: BIG Members free
Guests $40 in advance, $50 at the door
Registration: RSVP to terry@bigcouncil.com or register below.
Dress Attire: Business casual


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Pretty Little Liars actor Julian Morris comes out as gay
This actor posted heartwarming pictures and video to celebrate the 18th anniversary of his partner.
On Thursday on Thursday, Former star of Pretty Little Liars actor Julian Morris came out with a touching Instagram post where the actor also celebrated his 18th birthday with his partner.
"18 years together, and they were the best ones because they were with you," Morris posted in the caption of the carousel of photos along with videos of him and his partner, L.A. artist Landon Ross and added "I love you" along with the heart emoji.
A post shared by Julian Morris (@julianmorris)
Most well-known for his role as Wren Kingston on the Freeform mystery-thriller show Pretty Little Liars The actor has also appeared on television shows like 24 Once Upon a Time, and New Girl. Recently, Morris appeared on the Apple TV+ drama The Morning Show.
Also Read: Machine Gun Kelly Once Stabbed Himself to Look Cool For Megan Fox
The 38-year-old, who's been in love with his partner since he was just 20 years old, young, also sent an expression of gratitude to his fans on his Instagram account, writing "Really moved by all the love and support. Thanks guys."
In the midst of his Jennifer Lopez romance, Ben Affleck wants to 'ostensibly' be a 'good husband.'
Adele goes makeup-free in YouTube video and her skin is glowing


The Citadel's Department of Health, Exercise and Sport Science will dedicate the Dr. Hank Cross Human Performance Laboratory during Homecoming events this coming weekend.
The Dr. Hank Cross Human Performance Lab is what industry leaders need to perform cutting-edge research in exercise and health science.
The lab is a state-of-the-art facility that has more than doubled the exercise science research laboratory and teaching space in Deas Hall. At 1,400-square-feet, the lab has equipment for phlebotomy and exercise testing along with meeting space, offices, and locker room facilities.
Bite Tech Inc. provided the $200,000 that was used to build the Human Performance Lab. Hank Cross is director of research at Bite Tech, a Minneapolis-based technology company dedicated to improving performance and health. The company recently partnered with Under Armour to release custom performance mouthwear.
HESS professor Dena Garner is one of several professors researching how mouthpieces used during sports and exercise affect athletic performance. The lab will created enhanced opportunities for the department and students to investigate how mouthpieces affect hormones and muscles in the body and, more specifically, the effect on pistol shooting accuracy.
"The research conducted in the laboratory is innovative and is making significant contributions in the dental and the exercise science research," said Garner, who will direct the Cross lab.
Working with Garner on the research are HESS's Wesley Dudgeon and Erica McDivitt, and Timothy Scheett, Department of Health and Human Performance at the College of Charleston.


text:
About Dale Hurst
Lust & Liberty
Sin & Secrecy
You Can Hear Chopin
Ballad of a Godless Man
5 Authors Whose Work is Darker Than You Thought
From terrible regrets to deep hatred and prejudice… what inner darkness gets betrayed in the work of these writers?
The other day, I was chatting with a friend of mine about how I like to keep my writing at face value. I don't try to inject much deeper meaning behind my plots and characters. For one thing, I don't personally read like that, and I don't encourage it in my own readers, either. Stories are there to be enjoyed and to entertain, not to be analysed so hard that you take away the fun (and the fiction!) A sentiment I upheld even from my GCSE English days — did you ever feel like you were putting more thought into the book than the author?
Others have analysed the work of some much-loved authors and discovered that their work – some of which is already pretty dark – was actually channelling some dark inner feelings. I have put together a list of five; a couple on here won't surprise you, while others – I hope – will shock you!
Lisbeth Salander was based on a figure from Stieg Larsson's past. A character inspired by haunting guilt and terrible regret. (Image: Tori @Flickr)
Anyone read the Millennium books? The Girl with the Dragon Tattoo and its sequels. They were authored by a Swedish journalist by the name of Stieg Larsson, and published after his death in 2004. If you've read or even heard of the series, you will know that they deal with rather extreme themes, one of which is abuse and hatred against women. Most notably, sexual abuse, as is inflicted on Lisbeth Salander in The Girl with the Dragon Tattoo by her guardian, Nils Bjurman.
The character of Lisbeth and, by extension, the prevalence of the sexual violence theme, was inspired by Larsson's own regret and inability to forgive himself for failing to intervene during an incident when he was 15. When he witnessed three of his friends gang-raping a girl, after whom he named his heroine. He reportedly approached the victim days after the ordeal, begging for her forgiveness, which, rightfully, she did not give him. Lisbeth Salander is the manifestation of Larsson's guilt and his past haunting him.
What dark recesses did Roald Dahl channel in creating his universally-loved stories?
I bet you think I must be joking. One of the world's best-loved children's authors? I was surprised too, but if you look over Roald Dahl's work again, you will note there is actually some pretty dark stuff there. A lot of children punishing adults, which oddly enough – albeit inadvertently – is also present in the upcoming Berylford book, Sin & Secrecy. For example, we see James in James and the Giant Peach at the mercy of his cruel and abusive aunts, but he later kills them on the titular giant peach, before going on his adventure with his new, contrastingly loving friends.
But where did this all stem from? The macabre nature of his plots and his making villains out of adults is thought to have been inspired from Dahl's early childhood. He lost his father when he was only three, as well as his sister, and was soon sent away to boarding school, where he suffered at the hands of the teachers' brutality.
James and the Giant Peach also contains aspects of profanity and even racism, betraying some of Dahl's worst faults. He was, by all accounts, not a particularly nice man. Numerous infidelities, not to mention a self-confessed anti-Semite. He was once quoted to have said, "…even a stinker like Hitler didn't just pick on [the Jews] for no reason."
You can learn more about the darkness behind Roald Dahl in these articles here and here.
Rev. W. V. Awdry
How does Thomas the Tank Engine creator Rev. W. V. Awdry's relish for punishment come out in his stories?
Any Thomas the Tank Engine fans out there? Prepare yourselves — I'm about to ruin your childhood again. I mean, what can possibly be dark about a series of stories about anthropomorphic trains? A lot, it turns out — especially when you psychoanalyse the man behind them, the Reverend Wilbert Vere Awdry. Then it shows off the Isle of Sodor at its most tyrannical, governed by a severe, punitive, authoritarian regime that Awdry personally endorsed.
The example I have seen given by a lot of critics and academics is the story in which Henry, afraid that the rain will spoil his paintwork, refuses to come out a tunnel. Disrupting the day's workflow as a result, and of course, angering the Fat Controller. What is the Rev. Awdry's ideal punishment for not doing as you're told? In Henry's case, life imprisonment. He has his rails removed and the tunnel in which he is sitting is bricked up, to be left there, "for always and always." Ouch…
Many argue that the brutality of such punishments were the Reverend's way of emphasising what lay in store for anyone who strayed from the straight-and-narrow.
Read a little more about it in this article here.
Another childhood favourite – The Wind in the Willows. A pretty little story celebrating nature? Or a form of escapism for the author?
Like Roald Dahl, the early life of The Wind in the Willows author Kenneth Grahame was marred by sadness and grief.
He was a great nature-lover, as it might not be too hard to discern from his work. But the reason why he, in his own words, "like[d] most of [his] friends among the animals more than [he] like[d] most of [his] friends among mankind…" was in light of a turbulent childhood and subsequent family life. His father was an alcoholic and his mother died young.
Grief inspired his imaginary natural idyll with picnicking moles and water rats, furthered by his failed marriage and the suicide of his son Alastair. A form of escapism to a much happier, fantasy world.
And you can find out more about Kenneth Grahame in this article.
Maurice Sendak
Does Where the Wild Things Are portray the author's inner demons and turbulent relationship with his emotionally-distant mother? (Image: Scott Woods-Fehr @Flickr)
Author of Where the Wild Things Are Maurice Sendak grew up in Brooklyn, and fans of the story may already know that it is a self-expression of his early life experiences. And his relationships with the adults in his life. Even the title is a homage to what his mother used to call him. "Vilde chaya," means, "Wild animal," in Yiddish, which Sendak's mother often used to brand him, which in turn inspired the use of the phrase, "Wild thing," in the book.
Indeed, his mother seems to have cut an ambiguous or in some cases villainous figure in Sendak's life – being described as, "disturbed, chronically sad and emotionally unavailable." She and his father were of Polish descent, whose family had endured and, in the case of his father, perished in the Holocaust. Sendak and his mother's difficulties formed an overall theme in Where the Wild Things Are – it is about a boy who is trying to defeat his inner demons and rectify his relationship with his mother.
Can you find any inner darkness in my work?
Even though I do not promise to confirm or deny anything, I encourage people to read or re-read Lust & Liberty and, once it's released, Sin & Secrecy. See what meanings and emotions you can read between the lines. Does Lady Vyrrington represent the urge to carry on through one's grief, blaming oneself all the while. And are Abel and Rebecca Stirkwhistle an allegory for the cold and bullying education system that still exists in the UK today? Like I say, I'm not saying, "Yea," or, "Nay," on that score. But it gives you something to think about, and I'd be interested to see what you can suggest.
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Written by: Dale Hurst
This is Dale Hurst here. Novelist and journalist. Subscribe above to receive all updates.
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BRINSTON/SOUTH BRANCH/NORTH DUNDAS/NORTH STORMONT
Ottawa's "Energy Evolution": wind turbines coming to rural communities
Wind Concerns Ontario
Ottawa Wind Concerns
~ A safe environment for everyone
Tag Archives: LSARC
Is Ontario's push for giant wind farms killing the green energy movement?
Posted by ottawawindconcerns in Renewable energy, Wind power
Algoma, Amherst Island, at-risk species Ontario, endangered species Ontario, Environmental Review Tribunal, green energy, Green Energy Act, Kathleen Wynne, Lake Ontario, LSARC, Nature Canada, Ontario, Ontario environment, Ontario Ministry of the Environment and Climate Change, Ostrander Point, Prince Edward County Field Naturalists, Pronce Edward County, Robert Quaiff, wind farm, wind farm environmental damage, wind power
Is Ontario's rush to wind power killing the green energy movement?
TORONTO, CAN, September 2, 2015
Ontario's stance as an environmental activist province in Canada and would-be leader in climate change action is taking a beating after the government approved two controversial wind power projects, and continues to fight environmental groups and citizens on a third.
Last week, the Ontario Ministry of the Environment and Climate Change approved a 75-megawatt power project on tiny Amherst Island in Lake Ontario. The island is home to several species of wildlife declared endangered or at-risk by the same government, and is also a resting place for migrating birds. The birds attract eco-tourists from all over the world.
The threat of the wind power project to the heritage environment is so great that Heritage Canada's National Trust named the island one of Canada's Top Ten Endangered Places.
"There are some places where wind power projects shouldn't go," says Michele LeLay, spokesperson for the community group the Association to Protect Amherst Island. "This is one of them."
Abundance of birds at risk
Also on Lake Ontario, is Prince Edward County where the province recently approved another large wind power generation project for the South Shore. The environmental danger is undeniable, says Cheryl Anderson, of the Prince Edward County Field Naturalists: "Data gathered over 20 years confirms the South Shore is a major migratory pathway for an astonishing diversity and abundance of birds. This unique blend of ecosystems supports numerous varieties of rare plants, eight species of at-risk turtles, Monarch butterflies and many amphibian species. Because of its unique biodiversity, the value of Prince Edward County's South Shore is unparalleled as an ecotourism venue."
The Ontario government heads back to the quasi-judicial Environmental Review Tribunal in September, to hear the appeal of a wind power project at Ostrander Point, also in Prince Edward County, halted by the Tribunal in 2013 due to the danger to a rare species of turtle. After several sessions in court, the decision has been returned to the Tribunal where community groups are in the unusual position of spending hundreds of thousands to protect the environment from the Ministry of the Environment.
Prince Edward County Mayor Robert Quaiff is outraged at the approvals and has been trying to see the Premier of Ontario, so far with no luck. In a letter to her he said "efforts to implement the Green Energy Act [legislation pushing wind power] are becoming counter-productive through resulting negative impacts to endangered species, as well as the prosperity and well-being of rural Ontario Communities."
The concern about Ontario's pro-wind agenda and resulting environmental damage is not limited to the southern parts of the province. Canada is known around the world for its iconic landscapes in the Algoma region around Lake Superior, now also the site for unbridled wind power development. Hills and valleys made famous by Canada's Group of Seven artists are now scarred by clear-cutting of trees, flattening of ridges, and the construction of roads and turbine foundations.
George Browne of Lake Superior Action Research Conservation (LSARC) says the devastation to the wilderness is immense. Wilderness, he says, "is a rare and unique feature, understood by many to represent the grandeur of nature; vastness is an essential part of the aesthetic appeal of the landscape. It is worthy of conservation."
Approval of projects in fragile areas will tarnish green energy industry
Nature groups believe that Ontario's inappropriate choices for wind power development will actually harm the green energy movement. Ontario Nature and Nature Canada jointly stated: "We sincerely believe [approval of the Amherst Island project] will further tarnish Ontario's green energy industry, and ultimately undermine future projects in less controversial areas. The opposition of this project in the naturalist community is palpable. The risks of killing large numbers of raptors, swallows and bobolinks is high. Approval will further alienate a segment of Ontario's population from the green energy agenda and tip an already fragile balance."
Ontario is guilty of hypocrisy says Ontario's premier community coalition, Wind Concerns Ontario. "The government's recent decisions show they have lost their way," says President Jane Wilson. "Killing birds and despoiling wilderness is not the way to save the environment."
Contact: Wind Concerns Ontario www.windconcernsontario.ca
Email us here.
Prince Edward County South Shore: major pathway for migratory birds
Wind farms in Northern Ontario: massive change
appeals wind farm, Bow Lake, endangered species, George Brown, Goulais Bay, Lake Superior Action Research Conservation, LSARC, Northern Hoot, Northern Ontario, Ontario bats, Ontario Court of Appeal, Ontario Ministry of NAtural Resources, Steffanie Petroni, wind farm, wind farm environment, wind power northern Ontario
Northern Hoot is a new website run by journalist Steffanie Petroni on all things Northern Ontario. Devoted to "long-form" journalism, Petroni recently published an article on wind power development, of interest now because two very large projects–Bow Lake and Goulais Bay–will be proceeding. Appeals by First Nations groups and residents failed.
Of special interest in this posting are photos by Gary McGuffin, who is renowned for his depiction of Northern Ontario scenery.
In Ontario there have been 20 appeals in opposition to industrial wind turbine farms brought before the Ministry of the Environment (MOE) and 19 have been dismissed. An appeal by Prince Edward County Field Naturalists to kill the development of an industrial wind turbine farm on Ostrander Point was won before an ERT in July 2013. However, the decision has since been reversed by the Ontario Divisional Court and appellants are seeking an appeal before the Ontario Court of Appeal.
George [Brown, of the Lake Superior Action Research Conservation] commented, "The 240 Bow Lake appeal came close to winning. Based on the Ostrander Judicial Review decision the Tribunal found that in order to prove irreversible harm it was necessary for the appellant to know the size of the populations being harmed. Having found that the 240 appeal failed to prove irreversible harm the Tribunal declined to make a finding on the issue of serious harm, though it agreed with virtually all the arguments on bats submitted by the 240 appeal.
As a result the Tribunal imposed immediate and more stringent mitigation measures on the project – a tacit admission that species-at-risk bats would otherwise be killed, which would be a serious harm.
The Tribunal's decision is peculiar in that it allows these more stringent mitigation measures to be rescinded should they prove effective. Had the MNR required, or done, a baseline study, or had the 240 appeal had the time and money to do one, to determine the size of existing bat species populations in the project area, we would perhaps have had the final piece of the puzzle required to win."
For more information, go to http://www.lsarc.ca
City documents still say YES to wind turbines
Coldest day of the winter so far and wind power is NOWHERE
Ottawa planning docs show wind turbines for City
Ottawa "community" investor syndicate buys wind farm hundreds of kilometers away
Prepare for battle: Ottawa residents get opportunity to learn about city planning processes next week
Bob Chiarelli electricity bills Ontario Green Energy Act IESO Ontario Ottawa wind concerns wind farm wind farms wind power wind turbines
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Q: Logout from Facebook programmatically in iOS The problem is that Facebook use Safari when logged in, so when I try logout from my app it does keep credentials and cookies of Safari.
In other words, the only way to logout correctly is to open Safari and logout from Facebook.
My question is, is there any work around this way?
FBSDKLoginManager *loginManager = [FBSDKLoginManager new];
[loginManager logOut];

I also tried:
[FBSDKAccessToken setCurrentAccessToken:nil];
[FBSDKProfile setCurrentProfile:nil];

Although they are being called inside the logout method :(
Finally I tried removing cookies
NSHTTPCookieStorage* cookies = [NSHTTPCookieStorage sharedHTTPCookieStorage];
NSArray* facebookCookies = [cookies cookiesForURL:
                            [NSURL URLWithString:@"http://login.facebook.com"]];
for (NSHTTPCookie* cookie in facebookCookies) {
    [cookies deleteCookie:cookie];
}

No luck at all!


A gel eyeliner that offers the look of a liquid with the ease of a pencil.
Step 1: Place your left middle finger (or use your right if your left handed) just under the eyebrow at the outer corner and apply light pressure. Close your eye slightly to get a better view of the upper lid.
Step 2: Use short strokes to apply eyeliner from the inner corner to the outer edge of the upper lid. Start out with a thin line and let the line get a little thicker as you work your way across the lid.
Step 3: For your lower lid, apply eyeliner from the inner corner to the outer corner of the lower lid. Start where the lash begins, making sure to run the brush as close to the lash as possible. Blend any harsh lines when you're done.
Ingredients: Cyclopentasiloxane, Trimethylsiloxysilicate, Polyethylene, Candelilla Cera/Euphorbia Cerifera (Candelilla) Wax/Cire de Candelilla, Isododecane, Cera Microcristallina/Microcrystalline Wax/Cire Microcristalline, Nylon-12, Cera Alba/Beeswax/Cire d'abeille, Disteardimonium Hectorite, Propylene Carbonate, Butylparaben, BHT, Aqua /Water/Eau. May Contain: CI 77891/Titanium Dioxide, CI 77491, CI 77492, CI 77499/Iron Oxides.