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BMC Genomics
Transcriptomic analysis of sea cucumber (Holothuria leucospilota) coelomocytes revealed the echinoderm cytokine response during immune challenge
Xiaofen Wu1,2 na1,
Ting Chen1,3 na1,
Da Huo1,3,4,
Zonghe Yu1,3,4,
Yao Ruan1,2,
Chuhang Cheng1,2,
Xiao Jiang1,3,4 &
Chunhua Ren1,3,4
BMC Genomics volume 21, Article number: 306 (2020) Cite this article
The sea cucumber Holothuria leucospilota belongs to echinoderm, which is evolutionally the most primitive group of deuterostomes. Sea cucumber has a cavity between its digestive tract and the body wall that is filled with fluid and suspended coelomic cells similar to blood cells. The humoral immune response of the sea cucumber is based on the secretion of various immune factors from coelomocytes into the coelomic cavity. The aim of this study is to lay out a foundation for the immune mechanisms in echinoderms and their origins in chordates by using RNA-seq.
Sea cucumber primary coelomocytes were isolated from healthy H. leucospilota and incubated with lipopolysaccharide (LPS, 10 μg/ml), polyinosinic-polycytidylic acid [Poly (I:C), 10 μg/ml] and heat-inactived Vibrio harveyi (107 cell/ml) for 24 h, respectively. After high-throughput mRNA sequencing on an Illumina HiSeq2500, a de novo transcriptome was assembled and the Unigenes were annotated. Thirteen differentially expressed genes (DEGs) were selected randomly from our data and subsequently verified by using RT-qPCR. The results of RT-qPCR were consistent with those of the RNA-seq (R2 = 0.61). The top 10 significantly enriched signaling pathways and immune-related pathways of the common and unique DEGs were screened from the transcriptome data. Twenty-one cytokine candidate DEGs were identified, which belong to 4 cytokine families, namely, BCL/CLL, EPRF1, IL-17 and TSP/TPO. Gene expression in response to LPS dose-increased treatment (0, 10, 20 and 50 μg/ml) showed that IL-17 family cytokines were significantly upregulated after 10 μg/ml LPS challenge for 24 h.
A de novo transcriptome was sequenced and assembled to generate the gene expression profiling across the sea cucumber coelomocytes treated with LPS, Poly (I:C) and V. harveyi. The cytokine genes identified in DEGs could be classified into 4 cytokine families, in which the expression of IL-17 family cytokines was most significantly induced after 10 μg/ml LPS challenge for 24 h. Our findings have laid the foundation not only for the research of molecular mechanisms related to the immune response in echinoderms but also for their origins in chordates, particularly in higher vertebrates.
Holothuria leucospilota is a tropical sea cucumber that belongs to phylum Echinodermata, class Holothuroidea, order Aspidochirotida and family Holothuriidae. Naturally, H. leucospilota is distributed in the Indo-West Pacific, mostly from eastern Africa to the Hawaii islands and Society islands in the Pacific ocean, and from southern Japan to the Sark bay in Australia [1]. In recent years, H. leucospilota has become an emerging aquaculture species in southern China [2]. Evolutionally, echinoderms are positioned taxonomically at the base of deuterostomes, along with the higher-order hemichordate and chordate groups [3]. Therefore, studies on the biological processes in echinoderms, such as development, reproduction, metabolism and immunity, may provide new insights not only for echinoderms themselves, but also for the origins of these biological processes in chordates, particularly in higher vertebrates.
Given that lacking of the adaptive immunity, the innate immunity is the major mechanism for sea cucumber to defend the environmental pathogens. The innate immunity of sea cucumber includes multiple immune-related factors, such as, antimicrobial peptides (e.g. lectins, lysozyme, clotting protein and complement) [4, 5], antimicrobial reactive oxygen species [6], pattern recognition receptors, apoptosis [7, 8] and immune cytokines [9]. Sea cucumber has a cavity between its digestive tract and body wall that is filled with coelomic fluid and suspended coelomocytes that are similar to the hematocyts of vertebrates. In sea cucumber, the cellular immunity is executed by coelomocytes, and the humoral immunity is based on a variety of macromolecules in the coelomic cavity that secreted by coelomocytes [10,11,12]. When sea cucumbers are infected by pathogenic microbes, they rely on their cellular and humoral immune responses to identify and eliminate the invading microbes and repair the wounds [9].
Next-generation sequencing (NGS) technology is a revolutionary change to traditional sequencing technology. The high-throughput mRNA sequencing (RNA-seq) is a transcriptomic research method that provides information on transcript expression and has the advantages of being easy-handle and low-cost [13]. For non-model organisms, RNA-Seq is not limited to detecting transcripts that correspond to existing genomic sequence. RNA-Seq focus more on the coding region of genes and has very low background signal [14]. Therefore, RNA-Seq is particularly attractive for non-model organisms with genomic sequences that are yet to be determined.
To date, transcriptomic sequencing technology has been widely applied for analyzing the gene expression profiles of echinoderms in a variety of developmental and physiological processes, and under multiple infected- or stressed-conditions. In the sea urchin Heliocidaris erythrogramma, RNA-seq has been used to measure the mRNA expression profiles of larvae, metamorphism and post-metamorphism life cycle stages, to elucidate the evolutional and developmental mechanism of the radial adult body in echinoderms [15]. The expression dynamics across development has also been measured by RNA-seq in three sea urchin species: the lecithotroph H. erythrogramma, the closely related planktotroph H. tuberculata, and an outgroup planktotroph Lytechinus variegatus, to reveal how evolutionary changes in gene regulation contribute to phenotypic differences between different species [16]. In addition, RNA-seq has been used to analyze the differentially expressed genes (DEGs) in the body cavity cells between the viral-infected and normal starfish Pycnopodia helianthoides, to illustrate the immune and nervous responses to the sea star wasting disease [17]. Furthermore, the next-generation transcriptome data from 42 echinoderm specimens from 24 orders, 37 families have been collected to establish a web-based application (http://echinotol.org) to identify orthologs suitable for phylogenetic analysis in assembling the echinoderm tree of life [18].
In sea cucumber, transcriptomic sequencing has been performed together with genomic and proteomic analyses in Apostichopus japonicus, to facilitate the molecular underpinnings of visceral regeneration [19]. RNA-seq has been applied independently to analyze the DEGs between normal and regenerating radial nerve cord in Holothuria glaberrima, and revealed the key roles of extracellular matrix (ECM) remodeling and ECM-cell interactions in regeneration [20]. In addition, the applications of transcriptomic analyses in sea cucumber include identification of long noncoding RNA species [21], illustration of the mechanisms of aestivation [22], abnormal development [23], and body wall pigmentation [24]. Moreover, immune-related genes in the A. japonicus coelomocytes under Vibrio splendidus challenge have been identified, which are clustered into the immune pathways of endocytosis, lysosome, chemokine, and MAPK and ERBB signaling [25]. However, there is still limited research reports on the species and response of the cytokines secreted by coelomocytes of sea cucumber under challenge of immune stimuli.
In this study, high-throughput transcriptomic sequencing and bioinformatics analysis were performed on the primary coelomocytes isolated from the sea cucumber H. leucospilota and treated with three different immune stimuli including lipopolysaccharide (LPS), polyinosinic-polycytidylic acid [poly (I:C)] and heat-inactived Vibrio harveyi for 24 h. Based on the DEGs between different groups, the immune-related pathways were screened out and the responded cytokines were identified. This study provides evidences for the potential roles of cytokines in the innate immunity of sea cucumber.
Illumina draft reads and sequence assembly
Primary coelomocytes isolated from the sea cucumber H. leucospilota were respectively challenged with LPS, poly (I:C) and heat-inactivated V. harveyi for 24 h (Fig. 1a), and twelve cDNA libraries were constructed to perform Illumina sequencing. After assembly and redundancy removal, a transcriptome with twelve RNA-seq libraries in total of 6.69 GB with 73,472 identified Unigenes was obtained and submitted to GenBank under the BioProject accession No. PRJNA559679. The total length, average length, N50, and GC content of the Unigenes were 47,163,631 bp, 641 bp, 1015 bp and 39.54%, respectively. The number of transcripts and Unigenes decreased with increasing of length, and the majority of them were concentrated in 200–3000 bp (Fig. 1b). The Unigene sequences were annotated to seven functional databases, and 20,926 (28.48%) of them were significantly matched to at least one of the databases (Table 1), in which 19,156 (26.07%) to NR, 3990 (5.43%) to NT, 14803 (20.15%) to SwissProt, 13,615 (18.53%) to KOG, 15277 (20.79%) to KEGG, 7097 (9.66%) to GO and 15,624 (21.27%) to InterPro. The annotation results for five databases were further showed in a Venn diagram: 1584, 11, 88, 21 and 528 genes were independently annotated into the NR, KOG, KEGG, SwissProt and InterPro databases, respectively, and the intersection set of these five databases was 11,103 (Fig. 1c). For the NR database, the annotated Unigenes were majorly matched to Strongylocentrotus purpuratus (5552, 28.98%), Acanthaster planci (6117, 31.93%), Saccoglossus kowalevskii (883, 4.61%), Branchiostoma belcheri (457, 2.39%) and other species (6147, 32.09%, Fig. 1d).
Experimental design and transcriptome information. a Experimental design. Sea cucumber coelomocytes isolated from H. leucospilota were challenged with LPS (10 μg/ml), Poly (I:C) (10 μg/ml) and V.harveyi (107 cell/ml) for 24 h with three biological duplicates. b The length distribution of all-Unigene. The X-axis represents the sequence size, and the Y-axis represents the number of Mix-Unigene. The orange bar shows the number of unigene which is the representative sequences, and the blue bar shows the number of transcripts which include the rough sequences. c Venn diagram of Unigene annotation. The databases used for gene annotation include NR、KOG、KEGG、SwissProt and InterPro. d Species distribution of Unigene annotation in NR database
Table 1 Functional annotation analysis
Analysis of DEGs and validation of RNA-Seq data by RT-qPCR
The transcriptomic data obtained from the control group (CT), LPS-treatment group (LPS), Poly (I:C)-treatment group (PIC) and V. harveyi-treatment group (VH) were analyzed comparatively. The results showed that the co-expressed DEGs for the three groups were 1180, and the uniquely-expressed genes in the LPS-vs-CT, PIC-vs-CT and VH-vs-CT groups were 3846, 3869 and 2279, respectively (Fig. 2a). The significantly DEGs were acquired by comparing of the gene expression between the LPS, PIC and VH groups and the CT group with the following criteria: P ≤ 0.01, |log 2-fold-change| ≥ 1 and false discovery rate (FDR) ≤ 0.05. Finally, 7074 DEGs in the LPS-vs-CT comparison (666 upregulated and 6408 downregulated), 7737 DEGs in the PIC-vs-CT comparison (355 upregulated and 7382 downregulated), and 5481 DEGs in the VH-vs-CT comparison (387 upregulated and 5094 downregulated) were obtained (Fig. 2b).
Comparative transcriptome analysis of DEGs among different immune challenges. a Venn diagram of unique and common DEGs among the immune challenges of LPS、Poly (I:C) and V. harveyi. b the number of up- and down-regulated DEGs in each immune challenge group compared with control group (the first 3 combination bar) and the pairwise comparison of the three different immune challenge groups (the last 3 combination bar). c Volcano map of DEGs in LPS-vs-CT group: X-axis represents the fold change value after log2 conversion, and Y-axis represents the fold change after -log10 conversion. Red dots represent the up-regulated DEGs, blue dots represent the down-regulated DEGs, and gray dots represents the non-DEGs. d comparison results between RNA-Seq by RT-qPCR data in LPS-vs-CT group. X axis shows the fold changes of gene expression in RT-qPCR data, while Y axis represents the fold changes of gene expression in RNA-Seq data
To validate the gene expression results of the RNA-seq data, 13 significant DEGs from the LPS-vs-CT comparison with the criteria of P < 0.01, FDR < 0.05 and fold change of at least 4 were selected for RT-qPCR validation. The volcano plot for the CT-vs-LSP comparison was shown in Fig. 2c. The expression levels of the selected 13 genes in the LPS challenge group were normalized to the control group. As anticipated, the RT-qPCR data showed a positive linear relationship with the RNA-Seq data (Fig. 2d), and there was no statistically significant difference between the two datasets (P > 0.05). This result suggested that the RNA-Seq was a positively related reference for expression profiling study on the whole, and the assembly quality of the sequences was desirable.
Functional classification and enrichment analysis of GO terms and KEGG pathways for co-expressed DEGs
The co-expressed DEGs after the three different immune challenges may represent the essential genes for immune defense in sea cucumber. GO (gene ontology) is a major bioinformatics initiative to unify the classification of genes and gene product attributes across all species. Among the total 1180 co-expressed DEGs, 796 were annotated with GO terms, in which 321 were annotated as "biological process", 279 were annotated as "cellular components" and 196 were annotated as "molecular functions" (Fig. 3a).
Co-expressed DEGs of functional classification. a GO classification of a co-expressed gene. The X-axis represents the enrichment factor value, and the Y-axis represents the path name. b Functional classification of KEGG of co-expressed genes. c Pathway enrichment distribution of co-expressed genes. The X-axis represents the enrichment factor value, and the Y-axis represents the path name. The color represents q-value, which is corrected p-value ranging from 0~1, and less q-value means greater intensiveness. The size of the point represents the number of DEGs (the larger the point, the larger the number; the smaller the point, the smaller the number). Rich Factor refers to the enrichment Factor value, which is the quotient between the foreground value (number of DEGs) of a certain pathway on the annotation and the background value (number of all genes) of a certain pathway on the annotation. The larger the data is, the more obvious the enrichment result will be. d GO classification and enrichment of differentially expressed genes under different immune challenges. The Y axis (horizontal direction) represents the number of genes, and the X axis (vertical direction) represents the specific classification under the three functional categories of GO. The red bars represent the GO classification entries annotated by the DEGs in LPS compared to the control group, and accordingly, the purple and blue represent those of Poly (I:C) and V. harveyi
KEGG (Kyoto Encyclopedia of Genes and Genomes) is a reference database dealing with genomes, biological pathways, diseases, drugs, and chemical substances. In this case, 1003 co-expressed DEGs were annotated in KEGG, and the number of DEGs in cellular processes, environmental information processing, genetic information processing, human diseases, metabolism and organismal systems categories were 123, 113, 20, 344, 161 and 242, respectively (Fig. 3b).
Gene set enrichment analysis (GSEA, also called functional enrichment analysis) is a method to identify classes of genes or proteins that are overrepresented in a large set of genes or proteins and [26]. The co-expressed DEGs in this study were annotated into 273 KEGG pathways. The top 10 and top 20 significantly enriched pathways were shown in Table 2 and Fig. 3c, respectively. The pathways of Staphylococcus aureus infection, complement and coagulation cascades, and tubercuosis were the three most significantly enriched pathways, with a total of 15, 16 and 24 co-expressed DEGs annotation, respectively (Table 2). In addition, the top 10 immune-related KEGG pathways with the highest number of co-expressed genes were shown in Table 3. Within them, the most significant immune-related KEGG pathways were complement and coagulation cascades, which contained 84 annotated genes with 16 co-expressed DEGs. These results laid a foundation for discovering the immune-related genes and developing the immune responding mechanisms of sea cucumber. The functional classification and enrichment analysis of GO terms and KEGG pathways for the DEGs were further performed for the challenges of different immune stimuli (Fig. 3d). In this case, the totals of 2591, 3260 and 2585 Unigenes respectively for the comparisons of the LPS-vs-CT, PIC-vs-CT and VH-vs-CT were annotated in GO terms. Among three different immune challenge groups, the Poly (I:C) treatment group had the most DEGs annotated into the GO terms, indicating that more genes were involved in the administration of Poly (I:C) in the sea cucumber coelomocytes when compared to other two immune stimuli.
Table 2 The top 10 KEGG pathways with the highest enrichment of co-expressed DEGs
Table 3 The top 10 immune-related KEGG pathways with the highest number of co-expressed DEGs
The most significant enrichment pathway of DEGs in different immune challenges
A hypergeometric test was used for enrichment analysis of all signal pathways in the KEGG database. Compared to the control group, the number of DEGs annotated to specific pathways for the LPS, Poly (I:C) and V. harveyi treatment groups were 3415, 4261 and 3120, respectively. The top 10 most significantly enriched pathways for each challenge were shown in Table 4, in which the common pathways for the three treatments groups were the ABC transporter pathway, ubiquitin-mediated proteolysis pathway and inositol phosphate metabolism pathway. The glucagon signaling pathway was a common pathway for the Poly (I:C) and V. harveyi treatment groups, and other pathways were specific for each treatment. Analysis of the DEGs enriched KEGG pathways provides an effective basis for studying the immune defense process, biological function and metabolic pathways in the sea cucumber coelomocytes.
Table 4 The top 10 KEGG pathways with the highest enrichment of DEGs in groups with different immune challenges
Analysis of immune-related pathway in different immune challenges
Compared to the control group, the DEGs of the LPS, Poly (I: C) and V. harveyi treatment groups were categorized into 308, 316 and 305 annotated KEGG pathways, respectively. Accordingly, 18, 22 and 23 significantly enriched immune-related pathways for LPS, Poly (I: C) and V. harveyi treatments, respectively, were identified according to the immune system of KEGG pathways. The most significantly enriched immune-related pathways for LPS, PIC and V. harveyi treatments were the Th1 and Th2 cell differentiation signaling pathways, the intestinal immune network for IgA production pathway, and the Fc gamma R-mediated phagocytosis pathway, respectively. Other top 10 significantly enriched immune-related pathways for the three immune challenges are shown in Table 5. Base on the analysis of different regulatory pathways of the DEGs in KEGG, the mechanism of cellular immune response of the sea cucumber coelomocytes can be understood more directly.
Table 5 The top 10 immune-related KEGG pathways with the highest enrichment of DEGs in groups with different immune challenges
Identification of cytokines and their expression analysis after different challenges
Cytokines are a broad and loose category of small proteins (~ 5–20 kDa) that are important for cell signaling, especially the immune signaling. Twenty-one cytokines were selected according to the NR database annotation of the transcriptomic data. The identified cytokines belong to four cytokine families, namely, the B-cell lymphokine (BCL/CLL), erythroid differentiation-related factor 1-like (EPRF1), interleukin 17-like (IL-17) and thrombospondin-like (TSP/TPO) families. In our transcriptome for the sea cucumber H. leucospilota, the BCL/CLL family included CCL11A, BCL3-X3, BCL10, CLL7A, CLL9-X3a, CLL9-X3b, CLL9-X3c, and CLL9-X3d; the EPRF1 family included EDRF1a, EDRF1b, EDRF1c, and EDRF1d; the IL-17 family included IL-17, IL-17-2, IL-17B, and IL-17C/E; and the TSP/TPO family included TPO-I-7A1–3, TPO-I-7B1–3, TSP-1a-c, and TSP-4. Among these cytokine families, the expression levels of the four genes in the interleukin-17 family after LPS challenge were significantly higher than those of other cytokines (Fig. 4), indicating that IL-17 was an important family of the cytokines in the immune response of sea cucumber, and its effective mechanism needs to be further investigated.
Changes in expression of cytokine genes upon immune challenges of LPS, Poly (I: C) and V. harveyi. a The periphery of the circle is the X-axis which presents cytokine genes, while the radius of the circle is the Y-axis which presents the log 2 fold changes value of LPS, Poly (I:C) and V. harveyi immune challenges of corresponding cytokine genes. The closer from the edge of the circle the higher the up regulated expression of corresponding gene is, and the closer from the center of the circle the higher the down regulated expression of corresponding gene is. b two-dimensional hierarchical clustering performed on the clusters of cytokine genes FPKM under three different stimulis. The pink and blue color represents the up- and down-regulation, respectively
Phylogenetic and structural domain analysis of the selected cytokines
A phylogenetic tree was constructed using a maximum-likelihood (ML) method under MEGA7.0 with the deduced amino acid sequences from the selected cytokines, including BCL/CLL, EPRF1, IL-17 and the TSP/TPO family (Fig. 5a). The result showed that the cytokines were clustered into the corresponding branches.
Bioinformatics analysis of selected cytokines. a Phylogenetic analysis of the selected cytokines family with maximum-likelihood (ML) method. b The structural domains of some of the cytokines
The structural domains for some of the selected cytokines were predicted and shown in Fig. 5b. Specially, the members of TSP/TPO family contain repeated type 1 thrombospondin domains. The BCL/CLL family cytokines contain a conserved N-terminal domain that is found in the BCL7 family. The predicted IL-17, IL-17-2, IL-17B, and IL-17C/E in the IL-17 family commonly contain a cysteine knot fold domain.
Transcript expression of cytokines in the coelomocytes with dosage-increased LPS treatment
The selected cytokines were applied to study their expressing upon LPS treatments of increased concentrations of 10.0 μg/ml, 20.0 μg/ml and 50.0 μg/ml. As the result, the mRNA levels of IL-17 cytokines family were significantly upregulated after 10 μg/ml LPS challenge, and their expression levels generally showed a trend of first increasing, then decreasing, and finally stabilizing with the increasing of LPS concentration (Fig. 6). In the BCL/CLL family, with the increase of LPS concentration, BCL10, CLL7A, CLL9-X3b and CLL9-X3c showed a dose-dependent increasing expression pattern, similar to the expression patterns of TPO-I-7A2 and TPO-I-7A2 in the TSP/TPO family. In contrast, CCL11A, BCL3-X3, CLL9-X3a and CLL9-X3d were expressed in a dose-dependent descending pattern, similar to the expression trends of TPO-I-7B1–3 and TSP-4 in the TSP/TPO family. In the EPRF1 family, EDRF1a, EDRF1b, EDRF1c, and EDRF1d showed similar parallel expression patterns, which first increased, then decreased, and finally stabilized with increasing of LPS concentrations (Fig. 6).
Dose-increased expression of cytokine genes. Transcriptional expression of H. leucospilota cytokines gene, with the β-actin as reference gene, in the sea cucumber coelomocytes treated respectively with LPS concentration of 10.0 μg/ml, 20.0 μg/ml, 50.0 μg/ml for 24 h. The data presented are expressed as the mean ± S.E. (n = 3). The same letter in the experimental groups represent a similar level (p > 0.05, ANOVA followed by Fisher's LSD test)
Based on Illumina HiSeq2500 sequencing, a transcriptome for the sea cucumber primary coelomocytes under challenges of LPS, Poly (I: C) and V. harveyi was obtained in this study. Without referenced genomic data, the assembled Unigene sequences were annotated using the databases of nucleotides and genomes of other species. There are 28.98 and 31.91% of the unigenes that annotated to the NR database were matched to the sequences from S. purpuratus and A. planci, respectively. One of the possible reason is that the first two reference genomes databases were supplied in these two species [27, 28]. However, other two studies for the genome of the sea cucumber A. japonicas have been published recently [19, 29], which can provide more conveniences for annotating the genes in the transcriptome of other sea cucumber speices in the future.
A Unigene in the transcriptomic data is declared differentially expressed if a difference observed in read counts between the experimental and control conditions is statistically significant. Thirteen Unigenes were selected from the DEGs for RT-qPCR verification (Fig. 2c & d). The results showed that the trends of gene expression by RT-qPCR were consistent with those by RNA-seq analysis, verifying the credibility of the RNA- seq results.
The co-expressed DEGs in all three groups with different immune challenge [LPS, Poly (I: C) and V. harveyi] may speculated to be the essential genes of the immune defense in sea cucumber, while the DEGs unique to a kind of immune stimuli may specifically respond to the corresponding stimuli. LPS is the main component of the wall of gram-negative bacteria, as a typical endotoxin, can bind to the CD14/TLR4/MD2 complexes in many cell types, especially monocytes, dendritic cells, macrophages and B cells, which can promote the secretion of pro-inflammatory cytokines, nitric oxide and eicosenoic acid, leading to a series of immune responses in the host [30]. In the LPS-treatment group, the Th1 and Th2 cell differentiation pathway was the most significantly enriched immune-related pathway. Poly (I:C), structurally similar to double-stranded RNA [31], is an immune activator that is used to mimic viral infection, and it is known to interact with TLR3, which is expressed on the cell membrane of B cells, macrophages and dendritic cells. In the Poly (I:C) treatment group, the intestinal immune network for IgA production pathway was the most significantly enriched immune-related pathway. V. harveyi is a gram-negative marine bacterium that is pathogenic to commercial aquaculture species, including shrimp, fish, and sea cucumber [32]. In the V. harveyi treatment group, the Fc gamma R-mediated phagocytosis pathway was the most significantly enriched immune-related pathway. Annotation of the DEGs into the immune-related pathways provides a basis for the research of immune molecular mechanism of echinoderms.
For analysis of DEGs in the coelomocytes with different immune challenges, the results showed that compared with the control group, more down-regulated DEGs than up-regulated DEGs were presented in the groups challenged with three different immune stimuli (Fig. 2b), indicating that the expression for a majority of genes were suppressed by immune challenge. The top 10 significantly enriched KEGG pathways (Tables 2 and 4) and immune-related KEGG pathways (Tables 3 and 5) for common and specific immune challenged DEGs were further screened out. Based on the transcriptomic data in this study, the cDNAs of H. leucospilota Fas-associated death domain (FADD) have been cloned with functional characterization [33] It has been reported that there are many humoral immune factors in the coelomic fluid of echinoderms that can recognize and attack the invaded pathogens, including different types of lectin, lysozyme, hemolysin, hydrolase, phenol oxidase, superoxide dismutase, nitric oxide synthase and complement-like factor [6, 9, 34,35,36]. Similarly, a number of humoral immune factors in the corresponding immune-related pathways (Table 5) were identified in the current study.
The top 10 KEGG pathways for the most significant DEG enrichment after different immune challenges also included amino acid and carbohydrate metabolic pathways, such as alanine, aspartate and glutamate metabolism pathway, amino sugar and nucleotide sugar metabolism pathway, and glycosaminoglycan biosynthesis-heparan sulfate/heparin pathway (Table 4). A similar study performed in in the sea cucumber A. japonicas has reported that the arginine metabolic pathway is related to the pathogenic challenge with a dose-dependent manner [37]. Combined with our current study, it is speculated that the metabolism of some amino acids is related to the host immune response in echinoderm, and the regulatory network between immunity and metabolism is more complicated than known now.
Based on the DEGs identified in the transcriptome of sea cucumber coelomocytes challenged with immune stimuli, twenty-one candidate genes of immune cytokines were selected for analyzing the cytokine response of sea cucumber against invaded pathogens. The selected cytokines belong to four cytokine families, namely, the TSP/TPO, BCL/CLL, EPRF1 and IL-17 families (Fig. 5a). Among these gene families, the expression levels of 4 genes in the IL-17 family after LPS challenge were significantly higher than those of other cytokines. By further confirmation with RT- qPCR of the expression levels of 4 IL-17 s generally showed a trend of first increasing, then decreasing, and finally stabilizing with incubation of LPS with increasing concentrations (Fig. 6). IL-17 is a kind of potent proinflammatory cytokine produced by activated memory T cells in mammals [38]. The IL-17 family (of which there are 6 known members, termed IL-17A to IL-17F) is thought to represent a distinct signaling system that appears to highly conserved across vertebrate evolution [38]. IL-17 family members play an active role in inflammatory diseases, autoimmune diseases and cancer [39]. Based on the identification of differentially expressed cytokine genes in the sea cucumber coelomocytes under immune challenges, this study may provide an evidence that many kinds of cytokines interact with each other, and play a complex and essential role in the innate immunity of sea cucumber.
At present, more and more studies on the immune system of the sea cucumber have been developed at both the molecular and cellular levels, which were based on the basal information for immune-related genes that provided by the transcriptomic studies [25]. However, the specific immune defense mechanism in the effector coelomocytes is still unclear. In this study, high-throughput transcriptome sequencing and bioinformatics analysis were performed on coelomocytes treated with three different immune stimuli (LPS, Poly (I:C) and Vibrio harveyi). Analysis of the results could elucidate the molecular mechanism for the immune response in the sea cucumber coelomocytes to the pathogenic/immune challenge, laying a foundation for the future identification of immune-related genes and characterizations of immune-responded mechanisms of echinoderm.
In this study, comparative transcriptome analysis of DEGs among LPS (10 μg/ml), Poly (I:C) (10 μg/ml) and heat-inactivated Vibrio harveyi (107 cells/ml) immune stimuli of sea cucumber H. leucospilota coelomocytes revealed the DEGs and immune-related pathways that are crucial for research of molecular mechanisms related to the immune response in echinoderms. Twenty-one cytokine candidate DEGs were identified, which belong to 4 cytokine families, namely, BCL/CLL, EPRF1, IL-17 and TSP/TPO. Among them IL-17 family cytokines were significantly upregulated after 10 μg/ml LPS challenge for 24 h in response to LPS dose-increased treatment (0, 10, 20 and 50 μg/ml), which provide new insights for the echinoderm cytokine response during immune challenge.
Animals used in this research were obtained from commercial sea cucumber catches, therefore approval from any ethics committee or institutional review board was not necessary. A total of nine healthy H. leucospilota weighing 190–210 g were obtained from the Daya Bay in Guangdong province, China, and maintained in a seawater aquarium with aerated and filtrated seawater (30‰ salinity) at 32 °C for 1 week before the experiments were performed.
Isolation, primary culture and static incubation of coelomocytes
Given that coelomocytes are considered as the immune effector cells in echinoderms [10], the coelomocytes from H. leucospilota were applied to explore the response of sea cucumber after challenge of immune stimuli. Isolation of primary cells and static incubation of coelomocytes were performed as previously reported with some modifications [40, 41]. Briefly, three sea cucumbers were washed with sterile DEPC water three times before dissection on ice. The coelomic fluids were sieved through a 150-mesh cell sieve to remove large tissue debris, mixed with the prechilled anticoagulant solution (20 mM EGTA, 480 mM NaCl, 19 mM KCl and 68 mM Tris, pH 7.6) in a 1:1 volume ratio. The cell suspension was filtered through a 100-μm nylon mesh, and the cells were collected by centrifugation at 500×g at 4 °C for 10 min. The harvested cells were washed twice with 30 ml isotonic buffer (1 mM EGTA, 530 mM NaCl and 10 mM Tris, pH 7.6) and resuspended in 10 ml Leiboviz's L-15 cell culture medium (Gibco BRL, USA; containing 390 mM NaCl, 100 U/mL penicillin and 100 μg/ml streptomycin, pH 7.6) and filtered through a 40-μm nylon mesh to remove the cell clusters. The cell yield was counted with a 0.10-mm hematocytometer, and only the cell preparations with greater than 95% viability, assessed by trypan blue exclusion assay, were used in subsequent experiments.
The cell suspensions were diluted to 0.1 × 106 cells/ml and seeded onto two 24-well culture plates (1 ml/well) that were precoated with ploy D-lysine. The isolated coelomocytes were incubated at 28 °C for another 18 h for recovery. On the following day, 1 ml of immune stimulus prepared in Leiboviz's L-15 medium was gently overlaid onto the cells after removal of the old medium. The immune stimuli used in this study included: LPS (10 μg/ml), Poly (I:C) (10 μg/ml) and heat-inactivated Vibrio harveyi (107 cells/ml), and fresh Leiboviz's L-15 medium was used as a control. For RNA sequencing, the cells were harvested at 24 h after incubation of tested substrates. In this case, cells from four wells were pooled together as a biological duplicate, and three duplicates for each group were sequenced and analyzed.
RNA extraction, library construction and sequencing
Total RNA from each sample was extracted using TRIzol Reagent (Invitrogen, USA). The quality and concentration of total RNA were determined with NanoDrop (Thermo Scientific, USA), and the RNA integrity value (RIN) was checked using the RNA 6000 Pico LabChip on an Agilent 2100 Bianalyzer (Agilent, USA). The RNA-seq library was constructed in six steps: 1) ployA-tailed mRNAs were enriched by oligo (dT) selection; 2) the obtained mRNAs were fragmented and reverse transcribed into double-stranded cDNA (dscDNA) by N6 random primer; 3) the cDNA ends were repaired by 3′-adenylation and adaptor ligation; 4) the ligation products were amplified by PCR; 5) the PCR products were heat-denatured and the single-stranded DNAs were cyclized; and 6) sequencings were performed on an Illumina HiSeq2500 (Illumina, USA).
De novo assembly and Unigene functional annotation
The raw sequencing data were first filtered by using SOAPnuke (v1.5.2) software under the parameters of -l 15 -q 0.2 -n 0.1 to remove the reads in which unknown bases (N) are more than 5%. The low quality reads, which were defined by the base qualities for more than 20% of them were less than 15, were then removed. After reads filtering, de novo assembly was performed using Trinity2.0.6 with clean reads [42]. Tgicl2.0.6 was used on cluster transcripts to remove redundancy and get Unigenes [43]. After assembly, the Unigenes functional annotation were performed with seven functional databases, namely NR, NT, GO, KOG, KEGG, SwissProt and InterPro. Specifically, We use Blastn 2.2.23 [44], We use Blastx 2.2.23 [44] or Diamond 0.8.31 [45] align Unigenes to NT, NR, KOG, KEGG and SwissProt database to do the annotation, use Blast2GO 2.5.0 [46] with NR annotation to do the GO annotation, and use InterProScan5 5.11–51.0 [47] to do the InterPro annotation, under default parameter.
Quantification of gene expression level and identification of DEGs
Clean reads of each sample were mapped to the Unigenes with Bowtie2 2.2.5 [48], and the gene expression levels were calculated with RSEM 1.2.12 (RNA-Seq by Expectation-Maximization) to obtain the FPKM (Fragments Per Kilobase of exon model per Million mapped reads) value [49]. The DEGseq algorithm was used to detect the differences of gene expression between different groups. To improve the accuracy of DEGs, genes with over two-fold changes (log2 ratio ≥ 1) and q-value < 0.001 were considered as significantly DEGs.
Screening and analysis of immune-related pathways
Based on Wallenius non-central hyper-geometric distribution [50], GO enrichment analysis of the differentially expressed genes (DEGs) was implemented by the GOseqR packages 1.10.0, which can be adjusted for gene length bias in DEGs. KOBAS (KEGG Orthology-Based AnnotationSystem) software was used to test the statistical enrichment of DEGs in KEGG pathways. The FDR correction was performed for p-value, and pathways with FDR < 0.01 were regarded as significantly enriched pathways. The top 10 immune-related pathways significantly enriched in the groups treated with different stimuli were selected by the immune-related annotation of the KEGG pathways.
Validation of differentially expressed genes by RT-qPCR
Significant DEGs (P < 0.05, FDR < 0.05, fold change ≥8) were selected for RT-qPCR analysis to validate the transcriptomic data, and corresponding primers were designed by based on the Unigenes sequences (Table 1). The primary coelomocytes isolated from another three H. leucospilotas were then challenge by LPS (10 μg/ml) for 24 h. Total RNA from each sample was extracted using TRIzol Reagent and reverse transcribed into the first cDNA using Honorll 1st strand cDNA Synthesis SuperMix for qPCR kit (Novogene, China). Finally, quantitative real-time PCRs were performed using the Unique Aptamer qPCR SYBR probe kit (Novogene). In this case, the qPCRs were performed in triplicates for cells cultured in three individual wells.
Identification and bioinformatics analysis of cytokines responding to immune challenge
According to the NR annotation of the transcriptomic data from the coelomocytes challenged by LPS, Poly (I:C) and V. harveyi, Twenty-one candidate cytokine genes were selected. The log2-fold changes of their expression levels under different challenges were presented as heatmap by the Pheatmap package using R3.5 software [51, 52].
The CDSs of the candidate cytokine genes were translated to amino acid sequences to construct a maximum-likelihood phylogenetic tree with 1000 bootstrap replicates by using MEGA 7. The structural domains of the cytokines were predicted by using the SMART program.
Measurement of cytokines mRNA levels in coelomocytes after LPS challenge
Primers for the selected cytokine genes were designed by based on their Unigenes sequences (Table 6). The primary coelomocytes isolated from the other three H. leucospilotas were obtained after treatment of LPS in gradient increased concentrations (0, 10, 20 and 50 μg/ml) for 24 h. Total RNA extraction, first-strand cDNA synthesis and RT-qPCR were performed as described above. In this case, β-actin was used as a reference gene, and the RT-qPCRs were performed in triplicates for cells cultured in three individual wells.
Table 6 Primers designed for RT-qPCR of DEGs
RNA-seq data from this article have been deposited in NCBI under the accession number of STUDY: PRJNA559679, SAMPLE: D2CT (SAMN12551561), EXPERIMENT: C1_1 (SRX6693868), RUN: D2CT1_1.fq.gz (SRR9945372), SAMPLE: D2LPS (SAMN12551562), EXPERIMENT: L2_2 (SRX6693869), RUN: D2LPS1_1.fq.gz (SRR9945371), SAMPLE: D2PIC (SAMN12551563), EXPERIMENT: P3_3 (SRX6693870), RUN: D2PIC1_1.fq.gz (SRR9945370), SAMPLE: D2VH (SAMN12551564), EXPERIMENT: V4_4 (SRX6693871), RUN: D2VH1_1.fq.gz (SRR9945369).
ANOVA:
BCL/CLL:
B-cell lymphokine
Differentially expressed genes
EPRF1:
Erythroid differentiation-related factor 1
FPKM:
Fragments per kilobase of exon model per million mapped reads
GSEA:
Gene set enrichment analysis
IL-17:
Interleukin 17
Kyoto encyclopedia of genes and genomes
KOG:
Clusters of orthologous groups for eukaryotic complete genomes
LPS:
Lipopolysaccharide
MAPK:
ML:
Maximum-likelihood
NGS:
Nonredundant
PIC:
Polyinosinic:polycytidylic acid
Poly (I:C):
Polyinosinic-polycytidylic acid
RIN:
RNA integrity value
RSEM:
RNA-seq by expectation-maximization
RT-qPCR:
Real time quantitative polymerase chain reaction
TSP/TPO:
Thrombospondin
VH:
Vibrio harveyi
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We are thankful to Mr. Zezhong, Zhang (Sino-Danish College, University of Chinese Academy of Sciences) and Dr. Hongmei, Li (Institution of South China Sea Ecology and Environmental Engineering, Chinese Academy of Sciences) for help with submitting data.
This study was supported by the National Key R & D Program of China (2018YFD0901605), the Strategic Priority Research Program of the Chinese Academy of Sciences (XDA13020205), the Science & Technology Promoting Projects for Oceanic & Fishery in Guangdong Province (SDYY-2018-01) and the Guangdong Province Program (2017B030314052, 2018A030313857, 2015A030310120, A2015230). The National Natural Science Foundation of China (41676162), the Science and Technology Service Network Initiative of Chinese Academy of Sciences (KFJ-STS-ZDTP-055). The Science and Technology Research Programs of Guangdong Province (2018A030313857). The funding bodies had no role in the design of the study, the collection, analysis, and interpretation of data, and in writing the manuscript and the decision to submit the work for publication.
Xiaofen Wu and Ting Chen contributed equally to this work.
CAS Key Laboratory of Tropical Marine Bio-resources and Ecology (LMB), Guangdong Provincial Key Laboratory of Applied Marine Biology (LAMB), South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou, 510301, PR China
Xiaofen Wu, Ting Chen, Da Huo, Zonghe Yu, Yao Ruan, Chuhang Cheng, Xiao Jiang & Chunhua Ren
University of Chinese Academy of Sciences, Beijing, 100049, PR China
Xiaofen Wu, Yao Ruan & Chuhang Cheng
Institution of South China Sea Ecology and Environmental Engineering, Chinese Academy of Sciences, ISEE, CAS, Guangzhou, PR China
Ting Chen, Da Huo, Zonghe Yu, Xiao Jiang & Chunhua Ren
South China Sea Bio-Resource Exploitation and Utilization Collaborative Innovation Center, Guangzhou, 510301, People's Republic of China
Da Huo, Zonghe Yu, Xiao Jiang & Chunhua Ren
Xiaofen Wu
Ting Chen
Da Huo
Zonghe Yu
Yao Ruan
Chuhang Cheng
Xiao Jiang
Chunhua Ren
T C designed and conducted the experiments, and revised the primer manuscript. XW analyzed the data, wrote the manuscript and carried out the main experiments. D H, Y R, C C, X J and Z Y also participated in carry out a part of the experiments and data analysis. C R supervised, conceived the experiments and corrected the manuscript. All authors read and approved the final manuscript.
Correspondence to Chunhua Ren.
Animals used in this research were obtained from commercial sea cucumber catches, therefore approval from any ethics committee or institutional review board was not necessary. No ethics approval was required for the collection or experimentation of the animal used in this study, the Holothuria leucospilota.
Wu, X., Chen, T., Huo, D. et al. Transcriptomic analysis of sea cucumber (Holothuria leucospilota) coelomocytes revealed the echinoderm cytokine response during immune challenge. BMC Genomics 21, 306 (2020). https://doi.org/10.1186/s12864-020-6698-6
Coelomocytes
Transcriptome
Differentailly expressed genes
Comparative and evolutionary genomics
Submission enquiries: bmcgenomics@biomedcentral.com


Discussion in 'Rebuildable Mods and Devices' started by SteveVape, Jan 15, 2019.
Currently have a Goon Custom 528 which is ok but have to say not amazing so wondering what people would recommend. I know I'm being a bit picky but would love it in gold too!
I tried an original Goon in x-mass and was blown away by the flavour it produces, then bought a Kennedy 25mm clone from knucklevapes and couldn't believe how good and spot on - for my taste - it is. According to some dripping gurus I know, the CSMNT, Goon and Kennedy are the 101 of drippers. Heard good things about the dead rabbit as well. When it comes to single coils RDA I can't say much, it seems that about 9000 launched in 2018 and most of them should be good.
The Squid Industries Chief King is my favorite . As the owner of multiple Goons and Recoils I've let them sit for a good while due to this one.
I had a Goon v1 24mm and a Kennedy 24mm, both original - Goon was quickly moved on as for me the Kennedy kicked its arse (except for the squonk action where the Goon was better/less leaky due to the Kennedy being under coil airflow) I've got a Kennedy 25mm now and absolutely love it, my favourite cloudy bugger RDA.
24 or 25mm Kennedy is highly recommended.
The Drop is also good kit.
And don't over look the Oumier Wasp, totty wee thing but damned finest kind kit.
I would recommend the CSMNT if you can get hold of it. If not then id'd say the drop.


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My move from Denver to New Orleans was both exciting and tragic. After months of planning, my husband, beagles and I arrived Uptown in early August … 2005. My husband was eager to begin his new future at Tulane Law School. Our eyes were bright with possibility, yet we were completely unprepared for what was to come. I barely had enough time to unpack all of my treasured belongings before the storm hit; most of them were wedding gifts that would be enjoyed much less than the lifetime that was intended. We lost everything. Because we only had a few weeks here before the storm struck, the city we now know is the one that has since been rebuilt.
We are the reNew Orleans.
For me, falling in love with the city was not easy. In fact, there were times of disdain and loathing before love grew in its place. I look back now and think, if only I knew then…it would have been so much easier. I would have saved myself from so many puzzled looks and confused nights.
The top 10 things I wish that I would have know BEFORE I moved here!
Mardi Gras does not take place in one night on Bourbon Street! It's a several week long season dedicated to debauchery, excess and family time. Yes, FAMILY time. We place our young children atop huge ladders (with a seatbelt and parent to anchor it, of course) and let random strangers throw heavy, hard shiny beads at them. All while we sip a cocktail and enjoy the company of our fellow parade goers (whether we know them or not). At the end of the night, our kids had more fun here than at Christmas! And, the good news is, they get to do it again and again for weeks! There's football in the street, music, bright lights, good convo, sweet treasures and a fun FREE time had by all. It's extremely excessive and extremely fantastic. Schools are closed, workplaces run at a minimum and we, the good people of New Orleans, enjoy all that our great city has to offer!
Taking your kids to the Quarter during the day can be fun! When we go, we stick to Decatur Street and take a candy store tour. There are a bundle of local candy makers up and down Decatur Street that will be happy to provide your little one with a sweet treat to keep them happy while you shop the local markets. On a cool day, it's a mommy favorite! Personally, I can't not get a milk chocolate and cashew tortue. On the way back to the car, you can walk by the river and enjoy the sights and sounds.
Taking your kids to feed the nutria…I mean ducks…is an experience none of you will forget! There is an AWESOME park in Metairie called Lafreniere Park. It's a sanctuary for birds that the kids love. I take my kids there to feed the roving hordes of fearless ducks, chickens, swans, turtles, egrets and … nutria. It's a great place to exercise, let the kids play, and even take a ride on the carousel. This one will be a definite favorite for your family.
Get to know your grocers. New Orleans is blessed to still have several local, family-owned grocery stores for your shopping pleasure, with each having their own specialties. For example, Dorignac's is the best place to get alcohol, beef and bakery goods. Langenstein's is best for their prepared foods ("better cheddar" dip topped with some pepper jelly… yum), fried chicken and meat department. Don't waste all your time at the corporate grocers; you'll get that small town southern service at the local grocers. I've really come to enjoy knowing my deli person and my butcher, something I never knew I was missing before.
City Park has something fun year round. StoryLand, Casino Building, Playgrounds, Putt Putt, Carousel Gardens, fishing, boats, nature walks, golf…the list goes on and on. My favorite is the Celebration in the Oaks in December. It's romantic, fun and family oriented. You can get some tasty rum punch and walk through the park. My boys love the train display. Heck, I love the train display. If you've never been, go! It's truly something you must see!
Jazz Fest is the Super Bowl of festivals, but French Quarter Fest is free! New Orleans is NEVER in shortage of fun things to do, and Jazz Fest tops most locals' list of favorites. It's becoming a religious holiday, and you must go and perhaps, bring your children! However, French Quarter Fest should not be overlooked! You don't have to pay for a ticket (so you can leave freely in the event of a toddler meltdown) and you can roam the Quarter and get EXCELLENT festival-style food from top rated New Orleans restaurants all while listening to all the music you can handle! It may not be (insert fave national band here), but who needs them!? This is New Orleans! Let your kids dance, eat and enjoy the local sounds.
Mardi Gras parades are not random. It's actually one of the more organized things that New Orleans does. Parades are run by Krewes, each one having their own traditions and scheduled parade. You'll find your favorite! If given the opportunity, don't pass up on the opportunity to ride. It's like being a rock-star for a day. I happen to enjoy NYX the most!
The Saints and LSU are a religion, not a sport, so tread lightly if you disagree. Even if you don't get it now, chances are, you will. The Saints in particular were instrumental in the city's recovery, serving as a source of PRIDE, COMMUNITY, and a sign of REBIRTH. Don't mess with them boys. Note that kids often wear their Saints or LSU clothes to school on Fridays to show support! I was not a huge football fan prior to moving here, but the gathering of the entire region every Sunday en masse all in support of a common goal eventually won my support.
For my kids, the Zoo, Aquarium, Children's Museum and such are not just places for field trips. We go often, and since the city is geographically small, it's SO easy to hop from one place to the next! If you become a member of many of the local family venues, there are even members-only events at night that make for a great family date night! For example, the Zoo hosts a Halloween party called Boo at the Zoo that is incredible for all ages.
Last, but certainly not least, most of the world is separated by the proverbial six degrees of separation. In NOLA, it's more like two. This city brings new meaning to the old saying, " it's not what you know, it's who you know." Just this past weekend, I was at a hotel at the last minute, on a busy weekend, trying to get the room we wanted. As it turned out, I knew the hotel manager's mother. We got to talking, and ultimately he was able to work some magic to help my husband and me out! He even sent a bottle of wine and fruit basket to help complete our much needed stay-cation. AMAZING! Thanks, Bradley! Most people here are extremely friendly and truly willing to help. If there is something you're struggling with resolving, ask around. Someone will likely have a solution that you wouldn't have known to consider.
Living here has been tumultuous. I've had moments of disgust quickly followed by moments of awe and admiration. We live not in a city, but rather a spirit; a tryst of good and evil that is both exciting and tragic. It reminds us of what we have to be thankful for, and we live to eat, pray and party.
Wheel Fun Rentals is Hosting a Pumpkin Patch at City Park!
Awe.. I too have left New Orleans. I feel like once this city gets ahold of you, a piece of you will forever remain! Thank you for your comment!
How do you decide where to live? There are so many options and pros and cons to all. I have a 7 th grade boy who will be going to Newman. I've looked from the West Bank to the East Bank and haven't settled on a house yet. Please help?
Hi Maria! Welcome to NOLA! I'm sure you'll find the perfect place for your family. There are lovely neighborhoods on both sides of the river. I would really make your commute to/from Newman during drop off and pickup. With construction uptown, traffic can really be horrific.
The neighborhood around Newman can be great as well. I personally spent time walking the streets in each neighborhood where I could get a "feel" for the culture. Whatever you decide, this isn't a city where you can presume the neighborhood is safe simply based on location. You really need to see it in person.
Whether you stay Uptown, Old Metairie, or the West Bank, there are so many wonderful options that fit any family! 🙂 Hope this helps!
Love this! I want to go on a Candy shop tour with you! And you know, I've lived her all my life and visited Celebration in the Oaks more times than I can count, and I had NO IDEA they sold rum punch.
I lived in New Orleans for three years and I agree with most of this article — except the part about Mardi Gras parades. I tried watching in several locations from Uptown to the Quarter, and every time I was met with drunk teens or drunker adults acting a fool and being extremely rude. I would suggest Metairie parades for a truly family-friendly experience.
Thank you for your comment! Yes, Metairie parades are a lot more tame, but I do like to take the kids to a few Uptown each year. We have had the most luck at the beginning of the route. To us, it seems that the farther down the route you go, the crazier it becomes!
WHY Did you mention the Nutria! I love New Orleans but saw those things on Tv once and I have been terrified of crossing paths with one. When I'm in New Orleans, I freak out when ever a bush rustles. If I ever seen one in person, i'd faint (Southern Bell Style). They are my biggest fear about moving there!
Hahaha! Geneva, I feel ya. They are nasty. That being said, I've never seen one by my house. I only see them at the park and in the canals. They are nasty, but if you pretend they're beavers, it's not as bad. Just avoid looking at the rat like tail! Nutria need not be a worry!
I think the last bit, about NOLA being the epitome of six degrees of separation, is so true! Really, though… all of it. Living in New Orleans can definitely be a tumultuous experience at times, because we are a city so full of culture and different personalities, but it is so, SO worth it!
Love your article! It's a great to know from someone who lived there with their kids. My husband and I are planning our moving to New Orleans for the middle of next year and we are very excited about it. My parents and my husband's parents are living there for the last ten years and our kids are very excited for living so near to their grandparents. But they seem a bit nervous about the moving at all and I hope we'll have not too many problems and go through this as smooth as possible. Liked your list and I'll have it on mind wen we are there already, it seems to be very helpful for the kids to know their new city. Thank you for sharing!
Jamie, I see your article was written back in 2013. It is really good. I found it by searching "relocating to New Orleans". The same search revealed an article comparing NYC & NOLA. The article was good but the resulting venomous dialogue threads were disturbing. Thank you for writing a sensible and relevant article. It was completely refreshing.
Most of Southern Louisiana is a swampy stinking toilet (surely including New Orleans)! The buildings, bridges and overpasses there are often besmeared with foul smelling, green to black slime that is largely pervasive, persistent and putrid. The morning air is typically befouled by this and other forms of various animal and/or vegetable matter decomposing there.
It's Hot & Humid most of the year or cool-cold and wet much of the remainder thereof. One typically has to start relying on air conditioning in the month of March and it doesn't cool off sufficiently to manage without it until some time in October. Almost 7 months of summer annually is just too much! I was born there, lived roughly 26 years there and am VERY V E R Y glad not to be living there or anywhere even near there. While I do like Southern style cooking (even though it is seldom to never actually healthy) and at one time what was good fishing that once was readily available and free there. These 2 things are definitely insufficient to make most civilized and even moderately well educated people have any valid desire to live there. With the ongoing seepage of the offshore oil well(s) …which have POISONED the seafood deriving from The Gulf permanently and causing multiple-serious health problems for anyone who eats of them regularly, I cannot, for the life of me, understand why anyone, in their right mind, would ever want to actually live there if he/she doesn't ABSOLUTELY have to. I left there in 1972 and have no desire to ever return.
This does not even begin to address the large amounts of radioactive waste materials that have been 'stored' in underground salt caves in the State that shall, in time, bring about (via inevitable leakage) the poisoning of the natural water table and locally 'drinkable' water. .


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Johnny Furdal (born 4 May 1986) is a Norwegian former professional footballer who played as a midfielder.

Career
Furdal has spent most of his career playing for Nest-Sotra. He played for the club for 11 years. Nest-Sotra retired his shirt number, 15, when he left the club in 2018.

On 26 July 2018, Furdal signed a two-and-a-half-year contract with Viking FK. He was named player of the year in the 2018 1. divisjon, after scoring 12 goals and making 14 assists. On 5 June 2020, his contract with Viking was extended until the end of the 2021 season. On 12 July 2021, he announced that he would retire at the end of the 2021 season.

Career statistics

Notes
League appearances between 2009 and 2011 are missing.

Honours
Viking
 Norwegian First Division: 2018
 Norwegian Football Cup: 2019

Individual
 Norwegian First Division player of the season: 2018

References

External links
 Profile for Viking FK

1986 births
Living people
People from Kvinnherad
Norwegian footballers
FK Haugesund players
SK Vard Haugesund players
Nest-Sotra Fotball players
Viking FK players
Norwegian Second Division players
Norwegian First Division players
Eliteserien players
Association football midfielders
Sportspeople from Vestland

redpajama_set_name: RedPajamaWikipedia


It only takes one counter example to disprove a mathematical theorem. The same is true in finance. This makes counter examples particularly useful for investors. In previous posts, I discussed Bitcoin as a possible counter example to the efficient market hypothesis view that the market price always reflects fundamental valuation. My argument was the Bitcoin had essentially no fundamental value and yet traded as high $20,000 per coin. For stock investors, a more direct example is the recent behavior of Tilray, the Canadian marijuana company. The problem is not that Tilray has no fundamental value – it clearly does. The problem is that the market cannot seem to decide what that fundamental value is, even minute to minute.
To illustrate the point, Exhibit 1 plots the minute by minute price of Tilray (in terms of Eastern time) during trading on September 19, 2018. The close on September 18 is included as the first observation. The points where the line plunges down and jumps back up are trading halts. As the exhibit shows, there were five trading halts during the day.
Of course, Tilray is an extreme example, but its existence suggests that significant mispricing is always lurking out there. The stock could not have been fairly valued at $150 and $300 on the same day without the release of fundamental information. The hard part is identifying and exploiting mispricing. For example, in the case of Tilray was it too cheap at $150 or too expensive at $300? Or maybe it was overpriced in both cases? The only way to decide is with a fundamental valuation model.


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"Follow Us"
Former Judge William "Bill" McLeod for Harris County Civil Court At Law No. 4
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Judge William "Bill" McLeod
LEGAL BRIEF IN SUPPORT OF JUDGE WILLIAM MCLEOD
Hon. Lina Hidalgo, Harris County Judge
Hon. Rodney Ellis, Precinct One Commissioner
Hon. Adrian Garcia, Precinct Two Commissioner
Hon. Steve Radack, Precinct Three Commissioner
Hon. R. Jack Cagle, Precinct Four Commissioner
1001 Preston Street
Re: Judge William "Bill" McLeod Harris County Civil Court at Law No. 4
Dear Judge Hidalgo and Honorable Commissioners,
We would like to voice our support for Judge William "Bill" McLeod. We believe the "resign-to-run" rule set forth in Article XVI, Section 65 of the Texas Constitution, as it applies to a judge running for a judicial position, is not settled law. We will outline below the basis for this position.
1. Texas courts have never ruled on the meaning of "office of profit or trust" found in Article XVI, Section 65 of the Texas Constitution, to determine if such a term applies to the judiciary.
The Texas Supreme Court has never ruled on the issue of whether Article XVI, Section 65 of the Texas Constitution requires a judge seeking a judicial office to resign. Texas appellate court cases that consider Article XVI, Section 65 do not pertain to judges running for judicial office. [1] The issue of whether Judge McLeod has automatically resigned pursuant to Article XVI, Section 65 would be a case of first impression for the Texas Supreme Court.
2. The Texas Supreme Court could easily rule that Judge McLeod never resigned. The Texas Supreme Court has strongly indicated that Article XVI, Section 65 of the Texas Constitution does not apply to judges running for judicial positions. The Court has given the following three signals that it does not:
Canon 5(3) of the Texas Code of Judicial Conduct, often called a "resign-to-run" rule, directly contradicts "resign-to-run" rule Article XVI, Section 65 of the Texas Constitution, as it is being applied to Judge McLeod
[1] Ramirez v. Flores, 505 SW2d 406 (Tex. App—San Antonio 1973) (County Commissioner running for school district trustee); Standley v. Sansom, 367 SW3d 343 (Tex. App—San Antonio 2012) (Constable running for sheriff); Bianchi v. State of Texas, 444 SW3d 231 (Tex. App—Corpus Christi-Edinburg 2014) (County Attorney running for County Court at Law judge)
In Canon 5(3) of the Texas Code of Judicial Conduct, the Texas Supreme Court states:
A judge shall resign from judicial office upon becoming a candidate in a contested election for a non-judicial office either in a primary or in a general or in a special election. A judge may continue to hold judicial office while being a candidate for election to or serving as a delegate in a state constitutional convention or while being a candidate for election to any judicial office. (emphasis added)[2]
The Texas Supreme Court explained the reasons behind Canon 5(3) in its Per Curiam Opinion Concerning Amendments to Canons 5 and 6 of the Code of Judicial Conduct dated October 30, 1997, (hereinafter "Per Curiam Op."). Canon 5(3) comes from the American Bar Association Model Code of 1990, which states:
A judge shall resign from judicial office upon becoming a candidate [in a contested election] for a non-judicial office either in a primary or in a general election [or in a special election], except that the judge may continue to hold judicial office while being a candidate for election to or serving as a delegate in a state constitutional convention if the judge is otherwise permitted by law to do so [or while being a candidate of election to any judicial office]. Per Curiam Op., pp. 1-2.
The words appearing in brackets do not appear in the ABA model code but were added by the Texas Supreme Court to Canon 5(3). These additional words provide insight into the Court's objectives. The addition of "in a contested election" allows judges to remain in office even in a non-judicial race as long as the election is uncontested—a significant departure from the way Article XVI, Section 65 has been interpreted. The section of Canon 5(3) mandating that judges running in non-judicial (contested) elections must resign, implies that judges running in judicial elections need not resign. However, the Texas Supreme Court was not content to rely on inference to guide judges running for judicial office. To further clarify its intentions, the Court added the provision that a judge could stay in office "while being a candidate of election to any judicial office".
B. The Texas Supreme Court was well aware of Article XVI, Section 65 of the Texas Constitution when it promulgated Canon 5(3). This canon appears to embody the Court's interpretation of Article XVI, Section 65 as it applies to the judiciary.
[2]The Texas Code of Judicial Conduct is issued by the Texas Supreme Court and incorporated into the TEX. GOV'T CODE, Title 2, Sub. G, App'x B. This canon was originally numbered as 5(4) but was renumbered as 5(3) by amendment dated August 22, 2002. The Texas Supreme Court has never altered the text of the canon since its adoption.
The Texas Supreme Court specifically cites Article XVI, Section 65 in its per curiam opinion.[1] The Court recites the history of the provision and notes that it was adopted in 1954 to lengthen the terms of certain elective offices from two (2) to four (4) years. District and appellate court judges were not included in the restrictions imposed by this provision because they already served four-year terms. The Court notes the disparity in the restrictions on running for office placed on judges in different courts and concludes that "nothing in the language or history of this proviso suggests that it is a conscious policy choice by either the Legislature or the people to give some judges, but not others, free reign to seek non-judicial office. This Court is free to place ethical restrictions on judges which may have the effect of precluding their seeking non-judicial office while serving on the bench." Per Curiam Op., p. 8 (emphasis added)
In bringing all of the Texas judiciary under Canon 5(3), the Texas Supreme Court relies on the Texas Constitution for its authority to efficiently administer the judicial branch. TEX. CONST. art. V, Section 31(a). The Court further states that its actions are mandated by Art. V, Section 1-a. The Court sees itself as righting an unintentional discriminatory application of the Texas Constitution's resign-to-run provision on some judges, but not others. The Texas Supreme Court makes no mention of any restriction Art. XVI, Section 65 places on judges running for judicial office. The Court only seems to acknowledge the constitutional provision's restrictions on judges seeking non-judicial office.
In adopting Canon 5(3), the Court does not appear to believe it is contradicting Article XVI, Section 65, but merely imposing the provision's restrictions on all of the judiciary. The Court's careful and thorough analysis of the need for a "resign-to-run rule" for all judges running for non-judicial offices strongly implies the Court will not hold that judges are constitutionally required to resign when they run for judicial office.
C. The Texas Supreme Court strives to explain why it believes judges may run in judicial elections without resigning. The Court distinguishes between the effect of judicial and non-judicial elections on the integrity of the judiciary and expresses concern that judges running in contested non-judicial elections damage public confidence in the judiciary.
The Court sees three problems with judges running for non-judicial offices: 1) The Code of Judicial Conduct constrains judges from stating opinions on issues because judges must preserve the appearance of impartiality and fairness. These constraints are, nevertheless, "unfair to both candidates and voters in the non-judicial campaign" because they curb debate. Per Curiam Op., p. 4. 2) Running for office entails having to raise large amounts of money to finance a campaign. 3) Contested elections may interfere with the performance of judicial duties. Per Curiam Op., pp. 4-6.
By contrast, the Texas Supreme Court has no problem with judges who want to run for judicial positions. The Court sets out how a non-judicial campaign differs from a judicial one as follows:
[3] https://www.txcourts.gov/All_Archived_Documents/SupremeCourt/AdministrativeOrders/miscdocket/97/97-9186.pdf
"First, a non-judicial campaign generally involves more public scrutiny and more diverse demands than an average judicial campaign. Second, a judge's judicial performance is almost always a pre-eminent issue in a judicial campaign, but may play little or no role in a judge's campaign for non-judicial office. Finally, all judges must run in judicial elections, and a persuasive case can be made that a judge's campaign for higher judicial office is a contribution to improving the judiciary; but judicial campaigns for non-judicial office confer no such benefit on the administration of justice. Per Curiam Op., p. 6 (emphasis added)
In other words, judges who run for contested, non-judicial elections must resign because they are not free to engage in the kinds of campaigning and fund-raising running for office requires, without risking the reputation and integrity of the judiciary. Judges running for judicial office, by contrast, need not resign because they are limited to running on their judicial skills and actually improve the judiciary when seeking higher judicial office. Based on this reasoning, the Texas Supreme Court would seem to welcome Judge McLeod's intention to run for the Texas Supreme Court while he remains on the bench.
Over the last twenty (20) years, the Court has not altered one word of Canon 5(3). Its per curiam opinion offers us an inside look at how and why it may rule. Based on the language of Canon 5(3) and the supporting per curiam opinion issued by the Court, it is reasonable to believe the Texas Supreme Court could rule that Judge McLeod has not resigned his office and is well within his rights to actively run for justice of the Texas Supreme Court while he remains on the bench. In light of this canon and per curiam opinion, we respectfully request that the Commissioners' Court vote to keep Judge William "Bill" McLeod in his position as judge of Harris County Civil Court at Law No. 4 until the Texas Supreme Court has settled the law in this matter.
Karen Turbyfill Taylor
Jay A. Taylor
Political Advertising Paid For By Judge William "Bill" McLeod Campaign. In Compliance with the Voluntary Limits of The Judicial Campaign Fairness Act.
Houston GLBT Support

redpajama_set_name: RedPajamaCommonCrawl


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The Last Kilometer (original title: L'Ultimo Chilometro) is a 2012 Italian documentary film directed by Paolo Casalis.

Summary
The film is portrait of road cycling made through 4 characters: Ignazio Moser and his father Francesco Moser, Davide Rebellin, Gianni Mura, Didi Senft.

References

2012 films
Italian documentary films
2010s Italian-language films
Documentary films about cycling


The treatment needs of each individual will vary. There is a cohesive balance between group and individual therapy. Residents have approximately 5 to 7 weekly individual sessions with their primary therapist, dietitian, clinical director and psychiatrist, in addition to close to 20 hours of group therapy, plus expressive arts, yoga and movement, physical training, and therapeutic outings.
A listing here does not necessarily mean Eating Disorders Online endorses the Eating Disorders treatment facility or practice. What works for one person, may not work for another. We simply want to offer options to you.


text:
SYDNEY FILM FESTIVAL ANNOUNCES FAMILY-FRIENDLY FILMS
BY Genvin | 26-Apr-2019
The 66th Sydney Film Festival (5 – 16 June) announces seven family-friendly films will screen at HOYTS Entertainment Quarter as part of the Family Program.
Date: 5-16 June 2019
Buy / Ticket: https://www.sff.org.au/tickets/buying-tickets/
"Kids, tweens and teens have an excellent selection of wonderful family-friendly films to look forward to, including 'The Secret Life of Pets 2', voiced by an all-star cast including Kevin Hart, Tiffany Haddish, Harrison Ford, and Patton Oswalt, and The Elephant Queen, a stunning documentary narrated by Oscar-nominated actor Chiwetel Ejiofor, about an elephant mother leading her family on an epic journey across the African plains," Family Films Programmer Clare Sawyer said.
" 'Emu Runner' follows a young Indigenous girl who forms a very special bond with a wild emu, made in collaboration with the Indigenous community of Brewarrina; 'Ringside' shines a light on the travails and triumphs of two young aspiring professional boxers from Chicago's impoverished South Side; and 'Stupid Young Heart' is a fresh, edgy drama tackling first love and politics in Northern Europe," she said.
"Other animated hits include 'Tito and the Birds', about 10-year-old Tito and his feathered friends as they save the world from an epidemic of fear; and 'Minuscule: Mandibles From Far Away', diving into the delightful world of insects," she said.
Since 2016, changes to classification restrictions have made over 50% of the Festival program accessible to audiences 15+. The return of the Youth Pass provides cheaper tickets to film lovers aged 18-24. Festival films now cost young people just $75 for a bundle of six-tickets. Tickets for under 18s are just $14.
The full Sydney Film Festival program is announced on Wednesday 8 May 2019.
Flexipasses and subscriptions to Sydney Film Festival 2019 are on sale now.
Call 1300 733 733 or visit sff.org.au for more information.
Contemporary indigenous art at its finest...A rare exhibition and sale of works from the Hank Ebes collection.
One of Australia's foremost art collectors and authority on Contemporary Indigenous Art, Hank Ebes, offers some of his favorite paintings from his extensive and valuable private collection for exhibition and sale at Gallery Elysium.
THE LAST FOR 2019 , GREAT ITEMS FOR CHRISTMAS AND THE NEW YEAR AT AMANDA ADDAMS ON DECEMBER 15TH
Christmas Auction with something for everyone, a must to view and not to be missed, majority will be sold unreserved, it is the first time ever we are holding an auction at our office/shop at 75a Tucker Road Bentleigh.
Sunday 15th December 2019 at 1 pm. Important and outstanding onsite contents auction. Onsite at 75 a Tucker Road, Bentleigh, Victoria.Viewing: Saturday 14th December 11 am to 5 pm- Sunday 15th December 10am -1 pm. Under instructions from Estate of Helen Halpin, Brighton East, St Marks Church, Collingwood, Collections from homes in Bentleigh East, Kew, Brighton and Toorak


text:
Search New Republic Liberia
About New Republic Liberia
STATEMENT BY LIBERIAN WOMEN LEADERS NETWORK (LWLN) On RESOURCE ALLOCATION TO THE OFFICE OF THE VICE PRESIDENT OF LIBERIA
Share this...FacebookGoogle+TwitteremailLinkedinPinterestJanuary 17, 2020 The Strategic Steering Committee of the Liberian Women Leaders Network (LWLN) is in receipt ...
We Tell You Exactly About Sexual Difference and Ontology
Share this...FacebookGoogle+TwitteremailLinkedinPinterest We Tell You Exactly About Sexual Difference and Ontology To also suggest talking about intimate huge ...
New research finds homosexual and bisexual guys have actually diverse intimate repertoires
Share this...FacebookGoogle+TwitteremailLinkedinPinterest New research finds homosexual and bisexual guys have actually diverse intimate repertoires One of the primary ...
The SC430 gets the biggest boot associated with lot, the flexibility associated with foldable metal roof and also by far the most effective audio system
Share this...FacebookGoogle+TwitteremailLinkedinPinterestThe SC430 gets the biggest boot associated with lot, the flexibility associated with foldable metal roof and ...
Sen. Dillon Gives US$12k To Montserrado County
Share this...FacebookGoogle+TwitteremailLinkedinPinterest–Vows To Keep His Campaign Promises By Reuben Sei Waylaun Montserrado County Senator Abraham Darius Dillon has ...
Sime Darby Plantation Completes the Divestment of its Liberia Operations
Cape Mountainians Want Sierra Leonean Nationals Out Of Elections
Rep. Kargon Calls For Dialogue
A Joint Press Statement from the More4Education Coalition Partners
STATEMENT BY CIVIL SOCIETY LEADERS IN SIERRA LEONE ON THE ARREST OF LIBERIAN CIVIL RIGHTS ACTIVIST, HENRY P. COSTA
"Weed Them Out"
By Reporter on November 4, 2019
-Morlu Tells Weah Of Incompetent Public Officials
By Esau J. Farr
The Chairman of the ruling Coalition for Democratic Change, Mulbah K. Morlu has called on President George M. Weah to weed out people he referred to as unqualified and incompetent public officials in his government.
According to the CDC chairman, the party (CDC) has observed with grave concern that there are more competent and qualified men and women of the party yet to be placed in appointed positions compared to those 'privileged' to be appointed by President Weah and subsequently confirmed by the Liberian Senate for the first two years of the CDC led government.
"Mr. President, we want to take this time on behalf of the Coalition for Democratic Change (CDC) that it is about time that you weed out people who are not prepared to implement the mandate of the party because we believe that there are even more competent people on the 'bench' waiting to serve their country compared to some of the people who are currently holding public positions," Mr. Morlu said.
He made the disclosure over the weekend when he addressed scores of supporters of the CDC in Monrovia.
The assertions come in the wake of mounting pressure on the ruling establishment to deliver to the expectations of thousands of Liberians who turned out and voted the CDC in 2017 on the basis of making a change in the governance of the country perceived to have been wrongly governed under former Liberian President, Ellen Johnson-Sirleaf.
The administration of President Weah is said to be witnessing and or experiencing continual protests from Liberians from all sectors ranging from bad governance to delay in salary payment amongst others.
The declaration from the CDC Chairman also came days after President George M. Weah is quoted as officially responding to a popular Liberian music titled, "Photo" with the President allegedly telling people who are complaining that they are not in the photo that they are not in the photo because they 'jumped from one studio to another and therefore should blame themselves and check up with the many photographers who took their photos.
The music produced by a circular Liberian musician Jonathan Kofa popularly known as 'Takun J' is a political satire that ridicules politicians and supporters who supported the ascendency of the CDC to state power, but are reportedly barely feeling any impact of the government as expected.
The CDC Chairman also intoned that people who are not true partisans of the party should be replaced with what he described partisans who are still on 'the bench' for best results.
In recent times, critics of the government have branded the Weah led administration as a total failure to the vast majority of people who voted the government to power and a complete disappointment to thousands who believe in the ideology of the party party for the past thirteen years since its establishment in Liberia. TNR
Published in Politics
About US: The New Republic Newspaper is an independent newspaper established in 2009 by a Liberian journalist, Alphonso Toweh with many years of experience for the key purpose of reporting a balanced coverage of events as well as promoting Liberia's image locally and internationally. Toweh has been working for Reuters News Agency as its correspondent since 1998 to present. In addition to that, he has served as correspondents for the following magazines: West Africa New African, Africa Week and African Observer. More to that, he worked for Radio Deutche Welle radio in Germany, Radio Netherlands and contributed to CNN, BBC News hour, BBC TV as well as Africa Confidential and Sunday Times in London. The paper has no political affiliation nor ethic lineage. The focus and primary commitment is to ensure the sovereignty of Liberia and unity for Africa. It seeks to foster human rights and freedom of the press. The New Republic is a liberal paper dedicated to upholding the tenets of democracy. It believes that state can not only create the political, social, economic and cultural spirit, but also to ensure that all human beings, irrespective of any affiliation is able to achieve its highest human potentials. The paper strives for free speech and equal opportunity for all. Importantly, it believes that the nation must intervene judiciously in the economic life, in order to minimise the adverse effects of free enterprise and ensure that less privileged people have reasonable and fair access to the basic necessities of life. By this, it would help reduce some level of threat. New Republic brings huge commitment to its readers and offers the nation the type of media that will advocate for the people and nudge our nation on the path of development and social re-engineering
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At the latest when spring cleaning comes around, you notice all these documents only "wasting" space and "gathering dust". This article will tell you what you must keep and what you can get rid of.
supplementary documents needed to understand the monetary situation.
May I store these documents electronically?
Who cares what these rules what these rules might say. I don't want to keep that junk! What will be the consequences when I dump everything?
In every business day of life, you will not feel the consequences. Any consequences will be an issue when a wage tax audit is held and the officer wants to see certain details. The consequences are that you will have a lack of evidence. You bear the burden of proving your tax situation. Not so much will your income be questioned but much more the expenses. No bill, no deduction. It's simple as that! Payroll auditing typically happens every three years.
I have been audited? Can I now throw the stuff away?
It makes sense to store these "unnecessary" details in a special room (or rooms) called "archive".
Since the documents of each business year have a different storage period, it is recommendable to store everything sorted by year of archiving.
In order to maintain an overview, do not keep anything else in that room. Practice shows that documents are to be sorted by topic / contents and then in binders in shelves in the archive.
The storing period is purposely set to 10 years in this article even though some documents only need to be saved for six years. Since the exact differentiation is ambiguous, LG2G decided to play safe and took the strictest rule. When your cellar, um archive, is already to full to the brim then consider renting storage space or as an imposition assign somebody to digitalize everything. They do not have to be produced instantly. Some other statutes might have not as long storage periods but these periods do not influence the above mentioned rules. Tax requirements, when in doubt, prevail over other!
Oh, you working in an eOffice? Fine! Then just store the unneeded data on another hard disk of an external drive and put it in the shelf. . .


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121817 Szatmáry è un asteroide della fascia principale. Scoperto nel 2000, presenta un'orbita caratterizzata da un semiasse maggiore pari a 3,1764666 UA e da un'eccentricità di 0,1923639, inclinata di 15,98944° rispetto all'eclittica.

Collegamenti esterni

Szatmáry
Corpi celesti scoperti nel 2000


text:
Q: how to use (WebAssembly) SharedArrayBuffer, if it will require cross-origin isolation? I am implementing a web app, using microservices deployed in docker, then to get some data, I need to make requests to each microservices(different ports).
My problem now : is that I need to use WASM to paint in a canvas some information I am getting from a web-socket connection from different socket servers (different ip/port).
I am getting this error:

SharedArrayBuffer is not defined

Also an explanation that says:

SharedArrayBuffer will require cross-origin isolation as of M92,
around July 2021.

But I can't do my app cross-origin isolated(I think so because of microservices and web-socket are deployed in different domains/IPS/ports).
I am using java(spring-boot), and Angular in the front-end.

redpajama_set_name: RedPajamaStackExchange