PRMT5 links lipid metabolism to contractile function of skeletal muscles.

Skeletal muscle plays a key role in systemic energy homeostasis besides its contractile function, but what links these functions is poorly defined. Protein Arginine Methyl Transferase 5 (PRMT5) is a well-known oncoprotein but also expressed in healthy tissues with unclear physiological functions. As adult muscles express high levels of Prmt5, we generated skeletal muscle-specific Prmt5 knockout (Prmt5MKO ) mice. We observe reduced muscle mass, oxidative capacity, force production, and exercise performance in Prmt5MKO mice. The motor deficiency is associated with scarce lipid droplets in myofibers due to defects in lipid biosynthesis and accelerated degradation. Specifically, PRMT5 deletion reduces dimethylation and stability of Sterol Regulatory Element-Binding Transcription Factor 1a (SREBP1a), a master regulator of de novo lipogenesis. Moreover, Prmt5MKO impairs the repressive H4R3 symmetric dimethylation at the Pnpla2 promoter, elevating the level of its encoded protein ATGL, the rate-limiting enzyme catalyzing lipolysis. Accordingly, skeletal muscle-specific double knockout of Pnpla2 and Prmt5 normalizes muscle mass and function. Together, our findings delineate a physiological function of PRMT5 in linking lipid metabolism to contractile function of myofibers.

Hepatic Acat2 overexpression promotes systemic cholesterol metabolism and adipose lipid metabolism in mice.

AIMS/HYPOTHESIS: Acetyl coenzyme A acetyltransferase (ACAT), also known as acetoacetyl-CoA thiolase, catalyses the formation of acetoacetyl-CoA from acetyl-CoA and forms part of the isoprenoid biosynthesis pathway. Thus, ACAT plays a central role in cholesterol metabolism in a variety of cells. Here, we aimed to assess the effect of hepatic Acat2 overexpression on cholesterol metabolism and systemic energy metabolism. METHODS: We generated liver-targeted adeno-associated virus 9 (AAV9) to achieve hepatic Acat2 overexpression in mice. Mice were injected with AAV9 through the tail vein and subjected to morphological, physiological (body composition, indirect calorimetry, treadmill, GTT, blood biochemistry, cardiac ultrasonography and ECG), histochemical, gene expression and metabolomic analysis under normal diet or feeding with high-fat diet to investigate the role of ACAT2 in the liver. RESULTS: Hepatic Acat2 overexpression reduced body weight and total fat mass, elevated the metabolic rate, improved glucose tolerance and lowered the serum cholesterol level of mice. In addition, the overexpression of Acat2 inhibited fatty acid, glucose and ketone metabolic pathways but promoted cholesterol metabolism and changed the bile acid pool and composition of the liver. Hepatic Acat2 overexpression also decreased the size of white adipocytes and promoted lipid metabolism in white adipose tissue. Furthermore, hepatic Acat2 overexpression protected mice from high-fat-diet-induced weight gain and metabolic defects CONCLUSIONS/INTERPRETATION: Our study identifies an essential role for ACAT2 in cholesterol metabolism and systemic energy expenditure and provides key insights into the metabolic benefits of hepatic Acat2 overexpression. Thus, adenoviral Acat2 overexpression in the liver may be a potential therapeutic tool in the treatment of obesity and hypercholesterolaemia.

Proto-oncogene FAM83A contributes to casein kinase 1-mediated mitochondrial maintenance and white adipocyte differentiation.

Family with sequence similarity 83 A (FAM83A) is a newly discovered proto-oncogene that has been shown to play key roles in various cancers. However, the function of FAM83A in other physiological processes is not well known. Here, we report a novel function of FAM83A in adipocyte differentiation. We used an adipocyte-targeting fusion oligopeptide (FITC-ATS-9R) to deliver a FAM83A-sgRNA/Cas9 plasmid to knockdown Fam83a (ATS/sg-FAM83A) in white adipose tissue in mice, which resulted in reduced white adipose tissue mass, smaller adipocytes, and mitochondrial damage that was aggravated by a high-fat diet. In cultured 3T3-L1 adipocytes, we found loss or knockdown of Fam83a significantly repressed lipid droplet formation and downregulated the expression of lipogenic genes and proteins. Furthermore, inhibition of Fam83a decreased mitochondrial ATP production through blockage of the electron transport chain, associated with enhanced apoptosis. Mechanistically, we demonstrate FAM83A interacts with casein kinase 1 (CK1) and promotes the permeability of the mitochondrial outer membrane. Furthermore, loss of Fam83a in adipocytes hampered the formation of the TOM40 complex and impeded CK1-driven lipogenesis. Taken together, these results establish FAM83A as a critical regulator of mitochondria maintenance during adipogenesis.

Dynamic Correlation Adjacency-Matrix-Based Graph Neural Networks for Traffic Flow Prediction.

Modeling complex spatial and temporal dependencies in multivariate time series data is crucial for traffic forecasting. Graph convolutional networks have proved to be effective in predicting multivariate time series. Although a predefined graph structure can help the model converge to good results quickly, it also limits the further improvement of the model due to its stationary state. In addition, current methods may not converge on some datasets due to the graph structure of these datasets being difficult to learn. Motivated by this, we propose a novel model named Dynamic Correlation Graph Convolutional Network (DCGCN) in this paper. The model can construct adjacency matrices from input data using a correlation coefficient; thus, dynamic correlation graph convolution is used for capturing spatial dependencies. Meanwhile, gated temporal convolution is used for modeling temporal dependencies. Finally, we performed extensive experiments to evaluate the performance of our proposed method against ten existing well-recognized baseline methods using two original and four public datasets.

Reduced electron transport chain complex I protein abundance and function in Mfn2-deficient myogenic progenitors lead to oxidative stress and mitochondria swelling.

Mitochondrial remodeling through fusion and fission is crucial for progenitor cell differentiation but its role in myogenesis is poorly understood. Here, we characterized the function of mitofusin 2 (Mfn2), a mitochondrial outer membrane protein critical for mitochondrial fusion, in muscle progenitor cells (myoblasts). Mfn2 expression is upregulated during myoblast differentiation in vitro and muscle regeneration in vivo. Targeted deletion of Mfn2 gene in myoblasts (Mfn2MKO ) increases oxygen-consumption rates (OCR) associated with the maximal respiration and spare respiratory capacity, and increased levels of reactive oxygen species (ROS). Skeletal muscles of Mfn2MKO mice exhibit robust mitochondrial swelling with normal mitochondrial DNA content. Additionally, mitochondria isolated from Mfn2MKO muscles have reduced OCR at basal state and for complex I respiration, associated with decreased levels of complex I proteins NDUFB8 (NADH ubiquinone oxidoreductase subunit B8) and NDUFS3 (NADH ubiquinone oxidoreductase subunit S3). However, Mfn2MKO has no obvious effects on myoblast differentiation, muscle development and function, and muscle regeneration. These results demonstrate a novel role of Mfn2 in regulating mitochondrial complex I protein abundance and respiratory functions in myogenic progenitors and myofibers.

PTEN Inhibition Ameliorates Muscle Degeneration and Improves Muscle Function in a Mouse Model of Duchenne Muscular Dystrophy.

Duchenne muscular dystrophy (DMD) is caused by a mutation of the muscle membrane protein dystrophin and characterized by severe degeneration of myofibers, progressive muscle wasting, loss of mobility, and, ultimately, cardiorespiratory failure and premature death. Currently there is no cure for DMD. Herein, we report that skeletal muscle-specific knockout (KO) of the phosphatase and tensin homolog (Pten) gene in an animal model of DMD (mdx mice) alleviates myofiber degeneration and restores muscle function without increasing tumor incidence. Specifically, Pten KO normalizes myofiber size and prevents muscular atrophy, and it improves grip strength and exercise performance in mdx mice. Pten KO also reduces fibrosis and inflammation, and it ameliorates muscle pathology in mdx mice. Unbiased RNA sequencing reveals that Pten KO upregulates extracellular matrix and basement membrane components positively correlated with wound healing and suppresses negative regulators of wound healing and lipid biosynthesis, thus improving the integrity of muscle basement membrane at the ultrastructural level. Importantly, pharmacological inhibition of PTEN similarly ameliorates muscle pathology and improves muscle integrity and function in mdx mice. Our findings provide evidence that PTEN inhibition may represent a potential therapeutic strategy to restore muscle function in DMD.

Regulation of apoptosis by Pacific oyster Crassostrea gigas reveals acclimation strategy to CO2 driven acidification.

Ocean acidification (OA) has posed formidable threats to marine calcifiers. In response to elevated CO2 levels, marine calcifiers have developed multiple strategies to survive, such as taking advantage of apoptosis, but its regulation mechanism remains largely unknown. Here, we used the Pacific oyster Crassostrea gigas as model to understand the apoptotic responses and regulation mechanism at short- (7 d) to long-term (56 d) CO2 exposure (pH = 7.50). The apoptosis of hemocytes was significantly induced after short-term treatment (7-21 d) but was suppressed under long-term CO2 exposure (42-56 d). Similarly, caspase-3 and caspase-9 were also increased post short-term exposure and fell back to normal levels after long-term exposure. These data together indicated diverse regulation mechanisms of apoptosis through different exposure periods. Through analysis of the B-cell lymphoma 2 (Bcl-2) family mitochondrial apoptosis regulators, we showed that only CgBcl-XL's expression kept at high levels after 42- and 56-day CO2 exposure. CgBcl-XL shared sequence, and structural similarity with its mammalian counterpart, and knockdown of CgBcl-XL in hemocytes via RNA interference promoted apoptosis. The protein level of CgBcl-XL was significantly increased after long-term CO2 exposure (28-56 d), and its distribution in hemocytes became more concentrated and dense. Therefore, CgBcl-XL serves as an essential anti-apoptotic protein for tipping the balance of cell apoptosis, which may play a key role in survival under long-term CO2 exposure. These results reveal a potential adaptation strategy of oysters towards OA and the variable environment changes through the modulation of apoptosis.

Immune-related genes response to stimulation of miR-155 overexpression in CIK (ctenopharyngodon idella kidney) cells and zebrafish.

MiR-155 regulates the development of germinal-center and the generation of immunoglobulin class-switched plasma cells. However, whether miR-155 is involved in immune response in fish is still unclear. Here, CIK cells transfected with miR-155 overexpressed plasmid inhibited mRNA expression of mIg and Rag2 (P < 0.05). Interestingly, mIg was predicted as a potential target gene of miR-155 by RNAhybrid, with a putative binding site in its CDS. Further, mIg luciferase reporter vectors with successive deletions of mIg cDNA sequence were constructed and dual luciferase reporter assay showed that vectors containing the sequence from 318 to 347 in CDS exhibited lower relative luciferase activity than others without predicted binding region (P < 0.05), which indicated mIg is the target gene of miR-155 and reveal bona fide targeted binding site of mIg for miR-155 in fish. In vivo, the zebrafish were respectively injected with miR-155 overexpressed and empty vector, and showed that miR-155 efficiently expressed in zebrafish (P < 0.01), which consistently decreased mRNA level of immune-related genes, including mIg (P < 0.01), sIg (P < 0.05), AID (P < 0.01), PU.1 (P < 0.05) and Rag2 (P < 0.05) at d 3 and d 6 post injection, comparing to control. Collectively, this work indicates that overexpression of miR-155 suppresses the mRNA level of immune-related genes in CIK cells and zebrafish, and mIg is a novel target gene of miR-155 in fish. These findings provide an insight into the miR-155 modulating adaptive immunity in grass carp and zebrafish.

A novel LRR and Ig domain-containing protein could function as an immune effector in Crassostrea gigas.

A variety of combinations of leucine-rich repeat (LRR) and immunoglobulin-like (Ig) domains have been found and discovered in invertebrates and vertebrates, but the functions remain largely unexplored. In the present study, a novel LRR and Ig domain-containing protein (LRRIG), CgLRRIG-3, was identified and characterized from oyster Crassostrea gigas. It contained two typical LRR motifs, a LRRNT motif and an Ig domain and PSI-BALST and phylogeny analysis revealed that the sequence of CgLRRIG-3 was most related with leucine-rich repeat neuronal 1 proteins from vertebrate. Its mRNA transcripts were constitutively expressed in muscle, gill, hepatopancreas, mantle, gonad and hemocytes with the highest level in hepatopancreas. The mRNA expression level of CgLRRIG-3 in hemocytes could respond to the stimulations of variety PAMPs including lipopolysaccharide (LPS), peptidoglycan (PGN), glucan (GLU) and polyinosinic-polycytidylic acid (poly I:C). The recombinant proteins exhibited a wide PAMP binding repertoire to four typical PAMPs and could significantly induce the expression of CgTNF-1 and CgIL17-5 as well as increase phagocytosis in primary cultured oyster hemocytes. In hepatopancreas, CgLRRIG-3 was mainly distributed in the basolateral membrane of digestive tubule and the hemocoel sinusoid between the digestive tubules. And in hemocytes, the positive signal was mainly distributed in a special group of granulocytes. These results collectively indicated that CgLRRIG-3 could not only function as an immune effector.

The activated ß-integrin (CgßV) enhances RGD-binding and phagocytic capabilities of hemocytes in Crassostrea gigas.

Integrins are an important family of cell receptors that can bind foreign particles and promote cell phagocytosis after they are activated. In the present study, a novel ß integrin was identified from pacific oyster Crassostrea gigas with an extracellular domain, a single transmembrane segment, and a short cytoplasmic domain. It was phylogenetically clustered with phagocytosis-related insecta ßV, and designated as CgßV. CgßV shared a conserved NPX[Y/F] motif related to integrin activation with other phagocytosis-related ß integrins. The mRNA transcripts of CgßV were widely detected in oyster tissues including hemocytes, gonad, adductor muscle, mantle, gill, and hepatopancreas, and the expression level in hemocytes was significantly up-regulated at 12 h after lipopolysaccharide (LPS) stimulation (p < 0.05), which was 2.29-fold higher than that in the control group. CgßV proteins were mainly observed on the hemocytes surface. The oyster hemocytes were found to bind fluorescein isothiocyanate (FITC)-labeled Arg-Gly-Asp-containing peptides (RGDCPs), and the binding capability was significantly up-regulated with the peak percentage of 37.6% at 12 h post LPS stimulation, which was higher than that in the control group (8.8%, p < 0.05), suggesting the activation of RGD-binding integrins on oyster hemocytes surface. The label-free RGDCPs and anti-CgßV antibody inhibited the binding capability of hemocytes towards FITC-labeled RGDCPs, which were significant lower in RGD blocking group (7.4%, p < 0.05) and anti-CgßV blocking group (22.1%, p < 0.05) than that in the control group (37.6%), indicating that CgßV could be a RGD-binding integrin. Phagocytosis assay demonstrated that LPS could enhance the phagocytosis of hemocytes towards Escherichia coli and fluorescent beads with the phagocytic rate (PR) of 18.3% and 17.4%, and phagocytic index (PI) of 5.29 and 37.71, respectively, which were significant higher than that in the control group (11.0% and 3.65 for E. coli, 9.8% and 29.26 for fluorescent beads, respectively, p < 0.05). In addition, both the label-free RGDCPs and anti-CgßV antibody significantly hindered the phagocytosis of hemocytes towards E. coli and fluorescent beads. After the E. coli and fluorescent beads were opsonized by oyster serum, the label-free RGDCPs still inhibited the phagocytosis of hemocytes towards them, while the anti-CgßV antibody could only inhibit the phagocytosis of hemocytes towards E. coli, suggesting that only the activated CgßV was involved in the enhancing phagocytosis for bacteria in oyster. Moreover, the key components of conserved integrin-mediated phagocytosis pathway including GTPases, talin proteins, Ca2+ and cAMP were all induced by LPS in hemocytes of oyster. All these results suggested that the activated CgßV enhanced RGD-binding and phagocytic capabilities of hemocytes, shedding lights on the mechanisms of integrin-mediated phagocytosis in mollusks.

A single-CRD C-type lectin (CgCLec-3) with novel DIN motif exhibits versatile immune functions in Crassostrea gigas.

C-type lectins (CTLs), as important pattern recognition receptors (PRRs), are a superfamily of Ca2+-dependent carbohydrate-recognition proteins which participate in nonself-recognition and eliminating pathogens. In the present study, a novel CTL (designated as CgCLec-3) was identified from the Pacific oyster Crassostrea gigas. There was only one carbohydrate-recognition domain (CRD) of 151 amino acid residues within the deduced amino acid sequence of CgCLec-3. The deduced amino acid sequence of CgCLec-3 CRD shared low homology with the CRDs of other CTLs in oyster with the identities ranging from 12% to 22%. A novel DIN motif was found in Ca2+-binding site 2 of CgCLec-3. The relative expression level of CgCLec-3 in hemocytes was up-regulated significantly after the stimulations of bacteria and Pathogen Associated Molecular Patterns (PAMPs). Immunohistochemistry assay showed that CgCLec-3 protein was mainly distributed in gill and mantle, less in gonad, and could not be detected in adductor muscle and hepatopancreas. The recombinant protein (rCgCLec-3) could bind lipopolysaccharide (LPS), mannose (MAN) and peptidoglycan (PGN), but not poly (I:C). rCgCLec-3 exihibited strong binding ability to Vibrio anguillarum and V. splendidus, moderate binding activities to Escherichia coli, Pichia pastoris and Yarrowia lipolytica, weak binding affinity to Staphylococcus aureus and Micrococcus luteus. rCgCLec-3 could agglutinate microorganisms, in a Ca2+-dependent manner and its activity to agglutinate V. splendidus was remarkably higher than that to agglutinate E. coli, S. aureus and P. pastoris. The phagocytic activity of oyster hemocytes was significantly enhanced after incubation with rCgCLec-3. rCgCLec-3 also exhibited antibacterial activity against E. coli and S. aureus. The results clearly suggested that CgCLec-3 in Pacific oyster C. gigas not only served as a PRR involved in the PAMPs recognition and microbes binding, but also functioned as an immune effector participating in the clearance of invaders.

The modulation of Smac/DIABLO on mitochondrial apoptosis induced by LPS in Crassostrea gigas.

The mitochondrial pathway of apoptosis is well studied as the major mechanism of physiological cell death in vertebrates. In the present study, a second mitochondria-derived activator of caspases (Smac)/direct inhibitor of apoptosis-binding protein (IAP) with low pI protein (DIABLO) (designated as CgSmac) was identified from oyster Crassostrea gigas. The open reading frame of CgSmac was of 966 bp nucleotides encoding a predicted polypeptide of 321 amino acids with a conserved Smac/DIABLO domain containing a potential IAP-binding motif of VMPV. CgSmac proteins were distributed in hemocytes and co-localized with mitochondria. Western blotting analysis revealed that CgSmac proteins mainly existed in the dimer form in hemocytes, and the monomeric precursors and mature monomers were also detected. After lipopolysaccharide (LPS) stimulation, the mRNA expression of CgSmac in hemocytes was significantly up-regulated and peaked at 6 h (12.26-fold, p < 0.05), and the protein level of its dimers was significantly up-regulated at 6 h, 12 h, 24 h, and 48 h, while that of CgSmac monomers was up-regulated at 6 h, 12 h and down-regulated at 24 h, 48 h. The decrease of mitochondrial membrane potential indicated that the occurrence of early stage of apoptosis in primary cultured hemocytes was induced by LPS, and RNA interference (RNAi) of CgSmac could not rescue this decrease. The caspase-3 activity in primary cultured hemocytes was significantly suppressed after RNAi of CgSmac. Correspondingly, the total apoptotic rate of primary cultured hemocytes was also significantly suppressed in dsCgSmac + LPS group (31.57%) compared to dsEGFP + LPS group (40.27%, p < 0.05), which in turn demonstrated the conserved pro-apoptotic function of CgSmac. Furthermore, the early apoptotic rate (10.4% vs. 8.5%, p < 0.05) was significantly higher in dsCgSmac + LPS group than that of dsEGFP + LPS group, while the necrosis (7.7% vs. 10.0%, p < 0.05) and late apoptotic rates (13.4% vs. 21.9%, p < 0.05) were lower in dsCgSmac + LPS group than those of dsEGFP + LPS group. Collectively, CgSmac could activate mitochondrial apoptosis pathway by promoting caspase-3 activity in oyster hemocytes against exogenous LPS invasion. These results provided new insights on oyster apoptosis and the immune defense mechanisms in invertebrates.

A novel brown adipocyte-enriched long non-coding RNA that is required for brown adipocyte differentiation and sufficient to drive thermogenic gene program in white adipocytes.

The thermogenic activities of brown and beige adipocytes can be exploited to reduce energy surplus and counteract obesity. Recent RNA sequencing studies have uncovered a number of long noncoding RNAs (lncRNAs) uniquely expressed in white and brown adipose tissues (WAT and BAT), but whether and how these lncRNAs function in adipogenesis remain largely unknown. Here, we report the identification of a novel brown adipocyte-enriched LncRNA (AK079912), and its nuclear localization, function and regulation. The expression of AK079912 increases during brown preadipocyte differentiation and in response to cold-stimulated browning of white adipocytes. Knockdown of AK079912 inhibits brown preadipocyte differentiation, manifested by reductions in lipid accumulation and down-regulation of adipogenic and BAT-specific genes. Conversely, ectopic expression of AK079912 in white preadipocytes up-regulates the expression of genes involved in thermogenesis. Mechanistically, inhibition of AK079912 reduces mitochondrial copy number and protein levels of mitochondria electron transport chain (ETC) complexes, whereas AK079912 overexpression increases the levels of ETC proteins. Lastly, reporter and pharmacological assays identify Pparγ as an upstream regulator of AK079912. These results provide new insights into the function of non-coding RNAs in brown adipogenesis and regulating browning of white adipocytes.

The various components implied the diversified Toll-like receptor (TLR) signaling pathway in mollusk Chlamys farreri.

Toll-like receptor (TLR) signaling pathway, composed of various components, plays pivotal roles in host innate immune defense mechanism. In the present study, twenty-nine TLR signaling pathway components, including receptors, adaptors, transduction molecules and immune effectors, were identified in Zhikong scallop Chlamys farreri via assembling and screening public available transcriptomic data and expression sequence tags (ESTs). These identified TLR signaling pathway components were constitutively expressed and detectable in various tissues, and almost all of them were highly expressed in gill and hepatopancreas. These results indicated the presence of TLR signaling pathways in both MyD88-dependent and MyD88-independent forms in scallop, and implied the diversified TLR signaling pathway in mollusk C. farreri.

Identification of a clip domain serine proteinase involved in immune defense in Chinese mitten crab Eriocheir sinensis.

Clip-domain serine proteinase is an important serine proteinase family involved in many biological processes, which is only found in invertebrates. In the present study, the full-length cDNA of a clip domain serine proteinase (designed as EsCDSP) gene was cloned from Chinese mitten crab Eriocheir sinensis using rapid amplification of cDNA ends (RACE) technique. It was of 1488 bp with an open reading frame (ORF) of 1134 bp encoding a polypeptide of 377 amino acids. There were a signal peptide, a clip domain, and a Tryp_SPc domain in the deduced amino acid sequence of EsCDSP. Highly conserved cysteine residues were identified in the clip domain and Tryp_SPc domain. EsCDSP shared similarities of 40%-61% with CDSPs from Penaeus monodon (ACP19562.1), Scylla paramamosain (CCW43200.1), Drosophila melanogaster (NP_649734.2) and Delia antiqua (AAW57295.1). It was clustered with other CDSPs from crabs in the phylogenetic tree. EsCDSP transcript was highly expressed in hemocytes and it could response to the stimulations of Vibro anguillarum and Pichia pastoris. rEsCDSP could activate proPO system and significantly increase the PO activity of HLS. In addition, rEsCDSP could bond to Aeromonas hydrophila, Vibro anguillarum and Vibro alginolyticus, and reduced the mortality rate causing by pathogen infection. All the results suggested that EsCDSP was an important immune response participator involved in activation of the proPO system of crab.

Transcriptomic analysis of exosomal shuttle mRNA in Pacific oyster Crassostrea gigas during bacterial stimulation.

As marine invertebrates, oysters lack adaptive immunity and employ innate immunity as the front line and almost the solo defense mechanism to protect them against invaders. Accumulating research achievements demonstrated that exosomes could act as innate immune effectors that contribute to host defense mechanism. To better understand the immune functions of exosomes in Crassostrea gigas against bacterial stimulation, RNA-Seq was applied to explore the global expression changes of exosomes in oyster after Staphylococcus aureus and Vibrio splendidus stimulation. Totally 171573691 single end raw reads were yielded via Ion Torrent Proton sequencing, which were trimmed into 121988325 clean reads, and then 1505 abundant exosomal shuttle mRNAs (esmRNAs) were identified. Gene ontology (GO) analysis revealed that these abundant esmRNAs could be categorized into 15 cellular components, 12 molecular functions and 21 biological processes, and these abundant esmRNAs were mapped onto 62 biological signaling pathways by KEGG. In total, 68 significant differentially expressed genes (DEGs, Fold change ≥ 2, Q-value < 0.05) were identified between S. aureus stimulated group and control group, including 21 up-regulated and 47 down-regulated ones. While 99 significant DEGs between V. splendidus challenged group and control group were identified, including 42 up-regulated and 57 down-regulated ones. To validate the transcriptomic data, 24 DEGs were randomly selected and confirmed via quantitative real-time PCR (qRT-PCR) and the results showed that their expression patterns agreed well with the RNA-Seq analysis. This study would enrich the C. gigas transcriptome database and provide insight into the immune functions of oyster exosomes against bacterial infection.

A Prokineticin (PK)-like cytokine from Chinese mitten crab Eriocheir sinensis promotes the production of hemocytes via reactive oxygen species.

Astakine is a cytokine-like factor containing a prokineticin domain, which directly participates in hematopoiesis and blood cell differentiation. In the present study, a novel Astakine gene was identified from Chinese mitten crab Eriocheir sinensis (designated as EsAst). The full-length cDNA of EsAst was of 1163 bp, consisting of a 5' untranslated region (UTR) of 120 bp, a 3' UTR of 656 bp, and an open reading frame (ORF) of 387 bp encoding a polypeptide of 128 amino acids. There were a signal peptide and a prokineticin domain with nine conserved cysteine residues in the deduced amino acid sequence of EsAst. EsAst shared higher similarity with Astakines from Penaeus monodon and Pacifastacus leniusculus, and it was closely clustered with the Astakine from shrimp P. monodon in the phylogenetic tree. The EsAst mRNA transcript was higher expressed in hemocytes and hepatopancreas. The relative expression level of EsAst in hemocytes was continuously increased from 1.5 to 48 h after Vibro anguillarum challenge compared that in the untreated control group. After Pichia pastoris GS115 challenge, the relative expression level of EsAst in hemocytes was also up-regulated. After rEsAst injection, ROS levels in HPT cells were also increased at 12 and 24 h, and the total hemocyte counts were also significantly increased at 6, 9, 12, and 24 h post rEsAst injection. The interference of EsAst expression with dsRNA injection could delay the recovery of hemocytes production post A. hydrophila stimulation. When mitochondrial complexes I was knock down by dsRNA, ROS levels were decreased and THCs were also decreased. Recovery of hemocyte production inducing by A. hydrophila stimulation and rEsAst injection were delayed with dsEsbc1 injection. When ROS levels were increased after RNAi of Lon protease, THCs were also increased. The expression levels of five genes (EsJNK, EsSTAT, EsPI3K, EsAKT1, EsP70S6K) involved in SAPK-JNK and mTOR signaling pathways were up-regulated at 12 and 24 h in rEsAst group and EsLon dsRNA group compared with that in EGFP dsRNA group, and were similar to the trend of ROS levels. These results collectively suggested that EsAst should be a novel Astakine to promote the production of hemocytes in a ROS-dependent way in E. sinensis.

A novel GATA-like zinc finger transcription factor involving in hematopoiesis of Eriocheir sinensis.

GATA transcription factor is a family of DNA-binding proteins that can recognize and bind to sequence of (A/T) GATA (A/G). In the present study, a GATA-like protein (named as EsGLP) was characterized from Eriocheir sinensis, including an 834 bp full length open reading frame of EsGLP, encoding a polypeptide of 277 amino acids. The deduced amino acid sequence of EsGLP contained one conserved GATA-type zinc finger of the form Cys-X2-Cys-X17-Cys-X2-Cys, with four cysteine sites. The EsGLP mRNA transcripts were mainly detected in the hematopoietic tissue, hepatopancreas and gonad. The recombinant EsGLP protein was prepared for the antibody production. The EsGLP protein was mainly distributed in the edge of lobules in the HPT and the cytoplasm of hemocytes. The mRNA transcripts of EsGLP in hemocytes were significantly decreased at 24 h (0.39-fold and 0.27-fold, p < .05) and 48 h (0.35-fold and 0.16-fold, p < .05) after LPS and Aeromonas hydrophila stimulation, respectively. However, one peak of EsGLP mRNA transcripts were recorded at 24 h (8.71-fold, p < .05) in HPT after A. hydrophila stimulation. The expression level of EsGLP mRNA in HPT was significantly up-regulated at 2 h, 2.5 h and 9 h (41.74-fold, 45.38-fold and 26.07-fold, p < .05) after exsanguination stimulation. When EsGLP gene expression was inhibited by the injection of double-stranded RNA, both the total hemocytes counts and the rate of EdU-positive hemocytes were significantly decreased (0.32-fold and 0.56-fold compared to that in control group, p < .05). All these results suggested that EsGLP was an important regulatory factor in E. sinensis which involved in the hemocytes generation and the immune response against invading pathogens.

Broadband perfect infrared absorption by tuning epsilon-near-zero and epsilon-near-pole resonances of multilayer ITO nanowires.

We numerically investigate the broadband perfect infrared absorption by tuning epsilon-near-zero (ENZ) and epsilon-near-pole (ENP) resonances of multilayer indium tin oxide nanowires (ITO NWs). The monolayer ITO NWs array shows intensive absorption at ENZ and ENP wavelengths for p polarization, while only at the ENP wavelength for s polarization. Moreover, the ENP resonances are almost omnidirectional and the ENZ resonances are angularly dependent. Therefore, the absorption bandwidth is broader for p polarization than that for s polarization when polarized waves are incident obliquely. The ENZ resonances can be tuned by altering the doping concentration and volume filling factor of ITO NWs. However, the ENP resonances only can be tuned by changing the doping concentration of ITO NWs, and volume filling factor impacts little on the ENP resonances. Based on the strong absorption properties of each layer at their own ENP and ENZ resonances, the tuned absorption of the bilayer ITO NWs with the different doping concentrations can be broader and stronger. Furthermore, multilayer ITO NWs can achieve broadband perfect absorption by controlling the doping concentration, volume filling factor, and length of the NWs in each layer. This study has the potential to apply to applications requiring efficient absorption and energy conversion.

Functional characterization of hemocytes from Chinese mitten crab Eriocheir sinensis by flow cytometry.

Hemocytes comprise a diversity of cell types with functional and structural heterogeneity, and they play key roles in the host defense of invertebrates. In the present study, the hemocytes from Chinese mitten crab Eriocheir sinensis were directly separated into two groups by flow cytometry. The hemocytes in P1 group were full of round and abundant granules with deeply staining cytoplasm, while P2 hemocytes were more diverse with a wide range of sizes and less granularity. Both P1 and P2 hemocytes exhibited phagocytic ability, but the phagocytic rate of P1 hemocytes increased which was significantly higher than that of P2 hemocytes after LPS stimulations. The levels of ROS production and intracellular Calcium as well as lysosome content were higher in P1 hemocytes than that in P2 hemocytes under both normal and immune-activated situations. The genes involved in phagocytosis, antimicrobial and antioxidant activities were mainly expressed in P1 hemocytes, while the genes involved in proPO activation system were highly expressed in P2 hemocytes. These results collectively suggested that P1 hemocytes were the main immunocompetent hemocytes in Chinese mitten crab and P2 hemocytes mainly participated in proPO activation system.