PRMT5 mediates FoxO1 methylation and subcellular localization to regulate lipophagy in myogenic progenitors.

Development is regulated by various factors, including protein methylation status. While PRMT5 is well known for its roles in oncogenesis by mediating symmetric di-methylation of arginine, its role in normal development remains elusive. Using Myod1Cre to drive Prmt5 knockout in embryonic myoblasts (Prmt5MKO), we dissected the role of PRMT5 in myogenesis. The Prmt5MKO mice are born normally but exhibit progressive muscle atrophy and premature death. Prmt5MKO inhibits proliferation and promotes premature differentiation of embryonic myoblasts, reducing the number and regenerative function of satellite cells in postnatal mice. Mechanistically, PRMT5 methylates and destabilizes FoxO1. Prmt5MKO increases the total FoxO1 level and promotes its cytoplasmic accumulation, leading to activation of autophagy and depletion of lipid droplets (LDs). Systemic inhibition of autophagy in Prmt5MKO mice restores LDs in myoblasts and moderately improves muscle regeneration. Together, PRMT5 is essential for muscle development and regeneration at least partially through mediating FoxO1 methylation and LD turnover.

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.

Effects of exercise combined with diet intervention on body composition and serum biochemical markers in adolescents with obesity: a systematic review and meta-analysis.

BACKGROUND: This systematic review aims to evaluate the effects of exercise combined with diet (ECWD), exercise alone, diet alone, and no intervention on body composition and serum biochemical markers in adolescents with obesity to provide reference for solving the metabolic disorders of adolescents caused by obesity. CONTENTS: Studies published before January 5, 2021 were retrieved from PubMed, Web of Science, Ovid, Cochrane Library, China National Knowledge Infrastructure (CNKI), Wanfang data, VIP database, and SinoMed. Randomized controlled trials with an age between 10 and 20 years, body mass index (BMI) ≥28 kg/m2 or ≥95th percentiles, no history of endocrine and metabolic diseases, heart disease, hematologic disease, and so on before the trial were included. SUMMARY: Fifteen of the 50,155 studies met the criteria. Meta-analysis showed that ECWD was more effective in reducing BMI (kg/m2) (-2.45 kg/m2, 95% CI: -3.06; -1.85) and fat thickness of back (-13.77 mm, 95% CI: -15.92; -11.62), abdomen (-11.56 mm, 95% CI: -14.04; -9.09), and upper arm (-14.81 mm, 95% CI: -16.74; -12.89) than other interventions; in reducing body fat (-7.03 kg, 95% CI: -9.77; -4.29) and thigh circumference (-4.05 cm, 95% CI: -5.58; -2.52), ECWD and diet alone were more effective than exercise alone; ECWD and exercise alone were more effective in reducing waist circumference (-6.05 cm, 95% CI: -8.37; -3.72), waist-to-hip ratio (WHR; -0.06, 95% CI: -0.11; -0.01), upper arm circumference (-2.57 cm, 95% CI: -3.70; -1.45), triglycerides (TG; -0.30 mmol/L, 95% CI: -0.45; -0.14), total cholesterol (TC; -0.30 mmol/L, 95% CI: -0.59; -0.01), and low density lipoprotein (LDL; -0.22 mmol/L, 95% CI: -0.40; -0.04) than diet alone. ECWD also had effects on tumor necrosis factor-α (TNF-α), interleukin-6, leptin, and so on. OUTLOOK: ECWD is more effective than exercise alone, diet alone, or no intervention in solving the problems of body shape and metabolic disorder of adolescents with obesity.

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.

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.

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.

Protein Arginine Methyltransferase PRMT5 Regulates Fatty Acid Metabolism and Lipid Droplet Biogenesis in White Adipose Tissues.

The protein arginine methyltransferase 5 (PRMT5) is an emerging regulator of cancer and stem cells including adipogenic progenitors. Here, a new physiological role of PRMT5 in adipocytes and systemic metabolism is reported. Conditional knockout mice were generated to ablate the Prmt5 gene specifically in adipocytes (Prmt5AKO). The Prmt5AKO mice exhibit sex- and depot-dependent progressive lipodystrophy that is more pronounced in females and in visceral (than subcutaneous) white fat. The lipodystrophy and associated energy imbalance, hyperlipidemia, hepatic steatosis, glucose intolerance, and insulin resistance are exacerbated by high-fat-diet. Mechanistically, Prmt5 methylates and releases the transcription elongation factor SPT5 from Berardinelli-Seip congenital lipodystrophy 2 (Bscl2, encoding Seipin) promoter, and Prmt5AKO disrupts Seipin-mediated lipid droplet biogenesis. Prmt5 also methylates Sterol Regulatory Element-Binding Transcription Factor 1a (SREBP1a) and promotes lipogenic gene expression, and Prmt5AKO suppresses SREBP1a-dependent fatty acid metabolic pathways in adipocytes. Thus, PRMT5 plays a critical role in regulating lipid metabolism and lipid droplet biogenesis in adipocytes.

Identification of a Novel Pattern Recognition Receptor DM9 Domain Containing Protein 4 as a Marker for Pro-Hemocyte of Pacific Oyster Crassostrea gigas.

DM9 refers to an uncharacterized protein domain that is originally discovered in Drosophila melanogaster. Two proteins with DM9 repeats have been recently identified from Pacific oyster Crassostrea gigas as mannose-specific binding pattern-recognition receptors (PRRs). In the present study, a novel member of DM9 domain containing protein (designated as CgDM9CP-4) was identified from C. gigas. CgDM9CP-4, about 16 kDa with only two tandem DM9 domains, was highly enriched in hemocytes and gill. The transcripts level of CgDM9CP-4 in circulating hemocytes were decreased after LPS, PGN and Vibrio splendidus stimulations. The recombinant protein of CgDM9CP-4 (rCgDM9CP-4) displayed a broad binding spectrum towards various pathogen-associated molecular patterns (PAMPs) (LPS, PGN, ß-glucan and Mannose) and microorganisms (Staphylococcus aureus, Micrococcus luteus, V. splendidus, V. anguillarum, Escherichia coli, Pichia pastoris and Yarrowia lipolytica). CgDM9CP-4 was mostly expressed in gill and some of the hemocytes. Flow cytometry analysis demonstrated that the CgDM9CP-4-positive hemocytes accounted for 7.3% of the total hemocytes, and they were small in size and less in granularity. CgDM9CP-4 was highly expressed in non-phagocytes (~82% of total hemocytes). The reactive oxygen species (ROS) and the expression levels of cytokines in CgDM9CP-4-positive hemocytes were much lower than that in CgDM9CP-4-negative hemocytes. The mRNA expression level of CgDM9CP-4 in hemocytes was decreased after RNAi of hematopoietic-related factors (CgGATA, CgRunt, CgSCL, and CgNotch). In addition, CgDM9CP-4-positive cells were found to be much more abundant in hemocytes from gill than that from hemolymph, with most of them located in the gill filament. All these results suggested that CgDM9CP-4 was a novel member of PRR that expressed in undifferentiated pro-hemocytes to mediate immune recognition of pathogens.

A requirement of Polo-like kinase 1 in murine embryonic myogenesis and adult muscle regeneration.

Muscle development and regeneration require delicate cell cycle regulation of embryonic myoblasts and adult muscle satellite cells (MuSCs). Through analysis of the Polo-like kinase (Plk) family cell-cycle regulators in mice, we show that Plk1's expression closely mirrors myoblast dynamics during embryonic and postnatal myogenesis. Cell-specific deletion of Plk1 in embryonic myoblasts leads to depletion of myoblasts, developmental failure and prenatal lethality. Postnatal deletion of Plk1 in MuSCs does not perturb their quiescence but depletes activated MuSCs as they enter the cell cycle, leading to regenerative failure. The Plk1-null MuSCs are arrested at the M-phase, accumulate DNA damage, and apoptose. Mechanistically, Plk1 deletion upregulates p53, and inhibition of p53 promotes survival of the Plk1-null myoblasts. Pharmacological inhibition of Plk1 similarly inhibits proliferation but promotes differentiation of myoblasts in vitro, and blocks muscle regeneration in vivo. These results reveal for the first time an indispensable role of Plk1 in developmental and regenerative myogenesis.

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.

Molecular characterization of a cathepsin L1 highly expressed in phagocytes of pacific oyster Crassostrea gigas.

Cathepsin L1 (CTSL1) is a lysosomal cysteine protease with a papain-like structure. It is known to be implicated in multiple processes of immune response against pathogen infection based on the proteolytic activity. In the present study, a CTSL1 homologue (designated as CgCTSL1) was identified from Crassostrea gigas. It contained a typically single Pept_C1 domain with three conserved catalytically essential residues (Gln25, His135 and Asn178). The mRNA of CgCTSL1 was ubiquitously expressed in oyster tissues with the highest expression level in important immune tissues such as gill and hemocytes. CgCTSL1 proteins were mainly detected in gill and hepatopancreas by immunohistochemistry. Recombinant CgCTSL1 (rCgCTSL1) exhibited proteolytic activity to cleave the substrate Ac-FR-amino-4-trifluoromethyl coumarin (AFC) in a dose-dependent manner, and the inhibitor could reduce its proteolytic activity. After the interference of CgCTSL1 mRNA, the proteolytic activity of oyster hemocytes was significantly down-regulated with the released AFC fluorescence value decreasing from 375.84 to 179.21 (p < 0.05). Flow cytometry analysis revealed that the expression of CgCTSL1 protein was higher in phagocytes with the mean fluorescence intensity (MFI) value of 21,187 (4.13-fold, p < 0.01) compared to the MFI value of 5,130 in non-phagocytic hemocytes. The further confocal analysis demonstrated that the actively phagocytic hemocytes with green bead signals were co-localized with stronger CgCTSL1 positive signals. The mRNA expression levels of CgCTSL1 in phagocyte-like sub-populations of granulocytes and semi-granulocytes were 298.12-fold (p < 0.01) and 2.75-fold (p < 0.01) of that in agranulocytes, respectively. Western blotting analysis of the hemocyte proteins revealed that CgCTSL1 was relatively abundant in granulocytes and semi-granulocytes compared to that in agranulocytes. These results collectively suggested that CgCTSL1, a CTSL1 homologue highly expressed in phagocyte-like hemocytes, was possibly involved in cellular immune response dependent on its conserved proteolytic activity, which might provide clues for the divergence between phagocytes and non-phagocytic hemocytes as well as the identification of promising molecular markers for phagocytes in oyster C. gigas.

Transcriptomic and Quantitative Proteomic Analyses Provide Insights Into the Phagocytic Killing of Hemocytes in the Oyster Crassostrea gigas.

As invertebrates lack an adaptive immune system, they depend to a large extent on their innate immune system to recognize and clear invading pathogens. Although phagocytes play pivotal roles in invertebrate innate immunity, the molecular mechanisms underlying this killing remain unclear. Cells of this type from the Pacific oyster Crassostrea gigas were classified efficiently in this study via fluorescence-activated cell sorting (FACS) based on their phagocytosis of FITC-labeled latex beads. Transcriptomic and quantitative proteomic analyses revealed a series of differentially expressed genes (DEGs) and proteins present in phagocytes; of the 352 significantly high expressed proteins identified here within the phagocyte proteome, 262 corresponding genes were similarly high expressed in the transcriptome, while 140 of 205 significantly low expressed proteins within the proteome were transcriptionally low expressed. A pathway crosstalk network analysis of these significantly high expressed proteins revealed that phagocytes were highly activated in a number of antimicrobial-related biological processes, including oxidation-reduction and lysosomal proteolysis processes. A number of DEGs, including oxidase, lysosomal protease, and immune receptors, were also validated in this study using quantitative PCR, while seven lysosomal cysteine proteases, referred to as cathepsin Ls, were significantly high expressed in phagocytes. Results show that the expression level of cathepsin L protein in phagocytes [mean fluorescence intensity (MFI): 327 ± 51] was significantly higher (p < 0.01) than that in non-phagocytic hemocytes (MFI: 83 ± 26), while the cathepsin L protein was colocalized with the phagocytosed Vibrio splendidus in oyster hemocytes during this process. The results of this study collectively suggest that oyster phagocytes possess both potent oxidative killing and microbial disintegration capacities; these findings provide important insights into hemocyte phagocytic killing as a component of C. gigas innate immunity.

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 DM9-containing protein from oyster Crassostrea gigas (CgDM9CP-2) serves as a multipotent pattern recognition receptor.

DM9 is a novel protein domain with unknown function originally discovered in Drosophila melanogaster. Recently, a protein harboring DM9 repeats was identified as mannose-specific lectin (CgCGL1, renamed as CgDM9CP-1) from the Pacific oyster Crassostrea gigas. In the present study, another DM9 containing protein was identified from oyster C. gigas (designated as CgDM9CP-2). The open reading frame of CgDM9CP-2 gene was of 432 bp, encoding a polypeptide of 143 amino acids with two tandem DM9 repeats. The deduced amino acid sequence of CgDM9CP-2 shared 60.8% identity with that of CgDM9CP-1. In the unrooted phylogenetic tree, CgDM9CP-2 was closely clustered with CgDM9CP-1, and then assigned into the branch of invertebrate DM9CPs. The mRNA transcripts of CgDM9CP-2 were expressed in all the tested tissues, including mantle, gonad, gills, adductor muscle, hemocytes, and hepatopancreas, with the highest expression level in gills. CgDM9CP-2 protein was mainly distributed on the cytomembrane of oyster hemocytes. After mannose stimulation, the mRNA expression of CgDM9CP-2 in gills was up-regulated to the peak level (5.90-fold of that in SSW group, p < 0.05) at 24 h, and kept at a significantly higher level compared with that in control group at 6-48 h. It significantly increased at 6 h (2.33-fold, p < 0.05), and 12 h (3.08-fold, p < 0.05) post Vibrio splendidus stimulation, and then gradually decreased from 48 to 72 h (p < 0.05) with significant difference comparing with that in control group. The recombinant CgDM9CP-2 protein (rCgDM9CP-2) displayed higher binding affinity to D-(+)-mannose while lower binding affinity to lipopolysaccharide and peptidoglycan. rCgDM9CP-2 also exhibited binding activity towards fungi (Pichia pastoris and Yarrowia lipolytica), gram-positive bacteria (Staphylococcus aureus and Micrococcus luteus), and gram-negative bacteria (Escherichia coli, Vibrio anguillarum, Aeromonas hydrophila and V. splendidus). It could agglutinate fungi P. pastoris and Y. lipolytica, and inhibit the growth of P. pastoris, S. aureus, V. anguillarum, and V. splendidus. These results collectively indicated that CgDM9CP-2 not only served as a pattern recognition receptor with a broad range of recognition spectrum, but also involved in inhibiting the growth of invading microbe in the innate immune response of oyster, which would provide further evidence for the function of DM9 domain in the innate immune system.

Chinese mitten crab (Eriocheir sinensis) iron-sulphur cluster assembly protein 2 (EsIscA2) is differentially regulated after immune and oxidative stress challenges.

Iron-sulphur clusters (ISCs), one of the oldest and most versatile cofactors of proteins, are involved in catalysis reactions, electron transport reactions, regulation processes as well as sensing of ambient conditions. Iron-sulphur cluster assembly protein (IscA) is a scaffold protein member of ISC formation system, which plays a significant role in the assembly and maturation process of ISC proteins. In the present study, the cDNA sequence of iron-sulphur cluster assembly protein 2 (designated as EsIscA2) was cloned from Eriocheir sinensis. The open reading frame (ORF) of EsIscA2 was of 507 bp, encoding a peptide of 168 amino acids with a typically conserved Fe-S domain. A tetrameric form was predicated by the SWISS-MODEL prediction algorithm, and three conserved cysteine residues (Cys-93, Cys-158, Cys-160) from each IscA monomer were predicted to form a 'cysteine pocket'. The deduced amino acid sequence of EsIscA2 shared over 50% similarity with that of other IscAs. EsIscA2 was clustered with IscA2 proteins from invertebrates and vertebrates, indicating that the protein was highly conservative in the evolution. rEsIscA2 exhibited a high iron binding affinity in the concentration ranging from 2 to 200 µM. EsIscA2 transcripts were detected in all the tested tissues including gonad, hemocytes, gill, muscle, heart, hepatopancreas and eyestalk, and EsIscA2 protein was detected in the mitochondria of hemocytes. The highest mRNA expression level of EsIscA2 was detected in muscle and hepatopancreas, which was about 34.66-fold (p < 0.05) and 27.07-fold (p < 0.05) of that in hemocytes, respectively. After Aeromonas hydrophila and lipopolysaccharide (LPS) stimulations, the mRNA expression of EsIscA2 in hemocytes was down-regulated and reached the lowest level at 24 h (0.31-fold, p < 0.05) and 48 h (0.29-fold, p < 0.05) compared to control group, respectively. And the expression of EsIscA2 mRNA in hepatopancreas was repressed from 6 h to 48 h post stimulation (p < 0.05). When the primary cultured crab hemocytes were incubated with different concentrations of H2O2 for 15 min, the expression level of EsIscA2 mRNA was significantly repressed to the 0.34-0.44-fold of that in the control group. After A. hydrophila stimulation, the mRNA expression of EsGrx2 was up-regulated at 3 h (3.22-fold compared to control group, p < 0.05) and reached the peak at 12 h (4.88-fold, p < 0.05). All these results suggested that EsIscA2 had iron-binding capabilities as observed in IscA proteins from other organisms, supporting the role of EsIscA2 as a mitochondrial iron donor for ISC synthesis in Chinese mitten crab. Its differential mRNA expression after immune and oxidative stress challenges suggested the adaptations of ISC synthesis rates to these stress conditions.

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.

Transcriptome sequencing reveals the involvement of reactive oxygen species in the hematopoiesis from Chinese mitten crab Eriocheir sinensis.

Reactive oxygen species (ROS) produced in vivo during various electron transfer reactions are generally kept at a certain level since they are harmful to cells. However, it can sensitize hematopoietic progenitors to differentiation, and plays a signaling role in the regulation of hematopoietic cell fate. In the present study, the transcriptomes of crab HPT and hemocytes were sequenced using the Ion Torrent Proton sequencing platform. A total of 51,229,690 single end reads were obtained from six single-end libraries, which were assembled into 31346 unireads as reference. After mapping and transcript assembling, 362 differently expressed genes were identified and 301 of them were deemed to be more abundant in HPT. GO annotation revealed that they were mostly implicated in DNA, RNA and protein synthesis, cell division, mitochondria activities and energy metabolism. The expression level of mitochondrial complexes I (mitochondrial NADH-ubiquinone oxidoreductase) which was the main natural producers of mitochondrial ROS was found to be 8.6-fold (p < 0.01) higher in HPT than that in hemocytes. In hemocytes, the proteinase genes associated with proPO activation from the 61 up-regulated genes in hemocytes were the main up-regulated genes which might be the potential markers for mature hemocytes. ROS level in HPT cells was relatively higher which was confirmed with the high expression level of mitochondria related genes identified by transcriptome sequencing. After the ROS level was depressed by N-acetyl-l-cysteine (NAC), the production of hemocytes from HPT was inhibited, and the recovery of the total hemocytes counts was delayed. These results collectively indicated that the genes in redox system were more active in HPT, and ROS could function as an important modulator in the hematopoiesis of crab and promote the production of hemocytes from HPT.

A serotonin receptor (Cg5-HTR-1) mediating immune response in oyster Crassostrea gigas.

Serotonin receptors, including ligand-gated ion channel (LGICs) and G protein-coupled receptors (GPCR), play vital roles in modulating physiological processes and immunoreaction. In the present study, a homologue of serotonin (5-HT) receptor was identified from oyster Crassostrea gigas (designated Cg5-HTR-1). Its open reading frame (ORF) was of 1239 bp, encoding a polypeptide of 412 amino acids with a seven transmembrane region. Cg5-HTR-1 shared high similarity with the 5-HTRs from other animals. The cAMP contents in HEK293T cells decreased significantly after Cg5-HTR-1 transfection and 5-HT incubation (p < .05), while blocking Cg5-HTR-1 with specific receptor antagonist reversed this downtrend. The intracellular Ca2+ concentrations increased significantly (p < .05) after cell transfection and 5-HT incubation, and the antagonist treatment also arrested this process. Cg5-HTR-1 transcripts were widely distributed in various tissues, with the highest level in hepatopancreas and lowest level in mantle and gill. The mRNA expression of Cg5-HTR-1 in hemocyte increased significantly after lipopolysaccharide (LPS) stimulation and reached the peak level (6.47-fold, p < .05) at 6 h post treatment. The inhibition of Cg5-HTR-1 significantly reduced the expression of tumor necrosis factor (TNF) mRNA in hemocyte, down-regulated the superoxide dismutase (SOD) activity in serum, and induced the apoptosis of hemocyte (p < .05). These results suggested that Cg5-HTR-1 was a novel member of 5-HT1 receptor family and it mediated serotonergic immunomodulation on both cellular and humoral immune responses.

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.