Weiwei Song

Gene Transcriptional and Metabolic Profile Changes in Mimetic Aging Mice Induced by D-Galactose

D-galactose injection has been shown to induce many changes in mice that represent accelerated aging. This mouse model has been widely used for pharmacological studies of anti-aging agents. The underlying mechanism of D-galactose induced aging remains unclear, however, it appears to relate to glucose and 1ipid metabolic disorders. Currently, there has yet to be a study that focuses on investigating gene expression changes in D-galactose aging mice. In this study, integrated analysis of gas chromatography/mass spectrometry-based metabonomics and gene expression profiles was used to investigate the changes in transcriptional and metabolic profiles in mimetic aging mice injected with D-galactose. Our findings demonstrated that 48 mRNAs were differentially expressed between control and D-galactose mice, and 51 potential biomarkers were identified at the metabolic level. The effects of D-galactose on aging could be attributed to glucose and 1ipid metabolic disorders, oxidative damage, accumulation of advanced glycation end products (AGEs), reduction in abnormal substance elimination, cell apoptosis, and insulin resistance.

Development and characterization of EST-derived microsatellite makers for swimming crab, Portunus trituberculatus

In this study, sixteen polymorphic microsatellite makers were developed and characterized from expressed sequence tag sequence of the swimming crab, Portunus trituberculatus. The analysis of polymorphic was performed by using 30 specimens from the coastal waters of Xiangshan, Zhejiang province, China. The number of alleles at each locus ranged from 4 to 14 with an average of seven alleles per locus. The ranges of observed and expected heterozygosity were from 0.517–1.000 to 0.508–0.736, respectively. The polymorphism information content ranged from 0.375 to 0.710. Four loci were found deviate significantly from Hardy–Weinberg equilibrium. These microsatellite loci will be helpful for the genetic evaluation of P. trituberculatus enhancement.

Enhanced resistance of Portunus trituberculatus to Vibrio alginolyticus by selective breeding

We established a line (screened) of Portunus trituberculatus by selectively breeding individuals that survived from challenge with Vibrio alginolyticus, and compared the response of screened and unscreened (control) P. trituberculatus challenged with V. alginolyticus. We measured superoxide dismutase, catalase, acid phosphatase, alkaline phosphatase, and peroxidase activity and the content of hemocyanin in the plasma and phenoloxidase activity in serum. The cumulative survival rate after 24-h challenge with V. alginolyticus was significantly higher in the screened crabs than in the unscreened crabs (P <0.05). T-SOD and PO activity were significantly lower in the screened stock than in the unscreened stock (P <0.05). POD, CAT, and ACP activity and hemocyanin content were significantly higher in the screened stock than in the unscreened stock. Our results suggest that the screened stock was more resistant to infection. Furthermore, the indices we measured may be used to evaluate the health state of P. trituberculatus.

Characterization and functional analysis of a novel C-type lectin from the swimming crab Portunus trituberculatus

ABSTRACT C‐type lectins (CTLs) are a family of calcium‐dependent carbohydrate‐binding proteins. In the present study, a novel C‐type lectin (designated as PtCTL1) was identified and characterized from Portunus trituberculatus. The full‐length cDNA of PtCTL1 was of 702 bp, containing a 5′ untranslated region (UTR) of 91 bp, a 3′ UTR of 110 bp with a poly (A) tail, and an open reading frame (ORF) of 501 bp encoding a polypeptide of 166 amino acids with a putative signaling peptide of 21 amino acids. A C‐type lectin carbohydrate‐recognition domain (CRD) containing four conserved cysteines was identified in the amino acid sequence of PtCTL1. The cDNA fragment encoding the mature peptide of PtCTL1 was recombined into pET‐21a(+) with a C‐terminal hexa‐histidine tag fused in‐frame and expressed in Escherichia coli Origami (DE3). The recombinant PtCTL1 (rPtCTL1) can agglutinate all the tested bacteria, including three Gram‐positive bacterial strains and three Gram‐negative bacterial strains. In addition, erythrocyte agglutination and LPS‐binding activity were observed in a Ca2+‐dependent manner. The erythrocyte agglutination was inhibited by EDTA, indicating that PtCTL1 was Ca2+‐dependent. The mRNA transcripts of PtCTL1 were detected mainly in the tissues of hepatopancreas and hemocytes and its levels were significantly up‐regulated in hemocytes following Vibrio alginolyticus challenge. These results indicate that PtCTL1 may function as a pattern recognition receptor (PRR) for protecting P. trituberculatus from bacterial infection. Moreover, such findings also provide evidence for further understanding the innate immunology of invertebrate. HIGHLIGHTSA novel C‐type lectin from Portunus trituberculatus.The recombinant PtCTL1 (rPtCTL1) can agglutinateGram‐positiveGram‐negative bacterial.PtCTL1is Ca2+‐dependent.

An MBT domain containing anti-lipopolysaccharide factor (PtALF8) from Portunus trituberculatus is involved in immune response to bacterial challenge

Anti-lipopolysaccharide factors are effective antimicrobial peptides that can bind and neutralize lipopolysaccharide (LPS). In the present study, a new sequence encoding for ALF (designated as PtALF8) was cloned by suppression subtractive hybridization method using ovary of swimming crab Portunus trituberculatus as material. The full-length cDNA of PtALF8 consisted of 531 bp with an ORF of 348 bp encoding a peptide of 115 amino acids containing a putative signal peptide of 19 amino acids. The mature PtALF8 had a predicted molecular weight (MW) of 11.28 kDa and theoretical isoelectricpoint (pI) of 5.11. The PtALF8 contains an MBT domain which was not found in the other 7 isoforms of ALF reported in P. trituberculatus. Unlike most ALFs expressed in hemocytes, PtALF8 transcript was predominantly detected in hepatopancreas. After challenge with Vibrio alginolyticus, the temporal expression level of PtALF8 transcript in hemocytes reached the highest level at 3 h, then decreased to the lowest level at 24 h, and started to increase at 48 h. The recombinant protein showed antimicrobial and bactericidal activity against several bacteria, such as Gram-positive bacteria, Staphylococcus aureus, Micrococcus luteus and Gram-negative bacteria, V. alginolyticus, indicated that the PtALF8 isoform might play protective function against invading bacteria in P. trituberculatus.

Transcriptomic analysis of Portunus trituberculatus reveals a critical role for WNT4 and WNT signalling in limb regeneration

The swimming crab (Portunus trituberculatus) is among the most economically important seawater crustacean species in Asia. Despite its commercial importance and being well-studied status, genomic and transcriptomic data are scarce for this crab species. In the present study, limb bud tissue was collected at different developmental stages post amputation for transcriptomic analysis. Illumina RNA-sequencing was applied to characterise the limb regeneration transcriptome and identify the most characteristic genes. A total of 289,018 transcripts were obtained by clustering and assembly of clean reads, producing 150,869 unigenes with an average length of 956 bp. Subsequent analysis revealed WNT signalling as the key pathway involved in limb regeneration, with WNT4 a key mediator. Overall, limb regeneration appears to be regulated by multiple signalling pathways, with numerous cell differentiation, muscle growth, moult, metabolism, and immune-related genes upregulated, including WNT4, LAMA, FIP2, FSTL5, TNC, HUS1, SWI5, NCGL, SLC22, PLA2, Tdc2, SMOX, GDH, and SMPD4. This is the first experimental study done on regenerating claws of P. trituberculatus. These findings expand existing sequence resources for crab species, and will likely accelerate research into regeneration and development in crustaceans, particularly functional studies on genes involved in limb regeneration.

Molecular cloning of a C-type lectin from Portunus trituberculatus, which might be involved in the innate immune response

&NA; C‐type lectin plays an important role in the innate immune response of crustaceans including Portunus trituberculatus which is an important marine species. In the present study, we cloned the full length of a C‐type lectin (designated as PtCTL4) from P. trituberculatus via 3′RACE. The full length of the nucleic acid sequence has a length of 654 bp including an open reading frame (ORF) of 480 bp. PtCTL4 possesses conserved CTL features, while containing a CRD domain with Ca2+ binding site 2 and six conserved cysteine residues. Quantitative RT‐PCR analysis showed that PtCTL4 expression level was highest in the hepatopancreas, while it was relatively low in other tissues such as hemocytes, eyestalk, muscle, and gonad. The expression level of PtCTL4 reached a maximum at 3 h after challenge with Vibrio alginolyticus, then decreased to the lowest level at 12 h, and returned to normal level at 48 h. Hemagglutination analysis showed that the recombinant PtCTL4 (rPtCTL4) can agglutinate rabbit erythrocyte. The rPtCTL4 can agglutinate Gram‐positive bacteria (Bacillus aquimaris, Micrococcus lysodeik, and Staphylococcus aureus) and Gram‐negative bacteria (Aeromonas hydrophila, V. alginolyticus, and Chryseobacterium indologenes) in the presence of Ca2+. This study indicated that PtCTL4 acts as a pattern recognition receptor in the innate immune response of P. trituberculatus. HighlightsPtCTL4 possesses conserved CTL features.PtCTL4 involved in immune response of bacterial challenge.rPtCTL4 can agglutinate rabbit erythrocyte.rPtCTL4 can agglutinate Gram‐positive bacteria and Gram‐negative bacteria.

An effective method for identification of three mussel species and their hybrids based on SNPs

We described here the successful identification of three economic aquaculture mussel species in China and their hybrids by high resolution melting curve analysis (HRM) of single nucleotide polymorphisms (SNPs). The nuclear 18S ribosomal RNA gene sequences from Mytilus edulis, Mytilus coruscus , and Perna viridis were aligned and amplified. In total, 8 SNP sites were detected from the 79-bp amplicon. A total of 90 samples from the three mussel species were clearly separated, hybrids from M. edulis and M. coruscus could also be clearly identified. The melting profiles from HRM analysis were found to be distinct across the species tested. This new identification method will be a useful tool for discrimination of the three commercially important mussel species and their hybrids.

iTRAQ-based proteomic analysis identifies proteins involved in limb regeneration of swimming crab Portunus trituberculatus

The swimming crab (Portunus trituberculatus) has a striking capacity for limb regeneration, which has drawn the interest of many researchers. In this study, isobaric tag for relative and absolute quantitation (iTRAQ) approach was utilised to investigate protein abundance changes during limb regeneration in this species. A total of 1830 proteins were identified, of which 181 were significantly differentially expressed, with 94 upregulated and 87 downregulated. Our results highlight the complexity of limb regeneration and its regulation through cooperation of various biological processes including cytoskeletal changes, extracellular matrix (ECM) remodelling and ECM-receptor interactions, protein synthesis, signal recognition and transduction, energy production and conversion, and substance transport and metabolism. Additionally, real-time PCR confirmed that mRNA levels of differentially expressed genes were correlated with protein levels. Our results provide a basis for studying the regulatory mechanisms associated with crab limb regeneration.

Peroxiredoxin 1 from cuttlefish ( Sepiella maindroni ): Molecular characterization of development and its immune response against Vibrio alginolyticus

Abstract The aim of this work was constructive to understand the function of peroxiredoxin (PRDX) family member Peroxiredoxin 1 in Sepiella maindroni (SmPrx1) through molecular mechanisms of reproduction, embryonic development and immune responses to Vibrio alginolyticus. The full‐length cDNA of SmPrx1 was of 1062 bp, contains a 5′ untranslated region (UTR) of 79bp, a 3′ UTR of 359 bp, an open reading frame of 624 bp encoding 207 amino acids. The conserved peroxidase catalytic center “FYPLDFTFVCPTEI” and “GEVCPA” were observed in the sequence of SmPrx1; this indicated that it was a member of 2‐Cys Prx. Quantitative real‐time (qRT)‐PCR assays revealed that SmPrx1 was ubiquitously expressed in all examined tissues, muscle, ink sac, liver, ovary, testis, intestine, gill and totally blood cells, and showed high levels in testis. SmPrx1 mRNA was ubiquitously detected in all tested tissues, and the expression was comparatively high in testis, hemocyte, liver and ovary. Moreover, the SmPrx1 gene transcript was detected at all five stages of embryonic development phases that were respectively the zygote stage, the pre‐embryonic stage, the organogenesis stage, the morphological integrity stage, the pre‐hatching stage. The general tendency of expression was gradually increased and rapidly decreased. High expressed in progenitive tissues and embryonic development exhibit the proliferation‐associated protein characterization like in mammal. The expression levels of SmPrx1 in liver and hemocytes grew swiftly and quickly reached peak value after Vibrio alginolyticus challenge. As hours passed by, the expression level began to reduce and resumed to normal levels after 48 h. The antioxidant activity and peroxidase activity of SmPrx1 were 6.17 U/mg. The results showed that the recombined protein of SmPrx1 had antioxidant activity and was the importance part of the antioxidant system in Sepiella maindroni. This study provides useful information to help further understand the functional mechanism of Prx 1 in marine cephalopod immunity. HighlightsDetecting gene structure, evolution and temporal‐spatial expression of tissues and development.Examine the temporal response of SmPrx1 gene to immune responses to V. alginolyticus.Purify the recombinant protein and detect the antioxidant activity of the recombinant protein.