CSNK2A2 promotes hepatocellular carcinoma progression through activation of NF-κB pathway.

INTRODUCTION AND OBJECTIVES: Breast and non-small cell lung cancers harbor an upregulated CSNK2A2 oncogene that encodes the protein kinase CK2 alpha', a catalytic subunit of the highly conserved serine/threonine kinase CK2. However, its role and biological significance in hepatocellular carcinoma (HCC) remains unclear. MATERIALS AND METHODS: Western-blotting and immunohistochemistry were used to measure the expression of CSNK2A2 in HCC tumor tissues and cell lines. CCK8, Hoechst staining, transwell, tube formation assay in vitro and nude mice experiments in vivo were used to measure the effects of CSNK2A2 on HCC proliferation, apoptosis, metastasis, angiogenesis and tumor formation. RESULTS: In the study, we showed that CSNK2A2 was highly expressed in HCC comparison with matched control tissues, and was linked with lower survival of patients. Additional experiments indicated that silencing of CSNK2A2 promoted HCC cell apoptosis, while inhibited HCC cells migrating, proliferating, angiogenesis both in vitro and in vivo. These effects were also accompanied by reduced expression of NF-κB target genes, including CCND1, MMP9 and VEGF. Moreover, treatment with PDTC counteracted the promotional effects of CSNK2A2 on HCC cells. CONCLUSIONS: Overall, our results suggested that CSNK2A2 could promote HCC progression by activating the NF-κB pathway, and this could serve as a biomarker for future prognostic and therapeutic applications.

SIRT7-Induced PHF5A Decrotonylation Regulates Aging Progress Through Alternative Splicing-Mediated Downregulation of CDK2.

Dysregulation of protein posttranslational modification (PTM) can lead to a variety of pathological processes, such as abnormal sperm development, malignant tumorigenesis, depression, and aging process. SIRT7 is a NAD+-dependent protein deacetylase. Besides known deacetylation, SIRT7 may also have the capacity to remove other acylation. However, the roles of SIRT7-induced other deacylation in aging are still largely unknown. Here, we found that the expression of SIRT7 was significantly increased in senescent fibroblasts and aged tissues. Knockdown or overexpression of SIRT7 can inhibit or promote fibroblast senescence. Knockdown of SIRT7 led to increased pan-lysine crotonylation (Kcr) levels in senescent fibroblasts. Using modern mass spectrometry (MS) technology, we identified 5,149 Kcr sites across 1,541 proteins in senescent fibroblasts, and providing the largest crotonylome dataset to date in senescent cells. Specifically, among the identified proteins, we found SIRT7 decrotonylated PHF5A, an alternative splicing (AS) factor, at K25. Decrotonylation of PHF5A K25 contributed to decreased CDK2 expression by retained intron (RI)-induced abnormal AS, thereby accelerating fibroblast senescence, and supporting a key role of PHF5A K25 decrotonylation in aging. Collectively, our data revealed the molecular mechanism of SIRT7-induced k25 decrotonylation of PHF5A regulating aging and provide new ideas and molecular targets for drug intervention in cellular aging and the treatment of aging-related diseases, and indicating that protein crotonylation has important implications in the regulation of aging progress.

The Effects of Acarbose on Non-Diabetic Overweight and Obese Patients: A Meta-Analysis.

INTRODUCTION: This systematic review aims to verify the efficacy of acarbose monotherapy in treating obese or overweight patients without diabetes. METHODS: In the study, we conducted a systematic search of the Pub-Med, EMBASE, Cochrane and Science Citation Index Expanded databases in search of clinical trials on acarbose treatment, overweight and obesity. The crucial inclusion criteria were as follows: (1) patients were diagnosed as overweight or obese (BMI ≥ 25 kg/m2); (2) randomized controlled trials (RCTs); (3) patients had undergone acarbose monotherapy or placebo control; (4) acarbose treatment had been carried out for at least 3 months. Exclusion criteria were as follows: (1) patients diagnosed with diabetes mellitus (DM); (2) patients had received a weight loss medication or surgery in the past 3 months; (3) papers not published in English; (4) repeated research results of the same experiment or repeated published documents. RESULTS: A total of 7 studies involving 132 in the acarbose group and 137 in placebo group, 269 subjects in total, were included in this meta-analysis. From the selected seven papers, we extracted the following clinical parameters: systolic blood pressure (SBP), diastolic blood pressure (DBP), body weight (BW), body mass index (BMI), triglyceride (TG), total cholesterol (TC), low density lipoprotein (LDL), high density cholesterol (HDL) and fasting plasma glucose (FPG). An important finding of our research is that TG was the only significantly reduced parameter in the acarbose group. Weight mean difference (WMD) was - 0.21 (95% CI - 0.33, - 0.09) mmol/l between acarbose (P = 0.0006) and placebo patients. Reduction of BMI was also greater for acarbose than placebo subjects, although the discrepancy was not statistically significant (P = 0.56). Moreover, no hypoglycemia occurred in either the acarbose group or placebo group. A few subjects experienced gastrointestinal reactions, but these were mild and improved over time. Acarbose has no obvious influence on other metabolic indexes. CONCLUSION: Acarbose monotherapy is beneficial in reducing TG levels in obese or overweight patients and will not result in hypoglycemia during medication. The side effects of acarbose are mild.

Senescent Cell-Secreted Netrin-1 Modulates Aging-Related Disorders by Recruiting Sympathetic Fibers.

Cellular senescence is implicated in several lines of aging-related disorders. However, the potential molecular mechanisms by which cellular senescence modulates age-related pathologies remain largely unexplored. Herein, we report that the density of sympathetic fibers (SFs) is significantly elevated in naturally aged mouse tissues and human colon adenoma tissues compared to the SFs densities in the corresponding young mouse tissues and human non-lesion colon tissues. A dorsal root ganglion (DRG)-human diploid fibroblast coculture assay revealed that senescent cells promote the outgrowth of SFs, indicating that the senescent cells induce recruitment of SFs in vitro. Additionally, subcutaneous transplantation of 2BS fibroblasts in nude mice shows that transplanted senescent 2BS fibroblasts promote SFs infiltration. Intra-articular senolytic molecular injection can reduce SFs density and inhibit SFs infiltration caused by senescent cells in osteoarthritis (OA), suggesting senescent cells promote the infiltration of SFs in vivo in aged tissues. Notably, the elevated level of SFs contributes to impaired cognitive function in naturally aged mice, which can be reversed by treatment with propranolol hydrochloride, a non-selective ß receptor blocker that inhibits sympathetic nerve activity (SNA) by blocking non-selective ß receptors. Additionally, 6-hydroxydopamine (6-OHDA)-induced sympathectomy improved hepatic sympathetic overactivity mediated hepatic steatosis in high fat diet (HFD)-fed APOE knockout mice (APOE-/- mice) by reducing hepatic SNA. Taken together, this study concludes that senescent cell-secreted netrin-1 mediated SFs outgrowth and infiltration, which contributes to aging-related disorders, suggesting that clearing senescent cells or inhibiting SNA is a promising therapeutic strategy for improving sympathetic nervous system (SNS) hyperactivity-induced aging-related pathologies.

Circular RNA CircCCNB1 sponges micro RNA-449a to inhibit cellular senescence by targeting CCNE2.

Circular RNAs (CircRNAs) are a novel subset of non-coding RNA widely present in eukaryotes that play a central role in physiological and pathological conditions. Accumulating evidence has indicated that CircRNAs participated in modulating tumorigenesis by acting as a competing endogenous RNA (CeRNA). However, the roles and functions of CircRNAs in cellular senescence and aging of organisms remain largely obscure. We performed whole transcriptome sequencing to compare the expression patterns of circular RNAs in young and prematurely senescent human diploid fibroblast 2BS cells, and identified senescence-associated circRNAs (SAC-RNAs). Among these SAC-RNAs, we observed the significantly downregulated expression of CircRNAs originating from exons 6 and 7 circularization of the cyclin B1 gene (CCNB1), termed CircCCNB1. Reduced CircCCNB1 expression triggered senescence in young 2BS cells, as measured by increased senescence associated-beta-galactosidase (SA-ß-gal) activity, enhanced expression of cyclin-dependent kinase inhibitor 1A (CDKN1A)/P21 and tumor protein 53 (TP53) expression, and reduced cell proliferation. Mechanistically, reduced CircCCNB1 level inhibited cyclin E2 (CCNE2) expression by modulating micro RNA (miR)-449a activity, which repressed cellular proliferation. Our data suggested that CircCCNB1may serve as a sponge against miR-449a to delay cellular senescence by targeting CCNE2. Targeting CircCCNB1 may represent a promising strategy for aging and age-related disease interventions. Furthermore, we also identified and characterized several kinds of the CircCCNB1-binding proteins (CBPs), which may contribute to the degradation of CircCCNB1.

TALENs-mediated homozygous CCR5Δ32 mutations endow CD4+ U87 cells with resistance against HIV­1 infection.

Since evidence suggests that transplantation of bone marrow stem cells with the C­C chemokine receptor type 5 (CCR5)Δ32/Δ32 genotype may cure patients infected with human immunodeficiency virus (HIV)­1, the present study aimed to reproduce the CCR5Δ32 mutation in cluster of differentiation (CD)4+ U87 cells using genome engineering methods. A modified transcription activator­like effector nucleases (TALENs) technique, combined with homologous recombination for site­specific, size­controlled and homozygous DNA deletions, was used to reproduce the homozygous CCR5Δ32 mutation in CD4+ U87 cells. The results indicated that the frequency of the TALENs­targeted mutation reached 50.4% without any selection, whereas homologous recombination from CCR5 to CCR5Δ32 occurred in 8.8% of targeted cells. Notably, a HIV­1 challenge test demonstrated that CCR5Δ32/Δ32 CD4+ U87 cells were resistant to HIV infection. In conclusion, engineered CCR5Δ32/Δ32 mutations endowed CD4+ U87 cells with resistance against HIV­1 infection; this site­specific, size­controlled and homozygous DNA deletion technique was able to induce precise genomic editing, i.e., the deletion or insertion of a predetermined length of DNA sequence at a specific locus throughout the genome.

[The Effect of TALENs-mediated Downregulation Expression of Nanog on Malignant Behavior of Cervical Cancer HeLa Cells].

OBJECTIVE: To study the effect of downregulation expression of Nanog on malignant behavior of cervical cancer HeLa cells. METHODS: Gene editing tool TALENs was employed to induce downregulation expression of Nanog, and Nanog mutation was evaluated by sequencing. RT-PCR and Western blot was used to detect the mRNA and protein expression level, respectively. Colony-formation assay, Transwell invasion assay, and chemotherapy sensibility assay was carried out to assess the capacity of colony-formation, invasion, and chemoresistance, respectively. RESULTS: TALENs successfully induced Nanog mutation and downregulated Nanog expression. Nanog mRNA and protein expression of Nanog-mutated monoclonal HeLa cells downregulated 3 times compared to thoses of wild-type HeLa cells (P < 0.05). Additionally, significant weakened abilities of colony-formation, invasion, and chemoresistance in monoclonal HeLa cells were observed when compared to those of wild-type HeLa cells (P < 0.05). CONCLUSION: Nanog mutation attenuates the malignant behavior of HeLa cells. Importantly, downregulation or silencing of Nanog is promising to be a novel strategy for the treatment of cervical carcinoma.

Forced expression of Nanog with mRNA synthesized in vitro to evaluate the malignancy of HeLa cells through acquiring cancer stem cell phenotypes.

Nanog is a pluripotency-related factor. It was also found to play an important role in tumorigenesis. To date, the mechanisms underlying cervical tumorigenesis still need to be elucidated. In the present study, Nanog mRNA was synthesized in vitro and transfected into HeLa cells. After mRNA transfection, the forced expressed of Nanog in HeLa cells led to markedly increased invasion, migration, resistance to chemotherapeutic agents and dedifferentiation. In a subcutaneous xenograft assay, these cells had significantly increased tumorigenic capacity. Real-time PCR indicated that Nanog­induced dedifferentiation was associated with increased expression of endogenous Oct4, Sox2 and FoxD3. In addition, the dedifferentiated HeLa cells acquired features associated with cancer stem cells (CSCs), such as multipotent differentiation capacity, and expression of CSC markers such as CD133. These data imply that Nanog is a positive regulator of cervical cancer dedifferentiation.

Characterisation of a novel Type I crustin involved in antibacterial and antifungal responses in the red claw crayfish, Cherax quadricarinatus.

Antimicrobial peptides are important immune effectors involved in mediating innate immune responses against intruding pathogens. Here, we successfully isolated and characterized a novel Type I crustin from the red claw crayfish Cherax quadricarinatus. The full-length cDNA encoded by this gene, designated CqCrs, comprised 608 bp, containing a 5'-untranslated region (UTR) of 55 bp, a 3'-UTR of 229 bp with a poly (A) tail, and an open reading frame (ORF) of 324 bp encoding a polypeptide of 107 amino acids. The deduced amino acid sequence of CqCrs exhibited a configuration typical of other crustacean Type I crustin orthologs, including one signal peptide region at the N-terminus between residues 1 and 16 and a long whey acidic protein (WAP) domain at the C-terminus between residues 60 and 107, along with a WAP-type "four-disulfide core" motif. Phylogenetic analysis showed that CqCrs was clustered first with other crustacean Type I crustins, then with other crustacean Type II crustins, and finally with other crustacean Type III crustins. Transcription of CqCrs was detected in all tissues, especially in immune tissues and was differentially induced in hemocytes post-stimulation with ß-1, 3-glucan, lipopolysaccharides (LPS) and peptidoglycans (PG) at selected time-points. To clarify the biological activity of CqCrs, the recombinant CqCrs protein (rCqCrs) was constructed and expressed in Escherichia coli BL21 (DE3). Purified rCqCrs bound to diverse bacteria and inhibited the growth of different microbes to varying degrees. These findings suggest that CqCrs is involved in a specific innate immune recognition and defense mechanisms against bacterial and fungal in C. quadricarinatus.

TALEN-induced disruption of Nanog expression results in reduced proliferation, invasiveness and migration, increased chemosensitivity and reversal of EMT in HepG2 cells.

Accumulating evidence indicates that Nanog plays a central role in modulating the biological behaviors of human hepatocellular carcinoma (HCC). However, the underlying mechanisms remain unclear. In the present study, we employed transcription activator-like effector nucleases (TALEN) to disrupt Nanog expression in HepG2 cells and obtained subcloned cells with diallelic Nanog mutations. Significantly, we found that the expression of pluripotency factors Sox2, Oct4 and Klf4, as well as expression of cancer stem cell (CSC) marker CD133, in the Nanog-targeted HepG2 cells was markedly downregulated. This finding suggests that Nanog may play an important role in maintaining the pluripotency and malignancy of HepG2 cells. We also revealed that Nanog regulated cell proliferation by modulating the expression of cyclin D1/D3/E1 and CDK2, respectively. Additionally, the disruption of Nanog resulted in the downregulation of epithelial-mesenchymal transition (EMT) regulators Snail and Twist, which contributed to the elevated level of epithelial marker E-cadherin, and to the decreased level of mesenchymal markers N-cadherin and vimentin in the HepG2 cells. In addition, the Nanog-targeted HepG2 cells exhibited reduced ability of invasion, migration and chemoresistance in vitro. In conclusion, the disruption of Nanog expression results in less proliferation, invasiveness, migration, more chemosensitivity and reversal of EMT in HepG2 cells, by which Nanog plays crucial roles in influencing the malignant phenotype of HepG2 cells.

Antimicrobial activity of a novel hypervariable immunoglobulin domain-containing receptor Dscam in Cherax quadricarinatus.

Down syndrome cell adhesion molecule (Dscam) mediates innate immunity against pathogens in arthropods. Here, a novel Dscam from red claw crayfish Cherax quadricarinatus (CqDscam) was isolated. The CqDscam protein contains one signal peptide, ten immunoglobulin domains, six fibronectin type III domains, one transmembrane domain and cytoplasmic tail. CqDscam phylogenetically clustered with other invertebrate Dscams. Variable regions of CqDscam in N-terminal halves of Ig2 and Ig3 domains, complete Ig7 domain and TM domain can be reshuffled after transcription to produce a deluge of >37,620 potential alternative splice forms. CqDscam was detected in all tissues tested and abundantly expressed in immune system and nerve system. Upon lipopolysaccharides (LPS) and b-1, 3-glucans (Glu) challenged, the expression of CqDscam was up-regulated, while no response in expression occurred after injection with peptidoglycans (PG). Membrane-bound and secreted types of CqDscam were separated on the protein level, and were both extensively induced post LPS challenge. Membrane-bound CqDscam protein was not detected in the serum, but localized to the hemocyte surface by immuno-localization assay. In the antimicrobial assays, the recombinant LPS-induced isoform of CqDscam protein displayed bacterial binding and growth inhibitory activities, especially with Escherichia coli. These results suggested that CqDscam, as one of pattern-recognition receptors (PRRs), involved in innate immune recognition and defense mechanisms in C. quadricarinatus, possibly through alternative splicing.

Caspase-mediated apoptosis in crustaceans: cloning and functional characterization of EsCaspase-3-like protein from Eriocheir.

The caspase-3-like gene was cloned from Eriocheir sinensis, and its properties were characterized to identify the biological implications of this caspase in apoptosis in crab. Its deduced full-length protein sequence consists of 462 amino acid residues, including the prodomain and the large and small subunits. Moreover, several residues known to be critical in the caspase-3 catalytic center and binding pocket, as well as the active site pentapeptide motif Q(220)ACRG(224), were identically present in the deduced EsCaspase-3-like protein. Subsequently, the recombinant EsCaspase-3-like (rEsCaspase-3-like) protein was expressed from Escherichia coli and obtained via affinity purification. Results of the in vitro enzymatic activity assays indicated that the rEsCaspase-3-like protein is capable of hydrolyzing the substrate Ac-DEVD-pNA, suggesting a functional role in physiology. EsCaspase-3-like gene transcripts were found to be widely distributed in all tissues as detected by quantitative RT-PCR, being especially abundant in hemocytes and comparatively rare in muscles. Furthermore, EsCaspase-3-like, at both the mRNA and protein levels, was demonstrated to participate in the apoptotic process after stimulation by different pathogen-associated molecular patterns (PAMPs) in hemocytes. In conclusion, our findings suggest that the EsCaspase-3-like protein functions as an effector caspase and contributes to immune responses against pathogens.

TALEN-mediated Nanog disruption results in less invasiveness, more chemosensitivity and reversal of EMT in Hela cells.

Emerging evidence suggests that Nanog is involved in cervical tumorigenesis. However, the regulating role of Nanog in tumorigenesis and chemosensitivity are still poorly understood. In this study, Nanog was disrupted by transcription activator-like effector nucleases (TALEN) in Hela cells and its expression was significantly decreased in a single-cell derived sub-clone with biallelic mutations. The disruption of Nanog not only induced down regulation of some other core transcription factor genes for cell self-renewal, such as Oct4, Sox2 and FoxD3, but also led to the down regulation of some mesenchymal representative genes, vimentin and N-adherin, and up regulation of the epithelial gene, E-cadherin. In addition, the invasiveness and clonogenicity of the Hela cells were obviously affected, and surprisingly their sensitivities to anti-cancer drugs were also significantly increased in vitro. After Xenograft into nude mice, the growth volumes of the neoplasms from the Nanog disrupted Hela cells were significantly smaller compared with those from wild type ones. In conclusion, these results suggest that disruption of Nanog may reverse the status of epithelial-mesenchymal transition, which is critical in tumorigenesis, and alleviate chemoresistance, as well as their invasiveness, in cervical cancer cells.

Identification and characterization of Tube in the Chinese mitten crab Eriocheir sinensis.

As a key component of the Toll signaling pathway, Tube plays central roles in many biological activities, such as survival, development and innate immunity. Tube has been found in shrimps, but has not yet been reported in the crustacean, Eriocheir sinensis. In this study, we cloned the full-length cDNA of the adaptor Tube for the first time from E. sinensis and designated the gene as EsTube. The full-length cDNA of EsTube was 2247-bp with a 1539-bp open reading frame (ORF) encoding a 512-amino acid protein. The protein contained a 116-residue death domain (DD) at its N-terminus and a 272-residue serine/threonine-protein kinase domain (S_TKc) at its C-terminus. Phylogenetic analysis clustered EsTube initially in one group with other invertebrate Tube and Tube-like proteins, and then with the vertebrate IRAK-4 proteins, finally with other invertebrate Pelle proteins. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis results showed that EsTube was highly expressed in the ovary and testis, and moderately expressed in the thoracic ganglia and stomach. EsTube was expressed at all selected stages and was highly expressed in the spermatid stage (October, testis) and the stage III-2 (November, ovary). EsTube was differentially induced after injection of lipopolysaccharides (LPS), peptidoglycan (PG) or zymosan (ß-1,3-glucan). Our study indicated that EsTube might possess multiple functions in immunity and development in E. sinensis.

Association of a hepatopancreas-specific C-type lectin with the antibacterial response of Eriocheir sinensis.

Pattern recognition receptors (PPRs) are part of the initial step of a host defense against pathogens in detecting pathogen-associated molecular patterns. However, determinants of the specificity of this recognition by innate immune molecules of invertebrates remain largely unknown. In this study, we investigated the potential involvement of an invertebrate PRR C-type lectin in the antimicrobial response of the crustacean Eriocheir sinensis. Based on the initial expressed sequence tags (EST) of a hepatopancreatic cDNA library, the full-length EsLecF cDNA was cloned and determined to contain a 477-bp open reading frame encoding a putative 158-amino-acid protein. A comparison with other reported invertebrate and vertebrate C-type lectin superfamily sequences revealed the presence of a common carbohydrate recognition domain (CRD). EsLecF transcripts in E. sinensis were mainly detected in the hepatopancreas and were inducible by a lipopolysaccharide (LPS) injection. The recombinant EsLecF (rEsLecF) protein produced via a prokaryotic expression system and affinity chromatography was found to have a wide spectrum of binding activities towards various microorganisms, and its microbial-binding activity was calcium-independent. Moreover, the binding of rEsLecF induced the aggregation of microbial pathogens. Results of the microorganism growth inhibitory assay and antibacterial assay revealed capabilities of rEsLecF in suppressing microorganism growth and directly killing bacteria, respectively. Furthermore, rEsLecF could enhance cellular encapsulation in vitro. Collectively, the findings presented here demonstrated the successful isolation of a novel C-type lectin in a crustacean and highlighted its critical role in the innate immunity of an invertebrate.

A novel C-type lectin from Eriocheir sinensis functions as a pattern recognition receptor with antibacterial activity.

As pattern recognition receptors (PRRs), C-type lectins (CTLs) play significant roles in recognizing and eliminating pathogens in innate immunity. In this study, a novel CTL (EsLecD) was identified from the crustacean Eriocheir sinensis. The cloning of full-length EsLecD cDNA was based on the initial expressed sequence tags (ESTs) isolated from a hepatopancreatic cDNA library. The full-length EsLecD cDNA of 686 bp with an open reading frame of 468 bp encodes a putative protein of 155 aa residues, including an N-terminal signal peptide and a single carbohydrate-recognition domain (CRD). By quantitative RT-PCR analysis, the EsLecD transcript was mainly detected in the hepatopancreas but rarely in other tissues, and it was significantly upregulated in the hepatopancreas after immune challenge with lipopolysaccharides. The recombinant EsLecD protein (rEsLecD) exhibited the ability to bind to all tested microorganisms, including bacteria and yeast. Meanwhile, calcium significantly increased the binding affinity of rEsLecD toward microorganisms, but it was not essential. The binding of rEsLecD induced the aggregation of microbial pathogens. Moreover, rEsLecD was capable of inhibiting the growth of microorganisms and even directly killing bacteria. Interestingly, rEsLecD could stimulate cellular encapsulation in vitro. In conclusion, results of this study suggest that EsLecD acts as an antibacterial PRR participating in the innate immunity of invertebrates.

Two novel Toll genes (EsToll1 and EsToll2) from Eriocheir sinensis are differentially induced by lipopolysaccharide, peptidoglycan and zymosan.

Tolls/Toll-like receptors (TLRs) play an essential role in initiating innate immune responses against pathogens and are found throughout the insect kingdom but have not yet been reported in the crustacean, Eriocheir sinensis. For this purpose, we cloned two novel Toll genes from E. sinensis, EsToll1 and EsToll2. The full-length cDNA of EsToll1 was 3963 bp with a 3042-bp open reading frame (ORF) encoding a 1013-amino acid protein. The extracellular domain of this protein contains 17 leucine-rich repeats (LRRs) and a 139-residue cytoplasmic Toll/interleukin-1 receptor (TIR) domain. The cDNA full-length of EsToll2 was 4419 bp with a 2667-bp ORF encoding an 888-amino acid protein with an extracellular domain containing 10 LRRs and a 139-residue cytoplasmic TIR domain. By phylogenetic analysis, EsToll1 and EsToll2 clustered into one group together with Tolls from other crustaceans. Quantitative RT-PCR analysis demonstrated that a) both EsToll1 and EsToll2 were constitutively expressed in all tested crab tissues; b) EsToll1 and EsToll2 were differentially induced after injection of lipopolysaccharides (LPS), peptidoglycan (PG) or zymosan (GLU). Importantly, EsToll2 expression was significantly upregulated at almost all time intervals post-challenge with LPS, PG and GLU. Our study indicated that EsToll1 and EsToll2 are differentially inducibility in response to various PAMPs, suggesting their involvement in a specific innate immune recognition mechanism in E. sinensis.

A double WAP domain-containing protein Es-DWD1 from Eriocheir sinensis exhibits antimicrobial and proteinase inhibitory activities.

Whey acidic proteins (WAP) belong to a large gene family of antibacterial peptides, which are critical in the host immune response against microbial invasion. The common feature of these proteins is a single WAP domain maintained by at least one four-disulfide core (4-DSC) structure rich in cysteine residues. In this study, a double WAP domain (DWD)-containing protein, Es-DWD1, was first cloned from the Chinese mitten crab (Eriocheirsinensis). The full-length Es-DWD1cDNA was 1193 bp, including a 411 bp open reading frame (ORF) encoding 136 amino acids with a signal peptide of 22 amino acids in the N-terminus. A comparison with other reported invertebrate and vertebrate sequences revealed the presence of WAP domains characteristic of WAP superfamilies. As determined by quantitative real-time RT-PCR, Es-DWD1 transcripts were ubiquitously expressed in all tissues, but it was up-regulated in hemocytes post-challenge with pathogen-associated molecular patterns (PAMPs). The mature recombinant Es-DWD1 (rEs-DWD1) protein exhibited different binding activities to bacteria and fungus. Moreover, rEs-DWD1 could exert agglutination activities against Bacillus subtilis and Pichiapastoris and demonstrated inhibitory activities against the growth of Staphylococcus aureus, Aeromonas hydrophila and P. pastoris. Furthermore, rEs-DWD1 showed a specific protease inhibitory activity in B. subtilis. Coating of rEs-DWD1 onto agarose beads enhanced encapsulation of the beads by crab hemocytes. Collectively, the results suggest that Es-DWD1 is a double WAP domain containing protein with antimicrobial and proteinase inhibitory activities, which play significant roles in the immunity of crustaceans.

Molecular cloning and expression analysis of a dorsal homologue from Eriocheir sinensis.

Dorsal as a crucial component of Toll signaling pathway, played important roles in induction and regulation of innate immune responses. In this study, we cloned a NF-κB-like transcription factor Dorsal from Eriocheir sinensis and designated it as EsDorsal. The full-length cDNA of EsDorsal was 2493 bp with a 2022-bp open reading frame (ORF) encoding a 673-amino acid protein. This protein contained a 171-residue conserved Rel homology domain (RHD) and a 102-residue Ig-like, plexins and transcription factors domain (IPT). By phylogenetic analysis, EsDorsal was clustered into one group together with other invertebrate Dorsals or NF-κBs, and then clustered with vertebrate NF-κBs. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis results showed that (a) EsDorsal had higher expression level in immune organs; (b) EsDorsal differentially induced after injection of lipopolysaccharides (LPS), peptidoglycan (PG) or zymosan (GLU). Importantly, EsDorsal was more responsive to LPS than GLU and PG. Collectively, EsDorsal was differentially inducibility in response to various PAMPs, suggesting its involvement in a specific innate immune regulation in E. sinensis.