Design, synthesis and biological evaluation of 4-(indolin-1-yl)-6-substituted-pyrido[3,2-d]pyrimidine derivatives as Mnk1/2 inhibitors.

The Mnk-eIF4E axis plays a crucial role in tumor development, and inhibiting Mnk kinases is a promising approach for cancer therapy. Starting with fragment WS23, a series of 4-(indolin-1-yl)-6-substituted-pyrido[3,2-d]pyrimidine derivatives were designed and synthesized. Among these derivatives, compound 15b showed the highest potency with IC50 values of 0.8 and 1.5 nM against Mnk1 and Mnk2, respectively. Additionally, it demonstrated good selectivity among 30 selected kinases. 15b significantly suppressed MOLM-13 and K562 cell lines growth and caused cell cycle arrest. Furthermore, the Western blot assay revealed that 15b effectively downregulated the downstream proteins p-eIF4E, Mcl-1, and c-myc. Additionally, 15b exhibited remarkable stability in rat plasma and rat and human microsomes. In vivo anti-tumor activity study suggested that treatment with 15b suppressed tumor growth in LL/2 syngeneic models. These findings highlight the potential of 15b as a novel and potent Mnks inhibitor, which deserves further investigation.

Detection of an anti-angina therapeutic module in the effective population treated by a multi-target drug Danhong injection: a randomized trial.

It's a challenge for detecting the therapeutic targets of a polypharmacological drug from variations in the responsed networks in the differentiated populations with complex diseases, as stable coronary heart disease. Here, in an adaptive, 31-center, randomized, double-blind trial involving 920 patients with moderate symptomatic stable angina treated by 14-day Danhong injection(DHI), a kind of polypharmacological drug with high quality control, or placebo (0.9% saline), with 76-day following-up, we firstly confirmed that DHI could increase the proportion of patients with clinically significant changes on angina-frequency assessed by Seattle Angina Questionnaire (ΔSAQ-AF ≥ 20) (12.78% at Day 30, 95% confidence interval [CI] 5.86-19.71%, P = 0.0003, 13.82% at Day 60, 95% CI 6.82-20.82%, P = 0.0001 and 8.95% at Day 90, 95% CI 2.06-15.85%, P = 0.01). We also found that there were no significant differences in new-onset major vascular events (P = 0.8502) and serious adverse events (P = 0.9105) between DHI and placebo. After performing the RNA sequencing in 62 selected patients, we developed a systemic modular approach to identify differentially expressed modules (DEMs) of DHI with the Zsummary value less than 0 compared with the control group, calculated by weighted gene co-expression network analysis (WGCNA), and sketched out the basic framework on a modular map with 25 functional modules targeted by DHI. Furthermore, the effective therapeutic module (ETM), defined as the highest correlation value with the phenotype alteration (ΔSAQ-AF, the change in SAQ-AF at Day 30 from baseline) calculated by WGCNA, was identified in the population with the best effect (ΔSAQ-AF ≥ 40), which is related to anticoagulation and regulation of cholesterol metabolism. We assessed the modular flexibility of this ETM using the global topological D value based on Euclidean distance, which is correlated with phenotype alteration (r2: 0.8204, P = 0.019) by linear regression. Our study identified the anti-angina therapeutic module in the effective population treated by the multi-target drug. Modular methods facilitate the discovery of network pharmacological mechanisms and the advancement of precision medicine. (ClinicalTrials.gov identifier: NCT01681316).

Optimization of 4,6-Disubstituted Pyrido[3,2-d]pyrimidines as Dual MNK/PIM Inhibitors to Inhibit Leukemia Cell Growth.

Mitogen-activated protein kinase-interacting kinases (MNKs) and provirus integration in maloney murine leukemia virus kinases (PIMs) are downstream enzymes of cell proliferation signaling pathways associated with the resistance of tyrosine kinase inhibitors. MNKs and PIMs have complementary effects to regulate cap-dependent translation of oncoproteins. Dual inhibitors of MNKs and PIMs have not been developed. We developed a novel 4,6-disubstituted pyrido[3,2-d]pyrimidine compound 21o with selective inhibition of MNKs and PIMs. The IC50's of 21o to inhibit MNK1 and MNK2 are 1 and 7 nM and those to inhibit PIM1, PIM2, and PIM3 are 43, 232, and 774 nM, respectively. 21o inhibits the growth of myeloid leukemia K562 and MOLM-13 cells with GI50's of 2.1 and 1.2 µM, respectively. 21o decreases the levels of p-eIF4E and p-4EBP1, the downstream products of MNKs and PIMs, as well as cap-dependent proteins c-myc, cyclin D1, and Mcl-1. 21o inhibits the growth of MOLM-13 cell xenografts without causing evident toxicity. 21o represents an innovative dual MNK/PIM inhibitor with a good pharmacokinetic profile.

Design, Synthesis and Bioactivity Evaluation of 4,6-Disubstituted Pyrido[3,2-d]pyrimidine Derivatives as Mnk and HDAC Inhibitors.

Both HDACs and Mnks play important role in translating multiple oncogenic signaling pathways during oncogenesis. As HDAC and Mnk are highly expressed in a variety of tumors; thus simultaneous inhibit HDAC and Mnk can increase the inhibition of tumor cell proliferation and provide a new way of inhibiting tumor growth. Based on the previous work and the merge pharmacophore method; we designed and synthesized a series of 4,6-disubstituted pyrido[3,2-d]pyrimidine derivatives as HDAC and Mnk dual inhibitors. Among them; compound A12 displayed good HDAC and Mnk inhibitory activity. In vitro antiproliferative assay; compound A12 exhibited the best antiproliferative activity against human prostate cancer PC-3 cells. Docking study revealed that the pyrido[3,2-d]pyrimidine framework and hydroxamic acid motif of compound A12 were essential for maintaining the activity of HDAC and Mnk. These result indicated that A12 was a potent Mnk /HDAC inhibitor and will be further researched.

Oncogenic TRIM37 Links Chemoresistance and Metastatic Fate in Triple-Negative Breast Cancer.

The majority of clinical deaths in patients with triple-negative breast cancer (TNBC) are due to chemoresistance and aggressive metastases, with high prevalence in younger women of African ethnicity. Although tumorigenic drivers are numerous and varied, the drivers of metastatic transition remain largely unknown. Here, we uncovered a molecular dependence of TNBC tumors on the TRIM37 network, which enables tumor cells to resist chemotherapeutic as well as metastatic stress. TRIM37-directed histone H2A monoubiquitination enforces changes in DNA repair that rendered TP53-mutant TNBC cells resistant to chemotherapy. Chemotherapeutic drugs triggered a positive feedback loop via ATM/E2F1/STAT signaling, amplifying the TRIM37 network in chemoresistant cancer cells. High expression of TRIM37 induced transcriptomic changes characteristic of a metastatic phenotype, and inhibition of TRIM37 substantially reduced the in vivo propensity of TNBC cells. Selective delivery of TRIM37-specific antisense oligonucleotides using antifolate receptor 1-conjugated nanoparticles in combination with chemotherapy suppressed lung metastasis in spontaneous metastatic murine models. Collectively, these findings establish TRIM37 as a clinically relevant target with opportunities for therapeutic intervention. SIGNIFICANCE: TRIM37 drives aggressive TNBC biology by promoting resistance to chemotherapy and inducing a prometastatic transcriptional program; inhibition of TRIM37 increases chemotherapy efficacy and reduces metastasis risk in patients with TNBC.

Wnt-mediated endothelial transformation into mesenchymal stem cell-like cells induces chemoresistance in glioblastoma.

Therapeutic resistance remains a persistent challenge for patients with malignant tumors. Here, we reveal that endothelial cells (ECs) acquire transformation into mesenchymal stem cell (MSC)-like cells in glioblastoma (GBM), driving tumor resistance to cytotoxic treatment. Transcriptome analysis by RNA sequencing (RNA-seq) revealed that ECs undergo mesenchymal transformation and stemness-like activation in GBM microenvironment. Furthermore, we identified a c-Met-mediated axis that induces ß-catenin phosphorylation at Ser675 and Wnt signaling activation, inducing multidrug resistance-associated protein-1(MRP-1) expression and leading to EC stemness-like activation and chemoresistance. Last, genetic ablation of ß-catenin in ECs overcome GBM tumor resistance to temozolomide (TMZ) chemotherapy in vivo. Combination of Wnt inhibition and TMZ chemotherapy eliminated tumor-associated ECs, inhibited GBM growth, and increased mouse survival. These findings identified a cell plasticity-based, microenvironment-dependent mechanism that controls tumor chemoresistance, and suggest that targeting Wnt/ß-catenin-mediated EC transformation and stemness activation may overcome therapeutic resistance in GBM.

Discovery of novel (+)-Usnic acid derivatives as potential anti-leukemia agents with pan-Pim kinases inhibitory activity.

Usnic acid (UA) is the main secondary metabolite isolated from lichens, with moderate anticancer activity. A small group of (+)-UA derivatives characterized with flavanone moiety was designed and synthesized, and their anticancer activities were evaluated in leukemia cells. It was demonstrated that (+)-UA derivatives 6a-6g inhibited the proliferation of leukemia cells HL-60 and K562 with low micromolar IC50 values. Mechanisms of action were investigated to find that 6g induced apoptosis in HL-60 and K562 cell lines, and affected the expression of MNK/eIF4E axis-related proteins, such as Mcl-1, p-eIF4E, p-4E-BP1. Finally, kinase inhibition assay suggested 6g was a potential inhibitor of pan-Pim kinases. Meanwhile, the blocking of phosphorylation of BAD and 4E-BP1 by 6g, together with the proposed binding mode of 6g with Pim-1 further confirmed its Pim inhibition effects. Our finding provides the sight towards the potential mechanism of (+)-UA derivatives 6g as anti-leukemia agents.

Structure-Based Design of Novel Benzimidazole Derivatives as Pin1 Inhibitors.

Peptidyl-prolyl cis/trans isomerase Pin1 plays a key role in amplifying and translating multiple oncogenic signaling pathways during oncogenesis. The blockade of Pin1 provided a unique way of disrupting multiple oncogenic pathways and inducing apoptosis. Aiming to develop potent Pin1 inhibitors, a series of benzimidazole derivatives were designed and synthesized. Among the derivatives, compounds 6h and 13g showed the most potent Pin1 inhibitory activity with IC50 values of 0.64 and 0.37 µM, respectively. In vitro antiproliferative assay demonstrated that compounds 6d, 6g, 6h, 6n, 6o and 7c exhibited moderate antiproliferative activity against human prostate cancer PC-3 cells. Taken together, these unique benzimidazole derivatives exhibited great potential to be further explored as potent Pin1 inhibitors with improved potency.

Comparative Study of Different Maternal Zinc Resource Supplementation on Performance and Breast Muscle Development of their Offspring.

Maternal zinc supplementation has a pivotal role in enhancing breast muscle development of the offspring. What is poorly defined is the impact of supplemental zinc from different sources on the offspring. Broiler breeders at 24-week-old were randomly divided into three treatments with six replicates of 40 hens each and respectively fed for 8 weeks with supplemental 0-(group Zn/C), 100 mg/kg organic-(group Zn/O), and 100 mg/kg inorganic-(group Zn/I) zinc. The male offspring from each nutritional treatment were allocated into eight cages of 14 birds each, and a commercial diet supplemented with zinc from ZnSO4 at 20 mg/kg was fed to the offsprings. Results showed that eggs from Zn/O group had the highest zinc deposition (P < 0.05). Furthermore, maternal zinc supplementation promoted breast muscle yield; increased serum insulin and IGF-I concentration; upregulated AKT, mTOR, and P70S6K mRNA levels; and improved the phosphorylation of AKT at Serine 473 residue, mTOR at Serine 2448 residue, and FOXO at Serine 256 residue in the breast muscles of the offspring. In contrast, hens' diet supplemented with zinc could result in downregulation of atrogin-1 and MuRF1 mRNA levels in the breast muscle of the offspring. Additionally, no significant effect on breast muscle development post-hatch was observed between organic and inorganic zinc supplementation. In conclusion, maternal organic zinc supplementation improved zinc deposition in egg; however, no significant difference was detected in breast muscle development of the offspring of broiler breeder between organic and inorganic zinc supplementation.

Dietary Genistein Alleviates Lipid Metabolism Disorder and Inflammatory Response in Laying Hens With Fatty Liver Syndrome.

This study investigated the molecular mechanism underlying the effect of dietary genistein (GEN) on fatty liver syndrome (FLS) in laying hens. Hens in the control group (CG) were fed a high-energy and low-choline (HELC) diet to establish the FLS model. The livers of the FLS hens were friable and swollen from hemorrhage. Hepatic steatosis and inflammatory cell infiltration were present around the liver blood vessels. Hens in the low-genistein (LGE) and high-genistein (he) groups were fed GEN at 40 and 400 mg/kg doses, respectively, as supplements to the HELC diet. GEN at 40 mg/kg significantly increased gonadotropin-releasing hormone (GnRH) mRNA expression in the hypothalamus, the serum estrogen (E2) level, and the laying rate, whereas 400 mg/kg of GEN decreased GnRH expression and the laying rate without significantly affecting E2, suggesting that high-dose GEN adversely affected the reproductive performance. Either high- or low-dose GEN treatment could alleviate metabolic disorders and inflammatory responses in FLS hens. GEN significantly decreased the serum ALT, creatinine, triglyceride (TG), total cholesterol (TC), and free fatty acid (FFA) levels. Accordingly, the TG and long-chain fatty acid (LCFA) levels, including long-chain saturated fatty acids (LSFAs) and monounsaturated fatty acids (MUFAs), and the n-6:n-3 polyunsaturated fatty acid (PUFA) ratio in the liver were reduced after the GEN treatments, whereas the levels of C22:0, n-3 family fatty acids, C20:3n6, and C20:4n6 were increased. These results indicated that dietary GEN downregulated the expression of genes related to fatty acid synthesis [sterol regulatory element-binding protein 1 (SREBP1c), liver X receptor alpha (LXRα), fatty acid synthase (FAS), and acetyl coenzyme A synthetase (ACC)] and the fatty acid transporter (FAT). Furthermore, GEN treatments upregulated the transcription of genes related to fatty acid ß-oxidation [peroxisome proliferator-activated receptor (PPAR)α, PPARδ, ACOT8, ACAD8, and ACADs] in the liver and reduced PPARγ and AFABP expression in abdominal fat. Dietary GEN alleviated inflammatory cell infiltration in the livers of FLS hens and downregulated TNF-α, IL-6, and IL-1ß expression. Moreover, GEN treatment increased SOD activity and decreased malondialdehyde activity in the liver. In conclusion, GEN supplementation in the feed inhibited fatty acid synthesis and enhanced ß-oxidation in the liver through the PPAR-ACAD/ACOT and PPAR-LXRα-SREBP1c-ACC/FAS/FAT pathways. Dietary GEN alleviated metabolic disorder and inflammation in the FLS hens by improving the antioxidant capacity and fatty acid profile.

Intraventricular infusion of clusterin ameliorated cognition and pathology in Tg6799 model of Alzheimer's disease.

BACKGROUND: Alzheimer's disease (AD) is characterized by the deposition of amyloid-ß (Aß) in brain parenchyma and cerebral blood vessels as cerebral amyloid angiopathy (CAA). Clusterin, a chaperon protein associated with Aß aggregation, toxicity and transport through blood-brain barrier, may play a key role in the development of AD. Recently, clusterin peptide D-[113-122] was shown to mimic clusterin's function and exerted therapeutic effect in atherosclerosis. In this study, we investigated whether this clusterin peptide also affected (Aß) deposition in AD transgenic mouse. RESULTS: Using a micropump, synthetic peptide 113-122 of clusterin protein (20 µg/200 µl) was infused into the lateral ventricle of 8-month 5 × FAD transgenic mouse model (Tg6799), for 2 weeks. Water-maze testing showed an improved cognitive function of the Tg6799 mice treated with clusterin. Immunocytochemistry and quantitative analysis revealed that intraventricular (icv) administration of clusterin peptide in Tg6799 mouse reduced Aß plaques as well the severity of cerebral amyloid angiopathy. Enzyme-linked immunosorbent assay demonstrated a decreased in the soluble levels of Aß (Aß40 and Aß42) in the brain. Western-blot revealed an increased level of LRP-2 after clusterin peptide treatment. CONCLUSION: These results suggest that icv infusion of clusterin peptide D-[113-122] offers a promising therapeutic approach to reduce Aß deposition as well as CAA. The LRP2-mediated clearance system might be involved in the mechanism of these effects.

Down-regulation of Noggin and miR-138 coordinately promote osteogenesis of mesenchymal stem cells.

Mesenchymal stem cells (MSCs) can differentiate to osteocytes under suitable conditions. In recent years, micro-nucleotides have been progressively used to modulate gene expression in cells due to the consideration of safety. Our present study aimed to investigate whether co-delivery of Noggin-siRNA and antimiR-138 enhances the osteogenic effect of MSCs. Using a murine MSC line, C3H/10T1/2 cells, the delivery efficiency of Noggin-siRNA and antimiR-138 into MSCs was evaluated by quantitative real-time polymerase chain reaction (qRT-PCR). Cell phenotype and proliferation capacity was assessed by flow cytometry and MTT assay respectively. The osteogenesis of MSCs was tested by Alkaline Phosphatase (ALP) staining, qRT-PCR, and western blot analyses. Our results demonstrated that the expression of Noggin and miR-138 were significantly silenced in MSCs by Noggin-siRNA and/or antimiR-138 delivery, while the phenotype and proliferation capacity of MSCs were not affected. Down-regulation of Noggin and miR-138 cooperatively promoted osteogenic differentiation of MSCs. The ALP positive cells reached about 83.57 ± 10.18%. Compared with single delivery, the expression of osteogenic related genes, such as Alp, Col-1, Bmp2, Ocn and Runx2, were the highest in cells with co-delivery of the two oligonucleotides. Moreover, the protein level of RUNX2, and the ratios of pSMAD1/5/SMAD1/5 and pERK1/2/ERK1/2 were significantly increased. The activation of Smad, Erk signaling may constitute the underlying mechanism of the enhanced osteogenesis process. Taken together, our study provides a safe strategy for the clinical rehabilitation application of MSCs in skeletal deficiency.

Upregulation of CC Chemokine Receptor 7 (CCR7) Enables Migration of Xenogeneic Human Adipose-Derived Mesenchymal Stem Cells to Rat Secondary Lymphoid Organs.

BACKGROUND CC chemokine receptor 7 (CCR7) expression is vital for cell migration to secondary lymphoid organs (SLOs). Our previous work showed that inducing CCR7 expression enabled syngeneic mesenchymal stem cells (MSCs) to migrate into SLOs, resulting in enhanced immunosuppressive performance in mice. Given that human adipose-derived stem cells (hASCs) are widely used in clinical therapy, we further investigated whether upregulation of CCR7 enables xenogeneic hASCs to migrate to rat SLOs. MATERIAL AND METHODS hASCs rarely express CCR7; therefore, hASCs were transfected with lentivirus encoding rat CCR7 (rCCR7) plus green fluorescence protein (GFP) or GFP alone. CCR7 mRNA and cell surface expression of rCCR7-hASCs and GFP-hASCs were examined by reverse transcription-polymerase chain reaction (RT-PCR) and flow cytometry (FCM), respectively. The phenotype, differentiation, and proliferation capacity of each cell type was also determined. To examine migration, rCCR7-hASCs and GFP-hASCs were injected intravenously into Lewis rats, and the proportion of GFP-positive cells in the spleen and lymph nodes was determined with FCM. RESULTS mRNA and cell surface protein expression of CCR7 was essentially undetectable in hASCs and GFP-ASCs; however, CCR7 was highly expressed in rCCR7-ASCs. rCCR7-hASCs, GFP-hASCs, and hASCs shared a similar immunophenotype, and maintained the ability of multilineage differentiation and proliferation. In addition, the average proportion of GFP-positive cells was significantly higher following transplantation of rCCR7-hASCs compared with GFP-hASCs (p<0.01). CONCLUSIONS These results suggest that upregulation of rat CCR7 expression does not change the phenotype, differentiation, or proliferation capacity of hASCs, but does enable efficient migration of hASCs to rat SLOs.

Effect of rhBNP on renal function in STEMI-HF patients with mild renal insufficiency undergoing primary PCI.

This study aims to investigate the effect of recombinant human brain natriuretic peptide (rhBNP) on renal function and contrast-induced nephropathy (CIN) incidence in ST-segment elevation myocardial infarction and heart failure (STEMI-HF) patients with mild renal insufficiency undergoing primary percutaneous coronary intervention (PCI). A total of 116 participants were randomized into rhBNP (rhBNP, n = 57) and nitroglycerin group (NIT, n = 59), receiving intravenous rhBNP or nitroglycerin from admission to 72 h after PCI. Renal function was assessed by serum creatinine (SCr), estimated glomerular filtration rate (eGFR), Cystatin-C (Cys-C) and ß2-microglobulin before and after primary PCI, and calculated the incidence of CIN within 72 h after PCI. There were no significant differences in SCr, eGFR and ß2-microglobulin between the two groups (P > 0.05, respectively). Compared with the NIT group, the total urinary volume within 72 h was higher while the level of Cys-C at 24 and 72 h after PCI was lower in the rhBNP group. rhBNP was associated with a decline in the incidence of CIN (12.28 vs. 28.81 %, P < 0.05). No differences were detected in mortality and re-hospitalization in 3 months between the two groups. The incidence of renal injury was not different between rhBNP and nitroglycerin in STEMI-HF patients with mild renal insufficiency. However, infusion of rhBNP was associated with a decline in incidence of CIN.

Effects of liposomal prostaglandin E1 on periprocedural myocardial injury in patients with unstable angina undergoing an elective percutaneous coronary intervention.

OBJECTIVES: The aim of this study was to explore whether intravenous administration of liposomal prostaglandin E1 (lipo-PGE1) can reduce the incidence of periprocedural myocardial injury (PMI) in patients with unstable angina undergoing an elective percutaneous coronary intervention (PCI). PATIENTS AND METHODS: In this randomized-controlled study, a total of 219 patients were randomly assigned to a lipo-PGE1 group (n=110) and a control group (n=109). Patients in the lipo-PGE1 group received 20 µg/day of lipo-PGE1 diluted in 10 ml of normal saline through an intravenous injection over 5 min starting at 3 days before PCI and continuing for 4 days after PCI. In the control group, 10 ml of normal saline was administered using the same method. The primary end point was the occurrence of PMI defined as an elevation of cardiac troponin I above the upper limit of normal within 24 h after the procedure. The secondary end points were (i) changes in inflammatory factors including plasma high-sensitivity C-reactive protein, tumor necrosis factor α, and interleukin 6 before and at 24 h after PCI; (ii) the incidence of major adverse cardiac events in the patients during hospitalization and 30 days of follow-up after discharge, including cardiac deaths, severe heart failure, malignant arrhythmias, and target vessel revascularization. RESULTS: Within 24 h after PCI, the incidence of PMI was significantly lower in the lipo-PGE1 group compared with that in the control group (20 vs. 36.69%, P=0.009). Although the procedure induced a significant increase in high-sensitivity C-reactive protein, tumor necrosis factor α, and interleukin 6 levels, the values were significantly lower in the lipo-PGE1 group than those in the control group at 24 h after PCI (P<0.05). The proportion of thrombolysis in myocardial infarction grade 3 in the lipo-PGE1 group was higher than that in the control group (92.72 vs. 82.56%, P=0.037). There were no significant differences between the lipo-PGE1 group and the control group in the incidence of major adverse cardiac events during hospitalization and 30 days of follow-up (2.1 vs. 4%, P=0.72). Multivariate logistic analysis showed that lipo-PGE1 was an independent protective factor against PMI (odds ratio 0.385, 95% confidence interval 0.195-0.760, P=0.006). CONCLUSION: Intravenous lipo-PGE1 can reduce the incidence of PMI following elective PCI in patients with unstable angina. The benefit of lipo-PGE1 may be associated with the effects of anti-inflammation as well as improvement in coronary microvascular perfusion.

Fatty acid binding protein FABP3 from Chinese mitten crab Eriocheir sinensis participates in antimicrobial responses.

Intracellular fatty acid-binding proteins (FABPs) are members of the lipid-binding protein superfamily. Aside from the main functions of FABPs in the uptake and transport of fatty acids, they are also critical in innate immunology. In this work, the full-length cDNA for a Chinese mitten crab Eriocheir sinensis FABP (Es-FABP3) was cloned with an open reading frame of 402 bp encoding a 133 amino acid polypeptide. Analysis using quantitative real-time PCR (qPCR) revealed that Es-FABP3 transcripts were widely distributed in gills, muscle, intestine, hepatopancreas, eyestalk, heart, stomach, brain, thoracic ganglia and hemocytes. After challenge with pathogen associated molecular pattern molecules (PAMPs), the relative mRNA expression levels of Es-FABP3 increased in hepatopancreas, gills and hemocytes. Moreover, the mature recombinant Es-FABP3 protein exhibited different binding activities to bacteria and fungus and inhibited the growth of different microbes. These collective results demonstrated the role of Es-FABP3 in the immunoreactions of E. sinensis to PAMPs.

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, characterization and expression analysis of two apoptosis genes, caspase and nm23, involved in the antibacterial response in Chinese mitten crab, Eriocheir sinensis.

Apoptosis is a central regulatory feature of the immune system, and the most common form of death among immunological cells. However, the function of apoptosis, within the innate immune system of invertebrates, remains largely unknown. For this reason, we investigated the immune functionality of two apoptosis genes, caspase and nm23, in the Chinese mitten crab (Eriocheir sinensis), which is a commercially important and disease vulnerable aquaculture species. The entire length caspase and nm23 cDNA genes were cloned using PCR, based on an initial expressed sequence tag (EST) isolated from a hepatopancreatic cDNA library. The caspase cDNA contained an 1119 bp open reading frame that encoded a putative 372 amino acid protein, while nm23 cDNA contained a 456 bp open reading frame that encoded a putative 151 amino acid protein. Comparison, with other reported invertebrate and vertebrate sequences, revealed the presence of conserved enzyme active sites that were common among caspase and nm23 superfamilies. In brief, caspase and nm23 mRNA expression in E. sinensis were (a) both detected in all tissues, including the hemocytes, heart, hepatopancreas, gill, stomach, muscle, intestine, brain and eyestalk, and (b) responsive in hemocytes, gill and hepatopancreas to a Vibrio anguillarum immuno-challenge all appeared sharp increase. Collectively, the data presented here demonstrate the successful isolation of caspase and nm23 apoptosis genes from the Chinese mitten crab, and their role in the innate immune system of an invertebrate.

Molecular cloning and tissue expression of the fatty acid-binding protein (Es-FABP9) gene in the reproduction seasons of Chinese mitten crab, Eriocheir sinensis.

Fatty acid-binding proteins (FABPs), small cytosolic proteins that function in the uptake and utilization of fatty acids, have been extensively studied in higher vertebrates while invertebrates have received little attention despite similar nutritional requirements during periods of reproductive activity. Therefore, a cDNA encoding Eriocheir sinensis FABP (Es-FABP9) was cloned based upon EST analysis of a testis cDNA library. The full length cDNA was 898 bp and encoded a 136 aa polypeptide that was highly homologous to related genes reported in shrimp. Gene expression analysis, as determined by RT-PCR, revealed the presence of Es-FABP9 transcripts was widely distributed with high and detectable expression levels observed in intestine, ovary, testis and heart, while expression were comparable among hepatopancreas, hemolymphe, gills, muscle, stomach and brain. Real-time quantitative RT-PCR analysis revealed that Es-FABP9 expression in testis, hemolymphe, hepatopancreas and ovarian was dependent on the status of testis development. Evidence provided in the present report supports a role of Es-FABP9 in lipid transport during the period of rapid testis growth in E. sinensis, and indirectly confirms the participation of the hepatopancreas, testis, hemolymphe and ovarian in lipid nutrient absorption and utilization processes.

Molecular cloning, characterization, expression and activity analysis of cathepsin L in Chinese mitten crab, Eriocheir sinensis.

Cathepsins, a superfamily of hydrolytic enzymes produced and enclosed within lysosomes, function in immune response in vertebrates; however, their function within the innate immune system of invertebrates remains largely unknown. Therefore, we investigated the immune functionality of cathepsin L (catL) in Chinese mitten crab (Eriocheir sinensis), a commercially important and disease vulnerable aquaculture species. The full-length catL cDNA (1274 bp) was cloned via PCR based upon an initial expressed sequence tag (EST) isolated from a hepatopancreatic cDNA library. The catL cDNA contained a 978 bp open reading frame (ORF) that encoded a putative 325 amino acid (aa) protein. Comparisons with other reported invertebrate and vertebrate sequences revealed conserved gene structure and enzyme active sites common among papain-like cysteine proteases, and high percent identity among other invertebrate cathepsins. CatL mRNA expression in E. sinensis was (a) tissue-specific, with the highest expression observed in hepatotpancreas, gill, stomach, and hemocytes, and (b) responsive in hemocytes to a Vibrio anguillarum challenge, the catL expression level and enzyme activity both with peak exposure observed 8 h post-injection. Collectively, data demonstrate the successful isolation of catL from the Chinese mitten crab, and its involvement in the innate immune system of an invertebrate.