Chromosome-level genome assembly of the Chinese longsnout catfish Leiocassis longirostris.

The Chinese longsnout catfish ( Leiocassis longirostris Günther) is one of the most economically important freshwater fish in China. As wild populations have declined sharply in recent years, it is also a valuable model for research on sexual dimorphism, comparative biology, and conservation. However, the current lack of high-quality chromosome-level genome information for the species hinders the advancement of comparative genomic analysis and evolutionary studies. Therefore, we constructed the first high-quality chromosome-level reference genome for L. longirostris. The total genome was 703.19 Mb, with 389 contigs and contig N50 length of 4.29 Mb. Using high-throughput chromosome conformation capture (Hi-C) data, the genome sequences (685.53 Mb) were scaffolded into 26 chromosomes ranging from 17.36 to 43.97 Mb, resulting in a chromosomal anchoring rate for the genome of 97.44%. In total, 23 708 protein-coding genes were identified in the genome. Phylogenetic analysis indicated that L. longirostris and its closest related species P. fulvidraco diverged approximately 26.6 million years ago. This high-quality reference genome of L. longirostris should pave the way for future genomic comparisons and evolutionary research.

Design, synthesis and biological evaluation of 1H-indazole derivatives as novel ASK1 inhibitors.

Apoptosis signal-regulating kinase 1 (ASK1, MAP3K5), a member of the mitogen-activated protein kinase (MAPK) signaling pathway, is involved in cell survival, differentiation, stress response, and apoptosis. ASK1 kinase inhibition has emerged as a promising therapeutic strategy for inflammatory disease. A series of novel ASK1 inhibitors with 1H-indazole scaffold were designed, synthesized and evaluated for their ASK1 kinase activity and AP1-HEK293 cell inhibitory effect. Systematic structure-activity relationship (SAR) efforts led to the discovery of promising compound 15, which showed excellent in vitro ASK1 kinase activity and potent inhibitory effects on ASK1 in AP1-HEK293 cells. In a tumor necrosis factor-α (TNF-α)-induced HT-29 intestinal epithelial cell model, compound 15 exhibited a significantly protective effect on cell viability comparable to that of GS-4997; moreover, compound 15 exhibited no obvious cytotoxicity against HT-29 cells at concentrations up to 25 µM. Mechanistic research demonstrated that compound 15 suppresses phosphorylation in the ASK1-p38/JNK signaling pathway in HT-29 cells, and regulates the expression levels of apoptosis-related proteins. Altogether, these results show that compound 15 may serve as a potential candidate compound for the treatment of inflammatory bowel disease (IBD).

Structure-based discovery of 1H-indole-2-carboxamide derivatives as potent ASK1 inhibitors for potential treatment of ulcerative colitis.

Apoptosis signal-regulating kinase 1 (ASK1), a member of the mitogen-activated protein kinase (MAPK) family, is implicated in many human diseases. Here, we describe the structural optimization of hit compound 7 and conduct further structure-activity relationship (SAR) studies that result in the development of compound 19 with a novel indole-2-carboxamide hinge scaffold. Compound 19 displays potent anti-ASK1 kinase activity and stronger inhibitory effect on ASK1 in AP1-HEK293 cells than previously described ASK1 inhibitor GS-4997. Besides improved in vitro activity, compound 19 also exhibits an appropriate in vivo PK profile. In a dextran sulfate sodium (DSS)-induced mouse model of ulcerative colitis (UC), compound 19 shows significant anti-UC efficacy and markedly attenuates DSS-induced body weight loss, colonic shortening, elevation in disease activity index (DAI) and inflammatory cell infiltration in colon tissues. Mechanistically, compound 19 represses the phosphorylation of ASK1-p38/JNK signaling pathways and suppresses the overexpression of inflammatory cytokines. Together, these findings suggest that ASK1 inhibitors can potentially be used as a therapeutic strategy for UC.

Full-length transcript sequencing accelerates the transcriptome research of Gymnocypris namensis, an iconic fish of the Tibetan Plateau.

Gymnocypris namensis, the only commercial fish in Namtso Lake of Tibet in China, is rated as nearly threatened species in the Red List of China's Vertebrates. As one of the highest-altitude schizothorax fish in China, G. namensis has strong adaptability to the plateau harsh environment. Although being an indigenous economic fish with high value in research, the biological characterization, genetic diversity, and plateau adaptability of G. namensis are still unclear. Here, we used Pacific Biosciences single molecular real time long read sequencing technology to generate full-length transcripts of G. namensis. Sequences clustering analysis and error correction with Illumina-produced short reads to obtain 319,044 polished isoforms. After removing redundant reads, 125,396 non-redundant isoforms were obtained. Among all transcripts, 103,286 were annotated to public databases. Natural selection has acted on 42 genes for G. namensis, which were enriched on the functions of mismatch repair and Glutathione metabolism. Total 89,736 open reading frames, 95,947 microsatellites, and 21,360 long non-coding RNAs were identified across all transcripts. This is the first study of transcriptome in G. namensis by using PacBio Iso-seq. The acquisition of full-length transcript isoforms might accelerate the transcriptome research of G. namensis and provide basis for further research.

De novo assembly of Schizothorax waltoni transcriptome to identify immune-related genes and microsatellite markers.

Schizothorax waltoni (S. waltoni) is one kind of the subfamily Schizothoracinae and an indigenous economic tetraploid fish to Tibet in China. It is rated as a vulnerable species in the Red List of China's Vertebrates, owing to overexploitation and biological invasion. S. waltoni plays an important role in ecology and local fishery economy, but little information is known about genetic diversity, local adaptation, immune system and so on. Functional gene identification and molecular marker development are the first and essential step for the following biological function and genetics studies. For this purpose, the transcriptome from pooled tissues of three adult S. waltoni was sequenced and analyzed. Using paired-end reads from the Illumina Hiseq4000 platform, 83 103 transcripts with an N50 length of 2337 bp were assembled, which could be further clustered into 66 975 unigenes with an N50 length of 2087 bp. The majority of the unigenes (58 934, 87.99%) were successfully annotated by 7 public databases, and 15 KEGG pathways of immune-related genes were identified for the following functional research. Furthermore, 19 497 putative simple sequence repeats (SSRs) of 1-6 bp unit length were detected from 14 690 unigenes (21.93%) with an average distribution density of 1 : 3.28 kb. We identified 3590 unigenes (5.36%) containing more than one SSR, providing abundant potential polymorphic markers in functional genes. This is the first reported high-throughput transcriptome analysis of S. waltoni, and it would provide valuable genetic resources for the functional genes involved in multiple biological processes, including the immune system, genetic conservation, and molecular marker-assisted breeding of S. waltoni.

Involvement of Caveolin-1 in CD83 Internalization in Mouse Dendritic Cells.

To become potent T-cell stimulators, DCs need to mature. Treatment with soluble CD83 (sCD83) induces immune tolerance and protects against transplant rejection by maintaining dendritic cells in an immature, tolerogenic state. Until now, the mechanism through which sCD83 keeps DCs immature has not been investigated. The internalizing pathway of CD83 was screened by Western blot, and the direct interactions between internalized proteins were verified through coimmunoprecipitation (co-IP) and transmission electron microscopy (TEM). CD83 plasma membrane levels were detected by Western blot using a plasma membrane protein extraction protocol. The changes in CD83 surface levels in DCs were detected by flow cytometry. Caveolin-1 function was detected in a kidney transplant model. In this study, we demonstrated that caveolin-1 could affect CD83 level during endocytosis in mouse DCs. Caveolin-1 coprecipitates with CD83, as demonstrated by co-IP analysis. TEM morphometric analysis of the entire CD83 distribution associated with internalized caveolin-1 demonstrated a significant interaction in cellular vesicles. sCD83 reduces endogenous CD83 plasma membrane levels, and caveolin-1 knockdown reverts CD83 levels in plasma membrane. sCD83 treatment decreases CD83 surface levels in DCs. siRNA to caveolin-1 in DCs inhibits this effect of sCD83. The effects of sCD83-treated DCs were proved in CD1 mice. Knocking down caveolin-1 in DCs obstructs the effects of sCD83 on kidney transplant. In conclusion, our data indicated that a caveolin-dependent endocytic pathway is involved in CD83 internalization in DCs and that caveolin-1 is involved in the activity of DCs.

Hospital quality improvement initiative for patients with acute coronary syndromes in China: a cluster randomized, controlled trial.

Background- Substantial evidence-practice gaps exist in the management of acute coronary syndromes (ACS) in China. Clinical pathways are tools for improving ACS quality of care but have not been rigorously evaluated. Methods and Results- Between October 2007 and August 2010, a quality improvement program was conducted in 75 hospitals throughout China with mixed methods evaluation in a cluster randomized, controlled trial. Eligible hospitals were level 2 or level 3 centers routinely admitting >100 patients with ACS per year. Hospitals were assigned immediate implementation of the American Heart Association/American College of Cardiology guideline based clinical pathways or commencement of the intervention 12 months later. Outcomes were several key performance indicators reflecting the management of ACS. The key performance indicators were measured 12 months after commencement in intervention hospitals and compared with baseline data in control hospitals, using data collected from 50 consecutive patients in each hospital. Pathway implementation was associated with an increased proportion of patients discharged on appropriate medical therapy, with nonsignificant improvements or absence of effects on other key performance indicators. Conclusions- Among hospitals in China, the use of a clinical pathway for the treatment of ACS compared with usual care improved secondary prevention treatments, but effectiveness was otherwise limited. An accompanying process evaluation identified several health system barriers to more successful implementation. Clinical Trial Registration- URL: http://www.anzctr.org.au/default.aspx. Unique identifier: ACTRN12609000491268.

Comparison of cilostazol versus ticlopidine following coronary stenting in patients with coronary heart disease: A meta-analysis of randomized controlled trials.

Previous studies have shown that the combination of cilostazol and aspirin may be a more effective regimen than ticlopidine plus aspirin in the prevention of late restenosis and acute or subacute stent thrombosis following coronary stenting; however, individually published results are inconclusive. The aim of this meta-analysis was to compare the differences in late restenosis and stent thrombosis between cilostazol plus aspirin and ticlopidine plus aspirin for patients with coronary heart disease (CHD) following coronary stenting. A literature search of Pubmed, Embase, Web of Science and Chinese BioMedicine (CBM) databases was conducted from 1998 to March 1, 2013 and statistical analysis was performed using Stata statistical software, version 12.0. Twelve randomized controlled trials were included in the study, with a total of 2,708 patients with CHD following coronary stenting. The patient population comprised 1,371 patients treated with cilostazol plus aspirin and 1,337 patients treated with ticlopidine plus aspirin. The meta-analysis showed that cilostazol plus aspirin demonstrated a lower rate of restenosis than ticlopidine plus aspirin [odds ratio (OR)=0.83, 95% confidence interval (CI)=0.69-0.99, P=0.047]. A significant difference was also observed in the average percent diameter stenosis between cilostazol plus aspirin and ticlopidine plus aspirin [standardized weight difference (SMD)= -0.57, 95% CI=-0.92, -0.23, P=0.001). However, there were no significant differences in the rates of acute or subacute stent thrombosis between cilostazol plus aspirin and ticlopidine plus aspirin. The present meta-analysis suggests that cilostazol plus aspirin may result in a lower restenosis rate and percent diameter stenosis than ticlopidine plus aspirin for patients with CHD following coronary stenting.

Specific LPA receptor subtype mediation of LPA-induced hypertrophy of cardiac myocytes and involvement of Akt and NFkappaB signal pathways.

Lysophosphatidic acid (LPA) is a bioactive phospholipid with diverse functions mediated via G-protein-coupled receptors (GPCRs). In view of the elevated levels of LPA in acute myocardial infarction (MI) patients we have conducted studies aimed at identifying specific LPA receptor subtypes and signaling events that may mediate its actions in hypertrophic remodeling. Experiments were carried out in cultured neonatal rat cardiomyocytes (NRCMs) exposed to LPA and in a rat MI model. In NRCMs, LPA-induced hypertrophic growth was completely abrogated by DGPP, an LPA1/LPA3 antagonist. The LPA3 agonist OMPT, but not the LPA2 agonist dodecylphosphate, promoted hypertrophy as examined by 3[H]-Leucine incorporation, ANF-luciferase expression and cell area. In in vivo experiments, LPA1, LPA2 and LPA3 mRNA levels as well as LPA1 and LPA3 protein levels increased together with left ventricular remodeling (LVRM) after MI. In addition, LPA stimulated the phosphorylation of Akt and p65 protein and activated NF-kappaB-luciferase expression. Inhibitors of PI3K (wortmannin), mTOR (rapamycin), and NF-kappaB (PDTC or SN50) effectively prevented LPA-induced 3[H]-Leucine incorporation and ANF-luciferase expression. Furthermore, ERK inhibitors (U0126 and PD98059) suppressed LPA-stimulated activation of NF-kappaB and p65 phosphorylation whereas wortmannin showed no effect on NF-kappaB activation. Our findings indicate that LPA3 and/or LPA1 mediate LPA-induced hypertrophy of NRCMs and that LPA1 and LPA3 may be involved in LVRM of MI rats. Moreover, Akt and NF-kappaB signaling pathways independently implicate in LPA-stimulated myocardial hypertrophic growth.