Antibacterial Activity and Mechanisms of TroHepc2-22, a Derived Peptide of Hepcidin2 from Golden Pompano (Trachinotus ovatus).

Hepcidin, a cysteine-rich antimicrobial peptide, has a highly conserved gene structure in teleosts, and it plays an essential role in host immune response against various pathogenic bacteria. Nonetheless, few studies on the antibacterial mechanism of hepcidin in golden pompano (Trachinotus ovatus) have been reported. In this study, we synthesized a derived peptide, TroHepc2-22, from the mature peptide of T. ovatus hepcidin2. Our results showed that TroHepc2-22 has superior antibacterial abilities against both Gram-negative (Vibrio harveyi and Edwardsiella piscicida) and Gram-positive (Staphylococcus aureus and Streptococcus agalactiae) bacteria. Based on the results of a bacterial membrane depolarization assay and propidium iodide (PI) staining assay in vitro, TroHepc2-22 displayed antimicrobial activity by inducing the bacterial membrane depolarization and changing the bacterial membrane permeability. Scanning electron microscopy (SEM) visualization illustrated that TroHepc2-22 brought about membrane rupturing and the leakage of the cytoplasm for the bacteria. In addition, TroHepc2-22 was verified to have hydrolytic activity on bacterial genomic DNA in view of the results of the gel retardation assay. In terms of the in vivo assay, the bacterial loads of V. harveyi in the tested immune tissues (liver, spleen, and head kidney) were significantly reduced in T. ovatus, revealing that TroHepc2-22 significantly enhanced the resistance against V. harveyi infection. Furthermore, the expressions of immune-related genes, including tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), interleukin 1-ß (IL-1ß), IL-6, Toll-like receptor 1 (TLR1), and myeloid differentiation factor 88 (MyD88) were significantly increased, indicating that TroHepc2-22 might regulate inflammatory cytokines and activate immune-related signaling pathways. To summarize, TroHepc2-22 possesses appreciable antimicrobial activity and plays a vital role in resisting bacterial infection. The observation of our present study unveils the excellent application prospect of hepcidin as a substitute for antibiotics to resist pathogenic microorganisms in teleosts.

CpG ODN 1668 as TLR9 agonist mediates humpback grouper (Cromileptes altivelis) antibacterial immune responses.

Cromileptes altivelis (humpback grouper) is the main farmed species in the southern coastal area of China owing to its important economic value. Toll-like receptor 9 (TLR9) belongs to the toll-like receptor (TLR) family and functions as a pattern recognition receptor, recognising unmethylated oligodeoxynucleotides containing the CpG motif (CpG ODNs) in bacterial and viral genomes, thereby activating host immune response. In this study, the C. altivelis TLR9 (CaTLR9) ligand CpG ODN 1668 was screened and found to significantly enhance the antibacterial immunity of humpback grouper in vivo and head kidney lymphocytes (HKLs) in vitro. In addition, CpG ODN 1668 also promoted the cell proliferation and immune gene expression of HKLs and strengthened the phagocytosis activity of head kidney macrophages. However, when the CaTLR9 expression was knocked down in the humpback group, the expression levels of TLR9, myeloid differentiation factor 88 (Myd88), tumour necrosis factor-α (TNF-α), interferon γ (IFN-γ), interleukin-1ß (IL-1ß), IL-6, and IL-8 were significantly reduced, and the antibacterial immune effects induced by CpG ODN 1668 were mostly abolished. Therefore, CpG ODN 1668 induced antibacterial immune responses in a CaTLR9-dependent pathway. These results enhance the knowledge of the antibacterial immunity of fish TLR signalling pathways and have important implications for exploring natural antibacterial molecules in fish.

CpG ODN 2102 promotes antibacterial immune responses and enhances vaccine-induced protection in golden pompano (Trachinotusovatus).

CpG oligodeoxynucleotides (ODNs) are oligodeoxynucleotides containing CpG motifs and can be recognized by toll-like receptor 9 (TLR9), activating the host's immune responses. In this study, ten different CpG ODNs were designed and synthesized to study the antibacterial immune responses of CpG ODNs in golden pompano (Trachinotus ovatus). Results showed that CpG ODN 2102 significantly improved the immunity of golden pompano against bacteria. Besides, CpG ODN 2102 promoted the proliferation of head kidney lymphocytes and activated the head kidney macrophages. When TLR9-specific small interfering RNA (siRNA) was used to interfere with TLR9 expression, the immune responses were decreased. Moreover, the expression levels of myeloid differentiation primary response 88 (Myd88), p65, tumor necrosis factor receptor-associated factor 6 (TRAF6), and tumor necrosis factor-alpha (TNF-α) in the TLR9-knockdown golden pompano kidney (GPK) cells were significantly reduced. The nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) promoter activity of the TLR9-knockdown GPK cells was also significantly reduced. In vivo, the antibacterial immune effects induced by CpG ODN 2102 in golden pompano were mostly abolished when TLR9 expression was knocked down. These results suggested that TLR9 was involved in the immune responses induced by CpG ODN 2102. CpG ODN 2102 also enhanced the protective effect of the Vibrio harveyi vaccine pCTssJ, where the survival rate of golden pompano was significantly improved by 20%. In addition, CpG ODN 2102 enhanced the messenger RNA (mRNA) expression levels of TLR9, Myxovirus resistance (Mx), interferon γ (IFN-γ), TNF-α, interleukin (IL)-1ß, IL-8, major histocompatibility complex class (MHC) Iα, MHC IIα, Immunoglobulin D (IgD), and IgM. Therefore, TLR9 was involved in the antibacterial immune responses induced by CpG ODN 2102 and CpG ODN 2102 possessed adjuvant immune effects. These results enlarged our knowledge of the antibacterial immunity of fish TLRs signaling pathway and had important implications for exploring natural antibacterial molecules in fish and developing new vaccine adjuvants.

Molecular characterization and expression analysis of B-cell lymphoma-2 in Trachinotus ovatus and its role in apoptotic process.

Introduction: B-cell lymphoma-2 (Bcl-2) is the first identified member of the Bcl-2 family that performs an anti-apoptotic function in mammals. However, its role in teleosts is not fully understood. In this study, Bcl-2 of Trachinotus ovatus (TroBcl2) was cloned, and its role in apoptosis was investigated. Methods: In this study, Bcl-2 of Trachinotus ovatus (TroBcl2) was cloned by PCR. Quantitative real-time PCR (qRT-PCR) was used to detect its mRNA expression level in healthy condition and after LPS stimulation. Subcellular localization was performed by transfecting the pTroBcl2-N3 plasmid into golden pompano snout (GPS) cells and observed under an inverted fluorescence microscope DMi8 and further verified by immunoblotting. In vivo overexpression and RNAi knockdown method were performed to evaluate the role of TroBcl2 in apoptosis. The anti-apoptotic activity of TroBcl2 was detected by flow cytometry. The effect of TroBcl2 on the mitochondrial membrane potential (MMP) was measured by an enhanced mitochondrial membrane potential assay kit with JC-1. The terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) method was performed to evaluate the role of TroBcl2 in the DNA fragmentation. Immunoblotting was used to verify whether TroBcl2 inhibits the release of cytochrome c from mitochondria into the cytoplasm. The Caspase 3 and Caspase 9 Activity Assay Kits were used to investigate the effect of TroBcl2 on caspase 3 and caspase 9 activities. The effects of TroBcl2 on the expression of apoptosis-related and nuclear factor- κB (NF-κB) signaling pathway-related genes in vitro were evaluated by qRT-PCR and Enzyme linked immunosorbent assay (ELISA). Luciferase reporter assay was used to evaluate the activity in NF-κB signaling pathway. Results and discussion: The full-length coding sequence of TroBcl2 contains 687 bp and encodes a protein containing 228 amino acids. Four conserved Bcl-2 homology (BH) domains and one invariant "NWGR" motif located in BH1 were identified in TroBcl2. In healthy T. ovatus, TroBcl2 was widely distributed in the eleven tested tissues, and higher expression levels were found in immune-related tissues, such as spleen and head kidney tissues. After stimulation with lipopolysaccharide (LPS), the expression of TroBcl2 in the head kidney, spleen, and liver was significantly upregulated. In addition, subcellular localization analysis revealed that TroBcl2 was localized in both the cytoplasm and nucleus. Functional experiments showed that TroBcl2 inhibited apoptosis, possibly by reducing mitochondrial membrane potential loss, decreasing DNA fragmentation, preventing cytochrome c release into cytoplasm, and reducing the caspase 3 and caspase 9 activations. Moreover, upon LPS stimulation, overexpression of TroBcl2 suppressed the activation of several apoptosis-related genes, such as BOK, caspase-9, caspase-7, caspase-3, cytochrome c, and p53. Furthermore, knockdown of TroBcl2 significantly increased the expression of those apoptosis-related genes. In addition, TroBcl2 overexpression or knockdown induced or inhibited, respectively, the transcription of NF-κB and regulated the expression of genes (such as NF-κB1 and c-Rel) in the NF-κB signaling pathway as well as the expression of the downstream inflammatory cytokine IL-1ß. Overall, our study suggested that TroBcl2 performs its conserved anti-apoptotic function via the mitochondrial pathway and may serve as an anti-apoptotic regulator in T. ovatus.

Study on the application of optical coherence microscopy in Hirschsprung's disease.

To explore the clinical application value of optical coherence microscopy (OCM) in Hirschsprung's disease. 109 HSCR patients were recuited in a Chinese hospital from January 2018 to July 2021. All the recruited patients underwent barium enema angiography preoperatively and the resected diseased intestinal tubes were evaluated intraoperatively. The OCM and the histopathological examination were performed successively on the surgical specimens, and the OCM images were compared with the relevant tissue sections to characterize different lesions. 10 non-HSCR fetal colorectal tissues at the same period were retained for OCM, the characteristics of which with and without HSCR under OCM imaging were analyzed. In the OCM images of in vitro tissue, it can be clearly observed that the scattering degree of HSCR narrow segment mucosal is high, glands and crypt structures are reduced or even atrophy, and the scattering degree of submucosal and intermuscular is low; In the dilated segment, the low scattering and high scattering are complex, and the muscle layer is obviously hypertrophy and structural disorder. Compared with the pathological findings, the OCM sensitivity, Kappa value, and AUC area reached 92.66%, 0.63, and 0.91, respectively. OCM can quickly and clearly display the structure of all layers of colorectal tissue, which is highly consistent with the corresponding histopathological examination results and has high sensitivity. which will provide a more reliable basis for OCM diagnosis of early HSCR, targeted biopsy and location of operative treatment, and has a certain potential for clinical application.

TroTNFSF6, a tumor necrosis factor ligand superfamily member, promotes antibacterial immune response of golden pompano, Trachinotus ovatus.

Tumor necrosis factor ligand superfamily member 6 (TNFSF6), also known as FasL/CD95L, is essential for maintaining the body's immune homeostasis. However, the current reports on TNFSF6 in fish are relatively scarce. In the present study, we conducted functional analyses of a TNFSF6 (TroTNFSF6) from the teleost fish golden pompano (Trachinotus ovatus). TroTNFSF6 is composed of 228 amino acids and has a low similarity with other species (9.65%-58.79%). TroTNFSF6 was expressed in the 11 tissues tested and was significantly up-regulated after Edwardsiella tarda infection. In vivo, overexpression of TroTNFSF6 effectively stimulated the AKP and ACP activities, and reduced bacterial infection in fish tissues. Correspondingly, knockdown of TroTNFSF6 expression resulted in increasing bacterial dissemination and colonization in fish tissues. In vitro, recombinant TroTNFSF6 protein promoted the proliferation of T. ovatus head kidney lymphocytes (HKLs), and promoted the apoptosis of murine liver cancer cells (Hepa1-6). The results indicated that TroTNFSF6 plays an important role in the T. ovatus antibacterial immunity. These observations will facilitate the future in-depth study of teleost TNFSF6.

Primary repair of esophageal atresia gross type C via thoracoscopic magnetic compression anastomosis: A case report.

BACKGROUND: Esophageal atresia (EA) is a life-threatening congenital malformation in newborns, and the traditional repair approaches pose technical challenges and are extremely invasive. Therefore, surgeons have been actively investigating new minimally invasive techniques to address this issue. Magnetic compression anastomosis has been reported in several studies for its potential in repairing EA. In this paper, the primary repair of EA with magnetic compression anastomosis under thoracoscopy was reported. CASE SUMMARY: A full-term male weighing 3500 g was diagnosed with EA gross type C. The magnetic devices used in this procedure consisted of two magnetic rings and several catheters. Tracheoesophageal fistula ligation and two purse strings were performed. The magnetic compression anastomosis was then completed thoracoscopically. After the primary repair, no additional operation was conducted. A patent anastomosis was observed on the 15th day postoperatively, and the magnets were removed on the 23rd day. No leakage existed when the transoral feeding started. CONCLUSION: Thoracoscopic magnetic compression anastomosis may be a promising minimally invasive approach for repairing EA.

Cathepsin C (CTSC) contributes to the antibacterial immunity in golden pompano (Trachinotus ovatus).

Cathepsins, as a class of protein hydrolases, are widely found in the lysosomes of many tissues and play an essential role in various physiological activities. Cathepsin C (CTSC), a lysosomal cysteine protease, is an essential component of the lysosomal hydrolase family. In this study, we identified a CTSC from Trachinotus ovatus (TroCTSC) and analyzed its function. TroCTSC contained an ORF of 1368 bp and encoded 455 amino acids, which included three conserved catalytically active sites (Cys251, His397, and Asn419). It shares high homology (69.47%-90.77%) with the other known CTSC sequences of teleosts, which was most closely related to Seriola dumerili. TroCTSC was most abundant in the muscle, liver, and head kidney. After Vibrio harveyi infection, the expression levels of TroCTSC in liver, spleen, and head kidney were significantly up-regulated. TroCTSC was found in the cytoplasm with some of which were co-located with the lysosome. After V. harveyi stimulation, TroCTSC was translocated to nucleus in golden pompano snout (GPS) cells. In vitro, results revealed that the optimal hydrolase activity of the recombinant protein, rTroCTSC, was at 40 °C and pH 5.5. The activity of rTroCTSC was promoted by Zn2+ and Ca2+ but inhibited by Fe2+ and Cu2+. However, three mutant proteins, rTroCTSC-C251A, rTroCTSC-H397A, rTroCTSC-N419A, were dramatically reduced the proteolytic activity. Furthermore, in vivo results showed that overexpression of TroCTSC could significantly enhance body's ability to resist V. harveyi and promote the expression of proinflammatory cytokines, including interleukin 1-beta (IL-1ß), IL-6, IL-8, interferon-gamma (IFN-γ), and tumor necrosis factor-alpha (TNF-α). In contrast, the interference of TroCTSC expression induced a significant increase in the number of bacteria after V. harveyi infection. Our results suggested that TroCTSC was an essential effector of the innate immune system and played a pivotal role in antibacterial immunity.

Macrophage migration inhibitory factor (MIF) of golden pompano (Trachinotus ovatus) is involved in the antibacterial immune response.

Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine with a unique structure involved in immune regulation and inflammation. In the present study, we identified a MIF from Trachinotus ovatus (golden pompano) and analyzed its function. TroMIF shares high homology (58.26%-94.78%) with the other known MIF sequences of vertebrates. TroMIF is most closely related to large yellow croaker (Larimichthys crocea). The expression of TroMIF was most abundant in the liver and head kidney, and was significantly up-regulated after Edwardsiella tarda infection. The subcellular localization of TroMIF was mostly distributed in the cytoplasm. In vitro results revealed that the recombinant protein rTroMIF could inhibit the migration of head kidney lymphocytes (HKLs) and macrophages (HKMs) and enhance the phagocytic activity of HKMs. As a pro-inflammatory cytokine, rTroMIF could increase the expression levels of some pro-inflammatory cytokines, including tumor necrosis factor-alpha (TNF-α), interleukin 1-beta (IL-1ß), IL-6, IL-8, and interferon-gamma (IFN-γ) and decrease the expression of IL-10. The rTroMIF was proved to have enzymatic redox activity in vitro. Furthermore, overexpression of TroMIF in the head kidney cell line of golden pompano could significantly enhance its ability to resist E. tarda infection from 1 h to 4 h. The knockdown of TroMIF expression induced a significant increase in the number of bacteria after E. tarda infection at 1, 2, and 4 hpi. Our results suggest that TroMIF is an essential effector of the innate immune system and plays a pivotal role in antibacterial immunity.

Upregulated YAP promotes oncogenic CTNNB1 expression contributing to molecular pathology of hepatoblastoma.

BACKGROUND: Hepatoblastoma (HB) is one of the most common cancers in children. Recent studies have shown that the occurrence of nuclear accumulation of ß-catenin reaches 90%-100% because of the anomalous activation of the Wnt pathway in HB patients. Furthermore, emerging studies have shown that concomitant activated forms of YAP and ß-catenin trigger the formation and progression of HB. YAP might play a vital role in ß-catenin-mediated HB development. However, the molecular mechanisms by which YAP/TEAD4 transcription factor regulates CTNNB1 underlying HB pathogenesis are still unclear. PROCEDURE: YAP and CTNNB1 expression and correlation were analyzed by a combination of network enrichment analysis and gene set enrichment analysis of the public microarray datasets (GSE131329 and GSE81928). The protein levels of YAP and ß-catenin were further validated by Western blotting in paired patients' samples. The direct interplay between YAP/TEAD4 and the promoter region of CTNNB1 was proven by the combination of dual-luciferase report assay and chromatin immunoprecipitation assay. RESULTS: YAP-conserved signature and WNT signaling pathway were significantly enriched in HB patients, with upregulated expression of YAP and ß-catenin compared to non-HB patients. Further functional assays demonstrated that YAP/TEAD4 transcription factor complex could bind to the CTNNB1 promoter region directly to promote ß-catenin expression and cell proliferation. Targeting the YAP/TEAD4 complex with a specific small-molecule compound markedly suppressed HepaG2 cell proliferation. CONCLUSIONS: As the upstream transcription factor of CTNNB1, YAP/TEAD4 is a promising target for the treatment of HB patients with high levels of YAP and ß-catenin.

CC chemokine 1 protein from Cromileptes altivelis (CaCC1) promotes antimicrobial immune defense.

Chemokines are a family of small signaling proteins that are secreted by various cells. In addition to their roles in immune surveillance, localization of antigen, and lymphocyte trafficking for the maintenance of homeostasis, chemokines also function in induce immune cell migration under pathological conditions. In the present study, a novel CC chemokine gene (CaCC1) from humpback grouper (Cromileptes altivelis) was cloned and characterized. CaCC1 comprised a 435 bp open reading frame encoding 144 amino acid residues. The putative molecular weight of CaCC1 protein was 15 kDa CaCC1 contains four characteristic cysteines that are conserved in other known CC chemokines. CaCC1 also shares 11.64%-90.28% identity with other teleost and mammal CC chemokines. Phylogenetic analysis revealed that CaCC1 is most closely related to Epinephelus coioides EcCC1, both of which are in a fish-specific CC chemokine clade. CaCC1 was constitutively expressed in all examined C. altivelis tissues, with high expression levels in skin, heart, liver, and intestine. Vibrio harveyi stimulation up-regulated CaCC1 expression levels in liver, spleen, and head-kidney. Functional analyses revealed that the recombinant protein (rCaCC1) could induce the migration of head-kidney lymphocytes from C. altivelis. Moreover, rCaCC1 significantly enhanced phagocytosis in head-kidney macrophages from C. altivelis. In addition, rCaCC1 exhibited antimicrobial activities against Staphylococcus aureus, Edwardsiella tarda, and V. harveyi. In vivo, CaCC1 overexpression improved bacterial clearance in V. harveyi infected fish. Conversely, CaCC1 knockdown resulted in a significant decrease of bacterial clearance. These results demonstrate the important roles that CaCC1 plays in homeostasis and in inflammatory response to bacterial infection.

The added value of an artificial intelligence system in assisting radiologists on indeterminate BI-RADS 0 mammograms.

OBJECTIVES: To investigate the value of an artificial intelligence (AI) system in assisting radiologists to improve the assessment accuracy of BI-RADS 0 cases in mammograms. METHODS: We included 34,654 consecutive digital mammography studies, collected between January 2011 and January 2019, among which, 1088 cases from 1010 unique patients with initial BI-RADS 0 assessment who were recalled during 2 years of follow-up were used in this study. Two mid-level radiologists retrospectively re-assessed these BI-RADS 0 cases with the assistance of an AI system developed by us previously. In addition, four entry-level radiologists were split into two groups to cross-read 80 cases with and without the AI. Diagnostic performance was evaluated using the follow-up diagnosis or biopsy results as the reference standard. RESULTS: Of the 1088 cases, 626 were actually normal (BI-RADS 1 and no recall required). Assisted by the AI system, 351 (56%) and 362 (58%) normal cases were correctly identified by the two mid-level radiologists hence can be avoided for unnecessary follow-ups. However, they would have missed 12 (10 invasive cancers and 2 ductal carcinoma in situ cancers) and 6 (invasive cancers) malignant lesions respectively as a result. These missed lesions were not highly malignant tumors. The inter-rater reliability of entry-level radiologists increased from 0.20 to 0.30 (p < 0.005) by introducing the AI. CONCLUSION: The AI system can effectively assist mid-level radiologists in reducing unnecessary follow-ups of mammographically indeterminate breast lesions and reducing the benign biopsy rate without missing highly malignant tumors. KEY POINTS: • The artificial intelligence system could assist mid-level radiologists in effectively reducing unnecessary BI-RADS 0 mammogram recalls and the benign biopsy rate without missing highly malignant tumors. • The artificial intelligence system was capable of detecting low suspicion lesions from heterogeneously and extremely dense breasts that radiologists tended to miss. • The use of an artificial intelligence system may improve the inter-rater reliability and sensitivity, and reduce the reading time of entry-level radiologists in assessing potential lesions in BI-RADS 0 mammograms.

Do Various Treatment Modalities of Vesicoureteral Reflux Have Any Adverse Effects in Pediatric Patients? A Meta-Analysis.

PURPOSE: Vesicoureteral reflux (VUR) is a risk factor for various renal problems like recurrent urinary tract infections (UTIs), pyelonephritis, renal scarring, hypertension, and other renal parenchymal defects. The interventions followed by pediatricians include low-dose antibiotic treatment, surgical correction, and endoscopy. This meta-analysis aimed to assess the advantages and drawbacks of various primary VUR treatment options. SEARCH STRATEGY: The Cochrane Central Register of Controlled Trials, MEDLINE, EMBASE, reference lists of journals, and abstracts from conference proceedings were all used to find randomized controlled trials. The articles were retrieved from 1985 till 2020. Twenty articles were used for the data analysis. Criteria for Selection: Surgery, long-term antibiotic prophylaxis, noninvasive techniques, and any mix of therapies are also options for treating VUR. Collection and Interpretation of Data: Two authors searched the literature separately, determining research qualifications, assessing accuracy, and extracting and entering results. The odds ratio (OR) of these studies was used to construct the forest plot. The random-effects model was used to pool the data. Also, the random-effects model was used with statistical significance at a p value < 0.05 to assess the difference in side effects after treatment of VUR using different modalities. RESULTS: We found no statistically significant differences between surgery plus antibiotics and antibiotic alone-treated patients in terms of recurrent UTIs (OR = 0.581; 95% confidence interval [CI] 0.259-1.30), renal parenchymal defects (OR = 1.149; 95% CI 0.75-1.754), and renal scarring (OR = 1.042; 95% CI 0.72-1.50). However, the risk of developing pyelonephritis after surgical treatment of VUR was lesser than that in the conservative approach, that is, antibiotics (OR = 0.345; 95% CI 0.126-0.946.), positive urine culture (OR = 0.617; 95% CI 0.428-0.890), and recurrent UTIs were more common in the placebo group than in the antibiotic group (p < 0.05; OR = 0.639; 95% CI 0.436-0.936) which is statistically significant. CONCLUSION: Based on current research, we recommend that a child with a UTI and significant VUR be treated conservatively at first, with surgical care reserved for children who have issues with antimicrobials or have clinically significant VUR that persists after several years of follow-up.

Health-adjusted life expectancy (HALE) in Chongqing, China, 2017: An artificial intelligence and big data method estimating the burden of disease at city level.

BACKGROUND: A universally applicable approach that provides standard HALE measurements for different regions has yet to be developed because of the difficulties of health information collection. In this study, we developed a natural language processing (NLP) based HALE estimation approach by using individual-level electronic medical records (EMRs), which made it possible to calculate HALE timely in different temporal or spatial granularities. METHODS: We performed diagnostic concept extraction and normalisation on 13•99 million EMRs with NLP to estimate the prevalence of 254 diseases in WHO Global Burden of Disease Study (GBD). Then, we calculated HALE in Chongqing, 2017, by using the life table technique and Sullivan's method, and analysed the contribution of diseases to the expected years "lost" due to disability (DLE). FINDINGS: Our method identified a life expectancy at birth (LE0) of 77•9 years and health-adjusted life expectancy at birth (HALE0) of 71•7 years for the general Chongqing population of 2017. In particular, the male LE0 and HALE0 were 76•3 years and 68•9 years, respectively, while the female LE0 and HALE0 were 80•0 years and 74•4 years, respectively. Cerebrovascular diseases, cancers, and injuries were the top three deterioration factors, which reduced HALE by 2•67, 2•15, and 1•19 years, respectively. INTERPRETATION: The results demonstrated the feasibility and effectiveness of EMRs-based HALE estimation. Moreover, the method allowed for a potentially transferable framework that facilitated a more convenient comparison of cross-sectional and longitudinal studies on HALE between regions. In summary, this study provided insightful solutions to the global ageing and health problems that the world is facing. FUNDING: National Key R and D Program of China (2018YFC2000400).

MommiNet-v2: Mammographic multi-view mass identification networks.

Many existing approaches for mammogram analysis are based on single view. Some recent DNN-based multi-view approaches can perform either bilateral or ipsilateral analysis, while in practice, radiologists use both to achieve the best clinical outcome. MommiNet is the first DNN-based tri-view mass identification approach, which can simultaneously perform bilateral and ipsilateral analysis of mammographic images, and in turn, can fully emulate the radiologists' reading practice. In this paper, we present MommiNet-v2, with improved network architecture and performance. Novel high-resolution network (HRNet)-based architectures are proposed to learn the symmetry and geometry constraints, to fully aggregate the information from all views for accurate mass detection. A multi-task learning scheme is adopted to incorporate both Breast Imaging-Reporting and Data System (BI-RADS) and biopsy information to train a mass malignancy classification network. Extensive experiments have been conducted on the public DDSM (Digital Database for Screening Mammography) dataset and our in-house dataset, and state-of-the-art results have been achieved in terms of mass detection accuracy. Satisfactory mass malignancy classification result has also been obtained on our in-house dataset.

Functional characterization of cathepsin B and its role in the antimicrobial immune responses in golden pompano (Trachinotus ovatus).

Cathepsin B (CTSB) is one of the typical representatives of cysteine protease family. It has the activity of both exopeptidase and endopeptidase. It plays an important role in antigen presentation, degradation, apoptosis, inflammatory response and physiological process of many diseases. In this study, CTSB of Trachinotus ovatus (TroCTSB) was cloned, and its structure and function were analyzed. The results showed that the coding region of TroCTSB was 993 bp, encoding 330 amino acid residues. The homology analysis showed that the amino acid sequence of TroCTSB was similar to that in other teleosts and mammals (68.69%-88.48%). Under normal physiological conditions, TroCTSB was widely distributed in various tissues with the highest expression level in stomach, followed by liver, and the lowest expression level in blood. The optimal pH and temperature of purified recombinant protein rTroCTSB were 5.5 and 40 °C, respectively. The toxicity test of metal ions showed that Fe2+, Cu2+, Ca2+ and Zn2+ could all inhibit the activity of TroCTSB, with Zn2+ ranking the first. In addition, after Edwardsiella tarda infection, the expression of TroCTSB was significantly up-regulated in liver, spleen and head kidney. The overexpression of TroCTSB significantly inhibited the infection of E. tarda in golden pompano tissues, and the knockdown of TroCTSB remarkably promoted the reproduction of E. tarda in golden pompano tissues in vivo. This study suggests that TroCTSB was involved in the antibacterial immune response of T. ovatus, and provided a reference for further research in elucidating the resistance mechanism of TroCTSB.

Molecular characterization and antibacterial immunity functional analysis of liver-expressed antimicrobial peptide 2 (LEAP-2) gene in golden pompano (Trachinotus ovatus).

Liver-expressed antimicrobial peptide-2 (LEAP-2) is a member of the antimicrobial peptides family. Research has demonstrated that LEAP-2 contains a number of cations and plays a key role in the innate immune system of organism. In this study, we cloned and identified TroLEAP-2, from the golden pompano (Trachinotus ovatus), and analyzed its functions in vivo and in vitro. Results showed that TroLEAP-2 contains a 321 bp open reading frame (ORF) that encodes 106 putative amino acids with a molecular weight of 11.65 kDa. The mature TroLEAP-2 peptide possesses four conserved cysteine residues, which can form a core structure with two disulfide bonds between the cysteine residues in the relative 1-3 (Cys 77 and Cys 88) and 2-4 (Cys 83 and Cys 93) positions. It has a high amino acid sequence similarity (38.68%-83.02%) with the liver-expressed antimicrobial peptide -2 of other teleosts. Phylogenetic analysis showed that TroLEAP-2 clustered with the LEAP-2 of Paralichthys olivaceus and Miichthy milluy. TroLEAP-2 was most abundantly expressed in the liver, spleen, and kidney, and was significantly upregulated during Edwardsiella tarda and Streptococcus agalactiae infection. Purified recombinant TroLEAP-2 (rTroLEAP-2) could significantly inhibit the in vitro growth of E. tarda and S. agalactiae. Overexpression of TroLEAP-2 in vivo was shown to significantly reduce E. tarda and S. agalactiae colonization of tissues, whereas its knockdown resulted in an increase of bacteria in fish tissues. We also saw that TroLEAP-2 overexpression significantly improved macrophage activation in vivo. Moreover, TroLEAP-2 can induce the expression of nonspecific immune-related genes. These results showed that it might play a significant role in the innate immune system of golden pompano. In conclusion, our results indicate that TroLEAP-2 plays an important role in antibacterial immunity and provides a new avenue for protection against pathogenic infections in golden pompano.

Insulin-like growth factor binding protein 3 gene of golden pompano (TroIGFBP3) promotes antimicrobial immune defense.

Insulin-like growth factor binding protein 3 (IGFBP3), an important member of the IGFBP family, plays an important biological role in regulating cellular proliferation, differentiation, growth, apoptosis, and innate immunity. However, studies concerning IGFBP3 in teleosts are very limited and IGFBP3 function remains unclear. In this study, we conducted both in vivo and in vitro functional analyses of an IGFBP3 (TroIGFBP3) from the teleost fish golden pompano (Trachinotus ovatus). TroIGFBP3 is composed of 286 amino acid residues and shares a high amino acid sequence similarity (50.18%-93.71%) with other IGFBP3 sequences in humans and teleosts. TroIGFBP3 was widely distributed in various tissues, with the highest expression in the liver. TroIGFBP3 expression was significantly upregulated following Vibrio harveyi infection. The results of in vitro assays showed that TroIGFBP3 could stimulate macrophage activation and promote peripheral blood leukocytes (PBLs) proliferation. Meanwhile, TroIGFBP3 overexpression significantly inhibited bacterial infection in fish tissues, whereas TroIGFBP3 knockdown resulted in increased bacterial dissemination and colonization in golden pompano tissues in vivo. Furthermore, recombinant TroIGFBP3 could inhibit cellular proliferation and promote apoptosis of mouse tumor cells. Taken together, these results indicated that TroIGFBP3 plays a significant role in innate antibacterial immunity and provides a theoretical foundation for investigating the function of IGFBP3 in fish immune response.

TroCCL4, a CC chemokine of Trachinotus ovatus, is involved in the antimicrobial immune response.

CC chemokines are a large subfamily of chemokines that play an important role in the innate immune system. To date, several CC chemokines have been identified in fish species; however, the activities and functions of these putative chemokines remain ambiguous in teleosts, especially in the golden pompano, Trachinotus ovatus. Here, we characterized CC chemokine ligand 4 from T. ovatus (TroCCL4) and studied its functions. TroCCL4 contains a 294 bp open reading frame that encodes a putative peptide comprising 97 amino acids. TroCCL4 shares a high amino acid sequence similarity of 31.11%-78.35% with other CC chemokines sequences in humans and teleosts and has four cysteine residues that are conserved among other CC chemokines. TroCCL4 is also related to the macrophage inflammatory protein (MIP) group of CC chemokines. TroCCL4 expression was most abundant in immune organs and significantly upregulated in a time-dependent manner following Edwardsiella tarda infection. Recombinant TroCCL4 (rTroCCL4) induced the migration of peripheral blood leukocytes and the cellular proliferation of head kidney lymphocytes. In addition, rTroCCL4 inhibited the growth of Escherichia coli and E. tarda, indicating an antimicrobial function. Furthermore, the results of in vivo analysis showed that TroCCL4 overexpression in T. ovatus significantly enhanced macrophage activation; upregulated the gene expression of interleukin 1-ß (IL-1ß), interleukin 15 (IL15), interferon-induced Mx protein (Mx), tumor necrosis factor α (TNFα), complement C3, and major histocompatibility complex (MHC) class Iα and class IIα; and protected against bacterial infection in fish tissues. In contrast, knockdown of TroCCL4 expression resulted in increased bacterial dissemination and colonization in fish tissues. Taken together, our results provide evidence indicating that TroCCL4 has the ability to stimulate leukocytes and macrophages and enhance host immunity to defend against bacterial infection.

A ß-defensin gene of Trachinotus ovatus might be involved in the antimicrobial and antiviral immune response.

Defensins are a group of small cationic and cysteine-rich peptides that are important components of the innate immune system. However, studies on defensins in teleosts are very limited, particularly studies on defensin functions through in vivo assays. In this study, we cloned and identified one ß-defensin (TroBD) the golden pompano, Trachinotus ovatus, and analyzed the functions of TroBD in both in vivo and in vitro assays. TroBD is composed of 63 amino acids and shares high sequence identities (27.27-98.41%) with known ß-defensins of other teleosts. The protein has a signature motif of six conserved cysteine residues within the mature peptide. The expression of TroBD was most abundant in the head kidney and spleen and was significantly upregulated following infection by Vibrio harveyi and viral nervous necrosis virus (VNNV). Purified recombinant TroBD (rTroBD) inhibited the growth of V. harveyi, and its antimicrobial activity was influenced by salt concentration. TroBD was found to have a chemotactic effect on macrophages in vitro. The results of an in vivo study demonstrated that TroBD overexpression/knockdown in T. ovatus significantly reduced/increased bacterial colonization or viral copy numbers in tissues. Taken together, these results indicate that TroBD plays a significant role in both antibacterial and antiviral immunity and provide new avenues for protection against pathogen infection in the aquaculture industry.