A Video-based Automated Tracking and Analysis System of Plaque Burden in Carotid Artery Using Deep Learning: A Comparison with Senior Sonographers.

BACKGROUND AND OBJECTIVE: The incidence of stroke is rising, and it is the second major cause of mortality and the third leading cause of disability around the globe. The goal of this study was to rapidly and accurately identify carotid plaques and automatically quantify plaque burden using our automated tracking and segmentation US-video system. METHODS: We collected 88 common carotid artery transection videos (11048 frames) with a history of atherosclerosis or risk factors for atherosclerosis, which were randomly divided into training, test, and validation sets using a 6:3:1 ratio. We first trained different segmentation models to segment the carotid intima and adventitia, and calculate the maximum plaque burden automatically. Finally, we statistically analyzed the plaque burden calculated automatically by the best model and the results of manual labeling by senior sonographers. RESULTS: Of the three Artificial Intelligence (AI) models, the Robust Video Matting (RVM) segmentation model's carotid intima and adventitia Dice Coefficients (DC) were the highest, reaching 0.93 and 0.95, respectively. Moreover, the RVM model has shown the strongest correlation coefficient (0.61±0.28) with senior sonographers, and the diagnostic effectiveness between the RVM model and experts was comparable with paired-t test and Bland-Altman analysis [P= 0.632 and ICC 0.01 (95% CI: -0.24~0.27), respectively]. CONCLUSION: Our findings have indicated that the RVM model can be used in ultrasound carotid video. The RVM model can automatically segment and quantify atherosclerotic plaque burden at the same diagnostic level as senior sonographers. The application of AI to carotid videos offers more precise and effective methods to evaluate carotid atherosclerosis in clinical practice.

A deep learning-based calculation system for plaque stenosis severity on common carotid artery of ultrasound images.

OBJECTIVES: Assessment of plaque stenosis severity allows better management of carotid source of stroke. Our objective is to create a deep learning (DL) model to segment carotid intima-media thickness and plaque and further automatically calculate plaque stenosis severity on common carotid artery (CCA) transverse section ultrasound images. METHODS: Three hundred and ninety images from 376 individuals were used to train (235/390, 60%), validate (39/390, 10%), and test (116/390, 30%) on a newly proposed CANet model. We also evaluated the model on an external test set of 115 individuals with 122 images acquired from another hospital. Comparative studies were conducted between our CANet model with four state-of-the-art DL models and two experienced sonographers to re-evaluate the present model's performance. RESULTS: On the internal test set, our CANet model outperformed the four comparative models with Dice values of 95.22% versus 90.15%, 87.48%, 90.22%, and 91.56% on lumen-intima (LI) borders and 96.27% versus 91.40%, 88.94%, 91.19%, and 92.88% on media-adventitia (MA) borders. On the external test set, our model still produced excellent results with a Dice value of 92.41%. Good consistency of stenosis severity calculation was observed between CANet model and experienced sonographers, with Intraclass Correlation Coefficient (ICC) of 0.927 and 0.702, Pearson's Correlation Coefficient of 0.928 and 0.704 on internal and external test set, respectively. CONCLUSIONS: Our CANet model achieved excellent performance in the segmentation of carotid IMT and plaques as well as automated calculation of stenosis severity.

Role of the circular RNA regulatory network in the pathogenesis of biliary atresia.

Circular RNAs (circRNAs) serve an essential role in the occurrence and development of cholangiocarcinoma, but the expression and function of circRNA in biliary atresia (BA) is not clear. In the present study, circRNA expression profiles were investigated in the liver tissues of patients with BA as well as in the choledochal cyst (CC) tissues of control patients using RNA sequencing. A total of 78 differentially expressed circRNAs (DECs) were identified between the BA and CC tissues. The expression levels of eight circRNAs (hsa_circ_0006137, hsa_circ_0079422, hsa_circ_0007375, hsa_circ_0005597, hsa_circ_0006961, hsa_circ_0081171, hsa_circ_0084665 and hsa_circ_0075828) in the liver tissues of the BA group and control group were measured using reverse transcription-quantitative polymerase chain reaction. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis demonstrated that the identified DECs are involved in a variety of biological processes, including apoptosis and metabolism. In addition, based on the GO and KEGG pathway enrichment analyses, it was revealed that target genes that can be affected by circRNAs regulatory network were enriched in the TGF-ß signaling pathway, EGFR tyrosine kinase inhibitor resistance pathway and transcription factor regulation pathway as well as other pathways that may be associated with the pathogenesis of BA. The present study revealed that circRNAs are potentially implicated in the pathogenesis of BA and could help to find promising targets and biomarkers for BA.

Evaluation of Turbulence Index and Flow Pattern for Atherosclerotic Carotid Stenosis: A High-Frame-Rate Vector Flow Imaging Study.

OBJECTIVE: The emerging high-frame-rate vector flow imaging provides a new way of hemodynamic evaluation for complex blood flow. This study was aimed at exploring quantitatively the characteristics of complex flow with turbulence (Tur) index and analyzing flow patterns in atherosclerotic internal carotid artery stenosis (ICAS) using high-frame-rate vector flow imaging. METHODS: This study prospectively included 60 patients with ICAS. Tur values in different segments of stenosis and cardiac phases were compared. Spearman correlation analysis was performed between clinical plaque characteristics with turbulence grading by ln(Tur). Three complex flow patterns were qualitatively drawn on vector flow mode, and the rates of detection of flow patterns in different stenosis groups and ulceration groups were compared. RESULTS: Highly disordered blood flow was observed in the stenotic (Tur [M, QR] = 12.5%, 21.5%) and distal segment (15.4%, 27.2%), particularly during systole (21.0%, 30.7%, 33.3%, 38.7%, p < 0.05). Spearman correlation analysis revealed that stenosis rate was correlated with turbulence grading in the stenotic (ρ = 0.65, p < 0.05) and distal segment (ρ = 0.79, p < 0.05), and ulcer formation was correlated with turbulence grading in the stenotic segment (ρ = 0.58, p < 0.05). The overall rate of detection of three flow patterns was higher in the severe stenosis group (22/22) versus the mild to moderate stenosis group (21/38) (p < 0.001) and in the ulcer group (21/23) versus the non-ulcer group (23/37) (p < 0.001). CONCLUSION: High-frame-rate vector flow imaging was helpful in assessing the severity and characteristics of flow turbulence. Lumen geometric factors could affect flow turbulence and blood flow patterns around the plaque. This would provide important hemodynamic information for the detection of high-risk plaque.

Characteristics and Correlations of Wall Shear Stress and Flow Turbulence in the Carotid Bifurcation Evaluated Using an Ultrasound Vector Flow Imaging.

INTRODUCTION: The aim of the study was to evaluate characteristics and provide the normal values of wall shear stress (WSS) and flow turbulence (Tur), and the relationship between them in the carotid bifurcation based on an ultrasound vector flow imaging (V Flow) in healthy adults. METHODS: Max and mean WSS and Tur values at three segments (initial segments of internal and external carotid arteries [IICA and IECA]; distal segment of common carotid artery [DCCA]), both in anterior and posterior walls, were successfully obtained in 56 healthy adults, using ultrasound V Flow function. Relationship between mean WSS and Tur was further explored. RESULTS: The mean WSS value was 0.71 Pa, 0.86 Pa, and 0.96 Pa at IICA, IECA, and DCCA, respectively (IICA < IECA < DCCA, p < 0.05). The mean Tur value was 13.85%, 5.46%, and 4.17% at IICA, IECA, and DCCA, respectively (IICA > IECA > DCCA, p < 0.05). A cutoff value (WSS = 0.4 Pa) was selected and Tur values were significantly higher in group with WSS cutoff value <0.4 Pa than group with WSS cutoff value ≥0.4 Pa (p < 0.01). CONCLUSION: WSS and Tur are moderately negatively correlated, which can be used in the quantitative evaluation of carotid bifurcation and could be a potential dual-parameter tool in the clinical research for early detection of carotid atherosclerosis.

Acute Salinity Stress Disrupts Gut Microbiota Homeostasis and Reduces Network Connectivity and Cooperation in Razor Clam Sinonovacula constricta.

Accumulating evidence demonstrates that it is of great importance to maintain a stable and functional gut microbial community for host's growth and health. However, gut microenvironment is constantly affected by diverse environmental factors. Salinity can cause stress, including hypersaline or hyposaline stress to aquatic species, thereby affecting their growth conditions. Razor clam (Sinonovacula constricta), an economically important bivalve species, inhabits in intertidal and estuarine zones and constantly experiences salinity stress. Yet little is known about how and to what extent clam gut microbiota is affected by salinity stress, while this knowledge is fundamental for clam aquaculture health management. To address this concern, this study compared the temporal differences of gut bacterial signatures and community assembly of S. constricta under normal salinity (NS), low salinity (LS), and high salinity (HS) conditions. Acute salinity stress affected the compositions, structures, and functional potentials of clam gut microbial community, of which salinity stress, hours post stress, and their interaction respectively constrained 7.6%, 16.4%, and 7.9% of community variation. Phylogenetic bin-based null model result revealed that the gut bacterial assembly of three salinity groups seemed to be largely driven by stochastic processes. Network analysis indicated that gut bacterial interspecies interaction exhibited less connected and lower cooperative activity under the conditions of LS and HS compared with NS. Notably, some pathogenic bacteria, including Vibrio and Pseudoalteromonas, were identified as keystone taxa of gut microbial networks in LS and HS groups. Above findings suggest that the clams under LS and HS conditions might be at a higher risk of developing disease. Our findings enhance the mechanism understanding of gut microbial assembly in S. constricta under abiotic factor challenge, which has important implications for clam health control from a microbial ecological perspective.

Genome-Wide Identification of 5-HT Receptor Gene Family in Razor Clam Sinonovacula constricta and Their Circadian Rhythm Expression Analysis.

Serotonin (5-HT) is primarily distributed in the gastrointestinal and central nervous systems, where it plays a crucial role in regulating various physiological functions such as digestion, reproduction and establishing animal emotions. 5-HT is an effective oxytocin widely used in molluscan aquaculture, and its physiological functions are performed by binding to corresponding 5-HT receptors (5-HTRs). In this study, seven 5-HTR genes of Sinonovacula constricta (Sc5-HTRs) were identified and analyzed, and they were designated as Sc5-HT1A, Sc5-HT1D, Sc5-HT2-1, Sc5-HT2-2, Sc5-HT2-3, Sc5-HT4 and Sc5-HT6. Phylogenetic analysis showed that the seven Sc5-HTRs were conserved among mollusks, and the Sc5-HTRs were all transmembrane proteins. The seven Sc5-HTR genes were distributed on chromosome 1, 2, 13 and 14. After injecting 5-HT, there was a significant increase in mRNA expression levels of Sc5-HT1A (p < 0.05) and Sc5-HT2-3 (p < 0.01), while Sc5-HT4 decreased significantly (p < 0.01) compared to control groups which might be effective 5-HT receptors. Furthermore, two of the receptors (Sc5-HT2-3 and Sc5-HT4) were expressed in the circadian rhythm patterns, indicating their potential influence on the nocturnal spawning of S. constricta. Overall, these findings provide a theoretical basis for understanding the structures and functions of 5-HTR gene family members, and may facilitate the artificial propagation of mollusks.

Precise evaluation of blood flow patterns in human carotid bifurcation based on high-frame-rate vector flow imaging.

PURPOSE: To investigate the feasibility of high-frame-rate vector flow imaging (HiFR-VFI) compared to ultrasound color Doppler flow imaging (CDFI) for precisely evaluating flow characteristics in the carotid bifurcation (CB) of presumed healthy adults. METHODS: Forty-three volunteers were assessed for flow characteristics and their extensions using HiFR-VFI and CDFI in CBs. The flow patterns were classified according to the streamlines in HiFR-VFI and quantitatively measured using an innovative turbulence index (Tur-value). Interobserver agreement was also assessed. RESULTS: HiFR-VFI was consistent with CDFI in detecting laminar and nonlaminar flow in 81.4% of the cases; however, in 18.6% of the cases, only HiFR-VFI identified the nonlaminar flow. HiFR-VFI showed a larger extension of complex flow (0.37 ± 0.26 cm2 ) compared to CDFI (0.22 ± 0.21 cm2 ; p < 0.05). The flow patterns were classified into four types: 3 type-I (laminar flow), 35 type-II (rotational flow), 27 type-III (reversed flow), and 5 type-IV (complex flow). The Tur-value of type-IV (50.03 ± 14.97)% is larger than type-III (44.57 ± 8.89)%, type-II (16.30 ± 8.16)%, and type-I (1.48 ± 1.43)% (p < 0.05). Two radiologists demonstrated almost perfect interobserver agreement on recognizing the change of streamlines (κ = 0.81, p < 0.001). The intraclass correlation coefficient of the Tur-value was 0.98. CONCLUSION: HiFR-VFI can reliably characterize complex hemodynamics with quantitative turbulence measurement and may be an auxiliary diagnostic tool for assessing atherosclerotic arterial disease.

Expression and Functional Analysis of AMT1 Gene Responding to High Ammonia Stress in Razor Clam (Sinonovacula constricta).

Ammonium transporter 1 (AMT1), a member of ammonia (NH3/NH4+) transport proteins, has been found to have ammonia transport activity in plants and microorganisms. However, the functional characteristics and molecular mechanisms of AMT1 in mollusks remain unclear. The razor clam (Sinonovacula constricta) is a suitable model species to explore the molecular mechanism of ammonia excretion because of the high concentration of ambient ammonia it is exposed to in the clam-fish-shrimp polyculture system. Here, the expression of AMT1 in S. constricta (Sc-AMT1) in response to high ammonia (12.85 mmol/L NH4Cl) stress was identified by real-time quantitative PCR (qRT-PCR), Western blotting, RNA interference, and immunofluorescence analysis. Additionally, the association between the SNP_g.15211125A > T linked with Sc-AMT1 and ammonia tolerance was validated by kompetitive allele-specific PCR (KASP). A significant upregulated expression of Sc-AMT1 was observed during ammonia exposure, and Sc-AMT1 was found to be localized in the flat cells of gill. Moreover, the interference with Sc-AMT1 significantly upregulated the hemolymph ammonia levels, accompanied by the increased mRNA expression of Rhesus glycoprotein (Rh). Taken together, our findings imply that AMT1 may be a primary contributor to ammonia excretion in S. constricta, which is the basis of their ability to inhabit benthic water with high ammonia levels.

Feasibility study of combining wall shear stress and elastography to assess the vascular status of carotid artery.

INTRODUCTION: At present, early detection of the potential risk of atherosclerosis and prevention is of great significance to reduce the occurrence of stroke. AIM: This study aims to explore the value of combining the wall shear stress measured by ultrasound vector flow imaging technique and sound touch elastography of common carotid artery in normal adults using the Mindray Resona 7 ultrasound system. METHODS: Forty volunteers (mean age 39.5 y, 23 females, 17 males) were divided into four groups according to their age. All volunteers underwent ultrasound carotid artery examination, and the values of wall shear stress and elasticity on the posterior wall of the common carotid artery were measured using advanced imaging functions, vector flow imaging technique, and sound touch elastography. RESULTS: Different cut-off values of wall shear stress were used to investigate the significance between two groups with corresponding sound touch elastography values. It can be seen that the statistical difference could be found when the mean wall shear stress was larger than 1.5 Pa approximately (statistical significance was defined when P < 0.05), and the sound touch elastography value was positively correlated with the wall shear stress value. CONCLUSION: This study reveals that the combination of wall shear stress and sound touch elastography is an effective and feasible method for assessing carotid artery health. When the mean wall shear stress value is over 1.5 Pa, the corresponding sound touch elastography value increases significantly. The risk of atherosclerosis increases with the stiffness of blood vessel walls.

Comprehensive Analysis of Whole-Transcriptome Profiles in Response to Acute Hypersaline Challenge in Chinese Razor Clam Sinonovacula constricta.

The Chinese razor clam (Sinonovacula constricta) is an important for Chinese aquaculture marine bivalve that naturally occurs across intertidal and estuarine areas subjected to significant changes in salinity level. However, the information on the molecular mechanisms related to high salinity stress in the species remain limited. In this study, nine gill samples of S. constricta treated with 20, 30, and 40 ppt salinity for 24 h were used for whole-transcriptome RNA sequencing, and a regulatory network of competing endogenous RNAs (ceRNAs) was constructed to better understand the mechanisms responsible for adaptation of the species to high salinity. A total of 83,262 lncRNAs, 52,422 mRNAs, 2890 circRNAs, and 498 miRNAs were identified, and 4175 of them displayed differential expression pattern among the three groups examined. The KEGG analyses of differentially expressed RNAs evidenced that amino acid synthesis and membrane transport were the dominant factors involved in the adaptation of the Chinese razor clam to acute salinity increase, while lipid metabolism and signaling played only a supporting role. In addition, lncRNA/circRNA-miRNA-mRNA regulatory networks (ceRNA network) showed clearly regulatory relationships among different RNAs. Moreover, the expression of four candidate genes, including tyrosine aminotransferase (TAT), hyaluronidase 4 (HYAL4), cysteine sulfinic acid decarboxylase (CSAD), and ∆1-pyrroline-5-carboxylate synthase (P5CS) at different challenge time were detected by qRT-PCR. The expression trend of TAT and HYAL4 was consistent with that of the ceRNA network, supporting the reliability of established network. The expression of TAT, CSAD, and P5CS were upregulated in response to increased salinity. This might be associated with increased amino acid synthesis rate, which seems to play an essential role in adaptation of the species to high salinity stress. In contrast, the expression level of HYAL4 gene decreased in response to elevated salinity level, which is associated with reduction Hyaluronan hydrolysis to help maintain water in the cell. Our findings provide a very rich reference for understanding the important role of ncRNAs in the salinity adaptation of shellfish. Moreover, the acquired information may be useful for optimization of the artificial breeding of the Chinese razor clam under aquaculture conditions.

Genome-wide characterization of the cytosolic sulfotransferase 1B member 1 (SULT1B1) family and its expression responses to sulfide stress in the razor clam Sinonovacula constricta.

The razor clam (Sinonovacula constricta), a typical burrowing organism in the intertidal zones, is often exposed to sulfide environment and shows strong sulfide tolerance. Located downstream of the sulfur metabolism pathway, cytosolic sulfotransferase family 1B member 1 (SULT1B1) is a key enzyme catalysing the sulfonation reaction, and plays an important role in the biotransformation of endogenous substances such as thyroid hormones (THs). To investigate their roles in sulfide resistance, a systematic analysis of S. constricta SULT1B1s (ScSULT1B1s), including genomic distribution, phylogenetic relationships, gene structure, conserved motifs, and expression profiles under sulfide stress, was performed. A total of 10 ScSULT1B1 genes were found in the S. constricta genome. Sequence analysis showed that ScSULT1B1 gene family encoded 155-425 amino acids, containing four catalytic active sites (K, N, H, and S), one PAPS binding domain at the N-terminus, and one PAPS binding and dimerization domain at the C-terminus. The spatial-temporal expression patterns of ScSULT1B1s were further estimated by quantitative real-time PCR (qRT-PCR). Among them, partial ScSULT1B1s showed significantly high expression in the gill, hepatopancreas, and siphon. Furthermore, the response expression of certain ScSULT1B1s significantly fluctuated under sulfide stress. Together, our results suggest that ScSULT1B1s, by mediating the sulfonation reaction, may regulate THs levels to maintain basic metabolic and immune functions, making S. constricta highly sulfide tolerant.

Blood Flow Turbulence Quantification of Carotid Artery With a High-Frame Rate Vector Flow Imaging.

OBJECTIVES: To assess the feasibility and performance of Turbulence (Tur) index as a quantitative tool for carotid artery flow turbulence; to detect and compare the blood flow patterns of common carotid artery (CCA) and carotid bulb (CB) at different ages and cardiac phases in healthy adults, and thus interpret the evolvement of etiology difference between CCA and CB. METHODS: Carotid flow characteristics of 40 healthy volunteers were evaluated quantitatively by a high-frame rate vector flow imaging. Three types of flow patterns were defined depending on the distributive range of complex flow during systole in CB. Comparison of mean Tur value in CCA and CB at different age groups and cardiac phases was performed. And the correlation between Tur value and the diameter ratio of proximal internal carotid artery to common carotid artery (DRpro-ica/cca) was tested. RESULTS: Mean Tur values in CB were remarkably higher than that in CCA, whether during systole or diastole (P < .001). Meanwhile Tur values in CB during systole were significantly higher than that during diastole (P < .001). Flow complexity of CB showed variations among 40 participants especially in systole, whereas the flow pattern of CCA was relatively consistent. Mean Tur values were positively correlated with DRpro-ica/cca in CB (ρ = 0.69, P < .05). CONCLUSIONS: V Flow imaging provided a reliable method-Tur, for quantitative analysis of carotid blood flow. It had potential to be further applied in distinguishing complex hemodynamic characteristics in high-risk people of carotid diseases for the risk stratification of cardiovascular events.

Investigation on the differences of hemodynamics in normal common carotid, subclavian, and common femoral arteries using the vector flow technique.

Objectives: To investigate the feasibility of the vector flow imaging (V Flow) technique to measure peripheral arterial hemodynamic parameters, including wall shear stress (WSS) and turbulence index (Tur) in healthy adults, and compare the results in different arteries. Materials and methods: Fifty-two healthy adult volunteers were recruited in this study. The maximum and mean values of WSS, and the Tur values at early-systole, mid-systole, late-systole, and early diastole for total 156 normal peripheral arteries [common carotid arteries (CCA), subclavian arteries (SCA), and common femoral arteries (CFA)] were assessed using the V Flow technique. Results: The mean WSS values for CCA, SCA, and CFA were (1.66 ± 0.68) Pa, (0.62 ± 0.30) Pa, and (0.56 ± 0.27) Pa, respectively. The mean Tur values for CCA, SCA, and CFA were (0.46 ± 1.09%), (20.7 ± 9.06%), and (24.63 ± 17.66%), respectively. The CCA and SCA, as well as the CCA and CFA, showed statistically significant differences in the mean WSS and the mean Tur (P < 0.01). The mean Tur values had a negative correlation with the mean WSS; the correlation coefficient between log(Tur) and WSS is -0.69 (P < 0.05). Conclusion: V Flow technique is a simple, practical, and feasible quantitative imaging approach for assessing WSS and Tur in peripheral arteries. It has the potential to be a useful tool for evaluating atherosclerotic plaques in peripheral arteries. The results provide a new quantitative foundation for future investigations into diverse arterial hemodynamic parameters.

PDIA6 involves the thermal stress response of razor clam, Sinonovacula constricta.

Protein disulfide isomerases A6 (PDIA6), an oxidoreductase and isomerase, catalyzes the oxidation reduction and isomerization of disulfide bonds, and serves as molecular chaperone to prevent the buildup of misfolded proteins under various environmental insults. However, the role of PDIA6 in mollusks remains largely obscure, although its multifunctional protein has been reported in other species under adverse conditions. To fill this gap, we identified PDIA6 from the razor clam Sinonovacula constricta (ScPDIA6) and investigated its expression patterns in response to thermal stress. Tissue distribution showed that the mRNA transcript of ScPDIA6 was ubiquitously expressed in nine tested tissues. Temporal expression profiles by qPCR revealed that ScPDIA6 in the gill and mantle was significantly increased by hyper-thermic treatment. Further, Western blot and immunofluorescence indicated that ScPDIA6 was significantly upregulated by thermal treatment at the protein level. Additionally, the survival test demonstrated that the viability of E. coli cells expressing recombinant ScPDIA6 protein increased at 42 °C compared with empty vector. Overall, these findings suggested that ScPDIA6 may play a pivotal role in counteracting thermal stress. This study will provide valuable reference data resource for understanding the potential role of PDIA6 in mollusks.

Integrated RNA-seq and RNAi Analysis of the Roles of the Hsp70 and SP Genes in Red-Shell Meretrix meretrix Tolerance to the Pathogen Vibrio parahaemolyticus.

The "Wanlihong" Meretrix meretrix (WLH-M) clam is a new variety of this species that has a red shell and stronger Vibrio tolerance than ordinary M. meretrix (ORI-M). To investigate the molecular mechanisms responsible for the WLH-M strain's tolerance to Vibrio, we challenged clams with Vibrio parahaemolyticus and then assessed physiological indexes and conducted transcriptome analysis and RNA interference experiments. The mortality, tissue bacterial load, and hemocyte reactive oxygen species level of ORI-M were significantly higher than those of WLH-M, whereas the content and activity of lysozyme were significantly lower. Gene Ontology functional annotation analysis and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis revealed that immune and metabolic pathways were enriched in Vibrio-challenged clams. The expressions of the heat shock protein 70 (Hsp70) and serine protease (SP) genes, which are involved in antibacterial immunity, were significantly upregulated in WLH-M but not in ORI-M, while the expression of the kynurenine 3-monooxygenase gene, a proinflammatory factor, was significantly downregulated in WLH-M. RNA interference experiments confirmed that Hsp70 and SP downregulation could result in increased mortality of WLH-M. Therefore, we speculate that Hsp70 and SP may be involved in the antibacterial immunity of WLH-M in vivo. Our data provided a valuable resource for further studies of the antibacterial mechanism of WLH-M and provided a foundation for the breeding of pathogen-resistant strains.

Characteristics of glutathione peroxidase gene and its responses to ammonia-N stress in razor clam Sinonovacula constricta.

Glutathione peroxidase (GPX) is a crucial enzyme in the antioxidant defense system. However, the previous studies on the structure and functions of mollusk GPX genes are still very limited. Here, we investigated the GPX gene from Sinonovacula constricta (Sc-GPX), and its expression profiles, protein localization, gene function and association with ammonia tolerance. The full length of sequence of Sc-GPX was 1781 bp, containing an open reading frame (ORF) of 588 bp encoding 195 amino acids. Quantitative expression of seven adult tissues showed that Sc-GPX was most abundant in hepatopancreas, followed by gills. Furthermore, the enzyme activity of Sc-GPX in hepatopancreas increased significantly under different ammonia concentrations (100, 140, and 180 mg/L) (P < 0.01). Further, we explored the mRNA expression level, histological structure and histo-cellular localization in gills and hepatopancreas of Sc-GPX under 140 mg/L ammonia stress. The mRNA expression level in gills and hepatopancreas of Sc-GPX increased significantly (P < 0.05) and immunohistochemistry results suggested that the columnar cells of gills filaments and the endothelial cells of hepatopancreas were the major sites for the action of Sc-GPX protein. In addition, we performed western blotting (WB), RNA interference (RNAi) and single nucleotide polymorphisms (SNPs) in the hepatopancreas of Sc-GPX under ammonia stress (140 mg/L). WB results indicated that the protein expression of Sc-GPX increased significantly (P < 0.01) after ammonia challenge. In addition, expression of Sc-GPX mRNA were significantly downregulated at 24 and 48 h after RNAi (P < 0.01). The association analysis between ammonia-tolerance group and control group identified six SNPs in coding sequence (CDS) of Sc-GPX from 449 individuals. Among them, c.162A > C was missense mutation, which lead to the amino acid change from Lys to Asn. These findings revealed that Sc-GPX may play a critical role in clam ammonia detoxification.

Expression and functional characterization of peptidoglycan recognition protein-S6 involved in antibacterial responses in the razor clam Sinonovacula constricta.

It has been recognized that peptidoglycan recognition proteins (PGRPs), structurally conserved molecules, play crucial roles in the innate immunity of invertebrate. However, few studies have been taken to explore their potential functions. In this study, a novel PGRP from the razor clam Sinonovacula constrict designated as ScPGRP-S6 was identified and characterized. The open reading frame (ORF) of ScPGRP-S6 was 666 bp in length, encoding a protein of 221 amino acid with a signal peptide (1-30) and a typical PGRP domain (39-187). The sequence alignment combined with phylogenetic analysis collectively confirmed that ScPGRP-S6 was a novel member belonging to PGRP-S family. The mRNA transcript of ScPGRP-S6 in the hepatopancreases was significantly up-regulated after peptidoglycan (PGN) stimulation, while it was moderately up-regulated after lipopolysaccharide (LPS) stimulation. The result of immunofluorescence detection demonstrated that the positive signal enhanced obviously after Vibrio parahaemolyticus challenge. Notably, the recombinant protein of ScPGRP-S6 (designed as rScPGRP-S6) exhibited high agglutination activity towards V. parahaemolyticus but weak to Staphylococcus aureus. Furthermore, rScPGRP-S6 showed strong amidase and antibacterial activity in the presence of Zn2+. Collectively, our results manifested that ScPGRP-S6 could act as a scavenger in the innate immune response of S. constricta.

Research progress in gene editing technology.

As a tool for modifying the genome, gene editing technology has developed rapidly in recent years, especially in the past two years. With the emergence of new gene editing technologies, such as transposon editing tools, numerous advancements have been made including precise editing of the genome, double base editing, and pilot editing. This report focuses on the development of gene editing tools in recent years, elaborates the progress made in classic editing tools, base editor and other new editing tools, and provides insights into challenges and opportunities.