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.

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.

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.

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.

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.

The razor clam Sinonovacula constricta uses the strategy of conversion of toxic ammonia to glutamine in response to high environmental ammonia exposure.

High ammonia can inhibit the survival and growth, and even cause mortality of razor clam (S. constricta). The accumulation of ammonia to lethal concentrations in some invertebrates may be partially prevented by converting some of the ammonia into glutamine (Gln). Glutamine dehydrogenase (GDH) and glutamine synthetase (GS) have been widely implicated a central role in response to ammonia stress. However, the molecular and physiological response of GDH and GS to ammonia alterations has not yet been determined in clams. To investigate the possible participatory role of GDH and GS genes in altered ammonia conditions, we have cloned their gene sequences and examined the mRNA expression and western blotting under ammonia exposure in S. constricta (ScGDH and ScGS), and detected the levels of GS and GDH, and the content of glutamate (Glu) and Gln. The full-length cDNA of ScGDH was 3924 bp, with a 1629 bp open reading frame (ORF) encoding a 542 amino-acid polypeptide. The complete cDNA sequence for ScGS had 2739 bp with an ORF of 1110 bp encoding 369 amino acids. To investigate ammonia detoxification strategies, the clams were exposed to ammonia for 96 h at four different concentrations (0, 100, 140, and 180 mg/L). Exposure to ammonia resulted in a significant increase of glutamate concentration and Gln in the haemocytes. GDH activity, GDH relative mRNA and protein expression, GS activity, GS relative mRNA and protein expression increased significantly and showed a pronounced time and dosage interaction in the liver. The results suggested that the protective strategies of Gln formation existed in S. constricta, which could convert ammonia to non- or less toxic nitrogenous compounds on the exposure of ammonia. Glutamate content in the haemocytes increased significantly, which is to ensure sufficient Glu to meet the needs for GS to catalyze the conversion of ammonia to Gln. We proposed that the induction of Glu synthesis-related genes and the subsequent formation of the active protein occurred in preparation for the increased capacity of the body to convert ammonia, into Gln. The results of this study suggested that GDH and GS play an important role in the synthesis of Gln, emphasizing, the protective strategies of Gln formation in S. constricta convert ammonia to nontoxic or less toxic nitrogenous compounds upon exposure to ammonia.

Polymorphisms of LAP3 gene and their association with the growth traits in the razor clam Sinonovacula constricta.

Leucine aminopeptidase 3 (LAP3) is an important proteolytic enzyme that catalyzes the hydrolysis of leucine residues from the amino termini of protein or peptide substrates and plays a critical role in protein metabolism and growth. In the present study, we investigated the full-length cDNA sequence of the LAP3 gene in Sinonovacula constricta (ScLAP3) using expressed sequence tags and rapid amplification of cDNA ends. The full-length ScLAP3 cDNA was 2885 bp, with a 1560 bp open reading frame encoding 519 amino acids. Sequence analysis revealed that ScLAP3 shared 70.9% identity with LAP3 from the blood clam Tegillarca granosa and 62.0-68.0% with other species. ScLAP3 was expressed in all six tested tissues, with significantly higher expression levels in the foot compared with mantle, adductor muscle, liver, gills, and siphon tissues in adults (P < 0.01). In the eight developmental stages, ScLAP3 expression gradually increased, with significantly higher levels in D-shaped larvae compared with other developmental stages (P < 0.01), suggesting that it may be involved in the formation of certain organs during early development. Association analysis identified three shared single nucleotide polymorphisms (SNPs), c.1073A > G, c.1139C > T and c.1154A > G in exons of ScLAP3 gene from 177 individuals of two groups, one selective strain and one wild population, which had significant effects on growth traits of S. constricta. The results provided candidate genetic markers to assist selective breeding of razor clams toward improved growth.

Transcriptomics analysis revealing candidate genes and networks for sex differentiation of yesso scallop (Patinopecten yessoensis).

BACKGROUND: The Yesso scallop, Patinopecten (Mizuhopecten) yessoensis, is a commercially important bivalve in the coastal countries of Northeast Asia. It has complex modes of sex differentiation, but knowledge of the mechanisms underlying this sex determination and differentiation is limited. RESULTS: In this study, the gonad tissues from females and males at three developmental stages were used to investigate candidate genes and networks for sex differentiation via RNA-Req. A total of 901,980,606 high quality clean reads were obtained from 18 libraries, of which 417 expressed male-specific genes and 754 expressed female-specific genes. Totally, 10,074 genes differentially expressed in females and males were identified. Weighted gene co-expression network analysis (WGCNA) revealed that turquoise and green gene modules were significantly positively correlated with male gonads, while coral1 and black modules were significantly associated with female gonads. The most important gene for sex determination and differentiation was Pydmrt 1, which was the only gene discovered that determined the male sex phenotype during early gonadal differentiation. Enrichment analyses of GO terms and KEGG pathways revealed that genes involved in metabolism, genetic and environmental information processes or pathways are sex-biased. Forty-nine genes in the five modules involved in sex differentiation or determination were identified and selected to construct a gene co-expression network and a hypothesized sex differentiation pathway. CONCLUSIONS: The current study focused on screening genes of sex differentiation in Yesso scallop, highlighting the potential regulatory mechanisms of gonadal development in P. yessoensis. Our data suggested that WCGNA can facilitate identification of key genes for sex differentiation and determination. Using this method, a hypothesized P. yessoensis sex determination and differentiation pathway was constructed. In this pathway, Pydmrt 1 may have a leading function.

The Highly Conserved Barley Powdery Mildew Effector BEC1019 Confers Susceptibility to Biotrophic and Necrotrophic Pathogens in Wheat.

Effector proteins secreted by plant pathogens play important roles in promoting colonization. Blumeria effector candidate (BEC) 1019, a highly conserved metalloprotease of Blumeria graminis f. sp. hordei (Bgh), is essential for fungal haustorium formation, and silencing BEC1019 significantly reduces Bgh virulence. In this study, we found that BEC1019 homologs in B. graminis f. sp. tritici (Bgt) and Gaeumannomyces graminis var. tritici (Ggt) have complete sequence identity with those in Bgh, prompting us to investigate their functions. Transcript levels of BEC1019 were abundantly induced concomitant with haustorium formation in Bgt and necrosis development in Ggt-infected plants. BEC1019 overexpression considerably increased wheat susceptibility to Bgt and Ggt, whereas silencing this gene using host-induced gene silencing significantly enhanced wheat resistance to Bgt and Ggt, which was associated with hydrogen peroxide accumulation, cell death, and pathogenesis-related gene expression. Additionally, we found that the full and partial sequences of BEC1019 can trigger cell death in Nicotiana benthamiana leaves. These results indicate that Bgt and Ggt can utilize BEC1019 as a virulence effector to promote plant colonization, and thus these genes represent promising new targets in breeding wheat cultivars with broad-spectrum resistance.

MiR-122 inhibits epithelial mesenchymal transition by regulating P4HA1 in ovarian cancer cells.

Ovarian cancer is one of the most common gyneacologic malignancies, with high morbidity and high mortality. Hsa-miR-122-5p (miR-122) has been reported with tumor-suppressing roles in various cancers. In this study, miR-122 was overexpressed in ovarian cancer cells, and phenotypic experiments demonstrated that miR-122 inhibited migration and invasion in SKOV3 and OVCAR3 cells. MiR-122 also suppressed epithelial mesenchymal transition (EMT), evidenced by expression changes of E-cadherin, vimentin, matrix metalloproteinase (MMP)2, and MMP14. Prolyl-4-hydroxylase subunit alpha-1 (P4HA1) was identified as a target of miR-122, and downregulated by miR-122. MiR-122-induced the elevation of migration, invasion, and EMT were recovered by P4HA1. Additionally, miR-122 restrained the tumor metastasis of SKOV3 cells in peritoneal cavity of nude mice. In summary, we demonstrated that miR-122 inhibited migration, invasion, EMT, and metastasis in peritoneal cavity of ovarian cancer cells by targeting P4HA1 for the first time, which shed lights on the discovery of miR-122 and P4HA1 as possible potential diagnostic markers and therapeutic targets for ovarian cancer.

Two I84 family protease inhibitors from Chinese razor clams Sinonovacula constricta expressed in response to environmental challenges.

Protease inhibitors play critical roles in numerous biological processes including host defense in all multicellular organisms. Eighty three evolutionary families of protease inhibitors are currently accommodated in the MEROPS database and the I84 family currently consists of 3 novel serine protease inhibitors from the eastern oyster Crassostrea virginica. In this study, we identified 2 new I84 family members from the Chinese razor clam Sinonovacula constricta, scSI-1 and scSI-2, using cDNA cloning and sequencing. The scSI-1 cDNA consisted of 494 bp with a 282 bp ORF encoding a 93-amino acid polypeptide that was predicted to have a 19-amino acid signal peptide and a 74-residue mature protein with a calculated molecular mass of 8248.5 Da. The scSI-2 cDNA was 490 bp long with a 273 bp ORF encoding a 90-amino acid polypeptide that was predicted to have an 18-amino acid signal peptide and a 72-residue nature protein with a calculated molecular mass of 7528.4 Da. ScSI-1 and scSI-2 shared high sequence similarity with the 3 known members of I84 family and both expressed primarily in the clam digestive glands. Protease inhibitory activity in the clam plasma also exhibited the signature kinetic characteristics of the I84 members from the oyster. In addition, levels of scSI-1 and scSI-2 gene expression in digestive glands and the protease inhibitory activity in plasma elevated significantly in clams challenged by bacterial injections and Vibrio harveyi was more effective than Staphylococcus epidermidis in inducing the gene expression and plasma protease inhibitory activity. Moreover, drastic changes of salinity and temperature also caused significant changes in the gene expression and plasma activity. These results indicated that scSI-1 and scSI-2 represented 2 new members of the I84 family and they likely play a role in clam host defense against infections and in reactions against physiochemical stressors.

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.

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.

Polymorphism of the multiple hemoglobins in blood clam Tegillarca granosa and its association with disease resistance to Vibrio parahaemolyticus.

Hemoglobin (Hb) is the major protein component of erythrocytes in animals with red blood, but it can serve additional functions beyond the transport of oxygen. In this study, we identified polymorphism in the blood clam Tegillarca granosa Hb (Tg-Hb) genes and investigated the association of this polymorphism with resistance/susceptibility to Vibrio parahaemolyticus. Analysis of the 540 sequences revealed 28 SNPs in the coding region of three Tg-Hbs, corresponding to about one SNP per 48 bp. Three SNPS: HbIIA-E2-146, HbIIB-E2-23, HbIIB-E2-121 showed a significant association with resistance/susceptibility to V. parahaemolyticus (P < 0.05). To further demonstrate that three significant SNPs of Tg-Hbs is associated with resistance of clams to V. parahaemolyticus, SNPs were genotyped in V. parahaemolyticus resistant strain clams and the wild base population from which this strain was derived. The results indicated that the nonsynonymous mutation T allele at HbIIA-E2-146 and A allele at HbIIB-E2-23 are associated with V. parahaemolyticus resistance in the blood clam, and its association with disease resistance may be due to its cause changes in amino acid sequences to a functional polymorphism. Together with previous bacterial challenge study, these results provides direct evidence that variation at HbIIA-E2-146 and HbIIB-E2-23 are associated with disease resistance in the blood clam, and these two polymorphic loci could be potential gene markers for the future molecular selection of strains that are resistant to diseases caused by V. parahaemolyticus.

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.

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.

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.