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The Chinese tongue sole (Cynoglossus semilaevis) belonging to Pleuronectiformes, Cynoglossidae, Cynoglossus, is distributed in the sea areas of Korea, Japan and China. C. semilaevis has limited natural resources and no long-distance migration, which is suitable for the development of aquaculture in coastal areas. After more than ten years of artificial domestication, C. semilaevis has become one of the main mariculture species. Water temperature is an important environmental factor affecting the growth and development of fish. High temperature in summer can cause stress and even death of C. semilaevis, which is one of the important factors affecting factory farming and the promotion in the southern coast. However, the physiological and molecular changes of C. semilaevis in response to acute high temperature stress was still unclear to date.When fish are stimulated by high temperature, it can cause oxidative stress, resulting in a variety of toxic effects. It has been found that there is a significant correlation between high temperature and the activity of antioxidant enzymes in many aquatic animals. Therefore, changes in the activities of antioxidant enzymes can be used as an important indicator when fish are in a state of oxidative stress under high temperature stress. As a tissue related to detoxification metabolism and immunity of fish, liver plays an important role in response to high temperature stress, thus the tissue structure and apoptosis are also important indicators of liver health status after high temperature stress. Heat shock proteins (HSPs) are a class of common biological stress proteins, which have biological functions such as anti-stress, anti-oxidation, and regulation of apoptosis. HSPs are also involved in the resistance of fish to heat stress. Recently, it has been found that zebrafish (Danio rerio) is extremely sensitive to temperature changes after knockout of dual-specificity phosphatase 1 (dusp1) gene, and dusp1 has the possibility of maintaining redox homeostasis, so it was speculated that dusp1 gene may plays an important role in fish resistance to heat stress. In this study, heat shock protein family A member 1A (hspa1a) in the heat shock protein HSP70 family, heat shock protein 90 beta family member 1 (hsp90b1) in the HSP90 family, and dusp1 gene were selected to study the temporal expression characteristics under high temperature stress.In order to explore the effects of high temperature stress on physiological and molecular changes in the liver of C. semilaevis, a full-sib family of C. semilaevis was selected as the experimental object to detect the oxidative damage and heat stress-related gene expressions. After continuous heating to high temperature stress conditions (35 ℃), liver tissues were collected at 0 h, 3 h, 6 h, 12 h and 24 h, respectively. Hematoxylin and eosin (HE) staining and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) were used to observe cell damage, antioxidant enzyme activity and malondialdehyde (MDA) content were measured, and the expression changes of stress-related genes hspa1a, hsp90b1 and dusp1 were detected. The results showed that acute high temperature stress could cause obvious pathological changes and apoptosis in the liver tissue of C. semilaevis. The activity of antioxidant enzyme superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase (CAT) and the content of MDA in the high temperature stress group was significantly higher than that of the control group at 6 h, 12 h, 0 h and 24 h, respectively (P

The leopard coral grouper (Plectropomus leopardus) belongs to the family Epinephelinae, and genus Plectropomus. Vibrio harveyi is the main pathogen that causes "rot disease" in leopard coral grouper, which is a major threat to the sustainable development of its aquaculture industry. The disease is highly prevalent from June to August and severely affects aquaculture. Therefore, developing disease-resistant strains is a necessity. However, currently, artificial breeding techniques for leopard coral groupers cannot establish a family lineage through one-on-one artificial insemination, making traditional breeding methods that depend on a clear pedigree difficult. Considering the successful breeding of disease-resistant fish species with or without a pedigree, genome selection breeding technology are vital for cultivating disease-resistant leopard coral groupers.In genetic selection, the genetic parameters of target traits are important reference factors for specifying breeding programs. To evaluate the genetic parameters of leopard coral grouper resistance to V. harveyi, we constructed a genome-relatedness matrix based on high-density single-nucleotide polymorphisms using four models (binary linear model [BLM], binary threshold model [BTM], longitudinal linear model [LLM], and linear threshold model [LTM]) to fit two disease-resistant phenotypes (test-day trait, TDS; Bivariate survival trait, TS), and used restricted maximum likelihood [REML] to estimate variance components. Our findings illustrated that the genetic heritability of leopard coral grouper resistance to V. harveyi ranged from 0.182 to 0.486, which belongs to the medium-to-high genetic heritability range. The additive genetic variance ranged from 0.071 to 0.262. The genetic heritability estimated by the linear model was 0.382 and 0.476, whereas that estimated by the threshold model was 0.182 and 0.207, respectively. These results suggest that leopard coral groupers resistance to V. harveyi can be improved through genetic breeding.Herein, the linear models (BLM and LLM) obtained higher genetic heritability estimates and more accurate genomic estimated breeding value (GEBV) predictions than the threshold models (BTM and LTM). However, despite the model used, the correlation coefficient between the GEBV rankings under the same phenotype definition > 0.9, indicating that their impact on the GEBV ranking was not significant. Compared to the cross-sectional models (BLM and BTM), numerous leopard coral grouper GEBVs were rearranged in the LLM results. There was a strong correlation between the LTM and phenotype (TS), indicating that LLM has an excellent prediction effect. Therefore, when breeding leopard coral groupers for V. harveyi-resistant traits, a LLM should be considered.The study observed that using longitudinal models (LLM and LTM) to estimate genetic heritability produced higher results than the cross-sectional models (BLM and BTM), which may be due to the death time explaining different components of fish disease resistance. In longitudinal models, the genetic component influenced by the time of death is effectively harnessed. However, in cross-sectional models, this effect is inadvertently subsumed within the residuals. Consistent with the genetic heritability findings, the longitudinal models produced more precise GEBVs compared to cross-sectional models. Our results suggest that TDS might offer a more accurate measure for assessing the resistance of leopard coral groupers to V. harveyi than the TS.Compared with the threshold models, linear models performed better in GEBV prediction, and higher genetic heritability estimates were obtained. Although, most previous studies on disease resistance traits have reported inconsistent genetic heritability estimates between threshold and linear models, some studies support these conclusions. This result may be due to differences in information processing between the different models, which leads to different results. In this study, the additive genetic variance obtained using threshold models (BTM and LTM) was 0.222–0.262, and additive genetic variance obtained using linear models (LLM and BLM) was 0.071–0.086. It is expected that the additive genetic variance obtained using threshold models was higher than that obtained using linear models. Furthermore, the residual variance resulting from fitting linear models was notably low. We posit that when threshold traits are erroneously treated as normally distributed data and linear models are employed for analysis, the residual variance may be underestimated. This underestimation is likely due to the model's underfitting, which consequently leads to an inflated heritability estimate for the linear model.This study aimed to estimate the genetic parameters of leopard coral grouper resistance to V. harveyi using infection test data of leopard coral groupers injected with V. harveyi and to construct an individual genotype relationship matrix based on single-nucleotide polymorphisms. The genetic heritability of leopard coral grouper resistance to V. harveyi was estimated to be between 0.182 and 0.486 by comparing different models and phenotype definitions. The linear (0.382 and 0.476) and threshold models (0.182 and 0.207) were used to estimate genetic heritability. The estimated genetic heritability was within the medium genetic heritability range. Our findings were used to improve the target traits of leopard coral groupers, specifically their resistance to V. harveyi. This study supplements the genetic parameter estimation of leopard coral grouper resistance to V. harveyi and provides a reference for selecting V. harveyi-resistant leopard coral groupers for breeding.

Abstract Vibrio spp. are major pathogens responsible for mortality and disease in various marine aquaculture organisms. Effective disease control and genetic breeding strategies rely heavily on understanding host vibriosis resistance mechanisms. The Chinese tongue sole (Cynoglossus semilaevis) is economically vital but suffers from substantial mortalities due to vibriosis. Through continuous selective breeding, we have successfully obtained vibriosis-resistant families of this species. In this study, we conducted RNA-seq analysis on three organs, including liver, spleen and intestine from selected resistant and susceptible tongue soles. Additionally, we integrated these data with our previously published RNA-seq datasets of skin and gill, enabling the construction of organ-specific transcriptional profiles and a comprehensive gene co-expression network elucidating the differences in vibriosis resistance. Furthermore, we identified 12 modules with organ-specific functional implications. Overall, our findings provide a valuable resource for investigating the molecular basis of vibriosis resistance in fish, offering insights into target genes and pathways essential for molecular selection and genetic manipulation to enhance vibriosis resistance in fish breeding programs.

Leopard coral grouper (Plectropomus leopardus) is one of the most important cultured fish in the Pacific and Indian oceans. Vibrio harveyi is a serious pathogen causing serious skin ulceration and high mortality in P. leopardus. To gain more insight into the tissue-specific and dynamic immune regulation process of P. leopardus in response to V. harveyi infection, RNA sequencing (RNA-seq) was used to examine the transcriptome profiles in the spleen and liver at 0, 6, 12, 24, 48, and 72 h post-infection. The upregulated differentially expressed genes (DEGs) were predominantly involved in the immune response in the spleen and liver at the early infection stage (6–12 h), and downregulated DEGs were mainly involved in metabolic processes in the liver at the early and middle infection stage (6–48 h). Moreover, an overview of the immune response of P. leopardus against V. harveyi was exhibited including innate and adaptive immune-related pathways. Afterwards, the results of WGCNA analysis in the spleen indicated that TAP2, IRF1, SOCS1, and CFLAR were the hub genes closely involved in immune regulation in the gene co-expression network. This study provides a global picture of V. harveyi-induced gene expression profiles of P. leopardus at the transcriptome level and uncovers a set of key immune pathways and genes closely linked to V. harveyi infection, which will lay a foundation for further study the immune regulation of bacterial diseases in P. leopardus.

Cynoglossus semilaevis (half-smooth tongue sole) is an important aquaculture species that has a ZW/ZZ sex-determination system (females, ZW; males, ZZ). Some genotypic females may develop phenotypic males, and are known as pseudomales (ZW). These pseudomales can develop mature testes and produce sperm. However, previous studies have shown that pseudomales cannot produce sperms bearing W chromosome (W sperm). In this study, transcriptomic, proteomic, and ubiquitinated proteomic analyses were employed simultaneously to investigate the differences in gonads between pseudomales and normal males. Functional enrichment analysis of the differentially expressed genes/proteins showed that non-coding RNA metabolic processes and RNA degradation were more active in the gonads of pseudomales, and they showed higher levels of ubiquitination compared to males. In addition, most genes/proteins related to sperm morphogenesis expressed downwards in pseudomales. These genes included genes located exclusively on the Z chromosome, such as sperm flagellar protein 2 (spef2), doublesex and mab-3 related transcription factor 1 (dmrt1), doublesex and mab-3 related transcription factor 3 (dmrt3), and cilia- and flagella-associated protein 157 (cfap157). These findings characterized the transcriptional and proteomic profiles of the gonads of pseudomales and males, and suggested that W sperm were absent in pseudomales, possibly due to the disruption of sperm morphogenesis, resulting from W chromosome lacking spermatogenesis-related genes.

Abstract The assembly of W and Y chromosomes poses significant challenges in vertebrate genome sequencing and assembly. Here, we successfully assembled the W chromosome of Verasper variegatus with a length of 20.48 Mb by combining population and PacBio HiFi sequencing data. It was identified as a young sex chromosome and showed signs of expansion in repetitive sequences. The major component of the expansion was Ty3/Gypsy. The ancestral Osteichthyes karyotype consists of 24 protochromosomes. The sex chromosomes in four Pleuronectiformes species derived from a pair of homologous protochromosomes resulting from a whole-genome duplication event in teleost fish, yet with different sex-determination systems. V. variegatus and Cynoglossus semilaevis adhere to the ZZ/ZW system, while Hippoglossus stenolepis and H. hippoglossus follow the XX/XY system. Interestingly, V. variegatus and H. hippoglossus derived from one protochromosome, while C. semilaevis and H. stenolepis derived from another protochromosome. Our study provides valuable insights into the evolution of sex chromosomes in flatfish and sheds light on the important role of whole-genome duplication in shaping the evolution of sex chromosomes.

Ras-related associated with diabetes (RRAD) is a member of the Ras GTPase superfamily that plays a role in several cellular functions, such as cell proliferation and differentiation. In particular, the superfamily acts as an NF-κB signaling pathway inhibitor and calcium regulator to participate in the immune response pathway. A recent transcriptome study revealed that rrad was expressed in the spleen of disease-resistant Japanese flounder (Paralichthys olivaceus) individuals compared with disease-susceptible individuals, and the results were also verified by qPCR. Thus, the present study aimed to explore how rrad regulates antimicrobial immunity via the NF-κB pathway. First, the coding sequence of P. olivaceus rrad was identified. The sequence was 1092 bp in length, encoding 364 amino acids. Based on phylogenetic and structural relationship analyses, P. olivaceus rrad appeared to be more closely related to teleosts. Next, rrad expression differences between disease-resistant and disease-susceptible individuals in immune-related tissues were evaluated, and the results revealed that rrad was expressed preferentially in the spleen of disease-resistant individuals. In response to Edwardsiella piscicida infection, rrad expression in the spleen changed. In vitro, co-culture was carried out to assess the hypo-methylated levels of the rrad promoter in the disease-resistant spleen, which was consistent with the high mRNA expression. The siRNA-mediated knockdown of rrad performed with the gill cell line of P. olivaceus affected many rrad-network-related genes, i.e., dcp1b, amagt, rus1, rapgef1, ralbp1, plce1, rasal1, nckipsd, prkab2, cytbc-1, sh3, and others, as well as some inflammation-related genes, such as bal2 and Il-1β. In addition, flow cytometry analysis showed that rrad overexpression was more likely to induce cell apoptosis, with establishing a link between rrad‘s function and its potential roles in regulating the NF-κB pathway. Thus,. the current study provided some clarity in terms of understanding the immune response about rrad gene differences between disease-resistant and disease-susceptible P. olivaceus individuals. This study provides a molecular basis for fish rrad gene functional analysis and may serve as a reference for in-depth of bacterial disease resistance of teleost.

Gametogenesis, the intricate developmental process responsible for the generation of germ cells (gametes), serves as a fundamental prerequisite for the perpetuation of the reproductive cycle across diverse organisms. The g2e3 enzyme is a putative ubiquitin E3 ligase implicated in the intricate regulatory mechanisms underlying cellular proliferation and division processes. The present study delves into the function of G2/M phase-specific E3 ubiquitin protein ligase (Cs-g2e3) in gametogenesis in Chinese Tongue Sole (Cynoglossus semilaevis). Sequence analysis shows that the Cs-g2e3 mRNA spans 6479 bp, encoding a 733 amino acid protein characterized by three conserved structural domains: PHD, RING, and HECT—typical of HECT E3 ubiquitin ligases. The predominant expression of Cs-g2e3 in the gonad tissues is further verified by qPCR. The expression profile of Cs-g2e3 in the gonads of the Chinese Tongue Sole is analyzed at different ages, and the results show that its expression peaks at 8 months of age and then begins to decline and stabilize. It is noteworthy that the expression level remains significantly elevated compared to that observed during the juvenile period. In situ hybridization shows that the mRNA of Cs-g2e3 is mainly localized in the germ cells of the ovary and the testis. RNA interference experiments show that the knockdown of Cs-g2e3 in ovarian and testicular germ cell lines significantly downregulates the expression of key genes involved in oogenesis (e.g., sox9 and cyp19a) and spermatogenesis (e.g., tesk1 and piwil2), respectively. Furthermore, the analysis of mutations in the transcription factor binding sites reveals that mutations within the Myogenin, YY1, and JunB binding sites significantly impact the transcriptional activity of the Cs-g2e3 gene, with the mutation in the YY1 binding site exhibiting the most pronounced effect (p < 0.001). This study contributes to a deeper understanding of the tissue-specific expression patterns of Cs-g2e3 across various tissues in Cynoglossus semilaevis, as well as the potential regulatory influences of transcription factors on its promoter activity. These findings may facilitate future research endeavors aimed at elucidating the expression and functional roles of the Cs-g2e3 gene.

Vibriosis, caused by Vibrio, seriously affects the health of fish, shellfish, and shrimps, causing large economic losses. Teleosts are represent the first bony vertebrates with both innate and adaptive immune responses against pathogens. Aquatic animals encounter hydraulic pressure and more pathogens, compared to terrestrial animals. The skin is the first line of defense in fish, constituting the skin-associated lymphoid tissue (SALT), which belongs to the main mucosa-associated lymphoid tissues (MALT). However, little is known about the function of immunity related proteins in fish. Therefore, this study used iTRAQ (isobaric tags for relative and absolute quantitation) to compare the skin proteome between the resistant and susceptible families of Cynoglossus semilaevis. The protein integrin beta-2, the alpha-enolase isoform X1, subunit B of V-type proton ATPase, eukaryotic translation initiation factor 6, and ubiquitin-like protein ISG15, were highly expressed in the resistant family. The 16S sequencing of the skin tissues of the resistant and susceptible families showed significant differences in the microbial communities of the two families. The protein-microbial interaction identified ten proteins associated with skin microbes, including immunoglobulin heavy chain gene (IGH), B-cell lymphoma/leukemia 10 (BCL10) and pre-B-cell leukemia transcription factor 1 isoform X2 (PBX2). This study highlights the interaction between skin proteins and the microbial compositions of C. semilaevis and provides new insights into understanding aquaculture breeding research.

The tiger pufferfish (Takifugu rubripes), also known as fugu, has recently suffered from severe C. irritans infections under aquaculture environment, yet the underlying immune mechanisms against the parasite remain poorly understood. In this study, we conducted a comprehensive transcriptome analysis of the gill tissue from infected and uninfected fish using PacBio long-read (one pooled sample each for seriously infected and healthy individuals, respectively) and Illumina short-read (three pools for mildly infected, seriously infected, and healthy individuals, respectively) RNA sequencing technologies. After aligning sequence data to fugu’s reference genome, 47,307 and 34,413 known full-length transcripts were identified and profiled in healthy and infected fish, respectively. Similarly, we identified and profiled 1126 and 803 novel genes that were obtained from healthy and infected fish, respectively. Interestingly, we found a decrease in the number of alternative splicing (AS) events and long non-coding RNAs (lncRNAs) after infection with C. irritans, suggesting that they may be involved in the regulation of the immune response in fugu. There were 687 and 1535 differentially expressed genes (DEGs) in moderately and heavily infected fish, respectively, compared to uninfected fish. Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses showed that immune-related DEGs in the two comparison groups were mainly enriched in cytokine-cytokine receptor interactions, ECM-receptor interactions, T-cell receptor signaling pathways, Th1 and Th2 cell differentiation, and Th17 cell differentiation pathways. Further analysis revealed that a large number of immune-related genes were downregulated in infected fish relative to uninfected ones, such as CCR7, IL7R, TNFRSF21, CD4, COL2A1, FOXP3B, and ITGA8. Our study suggests that C. irritans is potentially a highly efficient parasite that may disrupt the defense mechanisms of fugu against it. In addition, in combination of short-read RNA sequencing and previous genome-wide association analyses, we identified five key genes (NDUFB6, PRELID1, SMOX, SLC25A4, and DENND1B) that might be closely associated with C. irritans resistance. This study not only provides valuable resources of novel genic transcripts for further research, but also provides new insights into the immune mechanisms underlying C. irritans infection response in farmed fugu.

The gut microbiota constitutes a complex ecosystem that has an important impact on host health. In this study, genetically engineered zebrafish with inducible nitric oxide synthase (iNOS or NOS2) knockout were used as a model to investigate the effects of nos2a/nos2b gene single knockout and nos2 gene double knockout on intestinal microbiome composition and function. Extensive 16S rRNA sequencing revealed substantial changes in microbial diversity and specific taxonomic abundances, yet it did not affect the functional structure of the intestinal tissues. Notably, iNOS-deficient zebrafish demonstrated a decrease in Vibrio species and an increase in Aeromonas species, with more pronounced effects observed in double knockouts. Further transcriptomic analysis of the gut in double iNOS knockout zebrafish indicated significant alterations in immune-related and metabolic pathways, including the complement and PPAR signaling pathways. These findings underscore the crucial interplay between host genetics and gut microbiota, indicating that iNOS plays a key role in modulating the gut microbial ecology, host immune system, and metabolic responses.

The healthcare industry is confronted with the challenge to offer an increasing variety of healthcare services while in the meantime controlling rapidly increasing healthcare costs. Mass customization has been proven to be an effective strategy to fulfill customers’ individual specific needs with high efficiency and low cost in the manufacturing industry. This paper investigates the theoretical feasibility and practical applicability of adopting mass customization as a conceptual framework for designing a healthcare service delivery system. The nature of healthcare delivery systems and their evolution are discussed relative to those of manufacturing systems. Recent research in personalized medicine, consumer-driven healthcare, consumer healthcare informatics, and integrated healthcare delivery is reviewed as enabling technologies towards mass customization of healthcare services. By synthesizing these scattered efforts in different streams of literature, this paper concludes that mass customization can contribute to the redesign of healthcare service systems, and delineates a roadmap for future research.

Edwardsiella tarda is a major causative pathogen of bacterial ascites in Japanese flounder, leading to massive economic losses, and the discovery of molecular markers linked to disease resistance is an effective strategy in resistance breeding programs. The Rho GTPase family comprises small proteins with a molecular weight of 20~30 kDa. Rho GTPase family members are involved in diverse cellular processes, such as cytoskeleton, cell adhesion, vesicle transport, and proliferation. In addition, they play pivotal roles in infection by different pathogens. Rho-related GTP-binding protein Rho6 (Rnd1), a member of the Rho-GTPase family, participates in various biological functions, including neural junction formation, axonal extension, tumorigenesis, neuronal function, and apoptosis. Some members of the Rho family, such as Rac1 and Rac2, regulate immune response in grass carp, large yellow croaker, zebrafish, and half-smooth tongue sole. However, the function of Rnd1 in fish is poorly understood. Japanese flounder (Paralichthys olivaceus) is greatly affected by E. tarda infections during the breeding process. In previous studies, whole-genome sequencing and assembly of Japanese flounder were performed, and subsequently, various disease resistance genes were screened to support the improvement of Japanese flounder germplasm resources. To study the role of Pornd1 in resistance against E. tarda infection in Japanese flounder, Pornd1 was cloned and identified using PCR. The full-length Pornd1 cDNA was 699 bp, containing an open reading frame encoding a 232-amino acid protein. The predicted molecular weight of PoRnd1 was 26 kDa. Sequence and homology analyses showed that the Rnd1 protein harbors a Rho-GTP superfamily structural domain, which is highly conserved in various species. PoRnd1 shares the highest homology with Rnd1 from Hippoglossus hippoglossus (98.28%). On phylogenetic tree, PoRnd1 was clustered with Rnd1 from other fish species. The single-nucleotide polymorphism (SNP) locus associated with E. tarda resistance is located at 4 575 720 bp on chromosome 14 of Japanese flounder. The frequency of the T allele in disease-resistant families (freqT=0.92) was significantly higher than that in susceptible families (freqT=0.20). The SNP was located at the 2nd intron of Pornd1. Real-time quantitative PCR was employed to characterize the expression profiles of Pornd1 in the tissues of healthy and E. tarda-infected fish. Pornd1 expression was the highest in the heart, followed by the liver, kidney, head kidney, and spleen, but its expression was low in the skin, blood, gills, and muscle. In E. tarda-infected fish, the expression of Pornd1 mRNA decreased after 6 h, then gradually increased, and subsequently reached the highest level after 48 h in the liver, kidney, and spleen. Pornd1 expression in the kidney and spleen in the 48 h group was significantly higher than that in the 6 and 12 h groups. Furthermore, Pornd1 expression in the liver of resistant families was significantly higher than that in susceptible families. Based on its His tag, the PoRnd1 recombinant protein was purified using an Ni column and subjected to SDS-PAGE. The target band of PoRnd1 at 32 kDa was observed in the gel after Coomassie Blue staining. The PoRnd1 recombinant protein (0.5 mg/mL) was used to study antibacterial activity through the Oxford cup assay. PoRnd1 significantly inhibited the growth of Staphylococcus aureus, Escherichia coli, E. tarda, and Vibrio harveyi. In summary, Pornd1 may be closely linked to disease resistance in Japanese flounder and can thus serve as an effective gene marker for disease resistance breeding. Our findings provide a theoretical basis for further elucidating the molecular mechanisms of immunity in Japanese flounder.

Xanthogranulomatous cystitis (XC) is a rare benign disease of chronic granulomatous inflammation. We report a 23-year-old woman with xanthogranulomatous cystitis. She was referred to our hospital with the chief complaint of a 1-year history of frequent, urgent dysuria with recurrent fever. An imaging examination showed bilateral ureteral reflux and a normal bladder. Urodynamic findings suggested bladder outlet obstruction and increased post-void residual urine. Finally, the patient underwent endoscopy, and bladder neck obstruction was confirmed. Additionally, we found multiple granulomatous masses in the bladder. Therefore, we performed transurethral resection of the tumor and bladder neck. A histopathological examination of resected tumor tissue showed xanthogranulomatous cystitis, and the patient received anti-infective therapy. Follow-up cystourethroscopic results and urination symptoms returned to normal, and the bilateral ureteral reflux was gradually reduced.

Interferon regulatory factor (irf7) is an immune regulatory factor that plays an important role in the antiviral process. To explore the role of irf7 in Oplegnathus punctatus (Temminck & Schlegel, 1844) under viral infection, we cloned the coding DNA sequence (CDS) region of irf7 through PCR and analyzed the expression patterns at both tissue and cell levels. The results showed that the CDS region of Opirf7 was 1 332 bp and encoded a peptide with 443 amino acids. The predicted molecular weight was 50.5 kDa and the theoretical isoelectric point was 5.546. Protein structure analysis showed that Opirf7 has three conserved domains: the DNA binding domain (DBD), IRF-associated domain (IAD), and serine-rich domain (SRD). Amino acid similarity analysis showed that OpIRF7 had the highest similarity to the IRF7 of Lates calcarifer, which was 82.92%. The similarity of Opirf7 with the IRF7 of Larimichthys crocea, Paralichthys olivaceus, and Cynoglossus semilaevis were 81.99%, 79.95%, and 73.74%, respectively. Phylogenetic analysis showed that Opirf7 and other fish irf7 genes were clustered into one branch, and irf7s from Gallus gallus, Mus musculus, Macaca mulatta, and Homo sapiens were clustered into another. Tissues from healthy O. punctatus were collected, including the liver, spleen, kidney, head kidney, intestine, gill, skin, muscle, brain, heart, and blood. After RNA extraction and cDNA synthesis, real-time PCR (qPCR) was performed to detect the expression level of Opirf7 using the comparative CT method (2−ΔΔCT method). The results of qPCR showed that Opirf7 was expressed in different tissues of healthy individuals and its expression was highest in the liver, followed by the skin and intestines. The lowest expression was observed in the head kidney. In this study, the expression profiles of Opirf7 before and after viral infection were determined at the tissue and cell levels. For the in vivo challenge study, fish were intraperitoneally injected with spotted knifejaw iridovirus, and the expression level of Opirf7 was tested in the spleen, kidney, and liver. Compared with the control group at 0 h, the expression level of Opirf7 was 15-fold in the spleen and 3-fold in the kidney 4 days after infection, and the expression peak was at 7 days after infection. However, the expression of Opirf7 was not significantly altered in the liver. A poly I: C-infected O. punctatus brain cell model was established, and the expression profiles of Opirf7 mRNA were detected before and after infection. The expression of Opirf7 mRNA in the low and medium concentration groups (50 μg/mL and 100 μg/mL, respectively) increased by 13 to 17 times, and the expression level of Opirf7 mRNA in the high concentration group (200 μg/mL) increased by approximately 8 times. It was speculated that the high concentration of 200 μg/mL caused some damage to the cells and that the expression level in the high concentration group was lower than that in the low and medium groups. In this study, the full-length open reading frame sequence of Opirf7 was cloned and characterized for the first time. The deduced amino acid sequence displayed a structure similar to those of other vertebrates. Further functional analysis showed that Opirf7 has a significant response to viral infection at both tissue and cell levels. This study demonstrated that the Opirf7 gene might play an important role in the antiviral response of O. punctatus and provide a potential molecular marker for antivirus breeding of O. punctatus.

Abstract In point-of-care testing (POCT), tests are performed near patients and results are given rapidly for timely clinical decisions. Immunodiagnostic assays are one of the most important analyses for detecting and quantifying protein-based biomarkers. However, existing POCT immunodiagnostics mainly rely on the lateral flow assay (LFA), which has limited sensitivity or quantification capability. Although other immunodiagnostic assays, such as enzyme-linked immunosorbent assays (ELISAs), offer more sensitive and quantitative results, they require complex liquid manipulations that are difficult to implement in POCT settings by conventional means. Here, we show the development of DropLab, an automated sample-in-answer-out POCT immunodiagnostic platform based on magnetic digital microfluidic (MDM) technology. DropLab performs microbead-based ELISA in droplets to offer more sensitive and quantitative testing results. The intricate liquid manipulations required for ELISA are accomplished by controlling droplets with magnetic microbeads using MDM technology, which enables us to achieve full automation and easy operations with DropLab. Four ELISAs (the sample in triplicates and a negative control) can be run in parallel on the thermoformed disposable chip, which greatly improves the throughput and accuracy compared to those of other POCT immunodiagnostic devices. DropLab was validated by measuring two protein targets and one antibody target. The testing results showed that the limit of detection (LOD) of DropLab matched that of the conventional ELISA in a microwell plate. DropLab brings MDM one step closer to being a viable medical technology that is ready for real-world POCT applications.

Measurement(s) genome assembly Technology Type(s) PacBio RS II

Blind-side hypermelanosis has emerged as a major concern in flatfish aquaculture worldwide, including tongue sole (Cynoglossus semilaevis) in China. The causative gene and the molecular basis are still unclear. In this study, comparative transcriptome analyses were performed using different skin tissues of tongue sole: ocular-side normal (pigmented) skin, blind-side normal (non-pigmented) skin and blind-side hypermelanotic (pigmented) skin. Finally, 60 key hypermelanosis-related genes were mined, providing potential candidate gene resources involved in blind-side hypermelanosis. These genes were selected based on the log2(FoldChange) and false discovery rate (FDR) values (with corresponding P-Values

Abstract Background Infectious diseases have caused huge economic loss and food security issues in fish aquaculture. Current management and breeding strategies heavily rely on the knowledge of regulative mechanisms underlying disease resistance. Though the intestinal microbial community was linked with disease infection, there is little knowledge about the roles of intestinal microbes in fish disease resistance. Cynoglossus semilaevis is an economically important and widely cultivated flatfish species in China. However, it suffers from outbreaks of vibriosis, which results in huge mortalities and economic loss. Results Here, we used C. semilaevis as a research model to investigate the host-microbiome interactions in regulating vibriosis resistance. The resistance to vibriosis was reflected in intestinal microbiome on both taxonomic and functional levels. Such differences also influenced the host gene expressions in the resistant family. Moreover, the intestinal microbiome might control the host immunological homeostasis and inflammation to enhance vibriosis resistance through the microbe-intestine-immunity axis. For example, Phaeobacter regulated its hdhA gene and host cyp27a1 gene up-expressed in bile acid biosynthesis pathways, but regulated its trxA gene and host akt gene down-expressed in proinflammatory cytokines biosynthesis pathways, to reduce inflammation and resist disease infection in the resistant family. Furthermore, the combination of intestinal microbes and host genes as biomarkers could accurately differentiate resistant family from susceptible family. Conclusion Our study uncovered the regulatory patterns of the microbe-intestine-immunity axis that may contribute to vibriosis resistance in C. semilaevis. These findings could facilitate the disease control and selective breeding of superior germplasm with high disease resistance in fish aquaculture. Video Abstract

Massive macroalgae blooms, primarily caused by the overgrowth of Ulva prolifera (U. prolifera) in the Yellow Sea of China, pose a severe risk to both marine organisms and the aquaculture industry. This study’s aim was to evaluate the impact of U. prolifera degradation on the growth performance and antioxidant capacity of Japanese flounder (Paralichthys olivaceus) and select some potential Japanese flounder families (labeled 2101–2108, established by crossbreeding) tolerating U. prolifera degradation conditions. Thus, a 60-day U. prolifera exposure experiment was conducted. The results showed that the contents of Na, K, Mg, and Fe elements in the U. prolifera degradation water were significantly increased. The specific growth rate and survival rate of flounder were significantly decreased under the U. prolifera degradation condition, while the 2101 and 2103 flounder families showed a better growth performance compared with the positive control (PC) group. Moreover, the results showed that activities of total antioxidant capacity (T-AOC), transaminases, and alkaline phosphatase (AKP) in serum were significantly decreased, while the 2103 flounder family showed higher activities. Furthermore, U. prolifera degradation significantly increased superoxide dismutase (SOD) activity and glutathione (GSH) content while decreasing catalase (CAT) activity and malondialdehyde (MDA) content in the liver. Specifically, SOD and CAT activities of the 2103 flounder family were higher than the 2101 flounder family and PC group. In addition, the gill SOD and CAT activities of the 2103 flounder family were significantly higher than the PC group. Similarly, the antioxidant-related gene (sod and cat) expressions were synchronously upregulated or downregulated in the liver and gills in response to U. prolifera degradation. These results revealed that U. prolifera degradation decreased the growth performance and influenced the antioxidant capacity of Japanese flounder, while the 2103 flounder family had better advantages in the U. prolifera degradation condition. Therefore, the 2103 flounder family could be regarded as the potential flounder family tolerating U. prolifera degradation. The increased Fe content in the U. prolifera degradation water may be one of the main causes of the physiological alterations observed in Japanese flounder.

Long non-coding RNAs (lncRNAs) play crucial roles in a variety of biological processes, including stress response. However, the number, characteristics and stress-related expression of lncRNAs in turbot are still largely unknown. In this study, a total of 12,999 lncRNAs were identified at the genome-wide level of turbot for the first time using 24 RNA-seq datasets. Sequence characteristic analyses of transcripts showed that lncRNA transcripts were shorter in average length, lower in average GC content and in average expression level as compared to the coding genes. Expression pattern analyses of lncRNAs in 12 distinct tissues showed that lncRNAs, especially lincRNA, exhibited stronger tissue-specific expression than coding genes. Moreover, 612, 1351, 1060, 875, 420 and 1689 differentially expressed (DE) lncRNAs under Vibrio anguillarum, Enteromyxum scophthalmi, and Megalocytivirus infection and heat, oxygen, and salinity stress conditions were identified, respectively. Among them, 151 and 62 lncRNAs showed differential expression under various abiotic and biotic stresses, respectively, and 11 lncRNAs differentially expressed under both abiotic and biotic stresses were selected as comprehensive stress-responsive lncRNA candidates. Furthermore, expression pattern analysis and qPCR validation both verified the comprehensive stress-responsive functions of these 11 lncRNAs. In addition, 497 significantly co-expressed target genes (correlation coefficient (R) > 0.7 and q-value < 0.05) for these 11 comprehensive stress-responsive lncRNA candidates were identified. Finally, GO and KEGG enrichment analyses indicated that these target genes were enriched mainly in molecular function, such as cytokine activity and active transmembrane transporter activity, in biological processes, such as response to stimulus and immune response, and in pathways, such as protein families: signaling and cellular processes, transporters and metabolism. These findings not only provide valuable reference resources for further research on the molecular basis and function of lncRNAs in turbot but also help to accelerate the progress of molecularly selective breeding of stress-resistant turbot strains or varieties.

Measurement(s) eye movement Technology Type(s) eye tracking Factor Type(s) word frequency • word length Sample Characteristic - Organism Human Sample Characteristic - Environment laboratory environment

Measurement(s) whole genome sequencing Technology Type(s) PacBio long-read and Illumina short-read sequencing technologies

The middle and lower reaches of the Yangtze River is an important iron, copper, gold and other polymetallic metallogenic belt in China. The southern part of the Yangtze River is the southeast Hubei ore concentration area. As an important source of iron and copper deposits in China, several iron-rich copper deposits are concentrated in this area. The Jinniu volcanic basin is located in the southeastern Hubei ore concentration area. The basin has the ore potential of iron, copper, gold and other polymetallic deposits with the similar metallogenic conditions of other mining areas in this metallogenic belt. However, the basin has a thick overlying strata of magnetic rock and sedimentary rock, which increases the difficulty of ore prospecting. Therefore, it is necessary to strengthen the application of technologies for improving the effect of ore prediction. In the adjacent ore concentration areas, the prospecting work mainly focuses on gravity and magnetic data and combined with other geophysical data for comprehensive analysis, which lays a good foundation for the study of structural characteristics and the guidance of prospecting prediction. These previous works has proved that by comprehensive analysis and interpretation of high-precision gravity and magnetic data combined with other geophysical data, the tectonic framework characteristics, buried rocks and physical property distribution of the study area can be extracted, so as to infer the tectonic characteristics of the basin and its basement and put forward prospecting prediction. For the lack of systematic research and demand for prospecting in Jinniu volcanic basin, based on measured and collected geological and geophysical data, comprehensive analysis and interpretation of gravity and magnetic data combined with MT data were carried out. The techniques of linear signal extraction, multi-scale analysis, inversion and resistivity imaging, etc., were utilized to estimate the depth of the basin basement, delineate volcanic mechanism and buried rocks, and speculate fractures, basin boundary, and some areas of prospecting prospect. The such research has the guidance or reference significance for the basic geological survey and deep ore prediction in this area.

Abstract Background Soybean-corn intercropping is widely practised by farmers in Southwest China. Although rhizosphere microorganisms are important in nutrient cycling processes, the differences in rhizosphere microbial communities between intercropped soybean and corn and their monoculture are poorly known. Additionally, the effects of cadmium (Cd) pollution on these differences have not been examined. Therefore, a field experiment was conducted in Cd-polluted soil to determine the effects of monocultures and soybean–corn intercropping systems on Cd concentrations in plants, on rhizosphere bacterial communities, soil nutrients and Cd availability. Plants and soils were examined five times in the growing season, and Illumina sequencing of 16S rRNA genes was used to analyze the rhizosphere bacterial communities. Results Intercropping did not alter Cd concentrations in corn and soybean, but changed soil available Cd (ACd) concentrations and caused different effects in the rhizosphere soils of the two crop species. However, there was little difference in bacterial community diversity for the same crop species under the two planting modes. Proteobacteria, Chloroflexi, Acidobacteria, Actinobacteria and Firmicutes were the dominant phyla in the soybean and corn rhizospheres. In ecological networks of bacterial communities, intercropping soybean (IS) had more module hubs and connectors, whereas intercropped corn (IC) had fewer module hubs and connectors than those of corresponding monoculture crops. Soil organic matter (SOM) was the key factor affecting soybean rhizosphere bacterial communities, whereas available nutrients (N, P, K) were the key factors affecting those in corn rhizosphere. During the cropping season, the concentration of soil available phosphorus (AP) in the intercropped soybean–corn was significantly higher than that in corresponding monocultures. In addition, the soil available potassium (AK) concentration was higher in intercropped soybean than that in monocropped soybean. Conclusions Intercropped soybean–corn lead to an increase in the AP concentration during the growing season, and although crop absorption of Cd was not affected in the Cd-contaminated soil, soil ACd concentration was affected. Intercropped soybean–corn also affected the soil physicochemical properties and rhizosphere bacterial community structure. Thus, intercropped soybean–corn was a key factor in determining changes in microbial community composition and networks. These results provide a basic ecological framework for soil microbial function in Cd-contaminated soil.

The mitogen-activated protein kinase (MAPK) gene family performs crucial roles in cell division, migration, development, apoptosis, inflammatory response, and abiotic and biotic stress responses. However, very little information is available about the MAPKs in turbot (Scophthalmus maximus). In this study, 15 turbot MAPKs (SmMAPKs) were identified throughout the whole genome, and their basic chemical and physical properties and subcellular localization were illustrated. All SmMAPKs contained the serine/threonine protein kinases, catalytic domain (S_TKc, SMART00220). Phylogenetic analysis revealed that SmMAPKs were classified into three subfamilies, namely, c-Jun NH2-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), and p38. Conserved motif and gene structure analysis revealed high levels of conservation within and between phylogenetic subfamilies. Expression patterns of MAPKs in distinct tissues and under diverse abiotic and biotic stresses were examined using the published available RNA-seq data sets. As a result, SmMAPKs showed obviously tissue-specific expression. Furthermore, 7 and 10 candidate stress-responsive MAPK genes were detected under abiotic and biotic stresses, respectively, among which five common MAPK genes, namely, SmMAPK4 (ERK4), SmMAPK6 (ERK3), SmMAPK11 (p38β), SmMAPK12b (p38γ), and SmMAPK15 (ERK7/8) showed extremely significant responses to both abiotic and biotic stresses, demonstrating their potential functions in comprehensive antistress. These results demonstrate that MAPKs might play vital roles in response to various abiotic and biotic stresses in turbot, which would contribute to making scientific preventive measures to environmental changes in the process of farming and promoting the development of selective breeding for comprehensive stress resistance in turbot.

Infectious diseases have caused dramatic production decline and economic loss for fish aquaculture. However, the poor understanding of fish disease resistance severely hampered disease prevention. Chinese tongue sole (Cynoglossus semilaevis) is an important economic flatfish suffering from vibriosis. Here we used genomic, transcriptomic and experimental approaches to investigate the molecular genetic mechanisms underlying fish vibriosis resistance. A genome-wide comparison revealed that the genes under selective sweeps were enriched for glycosaminoglycan (GAG) chondroitin sulfate (CS)/dermatan sulfate (DS) metabolism. Transcriptomic analyses prioritized synergic gene expression patterns in this pathway, which may lead to an increased CS/DS content in the resistant family. Further experimental evidence showed that carbohydrate sulfotransferases 12 (Chst12), a key enzyme for CS/DS biosynthesis, has a direct antibacterial activity. To the best of our knowledge, this is the first report that the chst12 gene has a bactericidal effect. In addition, CS/DS is a major component of the extracellular matrix (ECM) and the selection signatures and fine-tuned gene expressions of ECM-receptor interaction genes indicated a modification in the ECM structure with an enhancement of the barrier function. Furthermore, functional studies conducted on Col6a2, encoding a collagen gene which constitutes the ECM, pointed to that it may act as a cellular receptor for Vibrio pathogens, thus plays an important role for the Vibrio invasion. Taken together, these findings provide new insights into the molecular protective mechanism underlying vibriosis resistance in fish, which offers crucial genomic resources for the resistant germplasm breeding and infectious disease control in fish culturing.

Thanks to the advantages of low latency, high efficiency, and oneself-security, the edge computing (EC) paradigm is expected to widely be applied in a large number of scenarios. However, because EC equipment is closer to a wide variety of terminals in a realistic environment, it is also more vulnerable to terminal attacks. The security of edge computing equipment itself cannot be ignored. Docker is a lightweight container technology that closely fits the existing needs of edge computing for portability, security isolation, and convenience. In this paper, Docker technology is taken as an edge computing security support engine and a security monitoring system based on the Docker container is built. In addition, combined with container monitoring and objective weighting method, a node security judgment method is proposed. Finally, according to the results of the node security judgment, a method for evaluating the security of the unmonitored node is put forward. Consequently, an edge computing security monitoring system based on the Docker container is built, and its performance in the real environment of a smart grid system is implemented to verify the proposed method. The results prove the efficiency and security protection of the novel edge power system.

Construction of engineering structures in geomaterials with soil–rock mixture (S-RM) is often a challenging task for engineers. When analyzing the stability of the engineering structures, the mechanical properties of S-RM often receive the most attention. To study the mechanical damage evolution characteristics of S-RM under triaxial loading conditions, a modified triaxial apparatus was used to conduct shear test on S-RM, and the change of electrical resistivity was measured simultaneously. The stress–strain–electrical resistivity curve and stress–strain characteristics under different confining pressures were obtained and analyzed. Based on the electrical resistivity, a mechanical damage model was established and verified to analyze the damage evolution regularities of S-RM during shearing. The results show that the electrical resistivity of S-RM decreases with increasing axial strain and that the differences in decrease rates correspond to the different deformation stages of the samples. With the increase in loading confining pressure, the stress–strain curve characteristics change from a slight strain softening to a strong strain hardening. Additionally, an increase in rock content and confining pressure can enhance the bearing capacity of S-RM. Moreover, the derived damage evolution model based on electrical resistivity can accurately characterize the mechanical behavior of S-RM under triaxial shear. Based on the damage variable D, it is found that the damage evolution process of S-RM can be divided into a non-damage stage, a rapid damage stage and a stable damage stage. Furthermore, the structure enhancement factor, which is a model modification parameter for the effect of rock content difference, can accurately predict the stress–strain curves of S-RMs with different rock contents. This study sets the stage for an electrical-resistivity-based monitoring method for studying the evolution of internal damage in S-RM.

The Chinese tongue sole (Cynoglossus semilaevis) is a traditional, precious fish in China. Due to the large growth difference between males and females, the investigation of their sex determination and differentiation mechanisms receives a great deal of attention. Forkhead Box O (FoxO) plays versatile roles in the regulation of sex differentiation and reproduction. Our recent transcriptomic analysis has shown that foxo genes may participate in the male differentiation and spermatogenesis of Chinese tongue sole. In this study, six Csfoxo members (Csfoxo1a, Csfoxo3a, Csfoxo3b, Csfoxo4, Csfoxo6-like, and Csfoxo1a-like) were identified. Phylogenetic analysis indicated that these six members were clustered into four groups corresponding to their denomination. The expression patterns of the gonads at different developmental stages were further analyzed. All members showed high levels of expression in the early stages (before 6 months post-hatching), and this expression was male-biased. In addition, promoter analysis found that the addition of C/EBPα and c-Jun transcription factors enhanced the transcriptional activities of Csfoxo1a, Csfoxo3a, Csfoxo3b, and Csfoxo4. The siRNA-mediated knockdown of the Csfoxo1a, Csfoxo3a, and Csfoxo3b genes in the testicular cell line of Chinese tongue sole affected the expression of genes related to sex differentiation and spermatogenesis. These results have broadened the understanding of foxo’s function and provide valuable data for studying the male differentiation of tongue sole.

Heat shock proteins 70 (HSP70s) are known to play essential roles in organisms’ response mechanisms to various environmental stresses. However, no systematic identification and functional analysis has been conducted for HSP70s in the turbot (Scophthalmus maximus), a commercially important worldwide flatfish. Herein, 16 HSP70 genes unevenly distributed on nine chromosomes were identified in the turbot at the genome-wide level. Analyses of gene structure, motif composition, and phylogenetic relationships provided valuable data on the HSP70s regarding their evolution, classification, and functional diversity. Expression profiles of the HSP70 genes under five different stresses were investigated by examining multiple RNA-seq datasets. Results showed that 10, 6, 8, 10, and 9 HSP70 genes showed significantly up- or downregulated expression after heat-induced, salinity-induced, and Enteromyxum scophthalmi, Vibrio anguillarum, and Megalocytivirus infection-induced stress, respectively. Among them, hsp70 (hspa1a), hspa1b, and hspa5 showed significant responses to each kind of induced stress, and qPCR analyses further validated their involvement in comprehensive anti-stress, indicating their involvement in organisms’ anti-stress mechanisms. These findings not only provide new insights into the biological function of HSP70s in turbot adapting to various environmental stresses, but also contribute to the development of molecular-based selective breeding programs for the production of stress-resistant turbot strains in the aquaculture industry.

For real-time industrial wireless edge-enabled cyber-physical systems, this paper proposes an adaptive secure transmission mechanism under the large scale path loss to fight against randomly distributed illegal sensing devices. According to changes in the communication environment, the proposed scheme adaptively obtains the corresponding optimal power allocations and wiretap code rates. Specifically, by injecting artificial noise, an edge controller provides confidential control information for a legitimate wireless sensing device and achieves non-confidential data service for a service subscriber, simultaneously. Considering whether the secrecy and non-confidential service rate constraints are met, a dynamic secrecy transmission mechanism is introduced to minimize secrecy outage probability (SOP) based on random channel realizations. The optimal power allocation for minimizing the SOP is derived through the efficient alternating optimization algorithm with numerical methods. Numerical results show that the proposed adaptive secure transmission strategy can efficiently utilize transmit power to achieve non-confidential information traffic without compromising the secrecy performance.

Blind-side hypermelanosis is an emerging concern across the flatfish aquaculture industry including Chinese tongue sole (Cynoglossus semilaevis). Circular RNAs (circRNAs) as endogenous non-coding RNAs have been acknowledged to play important roles in various biological processes. However, the underlying regulatory mechanisms of circRNAs involved in flatfish blind-side hypermelanosis remain unclear. In this study, to profile the circRNA expression pattern and circRNA-microRNA-messenger RNA (mRNA) network, high-throughput sequencing was performed by using blind-side normal and hypermelanotic skins of tongue sole. A total of 73 differentially expressed circRNAs were identified, and the competing endogenous RNA (ceRNA) network was constructed. Furthermore, circRNA host genes and mRNAs involved in ceRNA network were subjected to Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes enrichment analyses. Several GO terms and pathways of biological significance were identified and well addressed the generation of blind-side hypermelanosis to some extent. These interesting results extend the understanding of the functional profile of circRNAs and yield valuable insights into the molecular regulatory mechanisms of hypermelanosis in flatfish.

In mammals, the mutation of nipped-B-like protein (nipbl) leads to Cornelia de Lange Syndrome (CdLS), characterized by low birth weight, short stature, and structural abnormalities of the skeleton, heart, and gut. In Chinese tongue sole (Cynoglossus semilaevis), a typical marine fish exhibiting sexual size dimorphism, the nipbl homolog gene (nipped-B-like protein A (nipbla)) was also screened with female higher expression level by somatotropic and reproductive tissues’ transcriptomic analysis. In this study, two nipbla genes, namely, nipbla-w and nipbla-z, were identified from the W and Z chromosomes of C. semilaevis, respectively. Similar to other mammalian and fish species nipbl, the two homolog proteins of C. semilaevis contained two conserved domains, namely, cohesion_HEAT and Nipped-B_C. The phylogenetic tree analysis showed that these two nipbla gametolog proteins were first clustered together and then grouped with other fish species. At least two types of alternative splicing sites were observed in exon 12 of the nipbla-z gene, which produced nipbla-z-tv1 and nipbla-z-tv2. Also, the sex-biased expression patterns of different nipbla-w and nipbla-z transcripts in female and male tissues were revealed by quantitative PCR (qPCR). The highest expression level of nipbla-w was observed in female gonad. While nipbla-z-tv1 exhibited relatively high expression in the muscle, liver, gonad, and brain, nipbla-z-tv2 only showed its expression superiority in the muscle of male individuals. The promoter regions of nipbla genes were amplified, and their transcription activity was successfully verified by a dual-luciferase reporter system. After nipbla-w and nipbla-z knockdown in the brain cell lines by RNA interference, a series of growth-related genes were influenced, including Bone Morphogenetic Protein 4 (bmp4), Wnt Family Member 11 (wnt11), and Sprouty Related EVH1 Domain Containing 2 (spred2). The prediction of transcription factors suggested that c-Jun, sex-determining region Y (SRY), POU Class 1 Homeobox 1 (POU1F1a), myogenic differentiation antigen (MyoD), signal transducer and activator of transcription 5a (STAT5A), and nuclear factor I C (Nfic) might be the putative upstream regulatory factors for nipbla; among them, c-Jun has been verified to effectively regulate the transcriptional activity of nipbla. The identification of two nipbla genes provided important data for interpreting the sexual size dimorphism in C. semilaevis.

Blind-side hypermelanosis has emerged as a major concern in commercial rearing environments of the flatfish aquaculture industry. To date, the underlying molecular mechanisms are not well understood. To fill this gap, in this study, whole transcriptomic sequencing and analyses were performed using normal skins and hypermelanic skins of the blind side of Chinese tongue sole (Cynoglossus semilaevis). Differentially expressed long non-coding RNAs (DElncRNAs), miRNAs (DEmiRNAs), and differentially expressed genes as well as their competing endogenous RNA (ceRNA) networks were identified. A total of 34 DElncRNAs, 226 DEmiRNAs, and 610 DEGs were identified. Finally, lncRNA–miRNA–mRNA regulatory networks (involving 29 DElncRNAs, 106 DEmiRNAs, and 162 DEGs) associated with blind-side hypermelanosis were constructed. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses of 162 DEGs in ceRNA networks identified DEGs (e.g., oca2, mc1r, and ihhb) in pigmentation-related biological processes and DEGs (e.g., ca4, glul, and fut9) in nitrogen metabolism, glycosphingolipid biosynthesis, and folate biosynthesis pathways, as well as their corresponding DElncRNAs and DEmiRNAs to potentially play key regulatory roles in blind-side hypermelanosis. In conclusion, this is the first study on the ceRNA regulatory network associated with blind-side hypermelanosis in flatfish. These new findings expand the spectrum of non-coding regulatory mechanisms underpinning blind-side hypermelanosis, which facilitates the further exploration of molecular regulatory mechanisms of malpigmentation in flatfish.

Chinese tongue sole (Cynoglossus semilaevis) is a flatfish species unique to Northeast Asia, exhibiting the typical female-biased sexual size dimorphism (SSD). To explore the possible regulatory roles of non-coding RNAs (ncRNAs) on this phenomenon, whole transcriptomic analysis was performed by using female, male, and pseudomale C. semilaevis to identify differentially expressed (DE) long ncRNAs (DE lncRNAs), microRNA (DE miRNAs), and differentially expressed genes (DEGs) from the brain, gonad, liver, and muscle tissues. Most of them were concentrated in the gonad and muscle, and the gene expression patterns of pseudomale individuals were similar to male individuals. The association of DE lncRNAs and target messenger RNAs (mRNAs) was predicted based on antisense, cis-, and trans-regulatory mechanisms, with enriched protein digestion and absorption, cyclic adenosine monophosphate (cAMP) signaling pathway, sulfur metabolism, cell cycle, and splicesome (p < 0.05). Furthermore, weighted gene co-expression network analysis (WGCNA) was employed to cluster the expression patterns of DE lncRNA, and two modules (greenyellow and blue) had the highest positive and negative correlations with growth traits, respectively. Importantly, the female-biased expression in the greenyellow module and the male- and pseudomale-biased expression in the blue module were observed in the gonad. The target gene analysis for DE miRNA revealed 3,034 mRNA-miRNA pairs with the opposite expression patterns. Finally, the lncRNA-miRNA-mRNA network, including 385 DE lncRNAs, 138 DE miRNAs, and 456 DEGs, was constructed. Among which, 78 DE lncRNAs, 12 DE miRNAs, and 13 DEGs involved in cell growth and death pathway were related to the SSD of C. semilaevis. This study described the lncRNA-miRNA-mRNA regulatory network in the SSD of C. semilaevis for the first time. The functional prediction analysis suggested that these DE lncRNAs and DE miRNAs might be involved in flatfish SSD by regulating several potential growth-related pathways (e.g., cell cycle, cAMP signaling, and Rap1 signaling). Further studies related to these ncRNAs will enlarge our understanding of the regulatory effects of ncRNAs on fish SSD.

Abstract Background Edwardsiella tarda causes acute symptoms with ascites in Japanese flounder (Paralichthys olivaceus) and is a major problem for China’s aquaculture sector. Genomic selection (GS) has been widely adopted in breeding industries because it shortens generation intervals and results in the selection of individuals that have great breeding potential with high accuracy. Based on an artificial challenge test and re-sequenced data of 1099 flounders, the aims of this study were to estimate the genetic parameters of resistance to E. tarda in Japanese flounder and to evaluate the accuracy of single-step GBLUP (ssGBLUP), weighted ssGBLUP (WssGBLUP), and BayesB for improving resistance to E. tarda by using three subsets of pre-selected single nucleotide polymorphisms (SNPs). In addition, SNPs that are associated with this trait were identified using a single-SNP genome-wide association study (GWAS) and WssGBLUP. Results We estimated a heritability of 0.13 ± 0.02 for resistance to E. tarda in Japanese flounder. One million SNPs at fixed intervals were selected from 4,978,724 SNPs that passed quality controls. GWAS identified significant SNPs on chromosomes 14 and 24. WssGBLUP revealed that the putative quantitative trait loci on chromosomes 1 and 14 contained SNPs that explained more than 1% of the genetic variance. Three 50 k-SNP subsets were pre-selected based on different criteria. Compared with pedigree-based prediction (ABLUP), the three genomic methods evaluated resulted in at least 7.7% greater accuracy of predictions. The accuracy of these genomic prediction methods was almost unchanged when pre-selected trait-related SNPs were used for prediction. Conclusions Resistance to E. tarda in Japanese flounder has a low heritability. GWAS and WssGBLUP revealed that the genetic architecture of this trait is polygenic. Genomic prediction of breeding values performed better than ABLUP. It is feasible to implement genomic selection to increase resistance to E. tarda in Japanese flounder with 50 k SNPs. Based on the criteria used here, pre-selection of SNPs was not beneficial and other criteria for pre-selection should be considered.

The purpose is to study the influence of Zhu Xi’s neo-Confucianism on the ideological and political education (IPE) of college students. Firstly, the aesthetic connotation of Zhu Xi’s Neo-Confucianism is studied. Secondly, an interview is performed with teachers of the IPE course in a university in Xi’an, and a questionnaire survey is conducted on some students of the IPE course in the school. It aims to investigate the current situation of college students’ IPE, and the impact of integrating aesthetic education into IPE of college students on the improvement of students’ overall quality and internalized psychology. Finally, the reasons for this situation are analyzed and solutions are given. The survey results reveal that the aesthetic education thoughts based on Zhu Xi’s neo-Confucianism are integrated into the construction of college students’ internalized psychological mechanism, and most students think that their mental health status has changed a lot. The proportion of boys who choose “improved” is 57.95%, and the proportion of girls is 53.45%. The aesthetic education is integrated into the IPE of college students, and the proportion of students who think “no effect” after integration and before integration has decreased by 2.01%. The proportion of the number of people who chose the three indicators of “increasing the innovation of teaching methods”, “advancing aesthetic ability”, and “improving the effectiveness of ideological education” increased by 5.88%, 5.5%, and 3.98%, respectively. It demonstrates that the effect of IPE on college students has been greatly improved after integration into aesthetic education.

Sexual size dimorphism (SSD) is the difference in segments or body size between sexes prevalent in various species. Understanding the genetic architecture of SSD has remained a significant challenge owing to the complexity of growth mechanisms and the sexual influences among species. The Chinese tongue sole (Cynoglossus semilaevis), which exhibits a female-biased SSD and sex reversal from female to pseudomale, is an ideal model for exploring SSD mechanism at the molecular level. The present study aimed to integrate transcriptome and methylome analysis to unravel the genetic and epigenetic changes in female, male, and pseudomale C. semilaevis. The somatotropic and reproductive tissues (brain, liver, gonad, and muscle) transcriptomes were characterized by RNA-seq technology. Transcriptomic analysis unravelled numerous differentially expressed genes (DEGs) involved in cell growth and death-related pathways. The gonad and muscle methylomes were further employed for screening differentially methylated genes (DMGs). Relatively higher DNA methylation levels were observed in the male and pseudomale individuals. In detail, hypermethylation of the chromosome W was pronounced in the pseudomale group than in the female group. Furthermore, weighted gene co-expression network analysis showed that turquoise and brown modules positively and negatively correlated with the female-biased SSD, respectively. A combined analysis of the module genes and DMGs revealed the female-biased mRNA transcripts and hypomethylated levels in the upstream and downstream regions across the cell cycle-related genes. Moreover, the male and pseudomale-biased gene expression in the hippo signaling pathway were positively correlated with their hypermethylation levels in the gene body. These findings implied that the activation of the cell cycle and the inhibition of the hippo signaling pathway were implicated in C. semilaevis female-biased SSD. In addition, the dynamic expression pattern of the epigenetic regulatory factors, including dnmt1, dnmt3a, dnmt3b, and uhrf1, among the different sexes correspond with their distinct DNA methylation levels. Herein, we provide valuable clues for understanding female-biased SSD in C. semilaevis.

A new cell line Japanese flounder spleen (JFSP) derived from the spleen of Japanese flounder (Paralichthys olivaceus) was established and characterized in this study. The JFSP cells grew rapidly at 29 °C, and the optimum fetal bovine serum concentration in the L-15 medium was 15%. Cells were subcultured for more than 80 passages. The JFSP cells have a diploid chromosome number of 2n = 68, which differs from the chromosome number of normal diploid Japanese flounder. The established cells were susceptible to Bohle virus (BIV), Viral hemorrhagic septicemia virus (VHSV), Hirame rhabdovirus (HIRRV), Infectious hematopoietic necrosis virus (IHNV), and Lymphocystis disease virus (LCDV), as evidenced by varying degrees of cytopathic effects (CPE). Replication of the virus in JFSP cells was confirmed by qRT-PCR and transmission electron microscopy. In addition, the expression of four immune-related genes, TRAF3, IL-1β, TNF-α, and TLR2, was differentially altered following viral infection. The results indicated that the cells underwent an antiviral immune response. JFSP cell line is an ideal tool in vitro for virology. The use of fish cell lines to study the immune genes and immune mechanism of fish and to clarify the immune mechanism of fish has important theoretical significance and practical application value for the fundamental prevention and treatment of fish diseases.

Sexual size dimorphism (SSD), which is the sexual differences in body size, has been widely reported in various species including fishes. For Chinese tongue sole (Cynoglossus semilaevis), a flatfish exhibiting typically female-biased SSD, little is known for its epigenetic regulation mechanism, especially the role of circRNAs. Here, we identified the differently expressed abundances of circRNAs in females, males, and pseudo-males to explore the potential functions of circRNAs in Chinese tongue sole SSD. In total, 14,745 novel circRNAs were screened, among which 1461 DE circRNAs were identified from the brain, gonad, liver, and muscle in female, male, and pseudo-male individuals. The ceRNA network was subsequently constructed, including 10 circRNAs, 26 mRNAs, and 11 miRNAs. These DE mRNAs were mainly related to the mRNA surveillance pathway, metabolic pathways, and cellular senescence. Importantly, the ceRNA network has revealed that several circRNAs such as novel_circ_004374 and novel_circ_014597 may regulate homeodomain interacting protein kinase 2 (hipk2) expression by sponging miR-130-x. It is also worth exploring whether or how novel_circ_008696 regulates SET Domain Containing 2, histone lysine methyltransferase (setd2), which in turn affects the epigenetic patterns of different sexual individuals. The present study not only enriches the knowledge on the potential roles of circRNA in the physiological process, but also provides new clues for the explanation of fish SSD. In future studies, the precise function and involvement of circRNAs in female-biased SSD will require more efforts.

As an RA-metabolizing enzyme, cyp26b1 has a substantial impact on RA-signaling pathways. The cyp26b1 gene from the Chinese tongue sole was cloned and identified in this investigation. The cyp26b1 ORF was 1536 bp in length and encoded a 512 amino acid protein. A quantitative real-time PCR (qPCR) indicated that the cyp26b1 expression is no significant sexual dimorphism in the gonads at the 80 days post-hatching (dph) stages. After 4 months post-hatching (mph), the expression of cyp26b1 showed sexual dimorphism and lower level of expression in the ovaries than in the testes. An in situ hybridization demonstrated that cyp26b1 mRNA was primarily located in the testis. Interestingly, the cyp26b1 mRNA probe was also detected in the ovaries. These results suggested that cyp26b1 participates in the sex-differentiation and gonadal development of the Chinese tongue sole.

Ewsr1 encodes a protein that acts as a multifunctional molecule in a variety of cellular processes. The full-length of Cs-ewsr1-w and Cs-ewsr1-z were cloned in Chinese tongue sole (Cynoglossus semilaevis). The open reading frame (ORF) of Cs-ewsr1-w was 1,767 bp that encoded 589 amino acids, while Cs-ewsr1-z was 1,794 bp that encoded 598 amino acids. Real-time PCR assays showed that Cs-ewsr1-w exhibited significant female-biased expression and could be hardly detected in male. It has the most abundant expression in ovaries among eight healthy tissues. Its expression in ovary increased gradually from 90 d to 3 y with C. semilaevis ovarian development and reached the peak at 3 y. After Cs-ewsr1-w knockdown with siRNA interference, several genes related to gonadal development including foxl2, sox9b and pou5f1 were down-regulated in ovarian cell line, suggesting the possible participation of Cs-ewsr1-w in C. semilaevis ovarian development. The dual-luciferase reporter assay revealed that the -733/-154 bp Cs-ewsr1-w promoter fragment exhibited strong transcription activity human embryonic kidney (HEK) 293T cell line. The mutation of a MAF BZIP Transcription Factor K (Mafk) binding site located in this fragment suggested that transcription factor Mafk might play an important role in Cs-ewsr1-w basal transcription. Our results will provide clues on the gene expression level, transcriptional regulation and knock-down effect of ewsr1 gene during ovarian development in teleost.

The aim of this study was to evaluate genetic parameters of growth traits for improving selective breeding in olive flounder (Paralichthys olivaceus). A total of 82 full-sib families from 2 year-classes with complete pedigree information were used to estimate heritabilities and genetic/phenotypic correlations for 4 growth performance traits (body weight BW, total length TL, body height BH and condition factor K) based on uni-trait and multi-trait linear mixed models, respectively. Data were analyzed by the restricted maximum likelihood (REML) method. The heritabilities of BW, TL and BH were 0.81, 0.70 and 0.61, which are relatively high, however, heritability for K is low (0.11). For all growth traits, the common full-sib effects (c2) were at a low to moderate level (0.14–0.20); the maternal effects (m2) of the traits (except K) were also detected (0.15–0.19). The phenotypic and genetic correlations among BW, TL and BH were highly positive (0.70–0.91), however, the correlations between K and the other 3 traits was low or negligible (−0.22–0.26). According to our results, these traits (except K) could be selected and enhanced simultaneously. In addition, direct selection based on TL or BH, which is an easily measurable trait, will be more favorable than selection on BW. Moreover, our results indicated that growth parameters estimated in the juvenile stage could be used as a predictor of growth performance at later growth stages. Keywords: Genetic parameter, Growth trait, Heritability, Genetic correlation, Olive flounder, Paralichthys olivaceus

In this paper, an edge computing system for IoT-based (Internet of Things) smart grids is proposed to overcome the drawbacks in the current cloud computing paradigm in power systems, where many problems have yet to be addressed such as fully realizing the requirements of high bandwidth with low latency. The new system mainly introduces edge computing in the traditional cloud-based power system and establishes a new hardware and software architecture. Therefore, a considerable amount of data generated in the electrical grid will be analyzed, processed, and stored at the edge of the network. Aided with edge computing paradigm, the IoT-based smart grids will realize the connection and management of substantial terminals, provide the real-time analysis and processing of massive data, and foster the digitalization of smart grids. In addition, we propose a privacy protection strategy via edge computing, data prediction strategy, and preprocessing strategy of hierarchical decision-making based on task grading (HDTG) for the IoT-based smart girds. The effectiveness of our proposed approaches has been demonstrated via the numerical simulations.

The understanding of the evolution of variable sex determination mechanisms across taxa requires comparative studies among closely related species. Following the fate of a known master sex-determining gene, we traced the evolution of sex determination in an entire teleost order (Esociformes). We discovered that the northern pike (Esox lucius) master sex-determining gene originated from a 65 to 90 million-year-old gene duplication event and that it remained sex linked on undifferentiated sex chromosomes for at least 56 million years in multiple species. We identified several independent species- or population-specific sex determination transitions, including a recent loss of a Y chromosome. These findings highlight the diversity of evolutionary fates of master sex-determining genes and the importance of population demographic history in sex determination studies. We hypothesize that occasional sex reversals and genetic bottlenecks provide a non-adaptive explanation for sex determination transitions.

The sustainable development of aquaculture has been impeded by infectious diseases worldwide. However, the genomic architecture and the genetic basis underlying the disease resistance remain poorly understood, which severely hampers both the understanding of the evolution of fish disease resistance traits and the prevention of these diseases in the aquaculture community. Cynoglossus semilaevis is a representative and commercially-important flatfish species. Here we combined genome-wide association study and Fst and nucleotide diversity filtration to identify loci important for the disease resistance. Based on 1,016,774 single-nucleotide polymorphisms (SNPs) identified from 650 Gb genome resequencing data of 505 individuals, we detected 33 SNPs significantly associated with disease resistance and 79 candidate regions after filtration steps. Both the allele frequencies and genotype frequencies of the associated loci were significantly different between the resistant and susceptible fish, suggesting a role in the genetic basis of disease resistance. The SNP with strongest association with disease resistance was located in Chr 17, at 145 bp upstream of fblx19 gene, and overlapped with the major quantitative trait locus previously identified. Several genes, such as plekha7, nucb2, and fgfr2, were also identified to potentially play roles in the disease resistance. Furthermore, the expression of some associating genes were likely under epigenetic regulations between the bacterial resistant and susceptible families. These results provide insights into the mechanism that enable variation of disease resistance to bacterial pathogen infection. The identified polymorphisms and genes are valuable targets and molecular resources for disease resistance and other traits, and for advanced breeding practice for superior germplasm in fish aquaculture.

Abstract Background RNA-Seq has become one of the most widely used applications based on next-generation sequencing technology. However, raw RNA-Seq data may have quality issues, which can significantly distort analytical results and lead to erroneous conclusions. Therefore, the raw data must be subjected to vigorous quality control (QC) procedures before downstream analysis. Currently, an accurate and complete QC of RNA-Seq data requires of a suite of different QC tools used consecutively, which is inefficient in terms of usability, running time, file usage, and interpretability of the results. Results We developed a comprehensive, fast and easy-to-use QC pipeline for RNA-Seq data, RNA-QC-Chain, which involves three steps: (1) sequencing-quality assessment and trimming; (2) internal (ribosomal RNAs) and external (reads from foreign species) contamination filtering; (3) alignment statistics reporting (such as read number, alignment coverage, sequencing depth and pair-end read mapping information). This package was developed based on our previously reported tool for general QC of next-generation sequencing (NGS) data called QC-Chain, with extensions specifically designed for RNA-Seq data. It has several features that are not available yet in other QC tools for RNA-Seq data, such as RNA sequence trimming, automatic rRNA detection and automatic contaminating species identification. The three QC steps can run either sequentially or independently, enabling RNA-QC-Chain as a comprehensive package with high flexibility and usability. Moreover, parallel computing and optimizations are embedded in most of the QC procedures, providing a superior efficiency. The performance of RNA-QC-Chain has been evaluated with different types of datasets, including an in-house sequencing data, a semi-simulated data, and two real datasets downloaded from public database. Comparisons of RNA-QC-Chain with other QC tools have manifested its superiorities in both function versatility and processing speed. Conclusions We present here a tool, RNA-QC-Chain, which can be used to comprehensively resolve the quality control processes of RNA-Seq data effectively and efficiently.

TFIIB‐related factor 1 (Brf1) modulates the transcription of RNA Pol III genes (polymerase‐dependent genes). Upregulation of Pol III genes enhances tRNA and 5S RNA production and increases the translational capacity of cells to promote cell transformation and tumor development. However, the significance of Brf1 overexpression in human breast cancer (HBC) remains to be investigated. Here, we investigate whether Brf1 expression is increased in the samples of HBC, and we explore its molecular mechanism and the significance of Brf1 expression in HBC. Two hundred and eighteen samples of HBC were collected to determine Brf1 expression by cytological and molecular biological approaches. We utilized colocalization, coimmunoprecipitation, and chromatin immunoprecipitation methods to explore the interaction of Brf1 with estrogen receptor alpha (ERα). We determined how Brf1 and ERα modulate Pol III genes. The results indicated that Brf1 is overexpressed in most cases of HBC, which is associated with an ER‐positive status. The survival period of the cases with high Brf1 expression is significantly longer than those with low levels of Brf1 after hormone treatment. ERα mediates Brf1 expression. Brf1 and ERα are colocalized in the nucleus. These results indicate an interaction between Brf1 and ERα, which synergistically regulates the transcription of Pol III genes. Inhibition of ERα by its siRNA or tamoxifen reduces cellular levels of Brf1 and Pol III gene expression and decreases the rate of colony formation of breast cancer cells. Together, these studies demonstrate that Brf1 is a good biomarker for the diagnosis and prognosis of HBC. This interaction of Brf1 with ERα and Brf1 itself are potential therapeutic targets for this disease.