GmANKTM21 Positively Regulates Drought Tolerance and Enhanced Stomatal Response through the MAPK Signaling Pathway in Soybean.

Drought stress is one of the significant abiotic stresses that limit soybean (Glycine max [L.] Merr.) growth and production. Ankyrin repeat (ANK) proteins, being highly conserved, occupy a pivotal role in diverse biological processes. ANK genes were classified into nine subfamilies according to conserved domains in the soybean genome. However, the function of ANK-TM subfamily proteins (Ankyrin repeat proteins with a transmembrane domain) in the abiotic-stress response to soybean remains poorly understood. In this study, we first demonstrated the subcellular localization of GmANKTM21 in the cell membrane and nucleus. Drought stress-induced mRNA levels of GmANKTM21, which encodes proteins belonging to the ANK-TM subfamily, Transgenic 35S:GmANKTM21 soybean improved drought tolerance at the germination and seedling stages, with higher stomatal closure in soybean, lower water loss, lower malondialdehyde (MDA) content, and less reactive oxygen species (ROS) production compared with the wild-type soybean (Dongnong50). RNA-sequencing (RNA-seq) and RT-qPCR analysis of differentially expressed transcripts in overexpression of GmANKTM21 further identified potential downstream genes, including GmSPK2, GmSPK4, and GmCYP707A1, which showed higher expression in transgenic soybean, than those in wild-type soybean and KEGG enrichment analysis showed that MAPK signaling pathways were mostly enriched in GmANKTM21 overexpressing soybean plants under drought stress conditions. Therefore, we demonstrate that GmANKTM21 plays an important role in tolerance to drought stress in soybeans.

Porcine reproductive and respiratory syndrome virus infection inhibits NF-κB signaling pathway through cleavage of IKKß by Nsp4.

Porcine reproductive and respiratory syndrome virus (PRRSV) is a highly contagious porcine pathogen that causes serious economic losses to the world swine industry. The inhibitor kappa B kinase ß (IKKß), a catalytic subunit of the IKK complex, plays multiple roles in regulating the nuclear transcription factor kappa B (NF-κB) activity and a variety of cytokines transcription involved in immune responses. Here, we reported that the nonstructural protein 4 (Nsp4) of PRRSV cleaved IKKß at the E378 site to inhibit the activation of NF-κB signaling pathway. Additionally, we clearly showed that cleavage of IKKß by PRRSV Nsp4 depends on the 3 C-like serine protease activity of Nsp4 because the catalytically inactivate mutants of Nsp4 lost the function to cleave IKKß. Furthermore, we found that hydrophobic patch at the KD-ULD junction of IKKß could be disrupted by PRRSV Nsp4 via the cleavage of the E378 site, resulting in disruption of NF-κB activity. Of note, the two cleavage fragments of IKKß lose their function to phosphorylate IκBα and activate NF-κB signaling pathway. Our findings provide a clue to better understand the pathogenic mechanism of PRRSV involved in PRRSV evasion of host antiviral innate immune responses.

Pseudorabies Virus Infection Activates the TLR-NF-κB Axis and AIM2 Inflammasome To Enhance Inflammatory Responses in Mice.

Pseudorabies virus (PRV) infection activates inflammatory responses to release robust proinflammatory cytokines, which are critical for controlling viral infection and clearance of PRV. However, the innate sensors and inflammasomes involved in the production and secretion of proinflammatory cytokines during PRV infection remain poorly studied. In this study, we report that the transcription and expression levels of some proinflammatory cytokines, including interleukin 1ß (IL-1ß), IL-6, and tumor necrosis factor alpha (TNF-α), are upregulated in primary peritoneal macrophages and in mice during PRV infection. Mechanistically, Toll-like receptor 2 (TLR2), TLR3, TLR4, and TLR5 were induced by the PRV infection to enhance the transcription levels of pro-IL-1ß, pro-IL-18, and gasdermin D (GSDMD). Additionally, we found that PRV infection and transfection of its genomic DNA triggered AIM2 inflammasome activation, apoptosis-related speckle-like protein (ASC) oligomerization, and caspase-1 activation to enhance the secretion of IL-1ß and IL-18, which was mainly dependent on GSDMD, but not GSDME, in vitro and in vivo. Taken together, our findings reveal that the activation of the TLR2-TLR3-TRL4-TLR5-NF-κB axis and AIM2 inflammasome, as well as GSDMD, is required for proinflammatory cytokine release, which resists the PRV replication and plays a critical role in host defense against PRV infection. Our findings provide novel clues to prevent and control PRV infection. IMPORTANCE PRV can infect several mammals, including pigs, other livestock, rodents, and wild animals, causing huge economic losses. As an emerging and reemerging infectious disease, the emergence of PRV virulent isolates and increasing human PRV infection cases indicate that PRV is still a high risk to public health. It has been reported that PRV infection leads to robust release of proinflammatory cytokines through activating inflammatory responses. However, the innate sensor that activates IL-1ß expression and the inflammasome involved in the maturation and secretion of proinflammatory cytokines during PRV infection remain poorly studied. In this study, our findings reveal that, in mice, activation of the TLR2-TLR3-TRL4-TLR5-NF-κB axis and AIM2 inflammasome, as well as GSDMD, is required for proinflammatory cytokine release during PRV infection, and it resists PRV replication and plays a critical role in host defense against PRV infection. Our findings provide novel clues to prevent and control PRV infection.

Genome-Wide Identification and Characterization of Copper Chaperone for Superoxide Dismutase (CCS) Gene Family in Response to Abiotic Stress in Soybean.

Copper Chaperone For Superoxide Dismutase (CCS) genes encode copper chaperone for Superoxide dismutase (SOD) and dramatically affect the activity of SOD through regulating copper delivery from target to SOD. SOD is the effective component of the antioxidant defense system in plant cells to reduce oxidative damage by eliminating Reactive oxygen species (ROS), which are produced during abiotic stress. CCS might play an important role in abiotic stress to eliminate the damage caused by ROS, however, little is known about CCS in soybean in abiotic stress regulation. In this study, 31 GmCCS gene family members were identified from soybean genome. These genes were classified into 4 subfamilies in the phylogenetic tree. Characteristics of 31 GmCCS genes including gene structure, chromosomal location, collinearity, conserved domain, protein motif, cis-elements, and tissue expression profiling were systematically analyzed. RT-qPCR was used to analyze the expression of 31 GmCCS under abiotic stress, and the results showed that 5 GmCCS genes(GmCCS5, GmCCS7, GmCCS8, GmCCS11 and GmCCS24) were significantly induced by some kind of abiotic stress. The functions of these GmCCS genes in abiotic stress were tested using yeast expression system and soybean hairy roots. The results showed that GmCCS7/GmCCS24 participated in drought stress regulation. Soybean hairy roots expressing GmCCS7/GmCCS24 showed improved drought stress tolerance, with increased SOD and other antioxidant enzyme activities. The results of this study provide reference value in-depth study CCS gene family, and important gene resources for the genetic improvement of soybean drought stress tolerance.

Characterization of the lipidomic profile of clam Meretrix petechialis in response to Vibrio parahaemolyticus infection.

Vibrio parahaemolyticus is a devastating pathogen of clam Meretrix petechialis, which brings about huge economic losses in aquaculture breeding industry. In our previous study, we have found that Vibrio infection is closely associated with lipid metabolism of clams. In this study, an untargeted lipidomics approach was used to explore the lipid profiling changes upon Vibrio infection. The results demonstrated that the hepatopancreas of clams was composed of five lipid categories including fatty acyls, glycerolipids, glycerophospholipids, sphingolipids and sterol lipids. And the content of lipid classes altered during Vibrio infection, implying that Vibrio infection altered intracellular lipid homeostasis in clams. Meanwhile, a total of 200 lipid species including 82 up-regulated and 118 down-regulated significantly were identified in response to Vibrio infection, of which ceramide (Cer), phosphatidylcholine (PC) and triglyceride (TG) accounted for the largest proportion. Notably, all Cers showed a significantly decreased trend while nearly all TG species were increased significantly during Vibrio infection, which suggested that Cer and TG could be determined as effective biomarkers. Furthermore, these differentially expressed lipid species were enriched in 20 metabolic pathways and sphingolipid metabolism was one of the most enriched pathways. These results evidenced how the lipid metabolism altered in the process of Vibrio infection and opened a new perspective on the response of marine bivalves to pathogen infection.

African swine fever virus pE301R negatively regulates cGAS-STING signaling pathway by inhibiting the nuclear translocation of IRF3.

African swine fever (ASF) is a highly contagious and lethal infectious disease of domestic pigs and wild boars by the African swine fever virus (ASFV). ASFV infects domestic pigs with the mortality rate approaching 100 % at acute stage of infection. The cGAS-STING-mediated antiviral responses are wildly accepted that cGAS acts as DNA sensor for sensing of viral DNA during DNA virus infection. However, the molecular mechanisms underlying negatively regulation of cGAS-STING signaling and type I IFN (IFN-I) production by ASFV proteins are not fully understood. In this study, we demonstrated that ASFV pE301R antagonize the activities of IFN-ß-, NF-κB-, ISRE-luciferase (Luc) reporters-induced by cGAS-STING in a dose dependent manner. Consistent with these results, the mRNA levels of Ifnb1, Isg15, Isg56 are attenuated by ASFV pE301R. Furthermore, ASFV pE301R executes its inhibitory function at the downstream of IFN-regulatory factor 3 (IRF3) phosphorylation. Mechanistically, pE301R interacts with IRF3 via its amino acid (aa) 1-200 region, resulting in inhibition of the nuclear translocation of IRF3 induced by cGAMP and poly(dA:dT). Overall, our findings reveal that pE301R acts as a negatively regulator to inhibit IFN-I production and to subvert host antiviral innate immunity during ASFV infection.

QNE1 is a key flowering regulator determining the length of the vegetative period in soybean cultivars.

The soybean E1 gene is a major regulator that plays an important role in flowering time and maturity. However, it remains unclear how cultivars carrying the dominant E1 allele adapt to the higher latitudinal areas of northern China. We mapped the novel quantitative trait locus QNE1 (QTL near E1) for flowering time to the region proximal to E1 on chromosome 6 in two mapping populations. Positional cloning revealed Glyma.06G204300, encoding a TCP-type transcription factor, as a strong candidate gene for QNE1. Association analysis further confirmed that functional single nucleotide polymorphisms (SNPs) at nucleotides 686 and 1,063 in the coding region of Glyma.06G204300 were significantly associated with flowering time. The protein encoded by the candidate gene is localized primarily to the nucleus. Furthermore, soybean and Brassica napus plants overexpressing Glyma.06G204300 exhibited early flowering. We conclude that despite their similar effects on flowering time, QNE1 and E4 may control flowering time through different regulatory mechanisms, based on expression studies and weighted gene co-expression network analysis of flowering time-related genes. Deciphering the molecular basis of QNE1 control of flowering time enriches our knowledge of flowering gene networks in soybean and will facilitate breeding soybean cultivars with broader latitudinal adaptation.

Cationic P,O-Coordinated Nickel(II) Catalysts for Carbonylative Polymerization of Ethylene: Unexpected Productivity via Subtle Electronic Variation.

Transition-metal-catalyzed copolymerization of ethylene with carbon monoxide affords polyketones materials with excellent mechanical strength, photodegradability, surface and barrier properties. Unlike the widely used and rather expensive Pd catalysts, Ni-catalyzed carbonylative polymerization is very difficult since the strong binding affinity of CO to Ni deactivates the highly electrophilic metal center easily. In this study, various cationic P,O-coordinated Ni complexes were synthesized using the electronic modulation strategy, and the catalyst with strong electron-donating substituents exhibits an excellent productivity of 104  g polymer (g Ni)-1 , which represents a rare discovery that a Ni complex could operate with such exceptional efficiency in comparison with Pd catalysts. Notably, those Ni catalysts were also efficient for terpolymerization of ethylene, propylene with CO for producing commercial polyketone materials with low melting temperatures and easy processibility.

Improvement of Lightweight Convolutional Neural Network Model Based on YOLO Algorithm and Its Research in Pavement Defect Detection.

To ensure the safe operation of highway traffic lines, given the imperfect feature extraction of existing road pit defect detection models and the practicability of detection equipment, this paper proposes a lightweight target detection algorithm with enhanced feature extraction based on the YOLO (You Only Look Once) algorithm. The BIFPN (Bidirectional Feature Pyramid Network) network structure is used for multi-scale feature fusion to enhance the feature extraction ability, and Varifocal Loss is used to optimize the sample imbalance problem, which improves the accuracy of road defect target detection. In the evaluation test of the model in the constructed PCD1 (Pavement Check Dataset) dataset, the mAP@.5 (mean Average Precision when IoU = 0.5) of the BV-YOLOv5S (BiFPN Varifocal Loss-YOLOv5S) model increased by 4.1%, 3%, and 0.9%, respectively, compared with the YOLOv3-tiny, YOLOv5S, and B-YOLOv5S (BiFPN-YOLOv5S; BV-YOLOv5S does not use the Improved Focal Loss function) models. Through the analysis and comparison of experimental results, it is proved that the proposed BV-YOLOv5S network model performs better and is more reliable in the detection of pavement defects and can meet the needs of road safety detection projects with high real-time and flexibility requirements.

GmMDE genes bridge the maturity gene E1 and florigens in photoperiodic regulation of flowering in soybean.

Soybean (Glycine max) is highly sensitive to photoperiod, which affects flowering time and plant architecture and thus limits the distribution range of elite soybean cultivars. The major maturity gene E1 confers the most prominent effect on photoperiod sensitivity, but its downstream signaling pathway remains largely unknown. Here, we confirm that the encoded E1 protein is a transcriptional repressor. The expression of seven GmMDE genes (Glycine max MADS-box genes downregulated by E1) was suppressed when E1 was overexpressed and promoted when E1 was knocked out through clustered regularly-interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9)-mediated mutagenesis. These GmMDEs exhibited similar tissue specificity and expression patterns, including in response to photoperiod, E1 expression, and E1 genotype. E1 repressed GmMDE promoter activity. Results for two GmMDEs showed that E1 epigenetically silences their expression by directly binding to their promoters to increase H3K27me3 levels. The overexpression of GmMDE06 promoted flowering and post-flowering termination of stem growth. The late flowering phenotype of E1-overexpressing soybean lines was reversed by the overexpression of GmMDE06, placing GmMDE06 downstream of E1. The overexpression of GmMDE06 increased the expression of the soybean FLOWERING LOCUS T orthologs GmFT2a and GmFT5a, leading to feedback upregulation of GmMDE, indicating that GmMDE and GmFT2a/GmFT5a form a positive regulatory feedback loop promoting flowering. GmMDE06 also promoted post-flowering termination of stem growth by repressing the expression of the shoot identity gene Dt1. The E1-GmMDEs-GmFT2a/5a-Dt1 signaling pathway illustrates how soybean responds to photoperiod by modulating flowering time and post-flowering stem termination.

A Hybrid Deep Learning Model for Recognizing Actions of Distracted Drivers.

With the rapid spreading of in-vehicle information systems such as smartphones, navigation systems, and radios, the number of traffic accidents caused by driver distractions shows an increasing trend. Timely identification and warning are deemed to be crucial for distracted driving and the establishment of driver assistance systems is of great value. However, almost all research on the recognition of the driver's distracted actions using computer vision methods neglected the importance of temporal information for action recognition. This paper proposes a hybrid deep learning model for recognizing the actions of distracted drivers. Specifically, we used OpenPose to obtain skeleton information of the human body and then constructed the vector angle and modulus ratio of the human body structure as features to describe the driver's actions, thereby realizing the fusion of deep network features and artificial features, which improve the information density of spatial features. The K-means clustering algorithm was used to preselect the original frames, and the method of inter-frame comparison was used to obtain the final keyframe sequence by comparing the Euclidean distance between manually constructed vectors representing frames and the vector representing the cluster center. Finally, we constructed a two-layer long short-term memory neural network to obtain more effective spatiotemporal features, and one softmax layer to identify the distracted driver's action. The experimental results based on the collected dataset prove the effectiveness of this framework, and it can provide a theoretical basis for the establishment of vehicle distraction warning systems.

Smyd1 is essential for myosin expression and sarcomere organization in craniofacial, extraocular, and cardiac muscles.

Skeletal and cardiac muscles are striated myofibers that contain highly organized sarcomeres for muscle contraction. Recent studies revealed that Smyd1, a lysine methyltransferase, plays a key role in sarcomere assembly in heart and trunk skeletal muscles. However, Smyd1 expression and function in craniofacial muscles are not known. Here, we analyze the developmental expression and function of two smyd1 paralogous genes, smyd1a and smyd1b, in craniofacial and cardiac muscles of zebrafish embryos. Our data show that loss of smyd1a (smyd1amb5) or smyd1b (smyd1bsa15678) has no visible effects on myogenic commitment and expression of myod and myosin heavy-chain mRNA transcripts in craniofacial muscles. However, myosin heavy-chain protein accumulation and sarcomere organization are dramatically reduced in smyd1bsa15678 single mutant, and almost completely diminish in smyd1amb5; smyd1bsa15678 double mutant, but not in smyd1amb5 mutant. Similar defects are also observed in cardiac muscles of smyd1bsa15678 mutant. Defective craniofacial and cardiac muscle formation is associated with an upregulation of hsp90α1 and unc45b mRNA expression in smyd1bsa15678 and smyd1amb5; smyd1bsa15678 mutants. Together, our studies indicate that Smyd1b, but not Smyd1a, plays a key role in myosin heavy-chain protein expression and sarcomere organization in craniofacial and cardiac muscles. Loss of smyd1b results in muscle-specific stress response.

Multilabel Image Classification Based Fresh Concrete Mix Proportion Monitoring Using Improved Convolutional Neural Network.

Proper and accurate mix proportion is deemed to be crucial for the concrete in service to implement its structural functions in a specific environment and structure. Neither existing testing methods nor previous studies have, to date, addressed the problem of real-time and full-scale monitoring of fresh concrete mix proportion during manufacturing. Green manufacturing and safety construction are hindered by such defects. In this study, a state-of-the-art method based on improved convolutional neural network multilabel image classification is presented for mix proportion monitoring. Elaborately planned, uniformly distributed, widely covered and high-quality images of concrete mixtures were collected as dataset during experiments. Four convolutional neural networks were improved or fine-tuned based on two solutions for multilabel image classification problems, since original networks are tailored for single-label multiclassification tasks, but mix proportions are determined by multiple parameters. Various metrices for effectiveness evaluation of training and testing all indicated that four improved network models showed outstanding learning and generalization ability during training and testing. The best-performing one was embedded into executable application and equipped with hardware facilities to establish fresh concrete mix proportion monitoring system. Such system was deployed to terminals and united with mechanical and weighing sensors to establish integrated intelligent sensing system. Fresh concrete mix proportion real-time and full-scale monitoring and inaccurate mix proportion sensing and warning could be achieved simply by taking pictures and feeding pictures into such sensing system instead of conducting experiments in laboratory after specimen retention.

Phosphatase AtDBP1 negatively regulates drought and salt tolerance through altering leaf surface permeability in Arabidopsis.

In our previous study, AtDBP1 encoding a DBP factor was identified as a putative abiotic stress candidate gene. DBP factors are important regulators that participate in both transcriptional regulation and post-translational regulation, but their roles in abiotic stress are still not well-understood. So we conducted a detailed study on the function of AtDBP1 in abiotic stress. It is found that expression of AtDBP1 could be induced by drought and salt, and the induction by salt was inhibited in ABA-deficient mutant aba2-3, indicating the expression of AtDBP1 was ABA-inducible. Overexpression of AtDBP1 resulted in a rapid stomatal closure, and elevated expression of drought/salt-responsive genes, which should help Arabidopsis to enhance the drought and salt tolerance. Unexpectedly, overexpression of AtDBP1 decreased the drought and salt tolerance of Arabidopsis. Further analysis suggested that AtDBP1 is involved in cuticle wax and cuticle membrane regulation. Overexpression of AtDBP1 showed increased cuticular conductance due to a decreased cuticle wax accumulation and cuticle membrane thickness. The cuticular wax provides an essential barrier for decreasing nonstomatal water loss during drought stress, so overexpression of AtDBP1 showed decreased drought tolerance possibly ascribed to the change of cuticle membrane structure. Our previous study elucidated that AtDBP1 was also involved in flowering time regulation. Taken together, the results above indicated that AtDBP1 was involved in both plant development and stress regulation. The mechanism of AtDBP1 in this study indicates that genes involved in both plant development and stress regulation might be not suitable for production application in breeding. Collectively, our results provide some new ideas on purposefully increasing the abiotic stress without influence on plant growth and development.

Physiological responses to cold and starvation stresses in the liver of yellow drum (Nibea albiflora) revealed by LC-MS metabolomics.

As global climate changes, mass mortality in farmed fish associated with the severely cold weather has aroused growing concerns. Yellow drum (Nibea albiflora) is an important maricultured fish in China, whereby its aquaculture suffered from overwinter mortality associated with cold and cold-induced-fasting stresses. Here, by using LC-MS metabolomics combined with transcriptomics, we investigated the physiological responses of yellow drum liver to cold and starvation stresses. The experiment involved four groups: 16 °C fed group (CG1), 16 °C unfed group (CG2), 8 °C fed group (EG1), and 8 °C unfed group (EG2). Under cold stress, a total of 308 and 257 differential metabolites were identified in EG1 vs. CG1 and EG2 vs. CG2, respectively, showing 5 overlapping significant pathways: glutathione metabolism, biosynthesis of unsaturated fatty acids, galactose metabolism, arginine and proline metabolism, and ABC transporters. Intersection analysis identified that glutamate, oxidized glutathione (GSSG), and eicosenoic acid were the common metabolites induced by cold stress. Under starvation stress, a total of 300 and 215 differential metabolites were identified in CG2 vs. CG1 and EG2 vs. EG1, respectively, showing 2 overlapping significant pathways: glutathione metabolism and galactose metabolism. Intersection analysis revealed that glutamate and GSSG were the common metabolites caused by fasting. Under cold and starvation combined stresses, 286 differential metabolites were identified in EG2 vs. CG1, showing 7 influenced pathways: glycerophospholipid metabolism, biosynthesis of unsaturated fatty acids, glutathione metabolism, sphingolipid metabolism, glycosylphosphatidylinositol (GPI)-anchor biosynthesis, autophagy, and purine metabolism. Interestingly, the glutamate and GSSG were induced by both single and combined stresses of cold and starvation treatments. These findings suggest that glutathione metabolism and its related metabolites (glutamate and GSSG) could be potential biomarkers of cold and starvation stresses in yellow drum. Overall, the results of this study provided insights into the physiological regulation in response to cold and starvation stresses in this fish.

Relationship between polymorphisms in -572G/C interleukin 6 promoter gene polymorphisms (rs1800796) and risk of rheumatoid arthritis: A meta-analysis.

AIMS: This meta-analysis was aimed to investigate the association between -572G/C interleukin (IL)-6 gene polymorphism and occurrence risk of rheumatoid arthritis (RA). METHODS: Literature search was conducted in PubMed, Embase, Springer and Google Scholar up to November 2018. The pooled odds ratios (ORs) and 95% confidence interval (CI) were calculated by Revman 5.3. RESULTS: A total of six case-control studies were included in this meta-analysis. In the allele model (G vs C), homozygous gene model (GG vs CC), recessive gene model (GG vs GC + CC), and dominant gene model (GG + GC vs CC), the pooled estimate indicated there was significant association between -572G/C IL-6 gene polymorphism and risk of RA. However, no significant statistical results were found in meta-analyses of heterozygote gene models. CONCLUSIONS: The -572G/C IL-6 gene polymorphism is associated with the risk of RA. The GG genotype may be the main contributor in increasing susceptibility to RA.

Roles of forkhead box protein L2 (foxl2) during gonad differentiation and maintenance in a fish, the olive flounder (Paralichthys olivaceus).

As an important maricultured fish, the olive flounder Paralichthys olivaceus shows sex-dimorphic growth. Thus, the molecular mechanisms involved in sex control in P. olivaceus have attracted researchers' attention. Among the sex-related genes, forkhead box protein L2 (foxl2) exhibits significant sex-dimorphic expression patterns and plays an important role in fish gonad differentiation and development. The present study first investigated the expression levels and promoter methylation dynamics of foxl2 during flounder gonad differentiation under treatments of high temperature and exogenous 17ß-oestradiol (E2). During high temperature treatment, the expression of flounder foxl2 may be repressed via maintenance of DNA methylation. Then, flounder with differentiated testis at Stages I-II were treated with exogenous 5ppm E2 or 5ppm E2+150ppm trilostane (TR) to investigate whether exogenous sex hormones could induce flounder sex reversal. The differentiated testis exhibited phenotypic variations of gonadal dysgenesis with upregulation of female-related genes (foxl2 and cytochrome P450 family 19 subfamily A (cyp19a)) and downregulation of male-related genes (cytochrome P450 family 11 subfamily B member 2 (cyp11b2), doublesex- and mab-3 related transcription factor 1 (dmrt1), anti-Mullerian hormone (amh) and SRY-box transcription factor 9 (sox9)). Furthermore, a cotransfection assay of the cells of the flounder Sertoli cell line indicated that Foxl2 was able alone or with nuclear receptor subfamily 5 group A member 2 (Nr5a2) jointly to upregulate expression of cyp19a. Moreover, Foxl2 and Nr5a2 repressed the expression of dmrt1. In summary, Foxl2 may play an important role in ovarian differentiation by maintaining cyp19a expression and antagonising the expression of dmrt1. However, upregulation of foxl2 is not sufficient to induce the sex reversal of differentiated testis.

Characterization and expression of androgen receptors in olive flounder.

Androgens are critical hormones that regulate sex differentiation, sexual maturation, and spermatogenesis in vertebrates, which is mainly mediated by androgen receptors (ARs). Reports on transcript variants of ar (AR encoding gene) in human are almost always associated with cancers and androgen insensitivity syndrome. However, the knowledge of ar variants in teleosts is scarce. In this study, arß and two transcript variants of arα (arα1 and arα2) in olive flounder (Paralichthys olivaceus) were cloned and analyzed. Their expression patterns were investigated in 16 adult female and male tissues by RT-PCR, respectively. arα1 was expressed in the majority of tissues excluding male liver, medulla oblongata and female cerebellum, with higher levels in male gonad, kidney, head kidney, intestine, stomach, spleen, heart and gill than in female. arα2 had similar expression patterns as arα1, with lower levels in general. arß was also widely expressed in various tissues excluding male spleen, female spleen and gill, with higher levels in male gonad, kidney, head kidney, intestine and lower levels in hypothalamus than in female. Compared with arß, much lower expression levels of arα1 and arα2 were detected in different brain areas. The real-time quantitative PCR (qPCR) results showed that the total arα expression level was relatively higher during olive flounder gonadal differentiation and before the onset of testis differentiation, whereas arß was expressed significantly higher during male gonadal differentiation period than female gonadal differentiation period. The in vitro transient transfection assays showed that ARα1, ARα2 and ARß could all suppress the activity of cyp19a (p450arom aromatase gene) promoter, and the inhibitory effect of ARα1 was dose dependent. Our results imply that arα1, arα2 and arß are sex-related genes and they might play important roles in gonadal differentiation in flounder.

Muscle fibre type composition in the lateral muscle of olive flounder Paralichthys olivaceus.

In this paper, a combined-method study has been made on the lateral muscle of the teleost olive flounder Paralichthys olivaceus in just-hatched and adult stages. In just-hatched stage, both slow and fast muscle fibres were detected: (1) in situ hybridization analysis indicated that slow and fast myosin heavy chain genes were specifically expressed in the superficial and deep part of the myotomal muscle, respectively; (2) immunohistochemistry analysis showed that fibres in the deep part reacted with anti-fast myosin antibody F310; (3) western blot analysis detected a weak expression of slow myosin and a strong expression of fast myosin. In adult stage, the slow and fast muscle fibres had their own distribution characteristics: (1) hematoxylin/eosin staining showed the histological characteristics of the muscle fibre composition; (2) histochemical observations showed that the deep muscle fibres, and some fibres near the epidermis, contain alkali-stable myofibrillar ATPase activity; (3) immunohistochemistry analysis indicated that all the deep muscle fibres reacted with F310 antibody and some fibres in the superficial layer of muscle also reacted with F310; (4) western blot analysis showed that fast myosin was expressed both in the blended muscles (the mix of superficial and deep muscles) and deep muscles, while slow myosin was mainly expressed in the blended muscles. These findings suggested that both slow and fast muscle fibres existed in the musculature of the olive flounder in just-hatched and adult stages. Notably, the adult fast fibres also exist in the superficial layer of the muscle.

Characteristics of Cyp11a during Gonad Differentiation of the Olive Flounder Paralichthys olivaceus.

The P450 side-chain cleavage enzyme, P450scc (Cyp11a) catalyzes the first enzymatic step for the synthesis of all steroid hormones in fish. To study its roles in gonads of the olive flounder Paralichthys olivaceus, an important maricultured fish species, we isolated the cyp11a genomic DNA sequence of 1396 bp, which consists of 5 exons and 4 introns. Semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) results indicated that the flounder cyp11a was exclusively expressed in gonad and head kidney tissues. Its expression level in the testis was higher than that in the ovary. According to the in situ hybridization patterns, cyp11a was mainly expressed in the Leydig cells of the testis, and the thecal cells of the ovary. Immunofluorescence analysis showed that Cyp11a was located in the cytoplasm of the cultured flounder testis cells. Further quantitative real-time PCR results presented the cyp11a differential expression patterns during gonad differentiation. Among different sampling points of the 17ß-estradiol (E2, 5 ppm) treatment group, cyp11a expression levels were relatively high in the differentiating ovary (30 and 40 mm total length, TL), and then significantly decreased in the differentiated ovary (80, 100 and 120 mm TL, p < 0.05). The pregnenolone level also dropped in the differentiated ovary. In the high temperature treatment group (HT group, 28 ± 0.5 °C), the cyp11a expression level fluctuated remarkably in the differentiating testis (60 mm TL), and then decreased in the differentiated testis (80, 100 mm TL, p < 0.05). In the testosterone (T, 5 ppm) treatment group, the cyp11a was expressed highly in undifferentiated gonads and the differentiating testis, and then dropped in the differentiated testis. Moreover, the levels of cholesterol and pregnenolone of the differentiating testis in the HT and T groups increased. The expression level of cyp11a was significantly down-regulated after the cultured flounder testis cells were treated with 75 and 150 µM cyclic adenosine monophosphate (cAMP), respectively (p < 0.05), and significantly up-regulated after treatment with 300 µM cAMP (p < 0.05). Both nuclear receptors NR5a2 and NR0b1 could significantly up-regulate the cyp11a gene expression in a dosage dependent way in the testis cells detected by cell transfection analysis (p < 0.05). The above data provides evidence that cyp11a would be involved in the flounder gonad differentiation and development.