Efficient Reinforcement Learning With the Novel N-Step Method and V-Network.

The application of reinforcement learning (RL) in artificial intelligence has become increasingly widespread. However, its drawbacks are also apparent, as it requires a large number of samples for support, making the enhancement of sample efficiency a research focus. To address this issue, we propose a novel N -step method. This method extends the horizon of the agent, enabling it to acquire more long-term effective information, thus resolving the issue of data inefficiency in RL. Additionally, this N -step method can reduce the estimation variance of Q -function, which is one of the factors contributing to estimation errors in Q -function estimation. Apart from high variance, estimation bias in Q -function estimation is another factor leading to estimation errors. To mitigate the estimation bias of Q -function, we design a regularization method based on the V-function, which has been underexplored. The combination of these two methods perfectly addresses the problems of low sample efficiency and inaccurate Q -function estimation in RL. Finally, extensive experiments conducted in discrete and continuous action spaces demonstrate that the proposed novel N -step method, when combined with classical deep Q -network, deep deterministic policy gradient, and TD3 algorithms, is effective, consistently outperforming the classical algorithms.

Mediating effect of psychological capital on the relationship between mental health literacy and coping styles among newly recruited nurses.

BACKGROUND: Newly recruited nurses face multiple sources of stress and their coping styles need to be focused on to ensure good mental health. This study aimed to examine the relationship among mental health literacy, psychological capital and coping styles in newly recruited nurses. METHODS: A cross-sectional study was conducted in August and September 2022. A total of 315 newly recruited nurses were recruited in a tertiary hospital in Henan Province, central China, employing the convenience sampling method. The self-reported questionnaires were sent through a QR code, including the Mental Health Literacy Scale for Healthcare Students, Psychological Capital Questionnaire, and Simplified Coping Style Questionnaire. Pearson correlation analysis was used to evaluate the relationships among the variables. Mediation analysis was performed to identify the mediating effect of psychological capital on the relationship between mental health literacy and coping styles. RESULTS: Positive coping showed a positive relationship with psychological capital and mental health literacy, while negative coping showed a negative relationship with psychological capital and mental health literacy. For positive coping, psychological capital was a partial mediator with an effect of 0.140, accounting for 62.8%. For negative coping, a full mediating effect was shown by psychological capital between mental health literacy and negative coping, with an indirect effect of -0.048. CONCLUSION: Psychological capital plays a partial and complete mediating role between mental health literacy and different coping styles among newly recruited nurses. Diversified training and personalized guidance in improving mental health literacy and increasing psychological capital simultaneously can be provided to newly recruited nurses continuously to adjust their coping styles.

Graphical modeling of causal factors associated with the postoperative survival of esophageal cancer subjects.

PURPOSE: To clarify the causal relationship between factors contributing to the postoperative survival of patients with esophageal cancer. METHODS: A cohort of 195 patients who underwent surgery for esophageal cancer between 2008 and 2021 was used in the study. All patients had preoperative chest computed tomography (CT) and positron emission tomography-CT (PET-CT) scans prior to receiving any treatment. From these images, high throughput and quantitative radiomic features, tumor features, and various body composition features were automatically extracted. Causal relationships among these image features, patient demographics, and other clinicopathological variables were analyzed and visualized using a novel score-based directed graph called "Grouped Greedy Equivalence Search" (GGES) while taking prior knowledge into consideration. After supplementing and screening the causal variables, the intervention do-calculus adjustment (IDA) scores were calculated to determine the degree of impact of each variable on survival. Based on this IDA score, a GGES prediction formula was generated. Ten-fold cross-validation was used to assess the performance of the models. The prediction results were evaluated using the R-Squared Score (R2 score). RESULTS: The final causal graphical model was formed by two PET-based image variables, ten body composition variables, four pathological variables, four demographic variables, two tumor variables, and one radiological variable (Percentile 10). Intramuscular fat mass was found to have the most impact on overall survival month. Percentile 10 and overall TNM (T: tumor, N: nodes, M: metastasis) stage were identified as direct causes of overall survival (month). The GGES casual model outperformed GES in regression prediction (R2  = 0.251) (p < 0.05) and was able to avoid unreasonable causality that may contradict common sense. CONCLUSION: The GGES causal model can provide a reliable and straightforward representation of the intricate causal relationships among the variables that impact the postoperative survival of patients with esophageal cancer.

XRayWizard: Reconstructing 3-D lung surfaces from a single 2-D chest x-ray image via Vision Transformer.

BACKGROUND: Chest x-ray is widely utilized for the evaluation of pulmonary conditions due to its technical simplicity, cost-effectiveness, and portability. However, as a two-dimensional (2-D) imaging modality, chest x-ray images depict limited anatomical details and are challenging to interpret. PURPOSE: To validate the feasibility of reconstructing three-dimensional (3-D) lungs from a single 2-D chest x-ray image via Vision Transformer (ViT). METHODS: We created a cohort of 2525 paired chest x-ray images (scout images) and computed tomography (CT) acquired on different subjects and we randomly partitioned them as follows: (1) 1800 - training set, (2) 200 - validation set, and (3) 525 - testing set. The 3-D lung volumes segmented from the chest CT scans were used as the ground truth for supervised learning. We developed a novel model termed XRayWizard that employed ViT blocks to encode the 2-D chest x-ray image. The aim is to capture global information and establish long-range relationships, thereby improving the performance of 3-D reconstruction. Additionally, a pooling layer at the end of each transformer block was introduced to extract feature information. To produce smoother and more realistic 3-D models, a set of patch discriminators was incorporated. We also devised a novel method to incorporate subject demographics as an auxiliary input to further improve the accuracy of 3-D lung reconstruction. Dice coefficient and mean volume error were used as performance metrics as the agreement between the computerized results and the ground truth. RESULTS: In the absence of subject demographics, the mean Dice coefficient for the generated 3-D lung volumes achieved a value of 0.738 ± 0.091. When subject demographics were included as an auxiliary input, the mean Dice coefficient significantly improved to 0.769 ± 0.089 (p < 0.001), and the volume prediction error was reduced from 23.5 ± 2.7%. to 15.7 ± 2.9%. CONCLUSION: Our experiment demonstrated the feasibility of reconstructing 3-D lung volumes from 2-D chest x-ray images, and the inclusion of subject demographics as additional inputs can significantly improve the accuracy of 3-D lung volume reconstruction.

Batch-balanced focal loss: a hybrid solution to class imbalance in deep learning.

Purpose: To validate the effectiveness of an approach called batch-balanced focal loss (BBFL) in enhancing convolutional neural network (CNN) classification performance on imbalanced datasets. Materials and Methods: BBFL combines two strategies to tackle class imbalance: (1) batch-balancing to equalize model learning of class samples and (2) focal loss to add hard-sample importance to the learning gradient. BBFL was validated on two imbalanced fundus image datasets: a binary retinal nerve fiber layer defect (RNFLD) dataset (n=7,258) and a multiclass glaucoma dataset (n=7,873). BBFL was compared to several imbalanced learning techniques, including random oversampling (ROS), cost-sensitive learning, and thresholding, based on three state-of-the-art CNNs. Accuracy, F1-score, and the area under the receiver operator characteristic curve (AUC) were used as the performance metrics for binary classification. Mean accuracy and mean F1-score were used for multiclass classification. Confusion matrices, t-distributed neighbor embedding plots, and GradCAM were used for the visual assessment of performance. Results: In binary classification of RNFLD, BBFL with InceptionV3 (93.0% accuracy, 84.7% F1, 0.971 AUC) outperformed ROS (92.6% accuracy, 83.7% F1, 0.964 AUC), cost-sensitive learning (92.5% accuracy, 83.8% F1, 0.962 AUC), and thresholding (91.9% accuracy, 83.0% F1, 0.962 AUC) and others. In multiclass classification of glaucoma, BBFL with MobileNetV2 (79.7% accuracy, 69.6% average F1 score) outperformed ROS (76.8% accuracy, 64.7% F1), cost-sensitive learning (78.3% accuracy, 67.8.8% F1), and random undersampling (76.5% accuracy, 66.5% F1). Conclusion: The BBFL-based learning method can improve the performance of a CNN model in both binary and multiclass disease classification when the data are imbalanced.

Pregnancy-related low back/pelvic girdle pain: Prevalence, severity, and risk factors in Zhengzhou, China.

BACKGROUND: Pregnancy-related low back pain (LBP) and pelvic girdle pain (PGP) significantly affect the quality of life of pregnant women. Understanding their severity and risk factors may help prevent and alleviate such pain and their resulting dysfunction. OBJECTIVE: This study investigated the prevalence, severity, and biopsychosocial risk factors of pregnancy-related LBP and PGP in Zhengzhou, China. METHODS: The Numeric Pain Rating Scale (NPRS), Chinese version of the Roland-Morris Disability Questionnaire (RMDQ), and other questionnaires were self-administered by 1020 pregnant women undergoing treatment at a tertiary hospital between July and December 2019. Binary logistic regression was used to identify factors associated with pregnancy-related LBP and/or PGP. RESULTS: The prevalence of LBP and/or PGP during pregnancy was 63.0%, and most participants (80.4%) had both. The mean NPRS and RMDQ disability scores were 2.44 ± 2.10 and 6.66 ± 4.65, respectively. A higher gestational body mass index, LBP and/or PGP during menstruation, history of pregnancy-related LBP and/or PGP, and constant pressure were significantly associated with pregnancy-related LBP and/or PGP. Subjective support was a protective factor against LBP and/or PGP. CONCLUSION: The prevalence of LBP and/or PGP was high. The risk factors should be included in routine prenatal care to identify patients at risk of LBP and/or PGP.

Thyroid Hormone Signaling Is Required for Dynamic Variation in Opsins in the Retina during Metamorphosis of the Japanese Flounder (Paralichthys olivaceus).

In the present study, we investigated the function of thyroid hormones (TH) in visual remodeling during Japanese flounder (Paralichthys olivaceus) metamorphosis through cellular molecular biology experiments. Our results showed that the expression of the five opsin genes of the flounder were highest in eye tissue and varied with the metamorphosis process. The expression of rh1, sws2aß and lws was positively regulated by exogenous TH, but inhibited by thiourea (TU) compared to the control group. In addition, there was a significant increase in sws2aß and lws in the rescue experiments performed with TU-treated larvae (p < 0.05). Meanwhile, T3 levels in flounder larvae were increased by TH and decreased by TU. Based on the differences in the expression of the three isoforms of the thyroid hormone receptor (TR) (Trαa, Trαb and Trß), we further hypothesized that T3 may directly or indirectly regulate the expression of sws2aß through Trαa. This study demonstrates the regulatory role of TH in opsins during flounder metamorphosis and provides a basis for further investigation on the molecular mechanisms underlying the development of the retinal photoreceptor system in flounders.

Moderating effect of organizational climate on the relationship between research motivation and learning engagement among nurses taking part-time master's program: A cross-sectional study.

AIMS: To investigate the moderating effects of perceived organizational climate on the relationship between research motivation and learning engagement in research among nurses pursuing a part-time master's degree. BACKGROUND: Research motivation positively affects learning engagement in research. However, the role of perceived organizational climate has not been explored in nurses taking part-time master's program in China. This study examined the relationships between various types of research motivation and learning engagement in research and whether the perceived organizational climate moderates the relationship between the other variables. METHODS: This cross-sectional one-center study was performed on 230 nurses by assessing learning engagement in research, research motivation and perceived organizational climate. RESULTS: The mean age of participants was 31.3 ± 3.5 years and 91.3% were female. Participants who had been assigned a supervisor showed higher learning engagement in research than those without a supervisor (3.65 ± 0.60 vs. 3.48 ± 0.61; P < 0.001). Participants who had experience conducting research reported higher learning engagement in research than those with no experience (3.30 ± 0.71 vs. 3.14 ± 0.83; P < 0.05). The mean score of learning engagement in research was 3.39 ± 0.63. The mean scores for intrinsic, extrinsic and failure-avoidance motivation were 3.72 ± 0.61, 3.71 ± 0.71 and 3.43 ± 0.70, respectively. As for perceived organizational climate, the mean score was 122.70 out of 148. Learning engagement in research was significantly positively correlated with intrinsic research motivation, extrinsic research motivation and perceived organizational climate scores (r = 0.441, 0.336, 0.307, p < 0.001, respectively). Perceived organizational climate moderated the correlation between intrinsic and extrinsic research motivation and learning engagement in research. CONCLUSION: Nurses taking the master's program perceived a higher level of perceived organizational climate could strengthen the relationship between intrinsic and extrinsic research motivation and learning engagement in research. More organizational support can enhance learning engagement in research by reinforcing intrinsic and extrinsic research motivation. IMPLICATIONS FOR NURSING MANAGEMENT: Universities can assign supervisors promptly. Universities and hospitals can provide education about the importance of research in nursing and reinforce their intrinsic and extrinsic research motivation. Hospitals can adjust policies based on the needs of nurses taking the master's program to provide organizational support, to facilitate the research learning process.

Strolling in Room-Scale VR: Hex-Core-MK1 Omnidirectional Treadmill.

The natural locomotion interface is critical to the development of many VR applications. For household VR applications, there are two basic requirements: natural immersive experience and minimized space occupation. The existing locomotion strategies generally do not simultaneously satisfy these two requirements well. This article presents a novel omnidirectional treadmill (ODT) system named Hex-Core-MK1 (HCMK1). By implementing two kinds of mirror-symmetrical spiral rollers to generate the omnidirectional velocity field, this proposed system is capable of providing real walking experiences with a full-degree of freedom in an area as small as 1.76 m 2, while delivering great advantages over several existing ODT systems in terms of weight, volume, latency and dynamic performance. Compared with the sizes of Infinadeck and HCP, the two best motor-driven ODTs so far, the 8 cm height of HCMK1 is only 20% of Infinadeck and 50% of HCP. In addition, HCMK1 is a lightweight device weighing only 110 kg, which provides possibilities for further expanding VR scenarios, such as terrain simulation. The system latency of HCMK1 is only 9ms. The experiments show that HCMK1 can deliver a starting acceleration of 16.00 m/s 2 and a braking acceleration of 30.00 m/s 2.

Integrative cytological analysis of the effects of Ca2+ and vitamin D3 on extracellular Ca2+ flux and intracellular Ca2+ reserves in the mantle of the pearl oyster (Hyriopsis cumingii Lea).

To examine Ca2+ absorption and transportation in the freshwater pearl oyster, Hyriopsis cumingii Lea, we studied the effects of different levels of either extracellular Ca2+ or 1,25(OH)2D3 on extracellular Ca2+ flux and intracellular Ca2+ concentrations in mantle cells using the non-invasive micro-test technique and laser scanning confocal microscopy. The inner and outer mantle (IM and OM) cells from mussels were cultured and then treated with different concentrations of Ca2+ and 1,25(OH)2D3. Extracellular Ca2+ flux and intracellular Ca2+ reserves were analyzed. The results showed that both extracellular Ca2+ and 1,25(OH)2D3 had significant effects on Ca2+ flux and reserves in mantle cells, especially in IM cells (P < .05). The increase in extracellular Ca2+ concentrations resulted in the conversion of extracellular Ca2+ flux into influx with an increase in flow rate (P < .05). The calcium ion fluorescence intensity of OM cells was higher than that of IM cells (P < .05). 1,25(OH)2D3 addition also significantly increased the influx rate of extracellular Ca2+, especially in IM cells, which were more sensitive to 1,25(OH)2D3 addition and had significantly higher Ca2+ influx rates than did OM cells (P < .05). Fluorescence intensities of intracellular Ca2+ first increased and then decreased with increasing 1,25(OH)2D3 levels. The study showed that IM cells play an important role in absorbing Ca2+ from the environment, while OM cells mainly function in the temporary storage and transportation of Ca2+ in the body. The current results suggested that high levels of extracellular Ca2+ (1.25 mM) or 1,25(OH)2D3 (over 100 IU/L) were favorable for Ca2+ uptake and maintenance in the body.

Proteomic variation in metamorphosing Paralichthys olivaceus induced by exogenous thyroid hormone.

Thyroid hormone (TH) is essential for Paralichthys olivaceus metamorphosis. Exogenous TH treatment induces premature metamorphosis in P. olivaceus larvae and a series of studies have been conducted to identify thyroid hormone-regulated functional genes and microRNAs involved in the metamorphosis of P. olivaceus; however, the proteins involved in this process remain to be fully clarified. In this study, the differential proteomic responses of P. olivaceus larvae to exogenous TH treatment were examined using tandem mass tags (TMT) for quantitation labeling followed by liquid chromatography tandem mass spectrometry (LC-MS/MS). The expression levels of 629 cellular proteins were identified to be significantly affected by TH treatment. The reliability of our TMT-labeled LC-MS/MS analysis was verified by examining the mRNA and protein levels of four selected proteins using quantitative real-time reverse-transcription PCR and western blot analyses. The possible biological significance of these proteins was further investigated by Gene Ontology (GO) enrichment, Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment, and protein-protein interaction analyses. Notably, we identified and described five groups of proteins involved in different important life events that were significantly regulated by exogenous TH treatment. Our study provides an improved understanding of the molecular mechanisms by which TH regulates the metamorphosis of P. olivaceus.

Characterization and expression of lin-28a involved in lin28/let-7signal pathway during early development of P. olivaceus.

Heterochronic lin-28 is a conserved RNA-binding protein that plays a key role in the timing of developmental events in organisms. As a crucial heterochronic gene, the protein controls developmental events of the second of four larval stages in Caenorhabditi elegans. Heterochronic let-7 miRNAs are often present in various species and highly conserved in sequence and biological function and are required for various biological processes. Previous studies showed that ten let-7 miRNAs were identified in the Japanese flounder (Paralichthys olivaceus) and that they were primarily expressed during metamorphosis. In this study, we clone and characterize the lin-28a gene from P. olivaceus and exhibit its dynamic expression pattern at different developmental stages and various adult tissues. The results show that the P. olivaceus lin-28a gene has high sequence similarity with other species and is highly expressed in the embryonic stage but weakly expressed in the larval stage. In addition, lin-28a overexpression causes cell proliferation and significantly promotes the levels of pre-let-7a and pre-let-7d while markedly depressing let-7a and let-7d expression in FEC (Flounder Embryonic Cell), which indicate that lin-28 possibly blocks the maturation of let-7 miRNAs. Additionally, lin-28a is identified as a target gene of let-7 miRNAs, and let-7 miRNAs directly regulate lin-28a expression by targeting its 3' UTR. Taken together, lin-28a along with let-7 miRNA participates in a lin-28/let-7 axis pathway that regulates cell division and timing of embryonic and metamorphic events in P. olivaceus.

Gene expression patterns regulating embryogenesis based on the integrated de novo transcriptome assembly of the Japanese flounder.

The Japanese flounder (Paralichthys olivaceus) is one of the most important commercial and biological marine fishes. However, the molecular biology involved during embryogenesis and early development of the Japanese flounder remains largely unknown due to a lack of genomic resources. A comprehensive and integrated transcriptome is necessary to study the molecular mechanisms of early development and to allow for the detailed characterization of gene expression patterns during embryogenesis; this approach is critical to understanding the processes that occur prior to mesectoderm formation during early embryonic development. In this study, more than 117.8 million 100bp PE reads were generated from pooled RNA extracted from unfertilized eggs to 41dph (days post-hatching) embryos and were sequenced using Illumina pair-end sequencing technology. In total, 121,513 transcripts (≥200bp) were obtained using de novo assembly. A sequence similarity search indicated that 52,338 transcripts show significant similarity to 22,462 known proteins from the NCBI non-redundant database and the Swiss-Prot protein database and were annotated using Blast2GO. GO terms were assigned to 44,627 transcripts with 12,006 functional terms, and 10,024 transcripts were assigned to 133 KEGG pathways. Furthermore, gene expression differences between the unfertilized egg and the gastrula embryo were analysed using Illumina RNA-Seq with single-read sequencing technology, and 24,837 differentially and specifically expressed transcripts were identified and included 5,286 annotated transcripts and 19,569 non-annotated transcripts. All of the expressed transcripts in the unfertilized egg and gastrula embryo were further classified as maternal, zygotic, or maternal-zygotic transcripts, which may help us to understand the roles of these transcripts during the embryonic development of the Japanese flounder. Thus, the results will contribute to an improved understanding of the gene expression patterns and signalling pathways that control the molecular mechanisms of early embryonic development.

The genome and transcriptome of Japanese flounder provide insights into flatfish asymmetry.

Flatfish have the most extreme asymmetric body morphology of vertebrates. During metamorphosis, one eye migrates to the contralateral side of the skull, and this migration is accompanied by extensive craniofacial transformations and simultaneous development of lopsided body pigmentation. The evolution of this developmental and physiological innovation remains enigmatic. Comparative genomics of two flatfish and transcriptomic analyses during metamorphosis point to a role for thyroid hormone and retinoic acid signaling, as well as phototransduction pathways. We demonstrate that retinoic acid is critical in establishing asymmetric pigmentation and, via cross-talk with thyroid hormones, in modulating eye migration. The unexpected expression of the visual opsins from the phototransduction pathway in the skin translates illumination differences and generates retinoic acid gradients that underlie the generation of asymmetry. Identifying the genetic underpinning of this unique developmental process answers long-standing questions about the evolutionary origin of asymmetry, but it also provides insight into the mechanisms that control body shape in vertebrates.

Coordinated expression and regulation of deiodinases and thyroid hormone receptors during metamorphosis in the Japanese flounder (Paralichthys olivaceus).

In vertebrates, thyroid hormone receptors (TRs) and deiodinases are essential for developmental events driven by the thyroid hormones (THs). However, the significance of deiodinases during the metamorphosis of the Japanese flounder (Paralichthys olivaceus) remains unclear. Moreover, regulation and response of the TRs and deiodinases to THs in this fish are poorly understood. Therefore, we detected the expression patterns of THs, deiodinases, and TRs in drug-treated larvae and untreated larvae of P. olivaceus by using enzyme-linked immunosorbent assay and quantitative real-time PCR during P. olivaceus metamorphosis. To further understand the roles of these elements, a rescue assay was performed. Our results show the importance of THs, TRs, and deiodinases in flatfish metamorphosis. Our results also confirm that D1 and D2 activate THs and D3 plays the opposite and complementary role. Moreover, we demonstrated that both TRα and TRß have important but different roles during P. olivaceus metamorphosis.

miR-17 is involved in Japanese Flounder (Paralichthys olivaceus) development by targeting the Cdc42 mRNA.

The expression patterns of 197 miRNAs during Japanese flounder metamorphic development were recently analyzed. miR-17 was differentially expressed during the metamorphic period of the Japanese flounder; however, the role of miR-17 in Japanese flounder development has remained elusive to date. Bioinformatics analysis showed that Cdc42 was a putative target of miR-17. Cdc42 is a gene related to cell adhesion, migration, polarity, cytokinesis, growth, actin cytoskeleton, microtubule dynamics and transcription factor activity; thus, Cdc42 may contribute to metamorphic development. In our study, overexpression of miR-17 in FEC cells suppressed Cdc42 expression. The luciferase reporter assay confirmed that Cdc42 was the target of miR-17. The Cdc42 cDNA from the Japanese flounder was cloned and characterized for the first time. The expression of miR-17 was found to be negatively correlated with Cdc42 mRNA expression during temporal development and in the tissues of adult Japanese flounders. These results indicated that the decrease in miR-17 contributed to the up-regulation of Cdc42 during Japanese flounder metamorphosis. Cdc42 gene expression was down-regulated by thyroid hormone during Japanese flounder metamorphosis, whereas miR-17 was significantly up-regulated by thyroid hormone during these stages. These results indicated that miR-17 was a negative regulator of Cdc42.

Identification and expression of the target gene emx2 of miR-26a and miR-26b in Paralichthys olivaceus.

MicroRNAs (miRNAs) can regulate specific gene expression by binding to target mRNA further involution to diverse biological processes. Our previous miRNA sequencing showed that pol-miR-26a and pol-miR-26b have a sex-biased expression in ovary and testis of Paralichthys olivaceus (P. olivaceus). And the gene empty spiracles homeobox 2 (emx2) was proposed to be a candidate target by bioinformatics prediction. In this study, we cloned the P. olivaceus emx2 cDNA, including a coding region of 741 bp and a 3'-untranslated region (UTR) of 912 bp and the 5'-UTR of 12 bp. The Emx2 protein is highly conserved and especially its homeodomain region is 100% identical from teleosts to mammals. Real-time PCR results showed that the emx2 is not only highly expressed in embryonic neurula stage and adult brain but also has abundant expression in adult gonad, moreover, it exhibits higher expression in ovary than testis. To determine the relationship between emx2 and miRNAs, a luciferase reporter assay was performed and verified that the emx2 is a common target gene of pol-miR-26a and pol-miR-26b. These data thus helps further clarify that miR-26a and miR-26b are involved in regulating gonad development partially through its target on emx2 expression in P. olivaceus.

Identification and expression of SRF targeted by miR-133a during early development of Paralichthys olivaceus.

Serum response factor (SRF) is a MADS-box transcription factor that regulates the expression of genes involved in development, metabolism, cell proliferation, and differentiation. In the present study, we cloned the full-length SRF cDNA which includes the coding region of 1503 bp, a 573-bp 5'untranslated region (UTR) and a 400-bp 3'-UTR. The deduced 501 amino acid sequence of the SRF protein contained a MADS domain and NLS at the N terminus, similar to other organisms, and it also is highly phylogenetically conserved. SRF mRNA is ubiquitously expressed in various tissues, with the highest level in the kidneys, and it is also highly expressed during the embryonic and metamorphic stages. During metamorphosis, the SRF mRNA levels are down-regulated by exogenous thyroid hormone (TH) at 17 dph and by thiourea (TU) at 29, 36, and 41 dph, whereas SRF mRNA levels were significantly up-regulated by the added exogenous TH to the TU-treated larvae at 41 dph, which indicates that thyroid hormone is essential for expression of SRF mRNA, so, higher levels of TH did not result in changes of SRF mRNA levels, while TH deficiency or inhibited by the non-specific TU toxicity cause down-regulation of SRF mRNA, which indicated that TH can indirectly affect the SRF mRNA levels. Meanwhile, using a luciferase reporter assay, we verified that SRF is a common target gene of miR-133a which is a muscle-specific microRNA (miRNA), which indicated that SRF may be involved in the signaling pathway of miRNA that regulates muscle development.

A key gene of the small RNA pathway in the flounder, Paralichthys olivaceus: identification and functional characterization of dicer.

Dicer is critical for producing mature microRNAs (miRNAs) from precursor molecules and small interfering RNAs and plays an important role in controlling development and metabolism. In the present study, we cloned the flounder dicer gene, which is 6585 nucleotides (nt), including a 5'-untranslated region (UTR) of 231 nt, a 3'-UTR of 663 nt and an open reading frame of 5691 nt encoding a polypeptide of 1897 amino acids, and analyzed the conservation and expression pattern of dicer. The tissue distribution analysis indicated that dicer is abundantly expressed in the brain, heart, liver, spleen, stomach, kidney, gill, muscle, intestine and gonad of adult fish. Temporal expression analysis indicated that dicer mRNA is highly expressed during the embryonic and early larval stages, and exhibits low expression during the metamorphic stages. Treatment with thyroid hormone (TH) or thiourea indirectly or directly up-regulated dicer mRNA levels at 17 and 23 dph, whereas treatment with TH down-regulated dicer mRNA levels at 36 dph. The dicer-specific siRNA significantly down-regulated dicer mRNA and pol-let-7d levels, while pol-let-7d precursor levels were not differentially changed compared with the control (NC). These results demonstrated that dicer plays a key role in development and metabolism through the production of mature miRNAs, providing basic information for further studies concerning the role of dicer in Paralichthys olivaceus development.

Identification and expression of HDAC4 targeted by miR-1 and miR-133a during early development in Paralichthys olivaceus.

Histone deacetylase 4, which is a class II histone deacetylase, plays a critical role in development, differentiation of muscle, cell proliferation and metabolism. In our study, we obtained the full-length HDAC4 cDNA, which included the coding region of 3171bp, a 180bp 5'untranslated region (UTR) and a 760bp 3'UTR. The deduced HDAC4 protein contained all known functional domains identified in other organisms. The phylogenetic analysis indicated that Paralichthys olivaceus HDAC4 had the highest identity with the Takifugu rubripes. Tissue distribution analysis indicated that HDAC4 is abundantly expressed in muscle, and its levels are significantly higher in muscle than in other tissues (P<0.01). HDAC4 mRNA levels at 3dph (days post hatching) and 36dph were higher than that in other stages. Exogenous thyroid hormones either directly or indirectly promoted the expression of HDAC4 mRNA during metamorphosis, indicating that HDAC4 might directly or indirectly be regulated by thyroid hormone during muscle development in metamorphosis. To identify the miRNAs targeting HDAC4, we performed a luciferase reporter assay and verified that HDAC4 is a common target gene of miR-1 and miR-133a indicating that HDAC4 might be involved in a signal pathway of microRNA regulating muscle development.