Use of All-Male cyp17a1-Deficient Zebrafish (Danio rerio) for Evaluation of Environmental Estrogens.

Natural and synthetic environmental estrogens (EEs) are widespread and have received extensive attention. Our previous studies demonstrated that depletion of the cytochrome P450 17a1 gene (cyp17a1) leads to all-testis differentiation phenotype in zebrafish and common carp. In the present study, cyp17a1-deficient zebrafish with defective estrogen biosynthesis were used for the evaluation of EEs, as assessed by monitoring vitellogenin (vtg) expression. A rapid and sensitive assessment procedure was established with the 3-day administration of estradiol (E2), followed by examination of the transcriptional expression of vtgs in our cyp17a1-deficient fish. Compared with the control fish, a higher E2-mediated vtg upregulation observed in cyp17a1-deficient zebrafish exposed to 0.1 µg/L E2 is known to be estrogen receptor-dependent and likely due to impaired in vivo estrogen biosynthesis. The more responsive vtg expression in cyp17a1-deficient zebrafish was observed when exposed to 200 and 2000 µg/L bisphenol A (BPA) and perfluoro-1-octanesulfonate (PFOS). The estrogenic potentials of E2, BPA, and PFOS were compared and assessed by the feminization effect on ovarian differentiation in cyp17a1-deficient zebrafish from 18 to 50 days postfertilization, based on which a higher sensitivity of E2 in ovarian differentiation than BPA and PFOS was concluded. Collectively, through the higher sensitivity to EEs and the capacity to distinguish chemicals with different estrogenic potentials exhibited by the all-male cyp17a1-deficient zebrafish with impaired estrogen biosynthesis, we demonstrated that they can be used as an excellent in vivo model for the evaluation of EEs. Environ Toxicol Chem 2024;43:1062-1074. © 2024 SETAC.

Androgen signaling inhibits de novo lipogenesis to alleviate lipid deposition in zebrafish.

Testosterone is closely associated with lipid metabolism and known to affect body fat composition and muscle mass in males. However, the mechanisms by which testosterone acts on lipid metabolism are not yet fully understood, especially in teleosts. In this study, cyp17a1-/- zebrafish ( Danio rerio) exhibited excessive visceral adipose tissue (VAT), lipid content, and up-regulated expression and activity of hepatic de novo lipogenesis (DNL) enzymes. The assay for transposase accessible chromatin with sequencing (ATAC-seq) results demonstrated that chromatin accessibility of DNL genes was increased in cyp17a1-/- fish compared to cyp17a1+/+ male fish, including stearoyl-CoA desaturase ( scd) and fatty acid synthase ( fasn). Androgen response element (ARE) motifs in the androgen signaling pathway were significantly enriched in cyp17a1+/+ male fish but not in cyp17a1-/- fish. Both androgen receptor ( ar)-/- and wild-type (WT) zebrafish administered with Ar antagonist flutamide displayed excessive visceral adipose tissue, lipid content, and up-regulated expression and activity of hepatic de novo lipogenesis enzymes. The Ar agonist BMS-564929 reduced the content of VAT and lipid content, and down-regulated acetyl-CoA carboxylase a ( acaca), fasn, and scd expression. Mechanistically, the rescue effect of testosterone on cyp17a1-/- fish in terms of phenotypes was abolished when ar was additionally depleted. Collectively, these findings reveal that testosterone inhibits lipid deposition by down-regulating DNL genes via Ar in zebrafish, thus expanding our understanding of the relationship between testosterone and lipid metabolism in teleosts.

The food safety assessment of all-female common carp (Cyprinus carpio) (cyp17a1+/-;XX genotype) generated using genome editing technology.

There are several technical challenges and public issues concerning genome editing applications before they become viable in commercial aquaculture. Recently, we developed a novel strategy to generate all-female (AF) common carp, which exhibited a growth advantage over the control carp, using genetic editing through single gene-targeting manipulation. Here, we found that the body weight of the AF common carp was higher by 22.58% than that of the control common carp. Because the genotype of the AF common carp was cyp17a1+/-;XX, the contents of sex steroids were normally synthesized, as they were comparable to that of the control female carp. To evaluate the food safety of the AF carp, Wistar rats were fed a diet containing control female carp (control, C) or all-female (AF) carp at an incorporation rate of 5, 10 and 20% (w/w) for 90 days. Compared with those fed control carp, the rats fed AF common carp exhibited no significant difference in body weight, food intake, feed conversion ratio, hematology, serum biochemistry, urine test, relative organ weight, gross necropsy, and histopathological examination. This is the first food safety assessment of the farmed fish strain cultured using CRISPR/Cas9, which will further advance the fishery development of genome-edited animals.

Effects of short-term water velocity stimulation on the biochemical and transcriptional responses of grass carp (Ctenopharyngodon idellus).

Since 2011, ecological operation trials of the Three Gorges Reservoir (TGR) have been continuously conducted to improve the spawning quantity of the four major Chinese carp species below the Gezhouba Dam. In particular, exploring the effects of short-term water velocity stimulation on ovarian development in grass carp (Ctenopharyngodon idellus) is essential to understand the response of natural reproduction to ecological flows. We performed ovary histology analysis and biochemical assays among individuals with or without stimulation by running water. Although there were no obvious effects on the ovarian development characteristics of grass carp under short-term water velocity stimulation, estradiol, progesterone, follicle-stimulating hormone (FSH), and triiodothyronine (T3) concentrations were elevated. Then, we further explored the ovarian development of grass carp under short-term water velocity stimulation by RNA sequencing of ovarian tissues. In total, 221 and 741 genes were up- or downregulated under short-term water velocity stimulation, respectively, compared to the control group. The majority of differentially expressed genes (DEGs) were enriched in pathways including ABC transporters, cytokine-cytokine receptor interaction, ECM-receptor interaction, and steroid hormone biosynthesis. Important genes including gpr4, vtg1, C-type lectin, hsd17b1, cyp19a1a, cyp17a1, and rdh12 that are involved in ovarian development were regulated. Our results provide new insights and reveal potential regulatory genes and pathways involved in the ovarian development of grass carp under short-term water velocity stimulation, which may be beneficial when devising further ecological regulation strategies.

Identification of molecular signatures in acute myocardial infarction based on integrative analysis of proteomics and transcriptomics.

BACKGROUND: Myocardial infarction (MI) is one of the most serious cardiovascular diseases, characterized by a rapid and irreversible decline in myocardial function. Early detection of patients with MI and prolonging the optimal therapeutic window of acute myocardial infarction (AMI) are particularly important. This study aimed to identify the diagnostic biomarkers and novel therapeutic targets for acute myocardial infarction. METHOD: We generated the AMI mouse models by ligating the proximal left anterior descending coronary artery. Six time points-Sham, AMI 10-min, 1-h, 6-h, 24-h, and 72-h-were chosen to examine the molecular changes that occur during the AMI process. RNA-seq and DIA-MS were performed on the infarcted left ventricular tissues of AMI mice at each time point. Co-expression pattern genes were screened from myocardial infarction samples at different time points by time-series analysis. Gene Ontology (GO) function and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were used to examine these genes. Using the Interactive Gene/Protein Retrieval Tool (STRING) database, the protein-protein interaction network (PPI) was constructed and the hub genes were identified. In order to evaluate the diagnostic value of hub genes, a receiver operating characteristic (ROC) curve was constructed. An independent data set, GSE163772, confirmed the diagnostic value of hub genes further. RESULT: We obtained the expression profiles at different time points after the occurrence of heart failure through high-throughput sequencing, and found 167 genes with similar expression patterns through time series analysis. The immune response and immune-related pathways had the greatest enrichment of these genes. Among them, Itgb2 Syk, Tlr4, Tlr2, Itgax, and Lcp2 may play key roles as hub genes. Combined with the results of proteomic analysis, it was found that the expression of Coro1a in both omics increased with time. The results of external validation showed that TLR2, ITGAX, and LCP2 had good predictive ability for AMI diagnosis. CONCLUSION: Itgb2, Syk, Tlr4, Tlr2, Itgax, Lcp2 and Coro1a are considered to be the seven key genes significantly associated with AMI. Our results may provide potential targets for the prevention of adverse ventricular remodeling and the treatment of AMI.

The effect of thickness and elastic modulus of the anterior talofibular ligament on anterior ankle joint stiffness: A subject-specific finite element study.

Ankle sprain is a frequent type of sports injury leading to lateral ligament injury. The anterior talofibular ligament (ATFL) is a primary ligamentous stabilizer of the ankle joint and typically the most vulnerable ligament injured in a lateral ankle sprain (LAS). This study aimed to quantitively investigate the effect of the thickness and elastic modulus of ATFL on anterior ankle joint stiffness (AAJS) by developing nine subject-specific finite element (FE) models under acute injury, chronic injury, and control conditions of ATFL. A 120 N forward force was applied at the posterior calcaneus leading to an anterior translation of the calcaneus and talus to simulate the anterior drawer test (ADT). In the results, the ratio of the forward force to the talar displacement was used to assess the AAJS, which increased by 5.85% in the acute group and decreased by 19.78% in the chronic group, compared to those of the control group. An empirical equation described the relationship between AAJS, thickness, and elastic modulus (R-square 0.98). The equation proposed in this study provided an approach to quantify AAJS and revealed the effect of the thickness and the elastic modulus of ATFL on ankle stability, which may shed light on the potential diagnosis of lateral ligament injury.

A finite element analysis of the carpal arch with various locations of carpal tunnel release.

Objective: The purpose of this study was to investigate the effects of the location of transverse carpal ligament (TCL) transection on the biomechanical property of the carpal arch structure. It was hypothesized that carpal tunnel release would lead to an increase of the carpal arch compliance (CAC) in a location-dependent manner. Methods: A pseudo-3D finite element model of the volar carpal arch at the distal carpal tunnel was used to simulate arch area change under different intratunnel pressures (0-72 mmHg) after TCL transection at different locations along the transverse direction of the TCL. Results: The CAC of the intact carpal arch was 0.092 mm2/mmHg, and the simulated transections ranging from 8 mm ulnarly to 8 mm radially from the center point of the TCL led to increased CACs that were 2.6-3.7 times of that of the intact carpal arch. The CACs after radial transections were greater than those ulnarly transected carpal arches. Conclusion: The TCL transection in the radial region was biomechanically favorable in reducing carpal tunnel constraint for median nerve decompression.

Enhanced insulin activity achieved in VDRa/b ablation zebrafish.

Introduction: 1α,25-dihydroxyvitamin D3 (1α,25[OH]2VD3) is a hormone known for its key roles in calcium absorption and nutrient metabolism. In teleost fishes, 1α,25(OH)2VD3 insufficiency causes impaired glucose metabolism and lipid oxidation. However, the cascade and mechanisms of 1α,25(OH)2VD3 and the vitamin d receptor (VDR) signaling are unclear. Results: In this study, two genes (vdra and vdrb) encoding paralogs of VDRs were genetically knocked out in zebrafish. Growth retardation and accumulated visceral adipose tissue have been observed in vdra -/-;vdrb -/- deficient line. In the liver elevated accumulation of triglycerides and suppressed lipid oxidation were detected. Morover significantly elevated 1α,25(OH)2VD3 levels were detected in vdra-/-;vdrb-/- zebrafish due to cyp24a1 transcription repression. Furthermore VDRs ablation Enhanced insulin signaling including elevated insulin/insra trancriptional levels, glycolysis, lipogenesis and promoted AKT/mTOR activity. Discussion: In conclusion, our present studies provides a zebrafish model with an elevated 1α,25(OH)2VD3 levels in vivo. The 1α,25(OH)2VD3/VDRs signaling promote lipid oxidation activity. However 1α,25(OH)2VD3 activity of regulation of glucose homeostasis through Insulin/Insr was independent of nuclear VDRs in teleosts.

The roles of ubiquitin-proteasome system and regulator of G protein signaling 4 in behavioral sensitization induced by a single morphine exposure.

AIMS: Opioid addiction is a major public health issue, yet its underlying mechanism is still unknown. The aim of this study was to explore the roles of ubiquitin-proteasome system (UPS) and regulator of G protein signaling 4 (RGS4) in morphine-induced behavioral sensitization, a well-recognized animal model of opioid addiction. METHODS: We explored the characteristics of RGS4 protein expression and polyubiquitination in the development of behavioral sensitization induced by a single morphine exposure in rats, and the effect of a selective proteasome inhibitor, lactacystin (LAC), on behavioral sensitization. RESULTS: Polyubiquitination expression was increased in time-dependent and dose-related fashions during the development of behavioral sensitization, while RGS4 protein expression was not significantly changed during this phase. Stereotaxic administration of LAC into nucleus accumbens (NAc) core inhibited the establishment of behavioral sensitization. CONCLUSION: UPS in NAc core is positively involved in behavioral sensitization induced by a single morphine exposure in rats. Polyubiquitination was observed during the development phase of behavioral sensitization, while RGS4 protein expression was not significantly changed, indicating that other members of RGS family might be substrate proteins in UPS-mediated behavioral sensitization.

Stiffening of the gluteal muscle increased the intramuscular stress: An in-silico implication of deep tissue injury.

Objectives: Deep tissue injury is a common form of pressure ulcers in muscle tissues under bony prominences caused by sustained pressure or shear, which has a great impact on patients with restricted mobility such as spinal cord injury. Frequent spasms in spinal cord injury patients featured by muscle stiffening may be one of the factors leading to deep tissue injury. The purpose of this study was to investigate the relationship between the gluteal muscle shear modulus and intramuscular compressive/shear stress/strain. Methods: A semi-3D finite element model of the human buttock was established using COMSOL software and the acquired biomechanical data were analyzed through Pearson correlation and Spearman correlation. Results: Results showed that the compressive stress, strain energy density, and average von Mises stress increased with the increase of the gluteal muscle shear modulus. Conclusion: These results may indicate muscle stiffening caused by muscle spasms could lead to higher deep tissue injury development risk as well as shed light on effective treatments for relieving muscular sclerosis mechanically.

An experimental study of physical intelligence teaching on sensory integration function of 4-5-year-old children / 中国学校卫生

Objective@#To examine the impact of physical intelligence teaching on the function of children s sensory integration, so as to provide reference for promoting the development of sensory integration system.@*Methods@#From February to May 2023, the intervention was implemented for 12 weeks among 136 children aged 4-5 (68 in the intervention group and 68 in the control group). The intervention group received situational and game based physical intelligence teaching, the control group received sports game teaching according to the original curriculum objectives of the kindergarten. Intervention was administered 3 times a week for 40 minutes each time. The sensory integration ability of the intervention group and the control group were evaluated before and after the intervention with Chi square test and t test.@*Results@#The vestibular sensation, proprioception and tactile sensation of between boys and girls in the intervention group were significantly improved compared with before intervention (boys：44.14±11.52 vs. 53.34± 9.49 ，44.57±12.76 vs. 50.54±11.86，49.31±12.18 vs. 55.00±10.24,girls：46.00±11.01 vs. 54.58±10.06，48.79±13.17 vs. 53.64±11.97，52.67±11.67 vs. 56.91±10.42, t =-3.24，-2.49，-2.09，-5.24，-12.94，-2.56， P <0.05). There was no significant difference in vestibular sensation between boys and girls in the control group (boys：45.91±11.66 vs. 46.31± 11.20，girls：48.27±13.56 vs. 48.45 ±13.54, t =-0.87，-0.07， P >0.05), but there was a significant improvement in proprioception and tactile sensation in both boys and girls (boys：46.63±11.76 vs. 48.06±11.69，51.63±11.98 vs. 52.40±12.18，girls：50.45±12.16 vs. 51.67± 12.03 ，53.36±12.48 vs. 54.39±12.57， t =-3.36，-2.08，-4.66，-2.86， P <0.05). After the intervention, compared with the control group, the vestibular sensation of both boys and girls significantly improved ( t=2.83, 2.08, P <0.05), with exception of proprioception and tactile sensation ( t =0.88,0.67,0.97,0.88, P >0.05). In the experimental group, the number of normal boys increased from 12 to 24, while the number of dysfunctional boys decreased from 23 to 11, with a statistically significant difference ( χ 2=11.53, P <0.01). There was no statistically significant difference in sensory integration in boys of the control group before and after the experiment ( χ 2= 1.10 , P >0.05). After intervention,the number of normal girls in the experimental group increased from 15 to 27, while the number of dysfunctional girls decreased from 18 to 6, with a statistically significant difference ( χ 2=10.39, P < 0.05 ). There was no statistically significant difference in sensory integration in girls from the control group before and after the experiment ( χ 2=2.08, P > 0.05 ).@*Conclusion@#Physical intelligence teaching can effectively improve children s sensory integration ability, especially for vestibular function.

Optimization of Ultrahigh-Throughput Screening Assay for Protein Engineering of d-Allulose 3-Epimerase.

d-Allulose is the corresponding epimer of d-fructose at the C-3 position, which exhibits a similar taste and sweetness to sucrose. As a low-calorie sweetener, d-allulose has broad application prospects in the fields of medicine, food, and so on. Currently, the production method of d-allulose is mainly the enzymatic conversion of d-fructose by d-allulose 3-epimerase (DAEase). However, the limited specific activity and thermal stability of DAEase restrict its industrial application. Herein, an ultrahigh-throughput screening assay based on the transcription factor PsiR was extensively optimized from the aspects of culture medium components, screening plasmid, and expression host, which enhanced the correction between the fluorescent readout and the enzyme activity. Then, the error-prone PCR (epPCR) library of Clostridium cellulolyticum H10 DAEase (CcDAEase) was screened through the above optimized method, and the variant I228V with improved specific activity and thermal stability was obtained. Moreover, after combining two beneficial substitutions, D281G and C289R, which were previously obtained by this optimized assay, the specific activity of the triple-mutation variant I228V/D281G/C289R reached up to 1.42-fold of the wild type (WT), while its half-life (T1/2) at 60 °C was prolonged by 62.97-fold. The results confirmed the feasibility of the optimized screening assay as a powerful tool for the directed evolution of DAEase.

Prognostic Ability of Enhancer RNAs in Metastasis of Non-Small Cell Lung Cancer.

(1) Background: Non-small cell lung cancer (NSCLC) is the most common lung cancer. Enhancer RNA (eRNA) has potential utility in the diagnosis, prognosis and treatment of cancer, but the role of eRNAs in NSCLC metastasis is not clear; (2) Methods: Differentially expressed transcription factors (DETFs), enhancer RNAs (DEEs), and target genes (DETGs) between primary NSCLC and metastatic NSCLC were identified. Prognostic DEEs (PDEEs) were screened by Cox regression analyses and a predicting model for metastatic NSCLC was constructed. We identified DEE interactions with DETFs, DETGs, reverse phase protein arrays (RPPA) protein chips, immunocytes, and pathways to construct a regulation network using Pearson correlation. Finally, the mechanisms and clinical significance were explained using multi-dimensional validation unambiguously; (3) Results: A total of 255 DEEs were identified, and 24 PDEEs were selected into the multivariate Cox regression model (AUC = 0.699). Additionally, the NSCLC metastasis-specific regulation network was constructed, and six key PDEEs were defined (ANXA8L1, CASTOR2, CYP4B1, GTF2H2C, PSMF1 and TNS4); (4) Conclusions: This study focused on the exploration of the prognostic value of eRNAs in the metastasis of NSCLC. Finally, six eRNAs were identified as potential markers for the prediction of metastasis of NSCLC.

Sex-specific differences in zebrafish brains.

In this systematic review, we highlight the differences between the male and female zebrafish brains to understand their differentiation and their use in studying sex-specific neurological diseases. Male and female brains display subtle differences at the cellular level which may be important in driving sex-specific signaling. Sex differences in the brain have been observed in humans as well as in non-human species. However, the molecular mechanisms of brain sex differentiation remain unclear. The classical model of brain sex differentiation suggests that the steroid hormones derived from the gonads are the primary determinants in establishing male and female neural networks. Recent studies indicate that the developing brain shows sex-specific differences in gene expression prior to gonadal hormone action. Hence, genetic differences may also be responsible for differentiating the brain into male and female types. Understanding the signaling mechanisms involved in brain sex differentiation could help further elucidate the sex-specific incidences of certain neurological diseases. The zebrafish model could be appropriate for enhancing our understanding of brain sex differentiation and the signaling involved in neurological diseases. Zebrafish brains show sex-specific differences at the hormonal level, and recent advances in RNA sequencing have highlighted critical sex-specific differences at the transcript level. The differences are also evident at the cellular and metabolite levels, which could be important in organizing sex-specific neuronal signaling. Furthermore, in addition to having one ortholog for 70% of the human gene, zebrafish also shares brain structural similarities with other higher eukaryotes, including mammals. Hence, deciphering brain sex differentiation in zebrafish will help further enhance the diagnostic and pharmacological intervention of neurological diseases.

Augmentation of progestin signaling rescues testis organization and spermatogenesis in zebrafish with the depletion of androgen signaling.

Disruption of androgen signaling is known to cause testicular malformation and defective spermatogenesis in zebrafish. However, knockout of cyp17a1, a key enzyme responsible for the androgen synthesis, in ar-/- male zebrafish paradoxically causes testicular hypertrophy and enhanced spermatogenesis. Because Cyp17a1 plays key roles in hydroxylation of pregnenolone and progesterone (P4), and converts 17α-hydroxypregnenolone to dehydroepiandrosterone and 17α-hydroxyprogesterone to androstenedione, we hypothesize that the unexpected phenotype in cyp17a1-/-;androgen receptor (ar)-/- zebrafish may be mediated through an augmentation of progestin/nuclear progestin receptor (nPgr) signaling. In support of this hypothesis, we show that knockout of cyp17a1 leads to accumulation of 17α,20ß-dihydroxy-4-pregnen-3-one (DHP) and P4. Further, administration of progestin, a synthetic DHP mimetic, is sufficient to rescue testicular development and spermatogenesis in ar-/- zebrafish, whereas knockout of npgr abolishes the rescue effect of cyp17a1-/- in the cyp17a1-/-;ar-/- double mutant. Analyses of the transcriptomes among the mutants with defective testicular organization and spermatogenesis (ar-/-, ar-/-;npgr-/- and cyp17a-/-;ar-/-;npgr-/-), those with normal phenotype (control and cyp17a1-/-), and rescued phenotype (cyp17a1-/-;ar-/-) reveal a common link between a downregulated expression of insl3 and its related downstream genes in cyp17a-/-;ar-/-;npgr-/- zebrafish. Taken together, our data suggest that genetic or pharmacological augmentation of the progestin/nPgr pathway is sufficient to restore testis organization and spermatogenesis in zebrafish with the depletion of androgen signaling.

Multi-omics perspective on studying reproductive biology in Daphnia sinensis.

Daphnia sinensis is a widespread freshwater microcrustacean. The assembled D. sinensis genome totaled 131.58 Mb with 92.23% of the assembly anchored onto 10 chromosomes. Based on the whole genome information, we further compared the transcriptomic and epigenomic characterization among parthenogenetic females, sexual females and males in D. sinensis. Transcriptomic analysis showed that the up-regulated genes in males were mainly grouped into the cuticle, sex differentiation and methyl farnesoate synthesis, which might play a pivotal role in steering development and reproduction processes. By comparison, the highly expressed genes in parthenogenetic females were mainly grouped into energy metabolism, mitosis, and DNA replication, which might contribute to maintaining rapid production of parthenogenetic females, and nutrient uptake for the growth of neonates. The whole-genome DNA methylation analysis showed that the methylation rate in parthenogenetic females was higher than that in sexual females and males, which might contribute to its rapid response to environment stress.

Identification of prognostic and bone metastatic alternative splicing signatures in bladder cancer.

Bladder cancer (BLCA), originating from the epithelium of the urinary bladder, was the second most common malignancy in the urinary system with a high metastasis rate and poor post-metastasis prognosis. Alternative splicing events (ASEs) were regarded as important markers of tumor progression and prognosis, however, their roles in bladder cancer bone metastasis have not been recognized. In this study, we constructed a predictive model based on ASEs and explored the molecular mechanism of ASEs in BLCA bone metastasis, based on data from the Cancer Genome Atlas (TCGA) and TCGASpliceSeq databases. We proposed the hypothesis that the splicing events of ITGB4 was regulated by the splicing factor JUP, and this regulation might play a key role in BLCA bone metastasis through the glycosphingolipid biosynthesis ganglio series pathway.

Phylogenetic analysis of eight species of Anomopoda based on transcriptomic and mitochondrial DNA sequences.

Anomopoda is the widespread planktonic microcrustacean, which plays a crucial role in aquatic ecosystem. There are few studies about the evolutionary relationships among various Anomopoda basing on molecular data. In the present study, phylogenetic analysis of eight Anomopoda was carried out. Firstly, the culture system was developed to breed cladocerans. By using this system, eight species (Daphnia magna, D. pulex, D. sinensis, Ceriodaphnia reticulata, Moina micrura, Scapholeberis kingi, Simocephalus vetulus and Eurycercus lamellatus) were purified and cultured stably in the laboratory. Then, transcriptomic sequences and partial mitochondrial DNA sequences were both used to reconstruct the phylogenetic tree among 8 species. Transcriptomic sequences were sequenced on Illumina Hiseq 2500 platform. After assembly and annotation, transcriptomic sequences were spliced together and aligned for phylogenetic analysis. Basing on the orthologous genes derived from transcriptomic sequences, the phylogenetic analysis showed that 4 genera of Daphniidae were clustered into one group, and among the 4 genera, Ceriodaphnia was closer to Daphnia than Simocephalus, while Scapholeberis was farthest from other species. In addition, Eurycercidae was closer to Daphniidae than Moinidae. The phylogenetic trees based on both 12S rRNA and 16S rRNA sequences were similar with that based on transcriptomic sequences. Meanwhile, the phylogenetic tree based on 16S rRNA sequences was more suitable than that based on 12S rRNA sequences. These results suggested that the phylogenetic analysis basing on the transcriptomic sequences was available in cladocerans, which will help us to effectively understand the phylogenetic relationships among various cladocerans.

ITRAQ Proteomic Analysis of Yellow and Black Skin in Jinbian Carp (Cyprinus carpio).

Colors are important phenotypic traits for fitness under natural conditions in vertebrates. Previous studies have reported several functional genes and genetic variations of pigmentation, but the formation mechanisms of various skin coloration remained ambiguous in fish. Jinbian carp, a common carp variant, displays two colors (yellow and black) in the skin, thus, it is a good model for investigating the genetic basis of pigmentation. In the present study, using the Jinbian carp as model, isobaric tags for relative and absolute quantification (ITRAQ) proteomics analysis was performed for yellow and black skin, respectively. The results showed that 467 differentially expressed proteins (DEPs) were identified between the yellow skin and the black skin. Similar to mammals, the up-regulated DEPs in black skin included UV excision repair protein RAD23 homolog A (Rad23a), melanoregulin (mreg), 5,6-dihydroxyindole-2-carboxylic acid oxidase5 (tyrp1) and melanocyte protein PMEL (PMEL), which were mainly grouped into melanogenesis pathway. However, several up-regulated DEPs in yellow skin were mainly enriched in nucleotide metabolism, such as GTPase IMAP family member 5 (GIMAP5), AMP deaminase 1 (AMPD1), adenosylhomocysteinase b (ahcy-b), and pyruvate kinase (PKM). In summary, several candidate proteins and their enrichment pathways for color variation in Jinbian carp were identified, which may be responsible for the formation of different colorations.

Pituitary Actions of EGF on Gonadotropins, Growth Hormone, Prolactin and Somatolactins in Grass Carp.

In mammals, epidermal growth factor (EGF) plays a vital role in both pituitary physiology and pathology. However, the functional role of EGF in the regulation of pituitary hormones has rarely reported in teleost. In our study, using primary cultured grass carp pituitary cells as an in vitro model, we examined the effects of EGF on pituitary hormone secretion and gene expression as well as the post-receptor signaling mechanisms involved. Firstly, we found that EGF significantly reduced luteinizing hormone (LHß) mRNA expression via ErbB1 coupled to ERK1/2 pathway, but had no effect on LH release in grass carp pituitary cells. Secondly, the results showed that EGF was effective in up-regulating mRNA expression of growth hormone (GH), somatolactin α (SLα) and somatolactin ß (SLß) via ErbB1 and ErbB2 and subsequently coupled to MEK1/2/ERK1/2 and PI3K/Akt/mTOR pathways, respectively. However, EGF was not effective in GH release in pituitary cells. Thirdly, we found that EGF strongly induced pituitary prolactin (PRL) release and mRNA expression, which was mediated by ErbB1 and subsequent stimulation of MEK1/2/ERK1/2 and PI3K/Akt/mTOR pathways. Interestingly, subsequent study further found that neurokinin B (NKB) significantly suppressed EGF-induced PRL mRNA expression, which was mediated by neurokinin receptor (NK2R) and coupled to AC/cAMP/PKA signal pathway. These results suggested that EGF could differently regulate the pituitary hormones expression in grass carp pituitary cells.