Selection and Verification of Standardized Reference Genes of Angelica dahurica under Various Abiotic Stresses by Real-Time Quantitative PCR.

In traditional Chinese medicine, Angelica dahurica is a valuable herb with numerous therapeutic applications for a range of ailments. There have not yet been any articles on the methodical assessment and choice of the best reference genes for A. dahurica gene expression studies. Real-time quantitative PCR (RT-qPCR) is widely employed as the predominant method for investigating gene expression. In order to ensure the precise determination of target gene expression outcomes in RT-qPCR analysis, it is imperative to employ stable reference genes. In this study, a total of 11 candidate reference genes including SAND family protein (SAND), polypyrimidine tract-binding protein (PTBP), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), actin (ACT), TIP41-like protein (TIP41), cyclophilin 2 (CYP2), elongation factor 1 α (EF1α), ubiquitin-protein ligase 9 (UBC9), tubulin ß-6 (TUB6), thioredoxin-like protein YLS8 (YLS8), and tubulin-α (TUBA) were selected from the transcriptome of A. dahurica. Subsequently, three statistical algorithms (geNorm, NormFinder, and BestKeeper) were employed to assess the stability of their expression patterns across seven distinct stimulus treatments. The outcomes obtained from these analyses were subsequently amalgamated into a comprehensive ranking using RefFinder. Additionally, one target gene, phenylalanine ammonia-lyase (PAL), was used to confirm the effectiveness of the selected reference genes. According to the findings of this study, the two most stable reference genes for normalizing the expression of genes in A. dahurica are TIP41 and UBC9. Overall, our research has determined the appropriate reference genes for RT-qPCR in A. dahurica and provides a crucial foundation for gene screening and identifying genes associated with the biosynthesis of active ingredients in A. dahurica.

[Exploration of cross-cultivar group characteristics of a new cultivar of Prunus mume 'Zhizhang Guhong Chongcui'].

'Zhizhang Guhong Chongcui' is a new cultivar of Prunus mume with cross-cultivar group characteristics. It has typical characteristics of cinnabar purple cultivar group and green calyx cultivar group. It has green calyx, white flower, and light purple xylem, but the mechanism remains unclear. In order to clarify the causes of its cross-cultivar group traits, the color phenotype, anthocyanin content and the expression levels of genes related to anthocyanin synthesis pathway of 'Zhizhang Guhong Chongcui', 'Yuxi Zhusha' and 'Yuxi Bian Lü'e' were determined. It was found that the red degree of petals, sepals and fresh xylem in branches was positively correlated with the total anthocyanin content. MYBɑ1, MYB1, and bHLH3 were the key transcription factor genes that affected the redness of the three cultivars of flowers and xylem. The transcription factors further promoted the high expression of structural genes F3'H, DFR, ANS and UFGT, thereby promoting the production of red traits. Combined with phenotype, anthocyanin content and qRT-PCR results, it was speculated that the white color of petals of 'Zhizhang Guhong Chongcui' were derived from the high expression of FLS, F3'5'H, LAR and ANR genes in other branches of cyanidin synthesis pathway, and the low expression of GST gene. The green color of sepals might be originated from the relatively low expression of F3'H, DFR and ANS genes. The red color of xylem might be derived from the high expression of ANS and UFGT genes. This study made a preliminary explanation for the characteristics of the cross-cultivar group of 'Zhizhang Guhong Chongcui', and provided a reference for molecular breeding of flower color and xylem color of Prunus mume.

Broad-spectrum resistance against multiple PVY-strains by CRSIPR/Cas13 system in Solanum tuberosum crop.

Potato virus Y (PVY) is a deadly environmental constraint that damages productivity of potato (Solanum tuberosum) around the globe. One of the major challenges is to develop resistance against PVY. Emerging clustered regularly short palindromic repeat (CRISPR)/Cas systems have the potential to develop resistance against PVY. In the current research, CRISPR-Cas13 has been exploited to target multiple strains of PVYN, PVYO, and PVYNTN. Multiple genes PI, HC-Pro, P3, Cl1, Cl2, and VPg genes of PVY were targeted by CRISPR/Cas13a. Multiplex gRNA cassettes were developed on the conserved regions of the PVY-genes. Three independent CRISPR/Cas13 transgenic potato lines were developed by applying an optimized concentration of trans-ribo zeatin and indole acetic acid at callus development, rooting, and shooting growth stages. The level of resistance in transgenic plants was confirmed through double-antibody sandwich enzyme-linked immunosorbent assay and real-time quantitative PCR. Our results have shown that efficiency of PVY inhibition was positively correlated with the Cas13a/sgRNA expression. Finding provides the specific functionality of Cas13 with specific gRNA cassette and engineering the potential resistance in potato crop against multiple strains of PVY.

[Reference gene screening for Real-time quantitative PCR in Polygonum multiflorum].

To select suitable references gene of Polygonum multiflorum for gene expression analysis in different tissues, five candidate reference genes like Actin,GAPDH,SAND,PP2A,TIP41 were selected from the transcriptome data of P. multiflorum, then the specific primers were designed. The expression stability of the five reference genes in different tissues of P. multiflorum was analyzed by Real-time quantitative PCR through avilable analysis methods such as geNorm, NormFinder, BestKeeper, Delta CT and RefFinder, to ensure the reliability of the analysis results. The results showed that there were significant differences in the expression levels and stability of candidate genes in different tissues of P. multiflorum. Ct distribution analysis of the expression levels of candidate genes showed that the expression levels of Actin and GAPDH genes were relatively high in different tissues, while the expression levels of SAND, PP2A and TIP41 were lower. The stability of each candidate gene was analyzed by different methods. The results of geNorm analysis showed that the expression of PP2A and GAPDH was the most stable, the expression stability of SAND was the worst, the stability of PP2A was the highest in both NormFinder and Delta CT, the stability of SAND was the lowest, and the stability of Actin was the most stable in BestKeeper analysis. Through the comprehensive evaluation and analysis of the stability of candidate genes by RefFinder, it is concluded that the stability of PP2A gene is the highest, followed by GAPDH, Actin, TIP41, SAND, and SAND gene is the worst. Therefore, the PP2A gene is an ideal reference gene for the analysis of gene expression in different tissues of P. multiflorum.

TaqMan real-time quantitative PCR for identification of antlers in tradition Chinese medicine.

In this study, a method was established for discriminating the true Cervus antlers from its counterfeits using TaqMan real-time quantitative PCR. The method combines the use of true Cervus antlers-specific primers, that amplify a 226 bp fragment from true Cervus antlers DNA, and mammalian-specific primers amplifying a 146 bp fragment from mammalian species DNA, which are used as endogenous control. A TaqMan probe that hybridizes in the 'Cervus antler' and also in the 'mammalian' DNA fragments is used to monitor the amplification of the target gene. The Cervus antler mitochondrial DNA was used as target gene to design the primers and TaqMan probes. The data revealed that the TaqMan real-time PCR-based assay can be used for identification of the true Cervus antlers from counterfeits in a single step. The limit of detection (LOD) was lower than 1 pg of DNA per reaction.

[Selection and validation of internal reference genes for qPCR in Polygonatum cyrtonema tubers at different development stages and in response to abiotic stress].

In order to analyze the expression of genes involved in steroidal saponin biosynthesis pathway in Polygonatum cyrtonema tubers, it is very important to select internal reference genes that are stably expressed at different development stages and in response to abiotic stress. According to the previously established P. cyrtonema transcriptome database and reported internal reference genes in plant, this study systematically analyzed eight candidate internal reference genes including histone H2 A, glyceraldehyde-3-phosphate dehydrogenase, ACTIN, ß-tubulin, ubiquitin-conjugating enzyme-E2-10, elongation factor 1-alpha isoform, 18 S rRNA and α-tubulin 4 for expression stability in P. cyrtonema tubers at different development stages and in response to methyl jasmonate(MeJA) stress by using Real time fluorescence quantitative PCR(qPCR). Based on the statistical analysis of qPCR results by using GeNorm, NormFinder and BestKeeper softwares, the expression of ubiquitin-conjugating enzyme-E2-10 and elongation factor 1-alpha isoform are the most stable in P. cyrtonema tubes at different development stages and in response to MeJA stress. The two internal reference genes were further validated by analyzing the expression of 4 genes involved in steroidal saponin biosynthesis pathways. In conclusion, ubiquitin-conjugating enzyme-E2-10 and elongation factor 1-alpha isoform can be used as the most appropriate internal reference genes for qPCR analysis in P. cyrtonema. This study also provide a foundation for future investigate the molecular mechanism of steroidal saponin biosynthesis pathways in P. cyrtonema.

[Effect of Co-composting of Chicken Manure with Chinese Medicinal Herbal Residues on Antibiotic Resistance Genes].

Livestock manure is an important pathway by which antibiotic resistance genes (ARGs) enter the environment. To reduce the occurrence of antibiotic resistance genes in manures, we studied the variations of ARGs and mobile gene elements (MGEs) during the 46-day co-composting of chicken manure with Chinese medicinal herbal residues. The abundance of 100 ARGs and 5 MGEs were measured by Real-Time Quantitative PCR with 0, 3, 7, 14, 28, and 46 d co-composting. We detected 21 ARGs, 2 integrase genes (intI1 and intI2), and 3 transposase genes (tnpA-01, tnpA-02, and tnpA-03). The abundance of 5 MGEs significantly declined with co-composting time, particularly tnpA-01 and tnpA-02, which were reduced by two orders of magnitude. The abundance of aacA/aphD and aadE were significantly reduced (P<0.05) in aminoglycoside resistance genes. In ß-lactam resistance genes, the strongest relationships were demonstrated between blaOXA1 and compost days (P=0.016), and the removal rate was 78.63%. The average removal rate was 90.39% for amide resistance genes, which decreased significantly with composting time. The removal ratios were different among tetracycline resistance genes. For example, the removal ratios of tetG and tetR were 99.77% and 31.72%, respectively. The highest removal rate of qnrD was 99.89%. The removal rate of sulⅢ was as high as 99.88%, while sulⅠ showed an increasing trend. Correlations between ARGs and MGEs were significant correlation for tnpA-01 and ARGs (P<0.05). The trend of ARGs with composting time indicates that the composting of Chinese medicine residues and chicken manure can significantly reduce the abundance of ARGs, thus reducing the risk of ARGs being distributed via livestock manure application.

[Marker genes of geniposide-induced hepatotoxicity based on genomic strategy].

The aim of this paper was to screen out relevant genes of geniposide-induced hepatotoxicity based on genomics,in order to provide a scientific basis for the non-clinical evaluation of drugs containing Gardeniae Fructus and geniposide. Fifty-five SD rats were randomly divided into normal control group,24 h group and 72 h group. The changes of appearance,behavior and weight of rats were observed after administration by gavage for 3 days. The activities of ALT and AST were detected. Molecular mechanism of geniposideinduced hepatotoxicity was investigated by Affymetrix miRNA 4. 0 and Affymetrix Rat Gene 2. 0 to examine the gene expression levels in Sprague-Dawley rat livers at 24 h and 72 h after administration of overdose-geniposide( 300 mg·kg-1 daily),and then verified by Realtime quantitative PCR. Compared with the normal control group,the activities of ALT and AST were markedly increased. In addition,experimental results indicated that 324 genes were differentially expressed,among which 259 were up-regulated and 65 down-regulated.Nine candidate genes were verified by qRT-PCR,including Bcl2,Il1 b,Tpm3,MMP2,Col1α1,Ifit1,Aldob,Nr0 b2,Cyp2 c23. And Bcl2,Col1α1,Aldob,Nr0 b2 and Cyp2 c23 were found to be correlated with geniposide-induced hepatotoxicity. This study provides an important clue for mechanism of geniposide-induced hepatotoxicity.

High-level secretive expression of a novel achieved Talaromyces cellulolyticus endo-polygalacturonase in Pichia pastoris by improving gene dosage for hydrolysis of natural pectin.

Pectin is a type of complex hydrophilic polysaccharide widely distributed in plant resources. Thermal stable pectinase has its advantage in bioapplication in the fields of food processing, brewing, and papermaking, etc. In this study, we enzymatically characterized a putative endo-polygalacturonase TcPG from a Talaromyces cellulolyticus, realized its high-level expression in Pichia pastoris by in vitro constructing of a series of multi-copy expression cassettes and real time quantitative PCR screening. The secretive expression level of TcPG was nonlinear correlated to the gene dosage. Recombinants with five-copy TcPG gene in the host genome showed the highest expression. After cultivation in a bioreactor for about 96 h, the enzyme activity reached 7124.8 U/mL culture. TcPG has its optimal temperature of 70 °C. Under the optimized parameters, the pectin could be efficiently hydrolyzed into oligosaccharides.

[Identification of stable reference gene by qRT-PCR in Senecio scandens].

As a traditional Chinese medicine, Senecio scandens is rich in important compounds such as flavonoid and sesquiterpenoid. Based on the transcriptome data of S. scandens, 15 candidate reference genes were selected including ABCT, ACT1, ACT2, ACT3, ACBP, ARF, ATPS, EF-H, EF-1α, ETIF, GAPDH, GTPB, MPS, UCE and 60S. Firstly, 9 candidate genes with relatively stable expressions such as ACT1, ACBP, ARF, ATPS, EF-1α, GAPDH, MPS, UCE and 60S were screened from different tissues of S. scandens by RT-PCR. Then, qRT-PCR was used to quantitatively analyze gene expression of these nine candidates in S. scandens with or without stress treatments. Further analysis of these gene expression data by geNorm and NormFinder showed that ACT1 exhibited the stablest expression in all samples and could serve as a reference gene for future study of S. scandens, and provide an endogenous control for gene expression analysis.

Selection of Reference Genes for Expression Analysis in Chinese Medicinal Herb Huperzia serrata.

Huperzine A (HupA) is a powerful and selective inhibitor of acetylcholinesterase. It has attracted widespread attention endangering the ultimate plant sources of Lycopodiaceae family. In this study, we used Huperzia serrata, extensively used in Traditional Chinese medicine (TCM), a slow growing vascular plant as the model plant of the Lycopodiaceae family to develop and validate the reference genes. We aim to use gene expression platform to understand the gene expression of different tissues and developmental stages of this medicinal herb. Eight candidate reference genes were selected based on RNA-seq data and evaluated with qRT-PCR. The expression of L/ODC and cytochrome P450s genes known for their involvement in lycopodium alkaloid biosynthesis, were also studied to validate the selected reference genes. The most stable genes were TBP, GAPDH, and their combination (TBP + GAPDH). We report for the first time the reference gene of H. serrata's different tissues which would provide important insights into understanding their biological functions comparing other Lycopodiaceae plants and facilitate a good biopharming approach.

Identification of stable reference gene by qRT-PCR in Senecio scandens / 中国中药杂志

As a traditional Chinese medicine, Senecio scandens is rich in important compounds such as flavonoid and sesquiterpenoid. Based on the transcriptome data of S. scandens, 15 candidate reference genes were selected including ABCT, ACT1, ACT2, ACT3, ACBP, ARF, ATPS, EF-H, EF-1α, ETIF, GAPDH, GTPB, MPS, UCE and 60S. Firstly, 9 candidate genes with relatively stable expressions such as ACT1, ACBP, ARF, ATPS, EF-1α, GAPDH, MPS, UCE and 60S were screened from different tissues of S. scandens by RT-PCR. Then, qRT-PCR was used to quantitatively analyze gene expression of these nine candidates in S. scandens with or without stress treatments. Further analysis of these gene expression data by geNorm and NormFinder showed that ACT1 exhibited the stablest expression in all samples and could serve as a reference gene for future study of S. scandens, and provide an endogenous control for gene expression analysis.

[Expression of saponin biosynthesis related genes in different tissues of Panax quinquefolius].

The relationship between saponin content of Panax quinquefolius in different parts of the organization and expression of ginsenoside biosynthesis related gene was obtained by the correlation analysis between saponin content and gene expression. The 14 tissue parts of P. quinquefolius were studied, six saponins in P. quinquefolius. Samples (ginsenoside Rg1, Re, Rb1, Rc, Rb2 and Rd), group saponins and total saponins were determined by high performance liquid chromatography and vanillin-sulfuric acid colorimetric method. Simultaneously, the expression levels of 7 ginsenoside biosynthesis related genes (SQS, OSC, DS, ß-AS, SQE, P450 and FPS) in different tissues of P. quinquefolius were determined by Real-time fluorescence quantitative PCR. Although 7 kinds of ginsenoside biosynthesis related enzyme gene in the P. quinquefolius involved in ginsenoside synthesis, the expression of ß-AS and P450 genes had no significant effect on the content of monosodium saponins, grouping saponins and total saponins, FPS, SQS, OSC, DS and SQE had significant or extremely significant on the contents of single saponins Re, Rg1, Rb1, Rd, group saponin PPD and PPT, total saponin TMS and total saponin TS (P<0.05 or P<0.01). The biosynthesis of partial saponins, grouping saponins and total saponins in P. quinquefolius was affected by the interaction of multiple enzyme genes in the saponin synthesis pathway, the content of saponins in different tissues of P. quinquefolius was determined by the differences in the expression of key enzymes in the biosynthetic pathway. Therefore, this study further clarified that FPS, SQS, OSC, DS and SQE was the key enzyme to control the synthesis of saponins in P. quinquefolius by correlation analysis, the biosynthesis of ginsenosides in P. quinquefolius was regulated by these five kind of enzymes in cluster co-expression of interaction mode.

Expression of saponin biosynthesis related genes in different tissues of / 中国中药杂志

The relationship between saponin content of in different parts of the organization and expression of ginsenoside biosynthesis related gene was obtained by the correlation analysis between saponin content and gene expression. The 14 tissue parts of were studied, six saponins in Samples (ginsenoside Rg₁, Re, Rb₁, Rc, Rb₂ and Rd), group saponins and total saponins were determined by high performance liquid chromatography and vanillin-sulfuric acid colorimetric method. Simultaneously, the expression levels of 7 ginsenoside biosynthesis related genes ( and ) in different tissues of were determined by Real-time fluorescence quantitative PCR. Although 7 kinds of ginsenoside biosynthesis related enzyme gene in the involved in ginsenoside synthesis, the expression of and P450 genes had no significant effect on the content of monosodium saponins, grouping saponins and total saponins, and had significant or extremely significant on the contents of single saponins Re, Rg1, Rb1, Rd, group saponin PPD and PPT, total saponin TMS and total saponin TS (<0.05 or <0.01). The biosynthesis of partial saponins, grouping saponins and total saponins in was affected by the interaction of multiple enzyme genes in the saponin synthesis pathway, the content of saponins in different tissues of was determined by the differences in the expression of key enzymes in the biosynthetic pathway. Therefore, this study further clarified that and was the key enzyme to control the synthesis of saponins in by correlation analysis, the biosynthesis of ginsenosides in was regulated by these five kind of enzymes in cluster co-expression of interaction mode.

Main predators of insect pests: screening and evaluation through comprehensive indices.

BACKGROUND: Predatory natural enemies play key functional roles in integrated pest management. However, the screening and evaluation of the main predators of insect pests has seldom been reported in the field. Here, we employed comprehensive indices for evaluating the predation of a common pest (Ectropis obliqua) by nine common spider species in Chinese tea plantations. RESULTS: We established the relative dominance of the spider species and their phenological overlap with the pest species, and analyzed DNA from the nine spider species using targeted real-time quantitative polymerase chain reaction to identify the residual DNA of E. obliqua. The predation rates and predation numbers per predator were estimated by the positive rates of target fragments and the residual minimum number of E. obliqua in predators' guts, respectively. The results showed that only four spider species preyed on E. obliqua, and the order of potential of the spiders to control E. obliqua from greatest to smallest was Neoscona mellotteei, Xysticus ephippiatus, Evarcha albaria and Coleosoma octomaculatum by the Z-score method. CONCLUSION: The orb-weaving spider N. mellotteei has the maximum potential as a biological control agent of E. obliqua in an integrated pest management strategy. An approach of screening and evaluating main predators of insect pests through comprehensive indices was preliminarily established. © 2017 Society of Chemical Industry.

Cloning and expression analysis of gibberellin 3-oxidase gene in Dendrobium officinale / 中草药

Objective: To clone the gibberellin 3-oxidase gene DoGA3ox from an important and endangered medicinal plant Dendrobium officinale (Orchidaceae), followed by bioinformatic and expression analysis. Methods: RACE and RT-PCR were used to isolate the full-length gene. The physicochemical properties, conserved domains, and subcellular localization of DoGA3ox protein were determined using a series of bioinformatic tools. The phylogenetic analyses were performed using DNASTAR 7.0 and MEGA 6.0 software. Real time quantitative PCR was employed for gene expression analysis. Results: The full-length cDNA of DoGA3ox (GenBank registration number KT597694) was 1 318 bp in size, and encoded a 353-amino acid peptide chain with a molecular weight of 39052.5 and an isoelectric point (pI) of 6.21; The deduced DoGA3ox protein without transmembrane or signal peptide residues, contained the gibberellin 3-oxidases conserved domains that DIOX_N (40-130) and 2OG-FeII_Oxy (197-299). DoGA3ox had 51%-56% similarity with GA3ox proteins from various plants, and was closely related to the monocot Allium fistulosum, Elaeis guineensis, and Phoenix dactylifera. The relative transcript levels of DoGA3ox were increased at stage 1, then decreased (stage 2), and increased again (stage 3) during D. officinale symbiotic/asymbiotic seed germination, the fold change to ungerminated seeds was 13.44 (symbiotic)/5.21 (asymbiotic), 7.28/2.32 and 9.40/6.21, respectively. Moreover, its transcript level was higher in symbiotic germination than that in the asymbiotic status. Conclusion: The full-length DoGA3ox gene is cloned for the first time, and its expression in different stages during seed germination indicates that DoGA3ox gene plays a crucial role in the regulation of seed germination in D. officinale.

Development and validation of a real-time quantitative PCR assay to detect Xanthomonas axonopodis pv. allii from onion seed.

Bacterial blight of onion is an emerging disease threatening world onion production. The causal agent Xanthomonas axonopodis pv. allii is seed transmitted and a reliable and sensitive tool is needed to monitor seed exchanges. A triplex quantitative real-time PCR assay was developed targeting two X. axonopodis pv. allii-specific markers and an internal control chosen in 5.8S rRNA gene from Alliaceae. Amplification of at least one marker indicates the presence of the bacterium in seed extracts. This real-time PCR assay detected all the 79 X. axonopodis pv. allii strains tested and excluded 85.2% of the 135 non-target strains and particularly all 39 saprophytic and pathogenic bacteria associated with onion. Cross-reactions were mainly obtained for strains assigned to nine phylogenetically related X. axonopodis pathovars. The cycle cut-off was estimated statistically at 36.3 considering a risk of false positive of 1%. The limit of detection obtained in at least 95% of the time (LOD 95%) was 5×10(3) CFU/g (colony forming unit/g). The sensitivity threshold was found to be 1 infected seed in 32,790 seeds. This real-time PCR assay should be useful for preventing the long-distance spread of X. axonopodis pv. allii via contaminated seed lots and determining the epidemiology of the bacterium.

Electroacupuncture in the repair of spinal cord injury: inhibiting the Notch signaling pathway and promoting neural stem cell proliferation.

Electroacupuncture for the treatment of spinal cord injury has a good clinical curative effect, but the underlying mechanism is unclear. In our experiments, the spinal cord of adult Sprague-Dawley rats was clamped for 60 seconds. Dazhui (GV14) and Mingmen (GV4) acupoints of rats were subjected to electroacupuncture. Enzyme-linked immunosorbent assay revealed that the expression of serum inflammatory factors was apparently downregulated in rat models of spinal cord injury after electroacupuncture. Hematoxylin-eosin staining and immunohistochemistry results demonstrated that electroacupuncture contributed to the proliferation of neural stem cells in rat injured spinal cord, and suppressed their differentiation into astrocytes. Real-time quantitative PCR and western blot assays showed that electroacupuncture inhibited activation of the Notch signaling pathway induced by spinal cord injury. These findings indicate that electroacupuncture repaired the injured spinal cord by suppressing the Notch signaling pathway and promoting the proliferation of endogenous neural stem cells.

An Optimal Medium Supplementation Regimen for Initiation of Hepatocyte Differentiation in Human Induced Pluripotent Stem Cells.

Human induced pluripotent stem (hiPS) cells are an ideal source for hepatocytes. Glucose and arginine are necessary for cells to survive. Hepatocytes have galactokinase (GALK), which metabolizes galactose for gluconeogenesis, and ornithine transcarbamylase (OTC), which converts ornithine to arginine in the urea cycle. Hepatocyte selection medium (HSM) lacks both glucose and arginine, but contains galactose and ornithine. Although human primary hepatocytes survive in HSM, all the hiPS cells die in 3 days. The aim of this study was to modify HSM so as to initiate hepatocyte differentiation in hiPS cells within 2 days. Hepatocyte differentiation initiating medium (HDI) was prepared by adding oncostatin M (10 ng/ml), hepatocyte functional proliferation inducer (10 nM), 2,2'-methylenebis (1,3-cyclohexanedione) (M50054) (100 µg/ml), 1× non-essential amino acid, 1× sodium pyruvate, nicotinamide (1.2 mg/ml), L-proline (30 ng/ml), and L-glutamine (0.3 mg/ml) to HSM. HiPS cells (201B7 cells) were cultured in HDI for 2 days. RNA was isolated, used as template for cDNA, and subjected to real-time quantitative polymerase chain reaction. Alpha-fetoprotein, γ-glutamyl transpeptidase, and delta-like 1 were upregulated. Expression of albumin was not observed. Expression of transcription factors specific to hepatocytes was upregulated. The expression of GALK2, OTC, and CYP3A4 were increased. In conclusion, differentiation of 201B7 cells to hepatoblast-like cells was initiated in HDI. Limitations were small number of cells were obtained, and the cells with HDI were not mature hepatocytes.

Molecular characterization, tissue distribution and feeding related changes of NUCB2A/nesfatin-1 in Ya-fish (Schizothorax prenanti).

The protein nucleobindin-2 (NUCB2) was identified over a decade ago and recently raised great interest as its derived peptide nesfatin-1 was shown to reduce food intake and body weight in rodents. However, the involvement of NUCB2 in feeding behavior has not well been studied in fish. In the present study, we characterized the structure, distribution, and meal responsive of NUCB2A/nesfatin-1 in Ya-fish (Schizothorax prenanti) for the first time. The full length cDNA of Ya-fish was 2140base pair (bp), which encoded a polypeptide of 487 amino acid residues including a 23 amino acid signal peptide. A high conservation in NUCB2 sequences was found in vertebrates, however the proposed propeptide cleavage site (Arg-Arg) conserved among other species is not present in Ya-fish NUCB2A sequence. Tissue distribution analysis revealed that Ya-fish NUCB2A mRNA was ubiquitously expressed in all test tissues, and abundant expression was detected in several regions including the hypothalamus, hepatopancreas, ovary and intestines. NUCB2A mRNA expression respond to feeding status change may vary and be tissue specific. NUCB2A mRNA levels significantly increased (P<0.05) in the hypothalamus and intestines after feeding and substantially decreased (P<0.01) during a week food deprivation in the hypothalamus. Meanwhile, NUCB2A mRNA in the hepatopancreas was significantly elevated (P<0.001) during food deprivation, and a similar increase was also found after short-time fasting. This points toward a potential hepatopancreas specific local role for NUCB2A in the regulation of metabolism during food deprivation. Collectively, these results provide the molecular and functional evidence to support potential anorectic and metabolic roles for NUCB2A in Ya-fish.