Characterization of the genomic and transcriptional structure of chicken NRG4 gene.

Neuregulin 4 (NRG4) is an important adipocytokine, which plays crucial roles in maintaining energy balance, regulating glucose and lipid metabolism, and preventing non-alcoholic fatty liver disease in mammals. At present, the genomic organization, transcript and protein isoforms of human NRG4 gene have been fully explored. Previous studies in our laboratory have shown that the NRG4 gene is expressed in chicken adipose tissue, but the chicken NRG4 (cNRG4) genomic structure, transcript and protein isoforms are still unknown. To this end, in this study, the genomic and transcriptional structure of the cNRG4 gene were systematically investigated using rapid amplification of cDNA ends (RACE) and reverse transcription-polymerase chain reaction (RT-PCR). The results showed that the coding region (CDS) of the cNRG4 gene was small, but it had a very complex transcriptional structure characterized by multiple transcription start sites, alternative splicing, intron retention, cryptic exons, and alternative polyadenylation, thus leading to production of four 5?UTR isoforms (cNRG4 A, cNRG4 B, cNRG4 C, and cNRG4 D) and six 3?UTR isoforms (cNRG4 a, cNRG4 b, cNRG4 c, cNRG4 d, cNRG4 e, and cNRG4 f) of the cNRG4 gene. The cNRG4 gene spanned 21,969 bp of genomic DNA (Chr.10:3,490,314~3,512,282) and consisted of 11 exons and 10 introns. Compared with the cNRG4 gene mRNA sequence (NM_001030544.4), two novel exons and one cryptic exon of the cNRG4 gene were identified in this study. Bioinformatics analysis, RT-PCR, cloning and sequencing analysis showed that the cNRG4 gene could encode three protein isoforms (cNRG4-1, cNRG4-2 and cNRG4-3). This study lays a foundation for further research on the function and regulation of the cNRG4 gene.

Genome-wide investigation and expression analysis of the AP2/ERF family for selection of agarwood-related genes in Aquilaria sinensis (Lour.) Gilg.

Aquilaria sinensis is an important non-timber tree species for producing high-value agarwood, which is widely used as a traditional medicine and incense. Agarwood is the product of Aquilaria trees in response to injury and fungal infection. The APETALA2/ethylene responsive factor (AP2/ERF) transcription factors (TFs) play important roles in plant stress responses and metabolite biosynthesis. In this study, 119 AsAP2/ERF genes were identified from the A. sinensis genome and divided into ERF, AP2, RAV, and Soloist subfamilies. Their conserved motif, gene structure, chromosomal localization, and subcellular localization were characterized. A stress/defense-related ERF-associated amphiphilic repression (EAR) motif and an EDLL motif were identified. Moreover, 11 genes that were highly expressed in the agarwood layer in response to whole-tree agarwood induction technique (Agar-Wit) treatment were chosen, and their expression levels in response to methyl jasmonate (MeJA), salicylic acid (SA), or salt treatment were further analyzed using the quantitative real time PCR (qRT-PCR). Among the 11 genes, eight belonged to subgroup B-3. All 11 genes were significantly upregulated under salt treatment, while eight genes were significantly induced by both MeJA and SA. In addition, the gene clusters containing these upregulated genes on chromosomes were observed. The results obtained from this research not only provide useful information for understanding the functions of AP2/ERF genes in A. sinensis but also identify candidate genes and gene clusters to dissect their regulatory roles in agarwood formation for future research.

[Panax notoginseng: a review on chemical components, chromatographic analysis, P. notoginseng extracts, and pharmacology in recent five years].

As a famous and precious Chinese medicinal material, Panax notoginseng(PN) has been commonly used for a long history in China. As reported, PN exhibits significant pharmacological actions in protecting cardiocerebral vascular system and nervous system and suppressing tumors. In recent years, with the innovation in ideas, as well as the development of methods and equipment, PN has been extensively investigated, and notable progress has been made. This paper reviewed the advancements of PN in recent five years from chemical components, chromatographic analysis, P. notoginseng extracts, and pharmacology, in which the application of PN extracts in quality control was first summarized. The present study aims to provide a theoretical basis for quality control, product development, and rational medication of PN.

Treatment with metformin and sorafenib alleviates endometrial hyperplasia in polycystic ovary syndrome by promoting apoptosis via synergically regulating autophagy.

This study aimed to investigate the molecular mechanisms underlying the roles of metformin (MET) and Sorafenib (SOR) in the treatment of endometrial hyperplasia (EH) in polycystic ovary syndrome (PCOS). Effects of MET and SOR on the area of endometrium and myometrium were detected. Western blot analysis and immunohistochemistry assays were carried out to detect the levels of mammalian target of rapamycin complex 1 (mTORC1), mTORC2, LC3-II, P62, and Caspase-3 in rats and cultured cells. Furthermore, cell counting kit-8 assay and flow cytometry analysis was carried out to determine the apoptotic profiles of treated cells. MET and SOR could apparently decrease the areas of endometrium and myometrium in PCOS. MET notably enhanced the expression of LC3-II and Caspase-3 in PCOS while substantially reducing the level of mTORC1 and P62. Similarly, SOR also enhanced the expression of LC3-II and Caspase-3 in PCOS while substantially reducing the level of mTORC2 and P62. Treatment with MET and SOR significantly inhibited the proliferation of HCC-94 and HEC-1-A cells while promoting their apoptosis by upregulating the expression of Caspase-3. In cells treated with MET, the expression of mTORC1 and LC3-II was upregulated while the expression of P62 was downregulated. Similarly, in cells treated with SOR, the expression of mTORC2 and LC3-II was also upregulated while the expression of P62 was also downregulated. Furthermore, MET showed no effect on mTORC2 expression, while SOR showed no effect on mTORC1 expression. In this study, we suggested that MET and SOR alleviated the risk of EH in PCOS via the mTORC1/autophagy/apoptosis axis and mTORC2/autophagy/apoptosis axis, respectively.

WRKY44 represses expression of the wound-induced sesquiterpene biosynthetic gene ASS1 in Aquilaria sinensis.

Agarwood is derived from wounds in Aquilaria trees and is widely used in traditional medicine, incense, and perfume. Sesquiterpenes are one of the main active components in agarwood and are known to be induced by wounding or injury; However, the molecular mechanisms by which wounding leads to sesquiterpene formation remain largely unknown. Agarwood sesquiterpene synthase 1 (ASS1) is one of key enzymes responsible for the biosynthesis of sesquiterpenes and is a crucial jasmonate (JA)-responsive wound-inducible synthase. However, it is not known why ASS1 is not expressed in healthy trees and how its expression is induced as a result of wounding. Here, we report that ASS1 is a wound-induced gene with a promoter in which a 242-bp region (-973 to -731bp) is identified as the core sequence for responding to wound signals. AsWRKY44 binds directly to this region and represses ASS1 promoter activity. Down-regulation or disruption of AsWRKY44 can relieve the inhibition and activate ASS1 expression. In addition, AsWRKY44 is degraded and the expression of ASS1 is significantly up-regulated in response to exogenous application of methyl jasmonate. Thus, AsWRKY44 is a crucial negative regulator of wound-induced ASS1 transcription, and is central to the mechanism of sesquiterpene biosynthesis in agarwood.

[Characterization of AOX family members from Aquilaria sinensis and their responses to wounding].

Aquilaria sinensis is a typical inducible medicinal plant, that can produce agarwood only after it is wounded by external stimuli. Alternative oxidase(AOX) is one of the terminal oxidases of the plant mitochondrial electron transport, which plays an important role in plants' response to environmental stress. In order to reveal the physiological function of AOX gene in the process of agarwood formation from A.sinensis induced by wounding, AOX gene was cloned based on the transcriptome database and then identified by the bioinformatics analysis, and their expression pattern in different tissues and under wounding stress were detected by qRT-PCR. The results as follows. Three AOX genes were cloned from A.sinensis for the first time. They were named AsAOX1a, AsAOX1d and AsAOX2, respectively. The tissue expression shown that AsAOX1a is mainly expressed in the stem and the seed, and the AsAOX1d and AsAOX2 genes are mainly expressed in the pulp and the stem. AsAOX1a and AsAOX1d genes are highly responsive to wounding stress, and their response time was different. In addition, the expression of AsAOX1a and AsAOX2 induced by wounding are reduced by H_2O_2 treatment, but promoted by AsA treatment. The cloning, bioinformatics analysis and expression characteristics of AOX genes from A.sinensis provided basic information for further study the function of AOX genes in the development of A.sinensis, especially in the process of agarwood formation of A. sinensis induced by wounding.

[Advances in study of plant type â ¢ polyketide synthases].

Polyketides are a large class of natural products with notable structural diversity and different biological activities. They have essential pharmacological value for human health. In plants, the enzymes responsible for the formation of phenolic metabolites backbone structures are collectively known as type Ⅲ polyketide synthases (PKSs), which are the key enzymes for the polyketides biosynthesis. The PKSs catalyze a series of condensation reactions of two-carbon acetate units with an acyl starter. A brief overview of this group of enzymes, including their reaction mechanisms, function modification, expression regulation, molecular evolution, and recent interesting findings are presented here.

Transcription Factor AsMYC2 Controls the Jasmonate-Responsive Expression of ASS1 Regulating Sesquiterpene Biosynthesis in Aquilaria sinensis (Lour.) Gilg.

Sesquiterpenes are one of the most important defensive secondary metabolite components of agarwood. Agarwood, which is a product of the Aquilaria sinensis response to external damage, is a fragrant and resinous wood that is widely used in traditional medicines, incense and perfume. We previously reported that jasmonic acid (JA) plays an important role in promoting agarwood sesquiterpene biosynthesis and induces expression of the sesquiterpene synthase ASS1, which is a key enzyme that is responsible for the biosynthesis of agarwood sesquiterpenes in A. sinensis. However, little is known about this molecular regulation mechanism. Here, we characterized a basic helix-loop-helix transcription factor, AsMYC2, from A. sinensis as an activator of ASS1 expression. AsMYC2 is an immediate-early jasmonate-responsive gene and is co-induced with ASS1. Using a combination of yeast one-hybrid assays and chromatin immunoprecipitation analyses, we showed that AsMYC2 bound the promoter of ASS1 containing a G-box motif. AsMYC2 activated expression of ASS1 in tobacco epidermis cells and up-regulated expression of sesquiterpene synthase genes (TPS21 and TPS11) in Arabidopsis, which was also promoted by methyl jasmonate. Our results suggest that AsMYC2 participates in the regulation of agarwood sesquiterpene biosynthesis in A. sinensis by controlling the expression of ASS1 through the JA signaling pathway.

EFFECT OF COLLA CORNUS CERVI COMBINED WITH LV-MEDIATED BMP7 TRANSFECTED BMSCs ON ANFH IN RATS.

In the present study, we investigated the combined effect of Colla Comus Cervi (CCC) and BMP7-overexpressing bone marrow-derived mesenchymal stem cells (BMSCs) on osteogenic induction and the treatment of avascular necrosis of the femoral head (ANFH). BMSCs were isolated from rats. BMP7-overexpressing BMSCs were generated by lentiviral-mediated gene transduction. Cell proliferation, alkaline phosphatase (ALP) activity, osteogenesis related gene expression, osteocalcin levels, and calcified nodules were quantified and compared between four groups: untreated controls, BMSCs cultured with CCC complex medium, BMP7-overexpressing BMSCs, and BMP7-overexpressing BMSCs cultured with CCC complex medium (CCC+BMP7). CCC+BMP7 BMSCs showed higher proliferation rate. ALP activity and osteaocalcin content were significantly increased in CCC+BMP7 BMSCs. The osteogenesis related genes, COLI, and integrin-α2, -α5, and -ß1, were expressed significantly higher in CCC+BMP7 BMSCs. The number of calcified nodules in the CCC+BMP7 group was significantly higher than that in other groups. For in vivo assays, ANFH was induced in rats, and BMSCs were injected into the femoral head of the lower left extremity. In rats with induced ANFH, general observation scores of the CCC+BMP7 injected group were significantly higher than the model group. X-ray and microscopic observations revealed that ANFH was significantly improved and femoral head cells gradually recovered in rats treated with CCC+BMP7 BMSCs. Our results suggest that CCC+BMP7 significantly promote the proliferation and osteogenic differentiation of BMSCs in vitm. CCC+BMP7 BMSCs promote the ability of repairing ANFH in rats, providing a new therapeutic paradigm for the treatment of ANFH.

Curative effect of paclitaxel and cisplatin combined chemotherapy on cervical cancer and its relation with tissue micro vascular and lymphatic vessels density.

This study was to discuss the curative effect of paclitaxel and cisplatin combined chemotherapy on cervical cancer and its relation with tissue micro vascular and lymphatic vessels density. The combined chemotherapy of paclitaxel 135 mg/m² and cisplatin 25mg/m² were taken to observe the clinical curative effect. The postoperative paraffin tissue had been collected, had performed the LYVE-1 (lymphatic endothelium specific hyaluronan receptor-1) and CD31 immunohistochemical staining. The complete remission rate of high micro lymphatic vessels density group (was or more 6.0) and high micro vascular density group were obviously higher than in low micro lymphatic vessels density group and low micro vascular density group, the difference was statistically significance (P<0.05). This study further analyzed the relation of MVD and LVD with clinical pathological parameters. The difference was statistically significant (P<0.01). The curative effect of paclitaxel and cisplatin combined therapy was promising, positive and was closely related with cervical cancer tissue LVD and MVD. The LVD and MVD could be one of the predictors of early cervical CIN and early cervical cancer development.

[Cloning and gene expression of acetyl-CoA C-acetyl transferase gene (AsAACT) from Aquilaria sinensis].

OBJECTIVE: This study aimed to clone the acetyl-CoA C-acetyl transferase (AACT) gene from Aquilaria sinensis and analyze the bioinformatics and expression of the gene. METHOD: One unique sequence containing partly AACT gene sequence was discovered in our previous transcriptome dataset of A. sinensis. AACT gene was cloned by RT-PCR and RACE strategy with the template of RNA extracted from A. sinensis stem. The bioinformatic analysis of this gene and its corresponding protein was performed. The AsAACT expression in calli was analyzed with GADPH gene as an internal control gene in wounded condition by qRT-PCR technique. RESULT: One unique sequence of AACT, named as AsAACT, was cloned from A. sinensis. The full length of AsAACT cDNA was containing a 1 236 bp ORF that encoded 411 amino acids. The result of qRT-PCR displayed that the highest expression level was at 4 h. which indicated that it was possibly involved in early-stage response to wound. CONCLUSION: Cloning and analyzing AsAACT gene from A. sinensis provided basic information for study the function and expression regulation of AsAACT in terpenoid biosynthesis.

[Study of production of sesquiterpenes of Aquilaria senensis stimulated by Lasiodiplodia theobromae].

To investigate the mechanism of agarwood formation in Aquilaria sinensis induced by Lasiodiplodia theobromae, the fermentation liquor of L. theobromae was analyzed qualitatively and quantitatively by gas chromatography-mass spectrometry (GC-MS). JAs were detected in the fermentation liquor. The effect of the fermentation liquor on the abundance of sesquiterpenes in the callus of A. sinensis was analyzed by solid phase microextraction-gas chromatography-mass spectrometry (SPME-GC-MS). And the fermentation liquor stimulated alpha-guaiene, alpha-humulene and delta-guaiene biosynthesis in calli. It was inferred that L. theobromae produced JAs, which resulted in a significant increase of sesquiterpenes in A. sinensis.

[Analysis of mutations in exon 7 of phenylalanine hydroxylase gene among children with phenylketonuria in Ningxia, China].

OBJECTIVE: To investigate the type and frequency of mutations in exon 7 of phenylalanine hydroxylase (PAH) gene among children with phenylketonuria (PKU) in Ningxia, China and to provide a basis for the genetic diagnosis and prenatal diagnosis of PKU in this region. METHODS: Direct sequencing of PCR product was performed to analyze the sequences of exon 7 and its flanking introns of 146 PAH alleles in 73 children with typical PKU (39 cases of Hui nationality and 34 cases of Han nationality) in Ningxia. RESULTS: Six mutations were detected, including R243Q (14.4%), R241C (6.8%), IVS7+2T→A (2.7%), L255S (0.7%), G247V (0.7%), and G247R (0.7%). The overall frequency of mutations (missense mutation and splice site mutation) in exon 7 was 26.0% (38/146). The detection rate of R241C mutation was significantly higher in children of Hui nationality than in children of Han nationality(10% vs 3%; P<0.05). CONCLUSIONS: In Ningxia, R243Q mutation in exon 7 of PAH gene is most common in children with PKU, followed by R241C. The frequency of R241C mutation in exon 7 of PAH gene varies between children with PKU of Hui and Han nationality.

Selection and validation of reference genes for studying stress-related agarwood formation of Aquilaria sinensis.

UNLABELLED: Agarwood is a high-valued woody material for medicine, perfume, and incense production in Asia, Middle East, and Europe. The wild resources of agarwood-producing tree species, e.g., Aquilaria sinensis have been greatly threatened. The formation of agarwood is considered to be associated with the plant stress and defensive responses, thus it would be urgent and significant to investigate the molecular mechanism of these species responding to a variety of stresses. This is the first report regarding the reference gene selection of Aquilaria species for studying the molecular mechanism of stress-related agarwood production. Candidate reference genes were selected according to previous reports and the sequences were obtained from the 454 EST library of A. sinensis. To obtain the robust genes, we applied three independent programs depending on distinct assumptions and combined these results by a rank aggregation algorithm. The result supports tubulin, ribosomal protein, and glyceraldehyde-3-phosphate dehydrogenase to be the most stable reference genes for quantification of target gene expression in the overall samples examined. Validation of these genes through normalizing the expression of a terpene synthase demonstrated that these three genes are reliable. The selective usage of three algorithms based on their characteristics was underlined. However, more robust genes could be identified if the results of all algorithms were combined by a proper method such as the rank aggregation algorithm. KEY MESSAGE: Reference genes which are critical in gene expression studies are recommended for future molecular studies of stress response and agarwood production in the endangered Aquilaria and other tree species.

[The mechanical wound transcriptome of three-year-old Aquilaria sinensis].

Chinese agarwood is formed in the aromatic resinous wood formed in Aquilaria sinensis (Lour.) Gilg (botanical family: Thymelaeaceae). Only when suffering stress of wound, etc, can A. sinensis produce sesquiterpenes etc. compounds of agarwood around wounds. However, little is known about how wound induced the biosynthesis pathway of sesquiterpenes. To reveal the molecular mechanism of wound-induced agarwood formation, RNA sequencing (RNA-seq) technology was used to investigate the profile of gene expression in A. sinensis treated by mechanical wounding and elucidate its functional gene. A total of 40,295 ESTs with an average read length of 305 bp were generated and 22 095 unigenes were formed by initial gene splicing. 61.6% of these unigenes (13 611) were annotated using BLAST searches against the SwissProt, KEGG, Nr and Nt databases. Twenty-six unigenes (encoding 7 enzymes) were found to be involved in sesquiterpene of agarwood biosynthesis by bioinformatic tools of Gene Ontology and KEGG. Novel genes that are potentially involved in sesquiterpenes biosynthesis were identified in A. sinensis, providing data for further sesquiterpenes biosynthesis pathway by molecular methods and the EST data establish a foundation for future studies in the molecular mechanisms of wound-induce agarwood formation in A. sinensis.

Genome-wide analysis of basic helix-loop-helix (bHLH) transcription factors in Aquilaria sinensis.

The basic helix-loop-helix (bHLH) transcription factors are involved in several biological processes both in plant development and stress responses. Agarwood, a major active and economical product, is only induced and accumulated when the roots, stems, or branches are wounded in Aquilaria sinensis. Although genome-wide comprehensive analyses of the bHLH family have been identified in many plants, no systematic study of the genes in this family has been conducted in A. sinensis. In this study, 105 bHLH genes were identified in A. sinensis through genome-wide analysis and named according to their chromosomal locations. Based on a phylogenetic tree, AsbHLH family proteins were classified into 18 subfamilies. Most of them were distributed on eight chromosomes, with the exception of two genes. Based on the tissue-specific expression characteristics and expression patterns in response to methyl jasmonate (MeJA) treatment, seven AsbHLH genes were likely involved in wound-induced agarwood formation. The results provide comprehensive information on AsbHLHs that can be used to elucidate the molecular functions and physiological roles of these proteins in A. sinensis.

Genome-wide analysis of WRKY transcription factors in Aquilaria sinensis (Lour.) Gilg.

The WRKY proteins are a superfamily of transcription factor that regulate diverse developmental and physiological processes in plants. Completion of the whole-genome sequencing of Aquilaria sinensis allowed us to perform a genome-wide investigation for WRKY proteins. Here, we predicted 70 WRKY genes from the A. sinensis genome and undertaken a comprehensive bioinformatic analysis. Due to their diverse structural features, the 70 AsWRKY genes are classified into three main groups (group I-III), with five subgroups (IIa-IIe) in group II, except two belong to none of them. Distinct expression profiles of AsWRKYs with RNA sequencing data revealed their diverse expression patterns among different tissues and in the process of whole-tree-inducing agarwood formation. Based on the expression characteristics, we predict some AsWRKYs are pseudogenes, and some may be involved in the biosynthesis of agarwood sesquiterpenes as activators or repressors. Among the tested genes treated with MeJA and H2O2, most of them are induced by H2O2, but downregulated by MeJA, implying the complexity of their involvement in signal transduction regulation. Our results not only provide a basic platform for functional identification of WRKYs in A. sinensis but important clues for further analysis their regulation role in agarwood formation.

The Separation of Antler Polypeptide and Its Effects on the Proliferation and Osteogenetic Differentiation of Bone Marrow Mesenchymal Stem Cells.

BACKGROUND: Colla Cornus Cervi (CCC) has been used as a traditional Chinese medicine in the treatment of osteoporosis and osteonecrosis of the femoral head. However, the bioavailability of CCC is seriously limited owing to its large molecular weight and complex ingredients. In the present study, antler polypeptide was separated from CCC, and the effects of antler polypeptide on rat bone marrow mesenchymal stem cells (BMSCs) were investigated. METHODS: Antler polypeptide was separated from Colla Cornus Cervi by ultrafiltration into different samples according to the molecular weight. The total peptide content of these samples was determined by the biuret method. The content of antler polypeptide in different samples was quantified by high-performance liquid chromatography (HPLC). The effects of antler polypeptide at different concentrations on the proliferation, cell cycle, alkaline phosphatase activity, and BMP7 expression of BMSCs were investigated. RESULTS: Antler polypeptide was separated by ultrafiltration into different samples: A (molecular weight <800 Da), B (molecular weight 800-1500 Da), and C (molecular weight >1500 Da). The total peptide contents of A, B, and C were 0.602 mg/mL, 8.976 mg/mL, and 38.88 mg/mL. Antler polypeptide B eluted at 14.279∼15.351 min showed that the content of antler polypeptide was significantly higher than that of A and C with a peak area of 933.80927. The BMSCs proliferation rate (84.66%) of polypeptide B was the highest at the concentration of 1.578 × 10-2 g/mL. Antler polypeptide B significantly promoted the proliferation of BMSCs with a proliferation index of 38.68%, which was significantly higher than that of the other groups. Antler polypeptide B significantly enhanced the activity of alkaline phosphatase in BMSCs compared to that of the blank group (P < 0.001). Antler polypeptide B increased the BMP7 protein expression in BMSCs. CONCLUSIONS: Results suggested that antler polypeptide may promote the proliferation and osteogenic differentiation of BMSCs. Our study lays an experimental foundation for the further development and application of antler polypeptide in medicine.

Selection and Validation of Reference Genes for Gene Expression Studies by RT-PCR in Dalbergia odorifera.

Perennial tree Dalbergia odorifera T. Chen could form the precious heartwood used to produce chinese traditional medicine, rosewood furniture and fragrances. However the formation of heartwood is time-consuming and low efficient, leading to the severe destruction of its wild resources. Thus, it is urgent to study the molecular mechanism of heartwood formation in D. odorifera. But till now, there is no report about the reference gene selection in this species. In this study, the expression stability of nine candidate reference genes were evaluated across different tissues and stems treated by wound and chemical stimulators. Four algorithms were applied to obtain the robust genes. The results support HIS2, GAPDH, and CYP to be the most stable reference genes in samples under different wound treatments while DNAj was the least stable. In different tissues, HIS2, UBQ, and RPL were the most stable reference genes while DNAj was the least stable. The selected reference genes were validated through the normalization of the qRT-PCR data of six heartwood related genes in terpene biosynthesis pathway and ethylene signal pathway. The results showed that their expression levels were accurate when they were normalized by the most stable reference gene HIS2, or by the combination of the two or three most stable reference genes. These results demonstrated that these selected reference genes are reliable.