Constructing the in vitro culture system of the sika deer (cervus nippon) antler periosteal cell to detect its function on antler regeneration.

Periosteum is essential for bone regeneration and damage repair in mammals. Most species of deer family (Cervidae) develop two kinds of special periosteum, antler periosteum and pedicle periosteum, both supporting the complete regeneration of antler. Antler is the bone organ with the fastest growth rate in mammals. Along with the fast growth of antler, its external tissues such as blood vessels, nerves and the covering skin also grow rapidly. Currently, it is still unclear whether antler periosteum contributes to the fast growth of antler and how. It is also unclear why the regenerative capacity of antler periosteum is weaker than that of pedicle periosteum. In this study, the in vitro culture system for antler periosteal cells (AnPC) was constructed for the first time using the mid-beam antler periostea during antler fast-growth period. According to our results, the cultured AnPC expressed classical MSC markers, consistent with the pedicle periosteal stem cells (PPSC). However, the fluorescence intensities of the MSC markers on AnPC were significantly weaker than those on PPSC. In addition, AnPC showed much lower proliferation rates than PPSC. The proliferation rates of the AnPC also gradually decreased after successive passages, while the proliferation rates of the pedicle periosteal stem cells remained unchanged. These findings may partially explain the weaker regenerative capacity of antler periosteum. Further comparative global gene analysis revealed clearly the different gene expressed patterns between AnPC and PPSC. AnPC may mainly function on promoting angiogenesis, nerve growth and intramembrane bone formation during antler regeneration, whereas PPSC may primarily be involved in androgen signaling receptor pathway and PI3K-Akt signaling pathway and function on maintaining stem cell renewal.

Omics analyses indicate the routes of lignin related metabolites regulated by trypsin during storage of pitaya (Hylocereus undatus).

The storage quality of Hylocereus undatus was significantly improved by trypsin, a novel preservative. The transcriptomic results revealed that antioxidant signal pathways were induced, while lignin catabolic process was impeded by trypsin. In addition, the results of protein-protein interaction (PPI) network networks suggested that flavone 3'-O-methyltransferase 1 (OMT1), ferulic acid 5-hydroxylase 1 (CYP84A1), cellulose synthase isomer (CEV1), and 4-coumarate-CoA ligase 3 (4CL3) act as hubs of peroxidases, lignin related proteins, and proteins involved in the phenylpropanoid metabolism (PLPs) induced by trypsin. Trypsin also regulated the biosynthesis of lignin, chlorogenic acid, and flavonoids. Caffeic acid might be the hub in the metabolic network of the early pathways of phenylpropanoid biosynthesis. It has been hypothesized that trypsin might quickly induce lignin biosynthesis and then up-regulated bioactive metabolites to enhance storage quality of H. undatus.

Detecting the Mechanism behind the Transition from Fixed Two-Dimensional Patterned Sika Deer (Cervus nippon) Dermal Papilla Cells to Three-Dimensional Pattern.

The hair follicle dermal papilla is critical for hair generation and de novo regeneration. When cultured in vitro, dermal papilla cells from different species demonstrate two distinguishable growth patterns under the conventional culture condition: a self-aggregative three dimensional spheroidal (3D) cell pattern and a two dimensional (2D) monolayer cell pattern, correlating with different hair inducing properties. Whether the loss of self-aggregative behavior relates to species-specific differences or the improper culture condition remains unclear. Can the fixed 2D patterned dermal papilla cells recover the self-aggregative behavior and 3D pattern also remains undetected. Here, we successfully constructed the two growth patterns using sika deer (Cervus nippon) dermal papilla cells and proved it was the culture condition that determined the dermal papilla growth pattern. The two growth patterns could transit mutually as the culture condition was exchanged. The fixed 2D patterned sika deer dermal papilla cells could recover the self-aggregative behavior and transit back to 3D pattern, accompanied by the restoration of hair inducing capability when the culture condition was changed. In addition, the global gene expressions during the transition from 2D pattern to 3D pattern were compared to detect the potential regulating genes and pathways involved in the recovery of 3D pattern and hair inducing capability.

Antitumor activity and mechanism of resistance of the novel HDAC and PI3K dual inhibitor CUDC-907 in pancreatic cancer.

PURPOSE: Pancreatic cancer is a highly malignant disease with an extremely poor prognosis. The benefit of chemotherapy treatment for pancreatic cancer is very limited. Therefore, new therapeutic targets and approaches are urgently needed for this deadly disease. Multi-target therapy is a potential and feasible treatment strategy. Given the important roles that histone deacetylases (HDACs) and phosphoinositide-3-kinase (PI3K) play in pancreatic cancer, we investigated the antitumor activity and mechanism of novel HDAC and PI3K dual inhibitor CUDC-907 in pancreatic cancer. METHODS AND RESULTS: MTT assay and flow cytometric analysis were used to examine the in vitro antitumor activity of CUDC-907. A BxPC-3-derived xenograft mouse model was used to determine CUDC-907 in vivo efficacy. The TUNEL assay as used to determine apoptosis in tumors in vivo post CUDC-907 treatment. Western blots were used to determine the effect of CUDC-907 on protein levels. Our results show that CUDC-907 decreased viable cells and induced cell death in a concentration-dependent manner. Furthermore, CUDC-907 showed promising in vivo antitumor activity in the BxPC-3-derived xenograft mouse model while exhibiting tolerable toxicity. Furthermore, long-term treatment with CUDC-907 induced phosphorylation of AKT, S6 (ribosomal protein S6), and ERK (extracellular regulated protein kinase), and inhibition of PI3K (phosphatidylinositol 3-kinase), mTOR (mammalian target of rapamycin), or ERK significantly enhanced CUDC-907-induced cell death in pancreatic cell lines. CONCLUSION: Taken together, these findings support the clinical development of CUDC-907 for the treatment of pancreatic cancer and identify compensatory activation of mTOR and MEK/ERK as a possible mechanism of resistance to CUDC-907.

Catechin gallate acts as a key metabolite induced by trypsin in Hylocereus undatus during storage indicated by omics.

Trypsin is a novel superoxide scavenger. The storage quality of H. undatus was significantly improved by trypsin. To investigate the mechanism of flavonoid metabolism regulated by trypsin, combined analysis of widely targeted metabolomic and transcriptome were performed. GO and KEGG enrichment analyses of the transcriptome profiles of H. undatus revealed that some of the flavonoid related biosynthesis pathways were regulated by up or down patterns with the treatment of trypsin. Correlation analysis of flavonoid related genes expression in H. undatus provided a rationale for the functional significance of them. Furthermore, it has been revealed that the most significantly regulated flavonoid was catechin gallate in metabolomic profiles of H. undatus. The major route of flavonoid biosynthesis regulated by trypsin was also illustrated by both transcriptomic and metabolomic data. Finally, the results of PPI network revealed that C4H, HCT, and CYP75B1 acted as hub proteins involved in flavonoid metabolism regulated by trypsin.

Functional characterization of a WRKY family gene involved in somatic embryogenesis in Panax ginseng.

As a perennial herbaceous species, Panax ginseng is widely cultivated and used as traditional herbal medicine. The root of Panax ginseng commonly remains expensive as conventional breeding of Panax ginseng is difficult. Somatic embryogenesis (S.E.) is a useful tool for plant propagation and optimal model for understanding the mechanisms of plant embryogenesis. In Panax ginseng, increasing studies have been widely performed to optimize the technology of S.E., while the underlying mechanism remains unclear. In this paper, we cloned and identified a WRKY family gene named PgWRKY6 which is upregulated in response to 2,4-D (2,4-dichlorophenoxyacetic acid)-induced embryogenic callus development. The silencing of PgWRKY6 obviously reduces the induction rate of embryogenic callus, indicating its crucial role in S.E. of Panax ginseng hairy root. The expressions of several ROS-scavenging genes are also inducible during embryogenic callus development, and the transcriptions of PgGST, PgAPX1, and PgSOD are demonstrated to be regulated by PgWRKY6. Recombinant PgWRKY6, an approximate 40-KDa protein purified from Escherichia coli, shows a specific DNA-binding activity with a potential recognition site of TTGAC(C/T). This work demonstrated that as a conserved WRKY family transcription factor, PgWRKY6 functions upstream of PgGST, PgAPX1, and PgSOD, and potentially mediated auxins -ROS signaling pathway in the process of S.E. in Panax species.

Inhibition of Bcl-2 Synergistically Enhances the Antileukemic Activity of Midostaurin and Gilteritinib in Preclinical Models of FLT3-Mutated Acute Myeloid Leukemia.

PURPOSE: To investigate the efficacy of the combination of the FLT3 inhibitors midostaurin or gilteritinib with the Bcl-2 inhibitor venetoclax in FLT3-internal tandem duplication (ITD) acute myeloid leukemia (AML) and the underlying molecular mechanism. EXPERIMENTAL DESIGN: Using both FLT3-ITD cell lines and primary patient samples, Annexin V-FITC/propidium iodide staining and flow cytometry analysis were used to quantify cell death induced by midostaurin or gilteritinib, alone or in combination with venetoclax. Western blot analysis was performed to assess changes in protein expression levels of members of the JAK/STAT, MAPK/ERK, and PI3K/AKT pathways, and members of the Bcl-2 family of proteins. The MV4-11-derived xenograft mouse model was used to assess in vivo efficacy of the combination of gilteritinib and venetoclax. Lentiviral overexpression of Mcl-1 was used to confirm its role in cell death induced by midostaurin or gilteritinib with venetoclax. Changes of Mcl-1 transcript levels were assessed by RT-PCR. RESULTS: The combination of midostaurin or gilteritinib with venetoclax potently and synergistically induces apoptosis in FLT3-ITD AML cell lines and primary patient samples. The FLT3 inhibitors induced downregulation of Mcl-1, enhancing venetoclax activity. Phosphorylated-ERK expression is induced by venetoclax but abolished by the combination of venetoclax with midostaurin or gilteritinib. Simultaneous downregulation of Mcl-1 by midostaurin or gilteritinib and inhibition of Bcl-2 by venetoclax results in "free" Bim, leading to synergistic induction of apoptosis. In vivo results show that gilteritinib in combination with venetoclax has therapeutic potential. CONCLUSIONS: Inhibition of Bcl-2 via venetoclax synergistically enhances the efficacy of midostaurin and gilteritinib in FLT3-mutated AML.See related commentary by Perl, p. 6567.

Functional regulation of ginsenoside biosynthesis by RNA interferences of a UDP-glycosyltransferase gene in Panax ginseng and Panax quinquefolius.

Panax ginseng (Asian ginseng) and Panax quinquefolius (American ginseng) have been used as medicinal and functional herbal remedies worldwide. Different properties of P. ginseng and P. quinquefolius were confirmed not only in clinical findings, but also at cellular and molecular levels. The major pharmacological ingredients of P. ginseng and P. quinquefolius are the triterpene saponins known as ginsenosides. The P. ginseng roots contain a higher ratio of ginsenoside Rg1:Rb1 than that in P. quinquefolius. In ginseng plants, various ginsenosides are synthesized via three key reactions: cyclization, hydroxylation and glycosylation. To date, several genes including dammarenediol synthase (DS), protopanaxadiol synthase and protopanaxatriol synthase have been isolated in P. ginseng and P. quinquefolius. Although some glycosyltransferase genes have been isolated and identified association with ginsenoside synthesis in P. ginseng, little is known about the glycosylation mechanism in P. quinquefolius. In this paper, we cloned and identified a UDP-glycosyltransferase gene named Pq3-O-UGT2 from P. quinquefolius (GenBank accession No. KR106207). In vitro enzymatic activity experiments biochemically confirmed that Pq3-O-UGT2 catalyzed the glycosylation of Rh2 and F2 to produce Rg3 and Rd, and the chemical structure of the products were confirmed susing high performance liquid chromatography electrospray ionization mass spectrometry (HPLC/ESI-MS). High sequence similarity between Pq3-O-UGT2 and PgUGT94Q2 indicated a close evolutionary relationship between P. ginseng and P. quinquefolius. Moreover, we established both P. ginseng and P. quinquefolius RNAi transgenic roots lines. RNA interference of Pq3-O-UGT2 and PgUGT94Q2 led to reduce levels of ginsenoside Rd, protopanaxadiol-type and total ginsenosides. Expression of key genes including protopanaxadiol and protopanaxatriol synthases was up-regulated in RNAi lines, while expression of dammarenediol synthase gene was not obviously increased. These results revealed that P. quinquefolius was more sensitive to the RNAi of Pq3-O-UGT2 and PgUGT94Q2 when compared with P. ginseng.

Functional analysis of ß-amyrin synthase gene in ginsenoside biosynthesis by RNA interference.

KEY MESSAGE: Down-regulation of ß-amyrin synthase gene expression by RNA interference led to reduced levels of ß-amyrin and oleanane-type ginsenoside as well as up-regulation of dammarane-type ginsenoside level. In the biosynthetic pathway of ginsenosides, ß-amyrin synthase catalyzes the reaction from oxidosqualene to ß-amyrin, the proposed aglycone of oleanane-type saponins. Here, RNAi was employed to evaluate the role of this gene in ginsenoside biosynthesis of Panax ginseng hairy roots. The results showed that RNAi-mediated down-regulation of this gene led to reduced levels of ß-amyrin and oleanane-type ginsenoside Ro as well as increased level of total ginsenosides, indicating an important role of this gene in biosynthesis of ginsenoside. Expression of key genes involved in dammarane-type ginsenoside including genes of dammarenediol synthase and protopanaxadiol and protopanaxatriol synthases were up-regulated in RNAi lines. While expression of squalene synthase genes was not significantly changed, ß-amyrin oxidase gene was down-regulated. This work will be helpful for further understanding ginsenoside biosynthesis pathway.

Enhancement of ginsenoside biosynthesis and secretion by Tween 80 in Panax ginseng hairy roots.

We evaluated the effect of Tween 80 permeabilization on ginsenoside secretion in Panax ginseng hairy roots. Tween 80 (1.2%, w/v) had no significant effect on hairy root vitality. After a 25-day treatment with Tween 80, approximately 76% of the total ginsenosides was released into the surrounding medium. In the case of control, the ginsenosides secreted into the medium were negligible. Furthermore, when compared with control, the level of total ginsenosides was enhanced by approximately threefold under Tween treatment. Additionally, secretion of the typical ginsenoside monomers including Rb1 , Rg1 , and Re was analyzed, indicating that the most of them were released into the medium. Moreover, it was observed that dammarenediol synthase, a key enzyme involved in ginsenoside biosynthesis, was upregulated at both gene expression and enzyme activity levels. The expression of genes CYP716A47 and CYP716A53v2 encoding Cyt P450 enzymes catalyzing the formation of protopanaxadiol from dammarenediol and protopanaxatriol from protopanaxadiol, respectively, was slightly upregulated. These results clearly demonstrated that Tween 80 could act not only as an efficient permeabilizer to enhance ginsenoside secretion from the hairy roots, but also as an elicitor to promote the biosynthesis of ginsenoside.

Identification of the protopanaxatriol synthase gene CYP6H for ginsenoside biosynthesis in Panax quinquefolius.

Panax quinquefolius is one of perennial herbs and well known for its outstanding pharmacological activity. Ginsenosides are thought to be the main active ingredients in P. quinquefolius and exist in many kinds of plant genus Panax (ginseng). Protopanaxatriol synthase, which is considered cytochrome P450 (CYP450) in ginsenoside biosynthesis pathway can convert protopanaxadiol into protopanaxatriol. However, the protopanaxatriol synthase gene in P. quinquefolius has not been identified. Here, we cloned and identified a protopanaxatriol synthase gene from P. quinquefolius (CYP6H, GenBank accession no. KC190491) at the first time, reverse transcription-PCR (RT-PCR) analysis showed no obvious transcription change of CYP6H in methyl jasmonate (MeJA)-induced hairy roots. Ectopic expression of CYP6H in Saccharomyces cerevisiae resulted in the production of protopanaxatriol with added exogenous protopanaxadiol and confirmed by liquid chromatography-atmospheric pressure chemical ionization mass spectrometry (LC/APCIMS). Moreover, high-performance liquid chromatography (HPLC) analysis shows that RNA interferences of CYP6H in transgenic hairy roots could increase the accumulation of protopanaxadiol-type ginsenosides and decrease the accumulation of protopanaxatriol-type ginsenosides, whereas the effect of overexpression CYP6H in transgenic hairy roots was contrary. Our study indicated that CYP6H is a gene encoding protopanaxadiol 6-hydroxylase which could convert protopanaxadiol into protopanaxatriol in P. quinquefolius ginsenoside biosynthesis, we also have confirmed the function of CYP6H on effect accumulation of ginsenosides.

The isolation and characterization of dammarenediol synthase gene from Panax quinquefolius and its heterologous co-expression with cytochrome P450 gene PqD12H in yeast.

Panax quinquefolius is one of perennial herbs and well known for its outstanding pharmacological activity. Ginsenosides are thought to be the main active ingredients in Panax quinquefolius and exist in many kinds of plant genus Panax (ginseng). Dammarenediol synthase, which is considered as a key enzyme in ginsenoside biosynthesis pathway can convert 2, 3-oxidosqualene into dammarenediol-II. However, the dammarenediol synthase gene in Panax quinquefolius has not been identified. Here, we cloned and identified a dammarenediol synthase gene from Panax quinquefolius (PqDS, GenBank accession No. KC316048) at the first time, and reverse transcription-PCR (RT-PCR) analysis also showed an obvious transcription increase of PqDS in the methyl jasmonate (MeJA)-induced hairy roots. Ectopic expression of PqDS in yeast resulted in the production of dammarenediol-II was confirmed by liquid chromatography-atmospheric pressure chemical ionization mass spectrometry (LC/APCIMS). Moreover, overexpression of PqDS in transgenic hairy roots could increase the transcription of gene PqDS and another P450 gene PqD12H (encoding protopanaxadiol synthase in Panax quinquefolius), the accumulation of ginsenosides also increased at the same time. In addition, both PqDS and PqD12H gene co-expressed in recombinant yeast result in the production of protopanaxadiol was detected by LC/APCIMS; this result also provides a new strategy for the abundant production of protopanaxadiol in vitro.

Both the mevalonate and the non-mevalonate pathways are involved in ginsenoside biosynthesis.

KEY MESSAGE: When one of them was inhibited, the two pathways could compensate with each other to guarantee normal growth. Moreover, the sterol biosynthesis inhibitor miconazole could enhance ginsenoside level. ABSTRACT: Ginsenosides, a kind of triterpenoid saponins derived from isopentenyl pyrophosphate (IPP), represent the main pharmacologically active constituents of ginseng. In plants, two pathways contribute to IPP biosynthesis, namely, the mevalonate pathway in cytosol and the non-mevalonate pathway in plastids. This motivates biologists to clarify the roles of the two pathways in biosynthesis of IPP-derived compounds. Here, we demonstrated that both pathways are involved in ginsenoside biosynthesis, based on the analysis of the effects from suppressing either or both of the pathways on ginsenoside accumulation in Panax ginseng hairy roots with mevinolin and fosmidomycin as specific inhibitors for the mevalonate and the non-mevalonate pathways, respectively. Furthermore, the sterol biosynthesis inhibitor miconazole could enhance ginsenoside levels in the hairy roots. These results shed some light on the way toward better understanding of ginsenoside biosynthesis.

Stimulation of saponin production in Panax ginseng hairy roots by two oligosaccharides from Paris polyphylla var. yunnanensis.

Two oligosaccharides, a heptasaccharide (HS) and an octasaccharide (OS), isolated from Paris polyphylla var. yunnanensis, stimulated the growth and saponin accumulation of Panax ginseng hairy roots at 5-30 mg l(-1). HS and OS at 30 mg l(-1), fed separately to hairy root cultures at 10 days post-inoculation, increased the root biomass dry weight by more than 70% to approximately 20 g l(-1) from 13 g l(-1) and the total saponin content of roots by more than 1-fold to approximately 3.5% from 1.6% (w/w). The results suggest that the two oligosaccharides may have plant growth-regulatory activity in plant tissue cultures.

[Induction of hairy roots of Panax ginseng and studies on suitable culture condition of ginseng hairy roots].

Ginseng is a valuable medicinal plant with ginsenosides as its mian effective components. Because ginseng is a perennial plant and has a very strict demand for soil conditions, the way of cultivating ginseng by cutting woods is still used in China at present and thus forest resources has been extremely destroyed. Increasing attention has been paid to the hairy roots induced by the infection of Agrobacterium rhizogenes in the production of plant secondary metabolic products for the hairy roots are characterized by rapid growth and stable hereditary and biochemical traits. That has opened a new way for the industrial production of ginseosides. However, there is little report for such studies from China. In this paper, hairy roots of ginseng were induced from the root explants of two-year-old ginseng by Agrobacterium rhizogenes A4 with directly inoculating. The transformed hairy roots could grow rapidly on MS medium and 1/2 MS medium without hormones. The cultured clones of the hairy roots were established on a solid 1/2 MS medium. After 4 - 5 subcultures the hairy roots still maintained a vigorous growth. A pair of primers were designed and synthesized according to the analytical results of RiA4TL-DNA sequence by Slightom et al . 0.8kb rolC was obtained by PCR using the genome DNA of hairy root of ginseng. Transformation was confirmed by PCR amplification of rolC genes from the hairy roots of P. ginseng. Growth rate of hairy roots on liquid medium increased by 2 times then that of the solid medium. The growth of the hairy roots can be divided into three stages: high speed in the first two weeks, middle speed in the 3 - 4 weeks and low speed hereafter. Changing the culture solution at 2 weeks regular intervals is conductive to maintaining the rapid growth of the hairy roots. By means of determination for specific growth rate and ginsenosides content, the high-yield hairy root clone R9923 was selected. The content of monomer gisenoside of Rg1, Re, Rf, Rbl, Rc, Rb2 and Rd in hairy root clone R9923 was determined by the HPLC. The total ginsenosides content in the hairy toot clone R9923 came up to 15.2 mg/g. The suitable culture conditions for ginseng hairy roots growing were 1/2 MS liquid medium (30 g/L glucose), in a shaker at 110 r/min, changing the culture solution at 2 weeks and subculture time 4 weeks. In the liquid fermented culture of 2L medium, the yield of the hairy roots could amount to 270.10 g in 4 weeks. The industrial production of ginsenosides has been preliminarily realized. Effect factors on biomass and ginsenosides content such as culture volume, inoculation, in steps cultural technology at the scale-up process of hairy roots culture were also explorated. Our results have laid a foundation for defining optimum culture manner for large-scale cultivation and large-scale production of ginsenosides.