Organ-specific, integrated omics data-based study on the metabolic pathways of the medicinal plant Bletilla striata (Orchidaceae).

BACKGROUND: Bletilla striata is one of the important species belonging to the Bletilla genus of Orchidaceae. Since its extracts have an astringent effect on human tissues, B. striata is widely used for hemostasis and healing. Recently, some other beneficial effects have also been uncovered, such as antioxidation, antiinflammation, antifibrotic, and immunomodulatory activities. As a key step towards a thorough understanding on the medicinal ingredient production in B. striata, deciphering the regulatory codes of the metabolic pathways becomes a major task. RESULTS: In this study, three organs (roots, tubers and leaves) of B. striata were analyzed by integrating transcriptome sequencing and untargeted metabolic profiling data. Five different metabolic pathways, involved in polysaccharide, sterol, flavonoid, terpenoid and alkaloid biosynthesis, were investigated respectively. For each pathway, the expression patterns of the enzyme-coding genes and the accumulation levels of the metabolic intermediates were presented in an organ-specific way. Furthermore, the relationships between enzyme activities and the levels of the related metabolites were partially inferred. Within the biosynthetic pathways of polysaccharides and flavonoids, long-range phytochemical transportation was proposed for certain metabolic intermediates and/or the enzymes. CONCLUSIONS: The data presented by this work could strengthen the molecular basis for further studies on breeding and medicinal uses of B. striata.

Myricetin relieves LPS-induced mastitis by inhibiting inflammatory response and repairing the blood-milk barrier.

Mastitis, an inflammation of mammary gland, is a serious disease that affects the health of dairy cows around the world. Myricetin, a flavonoid from Bayberry, has been reported to suppress various inflammatory response. The aim of this study was to evaluate the effect of myricetin on lipopolysaccharide (LPS)-induced in vivo and in vitro mastitis model and clarify the underlying mechanism. In vivo experiments, myricetin attenuated the severity of inflammatory lesion and neutrophil infiltration. Moreover, myricetin pretreatment induced a significant decrease in the activity of myeloperoxidase (MPO) and the production of TNF-α, IL-6, and IL-1ß triggered by LPS. Myricetin pretreatment could also increase the integrity of the blood-milk barrier and upregulate the tight junction proteins in LPS-induced mice mastitis. In vitro, myricetin inhibited LPS-induced inflammatory response in mice mammary epithelial cells (mMECs). In the further mechanism studies, we found that the anti-inflammatory effect of myricetin was mediated by inhibiting LPS-induced phosphorylation of AKT, IKK-α, IκB-α, and P65 in vivo and in vitro. Collectively, these data suggested that myricetin effectively ameliorated the inflammatory response by inhibiting the AKT/IKK/NF-κB signaling pathway and repairing the integrity of blood-milk barrier in LPS-induced mice mastitis.

Preparative Separation of Alkaloids from Stem of Euchresta tubulosa Dunn. by High-Speed Counter-Current Chromatography Using Stepwise Elution.

Euchresta tubulosa Dunn. is a Chinese herbal medicine with biological activity, but there are few studies on its components at present. Alkaloids in the stem of Euchresta tubulosa Dunn. were isolated and purified by high-speed counter-current chromatography (HSCCC) using stepwise elution. First of all, liquid-liquid extraction (methylene chloride-methanol-water, 5:1:4, v/v) was used for the preliminary enrichment. According to the partition coefficient (K) of a target compound in a series of different two-phase solvents, the final result was that carbon tetrachloride-methylene chloride-methanol-water (2:3:3:2, v/v) (1) and methylene chloride-methanol-water (5:3:2, v/v) (2) were suitable for the HSCCC using stepwise elution. As a result, the purity was all higher than 93% and matrine (1), oxymatrine (2), N-formyl cytisine (3), and N-acetyl cytisine (4) can be eluted at one time by this mode. Cytisine-type alkaloids were isolated for the first time in this plant. Finally, the applicability of the mode was verified.

Epidermal growth factor receptor (EGFR) signaling requires a specific endoplasmic reticulum thioredoxin for the post-translational control of receptor presentation to the cell surface.

The epidermal growth factor receptor (EGFR) is a well characterized receptor-tyrosine kinase that functions in development and serves a vital role in many human cancers. Understanding EGFR regulatory mechanisms, and hence approaches for clinical intervention, has focused on ligand-receptor interactions and tyrosine kinase activity. Here, we show using the NCI-H460 lung and A431 epidermoid human cancer cell lines that EGFR binding to anterior gradient homolog 2 (AGR2) in the endoplasmic reticulum is required for receptor delivery to the plasma membrane and thus EGFR signaling. Reduced AGR2 protein levels or mutation of an essential cysteine in the active site result in decreased cell surface EGFR and a concomitant decrease in signaling as reflected by AREG, EGR1, and FOS expression. Similar to previously described EGFR nulls, an AGR2 null also resulted in embryonic lethality. Consistent with its role in regulating EGFR-mediated signaling, AGR2 expression is also enhanced in many human cancers and promotes the transformed phenotype. Furthermore, EGFR-mediated signaling in NCI-H460 cells, which are resistant to the tyrosine kinase inhibitor AG1478, is also disrupted with reduced AGR2 expression. The results provide insights into why cancer prognosis or response to therapy often does not correlate with EGFR protein or RNA levels because they do not reflect delivery to the cell surface where signaling is initiated. AGR2, therefore, represents a novel post-translational regulator of EGFR-mediated signaling and a promising target for treating human cancers.

Is Argonaute 1 the only effective slicer of small RNA-mediated regulation of gene expression in plants?

To maintain normal growth and development in a plant, gene expression must be under strict surveillance. One of the post-transcriptional regulatory pathways involves small RNA (sRNA)-guided, Argonaute (AGO) protein complex-mediated target cleavages. MicroRNAs (miRNAs) are the well-known sRNA species participating in the cleavage-based regulation of gene expression. In plants, most miRNAs are incorporated into AGO1-associated silencing complexes. Thus, the AGO1 protein is considered to be the most important slicer for sRNA-mediated target cleavages. Previous phylogenetic analysis revealed that AGO1, AGO5, and AGO10 belonged to the same clade in Arabidopsis (Arabidopsis thaliana). In addition, experimental evidence pointed to the possibility that AGO2, AGO7, and AGO10 were implicated in specific miRNA-mediated regulatory pathways. To investigate the potential slicer activities of AGO2, AGO5, AGO7, and AGO10, a comprehensive search was performed for the sRNAs enriched in the four AGO proteins in Arabidopsis. A total of 3 499, 1 618, 4 632, and 63 sRNAs are enriched in AGO2, AGO5, AGO7, and AGO10, respectively. Interestingly, several miRNAs were found to be enriched in AGO2 or AGO5. Transcriptome-wide target identification based on degradome sequencing data uncovered that a number of sRNAs enriched in the four AGOs could perform target cleavages like AGO1-associated miRNAs in plants. Based on the above results, the opinion was put forward that not only AGO1, but also AGO2, AGO5, AGO7, and AGO10 might be essential for the sRNA-mediated regulation of gene expression in plants.

AGR2 gene function requires a unique endoplasmic reticulum localization motif.

Soluble proteins are enriched in the endoplasmic reticulum (ER) by retrograde transport from the Golgi that is mediated by the KDEL receptors. In addition to the classic carboxyl-terminal KDEL motif, a variety of sequence variants are also capable of receptor binding that result in ER localization. Although different ER localization signals that exhibit varying affinities for the KDEL receptors exist, whether there are functional implications was unknown. The present study determines whether AGR2 requires a specific ER localization signal to be functionally active. AGR2 is expressed in most human adenocarcinomas and serves a role in promoting growth and the transformed phenotype. Using two different cell lines in which AGR2 induces expression of either the EGFR ligand amphiregulin or the transcription factor CDX2, only the highly conserved wild-type carboxyl-terminal KTEL motif results in the appropriate outcome. Deletion of the KTEL motif results in AGR2 secretion and loss of AGR2 function. AGR2 function is also lost when ER residence is achieved with a carboxyl-terminal KDEL or KSEL instead of a KTEL motif. Thus variations in ER localization sequences may serve a specific functional role, and in the case of AGR2, this role is served specifically by KTEL.

The human adenocarcinoma-associated gene, AGR2, induces expression of amphiregulin through Hippo pathway co-activator YAP1 activation.

Anterior Gradient Homolog 2 (AGR2) is expressed by the normal intestine and by most human adenocarcinomas, including those derived from the esophagus, pancreas, lung, breast, ovary, and prostate. Xenografts of human adenocarcinoma cell lines in nude mice previously demonstrated that AGR2 supports tumor growth. In addition, AGR2 is able to induce in vitro a transformed phenotype in fibroblast and epithelial cell lines. The mechanism underlying the growth promoting effects of AGR2 is unknown. The present study shows that AGR2 induces expression of amphiregulin (AREG), a growth promoting EGFR ligand. Induced AREG expression in adenocarcinoma cells is able to rescue the transformed phenotype that is lost when AGR2 expression is reduced. Additional experiments demonstrate that AGR2 induction of AREG is mediated by activation of the Hippo signaling pathway co-activator, YAP1. Thus AGR2 promotes growth by regulating the Hippo and EGF receptor signaling pathways.

Diagnostic accuracy of cyst fluid amphiregulin in pancreatic cysts.

BACKGROUND: Accurate tests to diagnose adenocarcinoma and high-grade dysplasia among mucinous pancreatic cysts are clinically needed. This study evaluated the diagnostic utility of amphiregulin (AREG) as a pancreatic cyst fluid biomarker to differentiate non-mucinous, benign mucinous, and malignant mucinous cysts. METHODS: A single-center retrospective study to evaluate AREG levels in pancreatic cyst fluid by ELISA from 33 patients with a histological gold standard was performed. RESULTS: Among the cyst fluid samples, the median (IQR) AREG levels for non-mucinous (n = 6), benign mucinous (n = 15), and cancerous cysts (n = 15) were 85 pg/ml (47-168), 63 pg/ml (30-847), and 986 pg/ml (417-3160), respectively. A significant difference between benign mucinous and malignant mucinous cysts was observed (p = 0.025). AREG levels greater than 300 pg/ml possessed a diagnostic accuracy for cancer or high-grade dysplasia of 78% (sensitivity 83%, specificity 73%). CONCLUSION: Cyst fluid AREG levels are significantly higher in cancerous and high-grade dysplastic cysts compared to benign mucinous cysts. Thus AREG exhibits potential clinical utility in the evaluation of pancreatic cysts.

[Fluorescence microscopy and HPLC assay for rapid detection of distribution and content of resveratrol in Polygonum cuspidatum].

OBJECTIVE: To establish fluorescence microscopy combined with HPLC method for rapid detection the distribution and content of resveratrol tissues in different growth stages of Polygonum cuspidatum. METHODS: Used sequential experiment to design conditions of frozen and observe of the section by fluorescence microscopy; Resveratrol was extracted by ultrasonic-assisted extraction and its content was detected by HPLC. RESULTS: The results showed that frozen condition for concentration of gum Arabic was from 20% (dipping time was 5 - 6 h) to 40% (2 - 5 min), the freezer temperature was -5 degrees C, and the thickness was 15 microm. Resveratrol in polygonum cuspidatum was mainly accumulated in the organs, tissues and cells of fiber and cellulose, its content in rhizomes declined as the following sequence: spinal cord > xylem > phloem > periderm; Its content declined in organ as the following sequence: buds > rhizomes > ground stem > leaves; The content of resveratrol in root increased with age. CONCLUSION: The results of fluorescence microscopic observation is in accordance with the HPLC results, indicating that the method is simple, fast and reliable, and provides a fast and reliable detection method for the determination of optimum harvesting period of Polygonum cuspidatum and acquisition of quality.

An organ-specific transcriptomic atlas of the medicinal plant Bletilla striata: Protein-coding genes, microRNAs, and regulatory networks.

As one of the important species belonging to the Bletilla genus of Orchidaceae, Bletilla striata (Thunb.) Rchb. f., possesses both ornamental and medicinal values. Its dried tubers are used as a traditional Chinese medicine, and several secondary metabolites have been indicated to be the active ingredients. However, the molecular mechanisms related to the regulation of secondary metabolism have not been characterized in B. striata. In this study, integrated analysis of RNA sequencing (RNA-seq), small RNA sequencing (sRNA-seq), and degradome sequencing (degradome-seq) data from three organs (leaf, root, and tuber) of B. striata provided us with a comprehensive view of the microRNA (miRNA)-mediated regulatory network. Firstly, based on the RNA-seq data, the organ-specific expression patterns of the protein-coding genes, especially for those related to secondary metabolism, were investigated. Secondly, 342 conserved miRNA candidates were identified from B. striata. These miRNAs were assigned to 88 families, some of which were selected for expression pattern analysis. Additionally, 31 hairpin-structured precursors encoding 23 novel miRNAs were uncovered from the transcriptome assembly. Thirdly, based on the degradome signatures, 1,142 validated miRNA-target pairs (involving 167 conserved miRNAs and six novel miRNAs and 51 target genes) were included in the regulatory network. Organ-specific expression level comparison between the miRNAs and their targets revealed some interesting miRNA-target pairs. Fourthly, some valuable subnetworks were extracted for further functional studies. Additionally, some regulatory pathways were indicated to be monocot specific. Summarily, our results lay a solid basis for in-depth studies on the regulatory mechanisms underlying the production of the medicinal ingredients in B. striata.

RNA-, sRNA-, and degradome-seq were performed for three organs of B. striata. Organ-specific expression patterns of the protein-coding genes were analyzed. A total of 365 miRNAs were identified and subject to expression pattern analysis. A total of 1,142 miRNA-target pairs were validated for network construction. Some miRNA-mediated regulatory pathways were indicated to be monocot specific.

Anterior Gradient 2 (AGR2) Induced Epidermal Growth Factor Receptor (EGFR) Signaling Is Essential for Murine Pancreatitis-Associated Tissue Regeneration.

A recently published study identified Anterior Gradient 2 (AGR2) as a regulator of EGFR signaling by promoting receptor presentation from the endoplasmic reticulum to the cell surface. AGR2 also promotes tissue regeneration in amphibians and fish. Whether AGR2-induced EGFR signaling is essential for tissue regeneration in higher vertebrates was evaluated using a well-characterized murine model for pancreatitis. The impact of AGR2 expression and EGFR signaling on tissue regeneration was evaluated using the caerulein-induced pancreatitis mouse model. EGFR signaling and cell proliferation were examined in the context of the AGR2-/- null mouse or with the EGFR-specific tyrosine kinase inhibitor, AG1478. In addition, the Hippo signaling coactivator YAP1 was evaluated in the context of AGR2 expression during pancreatitis. Pancreatitis-induced AGR2 expression enabled EGFR translocation to the plasma membrane, the initiation of cell signaling, and cell proliferation. EGFR signaling and tissue regeneration were partially inhibited by the tyrosine kinase inhibitor AG1478, but absent in the AGR2-/- null mouse. AG1478-treated and AGR2-/- null mice with pancreatitis died whereas all wild-type controls recovered. YAP1 activation was also dependent on pancreatitis-induced AGR2 expression. AGR2-induced EGFR signaling was essential for tissue regeneration and recovery from pancreatitis. The results establish tissue regeneration as a major function of AGR2-induced EGFR signaling in adult higher vertebrates. Enhanced AGR2 expression and EGFR signaling are also universally present in human pancreatic cancer, which support a linkage between tissue injury, regeneration, and cancer pathogenesis.

Potent antitumor activity of oncolytic adenovirus expressing mda-7/IL-24 for colorectal cancer.

It has been demonstrated that interleukin 24 (IL-24, also called melanoma differentiation associated gene 7) exerts antitumor activity. In this study, we investigated whether oncolytic adenovirus-mediated gene transfer of IL-24 could induce strong antitumor activity. A tumor-selective replicating adenovirus expressing IL-24 (ZD55-IL-24) was constructed by insertion of an IL-24 expression cassette into the ZD55 vector, which is based on deletion of the adenoviral E1B 55-kDa gene. ZD55-IL-24 could express substantially more IL-24 than Ad-IL-24 because of replication of the vector. It has been shown that ZD55-IL-24 exerted a strong cytopathic effect and significant apoptosis in tumor cells with p53 dysfunction. Moreover, no cytotoxic and apoptotic effects could be seen in normal cells infected with ZD55-IL-24. Expression of IL-24 did not interfere with viral replication induced by oncolytic adenovirus. Activation of caspase 3 and caspase 9, and induction of bax gene expression, were involved in tumor cell apoptosis induced by ZD55-IL-24. Treatment of established tumors with ZD55-IL-24 showed much stronger antitumor activity than that induced by ONYX-015 or Ad-IL- 24. These data indicated that oncolytic adenovirus expressing IL-24 could exert potential antitumor activity and offer a novel approach to cancer therapy.

Blockade of Wnt signaling inhibits angiogenesis and tumor growth in hepatocellular carcinoma.

Aberrant activation of Wnt signaling plays an important role in hepatocarcinogenesis. In addition to direct effects on tumor cells, Wnt signaling might be involved in the organization of tumor microenvironment. In this study, we have explored whether Wnt signaling blockade by exogenous expression of Wnt antagonists could inhibit tumor angiogenesis and control tumor growth. Human Wnt inhibitory factor 1 (WIF1) and secreted frizzled-related protein 1 (sFRP1) were each fused with Fc fragment of human IgG1 to construct fusion proteins WIF1-Fc and sFRP1-Fc. The recombinant adenoviral vectors carrying WIF1-Fc and sFRP1-Fc driven by cytomegalovirus promoter were constructed. Ad-WIF1-Fc or Ad-sFRP1-Fc induced the expression and correct conformation of WIF1-Fc and sFRP1-Fc fusion proteins. These molecules caused down-regulation of E2F1, cyclin D1, and c-myc and promoted cell apoptosis in hepatocellular carcinoma cells. Treatment of established hepatocellular carcinoma tumors with Ad-WIF1-Fc and/or Ad-sFRP1-Fc resulted in significant inhibition of tumor growth and prolonged animal survival. The antineoplastic effect was associated with increased apoptosis of tumor cells, reduced microvessel density, and decreased expression of vascular endothelial growth factor and stromal cell-derived factor-1. Tube formation and migration of human microvascular endothelial cells and mouse endothelial progenitor cells (EPC) were significantly inhibited by both WIF1-Fc and sFRP1-Fc. In addition, these molecules blocked EPC differentiation and caused EPC apoptosis. Our data indicate that Wnt antagonists WIF1-Fc and sFRP1-Fc inhibit Wnt signaling and exert potent antitumor activity by increasing the apoptosis rate in tumor cells and by impairing tumor vascularization.

Regulation and pharmacokinetics of inducible recombinant TRAIL expression.

TRAIL is a potent antitumor agent, but its potential toxicity to normal human tissues limits its clinical applications in future. Therapy of human tumors might benefit from the use of vectors enabling tight control of TRAIL expression in vivo. To this aim, we constructed an adenoviral vector carrying the RU486-dependent gene switch system for the regulable expression of recombinant TRAIL. Only was apoptotic recombinant TRAIL expressed and cytotoxicity observed upon binding of RU 486 to the inducible promoter. Expression levels and kinetics of recombinant TRAIL expression could be achieved by modulating the concentration of the inducer. As a broad implication, our data provide an alternative approach to circumvent the potential toxicity of TRAIL in future human trials and this system may be utilized to treat human cancer using a long-term expression vector.

Impairment of redox state and dopamine level induced by alpha-synuclein aggregation and the prevention effect of hsp70.

One hypothesis for the etiology of Parkinson's disease (PD) is that the formation of proteinaceous inclusion, which is mainly composed of alpha-synuclein, may contribute to the selective loss of dopaminergic neurons. To further explore the role of alpha-synuclein in neurodegeneration of PD, we examined the possible effects of aggregated alpha-synuclein on the intracellular redox state, dopamine level, and cell death of SK-N-SH cells. Our present studies show that alpha-synuclein aggregation gives rise to both elevated intracellular oxidative state and dopamine level in SK-N-SH cells. Moreover, alpha-synuclein aggregation results in a higher ratio of apoptosis population (55.8%+/-SEM) in cells overexpressing alpha-synuclein aggregation, compared to their normal control groups (8.0%+/-SEM). In contrast, coexpression of hsp70 with alpha-synuclein suppresses the oxidative state shift, restores the normal dopamine levels and blocks neuron cell loss. Therefore, our data provided one possible mechanism by which the alpha-synuclein aggregation may lead to the neurodegeneration in PD via regulating the level of cytoplasmic dopamine and then disturbing the intracellular redox homeostasis. On the other hand, hsp70 can mitigate the degenerative effect conferred by alpha-synuclein, acting as a protective factor in treatment of PD.