Genome sequencing-based transcriptomic analysis reveals novel genes in Peucedanum praeruptorum.

BACKGROUND: Peucedanum praeruptorum Dunn, a traditional Chinese herbal medicine, contains coumarin and volatile oil components that have clinical application value. However, early bolting often occurs in the medicinal materials of Apiaceae plants. The rhizomes of the medicinal parts are gradually lignified after bolting, resulting in a sharp decrease in the content of coumarins. At present, the link between coumarin biosynthesis and early bolting in P. praeruptorum has not been elucidated. RESULTS: Combining the genome sequencing and the previous transcriptome sequencing results, we reanalyzed the differential transcripts of P. praeruptorum before and after bolting. A total of 62,088 new transcripts were identified, of which 31,500 were unknown transcripts. Functional classification and annotation showed that many genes were involved in the regulation of transcription, defense response, and carbohydrate metabolic processes. The main domains are the pentatricopeptide repeat, protein kinase, RNA recognition motif, leucine-rich repeat, and ankyrin repeat domains, indicating their pivotal roles in protein modification and signal transduction. Gene structure analysis showed that skipped exon (SE) was the most dominant alternative splicing, followed by the alternative 3' splice site (A3SS) and the alternative 5' splice site (A5SS). Functional enrichment of differentially expressed genes showed that these differentially expressed genes mainly include transmembrane transporters, channel proteins, DNA-binding proteins, polysaccharide-binding proteins, etc. In addition, genes involved in peroxisome, hexose phosphate pathway, phosphatidylinositol signaling system, and inositol phosphate metabolism pathway were greatly enriched. A protein-protein interaction network analysis discoverd 1,457 pairs of proteins that interact with each other. The expression levels of six UbiA genes, three UGT genes, and four OMT genes were higher during the bolting stage. This observation suggests their potential involvement in the catalytic processes of prenylation, glycosylation, and methylation of coumarins, respectively. A total of 100 peroxidase (PRX) genes were identified being involved in lignin polymerization, but only nine PRX genes were highly expressed at the bolting stage. It is worth noting that 73 autophagy-related genes (ATGs) were first identified from the KEGG pathway-enriched genes. Some ATGs, such as BHQH00009837, BHQH00013830, and novel8944, had higher expression levels after bolting. CONCLUSIONS: Comparative transcriptome analysis and large-scale genome screening provide guidance and new opinions for the identification of bolting-related genes in P. praeruptorum.

Preoperative administration of branched-chain amino acids reduces postoperative insulin resistance in rats by reducing liver gluconeogenesis.

BACKGROUND: Postoperative insulin resistance (PIR) represents an important characteristic of metabolic response following surgical injury. Clinical outcomes are negatively correlated to postoperative insulin resistance and hyperglycemia, indicating a novel treatment for reducing postoperative insulin resistance is urgently needed. The current work aimed to assess the protective effects of branched-chain amino acids (BCAA) on glucose metabolism disorders induced surgically in a rat model, and to explore the underpinning mechanism. METHODS AND RESULTS: Rats were randomly assigned to 2 groups, including the control and BCAA groups. Rats were given a compulsory oral 3 mL load by gavage two hours before surgery. The results showed that BCAA remarkably reduced glycemia by suppressing liver gluconeogenesis via reduction of cAMP-response element-binding protein-regulated transcription coactivator 2 (CRTC2) and glucose-6-phosphatase (G6PC) gene and protein expression levels (all Ps < 0.05). CONCLUSIONS: This study revealed that BCAA lower blood glucose levels by reducing liver gluconeogenesis without significant elevation of plasma insulin levels. We anticipate that preoperative BCAA supplementation may be a means for preventing postoperative insulin resistance.

Comprehensive analysis of microglia gene and subpathway signatures for glioma prognosis and drug screening: linking microglia to glioma.

Glioma is the most common malignant tumors in the brain. Previous studies have revealed that, as the innate immune cells in nervous system, microglia cells were involved in glioma pathology. And, the resident microglia displayed its specific biological roles which distinguished with peripheral macrophages. In this study, an integrated analysis was performed based on public resource database to explore specific biological of microglia within glioma. Through comprehensive analysis, the biological characterization underlying two conditions, glioma microglia compared to glioma macrophage (MicT/MacT) as well as glioma microglia compared to normal microglia (MicT/MicN), were revealed. Notably, nine core MicT/MicN genes displayed closely associations with glioma recurrence and prognosis, such as P2RY2, which was analyzed in more than 2800 glioma samples from 25 studies. Furthermore, we applied a random walk based strategy to identify microglia specific subpathways and developed SubP28 signature for glioma prognostic analysis. Multiple validation data sets confirmed the predictive performance of SubP28 and involvement in molecular subtypes. The associations between SuP28 score and microglia M1/M2 polarization were also explored for both GBM and LGG types. Finally, a comprehensive drug-subpathway network was established for screening candidate medicable molecules (drugs) and identifying therapeutic subpathway targets. In conclusions, the comprehensive analysis of microglia related gene and functional signatures in glioma pathobiologic events by large-scale data sets displayed a framework to dissect inner connection between microglia and glioma, and identify robust signature for glioma clinical implications.

Identification of the occurrence and potential mechanisms of heterotopic ossification associated with 17-beta-estradiol targeting MKX by bioinformatics analysis and cellular experiments.

BACKGROUND: Tendon heterotopic ossification (HO) is a common condition occurring secondary to tendon injury or surgical trauma that significantly affects the patient's quality of life. The treatment of tendon HO remains challenging due to a lack of clarity regarding the pathological mechanism. Mohawk (MKX) is a key factor in preventing tendon HO; however, its upstream regulatory mechanism remains to be understood. This study aimed to identify potential compounds that target and regulate MKX and explore their functional mechanisms. METHODS: Bioinformatics analysis of MKX-related compounds and proteins was performed based on data from the STITCH and OncoBinder databases. Subsequently, the SymMap database was used to study MKX-related traditional Chinese medicine drugs and symptoms. Next, the OncoBinder genomic and proteomic discovery model was applied to identify potential regulators of MKX. The analytical tool Expert Protein Analysis System for proteomics was used to predict the three-dimensional structure of MKX, and the AutoDockTools software was used to identify pockets of activity at potential sites for molecular docking. Furthermore, we evaluated the effect of different doses of 17-beta-estradiol on bone marrow-derived mesenchymal stem cells (BM-MSCs). RESULTS: By predicting the three-dimensional structure of MKX and simulating molecular docking, Pro-Tyr and 17-beta-Estradiol were found to target and bind to MKX. Analysis of the STITCH and OncoBinder databases showed that MKX had a significant regulatory correlation with suppressor interacting 3 A/histone deacetylase 1 (SIN3A/HDAC1). The GO and KEGG pathway enrichment analysis revealed that the functions of MKX and its associated proteins were mainly enriched in osteogenic-related pathways. Assessment of the proliferation of BM-MSCs revealed that 17-beta-estradiol possibly upregulated the mRNA expression of the HDAC1-SIN3A/BMP pathway-related RUNX2, thereby promoting the proliferation of BM-MSCs. CONCLUSIONS: The compounds Pro-Tyr and 17-beta-Estradiol may bind to MKX and thus affect the interaction of MKX with SIN3A/HDAC1.

Comparative transcriptomic analysis of two Saccharopolyspora spinosa strains reveals the relationships between primary metabolism and spinosad production.

Saccharopolyspora spinosa is a well-known actinomycete for producing the secondary metabolites, spinosad, which is a potent insecticides possessing both efficiency and safety. In the previous researches, great efforts, including physical mutagenesis, fermentation optimization, genetic manipulation and other methods, have been employed to increase the yield of spinosad to hundreds of folds from the low-yield strain. However, the metabolic network in S. spinosa still remained un-revealed. In this study, two S. spinosa strains with different spinosad production capability were fermented and sampled at three fermentation periods. Then the total RNA of these samples was isolated and sequenced to construct the transcriptome libraries. Through transcriptomic analysis, large numbers of differentially expressed genes were identified and classified according to their different functions. According to the results, spnI and spnP were suggested as the bottleneck during spinosad biosynthesis. Primary metabolic pathways such as carbon metabolic pathways exhibited close relationship with spinosad formation, as pyruvate and phosphoenolpyruvic acid were suggested to accumulate in spinosad high-yield strain during fermentation. The addition of soybean oil in the fermentation medium activated the lipid metabolism pathway, enhancing spinosad production. Glutamic acid and aspartic acid were suggested to be the most important amino acids and might participate in spinosad biosynthesis.

Alternative splicing perturbation landscape identifies RNA binding proteins as potential therapeutic targets in cancer.

Alternative splicing (AS) plays an important role in gene regulation, and AS perturbations are frequently observed in cancer. RNA binding protein (RBP) is one of the molecular determinants of AS, and perturbations in RBP-gene network activity are causally associated with cancer development. Here, we performed a systematic analysis to characterize the perturbations in AS events across 18 cancer types. We showed that AS alterations were prevalent in cancer and involved in cancer-related pathways. Given that the extent of AS perturbation was associated with disease severity, we proposed a computational pipeline to identify RBP regulators. Pan-cancer analysis identified a number of conserved RBP regulators, which play important roles in regulating AS of genes involved in cancer hallmark pathways. Our application analysis revealed that the expression of 68 RBP regulators helped in cancer subtyping. Specifically, we identified four subtypes of kidney cancer with differences in cancer hallmark pathway activities and prognosis. Finally, we identified the small molecules that can potentially target the RBP genes and suggested potential candidates for cancer therapy. In summary, our comprehensive AS perturbation landscape analysis identified RBPs as potential therapeutic targets in cancer and provided novel insights into the regulatory functions of RBPs in cancer.

Potential mechanisms of tremor tolerance induced in rats by the repeated administration of total alkaloid extracts from the seeds of Peganum harmala Linn.

ETHNOPHARMACOLOGICAL RELEVANCE: The seeds of Peganum harmala Linn have been widely used for the treatment of nervous, cardiovascular, gastrointestinal, respiratory, and endocrine diseases and many other human ailments. However, tremor toxicity occurs after overdose and is tolerated following multiple dosing. Thus far, little is known about the underlying mechanisms of tremors and tremor tolerance. AIM OF THE STUDY: To investigate the potential mechanisms of tremors and tremor tolerance induced in rats by the repeated administration of total alkaloid extracts from the seeds of P. harmala (TAEP). MATERIALS AND METHODS: A tremor model was induced in male Wistar rats by administering TAEP at a dose of 150 mg/kg/day. To evaluate tremor action, behavioral assessment was conducted by using a custom-built tremor acquisition and analysis system. To investigate the relationships between tremors and neurotransmitter levels in the brain, various neurotransmitters were simultaneously quantified by an ultra-performance liquid chromatography combined with electrospray ionization-tandem mass spectrometry (UPLC-ESI-MS/MS) system, and the association between these two parameters was analyzed using Pearson correlation coefficients. To further elucidate the potential mechanisms of the alterations of neurotransmitter levels in cortical tissues, the protein expression levels of several important enzymes and transporters that are closely related to neurotransmitter levels were investigated. In addition, neuropathological analysis was conducted to assess the effect of TAEP on neurons in the brain. To further clarify the potential mechanisms of TAEP-induced neurodegeneration in the brain, c-fos was subjected to immunohistochemical analysis, and oxidative stress markers were examined. RESULTS: Tremors initially occurred in rats after the oral administration of TAEP at a dose of 150 mg/kg/day. However, they were tolerated following repeated dosing. The levels of 5-hydroxytryptamine (5-HT) and glycine (Gly) in cortical tissues were most likely associated with the tremor response. Tremor tolerance also likely resulted from the degeneration of cerebellar Purkinje cells. Furthermore, the alteration of 5-HT levels was mainly attributed to the downregulated expression of monoamine oxidase A (MAO-A). The degeneration of Purkinje neurons might have resulted from the overexpression of c-fos and increased oxidative stress in the cerebellum after the multiple dosing of TAEP. CONCLUSION: The tremor response induced by TAEP at high doses is closely related to the concentrations of 5-HT and Gly in cortical tissues. Tremor tolerance may also be attributed to the degeneration of cerebellar Purkinje cells after the repeated dosing of TAEP. Further studies should be conducted to elucidate the interaction of the alkaloids on the neurotransmitter receptors, the expression of related neurotransmitter receptors, the specific signaling pathway involved in regulating MAO-A, and the mechanism of the loss and functional recovery of cerebellar Purkinje neurons.

Modulation of hippocampal dopamine and synapse-related proteins by electroacupuncture improves memory deficit caused by sleep deprivation.

BACKGROUND: Sleep is crucial for proper functioning of the brain, whereas lack of sleep is very common in modern society and can cause memory impairment. Hence, it is of great significance to find effective methods to intervene in the pathogenesis of memory impairment. OBJECTIVE: We designed this study to explore the mechanism underlying the therapeutic effects of electroacupuncture (EA) on the deficits caused by sleep deprivation (SD). METHODS: In this study, we first utilized the modified multiple platform method (MMPM) to establish a rat model of SD, which was followed by use of the Y-maze and Morris water maze (MWM) to assess the performance of rats following EA treatment. RESULTS: We found that EA at GV20 and ST36 significantly decreased the number of error reactions, increased the number of active avoidance responses in the Y-maze and shortened the latency of finding the platform in the MWM test in SD + EA versus untreated SD groups. Moreover, EA treatment partially restored SD-induced reductions in hippocampal dopamine (DA) content and significantly increased the levels of phosphorylated (p) synapsin I, calcium/calmodulin-dependent protein kinase (CaMK) II, and tyrosine hydroxylase, which are related to the synthesis and release of DA. CONCLUSIONS: In summary, we it appears that EA at GV20 and ST36 may improve SD-induced memory deficits by restoring the quantity of DA in the hippocampus, which is related to activation of CaMK II, synapsin I, and tyrosine hydroxylase. EA may have potential as an alternative therapy for SD and could improve learning and memory deficits among those suffering from sleep deficiency, although this needs verification by prospective clinical studies.

Corrigendum to "Tuina Massage Improves Cognitive Functions of Hypoxic-Ischemic Neonatal Rats by Regulating Genome-Wide DNA Hydroxymethylation Levels".

[This corrects the article DOI: 10.1155/2019/1282085.].

Rapid identification and pharmacokinetic studies of multiple active alkaloids in rat plasma through UPLC-Q-TOF-MS and UPLC-MS/MS after the oral administration of Zanthoxylum nitidum extract.

Zanthoxylum nitidum (Roxb.) DC. (ZN) belongs to the genus Zanthoxylum of Rutaceae and has various chemical ingredients and pharmacologic effects. Alkaloids are its main active constituents responsible for diverse pharmacologic effects, such as anti-tumor, anti-bacterial, anti-inflammatory, and analgesic activities. The chemical and pharmacological effects of ZN are well reported, but the in vivo pharmacokinetic profiles of its main active alkaloids are poorly investigated. This study aims to elucidate the absorbed constituents and pharmacokinetic behavior of main active ingredients in rat plasma after the oral administration of ZN extract. The absorbed constituents in rat plasma were qualitatively analyzed using ultra-high-performance liquid chromatography with quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS). Ultra-high-performance liquid chromatography with triple quadrupole mass spectrometry (UPLC-MS/MS) method was developed for the simultaneous determination and pharmacokinetic studies of dihydrochelerythrine (DHCHE), nitidine chloride (NIT), chelerythrine (CHE), sanguinarine (SAN), liriodenine (LIR), skimmianine (SKI), γ-fagarine (FAG), and dictamnine (DIC) in rat plasma. Eighteen prototypes and metabolites were identified according to exact mass, characteristic diagnostic fragment ions, and reference standards. The established UPLC-MS/MS quantitative method met the requirements of FDA for biological analysis methods. Method validation showed that this method has good linearity (r ≥ 0.9910), precision (RSD ≤ 18.63 %), accuracy (88.11 %-117.50 %), and stability. The limit of detection (LOD) could reach 1 ng/mL, and the limit of quantitation could reach 2 ng/mL. The plasma drug concentration of benzophenanthridine alkaloids, such as NIT, CHE, and DHCHE, were still low even after dose differences were deducted. For the furan quinoline alkaloids (such as SKI, FAG, and DIC), only SKI showed high plasma drug concentration, although SKI content comprised only approximately 1/6 of benzophenanthridine alkaloids. This study is the first to simultaneously determine the above-mentioned active alkaloids in rat plasma and would contribute to the comprehensive understanding of in vivo pharmacokinetic behavior on active alkaloids in ZN extract.

20(S)-ginsenoside Rh2 as agent for the treatment of LMN-CRC via regulating epithelial-mesenchymal transition.

The lymph node metastasis of colorectal cancer (LMN-CRC) seriously threatens the prognosis of patients. Chemotherapy, as the most common treatment, results in severe bone marrow suppression. 20(S)-ginsenoside Rh2 (SGRh2), a major effective constituent of ginseng, has demonstrated therapeutic effects on a variety of diseases, including some tumours. SGRh2 treatment had no effect on other organs. Therefore, ginsenosides are considered a safe and effective antineoplastic drug. However, the effects of SGRh2 on LMN-CRC remain unknown. The present study investigated the potential effect of SGRh2 on LMN-CRC in vitro and in vivo. SW480 and CoLo205 cell lines were treated with SGRh2. SGRh2 dose-dependently decreased CRC cell proliferation by CCK-8, colony formation and Edu assays. The Transwell and scratch assays revealed that SGRh2 inhibits the migratory and invasive abilities of CRC cells in a dose-dependent manner. Furthermore, the results of Western blotting revealed that SGRh2 decreased the expression of matrix metalloproteinase (MMP)-2 and MMP9. In terms of the underlying mechanisms, SGRh2 regulates CRC metastasis by affecting epithelial-mesenchymal transition (EMT), which significantly up-regulated epithelial biomarkers (E-cadherin) and down-regulated mesenchymal biomarkers (N-cadherin and vimentin) and EMT transcriptional factors (Smad-3, Snail-1, and Twist-1). In vivo, SGRh2 significantly inhibited LMN-CRC without affecting other normal organs. Immunohistochemical results showed that SGRh2 treats LMN-CRC by regulating EMT. These results demonstrate that SGRh2 has therapeutic potential for LMN-CRC.

Tuina Massage Improves Cognitive Functions of Hypoxic-Ischemic Neonatal Rats by Regulating Genome-Wide DNA Hydroxymethylation Levels.

In addition to abnormalities of motor and posture, children with cerebral palsy (CP) often have intellectual disability. As a complementary and alternative traditional Chinese medicine (TCM) therapy, Chinese Tuina massage, also called Tuina in China, has been widely applied in clinical treatment for CP in China for a long time. However, the molecular basis for this still remains largely unknown. Recently, DNA hydroxymethylation has been shown to be sensitive to environment and plays critical roles in some neurological disorders, whereas the research focusing on the relationship between 5 hmC and Tuina therapy for cerebral palsy is deficient. In our study, we first observed that Tuina improved learning and memory functions of hypoxic-ischemic (HI) rat pups. Meanwhile, 5 hmC level of the temporal lobe cortex in the HI neonatal rat model is decreased significantly compared to that of the rats in control and Tuina groups. Then, we used the hMeDIP-Seq method to explore whether and how DNA hydroxymethylation is involved in Tuina therapy for cerebral palsy. Genomic annotation of DhMRs of HI group's hypo-hydroxymethylation to genes revealed enrichment in multiple neurodevelopmental signaling pathways. Moreover, we found the depletion of 5 hmC modifications in genes associated with neuronal development was accompanied by reduced mRNA levels of these genes. Taken together, our results indicate that Tuina may regulate the expression of neurodevelopment-related genes by changing the status of DNA hydroxymethylation, thereby improving learning and memory functions of cerebral palsy.

Rutin promotes the formation and osteogenic differentiation of human periodontal ligament stem cell sheets in vitro.

Cell sheet technology is a novel tissue engineering technology that has been rapidly developed in recent years. As a novel technology, cell sheet technology is expected to become one of the preferred methods for cell transplantation. The present study investigated the biological effects of rutin on the formation of periodontal ligament stem cell (PDLSC) sheets and their resultant osteogenic properties. The results of Cell Counting Kit­8 (CCK­8) assay demonstrated that a concentration of 1x10­6 mol/l rutin promoted the proliferation of PDLSCs more effectively compared with other designed concentrations. Rutin­modified cell sheets could be induced by complete medium supplemented with 20 µg/ml vitamin C (VC) and 1x10­6 mol/l rutin. Rutin­modified cell sheets appeared thicker and more compact compared with the VC­induced PDLSC sheets, demonstrating more layers of cells (3 or 4 layers), which secreted a richer extracellular matrix (ECM). Furthermore, the improved cell sheets exhibited varying degrees of increases in the mRNA and protein expression of collagen type I (COL1), alkaline phosphatase (ALP), runt­related transcription factor 2 (RUNX2) and osteopontin (OPN). Combined treatment with VC and rutin promoted the formation of PDLSC sheets and enhanced the osteogenic differentiation potential of the cell sheets. Therefore, rutin­modified cell sheets of PDLSCs are expected to play an important role in the treatment of periodontal tissue regeneration by stem cells.

Subchronic toxicity and concomitant toxicokinetics of long-term oral administration of total alkaloid extracts from seeds of Peganum harmala Linn: A 28-day study in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: The seeds of Peganum harmala Linn, in which the most abundant active compounds are harmaline and harmine, have been widely used as a traditional medicine in various countries to treat a broad spectrum of diseases including asthma, cough, depression, Parkinson's and Alzheimer's diseases. However, few studies on long-term or subchronic toxicity of seeds of P. harmala were reported after overdose. AIM OF THE STUDY: To investigate the subchronic toxicity and concomitant toxicokinetics of total alkaloid extracts from seeds of P. harmala (TAEP) after oral administration for four weeks in rats. MATERIALS AND METHODS: The subchronic toxicity and concomitant toxicokinetics of TAEP were evaluated after 28-day oral administration in rats at daily dose levels of 15, 45, and 150 mg/kg. The signs of toxicity and mortality were monitored and recorded daily. The body weight and average food consumption were measured weekly. The analyses of hematology, biochemistry, urine, relative organ weights and histopathology were conducted at the termination of treatment and recovery phase. For concomitant toxicokinetics study, the plasma toxicokinetic parameters, tissue distribution, and excretion of predominant ingredients harmaline and harmine in TAEP and metabolites harmalol and harmol were tested. RESULTS: Following initial repeated exposure to high-dose (150 mg/kg/day) of TAEP excitotoxic reaction, such as tremor, was observed, but tolerated on the fourth day after multiple dosing. The significant alterations in blood glucose and lipid metabolism in liver were observed, but recovered after four weeks of drug withdrawal. The no-observed-adverse-effect level (NOAEL) of TAEP was considered to be 45 mg/kg/day under the present study conditions. There were no significant gender differences in most indexes of subchronic toxicity throughout the experimental period with the exception of food consumption and body weight. In concomitant toxicokinetics study, the alterations of dynamic characteristic for harmaline, harmine and metabolite harmol after multiple oral administration at three doses had been observed. Harmaline, harmine and metabolites harmalol and harmol were widely distributed in organs and there was no accumulation in the tissues examined. The reduction of harmaline and metabolite harmalol in brain after multiple dosing at dose of 150 mg/kg might be closely related to the tremor tolerance. The main excretory pathway for metabolites harmalol and harmol was urinary excretion via kidney. CONCLUSIONS: The results revealed that TAEP at doses of 15 and 45 mg/kg/day in rats might be safe. Excitotoxic reaction such as tremor occurred initially at dose of 150 mg/kg/day, however, the toxicity was tolerant and reversible. In addition, harmaline and harmine in TAEP had a quick absorption into blood and metabolized to harmalol and harmol, and there was no drug accumulation in the detected tissues. Further studies should be investigated to clarify the mechanisms of tremor tolerance and neurotoxicity of TAEP.

In vivo and in vitro metabolism and pharmacokinetics of cholinesterase inhibitor deoxyvasicine from aerial parts of Peganum harmala Linn in rats via UPLC-ESI-QTOF-MS and UPLC-ESI-MS/MS.

ETHNOPHARMACOLOGICAL RELEVANCE: Aerial parts of Peganum harmala Linn are a Uighur traditional medicinal herb in China used to treat amnesia, bronchial asthma, and cough. Deoxyvasicine (DVAS), a potent cholinesterase inhibitor exhibiting anti-senile dementia activity, is one of the chief active ingredients in aerial parts of P. harmala and plays a key role in mediating the pharmacological effects of P. harmala. However, the metabolic profiling and in vivo pharmacokinetic characteristics of DVAS still remain unknown. AIM OF THE STUDY: The aim of this present study was to investigate the metabolism and pharmacokinetic properties of DVAS in rats by using ultra-performance liquid chromatography combined with electrospray ionization quadrupole time-of-flight tandem mass spectrometry (UPLC-ESI-QTOF-MS) and ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-ESI-MS/MS) method. MATERIALS AND METHODS: The metabolic profiling of DVAS was evaluated in vitro and in vivo by rat liver microsomes (RLMs) incubation and by rat bio-specimens, such as urine, feces, plasma, and bile, after the oral administration of 45 mg/kg DVAS. An efficient and sensitive UPLC-ESI-MS/MS method was developed and validated to simultaneously determine DVAS and its major four metabolites, namely, vasicine, deoxyvasicinone, vasicinone, and 1,2,3,9-tetrahydropyrrolo[2,1-b]quinazolin-3-ß-D-glucuronide in rat plasma. For pharmacokinetic studies, 32 Sprague-Dawley rats were randomly divided into four groups, namely, intravenous dosage group (2 mg/kg DVAS) and three oral dosage groups (5, 15, and 45 mg/kg DVAS). In addition, the activity of the components in plasma after intravenous administration of DVAS was evaluated by in vitro anti-butyrylcholinesterase (BChE) assays. RESULTS: A total of 23 metabolites were found in RLMs, plasma, urine, feces, and bile by UPLC-ESI-QTOF-MS. The metabolic pathway of DVAS in vivo and in vitro mainly involved hydroxylation, dehydrogenation, acetylation, methylation, glucuronidation, and O-sulphate conjugation, and the C-3 and C-9 sites were the main metabolic soft spots. All 23 metabolites were detected in the urine sample, and 13, 8, 22, and 6 metabolites were identified from rat feces, plasma, bile, and RLMs, respectively. The standard curves of DVAS and four metabolites in rat plasma showed good linearity in the concentration range of 0.82-524.00 ng/mL with acceptable selectivity, precision, accuracy, recovery, and stability. DVAS exhibited linear dose-proportional pharmacokinetics at doses of 5, 15, and 45 mg/kg after oral administration, and the average oral absolute bioavailability of DVAS was 47.46%. The in vitro anti-BChE assays implied that the inhibitive activities were mainly due to the different concentrations of prototype DVAS. CONCLUSIONS: DVAS can be rapidly absorbed and excreted by blood, and it is also extensively metabolized in vivo, and the anti-BChE activity in blood is mainly attributed to DVAS. These findings can lay a foundation for new drug development for DVAS.

Identification and characterization of forced degradation products and stability-indicating assay for notoginsenosidefc by using UHPLC-Q-TOF-MS and UHPLC-MS/MS: Insights into stability profile and degradation pathways.

Notoginsenoside Fc, a protopanaxadiol-type saponin, shows multi-pharmacological activities. Chemical stability evaluation plays a crucial role in drug development. In this study, the forced degradation behavior of Notoginsenoside Fc was investigated under hydrolytic and oxidative conditions. A specific ultra high performance liquid chromatography with quadrupole time-of-flight mass spectrometry was developed for the separation, identification, and characterization of the degradation products of Notoginsenoside Fc. Fifty potential degradation products were formed via deglycosylation, dehydration, hydration, isomerization, side-chain cleaving, oxidation, and superoxidation. Notoginsenoside Fc was subjected to different pH solutions, temperatures, and time periods to assess its stability. A sensitive ultra high performance liquid chromatography-tandem mass spectrometry was developed for the quantification of Notoginsenoside Fc, notoginsenoside ST-4, notoginsenoside Ft1, and relative quantification of notoginsenoside Ft2, 20(R)-notoginsenoside Ft2, notoginsenoside SFt3, and notoginsenoside SFt4. The assay was linear over the concentration range (R2  > 0.997) with the lowest limit of quantification of 0.02 µg/mL for Notoginsenoside Fc, Notoginsenoside ST-4, and Notoginsenoside Ft1. The intra-day precision, inter-day precision, and accuracy of the three analytes were within accepted levels. The degradation kinetics of Notoginsenoside Fc in pH 1 and 3 solutions fits to first- and second-order kinetics, respectively. The degradation of Notoginsenoside Fc is pH-, temperature-, and time-dependent.

Antitumor activity of the plant extract morin in tongue squamous cell carcinoma cells.

Morin is a naturally occurring bioflavonoid originally isolated from members of the Moraceae family of flowering plants and it possesses antitumor activity in various human cancer cells. The present study explored the antitumor effects of morin in tongue squamous cell carcinoma (TSCC) cells in vitro and investigated the underlying molecular events. A TSCC cell line was treated with different doses of morin for up to 48 h. Analyses of cell viability, using Cell Counting Kit­8 (CCK­8), EdU incorporation, colony formation, flow cytometric analysis of cell cycle distribution and apoptosis, wound healing assay, western blot analysis and qRT­PCR assays, were then performed. The data revealed that morin treatment reduced Cal27 cell proliferation and reduced the migration capacity of tumor cells in a dose­dependent manner. Morin treatment also significantly upregulated mammalian sterile 20­like 1 (MST1) and MOB kinase activator 1 (MOB1) phosphorylation in CAL27 cells, but suppressed nuclear translocation of yes­associated protein (YAP) through the induction of YAP phosphorylation in Cal27 cells. Moreover, the expression of YAP­targeting genes, such as CTGF, CYR61 and ANKRD, was downregulated in morin­treated TSCC cells, indicating that morin was able to activate the Hippo signaling pathway to inhibit YAP nuclear translocation and YAP­related transcriptional activity in TSCC cells. In conclusion, the data from the present study demonstrated that morin produces anti­TSCC activity in vitro through activation of the Hippo signaling pathway and the downstream suppression of YAP activity in TSCC cells. Future studies should assess the clinical antitumor effects of morin.

SEECancer: a resource for somatic events in evolution of cancer genome.

Cancer cells progressively evolve from a premalignant to a malignant state, which is driven by accumulating somatic alterations that confer normal cells a fitness advantage. Improvements in high-throughput sequencing techniques have led to an increase in construction of tumor phylogenetics and identification of somatic driver events that specifically occurred in different tumor progression stages. Here, we developed the SEECancer database (http://biocc.hrbmu.edu.cn/SEECancer), which aims to present the comprehensive cancer evolutionary stage-specific somatic events (including early-specific, late-specific, relapse-specific, metastasis-specific, drug-resistant and drug-induced genomic events) and their temporal orders. By manually curating over 10 000 published articles, 1231 evolutionary stage-specific genomic events and 5772 temporal orders involving 82 human cancers and 23 tissue origins were collected and deposited in the SEECancer database. Each entry contains the somatic event, evolutionary stage, cancer type, detection approach and relevant evidence. SEECancer provides a user-friendly interface for browsing, searching and downloading evolutionary stage-specific somatic events and temporal relationships in various cancers. With increasing attention on cancer genome evolution, the necessary information in SEECancer will facilitate understanding of cancer etiology and development of evolutionary therapeutics, and help clinicians to discover biomarkers for monitoring tumor progression.

MPINet: metabolite pathway identification via coupling of global metabolite network structure and metabolomic profile.

High-throughput metabolomics technology, such as gas chromatography mass spectrometry, allows the analysis of hundreds of metabolites. Understanding that these metabolites dominate the study condition from biological pathway perspective is still a significant challenge. Pathway identification is an invaluable aid to address this issue and, thus, is urgently needed. In this study, we developed a network-based metabolite pathway identification method, MPINet, which considers the global importance of metabolites and the unique character of metabolomic profile. Through integrating the global metabolite functional network structure and the character of metabolomic profile, MPINet provides a more accurate metabolomic pathway analysis. This integrative strategy simultaneously captures the global nonequivalence of metabolites in a pathway and the bias from metabolomic experimental technology. We then applied MPINet to four different types of metabolite datasets. In the analysis of metastatic prostate cancer dataset, we demonstrated the effectiveness of MPINet. With the analysis of the two type 2 diabetes datasets, we show that MPINet has the potentiality for identifying novel pathways related with disease and is reliable for analyzing metabolomic data. Finally, we extensively applied MPINet to identify drug sensitivity related pathways. These results suggest MPINet's effectiveness and reliability for analyzing metabolomic data across multiple different application fields.