The coumarin component isofraxidin targets the G-protein-coupled receptor S1PR1 to modulate IL-17 signaling and alleviate ulcerative colitis.

OBJECTIVE: The increasing global prevalence of ulcerative colitis (UC) underscores the imperative to explore novel therapeutic approaches. Traditional Chinese medicine has historically shown potential in addressing this ailment. The current study aimed to elucidate the functional attributes and underlying mechanisms of isofraxidin, a coumarin derivative from Acanthopanax, in the context of UC. METHODS: A murine model of dextran sodium sulfate (DSS)-induced UC was established, and we conducted a comprehensive assessment of the influence of isofraxidin on UC symptomatology, colonic histopathological manifestations, the inflammatory response, and apoptosis. The potential receptor of isofraxidin was initially identified through the Target database and molecular docking analysis. Subsequent in vivo and in vitro experiments were conducted to determine the effects of isofraxidin on the identified receptor and associated signaling pathways. Transfection was used to examine the receptor's role in the regulatory mechanism of isofraxidin. RESULTS: Isofraxidin reduced UC symptoms and colonic histopathological impairments. Furthermore, isofraxidin ameliorated the DSS-induced inflammatory response and apoptosis in tissues. S1PR1 was identified as a target of isofraxidin and effectively suppressed activation of the IL-17 signaling pathway. Intriguingly, cellular experiments indicated that overexpression of S1PR1 counteracted the protective effect of isofraxidin. DISCUSSION: In summary, our investigation revealed that isofraxidin could modulate S1PR1 and regulate the IL-17 signaling pathway, thus ameliorating DSS-induced UC. These findings establish a robust foundation for considering isofraxidin as a prospective therapeutic intervention to treat UC.

Antibacterial and antibiofilm activity of Lagotis brachystachya extract against extended-spectrum ß-lactamases-producing Escherichia coli from broiler chickens.

Lagotis brachystachya Maxim (L. brachystachya) is an herb widely used in traditional Tibetan medicine. In the present study, the antibacterial activity of L. brachystachya extract to extended-spectrum-lactamases (ESBLs)-producing E. coli was determined by Kirby-Bauer disc diffusion, minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) methods as well as time-kill curve assay. Meanwhile, the biofilm inhibition and eradication effects of L. brachystachya extract on the ESBLs-producing E. coli were evaluated by crystal violet staining, and further confirmed by confocal laser scanning microscope (CLSM) and scanning electron microscopy (SEM). The results indicated that L. brachystachya extract exhibited moderate antibacterial activity, with diameter of inhibition zones varying from 15.4 to 20.3 mm, and the MIC and MBC values were 6.25 to 25 mg/mL and 12.5 to 100 mg/mL, respectively. Time-kill curve showed that 4 × MIC level of L. brachystachya extract concentration of was able to kill 99.9% of ESBLs-producing E. coli after 16 h treatment. The biofilm inhibition rate and eradication rate for the ESBLs-producing E. coli were 35.66 to 79.91% and 22.18 to 56.21% at MIC level of extract concentration, respectively. CLSM images showed that the biofilm became thinner as the ESBLs-producing E. coli isolate exposed to L. brachystachya extract with a concentration-dependent manner from 1/4 × MIC to MIC compared with the control isolate. SEM images indicated that L. brachystachya extract at 1/2 × MIC and MIC levels could evidently inhibit the biofilm formation or eradicate the mature biofilms. The effect of L. brachystachya highlights its potential of antibacterial and antibiofilm activities against the ESBLs-producing E. coli.

Exploring the Mechanism of Action of Canmei Formula Against Colorectal Adenoma Through Multi-Omics Technique.

Background: Canmei formula (CMF) is a traditional Chinese medicine compound with definite effect on the prevention and treatment of colorectal adenoma (CRA). CMF can prevent the transformation of intestinal inflammation to cancer. This study explored the mechanism of action of CMF in anti-CRA using multi-omics techniques. Method: The mice were randomly divided into four groups: blank group (Control), high-fat diet (HFD) + AOM/DSS colorectal adenoma model (ADH) groups, Canmei formula treatment group (ADH-CMF) and sulfasalazine treatment group (Sul). Except for the blank group, ADH model was established in the other three groups by intraperitoneal injection with AOM reagent, and then mice were given 2.5% DSS in free drinking water and high-fat diet. The mice in the blank group and ADH groups were intragastrically perfused with normal saline, and the mice in the other two groups were treated with corresponding drugs for 20 weeks. During this period, the changes of physical signs of mice in each group were observed. The differentially expressed genes and proteins in the Control group, ADH group and ADH-CMF group were detected by RNA-seq transcriptome sequencing and Tandem Mass Tags (TMT) quantitative proteomics. After the combined analysis and verification, the key targets were analyzed by gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). Moreover, the changes of intestinal flora in mice of the three groups were examined. Results: A total of 2,548 differential genes were obtained by transcriptomics analysis, and 45 differential proteins were obtained by proteomics analysis. The results of proteomics data and experimental verification showed that CMF mainly affected the Phospholysine Phosphohistidine Inorganic Pyrophosphate Phosphatase (LHPP) target. GO analysis showed that the targets of CMF were involved in the biological processes such as cellular process, metabolic process and biological regulation. KEGG analysis showed that those genes were involved in oxidative phosphorylation, cell senescence, and metabolic pathways. Studies have shown that LHPP overexpression impeded colorectal cancer cell growth and proliferation in vitro, and was associated with a change in PI3K/AKT activity. The results of 16S DNA high-throughput sequencing showed that CMF could effectively regulate the abundance of Bifidobacterium, Candidatus_Saccharimonas and Erysipelatoclostridium in the intestinal flora at the genus level. Conclusion: CMF regulates LHPP via the PI3K/AKT signaling pathway. CMF affects the abundance of specific intestinal flora and can regulate the disorder of intestinal flora to achieve the role of prevention and treatment of CRA.

Bioinformatics Analysis to Screen Key Targets of Curcumin against Colorectal Cancer and the Correlation with Tumor-Infiltrating Immune Cells.

PURPOSE: Curcumin is a potential drug for the treatment of colorectal cancer (CRC). Its mechanism of action has not been elucidated. This study aims to investigate the mechanism of action of curcumin in the treatment of CRC via bioinformatics methods such as network pharmacology and molecular docking. METHODS: The targets of curcumin and CRC were obtained from the public databases. The component-targets network of curcumin in the treatment of CRC was constructed by Cytoscape v3.7.2. Through protein-protein interaction (PPI), the Gene Ontology (GO), and the Kyoto Encyclopedia of Genes and Genomes (KEGG), important targets and signaling pathways related to CRC treatment were identified. Finally, the results were verified by molecular docking, and the correlation between the key targets and tumor-infiltrating immune cells (TICs) was analyzed. RESULTS: A total of 30 potential targets of curcumin for CRC treatment were collected. The GO function enrichment analysis showed 140 items, and the KEGG pathway enrichment analysis showed 61 signaling pathways related to the regulation of protein kinase activity, negative regulation of apoptosis process, cancer signaling pathway, and PI3K-Akt signaling pathway. The molecular docking results showed that curcumin could be combined with AKT1, EGFR, and STAT3 more stably, and AKT1 has the strongest binding to curcumin. Bioinformatics analysis discovered that the expression of core targets AKT1, EGFR, and STAT3 in CRC was related to TICs. CONCLUSION: This study explored the targets and pathways of curcumin in the treatment of CRC. The core targets are AKT1, EGFR, and STAT3. The study indicated that curcumin has preventive and treatment effects on CRC through multitarget and multipathway, which laid the foundation for follow-up research.

Bipolar plasmakinetic transurethral enucleation and resection versus bipolar plasmakinetic transurethral resection for surgically treating large (≥60 g) prostates: a propensity score-matched analysis with a 3-year follow-up.

BACKGROUND: There are extremely limited published studies comparing bipolar plasmakinetic transurethral resection of prostate (BP-TURP), bipolar plasmakinetic transurethral enucleation and resection of prostate (BP-TUERP) for enlarged prostates. Our purpose was to evaluate the safety and efficiency of BP-TUERP and BP-TURP for large (≥60 g) prostates with a 3-year follow-up. METHODS: We retrospectively identified 229 patients according to inclusion criteria between 2014 and 2016. After applying propensity score matching method, preoperative results and three-year follow-up outcomes in International Prostate Symptom Score (IPSS), urinary peek flow rate (Qmax), postvoid residual urine (PVRU) volume and quality of life (QoL) score were compared. RESULTS: Finally, within the well-balanced matched cohort, the BP-TUERP was significantly associated with longer mean operating time (OT) (P=0.039), shorter mean catheter time (CT) (P=0.001) and lower mean hemoglobin decrease (P=0.002) with more prostatic tissue removed (P=0.001) than the BP-TURP, but the median hospital stay lengths and the short- and long-term complication rates were similar between the two series. The patients in the BP-TUERP group had better long-term outcomes in IPSS, Qmax and PVRU volume than these in the BP-TURP group, but not in QoL score. CONCLUSIONS: For patients with large (≥60 g) prostates, BP-TUERP and BP-TURP are safe options, but the former is a more effective choice in long-term follow-up outcomes. BP-TUERP is related to reduced CT and hemoglobin decrease with more removal of prostatic tissue at the expense of longer OT than BP-TURP.

Ultrasensitive photoelectrochemical aptasensor for diclofenac sodium based on surface-modified TiO2-FeVO4 composite.

Herein, a photoelectrochemical (PEC) aptasensing platform was designed by integrating surface oxygen vacancy (OV) defects, Ti3+ self-doping, the heterojunction, and resonance energy transfer (RET) effect into one platform for the detection of diclofenac sodium (DCF). Briefly, OV defects were introduced on TiO2 nanospheres with simultaneous Ti3+ self-doping, followed by a well-separated deposition of FeVO4 nanoparticles on TiO2 to obtain a Ti3+-O-TiO2/FeVO4 heterojunction. The surface modification of OVs, Ti3+ doping, and deposition of FeVO4 were confirmed by SEM, XPS, EPR, DRS, and PEC measurements. The surface OVs and doping of Ti3+ species created a new donor (defect) energy level under the conduction band of TiO2, which minimized the bandgap and thereby improved the visible light absorption of TiO2. Moreover, the capture of photo-excited electrons by surface OVs could hinder the electron-hole recombination. Due to the intimate surface contact and perfect energy matching between TiO2 and FeVO4, the formation of heterojunction decreased the bandgap and facilitated the electron-hole separation of TiO2. All these above events contributed to the enhancement of the PEC signals, which were then quenched by the RET effect between Ti3+-O-TiO2/FeVO4 and Au nanoparticle (AuNP)-labeled cDNA that had been attached to its complementary DCF aptamer on Ti3+-O-TiO2/FeVO4|ITO. The addition of target-DCF detached AuNP-labeled cDNA from the electrode to recover the photocurrent, resulting in a "signal-on" PEC aptasensor that exhibited a 0.1-500-nM linear range and a detection limit of 0.069 nM for DCF, attributed to the excellent amplification of the proposed aptasensing platform.

Integrated Analysis of Seed microRNA and mRNA Transcriptome Reveals Important Functional Genes and microRNA-Targets in the Process of Walnut (Juglans regia) Seed Oil Accumulation.

Walnut (Juglans regia) is known as a promising woody oil crop with abundant polyunsaturated fatty acids in its kernel. However, the regulation mechanism of walnut oil accumulation and fatty acid metabolism is still poorly understood, which restricted the breeding and genetic improvement of high-quality oil-bearing walnuts. To reveal the molecular mechanism of walnut oil accumulation, considering the potential regulation of microRNA (miRNA) in seed development, in this study, the oil content of walnut kernel on the 80th, 100th and 120th day after flowering (DAF) was tested and the corresponding proportions are 11.51%, 40.40% and 53.20%. Between DAF of 80th~120th, the content of stearic acid and oleic acid tended to increase, but the proportion of other fatty acids tended to decrease. Meanwhile, comparative transcriptome and sRNA-seq analysis on three stages (80th, 100th and 120th DAF), found 204 conserved miRNAs and 554 novel miRNAs in walnut kernels, among which 104 key genes related to walnut oil accumulation were screened. The phospholipid:diacylglycerol acyltransferase metabolic pathway may contribute more to oil accumulation in walnut. 16 miRNA-mRNA regulatory modules related to walnut oil accumulation and fatty acid synthesis were constructed. 8 known miRNAs and 9 novel miRNAs regulate 28 genes involved in fatty acid (FA) metabolism and lipid synthesis. Among them, jre-miRn105, jre-miRn434, jre-miR477d and jre-miR156a.2 are key miRNAs that regulate walnut FA synthesis. Jre-miRn411 and jre-miR399a.1 are closely related to oil accumulation. These data provide new insights and lay the foundation for subsequent studies on walnut FA synthesis and oil accumulation.

Pharmacological Interventions for Bacterial Prostatitis.

Prostatitis is a common urinary tract condition but bring innumerable trouble to clinicians in treatment, as well as great financial burden to patients and the society. Bacterial prostatitis (acute bacterial prostatitis plus chronic bacterial prostatitis) accounting for approximately 20% among all prostatitis have made the urological clinics complain about the genital and urinary systems all over the world. The international challenges of antibacterial treatment (emergence of multidrug-resistant bacteria, extended-spectrum beta-lactamase-producing bacteria, bacterial biofilms production and the shift in bacterial etiology) and the transformation of therapeutic strategy for classic therapy have attracted worldwide attention. To the best of our knowledge currently, there is not a single comprehensive review, which can completely elaborate these important topics and the corresponding treatment strategy in an effective way. This review summarizes the general treatment choices for bacterial prostatitis also provides the alternative pharmacological therapies for those patients resistant or intolerant to general treatment.

Thermal Intravesical Chemotherapy Reduce Recurrence Rate for Non-muscle Invasive Bladder Cancer Patients: A Meta-Analysis.

Background: Non-muscle invasive bladder cancer accounts for nearly 80% of newly diagnosed bladder cancer cases, which often recur and progress. This meta-analysis was evaluated by the adverse events and recurrence rate of thermal intravesical chemotherapy vs. normal temperature intravesical chemotherapy in the treatment of non-muscle invasive bladder cancer. Methods: A systematic review and cumulative analysis of studies reporting adverse events and recurrence rate of thermal intravesical chemotherapy vs. normal temperature intravesical chemotherapy was performed through a comprehensive search of Pubmed, Embase, Cochranelibrary.com, CNKI, Wanfang Med Online database and VIP database. All analyses were performed using the Revman manager 5. Result: Twelve studies (11 randomized controlled trials and 1 retrospective study) including 888 patients, 445 in the thermal intravesical chemotherapy group, and 443 in the normal temperature intravesical chemotherapy group, met the eligibility criteria. Patients in the thermal intravesical chemotherapy group had a lower risk of disease recurrence than those who had normal temperature intravesical chemotherapy (24 months follow-up group: RR = 0.30, 95% CI: 0.21-0.43, P < 0.00001, I 2 = 0%; 36 months follow-up group: RR = 0.27, 95% CI: 0.14-0.54, P = 0.0002, I 2 = 0%) while no significant difference in adverse events rate (RR = 0.89, 95% CI = 0.53-1.52; P = 0.67, I 2 = 78%). Conclusions: When compared with normal temperature intravesical chemotherapy, thermal intravesical chemotherapy can reduce the recurrence rate without increasing incidence of adverse events in patients with non-muscle invasive bladder cancer.

Circadian clock associates with tumor microenvironment in thoracic cancers.

The application of cancer chronotherapy is to treat cancers based on at specific times during circadian rhythms. Previous studies have characterized the impact of circadian clock on tumorigenesis and specific immune cells. Here, by using multi-omics computation techniques, we systematically characterized the distinct roles of core circadian clock genes in thoracic cancers including lung adenocarcinoma, lung squamous cell carcinoma, and esophageal carcinoma. Strikingly, a wide range of core clock genes are epigenetically altered in lung adenocarcinomas and lung squamous cell carcinomas but not esophageal carcinomas. Further cancer hallmark analysis reveals that several core clock genes highly correlate with apoptosis and cell cycle such as RORA and PER2. Interestingly, our results reveal that CD4 and CD8 T cells are correlated with core clock molecules especially in lung adenocarcinomas and lung squamous cell carcinomas, indicating that chrono-immunotherapy may serve as a candidate option for future cancer management.

Accuracy of multiparametric magnetic resonance imaging for diagnosing prostate Cancer: a systematic review and meta-analysis.

BACKGROUND: The application of multiparametric magnetic resonance imaging (mpMRI) for diagnosis of prostate cancer has been recommended by the European Association of Urology (EAU), National Comprehensive Cancer Network (NCCN), and European Society of Urogenital Radiology (ESUR) guidelines. The purpose of this study is to systematically review the literature on assessing the accuracy of mpMRI in patients with suspicion of prostate cancer. METHOD: We searched Embase, Pubmed and Cochrane online databases from January 12,000 to October 272,018 to extract articles exploring the possibilities that the pre-biopsy mpMRI can enhance the diagnosis accuracy of prostate cancer. The numbers of true- and false-negative results and true- and false-positive ones were extracted to calculate the corresponding sensitivity and specificity of mpMRI. Study quality was assessed using QUADAS-2 tool. Random effects meta-analysis and a hierarchical summary receiver operating characteristic (HSROC) plot were performed for further study. RESULTS: After searching, we acquired 3741 articles for reference, of which 29 studies with 8503 participants were eligible for inclusion. MpMRI maintained impressive diagnostic value, the area under the HSROC curve was 0.87 (95%CI,0.84-0.90). The sensitivity and specificity for mpMRI were 0.87 [95%CI, 0.81-0.91] and 0.68 [95%CI,0.56-0.79] respectively. The positive likelihood ratio was 2.73 [95%CI 1.90-3.90]; negative likelihood ratio was 0.19 [95% CI 0.14,-0.27]. The risk of publication bias was negligible with P = 0.96. CONCLUSION: Results of the meta-analysis suggest that mpMRI is a sensitive tool to diagnose prostate cancer. However, because of the high heterogeneity existing among the included studies, further studies are needed to apply the results of this meta-analysis in clinic.

Biochemical characterization reveals the functional divergence of two tropinone reductases from Przewalskia tangutica.

Przewalskia tangutica is a traditional medicinal plant from Tibet used for the analgesic effect from the tropane alkaloids (TAs) produced by the plant. Its roots have the highest yield of hyoscyamine in all plant species and so have been overharvested becoming an endangered medicinal plant species. Metabolic engineering is a good way to improve the yield of TAs in plants. In our study, two functionally distinct tropinone reductases genes, PtTRI and PtTRII, were cloned from P. tangutica and the functional divergence were characterized. The enzyme kinetics of PtTRI and PtTRII were investigated. The phylogenetic analysis classified them into different clades: PtTRI and PtTRII were in the clade of tropine-forming reductase and pseudotropine-forming reductase, respectively. We found PtTRI to be expressed in the roots but less in leaves, whereas PtTRII was expressed in the roots at higher levels than in the leaves. The kinetic parameters (Km , Vmax , and Kcat ) were analyzed using purified recombinant enzymes at their optimum pH. Enzymatic analysis results showed that tropinone is a better substrate for PtTRII compared with PtTRI, suggesting that PtTRII might be a potential gene target for TA biosynthesis engineering. Compared with the reported TRIs, PtTRI exhibited a higher affinity for tropinone.

A highly sensitive electrochemical aptasensor for detection of microcystin-LR based on a dual signal amplification strategy.

In this work, a sensitive and selective electrochemical aptasensor for determination of microcystin-LR (MC-LR) was developed based on a dual signal amplification system consisting of a novel ternary composite and horseradish peroxidase (HRP). The ternary composite was prepared by depositing gold nanoparticles (AuNPs) on molybdenum disulfide (MoS2) covered TiO2 nanobeads (TiONBs). MoS2 nanosheet modified TiONBs provided a large surface area for immobilization of AuNPs and biomolecules. The ternary composite also possesses an improved electron transfer and catalytic capability. To construct the aptasensor, thiolated MC-LR aptamers were immobilized on the AuNP@MoS2-TiONB modified electrode through a gold-sulfur bond. Then, biotin-cDNA with a sequence complementary to the MC-LR aptamer competed with MC-LR for binding to the immobilized aptamer. The current signal catalyzed by avidin-HRP decreased with the increase of MC-LR, based on which a linear range of 0.005-30 nM and a detection limit of 0.002 nM were obtained.

Metabolic characterization of Hyoscyamus niger root-specific putrescine N-methyltransferase.

N-methylputrescine is the precursor of nicotine and pharmaceutical tropane alkaloids such as hyoscyamine. Putrescine N-methyltransferase (PMT) catalyzes the N-methylation of putrescine to form N-methylputrescine. While the role of PMT in nicotine biosynthesis is clear, knowledge of PMT in the biosynthesis of tropane alkaloids (TAs) and the regulation of polyamines remains limited. We characterized a PMT gene from Hyoscyamus niger, designated HnPMT that was specifically expressed in roots, especially in the secondary roots and dramatically induced by methyl jasmonate (MeJA). The GUS gene was specifically expressed in Arabidopsis roots or in the vascular tissues, including pericycles and endodermis, of the H. niger hairy root cultures, when it was driven by the 5'-flanking promoter region of HnPMT. The recombinant HnPMT was purified for enzymatic assays. HnPMT converted putrescine to form N-methylputrescine, as confirmed by LC-MS. The kinetics analysis revealed that HnPMT had high affinity with putrescine but low catalytic activity, suggesting that it was a rate-limiting enzyme. When HnPMT was suppressed in the H. niger plants by using the VIGS approach, the contents of N-methylputrescine and hyoscyamine were markedly decreased, but the contents of putrescine, spermidine and a mixture of spermine and thermospermine were significantly increased; this suggested that HnPMT was involved in the biosynthesis of tropane alkaloids and played a competent role in regulating the biosynthesis of polyamines. Functional identification of HnPMT facilitated the understanding of TA biosynthesis and thus implied that the HnPMT-catalyzed step might be a target for metabolic engineering of the TA production in H. niger.

Enhancing Tropane Alkaloid Production Based on the Functional Identification of Tropine-Forming Reductase in Scopolia lurida, a Tibetan Medicinal Plant.

Scopolia lurida, a native herbal plant species in Tibet, is one of the most effective producers of tropane alkaloids. However, the tropane alkaloid biosynthesis in this plant species of interest has yet to be studied at the molecular, biochemical, and biotechnological level. Here, we report on the isolation and characterization of a putative short chain dehydrogenase (SDR) gene. Sequence analysis showed that SlTRI belonged to the SDR family. Phylogenetic analysis revealed that SlTRI was clustered with the tropine-forming reductases. SlTRI and the other TA-biosynthesis genes, including putrescine N-methyltransferase (SlPMT) and hyoscyamine 6ß-hydroxylase (SlH6H), were preferably or exclusively expressed in the S. lurida roots. The tissue profile of SlTRI suggested that this gene might be involved in tropane alkaloid biosynthesis. By using GC-MS, SlTRI was shown to catalyze the tropinone reduction to yield tropine, the key intermediate of tropane alkaloids. With the purified recombinant SlTRI from Escherichiacoli, an enzymatic assay was carried out; its result indicated that SlTRI was a tropine-forming reductase. Finally, the role of SlTRI in promoting the tropane alkaloid biosynthesis was confirmed through metabolic engineering in S. lurida. Specifically, hairy root cultures of S. lurida were established to investigate the effects of SlTRI overexpression on tropane alkaloid accumulation. In the SlTRI-overexpressing root cultures, the hyoscyamine contents were 1.7- to 2.9-fold higher than those in control while their corresponding scopolamine contents were likewise elevated. In summary, this functional identification of SlTRI has provided for a better understanding of tropane alkaloid biosynthesis. It also provides a candidate gene for enhancing tropane alkaloid biosynthesis in S. lurida via metabolic engineering.

[Cloning and characterization of an aromatic amino acid aminotransferase(ArAT) gene involved in tropane alkaloid biosynthesis from Hyoscyamus niger].

Tropane alkaloids are anticholinergic drugs widely used clinically. Biosynthesis of tropane alkaloids in planta involves a step of transamination of phenylalanine. Based on the sequenced transcriptomes of lateral roots and leaves of Hyoscyamus niger, we found three annotated aromatic amino acid aminotransferases, which were respectively named HnArAT1, HnArAT2 and HnArAT3. Sequence analysis showed that HnArAT3 had highest similarity with the reported Atropa belladonna Ab Ar AT4, which was involved in tropane alkaloid(TA) to provide the precursor of the phenyllactic acid moiety. Tissue expression pattern analysis indicated that HnArAT3 was specifically expressed in lateral roots, where is the organ synthesizing tropane alkaloids. Then, method of virus induced gene silencing (VIGS) was used to characterize the function of HnArAT3 in H. niger. Gene expression analysis given by real-time quantitative PCR showed that all the transgenic lines had lower expression levels of HnArAT3 than the non-transgenic control, and HPLC analysis of alkaloids demonstrated significant decrease in the contents of hyoscyamine, anisodamine and scopolamine in planta. These results suggested that HnArAT3 was involved in the phenyllactic acid branch of TA biosynthetic pathway. Molecular cloning and functional identification of HnArAT3 laid the foundation for further understanding of TA biosynthesis and metabolic regulation, and also provided a new candidate gene for engineering biosynthetic pathway of tropane alkaloids.

Promoting scopolamine biosynthesis in transgenic Atropa belladonna plants with pmt and h6h overexpression under field conditions.

Atropa belladonna is one of the most important plant sources for producing pharmaceutical tropane alkaloids (TAs). T1 progeny of transgenic A. belladonna, in which putrescine N-methyltransferase (EC. 2.1.1.53) from Nicotiana tabacum (NtPMT) and hyoscyamine 6ß-hydroxylase (EC. 1.14.11.14) from Hyoscyamus niger (HnH6H) were overexpressed, were established to investigate TA biosynthesis and distribution in ripe fruits, leaves, stems, primary roots and secondary roots under field conditions. Both NtPMT and HnH6H were detected at the transcriptional level in transgenic plants, whereas they were not detected in wild-type plants. The transgenes did not influence the root-specific expression patterns of endogenous TA biosynthetic genes in A. belladonna. All four endogenous TA biosynthetic genes (AbPMT, AbTRI, AbCYP80F1 and AbH6H) had the highest/exclusive expression levels in secondary roots, suggesting that TAs were mainly synthesized in secondary roots. T1 progeny of transgenic A. belladonna showed an impressive scopolamine-rich chemotype that greatly improved the pharmaceutical value of A. belladonna. The higher efficiency of hyoscyamine conversion was found in aerial than in underground parts. In aerial parts of transgenic plants, hyoscyamine was totally converted to downstream alkaloids, especially scopolamine. Hyoscyamine, anisodamine and scopolamine were detected in underground parts, but scopolamine and anisodamine were more abundant than hyoscyamine. The exclusively higher levels of anisodamine in roots suggested that it might be difficult for its translocation from root to aerial organs. T1 progeny of transgenic A. belladonna, which produces scopolamine at very high levels (2.94-5.13 mg g(-1)) in field conditions, can provide more valuable plant materials for scopolamine production.

Functional characterisation of a tropine-forming reductase gene from Brugmansia arborea, a woody plant species producing tropane alkaloids.

Brugmansia arborea is a woody plant species that produces tropane alkaloids (TAs). The gene encoding tropine-forming reductase or tropinone reductase I (BaTRI) in this plant species was functionally characterised. The full-length cDNA of BaTRI encoded a 272-amino-acid polypeptide that was highly similar to tropinone reductase I from TAs-producing herbal plant species. The purified 29kDa recombinant BaTRI exhibited maximum reduction activity at pH 6.8-8.0 when tropinone was used as substrate; it also exhibited maximum oxidation activity at pH 9.6 when tropine was used as substrate. The Km, Vmax and Kcat values of BaTRI for tropinone were 2.65mM, 88.3nkatmg(-1) and 2.93S(-1), respectively, at pH 6.4; the Km, Vmax and Kcat values of TRI from Datura stramonium (DsTRI) for tropinone were respectively 4.18mM, 81.20nkatmg(-1) and 2.40S(-1) at pH 6.4. At pH 6.4, 6.8 and 7.0, BaTRI had a significantly higher activity than DsTRI. Analogues of tropinone, 4-methylcyclohexanone and 3-quinuclidinone hydrochloride, were also used to investigate the enzymatic kinetics of BaTRI. The Km, Vmax and Kcat values of BaTRI for tropine were 0.56mM, 171.62nkat.mg(-1) and 5.69S(-1), respectively, at pH 9.6; the Km, Vmax and Kcat values of DsTRI for tropine were 0.34mM, 111.90nkatmg(-1) and 3.30S(-1), respectively, at pH 9.6. The tissue profiles of BaTRI differed from those in TAs-producing herbal plant species. BaTRI was expressed in all examined organs but was most abundant in secondary roots. Finally, tropane alkaloids, including hyoscyamine, anisodamine and scopolamine, were detected in various organs of B. arborea by HPLC. Interestingly, scopolamine constituted most of the tropane alkaloids content in B. arborea, which suggests that B. arborea is a scopolamine-rich plant species. The scopolamine content was much higher in the leaves and stems than in other organs. The gene expression and TAs accumulation suggest that the biosynthesis of hyoscyamine, especially scopolamine, occurred not only in the roots but also in the aerial parts of B. arborea.

Determination of BmKCT-13, a chlorotoxin-like peptide, in rat plasma by LC-MS/MS: application to a preclinical pharmacokinetic study.

A novel chlorotoxin-like toxin derived from Buthus martensii Karsch, namely BmKCT-13, is a potential candidate for glioma therapy and highly homologous to the chlorotoxin (CTX) derived from the venom of the scorpion Leiurus quinquestriatus. In this study, a simple, sensitive, and robust analytical method based on liquid chromatography-tandem mass spectrometry has been developed for the determination of BmKCT-13 in rat plasma using CTX as internal standard (IS). After sample preparation by protein precipitation with 0.1% formic acid in methanol, chromatography was performed on a Hanbon Dubhe C18 column (150 mm × 2.1 mm, 5 µm, and 100 Å) using a gradient elution with 0.1% formic acid in water and methanol. Mass spectrometry involved positive electrospray ionization and multiple reaction monitoring of the transitions at m/z 780.2→69.9 for BmKCT-13 and m/z 800.2→69.7 for CTX. The method was linear over the concentration range 10-1000 ng/mL with a lower limit of quantification of 10 ng/mL. Intra- and inter-day precision (expressed as relative standard deviation, RSD) were ≤8.1 and ≤7.9%, respectively, with intra-and inter-day accuracy of 94.5-99.0%. Recoveries of BmKCT-13 and IS were more than 65% and matrix effects were not significant. Stability studies showed that BmKCT-13 was stable under a variety of storage conditions. The method was successfully applied to a pharmacokinetic study involving intravenous administration of BmKCT-13 to rats.

In vitro potency and efficacy favor later generation fluoroquinolones for treatment of canine and feline Escherichia coli uropathogens in the United States.

Information regarding in vitro activity of newer fluoroquinolones (FQs) is limited despite increasing resistance in canine or feline pathogenic Escherichia coli (E. coli). This study describes in vitro potency and efficacy toward E. coli of seven FQs grouped according to similarities in chemical structure: enrofloxacin, ciprofloxacin, orbifloxacin (first-group), levofloxacin, marbofloxacin (second-group) and pradofloxacin, moxifloxacin (third-group; latest S, S-pyrrolidino-piperidine at C-7). Potency measures included minimum inhibitory concentration (MIC) (geometric mean MIC, MIC(50), MIC(90)); and mutant prevention concentration (MPC) for FQ susceptible isolates only. In vitro efficacy measures included relative susceptibility (MIC(BP-S):MIC) or resistance (MIC:MIC(BP-R)) and mutant selection window (MSW) (MPC:MIC). For enrofloxacin susceptible isolates, mean MIC (µg/ml) was least for each third-group drug and ciprofloxacin and greatest for enrofloxacin and orbifloxacin (P = 0.006). For enrofloxacin susceptible isolates, MPC were below MIC:MIC(BP-R) and least for pradofloxacin (0.29 ± 0.16 µg/ml) and greatest for enrofloxacin (1.55 ± 0.55 µg/ml) (P = 0.006). MSW was least for pradofloxacin (55 ± 30) and greatest for ciprofloxacin (152 ± 76) (P = 0.0024). MIC(BP-S):MIC was greatest (P = 0.025) for pradofloxacin (190.1 ± 0.61) and least for enrofloxacin (23.53 ± 0.83). For FQ susceptible isolates, FQs MIC:MIC(BP-R) may serve as a surrogate for MPC. Because in vitro efficacy was greatest for pradofloxacin; it might be preferred for treatment of urinary tract infections (UTIs) associated with FQ susceptible E. coli uropathogens.