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The synthesis of 1-(trifluoromethyl)alkenes through nickel-catalyzed alkylation/arylation of 1-chloro-3,3,3-trifluoroprop-1-ene (HCFO-1233zd) with alkyl/aryl zinc reagents and cobalt-catalyzed reductive cross-coupling between HCFO-1233zd and aryl bromides has been developed. These approaches feature broad substrate scope, high functional group tolerance, and the use of industrial feedstocks and low-cost nickel/cobalt catalysts, providing facile routes to access 1-(trifluoromethyl)alkenes. Diverse transformations of the resulting 1-(trifluoromethyl)alkenes demonstrated the synthetic utility of the current approaches.

The semipinacol rearrangement (SPR) is highly useful in the asymmetric synthesis of complex compounds. In biological systems, only a few semi-pinacolases belonging to a few families have been ident...

Oritavancin is a new-generation semisynthetic lipoglycopeptide antibiotic used to prevent the spread of vancomycin-resistant Gram-positive bacteria. The glycopeptide A82846B is the direct precursor of oritavancin. Considering the structural similarity between A82846B and vancomycin, the vancomycin producer Amycolatopsis orientalis was used as a chassis for the construction of a strain producing high-quality A82846B. To construct the A82846B synthetic pathway, we established a highly efficient CRISPR-Cas12a system by optimizing the conditions of conjugation and by screening the regulatory elements in the A. orientalis, which is difficult to be genetically manipulated. The efficiency of gene knockout was almost 100%. The glycosyltransferases module (gtfDE) and glycosyl synthesis module (vcaAEBD) in the vancomycin gene cluster were replaced with the corresponding glycosyltransferases module (gtfABC) and glycosyl synthesis module (evaAEBD) in the A82846B cluster, respectively. A82846B was successfully produced by the artificially constructed synthetic pathway. Moreover, the titer of A82846B was increased 80% by expressing the pathway-specific regulatory strR. This strategy has excellent potential for remodification of natural products to solve antibiotic resistance.

Efficient biosynthesis of microbial bioactive natural products (NPs) is beneficial for the survival of producers, while self-protection is necessary to avoid self-harm resulting from over-accumulation of NPs. The underlying mechanisms for the effective but tolerable production of bioactive NPs are not well understood. Herein, in the biosynthesis of two fungal polyketide mycotoxins aurovertin E (1) and asteltoxin, we show that the cyclases in the gene clusters promote the release of the polyketide backbone, and reveal that a signal peptide is crucial for their subcellular localization and full activity. Meanwhile, the fungus adopts enzymatic acetylation as the major detoxification pathway of 1. If intermediates are over-produced, the non-enzymatic shunt pathways work as salvage pathways to avoid excessive accumulation of the toxic metabolites for self-protection. These findings provided new insight into the interplay of efficient backbone release and multiple detoxification strategies for the production of fungal bioactive NPs.

Liver fibrosis has accounted for liver diseases and overall mortality, but no relevant drug has been developed. Filamentous fungi are important resources of natural products for pharmaceutical development. Calcarisporium arbuscula is a mushroom endophytic fungus, which primarily produces aurovertins. Here, in an aurovertin null-production mutant, one silent gene cluster (mca17) was activated by overexpression of a pathway-specific zinc finger transcriptional regulator, and a tetramic acid-type compound (1, MCA17-1) was identified. Along with detailed structural characterization, its biosynthesis was proposed to be produced from the core PKS-NRPS hybrid enzyme. Moreover, 1 suppressed the activation of LX-2 upon transforming growth factor-β (TGF-β) challenge and had stronger bioactivity than the positive control obeticholic acid (OCA) against liver fibrosis. Our work suggested that this engineered fungus could be a producer of 1 for promising pharmaceutical development, and alternatively, it would be developed as a mushroom ingredient in dietary therapy to prevent liver fibrosis.

Fungal natural products are rich sources of clinical drugs. Particularly, the fungicolous fungi have a large number of biosynthetic gene clusters (BGCs) to produce numerous bioactive natural products, but most BGCs are silent in the laboratory. We have shown that a fungicolous fungus Calcarisporium arbuscula NRRL 3705 predominantly produces the highly reduced polyketide-type mycotoxins aurovertins. Here after evaluation of the aurovertin-null mutant ΔaurA as an efficient host, we further screened two strong promoters aurBp and A07068p based on RNA-Seq, and successfully activated an endogenous gene cluster from C. arbuscula as well as three additional exogenous BGCs from other fungi to produce polyketide-type natural products. Thus, we showed an efficient expression system from the fungicolous fungus C. arbuscula, which will be highly beneficial and complementary to the conventional Aspergillus and Penicillium fungal cell factories, and provides a useful toolkit for genome-wide mining of bioactive natural products from fungicolous fungi.

A nickel-catalyzed reductive aryldifluoroacetylation of N-vinylacetamide with ethyl chloro(difluoro)acetate and aryl iodides is described. This chelating amide carbonyl group-assisted strategy provides rapid access to a variety of protected α,α-difluoro-γ-amino acids that might have potential applications in peptide chemistry and protein engineering. An advantage of this method is its synthetic simplicity, with no preparation of organometallic reagents.

Daptomycin is a new lipopeptide antibiotic for treatment of severe infection caused by multi-drug-resistant bacteria, but its production cost remains high currently. Thus, it is very important to improve the fermentation ability of the daptomycin producer Streptomyces roseosporus. Here, we found that the deletion of proteasome in S. roseosporus would result in the loss of ability to produce daptomycin. Therefore, transcriptome and 4D label-free proteome analyses of the proteasome mutant (Δprc) and wild type were carried out, showing 457 differential genes. Further, five genes were screened by integrated crotonylation omics analysis. Among them, two genes (orf04750/orf05959) could significantly promote the daptomycin synthesis by overexpression, and the fermentation yield in shake flask increased by 54% and 76.7%, respectively. By enhancing the crotonylation modification via lysine site mutation (K-Q), the daptomycin production in shake flask was finally increased by 98.8% and 206.3%, respectively. This result proved that the crotonylation modification of appropriate proteins could effectively modulate daptomycin biosynthesis. In summary, we established a novel strategy of gene screen for antibiotic biosynthesis process, which is more convenient than the previous screening method based on pathway-specific regulators. KEY POINTS: • Δprc strain has lost the ability of daptomycin production • Five genes were screened by multi-omics analysis • Two genes (orf04750/orf05959) could promote the daptomycin synthesis by overexpression.

It is found that there are great differences in the efficacy of quetiapine at the same dose in many patients with bipolar disorders. Therefore, therapeutic drug monitoring (TDM) is a valuable tool for guiding treatment with quetiapine. The aims of this study were to assess the relationship between serum concentration and clinical response of quetiapine in adolescents and adults with bipolar disorders in acute stage.The study design was prospective and observational. Within the naturalistic setting of a routine TDM service at the First Affiliated Hospital, Zhejiang University School of Medicine. Psychiatric symptoms were assessed using the HAMD (Hamilton Depression Scale), YRMS (Young manic rating scale) and CUDOS-M (Clinically Useful Depression Outcome Scale-Mixed Subscale). The decline of HAMD and YMRS scores was were used to assess clinical outcome of bipolar disorders respectively.169 inpatients (23.7 % male, 76.3 % female) were enrolled in the study. We found that there was a strong correlation between quetiapine serum concentrations and clinical outcomes (rWe found a significant positive relationship between serum concentration and clinical outcome, and also determined the serum concentration of quetiapine for the treatment of bipolar depression.

Efficient enabling technology is required for synthetic biology in

Oritavancin is a new-generation semisynthetic lipoglycopeptide antibiotic used to prevent the spread of vancomycin-resistant Gram-positive bacteria. The glycopeptide A82846B is the direct precursor of oritavancin. Considering the structural similarity between A82846B and vancomycin, the vancomycin producer

Drugs have been largely inspired from natural products, while enzymes underlying their biosynthesis have enabled complex structures and diverse bioactivities. Nevertheless, the high enzyme specificity and limited in vivo precursor types have restricted the natural product reservoir, but Nature has imprinted natural products with active sites, which can be readily modified by chemosynthesis with various functional groups for more favorable druggability. Here in the less exploited fungal natural products, we introduced CtvA, a polyketide synthase for a mycotoxin citreoviridin biosynthesis in Aspergillus, into an endophytic fungus Calcarisporium arbuscula to expand tetrahydrofuran (THF) into a dioxabicyclo-octane (DBO) ring moiety based on versatility and promiscuity of the aurovertin biosynthetic enzyme. Alternative acylations on the hydroxyl groups essential for cell toxicity by chemosynthesis produced compounds with improved anti-tumor activities and pharmacokinetics. Thus, we showed an effective strategic way to optimize the fungal natural product efficiently for more promising drug development.

Objective To investigate if co-transfection of human bone morphogenetic protein 2 (BMP-2, BMP2) and human fibroblast growth factor 2 (FGF2, FGF2) via chitosan nanoparticles promotes osteogenesis in human adipose tissue-derived stem cells (ADSCs) in vitro. Materials and Methods Recombinant BMP2 and/or FGF2 expression vectors were constructed and packaged into chitosan nanoparticles. The chitosan nanoparticles were characterized by atomic force microscopy. Gene and protein expression levels of BMP-2 and FGF2 in ADSCs in vitro were evaluated by real-time polymerase chain reaction (PCR), western blot, and enzyme-linked immunosorbent assay. Osteocalcin (OCN) and bone sialoprotein (BSP) gene expression were also evaluated by real-time PCR to assess osteogenesis. Results The prepared chitosan nanoparticles were spherical with a relatively homogenous size distribution. The BMP2 and FGF2 vectors were successfully transfected into ADSCs. BMP-2 and FGF2 mRNA and protein levels were significantly up-regulated in the co-transfection group compared with the control group. OCN and BSP mRNA levels were also significantly increased in the co-transfection group compared with cells transfected with BMP2 or FGF2 alone, suggesting that co-transfection significantly enhanced osteogenesis. Conclusions Co-transfection of human ADSCs with BMP2/FGF2 via chitosan nanoparticles efficiently promotes the osteogenic properties of ADSCs in vitro.

Fungal natural products are rich sources of clinical drugs. Particularly, the fungicolous fungi have a large number of biosynthetic gene clusters (BGCs) to produce numerous bioactive natural products, but most BGCs are silent in the laboratory. We have shown that a fungicolous fungus

Due to the plethora natural products made by Streptomyces, the regulation of its metabolism are of great interest, whereas there is a lack of detailed understanding of the role of posttranslational modifications (PTM) beyond traditional transcriptional regulation. Herein with Streptomyces roseosporus as a model, we showed that crotonylation is widespread on key enzymes for various metabolic pathways, and sufficient crotonylation in primary metabolism and timely elimination in secondary metabolism are required for proper Streptomyces metabolism. Particularly, the glucose kinase Glk, a keyplayer of carbon catabolite repression (CCR) regulating bacterial metabolism, is identified reversibly crotonylated by the decrotonylase CobB and the crotonyl-transferase Kct1 to negatively control its activity. Furthermore, crotonylation positively regulates CCR for Streptomyces metabolism through modulation of the ratio of glucose uptake/Glk activity and utilization of carbon sources. Thus, our results revealed a regulatory mechanism that crotonylation globally regulates Streptomyces metabolism at least through positive modulation of CCR., Chen-Fan Sun et al. show that key enzymes in several metabolic pathways are crotonylated in Streptomyces roseosporus. This study suggests that crotonylation increases carbon catabolite repression by increasing glucose uptake while reducing the activity of glucose kinase.

Oct4 protein encoded by POU5F1 plays a pivotal role in maintaining the self‑renewal of pluripotent stem cells; however, its presence in cancer cells remains controversial. In the present study, we provided evidence that the transcripts of authentic OCT4 gene (OCT4A) and its multiple pseudogenes were detected in a variety of cancer cell lines. A few major bands were also detected by western blotting using an anti‑Oct4A monoclonal antibody. Moreover, an anti‑Oct4‑pT235 antibody was used to identify a band in the majority of the tested cancer cell lines that coincided with one of the anti‑Oct4A bands which was decreasable by a specific shRNA. The Oct4‑pT235 signals were also detected in human glioblastoma and liver cancer specimens by immunofluorescence microscopy and immunohistochemistry. U87 glioblastoma cells were cultured in a neural stem cell medium to induce the formation of neurospheres rich in stem‑like cancer cells. The levels of Oct4‑pT235 in the sphere cells were markedly increased compared to their monolayer parental cells, a result that was accompanied by upregulation of the PI3K‑Akt pathway. Akti‑1/2, a specific inhibitor of Akt, effectively reduced the level of Oct4‑pT235 and attenuated the proliferation of U87 sphere cells. ITE, an agonist of the aryl hydrocarbon receptor, also significantly attenuated the Akt‑mediated phosphorylation of Oct4 in glioblastoma and liver cancer cells, and reduced their tumorigenic potential in a xenograft tumor model. Taken together, we concluded that the Akt‑mediated phosphorylation of Oct4A or its homolog protein was associated with the proliferation of stem‑like cancer cells that may serve as a novel biomarker and drug target for certain types of cancer.

For efficient collection of speech recordings, the ability to search for spoken terms in the speech stream is an essential capability. Although the Chinese spoken term detection (STD) does not suffer the out-of-vocabulary (OOV) problem as English, it is still hard to retrieve the long spoken terms which contain four characters or more. In this paper, we details our approach for long Mandarin spoken term detection which combines the search on inverted index produced by speech recognizer and linear scan on syllable confusion network. First, we split the long spoken terms into syllables and search the syllables on the inverted index _le to get the segments which may contain the long spoken terms. Then we use a linear scan algorithm on syllable confusion networks (SCNs). On two Mandarin conversation telephone speech sets, we compare performance using the method proposed with that of the baseline syllable-based systems, and our approach gives satisfying performance gains over the others.

In this study, the possible molecular mechanisms involved in the estrogen-like activities of ethanol extracts from two common Chinese legumes, i.e. Adzuki bean (Phaseolus angularis Wight.) and Lima bean (Phaseolus lunatus L.) were investigated. Reverse transcription-polymerase chain reaction (RT-PCR) measurement and western blot analysis were conducted to detect the functional mRNA and protein expressions, while immunofluorescence was used to determine the estrogen receptor (ER) intracellular distribution in the MCF-7/BOS cell line. The results indicated that (1) both mRNA and protein levels of progesterone receptor (PR) in the cell line increased after 48 h treatment with the extracts compared with those in the control; (2) Adzuki and Lima bean extracts might alter the ER intracellular distributions in two different manners: for the Adzuki bean, the ER appeared in both cytoplasm and nucleus, while for the Lima bean, it exhibited the same pattern as that of estradiol in which the ER almost disappeared in the nucleus and mainly occurred in the cytoplasm. Above all, the results showed a novel pattern of ER intracellular distribution in the MCF-7/BOS cell line upon exposure to Adzuki bean extract, suggesting that different pathways were involved in the estrogen-like activities of the ethanol extracts of Adzuki bean and Lima bean.

Objective: XRCC1 polymorphism is a research hotpot in individual treatment for non-small cell lung cancer (NSCLC). To obtain the association between XRCC1 polymorphism and clinical outcome of platinum-based treatment for NSCLC, a meta-analysis was conducted. Methods: Databases including PubMed, Embase, Cochrane, and Chinese National Knowledge Infrastructure (CNKI) were searched for publications that met the inclusion criteria. A fixed effect model was used to estimate pooled odds ratio (OR) and hazard ratio (HR) with 95% confidence interval (CI) for the association between XRCC1 Arg399Gln and response or survival of platinum-based treatment for advanced NSCLC. A chi-squared-based Q-test was used to test the heterogeneity hypothesis. Egger’s test was used to check publication bias. Results: Seventeen published case-control studies that focus on the association between XRCC1 Arg399Gln and response or survival of platinum-based treatment for advanced NSCLC in 2 256 subjects were included in this meta-analysis, of whom 522 were AA genotypes (23.2% frequency), 916 AG genotypes (40.6% frequency), and 818 GG genotypes (36.2% frequency). The overall response rate (ORR) was 45.2% (110/243) for AA genotype patients, 29.9% for AG genotype (73/244), and 30.7% for GG genotype (124/403). The heterogeneity test did not show any heterogeneity and the Egger’s test did not reveal an obvious publication bias among the included studies. The meta-analysis indicated that AA genotype patients presented higher response rates toward platinum drug treatment compared with G model (GG+GA) patients (GG vs. AA model: OR=0.489, 95% CI 0.266–0.900, P=0.021; AG vs. AA model: OR=0.608, 95% CI 0.392–0.941, P=0.026; GA+AA vs. GG model: OR=1.259, 95% CI 0.931–1.701, P=0.135; GG+GA vs. AA model: OR=0.455, 95% CI 0.313–0.663, P=0.0001). However, no evidence validates XRCC1 associates with the survival following platinum drug therapy. Conclusions: Our meta-analysis suggested that XRCC1 Arg399Gln is related with the sensitivity of NSCLC patients to platinum-based treatment. AA genotype patients present more desirable curative effectiveness compared with other patients.

To explore a more effective therapy for acute gouty arthritis.Sixty cases were randomly divided into an observation group and a control group, 30 cases in eachgroup. On the basis of diet intervention, the observation group was treated with electroacupuncture at local points combined with blood-letting puncture and cupping, and the control group with oral administration of Probenecid. Their therapeutic effects were ob served.The effective rate was 96.7% in the observation group which was better than 86.7% in the control group (P0.01). After treatment, blood uric acid decreased significantly in the two groups (both P0.01), the observed group being lower than the control group (P0.01).On the basis of diet intervention, electroacupuncture plus blood-letting puncture and cupping is a better therapy for acute gouty arthritis.

To explore the estrogenic activity of ethanol extract from adzuki bean Phaseolus angularis and its effect on progesterone receptor (PR) level of human breast cancer MCF-7 cells.The modified MCF-7 cell proliferation assay was used to evaluate the estrogenic activity of adzuki bean extract. And its effect on PR mRNA and protein were addressed using RT-PCR measurements and western blotting analysis respectively in which pure estrogen receptor antagonist ICI 182,780 was employed as the tool.The estrogenic activity of adzuki bean extract at various concentrations (10-200 microg x mL(-1)), expressed as proliferative effect (PE) relative to that of solvent control, was examined. The results indicated that the extract of adzuki bean was able to induce MCF-7 cell growth with a maximum at 100 microg x mL(-1). The results of RT-PCR and western blotting showed that PR mRNA and protein could be significantly induced by adzuki bean extract in MCF-7 cells. Moreover, the specific estrogen receptor antagonist ICI 182,780 could block these reactions.Ethanol extract of adzuki bean has the estrogen-like activities through the estrogen response pathway.

To explore the estrogenic activity and its mechanism of ethanol extract from black soybean (BSE).The modified MCF-7 cell proliferation assay was used to evaluate the estrogenic activity of BSE. And the possible mechanisms were addressed using RT-PCR measurements in which pure estrogen receptor antagonist ICI182,780 was employed as a tool.The estrogenic activity of BSE at various concentrations (1-1000 microg x mL(-1)), expressed as proliferative effect (PE) relative to that of solvent control, was examined. The results indicated that, at low concentration range (10-200 microg x mL(-1)), BSE was able to induce MCF-7 cell growth with a maximum at 100 microg x mL(-1). The results of RT-PCR showed that pS2 and PR mRNA could be significantly induced by BSE in MCF-7 cells, which was relative to the concentration of BSE. Moreover, the specific estrogen receptor antagonist, ICI182,780 could block these reactions.Ethanol extract of black soybean can stimulate the growth of MCF-7 cells and increase the expression of estrogen receptor-responsive gene, suggesting that the estrogenic effects of BSE are mediated by the estrogen receptor.

Sai-Ping Jiang,1,* Jian Chen,2,* Xing-Guo Zhang,1 Xiao-Yang Lu,1 Qing-Wei Zhao1 1Department of Pharmacy, 2Intensive Care Unit, the First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou, People’s Republic of China *These authors contributed equally to this work Background: Pharmacist interventions and medication errors potentially differ between the People’s Republic of China and other countries. This study aimed to report interventions administered by clinical pharmacists and analyze medication errors in an intensive care unit (ICU) in a tertiary hospital in People’s Republic of China.Method: A prospective, noncomparative, 6-month observational study was conducted in a general ICU of a tertiary hospital in the People’s Republic of China. Clinical pharmacists performed interventions to prevent or resolve medication errors during daily rounds and documented all of these interventions and medication errors. Such interventions and medication errors were categorized and then analyzed.Results: During the 6-month observation period, a total of 489 pharmacist interventions were reported. Approximately 407 (83.2%) pharmacist interventions were accepted by ICU physicians. The incidence rate of medication errors was 124.7 per 1,000 patient-days. Improper drug frequency or dosing (n=152, 37.3%), drug omission (n=83, 20.4%), and potential or actual occurrence of adverse drug reaction (n=54, 13.3%) were the three most commonly committed medication errors. Approximately 339 (83.4%) medication errors did not pose any risks to the patients. Antimicrobials (n=171, 35.0%) were the most frequent type of medication associated with errors.Conclusion: Medication errors during prescription frequently occurred in an ICU of a tertiary hospital in the People’s Republic of China. Pharmacist interventions were also efficient in preventing medication errors. Keywords: pharmacist, medication error, preva­lence rate, type, severity, intensive care unit&nbsp