Discovery and synthesis of sulfur-containing 6-substituted 5,8-dimethoxy-1,4-naphthoquinone oxime derivatives as new and potential anti-MDR cancer agents.

Multi-drug resistance (MDR) to anticancer drugs is the primary impediment to successful treatment of cancer. Hunting for new compounds with potent anti-MDR activity is an effectual approach to conquer cancer drug resistance. In this work, 33 new sulfur-containing 1,4-naphthoquinone oxime derivatives were prepared and investigated for their cytotoxicity against a panel of tumor cell lines and fibroblast normal cell line. Cell-based assay showed that most of target compounds displayed more potent cytotoxic potency than positive controls. Meanwhile, all of compounds were non-toxic to normal cells. More importantly, the cytotoxic activity of these oxime derivatives toward drug-resistant cancer cell lines was found to be much stronger than that toward drug-susceptible cell lines (anti-drug resistance coefficient (ADRC) > 1). Of these, compound 12 m was identified as the most effective molecule with IC50 values in the range of 0.29 ±â€¯0.01 to 1.33 ±â€¯0.05 µM toward MDR sublines. Further mechanism studies demonstrated that 12 m could inhibit colony formation, cause G1 phase arrest and promote cell apoptosis mediated by augmenting Bax/Bcl-2 ratio of Bel7402/5-FU cells. Our findings provide promising start points for development of sulfur-containing 1,4-naphthoquinone oxime derivatives as potential anti-MDR agents.

Synthesis and biological evaluation of sulfur-containing shikonin oxime derivatives as potential antineoplastic agents.

As a continuation of our research on developing potent and potentially safe antineoplastic agents, a set of forty five sulfur-containing shikonin oxime derivatives were synthesized and evaluated for their in vitro cytotoxic activity against human colon cancer (HCT-15), gastric carcinoma (MGC-803), liver (Bel7402), breast (MCF-7) cancer cells and human skin fibroblast (HSF) cells. All the synthesized compounds exhibited potent cytotoxic activity selectively towards HCT-15 cells and did not display apparent toxicity to the normal HSF cells, some of which were more or comparatively effective to the parent compound against HCT-15, MGC-803 and Bel7402 cells. The most active agent 9m displayed high potency against human cancer cells with IC50 ranging from 0.27 ± 0.02 to 9.23 ± 0.12 µM. The structure-activity relationships (SARs) studies suggested that the nature of substituent group in the side chain is important for antitumor potency in vitro. Additionally, nitric oxide release studies revealed that the amount of nitric oxide generated from these oxime derivatives was relatively low. Furthermore, cellular mechanism investigations indicated that compound 9m could arrest cell cycle at G1 phase and induce a strong apoptotic response in HCT-15 cells. Moreover, western blot studies revealed that compound 9m induced apoptosis through the down-regulation of Bcl-2 and up-regulation of Bax, caspase 3 and 9. For all these reasons, compound 9m hold promising potential as antineoplastic agent.

Activity of compounds from Taxillus sutchuenensis as inhibitors of HCV NS3 serine protease.

This study aimed to isolate active compounds from traditional Chinese medicinal Taxillus sutchuenensis to inhibit hepatitis C virus (HCV) NS3 protease activity. Under the guidance of bioassay, 10 compounds were isolated from the EtOAc extract fraction, which were identified as inhibitors of HCV NS3 protease. IC50 values of these compounds were obtained, and a broad degree of anti-HCV activity was observed. The most active compounds were kaempferol-3,7-bisrhamnoside (19.4 µM) and (3S)-3-hydroxy-1,7-bis(4-hydroxy-phenyl)-6E-hepten-5-one (28.7 µM). In conclusion, flavonoids and diarylheptanoids were responsible for the anti-HCV constitution of Taxilli Herba. These inhibitors of HCV NS3 protease might serve as potential candidate of anti-HCV agents.

Enhanced production of shikimic acid using a multi-gene co-expression system in Escherichia coli.

Shikimic acid (SA) is the key synthetic material for the chemical synthesis of Oseltamivir, which is prescribed as the front-line treatment for serious cases of influenza. Multi-gene expression vector can be used for expressing the plurality of the genes in one plasmid, so it is widely applied to increase the yield of metabolites. In the present study, on the basis of a shikimate kinase genetic defect strain Escherichia coli BL21 (ΔaroL/aroK, DE3), the key enzyme genes aroG, aroB, tktA and aroE of SA pathway were co-expressed and compared systematically by constructing a series of multi-gene expression vectors. The results showed that different gene co-expression combinations (two, three or four genes) or gene orders had different effects on the production of SA. SA production of the recombinant BL21-GBAE reached to 886.38 mg·L(-1), which was 17-fold (P < 0.05) of the parent strain BL21 (ΔaroL/aroK, DE3).

H1-A, a compound isolated from Fusarium oxysporum inhibits hepatitis C virus (HCV) NS3 serine protease.

The present study was aimed to isolate the active compounds from the fermentation products of Fusarium oxysporum, which had hepatitis C virus (HCV) NS3 protease inhibitory activity. A bioactive compound was isolated by reverse-phase silica-gel column chromatography, silica-gel column chromatography, semi-preparative reverse-phase High Performance Liquid Chromatography (HPLC), and then its molecular structure was elucidated based on the spectrosopic analysis. As a result, the compound (H1-A, 1) Ergosta-5, 8 (14), 22-trien-7-one, 3-hydroxy-,(3ß, 22E) was isolated and identified. To the best of our knowledge, this was the first report on the isolation of H1-A from microorganisms with the inhibitory activity of NS3 protease.

De novo biosynthesis of resveratrol by site-specific integration of heterologous genes in Escherichia coli.

Resveratrol is a well-known triphenolic natural product present in red wine. For its contribution to human health, the demand for resveratrol as a food and nutrition supplement has increased significantly. In recent years, the rapid development of synthetic biology has promoted extensive work to increase the production of resveratrol in microbes. However, supplementation of expensive phenylpropanoic precursors was required in current engineered strains. Here, we first utilized the site-specific integration strategy to produce resveratrol in Escherichia coli The genes tal, 4cl and sts were site-specific integrated into the loci of genes tyrR and trpED in the chromosome of E. coli BW25113 (DE3). The final strain was capable of producing 4.612 mg L(-1) of resveratrol from glucose.

Comparative expression profiling of genes involved in primary metabolism in high-yield and wild-type strains of Acremonium chrysogenum.

Cephalosporin C (CPC) productivity of Acremonium chrysogenum has been improved significantly through classical strain improvement programs. Here, we used transcription and metabolite profiling to address mechanisms underlying CPC production in a high yield (HY) strain. Transcription and metabolite profiling indicated that enzymes involved in amino acid production are higher in abundance in the HY strain. Moreover, results indicate a higher flow of precursors from the glycolysis and gluconeogenesis pathways to serine synthesis at the late stage of fermentation in the HY strain. In addition, less pyruvate would enter the TCA cycle thus favoring valine synthesis. Amino acid production would also benefit from a more active pentose phosphate pathway and γ-amino butyric acid shunt both generating NADPH. Moreover the glyoxylate pathway seems to be more active in the HY strain. These results may provide new leads for CPC strain improvement in industry.

Study on genetic engineering of Acremonium chrysogenum, the cephalosporin C producer.

Acremonium chrysogenum is an important filamentous fungus which produces cephalosporin C in industry. This review summarized the study on genetic engineering of Acremonium chrysogenum, including biosynthesis and regulation for fermentation of cephalosporin C, molecular techniques, molecular breeding and transcriptomics of Acremonium chrysogenum. We believe with all the techniques available and full genomic sequence, the industrial strain of Acremonium chrysogenum can be genetically modified to better serve the pharmaceutical industry.

Site-specific integration and constitutive expression of key genes into Escherichia coli chromosome increases shikimic acid yields.

As the key starting material for the chemical synthesis of Oseltamivir, shikimic acid (SA) has captured worldwide attention. Many researchers have tried to improve SA production by metabolic engineering, yet expression plasmids were used generally. In recent years, site-specific integration of key genes into chromosome to increase the yield of metabolites showed considerable advantages. The genes could maintain stably and express constitutively without induction. Herein, crucial genes aroG, aroB, tktA, aroE (encoding 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase, dehydroquinate synthase, transketolase and shikimate dehydrogenase, respectively) of SA pathway and glk, galP (encoding glucokinase and galactose permease) were integrated into the locus of ptsHIcrr (phosphoenolpyruvate: carbohydrate phosphotransferase system operon) in a shikimate kinase genetic defect strain Escherichia coli BW25113 (ΔaroL/aroK, DE3). Furthermore, another key gene ppsA (encoding phosphoenolpyruvate synthase) was integrated into tyrR (encoding Tyr regulator protein). As a result, SA production of the recombinant (SA5/pGBAE) reached to 4.14 g/L in shake flask and 27.41 g/L in a 5-L bioreactor. These data suggested that integration of key genes increased SA yields effectively. This strategy is environmentally friendly for no antibiotic is added, simple to handle without induction, and suitable for industrial production.

Acthi, a thiazole biosynthesis enzyme, is essential for thiamine biosynthesis and CPC production in Acremonium chrysogenum.

BACKGROUND: The filamentous fungus Acremonium chrysogenum is an important industrial fungus and is used in the production of the ß-lactam antibiotic cephalosporin C. Little is known regarding the molecular and biological mechanisms of how this industrial strain was improved by mutagenesis and molecular breeding. Comparative proteomics is one of the most powerful methods to evaluate the influence of gene expression on metabolite production. RESULTS: In this study, we used comparative proteomics to investigate the molecular mechanisms involved in the biosynthesis of cephalosporin C between a high-producer (HY) strain and a wide-type (WT) strain. We found that the expression levels of thiamine biosynthesis-related enzymes, including the thiazole biosynthesis enzyme (Acthi), pyruvate oxidase, flavin adenine dinucleotide (FAD)-dependent oxidoreductase and sulfur carrier protein-thiS, were up-regulated in the HY strain. An Acthi-silencing mutant of the WT strain grew poorly on chemically defined medium (MMC) in the absence of thiamine, and its growth was recovered on MMC medium supplemented with thiamine. The intracellular thiamine content was changed in the Acthi silencing or over-expression mutants. In addition, we demonstrated that the manipulation of the Acthi gene can affect the hyphal growth of Acremonium chrysogenum, the transcription levels of cephalosporin C biosynthetic genes, the quantification levels of precursor amino acids for cephalosporin C synthesis and the expression levels of thiamine diphosphate-dependent enzymes. Over-expression of Acthi can significantly increase the cephalosporin C yield in both the WT strain and the HY mutant strain. CONCLUSIONS: Using comparative proteomics, four differently expressed proteins were exploited, whose functions may be involved in thiamine diphosphate metabolism. Among these proteins, the thiazole biosynthesis enzyme (ActhiS) may play an important role in cephalosporin C biosynthesis. Our studies suggested that Acthi might be involved in the transcriptional regulation of cephalosporin C biosynthesis. Therefore, the thiamine metabolic pathway could be a potential target for the molecular breeding of this cephalosporin C producer for industrial applications.

Development of a quantitative ELISA kit for human secreted CD306/LAIR-2 and its application.

AIM: A quantitative ELISA kit for the detection of human secreted CD306/LAIR-2 was developed using monoclonal and polyclonal antibodies which were raised against a highly purified recombinant human secreted CD306/LAIR-2. METHODS: Anti-human secreted CD306/LAIR-2 monoclonal and polyclonal antibodies were raised by immunizing mouse or rabbit with recombinant human secreted CD306/LAIR-2. The monoclonal antibody was purified by protein G affinity, whereas the polyclonal antibody was purified by protein A affinity. The best match pair of antibodies were found and used to develop a double antibody sandwich ELISA kit for the detection of human secreted CD306/LAIR-2 in human samples. RESULTS: A human secreted CD306/LAIR-2 ELISA kit was formulated with highly purified recombinant human secreted CD306/LAIR-2, highly specific monoclonal and polyclonal antibodies. This kit realized the quantitative measurement of recombinant human CD306/LAIR-2 and natural CD306/LAIR-2 in human serum samples. CONCLUSIONS: The developed human secreted CD306/LAIR-2 ELISA kit is a reliable quantitation immunoassay kit.

De novo comparative transcriptome analysis of Acremonium chrysogenum: high-yield and wild-type strains of cephalosporin C producer.

ß-lactam antibiotics are widely used in clinic. Filamentous fungus Acremonium chrysogenum is an important industrial fungus for the production of CPC, one of the major precursors of ß-lactam antibiotics. Although its fermentation yield has been bred significantly over the past decades, little is known regarding molecular changes between the industrial strain and the wild type strain. This limits the possibility to improve CPC production further by molecular breeding. Comparative transcriptome is a powerful tool to understand the molecular mechanisms of CPC industrial high yield producer compared to wild type. A total of 57 million clean sequencing reads with an average length of 100 bp were generated from Illumina sequencing platform. 22,878 sequences were assembled. Among the assembled unigenes, 9502 were annotated and 1989 annotated sequences were assigned to 121 pathways by searching against the Kyoto Encyclopedia of Genes and Genomes pathway (KEGG) database. Furthermore, we compared the transcriptome differences between a high-yield and a wild-type strain during fermentation. A total of 4329 unigenes with significantly different transcription level were identified, among which 1737 were up-regulated and 2592 were down-regulated. 24 pathways were subsequently determined which involve glycerolipid metabolism, galactose metabolism, and pyrimidine metabolism. We also examined the transcription levels of 18 identified genes, including 11 up-regulated genes and 7 down-regulated genes using reverse transcription quantitative -PCR (RT-qPCR). The results of RT-qPCR were consistent with the Illumina sequencing. In this study, the Illumina sequencing provides the most comprehensive sequences for gene expression profile of Acremonium chrysogenum and allows de novo transcriptome assembly while lacking genome information. Comparative analysis of RNA-seq data reveals the complexity of the transcriptome in the fermentation of different yield strains. This is an important public information platform which could be used to accelerate the research to improve CPC production in Acremonium chrysogenum.

Metabolic engineering of Escherichia coli to enhance shikimic acid production from sorbitol.

Shikimic acid (SA) is the key synthetic material of Oseltamivir, which is an effective drug for the prevention and treatment of influenza. In this study, to block the downstream metabolic pathway of SA, the shikimate kinase isoenzyme genes aroK and aroL were deleted by Red recombination. Moreover, the key enzyme genes aroG, aroB, tktA and aroE of SA pathway were co-expressed by constructing the recombinant vector pETDuet-GBAE. As a result, SA production of E. coli BW25113 (∆aroL/aroK, DE3)/pETDuet-GBAE reached 1,077.6 mg/l when low amounts of sorbitol (5 g/l) were fed in shake flasks. The yield was 3.7 times that when glucose was used (P < 0.05). The results showed that sorbitol was an optimized carbon source for the high efficient accumulation of SA for the first time, which was applicable to use in the industry for high yields and low consumption.

Bioconversion of deoxysugar moieties to the biosynthetic intermediates of daunorubicin in an engineered strain of Streptomyces coeruleobidus.

Daunorubicin (DNR) is a representative anthracycline with anti-tumor bioactivity. Its convergent biosynthetic pathway has promoted the research on pursuing novel anthracyclines by combinatorial biosynthesis. SnoaL is a special polyketide cyclase that catalyzes the closure of nogalonic acid methyl ester with the C9-S stereochemistry. In this study, the gene cluster of DNR was cloned, and snoaL was integrated into the DNR biosynthetic pathway for the substitution of dnrD in Streptomyces coeruleobidus DM, which resulted in the production of epi-aklaviketone. The biosynthetic pathway of NDP-4-deacetyl-L-chromose B was then expressed in the engineered strain, which led to the production of corresponding glycosylated anthracycline compounds. Finally, the bioactivities of these engineering strains were evaluated.

[Neonatal group B streptococcus infection in the Children's Hospital of Gansu Province through PCR array].

OBJECTIVE: To study neonatal Streptococcus agalactiae (GBS) infection in The Children's Hospital of Gansu Province through Polymerase Chain Reaction(PCR) Array. METHOD: After obtaining the informed consent from parents or guardians, blood samples of 286 neonates were collected and studied in The Children's Hospital of Gansu Province from June 2011 to January 2012. DNA of the selected samples was extracted through the method of 5% Chelex-100 + 0.5% NP40 solution. Twenty-five genes were ultimately selected and then 25 pairs of primers were designed respectively through primer-BLAST tool of NCBI database.For every primer, PCR conditions were optimized through the identified GBS, and 25 pairs of primers were arrayed as to be used to study neonatal GBS infection. RESULT: The results of PCR Array showed that the 14 samples were detected positive, accounting for 4.90% of all the selected specimens. As for neonatal GBS infection, the positive rate was 4.55% within 7 days after birth and 5.19% in those older than 7 days. The positive rate of 53 preterm infants was 5.66%. The follow-up survey showed that none of the cases died. CONCLUSION: In the Children's Hospital of Gansu Province neonatal GBS infection rate was 4.90%, which is similar to the previous domestic reports, but is lower than the reports from Europe and the United States.Studies have shown that the gene expression related to immune evasion has a higher frequency. The present study suggests that the strategy of GBS immune adaptation may play an important role in neonatal GBS infection.

[Research progress on strain improvement of Acremonium chrysogenum by genetic engineering].

Acremonium chrysogenum, cephalosporin C (CPC) producing strain, is an important industrial microorganism. CPC is used to produce 7-ACA, a major intermediate for manufacturing of many first-line anti-infectious cephalosporin-antibiotics. The fermentation level of CPC determines the production, quality and cost of its downstream products. Therefore, it is necessary to develop the strains of A. chrysogenum. Along with the development of molecular biology, genetic manipulation technique is becoming more and more important in the field of molecular breeding. This paper reviews the latest research progresses on CPC biosynthesis and its regulation. Genetic manipulations of A. chrysogenum were summarized and concluded. We suggested that strain improvement of A. chrysogenum by means of induction and expression of biosynthetic and regulatory genes, as well as exogenous genes, and further optimization could be applied to different aspects including CPC production enhancement and metabolic pathway elongation, etc. Future direction of this field is also proposed. We believed that incorporation of comparative proteomics and genomic shuffling with molecular breeding could lead the achievements close to industry promptly.

Improvement of antibiotic productivity by knock-out of dauW in Streptomyces coeruleobidus.

Daunorubicin (DNR) is an important anthracycline antibiotic. Its biosynthesis pathway has been well understood, however, the regulation of DNR biosynthesis needs further investigations. An ORF cloned between drrB and dnrX from the genome of a DNR producer, Streptomyces coeruleobidus DM, was named dauW and designated as an orthologous gene with dnrW and drrD. Several plasmids were constructed for over-expression and/or disruption of dauW in DM. Complete disruption of dauW can significantly increase the yield of DNR. We also found that the transcription level of dnrI, a major regulatory protein in the biosynthesis of DNR, and the self-resistance level were improved in dauW knock-out mutant. These results suggested that dauW may be a down-regulatory gene for DNR biosynthesis. Antibiotics productivity in S. coeruleobidus could be improved via regulation of the transcription of dnrI, a SARP regulator. The production of DNR in a high-producer and the yield of epi-DNR in an engineering strain were also increased by disruption of dauW.

Environmentally safe production of 7-ACA by recombinant Acremonium chrysogenum.

7-Amino cephalosporanic acid (7-ACA), which is currently obtained by chemical deacylation from cephalosporin C (CPC), is a major intermediate for industrial production of ß-lactam antibiotics. 7-ACA can also be produced from CPC by enzymatic route including two-step and one-step procedures. In our research, an ecs gene coding for CPC acylase was synthesized and cloned into pET-28a(+) to construct an E. coli expression plasmid pYG232. E. coli BL21(DE3) bearing pYG232 was induced by IPTG and successfully expressed the recombinant ECS (88.9 kDa). Under the optimal conditions: 0.5 mg/ml purified ECS protein, 5 mg/ml CPC, 100 mM Tris-Cl (pH 9.6), supplement with 7 mM Zn(2+), slightly shaking for 6 h at 25°C, the transformation productivity was 54.4%. Then, ecs was cloned downstream of an A. chrysogenum endogenous promotor, PpcbC, to construct pYG233 for expression in A. chrysogenum. pYG233 was introduced into a CPC high-producer via integrative transformation of protoplasts. Two independent bleomycin-resistant transformants were investigated by PCR, Southern blotting, quantitative RT-PCR, western blotting, and fermentation. Although these two transformants both have one copy of integrated ecs, they showed different expression level of ECS protein and 7-ACA production. When concentration of CaCO(3) was reduced to 50 mM, ZnSO(4) was increased to 7 mM, CuSO(4) was eliminated from the fermentation media, and the pH was adjusted to 8.0 at day 4 during fermentation, 7-ACA production of one of the transformants could reach 1701 µg/ml, indicated that more than 30% of CPC produced by this high-producer have been transformed into 7-ACA directly in vivo. This is the highest 7-ACA production by direct fermentation ever reported.

Health-associated infections in a pediatric nephrology unit in China.

BACKGROUND: Health care-associated infection (HAI) in children is associated with morbidity and mortality, prolonged hospital stay, and increased health care costs. We report the prevalence of HAIs in children admitted to the pediatric nephrology unit of a large tertiary care pediatric hospital in China between 2000 and 2008. METHODS: A prospective infection control surveillance program led by physicians identified HAIs in admitted patients and sent monthly summary data to the hospital's Nosocomial Infections Committee. Infections at any body site meeting the Center for Disease Control and Prevention's former National Nosocomial Infection Surveillance System definitions were eligible for inclusion. Over the study period, various infection prevention and control strategies were introduced, including education on hand hygiene, measures to ensure appropriate antimicrobial prophylaxis for patients, and a guideline for antibiotic use. RESULTS: Of the 971 patients admitted, 81 had a total of 89 episodes of HAI (9.16%; 89/971); 75 patients (92.6%) had one HAI. The percentage of children acquiring HAI decreased from 12% to 6% over the observation period, representing a statistically significant linear trend. The most common type of HAI was respiratory tract infection (65.16%; n = 58), followed by gastrointestinal tract infection (11.24%; n = 10), skin and soft tissue infection (8.99% (n = 8), bloodstream infection (7.87%; n = 7), and urinary tract infection (6.74%; n = 6). CONCLUSION: The incidence of HAI in a pediatric nephrology ward decreased over an 8-year period, associated with a surveillance program and education directed at hand hygiene and appropriate antibiotic use. Despite a strict visitor policy, respiratory tract infection was the most common HAI seen.