Functional characterization of a novel flavin reductase from a deep-sea sediment metagenomic library and its application for indirubin production.

Microbial synthesis is a desirable approach to produce indirubin but suffers from low synthetic efficiency. Insufficient supply of reduced flavins is one major factor limiting synthetic efficiency. To address this, a novel flavin reductase, MoxB, was discovered through screening of the metagenomic library. MoxB showed a strong preference for NADH over NADPH as the electron source for FMN/FAD reduction and exhibited the highest activity at pH 8.0 and 30°C. It displayed remarkable thermostability by maintaining 80% of full activity after incubation at 60°C for 1 h. Furthermore, MoxB showed great organic solvent tolerance and its activity could be significantly increased by bivalent metal ions. In addition, heterologous expression of the moxB gene in the indirubin-producing E. coli significantly improved indirubin production up to 15.12-fold. This discovery expands the understanding of flavin reductases and provides a promising catalytic tool for microbial indirubin production.IMPORTANCEMuch effort has been exerted to produce indirubin using engineered Escherichia coli, but high-level production has not been achieved so far. Insufficient supply of reduced flavins is one key factor limiting the catalytic efficiency. However, the flavin reductases involved in indirubin biosynthesis have not been hitherto reported. Discovery of the novel flavin reductase MoxB provides a useful tool for enhancing indirubin production by E. coli. Overexpression of MoxB in indirubin-producing E. coli increased indirubin production by 15.12-fold in comparison to the control strain. Our results document the function of flavin reductase that reduces flavins during indirubin biosynthesis and provide an important foundation for using the flavin reductases to improve indirubin production by engineered microorganisms.

Characterization of a novel monooxygenase originating from a deep-sea sediment metagenomic library.

Oxygenases are important biocatalysts to produce many industrially important biomolecules. Here, a novel oxygenase, named MoxA, was identified through screening of a deep-sea sediment metagenomic library. Sequence analysis showed MoxA contains 424 amino acid residues with a predicated molecular mass of 46.9 kDa. Multiple sequence alignment and phylogenetic analysis indicated the sequence might be a new member of monooxygenase subfamily. A recombinant MoxA was obtained through the functional expression of moxA gene in Escherichia coli. Characterization of the purified MoxA indicated that it is an alkaline oxygenase showing maximal activity at pH 8.0. The optimal temperature of MoxA was 37 ℃, and it retained more than 70% of its initial activity after 1 h at 20-50 ℃ exhibiting good thermostability. Furthermore, effect of metal ions and organic solvents on enzymatic activity was investigated, and the results showed that the activity of MoxA was enhanced by Cu2+, Zn2+, Co2+ and Mg2+ at 1 mM, and by Co2+, Ca2+ and Mg2+ at 5 mM. Moreover, the recombinant strain harboring MoxA was used as a whole-cell biocatalyst for the efficient biosynthesis of indigo showing promising conversion efficiency. The biochemical properties of MoxA indicated that it would provide great contribution for the indigo bioproduction. KEY POINTS: • A novel monooxygenase from a metagenomic library was characterized. • The activity of MoxA was enhanced by metal ions at 1 mM and 5 mM. • MoxA has an optimal temperature of 37 ℃ and exhibited high conversion capacity.

Catalpol induces apoptosis in breast cancer in vitro and in vivo: Involvement of mitochondria apoptosis pathway and post-translational modifications.

Breast cancer is a fatal cancer with the highest mortality in female. New strategies for anti-breast cancer are still urgently needed. Catalpol, an iridoid glycoside extracted from the traditional Chinese medicinal plant Rehmannia glutinosa, has shown anticancer efficacy in various cancer cells. However, its effect on breast cancer remains unclear. In this study, we aim to investigate the anti-breast cancer activity of catalpol and elucidate its underlying mechanism. Cell counting kit-8 (CCK-8) and morphology change showed that catalpol could inhibit the proliferation and viability of MCF-7 cells. Catalpol administration reduced the tumor volume in xenograft model. Catalpol induced apoptosis in MCF-7 cells confirmed by Hoechst 33342 staining and Annexin V-FITC/PI double staining. In vivo, catalpol also induced apoptosis as seen from the increased level of terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL) in tumor. According to JC-1 and Dichlorodi-hydrofluorescein Diacetate (DCFH-DA) staining, loss of mitochondrial membrane potential (MMP) and reactive oxygen species (ROS) generation was found in MCF-7 cells treated with catalpol. Furthermore, catalpol also increased the level of cytoplasmic cytochrome c and activity of caspase-3 in MCF-7 cells. Likewise, histopathological and immunohistochemical (IHC) assay also found that catalpol enhanced the levels of cytochrome c and caspase-3 in breast cancer tissues. Ultimately, acetylation, 2-hydroxyisobutyrylation and lactylation were dramatically increased, whereas succinylation, malonylation and phosphorylation were markedly decreased in the breast cancer tumor treated with catalpol. Taken together, catalpol inhibited breast cancer in vitro and in vivo through induction of apoptosis via mitochondria apoptosis pathway and regulation of protein post-translational modifications (PTMs). Thus, it can be considered as an excellent candidate compound for treatment of breast cancer.

Catalpol Ameliorates Neurotoxicity in N2a/APP695swe Cells and APP/PS1 Transgenic Mice.

Alzheimer's disease (AD) causes progressive decline of memory and cognitive deficits. Because of its complicated pathogenesis, the prevention and therapy of AD remain an enormous challenge. It has been reported that catalpol possessed neuroprotective effects against AD. However, the involved mechanism still needs to be intensively studied. Therefore, the effects of catalpol on N2a/APP695swe cells and APP/PS1 mice were identified in the current study. Catalpol could improve cytotoxicity according to CCK-8 assay and ameliorate cellular morphological changes in N2a/APP695swe cells. Neuronal structural damage in the hippocampal CA1 region of APP/PS1 AD mice was improved according to HE staining and immunohistochemistry of NeuN. Meanwhile, catalpol administration ameliorated cognitive deficits confirmed by behavior performance of APP/PS1 mice. Hoechst 33,342 staining and Annexin V-FITC/PI double staining demonstrated that catalpol could reduce apoptosis in N2a/APP695swe cells. Likewise, TUNEL staining also manifested that catalpol significantly reduced apoptosis in hippocampal CA1 region of APP/PS1 mice. Catalpol administration also could improve mitochondrial functions indicated by the ameliorative mitochondrial morphology, the decreased ROS generation, and the increased MMP in N2a/APP695swe cells. Subsequently, catalpol restrained oligomerization of Aß1-42, verified by a reduced ThT fluorescence dose- and time-dependently. Additionally, both Aß1-40 and Aß1-42 aggregation were decreased in N2a/APP695swe cells and APP/PS1 mice administrated with catalpol confirmed by ELISA and western blot. Western blot also showed that catalpol facilitated the phosphorylation of AKT and GSK3ß, and impeded the expression of BACE1 both in vivo and in vitro. Finally, a slight alteration in lactylation, 2-hydroxyisobutyrylation, and phosphorylation were found in N2a/APP695swe cells treated with catalpol. Together, these findings manifested that catalpol served a neuroprotective effect in AD and might be a novel and expecting prophylactic or curative candidate for AD or neurodegenerative diseases therapy.

Saikosaponin-D Mitigates Oxidation in SH-SY5Y Cells Stimulated by Glutamate Through Activation of Nrf2 Pathway: Involvement of PI3K.

Alzheimer's disease (AD) is a typical neurodegenerative disease. Well-established studies have shown an elevated level of ROS (reactive oxygen species) that induces oxidative stress in AD. Saikosaponin-D exhibited significant therapeutic effects on neurodegenerative diseases. However, its in-depth molecular mechanisms against neurotoxicity remain not fully uncovered. Herein, the possible protective effects of saikosaponin-D on glutamate-induced neurotoxicity in SH-SY5Y cells and the underlying mechanism were elucidated. Saikosaponin-D pretreatment could ameliorate glutamate-induced cytotoxicity according to MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay and depress apoptosis according to Hoechst 33,342 staining and Annexin V-FITC/PI double staining in SH-SY5Y cells. Additionally, saikosaponin-D administration suppressed oxidative stress in response to glutamate indicated by diminished intracellular ROS formation and reduced MDA (malondialdehyde) content in SH-SY5Y cells. These phenomena, appeared to correlate with the recovered cellular antioxidant enzyme activities and inducted HO-1 (heme oxygenase-1) expression accompanying the nuclear translocation of Nrf2 conduct by saikosaponin-D preconditioning which had been altered by glutamate, were correlated with its neuroprotective. Furthermore, addition of LY294002, a selective inhibitor of PI3K (phosphatidylinositol 3 kinase), blocked saikosaponin-D-caused Nrf2 nuclear translocation and reversed the protection of saikosaponin-D against glutamate in SH-SY5Y cells. Moreover, saikosaponin-D exhibited antioxidant potential with high free radical-scavenging activity as confirmed by a DPPH (2,2-diphenyl-1-picrylhydrazyl) and TEAC (Trolox equivalent antioxidant capacity) in a cell-free system in vitro. Taken together, our results indicated that saikosaponin-D enhanced cellular antioxidant capacity through not only intrinsic free radical-scavenging activity but also induction of endogenous antioxidant enzyme activities and HO-1 expression mediated, at least in part, by activating PI3K and subsequently Nrf2 nuclear translocation, thereby protecting the SH-SY5Y cells from glutamate-induced oxidative cytotoxicity. In concert, these data raise the possibility that saikosaponin-D may be an attractive candidate for prevention and treatment of AD and other diseases related to oxidation in the future.

Paeonol triggers apoptosis in HeLa cervical cancer cells: the role of mitochondria-related caspase pathway.

Paeonol is a biologically active component purified from the root bark of Cortex Moutan that exerts pharmacological effects on the cervical cancer. In this study, we aim to evaluate the anti-cervical cancer capacity of paeonol and to investigate the mechanism driving its anti-cervical cancer effect. Paeonol administration markedly restrained the proliferation and caused apoptosis in HeLa cells. Furthermore, paeonol treatment resulted in a mitochondrial dysfunction in HeLa cells, including the inducing of mitochondrial membrane potential (MMP), reactive oxygen species (ROS) production, and the release of cytochrome c. Moreover, the Bcl-2/Bax proportion was obviously downregulated and cleaved caspase-3 expression was evaluated through paeonol treatment. Additionally, the expression of p-PI3K and p-Akt was noticeably reduced in response to paeonol treatment in HeLa cells. Our findings indicated that paeonol exerts an anticancer potential in HeLa cells, at least in a manner, via triggering the mitochondrial pathway of cellular apoptosis by inhibiting PI3K/Akt signaling. Thus, paeonol has great potential as a promising therapeutic compound to resist human cervical cancer.

High prevalence of vitamin D deficiency in Shenzhen pregnant women.

BACKGROUND: Vitamin D deficiency is a public health problem worldwide. Vitamin D deficiency in pregnant women often leads to negative clinical consequence and has been distributed differently in certain latitudes. Here, we aimed to determine the prevalence of vitamin D deficiency in pregnant women in Shenzhen City and investigate the influencing factors. METHODS: A total of 27,166 healthy pregnant women, undergoing prenatal examinations in our hospital between July 2014 and December 2018, were enrolled in our study. Maternal characteristics, including the duration of pregnancy, age and enrollment time, were recorded. The concentrations of serum 25(OH)D in the blood samples were detected by immunochemistry assays. RESULTS: For the total study population, the median serum 25(OH)D concentration was 23.36 [17.98-29.51] ng/mL, and 34.3% and 42.4% of the participants exhibited vitamin D deficiency (serum 25(OH) D < 20 ng/mL) and insufficiency (serum 25(OH)D 21-29 ng/mL), respectively. Vitamin D deficiency decreased with gestation (37.83%, 33.8%, and 29.3% for the first trimester, second trimester and third trimester, respectively, p < .001) and decreased by age (36.03%, 35.20%, 31.86% and 29.83%, for the age groups 18-24, 25-29, 30-34 and 35-46 years, respectively, p < .001). This prevalence had conspicuous seasonality (winter vs. autumn, OR 3.69, 95% CI: 3.42-3.99, p < .001). Temperature was positively associated with women's serum 25(OH)D level (r = 0.48, p < .001). CONCLUSIONS: Overall, we demonstrated that vitamin D deficiency in pregnant women in Shenzhen was common and was affected by gestation, age and season/temperature.

Icariin and Icariside II Reciprocally Stimulate Osteogenesis and Inhibit Adipogenesis of Multipotential Stromal Cells through ERK Signaling.

Herba Epimedii is a famous Chinese herbal medicine for treating bone diseases. Icariin and icariside II, the main chemical constituents, have attracted great attention from scientists for their potential as antiosteoporosis agents. Our study aimed to evaluate their effects on the lineage commitment of multipotential stromal cells (MSCs). The osteogenesis and adipogenesis of MSCs were assessed by ALP activity, calcium deposition, and adipocyte formation. The expression profiles and levels of osteogenic and adipogenic specific genes were evaluated by cDNA microarray and quantitative real-time PCR. The involvement of extracellular signal-regulated kinase (ERK) signaling was studied by enzyme-linked immunosorbent assay. Icariin and icariside II significantly increased ALP activity and mineralization during osteogenic differentiation of MSCs. Runx2, Col1, and Bmp2 were upregulated in the presence of icariin and icariside II. Meanwhile, they downregulated Pparg, Adipsin, and Cebpb expression during adipogenic differentiation. cDNA microarray revealed 57 differentially expressed genes during lineage commitment of MSCs. In addition, icariin and icariside II enhanced the phosphorylation of ERK, and the above biological effects were blocked by ERK inhibitor U0126. Icariin and icariside II may drive the final lineage commitment of MSCs towards osteogenesis and inhibit adipogenesis through the ERK signaling pathway. Both of them exert multiple osteoprotective effects and deserve more attention for their medicinal and healthcare prospects.

Saikosaponin-A induces apoptosis of cervical cancer through mitochondria- and endoplasmic reticulum stress-dependent pathway in vitro and in vivo: involvement of PI3K/AKT signaling pathway.

Cervical cancer causes considerable mortality in women worldwide. Saikosaponin-A, a triterpenoid glycoside isolated from Bupleurum falcatum, has been proven to exert anti-cancer property. In this study, we evaluated the possibility of saikosaponin-A on cervical cancer in vitro and in vivo. The results showed that saikosaponin-A induced cell death and altered cellular morphology dose-dependently. Saikosaponin-A significantly induced apoptosis in HeLa cells, confirmed by Hoechst 33,342 staining and flow cytometry. Sequentially, saikosaponin-A triggered the mitochondrial-mediated apoptosis demonstrated by deficiency of MMP, induction of Bax/Bcl-2 ratio, leakage of cytochrome c to cytoplasm, and activation of caspase-3. Moreover, ER stress also participated in the apoptosis induced by saikosaponin-A in HeLa cells as indicated by the upregulation of GPR78, CHOP and caspase-12 expression. Furthermore, HeLa cells showed increased expressions of p-PI3K and p-AKT in response to saikosaponin-A treatment. Additionally, saikosaponin-A could inhibit HeLa tumor growth in nude mice and induce apoptosis, reflected by the induction of TUNEL and the expression of cytochrome c, caspase-3 and CHOP confirmed by immunohistochemistry. These findings at least to a certain extent suggested that saikosaponin-A triggered apoptosis through both mitochondrial pathway and ER stress pathway and inhibiting PI3K/Akt signaling, thereby contributing to against cervical cancer. This work provides a new understanding of saikosaponin-A on therapeutic application in treatment of cancer, which has the potential to be a promising candidate therapeutic agent for cervical cancer patients.

Protective effects of crocin against endogenous Aß-induced neurotoxicity in N2a/APP695swe cells.

Alzheimer's disease (AD) is a most common neurodegenerative disorder worldwide. Because of its complex pathogenesis, the prevention and therapies of AD still are a severe challenge. Evidence suggested that crocin, the major component of saffron, exhibited neuroprotective effects in AD. As such, in this study, N2a/APP695swe cells were enrolled to investigate the effects of crocin on endogenous Aß-induced neurotoxicity. Crocin (100 and 200 µM) could ameliorate cytotoxicity according to CCK-8 assay and reduce apoptosis in line with Hoechst 33,342 staining and Annexin V-FITC/PI double staining in N2a/APP695swe cells. Reduced ROS generation and elevated MMP were found in N2a/APP695swe cells treated with crocin (100 and 200 µM). Additionally, crocin at concentrations of 100 and 200 µM inhibited the release of cytochrome and attenuated caspases-3 activity in N2a/APP695swe cells. Furthermore, succinylation, crotonylation, 2-hydroxyisobutyrylation, malonylation, and phosphorylation were significantly reduced, while a slight increase of acetylation was found in 100-µM crocin treated N2a/APP695swe cells. Taken together, crocin may be a promising natural product candidate for the effective cure of AD.

The Associations of Genital Mycoplasmas with Female Infertility and Adverse Pregnancy Outcomes: a Systematic Review and Meta-analysis.

The roles of genital mycoplasmas including Mycoplasma genitalium (M. genitalium), Mycoplasma hominis (M. hominis), Ureaplasma urealyticum (U. urealyticum), and Ureaplasma parvum (U. parvum) in reproductive diseases are equivocal. To investigate whether genital mycoplasmas are risk factors of female infertility and adverse pregnancy outcomes, we performed a systematic review and meta-analysis. Electronic databases were searched for related studies. A random-effects model or fixed-effects model was employed to generate forest plots. Pooled odd ratios (ORs) with 95% confidence intervals (CIs) were applied to measure the strength of associations. Meanwhile, heterogeneity was evaluated by H statistic and I2 statistic, and publication bias was explored by funnel plots based on Egger's test and Begg's test. The search yielded 2054 relevant records, and 35 articles were ultimately included for meta-analysis. M. genitalium was a significant risk factor for female infertility (OR, 13.03 [95% CI, 3.46-48.98]) and preterm birth (PTB) (OR, 1.81 [95% CI, 1.17-2.80]), but not for spontaneous abortion (SA) (OR, 0.58 [95% CI, 0.25-1.35]). M. hominis can significantly increase the potential risk of female infertility (OR, 1.56 [95% CI, 1.02-2.38]), SA (OR, 9.14 [95% CI, 4.14-20.18]), stillbirth (OR, 3.98 [95% CI, 1.39-11.36]), and premature rupture of membranes (PROM) (OR, 1.79 [95% CI, 1.26-2.55]), but was not associated with PTB (OR, 1.29 [95% CI, 0.78-2.15]). U. urealyticum had no significant risk effect on female infertility (OR, 0.68 [95% CI, 0.42-1.11]). Coinfections of M. hominis and Ureaplasma were significantly associated with female infertility, SA, and stillbirth, but not with PROM. On the basis of current evidences, this meta-analysis supports that M. genitalium is a risk factor for female infertility and PTB; M. hominis is a potential risk factor for female infertility, SA, stillbirth, and PROM; U. urealyticum has no significant association with female infertility; and the relationship of U. parvum with female infertility and adverse pregnancy outcomes needs to be paid more attention to and remains to be further revealed.

Matrine exerts anti­breast cancer activity by mediating apoptosis and protective autophagy via the AKT/mTOR pathway in MCF­7 cells.

Matrine, a major alkaloid isolated from the traditional Chinese herb Sophora flavescens, has been used clinically to treat breast cancer in China. However, the effects of matrine on apoptosis and autophagy in breast cancer cells remain unclear. In the present study, the anti­breast cancer capacity of matrine was evaluated and its role in regulating apoptosis and autophagy in vitro was investigated. Matrine significantly inhibited the growth of MCF­7 cells. In addition, Hoechst 33342 staining and Annexin V/propidium iodide staining demonstrated that incubation with matrine induced apoptosis in MCF­7 cells. Furthermore, matrine induced autophagy in MCF­7 cells, manifesting as an accumulation of light chain 3 II and downregulation of p62. Additionally, matrine suppressed AKT and mammalian target of rapamycin (mTOR) phosphorylation, indicating that the AKT/mTOR pathway is involved in matrine­induced apoptosis and autophagy. Overall, the results of the present study indicated that matrine possesses anti­breast cancer activity by providing protective autophagy via inhibition of the AKT/mTOR pathway. These findings indicated that matrine may be a promising candidate for drug development targeting breast cancer.

Rapid and accurate diagnosis of Chlamydia trachomatis in the urogenital tract by a dual-gene multiplex qPCR method.

Introduction. Chlamydia trachomatis (C. trachomatis, CT) is an obligatory intracellular bacterium that causes urogenital tract infections and leads to severe reproductive consequences. Therefore, a rapid and accurate detection method with high sensitivity and specificity is an urgent requirement for the routine diagnosis of C. trachomatis infections.Aim. In this study, we aimed to develop a multiplex quantitative real-time PCR (qPCR) assay based on two target regions for accurate detection of C. trachomatis in urogenital tract infections.Methodology. Primers and probes based on the conserved regions of the cryptic plasmid and 23S rRNA gene were designed. Then, two qPCR assays were established to screen for the optimal probe and primers for each of the two target regions. Subsequently, the multiplex qPCR method was developed and optimized. For the diagnostic efficiency evaluation, 1284 urogenital specimens were tested by the newly developed multiplex qPCR method, an immunological assay and a singleplex qPCR assay widely used in hospitals.Results. The multiplex qPCR method could amplify both target regions in the range of 1.0×102-1.0×108 copies ml-1 with a strong linear relationship, and lower limits of detection (LODs) for both targets reached 2 copies PCR-1. For the multiplex qPCR method, the diagnostic sensitivity and specificity was 100.0 % (134/134) and 99.3 % (1142/1150), respectively. For the singleplex qPCR assay, the diagnostic sensitivity and specificity was 88.8 % (119/134) and 100.0 % (1150/1150), respectively. For the immunological assay, the diagnostic sensitivity and specificity was 47.0 % (63/134) and 100.0 % (1150/1150), respectively.Conclusion. In this study, a multiplex qPCR assay with high sensitivity and specificity for rapid (≤2.0 h) and accurate diagnosis of C. trachomatis was developed. The qPCR assay has the potential to be used as a routine diagnostic method in clinical microbiology laboratories.

Microbial production of cyanophycin: From enzymes to biopolymers.

Cyanophycin is an attractive biopolymer with chemical and material properties that are suitable for industrial applications in the fields of food, medicine, cosmetics, nutrition, and agriculture. For efficient production of cyanophycin, considerable efforts have been exerted to characterize cyanophycin synthetases (CphAs) and optimize fermentations and downstream processes. In this paper, we review the characteristics of diverse CphAs from cyanobacteria and non-cyanobacteria. Furthermore, strategies for cyanophycin production in microbial strains, including Escherichia coli, Pseudomonas putida, Ralstonia eutropha, Rhizopus oryzae, and Saccharomyces cerevisiae, heterologously expressing different cphA genes are reviewed. Additionally, chemical and material properties of cyanophycin and its derivatives produced through biological or chemical modifications are reviewed in the context of their industrial applications. Finally, future perspectives on microbial production of cyanophycin are provided to improve its cost-effectiveness.

Rapid diagnosis of human adenovirus B, C and E in the respiratory tract using multiplex quantitative polymerase chain reaction.

Human adenovirus (HAdV) is increasingly recognized as a major cause of human respiratory tract viral infections. Its outbreaks and epidemics in various populations resulted in considerable morbidity and mortality. Therefore, a rapid and specific assay for HAdV in clinical samples is of crucial importance to diagnosing HAdV infections. The present study aimed to develop and evaluate a multiplex quantitative polymerase chain reaction (qPCR) assay for the rapid detection and accurate quantification of HAdV B, C and E. The lower limit of detection for this assay was two genomic copies per reaction, and quantitative linearity ranged from 2 to 2x106 copies per reaction of the input viral DNA. Furthermore, 3,160 throat swab samples that tested HAdV negative by the immunofluorescence assay were collected and retested using the multiplex qPCR assay. The results showed that 2,906 samples were HAdV negative and the other 254 samples were HAdV positive. The HAdV species identified included B (184 samples), C (51 samples), and E (39 samples). Among the three HAdV species, HAdV B and E were detected from 8 samples, and HAdV C and E were detected from other 12 samples. The overall results demonstrated that the sensitivity and specificity of the proposed assay were 100% (254/254) and 99.6% (2894/2906), respectively. From the perspective of routine clinical diagnosis, this assay represented a rapid (≤1.5 h) and economic strategy, and had the potential to be used for the rapid and accurate diagnosis of human respiratory infections caused by HAdV B, C and E.

Metabolic engineering of Escherichia coli for the production of indirubin from glucose.

Indirubin is an indole alkaloid that can be used to treat various diseases including granulocytic leukemia, cancer, and Alzheimer's disease. Microbial production of indirubin has so far been achieved by supplementation of rather expensive substrates such as indole or tryptophan. Here, we report the development of metabolically engineered Escherichia coli strain capable of producing indirubin directly from glucose. First, the Methylophaga aminisulfidivorans flavin-containing monooxygenase (FMO) and E. coli tryptophanase (TnaA) were introduced into E. coli in order to complete the biosynthetic pathway from tryptophan to indirubin. Further engineering was performed through rational strategies including disruption of the regulatory repressor gene trpR and removal of feedback inhibitions on AroG and TrpE. Then, combinatorial approach was employed by systematically screening eight genes involved in the common aromatic amino acid pathway. Moreover, availability of the aromatic precursor substrates, phosphoenolpyruvate and erythrose-4-phosphate, was enhanced by inactivating the pykF (pyruvate kinase I) and pykA (pyruvate kinase II) genes, and by overexpressing the tktA gene (encoding transketolase), respectively. Fed-batch fermentation of the final engineered strain led to production of 0.056 g/L of indirubin directly from glucose. The metabolic engineering and synthetic biology strategies reported here thus allows microbial fermentative production of indirubin from glucose.

Decursin attenuates the amyloid-ß-induced inflammatory response in PC12 cells via MAPK and nuclear factor-κB pathway.

Decursin, the major bioactive component of Angelica gigas Nakai, exhibited neuroprotective properties. Our previous studies showed that decursin conferred neuroprotective effects in PC12 cells induced by Amyloid-ß (Aß)25-35 via antiapoptosis and antioxidant. In this study, the antiinflammatory effects of decursin against PC12 cells injury stimulated by Aß25-35 were assessed. Our results demonstrated that decursin suppressed the expression of cyclooxygenase-2 protein and prostaglandin E2 content which was stimulated by Aß25-35 in PC12 cells. Meanwhile, the nuclear translocation of nuclear factor-κB in Aß25-35 -treated PC12 cells was also inhibited by decursin. In addition, decursin suppressed phosphorylation of the two upstream pathway kinases, p38 and c-Jun N-terminal kinase. Overall, our findings indicate that decursin exerts protective effects against neuroinflammation stimulated by Aß25-35 in PC12 cells by abolishing cyclooxygenase-2 protein expression through inactivation of nuclear factor-κB via the upstream kinases including p38 and c-Jun N-terminal kinase. This work provides a new insight into the pharmacological mode of decursin and should facilitate its therapeutic application in treatment of inflammatory disorders.

TAT-conjugated chitosan cationic micelle for nuclear-targeted drug and gene co-delivery.

We developed a high-efficiency nucleus-targeted co-delivery vector that delivers genes and drugs directly into the nucleus of cancer cells. The system is based on grafted poly-(N-3-carbobenzyloxy-lysine) (CPCL) with transactivator of transcription (TAT)- chitosan on the surface. It is designed to perform highly efficient nucleus- targeted gene and drug co-delivery. Confocal laser scanning microscopy (CLSM) revealed that more TAT-CPCL entered the nucleus than does CPCL alone. The TAT-modified vector serves as a gene and drug co-delivery mechanism to achieve high gene transfection efficiency, high apoptosis and low viability in HeLa cells. TAT-CPCL may become a vector for cancer gene treatment and a template for designing better co-deliver systems.