Search

Cerberus is a multifunctional antagonist implicated in embryonic patterning through modulation of Nodal, BMP and Wnt signals. Although its function is largely conserved in chordates, certain activities have diverged, even among vertebrates. Moreover, the antagonistic action of Cerberus from the basal chordate amphioxus toward Nodal, BMP and Wnt signals remains elusive. Here, we compared the activity of amphioxus and Xenopus Cerberus proteins using cross-species assays. We found that amphioxus and Xenopus Cerberus proteins display similar activities in antagonizing Nodal-induced events, but they exhibit both shared and distinct activities in modulating BMP and Wnt signals. Amphioxus Cerberus has reduced neuralizing activity that is dependent on inhibition of BMP signaling, and it modulates the signals of a restricted subset of Wnt proteins. Furthermore, we revealed that Xenopus Cerberus interacts with Wnt4 and Wnt11 to activate canonical Wnt signaling, whereas amphioxus Cerberus lacks this activity. These differences may be correlated with the divergence in the N-terminal region of Cerberus proteins between amphioxus and Xenopus. Our results indicate that chordate Cerberus proteins have evolved sub-functionalities that depend not only on their concentrations, but also on the properties of BMP and Wnt signals. This may account for their evolutionary distinct functions in different patterning processes.

In this research, the fermentation broth produced from seven strains of endophytic fungi was sprayed on tomato plants at seedling stage. This process was carried out under Ca(NO3)2 stress, with comparison to the commercial microbial fertilizer to study the physiological and biochemical characteristics of tomato seedling. The results showed that most of the fermentation broths have no phytotoxicity and could improve salt resistance of tomato. The fermentation broth of strain Schizophyllum sp. DL12 displayed the best effects which increased the chlorophyll content (0.244 mg g−1 FW), soluble protein (31.48 µg g−1 FW), proline content (305.6 µg g−1 FW), peroxidase (21.78 µg g−1 FW min−1) and catalase (0.345 µg g−1 FW min−1) activities in leaves significantly as compared with control (CK) and commercial microbial fertilizer. These findings indicated that fermentation broth of Schizophyllum sp. DL12 has bright prospects of applications.

Lecanicillium attenuatum is an important nematophagous fungus with potential as a biopesticide for control of plant-pathogenic nematodes. However, relatively low fungicide-tolerance limits its application in the field. To improve the propamocarb-tolerance of L. attenuatum , a low-energy N + ion implantation-based mutagenesis system was established. In the first round of N + ion implantation, a proper conidial lethality rate (87.2–88.8%) and the highest positive mutation rate (11.2–11.4%) were achieved, and the highest propamocarb-tolerant mutant LA-I-R1-T7-M1 and LA-I-R1-T7-M2 were isolated in the experiment 1 and experiment 2 respectively, having a median effective concentration (EC 50 ) value of 1223.6 and 1173.6 μg/ml respectively. Multiple rounds of N + ion implantation were further employed, but fungal propamocarb-tolerance had not been significantly enhanced until the fifth-round of ion implantation. The LA-I-R5-M1 was the highest tolerant to propamocarb among all the mutants, whose EC 50 value was increased 2.54-fold compared to wild-type strain, reaching to the 1298.7 μg/ml. Its EC 50 value was significantly increased with continuous passaging, which was stable from the fifth- to fifteenth-generation, and then declined. However the EC 50 value of the twentieth-generation strain was still higher than the EC 50 (1298.7 μg/ml) of the first-generation strain. The colony growth, conidia yield and conidial germination of mutants on plates, and their parasitism in the nematode eggs of Meloidogyne incognita and Heterodera glycines were not significantly changed in both single- and multiple-round of N + ion implantation. In conclusion, we had succeeded in improving the propamocarb-tolerance of L. attenuatum through this low-energy N + ion implantation-based mutagenesis system. The improved strain LA-I-R5-M1 could be potentially applied in combination with propamocarb in the field.

Lecanicillium lecanii is an important entomopathogenic fungus which is used as a biopesticide for control of sucking insects. However, low fungicide-tolerance limits its application in the field. To improve the propamocarb-tolerance of L. lecanii , an optimized UV-light radiation-based mutagenesis system was established. The radiation time was optimized to 60 s for L. lecanii in the first round of UV-light radiation when a 20 Watt germicidal lamp was placed at a distance of 45 cm from plates. Both the positive mutation rate and EC 50 value of the UV-mutant were the highest when using this radiation time. The tolerance ratio (TR) was always lower than 1.13 in the first round of UV-light radiation. To further increase fungal propamocarb-tolerance, the highest propamocarb-tolerant mutant in the first round of UV-light radiation was used as a starting strain for multiple rounds of UV-light radiation in which a same germicidal lamp and radiation parameter were applied as described in the first round of UV-light radiation. TR was largely accumulated to 2.13 on the fourth round of radiation, and then began to level off and remained constant. A higher propamocarb-tolerant mutant was obtained in the sixth-round UV-light radiation, whose median effective concentration (EC 50 ) value was increased 2.15-fold compared to the wild-type strain, and it was mitotically stable in 20 passages on PDAY medium. This mutant did not show any significant changes of conidial yield on plates and virulence on aphids, although its colony growth was significantly lower than that of the wild-type strain. After the application of propamocarb at a recommended field dose of 550 μg/ml, the interval time of using L. lecanii was significantly shortened in this mutant compared to the wild-type strain. The results indicated that this improved strain could be potentially applied in combination with propamocarb in the field.

Multifunctional viral protein (VP35) encoded by the highly pathogenic Ebola viruses (EBOVs) can antagonize host double-stranded RNA (dsRNA) sensors and immune response because of the simultaneous recognition of dsRNA backbone and blunt ends. Mutation of select hydrophobic conserved basic residues within the VP35 inhibitory domain (IID) abrogates its dsRNA-binding activity, and impairs VP35-mediated interferon (IFN) antagonism. Herein the detailed binding mechanism between dsRNA and WT, single mutant, and double mutant were investigated by all-atom molecular dynamics (MD) simulation and binding energy calculation. R312A/R322A double mutations results in a completely different binding site and orientation upon the structure analyses. The calculated binding free energy results reveal that R312A, R322A, and K339A single mutations decrease the binding free energies by 17.82, 13.18, and 13.68 kcal mol-1 , respectively. The binding energy decomposition indicates that the strong binding affinity of the key residues is mainly due to the contributions of electrostatic interactions in the gas phase, where come from the positively charged side chain and the negatively charged dsRNA backbone. R312A, R322A, and K339A single mutations have no significant effect on VP35 IID conformation, but the mutations influence the contributions of electrostatic interactions in the gas phase. The calculated results reveal that end-cap residues which mainly contribute VDW interactions can recognize and capture dsRNA blunt ends, and the central basic residues (R312, R322, and K339) which mainly contribute favorable electrostatic interactions with dsRNA backbone can fix dsRNA binding site and orientation. Proteins 2017; 85:1008-1023. © 2017 Wiley Periodicals, Inc.

Transgenic cotton GK 12 producing Cry1Ab/Ac proteins can effectively control lepidopteran pests in agriculture. However, there are concerns that GK 12 has unintended side-effects on actinobacterial communities in the soil. Actinobacterial communities in the rhizosphere soil of cottons were analyzed by real-time PCR (qPCR) and denaturing gradient gel electrophoresis (DGGE) at different growth stages in three annual replications under field conditions. Population sizes and community structures of Actinobacteria were markedly affected by natural variations in years, growth stages and cultivars. No significant differences were found in population sizes (excepting at budding in 2009), community structures, and Shannon indices of Actinobacteria (excepting at budding in 2010) between the GK 12 and its non-transgenic parent Simian 3, although there were differences between the GK 12 and the conventional cotton DP 5415. Phylogenetic analysis of DGGE band sequences indicated that Cellulosimicrobium sp. and Rhodococcus sp. only occurred in the GK 12 and the Simian 3. The dominant Actinobacteria did not differ between the GK 12 and the Simian 3. In conclusion, the overall impacts of the GK 12 on actinobacterial communities in the rhizosphere soil, based on qPCR and DGGE, were not significant during three continuous years in northern China.

Conventional radiotherapy has a good killing effect on femoral echinococcosis. However, the sciatic nerve around the lesion is irreversibly damaged owing to bystander effects. Although intensity-modulated radiation therapy shows great advantages for precise dose distribution into lesions, it is unknown whether intensity-modulated radiation therapy can perfectly protect the surrounding sciatic nerve on the basis of good killing of femoral echinococcosis foci. Therefore, this study comparatively analyzed differences between intensity-modulated radiation therapy and conventional radiotherapy on the basis of safety to peripheral nerves. Pure-breed Meriones meridiani with bilateral femoral echinococcosis were selected as the research object. Intensity-modulated radiation therapy was used to treat left femoral echinococcosis of Meriones meridianus, while conventional radiotherapy was used to treat right femoral echinococcosis of the same Meriones meridianus. The total radiation dose was 40 Gy. To understand whether intensity-modulated radiation therapy and conventional radiotherapy can kill femoral echinococcosis, trypan blue staining was used to detect pathological changes of bone Echinococcus granulosus and protoscolex death after radiotherapy. Additionally, enzyme histochemical staining was utilized to measure acid phosphatase activity in the protoscolex after radiotherapy. One week after radiotherapy, the overall structure of echinococcosis in bilateral femurs of Meriones meridiani treated by intensity-modulated radiation therapy disappeared. There was no significant difference in the mortality rate of protoscoleces of Echinococcus granulosus between the bilateral femurs of Meriones meridiani. Moreover, there was no significant difference in acid phosphatase activity in the protoscolex of Echinococcus granulosus between bilateral femurs. To understand the injury of sciatic nerve surrounding the foci of femoral echinococcosis caused by intensity-modulated radiation therapy and conventional radiotherapy, the ultrastructure of sciatic nerves after radiotherapy was observed by transmission electron microscopy. Additionally, apoptosis of neurons was examined using a terminal-deoxynucleotidyl transferase-mediated dUTP nick end labeling assay, and expression of Bcl-2 and Bax in sciatic nerve tissue was detected by immunohistochemical staining and western blot assay. Our results showed that most neurons in the left sciatic nerve of Meriones meridiani with echinococcosis treated by intensity-modulated radiation therapy had reversible injury, and there was no obvious apoptosis. Compared with conventional radiotherapy, the number of apoptotic cells and Bax expression in sciatic nerve treated by intensity-modulated radiation therapy were significantly decreased, while Bcl-2 expression was significantly increased. Our findings suggest that intensity-modulated radiation therapy has the same therapeutic effect on echinococcosis as conventional radiotherapy, and can reduce apoptosis of the sciatic nerve around foci caused by radiotherapy. Experiments were approved by the Animal Ethics Committee of People's Hospital of Xinjiang Uygur Autonomous Region, China (Approval No. 20130301A41) on March 1, 2013.

Transgenic cotton GK 12 expressing insecticidal Cry1Ab/1Ac gene can effectively control the lepidopteran pests in agriculture, however it may have unintended side-effects on soil fungal communities. Therefore, the fungal community size and structure in rhizosphere soil of Bt and non-Bt cottons were analyzed by real-time polymerase chain reaction (qPCR) and denaturing gradient gel electrophoresis (DGGE) at growth stages and three annual replications in an intensive conventional cultivation system. The Cry1Ab/1Ac protein was undetectable or occasionally detectable at a low level (

Background Lecanicillium lecanii has been developed as a biopesticide and used in biological control of several agricultural insects. To improve fungal virulence, an optimised polyethylene glycol (PEG)-mediated protoplast transformation system was established for L. lecanii. Pr1A-like cuticle-degrading protease gene (Cdep1) from Beauveria bassiana was transferred into L. lecanii, and its resulting activity against Aphis gossypii was assessed. Results The optimised protoplast generation yielded 2.5 × 10(8) protoplasts g(-1) wet mycelium of fungi, and gave nearly 98% viability and 80% regeneration on plates. Protease activities were increased about fivefold in transformants expressing CDEP1. The median lethal concentration (LC50 ) for transformants expressing CDEP1 was twofold lower than that for the wild type (WT). The median survival time (LT50 ) for transformants expressing CDEP1 was also 14.2% shorter than that for WT, though no significant difference. There were no significant differences in conidial germination as colony growth and conidial yield on plates between transformants expressing CDEP1 and WT. The transformants expressing CDEP1 grew significantly quicker than WT in insects. The transformants expressing CDEP1 were lower in conidial yields on insect cadavers, but insignificantly different from WT. Conclusion The PEG-mediated protoplast transformation system was effective for L. lecanii, and the expression of CDEP1 significantly enhanced fungal virulence against cotton aphids. © 2016 Society of Chemical Industry.

Macrolide biosensor protein MphR(A) has been known as a key regulatory protein in metabolite sensing and genetic expression regulating. MphR(A) protein binds to macrolide antibiotic erythromycin (Ery) and releases the gene operon, thus activates expression of the mphA gene and initiates Ery resistance. The two mutant amino acid residues (V66L and V126L) might potentially disrupt Ery binding to MphR(A). In these studies, the binding of macrolide antibiotic Ery to wild type (Wt) MphR(A) and double mutant (V66L/V126L) MphR(A) are explored by molecular dynamics simulations. Compared to the Apo-MphR(A) protein and Wt-MphR(A)-Ery complex, many interesting effects owing to the double mutant (V66L/V126L) are discovered. In the case of Ery, Helix I which plays an important role in transcription shows itself a right-hand α helix in Wt-MphR(A)-Ery, whereas the activated helix is broken down in double mutant-V66L/V126L-MphR(A)-Ery. The calculated results exhibit that the double mutant V66L/V126L reduces the binding affinity of the V66L/V126L-MphR(A) to Ery, resulting in the block of Ery resistance. The binding free energy decomposition analysis reveals that the decrease of the binding affinity for the variant V66L/V126L-MphR(A)-Ery is mainly attributed to the gas phase electrostatic energies. The residue Leu66, Thr154, and Arg122 enhance the binding affinity of V66L/V126L-MphR(A) to Ery. The residues Tyr103 and His147 contributes mainly to binding energies in the Wt-MphR(A)-Ery complex, whereas the two residues have no contribution to the binding free energy inV66L/V126L-MphR(A)-Ery complex. Our study gives useful insights into the nature of amino acids mutation effect, the mechanism of blocking drug resistance at the atomic level and the characteristics in binding affinity for Ery to double mutant (V66L/V126L) MphR(A), which will contribute to the design of more effective macrolide antibiotics.

We have generated PUMCi001-A, an induced pluripotent stem cells (iPSC) line from dermal fibroblasts of a 13-year-old male Krabbe disease patient with two hemizygous (461C > A and 1244G > A) mutations in Galactocerebrosidase (GALC) gene using a Sendai viral delivery of OCT4, SOX2, KLF4, and c-MYC. The PUMCi001-A iPSC line carried the GALC mutations, displayed typical iPSC morphology, expressed pluripotent stem cell makers, exhibited a normal karyotype and differentiation capacity into three germ layers.

Purpose Tyrosine kinase inhibitors (TKI) of the epidermal growth factor receptor (EGFR) are becoming the standard treatments for Chinese patients with advanced non-small cell lung cancer (NSCLC) harboring an EGFR mutation. However, the economic impact is unclear yet in China. Materials and methods A decision-analytic model was developed to simulate 1-month patient transitions in a 10-year time horizon from Chinese heath care system perspective. The health and economic outcomes of four first-line strategies (pemetrexed plus cisplatin [PC], gefitinib, erlotinib, and afatinib) among NSCLC patients harboring EGFR mutations were estimated and assessed via indirect comparisons. Costs in the Chinese setting were estimated by using local hospital data and literatures. A 5% annual discount rate was applied to both costs and outcomes. The primary outcome was the incremental cost-effectiveness ratio (ICER). Sensitivity analyses were performed. Results Afatinib achieved additional 0.382, 0.216 and 0.174 quality-adjusted life-years (QALYs) with marginal $7930, $3680 and $2818 costs in comparison with PC, gefitinib and erlotinib, which resulted in the ICERs of $20,758, $17,693 and $16,197 per QALY gained, respectively. The hazard ratios (HR) of overall survival (OS) of afatinib against gefitinib, erlotinib and PC strategy had substantial influential parameters. Conclusions First-line afatinib is cost-effective compared with gefitinib, erlotinib and PC treatment for Chinese patients with EGFR mutation-positive NSCLC.

Monsanto’s Bt-cotton NC 33B, planted in northern China for more than one decade, effectively controls cotton bollworms and decreases the use of chemical insecticides. Because of the concern about undesirable ecological side-effects of transgenic Bt-cottons, it is important to assess Bt-cotton NC 33B’s effects on soil microorganisms in this zone. Microbial communities in the rhizosphere soil of Bt and non-Bt cottons were monitored under field conditions by polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) fingerprints of eubacteria, fungi and actinomycetes at six growth stages after three-year cultivation. Results showed that the population sizes and community structures of eubacteria, fungi and actinomycetes in rhizosphere soil were markedly affected by natural variations in the environment related to cotton growth stages. However, there was no significant difference in eubacterial, fungal and actinomycete population size and community structures in rhizosphere soil between NC 33B and its non-transgenic parent. In general, Bt-cotton NC 33B did not show evident effects on microbial communities in the rhizosphere soil under field conditions after three-year cultivation. This study provides a theoretical basis for environmental impact monitoring of transgenic Bt cottons.

Ebola virus (EBOV) is highly lethal due to virally encoded immune antagonists, and the combination of EBOV VP24 with karyopherin alpha (KPNA) will trigger anti-interferon (IFN) signaling. The crystal structure of VP24–KPNA5 has been proposed in recent studies, but the precise binding mechanisms are still unclear. In order to explore the VP24–KPNA5 protein binding micro-mechanisms, Molecular Dynamic (MD) simulations and Molecular Mechanics Generalized Born Surface Area (MM-GB/SA) energy calculation are performed. The obtained results show that EBOV VP24 binding to KPNA5 will rigidify their binding-face, and both proteins will be compacted during binding. According to the analyses of binding free energies of WT and the eight mutant systems, MUT3 makes the most effective contributions to the interaction; additionally MUT4, R398A and the double mutant have the second most effective influence. Hydrogen bond analysis demonstrates that inhibitors which can interfere with the formation of hydrogen bonds D480–T138, E483–R137 and D205–R396 will prevent the anti-IFN effect. Meanwhile, by combining the decomposition of binding free energies (DC) with computational alanine scanning (CAS) results, it is shown that VP24 residues R137 and T138 will be potential targets for EBOV VP24 inhibitors, and KPNA5 residues R396, R398, R480, Y477 and F484 will be potential targets to prevent KPNA5 binding to VP24, which will ultimately block anti-IFN signaling. Our investigations provide theoretical data to understand the binding modes of VP24–KPNA5. The precise binding mechanisms of the complex may shed light on the development of potential novel inhibitors against EBOV infection.

Ebola viruses (EBOV) will induce acute hemorrhagic fever, which is fatal to humans and nonhuman primates. The combination of EBOV VP35 peptide with nucleoprotein N-terminal (NPNTD) is proposed based on static crystal structures in recent studies, but VP35 binding mechanism and conformational dynamics are still unclear. This investigation, using Molecular Dynamic (MD) simulation and Molecular Mechanics Generalized Born Surface Area (MM-GB/SA) energy calculation, more convincingly proves the greater roles of the protein binding mechanisms than do hints from the static crystal structure observations. Conformational analysis of the systems demonstrate that combination with VP35 may lead to the conformational transition of NPNTD from “open” to “closed” state. According to the analyses of binding free energies and their decomposition, VP35 residue R37 plays a crucial role in wild type as well as mutant systems. Mutations of I29 and L33 to aspartate as well as M34 to proline affect binding affinity mainly through influencing electrostatic interaction, which is closely related to H-bonds formation. In addition, mutations mainly affect β-hairpin and loop regions, among which, M34P may have the greatest influence to the binding. This study may provide specific binding mechanisms between VP35 peptide and NPNTD, especially some important residues concerning binding.

Lecanicillium lecanii has been used in the biological control of several insects in agricultural practice. Since the gene manipulation tools for this entomopathogenic fungus have not been sufficiently developed, Agrobacterium tumefaciens-mediated transformation (ATMT) in L. lecanii was investigated in this study, using the wild-type isolate FZ9906 as a progenitor strain and the hygromycin B resistance (hph) gene as a selection marker. Furthermore, a field carbendazim-resistant (mrt) gene from Botrytis cinerea was expressed in L. lecanii FZ9906 via the ATMT system. The results revealed that the frequency of transformation surpassed 25transformants/10(6) conidia, most of the putative transformants contained a single copy of T-DNA, and the T-DNA inserts were stably inherited after five generations. All putative transformants had indistinguishable biological characteristics relative to the wild-type strain, excepting two transformants with altered growth habits or virulence. Moreover, the resistance of the putative transformants to carbendazim (MBC) was improved, and the highest one was 380-fold higher than the wild-type strain. In conclusion, ATMT is an effective and suitable system for L. lecanii transformation, and will be a useful tool for the basic and application research of gene functions and gene modifications of this strain.

Rapid, low-cost, and user-friendly strategies are urgently needed for early disease diagnosis and timely treatment, particularly for on-site screening of pathogens in aquaculture. In this study, we successfully developed a real-time fluorogenic loop-mediated isothermal amplification assay integrated on a microfluidic disc chip (on-chip LAMP), which was capable of simultaneously detecting 10 pathogenic bacteria in aquatic animals, i.e., Nocardia seriolae, Pseudomonas putida, Streptococcus iniae, Vibrio alginolyticus, Vibrio anguillarum, Vibrio fluvialis, Vibrio harveyi, Vibrio parahaemolyticus, Vibrio rotiferianus, and Vibrio vulnificus. The assay provided a nearly-automated approach, with only a single pipetting step per chip for sample dispensing. This technique could achieve limits of detection (LOD) ranging from 0.40 to 6.42pg per 1.414μL reaction in less than 30 min. The robust reproducibility was demonstrated by a little variation among duplications for each bacterium with the coefficient of variation (CV) for time to positive (Tp) value less than 0.10. The clinical sensitivity and specificity of this on-chip LAMP assay in detecting field samples were 96.2% and 93.8% by comparison with conventional microbiological methods. Compared with other well-known techniques, on-chip LAMP assay provides low sample and reagent consumption, ease-of-use, accelerated analysis, multiple bacteria and on-site detection, and high reproducibility, indicating that such a technique would be applicable for on-site detection and routine monitoring of multiple pathogens in aquaculture.

A simple spectrophotometric assay of H2O2 and glucose using Ag nanoparticles has been carried out. Relying on the synergistic effect of H2O2 reduction and ultraviolet (UV) irradiation, Ag nanoparticles with enhanced absorption signals were synthesized. H2O2 served as a reducing agent in the Ag nanoparticles formation in which Ag+ was reduced to Ag0 by O2− generated via the decomposition of H2O2 in alkaline media. On the other hand, photoreduction of Ag+ to Ag0 under UV irradiations also contributed to the nanoparticles formation. The synthesized nanoparticles were characterized by TEM, XPS, and XRD. The proposed method could determine H2O2 with concentrations ranging from 5.0 × 10−7 to 6.0 × 10−5 mol/L. The detection limit was estimated to be 2.0 × 10−7 mol/L. Since the conversion of glucose to gluconic acid catalyzed by glucose oxidase was companied with the formation of H2O2, the sensing protocol has been successfully utilized for the determination of glucose in human blood samples. The results were in good agreement with those determined by a local hospital. This colorimetric sensor thus holds great promises in clinical applications.

We aimed to evaluate the expression of microRNA-182 (miR-182) in triple-negative breast cancer (TNBC) tissues and the TNBC cell line MDA-MB-231 and to investigate the effects of mirR-182 on the cellular behavior of MDA-MB-231 and the expression of the target gene profilin 1 (PFN1), thus providing new methods and new strategies for the treatment of TNBC. Quantitative real-time PCR (qRT-PCR) was utilized to evaluate the expression of miR-182 in TNBC tissues, relatively normal tissues adjacent to TNBC and the TNBC cell line MDA-MB-231. Forty-eight hours after the MDA-MB-231 cells were transfected with the miR-182 inhibitor, qRT-PCR was utilized to detect the changes in miR-182 expression levels, and an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was utilized to determine the effects of miR-182 on cell viability. Flow cytometry was adopted to determine whether miR-182 affects the proliferation rates and apoptosis levels of the MDA-MB-231 cells. The transwell migration assay method was used to investigate the effects of miR-182 on the migration of the MDA-MB-231 cells. A luciferase reporter gene system was applied to validate that PFN1 was the target gene of miR-182. Western blot was used to measure the effects of miR-182 on the PFN1 protein expression levels in the cells. qRT-PCR results showed that compared with the relatively normal tissues adjacent to TNBC, miR-182 expression was significantly increased in the TNBC tissues and the MDA-MB-231 cells (p

The purposes of this study were to investigate the effects of B cell translocation gene 2 (BTG2) on the proliferation, apoptosis, and invasion of triple-negative breast cancer and to provide an experimental basis for the future treatment of human triple-negative breast cancer. A pcDNA3.1-BTG2 eukaryotic expression vector was constructed and transfected into the MDA-MB-231 human triple-negative breast cancer cell line using lipofection. Then, relevant changes in the biological characteristics of the BTG2-expressing cell line were analyzed using MTT (tetrazolium blue), flow cytometry, and Transwell invasion chamber assays. Additionally, the effects of BTG2 expression on cyclin D1, caspase 3, and matrix metalloproteinases 1/2 (MMP-1/-2) expression were analyzed. Cell proliferation was significantly lower in the pcDNA3.1-BTG2-transfected group compared to the empty vector and blank control groups (p < 0.05). There was no significant difference between the empty vector and blank control groups. FCM results demonstrated that there were significantly more cells in the G1 phase of the cell cycle and fewer S phase cells in the pcDNA3.1-BTG2 group than in the empty vector and blank control groups (p < 0.05). Additionally, the proportion of cells that migrated across the membrane was significantly lower in the pcDNA3.1-BTG2 group than in the empty vector and blank control groups (p < 0.05). Cyclin D1 and MMP-1/-2 expression were significantly lower in MDA-MB-231 cells transfected with pcDNA3.1-BTG2 as compared to the empty vector and blank control groups (p < 0.05). Caspase 3 expression was significantly higher in MDA-MB-231 cells from the pcDNA3.1-BTG2 group compared to the empty vector and blank control groups (p < 0.05). In conclusion, BTG2 may inhibit MDA-MB-231 proliferation and promote apoptosis. Additionally, BTG2 may also inhibit the invasion of MDA-MB-231 human triple-negative breast cancer cells.

CR6-interacting factor 1 (CRIF1) is ubiquitously expressed in human tissues. CRIF1 was first identified as a Gadd45γ (also known as CR6)-interacting protein, and it was also identified in a human colon cancer cell line stably transformed with p53. These results suggested that CRIF1 functions in the nucleus with p53 and Gadd45 family proteins in the suppression of cell growth and tumor development. Here, we found that CRIF1 could be recruited to a specific region in the promoter of the p53 gene, eliciting an increase in the mRNA and protein levels of p53 as well as p53 functional target genes. These functions required CRIF1 to interact with SNF5. CRIF1 was further recruited to the upstream promoter region of the p53 gene to suppress cell cycle progression in HCT116 cells. To our knowledge, this is the first evidence indicating that SNF5 is indispensable for CRIF1-enhanced p53 activity and its function in the suppression of cell cycle arrest in human cancer cells.

Lecanicillium attenuatum is an important nematophagous fungus with potential as a biopesticide against plant-parasitic nematodes. The Pr1A-like cuticle-degrading protease (Cdep1) gene originating from the entomopathogenic fungus Beauveria bassiana was transformed into the nematophagous fungus L. attenuatum using a polyethylene-glycol mediated protoplast-based transformation system. Protease activity was increased 0.64- to 1.63-fold 2–10 d after growth in the transformed L. attenuatum. Inhibition of egg-hatching and J2 motility of soybean cyst nematodes (Heterodera glycines) by cell-free fungal culture filtrates were enhanced by 17–76% 2–14 d and 43–152% 1–13 d after incubation, respectively.

Seasonal hair follicle activity and fibre growth in some Cashmere-bearing goats ( Caprus hircus ) is a cyclic process that is well characterized morphologically but understood incompletely at the molecular level. As an initial step in discovering regulators in hair-follicle activity and cycling, we used qPCR to investigate 19 genes expression in Cashmere goat side skin from 12 mon. Many of these genes may be associated with the hair follicle development-relevant genes (HFDRGs) in the literature. Here we show that Hoxc13/β-catenin gene associated with the follicle activity. In addition, Hoxc13 was found to be expressed with an drastic increase between July and November for melatonin treatments. To further investigate the role of Hoxc13 on HFDRGs, fibroblasts and keratinocytes from Cashmere goat skin were transfected with p-ECFP-Hoxc13. The result suggested that overexpression of Hoxc13 gene decreased HFDRGs with negative role for hair follicle development and increase HFDRGs with positive role for hair follicle development in vitro. These findings provide data on the Hoxc13 expression profile of normal Cashmere goat skin and Cashmere goat skin with melatonin treatment, and demonstrate hair-follicle-activity dependent regulation of Hoxc13 expression.

The purpose of this study was to evaluate the importance of biological markers to predict pathologic complete response (pCR) to neoadjuvant chemotherapy (NCT) in patients with locally advanced triple-negative breast cancers (TNBCs). Forty-one patients (18.6%) among 220 breast cancer patients were identified as TNBCs from March 2006 to 2009 were included in this prospective study. The pre-NCT treatment expression levels of Ki-67 proliferation index, estrogen receptor (ER), progesterone receptor (PgR), epidermal growth factor receptor 2 (HER-2), CK5/6, epidermal growth factor receptor (EGFR), cyclin D1, and nm23-H1 were detected by immunohistochemistry (IHC). A total of 180 cycles were administered with the median number of four cycles per patient (range, 4-6). The pCR rate was 34.1% (95% CI, 19.6-48.6%). In univariate analysis, early T stage, clinical response after 2 cycles, negative basal-like, negative EGFR, high Ki-67 proliferation index, and positive nm23-H1 were found to be significantly predictive of a pCR (P = 0.010, 0.040, 0.007, 0.001, 0.019, and 0.010, respectively). Basal-like status and nm23-H1 status were significant for pCR on multivariate analysis (P = 0.004 and 0.031, respectively). Basal-like status and nm23-H1 are independent predictive factors of pCR to neoadjuvant docetaxel plus epirubicin combination chemotherapy in patients with TNBCs.

The SWI/SNF chromatin-remodeling complexes utilize energy from ATP hydrolysis to reposition nucleosomes and regulate the expression of human genes. Here, we studied the roles of human Brahma (hBrm) and Brahma-related gene 1 (Brg1), the ATPase subunits of the SWI/SNF complexes, in regulating human genes. Our results indicate that both hBrm and Brg1 interact with Signal transducer and activator of transcription (Stat) 1 in vitro. However, Stat1 in its native form only recruits hBrm to IFNγ-activated sequences (GAS) of individual genes; by contrast, in a stress-induced phosphorylated form, Stat1 mainly binds to Brg1. Under basal conditions, hBrm is recruited by native Stat1 to the GAS and exists in a mSin3/HDAC co-repressor complex on the hsp90α gene, which shows a compact chromatin structure. Upon heat-shock, hBrm is acetylated by p300 and dissociates from the co-repressor complex, which the phosphorylated Stat1 is increased, and binds and recruits Brg1 to the GAS, leading to elevated induction of the gene. This hBrm/Brg1 switch also occurs at the GAS of all of the three examined immune genes in heat-shocked cells; however, this switch only occurs in specific cell types upon exposure to IFNγ. Regardless of the stimulus, the hBrm/Brg1 switch at the GAS elicits an increase in gene activity. Our data are consistent with the hypothesis that the hBrm/Brg1 switch is an indicator of the responsiveness of a gene to heat-shock or IFNγ stimulation and may represent an "on-off switch" of gene expression in vivo.

The purpose of this study was to evaluate the importance of biological markers to predict pathologic complete response (pCR) to neoadjuvant docetaxel plus epirubicin combination chemotherapy in patients with locally advanced breast cancer (LABC). Two hundred and twenty consecutive patients with LABC who had received neoadjuvant chemotherapy (NCT) with docetaxel and epirubicin from March 2006 to March 2009 were included in this retrospective study. The pre- and post-neoadjuvant chemotherapy (NCT) treatment expression levels and changes of Ki-67 proliferation index, estrogen receptor (ER), progesterone receptor (PgR), epidermal growth factor receptor 2 (HER-2), cyclin D1, and nm23-H1 were detected by immunohistochemistry (IHC). The pCR rate was 9.1% (95% CI, 5.3-12.9%). In univariate analysis, poor tumor differentiation, OR after 2 cycles of NCT, both negative of ER/PgR, negative HER-2, positive cyclin D1, and positive nm23-H1 were found to be significantly predictive of a pCR. Histological grade and ER/PgR status were significant for pCR on multivariate analysis (P = 0.023 and 0.003, respectively). The expression levels of cyclin D1 (median, 8% vs. 9%; P = 0.016) after NCT treatment increased significantly, while the median Ki-67 proliferation index was dramatically decreased after NCT treatment from 35 to 15% (P = 0.036). However, after a Bonferroni adjustment, only the difference of Ki-67 proliferation index was still significant (P = 0.026). Histological grade and ER/PgR status are independent predictive factors of pCR to neoadjuvant docetaxel plus epirubicin combination chemotherapy in locally advanced breast cancer. Expression of HER-2, Ki-67, cyclin D1, and nm23-H1 were not predictive for pCR.

Three new polyoxygenated cyclohexenes, named uvacanols F, G, H (1-3), were isolated from the roots of Uvaria calamistrata (Annonaceae). Their structures were determined to be 2-acetoxyl-5-chlorine-benzoyloxymethylcyclohex-1 (6)-ene-4-ol-3-benzoate (1), benzoyloxy-methylcyclohex-1 (6)-ene-2,3,4-triols-5-benzoate (2), 3-acetoxyl-benzoyloxymethylcyclohex-1 (6)-ene-4,5-diols-2-benzoate (3) by spectroscopic methods and chemical derivatization.

Fusarium graminearum (teleomorph, Gibberella zeae) causes head blight of cereals and contaminates grains with trichothecene mycotoxins that are harmful to humans and domesticated animals. Control of Fusarium head blight relies on carbendazim (MBC) in China, but resistance to MBC in F. graminearum is now widespread. Sixty-seven strains were evaluated for trichothecene production in shake culture or in the field. The strains included 60 wild-type strains (30 MBC-resistant and 30 MBC-sensitive), three MBC-resistant site-directed mutants at codon 167 in β2-tubulin, three MBC-sensitive site-directed mutants at codon 240 in β2-tubulin, and their MBC-sensitive wild-type progenitor strain ZF21. The incidence of infected spikelets and the amount of F. graminearum DNA in field grain (AFgDNA) also were evaluated for all strains. MBC resistance increased trichothecene production in shake culture or in the field. Although MBC resistance did not change the incidence of infected spikelets, it did increase AFgDNA. Tri5 gene expression increased in MBC-resistant strains grown in shake culture. We found a significant exponential relationship between trichothecene production and Tri5 gene expression in shake culture and a linear relationship between the incidence of infected spikelets or AFgDNA and trichothecene production in field grain.

The occurrence of trichothecene toxins was determined in different kinds of wheat-based foods (convenience noodles, biscuits, bread and cakes) in Nanjing, China. A total of 74 commercially available samples were collected from the supermarkets in Nanjing from September 2006 to April 2007. Three trichothecenes named deoxynivalenol (DON), 3-acetyldeoxynivalenol (3ADON) and 15-acetyldeoxynivalenol (15ADON) were detected and quantified by gas chromatography with electron capture detection. The incidence of DON, 3ADON and 15ADON were 73.0, 8.1 and 23.0%, respectively, the content ranges were 0.07–11.38, 0.25–1.47 and 0.03–2.06 mg kg−1, respectively, and the average contents were 2.47, 0.71 and 0.48 mg kg−1 in positive samples, respectively. The average DON contents in convenience noodles, biscuits, bread and cakes were 2.36, 2.79, 1.86 and 0.62 mg kg−1, respectively. Thirty-seven of 74 samples contained more than 1 mg of DON per kilogram, which is the regulatory limit defined by the Chinese government.

Hoxc13 has an important role in controlling hair formation. In this study, we examine the Hoxc13 RNA expression pattern of skin during embryo development. The result indicated that changes of the Hoxc13 gene expression and thickness of skin have a similar trend during hair follicle morphogenesis. In interpreting these results, we investigated whether the regulation motifs is in Hoxc13 intron, which is a 5.4 kb fragment. To blast with other mammals, we found a very conservative region in all mammal animals and two regions in livestock, such as cow, sheep, horse, dog, and so on, which are not in other Hox genes. We have examined putative pre-miRNA in this region, providing an entry point for elucidating currently unknown mechanisms that are required for regulating quantitative levels of Hoxc13 gene expression.

Cucumber downy mildew caused byPseudoperonospora cubensis (Berk. and Curt.) Rostov. limits crop production in Shandong Province of China. Since management of downy mildew is strongly dependent on fungicides, a rational design of control programs requires a good understanding of the fungicide resistance phenomenon in field populations of the pathogen. A total of 106 and 97 isolates ofP. cubensis were obtained in 2006 and 2007, respectively. The EC50 values for the growth of all the 106 isolates collected in 2006 were 0.0063–0.0688µg ml−1 (average: 0.0196±0.0048µg ml−1) azoxystrobin and these were therefore considered sensitive isolates. However, 57 field isolates ofP. cubensis of the 97 collected in 2007 with EC50 values that ranged from 0.609 to >51.2µg ml−1 were considered resistant to azoxystrobin. Fragments of the fungicide-targeted mitochondrial cytochromeb gene from total pathogen DNA were amplified using polymerase chain reaction and their sequences analyzed to elucidate the molecular mechanism of resistance. A single point mutation (GGT to GCT) in the cytochromeb gene, resulting in substitution of glycine by alanine at position 143, was found in the three selected azoxystrobin-resistant isolates of downy mildew. This substitution in cytochromeb exhibited different resistance levels, with the resistance factor from 21.15 to greater than 2618.9. In addition, the different resistance levels seemed to appear within 1 year (between 2006 and 2007). Therefore, growers of Shandong Province in China now are faced with a challenge in managing the azoxystrobin resistance in cucumber downy mildew.

In our ongoing investigation of the bioactive constituents from plants, two new lignans, magnolignan A-2-O-β-D-glucopyranoside and strebluslignanol were isolated from heartwood of Streblus asper, along with three known lignans, magnolignan A, magnolol, and magnaldehyde D. 1D and 2D NMR experiments, including COSY, HMQC, and HMBC, and other spectroscopic methods, including UV, IR, and MS were used for the determination of the structures and NMR assignments. Primary bioassays showed that magnolignan A-2-O-β-D-glucopyranoside and strebluslignanol have medium cytotoxic activity against HEp-2 and HepG2 cells, with IC50 of 13.3 µM, 46.4 µM and 10.1 µM, 21.7 µM, respectively. Copyright © 2008 John Wiley & Sons, Ltd.

Phylogenetic analysis of SARS coronavirus isolates based on the spike gene and protein sequence using Neighbor-Joining, maximum likelihood and Bayesian inference methods indicated that a recent human SARS-CoV isolate was closer to some human SARS-CoV isolates from earlier epidemic phase than to the SARS-CoV-like viruses isolated from wild animals during previous epidemic phase. A reasonable judgment based on phylogenetic relationship and sequence variations it is likely that the recent human SARS-CoV isolate is closer to an unknown SARS-CoV predecessor.

The entomopathogenic fungus L. lecanii has been developed as biopesticides and used widely for biological control of several insects in agricultural practice. Due to the lack of isolation/count methods for L. lecanii in soil, the persistence of this fungus in soil appears to have attracted no attention. A selective medium and count method for L. lecanii in soil based on cetyl trimethyl ammonium bromide (CTAB) was developed, and then the persistence and viability of this fungus in soil were investigated under field conditions between 2012 and 2014. The results showed that the rate of recovery for L. lecanii in soil on the selective CTAB medium was satisfactory. The minimum CFUs for L. lecanii on the selective medium (0.5 g/L CTAB) was about 102 conidia/g soil. The L. lecanii density in soil declined quickly in the first month after inoculation with fungal conidia, kept stable for 6 to 10 months, and then decreased gradually until undetectable. L. lecanii could persist for at least 14 months in the agricultural soil of northern China. The colony growth, conidia yield and germination rate on plates, as well as the median lethal concentration or times (LC50 or LT50) to aphids, mycelium growth in aphids and sporulation on aphids of L. lecanii did not change significantly during the persistence in soil. In general, the count method developed here was a very useful tool for monitoring the dynamics of natural or introduced L. lecanii populations in soil, and the data on the persistence of L. lecanii in soil reported here were helpful for biological control and environmental risk assessment.

Mammalian Sirtuin proteins (SIRTs) are homologs of yeast Sir2, and characterized as class III histone deacetylases of NAD(+) dependence. Unlike their lower counterparts that are directly involved in the extending of lifespan, mammalian SIRTs mainly function in metabolism and cellular homeostasis, among them, SIRT7 is the least understood. SIRT7 is localized in the nucleus and rich in nucleoli associated with RNA polymerase I, and correlated with cell proliferation. In contrast, SIRT7 has recently been demonstrated to specifically deacetylate H3K18ac in the chromatin, and in most cases represses proliferation. Although MicroRNA as miR-125b has been reported to down-regulate SIRT7 by binding to its 3'UTR, however, how SIRT7 gene is regulated remains unclear. Here, we identified the transcription initiation site of human SIRT7 gene at the upstream 23rd A nucleotide respective to the translational codon, and the SIRT7 is a TATA-less and initiator-less gene. The sequences in the upstream region between -256 and -129 bp are identical with important functions in the three species detected. A C/EBPα responding element is found that binds both C/EBPα and C/EBPβ in vitro. We showed TSA induced SIRT7 gene transcription and only the HDAC3, but not its catalytic domain depleted mutant, interacted with C/EBPα to occupy the C/EBPα element and repressed SIRT7 gene in the hepatocellular carcinoma cells. To our knowledge, this is the first report on the regulation mechanism of SIRT7 gene, in which, HDAC3 collaborated with C/EBPα to occupy its responding element in the upstream region of SIRT7 gene and repressed its expression in human cells.

Six new styrylpyrone derivatives, goniolactones A-F (1-6), have been isolated from the roots of Goniothalamus cheliensis. The structures and stereochemistry of the new compounds were elucidated by interpretation of spectroscopic data and chemical methods. The relative configuration of goniolactone A (1) was determined by X-ray crystallography analysis, and the absolute configurations of goniolactones A (1) and B (2) were established by Mosher's method. Goniolactone B (2) exhibited significant cytotoxicity against A2780, HCT-8, and KB cells with IC(50) values of 7.40, 4.43, and 7.23 microM, respectively.

Tacrolimus is a widely used immunosuppressive drug for preventing the rejection of solid organ transplants. The efficacy of tacrolimus shows considerable variability, which might be related to genetic variation among recipients. We conducted a retrospective study of 240 Chinese renal transplant recipients receiving tacrolimus as immunosuppressive drug. The retrospective data of all patients were collected for 40 days after transplantation. Seventeen SNPs of CYP3A5, CYP3A4, COMT, IL-10 and POR were identified by the SNaPshot assay. Tacrolimus blood concentrations were obtained on days 1-3, days 6-8 and days 12-14 after transplantation, as well as during the period of the predefined therapeutic concentration range. Kruskal-Wallis test was used to examine the effect of genetic variation on the tacrolimus concentration/dose ratio (C 0/D) at different time points. Chi-square test was used to compare the proportions of patients who achieved the target C 0 range in the different genotypic groups at weeks 1, 2, 3 and 4 after transplantation. After correction for multiple testing, there was a significant association of C 0/D with CYP3A5*3, CYP3A4*1G and CYP3A4 rs4646437 T>C at different time points after transplantation. The proportion of patients in the IL-10 rs1800871-TT group who achieved the target C 0 range was greater (p = 0.004) compared to the IL-10 rs1800871-CT and IL-10 rs1800871-CC groups at week 3 after transplantation. CYP3A5*3, CYP3A4 *1G, CYP3A4 rs4646437 T>C and IL-10 rs1800871 C>T might be potential polymorphisms affecting the interindividual variability in tacrolimus metabolism among Chinese renal transplant recipients.

Apoptosis of sinusoidal endothelial cells (SECs) is one of the initial events in the development of ischemia-reperfusion injury of the liver. Glycine has been shown to diminish ischemia-reperfusion injury in the liver and improve graft survival in the rat liver transplantation model. Here, we investigated the effect of glycine on apoptosis of primary cultured rat SECs induced by vascular endothelial growth factor (VEGF) deprivation. Isolated rat SECs were cultured in EBM-2 medium supplemented with 10% fetal bovine serum (FBS) and growth factors including 20 ng/mL VEGF for 3 days. SECs at 3 days of culture showed spindle-like shapes; however, cells started shrinking and detaching from dishes by VEGF deprivation. Apoptosis was detected by terminal deoxynucleotidyl transferase (TdT)-mediated d-uridine triphosphate (dUTP)-biotin nick end labeling (TUNEL) staining in these conditions. Control SECs contained only a few percent of TUNEL-positive cells; however, they started increasing 4 hours after VEGF deprivation, and the percentage of TUNEL-positive cells reached about 50% at 8 hours and almost 100% at 16 hours after VEGF deprivation. Interestingly, this increase in TUNEL-positive cells after VEGF deprivation was prevented significantly when glycine (1-10 mmol/L) was added to the medium, the levels being around 60% of VEGF deprivation without glycine. Furthermore, strychnine (1 micromol/L), a glycine receptor antagonist, inhibited this effect of glycine, suggesting the possible involvement of the glycine receptor/chloride channel in the mechanism. Moreover, Bcl-2 protein levels in SECs were decreased 8 hours after VEGF deprivation, which was prevented almost completely by glycine. It is concluded that glycine prevents apoptosis of primary cultured SECs under VEGF deprivation.

Five new annonaceous acetogenins, calamistrins C-G (1-5), were isolated from an ethanolic extract of the roots of Uvaria calamistrata. Compounds 1-3 were mono-THF ring acetogenins; compounds 4 and 5 were bis-THF acetogenins, with the THF rings from C-18 to C-25. The absolute configurations of 3, 4, and 5 as well as the partial absolute configurations of 1 and 2 were determined by (13)C NMR spectroscopy and advanced Mosher methodology.

Three new styryl-lactones 8-acetylgoniofufurone(1), 7-acetylgonio-pypyrone(3), and 5-acetylgoniopypyrone(4), along with ten known compounds, goniofufurone(2), goniopypyrone(5), goniothalamin, goniothalenol, (+)-isoaltholactone, goniodiol, 7-acetylgoniodiol, goniotriol, 8-acetylgoniotriol, 9-deoxygoniopypyrone were isolated from the rhizomes of Goniothalamus griffithii Hook f. et. Thoms. Their structures were elucidated by IR, MS, NMR spectra and chemical evidence. All compounds showed cytotoxic activities against human cancer cell lines.

Background The 6986A > G polymorphism for CYP3A5 and the − 129T > C , 1236C > T , 2677G > T/A , and 3435C > T polymorphisms for MDR1 are considered the major genetic factors affecting a range of drugs' metabolism and transport. Simultaneous genotyping of these five polymorphisms would be useful for estimating the therapeutic effects of their related drugs. Subjects and methods We have described a SNaPshot assay that can simultaneously detect all the five polymorphisms based on multiplex PCR and minisequencing reaction. A total of 168 unrelated Chinese DNA samples were used to establish and evaluate the assay. Results The different genotypes of the five polymorphisms could be determined by peak retention time and colors. DNA sequencing was performed on samples randomly selected from each of the genotype groups detected by SNaPshot assay, and the results indicated 100% concordance. Conclusion The SNaPshot assay for the CYP3A5 and MDR1 five polymorphisms detection was accurate, automated, and cost-effective.

Heparan sulfate proteoglycans (HSPGs) are involved in the regulation of cell growth, apoptosis and lipid metabolism in vitro. Syndecans are the primary form of HSPGs. Syndecan-1 is involved in the processes of cell growth, differentiation, adhesion, wound healing and inflammation. Additionally, as a sinusoidal transmembrane HSPG facing the plasma compartment, syndecan-1 is a promising target to be involved in lipoprotein physiology. We aimed to examine the possible correlation of syndecan-1 and lipid profile in type 2 diabetes mellitus. In this study, serum syndecan-1 was detected by ELISA, and potential correlations between syndecan-1 and triglyceride, cholesterol, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, lipoprotein a, apolipoprotein (apo) B, apoA1 and apoB/apoA1 were analyzed. Forty-one patients with type 2 diabetes and 31 age-matched, non-diabetic healthy subjects (controls) were enrolled. Syndecan-1 in patients with diabetes (26.15 ± 2.42 ng/ml) was significantly higher than that of the controls (16.85 ± 1.98 ng/ml, t = −2.98, P = 0.005). Serum syndecan-1 level correlated negatively with apoA1 (r = −0.46, P = 0.003). Multiple regression analysis showed that apoA1 (b = −0.43, P = 0.003) was a predictor of serum syndecan-1 levels in subjects with type 2 diabetes.

Three new alkaloids-griffithazanone A (1), griffithdione (2), and griffithinam (3)-were isolated from the roots of Goniothalamus griffithii, along with six known compounds, 4-methyl-2,9, 10-(2H)-1-azaanthracenetrione (4), velutinam, aristololactam BI, aristololactam BII, aristololactam AII, and norcepharanone B. Their structures were elucidated on the basis of spectral and chemical methods. The absolute configuration of griffithazanone A (1) was determined by the preparation of Mosher's esters.

Objective To observe the ultrastructure of human oocytes at different maturity stages and morphologies before and after in vitro maturation. Design Case report. Setting University-based center for human reproduction. Patient(s) A 28-year-old patient with polycystic ovary syndrome undergoing IVM/IVF-ET treatment. Intervention(s) Human chorionic gonadotropin was given to the patient 36 hours before her oocytes were retrieved by transvaginal aspiration. Main Outcome Measure(s) Ultrastructure of human oocytes matured in vitro. Result(s) Obvious ultrastructural differences were found in oocytes at different maturity stages between those cultured in vitro and those matured in vivo. Degenerate structures were observed in abnormal oocytes. Granulosa cell apoptosis rates from abnormal oocytes were higher than those from normal oocytes. Cumulus cells surrounding morphologically abnormal oocytes had a significantly higher frequency of cell apoptosis, as compared with morphologically normal oocytes. Conclusion(s) Obvious ultrastructural differences were found between the oocytes matured in vitro and in vivo.

In this study, we have identified 876 polymorphism sites in 145 complete or partial genomes of SARS-CoV available in the NCBI GenBank. One hundred and seventy-four of these sites existed in two or more SARS-CoV genome sequences. According to the sequence polymorphism, all SARS-CoVs can be divided into three groups: (I) group 1, animal-origin viruses (such as SARS-CoV SZ1, SZ3, SZ13 and SZ16); (II) group 2, all viruses with clinical origin during first epidemic; and (III) group 3, SARS-CoV GD03T0013. According to 10 special loci, group 2 again can be divided into genotypes C and T, which can be further divided into sub-genotypes C1–C4 and T1–T4. Positive Darwinian selections were identified between any pair of these three groups. Genotype C gives neutral selection. Genotype T, however, shows negative selection. By comparing the death rates of SARS patients in the different regions, it was found that the death rate caused by the viruses of the genotype C was lower than that of the genotype T. SARS-CoVs might originate from an unknown ancestor.

AIM: To construct a recombined human AFP eukaryotic expression vector for the purpose of gene therapy and target therapy of hepatocellular carcinoma (HCC). METHODS: The full length AFP-cDNA of prokaryotic vector was digested, and subcloned to the multi-clony sites of the eukaryotic vector. The constructed vector was confirmed by enzymes digestion and electrophoresis, and the product expressed was detected by electrochemiluminescence and immunofluorescence methods. RESULTS: The full length AFP-cDNA successfully cloned to the eukaryotic vector through electrophoresis, 0.9723 IU/mL AFP antigen was detected in the supernatant of AFP-CHO by electrochemiluminescence method. Compared with the control groups, the differences were significant (P < 0.05). AFP antigen molecule was observed in the plasma of AFP-CHO by immunofluorescence staining. CONCLUSION: The recombined human AFP eukaryotic expression vector can express in CHO cell line. It provides experimental data for gene therapy and target therapy of hepatocellular carcinoma.

p28, a 28kD protein from toad (Bufo bufo gargarizans) oocytes, was identified by using p13 suc1 -agaroseaffinity chromatography. Sequence homology analysis of the full-length cDNA of p28(Gene Bank accessionnumber: AF 314091) indicated that it encodes a protein containing 224 amino-acids with about 55% iden-tities and more than 70% positives to human, rat or mouse UCH-L1, and contains homological functionaldomains of UCH family. Anti-p28 monoclonal antibody, on injecting into the oocytes, could inhibit theprogesterone-induced resumption of meiotic division in a dose-dependent manner. The recombinant pro-tein p28 showed similar SDS/PAGE behaviors to the native one, and promoted ubiquitin ethyl ester hydrolysis,a classical catalytic reaction for ubiquitin carboxyl terminal hydrolases (UCHs). The results in this paperreveal that a novel protein, p28, exists in the toad oocytes, is a UCH L1 homolog, was engaged in the processof progesterone-induced oocyte maturation possibly through an involvement in protein turnover anddegradation.Key words: p28, cDNA clone, recombinant expression, ubiquitin carboxyl terminal hydrolase, oocytematuration.

Background & Aims: In this study, we investigated the role of leptin and its receptors (Ob-R) in profibrogenic responses in the liver using Zucker (fa/fa) rats, a natural occurring Ob-R–deficient animal. Methods: Male Zucker (fa/fa) rats and their lean (+/?) littermates were given intraperitoneal injections of thioacetamide (TAA) (200 mg/kg body wt, 3 times/wk) for 4–8 weeks, and progression of hepatic fibrosis was evaluated. In vitro transactivation of hepatic stellate cells (HSCs) isolated from Zucker rats was evaluated by Western blotting and immunocytochemistry for α-smooth muscle actin and type I collagen. Further, a long-form Ob-R (Ob-Rb) in sinusoidal endothelial cells (SECs) and Kupffer cells was identified by reverse-transcription polymerase chain reaction. Moreover, transforming growth factor (TGF)-β1 messenger RNA in LSE cells, a human SEC-derived cell line, was measured by Northern blotting. Results: Although the normal liver does not produce leptin, activated HSCs produced leptin in vivo during fibrogenesis caused by TAA. In Zucker rats, TAA-induced hepatic fibrosis was prevented almost completely, whereas induction of TGF-β1 and activation of HSCs were abolished. It is less likely, however, that leptin plays an essential role in the activation of HSCs as a strong autocrine regulator, because HSCs isolated from Zucker rats undergo normal transactivation process in vitro. In contrast, SECs and Kupffer cells contain Ob-Rb, through which leptin up-regulates the expression of matrix remodeling genes including TGF-β1. Conclusions: Collectively, these findings indicated that leptin and its functional receptors (Ob-Rb) play a pivotal role in profibrogenic responses in the liver. GASTROENTEROLOGY 2002;122:1399-1410

113 Background: PIK3CAgene mutations are the most common activating mutations in human breast cancer (BC). Its association with the resistance to herceptin in HER2-positive BC was reported, but whether it’s related to the efficacy of neoadjuvant chemotherapy is unknown. Methods: We reviewed records of 108 patients with BC treated with epirubicin plus docetaxel neoadjuvant chemotherapy between June 2005 and April 2011. 92 patients’ clinical and pathologic objective response data were collected. FFPE tumor biopsies obtained at the time of diagnoses (and before treatment) were collected. EGFR, KRAS, BRAF, PIK3CA mutations and HER2, PTEN, EGFR mRNA expression were analyzed by liquidchip technology. Chi-square test and Fisher’s exact test were used in statistical analysis. A p value less than 0.05 was considered statistically significant. Results: No mutation was detected in EGFR, KRAS and BRAF. The mutation rate of PIK3CA was 35.1% (38/108), and mutation rates of E542K, E545K, H1047L and H1047R were 3.7, 10.2, 3.7 and 17.6% respectively. More PIK3CA mutations were detected in HER2 high expression tumors (p=0.018). No correlation was found between PIK3CA mutations and PTEN, EGFR expression. Clinical response (CR+PR, 63/92) was associated with PIK3CA mutations (p=0.002). However, PIK3CA mutations rate between pathologic complete release (pCR, 7/92) and non-pCR group was not significantly different (p=0.422). Conclusions: PIK3CA mutation may predict better clinical response in BC patients treated with epirubicin plus docetaxel neoadjuvant chemotherapy. More studies are needed to validate these results and the correlation between PIK3CA mutations and pCR.