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Kushneria phosphatilytica YCWA18 T (= CGMCC 1.9149 T  = NCCB 100306 T ) was isolated from sediment collected in a saltern on the eastern coast of Yellow Sea in China. The genome was sequenced and comprised of one circular chromosome with the size of 3,624,619 bp and DNA G + C content of 59.13%. A total of 3267 protein-coding genes, 64 tRNA genes and 12 rRNA genes were obtained. Genomic annotation indicated that the genome of K. phosphatilytica YCWA18 T had 34 genes involved in phosphorus (P) solubilization/metabolism, e.g., gdh, pqq, phoA, phoD and phoX, which products can convert insoluble P-containing compounds to more bio-available dissolved inorganic P. Comparative genomic analysis of Kushneria strains revealed that gdh, pqq, phoA, phoD and phoX were widely distributed in these strains, indicating the genus Kushneria may play an important role in the P cycle. Additionally, a multitude of salt tolerance genes were detected in the genome of K. phosphatilytica YCWA18 T . This study and the genome sequence data will be available for further research and will provide insights into potential biotechnological and agricultural applications of Kushneria strains., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The phytochemical investigation of the aerial parts of Isodon japonica var. glaucocalyx afforded four undescribed (glaucocalyxin O-R, 1-4) and six known ent-kauranoids (5-10). Their structures were established using NMR and MS measurements. Compounds 1 and 2 are dimeric ent-kaurane-type diterpenoids. Moreover, the plausible biogenetic pathways for compounds 1 and 2 were proposed as Michael addition between two monomers. Eight compounds were assayed for their anti-inflammatory activity by evaluating NO production in LPS-induced RAW 267.4 cells, and compounds 7, 8 and 9 exhibited relatively remarkable anti-inflammatory activities at 10 μM., Competing Interests: Declaration of competing interest The authors declare they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Thirteen previously undescribed iridoids (1-13), together with five known iridoids (14-18) were isolated from the roots and rhizomes of Valeriana jatamansi Jones. Their structures with absolute configurations were elucidated by analysis of MS, NMR, optical rotation and their experimental and calculated electronic circular dichroism spectra. All of the isolated compounds were tested for their protective effects against α-hemolysin-induced cell death in A549 cells. Compounds 14, 16 and 17 showed moderate protective effects, and compounds 15 and 18 showed weak protective effects., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

A phytochemical investigation of the medicinal plant Callicarpa macrophylla resulted in the characterization of two rare rearrangement abietane-type diterpenoids, macrophypene F-G (1-2), and three abietane diterpenoids, named macrophypene H-J (3-5). Additionally, five known diterpenoids (6-10) were identified. The structures of the newly discovered compounds were fully established through extensive analysis of HRESIMS, 1D and 2D NMR data. The absolute configurations of the isolated compounds were determined using CD comparison, chemical methods, and X-ray crystal diffraction experiments. Subsequently, all isolated diterpenoids were evaluated for their inhibitory effects on extracellular PCSK9 protein levels by PCSK9 AlphaLISA screening. Jiadfenoic acid B (6, 56.80% inhibition at 20 μM) and holophyllin F (10, 43.18% inhibition at 20 μM) significantly decreased PCSK9 protein levels in medium of HepG2 cells., Competing Interests: Declaration of Competing Interest The authors declare no conflicts of interest., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Seven new hexasaccharide resin glycosides, named calysepins I-VII ( 1 - 7 ), with 27-membered rings, were obtained from the aerial parts of Calystegia sepium . Their structures with absolute configuration were established on the basis of spectroscopic data interpretation analysis and the use of chemical methods. They were defined as hexasaccharides composed of one d-quinovose, four d-glucose, and one l-rhamnose unit, and their sugar moieties were partially acylated by (2 S ) - methylbutanoic acid in 1 - 7 and (2 R ,3 R )-nilic acid in 1 - 5 and 7 , which mainly differed at the positions of acylation. Additionally, calysepin IV ( 4 ) exhibited cytotoxicity against A549 cells with an IC 50 value of 5.2 μM.

A Gram-stain-negative, motile, rod-shaped, non-endospore-forming, aerobic and halophilic bacterium, designated strain YCWA18 T , was isolated from the sediment of Jimo-Daqiao saltern in China. This strain was able to grow at NaCl concentrations in the range 0.5-20 % (w/v) with optimum growth at 6 % (w/v) NaCl. Growth occurred at temperatures of 4-40 °C (optimum 28 °C) and pH 4.0-9.0 (optimum 7.0). Phylogenetic analysis based on 16S rRNA gene sequences showed that strain YCWA18 T belonged to the genus Kushneria and shared the highest sequence similarity of 98.7 % with Kushneria sinocarnis DSM 23229 T . Moreover, the phylogenetic analysis based on the 23S rRNA gene sequence also confirmed the phylogenetic position of this novel strain. The predominant fatty acids were C 16 : 0 , C 17 : 0 cyclo and C 12 : 0 3-OH. The major isoprenoid quinone was Q-9 (94.2 %) and the polar lipids were diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), an unidentified aminolipid (AL), an unidentified phospholipids (PL) and two unidentified lipids (L). The complete genome of strain YCWA18 T consisted of a single, circular chromosome of 3 624 619 bp, with an average G+C content of 59.1 mol%. A genome-based phylogenetic tree constructed using an up-to-date bacterial core gene set (UBCG) showed that strain YCWA18 T formed a clade with K. sinocarnis DSM 23229 T . However, the level of the ANI and dDDH values between strain YCWA18 T and K. sinocarnis DSM 23229 T were 82.3 and 24.6 %, respectively, which were low enough to distinguish strain YCWA18 T from K. sinocarnis DSM 23229 T . Overall, based on the phenotypic, chemotaxonomic, phylogenetic and genomic analyses, strain YCWA18 T represents a novel species of genus Kushneria . The name Kushneria phosphatilytica sp. nov. is proposed, with the type strain YCWA18 T (=CGMCC 1.9149 T =NCCB 100306 T ).

Objective: To explore whether a radiomics signature based on diffusion tensor imaging (DTI) can detect early kidney damage in diabetic patients., Materials and Methods: Twenty-eight healthy volunteers (group A) and thirty type 2 diabetic patients (group B) with micro-normoalbuminuria, a urinary albumin-to-creatinine ratio (ACR) < 30 mg/g and an estimated glomerular filtration rate (eGFR) of 60-120 mL/(min 1.73 m 2 ) were recruited. Kidney DTI was performed using 1.5T magnetic resonance imaging (MRI).The radiologist manually drew regions of interest (ROI) on the fractional anisotropy (FA) map of the right kidney ROI including the cortex and medulla. The texture features of the ROIs were extracted using MaZda software. The Fisher coefficient, mutual information (MI), and probability of classification error and average correlation coefficient (POE + ACC) methods were used to select the texture features. The most valuable texture features were further selected by the least absolute shrinkage and selection operator (LASSO) algorithm. A LASSO regression model based on the radiomics signature was established. The diagnostic performance of the model for detecting early diabetic kidney changes was evaluated by the area under the receiver operating characteristic (ROC) curve (AUC). Empower (R), R, and MedCalc15.8 software were used for statistical analysis RESULTS: A total of 279 texture features were extracted from ROI of the kidney, and 30 most valuable texture features were selected from groups A and B using MaZda software. After LASSO-logistic regression, a diagnostic model of diabetic kidney damage based on texture features was established. Model discrimination evaluation: AUC = 0.882 (0.770 ± 0.952). Model calibration evaluation: Hosmer-Lemeshow X2 = 5.3611, P = 0.7184, P > 0.05, the model has good calibration., Conclusion: The texture features based on DTI could play a promising role in detecting early diabetic kidney damage.

Salinivibrio proteolyticus strain YCSC6 was isolated from a saturated saltpan and demonstrated to have strong insecticidal activity against turbot's pathogenic ciliate-Uronema marinum. In this study, we sequenced its complete genome. Results showed that it consists of two circular chromosomes: 2.49 Mbps and 0.74 Mbps, respectively. It encodes 3429 protein-coding sequences. Biosynthetic gene clusters predicted to synthesize bacteriocins and antimicrobial peptides were discovered, which might be the key factors to lyse and kill U. marinum. The complete genome sequence of strain YCSC6 provides insights into the fundamental genetic potential for elucidating its insecticidal mechanism against U. marinum.

Rivers and reservoirs are affected by human activities and are sources of the greenhouse gas nitrous oxide (N 2 O). Concentrations of N 2 O in the middle and lower reaches of the Yellow River and Xiaolangdi Reservoir, China, were measured in June and December 2017. Fluxes were estimated by boundary layer method to explore their controlling factors, especially the impact of damming and reservoir operation. In the middle and lower reaches of the Yellow River, N 2 O concentrations in surface waters were 26.65 ± 14.67 nmol L -1 in summer and 21.16 ± 5.35 nmol L -1 in winter. In comparison, the concentrations of N 2 O in the reservoir were 32.94 ± 17.32 nmol L -1 in summer and 23.73 ± 5.60 nmol L -1 in winter. The longitudinal distribution of N 2 O along the river exhibited different patterns with surface N 2 O decreasing downstream towards the dam in summer but increasing in winter. Vertical profiles of N 2 O concentrations in the reservoir showed an increase with depth in summer but were almost vertically uniform in winter. In winter, N 2 O that had accumulated in the bottom water in summer was transported to the surface by vertical mixing and released into the atmosphere. Dissolved oxygen (DO), water temperature, and in situ biological production were the main factors affecting the distribution of N 2 O. The mean emissions rates of N 2 O from the surface waters were 13.7 ± 8.8 μmol m -2 d -1 in summer and 13.2 ± 7.6 μmol m -2 d -1 in winter. Approximately 1.31 × 10 6  mol N 2 O was released from the reservoir surface in 2017, which represents 0.12% of the annual N 2 O emissions from global reservoirs. The construction of dams increased N 2 O emission from the lower reaches of the river by 4.53 × 10 5  mol and 1.22 × 10 5  mol due to the discharge of the bottom water and the water and sediment regulation, respectively. This study demonstrates that the construction of dams and reservoir operation practices have made the Xiaolangdi Reservoir a key area for N 2 O emissions., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Using an optical fiber to both excite the nitrogen-vacancy (NV) center in diamond and collect its fluorescence is essential to build NV-based endoscope-type sensor. Such endoscope-type sensor can reach inaccessible fields for traditional NV-based sensors built by bulky optical components and extend the application areas. Since single NV's fluorescence is weak and can easily be buried in fluorescence from optical fiber core's oxide defects excited by the green laser, fixing a micrometer size diamond containing high-density NVs rather than a nanodiamond containing single NV or several NVs on the apex of an optical fiber to build an endoscope-type sensor is more implementable. Unfortunately, due to small numerical aperture (NA), most of the optical fibers have a low fluorescence collection efficiency, which limits the sensitivity and spatial resolution of the NV-based endoscope-type sensor. Here, using a tapered optical fiber (TOF) tip, we significantly improve the efficiency of the laser excitation and fluorescence collection of the NV ensembles in diamond. This could potentially enhance the sensitivity and spatial resolution of the NV-based endoscope-type sensor. Numerical calculations show that the TOF tip delivers a high NA and has a high NV excitation and fluorescence collection efficiency. Experiments demonstrate that such TOF tip can obtain up to over 7-fold excitation efficiency and over 15-fold fluorescence collection efficiency of that from a flat-ended fiber (non-TOF) tip.

In acidic media, cationic phthalocyanine Alcian blue 8GX, has an efficient fluorescence quenching effect on anionic phthalocyanine tetrasulphoaluminium phthalocyanines (AlS₄Pc), forming an almost non-fluorescent associate. Based on this discovery, a red-emitting fluorescent probe consisted of AlS₄P C and Alcian blue 8GX has been developed through molecular assembly. Further studies indicated that the presence of Hg(II) ion has a significant fluorescence recovery effect of the probe. Notably, only Hg(II) can significantly restore the fluorescence of AlS₄Pc-Alcian blue 8GX system which was revealed from the screening experiments of common metal ions, which confirmed that the fluorescence recovery by other metal ions is very weak or even unrestored, showing high specificity and sensitivity AlS₄Pc-Alcian blue 8GX to Hg(II). Thus, a new fluorimetry for Hg(II) with high specificity and high sensitivity in a wide concentration range has been established using AlS₄P c -Alcian blue 8GX associate as a red-emitting fluorescent probe. It is more noteworthy that this study opens a new way for development and application of functional phthalocyanine based red-emitting fluorescent probes., Competing Interests: The authors declare no conflict of interests.

The physical mechanism of the radiation generation of all possible output modes of the relativistic magnetron (RM) with all cavity-magnetron axial extraction technique is theoretically analysed, and the necessary conditions for generating these modes are obtained respectively. Assuming that n 0 is the number of the electron spokes, N ≥ 4 as the total number of the cavities is an even number, and k is a nonnegative integer, some conclusions can be drawn as follows. If n 0  = kN is true, no mode can be excited in the coaxial waveguide; if n 0  = (2k + 1)N/4 is true, the linearly polarized modes can be excited in the coaxial waveguide; if n 0  = (4k + 2)N/4 is true, the TEM mode and the linearly polarized modes can be excited in the coaxial waveguide; if n 0 takes other value, the left and right circularly polarized modes can be excited in the coaxial waveguide and the directions of rotation of the circularly polarized modes can be reversed with the reversion of the direction of rotation of the electron spokes; in addition, some other regular characteristics of the corresponding mode excitation are presented in detail in this paper. Such unique attractive properties that have been verified by the cold and hot simulations in this paper make it possible for this type of RM to meet application requirements of various high power microwave (HPM) modes.

Lack of satisfactory specificity towards tumor cells and poor intracellular delivery efficacy are the major drawbacks with conventional cancer chemotherapy. Conjugated anticancer drugs to targeting moieties e.g. to peptides with the ability to recognize cancer cells and to cell penetrating peptide can improve these characteristics, respectively. Combining a tumor homing peptide with an appropriate cell-penetrating peptide can enhance the tumor-selective internalization efficacy of the carrying cargo molecules. In the present study, the breast cancer homing ability of SP90 peptide and the synergistic effect of SP90 with a cell-penetrating peptide(C peptide) were evaluated. SP90 and chimeric peptide SP90-C specifically targeted cargo molecule into breast cancer cells, especially triple negative MDA-MB-231 cell, in a dose- and time-dependent manner, but not normal breast cells and other cancer cells, while C peptide alone had no cell-selectivity. SP90-C increased the intracellular delivery efficiency by 12-fold or 10-fold compared to SP90 or C peptide alone, respectively. SP90 and SP90-C conjugation increased the anti-proliferative and apoptosis-inducing activity of HIV-1 Vpr, a potential novel anticancer protein drug, to breast cancer cell but not normal breast cell by arresting cells in G2/M phase. With excellent breast cancer cell-selective penetrating efficacy, SP90-C appears as a promising candidate vector for targeted anti-cancer drug delivery. SP90-VPR-C is a potential novel breast cancer-targeted anticancer agent for its high anti-tumor activity and low toxicity., (Copyright © 2016 Elsevier Masson SAS. All rights reserved.)

The effect of hyperoxygenation with carbogen (95% O 2 + 5% CO 2 ) and 100% oxygen inhalation on partial pressure of oxygen (pO 2 ) of radiation-induced fibrosarcoma (RIF-1) tumor was investigated. RIF-1 tumors were innoculated in C3H mice, and aggregates of oximetry probe, lithium phthalocyanine (LiPc), was implanted in each tumor. A baseline tumor pO 2 was measured by electron paramagnetic resonance (EPR) oximetry for 20 minutes in anesthetized mice breathing 30% O 2 and then the gas was switched to carbogen or 100 % oxygen for 60 minutes. These experiments were repeated for 10 days. RIF-1 tumors were hypoxic with a baseline tissue pO 2 of 6.2-8.3 mmHg in mice breathing 30% O 2 . Carbogen and 100% oxygen significantly increased tumor pO 2 on days 1 to 5, with a maximal increase at approximately 32-45 minutes on each day. However, the extent of increase in pO 2 from the baseline declined significantly on day 5 and day 10. The results provide quantitative information on the effect of hyperoxic gas inhalation on tumor pO 2 over the course of 10 days. EPR oximetry can be effectively used to repeatedly monitor tumor pO 2 and test hyperoxic methods for potential clinical applications., Competing Interests: The authors declare no conflicts of interest.

A novel nanoparticle with multilayer core-shell architecture for cell imaging is designed and synthesized by coating a fluorescent YVO4:Eu core with a diblock copolymer, MPEG-b-PMAA. The synthesis of YVO4:Eu core, which further makes MPEG-b-PMAA-YVO4:Eu NPs adapt for cell imaging, is guided by the model determined upon the evaluation of pH and CEu%. The PMAA block attached tightly on the YVO4:Eu core forms the inner shell and the MPEG block forms the biocompatible outermost shell. Factors including reaction time, reaction temperature, CEu% and pH are optimized for the preparation of the YVO4:Eu NPs. A precise defined model is established according to analyzing the coefficients of pH and CEu% during the synthesis. The MPEG-b-PMAA-YVO4:Eu NPs, with an average diameter of 24 nm, have a tetragonal structure and demonstrate luminescence in the red region, which lies in a biological window (optical imaging). Significant enhancement in luminescence intensity by MPEG-b-PMAA-YVO4:Eu NPs formation is observed. The capping copolymer MPEG-b-PMAA improves the dispersibility of hydrophobic YVO4:Eu NPs in water, making the NPs stable under different conditions. In addition, the biocompatibility MPEG layer reduces the cytotoxicity of the nanoparticles effectively. 95% cell viability can be achieved at the NPs concentration of 800 mgL(-1) after 24h of culture. Cellular uptake of the MPEG-b-PMAA-YVO4:Eu NPs is evaluated by cell imaging assay, indicating that the NPs can be taken up rapidly and largely by cancerous or non-cancerous cells through an endocytosis mechanism., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Interest in carbon nanotubes for detecting the presence of pathogens arises because of developments in chemical vapor deposition synthesis and progresses in biomolecular modification. Here we reported the facile synthesis of multi-walled carbon nanotubes (MWCNTs), which functioned as immuno-, magnetic, fluorescent sensors in detecting Vibrio alginolyticus (Va). The structures and properties of functionalized MWCNTs were characterized by ultraviolet (UV), Fourier transform infrared spectra (FT-IR), X-ray diffraction (XRD), vibrating sample magnetometer (VSM), magnetic property measurement system (MPMS) and fluorescent spectra (FL). It was found that the functionalized MWCNTs showed: (1) low nonspecific adsorption for antibody-antigen, (2) strong interaction with antibody, and (3) high immune-magnetic activity for pathogenic cells. Further investigations revealed a strong positive linear relationship (R=0.9912) between the fluorescence intensity and the concentration of Va in the range of 9.0 × 10(2) to 1.5 × 10(6) cfum L(-1). Moreover, the relative standard deviation for 11 replicate detections of 1.0 × 10(4) cfum L(-1) Va was 2.4%, and no cross-reaction with the other four strains was found, indicating a good specificity for Va detection. These results demonstrated the remarkable advantages of the multifunctional MWCNTs, which offer great potential for the rapid, sensitive and quantitative detection of Va in fishery and environmental samples., (Copyright © 2014 Elsevier B.V. All rights reserved.)

In the title mol-ecule, C(13)H(10)N(4), the dihedral angle between the triazole ring and the naphthalene ring system is is 56.1 (2)°. In the crystal, mol-ecules are connected by weak C-H⋯N hydrogen bonds into chains along [100]. A short intra-molecular C-H⋯N contact is also observed.

The asymmetric unit of the title compound, C(10)H(8)N(6)·2H(2)O, comprises half the organic species, the mol-ecule being completed by inversion symmetry, and one water mol-ecule. The dihedral angle between the 1,2,4-triazole ring and the central benzene ring is 32.2 (2)°. The water mol-ecules form O-H⋯N hydrogen bonds with N-atom acceptors of the triazole rings. C-H⋯N hydrogen bonds are also observed, giving a three-dimensional framework.

In the title compound, [Fe(2)(NCS)(4)(H(2)O)(6)]·3C(10)H(8)N(6), the centrosymmetric dinuclear complex contains two Fe(II) ions bridged by two aqua ligand O atoms, forming a four-membered ring. The slightly distorted octa-hedral coordination environment of the two Fe(II) ions is completed by two monodentate aqua ligands and two thio-cyanate ligands. One of the 1,4-bis-(4H-1,2,4-triazol-4-yl)benzene mol-ecules lies across an inversion center. In the crystal, O-H⋯N hydrogen bonds connect the components, forming a two-dimensional network parallel to (011). In addition, π-π stacking inter-actions involving the benzene and triazole rings, with centroid-centroid distances in the range 3.502 (5)-3.787 (6) Å, connect the two-dimensional hydrogen-bonded network into a three-dimensional network.

Time efficient measurement of the spectroscopic magneto-optical (MO) activity of materials has always been desirable. In conventional MO systems, the monochromator produces quasi-monochromatic light in a narrow wavelength window. Therefore, to measure the spectroscopic MO activity, a large number of measurements over the full spectra is required to obtain satisfactory wavelength resolution and thus is very time consuming. Here, we develop a novel system that is capable of fast measurement of the MO activity by only one white light source, two polarizers, one achromatic quarter-wave plate, and one spectrometer. This system is flexible from UV to IR region, only depending on the power spectra of light source and sensitivity of the detector at the corresponding wavelengths. As examples, we measured the intriguing optic and MO activity in glass, ferromagnetic thin film, and bulk GaAs in the visible to near infrared region. The results of glass demonstrated a minimum resolvable Faraday rotation angle of 0.004° by the currently equipped system.

Aim: To construct phage antibody library with predetermined CDR3 and to screen humanized Fab of anti-human integrin alphanubeta(3) monoclonal antibody (mAb) by epitope guided selection., Methods: LCDR3 gene of mAb E10 was inserted into human light chain variable region gene library. Hybrid phage antibody library was constructed by cloning E10 chimeric Fd gene and human light chain variable region gene into pComb3. Humanized light chain gene was obtained by screening against human integrin alphanubeta(3). Likewise, humanized Fab were gained by panning human phage antibody library, which was constructed by cloning humanized light chain gene and human heavy chain Fd gene with E10 HCDR3 into pComb3., Results: Three humanized Fab clones was obtained by screening hybrid phage antibody library and human phage antibody library, which contained 2.1x10(6), 2x10(7) colony forming units, respectively. Indirect ELISA and competitive inhibition ELISA analysis demonstrated that three humanized Fab antibody had specific binding activity with human integrin alphanubeta(3). The strongest anti-human integrin alphanubeta(3) reactive D5 clone was sequenced and sequencing analysis showed that the V(kappa) and V(H) were derived from VKIII and VHI, respectively., Conclusion: Humanized Fab of anti-human integrin alphanubeta(3) mAb has been successfully obtained by phage display technology which lays the foundation for further clinical research.

Aim: To construct single chain antibody (scFv) gene of mAb E10 against human integrin alphavbeta3., Methods: The VH and VL genes were amplified from hybridoma cells secreting mAb E10 by RT-PCR and connected with the use of linker (Gly4Ser)3 to assemble scFv gene. The scFv gene was cloned into prokaryotic expression vector pTIG-TRX and expressed in E. coli BL21 (DE3)., Results: SDS-PAGE analysis showed the expressed recombinant protein with relative molecular mass (Mr) being 31,000. Western blot confirmed that the protein was labeled with His6. scFv protein was expressed as soluble protein under the condition of a small amount of IPTG induction and culture at lower temperature. The purity of the protein purified through Ni-NTA agarose metal affinity resin column was over 91%. The purified protein could bind to the human integrin alphavbeta3 by ELISA confirmation., Conclusion: scFv against human integrin alphavbeta3 has been successfully constructed and expressed,which lays the foundation for further clinical research.

Aim: To produce the recombinant NS3 protease of hepatitis C virus with enzymatic activity in insect cells., Methods: The gene of HCV serine proteinase domain which encodes 181 amino acids was inserted into pFastBacHTc and the recombinant plasmid pFBCNS3N was transformed into DH10Bac competent cells for transposition. After the recombinant bacmids had been determined to be correct by both blue-white colonies and PCR analysis, the isolated bacmid DNAs were transfected into Sf9 insect cells. The bacmids DNA was verified to replicate in insect cells and packaged into baculovirus particles via PCR and electronic microscopic analysis. The insect cells infected with recombinant baculovirus were determined by SDS-PAGE and Western-blot assays. The recombinant protein was soluted in N-lauryl sarcosine sodium (NLS) and purified by metal-chelated-affinity chromatography, then the antigenicity of recombinant protease was determined by enzyme-linked immunoabsorbant assay and its enzymatic activity was detected., Results: The HCV NS3 protease domain was expressed in insect cells at high level and it was partially solved in NLS. Totally 0.2 mg recombinant serine proteinase domain with high purity was obtained by metal-chelated-affinity chromatography from 5 x 10(7) cells, and both antigenicity and specificity of the protein were evaluated to be high when used as antigen to detect hepatitis C patients' sera in indirect ELISA format. In vitro cleavage assay corroborated its enzymatic activity., Conclusion: The recombinant HCV NS3 proteinase expressed by insect cells is a membrane-binding protein with good antigenicity and enzymatic activity.

Objectives: To construct an eucaryotic expression plasmid carrying the BMP7 gene and express in MSCs., Methods: The BMP7 gene was cloned into the eucaryotic expression vector pcDNA3.1. At the same time, mesenchymal stem cells (MSCs) were isolated and cultured in vitro. The plasmid carrying the BMP7 gene was transfected into MSCs., Results: PCR and digesting demonstrated that the eucaryotic expression plasmid -pcDNA-BMP7 was obtained. RT-PCR and immunohistochemical methods showed that the BMP7 gene was expressed in MSCs., Conclusion: Construction of an eucaryotic expression plasmid carrying BMP7 gene and expression in MSCs provide a sound basis for gene therapy using the BMP7 gene and the ideal seeds for tissue engineering.

Aim: To establish a simple and convenient assay in vitro for the Hepatitis C virus NS3 serine protease based on the recombinant protease and substrate, and to evaluate its feasibility in screening the enzyme inhibitors., Methods: Based on the crystallographic structure of hepatitis C virus (HCV) serine protease, a novel single-chain serine protease was designed, in which the central sequence of cofactor NS4A was linked to the N-terminus of NS3 serine protease domain via a flexible linker GSGS. The fusion gene was obtained by two-step PCR that was carried out with three primers and then cloned into the prokaryotic expression vector pQE30, and the recombinant clone was verified by DNA sequencing. The single-chain recombinant protease was expressed when the E.coli was induced with IPTG and the expression conditions were optimized to produce large amount of soluble protease. The recombinant substrate NS5ab that covers the cleavage point NS5A/B was also expressed in E.coli. Both of the protease and substrate were purified by using Ni-NTA agarose metal affinity resin, then they were mixed together in a specific buffer, and the mixture was analyzed by SDS-PAGE. The cleavage system was used to evaluate some compounds for their inhibitory activity on serine protease., Results: The single-chain recombinant protease was over-expressed as soluble protein when the E.coli was induced at a low dosage of IPTG (0.2 mM) and cultured at a low temperature (15 degrees ). The protease was purified by using Ni-NTA agarose metal affinity resin (the purity is over 95 %). The recombinant substrate NS5ab was expressed in an insoluble form and could refold successfully after purification and dialysis. A simple and convenient assay in vitro was established, in which the purified single-chain serine protease could cleave the recombinant substrate NS5ab into two fragments that were visualized by SDS-PAGE. PMSF had an effect on inhibiting activity of serine protease, while EDTA had not., Conclusion: A simple and convenient assay in vitro for hepatitis C virus NS3 serine protease is based on recombinant substrate NS5ab and single-chain serine protease. This assay can be used in screening of enzyme inhibitors.