Effects of osthole on osteoporotic rats: a systematic review and meta-analysis.

CONTEXT: Cnidium monnieri Cusson (Apiaceae) has been used in traditional Asian medicine for thousands of years. Recent studies showed its active compound, osthole, had a good effect on osteoporosis. But there was no comprehensive analysis. OBJECTIVE: This meta-analysis evaluates the effects of osthole on osteoporotic rats and provides a basis for future clinical studies. METHODS: Chinese and English language databases (e.g., PubMed, Web of Science, Cochrane Library, Google Scholar, Embase, China National Knowledge Infrastructure, Wanfang Data Knowledge Service Platform, Weipu Chinese Sci-tech periodical full-text database, and Chinese BioMedical Literature Database) were searched from their establishment to February 2021. The effects of osthole on bone mineral density, osteoclast proliferation, and bone metabolism markers were compared with the effects of control treatments. RESULTS: To our knowledge, this is the first meta-analysis to evaluate osthole for the treatment of osteoporosis in rats. We included 13 randomized controlled studies conducted on osteoporotic rats. Osthole increased bone mineral density (standardized mean difference [SMD] = 3.08, 95% confidence interval [CI] = 2.08-4.09), the subgroup analysis showed that BMD significantly increased among rats in osthole <10 mg/kg/day and duration of osthole treatment >2 months. Osthole improved histomorphometric parameters and biomechanical parameters, also inhibited osteoclast proliferation and bone metabolism. CONCLUSIONS: Osthole is an effective treatment for osteoporosis. It can promote bone formation and inhibit bone absorption.

The protective effect of recombinant Lactococcus lactis oral vaccine on a Clostridium difficile-infected animal model.

BACKGROUND: Oral immunization with vaccines may be an effective strategy for prevention of Clostridium difficile infection (CDI). However, application of previously developed vaccines for preventing CDI has been limited due to various reasons. Here, we developed a recombinant Lactococcus lactis oral vaccine and evaluated its effect on a C. difficile-infected animal model established in golden hamsters in attempt to provide an alternative strategy for CDI prevention. METHODS: Recombinant L. lactis vaccine was developed using the pTRKH2 plasmid, a high-copy-number Escherichia coli-L. shuttle vector: 1) L. lactis expressing secreted proteins was constructed with recombinant pTRKH2 (secreted-protein plasmid) carrying the Usp45 signal peptide (SPUsp45), nontoxic adjuvanted tetanus toxin fragment C (TETC), and 14 of the 38 C-terminal repeats (14CDTA) of nontoxic C. difficile toxin A (TcdA); and 2) L. lactis expressing secreted and membrane proteins was constructed with recombinant pTRKH2 (membrane-anchored plasmid) carrying SPUsp45, TETC, 14CDTA, and the cell wall-anchored sequence of protein M6 (cwaM6). Then, 32 male Syrian golden hamsters were randomly divided into 4 groups (n = 8 each) for gavage of normal saline (blank control) and L. lactis carrying the empty shuttle vector, secreted-protein plasmid, and membrane-anchored plasmid, respectively. After 1-week gavage of clindamycin, the animals were administered with C. difficile spore suspension. General symptoms and intestinal pathological changes of the animals were examined by naked eye and microscopy, respectively. Protein levels of anti-TcdA IgG/IgA antibodies in intestinal tissue and fluid were analyzed by enzyme-linked immunosorbent assay (ELISA). A cell culture cytotoxicity neutralization assay was done by TcdA treatment with or without anti-TcdA serum pre-incubation or treatment. Apoptosis of intestinal epithelial cells was examined by flow cytometry (FL) assay. Expression of mucosal inflammatory cytokines in the animals was detected by polymer chain reaction (PCR) assay. RESULTS: After the C. difficile challenge, the animals of control group had severe diarrhea symptoms on day 1 and all died on day 4, indicating that the CDI animal model was established in hamster. Of the 3 immunization groups, secreted-protein and membrane-anchored plasmid groups had significantly lower mortalities, body weight decreases, and pathological scores, with higher survival rate/time than the empty plasmid group (P < 0.05). The tilter of IgG antibody directed against TcdA was significantly higher in serum and intestinal fluid of secreted-protein and membrane-anchored plasmid groups than in the empty plasmid group (P < 0.05) while the corresponding titer of IgA antibody directed against TcdA had no substantial differences (P > 0.05). The anti-TcdA serum of membrane-anchored plasmid group neutralized the cytotoxicity of 200 ng/ml TcdA with the best protective effect achieved by anti-TcdA serum pre-incubation. The incidences of TcdA-induced death and apoptosis of intestinal epithelial cells were significantly reduced by cell pre-incubation or treatment with anti-TcdA serum of membrane-anchored plasmid group (P < 0.05). MCP-1, ICAM-1, IL-6, and Gro-1 mRNA expression levels were the lowest in cecum tissue of the membrane-anchored groups compared to the other groups. CONCLUSION: Recombinant L. lactis live vaccine is effective for preventing CDI in the hamster model, thus providing an alternative for immunization of C. difficile-associated diseases.

[Effects of epigallocatechin-3-gallate on the proliferation of colonic cancer cell line SW620 and PAK1 gene expression].

OBJECTIVE: To investigate the effects of epigallocatechin-3-gallate (EGCG) on the proliferation of SW620 cells and the expression of PAK1 gene. METHODS: Human colonic cancer cell line SW620 was treated with EGCG at 40, 60 and 80 micromol/L and cultured in RPMI 1640 medium for 0, 24, 48 and 72 h. The proliferation of SW620 cells was observed by MTT assay before and after EGCG treatment, and the expression of PAK1 protein was observed by Western blotting. RESULTS: SW620 cells treated with EGCG displayed a slowed growth in comparison with the control cells, and the growth rate decreased with the increase of EGCG concentration. PAK1 protein expression was lowered in SW620 cells after EGCG treatment for 48 h. CONCLUSION: EGCG can inhibit the proliferation and partially reduce the expression of PAK1 protein in SW620 cells.

[Construction and identification of recombinant Lactococcus lactis highly expressing human granulocyte-macrophage colony stimulating factor].

OBJECTIVE: To transfer human granulocyte-macrophage colony stimulating factor (hGM-CSF) gene into Actococcus lactis and obtain recombinant Lactococcus lactis highly expressing hGM-CSF (LL-CSF). METHODS: The optimized hGM-CSF gene sequence capable of expression in Lactococcus lactis was cloned into the vector pNBC1000, which contained P59 promoter, RBS, MCS, USP45 signal peptide and USP45 stop codon, to generate the recombinant plasmid pNCSF. pNCSF was subcloned into a shuttle vector pTR1001c to acquire the plasmid pTRCSF, which was transferred into Lactococcus lactis to obtain LL-CSF by means of electroporation. SDS-PAGE was used to verify the expression of hGM-CSF protein by the constructed LL-CSF. RESULTS: DNA sequencing and restriction enzyme digestion indicated the successful construction of the recombinant plasmid pNCSF, pTRCSF and the recombinant bacterium LL-CSF that was capable of steady and efficient expression of hGM-CSF as shown by SDS-PAGE. CONCLUSION: The recombinant Lactococcus lactis LL-CSF has been successfully constructed, which can be valuable for studying the biological activity of recombinant hGM-CSF and for evaluating the potential clinical application of the protein.

[PCR-based assembly of the DNA sequence coding for tetanus toxin C fragment].

OBJECTIVE: To develop a PCR-based method for gene assembly of tetanus toxin C fragment (TETC) DNA sequence from a large number of oligodeoxyribonucleotides (oligos). METHODS: To allow for its cloning and expression in Lactococcus lactis, the TETC gene sequence was designed according to the known TETC gene sequence (GenBank accession number M12739, 367-1719) and the amino acid coding in Lactococcus lactis. The sequence contained 1383 nucleotides (nt) with Sal I site added to its 5' end and Xho I and Hind III sites to its 3' end. There were 209 synonymous codon substitutions in the designed gene sequence as compared with the sequence reported in GenBank for amino acid coding in Lactococcus lactis and elimination of the restriction site of EcoR I and Kpn I. The 1380 nt of the sequence was divided into 68 oligos designated as TETC 1 to TETC 68, each containing 40 nt. A 16 nt oligos designated as TETC 69 was designed as the downstream primer. The TETC 1-24 fragment was acquired using the oligos TETC 1 to TETC 24 by PCR-based gene assembly method, and the TETC 23-46 and TETC45-68 fragments were assembled similarly. The full-length TETC gene was assembled using TETC 1 and TETC 69 as the primers when the 3 fragments were mixed. The target gene was gel-purified and digested with Sal I and Hind III, followed by ligation to the pBluescript II SK(+) and digestion with the same enzymes. The positive clones were confirmed by restriction enzyme excision and sequencing. RESULTS: Three 500-bp fragments were acquired by PCR-based gene assembly, and the full-length TETC gene was obtained from the 3 fragment mixed at a equal concentration by a second PCR-based gene assembly using TETC 1 and TETC 69 as the primers. The target gene was cloned to pBluescript II SK(+) vector, and sequence analysis of the positive clones indicated that the assembled sequence was identical to the designed coding sequence of TETC gene. CONCLUSION: PCR-based assembly of the synthesized constitutive gene fragments into the complete sequence can be an effective strategy for synthesis of long DNA sequences in vitro.

[Study on heavy metals in soils contaminated by acid mine drainage from Dabaoshan mine, Guangdong].

Mining activities in the Dabaoshan area in the upper reach of the Hengshihe River have caused severe environmental changes, the waste water of milling and refining drained directly into the Hengshihe River, which contaminated the soils along the river severely. It is shown that Pb, Zn, Cd and Cu have contaminated the soil, the Cd contamination was more severe, and the contaminated level of Pb, Zn reached moderately to strongly polluted. The pH value of river and soil affected directly the heavy metals concentration of total and exchangeable ions, and presented negative pertinences. The levels of Pb, Zn, Cu and Cd in the surface soil of Shangbacun village in the lower reach of the river were found as high as 257.762, 350.235, 5.083 and 186.901 mg x kg(-1) respectively, which were relatively higher than those of the background values of soil 1.03, 1.75, 16.9 and 3.7 times respectively, and the result on the soil profiles showed that the contaminations have infiltrated into lower layer soil, ecological environment was harmed severely.

FG020326-loaded nanoparticle with PEG and PDLLA improved pharmacodynamics of reversing multidrug resistance in vitro and in vivo.

AIM: FG020326, a novel imidazole derivative, is a potent multidrug-resistance (MDR) modulator in vitro and in vivo. However, FG020326 is insoluble. PEDLLA-FG020326 is a FG020326-loaded nanoparticle formed with diblock copolymers of poly (ethylene glycol)-block-poly (D,L-lactic acid) (PEG:PDLLA, PEDLLA) that can solubilize FG020326. This work was intended to evaluate the pharmacodynamics of PEDLLA-FG020326 on reversing MDR in vitro and in vivo. METHODS: Cytotoxicity was determined by tetrazolium assay. The intracellular accumulation and efflux of doxorubicin (Dox) were detected by fluorescence spectrophotometry. The function of P-glycoprotein was examined by Rhodamine 123 (Rh123) accumulation detected by flow cytometry. The KBv200 cell xenograft model was established to investigate the effect of PEDLLA-FG020326 on reversing MDR in vivo. RESULTS: PEDLLA-FG020326 and FG020326 exhibited 56.4- and 35.9-fold activity in reversing KBv200 cells to vincristine (VCR) resistance, respectively and 14.98- and 7.64-fold to Dox resistance, respectively. PEDLLA-FG020326 was much stronger than FG020326, resulting in the increase of Dox and Rh123 accumulation and the decrease of intracellular Dox extrusion in KBv200 cells. Importantly, PEDLLA-FG020326 exhibited more powerful activity than FG020326 in enhancing the effect of VCR against KBv200 cell xenografts in nude mice, but did not appear more toxic. CONCLUSION: The pharmacodynamics of FG020326 was improved by incorporating it into a micellar nanoparticle formed with PEG-block-PDLLA copolymers.

[Construction of Lactococcus lactis expression vector of recombinant human trefoil factor family 2].

OBJECTIVE: To construct a Lactococcus lactis expression vector of c-myc-tagged human trefoil factor family 2 (hTFF2) fusion gene to prepare for genetic modification of Lactococcus lactis that can secrete bioactive c-myc-hTFF2 protein. METHODS: Based on the amino sequence of hTFF2 and optimal Lactococcus lactis codon usage, the cDNA of hTFF2 was designed and extended at their 5' ends with a sequence encoding c-myc as the molecular tag. According to the restriction sites of pBluescript II sk (+), the SalI and BamHI sites were arranged at the 5' and 3' ends of the fusion gene respectively. The sequence of the fusion gene c-myc-hTFF2 was designed as 14 oligonucleotides that overlapped with each other, and by means of PCR, all the oligonucleotides were spliced to complete the construction of c-myc-hTFF2 fusion gene. The target gene of c-myc-hTFF2 was inserted into pBluescript II sk (+) to construct the cloning vector pBS-hTFF2 of c-myc-hTFF2 followed by verification by enzyme digestion and DNA sequencing. By digestion of pBS-TFF2 with BamHI/SalI and of pNBC1000 with BamHI/XhoI, we connected c-myc-hTFF2 with pNBC1000 to construct the expression vector c-myc-hTFF2 in E. coli named as pNTFF2. After digestion of pNTFF2 and pTRKH2 with XbaI, the target gene was subcloned into pTRKH2 and the construction of the expression vector pTRTFF2 in Lactococcus lactis was completed. The constructed vector was identified by restriction enzyme digestion. RESULTS AND CONCLUSION: The expression vector pTRTFF2 of c-myc-hTFF2 fusion gene has been successfully constructed. Assembly of oligonucleotides in vitro is an effective means to synthesize the target fusion gene and this prepares the ground for constructing engineered bacterium of Lactococcus lactis.

[Construction and identification of exogenous gene expression system in Lactococcus lactis].

OBJECTIVE: To construct and characterize an exogenous gene expression system in Lactococcus lactis. METHODS: PCR-based gene assembly was used to synthesize the gene sequence containing P59 promoter, USP45 signal peptide, ribosome binding site and multiple cloning site. Plasmid pBS-pu was obtained after ligation of the assembled sequence with pBluescript II SK (+), and the terminator of USP45 protein was added to construct the recombinant plasmid pNBC1000. The gene coding for Streptococcus pyogenes M6 protein was amplified and cloned into pNBC1000 to obtain the plasmid pNBC2000. To characterize the expression system, enhanced green fluorescent protein (EGFP) gene was amplified from the plasmid pEGFP-N1 and cloned into pNBC2000. Laser scanning confocal microscope was used to observe the bacteria containing pNBC2000, pNBC2000-EGFP or pBS-EGFP. RESULTS: Green fluorescence was visualized in the bacteria containing pNBC2000-EGFP, but not in the bacteria containing pNBC2000 or pBS-EGFP. CONCLUSION: The plasmids pNBC1000 and pNBC2000 containing P59 promoter, USP45 signal peptide, ribosome binding site and multiple cloning sites are successfully constructed, which are capable of expressing exogenous gene in Lactococcus lactis.

[Method for segmenting medical image based on anatomical model].

OBJECTIVE: To improve the automatization and reliability of medical image segmentation. METHOD: An anatomical model was built and used to guide the low-level segmentation process. The system architecture was made up of an anatomical model, image processing routines and an inference engine, the interaction of which are governed by a blackboard. RESULT: The result of application of the segmentation for chest CT image was satisfactory and needs less operator intensive. CONCLUSION: This method improves automatization and reliability of the medical image segmentation. Because of the good expansibility, it may serve as a template for knowledge-based processing of medical image.

[Construction and identification of the cDNA phage expression library for human colorectal cancer antigens].

OBJECTIVE: To construct a cDNA phage expression library for human colorectal carcinoma antigens. METHODS: After the total RNA was extracted from human colorectal cancer tissues, the single-strand and double-strand cDNA were synthesized through reverse transcriptase PCR and long-distance PCR, with the cDNA fragments smaller than 500 bp removed and the remaining cDNA combined with the right and left arms of dephosphorylated lambdaTriplEx2 phage vector. The recombinant phage were then packaged in vitro by MaxPlax Packaging extract, and a small portion of the packaged phage was used to infect E.coli XL1-Blue. Titer measurement was performed so as to determine the capacity of the library. SfiI restriction endonucleases was used to cut the recombined phage DNA in order to identify the size of inserted cDNA. RESULTS: The constructed cDNA phage expression library for human colorectal cancer antigens consisted of 2.39 x 10(6) pfu/ml bacteriophages with a recombination rate of 97.5% and the length of the inserted cDNA fragment ranged from 600 to 4,000 bp with an average of 1,400 bp. CONCLUSION: The cDNA phage expression library of human colorectal cancer antigens is successfully constructed to meet the currently recognized standards, and can be well applicable in screening cDNA-cloned genes of human colorectal cancer-associated antigens by immunoscreening.

[PCR cloning of 3'-terminal repeat fragments of Clostridium difficile toxin A and toxin B gene].

OBJECTIVE: To explore a method to rapidly detect the presence of Clostridium difficile. METHODS: PCR was used to amplify DNA fragments from the highly conserved and repeated domains of the 3'-terminal of Clostridium difficile toxin A gene and toxin B genes. RESULTS: The fragments 960 bp and 1,851 bp in length were respectively amplified from toxin A and toxin B, while the control bacteria presented no distinct results. CONCLUSION: The 960 bp and 1,851 bp fragments are specific for Clostridium difficile and PCR can be used to detect Clostridium difficile from the stool. As the peptides encoded by these conserved domains have high hydrophobicity and strong antigenicity, manufacture of the protein vaccine by gene engineering is possible on the basis of these conserved domains.