Objective To clone a stage-specific novel cDNA from 5 day-old adult worm (ADS) of Trichinella spiralis. Methods The cDNA library of AD5 was screened by an AD5 stage-specific cDNA probe labeled with digoxigenin (DIG). The positive clones were sequenced and analysed. Results The positive clone contained a cDNA insert of 1 132 bp in length with a full length open reading frame (ORF) of 1 032 bp. The cDNA encoded a polypeptide of 343 amino acid residues(aa) with a molecular weight of 35.1 kDa and an isoelectric point (IP) of 4.8. InterProScan analysis showed that the 117 - 120 aa (SGYG) was a glycosaminoglycan attachment site, 27- 86 aa was nematode cuticle collagen N-terminal domain and 153-228 aa was collagen repeat (G-x-y) domain. Signal PV2.0 analysis indicated that the region of 1-43 aa was a singal peptide. Blastn homology analysis in Genbank revealed that the cDNA had no obvious homology to any other known gene sequence. Blastp analysis revealed high homology to cuticle collagen with identities more than 40 % . Conclusion A novel ADS stage-specific cDNA encoding a full length ORF was cloned and sequence analysis showed this gene encoded cuticle collagen of Trichinella spiralis.

Objective:To observe influence of statins on antiplatelet activity of clopidogrel and provide basis for ra‐tionality of statins combined clopidogrel treatment .Methods :According to random number table ,a total of 90 pa‐tients diagnosed as acute coronary syndrome were equally divided into clopidogrel group ,clopidogrel + simvastatin group and clopidogrel + pravastatin group . Three groups received corresponding routine medication treatment . Plasma levels of platelet αgranule membrane protein (CD62P) ,lysosomal granule membrane glycoprotein (CD63) and maximum platelet aggregation rate (MPAR) were measured and compared among three groups before and 3d af‐ter treatment .Results:Compared with before treatment ,after treatment ,there were significant reductions in plas‐ma levels of CD62P and CD63 and MPAR in three groups , P<0.01 all .After treatment ,there were no significant difference in plasma levels of CD62P [ (14.63 ± 3.45) ng/ml vs .(14.14 ± 4.32) ng/ml vs .(14.59 ± 4.23) ng/ml] , CD63 [ (26.32 ± 10.43) ng/ml vs .(27.04 ± 10.75) ng/ml vs .(27.29 ± 9.27) ng/ml] and MPAR [ (28.62 ± 17.68)% vs .(28.38 ± 16.43)% vs .(29.13 ± 14.23)% ] among clopidogrel group ,clopidogrel + simvastatin group and clopidogrel + pravastatin group ,P>0.05 all .Conclusion:Short‐term and routine dose of statins combined clo‐pidogrel is feasible in treatment of acute coronary syndrome .The combined use of them will not affect antiplatelet function of clopidogrel .

BACKGROUND: Emerging studies have focused on cell transplantation. Schwann cells (SCs) can secrete various neurotrophic factors and improve local environment around injury. Plenty of documents have demonstrated that SCs could promote functional recovery following spinal injury. Many transplanting methods are available for treating spinal cord injury, and the intravenous cell transplantation is profitable for easy operation and avoidance of additional trauma. OBJECTIVE: To investigate the effects of intravenous transplantation of SCs on spinal cord injury in rats. METHODS: The bilateral sciatic nerves of Wistar rats were separated in vitro, cultured by tissue clot method, identified by S-100 and labeled by Hoechst33342. Sixty rat models with T10 spinal cord injury were prepared using impactor model- II type weight drop apparatus. Then the injured rats were randomly divided into 3 groups: blank control, DMEM control and SCs transplantation groups. No treatment was performed in the blank control group. Totally 1 mL DMEM and or SCs was injected into rats of DMEM control and SCs transplantation groups by tail vein respectively. Basso Beattie Bresnahan (B6B) scores were performed at 1 day before and 1, 3 days, 1 week and weekly after operation. The migration of transplanted SCs was observed at 2 weeks and 4 after transplantation. The expressions of glial fibrillary acidic protein (GFAP) and neuron specific enolase (NSE) were detected by haematoxylin-eosin staining and immune-fluorescence staining.RESULTS AND CONCLUSION: The purity of SCs reached 95%. Hoechst33342 positive cells were observed throughout the injured and the nearby region of spinal cord at 1, 2, and 4 weeks after transplantation. The statistical difference of BBB score among the SCs transplantation, blank control, and the DMEM control groups displayed at 4 weeks after transplantation (P < 0.05), and the BBB scores of the SCs transplantation were higher than other groups. Haematoxylin-eosin staining showed the cavity formed in each group at 8 weeks after transplantation, but the area of SCs transplantation was smaller than that of the blank control and DMEM control groups. The immunofluorescence staining indicated that the expression of GFAP were more intense in the blank control group and DMEM control than SCs transplantation (P < 0.05), while the expression of NSE was more intense in SCs transplantation than other groups (P< 0.05). It implied that intravenous transplantation of SCs promotes regeneration of axon and improves neurological functions after spinal cord injury in rats.

BACKGROUND:Schwann cells can secrete various neurotrophic factors,and promote functional recovery of injured spinal cord.However,xenogenic Schwann cells transplantation may induce autoimmune response.Moreover,local transplantation results in secondary injury.Vein transplantation may reached injury site passing the blood spinal cord barrier,but the treatment concentration is not effective.OBJECTIVE:To investigate the therapeutic effects of transplantation of autologous activated Schwann Cells(AASCs)via subarachnoid space on spinal cord injury(SCI)in rats.METHODS:A total of 66 rats were used to establish SCI models,and the model rats were randomly divided into 3 groups.The unilateral saphenous nerves of rats were ligated directly for 1 week to activate Schwann cells,but inactivated and model control groups were not subjected to nerve ligation.1 cm nerve was excised from distal end of each group,and Schwann cells were isolated and cultured by tissue mass method.The AASCs,autologous Schwann cells(ASCs)were injected with corresponding Hoechst33342-labeted SCs suspension,but the model control group was injected with DMEM injection.The basso beattie bresnahan(BBB)score and footprint analysis,as well as HE and GFAP immunohistochemistry staining were performed to evaluate functional recovery of rat hind limbs.RESULTS AND CONCLUSION:On 4 weeks after injury,BBB scores of AASCs were significantly superior to the other groups (P＜0.05).Two weeks after transplantation,some SCs migrated to injured spinal cord.Compared with ASCs group,the center distance of forward and hind feet and extorsion angle of the third toe of hind limb were significantly reduced in the AASCs group at 5 weeks(P＜0.05),the glial scar area was significantly decreased at 13 weeks(P＜0.05),and the cavity area of injured region was signiflcentJy diminished(P＜0.05).Results show that AASCs transplantation via subarachnoid space promoted functional recovery after SCI in rats.

Four phthalocyanines (iron tetracarboxylphthalocyanine, copper tetracarboxylphthalocyanine, manganese tetracarboxylphthalocyanine, cobalt tetracarboxylphthalocyanine) were used as dual functional mimic enzymes of superoxide dismutase (SOD) and catalase (CAT). The first function, eliminating O2－, was proved by using riboflavine-methionine photoreduction method in the concentration range of 10－5 to 10－6 mol/L. The second function, clearing out H2O2, was demonstrated by means of spectrophotometry with the decomposing percentage being increased with the increase of the concentration of the imitating compounds. Measurements of metal phthalocyanines, SOD and CAT by the liver homogenate technique of mice showed that they had obvious action of decreasing the lipid peroxidation.

Four phthalocyanines (iron tetracarboxylphthalocyanine, copper tetracarboxylphthalocyanine, manganese tetracarboxylphthalocyanine, cobalt tetracarboxylphthalocyanine) were used as dual functional mimic enzymes of superoxide dismutase (SOD) and catalase (CAT). The first function, eliminating O2－, was proved by using riboflavine-methionine photoreduction method in the concentration range of 10－5 to 10－6 mol/L. The second function, clearing out H2O2, was demonstrated by means of spectrophotometry with the decomposing percentage being increased with the increase of the concentration of the imitating compounds. Measurements of metal phthalocyanines, SOD and CAT by the liver homogenate technique of mice showed that they had obvious action of decreasing the lipid peroxidation.

In order to screen antigenic genes of Clonorchis sinensis (C.sinensis).Firstly,a cDNA expression library from adult worms of C.sinensis was successfully constructed with lambda ZAP express vector.Total RNA of C.sinensis adult worms were extracted by the Trizol reagent and mRNA were further purified through oligo-dT cellulose.The first strand eDNA was synthesized by using MMLV reverse transcriptase.After the synthesis of the second strand,the cDNA were purified by CHROMA SPIN-400 kit and then ligated with lambda ZAP express vector,then packaged in vitro and amplified.The original library contained 1.5 × 106 pfu cDNA clones,the titer of amplified library reached 1.5 × 1010 pfu/mL,in which about 99% clones were recombinants and most of insert DNA fragments were 0.4-2.0 kb.Secondly,immunoscreened using the naturely infected man serum from C.sinensis.The positive clones were sequenced and analyzed.From 2.0 × 105 recombinant clones of the eDNA library,41 positive clones were obtained.Sequence analysis indicated that the cDNAs encoded proteins including glycine rich antigen 2,proline rich antigen 2 and antigen Cs44 from C.sinensis,anothers were lower similarity to predicted protein from Nematostella vectensis,transcription elongation factor GreA from Bartonella quintana str.Toulouse and NM_132090 CG3446 gene product in Drosophila melanogaster from Schistosoma japonicum.These data may form a foundation for identifying recombinant antigens that can be used in the diagnosis or vaccination against clonorchiasis.

This study was aimed to construct a prokaryotic expressing vector of Hap gene from Nontypeable Haemophilus influenzae, and express and identify the fusion proteins of Hap-His in E. coli. The gene encoding protein Hap was amplified from Nontypeable Haemophilus influenzae ATCC49247 chromosomal DNA by PCR, then it was cloned into prokaryotic expression plasmid pET-32a (+). The recombinant plasmid pET-32a(+)-Hap was transformed into E. coli BL21 and expression was induced by Isopropy-beta-D-thiogalatoside(IPTG). The Hap-His fusion protein expressed so was analyzed by SDS-PAGE and Western-blot. The results showed that the recombinant expressive plasmid pET-32a (+)-Hap was constructed successfully, and the recombinant plasmid expressed Hap-His fusion protein with relative molecule mass 176 000 and mainly existed in inclusion body. This fusion protein could combine with anti-His monoclonal antibody specifically through Western blot analysis. Successful expression of Hap-His fusion protein in prokaryotic cell could lay a basis for further study of immunocompetence of Hap protein and development of NTHi vaccine.
Bacterial Outer Membrane Proteins ; biosynthesis ; genetics ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; genetics ; Haemophilus influenzae ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Serine Endopeptidases ; biosynthesis ; genetics

Objective:To learn about the infection status of Anisakis larvae in the major economic marine products in the Yellow Sea and Bohai Sea, and provide baseline data for systematic monitoring of Anisakis and prevention and control of related diseases. Methods:From April 2016 to September 2020, the samples of marine products collected in the surrounding waters of 9 fishing sites in the Yellow Sea and Bohai Sea (Bohai Bay, the middle part of the Yellow Sea and Bohai Sea junction, the southern part of the Yellow Sea and Bohai Sea junction, the northern part of the Yellow Sea and the southern part of the Yellow Sea) in the coastal areas of Yantai City and Weihai City, Shandong Province were dissected and tested for worms. The infection and distribution of Anisakis larvae in different types of samples and different organs in the samples were compared, and the differences of the infection level of Anisakis larvae in marine fish among the surrounding waters of different fishing sites and different sampling sites in China were compared. At the same time, a survey on the awareness of health knowledge of anisakiasis was carried out among the residents near each fishing sites. Results:A total of 708 cases of 5 types of marine products were tested in the Yellow Sea and Bohai Sea, including 581 cases of marine fish, 22 cases of mollusks, 20 cases of echinodermata, 75 cases of crustaceans and 10 cases of shellfish. Anisakis larvae infection was detected only in marine fish (191 cases), and 4 723 Anisakis larvae were found. The infection rate was 32.87% (191/581) and the infection intensity was 24.73(4 723/191) larvae/case. They were mainly distributed in mesentery and intestinal wall (38.96%, 1 840/4 723), coelom (22.04%, 1 041/4 723) and gastric wall (17.95%, 848/4 723). The infection levels of Anisakis larvae in marine fish among the surrounding waters of different fishing sites were compared, the infection rate in the southern part of the Yellow Sea was the highest, and its infection intensity was significantly higher than that in the middle and southern part of the Yellow Sea and Bohai Sea junction ( P < 0.05). The infection levels of Anisakis larvae in marine fish among different sampling sites in China were compared, the infection rates of Zhoushan Port, the fish sold in Jinzhou, Yantai and Shantou were significantly higher than those in the Yellow Sea and Bohai Sea ( P < 0.05), and the infection rates of the fish sold in Dandong and Qingdao were significantly lower than those in the Yellow Sea and Bohai Sea ( P < 0.05). A total of 1 805 residents living near the Yellow Sea and Bohai Sea were investigated on the health knowledge of anisakiasis. Among them, 20.78% (375/1 805) residents had heard of anisakiasis, 15.73% (284/1 805) residents knew how to get it, 12.30% (222/1 805) residents knew the harm of anisakiasis to human body, and 16.68% (301/1 805) residents knew how to prevent it. Conclusions:The marine fish in the Yellow Sea and Bohai Sea are infected by the Anisakis larvae, and the level of infection is relatively high. In the future, we should strengthen the popularization of knowledge on prevention and control of anisakiasis.

Objective To investigate the risk factors of anastomotic leakage (AL) after laparoscopic surgery in rectal cancer patient with neoadjuvant therapy and construct a nomogram prediction model. Methods This study was a retrospective case?control study that collected and reviewed the clinicopathological data of 359 patients who underwent laparoscopic surgery from January 2012 to January 2018, including 202 patients from the Department of General Surgery, Nanfang Hospital of Southern Medical University and 157 patients from the Department of Gastrointestinal Surgery of Fujian Provincial Cancer Hospital. Inclusion criteria: (1) age ≥ 18 years old; (2) diagnosis as rectal cancer by biopsy before treatment; (3) distance from tumor to anus within 12 cm; (4) locally advanced stage (T3?T4 or N+) diagnosed by imaging (CT, MRI, PET or ultrasound); (5) standardized neoadjuvant therapy followed by laparoscopic radical operation. Exclusion criteria: (1) previous history of colorectal cancer surgery; (2) short?term or incomplete standardized neoadjuvant therapy; (3) Miles, Hartmann, emergency surgery, palliative resection; (4) conversion to open surgery. Clinicopathological data, including age, gender, body mass index (BMI), preoperative albumin, distance from tumor to anus, operation hospital, American Society of Anesthesiologists score (ASA score), operation time, T stage, N stage, M stage, TNM stage, pathological complete response (pCR) were analyzed with univariate analysis to identify predictors for AL after laparoscopic surgery in rectal cancer patient with neoadjuvant therapy. Then, incorporated predictors of AL, which were screened by multivariate logistic regression, were plotted by the "rms" package in R software to establish a nomogram model. According to the scale of the nomogram of each risk factor, the total score could be obtained by adding each single score, then the corresponding probability of postoperative AL could be acquired. The area under ROC curve (AUC) was used to evaluate the predictive ability of each risk factor and nomogram on model. AUC > 0.75 indicated that the model had good predictive ability. The Bootstrap method (1000 bootstrapping resamples) was applied as internal verification to show the robustness of the model. The discrimination of the nomogram was determined by calculating the average consistency index (C?index) whose rage was 0.5 to 1.0. Higher C?index indicated better consistency with actual risk. The calibration curve was used to assess the calibration of prediction model. The Hosmer?Lemeshow test yielding a non?significant statistic (P>0.05) suggested no departure from the perfect fit. Results Of 359 cases, 224 were male, 135 were female, 189 were ≥ 55 years old, 98 had a BMI > 24 kg/m2, 176 had preoperative albumin ≤ 40 g/L, 128 had distance from tumor to anus ≤ 5 cm, 257 were TNM 0?II stage, 102 were TNM III?IV stage, and 84 achieved pCR after neoadjuvant therapy. The incidence of postoperative AL was 9.5% (34/359). Univariate analysis showed that gender, preoperative albumin and distance from tumor to the anus were associated with postoperative AL (All P<0.05). Multivariate logistic regression analysis revealed that male (OR=2.480, 95% CI: 1.012?6.077, P=0.047), preoperative albumin≤40 g/L (OR=5.319, 95% CI: 2.106?13.433, P<0.001) and distance from tumor to anus≤5 cm (OR=4.339, 95% CI: 1.990?9.458, P<0.001) were significant independent risk factors for postoperative AL. According to these results, a nomogram prediction model was constructed. The male was for 55 points, the preoperative albumin≤40 g/L was for 100 points, and the distance from tumor to the anus ≤ 5 cm was for 88 points. Adding all the points of each risk factor, the corresponding probability of total score would indicated the morbidity of postoperative AL predicted by this nomogram modal. The AUC of the nomogram was 0.792 (95% CI: 0.729?0.856), and the C?index was 0.792 after internal verification. The calibration curve showed that the predictive results were well correlated with the actual results (P=0.562). Conclusions Male, preoperative albumin ≤ 40 g/L and distance from tumor to the anus≤5 cm are independent risk factors for AL after laparoscopic surgery in rectal cancer patient with neoadjuvant therapy. The nomogram prediction model is helpful to predict the probability of AL after surgery.