Objective To obtain the recombinant Argonaute protein(TtAgo)from Thermus thermophilus using gene cloning and pro-tein purification techniques and detect its endonuclease activity against the KRAS 12D mutation in vitro,thereby to provide experimental evidence for its subsequent application in the detection of gene mutations.Methods The TtAgo gene sequence was amplified from the genome of Thermus thermophilus by the PCR technology.Then,the amplified sequence was inserted into the prokaryotic expression vec-tor pET-28a to construct the recombinant vector pET-28a-TtAgo.After the recombinant TtAgo protein was induced to be expressed,it was purified sequentially by the nickel ion affinity chromatography(Ni-NTA),heparin affinity chromatography,and size-exclusion chromatography.The high-specificity nuclease activity of the recombinant TtAgo protein mediated by a short DNA guide strand(gDNA)was verified in vitro.Results The prokaryotic expression vector pET-28a-TtAgo was successfully constructed by the gene cloning tech-nique,and the soluble expression of the recombinant TtAgo protein was achieved.With the help of protein purification technology,the high-purity recombinant TtAgo protein was obtained.Under the mediation of gDNA,the recombinant TtAgo protein could specifically cleave wild-type single-stranded DNA(ssDNA),double-stranded DNA(dsDNA),and RNA molecules,while had no cleavage activi-ty for tumor KRAS 12D mutant molecules.Conclusion The high-purity recombinant TtAgo protein with nuclease activity is successful-ly obtained,and its targeted cleavage activity towards the KRAS 12D mutation site is detected in vitro.

Objective To obtain the recombinant Argonaute protein(TtAgo)from Thermus thermophilus using gene cloning and pro-tein purification techniques and detect its endonuclease activity against the KRAS 12D mutation in vitro,thereby to provide experimental evidence for its subsequent application in the detection of gene mutations.Methods The TtAgo gene sequence was amplified from the genome of Thermus thermophilus by the PCR technology.Then,the amplified sequence was inserted into the prokaryotic expression vec-tor pET-28a to construct the recombinant vector pET-28a-TtAgo.After the recombinant TtAgo protein was induced to be expressed,it was purified sequentially by the nickel ion affinity chromatography(Ni-NTA),heparin affinity chromatography,and size-exclusion chromatography.The high-specificity nuclease activity of the recombinant TtAgo protein mediated by a short DNA guide strand(gDNA)was verified in vitro.Results The prokaryotic expression vector pET-28a-TtAgo was successfully constructed by the gene cloning tech-nique,and the soluble expression of the recombinant TtAgo protein was achieved.With the help of protein purification technology,the high-purity recombinant TtAgo protein was obtained.Under the mediation of gDNA,the recombinant TtAgo protein could specifically cleave wild-type single-stranded DNA(ssDNA),double-stranded DNA(dsDNA),and RNA molecules,while had no cleavage activi-ty for tumor KRAS 12D mutant molecules.Conclusion The high-purity recombinant TtAgo protein with nuclease activity is successful-ly obtained,and its targeted cleavage activity towards the KRAS 12D mutation site is detected in vitro.

Humans ; Data Warehousing ; East Asian People ; Intracranial Hemorrhages/diagnostic imaging* ; Asian People ; Cerebral Hemorrhage/diagnostic imaging*

Objective To explore clinical efficacy of swallowing function training in reducing lung infection in craniocerebral injury patients with tracheotomy.Methods A total of 124 craniocerebral injury patients in the hospital were randomized into experimental group and control group, the control group received conventional treatment, while the experimental group took swallowing function training based on the control group, secretion samples of pulmonary infection patients were collected for bacterial culture, identification and susceptibility test, and swallowing, lung infection rate and catheter indwelling time were compared.Results There were 223 strains of pathogens detected in 43 patients with lung infection after culture and isolation, including 65 strains(29.15%) of gram-positive bacteria, 148(66.37%) of gram-negative bacteria, and 18 (8.07%) of fungus.The resistance rates of staphylococcus aureus to penicillin G, ampicillin were more than 87%, and was sensitive to vancomycin, rifampin, and clindamycin.Enterococcus faecalis was resistant to ciprofloxacin, and penicillin G.Staphylococcus epidermidis cocci was sensitive to vancomycin, rifampin, clindamycin.Acinetobacter baumannii, pseudomonas aeruginosa, Klebsiella pneumoniae had higher resistance to ceftazidime, cefotaxime, and ofloxacin, whereas was sensitive to imipenem, and meropenem.And resistance rates of Acinetobacter baumannii and Klebsiella pneumoniae to piperacillin/tazobactam, and cefoperazone/sulbactam were less than 30%;Pseudomonas aeruginosa had wide range of drug resistance, but was sensitive to imipenem, and meropenem only.After treatment, excellent rate in the experimental group was significantly better than the treatment before and the control group (P<0.05).Experimental group had less lung infection and less catheter indwelling time than the control group(P<0.05).Conclusion Swallowing training for craniocerebral injury patients with tracheotomy can effectively reduce lung infection, shorten catheter indwelling time, and improve the prognosis.

Objective To explore clinical efficacy of swallowing function training in reducing lung infection in craniocerebral injury patients with tracheotomy.Methods A total of 124 craniocerebral injury patients in the hospital were randomized into experimental group and control group, the control group received conventional treatment, while the experimental group took swallowing function training based on the control group, secretion samples of pulmonary infection patients were collected for bacterial culture, identification and susceptibility test, and swallowing, lung infection rate and catheter indwelling time were compared.Results There were 223 strains of pathogens detected in 43 patients with lung infection after culture and isolation, including 65 strains(29.15%) of gram-positive bacteria, 148(66.37%) of gram-negative bacteria, and 18 (8.07%) of fungus.The resistance rates of staphylococcus aureus to penicillin G, ampicillin were more than 87%, and was sensitive to vancomycin, rifampin, and clindamycin.Enterococcus faecalis was resistant to ciprofloxacin, and penicillin G.Staphylococcus epidermidis cocci was sensitive to vancomycin, rifampin, clindamycin.Acinetobacter baumannii, pseudomonas aeruginosa, Klebsiella pneumoniae had higher resistance to ceftazidime, cefotaxime, and ofloxacin, whereas was sensitive to imipenem, and meropenem.And resistance rates of Acinetobacter baumannii and Klebsiella pneumoniae to piperacillin/tazobactam, and cefoperazone/sulbactam were less than 30%;Pseudomonas aeruginosa had wide range of drug resistance, but was sensitive to imipenem, and meropenem only.After treatment, excellent rate in the experimental group was significantly better than the treatment before and the control group (P<0.05).Experimental group had less lung infection and less catheter indwelling time than the control group(P<0.05).Conclusion Swallowing training for craniocerebral injury patients with tracheotomy can effectively reduce lung infection, shorten catheter indwelling time, and improve the prognosis.

Objective To establish viral inactivation/removal techniques for blood products , and apply them to inacti-vation/removal process validation of blood products .Methods Enveloped and non-enveloped model viruses were propaga-ted.Viral inactivation/removal techniques for blood products ,including solvent/detergent (S/D) treatment, low pH incuba-tion, dry heat method, pasteurization,and nanofiltration, were established.The virus titer was evaluated using cytopathic effects ( CPE) and Spearman and Karber method .The viral inactivation/removal techniques were believed to be effective when LRV≥4.These techniques were used in viral inactivation /removal validation of blood products .Results Enveloped model viruses were inactivated through S/D treatment and the low pH incubation method .Enveloped and non-enveloped model viruses were inactivated through dry heat and pasteurization .Within a certain range of filtration capacity , PPV was removed through nanofiltration .Conclusion The established viral inactivation/removal techniques can be used in viral inactivation/removal process validation of blood products , which can improve viral safety of blood products .

Objective To explore the changes of gene and protein expressions of nuclear factor-κB(NF-κB) and tumor necrosis factor-α (TNF-α) in immature rats with pilocarpine-induced epilepsy treated with ghrelin. Methods The pllocarpine-induced epilepsy model in immatured rats were built, then the rats were divided into three groups: Ghrelin-treated group, saline-treated group and model group, meanwhile the normal control group was set. The NF-κB and TNF-α levels of gene and protein in the cerebral cortex of immature rats were detected. Results The expression levels of gene and protein of NF-κB and TNF-α were increased in model group,but decreased in the normal control group;NF-κB and TNF-α levels in Ghrelin treated group were obviously lower than those of saline-treated group and model group(P < 0. 05). Conclusion The protective mechanism of Ghrelin for nerve cell is cutting down the expressions of NF-κB and TNF-α in the cerebral cortex of immature rats with epilepsy and lessening inflammatory reaction in neurocytes.

Thirty-seven cases of brain glioma,confirmed by light microscopy and immuno-'histochemistry were studied with transmission electron microscopy.It was found that there were certain ultrastructural features for each type and each grade of glioma.In astrocytoma,there was certain amount of glial filaments in the tumor cells; Rosenthal fibers consisted of irregular osmiophilic masses surrounded with glial filaments,occasionally cytoplasmic annulate lamellae and intranuclear filaments could be seen,and the interstitial capillaries were characteristic.In typical oligodendroglioma,astrocytic processes containing glial filaments were quite present in different amounts.These findings suggest that observation on the ultrastructure of gliomas is of significance to establish the diagnosis,to assess the degree of differentiation,and to identify some rare structures which can reveal the essence of the tumor.In addition,ultrastructural observation is helpful for prognosis.