Objective To study the enzymatic progress of amyloid precursor protein (APP), and construct the recombinant eukaryotic expression plasmid encoding the double mutations of APP and 2 different types of fluorescent protein. Methods The last 300 bases of APP named as C99 containing APP717 mutation was amplified by polymerase chain reaction (PCR) with the template pcDNA3.0-APP.The cyan and yellow fluorescence sequences named as CFP and YFP respectively were obtained by PCR with the template of digestion of the plasmid pcDNA3.0-CFP-CaM-YFP.The 54 bases in the middle of APP named as 54 bp containingSwedish mutation were synthesized. The 4 fragments, CFP, YFP, C99 and 54 bp were inserted into the vector pcDNA3.0.By genetic engineering the recombinant plasmid pcDNA3.0-CFP-54bp-YFP-C99 and pcDNA3.0-CFP-54bp-YFP were constructed and finally identified by enzyme digestion, PCR and sequencing. Then the 2 constructs were transfected into SH-SY5Y cells respectively. The expression of the fusion gene was examined by multi-photon con-focal microscopy and the fluorescence resonance energy transfer (FRET) signal was collected. The amyloid beta (Aβ)deposition was detected by immunocytochemistry. Results (1) DNA sequencing showed that the sequence of the construct was correct.(2)The cyan and yellow fluorescence could be seen by the multi-photon con-focal fluorescence microscopy. The fusion gene could be correctly expressed.(3)FRET was present in the cells expressing CFP-54bp-YFP,but absent in the cells expressing CFP-54bp-YFP-C99.(4)A13 labeled by YFP was detected by multi-photon con-focal microscope and deposited in the cytoplasm, membrane and in the intercellular space.(5)It was shown that by immunocytochemistry, the CFP-54bp-YFP-C99 expressionproduct could generate Aβ. The Aβ deposition was detected in the cell membrane, cytoplasma and intercellular space. Conclusions (1) The fusion protein could generate Aβ peptide by β and γ proteolytic processing.(2)Aβ may be generated in the conrse of APP transportation from cell plasma to cell membrane.(3)C99 is very important for the correct cleavage of APP. Our test data strongly suggest that C99 may function as the signal-like peptide. It may guide and direct the APP to the right location for the cleavage.(4)Before the Aβ deposition is formed outside of the cell, Aβ intracellular accumulation results in the change of cell shape.

Objective To established a diagnostic methods to idenfity Niemann-Pick disease type C (NPC) in China. Methods Two patients aged 5 and 20 years respectively who presented progressive neurologic regression and splenomegaly were subjected to filipin staining of cultured skin fibroblasts and genetic analyses of NPC1 gene. Results Although there were differences in onset ages and clinical presentations, filipin staining of the cultured skin fibroblasts confirmed the clinical diagnosis of NPC, showing an intense punctate pattern of fluorescence concentrating around the nuclei, consistent with the accumulation of unesterified cholesterol in NPC cells. Genetic sequence analysis further verified the results of filipin staining. The case 1 was compound heterozygous for M1142T(3425T＞C),R1186H(3557T＞C)and case 2 for Q88H(264G＞T),469_470insGT.The latter 2 mutations were novel, and the possibility of polymorphisms was not supposed by analyzing 134 DNA samples obtained form normal controls. Conclusions Filipin stainning of the cultured skin fibroblasts is a reliable method to clinically diagnose NPC with a sensitivity. Genetic diagnose should be performed where genetic analysis is allowed.

ObjectiveTo investigate the protective effect of sevoflurane postconditioning (Sevo-Postcon)on the hearts isolated from rats with diabetes mellitus (DM) of different duration against ischemia-reperfusion (I/R)injury.MethodsSeventy-two pathogen-free male SD rats weighing 200-240 g were randomly divided into 3 groups ( n =24 each):group Ⅰ rats without DM (C) ; group Ⅱ rats with 2 week DM (2w DM) and group Ⅲ rats with 6 week DM (6w DM).DM was produced by intraperitoneal (IP) 1％ streptozocin (STZ) 60 mg/kg and confirmed by fasting blood glucose concentration ＞ 16.7 mmol/L in groups Ⅱ and Ⅲ.Hearts were isolated from rats and perfused with Krebs-Henseleit buffer (KHB) in a Langendorff apparatus.After a 15 min stabilization period,the isolated hearts were subjected to 30 min of global no-flow ischemia followed by 75 min of reperfusion.Twelve hearts in each group were perfused after ischemia with KHB saturated with 3％ Sevo for 15 min followed by perfusion with regular KHB for 60 min.LVEDP,LVDP, ± dp/dt and HR were measured and recorded after 15 min stabilization (T0,baseline) and at 15 and 75 min of reperfusion (T1,2 ).Myocardial specimens were obtained at 15 min of reperfusion (T1) for detection of p-Akt expression (by Western blot analysis).Infarct size was determined at 75 min of reperfusion (T2).ResultsSevo-Postcon significantly improved cardiac function,reduced infarct size and up-regulated p-Akt expression in groups Ⅰ (C) and Ⅱ (2w DM),while in group Ⅲ (6w DM) Sevo-Postcon did not cause any change in cardiac function,infarct size and p-Akt expression as compared with the isolated hearts without Sevo-Postcon.ConclusionThe cardioprotective effect of Sevo-Postcon can be attenuated with increasing duration of DM by impairing PI3K/Akt signaling pathway.

Objective To know the efficacy and safety of the blood sugar control by double-pump a-mong severe patients with duodenectomy. Methods Divided 60 severe patients with doudenectomy into the experimental group and the,control group randomly, there were 30 cases in each group. In the control group, pa-tients received intravenous infusion with the liquid containing glucose and neutral insulin (4g: 1 U), intravenous injection of 50U neutral insulin added into 50ml normal saline by micro-pump, the pump speed was In the ex-perimental group, sugar-containing liquid without insulin was infused within 24 hours by infusion pump with u-niform speed, a neutral insulin 50U mixed with 5Oral normal saline were injected by micro-pump according to the ratio fo glucose 4g: 1U neutral insulin through the same vein. Which also adjusted the pump speed according to maintaining blood sugar in the range of 4.4～8.3mmol/L. Compared the level of blood sugar control, treatment safety, efficacy and the workload of nurses between the two groups. Results The highperglycemic index and the time to achieve target values of blood sugar in the experimental group was shorter than in the control group respectively, while the time of keep blood sugar in idea level in the experimental group was longer than in the control group. There were no significant difference about incidence rale of hypoglycemia and workload of nurses between the two groups. Conclusions Double-pump can effective control blood sugar for severe patients with duodenectomy without burden nurses' workload, which is more reasonable than the traditional method.

Objective To summarize the nursing experience of orthophoria peripherally inserted central catheter(PICC).Methods Patients who failed percutaneous intravenous puncture(23 cases)underwent orthophoria HCC.The operation technique,nursing points and prevention of complications were summarized.Results All 23 patients received successful first-time puncture.Accurate anatomical position,strict aseptic manipulation and change of dressing were required during nursing.One patient failed in transmission of the tube after successful puncture.One patient appeared local red swelling of skin.Conclusions Orthophoria PICC is suitable for patients with colorectal cancer undergoing chemotherapy who failed percutaneous intravenous puncture.Selective nursing has pivotal significance for successful operation.

Objective To analyze the characteristics of TCM syndromes of children abnormal sweating;To investigate the correlation between diseases and primary and secondary syndromes. Methods Through a multi-center epidemiology investigation, 400 cases were collected to statistically analyze the five TCM syndromes of children abnormal sweating, and characteristics of each syndrome were analyzed. Results In the 400 cases of children abnormal sweating, there were 129 cases (32.25%) of dysfunction of lung-wei pattern, 50 cases (12.50%) of nutrient qi and defensive qi discordance, 84 cases (21.00%) of qi and yin deficiency pattern, 57 cases (14.25%) of dampness heat forced steamed pattern, and 80 cases (20.00%) of qi and yang deficiency pattern. Spontaneous sweating and night sweating were found in the four patterns besides qi and yin deficiency pattern, and night sweating can be mainly found in qi and yin deficiency. Each type is consistent in the syndrome differentiation and cluster analysis. Conclusion Spontaneous sweating and night sweating, each has the certification of yin and yang. Spontaneous sweating can be caused by yin deficiency while night sweating can be caused by yang deficiency. Each pattern of children abnormal sweating has its own syndrome characteristic, which can provide the basis for clinical diagnosis and treatment.

C8orf32 is a gene which has not been functionally characterized,the mRNA level of this gene is significantly higher in breast cancer tissues than that in normal breast tissues.The amplified cDNA fragment was inserted into the pGEX-6P1 vector fused with the upstream GST gene.The expression vector was transformed into the E.coli BL21(DE3) strain and expression of GST-C8orf32 fusion protein was induced by IPTG..After removal of GST tag by site-specific protease,the C8orf32 protein fused with an eight amino acid peptide tag was obtained.The purity of recombinant C8orf32 protein was about 95%.The identity of the purified protein was confirmed by N-terminal sequencing and tandem mass spectrometry.The polyclonal antibody was prepared by immunizing the New Zealand white rabbits with C8orf32 protein.The polyclonal antibody was proved to recognize the C8orf32 protein correctly.The purified C8orf32 protein can be used for structural and functional studies and the polyclonal antibody can be used for tissue specific protein expression profiling.

Abstract: Objective　To analyze the distribution and drug resistance evolution characteristics of pathogenic bacteria of bloodstream infection in nine tertiary hospitals in Yunnan Province from 2017 to 2021, so as to provide reliable basis for rational selection of antibiotics in clinic. Methods Using the drug sensitive paper method or instrument method, the bacteria identification and drug sensitivity test were carried out in nine tertiary hospitals in different regions according to the unified technical scheme. The results were judged according to the Clinical and Laboratory Standards Institute (CLSI) breakpoint standard in 2021, and use WHONET5.6 for data statistical analysis. Results A total of 12 003 strains of pathogenic bacteria were isolated from bloodstream infection samples in the past five years, including 7 442 strains of Gram-negative bacteria (62.0%) and 4562 strains of Gram-positive bacteria (38.0%), with an increasing trend in the number of isolated strains; of these, 163 strains (1.4%) were isolated from outpatients and 11 840 strains (98.6%) were isolated from inpatients. The top three gram-negative bacteria were Escherichia coli, Klebsiella pneumoniae and Acinetobacter baumannii, of which 309 strains (4.2%) were carbapenem-resistant Klebsiella pneumoniae (CR-KPN), 29 strains (0.4%) carbapenem-resistant Escherichia coli and 19 strains (0.3%) carbapenem-resistant Enterobacter cloacae, and the number of CR-KPN was on the rise year by year. The top three Gram-positive bacteria were coagulase-negative staphylococci, Staphylococcus aureus and Enterococcus faecium, of which methicillin-resistant Staphylococcus aureus (MRSA) was detected for 213 strains, accounting for 27.7%, and decreased from 40.0% in 2017 to 23.4% in 2021, showing a downward trend year by year. No vancomycin-resistant staphylococci and enterococci were found. Conclusions The detection and composition of bloodstream infection pathogenic bacteria in multicenter have not changed much in the past five years, but each hospital has its own characteristics. The number of carbapenem resistant Enterobacteriaceae increased year by year, which should be paid more attention.

Objective To study the vaginal microecology of the patients in the outpatient department of Obstetrics and gynecology in China.Methods A multicenter cross-sectional study was conducted in gynecologic clinic of 9 collaborative hospitals in China.200 consecutive patients were collected in each hospital and their vaginal microecology combined with related factors were analyzed.Results A total of 2 093 specimens were collected in this study.The detection rate of Trichomonas was 5.5%(115/2 093). The detection rate of Candida mycelia was 15.9%(333/2 093), with germinal spores was 4.1%(86/2093).The detection rate of bacterial vaginosis was 18.8%(394/2 093).The distribution results of vaginal flora in patients showed that the normal flora accounted for only 27.3%(571/2 093).The normal flora with the insufficiency of H2O2 accounted for 23%(480/2 093).The bacteria inhibiting flora accounted for 3.8%(79/2 093).The abnormal microflora(non BV type)accounted for 14.9%(312/2 093).The abnormal microflora(BV intermediate type)accounted for 13.4%(280/2 093).The abnormal microflora(BV type)accounted for 17.6%(369/2 093).The average pH of vaginal discharge was 4.58 ±0.495.There was no significant difference of the incidence of trichomonas and bacterial vaginosis between north and south of the Yangtze river, while the detection rate of fungal hyphae and the fungal spores is significantly higher in the south than that in the north.The analysis results of factors affecting the microecology showed that age and contraception methods were two important factors.The patients′age from bacteria inhibition group was 49.64 +16.68 which was significantly higher than that of the other microecology groups.The proportion of abnormal microflora of patients from the oral contraceptive group was 40%(20/50).The proportion of abnormal microflora of patients from IUD group was 36.6%(63/172).Compared with these two contraception methods, the proportion of abnormal microflora of patients from condoms usage group was 27.8%(91/327)which was significantly lower.The incidence of abnormal leucorrhea in the normal group was 37.7%, which was significantly lower than that of other abnormal groups.Conclusion This study showed the vaginal microecology status of the Chinese outpatient ′s clinic and found that the vagina microecology was related to age, region and contraceptive methods.The typical manifestation of microecological abnormality is the increase of leucorrhea.(Chin J Lab Med,2018, 41:287-291)

In view of that there is no report of west Nile virus infection cases in our country, evaluation the self-prepared anti-WNV-IgG diagnostic ELISA kit should be employed with the establishment of the serum sample panel collected from the entry personnel. All individuals of entry personnel were traveled from epidemic area of infectious west Nile disease. In our study, the serum samples were both detected by self-prepared anti-WNV-IgG diagnostic ELISA kit and the FDA certified kits ,which are FOCUS West Nile Virus IgG Dxselect and Panbio Dengue IgG Capture ELISA kits. The self-prepared kit and FDA certified kits were compared and assessed simultaneously. Furthermore, the specificity, repeatability and stability of the kits were also evaluated. The results indicated that no significant difference of detective rates (35. 6% for self-prepared kit vs. 32.5% for FOCUS kit, χ2 = 3. 05, P > 0.05) and good consistency (Kappa = 0.8372) between the self-prepared kit and FDA certified kits. Also, the positive coincidence rate, the negative coincidence rate and the total coincidence rate were calculated as 91.18%, 95.34% and 92.66%, respectively. The laboratory self-developed kit presented similar quality as the counterpart kits with FDA certificate. The development of our self-prepared anti-WNV-IgG diagnostic ELISA kit will provide technical support for the prevention and control of west Nile virus entry.
Endemic Diseases ; Enzyme-Linked Immunosorbent Assay ; Humans ; Immunoglobulin G ; blood ; Reagent Kits, Diagnostic ; West Nile Fever ; epidemiology ; West Nile virus ; immunology