Objective To construct the vector that expresses the fusion protein of HIV-Tat protein and red fluorescent protein(mCherry) in mammalian cells,and observe by fluorescence microscopy the intracellular transduction and localization of recombinant protein in cells,in order to obtain a useful tool for the study of the uptake mechanism and intracellular localization of HIV-TAT.Methods With the designed primer coding mCherry sequence,the mCherry gene was amplified by PCR with the vector pmCherry-C2 as template,and inserted into vector pET14b-His-TAT to construct the expression vector pET14b-His-TAT-mCherry.The constructed vector was then transformed into E.coli BL21(DE3),which had been identified by PCR and double digested with restriction endonuclease,followed by sequencing.After IPTG induction,the recombinant protein of His-TAT-mCherry was lyzed and analyzed with SDS-PAGE.Purified His-TAT-mCherry recombinant protein was added to Hela cells and the fluorescence was observed to evaluate the transduction efficiency.Results The results of identification by PCR,digestion with restriction endonuclease and sequencing indicated that the vector His-TAT-mCherry was correctly constructed.His-TAT-mCherry fusion protein was expressed in mammalian Hela cell line and purified successfully,and the fusion protein showed cellular transduction activity.It was found by fluorescence microscopy that the red fluorescence protein located mainly over the cytoplasm,and also the membrane to some extent.Conclusion The expression vector is successfully constructed for HIV-TAT labeled with mCherry sequence.Effective expression and purification of this fusion protein is achieved.It has been observed that the constructed vector may be expressed in mammalian Hela cell under active condition.Thus,it might be useful in the study of uptake mechanism and intracellular localization of HIV-TAT.

Objective To investigate the protein markers that specifically expressed in patients with acute rejection (ACR) after liver transplantation, and to explore preliminarily the mechanisms. Methods Serum samples from three patients with pathologically confirmed ACR after liver transplantation in Tianjin First Central Hospital were collected as ACR group. Three serum samples from patients with normal liver function indicators after liver transplantation were collected as No-ACR group. And six serum samples from healthy examination were mixed with equal amount as healthy control group. Isobaric Tags for Relative and Absolute Quantitation (iTRAQ) was employed to separate, screen and identify the differentially expressed proteins between three groups. KEGG and STRING software were applied to deeply analyze the data of three groups. Results A total of 88 differentially expressed proteins were found between ACR group and healthy control group. There were 39 differentially expressed proteins between No-ACR group and healthy control group. Ten differentially expressed proteins were acquired between ACR group and No-ACR group. Comparing 88 and 10 differentially expressed proteins, 9 proteins were the same. Among 88 differentially expressed proteins, 30 of them showed a direct interaction, and can be positioned in 13 signaling pathways based on KEGG and STRING software. Fourteen (46.67%) of the 30 proteins were located in the complement and coagulation cascade pathway. Among 39 differentially expressed proteins, which were detected between No-ACR group and control group, 10 proteins showed a direct interaction including 9 proteins concentrated in the complement and coagulation cascade pathway. Conclusion By proteomic analysis, nine differentially expressed proteins are obtained, which may be regarded as the candidate bio-markers for ACR early diagnosis after liver transplantation. The complement and coagulation cascades system is significantly adjusted after liver transplantation, indicating this pathway plays an important role in the occurrence of ACR.

Objective To fractionate phosphoproteome of mouse liver by two-dimensional (2D) liquid phase chromatography fractionation. Methods Phosphoproteins were extracted from lysates of normal mice livers by phosphate metal affinity chromatography (PMAC) resin. The phosphoproteins were exchanged by start buffer and separated by chromatofocusing in the first dimension. Then the fractions between pH 8.5 and pH 4.0 were separated by non-porous silica (NPS) reverse-phase high performance liquid chromatography (RP-HPLC). Finally, the UV maps were converted into gel-like maps by ProteoVue software. Results Phosphoproteins of mouse liver were successfully extracted and fractionated by two dimensional liquid phase chromatographic fractionation after concentration and desalt. Then pI/UV map of mouse liver phosphoproteome was successfully set-up. There are 16 fractions between pH 8.5 and pH 4.0 after chromatofocusing in the first dimension and the UV maps of each fraction were converted into pI/UV gel-like maps. Conclusions Combination of technique of phosphoproteins enrichment and 2-D liquid phase chromatographic fractionation is an effective approach to research phosphoproteome and the key base for further identification and investigation of phosphoproteins.

AIM: To construct the prokaryotic expression plasmid of His-tagged human high mobility group box 1 fusion protein (hHMGB1) and to express the fusion protein in E. coli for the affinity purification. METHODS: The cDNA coding region of HMGB1 was amplified by PCR from pGEX4T-HMGB1 and cloned into a modified pET14b vector following the routine procedure. After identification by enzyme digestion, PCR and sequencing, the plasmid was transformed into BL21 (DE_3) competent cells, and the His-HMGB1 fusion protein was induced for expression with isopropyl-beta-D-thiogalactopyranoside (IPTG), and further purified by Ni-NTA affinity chromatography. The protein was filtered for sterilization and used to stimulate human umbilical vein endothelial cells (HUVECs). 24 hours later, the cultured supernatant of HUVECs was collected for the detection of cytokines/chemokines with LiquiChip system. RESULTS: The His-HMGB1 fusion protein expression plasmid was identified by enzyme digestion and sequencing. The purified His-tagged fusion protein was analyzed by SDS-PAGE and Western blotting with specific anti-His antibody. It was found that the production of IL-8 from HUVECs was highly induced in a dose-dependent manner by HMGB1. CONCLUSION: The His-tagged HMGB1 fusion protein expression plasmid was successfully constructed, and purified. Recombinant HMGB1 protein has a high bioactivity on the induction of cytokines in HUVECs, which may significantly facilitate the future study of HMGB1 biological functions.

TLR4-MD-2 complex plays a key role in LPS recognition and its signal transduction. These steps are the vital elements of the host's defensive reaction. Studying the functional domain of TLR4 and MD-2 is very important to further understand the mechanism of LPS signal transduction. It was studied the interaction domain of TLR4 and MD-2 in living cells based on gene mutation, gene transfection and fluorescence resonance energy tramsfer(FRET) which is considered as one of the best methods used for intracellular protein-protein interaction study. CY-15P which was fused by CFP and YFP through 15 neutral amino acids was used as positive control, while co-expressed CFP and YFP proteins were used as negative control. The results showed that the ability of TLR4 binding to MD-2 decreased dramatically after the deletion of Glu~(24) ～ Met~(41) in N terminal of TLR4. Aggregation of TLR4 to LPS stimulation was observed, however, TLR4 without the Glu~(24)～ Met~(41) mutation did not aggregate. All these results indicated that TLR4 Glu~(24)～ Met~(41) might be the interaction domain of TLR4 binding to MD-2 and participate in the aggregation effect of TLR4 upon LPS stimulation.

Objective To explore the long-term predictive value of serum concentration of N-terminal prosoma brain natriuretic peptide (NT-proBNP) in the early acute coronary syndrome (ACS). Methods The 164 patients firstly hospitalized and finally diagnosed as acute myocardial infarction (AMI) were selected, and then the serum concentration of NT-proBNP was determined in less than 12 hours. According to the 75 percentage points of serum concentration of NT-proBNP, the patients were divided into two groups: low concentration group (n = 123) and high concentration group (n = 41 ). The major adverse cardiac events (MACEs) were followed and compared at one month, six months and twelve months between low group and high group. Results At 1-, 6-, 12-month follow-up, the odds ratio (OR) of death event were 4.1, 5.6 and 4.0 in high group respectively, and the nonfatal heart failure occurred in 4, 4 and 7 patients in high group. Multiple factor logistic regression analysis showed that NT-proBNP was an independent risk factor of the MACEs at different periods including short time, middle time and long time in ACS patients (P＜0. 05). Conclusions NT-proBNP is a strong predictor of the long-term MACEs in patients with early ACS.

Objective To examine the practical value of early detection of heart-type fatty acid binding protein (H-FABP)for risk stratification and prognosis assessment in cardiac troponin T (cTnT)-negative acute coronary syndrome(ACS)patients.Methods From March 2010 to March 2012,55 patients with chest pain and negative cTnT were selected from 232 ACS patients at the General Hospital of PLA.Expression levels of cTnT and H-FABP were detected within 6 h of the onset of clinical symptoms.H-FABP and cTnT values at 12,24,and 48 h from the onset of clinical symptoms were continuously measured to monitor the dynamic changes.Based on prognosis,patients were divided into two groups,levels of H-FABP were compared,and its predictive value for prognosis was assessed with the ROC curve.Results Within 6 h of the onset of clinical symptoms,cTnT levels in cTnT-negative ACS patients increased gradually as disease progressed and reached the peak value at 12 h before decreasing slowly and arriving at 50％ of the peak value at 48 h.Meanwhile,HFABP levels reached the peak within 6 h,decreased slightly(12.8％) at 12 h,and then decreased rapidly at 48 h (about 79％).Of 55 patients,24 had acute myocardial infarction during hospitalization.The H-FABP level within 6 h was a good predictor for cTnT-negative ACS patients.The area under ROC curve was 0.946 and the cutoff value was 15.47 μg/L.The prediction sensitivity was 87.5 ％,with a specificity of 90.3％.Eleven patients had cardiovascular events after a 12-month follow-up.Levels of H-FABP were different in patients with or without cardiovascular events,[(38.08±8.43) μg/L vs.(18.96 ± 2.85) μg/L (t =2.438,P＜0.05)].ROC curve analysis showed that the area under the curve was 0.772 and the prediction cutoff value was 44.71 μg/L.The rates of cardiovascular events were markedly different between patients with high(≥44.71 μg/L)and those with 1ow(＜44.71 μg/L)H-FABP levels(54.5％ vs.11.4％).Conclusions For ACS patients with negative cTnT,H-FABP is a good index for early risk stratification and prognosis assessment.

Objective To investigate the factors affecting the door-to-needle (DTN) time for intravenous thrombolytic therapy in patients with acute ischemic stroke. Methods Patients with acute ischemic stroke treated with intravenous thrombolysis were enrolled prospectively. The demography, vascular risk factors, and other clinical data were collected. According to DTN time 60 min as a standard, the patients were divided into either a non-delay group or a delay group. The factors affecting DTN delay were analyzed. Results A total of 78 patients with acute ischemic stroke treated with intravenous thrombolysis were enrolled, 46 (59.0%) were males with an average age of 64.24 ± 10.06 years. The average National Institutes of Health Stroke Scale(NIHSS)score was 12.13 ± 3.17.The average DTN time was 79.77 ± 20.51 min, and the DTN time in 55 patients (70.51%) was >60 min. The age (66.3 ± 9.7 years vs. 59.3 ± 9.4 years; t=2.939, P=0.004), door-to-CT initiation time (32.7 ± 11.3 min vs. 24.6 ± 7.1 min; t= 3.183, P= 0.002), door-to-CT interpretation time (50.8 ± 16.8 min vs. 35.5 ± 8.8 min; t= 4.383, P< 0.001), and door-to-biochemistry result time (62.6 ± 11.0 min vs. 44.8 ± 5.6 min;t=7.377, P<0.001) in the delay group were all significantly higher or longer than those in the non-delay group.The proportions of patients with hypertension(58.2% vs. 26.1%;χ2=6.687,P=0.010) and using antihypertensive drugs before onset (41.8% vs. 13.0%; χ2=6.043, P=0.014) in the delay group were also higher than those in the non-delay group. Multivariate logistic regression analysis showed that the door-to-biochemistry result time (odds ratio 1.725, 95% confidence interval 1.058-2.814; P=0.029)was an independent influencing factor for DTN delay.Conclusions The delay of door-to-biochemistry result time is an independent factor for DTN time delay in patients with acute ischemic stroke treated with intravenous thrombolytic therapy.

OBJECTIVETo evaluation the performance of a heart-type fatty acid-binding protein (H-FABP) ELISA detection kit in the clinical diagnosis of acute coronary syndrome (ACS).

METHODSPlasma or serum samples from 160 suspected ACS patients hospitalized in General Hospital of PLA were examined using Lanzhou H-FABP reagent kit and Holland H-FABP kit. Correlation of the two kits was evaluated and Kappa test was used to examine the consistency of the results of the two products.

RESULTSThe sensitivity of H-FABP diagnosis of ACS detection Lanzhou kit was 91.8%, the specificity was 88.7%, and the total diagnostic rate was 90.42%. The sensitivity of H-FABP diagnosis of ACS detection Holland kit was 90.3%, the specificity was 86.8%, and the total diagnostic rate was 88.75%. The test results showed that two products yielded comparable results (P=0.668, >0.05) with a good consistency (Kappa=0.726, P<0.01).

CONCLUSIONSH-FABP ELISA detection kit produccted by LanZhou biological research institute has a good correlation with H-FABP detection kit produced by HBT company of Holland and has the potential for clinical application.

Acute Coronary Syndrome ; diagnosis ; Biomarkers ; blood ; Enzyme-Linked Immunosorbent Assay ; Fatty Acid Binding Protein 3 ; Fatty Acid-Binding Proteins ; blood ; Humans ; Reagent Kits, Diagnostic ; Reproducibility of Results ; Sensitivity and Specificity

Cardiopulmonary resuscitation (CPR) guidelines emphasize that external chest compressions should be started as soon as possible when CPR is performed in patients with cardiac arrest. Moreover, those guidelines stress on fast and hard compressions to make the chest fully rebound and minimize non-pressing time. Current mechanical recovery device has several problems such as displacement of the pressing position, high price, difficult to move, and easy dislocation of piston. Because of the physical loss of high-intensity unarmed CPR, the depth and frequency of external chest compression will decrease with the extension of CPR time, leading to CPR failure. Besides, there are other problems caused by non-professional staff, such as the deviation of compression position, the inaccuracy of compression depth and the unsatisfactory rebound of the chest wall. Based on the above factors, the medical staff from the intensive care unit of the Eighth Medical Center of the Chinese People's Liberation Army General Hospital designed a portable external chest cardiac compressor based on international CPR guidelines which obtained the National Utility Model Patent of China (ZL 2018 2 1173254.3). The portable external chest cardiac compressor is composed of a positioning sucker, elastic body, mounting shell, and pressing components. Rapid and accurate compression positioning, visible compression depth and full chest rebound can be achieved. This device is mobile, easy to operate, and suitable for a broad crowd and various occasions.