According to the designed specific primers of gene fragment based on the Salvia miltiorrhiza transcriptome data, a full-length cDNA sequence of SQS2 from S. miltiorrhiza f. alba was cloned by the method of reverse transcription polymerase chain reaction (RT-PCR). The SmSQS2 cDNA sequence was obtained, this sequence is named SmSQS2 and its GenBank registration number is KM244731. The full length of SmSQS2 cDNA was 1245 bp, encoding 414 amino acids including 5'UTR 115 bp and 3'UTR 237 bp. Sequence alignment and phylogenetic analysis demonstrated that SmSQS2 had relative close relationship to the SQS2 of S. miltiorrhiza. The induction of E. coli [pET28-SQS2] in different temperature, induction time, IPTG concentrations and density of inducing host bacterium (A600) were performed, Shaking the culture at 30 degrees C until the A600 is approximately 0.6 and add IPTG to final concentration of 0.2 mmol x L(-1), and then the optimal expression of SmSQS2 recombinant protein were accumulated after the induction time of 20 h. The research provided important base for the study of sterol and terpene biosynthesis of SQS2 in S. miltiorrhiza f. alba.
Cloning, Molecular ; Farnesyl-Diphosphate Farnesyltransferase ; chemistry ; genetics ; metabolism ; Models, Molecular ; Molecular Sequence Data ; Phylogeny ; Plant Proteins ; chemistry ; genetics ; metabolism ; Salvia miltiorrhiza ; chemistry ; classification ; enzymology ; genetics ; Sequence Alignment

OBJECTIVE: To analyze the indications of the therapies for rheumatoid arthritis (RA) with neural network model analysis. METHODS: Three hundred and ninety-seven patients were included in the clinical trial from 9 clinical centers. They were randomly divided into Western medicine (WM) treated group, 194 cases; and traditional Chinese herbal medicine (CM) treated group, 203 cases. A complete physical examination and 18 common clinical manifestations were prepared before the randomization and after the treatment. The WM therapy included voltaren extended action tablet, methotrexate and sulfasalazine. The CM therapy included Glucosidorum Tripterygii Totorum Tablet and syndrome differentiation treatment. The American College of Rheumatology 20 (ACR20) was taken as efficacy evaluation. All data were analyzed on SAS 8.2 statistical package. The relationships between each variable and efficacy were analyzed, and the variables with P<0.2 were included for the data mining analysis with neural network model. All data were classified into training set (75%) and verification set (25%) for further verification on the data-mining model. RESULTS: Eighteen variables in CM and 24 variables in WM were included in the data-mining model. In CM, morning stiffness, swollen joint number, peripheral immunoglobulin M (IgM) level, tenderness joint number, tenderness, rheumatoid factor (RF), C-reactive protein (CRP) and joint pain were positively related to the efficacy, and disease duration and more urination at night negatively related to the efficacy. In WM, erythrocyte sedimentation rate (ESR), weak waist, white fur in tongue, joint pain, joint stiffness and swollen joint were positively related to the efficacy, and yellow fur in tongue, red tongue, white blood negatively related to the efficacy. In the analysis with the neural network model in the patients of verification set, the predictive response rates of 20% patients would be 100% and 90% in the treatment with CM and WM, respectively. CONCLUSION: Neural network model analysis, based on the full clinical trial data with collection of both traditional Chinese medicine and modern medicine diagnostic information, shows a good predictive role for the information in the efficacy in rheumatoid arthritis.

OBJECTIVE: To evaluate the correlations between clinical symptoms and treatment efficacy in patients with rheumatoid arthritis (RA). METHODS: Four hundred and thirteen patients were included in the clinical trial from 9 clinical centers. They were randomly divided into Western medicine-treated group with 204 cases and Chinese herbal drug-treated group with 209 cases. Eighteen clinical symptoms were evaluated before and after treatment. The Western medicine therapy included voltaren extended release tablets, methotrexate and sulfasalazine. The Chinese herbal drug therapy included glucosidorum Tripterygii totorum tablets and Yishen Juanbi Tablets combined with treatment based on syndrome differentiation. The American College of Rheumatology 20 (ACR20) was used as efficacy evaluation criteria. RESULTS: In the Chinese herbal drug-treated group, clinical symptoms such as arthralgia and tenderness of joints were positively correlated with the efficacy after 12-week treatment, while frequent urination at night was negatively correlated. In the same group, tenderness of joints and fever were positively correlated with the efficacy after 24-week treatment, while deep-colored and turbid urine was negatively correlated. In the Western medicine-treated group, tenderness of joints and thirst were positively correlated with the efficacy after 12-week treatment, while vertigo was negatively correlated. And in the same group, tenderness of joints was positively correlated with the efficacy after 24-week treatment, while heaviness of limbs was negatively correlated to the efficacy. The statistical results showed that the treatment efficacy was improved when the correlated symptoms were included in the indications. CONCLUSION: The treatment efficacy of RA is correlated with some symptoms, so further studies should proceed on these correlations in order to achieve better treatment outcome.

HLA-A* 2402 is one of the most frequently encountered HLA-A alleles in East Asian populations. In order to study the CD8+ T cell responses in Chinese populations, we have described the generation and functional test of HLA-A* 2402 tetramer loaded with HCMV pp65(341-349) peptide (QYDPVAALF, QYD). The cDNA of HLA-A* 2402 heavy chain was cloned by RT-PCR from one of the donors. DNA fragment encoding the ectodomain of HLA-A* 2402 heavy chain fused at its carboxyl-terminal a BirA substrate peptide (BSP) was amplified by PCR with the cloned heavy chain cDNA as a template. The wild-type gene of HLA-A* 2402-BSP was not expressed in Escherichia coli (E. coli), while mutant HLA-A* 2402-BSP gene with optimized codons was overexpressed as inclusion bodies in E. coli. Furthermore, the soluble HLA-A* 2402-QYD monomers were generated by in vitro refolding of washed inclusion bodies in the presence of beta2-microglobulin and QYD peptide. The tetramer was subsequently formed by mixing HLA-A* 2402-QYD monomers with streptavidin-PE at a molar ratio of 4:1. Flow cytometry analysis indicated that this tetramer possessed binding activity with specific CTL from HLA-A24+ donors and the frequencies of tetramer-binding CTL were 0.09% - 0.37% within total CD8+ T cells. This tetrameric agent provides a powerful tool to explore the secrets of CTL responses against HCMV antigens in HLA-A* 2402 individuals.
Amino Acid Sequence ; CD8-Positive T-Lymphocytes ; cytology ; metabolism ; Carbon-Nitrogen Ligases ; metabolism ; Electrophoresis, Polyacrylamide Gel ; Escherichia coli ; genetics ; Escherichia coli Proteins ; metabolism ; Flow Cytometry ; Gene Expression ; HLA-A Antigens ; chemistry ; genetics ; metabolism ; HLA-A24 Antigen ; Humans ; Oligopeptides ; genetics ; metabolism ; Phosphoproteins ; chemistry ; genetics ; metabolism ; Protein Multimerization ; Recombinant Fusion Proteins ; chemistry ; genetics ; metabolism ; Repressor Proteins ; metabolism ; Substrate Specificity ; T-Lymphocytes, Cytotoxic ; cytology ; metabolism ; Viral Matrix Proteins ; chemistry ; genetics ; metabolism

The aim of this study was to establish a stable subline of K562 cells expressing the HLA-A(*)1101 protein, which was expected to provide target cells for characterizing the HLA-I restrictive antigen specific cytotoxic T lymphocyte (CTL) effects against chronic myeloid leukemia (CML). The HLA-A(*)1101 protein encoding gene was amplified from peripheral blood mononuclear cell (PBMNC) of CML patient by RT-PCR; the 2A peptide linker (D-V-E-X-N-P-G-P) gene was linked to the 3'terminal of the HLA-A(*)1101 gene by recombinant PCR, then the recombinant was cloned into the pEGFP-N3 plasmid which contains an enhanced green fluorescent protein gene, and the eukaryotic recombinant expression vector containing HLA-A(*)1101-T2A-EGFP transcription box was constructed; the pEGFP-N3 vector and recombinant vector was separately electroporated into K562 cells. The expression of GFP was monitored by fluorescence microscopy, finally stably transfected sublines of K562 cells containing HLA-A(*)1101 gene, and of K562 containing pEGFP-N3 vector were obtained by G418 selection; the transcriptional or translational expression of HLA-A(*)1101 gene was detected with RT-PCR and flow cytometry respectively. The results indicated that the eukaryotic expression vector HLA-A(*)1101-T2A-EGFP plasmid was successfully constructed; after G418 selection for 2 months, two sublines of K562 cells (HLA-A(*)1101(+)K562, pEGFP-N3(+)K562) expressing GFP were constructed. The expression of HLA-A*A1101 gene could be determined in HLA-A(*)1101(+)K562 cell line by RT-PCR, while the pEGFP-N3(+)K562 cells could not express HLA-A*A1101 gene. HLA-A(*)1101 protein and GFP double positive HLA-A(*)1101(+)K562 cells were up to 88.5%, which was obviously higher than pEGFP-N3(+)K562 cells (0.698%) by flow cytometric analysis. It is concluded that a simple and effective method to select HLA-A(*)1101(+)K562 cells has been established and a subline of K562 cell expressing HLA-A(*)1101 protein on its cell membrane was successfully constructed, which provides the tool cells for further studying the specific cellular immunity against-CML.
Genetic Vectors ; HLA-A11 Antigen ; genetics ; Humans ; K562 Cells ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; Leukocytes, Mononuclear ; Plasmids ; Transfection

AIM: To compare the efficacy and safety of tigecycline with polymyxin B in the treatment of carbapenem resistant enterobacteriaceae (CRE) pneumonia in critically ill patients. METHODS: A retrospective analysis was performed on the clinical data of patients with CRE pneumonia who received tigecycline or polymyxin B therapy from January 1, 2018 to Jun 30, 2023 in the Intensive Care Unit (ICU). Primary outcomes included the 28-day all-cause mortality and clinical cure rate within 28days. Secondary outcomes included the ICU mortality, in-hospital mortality, the length of hospital stay and ICU stay, microbial eradication, duration of mechanical ventilation. Independent predictors affecting 28-day clinical cure rate were tested using Cox regression analyses. RESULTS: A total of 83 eligible patients were included in the final analysis after propensity score matching, 54 in the tigecycline group and 29 in the polymyxin B group. The 28-day all-cause mortality was 31.5% (17/54) in the tigecycline group and 37.9% (11/29) in the polymyxin B group, the difference was not statistically significant (P=0.554); the clinical cure rate was 63% (34/ 54) in the tigecycline group, which was significantly higher than that of the polymyxin B group of 34.5% (10/29) (P = 0.013). There were no statistical differences between the two groups in terms of secondary outcomes. Multivariate logistic regression analysis found that the use of tigecycline was an independent predictor of the 28-day clinical cure rate (HR 2.083, 95%CI 1.018-4.263, P = 0.045). However, activated partial thromboplastin time (APTT) and prothrombin time (PT) were significantly prolonged in the tigecycline group compared with the polymyxin B group (P=0.047; P=0.027), and fibrinogen (FIB) was significantly decreased (P < 0.001) after drug administration. CONCLUSION: There was no significant difference in 28-day all-cause mortality between the tigecycline and polymyxin groups; tigecycline might be associated with a higher 28-day clinical cure rate compared with polymyxin B. It should be noted that tigecycline may increase the risk of coagulation abnormalities.

OBJECTIVETo investigate the clinical results of the proximal femoral nail antirotation (PFNA) system in the treatment of unstable intertrochanteric femoral fractures.

METHODSFrom September 2006 to September 2009, 90 patients (40 males and 50 females, ranged in age from 64 to 95 years with an average of 73.2 years with unstable intertrochanteric femoral fractures were surgically treated with PFNA. Fifty patients had the fractures in the right hip, and 40 patients had the fractures in the left hip. The fractures were classified according to the AO classification: 11 patients were type A2.1, 21 patients were type A2.2,25 patients were type A2.3 9 patients were type A3.1,6 patients were A3.2 and 18 patients were A3.3. The patients underwent surgery within a mean of 3.2 days(ranged,2 to 20.1 days) from injury. The mean hospital stay was 12.8 days(ranged,7 to 24 days). Closed reduction was achieved in all the patients. Harris hip score were used for the evaluation of clinical effects.

RESULTSThe mean operation time was 36.8 min (ranged, 23 to 110 min) and the mean blood loss was 150 mi (ranged, 100 to 500 ml). The mean follow-up period was 12 months (ranged, 6 to 24 months). All the patients had fracture union. Sixty-nine patients got excellent reduction, 14 good and 7 bad. The mean collodiaphysial angle was 135.60 (ranged, 1260 to 1470). Postoperative complications included secondary varus in 2 patients,calcification at the tip of the greater trochanter in 5 patients, medial thigh pain in 7 patients,and screw cut-out in 1 petient. Ten patients had femoral shortness (mean 9.3 mm,ranging from 8 to 14 mm). The mean Harris hip score was (80.5 +/- 9.8). According to Harris hip scores evaluation system, 26 patients reached an excellent result, 37 good, 18 poor and 9 bad.

CONCLUSIONDue to advantages of high union rate, short operation time, and early postoperative mobilization, PFNA osteosynthesis is an idea method for surgical treatment of unstable intertrochanteric femoral fractures.

Aged ; Aged, 80 and over ; Bone Nails ; Female ; Fracture Fixation, Internal ; methods ; Hip Fractures ; diagnostic imaging ; surgery ; Humans ; Male ; Middle Aged ; Radiography

This study reports the preparation and identification of soluble programmed death-1 (PD-1) ligand-1 (sPD-L1) and its antibodies of mouse origin. Immobilized metal ion affinity chromatography was used to perform on-column refolding with simultaneous purification of denatured sPD-L1, and soluble sPD-L1 with purity of 95% was obtained. The purified sPD-L1 was verified by immunoblotting using a commercial goat-anti-human PD-L1 antibody. An ELISA-based assay showed that it also had high binding activity for its cognate receptor PD-1. Furthermore, mouse anti-sPD-L1 antiserum of high titer was raised using the purified sPD-L1 as an immunogen, and the specific IgG antibodies were purified using sPD-L1-HiTrap affinity chromatography. In addition, a sensitive sandwich ELISA was established using the purified IgG antibodies together with the commercial goat antibodies. In conclusion, the preparation of soluble sPD-Ll and its antibodies provide the basis for detection of the potential anti-PD-L1 antibodies and soluble PD-L1 in humans as well as for further investigation of its in vivo bioactivities and characterization of its potential receptors.
Animals ; Antibodies ; immunology ; isolation & purification ; Antigens, CD ; genetics ; immunology ; metabolism ; B7-H1 Antigen ; Chromatography, Affinity ; Electrophoresis, Polyacrylamide Gel ; Enzyme-Linked Immunosorbent Assay ; Escherichia coli ; genetics ; Female ; Humans ; Immune Sera ; immunology ; Immunoblotting ; Mice ; Mice, Inbred C57BL ; Recombinant Proteins ; immunology ; isolation & purification ; metabolism ; Solubility

Objective:To explore the effect of combined administration of intravenous and topical tranexamic acid on perioperative blood loss in elbow arthrolysis.Methods:A retrospective analysis was conducted of 31 patients who had undergone elbow arthrolysis due to elbow stiffness from April 2019 to November 2020 at Department of Orthopaedic Trauma, Beijing Jishuitan Hospital. An observational group of 15 patients were subjected to combined administration of intravenous and topical tranexamic acid while a control group of 16 patients to no administration of tranexamic acid. In the observational group, 15 mg/kg of tranexamic acid was injected intravenously 5 to 10 minutes before surgery and 1.0 g of tranexamic acid was injected locally in the area of anterior and posterior joint capsules after incision was closed while drainage tubes were clamped for 2 hours before release. In the control group, there was no special operative procedure while drainage tubes were also clamped for 2 hours before release. The 2 groups were compared in terms of blood loss on day 1 and day 3 after operation, drainage volume on day 1 after operation, total drainage volume, time for indwelling drainage tube, complications, and Mayo elbow performance score (MEPS) at 3 months after operation.Results:There were no statistically significant difference in preoperative general data between the 2 groups, showing they were comparable ( P>0.05).On day 1 and day 3 after operation, the blood loss was respectively (533.4±318.3) mL and (792.0±375.6) mL in the observational group, and respectively (866.4±480.5) mL and (1,403.0±636.5) mL in the control group, showing significantly differences between the 2 groups ( P<0.05). The drainage volume on day 1 after operation was (151.3±90.1) mL in the observational group and (235.0±126.1) mL in the control group, showing a significant difference between the 2 groups ( P<0.05). There was no statistically significant difference in total drainage volume or time for indwelling drainage tube between the 2 groups ( P>0.05). There were no such complications as thromboembolic events in either group. There was no significant difference in MEPS between the 2 groups at 3 months after operation ( P>0.05). Conclusions:Combined administration of intravenous 15 mg/kg and topical 1.0 g tranexamic acid may reduce blood loss on day 1 and day 3 after operation and drainage volume on day 1 after operation, and may not increase the risk of thromboembolic events, but cannot reduce total drainage volume or time for indwelling drainage tube. Application of tranexamic acid may not affect early elbow joint function after operation.

BACKGROUND:As a routine method after lumbar spine surgery,a drainage tube is convenient for postoperative bleeding drainage and management,and there is still no consensus on the choice of postoperative removal time for short-segment lumbar spine surgery with less risk. OBJECTIVE:To explore the effect of different drainage times on early clinical efficacy after short-segment lumbar fusion. METHODS:A prospective randomized controlled study was performed on 220 patients in the Affiliated Hospital of Southwest Medical University who underwent posterior lumbar interbody fusion for lumbar degenerative diseases from March 2017 to April 2021.According to the different drainage times,the patients were randomly divided into removal on the second day after operation(group A),removal on the third day after operation(group B),and removal after the observation method 24-hour drainage volume<30 mL(group C).The perioperative indicators and follow-up results of the three groups of patients were observed and compared. RESULTS AND CONCLUSION:(1)Because 7 patients were lost to follow-up,2 patients were excluded,and 211 patients were finally included(72 patients in group A,71 patients in group B,and 68 patients in group C).(2)The average drainage time of group C was 2.91 days.The postoperative drainage volume in group A was significantly less than that in groups B and C,and the difference was statistically significant(P<0.05).On day 3 after operation,the hematocrit value of group C was lower than that of group A and group B,and the difference was statistically significant(P<0.05).Postoperative activity time and hospital stay in group A were shorter than those in groups B and C,and the difference was statistically significant(P<0.05).(3)Four patients in group A,two patients in group B and three patients in group C received an allogeneic blood transfusion.There was no significant difference among the groups(P>0.05).(4)In terms of postoperative complications,there were no statistical differences in postoperative wound leakage and surgical site infection in all three groups(P>0.05).(5)All patients were followed up for more than 12 months.Visual analog scale score and Oswestry dysfunction index of the three groups of patients before discharge and at the last follow-up were significantly improved compared with those before surgery(P<0.05).There was no statistical significance among the groups(P>0.05).(6)It is indicated that the removal of the drainage tube on the second day after a posterior lumbar fusion can effectively reduce the time to get out of bed and hospital stay,without increasing the postoperative blood loss and the risk of complications.