Staphylococcus aureus is a major cause of hospital-acquired infection. Because the bacteria are very easy to become resistant to antibiotics, vaccination is a main method against S. aureus infection. Clumping factor B (ClfB) is an adhesion molecule essential for S. aureus to colonize in the host mucosa and is regarded as an important target antigen. In this study, we successfully used Escherichia coli to express a segment encoding the N1-N3 regions of ClfB protein (Truncated-ClfB) cloned from S. aureus. The protein was purified by affinity and ion exchange chromatographies and gel filtration. Rabbits were immunized three times with purified Truncated-ClfB. After that, blood was collected to prepare serum which were then used for measurement of antibody level. Phagocytosis of S. aureus opsonized by the serum was determined by a flow cytometry. Results show that the serum IgG titer reached 1:640 000. Phagocytosed S. aureus by polymorphonuclear leukocytes were significantly more when the bacteria were opsonized by the serum from Truncated-ClfB immunized rabbits than those from no immunized group (P < 0.01). Therefore, the results indicated that Truncated-ClfB could be a promising vaccine candidate against S. aureus infection.
Adhesins, Bacterial ; immunology ; Animals ; Antibodies, Bacterial ; blood ; Escherichia coli ; Flow Cytometry ; Immune Sera ; Immunoglobulin G ; blood ; Opsonin Proteins ; immunology ; Phagocytosis ; Rabbits ; Staphylococcal Infections ; immunology ; Staphylococcus aureus

Objective To construct shifting mutant of cysteine-rich region to 3?@terminal of Tat gene of human immunodeficiency virus type-1 (HIV-1) HXB2 strain, and to analyze the immunogenicity of mutant protein (Tat-cct) after prokaryotically expressed and purified. Methods The cysteine-rich region (nucleotides 64--111) of Tat gene was shifted to 3'terminal of Tat of HIV-1 HXB2 strain by polymerase chain reaction (PCR) and Tat mutant DNA sequence was obtained. Prokaryotie express plasmid pET32a-Tat-cct was constructed and transformed into E. coli BL21 (DE3), then Tat-cct protein was expressed and purified. BALB/c mice were immunized with the fusion protein Tat-cct, and immunogenicity of the immunized serum was detected by enzyme-linked immunosorbent assay (ELISA). Results The recombinant plasmid pET32a-Tat-cct expressed in E. coli BL21 (DE3) and the relative molecular mass of the purified fusion protein was 31 000. The serum antibody titer of mice immunized with Tat-cct recombinant protein was 1 : 1600, which binded specifically with both Tat-ect protein and Tat protein (amino acids 1-101). Conclusions The recombinant protein Tat-cct of Tat mutant strain can be expressed efficiently in E. coli and well retains immunogenicity, which provides valuable information for basic research of HIV-1 Tat vaccine.

Objective Deleting the cysteine-rich region (22-37 amino acids)of HIV-1 HXB2 Tat protein(whole length is 101 amino acids) to improve its stability and expression level in E.coli and to analyze the immunogenicity of Tat protein without the cystein-rich region [Tat(△C)protein]. Methods Tat DNA deleted the cysteine-rich region (64-111 nucleotides), named as Tat(△C)DNA, was obtained in vitro by PCR and cloned into pET-32a vector. pET-32a-Tat(△C)plasmid and the pET-32a-Tat plasmid were established and transformed into E.coli BL21(DE3) strains respectively to express and purify the protein. Three rabbits were vaccinated with pET-32a-Tat(△C)protein, then testify the reactivity of sera from rabbits by ELISA and Western blot. Results The dense of the purified pET-32a-Tat(△C)protein was 7.12 mg/ml,which was greatly more than pET-32a-Tat protein(1.50 mg/ml). Dimer of pET-32a-Tat protein can be observed just after the protein purification and stored at 25℃ and 4℃ for 7 days, but dimer of pET-32a-Tat(△C)protein was not formed at the same condition. Experimental rabbits were immunized with pET-32a-Tat(△C)protein and produced high titre of anti-pET-32a-Tat(△C)serum(1∶320 000), the antibody can react specifically with Tat(△C)protein, Tat protein (1-101 AA)and synthetic Tat(1-86 AA) protein. Deletion mutation of the cysteine-rich region of Tat protein was first performed in the study. Conclusion The expression level in E.coli and the stability of Tat protein deleted the cysteine-rich region can be increased greatly, and the protein remains good immunogenicity. The results may provide a novel antigen for further development of HIV-1 Tat vaccine.

Objective:To study on the effect of ultrasound-guided thoracic paravertebral nerve (TPVB) block on quality of recovery from general anesthesia in tuberculosis patients with fiberboard exfoliation in post anesthesia recovery unit (PACU).Methods:From May 2018 to December 2019, 40 tuberculosis patients in Changsha Central Hospital with pulmonary fibreboard exfoliation and focal abscess lesions cleaning were randomly divided into two groups, with 20 patients in each group. The patients in group A received endobronchial general anesthesia and in group B received ultrasound-guided TPVB combined with endobronchial general anesthesia. Patients in the two groups were maintained under anesthesia by propofol, and the bispectral index (BIS) was maintained within the range of 40-50. The dosage of propofol and sufentanil was adjusted according to changes in BIS and hemodynamics. The mean arterial pressure (MAP), heart rate (HR) in two groups of patients were recorded at before anesthesia induction (T 0), before cutting leather (T 1), cut skin after (T 2), the end of operation (T 3), extubation time (T 4), and T 5 (time of leaving PACU). The visual analogue scale (VAS) of all patients in resting and cough state was recorded at 5, 30 min after extubation and the time of leaving PACU. The dosage of propofol and sufentanil in the operation and the additional dosage of sufentanil in PACU were recorded in both two groups. And the respiratory recovery time, consciousness recovery time, extubation time and sedation agitation scale(SAS) were observed. The adverse reactions such as nausea, vomiting, drowsiness and hypotension were observed in PACU. Results:Compared with group A, MAP and HR of patients at T 2, T 3, T 4, T 5 in group B were more stable during anesthesia, and VAS of patients in group B were lower than that in group A at each time point after extubation ( P<0.05). The dosage of sufentanil and propofol in group B were (35.92±8.12)μg and (749.56±95.30)mg respectively, which were significantly lower than those in group A [(45.74±4.42)μg and (862.83±105.34)mg, P<0.05]; the dosage of sufentanil in postoperative anesthesia recovery room of group B was (5.26±2.10)μg, significantly less than that of group A (10.35±5.86)μg ( P<0.05). The respiratory recovery time, consciousness recovery time and extubation time in group B were (12.92±5.12) min, (20.56±5.10) min and (26.87 ± 6.16) min, which were shorter than those in group A [(15.74±4.72)min, (25.83±5.34)min and (35.35±5.80)min, P<0.05]. The incidence of postoperative nausea, vomiting, lethargy and hypotension in group B were 10%, 10%, 35% and 20%, which were significantly lower than those in group A (30%, 20%, 75% and 45%, P<0.05). Conclusions:Ultrasound-guided paravertebral nerve block may significantly reduce the dosage of opioid analgesics for general anesthesia in tuberculosis patients with fiberboard exfoliation, accelerate the speed of anesthesia recovery, reduce the agitation during recovery, and improve the quality of anesthesia recovery.

Objective To evaluate the safety,efficacy and comfort of dexmedetomidine sedation combined with ultrasound-guided ilioinguinal and ilioinguinal nerve block in open inguinal hernia surgery for elderly patients with underlying diseases.Methods 40 elderly patients for open inguinal hernia surgery with underlying diseases were selected and randomly divided into group A and group B.Group A recived simple nerve block,while group B recived dextropyrimidine sedation combined with nerve block.The heart rate (HR),mean arterial pressure (MAP),SpO2,Ramsay sedation score and visual analogue score (VAS) at T0 (entering operating room),T1 (anesthesia puncture),T2 (15 min after anesthesia),T3 (surgical incision),T4 (hernia sac free),T5 (operation ending) in both two groups were recorded,and the intraoperative anesthesia effect were evaluated.The satisfaction rate,incidence of bradycardia,bradycardia and respiratory depression were compared between the two groups;and observed the sufentanil using,lidocaine using and the occurrence of adverse reactions.Results There was no significant difference of SpO2 at each time point between the two groups (P ＞ 0.05);HR,MAP and Ramsay sedation scores at T0 between the two groups had no significant difference (P ＞ 0.05);compared with group A,HR,MAP and Ramsay scores at T1-T5 in group B decreased and Ramsay scores increased,while VAS at T1,T3,T4 and T5 in group B were lower (P ＜ 0.05);in group B,local anesthesia with lidocaine at puncture point,intraoperative sufentanil and body movement were significantly reduced,and the overall satisfaction rate of patients was high (P ＜ 0.05).There were no obvious adverse reactions in both groups.Conclusions Dexmedetomidine sedation combined with ilioinguinal/iliohypogastric nerve block is safe and effective in elderly patients with open hernia surgery combined with basic diseases,and it can improve the comfort of patients during anesthesia puncture and operation more than simple nerve block.

This study was aimed to investigate the effect of Nan-She-Teng (Celastrus orbiculatus) extract on epithe-lial-mesenchymal transition(EMT) in HepG2 cells. Except the control group, human hepatocellular carcinoma HepG2 cells in other groups were treated with Celastrus Orbiculatus extract in different concentrations (10, 20, 40, 80, and 160 μg·mL-1). The protein expression levels related to EMT were detected by western blotting. At 48 h after fertiliza-tion, the zebrafish embryos were randomly assigned to 7 groups as follows: untreated control group (E3 buffer), DMSO group (E3 buffer with 1% DMSO), and different dosages treatment of C.orbiculatus extract (10, 20, 40, 80, and 160μg·mL-1) for 24 h. The protein expressions of mTOR signaling pathways were detected by western blotting. The re-sults showed that compared with the control group, C.orbiculatus extract significantly increased E-cadherin protein expression. Simultaneously, C.orbiculatus extract inhibited vimentin and mTOR signaling pathways at protein levels. It was concluded that to a certain extent, C.orbiculatus extract prevented EMT in HepG2 cells by modulating the mTOR signaling pathway. Therefore, it suggested that mTOR can be chosen as a new therapeutic target for clinical treatment of hepatic carcinoma.

OBJECTIVE:To investigate clinical efficacy of Sofren injection combined with Vinpocetine injection in the treatment of acute massive cerebral infarction,and its effects on hemorheological indexes and serum NOS.METHODS:A total of 60 patients with acute massive cerebral infarction in our hospital during Jan.2014-Jun.2016 were selected as research objects and divided into trial group and control group according to random number table,with 30 cases in each group.Control group was given Citicoline injection 0.5 g,ivgtt,qd.Trial group was additionally given Vinpocetine injection 20 mg added into 0.9％ Sodium chloride injection 250 mL,ivgtt,qd;1 h later washing tube,they were given Sofren injection 10 mL added into 0.9％ Sodium chloride injection 250 mL,ivgtt,for consecutive 14 d.Clinical efficacies and safety of 2 groups were observed,and hemorheological indexes and NOS levels were observed before and after treatment.RESULTS:The total response rate (83.33％)of trial group was significantly higher than that (50.00％) of control group,with statistical significance (P＜0.05).Before treatment,there was no statistical significance in hemorheological indexes or serum NOS levels between 2 groups (P＞0.05).After treatment,hemorheological indexes of 2 groups were decreased significantly,and the trial group was significantly lower than the control group.The level of serum NOS in 2 groups were increased significantly,and the trial group was significantly higher than the control group,with statistical significance (P＜0.05).No obvious ADR was found in 2 groups.CONCLUSIONS:Sofren injection combined with Vinpocetine injection show significant therapeutic efficacy for acute massive cerebral infarction,can reduce blood viscosity and increase blood perfusion with good safety.

Objective: To investigate and compare the advantages of surgical treatment of ingrown toenail with the traditional Winograd method. Methods: From Aug. 2013 to Jun. 2015, 77 cases (89 toes) with 15-58 years were involved in the study. 37 cases (42 toes) were treated with Winograd method and other 40 cases (47 toes) were treated with a method using lateral incision without cutting the nail fold. The time of returning to normal activities, infection rate, recurrence rate, pain VAS score and shape satisfaction VAS score were evaluated to display the aesthetic and functional result of the both methods. Results: All patients were followed up with an average of 8.6 months. The patients treated with the novel method returned to normal activities after (5.3±0.5) d post-surgery, which was much shorter than that (7.3±0.6) d in patients with the Winograd method (P<0.05). And the infection rate in the lateral incision group was also lower than that in the Winograd method group (P<0.05). At 3 days post-surgery, the pain VAS score in Winograd method group was higher (P<0.05), but the difference of pain VAS score disappeared after 1 and 6 months post-surgery (P>0.05). The shape satisfaction VAS score were 9.2±0.6 in the lateral incision group and 7.1±0.7 in the Winograd group, showing significant difference (P<0.05). Conclusions The lateral incision method without incising the nail fold reserves the nail groove and brings faster recovery to normal activities with less pain, lower infection rate and more satisfactory toenail shape.

Objective:To investigate the analgesic effect of ultrasound-guided pericapsular nerve group (PENG) block combined with laryngeal mask general anesthesia and internal fixation of proximal femoral nail antirotation (PFNA) under ERAS concept in elderly patients.Methods:A total of 50 elderly patients with femoral trochanteric fractures treated with PFNA internal fixation who were admitted to Changsha Central Hospital from January 2021 to March 2022 were selected and according to the random number table method, they were divided into groups P and F, with 26 cases in group P (ultrasound-guided PENG block combined with laryngeal mask general anesthesia group) and 24 cases in group F [ultrasound-guided fascia iliaca compartment block (FICB)] combined with laryngeal mask general anesthesia group]. The bispectral index (BIS) of the two groups of patients was maintained within the range of 40-60, and the dosage of cyclopofol and remifentanil was adjusted according to the BIS and hemodynamic changes. Postoperative oxycodone was used for patient controlled intravenous analgesia (PCIA). When the Visual Analogue Scale (VAS) score ≥4, 1 mg intravenous oxycodone was administered as rescue analgesia. The VAS score was observed and recorded before nerve block (T 0), 10 minutes after nerve block (T 1), 20 minutes after nerve block (T 2), when the patients were placed in body position after bed (T 3), after laryngeal mask removal (T 4), 12 h after operation (T 5), 24 h after operation (T 6) and 48 h after operation (T 7). The mean arterial pressure (MAP), heart rate (HR) and blood oxygen saturation (SpO 2) at each time point from T 0 to T 4 were observed and recorded; the intraoperative dose of remifentanil, the rescue dose of oxycodone at postoperative T 6 and T 7, Lovett muscle strength score, anesthesia operation time, anesthesia recovery time and postoperative anesthesia-related complications were recorded. Results:There was no significant difference between the two groups in VAS scores at T 0, T 4, T 5 and T 6, MAP, HR and SpO 2 at T 0, T 1, T 2 and T 4, the dosage of remifentanil during operation, the remedial dosage of oxycodone at T 6 after operation, anesthesia operation time and anesthesia recovery time (all P>0.05). Compared with group F, the VAS scores of group P at T 1, T 2, T 3 and T 7 were lower than those of group F (all P<0.05); At T 3, there was no significant difference in SpO 2 between the two groups ( P>0.05), but the MAP and HR in group P was lower than that in group F (all P<0.05); The remedial dose of oxycodone at T 7 in group P was lower than that in group F ( P<0.05), and the analgesic effect was longer; Lovett muscle strength score at T 6 and T 7 in group P was better than that in group F (all P<0.05). There was no significant difference in the incidence of postoperative complications between the two groups ( P>0.05). Conclusions:Compared with FICB, ultrasound-guided PENG block used in PFNA internal fixation in elderly patients has faster effect, lower dosage of opioid analgesics, longer duration of analgesia, and less impact on postoperative lower limb muscle strength.

We constructed a phage-displayed random mutation library of Tat38-61(51N/55N), for studying the molecular evolution screening of HIV-1 Tat38-61 epitope. We used primers containing the random nucleotide sequences, and introduced the random mutations at the sites of 51 and 55 amino acids coding sequences into full-length Tat sequences by overlapping PCR. With the randomly mutated full-length Tat as template, the Tat38-61(51N/55N) mutants which contained recognition sequences for the Xba I in both ends were amplified by PCR using the designed primers. The mutants were cloned into Xba I site in the phagemid vector pCANTAB5S, then the recombinants were transformed into E. coli TG1, a phage-displayed the random mutation library of Tat38-61(51N/55N) was constructed by the rescue of help virus M13KO7. The results showed that the library consisted of about 5.0 x 10(6) colonies and the phage library titer was 2.65 x 10(12) TU/mL. More than 56.50% colonies in the library were positive for insertion. Sequence analysis showed that the nucleotides encoding amino acids at the sites of 51 and 55 distributed randomly. The constructed mutation library could meet the requirements for the following molecular evolution screening, and might prepare the Tat mutants for the further study of new Tat vaccine candidates.
AIDS Vaccines ; immunology ; Escherichia coli ; genetics ; metabolism ; HIV-1 ; genetics ; Humans ; Mutation ; Peptide Fragments ; biosynthesis ; genetics ; immunology ; Peptide Library ; Recombinant Proteins ; biosynthesis ; genetics ; immunology ; tat Gene Products, Human Immunodeficiency Virus ; biosynthesis ; genetics ; immunology