Aim To explore the concentration range of organic solvent which can both effectively increase solubility of the difficult soluble medicine monomer, and have low toxicity to cells, and to clarify the influence of different concentration of ethanol on curcumol efficacy.Methods Different DMSO and ethanol concentrations were diluted in culture medium and incubated with cells A549, NCI-H460, NCI-H1299, NCI-1650, LTEP-a2 and SPC-A1 for 12 h, 24 h, or 48 h, cell viability was tested by a colorimetric assay with 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide(MTT), and different concentrations of ethanol with/without different concentrations of curcumol were also prepared with culture medium, then incubate with A549 and NCI-H460 cells for 24 h, and cell viability was tested by MTT as well.Results Within 48 h the solution with 0.008(V/V) DMSO or less had no significant effect on the cell A549 compared with control group, for NCI-H1650 the concentration was 0.004(V/V) or less, for NCI-H460 the result turned to be 0.002(V/V) or less, and the solution with DMSO below 0.001(V/V) had significant effect on the three other cells, NCI-H1299, LTEP-a2 and SPC-A1.While within 48 h, the liquor with 0.004(V/V) ethanol or less did not exhibit significant cytotoxic effect on the cell A549, for NCI-H460 and NCI-H1650 the result of ethanol concentration became 0.002(V/V) or less, for NCI-H1299 the data was 0.001(V/V) or less, and the liquor with ethanol below 0.001(V/V) showed significant cytotoxic effect on LTEP-a2and SPC-A1.When the proportion of ethanol in solution was below 0.01, it has no cytotoxic on cell A549 and NCI-H460, and the curcumol solution prepared with this kind of ethanol solution only represented the efficacy of curcumol on the cells.Since the solution with 0.01(V/V) ethanol dissolved the curcumol better, the cell viability changed from about 100% to about 35%.Conclusions Different organic solvent expressed different toxicity to the same cell, and the sensitivity of different cells to one organic solvent is dissimilar.and DMSO could be the optimum solvent for A549 and NCI-H1650, while the optimum solvent of NCI-H1299 is ethanol, for NCI-H460 it could be both and DMSO and ethanol, and DMSO and ethanol is not suitable for LETP-a2, SPC-A1 to be solvent.

Objective To analyze the platelet activating factor acetylhydrolase ( PAF-AH) activity of a gene product encoded by LA2144 gene of Leptospira interrogans ( L. interrogans) , to investigate the ex-pression and secretion of LA2144 protein in various cell cultures and to further understand its function in in-ducing internal hemorrhage in an animal model. Methods The DNA sample containing LA2144 gene was extracted from L. interrogans serogroup Icterohaemorrhagiae serovar Lai strain Lai and used as the template for gene cloning by PCR. The LA2144 gene without the signal sequence coding region was amplified by PCR and inserted into a prokaryotic expression construct for the protein expression. The expressed recombinant protein, rLep-PAF-AH, was purified by Ni-NTA affinity chromatography. Spectrophotometry was used to measure the hydrolytic activity, hydrolytic efficiency, Km and Kcat values of the rLep-PAF-AH protein in hydrolyzing PAF substrate. Real-time fluorescent quantitative RT-PCR ( qRT-PCR) and Western blot assay were performed to measure the expression of LA2144 gene at mRNA and protein levels in human umbilical vein endothelial cells (HUVEC), human monocytes (THP-1) and murine macrophages (J774A. 1) with L. interrogans strain Lai infection, respectively. Each syrian hamster was intravenously injected with 100 μg of LPS-free rLep-PAF-AH for two times. Hemorrhage in the lungs, livers and kidneys were observed in three days after the injection. Results The constructed prokaryotic expression system for LA2144 gene of L. inter-rogans strain Lai could highly express the rLep-PAF-AH upon the induction of IPTG. The purified rLep-PAF-AH showed high purity with a single protein band in gel as indicated by SDS-PAGE. The efficiency of 5 μg of rLep-PAF-AH in hydrolyzing PAF substrate was 26. 6 U/L with a Km value of 82. 79 μmol/L and a Kcat value of 0. 24 S-1 . The expression of Lep-PAF-AH at mRNA level in HUVEC, THP-1 and J774A. 1 cells were significantly elevated after co-culture with L. interrogans strain Lai for 1 or 2 hours (P<0. 05). A large amount of Lep-PAF-AH were detected in the supernatants from co-cultures of L. interrogans strain Lai with the three cell lines, but not from the culture of the spirochete in EMJH medium. The signs of hemor-rhage were observed in the lung of hamsters injected with rLep-PAF-AH, but not in tissue samples from liver and kidney. Conclusion The LA2144 gene product was characterized by a stronger PAF-AH activity. The expression of LA2144 gene at mRNA and protein levels in various cell lines were enhanced during L. interro-gans infection. Moreover, the rLep-PAF-AH could induce the pulmonary hemorrhage in hamsters. This stud-y indicated that the protein encoded by LA2144 gene was an important virulence factor causing hemorrhage in hosts during L. interrogans infection.

Objective: To establish a rapid bacterial identification and antimicrobial susceptibility testing assay in positive blood cultures, so as to provide insights into timely diagnosis and treatment of bloodstream infections. Methods: A total of 1 154 blood culture samples were collected from inpatients in Zhejiang Hospital from February to May, 2022. The bacterial isolates were enriched and purified using improved separation gel method, and bacterial identification and antimicrobial susceptibility tests were performed using VITEK2 mass spectrometry system and VITEK2 Compact automated microbiology system. The accuracy of the new assay for bacterial identification and antimicrobial susceptibility tests was evaluated with the conventional VITEK 2 compact system as the standard. Results: Of 1 154 blood culture specimens, the conventional VITEK 2 compact system detected 174 positives and 980 negatives. The new assay and the conventional VITEK 2 compact system identified consistent bacterial isolates in 165 out of 174 positive blood culture samples, and the accuracy of bacterial identification was 94.83% for the new assay, with a 99.21% accuracy for identifying Gram-negative bacteria and 82.22% for Gram-positive bacteria. Antimicrobial susceptibility tests were performed in 158 bacterial isolates, and the new assay presented a 90.17% accuracy, with a 90.27% accuracy for Gram-negative bacteria and 89.74% for Gram-positive bacteria. The conventional VITEK 2 compact system required 30 hours and longer to complete bacterial identification and antimicrobial susceptibility tests, and the new assay required 9 to 18 hours. Conclusions The new rapid bacterial identification and antimicrobial susceptibility testing assay shortens the time of bacterial culture, achieves rapid bacterial identification and antimicrobial susceptibility testing in blood culture specimens and has a high accuracy that meets clinical needs, which facilitates rapid diagnosis and treatment of bloodstream infections.

Objective:To evaluate the role of Sigma-1 receptor (Sigma-1R) in pentazoxine-induced reduction of oxygen-glucose deprivation and restoration (OGD/R) injury in SH-SY5Y cells and the relationship with endoplasmic reticulum stress (ERS).Methods:The well-growing SH-SY5Y cells were divided into 4 groups ( n=6 each) using a random number table method: control group (group C), OGD/R group (group O), OGD/R+ pentazoxin group (group OP) and OGD/R+ pintazoxin+ BD1047 group (group OPB). The cells in group C were normally cultured. In O group, OP group and OPB group, the culture medium was replaced with EBSS medium, and then the cells were cultured in an incubator of 5% CO 2-95% N 2 at 37 ℃ for 4 h, then replaced with DMEM/F12 medium containing 10% fetal bovine serum for restoration of O 2-glucose supply for 18 h, and in addition pentazoxin (final concentration 10 μmmol/L) was added during restoration in OP group, and pentazoxin (final concentration 10 μmmol/L) and Sigma-1R blocker BD1047 (final concentration 20 μmol/L) were added during restoration in OPB group. The apoptosis rate was detected by flow cytometry at the end of restoration, and the expression of Sigma-1R, C/EBP homologous protein (CHOP), phosphorylated inositol-requiring enzyme 1 (p-IRE1), spliced X-box binding protein 1 (XBP1s), and activated caspase-3 (c-cas-3) was detected by Western blot. Results:Compared with group C, the apoptosis rate was significantly increased, and the expression of CHOP and p-IRE1 was up-regulated in O group, OP group and OPB group, the expression of XBP1s and c-cas-3 was significantly up-regulated in O group and OPB group ( P<0.05), and no significant change was found in the expression of Sigma-1R, XBP1s and c-cas-3 in OP group ( P>0.05). Compared with O group, the apoptosis rate was significantly decreased, the expression of Sigma-1R was up-regulated, and the expression of CHOP, p-IRE1, XBP1s and c-cas-3 was down-regulated in OP group ( P<0.05). Compared with OP group, the apoptosis rate was significantly increased, the expression of Sigma-1R was down-regulated, and the expression of CHOP, p-IRE1, XBP1s and c-cas-3 was up-regulated in OPB group ( P<0.05). Conclusions:Sigma-1R is involved in pentazoxine-induced reduction of OGD/R injury in SH-SY5Y cells, and the mechanism may be related to inhibition of endoplasmic reticulum stress.

Objective To retrospectively analyze the factors of postoperative recurrence of stage pT1-3NoM0 esophageal squamous cell carcinoma.Methods A total of 488 patients who underwent two-field R0 esophagectomy,pathologically classified as stage pT1-3N0M0,without adjuvant radiotherapy and/or chemotherapy before or after surgery and postoperative survival time ≥ 3 months were enrolled in this study.Multivariate analysis was performed by using Cox model.Results At the end of follow-up,the overall recurrence rate was 36.9％(180/488);the local recurrence rate was 21.5％ (105/488),the distant metastasis rate was 6.8％ (33/488) and the local recurrence rate complicated with the distant metastasis rate was 8.6％ (42/488).Cox multivariate analysis demonstrated that tumor site and pT staging were the factors affecting the overall/local recurrence rate and distant metastasis.The recurrence rate in patients with the upper esophageal squamous cell carcinoma and stage pT3 was the highest,followed by those with the middle esophageal squamous cell carcinoma or stage pT2 and the lowest recurrence rate was observed in patients with the lower esophageal squamous cell carcinoma or stage pT1.Conclusions Tumor site and pT staging are the pivotal factors for postoperative recurrence of stage pT1-3 NoM0 esophageal squamous cell carcinoma after two-field R0 esophagectomy,which contributes to offer guidance to the selection of indications for postoperative adjuvant radiotherapy.

OBJECTIVES: This study aimed to build a surgical navigation system based on mixed reality (MR) and optical positioning technique and evaluate its clinical applicability in craniomaxillofacial trauma bone reconstruction. Me-thods We first integrated the software and hardware platforms of the MR-based surgical navigation system and explored the system workflow. The systematic error, target registration error, and osteotomy application error of the system were then analyzed via 3D printed skull model experiment. The feasibility of the MR-based surgical navigation system in craniomaxillofacial trauma bone reconstruction was verified via zygomatico-maxillary complex (ZMC) reduction experiment of the skull model and preliminary clinical study. RESULTS: The system error of this MR-based surgical navigation system was 1.23 mm±0.52 mm, the target registration error was 2.83 mm±1.18 mm, and the osteotomy application error was 3.13 mm±1.66 mm. Virtual surgical planning and the reduction of the ZMC model were successfully conducted. In addition, with the guidance of the MR-based navigation system, the frontal bone defect was successfully reconstructed, and the clinical outcome was satisfactory. CONCLUSIONS The MR-based surgical navigation system has its advantages in virtual reality fusion effect and dynamic navigation stability. It provides a new method for doctor-patient communications, education, preoperative planning, and intraoperative navigation in craniomaxillofacial surgery.
Humans ; Surgical Navigation Systems ; Augmented Reality ; Plastic Surgery Procedures ; Skull/surgery*