OBJECTIVETo determine whether a novel compound, 3-(4-bromophenyl)-2-(ethyl sulfonyl)-6-methylquinoxaline 1, 4-dioxide (Q39), induces apoptosis in human leukemia cell line k562 in hypoxic environment.

METHODSMTT assay was used to determine the 50% inhibitory concentrations (IC50). Flow cytometry and DAPI staining were employed to determine the apoptosis; JC-1 staining was used to determine mitochondria membrane potential (DeltaPsim); Western-blotting was used to determine protein expression of procaspase-3, cleaved caspase-3, PARP, Bax, Bcl-2 and HIF-1alpha.

RESULTSIn hypoxic environment, Q39 exerted higher antiproliferative activity in K562 cells, and the IC50 value was (0.21+/- 0.05) micromol/L. The apoptotic phenomenon was observed at 6 h after cells exposed to Q39, and apoptotic body emerged as exposure time increased. After K562 cells were incubated with Q39 for 0, 6, 12 and 24 h, the ratio of apoptotic cells was 2.8%, 3.2%, 5.9% and 19.2%, respectively. By fluorescence stain assay, an significant Delta Psim loss in K562 cells induced by Q39 was shown in a time-dependent manner. Western blot assay demonstrated that Q39 decreased the protein expression of Bcl-2, procaspase-3, and HIF-1alpha, meanwhile increased protein expression of Bax and cleaved caspase-3, and induced the cleavage of PARP.

CONCLUSIONSThe novel compound Q39 exhibits great anticancer activity against K562 cells in hypoxic environment. Q39 can downregulate the protein expression of HIF-1alpha, and regulate the apoptosis-related protein expression to cause a drop of DeltaPsim, suggesting that mitochondria and HIF-1alpha pathway might be involved in the antiproliferative effect of Q39.

Antineoplastic Agents ; pharmacology ; Apoptosis ; drug effects ; Blotting, Western ; Caspase 3 ; metabolism ; Cell Hypoxia ; Flow Cytometry ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit ; metabolism ; K562 Cells ; Membrane Potential, Mitochondrial ; drug effects ; Poly(ADP-ribose) Polymerases ; metabolism ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Quinoxalines ; pharmacology ; bcl-2-Associated X Protein ; metabolism

Objectives To investigate the clinical features,prognosis and risk factors of patients with cryptococcal meningitis.Methods Totally 146 patients with cryptococcal meningitis who were hospitalized in Huashan Hospital from January 2000 to December 2006 were enrolled in this study.The clinical data including diagnosis and misdiagnosis,experimental and etiology tests,treat- ments and prognosis from all the patients were analyzed retrospectively.Results Among the 146 patients enrolled in the study,78 patients(53.4%)had concomitant diseases.The misdiagnosis rate of all patients was 72.6%(106/146).The positive rate of cerebrospinal fluid(CSF)India ink smear was 59.6%(87/106),while 43.2%(63/146)cases of cryptococcus neoformans culture in CSF was positive.The positive rate of Latex agglutination test(LAT)was 91.7%(134/146)in CSF among all patients.The treatments were as follows:combination of Amphotericin B(AmpB)or its lipid formula- tions with flucytosine(5-FC)(98 cases),including combination with Fluconazole initally(62 cases), single therapy of Fluconazole(13 cases).Ommaya implanted for lateral cerebral ventricle drainage(53 cases)and AmpB intrathecal injection(53 cases).The average dose of AmpB is 3.06 g.The course of treatment lasted from 12 weeks to 20 months.There were 104 patients(71.2%)cured,27(18.5%) improved,15(10.3%)died and 34(23.3%)relapsed.Conclusions High misdiagnosis rate is common in patients with cryptococcal meningitis.Immunodeficiency is the major risk factor for cryp- tococcal meningitis.CSF LAT is the most sensitive diagnostic test.Early diagnosis,combination of AmpB with 5-FC antifungal therapy and control of acute intracranial hypertension are the keys to im- prove prognosis of cryptococcal meningitis.

AKT/mTOR/STATs signaling pathway not only plays an important role in tumor growth, but also in the regulation of immune system. Activated AKT promotes the activation of downstream signaling pathways mTORC1 and mTORC2 through phosphorylation. mTOR is currently being considered as an important regulator of immune system and plays an important role in regulating the function and metabolism of various immune cells. Multiple sclerosis (MS) is an autoimmune deficiency disease whose pathogenesis has not been fully elucidated. This article focuses on the regulation of AKT/mTOR/STATs signaling pathways in various immune cells such as macrophage M1/M2 polarization, B cell proliferation and differentiation, helper T lymphocyte (Th cell) proliferation and differentiation, and Treg cell proliferation and differentiation, which would be helpful to illustrate the role of the AKT/mTOR/STATs signaling pathway in mediating the regulation of immune cells in MS.

Objective Metabolites produced by metabolic imbalance such as free fatty acids and lipopolysaccharides can result in a state of chronic low-grade inflammation, or metabolic inflammation, which plays an important role in the pathogenesis of atherosclerosis, type 2 diabetes, non-alcoholic fatty liver disease, and obesity. The above metabolic disorders are closely related with the metabolic inflammation, which always coexist. Therefore, we proposed the concept ofmetabolic inflammatory syndrome ( MIS). According to our study, patients with two or more metabolic disorders above could be diagnosed as MIS. The current research is aimed to investigate the prevalence of MIS and its components, and to compare the clinical values of MIS and metabolic syndrome ( MS) . Methods 2 001 in patients with type 2 diabetes from 6 hospitals in Shanghai were recruited in the current multi-center cross-sectional study. The diagnostic rates of MIS and MS and their components of both syndromes were compared. Results In the patients with type 2 diabetes, the detective rate of MIS was 96. 2%, which was higher than that of MS (71. 3%). Among 4 components of MIS, atherosclerosis showed the highest detective rate (75.6%). MIS[OR=2.252(95%CI1.026-4.942),P=0.043],atherosclerosis[OR=2.726(95% CI1.953-3. 804),P<0. 001], and MS[OR=1. 915 (95%CI 1. 444-2. 540),P<0. 01] were the risk factors of coronary heart disease. Conclusion With atherosclerosis, type 2 diabetes mellitus, non-alcoholic fatty liver disease, and obesity as its 4 components, MIS has a high detective rate in patients with metabolic disorders, and seems to be more sensitive than MS to distinguish inflammation-related metabolic diseases. The concept of MIS will promote the screening and prevention of atherosclerosis in its early stage.

OBJECTIVETo assess the operative technique and results with the usage of cementless prosthesis in hip revision.

METHODSRetrospective study was done on revision of total hip arthroplasty with cementless prosthesis in 72 patients (41 males and 31 females) with an average age of 65.7 years (28-82 years) from January 2004 to December 2009. The reason for revision was 2 infection, 54 aseptic loosening, 4 periprosthetic fractures, 5 fracture of femoral stems and 7 cases of acetabular abrasion after hemi-arthroplasty. The operation time, bleeding loss, complications of infection, dislocation, periprosthetic fractures and loosening were evaluated. The Harris score were used for hip function evaluation.

RESULTSThe average operation time was (146±47) minutes (70-280 minutes) and bleeding loss during the operation was (970±540) ml (200-2500 ml). Bacterium cultivation during operation demonstrated infection in 2 patients. Bone windows at the lateral femoral were opened in 4 patients and extend trochanteric osteotomy was done in 7 patients. Fracture of the proximal femur occurred in 8 cases. Twenty-nine patients were treated with bone graft including 18 autografts and 11 allografts. Sixty-seven patients were followed up for an average time of 66 months (20-92 months). Additional revisions were performed in 3 cases including 2 dislocations and 1 infection. There were no death, no damage of major blood vessels and nerves. The bone graft healed during 3-5 months. The survival rates of the femoral prosthesis and the acetabulum prostheses were 95.5% and 98.4%. The mean Harris score was 86±8 (55-95 points). Osteolysis were seen in 13 hips but migration was seen in only 1 patient.

CONCLUSIONSThe cementless prosthesis is useful in revision total hip arthroplasty and the perfect clinical results are related to the reliable primary fixation.

Adult ; Aged ; Aged, 80 and over ; Arthroplasty, Replacement, Hip ; instrumentation ; Bioprosthesis ; Bone Transplantation ; Female ; Follow-Up Studies ; Hip Prosthesis ; Humans ; Male ; Middle Aged ; Retrospective Studies

Objective: To construct the expression vector pLCK-CD69-IRES-EGFP that contains mouse cell surface activation protein CD 69 and enhanced green fluorescent protein ( EGFP ) , and to generate CD69 transgenic mice based on this vector . Methods:First, RNA was extracted from mouse lung tissue and cDNA was synthesized via reverse transcription .PCR primer was designed through the PubMed searching , then mouse CD69 DNA fragment was amplified with PCR .Second, this DNA fragment was subcloned to the pInsulater-LCK-IRES-EGFP plasmid and constructed the transgenic vector after the verification of nucleotide sequence .Third, the expression vector was then transfected into 293 T cells and its expression in 293 T cells was observed under fluorescence microscope .Last , microinjection was performed to transfer the expression vector pLCK-CD69-IRES-EGFP into fertilized eggs , which were implanted into pseudo-pregnant recipient mice .After birth the tail samples of the pups were obtained for the purpose of genotyping to determine the transgenic founders . Fluorescence microscope and flow cytometer were used to measure the expression of CD69 on cells.Results: The construction of the expression vector pLCK-CD69-IRES-EGFP was verified by enzyme digestion and DNA sequencing .The transfected 293 T cell showed expression of the protein under fluorescence microscope .Identification of PCR for the tail tissue of the pups confirmed the present of CD 69 transgene and resting lymphocytes demonstrated the expression of CD 69 .Conclusion: The construction of expression vector pLCK-CD69-IRES-EGFP and generation of CD69 transgenic mice have been successfully processed , which lays a foundation of the solid pattern studies in inflammatory diseases .

Aim To establish human U87-MG glioma model in nude mice brain and to observe the characteristics of the tumor growth. Methods Human U87-MG glioma cells were cultured in vitro. 5 μL of cell suspension containing 3.0 ×1010·L-1, 4.0×1010·L-1and 5.0×1010·L-1respectively was inocula-ted into the right caudate nucleus of 18 male nude mice brain un-der the guidance of stereotaxic apparatus, separately, whereas another 6 nude mice as the control group, were inoculated into the same volume of Hanks solution. The moving and survival state of rats with gliomas were observed. The examinations of the tumors formation, volumes, metastasis and histopathology were performed and the obtained brain samples were stained with HE and immunohistochemistry. Results All the tested rats of dif-ferent inoculation doses developed brain tumors without extracra-nial metastasis. The mean survival time of three groups was (46.50 ± 3.27) d,(38.50 ± 3.28) d and (30.67 ± 3.51) d,respectively. The tumors showed the similar morphological fea-tures and immunophenotype to human glioma. There was positive expression of GFAP and S-100 in the tumors. Conclusions The orthotopic implantation model of human U87-MG glioma, by in-oculating quantitative U87-MG cells stereotaxically into the brains of the nude mice, is successfully established with 100 yield of intracranial tumor and no extracranial growth extension. It resembles the histopathological and morphological features of human glioma,which can be used as a reliable animal model for the study of the tumorigenesis, pathogenesis, biological charac-teristics and therapy of glioma.

Objective To evaluate the detection accuracy of the biomarkers dickkopf-1, DCP and AFP as a serum biomarker panel by comparing the sensitivity of the panel with those of the individual biomarkers. Methods The study was composed of three groups, one with HCC patients, one with non-HCC liver diseases and one with healthy controls. Serum AFP was measured using a chemiluminescence assay and serum dickkopf-1 and DCP were measured with ELISA. The sensitivity and specificity of the biomarkers were analyzed as single parameters and as a serum panel. Results The HCC group showed higher levels of dickkopf-1, DCP and AFP than the other two groups (P < 0.05). Dickkopf-1 showed better sensitivity (73.26% vs. 58.13%, P < 0.05) and better specificity (44.0% vs. 29.0%, P < 0.05) than AFP. DCP also had better sensitivity (74.42% vs. 58.13%, P < 0.05) than AFP, but their specificity was similar (30.00% vs. 29.00%, P > 0.05). The combination of the biomarkers as a serum panel produced much better sensitivity (93.02%) and specificity (78.00%) than each of the markers individually (P < 0.05). Conclusion The combination of AFP, DCP and dickkopf-1 as a biomarker panel can significantly improve the detection power with much higher sensitivity and specificity for HCC than any of the biomarkers alone. The tests are convenient and inexpensive, and may serve as a valuable addition to current options for the diagnosis of HCC.

OBJECTIVETo construct the expression vector pLCK-CD69-IRES-EGFP that contains mouse cell surface activation protein CD69 and enhanced green fluorescent protein(EGFP),and to generate CD69 transgenic mice based on this vector.

METHODSFirst, RNA was extracted from mouse lung tissue and cDNA was synthesized via reverse transcription. PCR primer was designed through the PubMed searching, then mouse CD69 DNA fragment was amplified with PCR. Second, this DNA fragment was subcloned to the pInsulater-LCK-IRES-EGFP plasmid and constructed the transgenic vector after the verification of nucleotide sequence. Third, the expression vector was then transfected into 293 T cells and its expression in 293 T cells was observed under fluorescence microscope. Last, microinjection was performed to transfer the expression vector pLCK-CD69-IRES-EGFP into fertilized eggs, which were implanted into pseudo-pregnant recipient mice. After birth the tail samples of the pups were obtained for the purpose of genotyping to determine the transgenic founders. Fluorescence microscope and flow cytometer were used to measure the expression of CD69 on cells.

RESULTSThe construction of the expression vector pLCK-CD69-IRES-EGFP was verified by enzyme digestion and DNA sequencing. The transfected 293 T cell showed expression of the protein under fluorescence microscope. Identification of PCR for the tail tissue of the pups confirmed the present of CD69 transgene and resting lymphocytes demonstrated the expression of CD69.

CONCLUSIONThe construction of expression vector pLCK-CD69-IRES-EGFP and generation of CD69 transgenic mice have been successfully processed, which lays a foundation of the solid pattern studies in inflammatory diseases.

Animals ; Antigens, CD ; genetics ; Antigens, Differentiation, T-Lymphocyte ; genetics ; DNA, Complementary ; Genetic Vectors ; Genotype ; Green Fluorescent Proteins ; genetics ; Lectins, C-Type ; genetics ; Mice ; Mice, Transgenic ; Plasmids ; Polymerase Chain Reaction ; Sequence Analysis, DNA ; Transfection

Pneumonia caused by SARS-CoV-2 has seriously threatened human life and health worldwide and caused a large number of deaths. Viral infection and acute inflammation are important causes of death, so it is particularly important to combine antiviral therapy with anti-inflammatory therapy. Glycyrrhizic acid, the main component of the glycyrrhizic root extract, has a wide range of pharmacological effects as well as high efficiency and low toxicity, its preparation has been widely used in the treatment of chronic hepatitis and other diseases. Glycyrrhizic acid can regulate the expression and release of a variety of cytokines and play a significant anti-inflammatory effect. At the same time, glycyrrhizic acid also showed significant inhibition towards a variety types of viruses. Therefore, the potential application of glycyrrhizic acid as COVID-19 treatment should be explored.