An electrochemical sensor was fabricated for determination of cholesterol ( ChO ) based on the molecularly imprinting and signal amplification of graphene. The sensor was prepared by electropolymerizing phenol on the surface of graphene modified glassy carbon electrode with ChO as template. The structure, properties and molecular imprinting effect of the imprinted membrane were studied by SEM, CV and DPV. The results showed that the sensor had good selectivity and high sensitivity to cholesterol. The calibration graph for the determination of cholesterol was linear in the range of 8. 0 × 10-8-2. 0 × 10-4 mol/L, with the detection limit (S/N=3) of 5. 6×10-8 mol/L. The sensor was applied to the determination of cholesterol in human serum samples with satisfactory results.

OBJECTIVETo explore the role of CD44 in monocyte adhesion to human brain microvascular endothelial cells (HBMECs) and monocyte migration across an in vitro model of blood-brain barrier (BBB) infected by Cryptococcus neoformans (Cn).

METHODSAn in vitro blood-brain barrier model was constructed using a transwell chamber covered with a HBMEC monolayer. The wild-type strain of Cn B4500FO2, TYCC645#32 strain with CPS1 gene deletion and PCIP strain with CPS1 complementation were chosen to infect the monolayer HBMECs. THP-1 cells were added to the upper chamber of transwell, and the relative migration rate was determined by counting the number of the cells entering the lower chambers. The inhibitory effects of anti-CD44 monoclonal antibody and the CD44 inhibitor bikunin were examined on THP-1 binding to and migration across HBMECs.

RESULTSCn infection of the HBMECs caused markedly enhanced THP-1 cell adhesion and migration across the monolyers (P<0.01) dependent on Cn concentration and exposure time. Addition of anti-CD44 monoclonal antibody and bikunin significantly lowered THP-1 adhesion and migration rates in the BBB model with Cn-infected HBMECs (P<0.01) with a dose dependence of the antibody (within 0-1 µg) and inhibitor (within 0-20 nmol/L). Both THP-1 adhesion rate and migration rate were lowered in the BBB model infected with CPS1 gene-deleted Cn but increased in the model infected with the complemented strain compared with those in the wild-type strain-infected model.

CONCLUSIONIn the in vitro BBB model, CD44 expressed on HBMECs may play an essential role in monocyte adhesion to and migration across the BBB. The capsular hyaluronic acid may mediate Cn-induced monocyte adhesion and migration.

Blood-Brain Barrier ; immunology ; microbiology ; Brain ; cytology ; microbiology ; Cell Line ; Cryptococcosis ; immunology ; Cryptococcus neoformans ; Endothelial Cells ; microbiology ; Humans ; Hyaluronan Receptors ; metabolism ; Monocytes ; cytology

OBJECTIVETo investigate the effect of c-Met inhibitor cabozantinib (XL-184) in inhibiting Listeria monocytogenes (LM) from invading Caco-2 cells to reduce the cell injury.

METHODSThe cell invasion capacity of LM was assayed in Caco-2 cells incubated with different doses of XL-184 for different durations. Caco-2 cells incubated with XL-184 were seeded on the upper room of the transwell chamber, and the cell monolayer was exposed to LM infection followed by addition of horseradish peroxidase (HRP). The trans-epithelial electric resistance (TEER), HRP concentration and LM colony-forming unit (CFU) were measured in the cell monolayer. Fluorescent staining was used to evaluate the cell viability, and LDH release from the cells was examined to assess the changes in cell membrane permeability.

RESULTSXL-184 significantly decreased LM invasion rate in Caco-2 cells in a dose- and time-dependent manner (P=0.000), and this effect was enhanced by co-incubation of the cells with ampicillin (P<0.05). In the cell membrane permeability assay in the monolayer cells, XL-184 markedly inhibited LM-induced reduction of TEER (P<0.05) and significantly suppressed LM-induced enhancement of cell membrane permeability shown by reduced HRP concentration and LM count in the lower chamber (P=0.000). The cells infected with LM showed significantly lowered cell viability, which was rescued by XL-184 (P<0.01); XL-184 also dose-dependently reduced LDH release from the cells (P<0.05).

CONCLUSIONSXL-184 can suppress LM invasion in Caco-2 cells to reduce the cell injury, suggesting its value as a promising candidate agent for prevention and treatment of LM infections.

Anilides ; pharmacology ; Caco-2 Cells ; Cell Membrane Permeability ; drug effects ; Cell Survival ; Humans ; Listeria monocytogenes ; drug effects ; Pyridines ; pharmacology

OBJECTIVETo investigate the effects of Lactobacillus rhamnosus GG (LGG) for inhibiting E.coli K1 (E44) adhesion and invasion of an intestinal epithelial cell model with Muc2 gene knockdown established using CRISPR-Cas9 system.

METHODSTwo 20-25 bp sgRNAs targeting Muc2 were chemically synthesized to construct CRISPR expression vectors for transfection in wild-type human colonic cancer cell line Ht29. The efficiency of Muc2 knockdown was determined using Western blotting. After assessment of the viability and proliferation of the transfected cells with MTT assay, we evaluated the effects of the probiotics against E44 adhesion and invasion of the cells through a competitive exclusion assay.

RESULTSTransfection of the cells with Lenticrisprv2 plasmid vectors resulted in a cell line with stable Muc2 knockdown by 81%. The inhibitory effects of probiotics against E44 adhesion and invasion of the transfected cells were markedly attenuated, and the relative adhesion and invasion rates of E44 were 72.23% (P<0.05) and 81.49% (P<0.05), respectively.

CONCLUSIONMuc2 knockdown causes attenuation of the inhibitory effects of probiotics against E44 adhesion and invasion of the intestinal epithelial cells, suggesting that up-regulation of Muc2 may serve as an important mechanism for the probiotics to reinforce the intestinal barrier and antagonize the pathogenic bacteria, which sheds light on a new strategy for prevention and treatment of bacterial intestinal infections.

Bacterial Adhesion ; CRISPR-Cas Systems ; Epithelial Cells ; cytology ; microbiology ; Escherichia coli ; pathogenicity ; Gene Knockdown Techniques ; HT29 Cells ; Humans ; Intestines ; cytology ; Lactobacillus rhamnosus ; Mucin-2 ; genetics ; Probiotics ; Transfection ; Up-Regulation

The study was aimed to investigate the factor V coagulation activity (FV:C), and to evaluate FVgene polymorphisms and activated protein C resistance (APCR) in the patients with venous thromboembolism (VTE). 95 patients with VTE and 95 normal controls were investigated for FV gene polymorphisms. FV Leiden, FVCambridge, and FVHong Kong were detected by PCR, MnlI and BstNI digestion respectively. FVAsp79His and FVI359T were detected by MassARRAY. FV:C and APCR in 65 patients with VTE and 60 normal controls were determined by a one-stage clotting method and the APTT-based assays respectively. The results showed that the mean levels of plasma FV:C were significantly higher in VTE group than that in controls (108.03% +/- 28.29% vs 95.17% +/- 29.75%) (P = 0.008), the incidence of APCR were 20.0% (13 of 65 cases) in patients with VTE and 5.0% (3 of 60 cases) in normal controls (P = 0.012). FV Leiden, FVCambridge, FVHong Kong, FVAsp79His and FVI359T mutations were not found in two groups. It is concluded that the increased plasma level of FV:C is a risk factor for VTE. There is APCR in both groups, APCR is also a risk factor to VTE. APCR may not be associated with mutations of FV Leiden, FVCambridge, FVHong Kong, FVAsp79His and FV I359T polymorphisms, other factors need to study further in APCR.
Activated Protein C Resistance ; complications ; genetics ; Adolescent ; Adult ; Aged ; Aged, 80 and over ; Factor V ; genetics ; metabolism ; Female ; Humans ; Male ; Middle Aged ; Polymorphism, Genetic ; Protein C ; metabolism ; Venous Thromboembolism ; blood ; complications ; genetics ; Young Adult

OBJECTIVE: To investigate the gene mutation types and spectrum of α, β-thalassemia in Fuzhou area of China. METHODS: Thalassemia gene screening was performed in the women receiving physical, prenatal, and pre-pregnancy examination, and the patients with suspected thalassemia in our hospital from July 2013 to March 2018.Genotypes of thalassem were detected by Gap-PCR and RDB-PCR. RESULTS: 1042 were positive among 2074 suspected cases with a positive rate of 50.24%; 618 cases were confirmed to be α-thalassemia and with a positive rate of 29.8%; 409 cases were confirmed to be β-thalassemia with a positive rate of 19.72%. 15 cases were confirmed to be α-β complex thalassemia with a positive rate of 0.72%. the --/αα(76.54%) was the most common genotype among α-thalassemia, -α/αα(10.03%) and -α/αα(2.91%) in hot pursuit. In addition, IVS-II-55 (T->G) and IVS-II-119 (-G, +CTCGGCCC) were newly found alpha mutations; the IVS-2-654 (C→T) (40.83%) was the most common genotype among β-thalassemia, CD41-42 (-TCTT) (35.94%) and CD17 (A→T) (9.78%) in hot pursuit. CONCLUSION The genotype of thalassemia in Fuzhou area is highly heterogenic, --/αα is the most common genotype among α-thalassemia, IVS-2-654 (C→T) is the most common genotype among β-thalassemia, Meanwhile, two α-mutation sites are found in this study which were not reported in the Database of Human Hemoglobin Variants and Thalassemias.
China ; Female ; Genotype ; Humans ; Mutation ; Pregnancy ; alpha-Thalassemia ; beta-Thalassemia

OBJECTIVETo investigate the expression of N-cadherin in bone marrow leukemic cells derived from acute leukemia patients and its clinical significances.

METHODSA total of 113 patients with acute leukemia were enrolled in this study. Flow cytometry was employed to detect the expression of N-Cadherin in bone marrow leukemic cells from acute leukemia patients and the relationships between the N-cadherin expression and the clinical characteristics of patients with acute leukemia were analyzed.

RESULTSThe expression of N-Cadherin in bone marrow leukemic cells deriveted from patients with acute leukemia was variable with 0%-99.7%. For adult AML patients, the positive rate of CD34 in N-cadheringroup was significantly higher than that in N-cadheringroup(67.39% vs 33.33%)(P=0.013), while the differences of total CR rate and rate of CR after 1 cycle of induction treatment were not significant between these 2 groups(P>0.05). As to ALL patients, N-cadheringroup had significant lower WBC count (21.31±7.07 vs 51.10±23.69)(P=0.008) and lower percentage of peripheral blood blast (43.22±5.75% vs 66.45±5.65%)(P=0.015). The CR rate after 1 cycle of induction treatment and rate of overall CR were lower and the relapse rate was higher in N-cadherinALL group than those in N-cadherinALL group, but the differences were not significant (P>0.05). For childhood ALL, the positive rate of CD33 in N-cadheringroup was significantly higher than that in N-cadheringroup(47.62% vs 0%)(P=0.012). The relapse rate was higher in N-cadheringroup than that in N-cadheringroup (30.00% vs 0%)(P=0.115). The median survival time, 3-year overall OS rate and 3-year relapse-free survival rate in N-cadheringroups of adult AML, non-M3 AML, ALL and chidhood ALL paients were superior to N-cadheringroups, but the differences were not significant.

CONCLUSIONThe expression of N-cadherin in bone marrow leukemic cells relates to some clinical features of patients with acute leukemia and to some extent has inferior effect on survival of patients with acute leukemia.

OBJECTIVEThis study was to investigate the expression of miR-10a in the different FAB subtype of acute myeloid leukemia (AML) and its relationship with drug resistance.

METHODSForty de novo patients with AML, 16 patients with non-malignant hematologic disease and three AML cell lines HL-60, U937 and HL-60/ADR were enrolled in this study, the MiR-10a expression in bone marrow mononuclear cells of above-mentioned patients and 3 AML cell lines was detected by TaqMan RT-PCR. The correlation of miR-10a with clinicopathological factors of AML patients was analyzed.

RESULTSThe miR-10a expression level in HL-60 cell line was higher than that in U937 cell line (P = 0.039). And its expression level in de novo AML patients was higher than that in patients with non-malignant hematologic disease (P < 0.01). FAB-AML-M3 patients exhibited higher expression of miR-10a than that in M1, M2 and M4 (P < 0.05); HL-60/ADR cell line showed higher miR-10a expression than that in HL-60 cell line (P < 0.01) . Except M3, the patients without CR (non-CR) after the first cycle of chemotherapy showed a higher level of miR-10a as compared with CR patients (P < 0.01).

CONCLUSIONThe high expression of miR-10a may be closely related to over-proliferation of promyelocyte and drug resistance of acute myeloid leukemia cells, except M3.

Cell Line, Tumor ; Drug Resistance, Neoplasm ; Humans ; Leukemia, Myeloid, Acute ; MicroRNAs