This study aims to analyse the value of CODEHOP RT-PCR in the detection of Flavivirus. According to the amino acid sequences of polyproteins of different flaviviruses published in GenBank, a pair of primers was designed using the CODEHOP method. One-step RT-PCR was used to detect Japanese encephalitis virus strain JEV1201, Dengue virus strain JKD001, and yellow fever virus vaccine YV6161. BLAST analysis and phylogenetic analysis were performed after the RT-PCR products of nucleocapsid genes were sequenced. The results showed that this method could amplify Flavivirus specifically, and the size and sequence of the target fragment accorded with the anticipated result. JEV1201 had the highest homology to Japanese encephalitis virus strain YL2009-4/YC2009-3, belonging to the branch of the phylogenetic tree of Japanese encephalitis virus strains. JKD001 had the highest homology to Dengue virus strain DENV-2/ID/1022DN/1975, belonging to the branch of the phylogenetic tree of Dengue virus strains. YV6161 had the highest homology to Yellow fever virus strain 17D, belonging to the branch of the phylogenetic tree of Yellow fever virus strains. In conclusion, the method of CODEHOP RT-PCR can be effectively used to detect, identify, and phylogenetically analyse Flavivirus.
DNA Primers ; genetics ; Flavivirus ; classification ; genetics ; isolation & purification ; Flavivirus Infections ; virology ; Humans ; Molecular Sequence Data ; Phylogeny ; Reverse Transcriptase Polymerase Chain Reaction ; methods ; Viral Nonstructural Proteins ; genetics

OBJECTIVETo investigate the expression of beta-dystroglycan (beta-DG) and the roles of tissue inhibitor of metalloproteinases (TIMPs) on beta-DG in salivary adenoid cystic carcinoma (SACC).

METHODSbeta-DG in highly lung metastatic cell line ACC-M and lowly lung metastatic one ACC-2 was tested by immunocytochemistry with different concentrations (10, 15, 20, 25 micromol x L(-1)) of TIMPs, and that without the regulation of TIMPs was served as controls. beta-DG was detected in seven specimens of SACC and ten cases of normal salivary gland tissues which were considered as a comparison group by immunohistochemistry.

RESULTSThere was no positive beta-DG immune-staining at the ACC-2 and ACC-M cell lines without TIMPs in the cell culture. beta-DG expressed after the regulation of TIMPs. beta-DG expression was localized predominantly in basement membrane of the acinus, while the negative results were distributed in the carcinoma cells and around the cancer cell nests.

CONCLUSIONBeta-DG is widely expressed by transmembrane protein that plays important roles in connecting the extracellular matrix to the cytoskeleton, the fracture of this structure means that it is easy to invade and transfer, so restoration of beta-DG expression by TIMPs is considered to be critical for successful treatment of SACC.

Carcinoma, Adenoid Cystic ; Cell Line, Tumor ; Dystroglycans ; Humans ; Immunohistochemistry ; Salivary Gland Neoplasms ; Tissue Inhibitor of Metalloproteinases

OBJECTIVETo investigate the methods of culturing and identifying mouse myeloid semimature dendritic cell (smDC) in vitro.

METHODSMyeloid monocytes derived from 6-week-old C57 BL/6 mice were cultured in RPMI-1640 medium containing 10% fetal bovine serum, 2 ng/ml recombinant murine granulocyte macrophage-colony stimulating factor (GM-CSF), and 20 ng/ml recombinant murine interleukin (IL)-4 for 9 days. Then cells were incubated with 40 ng/ml tumor necrosis factor-alpha (TNF-alpha) for 24 hours to obtain smDC. Meanwhile, smDC was differentiated into mature dendritic cell (mDC) or immature dendritic cell (iDC) by treatment with 1 micro/m1 lipopolysaccharide (LPS) or without LPS. The morphological features of smDC were assayed by inverted microscopy and scanning electron microscopy. Surface markers such as CD11c, CD4O, CD8O, CD86, and MHC-II were tested by flow cytometry. IL-1beta, IL-6, IL-12, and IL-10 in the supernatant were tested by ELISA. The activation of allogene lymphocyte (BALB/c mice) stimulated by C57BL/6 myeloid smDC in mixed lymphocyte reaction was examined by Cell Counting Kit-8 in vitro.

RESULTSThe shape of smDC was round or oval-shaped, and the diameter of smDC was about 15 microm. The length of smDC dendrite was between 5 to 10 microm. smDC, iDC, and mDC all expressed high level of CD11 c. The expressions of MHC-II, CD40, CD80, and CD86 on smDC were higher than those of iDC and lower than those of mDC. IL-1beta, IL-6, and IL-12 secretion of smDC was significantly lower than that of mDC (P < 0.01), and IL-12 was significantly lower than that of iDC (P < 0.05), while no significant difference of IL-1beta and IL-6 secretion was found between smDC and iDC (P > 0.05). Furthermore, IL-10 secretion was not significantly different among these three kinds of DCs (P > 0.05). The effect of allogene lymphocytes activation on smDC was significantly lower than that of mDC and positive control (P < 0.01), but had no significant difference when compared with that of iDC and negative control (P > 0.05).

CONCLUSIONSsmDC may be a relatively independent dendritic cell sub-population in terms of function and morphology. It is a feasible way to induce myeloid monocytes to differentiate into smDC using GM-CSF, IL-4, and TNF-alpha in vitro.

Animals ; Cell Culture Techniques ; Cell Differentiation ; Cells, Cultured ; Cytokines ; immunology ; Dendritic Cells ; cytology ; immunology ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Monocytes ; cytology ; immunology

OBJECTIVETo investigate the operative method and effect of the reversed sural neurocutaneous flap in repairing soft tissue defects of the anterior foot and the ankle.

METHODSFrom March 1999 to November 2004, 19 cases (17 male, 2 female) of soft tissue defects of the anterior foot and the ankle were repaired with the reversed sural neurocutaneous flaps. The flap sizes range from 18 cm x 9 cm to 30 cm x 18 cm.

RESULTSAll flaps survived well, while 1 flap had distal partial necrosis and healed after dressing change. The follow-up from 6 months to 2 years showed that all flaps were satisfactory.

CONCLUSIONSural neurocutaneous flap is easy to get and to survive. It is an effective method to repair the soft tissue defects of the anterior foot and the ankle.

Adolescent ; Adult ; Ankle ; blood supply ; surgery ; Female ; Foot ; blood supply ; surgery ; Humans ; Male ; Middle Aged ; Reconstructive Surgical Procedures ; Soft Tissue Injuries ; surgery ; Surgical Flaps

This study was aimed to investigate the clinical features of CD56(+) patients with acute monocytic leukemia (AML-M5) and their prognostic significance. The data of 76 newly-diagnosed patients from our hospital were analyzed retrospectively. Patients were divided into two groups: CD56(+) group (21 patients) and CD56(-) group (55 patients). The clinical features, CR rate, relapse rate, the duration of CR, and survival time of patients between the two groups were compared. The results indicated that the CD56(+) antigen was observed in 21 patients (27.6%), their median age was 51.5 years and with a range 16 - 70 years. Of the 21 CD56(+) patients, the high WBC count was found in 57.1% CD56(+) patients (12/21), but it only in 15% CD56(-) patients (P < 0.05). The extramedullary infiltration was seen in 13 CD56(+) patients, and accounted for 62% (13/21), meanwhile this infiltration was found in 18 CD56(-) patients (18/55) and accounted for 33% (P < 0.05). All cases immunophenotypically highly expressed CD13, CD33, CD64, CD11b, cMPO, CD38, in which only the expression frequency of CD11b was positively related with CD56 (r = 0.59, P < 0.05). The CR rate in CD56(+) group accounted for 60.0%, and had no significant difference in comparison with that in CD56(-) group. In CD56(+) group the relapse rate was 75% (P = 0.042), the mean duration of CR was 5.5 months (95%CI, 3.1 - 8.6, P = 0.002), the median overall survival time was 10.1 months (95%CI, 2.3 - 16.3, P = 0.001). and all these had statistical significance as compared with that in CD56(-) group. It is concluded that CD56(+) AML-M5 patients always complicate with high WBC count and extramedullary infiltration, their CR rate and duration of CR are lower and shorter respectively, their relapse rate and prognosis are high and poor respectively.
Adolescent ; Adult ; Aged ; CD56 Antigen ; metabolism ; Female ; Humans ; Immunophenotyping ; Leukemia, Monocytic, Acute ; diagnosis ; immunology ; Male ; Middle Aged ; Prognosis ; Young Adult

OBJECTIVETo study whether Ca(2+)-activated Cl(-) current (I(to2)) contributes to the functional remodeling of the failing heart.

METHODSThe cardiac myocytes were isolated enzymatically from rapidly pacing-induced failing canine hearts (HF) at room temperature. Patch-Clamp whole cell recording technique was employed to record the I(to2). The Cl(-) transport blocker 4, 4'-diisothiocyanostilbene-2, 2'-disulfonic acid (DIDS, 200 micromol) was used to isolated the I(to2). The relations of I(to2) to L-type Ca(2+) current (I(Ca-L)) and to the membrane voltage under the constant intracellular [Ca(2+)]i were evaluated in HF and the normal hearts.

RESULTSWe found that the current density of I(to2) was significantly decreased in HF cells compared with the controls. At membrane voltage of 20 mV, for example, the I(to2) density was (3.02 +/- 0.54) pA/pF in control cells (n = 7) vs. (1.31 +/- 0.25) pA/pF in HF (n = 8) cells, P < 0.05. While the averaged I(Ca-L) density did not show difference between two groups. The time constant of current decay of I(to2) was similar in both types of cells. However, in intracellular Ca(2+) clamped mode with 100 micromol [Ca(2+)]i, I(to2) density was increased significantly in HF cells at membrane voltage of +30 mV or higher.

CONCLUSIONSOur results suggest that the decrease of I(to2) density may contribute to the prolongation of the action potential in failing heart. I(to2) density abnormality may cause cardiac arrhythmia and a delayed after-depolarization. Impaired Ca(2+) handing in HF cells rather than reduced CLCA function itself may result in this abnormality.

Animals ; Calcium ; physiology ; Calcium Channels, L-Type ; physiology ; Chloride Channels ; physiology ; Dogs ; Heart Failure ; physiopathology ; Patch-Clamp Techniques ; Ventricular Remodeling ; physiology

BACKGROUNDCongenital cataract is a highly heterogeneous disorder at both the genetic and phenotypic levels. This study was conducted to identify disease locus for autosomal dominant congenital cataracts in a four generation Chinese family.

METHODSFamily history and clinical data were recorded. All the members were genotyped with microsatellite markers which are close to the known genetic loci for autosomal congenital cataracts. Two-point Lod scores were obtained using the MLINK of the LINKAGE program package (ver 5.1). Candidate genes were amplified by polymerase chain reaction (PCR) and direct cycle sequencing.

RESULTSThe maximum Lod score of Zmax-2.11 was obtained with three microsatellite markers D22S258, D22S315, and D22S1163 at recombination fraction theta=0. Haplotype analysis showed that the disease gene was localized to a 18.5 Mbp region on chromosome 22 flanked by markers D22S1174 and D22S270, spanning the beta-crystallin gene cluster. A c.752T-->C mutation in exon 6 of CRYBB1 gene, which resulted in a heterozygous S228P mutation in predicted protein, was found to cosegregate with cataract in the family.

CONCLUSIONSThis study identified a novel mutation in CRYBB1 gene in a Chinese family with autosomal dominant congenital cataract. These results provide strong evidence that CRYBB1 is a pathogenic gene for congenital cataract.

Amino Acid Sequence ; Cataract ; congenital ; genetics ; Female ; Genes, Dominant ; Genetic Linkage ; Humans ; Male ; Molecular Sequence Data ; Mutation, Missense ; beta-Crystallin B Chain ; genetics

BACKGROUNDAtrial fibrillation is a common arrhythmia with multi-factorial pathogenesis. Recently, a single nucleotide polymorphism (G/T) at position 1057 in the KCNE4 gene, resulting in a glutamic acid (Glu, E)/aspartic acid (Asp, D) substitution at position 145 of the KCNE4 peptide, was found in our laboratory to be associated with idiopathic atrial fibrillation (atrial fibrillation more frequent with KCNE4 145D). However, the functional effect of the KCNE4 145E/D polymorphism is still unknown.

METHODSWe constructed KCNE4 (145E/D) expression plasmids and transiently co-transfected them with the KCNQ1 gene into Chinese hamster ovary-K1 cells and performed whole-cell patch-clamping recording to identify the possible functional consequences of the single nucleotide polymorphism. Quantitative data were analyzed by Student;s t test. Probability values less than 0.05 were considered statistically significant.

RESULTSA slowly activating, non-inactivating voltage-dependent current ((24.0 +/- 2.9) pA/pF, at +60 mV)) could be recorded in the cells transfected with KCNQ1 alone. Co-expression of wild type KCNE4 inhibited the KCNQ1 current ((7.3 +/- 1.1) pA/pF)). By contrast, co-expression of KCNE4 (145D) augment the KCNQ1 current ((42.9 +/- 7) pA/pF)). The V(1/2) of activation for the KCNQ1/KCNE4 (145D) current was shifted significantly towards the depolarizing potential compared to that for the KCNQ1 current ((-2.3 +/- 0.2) mv vs (-13.0 +/- 1.5) mv, P < 0.01)) without changing the slope factorkappa. Furthermore, KCNE4 (145D) also affected the activation and deactivation kinetics of KCNQ1 channels.

CONCLUSIONWe provide experimental evidence that the KCNE4 (145E/D) polymorphism exerts the effect of "gain of function" on the KCNQ1 channel. It may underlie the genetic mechanism of atrial fibrillation. Further studies on the functional association between I(Ks) and KCNE4 (145D) polymorphism in cardiac myocytes are suggested.

Animals ; CHO Cells ; Cricetinae ; Cricetulus ; Humans ; KCNQ1 Potassium Channel ; physiology ; Polymorphism, Single Nucleotide ; Potassium Channels, Voltage-Gated ; analysis ; genetics ; physiology

Objective To knockout the exon51 of DMD gene in HEK293T cells using the CRISPR/Cas9 system. Methods Design the target sequences of sgRNA and clone them into plasmid PX459 respectively; transfer these plasmids into HEK293T cell and extract the total genome DNA; test the activity of sgRNAs with surveyor assay, choose the most efficient one in each end;construct plasmid PX459-2sgRNA and transfer it into HEK293T cells;check whether the exon51 has been knocked known with PCR and T vector sequencing. Results 50% of HEK293T cells' DMD gene exon51 were knocked out,showing a high gene editing efficiency. Conclusions We successfully establish a platform to target knockout the exon51 of DMD gene and provide an important experimental basis for the treatment of DMD and other genetic diseases.