OBJECTIVE: To study the effects and mechanisms of miR-181a-5p on the proliferation, cycle and migration of HOS osteosarcoma cells. METHODS: Real-time quantitative PCR was used to detect the expression of miR-181a-5p and HOXB4 in osteoblast hFOB1.19 cell line and osteosarcoma cell lines (HOS, U2OS, MG63). miR-181a-5p mimics and miR-181a-5p inhibitors were respectively transfected into HOS cells by Lipofectamine 2000, and miR NC group was set as control group. CCK-8 method was used to detect the change in cell proliferation. Flow cytometry was used to detect the changes in cell cycles. Wound healing experiments and Transwell migration experiments were used to detect the changes in cell migration ability. The target gene of miR-181a-5p was predicted by Targetscan website and validated by Dual-luciferase reporter gene system and Western blot. RESULTS: Compared with osteoblast hFOB1.19, miR-181a-5p was low expressed in osteosarcoma cells HOS, U2OS, and MG63(P<0.05), while HOXB4 was high expressed in osteosarcoma cells HOS, U2OS, and MG63(P<0.05). Compared with the miR NC group, over expression of miR-181a-5p inhibited the proliferation and migration of osteosarcoma HOS cells, and the number of cells in S phase decreased(P<0.05). However, knockdown miR-181a-5p promoted the proliferation and migration of osteosarcoma HOS cells, the cells in S phase increased(P<0.05). Bioinformatics prediction and Dual-luciferase reporter gene system validate HOXB4 as a downstream target gene of miR-181a-5p(P<0.05). Western blot showed that miR-181a-5p over expression or knockdown significantly down-regulated or up-regulated HOXB4 expressions in the HOS cells respectively(P<0.05). CONCLUSION miR-181a-5p is down expressed in osteosarcoma cells, and over-expression miR-181a-5p inhibits the proliferation, cell cycle and migration ability of osteosarcoma cells by targeting HOXB4.
Humans ; Apoptosis ; Bone Neoplasms/genetics* ; Cell Line, Tumor ; Cell Proliferation/genetics* ; Homeodomain Proteins/genetics* ; MicroRNAs/metabolism* ; Osteosarcoma/genetics* ; Transcription Factors/genetics*

OBJECTIVE: To investigate the biological function of Cysteine rich (CysR) domain of a disintegrin and metalloprotease with thrombospondin type 1 repeats-13 (ADAMTS13) on cleavage of von Willebrand factor (vWF) and provide experimental evidence for exploring the pathogenesis of thrombotic thrombocytopenic purpura (TTP). METHODS: The six amino acids (EDGTLS) in ADAMTS13 CysR domain were point mutated one by one, and the mutant ADAMTS13 proteins were expressed and purified. The cleavage products of vWF polymer by wild-type or mutant ADAMTS13 under denaturing condition or shear stress were separated by 1% SeaKem HGT agarose gel and detected by Western blot. RESULTS: The mutant ADAMTS13 plasmids (M1: Glu515Ala; M2: Asp516Ala; M3: Gly517Ala; M4: Thr518Ala; M5: Leu519Ala; M6: Ser520Ala) were successfully constructed and the proteins of wild-type and mutant ADAMTS13 were purified. Wild-type ADAMTS13 almost completely cleaved the vWF polymer under denaturing condition, while the cleavage activity of M1 mutant was significantly reduced in the same condition (P<0.01). The cleavage activity of M1 mutant of ADAMTS13 was also significantly reduced compared with that of the wild-type under shear stress (P<0.01). The activity of M1 mutant to cleave the FRETS-vWF73 was dramatically reduced compared with that of wild-type ADAMTS13. However, the binding ability of M1 mutant to vWF was similar with that of wild-type ADAMTS13. CONCLUSION The CysR domain of ADAMTS13 plays an important role in the digestion of vWF under denaturing condition and shear stress. The Glu515 amino acid residue might be an important site for substrate recognition.
ADAM Proteins ; ADAMTS13 Protein/genetics* ; Humans ; Purpura, Thrombotic Thrombocytopenic/genetics* ; von Willebrand Factor/genetics*

BACKGROUND AND OBJECTIVES: Coronary artery calcium (CAC) scoring in the asymptomatic population can improve cardiovascular risk prediction. We aimed to assess CAC progression and the impact of coronary risk factors on the CAC progression rate in asymptomatic Korean individuals with a baseline CAC score of zero. METHODS: The study population was derived from the Korea Initiatives on Coronary Artery Calcification (KOICA) registry: a retrospective, single ethnicity, multicenter registry of asymptomatic individuals who underwent CAC scoring as a part of a health checkup. Individuals with at least two CAC scores and an initial score of zero were included. CAC progression was defined as [√CAC score (follow-up) − √CAC score (baseline)] ≥2.5. The 10-year atherosclerotic cardiovascular disease (ASCVD) risk was calculated. RESULTS: Among 6,268 participants (mean age, 48.0±7.1 years; male, 80.5%), 719 (11.5%) experienced CAC progression during follow-up (median, 109 months; interquartile range, 78–208 months). The CAC progression rate was 0.3%, 1.9%, 4.3%, 8.6%, and 16.7% in years 1–5, respectively. The chance of CAC progression at 5 years was 13.1%, 22.0%, and 27.9% for individuals with a 10-year ASCVD risk of <5%, ≥5% but <7.5%, and ≥7.5%, respectively. A multivariable analysis revealed age, male sex, waist circumference, diabetes, and low-density lipoprotein cholesterol level as independently associated with annualized CAC progression (p<0.001, p=0.017, p=0.025, p=0.032, and p=0.003, respectively). CONCLUSIONS: The probability of CAC progression is very low in Korean individuals with a CAC score of zero. However, the risk of CAC progression increases nonlinearly over time, and increases as the 10-year ASCVD risk increases.
Calcium ; Cardiovascular Diseases ; Cholesterol ; Coronary Vessels ; Follow-Up Studies ; Humans ; Korea ; Lipoproteins ; Male ; Retrospective Studies ; Risk Assessment ; Risk Factors ; Waist Circumference

A 6-year-old intact male Maltese dog presented with a history of blindness and ataxia. Neuro-ophthalmic examination revealed dilated pupils with absent pupillary light reflexes and menace response in both eyes. Mild peripapillary edema was noted in the fundus of the right eye. After magnetic resonance imaging, the dog was provisionally diagnosed with meningoencephalitis of unknown etiology. Follow-up funduscopy was performed to monitor the condition of the optic discs for three years. Despite of the treatment with prednisolone, the optic nerve progressed to atrophy and the dog couldn't restore vision.
Animals ; Ataxia ; Atrophy ; Blindness ; Child ; Dogs ; Edema ; Follow-Up Studies ; Humans ; Magnetic Resonance Imaging ; Male ; Meningoencephalitis ; Optic Nerve ; Optic Neuritis ; Prednisolone ; Pupil ; Reflex

OBJECTIVE: To obtain the recombinant protein of spacer domain in von Willebrand factor cleaving protease (ADAMTS13), and further study its biological function in ADAMTS13. METHODS: The prokaryotic expression vector was constructed by using the template of plasmid with full-length ADAMTS13, and then transfected into E coli., following the induction of IPTG with the low temperature (30 ℃). The recombinant protein was purified with Ni-NTA agarose column by gradient imidazole. The purity and immune activity of purified products were identified with SDS-PAGE and Western blot respectively. By Adding the recombinant protein to the plasma of immune-mediated thrombotic thrombocytopenic purpura (iTTP) patients, the activity of ADAMTS13 was tested. RESULTS: The prokaryotic expression vector was successfully constructed and the protein of spacer domain with the high purity was obtained. Western blot showed that the recombinant fragment could both react with monoclonal antibody against 6×His and polyclonal sheep IgG against ADAMTS13 (Gln34-Trp688). The protein formed a main lane at the position of 15 kDa with SDS-PAGE. It was demonstrated that the recombinant protein could efficiently elevate the ADAMTS13 activity in plasma of iTTP patients to reach normal level by functional experiment. CONCLUSION The recombinant protein has high purity and immune activity, which provides the experimental basis for further research on mechanism of iTTP involved in spacer domain.
ADAM Proteins ; ADAMTS13 Protein ; Animals ; Escherichia coli ; Humans ; Purpura, Thrombotic Thrombocytopenic ; Recombinant Proteins ; Sheep ; von Willebrand Factor

BACKGROUND AND OBJECTIVES: Coronary artery calcium (CAC) scoring in the asymptomatic population can improve cardiovascular risk prediction. We aimed to assess CAC progression and the impact of coronary risk factors on the CAC progression rate in asymptomatic Korean individuals with a baseline CAC score of zero. METHODS: The study population was derived from the Korea Initiatives on Coronary Artery Calcification (KOICA) registry: a retrospective, single ethnicity, multicenter registry of asymptomatic individuals who underwent CAC scoring as a part of a health checkup. Individuals with at least two CAC scores and an initial score of zero were included. CAC progression was defined as [√CAC score (follow-up) −√CAC score (baseline)] ≥2.5. The 10-year atherosclerotic cardiovascular disease (ASCVD) risk was calculated. RESULTS: Among 6,268 participants (mean age, 48.0±7.1 years; male, 80.5%), 719 (11.5%) experienced CAC progression during follow-up (median, 109 months; interquartile range, 78–208 months). The CAC progression rate was 0.3%, 1.9%, 4.3%, 8.6%, and 16.7% in years 1–5, respectively. The chance of CAC progression at 5 years was 13.1%, 22.0%, and 27.9% for individuals with a 10-year ASCVD risk of <5%, ≥5% but <7.5%, and ≥7.5%, respectively. A multivariable analysis revealed age, male sex, waist circumference, diabetes, and low-density lipoprotein cholesterol level as independently associated with annualized CAC progression (p<0.001, p=0.017, p=0.025, p=0.032, and p=0.003, respectively). CONCLUSIONS The probability of CAC progression is very low in Korean individuals with a CAC score of zero. However, the risk of CAC progression increases nonlinearly over time, and increases as the 10-year ASCVD risk increases.

A 6-year-old intact male Maltese dog presented with a history of blindness and ataxia. Neuro-ophthalmic examination revealed dilated pupils with absent pupillary light reflexes and menace response in both eyes. Mild peripapillary edema was noted in the fundus of the right eye. After magnetic resonance imaging, the dog was provisionally diagnosed with meningoencephalitis of unknown etiology. Follow-up funduscopy was performed to monitor the condition of the optic discs for three years. Despite of the treatment with prednisolone, the optic nerve progressed to atrophy and the dog couldn't restore vision.

OBJECTIVETo investigate the metabolism characteristics, to search for potential biomarkers associated with disease and to explore related metabolic pathways by analyzing the plasma metabolic profile of patients with chronic myelogenous leukemia (CML) through metabolomies.

METHODSTwenty-six newly diagnosed CML patients in the First Affilated Hospital of Soochow University from February 2015 to April 2015, 26 allogeneic hematopoietic stem cell donors as healthy controls and 26 patients treated with tyrosine kinase inhibitors (TKI) to obtain the best efficacy as post-treatment controls were enrolled in this study. All the metabolites of plasma were extracted by Gas Chromatography-Mass Spectrometer(GC-MS) to collect metabolic fingerprint. Multivariate pattern recognition analysis and t test were combined to screen out the metabolic biomarkers at different time points. The receiver operating characteristic curve analysis was used to evaluate the clinical efficacy of metabolites, and the metabolic pathway analysis was performed.

RESULTSSignificantly different metabolite expression mode was found seen between CML and healthy control groups. Six changed metabolites in CML were confirmed by multivariate and variate statistical analyses. Compared with the healthy controls, the levels of tetradecanoic acid and glycerol were decreased, the lactic acid, myo-inositol, d-galactose and glycine in CML patients also increased (all VIP>1,P<0.05, AUC>0.7). The plasma metabolites in CML patients after treatment with tyrosine kinase inhibitors (TKI) showed a recovery trend toward to normal levels. The plasma metabolic pathways of CML were mainly related with galactose, pyruvate, glycerolipid, inositol phosphate and glycine, serine and threonine metabolism (all impact value>0.10).

CONCLUSIONSignificant changes in plasma metabolite levels were found in CML patients. Metabolomics combined with multivariate pattern recognition analysis may be a new tool to assist diagnosis.

OBJECTIVETo investigate the potential signaling pathway that regulates the proliferation of human CD34cells stimulated by prostaglandin E2 receptor 4 agonist (EP4A) in vitro.

METHODSTwenty samples of peripheral blood containing stem cells were collected from the G-CSF mobilized healthy donors in our department of hematology. Human CD34cells were isolated by magnetic activated cell sorting (MACS) microbeads kit. The Cell Counting Kit-8 (CCK8) assay was used to determine the optimal concentration and time of EP4A to promote human CD34cell proliferation in vitro. Under the optimal condition, quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect mRNA level of β-catenin, and Western blot was used to assay protein expression of β-catenin and P-GSK-3β in human CD34cells treated with EP4A.

RESULTSCulturing with 10 µmol/L EP4A for 72 h, it was found that EP4A promoted human CD34cell proliferation significantly, and the proliferation rate of human CD34cells was 1.36 times higher than that of the control(P=0.002). Under the optimal condition, it was also found that EP4A enhanced the β-catenin expression at both mRNA and protein levels, and up-regulated phosphorylation of GSK-3β in human CD34cells, but these effects could be inhibited by the EP4A antagonist EP4AA.

CONCLUSIONEP4A can enhance human CD34cell proliferation in vitro by activating Wnt/β-catenin signaling pathway.

Adverse reactions of subcutaneous low molecular weight heparin or unfractionated heparin could be complications by bleeding, heparin-induced thrombocytopenia, drug-induced liver injury, osteoporosis, and cutaneous reactions. Heparin-induced skin lesions vary from allergic reactions like erythema, urticaria, eczema to intradermal microvascular thrombosis associated with heparin-induced thrombocytopenia. There is a rare cutaneous complication, called bullous hemorrhagic dermatosis. We experienced this rare case of the cutaneous complication caused by enoxaparin. Several tense bullous hemorrhagic lesions occurred after 3 days of enoxaparin in a known bullous pemphigoid patient who had aortic valve replacement surgery with a mechanical prosthesis. The bullous hemorrhagic lesions were regressed after the discontinuation of enoxaparin but recurred after re-administration. The lesions were controlled by the administration of systemic corticosteroid and alternative anticoagulant. To date, less than 20 cases have been reported worldwide. This is the first case of bullous hemorrhagic dermatosis induced by enoxaparin, a low-molecular-weight heparin in Korea. This is also the first case of bullous hemorrhagic dermatosis in a known bullous pemphigoid patient.
Aortic Valve ; Drug-Induced Liver Injury ; Eczema ; Enoxaparin ; Erythema ; Hemorrhage ; Heparin ; Heparin, Low-Molecular-Weight ; Humans ; Hypersensitivity ; Korea ; Osteoporosis ; Pemphigoid, Bullous ; Prostheses and Implants ; Skin ; Skin Diseases ; Skin Diseases, Vesiculobullous ; Thrombocytopenia ; Thrombosis ; Transcutaneous Electric Nerve Stimulation ; Urticaria