Synthesis of ponatinib analogues as novel inhibitors of leukemia stem cells.

Aim: To synthesize new analogues of ponatinib and evaluate anti-leukemia cells and cytotoxicity. Methodology & results: The inhibitory activity of compounds 13a and 13c against K562 and HL60 cells was comparable to that of ponatinib (IC50 = 0.74, 0.88 vs 0.64 nM and 0.59, 0.77 vs 0.39 nM, respectively). Compounds 13a and 40b were 34- and 77-fold more potent than ponatinib against KG1a cells (IC50 = 0.091 and 0040 vs 3.6 µM, respectively). Compounds 13a, 13c and 40b also decreased the Abl protein level in the K562 cells, inhibited colony formation in MCF-7 cells and inhibited cell migration in B16BL6 cells. Compound 13a showed low cytotoxicity in 293 cells. Conclusion: Compound 13a was the best lead compound.

[Clinical analysis of sarcoidosis of ocular adnexa].

OBJECTIVE: To study the diagnosis and treatment of 17 patients with sarcoidosis in ocular adnexa. METHODS: The clinical data of 17 cases with sarcoidosis in ocular adnexa treated during 1993 and 2008 were retrospectively analyzed. All patients underwent surgical treatment, and the diagnosis was proven histopathologically. RESULTS: Among the 17 cases, 4 were male, and 13 were female. The patients' age ranged from 15 to 70 years, with a mean of 46.9 years. The lesions were located at the orbit (8 cases), lacrimal grand (5 cases) and eyelids (4 cases). Fourteen cases complained of the presence of a local mass, 2 cases complained of exophthalmos and 1 swelling of eyelids. Concurrent systemic sarcoidosis was present in 7 cases. Three cases coincided with lung sarcoidosis, 3 cases with uveitis and 1 case with dermatopathy. Angiotensin converting enzyme analysis was performed in 6 cases; 4 of them were elevated. Computer tomography was performed in 12 cases; in 11 cases it presented as moderate density parenchymatous mass, and in the remaining one it presented as hypodensity cystic mass. B scan of 5 cases showed hypoechoic parenchymatous homogeneous mass. None of 14 cases relapsed after 1 to 15 years follow-up. CONCLUSIONS: Ocular adnexal sarcoidosis usually presents as local mass and it should be included in the differential diagnosis of orbital and ocular adnexal lesions. Excision of localized mass alone could achieve satisfactory outcomes for isolated lesions, while for diffuse or systematic lesions, corticosteroid treatment should be prescribed routinely.

[Effect of insulin on VEGF expression in bovine retinal microvascular endothelial cells exposed to normal or high glucose].

OBJECTIVE: To investigate the effect of insulin or (and) high glucose on the vascular endothelial growth factor (VEGF) expression in bovine retinal microvascular endothelial cells (BREC). METHODS: It was a experimental study. BREC were cultured with selective culture media. Passaged cells were cultured in normal (5 mmol/L) or high glucose (30 mmol/L) medium for 3 days. Equimolar mannitol was supplemented for osmotic controls. Cells were serum-deprived overnight in Dulbecco's modified Eagle's medium containing 0.2% w/v bovine serum albumin and then incubated in the absence or presence of 100 nmol/L insulin for 24 hrs. Cells were harvested and the expression of VEGF mRNA was analyzed by Real-time PCR. VEGF protein level was determined by HUVEC proliferation assay, immunofluorescence and western-blot RESULTS: Insulin or (and) high glucose significantly increased VEGF mRNA (F = 5.67, 9.04; P <0.05) and protein (F = 5.50, 5.57; P <0.05) expression. However, the combined effect of insulin and high glucose is weak in contrast to insulin or high glucose alone. CONCLUSIONS: High glucose attenuated insulin-induced VEGF expression. Therefore, VEGF may be not the major factor that lead to transient worsening of diabetic retinopathy after acute insulin therapy in diabetes.

[Human mesenchymal stem cells differentiate into neuron-like cells and show SMN protein expression].

OBJECTIVES: To investigate the mRNA and protein expression of SMN gene in neuron-like cells by inducing MSC to differentiate into neuron-like cells. METHODS: Human MSC (hMSC) were isolated and purified. Human MSC were treated with basic fibroblast growth factor (bFGF) before inducing hMSC to differentiate into neuron-like cells with dimethylsulfoxide (DMSO) and butylated hydroxyanisole (BHA). The expression of NSE, NF, and SMN protein in neuron-like cells were detected by immunohistochemistry. The expression of SMN mRNA were detected with reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: Human MSC progressively assumed neuronal morphological characteristics after being induced for 3 hours. Cell bodies had long process contacting with each other. The neuronal marker of NSE and NF was positive in neuron-like cells. Both hMSC and neuron-like cells expressed the mRNA of SMN gene. Compared with hMSC the latter expressed highly the mRNA of SMN gene and it was significance (P < 0.01). SMN protein was only expressed in neuron-like cells. CONCLUSIONS: Human MSC are able to differentiate into neuron-like cells with expressing the neuronal marker. Neuron-like cells can express highly the mRNA of SMN gene and express SMN protein.

[Epidemiologic analysis on severe fever with thrombocytopenia syndrome in Hubei province, 2010].

OBJECTIVE: To identify the epidemic characteristics and risk factors of an emerging infectious disease-severe fever with thrombocytopenia syndrome (SFTS) in Hubei province. METHODS: Active surveillance program on SFTS was set up in monitoring sites-hospitals, at the township level or above, in Suizhou, Huanggang and Wuhan from January to December, 2010. Specific surveillance program on SFTS was launched across the province in hospitals above the county level. Cases that matched the definition of surveillance case were identified and reported to Centers for Disease Control and Prevention (CDCs). Cases were interviewed and their blood samples collected and detected using PCR and virus isolation. We also conducted serum antibody surveys among healthy population and livestock and surveillance on vector ticks in those high-epidemic areas. RESULTS: 188 cases that matched the definition of surveillance case and 21 deaths were reported in 11 cities, 32 countries and 100 towns in 2010, with an incidence rate of 0.33/10(6). The fatality rate was 11.2%. Data showed that the patients were from hilly areas at the altitude elevated between 28-940 meters. The epidemic period was between April and December with the peak from May to September. The youngest case was an 11-year old, while the eldest was 81 with median age as 56-year old. 95.3% of the patients were farmers. All Patients did not have the history of traveling, two weeks before the onset of SFTS. 93.6% of the patients engaged in different kind of work which was associated with agriculture. 52.8% of the patients had been exposed to ticks. 22.0% of the patients had been bitten by ticks. Skin injury was found in 64.2% of the patients. Samples from 129 cases (68.6%) were collected and detected, with 67.4% of them (87 cases) showed positive by Real time-PCR for SFTS virus. An elevation in antibody titer by a factor of four or evidence of sero-conversion was observed in 11 patients; SFTS virus was isolated from 2 patients. The total antibody positive rates were 3.8%, 55.0% (6/11), 36.7% (2/3) and 80.0% (4/5) respectively in healthy population, dogs, sheep and cows. Ticks from grass, cattle and sheep were detected positive by Real time-PCR. CONCLUSION: Most cases of SFTS in Hubei were infected by SFTS virus, and cases of livestock were infected by SFTS virus. Ticks might serve as an important vector. Skin injury, exposure to tick bites seemed to be the risk factors.

Generation of trans-arachidonic acid under nitrative stress is associated with upregulation of thromponsdin-1 in diabetic rats.

BACKGROUND: Trans-arachidonic acids (TAAs), newly discovered markers of nitrative stress and the major products of nitrogen dioxide (NO2(·))-mediated isomerization of arachidonic acid (AA), represent a new mechanism of NO2(·)-induced toxicity. It has been reported that TAAs were generated in oxygen-induced microvascular degeneration model and TAAs were also generated in a diabetic retinopathy (DR) model. In this study, we examined high glucose-induced nitrative stress damage and TAAs levels and explored the possible mechanisms for DR caused by reactive nitrogen species. METHODS: Diabetic rats were induced by intraperitoneal injection of streptozotocin (STZ) at 60 mg/kg. Bovine retinal capillary endothelial cells (BRECs) were selectively cultured and incubated with normal or high glucose. The serum TAAs and AA in diabetic rats were measured by the gas chromatography and mass spectrometry (GC/MS) method. The ratio of peak area of TAAs to AA with selected ion of 79 was estimated by a group t-test. Thrombospondin-1 (TSP-1) in the rat retinas and BRECs extracts were examined by Western blotting. The phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) protein was examined by Western blotting in BRECs incubated with high glucose. RESULTS: The TAAs to AA ratio (TAAs/AA) was significantly increased in the serum at 8, 12 and 16 weeks after STZ injection (P < 0.05), with no noticeable change found at 2 or 4 weeks (P > 0.05). Expression of TSP-1 in the retina of diabetic rats was progressively elevated according to the duration of diabetes. TSP-1 expression was increased in BRECs incubated with high glucose at 48 hours. Moreover, high glucose also increased ERK1/2 expression, which peaked at 30 minutes and then decreased in the following 48 hours. CONCLUSION: An elevation of TAAs/AA is associated with high glucose-induced nitrative stress, which probably involves upregulation of TSP-1 through activating ERK1/2.

De novo RNA synthesis by a recombinant classical swine fever virus RNA-dependent RNA polymerase.

Classical swine fever virus nonstructural protein 5B (NS5B) encodes an RNA-dependent RNA polymerase, a key enzyme of the viral replication complex. To better understand the initiation of viral RNA synthesis and to establish an in vitro replication system, a recombinant NS5B protein, lacking the C-terminal 24-amino acid hydrophobic domain, was expressed in Escherichia coli. The truncated fusion protein (NS5Bdelta24) was purified on a Ni-chelating HisTrap affinity column and demonstrated to initiate either plus- or minus-strand viral RNA synthesis de novo in a primer-independent manner but not by terminal nucleotidyle transferase activity. De novo RNA synthesis represented the preferred mechanism for initiation of classical swine fever virus RNA synthesis by RNA-dependent RNA polymerase in vitro. Both Mg2+ and Mn2+ supported de novo initiation, however, RNA synthesis was more efficient in the presence of Mn2+ than in the presence of Mg2+. De novo initiation of RNA synthesis was stimulated by preincubation with 0.5 mm GTP, and a 3'-terminal cytidylate on the viral RNA template was preferred for de novo initiation. Furthermore, the purified protein was also shown, by North-Western blot analysis, to specifically interact with the 3'-end of both plus- and minus-strand viral RNA templates.