A Strategy for the Molecular Diagnosis in Hemophilia A in Chinese Population

Hemophilia A is an x-linked recessive inherited bleeding disorder. So far, more than 1,885 disease-causing mutations of factor VIII gene have been identified. Clinic confers a great challenge for the molecular diagnosis. We aim to make a better strategy for the molecular diagnosis in Hemophilia A. First, factor VIII intron 22 inversion and intron 1 inversion mutations were detected using Inversion-PCR and double-tube multiple PCRs. And then, non-inversion mutations were analyzed by denaturing high performance liquid chromatography and/or direct sequencing. Novel mutations were further analyzed the conservation and 3D structures by a B domain deleted crystallographic model and bioinformatics. Finally, we can indirectly confirm the diagnosis by linkage analysis for the patients with the confusing diagnosis by the techniques mentioned above. Eleven patients with the factor VIII Inv 22 were found, and the remaining 16 patients were found with 11 different mutations, of which 3 was novel mutations affecting A1, B domains and splicing site. Moreover, the prenatal diagnosis was performed on 14 fetuses. Ten fetuses were successfully confirmed to be normal, 1 fetus to be a heterozygote with factor VIII c.3275–3276 ins A and 3 fetuses to be hemizygotes with factor VIII Inv 22 mutation.     

Comparative Study of 1,064-nm Laser-Induced Skin Burn and Thermal Skin Burn

Infrared lasers are widely used in medicine, industry, and other fields. While science, medicine, and the society in general have benefited from the many practical uses of lasers, they also have inherent safety issues. Although several procedures have been put forward to protect the skin from non-specific laser-induced damage, individuals receiving laser therapy or researchers who use laser are still at risk for skin damage. This study aims to understand the interaction between laser and the skin, and to investigate the differences between the skin damage caused by 1,064-nm laser and common thermal burns. Skin lesions on Wistar rats were induced by a 1,064-nm CW laser at a maximum output of 40 W and by a copper brass bar attached to an HQ soldering iron. Histological sections of the lesions and the process of wound healing were evaluated. The widths of the epidermal necrosis and dermal denaturalization of each lesion were measured. To observe wound healing, the epithelial gap and wound gap were measured. Masson’s trichrome and picrosirius red staining were also used to assess lesions and wound healing. The thermal damage induced by laser intensified significantly in both horizontal dimension and in vertical depth with increased duration of irradiation. Ten days after wounding, the dermal injuries induced by laser were more severe. Compared with the laser-induced skin damage, the skin burn induced by an HQ soldering iron did not show a similar development or increased in severity with the passage of time. The results of this study showed the pattern of skin damage induced by laser irradiation and a heated brass bar. This study also highlighted the difference between laser irradiation and thermal burn in terms of skin damage and wound healing, and offers insight for further treatment.     

Study on the Interaction Mechanism of 2‐Aminoanthraquinone with Calf Thymus DNA

By utilizing multispectrosopic techniques, the toxic interaction of 2‐aminoanthraquinone (2‐AAQ) with calf thymus deoxyribonucleic acid (ctDNA) was investigated in vitro under simulated physiological conditions. The experimental results proved that 2‐AAQ has a toxic interaction with ctDNA. The binding capacity of DNA with 2‐AAQ is diminishing as the pH value of system increasing in the optimization of experimental condition. Moreover we selected pH 7.4, which is nearly physiological condition to enhance the practical significance. According to the Stern–Volmer equation, the quenching was the static quenching process. And the quenching constant can be derived from the fluorescence quenching spectrogram. Ultraviolet absorption spectra and the change in the fluorescence intensity at different ionic strengths further indicated that there was electrostatic binding between 2‐AAQ and ctDNA. The circular dichroism experiment showed that the DNA conformation varied from B to A conformation. The basic group enhanced after 2‐AAQ embedding. The double helix is more compact, and the DNA conformation changes. © 2013 Wiley Periodicals, Inc. J BiochemMol Toxicol 27:272‐278, 2013; View this article online at wileyonlinelibrary.com . DOI 10.1002/jbt.21487     

Increased invasiveness of osteosarcoma mesenchymal stem cells induced by bone-morphogenetic protein-2

To evaluate the different traits of mesenchymal stem cell (MSC) isolated from osteosarcoma (OS) and normal bone marrow (BM) induced by bone-morphogenetic protein-2 (BMP-2). MSCs from implanted osteosarcoma or femur bone marrow were isolated and cultured. Differentiation potency was verified and phenotypes were evaluated by flow cytometry. Increased or decreased expressions of BMP-2 were delivered by adenovirus and lentivirus vector, respectively. Expressions of VEGF, EMMPRIN, and MMP-9 were examined. Cell cycle, apoptosis, invasiveness, and proliferation assays were performed between the transfected groups and controls. Increased BMP-2 induced over-expression of VEGF, EMMPRIN, and MMP-9 in OS- and BM-MSCs both intra- and extra-cellularly. Decreased BMP-2 expression induced inhibition of the factors. Increased BMP-2 also induced less population of cells at G1 phase, more apoptotic cells, more cells that invade through Transwell membrane, and faster proliferation in OSMSC compared to those in BMMSC. BMP-2 induced higher expression of tumorigenic factors, which could be responsible for promoting the proliferation and aggressiveness of OSMSC over BMMSC.     

Suppression of Grb2 expression improved hepatic steatosis, oxidative stress, and apoptosis induced by palmitic acid in vitro partly through insulin signaling alteration

In this study, we aimed to study the role of growth factor receptor-bound protein 2 (Grb2) in palmitic acid-induced steatosis and other “fatty liver” symptoms in vitro. HepG2 cells, with or without stably suppressed Grb2 expression, were incubated with palmitic acid for 24 h to induce typical clinical “fatty liver” features, including steatosis, impaired glucose metabolism, oxidative stress, and apoptosis. MTT and Oil Red O assays were applied to test cell viability and fat deposition, respectively. Glucose uptake assay was used to evaluate the glucose utilization of cells. Quantitative polymerase chain reaction and Western blot were used to measure expressional changes of key markers of insulin signaling, lipid/glucose metabolism, oxidative stress, and apoptosis. After 24-h palmitic acid induction, increased fat accumulation, reduced glucose uptake, impaired insulin signaling, enhanced oxidative stress, and increased apoptosis were observed in HepG2 cells. Suppression of Grb2 in HepG2 significantly reduced fat accumulation, improved glucose metabolism, ameliorated oxidative stress, and restored the activity of insulin receptor substrate-1/Akt and MEK/ERK pathways. In addition, Grb2 deficiency attenuated hepatic apoptosis shown by reduced activation of caspase-3 and fluorescent staining. Modulation of Bcl-2 and Bak1 also contributed to reduced apoptosis. In conclusion, suppression of Grb2 expression in HepG2 cells improved hepatic steatosis, glucose metabolism, oxidative stress, and apoptosis induced by palmitic acid incubation partly though modulating the insulin signaling pathway.     

MFG-E8 and HMGB1 Are Involved in the Mechanism Underlying Alcohol-Induced Impairment of Macrophage Efferocytosis

Efferocytosis is a unique phagocytic process for macrophages to remove apoptotic cells in inflammatory loci. This event is maintained by milk fat globule-EGF factor 8 (MFG-E8), but attenuated by high mobility group box 1 (HMGB1). Alcohol abuse causes injury and inflammation in multiple tissues. It alters efferocytosis, but precise molecular mechanisms for this effect remain largely unknown. Here, we showed that acute exposure of macrophages to alcohol (25 mmol/L) inhibited MFG-E8 gene expression and impaired efferocytosis. The effect was mimicked by hydrogen peroxide. Moreover, N-acetylcysteine (NAC), a potent antioxidant, blocked acute alcohol effect on inhibition of macrophage MFG-E8 gene expression and efferocytosis. In addition, recombinant MFG-E8 rescued the activity of alcohol-treated macrophages in efferocytosis. Together, the data suggest that acute alcohol exposure impairs macrophage efferocytosis via inhibition of MFG-E8 gene expression through a reactive oxygen species dependent mechanism. Alcohol has been found to suppress or exacerbate immune cell activities depending on the length of alcohol exposure. Thus, we further examined the role of chronic alcohol exposure on macrophage efferocytosis. Interestingly, treatment of macrophages with alcohol for seven days in vitro enhanced MFG-E8 gene expression and efferocytosis. However, chronic feeding of mice with alcohol caused increase in HMGB1 levels in serum. Furthermore, HMGB1 diminished efferocytosis by macrophages that were treated chronically with alcohol, suggesting that HMGB1 might attenuate the direct effect of chronic alcohol on macrophage efferocytosis in vivo. Therefore, we speculated that the balance between MFG-E8 and HMGB1 levels determines pathophysiological effects of chronic alcohol exposure on macrophage efferocytosis in vivo.     

Oxaliplatin induces immunogenic cells death and enhances therapeutic efficacy of checkpoint inhibitor in a model of murine lung carcinoma

Abstract

Background: Platinum compounds are commonly used for lung cancer treatment. However, the severe side effects and relatively poor prognosis limit their therapeutic effect. Therefore, developing novel platinum derivative and treatment strategy are critical for current lung cancer therapy.

Methods: Flow cytometry, HMGB1 and ATP release, and immunoblotting were performed to evaluate the Oxaliplatin-induced immunogenic cell death (ICD) in two lung carcinoma cells. Vaccination approach and subcutaneous tumor models were created to analyze the tumor regression effect of Oxaliplatin. PD-L1 mRNA and protein levels were detected in LLC (Lewis lung carcinoma). Enhanced therapeutic efficacy of LLC was assessed by co-administration Oxaliplatin and aPD-L1 in murine lung tumor model.

Results: Oxaliplatin induced robust ICD in LLC cells, activated dendritic cells (DCs, CD80⁺CD86⁺) and enhanced cytotoxic T cells (CD8⁺) in LLC tumor tissues, which resulted in tumor regression. Co-administration of Oxaliplatin and checkpoint inhibitor, aPD-L1, could enhance the therapeutic efficacy of LLC in murine lung carcinoma.

Conclusion: This study reveals Oxaliplatin can induce robust ICD in tumor tissues and suppress tumor growth by activating DCs and enhancing T-cell infiltration. Notably, the Oxaliplatin-induced ICD provides an immunogenic microenvironment, which enhances the checkpoint inhibitor therapeutic efficacy of LLC.     

Copper Nitrate Catalyzed Three-Component One-Pot Synthesis of 3,4-Dihydropyrimidin-2(1H)-Ones

Abstract

An efficient synthesis of 3,4-dihydropyrimidin-2(1H)-ones from aldehydes, 1,3-dicarbonyl compounds, and urea using copper nitrate under refluxing temperature in ethanol was described. Compared with other Lewis copper salts, copper nitrate proved to be the most efficient. The advantages of the new method were good yields (61–93%), short reaction time (0.4–3 h), and inexpensive catalyst.