Design of a zinc finger protein binding a sequence upstream of the A20 gene

<p>Abstract</p> <p>Background</p> <p>Artificial transcription factors (ATFs) are composed of DNA-binding and functional domains. These domains can be fused together to create proteins that can bind a chosen DNA sequence. To construct a valid ATF, it is necessary to design suitable DNA-binding and functional domains. The Cys₂-His₂zinc finger motif is the ideal structural scaffold on which to construct a sequence-specific protein. A20 is a cytoplasmic zinc finger protein that inhibits nuclear factor kappa-B activity and tumor necrosis factor (TNF)-mediated programmed cell death. A20 has been shown to prevent TNF-induced cytotoxicity in a variety of cell types including fibroblasts, B lymphocytes, WEHI 164 cells, NIH 3T3 cells and endothelial cells.</p> <p>Results</p> <p>In order to design a zinc finger protein (ZFP) structural domain that binds specific target sequences in the A20 gene promoter region, the structure and sequence composition of this promoter were analyzed by bioinformatics methods. The target sequences in the A20 promoter were submitted to the on-line ZF Tools server of the Barbas Laboratory, Scripps Research Institute (TSRI), to obtain a specific 18 bp target sequence and also the amino acid sequence of a ZFP that would bind to it. Sequence characterization and structural modeling of the predicted ZFP were performed by bioinformatics methods. The optimized DNA sequence of this artificial ZFP was recombined into the eukaryotic expression vector pIRES2-EGFP to construct pIRES2-EGFP/ZFP-flag recombinants, and the expression and biological activity of the ZFP were analyzed by RT-PCR, western blotting and EMSA, respectively. The ZFP was designed successfully and exhibited biological activity.</p> <p>Conclusion</p> <p>It is feasible to design specific zinc finger proteins by bioinformatics methods.</p

Alkylating HIV-1 Nef - a potential way of HIV intervention

<p>Abstract</p> <p>Background</p> <p>Nef is a 27 KDa HIV-1 accessory protein. It downregulates CD4 from infected cell surface, a mechanism critical for efficient viral replication and pathogenicity. Agents that antagonize the Nef-mediated CD4 downregulation may offer a new class of drug to combat HIV infection and disease. TPCK (N-α-p-tosyl-L-phenylalanine chloromethyl ketone) and TLCK (N-α-p-tosyl-L-lysine chloromethyl ketone) are alkylation reagents that chemically modify the side chain of His or Cys residues in a protein. In search of chemicals that inhibit Nef function, we discovered that TPCK and TLCK alkylated HIV Nef.</p> <p>Methods</p> <p>Nef modification by TPCK was demonstrated on reducing SDS-PAGE. The specific cysteine residues modified were determined by site-directed mutagenesis and mass spectrometry (MS). The effect of TPCK modification on Nef-CD4 interaction was studied using fluorescence titration of a synthetic CD4 tail peptide with recombinant Nef-His protein. The conformational change of Nef-His protein upon TPCK-modification was monitored using CD spectrometry</p> <p>Results</p> <p>Incubation of Nef-transfected T cells, or recombinant Nef-His protein, with TPCK resulted in mobility shift of Nef on SDS-PAGE. Mutagenesis analysis indicated that the modification occurred at Cys55 and Cys206 in Nef. Mass spectrometry demonstrated that the modification was a covalent attachment (alkylation) of TPCK at Cys55 and Cys206. Cys55 is next to the CD4 binding motif (A₅₆W₅₇L₅₈) in Nef required for Nef-mediated CD4 downregulation and for AIDS development. This implies that the addition of a bulky TPCK molecule to Nef at Cys55 would impair Nef function and reduce HIV pathogenicity. As expected, Cys55 modification reduced the strength of the interaction between Nef-His and CD4 tail peptide by 50%.</p> <p>Conclusions</p> <p>Our data suggest that this Cys55-specific alkylation mechanism may be exploited to develop a new class of anti HIV drugs.</p

Effect of the heating rate on the thermal explosion behavior and oxidation resistance of 3D-structure porous NiAl intermetallic

Porous NiAl intermetallic compounds demonstrate great potential in various applications by their high porosity and excellent oxidation resistance. However, to obtain a controllable NiAl intermetallic structure by tuning different process parameters remains unclear. In this work, porous NiAl intermetallic compounds were fabricated by economic and energy-saving thermal explosion (TE) reaction. The relationship between microstructure and process parameters was revealed using three-dimensional X-ray microscopy (3D-XRM) with high resolution and non-destructive characteristics. The geometrical features and quantitative statistics of the porous NiAl obtained at different heating rates (2, 10, 20 \ub0C min−1) were compared. The result of the closed porosity calculation showed that a lower heating rate (2 \ub0C min−1) promoted the Kirkendall reaction between Ni and Al, resulting in a high closed porosity (5.25%). However, at a higher heating rate (20 \ub0C min−1), a homogeneous NiAl phase was observed using the threshold segmentation method, indicating uniform and complete TE reaction can be achieved at a high heating rate. The result of the 3D fluid simulation showed that the sample heated at 10 \ub0C min−1 had the highest permeability (2434.6 md). In this study, we systematically investigated the relationship between the heating rates and properties of the porous NiAl intermetallic, including the phase composition, porosity, exothermic mechanism, oxidation resistance, and compression resistance. Our work provides constructive directions for designing and tailoring the performance of porous NiAl intermetallic compounds

The influence of admixtures on vibration viscosity coefficient of pavement concrete

Abstract: In this paper, the influences of common admixtures including air entraining agent, superplasticizer, shrinkage-reducing agent, retarder, fly ash and slag on vibration viscosity coefficient of fresh concrete were analyzed. The results showed that the vibration viscosity coefficient of fresh concrete was markedly decreased when using UNF-5 naphthalene formaldehyde sulphonated superplasticizer. When SJ-2 air entraining agent, SZ103 shrinkage-reducing agent, retarder and slag were used, the vibration viscosity coefficient of fresh concrete was also reduced. But when fly ash was added, the vibration viscosity coefficient of fresh concrete was increased

UCP2 Inhibits ROS-Mediated Apoptosis in A549 under Hypoxic Conditions

The Crosstalk between a tumor and its hypoxic microenvironment has become increasingly important. However, the exact role of UCP2 function in cancer cells under hypoxia remains unknown. In this study, UCP2 showed anti-apoptotic properties in A549 cells under hypoxic conditions. Over-expression of UCP2 in A549 cells inhibited reactive oxygen species (ROS) accumulation (P<0.001) and apoptosis (P<0.001) compared to the controls when the cells were exposed to hypoxia. Moreover, over-expression of UCP2 inhibited the release of cytochrome C and reduced the activation of caspase-9. Conversely, suppression of UCP2 resulted in the ROS generation (P = 0.006), the induction of apoptosis (P<0.001), and the release of cytochrome C from mitochondria to the cytosolic fraction, thus activating caspase-9. These data suggest that over-expression of UCP2 has anti-apoptotic properties by inhibiting ROS-mediated apoptosis in A549 cells under hypoxic conditions

Efficiency of electrochemical chloride removal from concrete at different environmental temperatures

Electrochemical chloride removal (ECR) is an effective and curative method to treat existed reinforced concrete structures about to suffer or already suffering from chloride attack, however, its application is still limited due to its side effect and efficiency, including the velocity and maximum capacity of chloride removal. This paper presents a temperature related numerical transport model to study the effect of temperature on efficiency of electrochemical chloride removal from concrete. Based on Fick’s law and Nernst-Planck equation with Gauss’ Law, temperature effect, chloride binding, multi-species coupling, electrochemical reactions were taken into account in this model. Temperature effect was considered on diffusion coefficient, chloride binding, ions migration capacity as well as electrolyte concentration. The model was validated by the comparison between the calculated results and experimental data. The results indicate that temperature dose have a considerable influence on electrochemical chloride removal and controlling temperature during treatment is a practical method to improve the electrochemical chloride removal when applied current density is not amplified

An equity evaluation in stroke inpatients in regard to medical costs in China: a nationwide study.

BackgroundStroke has always been a severe disease and imposed heavy financial burden on the health system. Equity in patients in regard to healthcare utilization and medical costs are recognized as a significant factor influencing medical quality and health system responsiveness. The aim of this study is to understand the equity in stroke patients concerning medical costs and healthcare utilization, as well as identify potential factors contributing to geographic variation in stroke patients' healthcare utilization and costs.MethodsCovering 31 provinces in mainland China, our main data were a 5% random sample of stroke claims from Urban Employees Basic Medical Insurance (UEBMI) and Urban Residents Basic Medical Insurance (URBMI) from 2013 to 2016. The Theil index was employed to evaluate the equity in stroke patients in regard to healthcare utilization and medical costs, and the random-effect panel model was used to explore the impact of province-level factors (health resource factors, enabling factors, and economic factors) on medical costs and health care utilization.ResultsStroke patients' healthcare utilization and medical costs showed significant differences both within and between regions. The UEBMI scheme had an overall lower Theil index value than the URBMI scheme. The intra-region Theil index value was higher than the inter-region Theil index, with the Theil index highest within eastern China, China's richest and most developed region. Health resource factors and enabling factors (represented by reimbursement rate and education attainment years) were identified significantly associated with medical costs (P ConclusionsChina's fragmented urban health insurance schemes require further reform to ensure better equity in healthcare utilization and medical costs for stroke patients. Improving education attainment, offering equal access to healthcare, allocating health resources reasonably and balancing health services prices in different regions also count

Medical costs and hospital utilization for hemophilia A and B urban inpatients in China: a national cross-sectional study

BACKGROUND: Hemophilia care in mainland China has been greatly improved since the establishment of the Hemophilia Treatment Center Collaborative Network of China (HTCCNC), and most of drugs for hemophilia have been covered by basic medical insurance schemes. This study assesses whether medical costs and hospital utilization disparities exist between hemophilia A and hemophilia B urban inpatients in China and, second, whether the prescription of coagulation factor concentrates for hemophilia A and hemophilia B inpatients was optimal, from the third payer perspective. METHODS: We conducted a retrospective nationwide analysis based on a 5% random sample from claims data of China Urban Employees’ Basic Medical Insurance (UEBMI) and Urban Residents’ Basic Medical Insurance (URBMI) schemes from 2010 to 2016. Univariate analysis and multiple regression analysis based on a generalized linear model were conducted. RESULT: A total of 487 urban inpatients who had hemophilia were identified, including 407 inpatients with hemophilia A and 80 inpatients with hemophilia B. Total medical cost for hemophilia B inpatients was significantly higher than for hemophilia A inpatients (USD 2912.81 versus USD 1225.60, P < 0.05), and hemophilia B inpatients had a significantly longer length of hospital stay than hemophilia A inpatients (9.00 versus 7.00, P < 0.05). Total medical costs were mostly allocated to coagulation factor products (76.86-86.68%), with coagulation factor cost of hemophilia B significantly higher than hemophilia A (P < 0.05). Both hemophilia cohorts utilized greatest amount of plasma-derived Factor VIII, followed by recombinant Factor VIII and prothrombin complex concentrates. CONCLUSIONS: Patients with hemophilia B experienced significantly higher inpatient cost, coagulation factor cost and longer length of hospital stay than patients with hemophilia A. Our findings revealed the suboptimal use of coagulation factor concentrate drugs and a higher drug cost burden incurred by hemophilia B than hemophilia A inpatients. Our results call for efforts to strengthen drug regulatory management for hemophilia and to optimize medical insurance schemes according to hemophilia types

LongSAGE analysis of skeletal muscle at three prenatal stages in Tongcheng and Landrace pigs

Transcriptional profiling of Tongcheng and Landrace pigs using long serial analysis of gene expression provides insight into the molecular mechanism underlying differences in muscle growth

A novel small molecule ameliorates ocular neovascularisation and synergises with anti-VEGF therapy

Ocular neovascularisation underlies blinding eye diseases such as retinopathy of prematurity, proliferative diabetic retinopathy, and wet age-related macular degeneration. These diseases cause irreversible vision loss, and provide a significant health and economic burden. Biologics targeting vascular endothelial growth factor (VEGF) are the major approach for treatment. However, up to 30% of patients are non-responsive to these drugs and they are associated with ocular and systemic side effects. Therefore, there is a need for small molecule ocular angiogenesis inhibitors to complement existing therapies. We examined the safety and therapeutic potential of SH-11037, a synthetic derivative of the antiangiogenic homoisoflavonoid cremastranone, in models of ocular neovascularisation. SH-11037 dose-dependently suppressed angiogenesis in the choroidal sprouting assay ex vivo and inhibited ocular developmental angiogenesis in zebrafish larvae. Additionally, intravitreal SH-11037 (1 μM) significantly reduced choroidal neovascularisation (CNV) lesion volume in the laser-induced CNV mouse model, comparable to an anti-VEGF antibody. Moreover, SH-11037 synergised with anti-VEGF treatments in vitro and in vivo. Up to 100 μM SH-11037 was not associated with signs of ocular toxicity and did not interfere with retinal function or pre-existing retinal vasculature. SH-11037 is thus a safe and effective treatment for murine ocular neovascularisation, worthy of further mechanistic and pharmacokinetic evaluation