Journal: PLoS ONE

Article Title: Tissue Kallikrein Mediates Pro-Inflammatory Pathways and Activation of Protease-Activated Receptor-4 in Proximal Tubular Epithelial Cells

doi: 10.1371/journal.pone.0088894

Figure Lengend Snippet: Cells were incubated with 0.1 or 0.5/ml AGE-BSA for 6 h, and gene expression was determined by real-time PCR analysis. AGE induced KLK1 mRNA expression in PTEC (A). Cells were transfected with KLK1-specific siRNA, and the endogenous protein level was determined by Western blot analysis (B). Transfected cells were incubated with 0.5 mg/ml AGE for 48 h, and protein expression of cytokine was detected in culture medium by ELISA. AGE -induced IL-8 (C) and ICAM-1 (D) protein expression was inhibited by KLK1 gene silencing. ***p<0.001 compared with control, δ P<0.05 compared with mock transfection and # p<0.05; ## p<0.01 compared with mock transfection incubated with AGE.

Article Snippet: PTEC were transfected with KLK1 or negative control of Silence®Select Pre-designed siRNA (Applied Biosystems) using Lipofectamine™ 2000 according to manufacturer’s instructions.

Techniques: Incubation, Expressing, Real-time Polymerase Chain Reaction, Transfection, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Human Mutation

Article Title: Weaver Syndrome‐Associated EZH2 Protein Variants Show Impaired Histone Methyltransferase Function In Vitro

doi: 10.1002/humu.22946

Figure Lengend Snippet: Weaver syndrome mutants are impaired in their histone methyltransferase activity in vitro . Histone methyltransferase reactions were performed using 2 μg purified core histones and 0.67 μM 3 H‐S‐adenosyl‐methionine ( 3 H‐SAM). Each reaction was incubated with 250 ng of either wild‐type (WT) or a mutant HMTase complex (or no enzyme controls). Histone methyltransferase activity was measured based on the incorporation of 3 H‐labeled methyl groups, represented in scintillation counts per minute. Counts were normalized by subtracting background counts (i.e., no enzyme) from the total counts. A : Incorporation of tritiated methyl groups from 3 H‐SAM onto core histones is shown for each complex: EZH2 WT • , p.(Phe672Ile) × , p.(Pro132Ser) ★ , p.(Tyr153del) △, p.(His694Tyr) ▽, p.(Glu745Lys) ▴, p.(Ala682Thr) ▾, p.(Arg684Cys) ▪, p.(Tyr133Cys) □, and p.(Asp185His) ◇. Error bars represent standard deviation (SD) within the groups “EZH2 WT” and “EZH2 mutants.” Unpaired t‐test showed statistically significant difference between the two groups (P value < 0.0001). B : Incorporation of tritiated methyl groups from 3 H‐SAM onto core histones is shown for the positive control EZH2 WT, the negative control EZH2 (p.Phe672Ile), and the mutant complex with activity closest to WT, namely, EZH2 (p.Pro132Ser). Error bars represent SD of four independent replicates for the controls, and three independent replicates for the mutant EZH2 (p.Pro132Ser). One‐way ANOVA showed statistically significant difference between all groups (overall P value < 0.0001; P values between WT and p.(Phe672Ile), between p.(Phe672Ile) and p.(Pro132Ser), and between WT and p.(Pro132Ser) were all <0.05).

Article Snippet: To test our hypothesis, we designed recombinant human EZH2 proteins, had them preassembled into PRC2 complexes (BPS Bioscience, San Diego CA), and tested their activity in vitro using a well‐accepted in vitro assay [Ernst et al., ; Yap et al., ; Score et al., ].

Techniques: Activity Assay, In Vitro, Purification, Incubation, Mutagenesis, Labeling, Standard Deviation, Positive Control, Negative Control

Journal: European Journal of Translational Myology

Article Title: A New Semi-Automatic Approach to Find Suitable Virtual Electrodes in Arrays Using an Interpolation Strategy

doi: 10.4081/ejtm.2016.6029

Figure Lengend Snippet: Overview of the closed-loop system. A controller K adjusts the global intensity u based on the error e between the angular output of one DoF y and the reference angle r. The interpolation function f i (u, p ,d) assigns an individual intensity q i to the elements in the plant G, which consists of the FES system, the array, the patient, and a motion measurement system. The motion in the remaining DoF y' can be observed by the therapist/patient who can adjust the position p and diameter d of the VE.

Article Snippet: The controller design and implementation were performed in MATLAB/Simulink.

Techniques: