Table_1_The association between tyrosine kinase inhibitors and fatal arrhythmia in patients with non-small cell lung cancer in Taiwan.docx

ObjectiveAs a standard therapy, tyrosine kinase inhibitors (TKIs) improved survival in patients with non-small cell lung cancer (NSCLC) and epidermal growth factor receptor (EGFR) mutation. However, treatment-related cardiotoxicity, particularly arrhythmia, cannot be ignored. With the prevalence of EGFR mutations in Asian populations, the risk of arrhythmia among patients with NSCLC remains unclear.MethodsUsing data from the Taiwanese National Health Insurance Research Database and National Cancer Registry, we identified patients with NSCLC from 2001 to 2014. Using Cox proportional hazards models, we analyzed outcomes of death and arrhythmia, including ventricular arrhythmia (VA), sudden cardiac death (SCD), and atrial fibrillation (AF). The follow-up duration was three years.ResultsIn total, 3876 patients with NSCLC treated with TKIs were matched to 3876 patients treated with platinum analogues. After adjusting for age, sex, comorbidities, and anticancer and cardiovascular therapies, patients receiving TKIs had a significantly lower risk of death (adjusted HR: 0.767; CI: 0.729–0.807, p ConclusionsCollectively, we highlighted a higher risk of VA/SCD in TKI users than in patients receiving platinum analogues. Further research is needed to validate these findings.</p

Image_1_The association between tyrosine kinase inhibitors and fatal arrhythmia in patients with non-small cell lung cancer in Taiwan.tif

ObjectiveAs a standard therapy, tyrosine kinase inhibitors (TKIs) improved survival in patients with non-small cell lung cancer (NSCLC) and epidermal growth factor receptor (EGFR) mutation. However, treatment-related cardiotoxicity, particularly arrhythmia, cannot be ignored. With the prevalence of EGFR mutations in Asian populations, the risk of arrhythmia among patients with NSCLC remains unclear.MethodsUsing data from the Taiwanese National Health Insurance Research Database and National Cancer Registry, we identified patients with NSCLC from 2001 to 2014. Using Cox proportional hazards models, we analyzed outcomes of death and arrhythmia, including ventricular arrhythmia (VA), sudden cardiac death (SCD), and atrial fibrillation (AF). The follow-up duration was three years.ResultsIn total, 3876 patients with NSCLC treated with TKIs were matched to 3876 patients treated with platinum analogues. After adjusting for age, sex, comorbidities, and anticancer and cardiovascular therapies, patients receiving TKIs had a significantly lower risk of death (adjusted HR: 0.767; CI: 0.729–0.807, p ConclusionsCollectively, we highlighted a higher risk of VA/SCD in TKI users than in patients receiving platinum analogues. Further research is needed to validate these findings.</p

Dopamine release by optogenetic stimulation.

<p>(A) Extracellular dopamine level recording with blue light illumination for 10 seconds on EGFP- and ChR2-EGFP-expressing PC12 cells. (C) ChR2-mediated dopamine release under different illumination durations, from 5 seconds to 20 seconds. Arrow indicates the start of illumination. (A) and (C) are dopamine signals in current vs. time. (B) and (D) are integrations of evoked currents with time during blue light illumination. Each column represents mean ± SEM from five independent experiments. ***, <i>P</i><0.001.</p

The <i>in vitro</i> real-time dopamine recording system.

<p>(A) Schematic diagram of working electrode (WE), counter electrode (CE), and reference electrode (RE) for dopamine sensing of ChR2-transfected PC12 cells under optogenetic stimulation at 473 nm. (B) Fluorescence and (C) phase-contrast images of ChR2-EGFP-expressing PC12 cells and electrode (arrows) under wide-field fluorescence microscope.</p

Extracellular calcium influx during optogenetic stimulation.

<p>(A) Relative intracellular Ca²⁺ levels of ratiometric fura-2 fluorescence intensity (ratio 340/380) for EGFP- and ChR2-EGFP-expressing PC12 cells in 1.8 mM Ca²⁺ buffer with or without 10 µM Nifedipine, or Ca²⁺-free buffer under illumination of blue light for 10 seconds. The Ca²⁺ signals during light illumination that could not be read out are depicted as dotted lines. (B) The changes of relative intracellular Ca²⁺ levels (mean ± SEM) by the fura-2 ratiometric imaging (Δ ratio 340/380) from at least 50 cells of four independent experiments. ***, <i>P</i><0.001.</p

Effect of stimulation parameters of blue light on calcium influx and dopamine release.

<p>Optogenetic stimulation at frequencies of 10, 20, and 30(CI) of blue laser light were applied for 10 seconds. (A) Changes of relative intracellular Ca²⁺ levels by the fura-2 ratiometric imaging (Δ ratio 340/380). Each column represents mean ± SEM from at least 60 cells of three independent experiments. *: significant difference within the same group; #: significant difference between groups. # and *, <i>P</i><0.05; **, <i>P</i><0.01. (B) Background current of stimulatory artifacts and (C) dopamine release under 10 Hz/10 ms, 30 Hz/30 ms, and CI of blue light.</p

Characterization of Au-NP/SAM modified electrodes.

<p>(A) CV curves of 10 µM dopamine recorded by native (-Δ-) and Au-NP/SAM (-•-) platinum microelectrodes at a scan rate of 10 V s⁻¹. The inserted picture represents the CV curve after background subtraction. SEM images showing the surface morphologies of (B) native and (C) Au-NP/SAM platinum electrodes. (D) Amperometric <i>i-t</i> curves of Au-NP/SAM microelectrodes recorded in PBS for dopamine calibration at a scan potential of 0.25 V (<i>vs.</i> Ag/AgCl).</p

Effect of calcium influx on ChR2-mediated dopamine release.

<p>(A) Simultaneous optical stimulation, dopamine current recording and Ca²⁺ imaging. Blue light illumination on ChR2-EGFP-expressing cells in controlled 1.8 mM Ca²⁺ buffer without or with 10 µM Nifedipine, and Ca²⁺-free buffer. The light-related artifact is not removed. (B) Significant inhibition in dopamine current during blue light illumination in Ca²⁺-free and Ca²⁺ buffer with Nifedipine. Each column represents mean ± SEM from five independent experiments. ***<i>P</i><0.001.</p

Viral load, but not serotype, affects DENV-induced IL-10 production.

<p>An ELISA showing IL-10 expression in THP-1 cells infected with DENV serotypes 1–4 (MOI  = 1) for 48 h (A) and with DENV serotype 2 PL046 (DENV 2) at the indicated MOI for 48 h in the presence or absence of anti-E (clone 50–2) mAb (C). The data are shown as the mean ± SD values from three independent experiments. ***P<0.001. N.S., not significant. B: A plaque assays showed viral replication in a MOI-dependent manner. The data are shown as the mean ± SD values from three independent experiments. *** P<0.001, compared with untreated cells. D: Western blots showing the expression of the DENV NS4B proteins, phosphorylation of PKB (pPKB Ser473), PKB, phosphorylation of GSK-3β (pGSK-3β Ser9), and GSK-3β in THP-1 cells infected with the indicated MOI of DENV 2 for 48 h. β-actin was the internal control. One set of representative data obtained from three independent experiments is shown. The ratios of phosphorylated PKB and GSK-3β to total proteins and the relative densities of NS4B are shown.</p

DENV infection induces PKA/PI3K-regulated GSK-3β inactivation, which facilitates CREB-mediated IL-10 production.

<p>A: A Western blot showing the time-kinetic phosphorylation of GSK-3β (pGSK-3β Ser9) and the expression of GSK-3β, β-catenin, and Mcl-1 in THP-1 cells infected with DENV serotype 2 PL046 (DENV 2, MOI  = 1). ** P<0.01 and *** P<0.001, compared with untreated cells. THP-1 cells were pre-treated with or without the GSK-3 inhibitor BIO, and then infected with DENV 2 (MOI  = 1) for 48 h. ELISA (B) and Western blotting (C) analyses were used to detect IL-10 production and CREB phosphorylation (pCREB Ser133), respectively. For the ELISA analyses, the data shown represent mean ± SD values of three independent experiments. ***P<0.001, compared with untreated cells; ###P<0.001, compared with DENV-infected cells. Western blots showing the expression of the indicated proteins in THP-1 cells transfected with lentiviral-based shGSK-3β (D) shRNA-transfected THP-1 cells pre-treated with or without H-89 or LY294002 for 0.5 h, and then infected with DENV 2 (MOI  = 1) for 48 h (E) and THP-1 cells pre-treated with or without H-89, LY294002, or Bis for 0.5 h, and then infected with DENV 2 (MOI  = 1) for 48 h (F). shLuc was used as a negative control. For Western blotting results, β-actin was the internal control. One set of representative data obtained from three independent experiments is shown. The ratios of phosphorylated GSK-3β and CREB to total proteins and the relative densities of β-catenin and Mcl-1 are shown. For all experiments, the quantitative data shown represent mean ± SD values of three independent experiments.</p