The Study of an Alcoholysis Reaction of Silicon Tetrafluoride with Alcohols and Magnesium to Prepare Tetraalkoxysilanes and Magnesium Fluoride

<div><p>GRAPHICAL ABSTRACT</p><p></p></div

Space-Confined Electrochemical Aptasensing with Conductive Hydrogels for Enhanced Applicability to Aflatoxin B1 Detection

Aflatoxin B1 (AFB1) contamination has received considerable attention for the serious harm it causes and its wide distribution. Hence, its efficient monitoring is of great importance. Herein, a space-confined electrochemical aptasensor for AFB1 detection is developed using a conductive hydrogel. Plasmonic gold nanoparticles (AuNPs) and methylene blue-embedded double-stranded DNA (MB-dsDNA) were integrated into the conductive Au-hydrogel by ultraviolet (UV) polymerization. Specific recognition of AFB1 by the aptamer released MB from MB-dsDNA in the matrix. The free DNA migrated to the outer layer due to electrostatic repulsion during the Au-hydrogel formation. The electrochemical aptasensor based on this Au-hydrogel offered a twofold enlarged oxidation current of MB (IMB) compared with that recorded in the homogeneous solution for AFB1 detection. Upon light illumination, this IMB was further enlarged by the local surface plasmon resonance (LSPR) of the AuNPs. Ultimately, the Au-hydrogel-based electrochemical aptasensor provided a detection limit of 0.0008 ng mL–1 and a linear range of 0.001–1000 ng mL–1 under illumination for AFB1 detection. The Au-hydrogel allowed for space-confined aptasensing, favorable conductivity, and LSPR enhancement for better sensitivity. It significantly enhanced the applicability of the electrochemical aptasensor by avoiding complicated electrode fabrication and signal loss in a bulk homogeneous solution

PKR knockdown enhanced IFN expression and activation of MAPKs, IRF3 and NF-κB pathways induced by poly(I:C).

<p>A549 cells were transfected with siPKR1 or siNC for 48 h, and then transfected with poly(I:C) for 1 h. Expression levels of IFN-β (A) were tested by real-time PCR and normalized to GAPDH. Phosphorylation form and total protein of p38 and JNK MAPKs, IRF3 and PKR were detected by Western blot (B). Immunoflurescence microscopy images to show subcellular localization of NF-κB p65 (C). PicCnt 100x was used to determine the percentages of cells stained with nuclei NF-κB (D). Data are shown as mean ± SEM for at least three independent experiments. *p<0.05,**p<0.01.</p

Pretreatment of poly(I∶C) suppresses the DENV2 replication in HepG2 cells.

<p>HepG2 cells were mock- or pretreated with 5 µg poly (I∶C) for 9 h, then infected with DENV2. (A) Real-time PCR to measure viral mRNA levels. Total RNAs were harvested at 2, 6, 12, and 24 h p.i., and used for real-time RT-PCR. (B) Immunofluorescence microscopy. Infected cells were fixed at 24 h p.i. and incubated with DENV2 prM antibody. (C) Percentages of positive-stained cells determined by PicCnt 100×. Error bars represent the standard error of mean from the average of three experiments. Student's <i>t</i> test, *, p<0.05; ***, p<0.001.</p

PKR depletion barely affected DENV2 replication in A549 cells.

<p>A549 cells were transfected with siNC, siPKR1, siRIG-I and siIPS-1, followed by DENV2 infection. Cells and culture supernatants were harvested for real-time PCR and viral titration respectively. Total cellular RNA was analyzed for IFN-β (A) or DENV2 RNA (B) level by real-time PCR and normalized to that of GAPDH in each sample. Virus yields were determined by TCID50 assay (C). DENV2 RNA levels in siNC- or siPKR-transfected HepG2 and THP-1 cells were measured by real-time PCR (D). Data are shown as mean ± SEM at least three independent experiments. Phosphorylation form and total protein levels of eIF-2α in DENV2-infected A549 cells were detected by Western blot (E). *p<0.05,**p<0.01, ***p<0.001.</p

DENV2 replication levels in IFN-β neutralizing antibody treated HepG2 cells.

<p>500 unit/ml IFN-β neutralizing antibody was added at 1 h before poly(I∶C) treatment and kept through poly(I∶C) treatment and virus infection. (A) Real-time PCR detecting the expression of viral mRNA; (B) Extracellular viral production determined by TCID50. Supernatants were harvested at 24 h p.i. and titered on C6/36 cells. Error bars represent the standard error of mean from the average of three experiments. Student's <i>t</i> test, **, p<0.01; ***, p<0.001.</p

The IFN induction mediated PKR knockdown was dependent on RIG-I and IPS-1, but not MDA-5.

<p>A549 cells were transfected with siRNA against RIG-I (siRIG-I), MDA-5 (siMDA-5) or IPS-1 (siIPS-1). Protein levels of RIG-I (A), MDA-5 (B) and IPS-I (C) were detected by Western blot. A549 cells were cotransfected with siRIG-I, siMDA-5 or siIPS-1 together with siPKR1 or siNC for 48 h followed by DENV2 infection (D, E). Total cellular RNA was analyzed for IFN-β using real-time PCR and normalized to that of GAPDH in each sample. Data are shown as the mean ± SEM and represent at least three independent experiments. *p<0.05, **p<0.01.</p

Expression of RIG-I in different cell lines.

<p>A549, HepG2 and THP-1 cells were harvested for real-time PCR analysis (A) or for Western blot analysis (B). Total cellular RNA was analyzed for RIG-I expression by real-time PCR and normalized to that of GAPDH in each sample. Data are shown as mean ± SEM at least three independent experiments.</p

Suppression of poly(I:C) induced IFN by PKR required RIG-I/IPS-I and dsRNA binding activity of PKR.

<p>(A, B) A549 cells were co-transfected with siRIG-I, siMDA-5 or siIPS-1 together with siPKR1 or siNC followed by poly(I:C). Total cellular RNA levels were analyzed for IFN-β using real-time PCR and normalized to those of GAPDH in each sample. (C) Cells were transfected with pcDNA6, pcDNA6-K64E, pcDNA6-K296R or pcDNA6-K64EK296R followed by siNC or siPKR1 transfection, and were then stimulated by poly(I:C). Total cellular RNA was analyzed for IFN-β expression by real-time PCR and normalized to that of GAPDH in each sample. Data are shown as mean ± SEM at least three independent experiments. *p<0.05, **p<0.01.</p

The dsRNA binding activity of PKR was important for downregulation of IFN induction.

<p>A549 cells were transfected with pcDNA6, pcDNA6-K64E, pcDNA6-K296R or pcDNA6-K64EK296R followed by siNC or siPKR1 transfection. Cells were then harvested for Western blot analysis (A). A549 cells transfected with different PKR mutants and siPKR1 were stimulated by DENV2 infection. Total cellular RNA was harvested for real-time PCR analysis (B). Data are shown as the mean ± SEM and represent for at least three independent experiments. *p<0.05.</p