Inhibition of Immunoproteasome Attenuates NLRP3 Inflammasome Response by Regulating E3 Ubiquitin Ligase TRIM31.

Excessive secretion of pro-inflammatory cytokines leads to the disruption of intestinal barrier in inflammatory bowel disease (IBD). The inflammatory cytokine tumor necrosis factor alpha (TNFα) induces the assembly of the NLRP3 inflammasome, resulting in the augmented secretion of inflammatory cytokines implicated in the pathogenesis of inflammatory bowel disease (IBD). TNFα has also been known to induce the formation of immunoproteasome (IP), which incorporates immunosubunits LMP2, LMP7, and MECL-1. Inhibition of IP activity using the IP subunit LMP2-specific inhibitor YU102, a peptide epoxyketone, decreased the protein levels of NLRP3 and increased the K48-linked polyubiquitination levels of NLRP3 in TNFα-stimulated intestinal epithelial cells. We observed that inhibition of IP activity caused an increase in the protein level of the ubiquitin E3 ligase, tripartite motif-containing protein 31 (TRIM31). TRIM31 facilitated K48-linked polyubiquitination and proteasomal degradation of NLRP3 with an enhanced interaction between NLRP3 and TRIM31 in intestinal epithelial cells. In addition, IP inhibition using YU102 ameliorated the symptoms of colitis in the model mice inflicted with dextran sodium sulfate (DSS). Administration of YU102 in the DSS-treated colitis model mice caused suppression of the NLRP3 protein levels and accompanied inflammatory cytokine release in the intestinal epithelium. Taken together, we demonstrated that inhibiting IP under inflammatory conditions induces E3 ligase TRIM31-mediated NLRP3 degradation, leading to attenuation of the NLRP3 inflammatory response that triggers disruption of intestinal barrier.

Fluorometric Detection of SARS-CoV-2 Single-Nucleotide Variant L452R Using Ligation-Based Isothermal Gene Amplification.

Since the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variant was first discovered, several variants showing different infectivity and immune responses have emerged globally. As the conventional method, whole-genome sequencing following polymerase chain reaction (PCR) is currently used for diagnosis of SARS-CoV-2 mutations. However, these conventional PCR-based direct DNA sequencing methods are time-consuming, complicated, and require expensive DNA sequencing modules. Here, we developed a fluorometric method for the accurate detection of a single missense mutation of U to G in the spike (S) gene that changes leucine to arginine (L452R) in SARS-CoV-2 genomic RNA. Our method for the detection of single-nucleotide mutations (SNM) in the viral RNA genome includes RNA sequence-dependent DNA ligation and tandem isothermal gene amplification methods, such as strand displacement amplification (SDA) and rolling circle amplification (RCA) generating G-quadruplex (GQ). In the presence of SNM in the viral RNA, ligation of both ends of the probe DNAs occurs between 5'-phosphorylated hairpin DNA and linear probe DNA that can discriminate a single base mismatch. The ligated DNAs were then extended to generate long-stem hairpin DNAs that are subjected to the first isothermal gene amplification (SDA). SDA produces multitudes of short ssDNA from the long-stem hairpin DNAs, which then serve as primers by annealing to circular padlock DNA for the second isothermal gene amplification (RCA). RCA produces a long stretch of ssDNA containing GQ structures. Thioflavin T (ThT) is then intercalated into GQ and emits green fluorescence, which allows the fluorometric identification of SARS-CoV-2 variants. This fluorometric analysis sensitively distinguished SNM in the L452R variant of SARS-CoV-2 RNA as low as 10 pM within 2 h. Hence, this fluorometric detection method using ligation-assisted tandem isothermal gene amplification can be applied for the diagnosis of SARS-CoV-2 SNM variants with high accuracy and sensitivity, without the need for cumbersome whole-genome DNA sequencing.

Mesoporous Silica Particle as an RNA Adsorbent for Facile Purification of In Vitro-Transcribed RNA.

Messenger RNA vaccines against SARS-CoV-2 hold great promise for the treatment of a wide range of diseases by using mRNA as a tool for generating vaccination antigens as well as therapeutic proteins in vivo. Increasing interest in mRNA preparation warrants reliable methods for in vitro transcription (IVT) of mRNA, which must entail the elimination of surplus side products such as immunogenic double-stranded RNA (dsRNA). We developed a facile method for the removal of dsRNA from in vitro transcribed RNA with mesoporous silica particles as RNA adsorbents. Various polyamines were tested for the facilitation of RNA adsorption onto mesoporous silica particles in the chromatography. Among the polyamines tested for RNA adsorption, spermidine showed a superior capability of RNA binding to the silica matrix. Mesoporous silica-adsorbed RNA was readily desorbed with elution buffer containing either salt, EDTA, or urea, possibly by disrupting electrostatic interaction and hydrogen bonding between RNA and the silica matrix. Purification of IVT RNA was enabled with the adsorption of RNA to mesoporous silica in a spermidine-containing buffer and subsequent elution with EDTA. By differing EDTA concentration in the eluting buffer, we demonstrated that at least 80% of the dsRNA can be removed from the mesoporous silica-adsorbed RNA. When compared with the cellulose-based removal of dsRNA from IVT RNA, the mesoporous silica-based purification of IVT RNA using spermidine and EDTA in binding and elution, respectively, exhibited more effective removal of dsRNA contaminants from IVT RNA. Thus, mRNA purification with mesoporous silica particles as RNA adsorbents is applicable for the facile preparation of nonimmunogenic RNA suitable for in vivo uses.

Facilitation of Dye-Based Quantitative Real-Time Polymerase Chain Reaction with Poly(ethylene glycol)-Engrafted Graphene Oxide.

Quantitative real-time polymerase chain reaction (qPCR) is an important and extensively utilized technique in medical and biotechnological applications. qPCR enables the real-time detection of nucleic acid during amplification, thus surpassing the necessity of post-amplification gel electrophoresis for amplicon detection. Despite being widely employed in molecular diagnostics, qPCR exhibits limitations attributed to nonspecific DNA amplification that compromises the efficiency and fidelity of qPCR. Herein, we demonstrate that poly(ethylene glycol)-engrafted nanosized graphene oxide (PEG-nGO) can significantly improve the efficiency and specificity of qPCR by adsorbing single-stranded DNA (ssDNA) without affecting the fluorescence of double-stranded DNA binding dye during DNA amplification. PEG-nGO adsorbs surplus ssDNA primers in the initial phase of PCR, having lower concentrations of DNA amplicons and thus minimizing the nonspecific annealing of ssDNA and false amplification due to primer dimerization and erroneous priming. As compared to conventional qPCR, the addition of PEG-nGO and the DNA binding dye, EvaGreen, in the qPCR setup (dubbed as PENGO-qPCR) significantly enhances the specificity and sensitivity of DNA amplification by preferential adsorption of ssDNA without inhibiting DNA polymerase activity. The PENGO-qPCR system for detection of influenza viral RNA exhibited a 67-fold higher sensitivity than the conventional qPCR setup. Thus, the performance of a qPCR can be greatly enhanced by adding PEG-nGO as a PCR enhancer as well as EvaGreen as a DNA binding dye to the qPCR mixture, which exhibits a significantly improved sensitivity of the qPCR.

Effects of time of bed rest on vascular complications after cardiac catheterization in pediatric patients with congenital heart disease: A randomized controlled trial.

BACKGROUND: Pediatric cardiac catheterization, which is performed by accessing the femoral vessel, requires immobilization and bed rest for 4-6 h to prevent vascular complications. Studies in adults suggest that the immobilization time for the same access can be safely reduced to approximately 2 h after catheterization. However, it is unclear whether bed-rest time can be safely decreased after catheterization in children. OBJECTIVE: To assess the effects of bed-rest duration on bleeding, vascular complications, pain level, and the use of additional sedatives after transfemoral cardiac catheterization in children with congenital heart disease. METHODS: This study was an open-label, randomized, controlled, posttest-only design, including 86 children who underwent cardiac catheterization. Children were allocated to receive either 2 h of bed rest (n = 42) in the experimental group or 4 h of bed rest (n = 42) in the control group following catheterization. RESULTS: The mean age of children was 3.93 (±3.82) years in the experimental group and 5.63 (±3.97) years in the control group. There was no difference in site bleeding incidence (P = 0.214), vascular complication score (P = 0.082), pain level (P = 0.445), or additional sedation use (P = 1.000) between the two groups. CONCLUSIONS: There were no significant hemostatic complications after 2 h of bed rest following pediatric catheterization; therefore, 2 h of bed rest was as safe as 4 h of bed rest. (Trial registration: KCT0007737).

Nonhalogenated Solvent-Processed High-Performance Indoor Photovoltaics Made of New Conjugated Terpolymers with Optimized Monomer Compositions.

Conjugated random terpolymers, PJ-25, PJ-50, and PJ-75 were successfully synthesized from three different monomers. Fluorine-substituted benzotriazole (2F-BTA) was incorporated into 4,8-bis(4-chlorothiophen-2-yl)benzo[1,2-b:4,5-b']dithiophene (BDT-T-Cl) and a 1,3-bis(4-(2-ethylhexyl)thiophen-2-yl)-5,7-bis(2-alkyl)benzo[1,2-c:4,5-c']dithiophene-4,8-dione (BDD)-based alternating copolymer PM7 as a third monomeric unit. The solubility of the random terpolymers in nonhalogenated solvents increased with the number of 2F-BTA units in PM7. The random terpolymers were mixed with 3,9-bis(2-methylene-((3-(1,1-dicyanomethylene)-6,7-difluoro)-indanone))-5,5,11,11-tetrakis(4-hexylphenyl)-dithieno[2,3-d:2',3'-d']-s-indaceno[1,2-b:5,6-b']dithiophene (IT-4F) to fabricate organic photovoltaic (OPV) cells. Among the three terpolymers and two related binary copolymers (e.g., PM7 and J52-Cl), outdoor photovoltaic (PV) cells (AM 1.5G) based on the PJ-50:IT-4F blend showed a high power conversion efficiency (PCE) of 11.34%. In addition, PJ-50 was employed as a donor in indoor PV (IPV) cells and was blended with nonfullerene acceptors, which have different absorption ranges. Among them, the PJ-50:IT-4F-based IPV device had the highest PCE of 17.41% with a Jsc of 54.75 µA cm-2 and an FF of 0.77 under 160 µW cm-2 light-emitting diode (LED) light. The terpolymer introduced in this study can be regarded as a promising material for the fabrication of outdoor PV and IPV cells with excellent performance involving the use of an eco-friendly solvent.