RNase Protection Assay Research Articles

The Rbfox1/LASR complex controls alternative pre-mRNA splicing by recognition of multi-part RNA regulatory modules.

The Rbfox proteins regulate alternative pre-mRNA splicing by binding to the RNA element GCAUG. In the nucleus, most of Rbfox is bound to LASR, a complex of RNA-binding proteins that recognize additional RNA motifs. However, it remains unclear how the different subunits of the Rbfox/LASR complex act together to bind RNA and regulate splicing. We used a nuclease-protection assay to map the transcriptome-wide footprints of Rbfox1/LASR on nascent cellular RNA. In addition to GCAUG, Rbfox1/LASR binds RNA containing motifs for LASR subunits hnRNPs M, H/F, C, and Matrin3. These elements are often arranged in tandem, forming multi-part modules of RNA motifs. To distinguish contact sites of Rbfox1 from the LASR subunits, we analyzed a mutant Rbfox1(F125A) that has lost RNA binding but remains associated with LASR. Rbfox1(F125A)/LASR complexes no longer interact with GCAUG but retain binding to RNA elements for LASR. Splicing analyses reveal that in addition to activating exons through adjacent GCAUG elements, Rbfox can also stimulate exons near binding sites for LASR subunits. Mini-gene experiments demonstrate that these diverse elements produce a combined regulatory effect on a target exon. These findings illuminate how a complex of RNA-binding proteins can decode combinatorial splicing regulatory signals by recognizing groups of tandem RNA elements.

Improving functional substrate delineation for ablation of ventricular tachycardia using high-density electroanatomic mapping and double ventricular extra stimuli protocol

Abstract Background A partial delineation of targets for ventricular tachycardia (VT) ablation may contribute to a suboptimal success rate. Abnormal electrograms (EGMs) with low-voltage near-field components might be concealed within high-amplitude far-field signals. Our hypothesis posited that acquiring functional mapping after double ventricular extrastimuli (S3 protocol) could reveal relevant areas of functional substrate overlooked during sinus or S1 mapping. Purpose To assess the benefit and feasibility of functional substrate mapping using a full ventricle S3 protocol and to evaluate its colocalization with conducting channels (CCs) identified in late gadolinium enhancement cardiac magnetic resonance (LGE-CMR). Methods (Figure 1) - An S3 mapping protocol with a drive train of S1 followed by S2 (effective refractory period (ERP) +30ms) and S3 (ERP+50ms) from the RV apex was conducted in 40 consecutive patients undergoing scar-related VT ablation. Deceleration zones (DZs) and areas of late potentials (LPs) were identified for all maps (S1, S2, and S3). Pre-procedural non-invasive substrate assessment was performed with LGE-CMR and post-processing using a commercially available system with automated CC identification. Results The S3 protocol was completed in 34 of the 40 procedures (85.0%). Repeated induction of ventricular arrhythmias prevented the full S3 protocol in 5 patients (15.0%). Functional substrate identified during S3 activation mapping was significantly more extensive than that identified using S2 and S1, including a higher number of DZs (2.94, 2.47, and 1.82, respectively; p<0.01) and a wider area of LPs (44.1cm2, 38.2cm2, and 29.4cm2, respectively; p<0.001). The percentage of CCs unmasked by DZs and LPs using S3 maps was significantly higher than the ones using S2 and S1 maps(78%, 65%, and 48%; p<0.001, and 88%, 81%, and 68%; p<0.01, repectively). After VT ablation based on an S3 protocol, 77.9% of patients have been VT free during a median follow-up of 13.6 months. Conclusions (Graphical abstract -Figure 2) - This novel substrate-based protocol for assessing functional substrate is feasible in most patients, enables better identification of ablation targets, and has the potential to improve patient prognosis post-ablation.Methods - The S3 protocolConclusion - Graphical abstract

The benefit of a double ventricular extra stimuli protocol to improve functional substrate mapping during ablation of ventricular tachycardia. - The S3-VT-FREE project

Abstract Introduction Ablation of ventricular tachycardia (VT) based on substrate-based mapping has been proposed to itemize areas of interest identifying areas of low-voltage, late potentials (LPs), and deceleration zones (DZs). However, partial delineation of targets for ablation is likely responsible for a suboptimal long-term success rate. The abnormal electrograms (EGMs), featuring low-voltage near-field components may be hidden within the high-amplitude far-field signal. We hypothesized that the acquisition of functional mapping after double ventricular extrastimuli (S3 protocol) would uncover relevant areas of functional substrate that could be missed during sinus or S1 map. We aim to evaluate the potential benefit and the feasibility of functional substrate mapping using an S3 protocol for VT ablation and to assess its colocalization with detected corridors using late gadolinium enhancement magnetic resonance imaging (LGE-MRI). Methods We included consecutively 29 patients with structural cardiomypathy who underwent a VT ablation. The S3 protocol included: (1) assessment of the ventricular effective refractory period (ERP), (2) pacing protocol from the apex of the right ventricle with S1 x 6 (cycle length: 600ms) followed by S2 (ERP+30ms) and S3 (ERP+50ms). Electroanatomical mapping was acquired with the EnsiteX system (Abbott, IL) using HD Grid catheter and omnipolar signals. ILAMs were used to identify deceleration zones and areas of late potentials were identified for all maps. A non-invasive substrate assessment was done with LGE-MRI and post-processing with a commercially available system (ADAS3D, Spain) with automated identification of corridors. Results The S3 protocol was completed in 25 of the 29 procedures (86.2%). The repeated induction of ventricular arrhythmias did not allow for full protocol of the S3 protocol in 4 patients (13.8%). The functional substrate identified during the S3 activation mapping was significantly more extensive than the one identified using S1 and S2 activation mappings (p<0.05) (Table). The S3 protocol unmasked a higher number of DZs (p<0.01), a wider area of LPs (p<0.01), and a longer duration of the DZs (p=0.01). All DZ maps were compared with preprocedural cardiac magnetic resonance. The number of DZs matching with conduction corridors identified by LGE-MRI was higher in the activation mapping of S3 compared with the S1 and S2 ones (p=0.02). After VT ablation based on an S3 protocol, 88% of patients have been VT free during a median follow-up of 9 months. Conclusions This novel substrate-based protocol to assess the ventricular functional substrate is feasible in the majority of patients (86.2%) and allows for a better identification of targets for ablation and could facilitate VT ablation, and improve the prognosis of the patients after ablation.

Usefulness of an RNA extraction-free test for the multiplexed detection of ALK, ROS1, and RET Gene Fusions in Real Life FFPE Specimens of Non-Small Cell Lung Cancers

ABSTRACT Background ALK, ROS1 and RET rearrangements occur, respectively, in 5%, 2%, and 1% non-small cell lung cancers (NSCLC). ALK and ROS1 fusion proteins detection by immunohistochemistry (IHC) has been validated for rapid patient screening, but ROS1 fusions need to be confirmed by another technique and no RET IHC test is available for clinical use. Research design and methods We report herein the usefulness of the HTG EdgeSeq Assay, an RNA extraction-free test combining a quantitative nuclease protection assay with NGS, for the detection of ALK, ROS1 and RET fusions from ‘real-life’ small NSCLC samples. A total of 203 FFPE samples were collected from 11 centers. They included 143 rearranged NSCLC (87 ALK, 39 ROS1, 17 RET) and 60 ALK-ROS1-RET negative controls. Results The assay had a specificity of 98% and a sensitivity for ALK, ROS1 and RET fusions of 80%, 94% and 100% respectively. Among the 19 HTG-assay false negative samples, the preanalytical conditions were identified as the major factors impacting the assay efficiency. Conclusions Overall, the HTG EdgeSeq assay offers comparable sensitivities and specificity than other RNA sequencing techniques, with the advantage that it can be used on very small and old samples collected multicentrically.

A novel EGFR variant EGFRx maintains glioblastoma stem cells through STAT5.

Glioblastomas are universally lethal brain tumors containing tumor-propagating glioblastoma stem cells (GSCs). EGFR gene amplification or mutation is frequently detected in GBMs and is associated with poor prognosis. However, EGFR variants in GSCs and their role in the maintenance of GSCs and progression of GBM are unclear. EGFR variants were detected through bioinformatic HISAT-StringTie-Ballgown pipeline and verified through 5' RACE, RT-PCR, ribonuclease protection, and northern blotting assays. EGFRx function was investigated through neurosphere, cell viability, intracranial xenograft and RNA-seq assays. EGFRx-STAT5 signaling was investigated through western blotting, coimmunoprecipitation, immunofluorescence, luciferase reporter, RT-PCR and CUT&Tag assays. We identified a novel EGFR variant (EGFRx), that is specifically expressed in GSCs. Unlike the EGFRvIII variant, which lacks exons 2-7, EGFRx is characterized by the absence of exons 2-14, and encodes an EGFR protein that does not possess the entire extracellular ligand-binding domain. We observed that EGFRx exhibits significant glycosylation, is required for GSC self-renewal, proliferation, and tumorigenesis, and highly active in glioblastomas compared to normal brain tissue. Mechanistically, EGFRx constitutively and specifically activates STAT5 in GSCs through spontaneous asymmetric dimerization of the kinase domain. EGFRx plays essential roles in the maintenance of the GSC phenotype through constitutive activation of STAT5 and promotes GBM progression, suggesting that EGFRx-STAT5 signaling represents a promising therapeutic target for GBM.

The long non-coding RNA PILNCR2 increases low phosphate tolerance in maize by interfering with miRNA399-guided cleavage of ZmPHT1s

The open reading regions of ZmPHT1s (inorganic phosphate [Pi] transporters) in maize possess target sites of microRNA399 (miR399). However, the relationship between miR399 and ZmPHT1s and its functional importance in response to Pi deficiency remain to be explored. We show here that ZmPHT1;1, ZmPHT1;3, and ZmPHT1;13 are the targets of ZmmiRNA399. We found that a long non-coding RNA, PILNCR2 (Pi-deficiency-induced lncRNA 2), is transcribed from the opposing DNA strand of ZmPHT1;1 and predominantly localized in the cytoplasm. A ribonuclease protection assay and an RNA–RNA binding assay showed that PILNCR2 and ZmPHT1s could form the RNA/RNA duplexes in vivo and in vitro. A co-expression assay in N. benthamiana revealed that the PILNCR2/ZmPHT1 RNA/RNA duplexes interfere with miR399-guided cleavage of ZmPHT1 mRNAs. Overexpression of PILNCR2 increased low-Pi tolerance in maize, whereas its knockout and knockdown decreased low-Pi tolerance in maize. Consistently, ZmPHT1;3 and ZmPHT1;13 mRNA abundance was increased in transgenic plants overexpressing PILNCR2 but reduced in its knock-out mutants, suggesting that PILNCR2 positively regulates the mRNA abundance of ZmPHT1;3 and ZmPHT1;13 in maize. Collectively, these results indicate that PILNCR2 plays an important role in maize Pi homeostasis by interfering with miRNA399-guided cleavage of ZmPHT1 mRNAs.

TDO2‐augmented fibroblasts secrete EVs enriched in immunomodulatory Y‐derived small RNA

AbstractMounting evidence implicates extracellular vesicles (EVs) factors as mediators of cell therapy. Cardiosphere‐derived cells are cardiac‐derived cells with tissue reparative capacity. Activation of a downstream target of wnt/β‐catenin signalling, tryptophan 2,3 dioxygenase (TDO2) renders therapeutically inert skin fibroblasts cardioprotective. Here, we investigate the mechanism by which concentrated conditioned media from TDO2‐augmented fibroblasts (TDO2‐CCM) exert cardioprotective effects. TDO2‐CCM is cardioprotective in a mouse model of MI compared to CCM from regular fibroblasts (HDF‐CCM). Transcriptomic analysis of cardiac tissue at 24 h demonstrates broad suppression of inflammatory and cell stress markers in animals given TDO2‐CCM compared to HDF‐CCM or vehicle. Sequencing analysis of TDO2‐EV RNA demonstrated abundance of a small Y‐derived small RNA dubbed ‘NT4’. Purification of TDO2‐EVs by size‐exclusion chromatography and RNAse protection assays demonstrated that NT4 is encapsulated inside EVs. Consistently with TDO2‐CCM, macrophages exposed to NT4 showed suppression of the inflammatory and cell stress mediators, particularly p21/cdkn1a. NT4‐depleted TDO2‐CCM resulted in diminished immunomodulatory capacity. Finally, administration of NT4 alone was cardioprotective in an acute model of myocardial infarction. Taken together, these findings elucidate the mechanism by which TDO2 augmentation mediates potency in secreted EVs through enrichment of NT4 which suppresses upstream cell stress mediators including p21/cdkn1a.

Characterization of PAMAM Dendrimers for the Delivery of Nucleic Acid Nanoparticles.

The protocols described in this book chapter are meant to be used as an outline and guideline to explore the use of a cationic, polymeric, and synthetic carrier-poly (amidoamine) (PAMAM) dendrimers. The amine-terminated, hyperbranched structures have been identified as a vehicle for the delivery of nucleic acids. As such, clear protocols for the optimization of dendrimer usage should be set in place. This chapter details the experiments used to determine the ratio that dendrimers and nucleic acids should be complexed at through the use of binding assays, nuclease protection assays, and competitive binding assays.

A Personal Perspective on My Scientific Career

A Personal Perspective on My Scientific Career

Development of a High-Density Genetic Map for Muscadine Grape Using a Mapping Population from Selfing of the Perfect-Flowered Vine ‘Dixie’

Intraspecific diversity of the immune grape Muscadinia rotundifolia Michaux. can serve as a rich source of valuable resistance loci to the most widespread pathogens and pests of grapevine. While only one Run1/Rpg1 resistance locus has been introgressed from M. rotundifolia to the Vitis vinifera gene pool, a number of other genes conferring resistance to powdery mildew and downy mildew have been identified in various Muscadinia cultivars. A larger introduction of Muscadinia varieties to the European continent would greatly facilitate experiments of interspecific crosses as well as stimulate biotechnological efforts to overcome the main barrier to F1 fertility caused by the differences in chromosome number. For the successful introduction of Muscadinia into the new European environment, it is necessary to overcome the difficulties associated with the physiological characteristics of the species, such as insufficient cold tolerance and very late fruit ripening. To facilitate the further discovery of valuable loci in Muscadinia and their transfer to grapevine breeding programs, we constructed a high-density linkage map using an S1 mapping population obtained from the self-pollination of M. rotundifolia cv. Dixie maintained on the southern coast of Crimea. Using ddRADseq, 3730 SNPs were ordered across 20 linkage groups spanning 2753.6 cM of the total map length. No segregation in resistance to diseases and pests was observed among the 'Dixie' S1 population, suggesting the presence of homozygous non-segregating resistant loci in the genetic background of 'Dixie'. Markers with high segregation distortion showed a bias towards chromosomal intervals on linkage groups 10 and 20, where loci affecting the survival of 'Dixie' S1 progeny may be localized. QTLs with significant additive and dominance effects were discovered on LG14 and LG18, affecting the morphological traits associated with the vigor of growth and adaptability of young Muscadinia vines in the conditions of Crimea.

Rapid detection of Prorocentrum donghaiense using nuclease protection assay integrated with dot nucleic acid chromatography strip

Harmful algal bloom (HAB), caused by harmful microalgae, is a global marine ecological disaster, and its frequency and damage have increased significantly in recent years. For effective monitoring of HAB, there is an urgent need to establish rapid and easy detection techniques for harmful microalgae. For this purpose, here a nuclease protection assay integrated with dot nucleic acid chromatography strip (NPA-dNACS) detection technology was proposed for the first time. Using Prorocentrum donghaiense that is one of the most typical HAB-forming species in the East China Sea as a target, the application of NPA-dNACS in the detection of harmful microalgae was explored in this study. NPA-dNACS does not require nucleic acid extraction and amplification. Instead, it directly targets high-copy rRNA in cells and employs the hand-made dNACS to automatically determine the detection results, making the detection process rather simple and fast. First, specific probes targeting the D1-D2 region of ribosomal large subunit DNA of P. donghaiense were designed, and the specificity of the probes for the target species was confirmed by the cross-test with 19 other microalgae. Next, all of the main parameters or conditions associated with NPA-dNACS were optimized separately. In addition, the sensitivity of NPA-dNACS was also evaluated, and the developed NPA-dNACS displayed a detection limit of 7 × 10−1 ng of target RNA. The test with spiked samples showed that NPA-dNACS can meet the demand for early warning of P. donghaiense-forming HAB. In conclusion, the proposed NPA-dNACS are specific, rapid, simple, and visualizable, providing a new alternative method for the rapid detection of harmful microalgae like P. donghaiense.

Transcriptome-Wide Gene Expression Profiles from FFPE Materials Based on a Nuclease Protection Assay Reveals Significantly Different Patterns between Synovial Sarcomas and Morphologic Mimickers.

Simple SummaryThe following study aimed to validate and test the feasibility, reliability, technical applicability robustness, and reliability of a new commercially available transcriptome profiling assay, being performed on formalin-fixed, paraffin-embedded (FFPE) patient samples. The patients were suffering from either synovial sarcoma or spindle cell sarcoma, which are morphologically similar tumors, but differ in molecular characteristics. Transcriptome analysis of FFPE material is still challenging. However, it is the most available material in pathological routine and therefore valuable for translational research approaches. This new commercially available assay is based on a nuclease protection assay and has shown to be a feasible method for adequate transcriptome profiling with low sample input and therefore is suitable for further research of biomarkers. Background: Transcriptome profiling provides large data on tumor biology, which is particularly valuable in translational research and is becoming more and more important for clinical decision-making as well. RNA sequencing is considered to be the gold standard for this. However, FFPE material, as the most available material in routine pathology, has been an undefeatable obstacle for RNAseq. Extraction-free nuclease protection assays have the potential to be a reliable alternative method for large-scale expression profiling. The aim of this study was to validate and test the basic feasibility, technical applicability robustness, and reliability of the HTG transcriptome profiling (HTP) assay on clinical tumor samples. Methods: FFPE samples from 44 synovial sarcomas (SyS) and 20 spindle cell sarcomas (SpcS) were used. The HTP assay was performed on 10 µm thin FFPE slides. After nuclease protection in the HTG Edge Seq System, libraries were generated for sequencing on an Illumina NextSeq 500 platform. Fastq data were parsed and then analyzed by using the HTG analysis platform EdgeSeq REVEAL. Immunohistochemistry was performed to validate the expression of TLE1. Results: The technical application of the HTP Panel revealed robust and reliable results with 62 samples, and only 2 samples failed due to an incomplete digestion of gDNA. The analysis, performed at the analysis platform REVEAL, showed 5964 genes being significantly differentially expressed between SpcS and SyS. In particular, overexpression of the known marker TLE1 in synovial sarcoma could be recovered, which underlines the reliability of this system. Discussion: Transcriptome profiling gets more and more important for tumor research and diagnostics. Among other established technologies, the HTP Panel has shown to be a feasible method to get robust and reliable results. Thereby, this method needs very few sample-input by getting a success-rate of 96.88%, which indicates the upper average range, compared to other technologies working with FFPE tissue. Conclusion: The nuclease protection assay-based HTP Panel is a feasible method for adequate transcriptome profiling with low sample input and therefore is suitable for further research of biomarkers.

Optimization of Anti-SARS-CoV-2 Treatments Based on Curcumin, Used Alone or Employed as a Photosensitizer.

Curcumin, the bioactive compound of the spice Curcuma longa, has already been reported as a potential COVID-19 adjuvant treatment due to its immunomodulatory and anti-inflammatory properties. In this study, SARS-CoV-2 was challenged with curcumin; moreover, curcumin was also coupled with laser light at 445 nm in a photodynamic therapy approach. Curcumin at a concentration of 10 μM, delivered to the virus prior to inoculation on cell culture, inhibited SARS-CoV-2 replication (reduction >99%) in Vero E6 cells, possibly due to disruption of the virion structure, as observed using the RNase protection assay. However, curcumin was not effective as a prophylactic treatment on already-infected Vero E6 cells. Notably, when curcumin was employed as a photosensitizer and blue laser light at 445 nm was delivered to a mix of curcumin/virus prior to the inoculation on the cells, virus inactivation was observed (>99%) using doses of curcumin that were not antiviral by themselves. Photodynamic therapy employing crude curcumin can be suggested as an antiviral option against SARS-CoV-2 infection.

Rapid Determination of RNA Modifications in Consensus Motifs by Nuclease Protection with Ion-Tagged Oligonucleotide Probes and Matrix-Assisted Laser Desorption Ionization Mass Spectrometry.

The reversible and substoichiometric modification of RNA has recently emerged as an additional layer of translational regulation in normal biological function and disease. Modifications are often enzymatically deposited in and removed from short (~5 nt) consensus motif sequences to carefully control the translational output of the cell. Although characterization of modification occupancy at consensus motifs can be accomplished using RNA sequencing methods, these approaches are generally time-consuming and do not directly detect post-transcriptional modifications. Here, we present a nuclease protection assay coupled with matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) to rapidly characterize modifications in consensus motifs, such as GGACU, which frequently harbor N6-methyladenosine (m6A). While conventional nuclease protection methods rely on long (~30 nt) oligonucleotide probes that preclude the global assessment of consensus motif modification stoichiometry, we investigated a series of ion-tagged oligonucleotide (ITO) probes and found that a benzylimidazolium-functionalized ITO (ABzIM-ITO) conferred significantly improved nuclease resistance for GGACU targets. After optimizing the conditions of the nuclease protection assay, we applied the ITO and MALDI-MS-based method for determining the stoichiometry of GG(m6A)CU and GGACU in RNA mixtures. Overall, the ITO-based nuclease protection and MALDI-MS method constitutes a rapid and promising approach for determining modification stoichiometries of consensus motifs.

Quantitative Trait Loci Analysis Based on High-Density Mapping of Single-Nucleotide Polymorphisms by Genotyping-by-Sequencing Against Pine Wilt Disease in Japanese Black Pine (Pinus thunbergii).

Identifying genes/loci for resistance to pine wilt disease (PWD) caused by the pine wood nematode (PWN) is beneficial for improving resistance breeding in Pinus thunbergii, but to date, genetic information using molecular markers has been limited. Here, we constructed a high-density linkage map using genotyping-by-sequencing (GBS) and conducted quantitative trait loci (QTL) analysis for PWD resistance for the self-pollinated progeny of “Namikata 73,” which is the most resistant variety among resistant varieties of P. thunbergii, following inoculation tests with PWN. An S1 mapping population consisting of the 116 progenies derived from self-pollination of the resistant variety, “Namikata 73” (resistance rank 5 to PWN), was inoculated with PWN isolate Ka-4 and evaluated for disease symptoms. To construct a high-density linkage map, we used single-nucleotide polymorphisms (SNPs) identified by GBS based on next-generation sequencing technology and some anchor DNA markers, expressed sequence tag (EST)-derived SNP markers and EST-derived simple sequence repeat (SSR) markers, and genomic SSR markers. The linkage map had 13 linkage groups (LGs) consisting of 2,365 markers including 2,243 GBS-SNP markers over a total map distance of 1968.4 centimorgans (cM). Results from QTL analysis using phenotype data and the linkage map indicated that PWD resistance is controlled by a single locus located on LG-3, as identified in a previous study. This locus showed overdominant genetic action in the present study. With the confirmation of PWD1 in two different mapping populations (present study and a previous study), the locus associated with this region is thought to be a good target for marker-assisted selection in P. thunbergii breeding programs in order to obtain high levels of resistance to PWD caused by PWN.

Glucocorticoid Induces Atrial Arrhythmogenesis via Modification of Ion Channel Gene Expression in Rats.

Excess psychological stress is one of the precipitating factors for paroxysmal atrial fibrillation (AF), although the involved mechanisms are still uncertain. To test a hypothesis that one of the stress-induced hormones, glucocorticoid, is involved in the pathogenesis of stress-induced AF, we investigated whether the glucocorticoid could alter the temporal profile of cardiac ion channels gene expression, thereby leading to atrial arrhythmogenesis.Dexamethasone (DEX, 1.0 mg/kg) was injected subcutaneously in Sprague-Dawley rats. At predetermined times after DEX injection (0, 1, 3, 6, 12, and 24 hours), the mRNA levels of cardiac ion channel genes (erg, KvLQT1, Kv4.3, Kv4.2, Kv2.1, Kv1.5, Kv1.4, Kv1.2, SUR2A, Kir6.2, Kir3.4, Kir3.1 Kir2.2, Kir2.1, SCN5A, and α1C) were determined using RNase protection assay. DEX induced immediate and transient increase in the mRNA level of Kv1.5 and Kir2.2 with peaks at 6 (5.0 fold) and 3 hours (3.3 fold) after DEX injection, respectively. Patch-clamp studies revealed a significantly increased current density of the corresponding current, IKur and IK1 at 6 hours after DEX injection. Simultaneously, electrophysiological study in isolated perfused hearts showed significantly increased number of repetitive atrial responses induced by single atrial extrastimulus (3.2 ± 2.4 to 26.7 ± 16.4, P = 0.004) with shorting of the refractory period (36.4 ± 4.6 to 27.4 ± 5.5 ms, P = 0.049) after DEX injection.Glucocorticoid immediately modified Kv1.5 and Kir2.2 gene expression at pretranslational levels, thus leading to effective refractory period shortening that could be arrhythmogenic. These results implied that transient glucocorticoid-induced biochemical modification of cardiac ion channels might be one of the mechanisms underlying the stress-induced paroxysmal AF.

Photodynamic Inactivation of Human Coronaviruses.

Photodynamic inactivation (PDI) employs a photosensitizer, light, and oxygen to create a local burst of reactive oxygen species (ROS) that can inactivate microorganisms. The botanical extract PhytoQuinTM is a powerful photosensitizer with antimicrobial properties. We previously demonstrated that photoactivated PhytoQuin also has antiviral properties against herpes simplex viruses and adenoviruses in a dose-dependent manner across a broad range of sub-cytotoxic concentrations. Here, we report that human coronaviruses (HCoVs) are also susceptible to photodynamic inactivation. Photoactivated-PhytoQuin inhibited the replication of the alphacoronavirus HCoV-229E and the betacoronavirus HCoV-OC43 in cultured cells across a range of sub-cytotoxic doses. This antiviral effect was light-dependent, as we observed minimal antiviral effect of PhytoQuin in the absence of photoactivation. Using RNase protection assays, we observed that PDI disrupted HCoV particle integrity allowing for the digestion of viral RNA by exogenous ribonucleases. Using lentiviruses pseudotyped with the SARS-CoV-2 Spike (S) protein, we once again observed a strong, light-dependent antiviral effect of PhytoQuin, which prevented S-mediated entry into human cells. We also observed that PhytoQuin PDI altered S protein electrophoretic mobility. The PhytoQuin constituent emodin displayed equivalent light-dependent antiviral activity to PhytoQuin in matched-dose experiments, indicating that it plays a central role in PhytoQuin PDI against CoVs. Together, these findings demonstrate that HCoV lipid envelopes and proteins are damaged by PhytoQuin PDI and expands the list of susceptible viruses.

MicroRNA detection using light-up aptamer amplification based on nuclease protection transcription.

The quantification of microRNAs (miRNAs) is important because the miRNA expression level is closely associated with the occurrence and development of diseases. Here, we report a simple nuclease protection transcription assay which combines nuclease protection assays and transcription-assisted light-up aptamer amplification for detecting miRNAs with great sensitivity.

Mapping of the Chemical Modifications of rRNAs.

Cellular RNAs, both coding and noncoding, contain several chemical modifications. Both ribose sugars and nitrogenous bases are targeted for these chemical additions. These modifications are believed to expand the topological potential of RNA molecules by bringing chemical diversity to otherwise limited repertoire. Here, using ribosomal RNA of yeast as an example, a detailed protocol for systematically mapping various chemical modifications to a single nucleotide resolution by a combination of Mung bean nuclease protection assay and RP-HPLC is provided. Molar levels are also calculated for each modification using their UV (254nm) molar response factors that can be used for determining the amount of modifications at different residues in other RNA molecules. The chemical nature, their precise location and quantification of modifications will facilitate understanding the precise role of these chemical modifications in cellular physiology.

CircRNF13 Promotes the Malignant Progression of Pancreatic Cancer through Targeting miR-139-5p/IGF1R Axis.

Background Mounting evidence has shown circular RNAs (circRNAs) play an important role in the initiation and progression of pancreatic cancer (PC). Meanwhile, circRNAs may serve as the biomarkers for the diagnosis, treatment, and prognosis of PC. Therefore, it is urgent to elucidate the function and underlying mechanism of circRNAs in the development of PC. Methods The Cancer-Specific CircRNA Database (CSCD), Circular RNA Interactome database (circinteractome database), and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) were used to verify the expression level of circRNF13 in PC cell lines. Fluorescence in situ hybridization (FISH) and RNase protection assay were used to detect the localization and structure of circRNF13. Then, cell functional experiments were employed to estimate the proliferated, migrated, and invasive abilities in PC. Furthermore, bioinformatic tools, luciferase dual reporter assay, and RT-qPCR were used to investigate the interaction among circRNF13, miR-139-5p, and IGF1R. Eventually, the rescue functional experiments were employed to confirm that circRNF13 targeted the miR-139-5p/IGF1R axis to participate in the development of PC. Results CircRNF13 was overexpressed in PC cell lines compared with the normal pancreatic duct cell line. Additionally, inhibition of circRNF13 impaired the proliferation, migration, and invasion of PC cells. CircRNF13 could serve as the molecular sponge of miR-139-5p to inhibit its association with IGF1R that eventually accelerated the malignant progression of PC. Conclusion CircRNF13 serves as a competitive endogenous RNA of IGF1R to inhibit the function of miR-139-5p that eventually reinforces the malignant phenotype of PC.