A severe respiratory ongoing outbreak of pneumonia associated with 2019 novel coronavirus was recently emerged in China. Here, we reported the epidemiological, clinical, laboratory and radiological characteristics of 19 suspect cases. We compared J o u r n a l P r e -p r o o f
RNA polymerase (RNAP) is one of the best characterized motor proteins which transcribes on a DNA template while presenting unique motor properties. 1 The powerful tracking ability of RNAP underlies devices supporting nanoscale motion control on planar substrates. 2 Nevertheless existing experimental systems that track protein motor activities are restricted to tethering the substrate or the motor, therefore cannot support observation of autonomous movement modulated by the motor in free solution. Although autonomous control has been reported by metallic, reactive colloids, 3 it has not been demonstrated with biomolecules under physiological conditions. Here we show the first experimental evidence that a molecular complex consisting of just a DNA template and associated RNAPs displays chemokinetic motion driven by transcription substrate NTPs. Furthermore this molecular complex exhibits biased migration into a concentration gradient of NTPs for mimicking cellular chemotaxis.We studied a robust transcription system using T7 RNAP and a rod-like 310 bp DNA template bearing a T7 transcription promoter sequence. For detection, the upstream portion from the promoter of the template was fluorescently labeled. Apparent diffusion of DNA was quantified by Fluorescence Recovery After Photobleaching (FRAP). 4 The diffusion coefficient (D) for the 310 bp DNA in transcription buffer without Mg 2+ was 15.3 μm 2 /s, which is 18.6% slower than the 18.8 μm 2 /s value obtained from theoretical calculations via Broersma's equations. 5 We attribute this difference to impedance of DNA motion by the gel matrix (added to eliminate convection) and fluorochrome labels attached to the template.We evaluated motility of the DNA-RNAP complex in the presence of substrate NTPs as compared to controls prepared from same aliquots of reagents, but without NTPs substrates ( Figure 1). We observed a systematic increase (25.2% on average) of apparent diffusion of DNA when transcription is enabled. Our control showed that DNA diffusion is invariant over the ionic strength range examined in our experiments. Increase in ionic strength by monovalent Li + (included in added NTPs) should not significantly affect diffusivity. Therefore, RNAP motor activity must be the major cause that renders faster movement of DNA -a phenomenon we describe as "Molecular Propulsion." While transcribing on the DNA track, the RNAP motor vigorously pushes and pulls on the template DNA against fluid viscous drag, affecting hydrodynamic interactions between DNA and surrounding water layers. We further examined transcription elongation and initiation as possible governing factors in Molecular Propulsion (Figure 2). By adding 3'-dNTPs to the NTPs at 1:100 ratio, the length of transcription elongation is truncated from the runoff full length (227 nt) to roughly 100 nt.Comparisons of apparent DNA diffusion between runoff transcription and 100 nt transcription showed no significant difference beyond experimental fluctuation. Increasing the ratio of 3'-dNTP vs. NTP to 1:10 traps the m...
We describe the incorporation of multiple fluorophores into a single stranded DNA (ssDNA) chain using terminal deoxynucleotidyl transferase (TdT), a template-independent DNA polymerase that catalyzes the sequential addition of deoxynucleotides (dNTPs) at the 3'-OH group of an oligonucleotide primer; we term this methodology surface initiated enzymatic polymerization (SIEP) of DNA. We found that long (>1 Kb) ssDNA homopolymer can be grown by SIEP, and that the length of the ssDNA product is determined by the monomer to oligonucleotide initiator ratio. We observed efficient initiation (≥50%) and narrow polydispersity of the extended product when fluorescently labeled nucleotides are incorporated. TdT's ability to incorporate fluorescent dNTPs into a ssDNA chain was characterized by examining the effect of the molar ratios of fluorescent dNTP to natural dNTP on the degree of fluorophore incorporation and the length of the polymerized DNA strand. These experiments allowed us to optimize the polymerization conditions to incorporate up to ~50 fluorescent Cy3-labeled dNTPs per kilobase into a ssDNA chain. With the goal of using TdT as an on-chip labeling method, we also quantified TdT mediated signal amplification on the surface by immobilizing ssDNA oligonucleotide initiators on a glass surface followed by SIEP of DNA. The incorporation of multiple fluorophores into the extended DNA chain by SIEP translated to a ~45 fold signal amplification compared to the incorporation of a single fluorophore. SIEP was then employed to detect hybridization of DNA, by the posthybridization, on-chip polymerization of fluorescently labeled ssDNA that was grown from the 3'-OH of target strands that hybridized to DNA probes that were printed on a surface. A dose-response curve for detection of DNA hybridization by SIEP was generated, with a ~1 pM limit of detection and a linear dynamic range of 2 logs.
This study aimed to evaluate the potential of long noncoding RNAs (lncRNAs) as biomarkers for coronary artery disease (CAD). We measured the levels of three atherosclerosis‐ or cardiac‐related lncRNAs in peripheral blood monocyte cells (PBMCs) from 20 CAD patients and 20 non‐CAD control participants using real‐time reverse transcription–polymerase chain reaction (real‐time RT–PCR) methods. We found that the levels of lncRNA KCNQ1 opposite strand/antisense transcript 1 (KCNQ1OT1), hypoxia‐inducible factor 1 alpha‐antisense RNA 2 (HIF1A‐AS2) and apolipoprotein A‐1 antisense RNA (APOA1‐AS) were significantly increased in CAD patients (KCNQ1OT1 increased by 2.38‐fold, P = 0.00042; HIF1A‐AS2 increased by 2.00‐fold, P = 0.0001; APOA1‐AS increased by 4.52‐fold, P = 0.000048). The area under the ROC curve was 0.865 for KCNQ1OT1, 0.852 for HIF1A‐AS2, and 0.967 for APOA1‐AS. Furthermore, the combination of lncRNAs resulted in a much higher AUC value of 0.990 for the prediction of CAD. Spearman's correlation analysis showed that APOA1‐AS was positively correlated with NT‐proBNP, CKMB, MYO and HsTnT, whereas HIF1A‐AS2 was correlated with NT‐proBNP and HsTnT. Hence, the elevation of KCNQ1OT1, HIF1A‐AS2 and APOA1‐AS predicts CAD and these molecules may be considered as novel biomarkers of CAD.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.