scholarly journals Dynamic regulation of translation quality control associated with ribosome stalling

2020 ◽  
Author(s):  
Daniel H. Goldman ◽  
Nathan M. Livingston ◽  
Jonathan Movsik ◽  
Bin Wu ◽  
Rachel Green
Keyword(s):  
Quality Control ◽  
Real Time ◽  
Single Molecule ◽  
Fold Increase ◽  
Mrna Levels ◽  
Dynamic Regulation ◽  
Ribosome Stalling ◽  
Subsequent Degradation ◽  
Human Quality ◽  
Control Apparatus

AbstractTranslation of problematic mRNA sequences induces ribosome stalling. Collided ribosomes at the stall site are recognized by cellular quality control machinery, resulting in dissociation of the ribosome from the mRNA and subsequent degradation of the nascent polypeptide and in some organisms, decay of the mRNA. However, the timing and regulation of these processes are unclear. We developed a SunTag-based reporter to monitor translation in real time on single mRNAs harboring difficult-to-translate poly(A) stretches. This reporter recapitulates previous findings in human cells that an internal poly(A) stretch reduces protein output ∼10-fold, while mRNA levels are relatively unaffected. Long-term imaging of translation indicates that poly(A)-containing mRNAs are robustly translated in the absence of detectable mRNA cleavage. However, quantification of ribosome density reveals a ∼3-fold increase in the number of ribosomes on poly(A)-containing mRNAs compared to a control mRNA, consistent with queues of many stalled ribosomes. Using single-molecule harringtonine runoff experiments, we observe the resolution of these queues in real-time by the cellular quality control machinery, and find that rescue is very slow compared to both elongation and termination. We propose that the very slow clearance of stalled ribosomes provides the basis for the cell to distinguish between transient and deleterious stalls, and that the human quality control apparatus predominantly targets the nascent protein rather than the mRNA.

scholarly journals Enabling metagenomic surveillance for bacterial tick-borne pathogens using nanopore sequencing with adaptive sampling

2021 ◽  
Author(s):  
Evan J. Kipp ◽  
Laramie L. Lindsey ◽  
Benedict S. Khoo ◽  
Christopher Faulk ◽  
Jonathan D. Oliver ◽  
...  
Keyword(s):  
Real Time ◽  
Single Molecule ◽  
Adaptive Sampling ◽  
Sequence Data ◽  
Ixodes Scapularis ◽  
Fold Increase ◽  
Borrelia Miyamotoi ◽  
Nucleotide Sequence Data ◽  
Oxford Nanopore ◽  
Sequencing Platforms

Technological and computational advancements in the fields of genomics and bioinformatics are providing exciting new opportunities for pathogen discovery and surveillance. In particular, single-molecule nucleotide sequence data originating from Oxford Nanopore Technologies (ONT) sequencing platforms can be bioinformatically leveraged, in real-time, for enhanced biosurveillance of a vast array of zoonoses. The recently released nanopore adaptive sampling (NAS) pipeline facilitates immediate mapping of individual nucleotide molecules (i.e., DNA, cDNA, and RNA) to a given reference as each molecule is sequenced. User-defined thresholds then allow for the retention or rejection of specific molecules, informed by the real-time reference mapping results, as they are physically passing through a given sequencing nanopore. Here, we show how NAS can be used to selectively sequence entire genomes of bacterial tick-borne pathogens circulating in wild populations of the blacklegged tick vector, Ixodes scapularis. The NAS method provided a two-fold increase in targeted pathogen sequences, successfully enriching for Borrelia (Borreliella) burgdorferi s.s.; Borrelia (Borrelia) miyamotoi; Anaplasma phagocytophilum; and Ehrlichia muris eauclairensis genomic DNA within our I. scapularis samples. Our results indicate that NAS has strong potential for real-time sequence-based pathogen surveillance.

scholarly journals Quantitative Real-Time RT—PCR Analysis of Inflammatory Gene Expression Associated with Ischemia—Reperfusion Brain Injury

2002 ◽  
Vol 22 (9) ◽  
pp. 1068-1079 ◽  
Author(s):  
Rossana Berti ◽  
Anthony J. Williams ◽  
John R. Moffett ◽  
Sarah L. Hale ◽  
Luisa C. Velarde ◽  
...  
Keyword(s):  
Gene Expression ◽  
Brain Injury ◽  
Real Time ◽  
Adhesion Molecules ◽  
Ischemia Reperfusion ◽  
Fold Increase ◽  
Pcr Analysis ◽  
Mrna Levels ◽  
Time Points ◽  
Time Point

Ischemia-reperfusion brain injury initiates an inflammatory response involving the expression of adhesion molecules and cytokines, some of which are regulated by the nuclear transcription factor NF-κB. In this study the authors examined mRNA expression levels for several important genes associated with inflammation at five time points (3, 6, 12, 24, and 72 hours) after transient middle cerebral artery occlusion (MCAO) in Sprague-Dawley rats. A sensitive and quantitative technique (TaqMan real-time QRT-PCR) was used to simultaneously measure mRNA levels for key cell adhesion molecules and inflammatory cytokines. Gene expression increased significantly in the injured hemisphere for interleukin (IL)-1β (12-fold increase at 24 hours), IL-6 (25-fold increase at 6 hours) and ICAM-1 (4-fold increase at 24 hours), and the in-terhemispheric differences for these genes were significant for every time point examined ( P < 0.05 for all values). Tumor necrosis factor-α mRNA was upregulated in the injured versus uninjured hemisphere from 3 to 24 hours (5-fold increase at 6 hours), while E-selectin showed a significant increase in mRNA levels from 6 to 24 hours after MCAO (10-fold increase at 6 hours) ( P < 0.05 for all values). VCAM-1 mRNA levels did not respond differentially to injury at any time point between the two brain hemispheres. At all time points examined, activated NF-κB immunoreactivity was observed in cells throughout the infarct-damaged tissue. These results are consistent with the proinflammatory properties of the induced molecules, which are involved in the initiation of the inflammatory cascade, and may thus contribute to secondary cellular responses that lead to further brain damage.

Involvement of Heparanase in Early Pregnancy Losses

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4081-4081
Author(s):  
Yona Nadir ◽  
Israel Henig ◽  
Israel Vlodavsky ◽  
Benjamin Brenner
Keyword(s):  
Real Time ◽  
Tissue Factor ◽  
Early Pregnancy ◽  
Fold Increase ◽  
Detectable Effect ◽  
Mrna Levels ◽  
Rt Pcr ◽  
Coagulation Activity ◽  
Active Release ◽  
Endothelial Growth Factor

Abstract Heparanase activity is implicated in cell invasion, tumor metastasis and angiogenesis. Recently, we have reported that heparanase stimulates tissue factor (TF) expression in endothelial and cancer cells, resulting in elevation of coagulation activity (Nadir et al. JTH, 2006). We also found that heparanase releases tissue factor pathway inhibitor (TFPI) from endothelial and tumor cells, contributing to the local procoagulant balance (Nadir et al. TH, 2008). Heparanase was cloned from and is abundant in the placenta. We investigated the role of heparanase in the placenta, focusing on its effect on TF, TFPI, TFPI-2, and vascular endothelial growth factor-A (VEGF-A). Twenty formalin embedded samples of early pregnancy losses (weeks 5–10) were studied. Ten cases were miscarriages of women with thrombophilia and recurrent fetal losses, and ten control cases were pregnancy terminations. Applying real time RT-PCR and immunostaining for heparanase, TF, TFPI, TFPI-2, and VEGF-A it was found that in fetal losses related to thrombophilia the levels of heparanase, VEGF-A and TFPI-2 were significantly increased (2–3 fold) compared to placentas from controls. Next, JAR (human choriocarcinoma throphoblast) cells were transfected to overexpress the full length (65 kDa) heparanase or incubated with active (50+8 kDa) recombinant heparanase. Immunoblotting and real time RT-PCR showed that throphoblasts overexpressing or incubated with exogenous heparanase exhibited a 2–3 fold increase in TFPI and TFPI-2 in cell lysates both at the protein and mRNA levels, with no detectable effect on VEGF-A and TF. Accumulation of TFPI and TFPI-2 in the cell culture medium was increased 5–6 fold, exceeding the observed induction of TFPI and TFPI-2 gene transcription. Extracellular accumulation of TFPI and TFPI-2 was already evident 60 min following heparanase addition, prior to protein induction, indicating an active release of preformed proteins. These results indicate a regulatory effect of heparanase on TFPI and TFPI-2 in throphoblasts, suggesting a potential involvement in early miscarriages.

Development and Application of Quality Control System for Highway Subgrade Construction

2019 ◽  
Vol 2 (5) ◽  
Author(s):  
Tong Wang
Keyword(s):  
Quality Control ◽  
Real Time ◽  
Detection Method ◽  
System Development ◽  
Effective Control ◽  
Construction Process ◽  
Development Mode ◽  
The Road ◽  
Whole Process

The compaction quality of the subgrade is directly related to the service life of the road. Effective control of the subgrade construction process is the key to ensuring the compaction quality of the subgrade. Therefore, real-time, comprehensive, rapid and accurate prediction of construction compaction quality through informatization detection method is an important guarantee for speeding up construction progress and ensuring subgrade compaction quality. Based on the function of the system, this paper puts forward the principle of system development and the development mode used in system development, and displays the development system in real-time to achieve the whole process control of subgrade construction quality.

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