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The Primer Hypothesis for the Regulation of Eukaryotic Gene Expression
Published in M. Gerald, M.D. Kolodny, Eukaryotic Gene Regulation, 2018
Published studies from this laboratory”3 have described the passage of RNA out of cells into the extracellular media. Other studies (reviewed in reference 115) have documented the uptake of exogenously added RNA with and without the addition of polyanions. Since the cell membrane was therefore permeable to RNA, it seemed worthwhile to attempt to demonstrate cellular uptake of labeled oligonucleotides and to examine the intracellular RNA for evidence of en bloc incorporation of these oligonucleotides. Labeled RNA oligonucleotides were therefore added to whole cells in the presence of a large excess of nonlabeled mononucleotides to limit the incorporation of labeled mononucleotide breakdown products. After incubation, the RNA was fractionated and evidence sought for incorporation of radioactive label in high molecular weight RNA.135
Anti-Infective Agents
Published in Keith Struthers, Clinical Microbiology, 2017
Transcription of the whole genome is done by the host RNA polymerase apparatus. Initially the viral RNA is highly spliced by the post-transcription splicing machinery of the nucleus, and amongst others, the mRNA for two ‘early’ proteins are produced, translated in the cytoplasm (7) and return to the nucleus (8). Tat binds to the host RNA polymerase complex, increasing the rate of transcription of the provirus by 200-fold or more, while Rev binds to specific Rev binding segments on the viral RNA, and actively moves viral RNA out to the cytoplasm, reducing their ‘splicing time’ in the nucleus (9). Rev supplies a shuttle service, ensuring that full length viral RNA (the genome for new virus), mRNA for the capsid (gag)/polymerase (pol) proteins, as well as the subgenomic mRNA for the envelope (env) proteins reach the cytoplasm (10).
Methods in molecular exercise physiology
Published in Adam P. Sharples, James P. Morton, Henning Wackerhage, Molecular Exercise Physiology, 2022
Adam P. Sharples, Daniel C. Turner, Stephen Roth, Robert A. Seaborne, Brendan Egan, Mark Viggars, Jonathan C. Jarvis, Daniel J. Owens, Jatin G. Burniston, Piotr P. Gorski, Claire E. Stewart
When wishing to isolate RNA, samples stored at −80°C (or in buffers that maintain RNA integrity) are immersed in Tri-Reagent (also termed TRIzol) or in the lysis buffers provided in commercially available kits. The Tri-Reagent method is popular, as it is relatively inexpensive (compared with commercially available kits). Tri-Reagent is a monophasic solution containing two chemicals (phenol and guanidine isothiocyanate) which maintains the ‘quality’ of the RNA by inhibiting the activity of RNA-specific degrading enzymes, RNases (enzymes that degrade RNA), while disrupting and dissolving cellular components during the lysis and homogenisation steps. The addition of the third chemical, chloroform, and centrifugation allow the RNA to become soluble in the upper ‘aqueous phase’ in a process called phase separation. Careful removal of this aqueous phase followed by the addition of isopropanol precipitates the RNA out of the solution, and after centrifugation, the resultant RNA pellet is washed with 75% ethanol to remove any remaining chemicals from the previous steps. RNA pellets are resuspended in specific RNA storage solutions or TE (Tris-EDTA) buffers to help maintain the integrity of the much less stable RNA. Finally, the quantity and quality of the RNA are then analysed in the same way as DNA described, although the optimal A260/A280 nM ratio for RNA being 2.0–2.2 (compared with 1.8–2 for DNA). Given DNA absorbs light at a similar wavelength to RNA, the UV spectrophotometry (e.g. Nanodrop, ThermoFisher Scientific) method does not identify DNA contamination within the RNA sample. Therefore, samples isolated by the Tri-Reagent method are often treated with DNase enzymes to degrade any DNA contaminants within the RNA sample. Often, commercially available column-based kits for RNA isolation contain DNase enzymes within the extraction protocol and therefore do not need to be repeated when quantifying RNA quantity and quality. Finally, as with DNA described above, fluorometry can also be used for RNA quantification using dyes that only bind to RNA to give an accurate concentration of the RNA material in a given sample.
Compliance with laboratory monitoring guidelines in outpatient HIV care: a qualitative study in the Netherlands
Published in AIDS Care, 2019
Dieuwke C. M. Toxopeus, Christopher L. Pell, Nadine Blignaut-van Westrhenen, Colette Smit, Ferdinand W. N. M. Wit, Pascale Ondoa, Peter Reiss, T. Sonia Boender
Furthermore, in line with previous research, costs also played a role in guideline compliance, especially for the HCV tests (Adams & Balderson, 2016), with some physicians monitoring liver enzymes rather than HCV antibody or HCV RNA, out of concern for cost-effectiveness. However, HCV infection is not necessarily accompanied by elevated ALAT levels. Therefore, this can result in cases of hepatitis being missed (Gholson et al., 1997; Giannini, Testa, & Savarino, 2005; Mofrad et al., 2003). The perceived cost-effectiveness of monitoring liver enzymes conflicts with the guideline recommendations, which state that it is cost-effective to monitor HCV because MSM are at risk of HCV, which has high treatment costs and a high risk of transmission (Götz et al., 2005; Kaplan-Lewis & Fierer, 2015; Linas, Wong, Schackman, Kim, & Freedberg, 2012; Popping, 2016; Woolf et al., 1999).
Oxidative stress implications for therapeutic vaccine development against Chagas disease
Published in Expert Review of Vaccines, 2021
Subhadip Choudhuri, Lizette Rios, Juan Carlos Vázquez-Chagoyán, Nisha Jain Garg
History of attenuated T. cruzi and recombinant antigen-based subunit vaccines development and their efficacy as prophylactic experimental vaccines is discussed in excellent recent reviews. Initial efforts to vaccine development utilized live, killed, or attenuated parasite, cell fraction, purified protein, recombinant protein etc. (reviewed in [97]). Many investigators, including us, have demonstrated outstanding prophylactic efficacy of subunit vaccines in regulating infection and concomitant pathologies in murine models of T. cruzi infection (reviewed in [98,99]). For the delivery of subunit vaccines, most investigators have utilized DNA-based platform as DNA vaccines are cost effective, stable at room temperature, and have demonstrated clinical safety in animal models and early-phase clinical trials [100]. DNA vaccines were shown to provide antigenic peptides for MHC I and MHC II (major histocompatibility complex) presentation, and elicit antigen-specific antibodies, type I cytokines, and cytotoxic CD8+ T lymphocyte response to provide protection from T. cruzi infection [100,101]. Yet, there is a concern regarding the antibiotic resistance genes in the plasmid DNA backbone. Antibiotic resistance genes can potentially be taken up by bacteria and may also be expressed in mammalian host after insertion into the genome [102,103]. To alleviate this concern, the nanoplasmid DNA vaccine was developed. The prototype nanoplasmid utilizes an antibiotic-free selection method based on sucrose selection vector using a small antisense RNA known as RNA-OUT. Another advantage of the nanoplasmid DNA vaccine is the reduced plasmid size that improves in vivo level and duration of expression [104].