The purpose of this method is to generate comprehensive single-site saturation mutagenesis libraries. The required input for Nicking Mutagenesis is double-stranded plasmid DNA, and any plasmid dsDNA can be used provided that it contains a 7-base pair BbvCI recognition site. The method works by first creating an ssDNA template using a site and strand specific nicking endonuclease (Nt.BbvCI) followed by exonuclease digestion of the nicked strand. After creation of a heteroduplex by thermal cycling template with mutagenic oligos, the parental template ssDNA is destroyed by employing the opposite strand nicking endonuclease (Nb.BbvCI) followed by exonuclease digestion. A schematic of the method is outlined in Figure 1. The protocol can be completed in a single day (with transformation) and libraries can be harvested the following day.
“Exhaustion” is a term used to describe a state of native and redirected T-cell hypo-responsiveness resulting from persistent antigen exposure during chronic viral infections or cancer. Although a well-established phenotype across mice and humans, exhaustion at the molecular level remains poorly defined and inconsistent across the literature. This is, in part, due to an overreliance on surface receptors to define these cells and explain exhaustive behaviours, an incomplete understanding of how exhaustion arises, and a lack of clarity over whether exhaustion is the same across contexts, e.g., chronic viral infections versus cancer. With the development of systems-based genetic approaches such as single-cell RNA-seq and CRISPR screens applied to in vivo data, we are moving closer to a consensus view of exhaustion, although understanding how it arises remains challenging given the difficulty in manipulating the in vivo setting. Accordingly, producing and studying exhausted T-cells ex vivo is burgeoning, allowing experiments to be conducted at scale up and with high throughput. Here, we first review what is currently known about T-cell exhaustion and how it’s being studied. We then discuss how improvements in their method of isolation/production and examining the impact of different microenvironmental signals and cell interactions has now become an active area of research. Finally, we discuss what the future holds for the analysis of this physiological condition and, given the diversity of ways in which exhausted cells are now being generated, propose the adoption of a unified approach to clearly defining exhaustion using a set of metabolic-, epigenetic-, transcriptional-, and activation-based phenotypic markers, that we call ‘M.E.T.A.’.
A major concern of business with respect to transport-charging interventions is the context of revenue-investment policy, particularly how the timing of improvements may alter the time lags between fewer car journeys and more public transport journeys, and the problems for business in the intervening periods. The authors present a conceptual framework and case study of the whole-life effects on business performance. The impacts of charging occur as a sequence of gradually interacting changes, rather than as a single set of impacts, and positive amenity effects brought about through revenue hypothecation occur incrementally, taking years to achieve full effect. In the case study, a Delphi panel of business leaders predicted the time-marching effects of workplace-parking levies and road-user charging over a 24-year period in Nottingham. The findings revealed that the temporal nature of hypothecation results in minor fluctuations in performance for some business sectors in the first few years, but that these tail off as benefits gradually overwhelm disbenefits resulting in modest increases in performance for most sectors in the medium to long term. Many local authorities are reluctant to implement charging interventions due to concerns about economic vitality; it is expected that the results will inform policy and future research in this area.
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