Precise cell targeting is challenging because most mammalian cell types lack a single surface marker that distinguishes them from other cells. A solution would be to target cells using specific ...combinations of proteins present on their surfaces. In this study, we design colocalization-dependent protein switches (Co-LOCKR) that perform AND, OR, and NOT Boolean logic operations. These switches activate through a conformational change only when all conditions are met, generating rapid, transcription-independent responses at single-cell resolution within complex cell populations. We implement AND gates to redirect T cell specificity against tumor cells expressing two surface antigens while avoiding off-target recognition of single-antigen cells, and three-input switches that add NOT or OR logic to avoid or include cells expressing a third antigen. Thus, de novo designed proteins can perform computations on the surface of cells, integrating multiple distinct binding interactions into a single output.
Genetic circuit design automation Nielsen, Alec A. K.; Der, Bryan S.; Shin, Jonghyeon ...
Science (American Association for the Advancement of Science),
04/2016, Letnik:
352, Številka:
6281
Journal Article
Recenzirano
Computation can be performed in living cells by DNA-encoded circuits that process sensory information and control biological functions. Their construction is time-intensive, requiring manual part ...assembly and balancing of regulator expression. We describe a design environment, Cello, in which a user writes Verilog code that is automatically transformed into a DNA sequence. Algorithms build a circuit diagram, assign and connect gates, and simulate performance. Reliable circuit design requires the insulation of gates from genetic context, so that they function identically when used in different circuits. We used Cello to design 60 circuits forEscherichia coli(880,000 base pairs of DNA), for which each DNA sequence was built as predicted by the software with no additional tuning. Of these, 45 circuits performed correctly in every output state (up to 10 regulators and 55 parts), and across all circuits 92% of the output states functioned as predicted. Design automation simplifies the incorporation of genetic circuits into biotechnology projects that require decision-making, control, sensing, or spatial organization.
How the immune system readies for battleAlthough gene expression is tightly controlled at both the RNA and protein levels, the quantitative contribution of each step, especially during dynamic ...responses, remains largely unknown. Indeed, there has been much debate whether changes in RNA level contribute substantially to protein-level regulation. Jovanovic et al. built a genome-scale model of the temporal dynamics of differential protein expression during the stimulation of immunological dendritic cells (see the Perspective by Li and Biggin). Newly stimulated functions involved the up-regulation of specific RNAs and concomitant increases in the levels of the proteins they encode, whereas housekeeping functions were regulated posttranscriptionally at the protein level.Science, this issue 10.1126/science.1259038; see also p. 1066 Protein expression is regulated by the production and degradation of messenger RNAs (mRNAs) and proteins, but their specific relationships remain unknown. We combine measurements of protein production and degradation and mRNA dynamics so as to build a quantitative genomic model of the differential regulation of gene expression in lipopolysaccharide-stimulated mouse dendritic cells. Changes in mRNA abundance play a dominant role in determining most dynamic fold changes in protein levels. Conversely, the preexisting proteome of proteins performing basic cellular functions is remodeled primarily through changes in protein production or degradation, accounting for more than half of the absolute change in protein molecules in the cell. Thus, the proteome is regulated by transcriptional induction for newly activated cellular functions and by protein life-cycle changes for remodeling of preexisting functions.
Thematic analysis is an increasingly popular method for analysing qualitative data within the social sciences. Current guides for conducting thematic analysis promote the method’s broad flexibility ...across research designs, which has resulted in criticism that the method lacks the rigour and structure necessary for credible academic research. Recognizing the challenges faced by qualitative scholars, I developed and present an 8-step prescriptive approach specific to abductive methodologies incorporating many concepts from seminal works in thematic analysis, and importantly maintaining the balance of theorising with empirical data, which is the core of abductive research.
T cell exhaustion limits immune responses against cancer and is a major cause of resistance to chimeric antigen receptor (CAR)-T cell therapeutics. Using murine xenograft models and an in vitro model ...wherein tonic CAR signaling induces hallmark features of exhaustion, we tested the effect of transient cessation of receptor signaling, or rest, on the development and maintenance of exhaustion. Induction of rest through enforced down-regulation of the CAR protein using a drug-regulatable system or treatment with the multikinase inhibitor dasatinib resulted in the acquisition of a memory-like phenotype, global transcriptional and epigenetic reprogramming, and restored antitumor functionality in exhausted CAR-T cells. This work demonstrates that rest can enhance CAR-T cell efficacy by preventing or reversing exhaustion, and it challenges the notion that exhaustion is an epigenetically fixed state.
Inductive/deductive hybrid thematic analysis offers significant opportunities for researchers, but its application within integrative mixed methods research has yet to be fully explored. Firstly, ...this article contributes by demonstrating the compatibility of inductive/deductive hybrid thematic analysis with quantitative work in a mixed methods approach to research. Secondly, the article then innovates by highlighting the value of this approach within a critical realist meta-theoretical perspective. Here, the critical realist concepts of abduction and retroduction are crucial, both in terms of facilitating the convergence of methods and in the generation of new theory. This article will be of relevance to researchers interested in integrating inductive/deductive hybrid thematic analysis with quantitative methods within a coherent and enabling philosophical paradigm.
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