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Influence of Self-Compacting Concrete Admixtures in Snow Resistance and Compression Strength-Commensurability involving Snow Opposition Criteria.
Ultrasound is acoustic waves that can penetrate deeply into tissue in a focused manner with limited adverse effects on cells. As such, ultrasound has been widely used for clinical diagnosis for several decades. Ultrasound induces bioeffects in tissues, providing potential value in therapeutic applications. However, the intrinsic millimeter scale of the ultrasound focal zone represents a challenge with respect to minimizing the illuminated regions to perturb target cells in a precise manner. To control a specific cell population or even single cells, sonogenetic tools that combine ultrasound and genetic methods have been recently developed. With these approaches, several ultrasound-responsive proteins are heterologously introduced into target cells, which enhances the cells' ability to respond to ultrasound stimulation. With optimization of the ultrasound parameters, these tools can specifically manipulate activities in genetically defined cells but not in unmodified cells present in the ultrasound-illuminated regions. These approaches provide new strategies for noninvasive modulation of target cells in various therapeutic applications.The quest to engineer increasingly complex synthetic gene networks in mammalian and plant cells requires an ever-growing portfolio of orthogonal gene expression systems. To control gene expression, light is of particular interest due to high spatial and temporal resolution, ease of dosage and simplicity of administration, enabling increasingly sophisticated man-machine interfaces. However, the majority of applied optogenetic switches are crowded in the UVB, blue and red/far-red light parts of the optical spectrum, limiting the number of simultaneously applicable stimuli. This problem is even more pertinent in plant cells, in which UV-A/B, blue, and red light-responsive photoreceptors are already expressed endogenously. To alleviate these challenges, we developed a green light responsive gene switch, based on the light-sensitive bacterial transcription factor CarH from Thermus thermophilus and its cognate DNA operator sequence CarO. The switch is characterized by high reversibility, high transgene expression levels, and low leakiness, leading to up to 350-fold induction ratios in mammalian cells. In this chapter, we describe the essential steps to build functional components of the green light-regulated gene switch, followed by detailed protocols to quantify transgene expression over time in mammalian cells. In addition, we expand this protocol with a description of how the optogenetic switch can be implemented in protoplasts of A. thaliana.Pluripotent stem cells have the potential to differentiate into various cell types that can be used for basic biological studies, drug discovery, and regenerative medicine. To obtain reliable results using the differentiated cells, the contamination of nontarget cells should be avoided. microRNAs (miRNAs) can serve as indicators to distinguish target and nontarget cells, because the activities of miRNAs are different among cell types.In this chapter, we introduce a method to purify target cells using synthetic messenger RNAs (mRNAs) that respond to cell-specific miRNAs. The method is composed of five steps mRNA sequence design, template DNA preparation by PCR, in vitro mRNA transcription, mRNA transfection into cells, and fluorescence-activated cell sorting. This synthetic mRNA-based cell purification method will advance various applications of pluripotent stem cells.Antibodies have been attracting attention as therapeutic tools owing to their high affinity and specificity. To develop potent antibodies, affinity maturation, epitope regulation, and using target antigens in native form are pivotal requirements. Here we describe a method to conduct epitope-directed affinity maturation of antibodies using engineered mammalian cells. This method utilizes protein chimeras that transduce cell death signaling in response to antibody binding. As the competition of antibody binding inhibits the cell death signaling, only affinity-matured antibodies retaining the same epitope as an original one can be selected using cell survival as readout.Diabetes mellitus is a complex metabolic disease characterized by chronically deregulated blood-glucose levels. Selleck Repertaxin To restore glucose homeostasis, therapeutic strategies allowing well-controlled production and release of insulinogenic hormones into the blood circulation are required. In this chapter, we describe how mammalian cells can be engineered for applications in diabetes treatment. While closed-loop control systems provide automated and self-sufficient synchronization of glucose sensing and drug production, drug production in open-loop control systems is engineered to depend on exogenous user-defined trigger signals. Rational, robust, and reliable manufacture practices for mammalian cell engineering are essential for industrial-scale mass-production in view of clinical and commercial applications.Synthetic receptors control cell behavior in response to environmental stimuli for applications in basic research and cell therapy. However, the integration of synthetic receptors in unexplored contexts is cumbersome, especially for nonspecialist laboratories. Here, I provide a detailed protocol on how to use receptors of the generalized extracellular molecule sensor (GEMS) platform. GEMS is a modular receptor system that can be adapted to sense molecules of choice by using affinity domains that dimerize in response to the target. GEMS consist of an erythropoietin receptor scaffold that has been mutated to no longer bind to erythropoietin. N-terminal fusions with affinity domains, such as single chain variable fragments (scFvs), that bind to two epitopes on the same target activate the receptor. The intracellular receptor domain can be chosen from several signal transduction domains of single-pass transmembrane receptors to activate endogenous signaling pathways. As of now, GEMS have been used for sensing proors in new research contexts.CAR-T cell therapy is one of the most successful cell-based therapies. T cells are the most common cells to be genetically modified for cancer therapy, not only because T cells have cytotoxicity but also because they are easily cultured ex vivo and genetically modified with viral vectors. Hence, for nonexperts, T cell engineering is an ideal starting point for mammalian cell engineering or for development of therapeutics. Here, we have described a basic procedure for lentiviral transduction of human primary T cells to generate a CAR-T cell and assays to confirm CAR expression and function.
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