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Cell-free transcription and translation (TXTL) system is a cell extract-based system for rapid in vitro protein expression. The system bypasses routine laboratory processes such as bacterial transformation, clonal screening and cell lysis, which allows more precise and convenient control of reaction substrates, reduces the impact of bacteria on protein production, and provides a high degree of versatility and flexibility. In recent years, TXTL has been widely used as an emerging platform in clusterd regularly interspaced short palindromic repeat (CRISPR) technologies, enabling more rapid and convenient characterization of CRISPR/Cas systems, including screening highly specific gRNAs as well as anti-CRISPR proteins. Furthermore, TXTL-based CRISPR biosensors combined with biological materials and gene circuits are able to detect pathogens through validation of related antibiotics and nucleic acid-based markers, respectively. The reagents can be freeze-dried to improve portability and achieve point-of-care testing with high sensitivity. In addition, combinations of the sensor with programmable circuit elements and other technologies provide a non-biological alternative to whole-cell biosensors, which can improve biosafety and accelerate its application for approval. Here, this review discusses the TXTL-based characterization of CRISPR and their applications in biosensors, to facilitate the development of TXTL-based CRISPR/Cas systems in biosensors.
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CRISPR-Cas Systems , BacteriaABSTRACT
As a powerful tool to advance drug discovery, molecular imaging may provide new insights into the process of drug effect and therapy at cellular and molecular levels. When compared with other detection methods, fluorescence-based strategies are highly attractive and can be used to illuminate pathways of drugs' transport, with multi-color capacity, high specificity and good sensitivity. The conjugates of fluorescent molecules and therapeutic agents create exciting avenues for real-time monitoring of drug delivery and distribution, both in vitro and in vivo. In this short review, we discuss recent developments of small molecule-based fluorophore-drug conjugates, including non-cleavable and cleavable ones, that are capable of visualizing drug delivery.
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Objective:To investigate the expression of activin A in paraventricular nucleus (PVN)of the WKY rats and its influence in arterial blood pressure,and to clarify the mechanism of activin A in the regulation of arterial blood pressure by PVN.Methods:The WKY rats were selected.The expressions of activin A,ActRⅡA,ActRⅡB,and Smads mRNA in PVN of the WKY rats were measured by RT-PCR.The expression of ActRⅡA protein in PVN was detected by immunohistochemical staining.The microinjection of exogenous activin A into PVN was used to observe the changes of arterial blood pressure.The primary cultured PVN neurons from the WKY rats were divided into control group and activin A group.The mRNA expression levels of ActRⅡA,ActRⅡB,and Smads in the PVN neurons were analyzed by RT-PCR.Results:Activin A,ActRⅡA,ActRⅡB,Smad2 and Smad3 mRNA were expressed in PVN of the WKY rats.The ActRⅡ A protein expression in PVN was further confirmed by immunohistochemical staining.After microinjection of activin A or angiotensin Ⅱ (AgⅡ)into PVN,the mean arterial blood pressure was increased obviously compared with before treatment (P <0.05).Moreover,compared with control group,the expression levels of ActRⅡA and Smad3 mRNA in primary cultured PVN neurons of the rats in vitro were significantly increased (P <0.05).Conclusion:Activin A can regulate the arterial blood pressure in PVN in an autocrine or paracrine manner,which is related to ActRⅡA-Smad3 signal pathway.
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Objective:To investigate the effect of activin A and nerve growth factor ( NGF) NGF on stimulating neurite outgrowth of dorsal root ganglia(DRG)of the embryonic chicken.Methods:In this study,we observed that activin A and NGF together induced neurite outgrowth of DRG and kept survival of DRG neurons by the primary cultured DRGs from embryonic day 8 ( E8 ) chicken.calcitonin gene-related peptide(CGRP)CGRP mRNA expressions were analyzed by RT-PCR.Results: The DRG treated with activin A +NGF had obvious neurite outgrowth ,compared with only NGF group on day 3,and the number of living DRG neurons also increased.Activin A +NGF up-regulated the mRNA expressions of CGRP in DRG.Conclusion:The Data demonstrated that activin A with NGF can synergistically stimulate DRG neurite outgrowth and maintain the DRG neurons survival , suggesting that it is more effective that NGF and activin A together treat the associated disease of nerve system.
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Objective:To study the role of activin A in regulation of neutrophil function by detecting activin receptor expression and cellular activities.Methods:Peritoneal neutrophils were isolated in mouse.After the neutrophils were stimulated with activin A,the expression of ActRⅡA on neutrophils was examined by immunofluorescence and flow cytometry.Expression of Smad3 in neutrophils was analyzed by Western blot.Assays of neutrophils function were performed by detecting respiratory burst, production of NO and phagocytosis.Results:The isolated cells were composed of more than 90% peritoneal neutrophils.ActRⅡA was expressed on mouse neutrophils and Gr-1/ActRⅡA double-positive cells were 41.1%.Activin A promoted Smad3 phosphorylation in neutrophils,increased the production of ROS and O2-(P<0.05),enhanced secretion of NO and phagocytosis of mouse neutrophils(P<0.01),and promoted fluorescent microsphere phagocytosis of neutrophils by flow cytometry ( P<0.01 ) .Conclusion: Activin receptor and activin signaling protein were expressed on mouse neutrophils,activin A might play an important regulatory role in activation and function of neutrophils.
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Objective To prepare anti-activin receptor-interacting protein 1 (ARIP1) polyclonal antibody.Methods The GST-ARIP1 fusion protein was expressed in E.coli BL21.A polyclonal antibody against ARIP1 was obtained by immunizing a rabbit with the purified GST-ARIP1 and the localization of mature ARIP1 protein in mouse brain tissues was detected by immunohistochemistry using the prepared anti-ARIP1 antibody.Results Anti-ARIP1 polyclonal antibody could bind specifically with recombinant ARIP1,but not recombinant ARIP2.Immunohistochemical staining showed that ARIP1 mainly expressed in hypothalamus and hippocampus using the prepared anti-ARIP1 antibody.Conclusion The polyclonal antibody against ARIP1 has been successfully prepared and can be used to do immunohistochemical analysis for ARIP1 protein expression.
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Objective To investigate the effect and mechanism of sphingosine 1-phosphate(S1P)on action potential(AP)and voltage-dependent potassium current(KV)in isolated guinea pig ventricular myocytes.Methods The ventricular myocytes were isolated by using Langendorff perfusion method.The AP and KV were recorded by whole cell patch-clamp recording technique.Results S1P prolonged the 50% and 90% action potential duration(APD50 and APD90)and decreased KV which were blocked by pertussis toxin(PTX)or Calphostin C.Conclusion S1P decreased KV in a PKC pathway and prolonged action potential duration in guinea-pig ventricular myocytes.
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Objective:Effects of oxymatrine on the sodium current were studied .Methods:The whole cell voltage clamp technique was used in isolated ventricular cells of guinea-pig.Results:Oxymatrine (0.1,0.3 and 1 mmol/L) showed inhibition to the sodium current in dose-dependence.Conclusion:These results indicate that inhibition of oxymatrine to the sodium current may be one of mechanisms of its antiarrhythmic action.
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Objective:To investigate the effect of the BmK-9-(2) on the sodium current in guinea-pigventricular myocytes. Methods:The whole cell patch-clamp technique was used. Results:The BmK-9-(2)increased the magnitude of the peak inward sodium current (27 ％) ,peak sodium conductace (17 %) and theprolong time course of sodium channel inactivation (8％) and there was no effects on calcium channels.Conclusion:BmK-9-(2) can activate sodium channel and prolong the inactivated course of myocytes.