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Methods in Molecular Biology (Clifton,... 2020Reverse-transcription quantitative real-time polymerase chain reaction (RT-qPCR) using fluorescent DNA-binding dyes is now a gold-standard methodology to study bacterial...
Reverse-transcription quantitative real-time polymerase chain reaction (RT-qPCR) using fluorescent DNA-binding dyes is now a gold-standard methodology to study bacterial gene expression through relative quantitation of target mRNAs under specific experimental conditions, and recent developments in the technology allow for gene expression analysis in single cells. Nevertheless, several critical steps of the RT-qPCR protocol need to be carefully addressed in order to obtain reliable results, particularly regarding RNA sample quality and appropriate choice of reference genes. Besides, accurate reporting of study conditions is essential, as recommended by the MIQE guidelines. Herein, we provide a practical approach to quantitation of the transcript levels of bacterial genes using RT-qPCR, including a general protocol for obtaining good-quality bacterial RNA and a discussion on the selection and validation of candidate bacterial reference genes for data normalization.
Topics: Bacteria; Fluorescent Dyes; Gene Expression Profiling; Gene Expression Regulation, Bacterial; Genes, Essential; Guidelines as Topic; Molecular Probe Techniques; Molecular Probes; RNA, Bacterial; Real-Time Polymerase Chain Reaction; Reference Standards; Reproducibility of Results
PubMed: 31578692
DOI: 10.1007/978-1-4939-9833-3_10 -
Advances in Experimental Medicine and... 2015Ion channel complexes are challenging to study by traditional biochemical methods due to their membranous lipid environment and large size. Bioreactive tethers are... (Review)
Review
Ion channel complexes are challenging to study by traditional biochemical methods due to their membranous lipid environment and large size. Bioreactive tethers are specialized chemical probes that have been used in electrophysiological experiments to provide unique insight into ion channel structure and function. Because bioreactive tethers are small molecular probes, they can be used to manipulate ion channel function in heterologous expression systems, native cells and animal models. This chapter covers three classes of tethers: photoswitchable, molecular rulers, and chemically reactive. The modular nature of bioreactive tethers enables the facile synthesis of next generation reagents with enhanced functionalities to interrogate and control ion channels in novel and multifarious ways.
Topics: Animals; Binding Sites; Humans; Ion Channel Gating; Ion Channels; Ion Transport; Membrane Potentials; Models, Molecular; Molecular Probe Techniques; Molecular Probes; Protein Binding; Protein Conformation; Structure-Activity Relationship
PubMed: 26381941
DOI: 10.1007/978-1-4939-2845-3_5 -
Biological & Pharmaceutical Bulletin 2017In vivo molecular imaging is the visualization, characterization, and measurement of biological processes at the molecular and cellular levels in humans and other living... (Review)
Review
In vivo molecular imaging is the visualization, characterization, and measurement of biological processes at the molecular and cellular levels in humans and other living systems. Among the methodologies used in in vivo molecular imaging, two methodologies are of great interest from the view of high sensitivity. One is nuclear medical imaging, and distribution and kinetics of a radiolabeled molecular probe are measured using positron emission tomography (PET) and single-photon emission computed tomography (SPECT). The other is optical molecular imaging, and distribution and kinetics of a fluorescent probe are measured using a fluorescent imaging instrument. In this review, the development of imaging probes for these two methodologies is briefly discussed. In nuclear medical molecular imaging, based on structure-activity-biodistribution relationship studies for small molecule and the concept of "functional unit-binding multifunctional molecular probe" containing 3 functional units (target recognition unit, signal-releasing unit, linker unit) for peptides and proteins, we developed radiolabeled probes with high and specific accumulation to the target for neuroreceptors, β-amyloid plaques, and tau aggregates in the brain, tumors, atherosclerotic plaques, pancreatic β-cell, myocardial sympathetic nerves, and so on. We also discuss the progression of molecular imaging toward therapy (radiotheranotics). In in vivo optical molecular imaging, taking into account the characteristics of optical imaging, we designed tumor-specific optical imaging probes with characteristic imaging mechanism, including near-infrared (NIR) fluorescent probes and activatable probes. Furthermore, we developed a photoacoustic imaging probe, which enables highly sensitive and high-resolution imaging in deep tissues.
Topics: Animals; Fluorescent Dyes; Humans; Molecular Imaging; Molecular Probes
PubMed: 28966233
DOI: 10.1248/bpb.b17-00505 -
Cell Chemical Biology Aug 2020Dynamic proteins perform critical roles in cellular machines, including those that control proteostasis, transcription, translation, and signaling. Thus, dynamic... (Review)
Review
Dynamic proteins perform critical roles in cellular machines, including those that control proteostasis, transcription, translation, and signaling. Thus, dynamic proteins are prime candidates for chemical probe and drug discovery but difficult targets because they do not conform to classical rules of design and screening. Selectivity is pivotal for candidate probe molecules due to the extensive interaction network of these dynamic hubs. Recognition that the traditional rules of probe discovery are not necessarily applicable to dynamic proteins and their complexes, as well as technological advances in screening, have produced remarkable results in the last 2-4 years. Particularly notable are the improvements in target selectivity for small-molecule modulators of dynamic proteins, especially with techniques that increase the discovery likelihood of allosteric regulatory mechanisms. We focus on approaches to small-molecule screening that appear to be more suitable for highly dynamic targets and have the potential to streamline identification of selective modulators.
Topics: Allosteric Regulation; CREB-Binding Protein; HSP70 Heat-Shock Proteins; Models, Molecular; Molecular Probes; Protein Binding; Proteins; Small Molecule Libraries
PubMed: 32783965
DOI: 10.1016/j.chembiol.2020.07.019 -
Journal of the American Chemical Society Jul 2020DNA-based probes constitute a versatile platform for making biological measurements due to their ability to recognize both nucleic acid and non-nucleic acid targets,... (Review)
Review
DNA-based probes constitute a versatile platform for making biological measurements due to their ability to recognize both nucleic acid and non-nucleic acid targets, ease of synthesis and chemical modification, amenability to be interfaced with signal amplification schemes, and inherent biocompatibility. Here, we provide a historical perspective of how a transition from linear DNA structures toward more structurally complex nanostructures has revolutionized live-cell analysis. Modulating the structure gives rise to probes that can enter cells without the aid of transfection reagents and can detect, track, and quantify analytes in live cells at the single-organelle, single-cell, tissue section, and whole organism levels. We delineate the advantages and disadvantages associated with different probe architectures and describe the advances enabled by these structures for elucidating fundamental biology as well as developing improved diagnostic and theranostic systems. We also discuss the outstanding challenges in the field and outline potential solutions.
Topics: Animals; Cell Survival; Cells; DNA; Fluorescence Resonance Energy Transfer; Humans; Molecular Probes; Nanostructures; Neoplasms; Neoplasms, Experimental
PubMed: 32573219
DOI: 10.1021/jacs.0c04978 -
Analytical and Bioanalytical Chemistry Jul 2016A label-free method for determining the 5'-end cap identity and orientation of a messenger RNA (mRNA) is described. Biotin-tagged probes that were complementary to the...
A label-free method for determining the 5'-end cap identity and orientation of a messenger RNA (mRNA) is described. Biotin-tagged probes that were complementary to the 5' end of target mRNA were used with RNase H to cleave the 5' end of the mRNA. The cleaved end sequence was isolated using streptavidin-coated magnetic beads and then analyzed by LC-MS. Quantitative and qualitative information on the 5' cap was determined from the unique mass of the isolated cleaved sequence. This approach, combined with the use of 5' RNA pyrophosphohydrolase, was also used to ascertain the orientation of the 5' cap. The assay showed low-picomole sensitivity for detecting capping reaction impurities. Uncapped triphosphate mRNA, spiked into 100 pmol of capped mRNA, could be detected over the tested range of 0.5 to 25 % with a linear response. The capping efficiency of several vaccinia-capped mRNA preparations was determined to be between 88 and 98 % depending on the modification type and length of the mRNA. mRNA of 2.2K and 9K nucleotides in length and containing the modified nucleotides pseudouridine and 5-methylcytidine were all successfully analyzed, demonstrating the utility of the technique to study mRNA capping. Graphical abstract mRNA 5' end analysis with RNAse H cleavage and capture probe.
Topics: Chromatography, Liquid; Mass Spectrometry; Molecular Probe Techniques; Molecular Probes; RNA Caps; RNA, Messenger; Reproducibility of Results; Ribonuclease H; Sensitivity and Specificity; Sequence Analysis, RNA; Staining and Labeling
PubMed: 27193635
DOI: 10.1007/s00216-016-9605-x -
Wiley Interdisciplinary Reviews.... Jul 2020Fluorescent carbon dot has emerged as promising alternative of conventionally known quantum dot or molecular probe as potential intracellular imaging probe. In... (Review)
Review
Fluorescent carbon dot has emerged as promising alternative of conventionally known quantum dot or molecular probe as potential intracellular imaging probe. In particular, <10 nm size, tunable and bright fluorescence of carbon dot deserve the application potential as intracellular imaging probe. However, synthesis of carbon dot with narrow particle size distribution, preparation of high-quality red/near-infrared emitting carbon dot and appropriate design of functional carbon dot for subcellular targeting are most critical issues. This advanced review focus on the application potential of fluorescent carbon dot as intracellular imaging probe. At first, we briefly discuss different types of fluorescent carbon dots and origin of their fluorescence. Next, we focus on surface chemistry and functionalization which are relevant to intracellular probe development. Finally we have summarized various types of intracellular nanoprobes that are developed from fluorescence carbon dot. This article is categorized under: Diagnostic Tools > in vitro Nanoparticle-Based Sensing.
Topics: Animals; Carbon; Diagnostic Imaging; Fluorescence; Humans; Intracellular Space; Molecular Probes; Quantum Dots
PubMed: 32040882
DOI: 10.1002/wnan.1617 -
Advances in Experimental Medicine and... 2023With the development of cellular biology, molecular biology, and other subjects, targeted molecular probe was combined with medical imaging technologies to launch a new...
With the development of cellular biology, molecular biology, and other subjects, targeted molecular probe was combined with medical imaging technologies to launch a new scientific discipline of molecular imaging that is a research discipline to visualize, characterize, and analyze biological process at the cellular and molecular levels for real-time tracking and precision therapy, also termed as the medical imaging in the twenty-first century. An array of imaging techniques has been developed to image specific targets of living cells or tissues by molecular probes, including optical molecular imaging (OI), magnetic resonance molecular imaging, ultrasound (US) molecular imaging, nuclear medicine molecular imaging, X-ray molecular imaging, and multi-mode molecular imaging. These imaging techniques make the early diagnosis of various diseases possible by means of visualization of gene expression, interactions between proteins, signal transduction, cell metabolism, cell traces, and other physiological or pathological processes in the living system, which bridge the gap between molecular biology and clinical medicine. This chapter will lay the emphasis on the early-stage diagnosis of fatal diseases, such as malignant tumors, cardio- or cerebrovascular diseases, digestive system disease, central nervous system disease, and other diseases employing molecular imaging in a real-time visualized manner.
Topics: Humans; Neoplasms; Ultrasonography; Magnetic Resonance Imaging; Optical Imaging; Molecular Imaging; Molecular Probes
PubMed: 37460726
DOI: 10.1007/978-981-32-9902-3_3 -
Theranostics 2022Exploring and understanding the interaction of changes in the activities of various enzymes, such as proteases, phosphatases, and oxidoreductases with tumor invasion,... (Review)
Review
Exploring and understanding the interaction of changes in the activities of various enzymes, such as proteases, phosphatases, and oxidoreductases with tumor invasion, proliferation, and metastasis is of great significance for early cancer diagnosis. To detect the activity of tumor-related enzymes, various molecular probes have been developed with different imaging methods, including optical imaging, photoacoustic imaging (PAI), magnetic resonance imaging, positron emission tomography, and so on. In this review, we first describe the biological functions of various enzymes and the selectively recognized chemical linkers or groups. Subsequently, we systematically summarize the design mechanism of imaging probes and different imaging methods. Finally, we explore the challenges and development prospects in the field of enzyme activity detection. This comprehensive review will provide more insight into the design and development of enzyme activated molecular probes.
Topics: Humans; Molecular Imaging; Molecular Probes; Neoplasms; Optical Imaging; Photoacoustic Techniques; Tomography, X-Ray Computed
PubMed: 35154500
DOI: 10.7150/thno.66676 -
Journal of Nuclear Cardiology :... Aug 2016Molecular probes provide imaging signal and contrast for the visualization, characterization, and measurement of biological processes at the molecular level. These... (Review)
Review
Molecular probes provide imaging signal and contrast for the visualization, characterization, and measurement of biological processes at the molecular level. These probes can be designed to target the cell or tissue of interest and must be retained at the imaging site until they can be detected by the appropriate imaging modality. In this article, we will discuss the basic design of molecular probes, differences among the various types of probes, and general strategies for their evaluation of cardiovascular disease.
Topics: Angiography; Cardiac Imaging Techniques; Cardiovascular Diseases; Humans; Image Enhancement; Molecular Imaging; Molecular Probe Techniques; Molecular Probes
PubMed: 27189171
DOI: 10.1007/s12350-016-0501-8