Many genetically encoded probes that employ fluorescent proteins and fluorescence resonance energy transfer (FRET) have been developed to better understand the spatiotemporal regulation of various ...cellular processes. The different types of FRET and measurement techniques necessitate characterization of their specific features. Here I provide theoretical and practical comparisons of bimolecular and unimolecular FRET constructs, intensity-based and lifetime-based FRET measurements, FRET imaging using live- and fixed-cell samples, green fluorescent protein-based and chemical fluorophore-based FRET, and FRET efficiency and indices. The potential benefits and limitations of a variety of features in the technologies using fluorescent proteins and FRET are discussed.
Background
Research suggests that preventive measures are critical to reducing the spread of coronavirus disease 2019 (COVID-19), but evidence regarding the association between trust in government ...and the practice of preventive measures is limited.
Objective
To examine whether the practice of preventive measures against COVID-19 differs by one’s level of trust in government.
Design
A cross-sectional analysis using the Japan COVID-19 and Society Internet Survey (JACSIS) conducted in August and September 2020.
Participants
A nationally representative sample of Japanese individuals aged 15 through 79 years.
Main Measures
The primary outcome was the composite score for COVID-19 preventive measures, defined as the percentage of preventive measures an individual reported to be practicing (out of nine measures: social distancing, wearing masks, avoiding closed spaces, avoiding crowded spaces, avoiding close contact settings, hand washing, avoiding touching one’s face, respiratory hygiene, and surface disinfection). The secondary outcomes were (1) support for stay-at-home requests, (2) use of a contact-tracing app, and (3) receipt of the influenza vaccine in the previous season.
Key Results
Our analysis included a total of 25,482 individuals. After adjusting for potential confounders, we found that individuals with high trust in government were likely to practice preventive measures more frequently compared to those with low trust (adjusted composite scores, 83.8% for high- vs. 79.5% for low-trust individuals; adjusted difference, +4.3 percentage points pp; 95% CI, +2.4 to +6.2pp; P<0.001). We also found that high trust in government was associated with higher likelihoods of support for stay-at-home requests, use of a contact-tracing app, and receipt of the influenza vaccine in the previous season.
Conclusions
High trust in government was associated with a higher intensity of practicing COVID-19 preventive measures among Japanese individuals at the national level. Our findings may provide useful information to develop and design effective public health interventions.
In recent years, our ability to unravel the finer details of intracellular signaling has improved remarkably. Technological innovations resulting from the introduction of green fluorescent protein ...(GFP) have played a significant role in these advances. Fluorescent indicators allow us to visualize events within a cell in real-time and space. This review focuses on indicators that use GFP-based fluorescence resonance energy transfer (FRET) technologies and discusses how these methodologies have given insights into biological questions relating to the spatiotemporal patterns of signaling by cAMP, calcium, receptor tyrosine kinases, and other molecules.
Casting Light on Life Miyawaki, Atsushi
Keio journal of medicine,
2020, 2020-00-00, 20200101, Letnik:
69, Številka:
4
Journal Article
Recenzirano
Odprti dostop
The behavior of biochemical molecules moving around in cells makes me think of a school of whales wandering in the ocean, captured by the Argus system on the artificial satellite. When bringing a ...whale back into the sea --- with a transmitter on its dorsal fin, every staff member hopes that it will return safely to a school of its species. A transmitter is now minute in size, but it was not this way before. There used to be some concern that a whale fitted with a transmitter could be given the cold shoulder and thus ostracized by other whales for “wearing something annoying.” How is whale’s wandering related to the tide or a shoal of small fish? What kind of interaction is there among different species of whales? We human beings have attempted to fully understand this fellow creature in the sea both during and since the age of whale fishing.In a live cell imaging experiment, a luminescent probe replaces a transmitter. We put a luminescent probe on a specific region of a biological molecule and bring it back into a cell. We can then visualize how the molecule behaves in response to external stimulation. Since luminescence is a physical phenomenon, we can extract various kinds of information by making full use of its characteristics.Cruising inside cells in a supermicro corps, gliding down in a microtubule like a roller coaster, pushing our ways through a jungle of chromatin while hoisting a flag of nuclear localization signal --- we are reminded to retain a playful and adventurous perspective at all times. What matters is mobilizing all capabilities of science and giving full play to our imagination. We believe that such serendipitous findings can arise out of such a sportive mind, a frame of mind that prevails when enjoying whale-watching.
Bioluminescence is a natural light source based on luciferase catalysis of its substrate luciferin. We performed directed evolution on firefly luciferase using a red-shifted and highly deliverable ...luciferin analog to establish AkaBLI, an all-engineered bioluminescence in vivo imaging system. AkaBLI produced emissions in vivo that were brighter by a factor of 100 to 1000 than conventional systems, allowing noninvasive visualization of single cells deep inside freely moving animals. Single tumorigenic cells trapped in the mouse lung vasculature could be visualized. In the mouse brain, genetic labeling with neural activity sensors allowed tracking of small clusters of hippocampal neurons activated by novel environments. In a marmoset, we recorded video-rate bioluminescence from neurons in the striatum, a deep brain area, for more than 1 year. AkaBLI is therefore a bioengineered light source to spur unprecedented scientific, medical, and industrial applications.
Brain/MINDS: brain-mapping project in Japan Okano, Hideyuki; Miyawaki, Atsushi; Kasai, Kiyoto
Philosophical transactions - Royal Society. Biological sciences,
05/2015, Letnik:
370, Številka:
1668
Journal Article
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There is an emerging interest in brain-mapping projects in countries across the world, including the USA, Europe, Australia and China. In 2014, Japan started a brain-mapping project called Brain ...Mapping by Integrated Neurotechnologies for Disease Studies (Brain/MINDS). Brain/MINDS aims to map the structure and function of neuronal circuits to ultimately understand the vast complexity of the human brain, and takes advantage of a unique non-human primate animal model, the common marmoset (Callithrix jacchus). In Brain/MINDS, the RIKEN Brain Science Institute acts as a central institute. The objectives of Brain/MINDS can be categorized into the following three major subject areas: (i) structure and functional mapping of a non-human primate brain (the marmoset brain); (ii) development of innovative neurotechnologies for brain mapping; and (iii) human brain mapping; and clinical research. Brain/MINDS researchers are highly motivated to identify the neuronal circuits responsible for the phenotype of neurological and psychiatric disorders, and to understand the development of these devastating disorders through the integration of these three subject areas.
Brain/MINDS (Brain Mapping by Integrated Neurotechnologies for Disease Studies) is a national brain project started by Japan in 2014. With the goal of developing the common marmoset as a model animal ...for neuroscience, the project aims to build a multiscale marmoset brain map, develop new technologies for experimentalists, create transgenic lines for brain disease modeling, and integrate translational findings from the clinical biomarker landscape. Brain/MINDS will collaborate with global brain projects to share technologies and resources.
The Japanese national brain initiative called Brain Mapping by Integrated Neurotechnologies for Disease Studies (Brain/MINDS) initiative was launched in 2014 with a focus on the common marmoset as a model system.
The observation of the regulation of fast protein dynamics in a cellular context requires the development of reliable technologies. Here, a signal regulation cascade reliant on the stimulus-dependent ...acceleration of the bidirectional flow of mitogen-activated protein kinase (extracellular signal-regulated kinase) across the nuclear envelope was visualized by reversible protein highlighting. Light-induced conversion between the bright and dark states of a monomeric fluorescent protein engineered from a novel coral protein was employed. Because of its photochromic properties, the protein could be highlighted, erased, and highlighted again in a nondestructive manner, allowing direct observation of regulated fast nucleocytoplasmic shuttling of key signaling molecules.
Over the past 20 years, protein engineering has been extensively used to improve and modify the fundamental properties of fluorescent proteins (FPs) with the goal of adapting them for a fantastic ...range of applications. FPs have been modified by a combination of rational design, structure-based mutagenesis, and countless cycles of directed evolution (gene diversification followed by selection of clones with desired properties) that have collectively pushed the properties to photophysical and biochemical extremes. In this review, we provide both a summary of the progress that has been made during the past two decades, and a broad overview of the current state of FP development and applications in mammalian systems.
Monomeric red and far-red FPs and indicators now perform nearly as well as the best green FPs (and indicators).
Reversible and irreversible photochromism in FPs can be exploited to increase optical resolution and improve contrast compared with traditional fluorescence microscopy.
Infrared FPs (IFPs) are becoming ever more useful as labels for various proteins that allow correct localization and whole-animal imaging. IFPs can serve as an additional fluorescent ‘color’ for simultaneous imaging with visible FP-labeled proteins.
Bacterial phytochrome (BphP)-based IFPs provide a new scaffold for engineering fluorogenic indicators, which are ideal to visualize spatiotemporal dynamics of cell signaling in vivo.
Small ultra-red FP (smURFP) is the brightest far-red nonprototypical FP (comparable with EGFP) and is extremely photostable. smURFP may prove particularly useful as a photostable FP for super-resolution imaging and as a FRET acceptor for biosensing applications.
The engineering of new fluorescent indicators that combine features of prototypical FP-based indicators with photochromic proteins can reveal the cellular maps of biochemical activities in super-resolution.
FPs can be used as optogenetic actuators to manipulate cellular and protein functions through chromophore-assisted light inactivation or light-controlled protein oligomerization.
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