Blue-shifted genetically encoded Ca2+ indicator with enhanced two-photon absorption.

Significance: Genetically encoded calcium ion (Ca2+) indicators (GECIs) are powerful tools for monitoring intracellular Ca2+ concentration changes in living cells and model organisms. In particular, GECIs have found particular utility for monitoring the transient increase of Ca2+ concentration that is associated with the neuronal action potential. However, the palette of highly optimized GECIs for imaging of neuronal activity remains relatively limited. Expanding the selection of available GECIs to include new colors and distinct photophysical properties could create new opportunities for in vitro and in vivo fluorescence imaging of neuronal activity. In particular, blue-shifted variants of GECIs are expected to have enhanced two-photon brightness, which would facilitate multiphoton microscopy. Aim: We describe the development and applications of T-GECO1-a high-performance blue-shifted GECI based on the Clavularia sp.-derived mTFP1. Approach: We use protein engineering and extensive directed evolution to develop T-GECO1. We characterize the purified protein and assess its performance in vitro using one-photon excitation in cultured rat hippocampal neurons, in vivo using one-photon excitation fiber photometry in mice, and ex vivo using two-photon Ca2+ imaging in hippocampal slices. Results: The Ca2+-bound state of T-GECO1 has an excitation peak maximum of 468 nm, an emission peak maximum of 500 nm, an extinction coefficient of 49,300 M-1 cm-1, a quantum yield of 0.83, and two-photon brightness approximately double that of EGFP. The Ca2+-dependent fluorescence increase is 15-fold, and the apparent Kd for Ca2+ is 82 nM. With two-photon excitation conditions at 850 nm, T-GECO1 consistently enabled the detection of action potentials with higher signal-to-noise (SNR) than a late generation GCaMP variant. Conclusions: T-GECO1 is a high-performance blue-shifted GECI that, under two-photon excitation conditions, provides advantages relative to late generation GCaMP variants.

A blue-shifted genetically encoded Ca2+ indicator with enhanced two-photon absorption.

Significance: Genetically encoded calcium ion (Ca2+) indicators (GECIs) are powerful tools for monitoring intracellular Ca2+ concentration changes in living cells and model organisms. In particular, GECIs have found particular utility for monitoring the transient increase of Ca2+ concentration that is associated with the neuronal action potential. However, the palette of highly optimized GECIs for imaging of neuronal activity remains relatively limited. Expanding the selection of available GECIs to include new colors and distinct photophysical properties could create new opportunities for in vitro and in vivo fluorescence imaging of neuronal activity. In particular, blue-shifted variants of GECIs are expected to have enhanced two-photon brightness, which would facilitate multiphoton microscopy. Aim: We describe the development and applications of T-GECO1 - a high-performance blue-shifted GECI based on the Clavularia sp.-derived mTFP1. Approach: We used protein engineering and extensive directed evolution to develop T-GECO1. We characterize the purified protein and assess its performance in vitro using one-photon excitation in cultured rat hippocampal neurons, in vivo using one-photon excitation fiber photometry in mice, and ex vivo using two-photon Ca2+ imaging in hippocampal slices. Results: The Ca2+-bound state of T-GECO1 has an excitation peak maximum of 468 nm, an emission peak maximum of 500 nm, an extinction coefficient of 49,300 M-1cm-1, a quantum yield of 0.83, and two-photon brightness approximately double that of EGFP. The Ca2+-dependent fluorescence increase is 15-fold and the apparent Kd for Ca2+ is 82 nM. With two-photon excitation conditions at 850 nm, T-GECO1 consistently enabled detection of action potentials with higher signal-to-noise (SNR) than a late generation GCaMP variant. Conclusion: T-GECO1 is a high performance blue-shifted GECI that, under two-photon excitation conditions, provides advantages relative to late generation GCaMP variants.

Biosensor Optimization Using a Förster Resonance Energy Transfer Pair Based on mScarlet Red Fluorescent Protein and an mScarlet-Derived Green Fluorescent Protein.

Genetically encoded biosensors based on Förster resonance energy transfer (FRET) are indispensable tools for monitoring biochemical changes in cells. Green and red fluorescent protein-based FRET pairs offer advantages over the classically employed cyan and yellow fluorescent protein pairs, such as better spectral separation, lower phototoxicity, and less autofluorescence. Here, we describe the development of an mScarlet-derived green fluorescent protein (designated as mWatermelon) and its use as a FRET donor to the red fluorescent protein mScarlet-I as a FRET acceptor. We tested the functionality of this FRET pair by engineering biosensors for the detection of protease activity, Ca2+, and K+. Furthermore, we described a strategy to enhance the FRET efficiency of these biosensors by modulating the intramolecular association between mWatermelon and mScarlet-I.

Complete genome sequence of piezotolerant Stutzerimonas kunmingensis 7850S isolated from the sediment of the Mariana Trench.

Stutzerimonas kunmingensis 7850S is a piezotolerant bacterium isolated from the sediment of the Mariana trench. Here, we described the complete genome of strain 7850S, which contains a single circular chromosome of 4,775,870 base pairs with 62.66% G + C content, and harbors 4494 protein-coding genes, 65 transfer RNA genes, and 12 ribosomal RNA genes. The experimental results showed that strain 7850S could grow under hydrostatic pressure of 0.1-70 MPa. Genomic analyses led to identifications of numbers of high hydrostatic pressure-associated genes, such as the ones associated with unsaturated fatty acids, betaine, and ectoine. A complete set of denitrification genes and some heavy metal detoxification genes were also found in this strain. The presence of these genes suggests metabolic characteristics for adaption to hadal environments and provides insights to further understand adaption strategies and ecological roles of Stutzerimonas in hadal environments.

A sensitive and specific genetically-encoded potassium ion biosensor for in vivo applications across the tree of life.

Potassium ion (K+) plays a critical role as an essential electrolyte in all biological systems. Genetically-encoded fluorescent K+ biosensors are promising tools to further improve our understanding of K+-dependent processes under normal and pathological conditions. Here, we report the crystal structure of a previously reported genetically-encoded fluorescent K+ biosensor, GINKO1, in the K+-bound state. Using structure-guided optimization and directed evolution, we have engineered an improved K+ biosensor, designated GINKO2, with higher sensitivity and specificity. We have demonstrated the utility of GINKO2 for in vivo detection and imaging of K+ dynamics in multiple model organisms, including bacteria, plants, and mice.

The Effectiveness of Teacher Support for Students' Learning of Artificial Intelligence Popular Science Activities.

The burgeoning of new technologies is increasingly affecting people's lives. One new technology that is heatedly discussed is artificial intelligence (AI) in education. To allow students to understand the impact of emerging technologies on people's future lives from a young age, some popular science activities are being progressively introduced into elementary school curricula. Popular science activities are informal education programs and practices of universal education. However, two issues need to be discussed in the implementation of these activities. First, because these informal curricula are usually short in duration, the question of whether they only serve to generate motivation or actually enhance learning outcomes requires examination. Second, the role of teacher support in popular science activities and its impact on students' learning results need to be further investigated. To this end, this study aims to explore the effectiveness of popular AI science activities in informal curricula on students' AI achievement and the interrelationship between students' learning outcomes in popular AI science activities with and without teacher support. A 6-h-long AI popular science activity was conducted with 22 fifth- and sixth-grade students in elementary school. This study was conducted using a one-group pretest and posttest design, and the data collection tools included AI achievement pre- and posttests and an artifact scoring rubric. The results showed that with regard to learning outcomes, popular science activities were helpful for cognitive enhancement of AI concepts, but more time was needed for skills to improve. Additionally, this study found that students' learning performance was different with and without teacher support. Activities with teacher support can enhance students' learning outcomes, but students become accustomed to relying on their teachers. In contrast, activities without teacher support seem to be more effective in fostering students' independent computational thinking and problem-solving abilities.

Fluorescent Indicators For Biological Imaging of Monatomic Ions.

Monatomic ions play critical biological roles including maintaining the cellular osmotic pressure, transmitting signals, and catalyzing redox reactions as cofactors in enzymes. The ability to visualize monatomic ion concentration, and dynamic changes in the concentration, is essential to understanding their many biological functions. A growing number of genetically encodable and synthetic indicators enable the visualization and detection of monatomic ions in biological systems. With this review, we aim to provide a survey of the current landscape of reported indicators. We hope this review will be a useful guide to researchers who are interested in using indicators for biological applications and to tool developers seeking opportunities to create new and improved indicators.

Applying data mining techniques to explore user behaviors and watching video patterns in converged IT environments.

Comfortable leisure and entertainment is expected through multimedia. Web multimedia systems provide diversified multimedia interactions, for example, sharing knowledge, experience and information, and establishing common watching habits. People use information technology (IT) systems to watch multimedia videos and to perform interactive functions. Moreover, IT systems enhance multimedia interactions between users. To explore user behaviors in viewing multimedia videos by key points in time, multimedia video watching patterns are analyzed by data mining techniques. Data mining methods were used to analyze users' video watching patterns in converged IT environments. After the experiment, we recorded the processes of clicking the Web multimedia video player. The system logs of using the video player are classified into four variables, playing time, active playing time, played amount, and actively played amount. To explore the four variables, we apply the k-means clustering technique to organize the similar playing behavior patterns of the users into three categories: actively engaged users, watching engaged users, and long engaged users. Finally, we applied statistical analysis methods to compare the three categories of users' watching behaviors. The results showed that there were significant differences among the three categories.

Genetically encoded fluorescent indicators for imaging intracellular potassium ion concentration.

Potassium ion (K+) homeostasis and dynamics play critical roles in biological activities. Here we describe three genetically encoded K+ indicators. KIRIN1 (potassium (K) ion ratiometric indicator) and KIRIN1-GR are Förster resonance energy transfer (FRET)-based indicators with a bacterial K+ binding protein (Kbp) inserting between the fluorescent protein FRET pairs mCerulean3/cp173Venus and Clover/mRuby2, respectively. GINKO1 (green indicator of K+ for optical imaging) is a single fluorescent protein-based K+ indicator constructed by insertion of Kbp into enhanced green fluorescent protein (EGFP). These indicators are suitable for detecting K+ at physiologically relevant concentrations in vitro and in cells. KIRIN1 enabled imaging of cytosolic K+ depletion in live cells and K+ efflux and reuptake in cultured neurons. GINKO1, in conjunction with red fluorescent Ca2+ indicator, enable dual-color imaging of K+ and Ca2+ dynamics in neurons and glial cells. These results demonstrate that KIRIN1 and GINKO1 are useful tools for imaging intracellular K+ dynamics.

Monomerization of far-red fluorescent proteins.

Anthozoa-class red fluorescent proteins (RFPs) are frequently used as biological markers, with far-red (λem ∼ 600-700 nm) emitting variants sought for whole-animal imaging because biological tissues are more permeable to light in this range. A barrier to the use of naturally occurring RFP variants as molecular markers is that all are tetrameric, which is not ideal for cell biological applications. Efforts to engineer monomeric RFPs have typically produced dimmer and blue-shifted variants because the chromophore is sensitive to small structural perturbations. In fact, despite much effort, only four native RFPs have been successfully monomerized, leaving the majority of RFP biodiversity untapped in biomarker development. Here we report the generation of monomeric variants of HcRed and mCardinal, both far-red dimers, and describe a comprehensive methodology for the monomerization of red-shifted oligomeric RFPs. Among the resultant variants is mKelly1 (emission maximum, λem = 656 nm), which, along with the recently reported mGarnet2 [Matela G, et al. (2017) Chem Commun (Camb) 53:979-982], forms a class of bright, monomeric, far-red FPs.

Pyranopterin Coordination Controls Molybdenum Electrochemistry in Escherichia coli Nitrate Reductase.

We test the hypothesis that pyranopterin (PPT) coordination plays a critical role in defining molybdenum active site redox chemistry and reactivity in the mononuclear molybdoenzymes. The molybdenum atom of Escherichia coli nitrate reductase A (NarGHI) is coordinated by two PPT-dithiolene chelates that are defined as proximal and distal based on their proximity to a [4Fe-4S] cluster known as FS0. We examined variants of two sets of residues involved in PPT coordination: (i) those interacting directly or indirectly with the pyran oxygen of the bicyclic distal PPT (NarG-Ser(719), NarG-His(1163), and NarG-His(1184)); and (ii) those involved in bridging the two PPTs and stabilizing the oxidation state of the proximal PPT (NarG-His(1092) and NarG-His(1098)). A S719A variant has essentially no effect on the overall Mo(VI/IV) reduction potential, whereas the H1163A and H1184A variants elicit large effects (ΔEm values of -88 and -36 mV, respectively). Ala variants of His(1092) and His(1098) also elicit large ΔEm values of -143 and -101 mV, respectively. An Arg variant of His(1092) elicits a small ΔEm of +18 mV on the Mo(VI/IV) reduction potential. There is a linear correlation between the molybdenum Em value and both enzyme activity and the ability to support anaerobic respiratory growth on nitrate. These data support a non-innocent role for the PPT moieties in controlling active site metal redox chemistry and catalysis.