Cyan Fluorescent Protein Research Articles

Imaging Transgenic Nude Mice Expressing Spectrally-distinct Fluorescent Reporters Emitting From Blue to Far Red Light With One Instrument With Single-nanometer-tuning of Laser-excitation Fluorescence.

Transgenic nude mice expressing green fluorescent protein (GFP), red fluorescent protein (RFP), or cyan fluorescent protein (CFP) were previously developed by our laboratory, AntiCancer Inc. In the present study, we demonstrate imaging of the GFP, RFP, or CFP nude mice with single-nanometer-tuning laser fluorescence excitation with a single instrument. Female transgenic C57/B6 nude GFP, RFP, and CFP mice aged six weeks were used. The images were obtained using the UVP Biospectrum Advanced system (Analytik Jena US LLC) with excitation at 480 nm and peak emission at 513 nm for GFP; 520 nm and 605 nm, respectively, for RFP; and 405 nm and 480 nm, respectively, for CFP. For each color transgenic fluorescent mouse, images without background could be obtained individually with the UVP Biospectrum Advanced system. Using a single instrument, brilliant and well-defined images of GFP, RFP, and CFP mice were obtained with single-nanometer-tuning laser fluorescence excitation. This imaging system will be used in future studies to analyze cancer cells in the colored mice that are spectrally distinct in order to determine how stromal cells and cancer interact in the tumor microenvironment.

Extremely Sensitive Genetically Encoded Temperature Indicator Enabling Measurement at the Organelle Level.

Intracellular temperature is a fundamental parameter in biochemical reactions. Genetically encoded fluorescent temperature indicators (GETIs) have been developed to visualize intracellular thermogenesis; however, the temperature sensitivity or localization capability in specific organelles should have been further improved to clearly capture when and where intracellular temperature changes at the subcellular level occur. Here, we developed a new GETI, gMELT, composed of donor and acceptor subunits, in which cyan and yellow fluorescent proteins, respectively, as a Förster resonance energy transfer (FRET) pair were fused with temperature-sensitive domains. The donor and acceptor subunits associated and dissociated in response to temperature changes, altering the FRET efficiency. Consequently, gMELT functioned as a fluorescence ratiometric indicator. Untagged gMELT was expressed in the cytoplasm, whereas versions fused with specific localization signals were targeted to the endoplasmic reticulum (ER) or mitochondria. All gMELT variations enabled more sensitive temperature measurements in cellular compartments than those in previous GETIs. The gMELTs, tagged with ER or mitochondrial targeting sequences, were used to detect thermogenesis in organelles stimulated chemically, a method previously known to induce thermogenesis. The observed temperature changes were comparable to previous reports, assuming that the fluorescence readout changes were exclusively due to temperature variations. Furthermore, we demonstrated how macromolecular crowding influences gMELT fluorescence given that this factor can subtly affect the fluorescence readout. Investigating thermogenesis with gMELT, accounting for factors such as macromolecular crowding, will enhance our understanding of intracellular thermogenesis phenomena.

Viable protoplast isolation, organelle visualization and transformation of the globally distributed plant pathogen Phytophthora cinnamomi

Phytophthora cinnamomi is an oomycete plant pathogen with a host range of almost 5000 plant species worldwide and therefore poses a serious threat to biodiversity. Omics technology has provided significant progress in our understanding of oomycete biology, however, transformation studies of Phytophthora for gene functionalisation are still in their infancy. Only a limited number of Phytophthora species have been successfully transformed and gene edited to elucidate the role of particular genes. There is a need to escalate our efforts to understand molecular processes, gene regulation and infection mechanisms of the pathogen to enable us to develop new disease management strategies. The primary obstacle hindering the advancement of transformation studies in Phytophthora is their challenging and unique nature, coupled with our limited comprehension of why they remain such an intractable system to work with. In this study, we have identified some of the key factors associated with the recalcitrant nature of P. cinnamomi. We have incorporated fluorescence microscopy and flow cytometry along with the organelle-specific dyes, fluorescein diacetate, Hoechst 33342 and MitoTracker™ Red CMXRos, to assess P. cinnamomi-derived protoplast populations. This approach has also provided valuable insights into the broader cell biology of Phytophthora. Furthermore, we have optimized the crucial steps that allow transformation of P. cinnamomi and have generated transformed isolates that express a cyan fluorescent protein, with a transformation efficiency of 19.5%. We therefore provide a platform for these methodologies to be applied for the transformation of other Phytophthora species and pave the way for future gene functionalisation studies.

Improvement of the Green-Red Förster Resonance Energy Transfer-Based Ca2+ Indicator by Using the Green Fluorescent Protein, Gamillus, with a Trans Chromophore as the Donor.

To monitor the Ca2+ dynamics in cells, various genetically encoded Ca2+ indicators (GECIs) based on Förster resonance energy transfer (FRET) between fluorescent proteins are widely used for live imaging. Conventionally, cyan and yellow fluorescent proteins have been often used as FRET pairs. Meanwhile, bathochromically shifted indicators with green and red fluorescent protein pairs have various advantages, such as low toxicity and autofluorescence in cells. However, it remains difficult to develop them with a similar level of dynamic range as cyan and yellow fluorescent protein pairs. To improve this, we used Gamillus, which has a unique trans-configuration chromophore, as a green fluorescent protein. Based on one of the best high-dynamic-range GECIs, Twitch-NR, we developed a GECI with 1.5-times higher dynamic range (253%), Twitch-GmRR, using RRvT as a red fluorescent protein. Twitch-GmRR had high brightness and photostability and was successfully applied for imaging the Ca2+ dynamics in live cells. Our results suggest that Gamillus with trans-type chromophores contributes to improving the dynamic range of GECIs. Therefore, selection of the cis-trans isomer of the chromophore may be a fundamental approach to improve the dynamic range of green-red FRET indicators, unlimited by GECIs.

A dual fluorescence protein expression system detects cell cycle dependent protein noise

Inherently identical cells exhibit significant phenotypic variation. It can be essential for many biological processes and is known to arise from stochastic, ‘noisy’, gene expression that is determined by intrinsic and extrinsic components. It is now obvious that the noise varies as a function of inducer concentration. However, its fluctuation over the cell cycle is limited. Applying dual colour fluorescence protein reporter system, Cyan Fluorescent Protein (CFP) and Yellow fluorescent protein (YFP) tagged multi-copy plasmids, we determine variation of the noise components over the phases in lac promoter induced by Isopropyl β-D-1-thiogalactopyranoside (IPTG) and in presence of additional Magnesium, Mg2+ ion. We, also, estimate the how such system deviates from observations of single-copy plasmid. Found 25 % difference between multi-copy system and single-copy system clarifies that observed noise is considerable and estimates population behaviour during the cell cycle. We show that total variation in cells induced with IPTG is determined by higher extrinsic than intrinsic noise. It increases from Lag to Exponential phase and decreases from Retardation to Stationary phase. By observing slow and fast dividing cells, we show that 5 mM Mg2+ increases population homogeneity compared to 2.5 mM Mg2+ in the environment. The experimental data obtained using dual colour fluorescence protein reporter system demonstrates that protein expression noise, depending on intra cellular ionic concentration, is tightly controlled by phase of the cell.

Generation of DNAzyme in Bacterial Cells by a Bacterial Retron System.

DNAzymes are catalytically active single-stranded DNAs in which DNAzyme 10-23 (Dz 10-23) consists of a catalytic core and a substrate-binding arm that reduces gene expression through sequence-specific mRNA cleavage. However, the in vivo application of Dz 10-23 depends on exogenous delivery, which leads to its inability to be synthesized and stabilized in vivo, thus limiting its application. As a unique reverse transcription system, the bacterial retron system can synthesize single-stranded DNA in vivo using ncRNA msr/msd as a template. The objective of this work is to reduce target gene expression using Dz 10-23 generated in vivo by the retron system. In this regard, we successfully generated Dz 10-23 by cloning the Dz 10-23 coding sequence into the retron msd gene and tested its ability to reduce specific gene expression by examining the mRNA levels of cfp encoding cyan fluorescence protein and other functional genes such as mreB and ftsZ. We found that Dz had different repressive effects when targeting different mRNA regions, and in general, the repressive effect was stronger when targeting downstream of mRNAs. Our results also suggested that the reduction effect was due to cleavage of the substrate mRNA by Dz 10-23 rather than the antisense effect of the substrate-binding arm. Therefore, this study not only provided a retron-based method for the intracellular generation of Dz 10-23 but also demonstrated that Dz 10-23 could reduce gene expression by cleaving target mRNAs in cells. We believe that this new strategy would have great potential in the regulation of gene expression.

Specific binding of G-quadruplex in SARS-CoV-2 RNA by RHAU peptide

G-quadruplexes (G4s) are reported to present on the SARS-CoV-2 RNA genome and control various viral activities. Specific ligands targeting those viral nucleic acid structures could be investigated as promising detection methods or antiviral reagents to suppress this menacing virus. Herein, we demonstrate the binding between a G4 structure in the RNA of SARS-CoV-2 and a fluorescent probe created by fusing a parallel-G4 specific RHAU53 and a cyan fluorescent protein. The specific binding of G4 in SARS-CoV-2 by RHAU peptide was easily detected under the fluorescence spectrometer. The drawbacks of this approach and potential solutions are also discussed.

A bright cyan fluorescence calcium indicator for mitochondrial calcium with minimal interference from physiological pH fluctuations.

Genetically Encoded Calcium (Ca2+) indicators (GECIs) are indispensable tools for dissecting intracellular Ca2+ signaling and monitoring cellular activities. Mitochondrion acts as a Ca2+ sink and a central player for maintaining Ca2+ homeostasis. Accurately monitoring Ca2+ transients within the mitochondrial matrix that undergo constant pH fluctuations is challenging, as signals of most currently available GECIs suffer from artifacts induced by physiological pH variations. Multiplexed monitoring of optophysiology is also hindered by the limited availability of GECIs with cyan fluorescence. Based on the bright variant of cyan fluorescence protein (CFP), mTurquoise2, we developed a GECI designated as TurCaMP. Results from molecular dynamics simulations and ab initio calculations revealed that the deprotonation of the chromophore may be responsible for the Ca2+-dependent changes in TurCaMP signals. TurCaMP sensors showed inverse response to Ca2+ transients, and their responses were not affected by pH changes within the range of pH 6-9. The high basal fluorescence and insensitivity to physiological pH fluctuations enabled TurCaMP to faithfully monitor mitochondrial Ca2+ responses with a high signal-to-noise ratio. TurCaMP sensors allow simultaneous multi-colored imaging of intracellular Ca2+ signals, expanding the possibility of multiplexed monitoring of Ca2+-dependent physiological events.

Bioengineering Phaeodactylum tricornutum, a marine diatom, for cannabinoid biosynthesis

Over the last few years, phytocannabinoids have been studied for their bioactive properties as potential drug candidates to treat or alleviate symptoms of several diseases. The isolation of single and pure cannabinoid (CB) from their natural source, i.e., Cannabis plants results in a low yield, diluted among hundreds of other plant metabolites. The use of biotechnological platforms to produce single CBs is appealing to the pharmaceutical industry. Therefore, our aim was to develop a sustainable system using the model diatom Phaeodactylum tricornutum to produce cannabigerolic acid (CBGA), the precursor to several CBs such as well-known cannabidiol (CBD) and delta-9-tetrahydrocannabinol (THC). We engineered P. tricornutum to express a mutant version of the Streptomyces sp. strain CL190's naptherpin biosynthetic cluster gene B NphB (Y288A/G286S, nphB), a non-Cannabis aromatic prenyltransferase enzyme, either by random integrated chromosomal expression (RICE) or extrachromosomal expression (EE), to maximize the success of protein production. The gene of nphB was linked to the reporter cyan fluorescent protein (CFP) and introduced in P. tricornutum. Clones were characterized by CFP fluorescence intensity, protein synthesis, enzymatic activity, and production of CBGA. We present, for the first time in diatoms, the successful production of a CB, CBGA up to 4.1 (± 0.2) mg/kg of microalgal fresh biomass weight. This work shows the potential of P. tricornutum as a sustainable controlled heterologous platform for CBs production, plant bioactive compounds, and relevant pharmaceuticals.

Longitudinal intravital imaging of the bone marrowfor analysis of the race for the surface in a murine osteomyelitis model.

Critical knowledge gaps of orthopedic infections pertain to bacterial colonization. The established dogma termed the Race for the Surface posits that contaminating bacteria compete with host cells for the implant post-op, which remains unproven without real-time in vivo evidence. Thus, we modified the murine longitudinal intravital imaging of the bone marrow (LIMB) system to allow real-time quantification of green fluorescent protein(GFP+) host cells and enhanced cyan fluorescent protein(ECFP+) or red fluorescent protein(RFP+) methicillin-resistant Staphylococcus aureus (MRSA) proximal to a transfemoral implant. Following inoculation with ~105 CFU, an L-shaped metal implant was press-fit through the lateral cortex at a 90° angle ~0.150 mm below a gradient refractive index (GRIN) lens. We empirically derived a volume of interest (VOI) = 0.0161 ± 0.000675 mm3 during each imaging session by aggregating the Z-stacks between the first (superior) and last (inferior) in-focus LIMB slice. LIMB postimplantation revealed very limited bacteria detection at 1 h, but by 3 h,56.8% of the implant surface was covered by ECFP+ bacteria, and the rest were covered by GFP+ host cells. 3D volumetric rendering of the GFP+ and ECFP+ or RFP+ voxels demonstrated exponential MRSA growth between 3 and 6 hin the Z-plane, which was validated with cross-sectional ex vivo bacterial burden analyses demonstrating significant growth by ~2 × 104 CFU/hon the implant from 2 to 12 hpost-op (p < 0.05; r2 > 0.98). Collectively, these results show the competition at the surface is completed by 3 hin this model and demonstrate the potential of LIMB to elucidate mechanisms of bacterial colonization, the host immune response, and the efficacy of antimicrobials.

Development of Zika Virus Mini-Replicon Based Single-Round Infectious Particles as Gene Delivery Vehicles.

Zika virus (ZIKV) is a type of RNA virus that belongs to the Flaviviridae family. We have reported the construction of a DNA-launched replicon of the Asian-lineage Natal RGN strain and the production of single-round infectious particles (SRIPs) via the combination of prM/E virus-like particles with the replicon. The main objective of the study was to engineer the ZIKV replicon as mammalian expression vectors and evaluate the potential of ZIKV mini-replicon-based SRIPs as delivery vehicles for heterologous gene expression in vitro and in vivo. The mini-replicons contained various genetic elements, including NS4B, an NS5 methyltransferase (MTase) domain, and an NS5 RNA-dependent RNA polymerase (RdRp) domain. Among these mini-replicons, only ZIKV mini-replicons 2 and 3, which contained the full NS5 and NS4B-NS5 genetic elements, respectively, exhibited the expression of reporters (green fluorescent protein (GFP) and cyan fluorescent protein-yellow fluorescent fusion protein (CYP)) and generated self-replicating RNAs. When the mini-replicons were transfected into the cells expressing ZIKV prM/E, this led to the production of ZIKV mini-replicon-based SRIPs. ZIKV mini-replicon 3 SRIPs showed a significantly higher yield titer and a greater abundance of self-replicating replicon RNAs when compared to ZIKV mini-replicon 2 SRIPs. Additionally, there were disparities in the dynamics of CYP expression and cytotoxic effects observed in various infected cell types between ZIKV mini-replicon 2-CYP and 3-CYP SRIPs. In particular, ZIKV mini-replicon 3-CYP SRIPs led to a substantial decrease in the survival rates of infected cells at a MOI of 2. An in vivo gene expression assay indicated that hACE2 expression was detected in the lung and brain tissues of mice following the intravenous administration of ZIKV mini-replicon 3-hACE2 SRIPs. Overall, this study highlights the potential of ZIKV mini-replicon-based SRIPs as promising vehicles for gene delivery applications in vitro and in vivo.

FRET-based probe for ratiometric detection and imaging of folic acid in real-time

Inadequate folic acid intake is linked to diseases such as megaloblastic anemia, neural tube defects, and hyperhomocysteinemia, increasing the risk of vascular disease and thrombosis. Folic acid, a cofactor in various enzymes, can be produced by plants and bacteria, but not by humans and other animals. L-5-methyl-tetrahydrofolate (L-5-methyl-THF) is the primary dietary folate form, transported in circulation for cellular metabolism. Traditional methods of determining folic acid levels are unreliable and time-consuming. SenFol (Sensor for folic acid) is a fluorescence resonance energy transfer (FRET)-based nanosensor that we have developed by inserting folic acid-binding protein (FolT) as the folate detecting domain between the pair of enhanced cyan fluorescent protein (ECFP) and Venus. The developed sensor is highly specific, produces a quick signal, which is pH stable, and delivers precise, ratiometric readings in cell-based experiments. The projected affinity score of folic acid with FolT was −7.4 kcal/mol. The apparent affinity (Kd) of SenFol for folic acid is 28.49 × 10−9 M, with a detection range of 5 × 10−9 M to 5 × 10−7 M, and a maximum FRET ratio change of 0.45. WT SenFol, a highly efficient folic acid nanosensor, can dynamically detect intracellular folic acid content in E. coli, yeast, and HEK-293 T cells, confirming its potential.

Fate mapping reveals compartment-specific clonal expansion of mononuclear phagocytes during kidney disease

Fate mapping reveals compartment-specific clonal expansion of mononuclear phagocytes during kidney disease

Diverse Fluorescence Resonance Energy Transfer Processes Originating from the Conformational Heterogeneity of the Calcium Indicator Yellow Cameleon YC3.60.

By combining time-resolved fluorescence spectroscopy and molecular biology, we have clearly elucidated the fluorescence mechanism of yellow cameleon YC3.60. YC3.60 is one of the most widely used fundamental calcium ion indicator proteins and is a tandem protein consisting of cyan fluorescent protein, calmodulin, and yellow fluorescent protein. Our results show that the conformational heterogeneity of YC3.60 leads to multiple FRET (fluorescence resonance energy transfer) processes occurring in a variety of structures. In the presence of calcium ions, FRET occurs in 75% of all YC3.60 molecules, and the intramolecular FRET faster than 20 ps is dominant among the three different FRET processes. Even in the absence of calcium ions, FRET occurs in 28% of YC3.60, where intra- and intermolecular FRET with a time constant of 160 ps is dominant among the four different FRET processes. YC3.60 with the immature chromophore, which is 25% of the total, would result in lower contrast on imaging. Conformational heterogeneity arises from the specific association of the two fluorescent proteins. It was clearly shown that the association of fluorescent proteins has a significant impact on the fluorescence mechanism. These observations lead to the promotion of research that elucidates the relationship between the higher-order structure and function of proteins.

PD25-10 A NEWLY DISCOVERED COLLECTIVE MIGRATION OF UROTHELIAL CELLS: A POTENTIAL DEFENSE MECHANISM AGAINST BLADDER CANCER DEVELOPMENT

You have accessJournal of UrologyCME1 Apr 2023PD25-10 A NEWLY DISCOVERED COLLECTIVE MIGRATION OF UROTHELIAL CELLS: A POTENTIAL DEFENSE MECHANISM AGAINST BLADDER CANCER DEVELOPMENT Zhang Ning, Takeshi Sano, Ryosuke Ikeuchi, Hideaki Takada, Kenji Nakamura, Toru Sakatani, Akihiro Hamada, Yuki Kita, Michiyuki Matsuda, and Takashi Kobayashi Zhang NingZhang Ning More articles by this author , Takeshi SanoTakeshi Sano More articles by this author , Ryosuke IkeuchiRyosuke Ikeuchi More articles by this author , Hideaki TakadaHideaki Takada More articles by this author , Kenji NakamuraKenji Nakamura More articles by this author , Toru SakataniToru Sakatani More articles by this author , Akihiro HamadaAkihiro Hamada More articles by this author , Yuki KitaYuki Kita More articles by this author , Michiyuki MatsudaMichiyuki Matsuda More articles by this author , and Takashi KobayashiTakashi Kobayashi More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003303.10AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Imaging of live animals at microscopic resolution (intravital imaging) found that bladder tumor (BT) development caused a unique collective migration of urothelial cells in a mouse orthotopic bladder cancer model. This study aimed to investigate the relationship between this newly discovered migration and BT growth. METHODS: MB49 cells (a mouse bladder cancer cell line) were labeled with a red fluorescent protein to distinguish them from urothelial cells of C57BL/6 mice expressing a cyan fluorescent protein. Labeled MB49 cells were then injected into mouse bladders pretreated with poly-l-lysine (PLL) to initiate BTs. While mice underwent general anesthesia using isoflurane, two-photon excitation microscopy, which enables deep tissue imaging, was performed to visualize the urothelium and the attached MB49 cells. Dasatinib, a Src/focal adhesion kinase signaling inhibitor, was intravenously or orally administered. RESULTS: A collective migration of urothelial cells was observed four days after intravesical injection of 1×106 MB49 cells (Figure 1). Pretreatment with PLL alone did not cause this migration. Intravenous and oral administration of dasatinib each remarkably retarded MB49-induced migration (Figure 2a). Surprisingly, inhibition of this migration by daily oral administration of dasatinib starting 24 hours after MB49 injection significantly enhanced BT growth despite dasatinib generally inhibiting cell-growth (Figure 2b). In a subcutaneous xenograft mouse model using MB49 cells, dasatinib slightly suppressed tumor growth. Intriguingly, transfer of urine from which cells had been removed by filtration from an MB49-injected bladder to another C57BL/6 mouse bladder caused the urothelial cell migration (Figure 2c). CONCLUSIONS: Cancer cell injection into the bladder caused a unique collective migration of urothelial cells. Inhibition of this migration enhances BT growth, suggesting that this migration may play a defensive role against BT development. We are currently working to identify the molecule(s) that trigger the migration. Enhancement of this migration may be a novel strategy for preventing BT recurrence after transurethral resection of BTs. Source of Funding: None © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e733 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Zhang Ning More articles by this author Takeshi Sano More articles by this author Ryosuke Ikeuchi More articles by this author Hideaki Takada More articles by this author Kenji Nakamura More articles by this author Toru Sakatani More articles by this author Akihiro Hamada More articles by this author Yuki Kita More articles by this author Michiyuki Matsuda More articles by this author Takashi Kobayashi More articles by this author Expand All Advertisement PDF downloadLoading ...

Designing of nanosensor and its applications for spatiotemporal visualization and real time quantification of biotin in biological samples

Biotin is a 244 Da water-soluble vitamin involved in vital metabolic processes by acting as an essential cofactor for several enzymes. When biotin levels are low, critical enzymes such as pyruvate carboxylase, 3-methylcrotonyl-CoA, and propionyl-CoA carboxylase become dysfunctional leading to fatal human disease. Traditional methods for measuring biotin in biological fluids yield valuable results for particular applications, but the majority of them are intrusive, expensive, and lack sensitivity and specificity. A highly effective genetically encoded FRET-based nanosensor OSenB (Optical Sensor for Biotin) is developed using FRET pair enhanced cyan fluorescent protein (ECFP) and Venus attached to the N- and C-termini of the avidin module, allowing real-time monitoring and quantification of biotin at a trace level. OSenB exhibits discernible FRET ratio shifts, measures biotin with excellent specificity and selectivity, provides a rapid detection response, is resilient to pH fluctuations, and produces a very precise FRET signal with a detection range of 12 pM to 165 nM biotin quantification. Developed mutant sensor OSenB-44 with 0.717 nM apparent affinity (Kd), was found to be the most successful nanosensor. Confocal images show that OSenB-44 was successfully expressed in vivo and dynamically measured biotin in Escherichia coli, yeast, and human embryonic kidney (HEK)− 293T in real-time.

Uncovering the link between environmental factors and coral immunity: A study of fluorescent protein expression and phenoloxidase activity in Acropora cervicornis

Worsening environmental conditions due to climate change have profoundly affected the health of coral reefs worldwide. Thus, understanding how corals respond to fluctuating and/or extreme levels of temperature and solar irradiation will guide future protection and restoration efforts of this valuable ecosystem. Herein, we present a study of the immune responses of the endangered coral Acropora cervicornis to seasonal fluctuations in water temperature (WT), light intensity (LI), and water depth. Immune responses were observed by measuring the concentration of green and cyan fluorescent proteins (GFP and CyFP) and the activity of phenoloxidase (PO), an enzyme involved in the biosynthesis of the photoprotective protein melanin. To study these responses, visually healthy A. cervicornis fragments were placed at 8, and 12 m depth, and GFP, CyPF, and PO activity were measured at three-month intervals over a 12-month period. Seawater temperature and light intensity were also measured at each depth during this period. A general linear mixed model was used to determine the effects of seasonal variations of WT, LI, and water depth on the immune proteins. GFP, CyFP, and PO activity varied significantly across time – all higher in late summer/early fall and lower in late winter/early spring. Likewise, WT and LI significantly affected GFP, CyFP, and PO activity. On the other hand, water depth only had a significant effect on fluorescent protein concentrations but not PO activity. Our study demonstrates that corals can modulate these key immune-related proteins throughout natural seasonal fluctuations. That is, increasing in months of higher thermal and light conditions while decreasing in months with mild thermal and light conditions. The phenotypic plasticity of A. cervicornis in adapting to a changing environment underscores the importance that in future studies time of the year should be a meaningful consideration when evaluating the responses of A. cervicornis to the environment.

Real-Time Monitoring of Selenium in Living Cells by Fluorescence Resonance Energy Transfer-Based Genetically Encoded Ratiometric Nanosensors.

Selenium is a component of selenoproteins, which plays a crucial role in cellular redox homeostasis, thyroid metabolism, and DNA synthesis. Selenium has pleiotropic effects like antioxidant and anti-inflammatory activities; however, excess intake of selenium can imbalance such processes. The effects of selenium on human health are numerous and complex, demanding additional research to monitor the flux rate of selenium. Here, we have created a noninvasive and highly efficient genetically encoded fluorescence resonance energy transfer (FRET)-based nanosensor, SelFS (Selenium FRET-Sensor), for real-time monitoring of selenium at the cellular and subcellular levels. The construct of the nanosensor contains a selenium-binding protein (SeBP) as the selenium-detecting element inserted between the green fluorescent protein variants enhanced cyan fluorescent protein and Venus. In the presence of selenium, SelFS brings a conformational change, which is seen in the form of FRET. In vitro studies showed that SelFS is highly specific and selective for selenium and stable at an altered pH range from 5.0 to 8.0. SelFS is a flexible and dynamic tool for the detection of selenium in both prokaryotes and eukaryotes in a noninvasive way, with a binding constant (K d) of 0.198 × 10-6 M as compared to its mutants. The developed nanosensor can provide us a reporter tool for a wide range of industrial and environmental applications, which will help us to understand its functions in biological systems.

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.

Monitoring lipid-protein interactions in planta using Förster resonance energy transfer.

Phospholipids are not only the major structural components of cellular membranes but also important signaling molecules regulating various cellular and physiological processes. One mode of action by lipid mediators is via lipid-protein interactions to modulate the downstream cellular events. An increasing number of lipid-binding proteins have been identified using in vitro lipid-protein binding assays, but it has been challenging to monitor lipid-protein interactions in vivo. Here we describe one Förster resonance energy transfer (FRET)-based method using the cyan fluorescence protein (CFP)-tagged protein cytosolic glyceraldehyde-3-phosphate dehydrogenase (GAPC) and TopFluor TMR-labeled lipid phosphatidic acid (PA) to monitor the lipid-protein interaction in planta. This approach permits detection of the subcellular localization of lipid-protein interactions and dynamics of the interactions in planta in response to different cues.