Chemical Control of Fluorescence Lifetime towards Multiplexing Imaging.

Fluorescence lifetime imaging has been a powerful tool for biomedical research. Recently, fluorescence lifetime-based multiplexing imaging has expanded imaging channels by using probes that harbor the same spectral channels and distinct excited state lifetime. While it is desirable to control the excited state lifetime of any given fluorescent probes, the rational control of fluorescence lifetimes remains a challenge. Herein, we chose boron dipyrromethene (BODIPY) as a model system and provided chemical strategies to regulate the fluorescence lifetime of its derivatives with varying spectral features. We find electronegativity of structural substituents at the 8' and 5' positions is important to control the lifetime for the green-emitting and red-emitting BODIPY scaffolds. Mechanistically, such influences are exerted via the photo-induced electron transfer and the intramolecular charge transfer processes for the 8' and 5' positions of BODIPY, respectively. Based on these principles, we have generated a group of BODIPY probes that enable imaging experiments to separate multiple targets using fluorescence lifetime as a signal. In addition to BODIPY, we envision modulation of electronegativity of chemical substituents could serve as a feasible strategy to achieve rational control of fluorescence lifetime for a variety of small molecule fluorophores.

Photoreversible Aggregation of the Biliprotein Containing the First and Second GAF Domains of a Cyanobacteriochrome All2699 in Nostoc sp. PCC7120.

As plant photoreceptors, phytochromes are capable of detecting red light and far-red light, thereby governing plant growth. All2699 is a photoreceptor found in Nostoc sp. PCC7120 that specifically responds to red light and far-red light. All2699g1g2 is a truncated protein carrying the first and second GAF (cGMP phosphodiesterase/adenylyl cyclase/FhlA) domains of All2699. In this study, we found that, upon exposure to red light, the protein underwent aggregation, resulting in the formation of protein aggregates. Conversely, under far-red light irradiation, these protein aggregates dissociated. We delved into the factors that impact the aggregation of All2699g1g2, focusing on the protein structure. Our findings showed that the GAF2 domain contains a low-complexity (LC) loop region, which plays a crucial role in mediating protein aggregation. Specifically, phenylalanine at position 239 within the LC loop region was identified as a key site for the aggregation process. Furthermore, our research revealed that various factors, including irradiation time, temperature, concentration, NaCl concentration, and pH value, can impact the aggregation of All2699g1g2. The aggregation led to variations in Pfr concentration depending on temperature, NaCl concentration, and pH value. In contrast, ΔLC did not aggregate and therefore lacked responses to these factors. Consequently, the LC loop region of All2699g1g2 extended and enhanced sensory properties.

The anti-apoptotic role of Ginkgolide B via mitochondrial permeability transition pore inhibition in retinal ischemia-reperfusion.

This research delves into the effectiveness of Ginkgolide B (GB), a compound from Ginkgo biloba, in combating cell death caused by glaucoma, with a focus on mitochondrial impairment and the mitochondrial permeability transition pore (mPTP). Utilizing models of high intraocular pressure and in vitro glaucoma simulations, the study investigates GB's impact on retinal progenitor cells (RPCs) under oxygen-glucose deprivation/reperfusion (OGD/R) and in a rat glaucoma model. The study methodologies included apoptosis assessment, apoptotic marker analysis via Western blot, and mitochondrial structure and function evaluation. The findings reveal that GB notably decreases apoptosis in RPCs exposed to OGD/R in vitro, and reduces ischemia-reperfusion damage in vivo. GB's protective role is attributed to its ability to preserve mitochondrial integrity, maintain membrane potential, regulate calcium levels, and inhibit mPTP opening. These results underscore GB's potential as a therapeutic agent for acute primary angle-closure glaucoma, highlighting its capability to alleviate mitochondrial damage and apoptosis in RPCs and retinal nerve fiber layer cells.

Detecting protein-protein interaction during liquid-liquid phase separation using fluorogenic protein sensors.

The formation of cellular condensates, akin to membraneless organelles, is typically mediated by liquid-liquid phase separation (LLPS), during which proteins and RNA molecules interact with each other via multivalent interactions. Gaining a comprehensive understanding of these interactions holds significance in unraveling the mechanisms underlying condensate formation and the pathology of related diseases. In an attempt toward this end, fluorescence microscopy is often used to examine the colocalization of target proteins/RNAs. However, fluorescence colocalization is inadequate to reliably identify protein interaction due to the diffraction limit of traditional fluorescence microscopy. In this study, we achieve this goal through adopting a novel chemical biology approach via the dimerization-dependent fluorescent proteins (ddFPs). We succeeded in utilizing ddFPs to detect protein interaction during LLPS both in vitro and in living cells. The ddFPs allow us to investigate the interaction between two important LLPS-associated proteins, FUS and TDP-43, as cellular condensates formed. Importantly, we revealed that their interaction was associated with RNA binding upon LLPS, indicating that RNA plays a critical role in mediating interactions between RBPs. More broadly, we envision that utilization of ddFPs would reveal previously unknown protein-protein interaction and uncover their functional roles in the formation and disassembly of biomolecular condensates.

Structure Reveals the Impact of Surface Charge Distribution on the Phase Separation and Aggregation of Slr0280.

Liquid-liquid phase separation (LLPS) plays a key role in the regulation of life activities. Here, we reported a protein from Synechocystis sp. PCC 6803 and annotated as Slr0280. To obtain a water-soluble protein, we deleted the N-terminus transmembrane domain and named it Slr0280Δ. Slr0280Δ with high concentration can undergo LLPS at a low temperature in vitro. It belongs to the phosphodiester glycosidase family of proteins and has a segment of a low-complexity sequence region (LCR), which is thought to regulate the LLPS. Our results show that electrostatic interactions impact the LLPS of Slr0280Δ. We also acquired the structure of Slr0280Δ, which has many grooves on the surface with a large distribution of positive and negative charges. This may be advantageous for the LLPS of Slr0280Δ through electrostatic interactions. Furthermore, the conserved amino acid (arginine at position 531) located on the LCR is important for maintaining the stability of Slr0280Δ as well as LLPS. Our research indicated that the LLPS of proteins can be transformed into aggregation by changing the surface charge distribution.

Microglia depletion exacerbates retinal ganglion cell loss in a mouse model of glaucoma.

To test whether depletion of microglia in the optic nerve head has a beneficial effect on retinal ganglion cell numbers and function, we depleted microglia by oral administration of the CSF1R antagonist PLX5622. Then, ocular hypertension was induced by unilateral injection of magnetic microbeads into the anterior chamber. Visual function was assessed with pattern electroretinography and measurement of the optomotor reflex. Retinal ganglion cell bodies and axons were counted and gene expression patterns in optic nerve head astrocytes were tested on freshly dissociated astrocytes. PLX5622 efficiently depleted microglia in the retina and the optic nerve head, but about 20% of microglia persisted in the myelinated optic nerve proper even after prolonged exposure to the drug. PLX5622 did not affect ganglion cell function by itself. Elevation of the IOP for four weeks led to the expected decrease in visual acuity and pattern ERG amplitude. Microglia ablation did not affect these parameters. Ganglion cell and axon numbers were counted histologically post mortem. Mice in the microglia depletion group showed a moderate but significantly greater loss of ganglion cells than the control group. At four weeks post microbead injection, gene expression patterns in optic nerve head astrocytes are consistent with an A2 (or neuroprotective) pattern. Microglia depletion blunted the up-regulation of A2 genes in astrocytes. In conclusion, microglia depletion is unlikely to protect retinal ganglion cells in early glaucoma.

New Far-Red and Near-Infrared Fluorescent Phycobiliproteins with Excellent Brightness and Photostability.

Far-red and near-infrared fluorescent proteins can be used as fluorescence biomarkers in the region of maximal transmission of most tissues and facilitate multiplexing. Recently, we reported the generation and properties of far-red and near-infrared fluorescent phycobiliproteins, termed BeiDou Fluorescent Proteins (BDFPs), which can covalently bind the more readily accessible biliverdin. Far-red BDFPs maximally fluoresce at ∼670 nm, while near-infrared BDFPs fluoresce at ∼710 nm. In this work, we molecularly evolved BDFPs as follows: (a) mutations L58Q, S68R and M81K of BDFPs, which can maximally enhance the effective brightness in vivo by 350 %; (b) minimization and monomerization of far-red BDFPs 2.1, 2.2, 2.3, and near-infrared BDFPs 2.4, 2.5 and 2.6. These newly developed BDFPs are remarkably brighter than the formerly reported far-red and near-infrared fluorescent proteins. Their advantages are demonstrated by biolabeling in mammalian cells using super-resolution microscopy.

Engineering Bacteria to Monitor the Bleeding of Animals Using Far-Red Fluorescence.

Microorganisms living in animals can function as drug delivery systems or as detectors for some diseases. Here, we developed a biosensor constructed by the deletion of hemF and harboring ho1, chuA, and bdfp1.6 in Escherichia coli. HemF is an enzyme involved in heme synthesis in E. coli. ChuA and HO1 can transfer extracellular heme into cells and generate biliverdin (BV). BDFP1.6 can bind BV autocatalytically, and it emits a far-red fluorescence signal at 667 nm. Therefore, we named this biosensor as the far-red light for bleeding detector (FRLBD). Our results indicated that the FRLBD was highly efficient and specific for detecting heme or blood in vitro. Moreover, the FRLBD could be used to detect bleeding in the zebrafish induced by aspirin, and a convolutional neural network was an appropriate model to identify the fluorescence features in the images.

LncRNA AK148321 alleviates neuroinflammation in LPS-stimulated BV2 microglial cell through regulating microRNA-1199-5p/HSPA5 axis.

AIMS: Dysregulated long non-coding RNA (lncRNA) expression is closely related to neuroinflammation, leading to multiple neurodegenerative diseases. In this study, we investigated the function and regulation of lncRNA AK148321 in neuroinflammation using an in vitro lipopolysaccharide (LPS)-stimulated BV2 microglial cell system. METHODS: Expression of AK148321 was analyzed by qPCR. Inflammatory cytokine expression levels were determined by ELISA assay. The interaction between AK148321, microRNA (miRNA), and its target gene was validated by luciferase reporter assay and RNA immunoprecipitation (RIP). Cell apoptosis was analyzed by Annexin V/PI staining. RESULTS: LPS treatment suppressed AK148321 expression in BV2 cells. Overexpression of AK148321 inhibited LPS-induced BV2 microglial cell activation and decreased the expression of inflammatory cytokine TNF-α and IL-1ß. AK148321 function as a competing endogenous RNA (ceRNA) by sponging microRNA-1199-5p (MiR-1199-5p). In LPS-stimulated BV2 cells, AK148321 exerted its inhibitory function via negatively modulating miR-1199-5p expression. Moreover, we identified that Heat Shock Protein Family A Member 5 (HSPA5) was a direct target of miR-1199-5p. RIP assay using the anti-Ago2 antibody further validated the relationship among AK148321, miR-1199-5p and HSPA5. The AK148321/miR-1199-5p/HSPA5 axis regulated the neuroinflammation in LPS-induced BV2 microglial cells. Microglial cell culture supernatant from LPS-stimulated, AK148321-overexpressing BV2 cells suppressed the cell apoptosis of mouse hippocampal neuronal cell HT22, while HSPA5 knockdown abrogated the suppression effect. CONCLUSION: Our findings suggest that AK148321 alleviates neuroinflammation in LPS-stimulated BV2 microglial cells through miR-1199-5p/HSPA5 axis.

Design of small monomeric and highly bright near-infrared fluorescent proteins.

Due to the low absorbance in the far-red (FR) and near-infrared (NIR) "optical window", NIR fluorescent proteins (FPs) are powerful tools for deep imaging. Here, we report three new, highly bright NIR FPs termed BDFP1.8, BDFP1.8:1.8 (tandem BDFP1.8) and BDFP1.9, which evolved from a previously reported FR FP, BDFP1.6: a derivative of ApcF2 from Chroococcidiopsis thermalis sp. PCC7203. ApcF2 binds phycocyanobilin (PCB) non-covalently, while BDFPs, the derivatives of ApcF2, can bind biliverdin (BV) covalently. We identified that dimeric BDFP1.8 and monomeric BDFP1.8:1.8 have a 2.4-and 4.4-fold higher effective brightness, respectively, than iRFP720, which has the highest effective brightness among the reported NIR FPs. Monomeric DBFP1.9 (17 kDa) has one of the smallest masses among highly bright FPs in the FR and NIR regions. Enhancing the affinity between the apo-proteins and the BV chromophore is an effective method to improve the effective brightness of biliprotein FPs. Moreover, BDFP1.8 and 1.9 exhibit higher stability to temperature, pH and light than iRFP720. Finally, the highly bright NIR BDFP1.8 together with FR BDFP1.6 could effectively biolabel cells in dual colors.

Very Bright Phycoerythrobilin Chromophore for Fluorescence Biolabeling.

Biliproteins have extended the spectral range of fluorescent proteins into the far-red (FR) and near-infrared (NIR) regions. These FR and NIR fluorescent proteins are suitable for the bioimaging of mammalian tissues and are indispensable for multiplex labeling. Their application, however, presents considerable challenges in increasing their brightness, while maintaining emission in FR regions and oligomerization of monomers. Two fluorescent biliprotein triads, termed BDFP1.2/1.6:3.3:1.2/1.6, are reported. In mammalian cells, these triads not only have extremely high brightness in the FR region, but also have monomeric oligomerization. The BDFP1.2 and BDFP1.6 domains covalently bind to biliverdin, which is accessible in most cells. The BDFP3.3 domain noncovalently binds phycoerythrobilin that is added externally. A new method of replacing phycoerythrobilin with proteolytically digested BDFP3.3 facilitates this labeling. BDFP3.3 has a very high fluorescence quantum yield of 66 %, with maximal absorbance at λ=608 nm and fluorescence at λ=619 nm. In BDFP1.2/1.6:3.3:1.2/1.6, the excitation energy that is absorbed in the red region by phycoerythrobilin in the BDFP3.3 domain is transferred to biliverdin in the two BDFP1.2 or BDFP1.6 domains and fluoresces at λ≈670 nm. The combination of BDFP3.3 and BDFP1.2/1.6:3.3:1.2/1.6 can realize dual-color labeling. Labeling various proteins by fusion to these new fluorescent biliproteins is demonstrated in prokaryotic and mammalian cells.

A Large Stokes Shift Fluorescent Protein Constructed from the Fusion of Red Fluorescent mCherry and Far-Red Fluorescent BDFP1.6.

Phycobiliproteins are constituents of phycobilisomes that can harvest orange, red, and far-red light for photosynthesis in cyanobacteria and red algae. Phycobiliproteins in the phycobilisome cores, such as allophycocyanins, absorb far-red light to funnel energy to the reaction centers. Therefore, allophycocyanin subunits have been engineered as far-red fluorescent proteins, such as BDFP1.6. However, most current fluorescent probes have small Stokes shifts, which limit their applications in multicolor bioimaging. mCherry is an excellent fluorescent protein that has maximal emittance in the red spectral range and a high fluorescence quantum yield, and thus, can be used as a donor for energy transfer to a far-red acceptor, such as BDFP1.6, by FRET. In this study, mCherry was fused with BDFP1.6, which resulted in a highly bright far-red fluorescent protein, BDFP2.0, with a large Stokes shift (≈79 nm). The excitation energy was absorbed maximally at 587 nm by mCherry and transferred to BDFP1.6 efficiently; thus emitting strong far-red fluorescence maximally at 666 nm. The effective brightness of BDFP2.0 in mammalian cells was 4.2-fold higher than that of iRFP670, which has been reported as the brightest far-red fluorescent protein. The large Stokes shift of BDFP2.0 facilitates multicolor bioimaging. Therefore, BDFP2.0 not only biolabels mammalian cells, including human cells, but also biolabels various intracellular components in dual-color imaging.

Bright near-infrared fluorescence bio-labeling with a biliprotein triad.

Biliproteins have extended the spectral range of fluorescent proteins into the near-infrared region (NIR, 700-770 nm) of maximal transmission of most tissues and are also favorable for multiplex labeling. Their application, however, presents considerable challenges to increase their stability under physiological conditions and, in particular, to increase their brightness while maintaining the emission in near-infrared regions: their fluorescence yield generally decreases with increasing wavelengths, and their effective brightness depends strongly on the environmental conditions. We report a fluorescent biliprotein triad, termed BDFP1.1:3.1:1.1, that combines a large red-shift (722 nm) with high brightness in mammalian cells and high stability under changing environmental conditions. It is fused from derivatives of the phycobilisome core subunits, ApcE2 and ApcF2. These two subunits are induced by far-red light (FR, 650-700 nm) in FR acclimated cyanobacteria. Two BDFP1.1 domains engineered from ApcF2 covalently bind biliverdin that is accessible in most cells. The soluble BDFP3 domain, engineered from ApcE2, binds phytochromobilin non-covalently, generating BDFP3.1. This phytochromobilin chromophore was added externally; it is readily generated by an improved synthesis in E. coli and subsequent extraction. Excitation energy absorbed in the FR by covalently bound biliverdins in the two BDFP1.1 domains is transferred via fluorescence resonance energy transfer to the non-covalently bound phytochromobilin in the BDFP3.1 domain fluorescing in the NIR around 720 nm. Labeling of a variety of proteins by fusion to the biliprotein triad is demonstrated in prokaryotic and mammalian cells, including human cell lines.

Far-red acclimating cyanobacterium as versatile source for bright fluorescent biomarkers.

Far-red and near-infrared emitting chromophores extend applications of fluorescent proteins to regions of maximal transmission of most tissues, but present considerable engineering challenges. Far-red adapting cyanobacteria generate a novel set of biliproteins. One of them, ApcF2, from a thermophilic cyanobacterium was subjected to structure-guided, site-directed random and specific mutagenesis, and was screened for bright far-red emission. We report the generation of chromoproteins, termed BDFPs, that are small, bind auto-catalytically the ubiquitous biliverdin as chromophore, express well, and retain their fluorescence in mammalian cells and in the nematode, C. elegans. They are, moreover, photostable and tolerate high temperature, low pH and chemical denaturation. Homo-bichromophoric tandems of these proteins improve labeling, while hetero-bichromophoric systems with large Stokes shifts are suitable for applications like FRET, multi-channel or super-resolution microscopy. The BDFPs compare favorably to other biliproteins and provide a novel, extremely versatile labeling tool-box.

Novel NADPH oxidase inhibitor VAS2870 suppresses TGF­ß­dependent epithelial­to­mesenchymal transition in retinal pigment epithelial cells.

NADPH oxidases (NOXs) are important in the pathophysiology of fibrotic diseases. The expression and activity of NOXs are regulated by growth factors, including transforming growth factor (TGF­ß). The proliferation of retinal pigment epithelial (RPE) cells following epithelial­ to­mesenchymal transition (EMT) is a major pathological change involved in proliferative vitreoretinopathy (PVR). The aim of the present study was to determine the effects of the novel NOX inhibitor VAS2870 on the TGF­ß­dependent expression of NOX4 and associated cellular events in RPE cells. Cell viability was examined using a Cell Counting Kit­8 assay and cell cycle progression was detected by flow cytometric analysis. Immunofluorescence analysis and western blot analysis were performed to assess EMT. It was found that TGF­ß increased the expression of NOX4 and that pre­incubation with VAS2870 eliminated this effect. Additionally, TGF­ß promoted RPE migration and increased EMT. Pre­incubation with VAS2870 significantly prevented TGF­ß2­induced EMT by decreasing the levels of α­smooth muscle actin and E­cadherin, and also inhibited the migratory ability of the RPE cells, as demonstrated by scratch assays. Finally, VAS2870 suppressed the proliferation of RPE cells, and led to G1­phase cell cycle arrest and a significant downregulation of the expression of cyclin D1. In conclusion, the pharmacological inhibition of NOX may be a promising tool for the treatment of PVR.