Green Fluorescent Protein Research Articles

The effect of enhancers on the lentiviral transduction efficiency in the human RPE cells: Insights for advancing retinal gene therapies.

The effect of enhancers on the lentiviral transduction efficiency in the human RPE cells: Insights for advancing retinal gene therapies.

A high affinity Sybody blocks Cofilin-1 binding to F-actin in vitro and in cancer cells.

A high affinity Sybody blocks Cofilin-1 binding to F-actin in vitro and in cancer cells.

Photobiomodulation therapy facilitates transplantation of dental pulp stem cells for spinal cord injury.

Photobiomodulation therapy facilitates transplantation of dental pulp stem cells for spinal cord injury.

Cellulophaga algicola alginate lyase and Pseudomonas aeruginosa Psl glycoside hydrolase inhibit biofilm formation by Pseudomonas aeruginosa CF2843 on three-dimensional aggregates of lung epithelial cells.

Cellulophaga algicola alginate lyase and Pseudomonas aeruginosa Psl glycoside hydrolase inhibit biofilm formation by Pseudomonas aeruginosa CF2843 on three-dimensional aggregates of lung epithelial cells.

Celebrating 50years of fluorescence correlation spectroscopy (FCS): Advancing live-cell massively parallel FCS studies with photostable GFPs, mStayGold and StayGold/E138D.

Celebrating 50years of fluorescence correlation spectroscopy (FCS): Advancing live-cell massively parallel FCS studies with photostable GFPs, mStayGold and StayGold/E138D.

The promoter of Zymomonas mobilis respiratory NADH dehydrogenase (ndh) is induced by oxygen.

The promoter of Zymomonas mobilis respiratory NADH dehydrogenase (ndh) is induced by oxygen.

Hair-follicle-associated pluripotent (HAP) stem-cell-sheet implantation accelerates cutaneous wound closure and suppresses scar formation in a mouse model

ABSTRACT Patients frequently experience physical, mental, and even financial distress because of acute or chronic skin wounds. In severe situations, scarring on the skin can be quite noticeable, cause persistent discomfort, restrict joint motion, or be mentally taxing. Hair-follicle-associated pluripotent (HAP) stem cells were discovered by our laboratory, in the bulge area of the hair follicle and can differentiate to neurons, glia, beating cardiomyocytes, keratinocytes and nascent vessels. In the present study, HAP stem cell sheets were formed by culturing the upper part of hair follicles and implanting into mice with skin ulcers. The HAP stem cell sheets contained keratinocytes, endothelial cells and neurons. Autologous HAP stem cell sheet implantation to the dorsal wound in C57BL/6J mice significantly accelerated wound closure compared with non-implanted control mice. HAP-stem-cell sheets expressing green fluorescent protein (GFP) implanted into nude mice differentiated into keratinocytes in the epidermis, and neurons and endothelial cells in the dermis. The thicknesses of the epidermis and dermis and M2 macrophage and myofibroblast infiltration into the wound were significantly decreased in HAP-stem cell-implanted mice compared with non-implanted control mice. Expression levels of TGF-β1, COL1A2 and COL3A1 mRNA in the wound were significantly decreased in HAP stem cell-implanted mice compared with non-implanted control mice. These results suggest that implanting HAP stem cell sheets accelerates cutaneous wound closure and suppresses scar formation. The HAP stem cells used in the present study thus have potential as a future clinical strategy for accelerating wound healing.

Light-Controllable PEG Hydrogel Cross-Linked by Reversibly Photodissociable Dimeric Green Fluorescent Protein pdDronpa for Drug Delivery.

Hydrogel has been widely studied as a carrier model system for drug delivery. Efficient encapsulation of drug molecules and their controlled release are important factors in the design of hydrogels to control drug release at a desired time and location. In this study, we propose a photoresponsive hydrogel with tunable mechanical properties for drug delivery. The hydrogel was synthesized by cross-linking maleimide-functionalized 4-armed polyethylene glycol (4-armed PEG-Mal) with reversibly photodissociable green fluorescent protein pdDronpa. Transitions in the physical state and/or mechanical strength of the hydrogel occurred rapidly when the cross-linking agent pdDronpa was switched off and on between the monomer and dimer states using 500 and 400 nm illumination, respectively. Optically controlled release of fluorescently labeled insulin was investigated, demonstrating the ability of this hydrogel as a potent drug delivery system.

Stromal Gene Therapy Mediates Prolonged Protection Against Corneal Neovascularization Induced by an Aggressive Angiogenic Insult.

Corneal neovascularization (CoNV) is both a sight-threatening condition in and of itself and a major risk factor associated with corneal graft failure. Here, we determine the effectiveness of an adeno-associated viral vector (AAV)-based gene therapy targeting both hematic and lymphatic neovascularization in a murine model of severe CoNV. We first assessed the profile of transgene expression mediated by intrastromal injection of AAV2/8[Y733F] via longitudinal visualization of an enhanced Green Fluorescent Protein (eGFP) transgene and found that this serotype mediates a temporary (∼18 day) transduction of the corneal epithelium and sustained (≥148 day) transduction within the stroma. Constitutively expressed sFlt1 or sFlt4 were prophylactically delivered via intrastromal injection of AAV2/8[Y733F] vector at various intervals prior to aggressive induction of CoNV in a murine model. The extent of CoNV induced was quantified by fluorescein angiography and immunohistochemistry 17 days after induction. AAV2/8[Y733F]-CMV-sFlt1 was highly effective in the prevention of hemangiogenesis (HA) induced at 3, 28, and 210 days after intrastromal injection, but ineffective in the prevention of lymphangiogenesis. Two variants of AAV2/8[Y733F]-CMV-sFlt4 were ineffective in the prevention of angiogenesis when delivered alone, but combined delivery of AAV2/8[Y733F]-CMV-sFlt1 and AAV2/8[Y733F]-CMV-sFlt4 suggested a synergistic effect. Our results show that a single intrastromal injection of AAV2/8[Y733F]-CMV-sFlt1 is sufficient to protect against a robust stimulus for corneal HA over the long term. This technique could also be applied ex vivo to reduce the risk of failure in cases of "high-risk" corneal transplantation.

Gene Delivery with Dual pH- and GSH-Responsive Lipoamide Polymers from Controlled Polymerization of Protected Lipoic Acid.

Gene delivery has great therapeutic potential, but there remain significant barriers for the effective use of this technology, including efficient transportation of the genetic payloads to the region of the cell where they are therapeutically active. Nanoparticle delivery systems have emerged as an important tool to improve the targeted delivery of nucleic acid cargo inside cells. Stimuli-responsive systems have generated particular interest for this application due to their capability to tailor the packing and effective release of nucleic acid cargo. Herein, we report dual pH- and glutathione (GSH)-responsive poly(lipoamide) (PLpAm) nanoparticles for the delivery of plasmid DNA (pDNA). PLpAm polymers were synthesized via a controlled polymerization method, followed by postpolymerization modification with pH-responsive charge-shifting pendent groups to optimize the endosomal escape capability. Our polymers efficiently complexed pDNA that encodes for an enhanced green fluorescent protein (eGFP), and the corresponding nanoparticles were shown to exhibit a dual response to pH and redox potential. A range of nanoparticles were synthesized with different N/P ratios. All nanoparticles demonstrated disassembly under acidic conditions, followed by efficient decomplexation to release pDNA upon spontaneous depolymerization of the disulfide backbone at the cytosolic GSH level. The results showed strong transfection efficiency, with the particles at the highest N/P ratio demonstrating an average transfection efficiency of 74 vs 34% for the Lipofectamine 3000 control at the same pDNA concentration. This nanoparticle library provides a promising platform for further optimization as nucleic acid delivery systems.

Tumor Immunology Visualized in Realtime in In-vivo-like 3-D Gelfoam® Histoculture by Color-coded Imaging.

Tumor immunology and immunotherapy have been intensely researched recently, especially with the development of immune checkpoint inhibitors (ICIs). However, only a small percentage of patients respond to ICIs, and this response is limited to a minority of cancer types, such as colon cancer, lung cancer, and melanoma. The aim of the present study was to develop an imageable in-vivo-like in vitro system to directly visualize tumor immunology and immunotherapy in real-time, to further understand tumor immunology and develop improved tumor immunotherapy. Lewis lung carcinoma cells labeled with red fluorescent protein (LLC-RFP) and peripheral blood mononuclear cells labeled with green fluorescent protein (PBMCs-GFP), derived from transgenic GFP mice, were cultured in two dimensions (2-D) on plastic or in three dimensions (3-D) in Gelfoam® histoculture, using RPMI-1640 culture medium with 10% fetal bovine serum, and 1% penicillin/streptomycin, and with concanavalin-A. LLC-RFP cells seeded on Gelfoam® formed glandular-like structures after 2 days of histoculture. PBMCs-GFP were seeded on Gelfoam® with LLC-RFP after one day to establish a co-culture. The PBMCs-GFP co-localized with many of the structures formed by the LLC-RFP cells after one day of seeding PBMCs-GFP, suggesting a very specific interaction necessary for an immunological interaction. In contrast, only a few randomized co-localizations of PBMCs-GFP and the LLC-RFP cells were seen on plastic in 2-D culture. The present results demonstrate a new 3-D co-culture color-coded system to visualize in real-time the interaction of PBMCs and cancer cells using fluorescence color-coded imaging. Future experiments will test drugs that can stimulate immune reactions between the PBMCs and cancer cells.

7,8-dihydroxyflavone and daphnetin have virucidal and neuraminidase inhibition activities against influenza A virus in vitro

Influenza A virus remains a major global health concern, underscoring the need for novel antiviral agents. This study investigated the antiviral potential of two natural compounds, 7,8-dihydroxyflavone (DHF) and daphnetin (DAP), against influenza A viruses in vitro. Following in vitro cytotoxicity and antioxidative activity assessments, the antiviral effects of DHF and DAP were evaluated, with a particular focus on their direct viral inhibition. DHF and DAP demonstrated complete virucidal activity influenza A virus at concentrations of 50 µM and 100 µM, respectively. However, neither compound inhibited influenza surface protein hemagglutination (HA), suggesting that their virucidal effects are independent of HA receptor binding. Both compounds exhibited neuraminidase (NA) inhibition, with DAP showing stronger activity compared to DHF. Furthermore, DHF and DAP suppressed influenza virus replication in cells, as evidenced by a reduction in green fluorescence protein (GFP) reporter expression in virus-infected cells. Growth kinetics analysis revealed that both compounds significantly reduced viral replication when applied to cells before or after viral infection. These findings demonstrate that DHF and DAP exhibit multifaceted antiviral activity, including direct virucidal action, NA inhibition, and suppression of viral replication. Our results suggest that DHF and DAP are promising candidates for the development of novel influenza therapeutics.

PEG-Mediated Protoplast Transformation of Penicillium sclerotiorum (scaumcx01): Metabolomic Shifts and Root Colonization Dynamics.

Protoplast-based transformation is a vital tool for genetic studies in fungi, yet no protoplast method existed for P. sclerotiorum-scaumcx01 before this study. Here, we optimized protoplast isolation, regeneration, and transformation efficiency. The highest protoplast yield (6.72 × 106 cells/mL) was obtained from liquid mycelium after 12 h of enzymatic digestion at 28 °C using Lysing Enzymes, Yatalase, cellulase, and pectinase. Among osmotic stabilizers, 1 M MgSO4 yielded the most viable protoplasts. Regeneration occurred via direct mycelial outgrowth and new protoplast formation, with a 1.02% regeneration rate. PEG-mediated transformation with a hygromycin resistance gene and GFP tagging resulted in stable GFP expression in fungal spores and mycelium over five generations. LC/MS-based metabolomic analysis revealed significant changes in glycerophospholipid metabolism, indicating lipid-related dynamics influenced by GFP tagging. Microscopy confirmed successful colonization of tomato roots by GFP-tagged scaumcx01, with GFP fluorescence observed in cortical tissues. Enzymatic (cellulase) seed pretreatment enhanced fungal colonization by modifying root surface properties, promoting plant-fungal interaction. This study establishes an efficient protoplast transformation system, reveals the metabolic impacts of genetic modifications, and demonstrates the potential of enzymatic seed treatment for enhancing plant-fungal interactions.

Sensitive and Broadly Compatible Transcription Factor-Based Biosensor for Monitoring c-di-GMP Dynamics in Biofilms.

Biofilms are ubiquitous and have many negative effects, for example, in infections or biocorrosion. Given the critical role of the second messenger cyclic di-GMP (c-di-GMP) in biofilm formation, targeting a reduction in intracellular concentrations of c-di-GMP is believed to be a key aspect in the development of biofilm mitigation strategies. To facilitate this effort, here, we developed a transcription factor (TF)-based biosensor that integrates the TF FleQ from Pseudomonas aeruginosa with a PR-Ppel tandem promoter. The dynamic range of the biosensor was optimized by fine-tuning the TF expression. The biosensor exhibited broad compatibility and effectiveness in detecting decreases in c-di-GMP levels across various biofilm model organisms, including strains lacking FleQ or its homologues, such as Escherichia coli, Shewanella oneidensis, Comamonas testosteroni, and Acinetobacter baumannii, as well as P. aeruginosa containing FleQ. Additionally, we monitored c-di-GMP levels in biofilms formed by P. aeruginosa and S. oneidensis through a ratiometric, image-based quantification method. The methodology used the green fluorescence protein (GFP) as a reporter for c-di-GMP levels and 4',6-diamidino-2-phenylindole (DAPI) or the monomeric red fluorescence protein (mRFP) as the indicator for biofilm biomass. The GFP/DAPI or GFP/mRFP ratio gives effective c-di-GMP per unit of biomass. This TF-based biosensor provides an important tool to study c-di-GMP dynamics, which facilitates efforts in developing biofilm control strategies and understanding regulatory networks for biofilm development.

Quantitative comparison of fluorescent proteins using protein nanocages in live cells.

To standardize comparison of fluorescent protein performance on a molecule-by-molecule basis in a physiological intracellular environment, we constructed fluorescent protein-tagged I3-01 peptides that self-assemble into stable 60-subunit dodecahedrons inside live mammalian cells. We were especially interested in determining which of the recently published monomeric StayGold variants is best for live microscopy in mammalian cells. Combining nanocage brightness and photobleaching measurements into a single metric, mStayGold stood out as far superior to all other green and red fluorescent proteins we tested with a functional lifetime that is at least 8-10-fold longer compared with EGFP or mEmerald. Analysis of intracellular nanocage diffusion further confirmed the monomeric nature of mStayGold, and we demonstrate that mStayGold-tagged nanocages can serve as highly photostable nanoparticles to analyze intracellular biophysical properties. Analysis of frequently used red fluorescent proteins was less encouraging and recent mScarlet or mRuby variants did not perform substantially better than mCherry on a typical spinning disc confocal microscope system, highlighting the importance of a standardized method to benchmark fluorescent proteins to make optimal choices for specific experimental setups.

Quantitative Assessment of Hormogonia Induction in Nostoc punctiforme by a Fluorescent Reporter Strain.

While symbiotic plant-cyanobacteria interactions hold significant potential for revolutionizing agricultural practices by reducing the application of artificial nitrogen fertilizers, the genetic underpinnings of the symbiotic interaction between the plant host and the cyanobiont remain poorly understood. In particular, the molecular mechanisms through which host plants induce the formation of motile cyanobacterial filaments (hormogonia), essential for colonization and initiation of symbiosis, are not well characterized. In this study, we present a novel yet objective method for quantifying hormogonia induction, addressing limitations of traditional qualitative approaches. We have developed a reporter strain of Nostoc punctiforme PCC 73102 capable of quantifying hormogonia induction in response to diverse biotic and abiotic stimuli. This reporter strain, generated via triparental mating conjugation transformation, contains the promoter sequence of prepilin pilA fused to a green fluorescent protein (GFP) and enables quantitative and high throughput monitoring of hormogonia induction using a microplate reader. Our innovative approach, using a cyanobacterial hormogonia reporter strain, allows high-throughput screening of the hormogonia-inducing effect of a wide array of environmental and plant signals. This method is expected to greatly advance our understanding of the genetic determinants underpinning plant-cyanobacteria symbioses.

Optimization of a High-Throughput Screen for Monitoring Disease-Associated Protein Misfolding and Aggregation in Bacteria.

Protein misfolding and aggregation are central features of a wide range of diseases, including neurodegenerative disorders, systemic amyloidoses, and cancer. The identification of compounds that can modulate protein folding and aggregation is a key step toward developing effective therapies. High-throughput screening methods are essential for efficiently identifying such compounds. In this study, we optimized a previously developed high-throughput genetic screen for monitoring protein misfolding and aggregation in bacteria. This system is based on monitoring the fluorescence of Escherichia coli cells expressing fusions of human misfolding-prone and disease-related proteins (MisPs) with the green fluorescent protein. We systematically tested a variety of experimental conditions, such as overexpression conditions and MisP-GFP fusion formats, to identify key parameters that affect the sensitivity and dynamic range of the assay. Using misfolding-prone, cancer-associated variants of human p53 as a model system, we found that strong overexpression conditions, such as high copy number vectors, strong promoters, high inducer concentrations, and high overexpression temperatures, can yield optimal assay performance. These optimized assay conditions were also validated with additional MisPs, such as the Alzheimer's disease-associated amyloid-β peptide and variants of superoxide dismutase 1 associated with amyotrophic lateral sclerosis. At the same time, we observed that certain conditions, such as inducer concentrations and overexpression temperature, may need to be precisely fine-tuned for each new MisP target to yield optimal assay performance. Our findings provide a framework for standardizing MisP-GFP screening assays, facilitating their broad application in the discovery of therapeutic agents targeting protein misfolding and aggregation.

MoSec13 combined with MoGcn5b modulates MoAtg8 acetylation and regulates autophagy in Magnaporthe oryzae

ABSTRACT Macroautophagy/autophagy is an evolutionarily conserved cellular degradation process that is crucial for cellular homeostasis in Magnaporthe oryzae. However, the precise regulatory mechanisms governing autophagy in this organism remain unclear. In this study, we found a multiregional localization of MoSec13 to the vesicle membrane, endoplasmic reticulum, nucleus, and perinucleus. MoSec13 negatively regulated autophagy through specific amino acid residues in its own WD40 structural domain by interacting with MoAtg7 and MoAtg8. We also found that the histone acetyltransferase MoGcn5b mediated the acetylation of MoAtg8 and regulated autophagy activity. Subsequently, we further determined that MoSec13 regulated the acetylation status of MoAtg8 by controlling the interaction between MoGcn5b and MoAtg8 in the nucleus. In addition, MoSec13 maintained lipid homeostasis by controlling TORC2 activity. This multilayered integration establishes MoSec13 as an essential node within the autophagic regulatory network. Our findings fill a critical gap in understanding the role of Sec13 in autophagy of filamentous fungi and provide a molecular foundation for developing new therapeutic strategies against rice blast fungus. ABBREVIATIONS BFA: brefeldin A; BiFC: bimolecular fluorescence complementation; CM: complete medium; CMAC: 7-amino-4-chloromethylcoumarin; Co-IP: co-immunoprecipitation; COPII: coat complex II; GFP: green fluorescent protein; HPH: hygromycin phosphotransferase; MM-N: nitrogen-starvation conditions; NPC: nuclear pore complex; PAS: phagophore assembly site; PE: phosphatidylethanolamine; UPR: unfolded protein response.

Epigenetic regulation of histone methyltransferase SUV39H1 on the expression of recombinant protein in CHO cells

Histone methylation–mediated epigenetic modification significantly influences gene transcription and expression regulation. This study examined the effects of histone 3 lysine 9 trimethylation (H3K9me3) methyltransferase SUV39H1 and its specific inhibitor chaetocin on recombinant protein expression in Chinese hamster ovary (CHO) cells. Results indicated that stable SUV39H1-knockdown CHO cells exhibited reduced H3K9me3 levels while showing increased expression of recombinant adalimumab (rADM) and human serum albumin (rHSA) by approximately 45% and 136%, respectively. Furthermore, treatment with 20 nM chaetocin, a SUV39H1-specific inhibitor, enhanced expression of enhanced green fluorescent protein (EGFP), rADM, and rHSA in CHO cells. These findings demonstrate that both stable SUV39H1 knockdown and pharmacological inhibition through chaetocin effectively reduce H3K9me3 modification levels in CHO cells while significantly boosting recombinant protein production. The results strongly suggest SUV39H1’s critical regulatory role in recombinant protein expression within CHO cell systems. This research establishes a methodological foundation for developing engineered cell lines and optimizing high-efficiency CHO expression systems through cell engineering approaches.Key points•SUV39H1 knockdown boosted recombinant protein expression and decreased H3 K9 me3 levels.•Treatment with the SUV39H1-specific inhibitor chaetocin (20 nM) enhanced recombinant protein expression.•It provides a basis for developing efficient epigenetically regulated CHO expression systems.

Cellular senescence promotes macrophage-to-myofibroblast transition in chronic ischemic renal disease

Cellular senescence participates in the pathophysiology of post-stenotic kidney damage, but how it regulates tissue remodeling is incompletely understood. Macrophage-myofibroblast transition (MMT) contributes to the development of tissue fibrosis. We hypothesized that cellular senescence contributes to MMT and renal fibrosis in mice with renal artery stenosis (RAS). INK-ATTAC mice expressing p16INK-4a and green fluorescent protein in senescent cells were assigned to control or unilateral RAS, untreated or treated with AP20187 (an apoptosis inducer in p16INK-4a-expressing cells) for 4 weeks. Renal perfusion was studied in vivo using micro-MRI, and kidney morphology, senescence, and MMT ex vivo. Cellular senescence was induced in human renal proximal tubular epithelial cells (HRPTEpiC) in vitro, and interferon-induced transmembrane protein-3 (IFITM3), a cellular senescence vector, was silenced (siRNA) or over-expressed (plasmid). HRPTEpiC were then co-incubated with macrophages with silenced integrin-3 (ITGB3), a regulator of mesenchymal transitions. CD68/p16INK-4a/α-SMA co-expression and senescence markers were studied. Murine RAS kidneys showed increased expression of p16INK-4a and MMT markers (F4/80, α-SMA) vs. controls, which decreased after AP20187, as did renal fibrosis and plasma creatinine, whereas renal perfusion increased. IFITM3 and ITGB3 expression were upregulated in senescent HRPTEpiC or co-cultured macrophages, respectively. MMT markers and TGF-β/Smad3 expression also rose in these macrophages and decreased after IFITM3 or ITGB3 silencing. p16INK-4a-expressing macrophages may regulate interstitial fibrosis in RAS via MMT. This process is associated with elevated expression of ITGB3 and TGF-β/Smad3 pathway activation through neighboring senescent cell-derived IFITM3. These findings may implicate MMT as a therapeutic target in ischemic kidneys.