Green Fluorescent Protein Research Articles

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.

Protein Synthesis via in vitro Transcription and Translation System inside Monodisperse Vesicles Fabricated by Microfluidics

ABSTRACTIn this study, we investigated the conditions for protein synthesis by an in vitro transcription and translation (IVTT) system within giant unilamellar vesicles (GUVs) produced with a microfluidic channel. The commercial IVTT system consisted of purified components (PURE system), and DNA encoding target protein was encapsulated in GUVs and incubated to synthesize the proteins. Synthesis of green fluorescent protein (GFP) and nanopore‐forming α‐hemolysin were tested as the models of water‐soluble protein and membrane protein, respectively. The stability of the GUVs and the efficiency of protein synthesis were assessed, focusing on variations in the concentration of the PURE system and the size of the GUVs. Our findings contribute to the development of homogeneous bioreactors and biosensors based on GUV technology.

Transcriptomic insights into methanol utilization in Pichia pastoris lacking AOX genes under co-feeding conditions.

The methylotrophic yeast Pichia pastoris (P. pastoris) exhibits remarkable capability for methanol-driven protein biosynthesis, positioning it as an attractive platform for carbon-neutral biomanufacturing utilizing methanol as a renewable feedstock. However, challenges arising from methanol metabolism, particularly the accumulation of toxic formaldehyde intermediates, significantly hinder efficient methanol biotransformation. To address this limitation, we implemented a metabolic engineering strategy involving dual knockout of alcohol oxidase genes (aox1 and aox2) combined with glycerol co-substrate supplementation. Using enhanced green fluorescent protein (EGFP) as a model heterologous product, we demonstrated that the ΔAOX1/2 strain achieved superior protein productivity in glycerol-methanol co-feeding cultures. Under optimized conditions (0.5% methanol + 0.4% glycerol), the engineered strain attained a biomass density of 38.5 (OD600) and EGFP fluorescence intensity of 494,723 units, representing improvements of 32.8% and 53.6%, respectively, compared to the wild-type (WT) strain cultivated with 1% methanol alone. Transcriptome profiling revealed that the observed enhancement in protein synthesis originated from optimized methanol utilization through coordinated upregulation of both assimilatory and dissimilatory metabolic modules. This study demonstrates that alcohol oxidase suppression coupled with glycerol co-metabolism constitutes an effective strategy to alleviate methanol-derived metabolic stress while enhancing heterologous protein yields in P. pastoris.

Therapeutic effect of hUC-MSCs from different transplantation routes on acute liver failure in rats

ObjectiveAcute liver failure (ALF) is a rare yet serious clinical syndrome. Recent studies have indicated that stem cells can effectively treat this condition. However, the optimal route for stem cell transplantation in the treatment of ALF remains unclear. This study aims to investigate the most effective transplantation route for stem cell therapy in ALF.MethodsHuman umbilical cord mesenchymal stem cells (hUC-MSCs) expressing both luciferase and green fluorescent protein were generated using a lentiviral vector. The hUC-MSCs were transplanted via the tail vein, portal vein, and abdominal cavity. The survival and distribution of the transplanted hUC-MSCs in rats were assessed through in vivo imaging and immunofluorescence. Furthermore, the therapeutic effects of hUC-MSCs transplanted via different routes on ALF were compared.ResultsThe survival time of hUC-MSCs transplanted via the tail vein and portal vein was shorter compared to those transplanted intraperitoneally. The distribution of hUC-MSCs varied by transplantation route: those injected via the tail vein and portal vein were primarily found in the lungs and liver, respectively, while intraperitoneally transplanted hUC-MSCs predominantly localized in the abdominal cavity. In ALF rats, hUC-MSCs transplanted via the tail vein and portal vein improved survival rates, enhanced liver pathology, and reduced levels of inflammatory cytokines in liver tissue. In contrast, abdominal transplantation of hUC-MSCs showed no significant therapeutic effect.ConclusionhUC-MSCs transplanted via the tail vein and portal vein exhibited similar therapeutic effects on ALF; however, abdominal transplantation of hUC-MSCs showed no significant effect.

Advancing Yarrowia lipolytica sub-organelle engineering with endogenous mitochondrial targeting sequence.

The aim of the study was identification and validation of an endogenous mitochondrial targeting signal (MTS) sequence of Yarrowia lipolytica, for efficient compartmentalization of a target protein to mitochondria. MTS from citrate synthase of Y. lipolytica (YlCISY-MTS) was identified, isolated and fused with green fluorescent protein (GFP) to direct it to the mitochondrial matrix. The efficiency of localization of GFP to mitochondrial matrix with YlCISY-MTS was compared with currently used MTS from Saccharomyces cerevisiae's cytochrome oxidase subunit IV. Confocal microscopy confirmed the targeted and greater GFP localization, underlining the potential of endogenous YlCISY-MTS for mitochondrial engineering in Y. lipolytica. The availability of endogenous MTS will evade the need of codon optimization of S. cerevisiae MTS for mitochondrial engineering in Y. lipolytica. This is the first report of an endogenous MTS of Y. lipolytica. An endogenous MTS of Y. lipolytica has been identified to facilitate the targeted delivery of a protein in the mitochondria enabling future advancements through leveraging the unique subcellular environment for metabolic engineering applications.

Transduction of Lentiviral Vectors and ADORA3 in HEK293T Cells Modulated in Gene Expression and Alternative Splicing

For steady transgenic expression, lentiviral vector-mediated gene delivery is a commonly used technique. One question that needs to be explored is how external lentiviral vectors and overexpressed genes perturb cellular homeostasis, potentially altering transcriptional networks. In this study, two Human Embryonic Kidney 293T (HEK293T)-derived cell lines were established via lentiviral transduction, one overexpressing green fluorescent protein (GFP) and the other co-overexpressing GFP and ADORA3 following puromycin selection to ensure stable genomic integration. Genes with differentially transcript utilization (gDTUs) and differentially expressed genes (DEGs) across cell lines were identified after short-read and long-read RNA-seq. Only 31 genes were discovered to have changed in expression when GFP was expressed, although hundreds of genes showed variations in transcript use. In contrast, even when co-overexpression of GFP and ADORA3 alters the expression of more than 1000 genes, there are still less than 1000 gDTUs. Moreover, DEGs linked to ADORA3 overexpression play a major role in RNA splicing, whereas gDTUs are highly linked to a number of malignancies and the molecular mechanisms that underlie them. For the analysis of gene expression data from stable cell lines derived from HEK293T, our findings provide important insights into changes in gene expression and alternative splicing.

Lipid Nanoparticles-Mediated mRNA Delivery to the Eye Affected by Ionizable Cationic Lipid.

Ionizable lipid serves as the key functional component in lipid nanoparticles (LNPs) for efficient mRNA delivery. This study aims to systematically evaluate clinically approved ionizable lipid DLin-MC3-DMA and SM102-based LNPs for ocular mRNA delivery, with a comprehensive assessment of their physicochemical characteristics, delivery efficiency, and biodistribution patterns within the ocular microenvironment. Enhanced green fluorescence protein or Luc encoding mRNA-loaded LNPs were formulated using microfluidic mixing technology and characterized by dynamic light scattering, ζ-potential measurements, and cryogenic transmission electron microscopy imaging. The two LNP systems with different ionizable cationic lipids demonstrated distinct capabilities for in vitro mRNA transfection and intraocular mRNA delivery following intravitreal administration. Notably, the SM102-LNPs exhibited superior performance compared to the MC3-LNPs, characterized by significantly higher transfection efficiency in retinal cells in vitro, and more efficient ocular expression with minimal systemic distribution in vivo. Safety assessment demonstrated that intravitreal administration of SM102-LNPs maintained excellent long-term biocompatibility throughout a five-month study period. The superior performance of SM102-LNPs establishes a promising platform for ocular mRNA therapeutics.

Induced Neural Progenitor Specification from Human Pluripotent Stem Cells by a Refined Synthetic Notch Platform.

Historically, studying the development of brain and central nervous system (CNS) tissues has been challenging. Human pluripotent stem cell (hPSC) technology has allowed for the in vitro reconstitution of relevant, early cell trajectories by using small molecules and recombinant proteins to guide differentiation of cells toward relevant brain and CNS phenotypes. However, many of these protocols fail to recapitulate the cell-guided differentiation programs intrinsic to embryonic development, particularly the signaling centers that emerge within the neural tube during brain formation. Located on the ventral end of the neural tube, the floor plate acts as one such signaling center to pattern the dorsal/ventral axis by secreting the morphogen Sonic Hedgehog (SHH). Here, we present a method for cell-guided differentiation using the synthetic Notch (synNotch) receptor platform to regulate SHH production and subsequent cell fate specification. We show that the widely used configuration of the orthogonal synNotch ligand green fluorescent protein (GFP) mounted on a platelet-derived growth factor receptor-β transmembrane chassis does not allow for robust artificial signaling in synNotch-hPSCs ("receivers") cocultured with ligand-presenting hPSCs ("senders"). We discovered that refined designs of membrane-bound GFP-ligand allow for efficient receptor activation in hPSC receivers. A variant of this enhanced synNotch system drives the production of SHH in hPSC sender:hPSC receiver cocultures and gives rise to floor plate-like cell types seen during neural tube development. This revised synNotch platform has the potential to pattern hPSC differentiation programs in synthetic morphogenesis studies designed to uncover key paradigms of human CNS 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 sixty 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 disk confocal microscope system, highlighting the importance of a standardized method to benchmark fluorescent proteins to make optimal choices for specific experimental setups.

Photophysics of a Methylated GFP Chromophore Anion in Vacuo.

The photophysical properties of the isolated chromophore anion from the green fluorescent protein have been extensively studied over the years to understand the factors influencing transition energies, excited-state lifetimes, and fluorescence. A commonly used model for the protein chromophore is 4'-hydroxybenzylidene-2,3-dimethyl-imidazolinone (p-HBDI). In this work, we have spectroscopically characterized a derivative, brMe-p-HBDI, which features methylation on the carbon bridging the phenol and imidazolinone rings. Experiments were conducted on the anionic form in the gas phase and at cryogenic temperatures using the SAPHIRA ion-storage ring and the LUNA2 fluorescence mass spectrometer, both located in Aarhus. Photoinduced action spectra reveal that brMe-p-HBDI- cooled to about 20-30 K exhibits maximum absorption at 496.0 ± 0.5 nm. Vibrationally resolved bands appear at shorter wavelengths, while a featureless absorption tail extends toward longer wavelengths, up to approximately 520 nm. The methyl substituent induces a clear redshift (75 meV) in absorption as p-HBDI- absorbs maximally at 481.51 ± 0.15 nm. The excited-state lifetime of brMe-p-HBDI- is determined to be 51 ± 3 ps following 495 nm photoexcitation and probing at 800 nm, which is significantly shorter than the nanosecond lifetime previously reported for p-HBDI-. Consistent with this, no fluorescence was detected from brMe-p-HBDI- at 100 K, in contrast to p-HBDI- that is strongly fluorescent according to recent work. These findings are corroborated by (time-dependent) density-functional theory calculations: A methyl substituent at the bridge carbon is predicted to cause a redshift of 77 meV, in excellent agreement with the experimental shift. We find that brMe-p-HBDI- is planar in the ground state (S0) but undergoes a twist motion in the S1 state, leading to a lower-energy nonplanar form where the angle between the two rings is 90°. Our work reveals that even a minor alteration in molecular structure can have a significant impact on the photophysics.

Adaptive Restraints to Accelerate Geometry Optimizations of Large Biomolecular Systems.

Quantum mechanical/molecular mechanical geometry optimizations of large-scale biological systems, such as enzymes, proteins, membranes, and solutions, are typically computationally expensive to the point of being cost-prohibitive. By convention, an approximation is made to such calculations that atoms beyond a certain distance from the QM region provide only negligible improvements to the resulting optimization energy and geometry, and as such are restrained to reduce the number of degrees of freedom. These constraints are normally applied beyond a user-defined radius. Here we describe a new method of geometry optimization acceleration and automation which generates adaptive gradient-based restraints for QM/MM optimizations, leading to significantly faster optimizations and generally lower relative energies. The restraints are determined by an algorithm rather than a user, and can adapt to directional optimizations as well as differences in starting geometry. This flexibility is key to finding excited state minima and minimum energy conical intersections (MECIs) in complex protein environments. This algorithm was implemented as an external Python tool for use alongside TeraChem, with a modular interface that can be straightforwardly applied to other QM/MM packages. We tested on a green fluorescent protein (rsEGFP2) and two red fluorescent proteins (FusionRed, mScarlet) in water and a proton-swapping aspartic acid pair in explicit water. We are able to produce a nearly 50% reduction in computational time while maintaining appropriately optimized geometries and relative energies.

A toolkit for facilitating markerless integration of expression cassettes in Komagataella phaffii via CRISPR/Cas9

BackgroundThe yeast Komagataella phaffii (formerly known as Pichia pastoris) has been widely used for functional expression of recombinant proteins, including plant and animal food proteins. CRISPR/Cas9 genome editing systems can be used for insertion of heterologous genes without the use of selection markers. The study aimed to create a convenient markerless knock-in method for integrating expression cassettes into the chromosome of K. phaffii using CRISPR/Cas9 technology. The approach was based on the hierarchical, modular, Golden Gate assembly employing the GoldenPiCS toolkit. Furthermore, the aim was to evaluate the system’s efficiency and suitability for producing secreted recombinant food proteins.ResultsThree Cas9/sgRNA plasmids were constructed, along with corresponding donor helper plasmids containing homology regions for chromosomal integration via homology-directed repair. The integration efficiency of an enhanced green fluorescent protein (eGFP) expression cassette was assessed at three genomic loci (04576, PFK1, and ROX1). The 04576 locus showed the highest integration efficiency, while ROX1 had the highest transformation efficiency. Whole genome sequencing revealed variable copy numbers of eGFP expression cassettes among clones, corresponding with increasing levels of fluorescence. Furthermore, the system’s applicability for producing recombinant food proteins was validated by successfully expressing and secreting chicken ovalbumin. This constitutes the first report of CRISPR/Cas9 applied to produce recombinant chicken ovalbumin.ConclusionsThe adapted GoldenPiCS toolkit combined with CRISPR/Cas9 technology enabled efficient and precise genome integration in K. phaffii. This approach holds promise for expanding the production of high-value recombinant proteins. Future research should focus on optimizing integration sites and improving cloning procedures to enhance the system’s efficiency and versatility.

Herpes simplex virus type-1 infection and spread in a novel porcine corneal explant model is restricted to the epithelium.

Herpes Keratitis (HK) is a debilitating infection of the cornea that remains the leading cause of infectious blindness in developed countries. Caused primarily by herpes simplex virus type 1 (HSV-1), it is associated with recurrent inflammation, leading to corneal scarring. This study investigated the initial events during acute HSV-1 infection in the cornea by adapting our human anogenital mucosal explant model to a HSV-1 infected porcine corneal explant model, We infected these corneas topically via high-density microarray patches (HD-MAPs) dipped in GFP-labelled HSV-1. Virus infection and spread was detected by both GFP protein and RNAscope, adapted for HSV-1 DNA. The punctures were consistent, usually in the epithelium but some extended into the underlying stroma. However, HSV-1 was restricted to the corneal epithelium, without spread through the anterior limiting membrane (ALM) or Bowman's layer into the stroma nor to the uppermost epithelial layer. This layer expressed SPRR1A similarly to the stratum granulosum of skin which is refractory to HSV-1 infection. In corneas where infected epithelial cells extended to the ALM, SPRR1A was also observed in this layer, suggesting it may contribute to its barrier function. Such studies of HSV-1 infection and spread will help improve therapy for HK and vaccine design to prevent it.

Creating a multifunctional degrader for co-mineralization of p-nitrophenol and 1,2-dichloroethane and its application in wastewater bioremediation.

Creating a multifunctional degrader for co-mineralization of p-nitrophenol and 1,2-dichloroethane and its application in wastewater bioremediation.

Niuhuang jiedu prescription alleviates realgar-induced dopaminergic and GABAergic neurotoxicity in Caenorhabditis elegans.

Niuhuang jiedu prescription alleviates realgar-induced dopaminergic and GABAergic neurotoxicity in Caenorhabditis elegans.

Development of a highly sensitive PbrR-based biosensor via directed evolution and its application for lead detection.

Development of a highly sensitive PbrR-based biosensor via directed evolution and its application for lead detection.

Expression, purification, and immunogenicity study of human papillomavirus type 52 virus-like particles produced in Hansenula polymorpha.

Expression, purification, and immunogenicity study of human papillomavirus type 52 virus-like particles produced in Hansenula polymorpha.