Fluorescent proteins have been extensively engineered and applied as optical indicators for chloride in a variety of biological contexts. Surprisingly, given the biodiversity of fluorescent proteins, a naturally occurring chloride sensor has not been reported to date. Here, we present the identification and spectroscopic characterization of the yellow fluorescent protein from the jellyfish Phialidium sp. (phiYFP), a rare example of a naturally occurring, excitation ratiometric, and turn-on fluorescent protein sensor for chloride. Our results show that chloride binding tunes the pKa of the chromophore Y66 and shifts the equilibrium from the fluorescent phenolate form to the weakly fluorescent phenol form. The latter likely undergoes excited state proton transfer to generate a turn-on fluorescence response that is pH dependent. Moreover, anion selectivity and mutagenesis in the chloride binding pocket provide additional evidence for the proposed chloride sensing mechanism. Given these properties, we anticipate that phiYFP, with further engineering, could be a new tool for imaging cellular chloride dynamics.

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Living cells rely on a finely tuned symphony of inorganic ion gradients composed of both cations and anions. This delicate balance is maintained by biological receptors all acting in concert to selectively recognize and position ions for homeostasis. These dynamic processes can be intercepted and visualized with optical microscopy at the organismal, tissue, cellular and subcellular levels using fluorescent protein-based biosensors. Since the first report of such tool for calcium (Ca2+) in 1997, outstanding biological questions and innovations in protein engineering along with associated fields have driven the development of new biosensors for Ca2+ and beyond. In this Review, we summarize a workflow that can be used to generate fluorescent protein-based biosensors to study monoatomic ions in biology. To showcase the scope of this approach, we highlight recent advances reported for Ca2+ biosensors and in detail discuss representative case studies of biosensors reported in the last four years for potassium (K+), magnesium (Mg2+), copper (Cu2+/+), lanthanide (Ln3+) and chloride (Cl−) ions.

Brain metastases originating from lung adenocarcinoma (LAD) occur frequently. The aim of the current study was to assess potential biomarkers for the prognosis of lung adenocarcinoma brain metastasis (LAD-BM) through the analysis of gene expression microarrays. The current study downloaded two gene expression datasets, GSE14108 and GSE10245, from the Gene Expression Omnibus database. From GSE14108 and GSE10245, 19 LAD-BM samples and 40 primary LAD samples were selected for analysis. To identify the differentially expressed genes (DEGs), the current study compared the two sample groups, using the limma R package. Subsequently, pathway enrichment analysis was conducted using the Cluster Profiler R package, and the construction of the protein-protein interaction (PPI) network was executed utilizing the Search Tool for the Retrieval of Interacting Genes database. The microRNA-target network was built using the TargetScore R package. Then, these networks were established and visualized using Cytoscape software. An array of 463 DEGs was identified in the LAD-BM samples, including 256 upregulated and 207 downregulated genes. Based on functional term enrichment analysis using the Gene Ontology database and signaling pathway enrichment analysis using the Kyoto Encyclopedia of Genes and Genomes database, it was identified that the overlapping DEGs were primarily involved in chemokine-associated signal transduction, which may mediate lung cancer cell metastasis to the brain. Chemokine ligand 2, lysozyme, matrix metalloproteinase-2 (MMP-2), lysyl oxidase (LOX) and granzyme B were identified as potential biomarkers according to a topological analysis of the PPI networks. Two notable nodes, MMP-2 and LOX, appeared in the PPI network and were key points in the microRNA-target network, as they were regulated by hsa-let-7d. Many DEGs and microRNAs were regarded as prognostic biomarkers for lung adenocarcinoma metastasis in the current study. These DEGs were primarily associated with chemokine-mediated signaling pathways. In addition, MMP-2 and LOX were predicted to be targets of hsa-let-7d.

Chloride is an essential anion for all forms of life. Beyond electrolyte balance, an increasing body of evidence points to new roles for chloride in normal physiology and disease. Over...

Background: Glioma is one of the important diseases that threaten human survival in today's society. WD repeat domain 5 (WDR5) belongs to the components of the lysine methyltransferase complex. WDR5 is involved in gene transcription regulation, cell senescence, cancer and other biological events through methylation modification. However, its expression and function in glioma are still unclear. Materials and Methods: The expression of WDR5 was observed in glioma cells, and then a glioma cell line SW1783 knocked down WDR5 was established. The effects of the decrease of WDR5 expression on proliferation, migration, invasion and EMT of glioma cells were detected, respectively. The downstream regulators of WDR5 were identified by gene expression profiling technology, and the possible molecular mechanisms were identified. Results: In this study, we found that WDR5 could promote glioma cell's proliferation, migration, invasion and tumor metastasis. In glioma, especially in metastatic glioma tissues, WDR5 levels were significantly increased, the higher expression level could also cause a significant reduction in overall survival of glioma patients. Second, the ability of cells' proliferation, migration, invasion and tumor metastasis was significantly reduced in WDR5 knockdown cell lines. We also found a significant change in the expression level of epithelial and mesenchymal markers in WDR5 knockdown cell lines. Furthermore, we found that knockdown of WDR5 could inhibit the expression of zinc finger E-box binding homeobox 1 (ZEB1), and knockdown of ZEB1 could inhibit invasion, migration and epithelial-mesenchymal transformation (EMT) in WDR5 over-expression cell line. We also found that WDR5 may regulate ZEB1's expression through H3K4me3. Conclusion:In summary, in this study, we have studied the relationship between WDR5 and glioma, and found that WDR5's expression is positively correlated with the proliferation, migration, and invasion of glioma cells, which will help find the potential therapeutic target for glioma patients.

Chloride is a ubiquitous ion essential for regulating biological processes. Long thought to be just a counterion in biological systems, chloride is emerging as a vital anion that plays a major role in a range of diseases. The ability to visualize intracellular chloride has been enabled by fluorescent protein-based sensors. Although the yellow fluorescent protein from Aequorea victoria, and variants thereof, quench in the presence of chloride and other anions, they have provided valuable insights in biological studies and have established the benchmark for applying protein-based sensors for chloride. However, a single domain fluorescent protein sensor that generates a turn-on response to provide a spatial and temporal map of endogenous chloride has yet to be reported. Here, we highlight how mutagenesis of non-coordinating residues in the binding pocket of mNeonGreen has unlocked this new sensing capability. Our protein engineering efforts coupled with in vitro spectroscopy have led us to discover the ChlorON series as turn-on fluorescent sensors for chloride that operate at physiological pH. Fluorescence microscopy experiments with the ChlorON sensors reveal that cells have baseline levels of chloride, which could not have been directly observed with turn-off sensors. These advancements set the stage for visualizing endogenous chloride dynamics in real-time and uncovering new roles for chloride in biology.