FGF23 contains two distinct high-affinity binding sites enabling bivalent interactions with α-Klotho

The three members of the endocrine-fibroblast growth factor (FGF) family, FGF19, 21, and 23 are circulating hormones that regulate critical metabolic processes. FGF23 stimulates the assembly of a signaling complex composed of α-Klotho (KLA) and FGF receptor (FGFR) resulting in kinase activation, regulation of phosphate homeostasis, and vitamin D levels. Here we report that the C-terminal tail of FGF23, a region responsible for KLA binding, contains two tandem repeats, repeat 1 (R1) and repeat 2 (R2) that function as two distinct ligands for KLA. FGF23 variants with a single KLA binding site, FGF23-R1, FGF23-R2, or FGF23-wild type (WT) with both R1 and R2, bind to KLA with similar binding affinity and stimulate FGFR1 activation and MAPK response. R2 is flanked by two cysteines that form a disulfide bridge in FGF23-WT; disulfide bridge formation in FGF23-WT is dispensable for KLA binding and for cell signaling via FGFRs. We show that FGF23-WT stimulates dimerization and activation of a chimeric receptor molecule composed of the extracellular domain of KLA fused to the cytoplasmic domain of FGFR and employ total internal reflection fluorescence microscopy to visualize individual KLA molecules on the cell surface. These experiments demonstrate that FGF23-WT can act as a bivalent ligand of KLA in the cell membrane. Finally, an engineered Fc-R2 protein acts as an FGF23 antagonist offering new pharmacological intervention for treating diseases caused by excessive FGF23 abundance or activity.

The large family of fibroblast growth factors (FGFs) has been known for its important roles in regulating critical cellular processes during embryonic development and homeostasis of normal tissues (1–3). While most FGFs act as cytokines or hormonelike proteins that mediate their pleiotropic cellular processes by binding to cell surface receptors endowed with intrinsic tyrosine kinase activity (FGFRs), a subfamily of FGFs (FGF 11–14) was shown to be uniquely expressed intracellularly. The mechanism of action and physiological roles of intracellular FGFs are poorly understood (4–6).In contrast to most receptor tyrosine kinases (RTKs) that are activated by a single ligand molecule that binds with high affinity to the extracellular domain of its cognate RTK with a dissociation constant in the subnanomolar range, the binding affinities of FGFs to FGFRs are, at least, 1,000–10,000 fold weaker with dissociation constants in the submicromolar range (7–9). The weak binding affinities toward FGFRs of the largest subfamily of FGF molecules designated canonical FGFs are offset by interactions with cell surface heparan sulfate proteoglycans (HSPGs). Both biochemical and structural studies revealed how multiple interactions between heparin or HSPG with both FGF and FGFR mediate tight association enabling robust receptor dimerization and tyrosine kinase activation (10, 11).The three endocrine FGFs, FGF19, 21, and 23 are part of an additional subfamily of FGF molecules. Endocrine FGFs function as circulating hormones that play essential roles in the control of various metabolic processes (12). In addition to the conserved FGF-domain found in all FGF ligands, endocrine FGFs contain unique C-terminal tails (CTs) composed of 46 (FGF19), 34 (FGF21), or 89 (FGF23) amino acids that serve as specific and high-affinity ligands for the two members of the Klotho family of surface receptors. It was shown that KLA serves as a high-affinity receptor for FGF23 while β-Klotho (KLB) functions as a high-affinity surface receptor for both FGF19 and FGF21 (13–16). Structural analyses of free and ligand-occupied Klotho proteins revealed the molecular basis underlying the specificity and high affinity of KLA and KLB toward endocrine FGFs. It also showed that Klotho proteins function as the primary receptors for endocrine FGFs whereas FGFR functions as a catalytic subunit that mediates cell signaling via its tyrosine kinase domain (8, 17, 18). Accordingly, endocrine FGFs stimulate their cellular responses by forming a ternary complex with Klotho proteins and FGFRs to induce receptor dimerization, tyrosine kinase activation, and cell signaling. Unlike FGFRs that are ubiquitously expressed, the expressions of KLA and KLB are restricted to specific tissues and organs to enable precise targeting of endocrine FGFs to stimulate their physiological responses in specific cells and tissues (19–22). The ability of endocrine FGFs to circulate is attributed to the loss of conserved heparin binding sites that are essential for the function of canonical FGFs (23).FGF23 is a 32-kDa glycoprotein, mainly produced in the bone by osteoblasts and osteocytes, that serve as a key hormone in regulating phosphate homeostasis, vitamin D, and calcium metabolism (24, 25). Circulating levels of physiologically active FGF23 are regulated by proteolytic cleavage to produce a FGF23 molecule lacking its unique CT (26, 27). The cleavage resulting in FGF23 inactivation prevents assembly and stimulation of the FGF23/FGFR/KLA complex. Additionally, the processing of FGF23 includes several posttranslational modifications which affect its stability and susceptibility toward proteolysis. Secreted FGF23 was shown to be O-glycosylated in its C-terminal cleavage site (28, 29) to protect the protein from C-terminal cleavage. In order for the cleavage site to be exposed, FGF23 has to be first phosphorylated in this region (30). Phosphorylation prevents glycosylation and exposes the cleavage site to proteolysis.In this paper, we demonstrate that the CT of FGF23 contains two tandem repeats and that each repeat binds with high affinity to KLA. This contrasts with FGF19 and FGF21, whose CTs contain a single binding site to KLB. Engineered FGF23 variants containing each of the two repeats individually or both repeats bind specifically to KLA and stimulate cell signaling to a similar extent. We also demonstrate that two cysteine residues flanking the second repeat form a disulfide bridge in FGF23 secreted by mammalian cells. However, both oxidized or unbridged forms of FGF23 exhibit similar KLA binding characteristics and similar cellular stimulatory activities. We also show that FGF23-WT induces mitogen-activated protein kinase (MAPK) activation in cells expressing chimeric KLA-FGFR proteins and use TIRFM imaging of individual KLA molecules on the cell surface to demonstrate that FGF23 has the capacity for simultaneous binding to two KLA molecules. These insights reveal the complexity of FGF23 regulation and its role in assembling the FGF23/FGFR/KLA signaling complex.     

The Effects of Matrigel® on the Survival and Differentiation of a Human Neural Progenitor Dissociated Sphere Culture

We have previously developed an in vitro organotypic culture setting in order to investigate the performance of cellular substrates transplanted to the auditory nervous system. We have utilized this system to predict the efficacy of human neural progenitor cells (HNPCs) in transplantation to the auditory nerve to facilitate regeneration of sensory auditory nerve structures in vivo and in vitro. To optimize the growth and differentiation of HNPCs we have introduced an expansion of our in vitro system, exploring the impact of a growth factor-altered microenvironment. Here, we seeded HNPCs as a dissociated sphere culture on a hydrogel matrix coating (Matrigel®). We evaluated the performance of HNPCs by studying their survival, differentiation, and their axon-forming capacity. In identical culture conditions, we found that the overall survival rate of HNPCs on Matrigel coated surfaces was better than that on surfaces that were not coated with Matrigel. Furthermore, cells on Matrigel differentiated into neuronal cells to a far greater extent leading to strong synaptic marker signatures. Overall, our findings show that the present Matrigel matrix setting offers an experimental environment for the HNPCs to grow where these cells show novel and promising phenotypic characteristics suitable for further in vivo transplantation to the auditory nerve. Anat Rec, 303:441–450, 2020. © 2019 American Association for Anatomy     

Inkjet assisted fabrication of planar biocompatible memristors

In this study we address a novel design of a planar memristor and investigate its biocompatibility. An experimental prototype of the proposed memristor assembly has been manufactured using a hybrid nanofabrication method, combining sputtering of electrodes, patterning the insulating trenches, and filling them with a memristive substance. To pattern the insulating trenches, we have examined two nanofabrication techniques employing either a focused ion beam or a cantilever tip of an atomic force microscope. Inkjet printing has been used to fill the trenches with the functional titania ink. The experimental prototypes have qualitatively demonstrated memristive current–voltage behavior, as well as high biocompatibility.

A planar memristor was fabricated by a hybrid method combining AFM patterning and inkjet printing.     

The influence of the sacrificial agent nature on transformations of the Zn(OH)2/Cd0.3Zn0.7S photocatalyst during hydrogen production under visible light

Photocatalysts based on zinc hydroxide and a solid solution of CdS and ZnS were prepared via the precipitation method and used for photocatalytic hydrogen production from aqueous solutions of inorganic (Na₂S/Na₂SO₃) and organic (ethanol) sacrificial agents. The photocatalysts were tested in cyclic experiments for hydrogen evolution and studied using X-ray diffraction (XRD), UV-Vis diffuse reflectance spectroscopy, high-resolution transmission electron microscopy (HRTEM), energy-dispersive X-ray spectroscopy (EDX), and X-ray photoelectron spectroscopy (XPS) techniques. Different transformations of the β-Zn(OH)₂ co-catalyst were observed in the presence of inorganic and organic sacrificial agents; namely, ZnS was formed in Na₂S/Na₂SO₃ solution, whereas the formation of ε-Zn(OH)₂ was detected in solution with ethanol. The composite Zn(OH)₂/Cd_1−xZn_xS photocatalysts have great potential in various photocatalysis processes (e.g., hydrogen production, CO₂ reduction, and the oxidation of organic contaminants) under visible light.

The nature of the sacrificial agent affects the transformations of a Zn(OH)₂ co-catalyst during photocatalytic hydrogen production.     

Determination of sodium and potassium ions in patients with SARS-Cov-2 disease by ion-selective electrodes based on polyelectrolyte complexes as a pseudo-liquid contact phase

Nowadays, there are several methods for the detection of various bioelements during SARS-CoV-2. Many of them require special equipment, high expenses, and a long time to obtain results. In this study, we aim to use polyelectrolyte multilayers for robust carbon fiber-based potentiometric sensing to determine the ion concentration in human biofluids of COVID-19 patients. The polyethyleneimine/polystyrene sulfonate complex is hygroscopic and has the ability to retain counterions of inorganic salts. This fact makes it possible to create a flexible ionometric system with a pseudo-liquid connection. The formation of the polyethyleneimine/polystyrene sulfonate complex allows for the adhesion of a hydrophobic ion-selective membrane, and creates a Nernst response in a miniature sensor system. This approach discloses the development of miniaturized ion-selective electrodes and their future application to monitor analyte changes as micro and macroelement ions in the human body to identify correlation to SARS-CoV-2. An imbalance in the content of potassium and sodium in urine and blood is directly related to changes in the zinc content in patients with coronavirus. The proposed method for assessing the condition of patients will allow fast determination of the severity of the course of the disease.

Supramolecular assemblies based on polyelectrolyte complexes made it possible to create complex interfaces with predictable properties. Polyelectrolyte complexes serve as a pseudo-liquid contact in ion-selective electrodes.     

Improving the Conductivity of the PEDOT:PSS Layers in Photovoltaic Cells Based on Organometallic Halide Perovskites

Among conductive polymers, PEDOT films find the widest application in electronics. For photovoltaic applications, studies of their optical properties, stability, and electrical conductivity are of greatest interest. However, the PEDOT:PSS transport layers, when used in photovoltaic cells, have a high electrical resistance, which prevents solar cells from increasing their efficiency. One of the promising ways to improve their electrical properties is the use of composite materials based on them, in which the conductivity can be increased by introducing various additives. In this work, conductive polymer films PEDOT:PSS (poly (3,4-ethylenedioxythiophene):polystyrene sulfonate acid) doped with a number of amines (Pentylamine, Octylamine, Diethylamine, Aniline with carbon nanotubes) were obtained and studied. It is shown that, depending on the concentration of dopants, the electrical conductivity of PEDOT:PSS films can be significantly improved. In this case, the light transmission of the films practically does not change. The process of improving the conductivity by treating the surface of the finished film with amines, followed by heat treatment, was studied. It is assumed that the improvement in conductivity is the result of the self-assembly of monolayers of organic molecules on the surface of the PEDOT:PSS film leading to its p-doping due to intermolecular interaction.