The SpACE-CCM: A facile and versatile cell culture medium-based biosensor for detection of SARS-CoV-2 spike-ACE2 interaction.

The COVID-19 pandemic is an ongoing global public health threat. COVID-19 is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, and binding of the SARS-CoV-2 spike to its receptor, angiotensin-converting enzyme 2 (ACE2), on host cells is critical for viral infection. Here, we developed a luminescent biosensor that readily detects interactions of the spike receptor-binding domain (RBD) and ACE2 in cell culture medium ('SpACE-CCM'), which was based on bimolecular complementation of the split nanoluciferase-fused spike RBD and ectodomain of ACE2 and further engineered to be efficiently secreted from cells by adding a heterologous secretory signal peptide (SSP). Screening of various SSPs identified 'interferon-α+alanine-aspartate' as the SSP that induced the highest activity. The SpACE-CCM biosensor was validated by observing a marked reduction of the activity caused by interaction-defective mutations or in the presence of neutralizing antibodies, recombinant decoy proteins, or peptides. Importantly, the SpACE-CCM biosensor responded well in assay-validating conditions compared with conventional cell lysate-based NanoLuc Binary Technology, indicating its advantage. We further demonstrated the biosensor's versatility by quantitatively detecting neutralizing activity in blood samples from COVID-19 patients and vaccinated individuals, discovering a small molecule interfering with the spike RBD-ACE2 interaction through high-throughput screening, and assessing the cross-reactivity of neutralizing antibodies against SARS-CoV-2 variants. Because the SpACE-CCM is a facile and rapid one-step reaction biosensor that aptly recapitulates the native spike-ACE2 interaction, it would be advantageous in many experimental and clinical applications associated with this interaction.

Development of Bisphenol-A-Glycidyl-Methacrylate- and Trimethylolpropane-Triacrylate-Based Stereolithography 3D Printing Materials.

The main advantages of the three-dimensional (3D) printing process are flexible design, rapid prototyping, multi-component structures, and minimal waste. For stereolithography (SLA) 3D printing, common photocurable polymers, such as bisphenol-A glycidyl methacrylate (Bis-EMA), trimethylolpropane triacrylate (TMPTMA), as well as urethane oligomers, have been widely used. For a successful 3D printing process, these photocurable polymers must satisfy several requirements, including transparency, a low viscosity, good mechanical strength, and low shrinkage post-ultraviolet curing process. Herein, we investigated SLA-type photocurable resins prepared using Bis-EMA, TMPTMA, and urethane oligomers. The flexural strength, hardness, conversion rate, output resolution, water absorption, and solubility of the printed materials were investigated. The degree of conversion of the printed specimens measured by infrared spectroscopy ranged from 30 to 60%. We also observed that 64-80 MPa of the flexural strength, 40-60 HV of the surface hardness, 15.6-29.1 MPa of the compression strength, and 3.3-14.5 MPa of the tensile strength. The output resolution was tested using three different structures comprising a series of columns (5-50 mm), circles (0.6-6 mm), and lines (0.2-5 mm). In addition, we used five different pigments to create colored resins and successfully printed complex models of the Eiffel Tower. The research on resins, according to the characteristics of these materials, will help in the design of new materials. These results suggests that acrylate-based resins have the potential for 3D printing.

Smithing Processes Based on Hammer Scale Excavated from the Third- to Fourth-Century Ancient Iron-Making Sites of the Korean Peninsula.

The by-products of iron smelting and smithing include slag, flake hammer scale, and spheroidal hammer scale. The analysis of such iron-making by-products reveals critical information regarding the development of iron culture and the process characteristics. Using a metallographic microscope, SEM-EDS, and Raman micro-spectroscopy, we investigated the manufacturing process by examining the microstructure and determining the composition of the flake hammer scale and spheroidal hammer scale excavated from Korean Peninsula sites of iron manufacture during the Proto-Three Kingdoms Period, in the third and fourth centuries CE. Microstructure analysis confirmed that as the process progressed, the flake hammer scale's thickness decreased owing to forging, which flattened the structure. Additionally, three layers were observed, with the surface layer identified as hematite (Fe2O3), the middle layer identified as magnetite (Fe3O4), and the inner layer identified as wüstite (FeO). The analysis of hammer scales revealed that the forging process to create iron bars required repeated working, following a refining process to remove impurities, confirming the division of labor in the smithing process. Correspondingly, the smithing process stages can be deduced from the structural shape and thickness of the hammer scale produced during the iron manufacturing process. Thus, the findings of this study are expected to be invaluable in furthering our understanding of the smithing process in detail, through future research on hammer scale.

Nuclear Localization of Fibroblast Growth Factor Receptor 1 in Breast Cancer Cells Interacting with Cancer Associated Fibroblasts.

Cancer-associated fibroblasts (CAFs) represent a major component of the tumor microenvironment and interplay with cancer cells by secreting cytokines, growth factors and extracellular matrix proteins. When estrogen receptor-negative breast cancer MDA-MB-231 cells were treated with the CAF-conditioned medium (CAF-CM), Akt and STAT3 involved in cell proliferation and survival were activated through phosphorylation. CAFs secrete fibroblast growth factor 2 (FGF2), thereby stimulating breast cancer cell progression. Akt activation induced by CAF-CM in MDA-MB-231 cells was abolished when FGF2-neutralizing antibody was added. Treatment of MDA-MB-231 cells directly with FGF2 enhanced the phosphorylation of Akt and the FGF receptor (FGFR) substrate, FRS2α. These events were abrogated by siRNA-mediated silencing of FGFR1. In a xenograft mouse model, co-injection of MDA-MB-231 cells with activated fibroblasts expressing FGF2 dramatically enhanced activation of Akt. Stable knockdown of FGFR1 blunted Akt phosphorylation in xenograft tumors. MDA-MB-231 cells co-cultured with CAFs or directly stimulated with FGF2 exhibited enhanced nuclear localization of FGFR1. Notably, FGF2 stimulation produced reactive oxygen species (ROS) accumulation in MDA-MB-231 cells, and FGF2-induced nuclear accumulation of FGFR1 was abrogated by the ROS scavenging agent, N-acetylcysteine.

Improving the Stability of Ball-Milled Lead Halide Perovskites via Ethanol/Water-Induced Phase Transition.

Recently, lead halide perovskite nanocrystals have been considered as potential light-emitting materials because of their narrow full width at half-maximum (FWHM) and high photoluminescence quantum yield (PLQY). In addition, they have various emission spectra because the bandgap can be easily tuned by changing the size of the nanocrystals and their chemical composition. However, these perovskite materials have poor long-term stability due to their sensitivity to moisture. Thus far, various approaches have been attempted to enhance the stability of the perovskite nanocrystals. However, the required level of stability in the mass production process of perovskite nanocrystals under ambient conditions has not been secured. In this work, we developed a facile two-step ball-milling and ethanol/water-induced phase transition method to synthesize stable CsPbBr3 perovskite materials. We obtained pure CsPbBr3 perovskite solutions with stability retention of 86% for 30 days under ambient conditions. Our materials show a high PLQY of 35% in solid films, and excellent thermal stability up to 80 °C. We believe that our new synthetic method could be applicable for the mass production of light-emitting perovskite materials.

STAT3 as a Potential Target for Tumor Suppressive Effects of 15-Deoxy-Δ12,14-prostaglandin J2 in Triple Negative Breast Cancer.

STAT3 plays a prominent role in proliferation and survival of tumor cells. Thus, STAT3 has been considered to be a prime target for development of anti-cancer therapeutics. The electrophilic cyclopentenone prostaglandin,15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) has been well recognized for its capability to modulate intracellular signaling pathways involved in cancer cell growth and progression. We previously reported that 15d-PGJ2 had potent cytotoxicity against harvey-ras transformed human mammary epithelial cells through direct interaction with STAT3. In this study, we have attempted to verify the inhibitory effects of 15d-PGJ2 on STAT3 signaling in human breast tumor cells. The triple negative breast cancer cell lines, MDA-MB-231 and MDA-MB-468 displaying constitutive phosphorylation of STAT3 on the tyrosine 705 (Tyr705) residue, underwent apoptosis upon inhibition of STAT3 by 15d-PGJ2. In contrast, estrogen receptor positive MCF-7 breast cancer cells that do not exhibit elevated STAT3 phosphorylation were much less susceptible to 15d-PGJ2-induced apoptosis as assessed by PARP cleavage. Furthermore, 15d-PGJ2 inhibited interleukin-6-induced tyrosine phosphorylation of STAT3 in LNCaP cells. According to molecular docking studies, 15d-PGJ2 may preferentially bind to the cysteine 259 residue (Cys259) present in the coiled-coil domain of STAT3. Site-directed mutagenesis of STAT3 identified Cys259 to be the critical amino acid for the 15d-PGJ2-induced apoptosis as well as epithelial-to-mesenchymal transition. Taken together, these findings suggest STAT3 inactivation through direct chemical modification of its Cys259 as a potential therapeutic approach for treatment of triple negative breast cancer treatment.

Aberrant activation of the CD45-Wnt signaling axis promotes stemness and therapy resistance in colorectal cancer cells.

Rationale: Chemoradiation (CRT) is commonly used as an adjuvant or neoadjuvant treatment for colorectal cancer (CRC) patients. However, resistant cells manage to survive and propagate after CRT, increasing the risk of recurrence. Thus, better understanding the mechanism of resistant cancer cells is required to achieve better clinical outcomes. Methods: Here, we explored gene expression profiling of CRC patient tumors to identify therapy resistance genes and discovered that protein tyrosine phosphatase receptor type C (PTPRC), which encodes CD45, was increased in remnant tumor tissues after CRT and correlated with metastasis. Through multiple validations using patient tumors and CRC cell lines, we found for the first time the increase of CD45 expression in CRC (EpCAM+) epithelial cells surviving after CRT. Thus, we investigated the biological role and downstream events of CD45 were explored in human CRC cells and CRC mouse models. Results: Increased CD45 expression in cancer cells in pretreated primary tumors accounts for poor regression and recurrence-free survival in CRT-treated patients. High CD45 expression promotes CRC cell survival upon 5-fluorouracil or radiation treatment, while CD45 depletion sensitizes CRC cells to CRT. Intriguingly, CD45 is preferentially expressed in cancer stem-like cells (CSCs), as determined by spheroid culture and the expression of CSC markers, and is required for the distinct functions of CSCs, such as cancer initiation, repopulation, and metastasis. Mechanistically, CD45 phosphatase activity promotes Wnt transcriptional activity by stabilizing the ß-catenin protein, which collectively enhances stemness and the therapy-resistant phenotype. Conclusions: Our results highlight a novel function of CD45 as a mediator of CRT resistance and provide a potential therapy strategy for CRC therapy.

15-Deoxy-Δ12,14 -prostaglandin J2 binds and inactivates STAT3 via covalent modification of cysteine 259 in H-Ras-transformed human breast epithelial cells.

Signal transducer and activator of transcription 3 (STAT3) has been considered as a potential target for development of anticancer therapeutics. Here, we report a novel mechanism by which the cyclopentenone prostaglandin, 15-deoxy-Δ12,14 -prostaglandin J2 (15d-PGJ2 ) functions as an allosteric inhibitor of STAT3. 15d-PGJ2 inhibits phosphorylation, dimerization, nuclear translocation, and transcriptional activity of STAT3 in H-Ras-transformed human mammary epithelial cells (MCF10A-Ras) through the Michael addition reaction at cysteine 259 of STAT3. Comparative studies with 15d-PGJ2 analogues reveal that both C12-C13 and C9-C10 double bonds conjugated to the carbonyl group in the cyclopentenone ring of 15d-PGJ2 are essential for STAT3 binding. Antiproliferative and pro-apoptotic activities of 15d-PGJ2 in MCF10A-Ras cells are attributable to covalent modification of STAT3 on Cys259, and mimic the effects induced by mutation of this amino acid.

Efficient Inverted Solar Cells Using Benzotriazole-Based Small Molecule and Polymers.

We synthesized medium-band-gap donor-acceptor (D-A) -type conjugated polymers (PBTZCZ-L and PBTZCZ-H) consisting of a benzotriazole building block as an acceptor and a carbazole unit as a donor. In comparison with the polymers, a small conjugated molecule (BTZCZ-2) was developed, and its structural, thermal, optical, and photovoltaic properties were investigated. The power conversion efficiency (PCE) of the BTZCZ-2-based solar cell devices was less than 0.5%, considerably lower than those of polymer-based devices with conventional device structures. However, inverted solar cell devices configured with glass/ITO/ZnO:PEIE/BTZCZ-2:PC71BM/MoO3/Ag showed a tremendously improved efficiency (PCE: 5.05%, Jsc: 9.95 mA/cm2, Voc: 0.89 V, and FF: 57.0%). We believe that this is attributed to high energy transfer and excellent film morphologies.

Interaction of Nrf2 with dimeric STAT3 induces IL-23 expression: Implications for breast cancer progression.

Persistent activation of STAT3 and Nrf2 is considered to stimulate the aggressive behavior of basal-like breast cancer (BLBC). However, the precise mechanism underlying sustained overactivation of these transcription factors and their roles in breast cancer progression remain elusive. Analysis of the TCGA multi-omics data showed that high levels of STAT3 and Nrf2 mRNA were correlated with elevated expression of P-STAT3Y705 and Nrf2 target proteins in breast cancer patients. Our present study demonstrates a unique interaction between Nrf2 and STAT3 in the maintenance and progression of BLBC. RNA sequencing analysis identified the gene encoding IL-23A upregulated by concurrent binding of STAT3 and Nrf2 to its promoter. IL-23A depletion also showed the similar phenotypic changes to those caused by double knockdown of both transcription factors. In conclusion, the STAT3-Nrf2 interaction accelerates BLBC growth and progression by augmenting IL-23A expression, which underscores the importance of subtype-specific molecular pathways in human breast cancer.

An Electrophilic Deguelin Analogue Inhibits STAT3 Signaling in H-Ras-Transformed Human Mammary Epithelial Cells: The Cysteine 259 Residue as a Potential Target.

Signal transducer and activator of transcription 3 (STAT3) is a point of convergence for numerous oncogenic signals that are often constitutively activated in many cancerous or transformed cells and some stromal cells in the tumor microenvironment. Persistent STAT3 activation in malignant cells stimulates proliferation, survival, angiogenesis, invasion, and tumor-promoting inflammation. STAT3 undergoes activation through phosphorylation on tyrosine 705, which facilitates its dimerization. Dimeric STAT3 translocates to the nucleus, where it regulates the transcription of genes involved in cell proliferation, survival, etc. In the present study, a synthetic deguelin analogue SH48, discovered by virtual screening, inhibited the phosphorylation, nuclear translocation, and transcriptional activity of STAT3 in H-ras transformed human mammary epithelial MCF-10A cells (MCF10A-ras). We speculated that SH48 bearing an α,ß-unsaturated carbonyl group could interact with a thiol residue of STAT3, thereby inactivating this transcription factor. Non-electrophilic analogues of SH48 failed to inhibit STAT3 activation, lending support to the above supposition. By utilizing a biotinylated SH48, we were able to demonstrate the complex formation between SH48 and STAT3. SH48 treatment to MCF10A-ras cells induced autophagy, which was verified by staining with a fluorescent acidotropic probe, LysoTracker Red, as well as upregulating the expression of LC3II and p62. In conclusion, the electrophilic analogue of deguelin interacts with STAT3 and inhibits its activation in MCF10A-ras cells, which may account for its induction of autophagic death.

Curcumin induces stabilization of Nrf2 protein through Keap1 cysteine modification.

The present study was aimed to investigate the effects of curcumin, a representative chemopreventive phytochemical with pronounced antioxidant and anti-inflammatory properties, on activation of Nrf2 and expression of its target protein heme oxygenase-1 (HO-1) in mouse skin in vivo and in cultured murine epidermal cells. Treatment of mouse epidermal JB6 cells with curcumin resulted in the induction of HO-1 expression, and this was abrogated in cells transiently transfected with Nrf2 siRNA. While curcumin treatment increased protein expression of Nrf2, it did not alter the steady-state level of the Nrf2 mRNA transcript. Treatment of cells with curcumin stabilized Nrf2 by inhibiting ubiquitination and subsequent 26S proteasomal degradation of this transcription factor. Tetrahydrocurcumin, a non-electrophilic analogue of curcumin that lacks the α,ß-unsaturated carbonyl group, failed to induce HO-1 expression as well as nuclear translocation of Nrf2 and its binding to the antioxidant/electrophile response elements. Cells transfected with a mutant Keap1 protein in which cysteine 151 (Cys151) is replaced by serine exhibited marked reduction in curcumin-induced Nrf2 transactivation. Mass spectrometric analysis revealed that curcumin binds to Keap1 Cys151, supporting that this amino acid is a critical target for curcumin modification of Keap1, which facilitates the liberation of Nrf2. Thus, it is likely that the α,ß-unsaturated carbonyl moiety of curcumin is essential for its binding to Keap1 and stabilization of Nrf2 by hampering ubiquitination and proteasomal degradation.

17-Oxo-docosahexaenoic acid induces Nrf2-mediated expression of heme oxygenase-1 in mouse skin in vivo and in cultured murine epidermal cells.

Recently, growing attention has been given to new classes of bioactive lipid mediators derived from ω-3 polyunsaturated fatty acids, such as docosahexaenoic acid (DHA), especially in the context of their role as endogenous signal modulators. One such molecule is 17-oxo-DHA, generated from DHA by the action of COX2 and a dehydrogenase. The redox-sensitive transcription factor, Nrf2 plays a key role in cellular stress responses. In the present study, the effects of 17-oxo-DHA on Nrf2-mediated expression of cytoprotective enzymes were examined in mouse skin in vivo and cultured murine epidermal JB6 cells. Topical application of 17-oxo-DHA markedly elevated the nuclear localization of Nrf2 and expression of heme oxygenase-1 (HO-1) and NAD(P)H:quinone oxidoreductase-1 in hairless mouse skin. In contrast to 17-oxo-DHA, the non-electrophilic metabolic precursor 17-hydroxy-DHA was a much weaker inducer of Nrf2 activation and its target protein expression. Likewise, 17-oxo-DHA significantly enhanced nuclear translocation and transcriptional activity of Nrf2 with concomitant upregulation of HO-1 expression in cultured JB6 cells. 17-Oxo-DHA was a much stronger inducer of Nrf2-mediated antioxidant response than its parent molecule, DHA. HO-1 expression was abolished in Nrf2 knockdown JB6 cells or embryo fibroblasts from Nrf2 knock out mice. 17-Oxo-DHA also markedly reduced the level of Keap1 protein by inducing ubiquitination. Mutation of Cys151 and Cys273 in Keap1 abrogated 17-oxo-DHA-induced ubiquitination and proteasome-mediated degradation of Keap1 as well as HO-1 expression, suggesting that these cysteine residues are putative sites for 17-oxo-DHA binding. Further, Keap1 degradation stimulated by 17-oxo-DHA coincided with accumulation of the autophagy substrate, p62/SQSTM1.

High Lithium Ion Transport Through rGO-Wrapped LiNi0.6Co0.2Mn0.2O2 Cathode Material for High-Rate Capable Lithium Ion Batteries.

In this work, we show an effective ultrasonication-assisted self-assembly method under surfactant solution for a high-rate capable rGO-wrapped LiNi0.6Co0.2Mn0.2O2 (Ni-rich cathode material) composite. Ultrasonication indicates the pulverization of the aggregated bulk material into primary nanoparticles, which is effectively beneficial for synthesizing a homogeneous wrapped composite with rGO. The cathode composite demonstrates a high initial capacity of 196.5 mAh/g and a stable capacity retention of 83% after 100 cycles at a current density of 20 mA/g. The high-rate capability shows 195 and 140 mAh/g at a current density of 50 and 500 mA/g, respectively. The high-rate capable performance is attributed to the rapid lithium ion diffusivity, which is confirmed by calculating the transformation kinetics of the lithium ion by galvanostatic intermittent titration technique (GITT) measurement. The lithium ion diffusion rate (D Li) of the rGO-wrapped LiNi0.6Co0.2Mn0.2O2 composite is ca. 20 times higher than that of lithium metal plating on anode during the charge procedure, and this is demonstrated by the high interconnection of LiNi0.6Co0.2Mn0.2O2 and conductive rGO sheets in the composite. The unique transformation kinetics of the cathode composite presented in this study is an unprecedented verification example of a high-rate capable Ni-rich cathode material wrapped by highly conductive rGO sheets.

Perovskite-Based Artificial Multiple Quantum Wells.

Semiconductor quantum well structures have been critical to the development of modern photonics and solid-state optoelectronics. Quantum level tunable structures have introduced new transformative device applications and afforded a myriad of groundbreaking studies of fundamental quantum phenomena. However, noncolloidal, III-V compound quantum well structures are limited to traditional semiconductor materials fabricated by stringent epitaxial growth processes. This report introduces artificial multiple quantum wells (MQWs) built from CsPbBr3 perovskite materials using commonly available thermal evaporator systems. These perovskite-based MQWs are spatially aligned on a large-area substrate with multiple stacking and systematic control over well/barrier thicknesses, resulting in tunable optical properties and a carrier confinement effect. The fabricated CsPbBr3 artificial MQWs can be designed to display a variety of photoluminescence (PL) characteristics, such as a PL peak shift commensurate with the well/barrier thickness, multiwavelength emissions from asymmetric quantum wells, the quantum tunneling effect, and long-lived hot-carrier states. These new artificial MQWs pave the way toward widely available semiconductor heterostructures for light-conversion applications that are not restricted by periodicity or a narrow set of dimensions.

Correction: Investigation of high contrast and reversible luminescence thermochromism of the quantum confined Cs4PbBr6 perovskite solid.

Correction for 'Investigation of high contrast and reversible luminescence thermochromism of the quantum confined Cs4PbBr6 perovskite solid' by Jong H. Kim et al., Nanoscale, 2019, DOI: 10.1039/c8nr10223f.

Investigation of high contrast and reversible luminescence thermochromism of the quantum confined Cs4PbBr6 perovskite solid.

Thermochromism of organic/inorganic halide perovskites has attracted particular interest due to their potential applications as photoluminescence (PL)-based temperature sensors. However, despite the outstanding PL characteristics, their use as a thermochromic material in practical temperature ranges has been limited because of their poor thermal stability. In this study, we used the quantum confinement effect and exceptional PL quantum efficiency of the Cs4PbBr6 perovskite to demonstrate their high on/off ratio (20) and reversible PL thermochromism in the solid state in practical temperature ranges including room temperature (RT). Systematic photophysical and optical characterization studies, including exciton-phonon scattering, exciton binding energy, exciton decay dynamics, and crystal structure change, were performed to investigate the origin of this unique thermochromic PL property. The results showed that the efficient and highly reversible thermochromic PL emission of the Cs4PbBr6 perovskite is due to its desirable optical properties such as highly luminescent emission, efficient PL quenching at high temperatures, and thermally reversible structural changes.

Acetazolamide-based [18F]-PET tracer: In vivo validation of carbonic anhydrase IX as a sole target for imaging of CA-IX expressing hypoxic solid tumors.

Carbonic anhydrase IX is overexpressed in many solid tumors including hypoxic tumors and is a potential target for cancer therapy and diagnosis. Reported imaging agents targeting CA-IX are successful mostly in clear cell renal carcinoma as SKRC-52 and no candidate was approved yet in clinical trials for imaging of CA-IX. To validate CA-IX as a valid target for imaging of hypoxic tumor, we designed and synthesized novel [18F]-PET tracer (1) based on acetazolamide which is one of the well-known CA-IX inhibitors and performed imaging study in CA-IX expressing hypoxic tumor model as 4T1 and HT-29 in vivo models other than SKRC-52. [18F]-acetazolamide (1) was found to be insufficient for the specific accumulation in CA-IX expressing tumor. This study might be useful to understand in vivo behavior of acetazolamide PET tracer and can contribute to the development of successful PET imaging agents targeting CA-IX in future. Additional study is needed to understand the mechanism of poor targeting of CA-IX, as if CA-IX is not reliable as a sole target for imaging of CA-IX expressing hypoxic solid tumors.

Direct interaction with 14-3-3γ promotes surface expression of Best1 channel in astrocyte.

BACKGROUND: Bestrophin-1 (Best1) is a calcium-activated anion channel (CAAC) that is expressed broadly in mammalian tissues including the brain. We have previously reported that Best1 is expressed in hippocampal astrocytes at the distal peri-synaptic regions, called microdomains, right next to synaptic junctions, and that it disappears from the microdomains in Alzheimer's disease mouse model. Although Best1 appears to be dynamically regulated, the mechanism of its regulation and modulation is poorly understood. It has been reported that a regulatory protein, 14-3-3 affects the surface expression of numerous membrane proteins in mammalian cells. METHODS: The protein-protein interaction between Best1 and 14-3-3γ was confirmed by yeast-two hybrid assay and BiFC method. The effect of 14-3-3γ on Best1-mediated current was measured by whole-cell patch clamp technique. RESULTS: We identified 14-3-3γ as novel binding partner of Best1 in astrocytes: among 7 isoforms of 14-3-3 protein, only 14-3-3γ was found to bind specifically. We determined a binding domain on the C-terminus of Best1 which is critical for an interaction with 14-3-3γ. We also revealed that interaction between Best1 and 14-3-3γ was mediated by phosphorylation of S358 in the C-terminus of Best1. We confirmed that surface expression of Best1 and Best1-mediated whole-cell current were significantly decreased after a gene-silencingof 14-3-3γ without a significant change in total Best1 expression in cultured astrocytes. Furthermore, we discovered that 14-3-3γ-shRNA reduced Best1-mediated glutamate release from hippocampal astrocyte by recording a PAR1 receptor-induced NMDA receptor-mediated current from CA1 pyramidal neurons in hippocampal slices injected with adenovirus carrying 14-3-3γ-shRNA. Finally, through a structural modeling, we found critical amino acid residues containing S358 of Best1 exhibiting binding affinities to 14-3-3γ. CONCLUSIONS: 14-3-3γ promotes surface expression of Best1 channel in astrocytes through direct interaction.