Freestanding Penta-Twinned Palladium Nanosheets.

Control over the morphology of nanomaterials to have a 2D structure and manipulating the surface strain of nanostructures through defect control have proved to be promising for developing efficient catalysts for sustainable chemical and energy conversion. Here a one-pot aqueous synthesis route of freestanding Pd nanosheets with a penta-twinned structure (PdPT NSs) is presented. The generation of the penta-twinned nanosheet structure can be succeeded by directing the anisotropic growth of Pd under the controlled reduction kinetics of Pd precursors. Experimental and computational investigations showed that the surface atoms of the PdPT NSs are effectively under a compressive environment due to the strain imposed by their twin boundary defects. Due to the twin boundary-induced surface strain as well as the 2D structure of the PdPT NSs, they exhibited highly enhanced electrocatalytic activity for oxygen reduction reaction compared to Pd nanosheets without a twin boundary, 3D Pd nanocrystals, and commercial Pd/C and Pt/C catalysts.

Cell-engineered virus-mimetic nanovesicles for vaccination against enveloped viruses.

Enveloped viruses pose a significant threat to human health, as evidenced by the recent COVID-19 pandemic. Although current vaccine strategies have proven effective in preventing viral infections, the development of innovative vaccine technologies is crucial to fortify our defences against future pandemics. In this study, we introduce a novel platform called cell-engineered virus-mimetic nanovesicles (VNVs) and demonstrate their potential as a vaccine for targeting enveloped viruses. VNVs are generated by extruding plasma membrane-derived blebs through nanoscale membrane filters. These VNVs closely resemble enveloped viruses and extracellular vesicles (EVs) in size and morphology, being densely packed with plasma membrane contents and devoid of materials from other membranous organelles. Due to these properties, VNVs express viral membrane antigens more extensively and homogeneously than EVs expressing the same antigen. In this study, we produced severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) VNVs expressing the SARS-CoV-2 Spike glycoprotein (S) on their surfaces and assessed their preclinical efficacy as a COVID-19 vaccine in experimental animals. The administration of VNVs successfully stimulated the production of S-specific antibodies both systemically and locally, and immune cells isolated from vaccinated mice displayed cytokine responses to S stimulation.

Isolation of Phytochrome B Photobodies.

Phytochrome B (phyB), a plant photoreceptor, forms a membraneless organelle known as a photobody. Here, we present a protocol for the isolation of phyB photobodies through fluorescence-activated particle sorting from mature transgenic Arabidopsis leaves expressing phyB-GFP. This protocol involves the isolation of nuclei from frozen ground leaves using sucrose gradient centrifugation, the disruption of nuclear envelopes by sonication, and the subsequent isolation of phyB photobodies through fluorescence-activated particle sorting. We include experimental tips and notes for each step.

Phytochrome B photobody components.

Phytochrome B (phyB) is a red and far-red photoreceptor that promotes light responses. Upon photoactivation, phyB enters the nucleus and forms a molecular condensate called a photobody through liquid-liquid phase separation. Phytochrome B photobody comprises phyB, the main scaffold molecule, and at least 37 client proteins. These clients belong to diverse functional categories enriched with transcription regulators, encompassing both positive and negative light signaling factors, with the functional bias toward the negative factors. The functionally diverse clients suggest that phyB photobody acts either as a trap to capture proteins, including negatively acting transcription regulators, for processes such as sequestration, modification, or degradation or as a hub where proteins are brought into close proximity for interaction in a light-dependent manner.

Impaired BCAA catabolism in adipose tissues promotes age-associated metabolic derangement.

Adipose tissues are central in controlling metabolic homeostasis and failure in their preservation is associated with age-related metabolic disorders. The exact role of mature adipocytes in this phenomenon remains elusive. Here we describe the role of adipose branched-chain amino acid (BCAA) catabolism in this process. We found that adipocyte-specific Crtc2 knockout protected mice from age-associated metabolic decline. Multiomics analysis revealed that BCAA catabolism was impaired in aged visceral adipose tissues, leading to the activation of mechanistic target of rapamycin complex (mTORC1) signaling and the resultant cellular senescence, which was restored by Crtc2 knockout in adipocytes. Using single-cell RNA sequencing analysis, we found that age-associated decline in adipogenic potential of visceral adipose tissues was reinstated by Crtc2 knockout, via the reduction of BCAA-mTORC1 senescence-associated secretory phenotype axis. Collectively, we propose that perturbation of BCAA catabolism by CRTC2 is critical in instigating age-associated remodeling of adipose tissue and the resultant metabolic decline in vivo.

Phytochrome B photobodies are comprised of phytochrome B and its primary and secondary interacting proteins.

Phytochrome B (phyB) is a plant photoreceptor that forms a membraneless organelle called a photobody. However, its constituents are not fully known. Here, we isolated phyB photobodies from Arabidopsis leaves using fluorescence-activated particle sorting and analyzed their components. We found that a photobody comprises ~1,500 phyB dimers along with other proteins that could be classified into two groups: The first includes proteins that directly interact with phyB and localize to the photobody when expressed in protoplasts, while the second includes proteins that interact with the first group proteins and require co-expression of a first-group protein to localize to the photobody. As an example of the second group, TOPLESS interacts with PHOTOPERIODIC CONTROL OF HYPOCOTYL 1 (PCH1) and localizes to the photobody when co-expressed with PCH1. Together, our results support that phyB photobodies include not only phyB and its primary interacting proteins but also its secondary interacting proteins.

Directing Energy Flow in Core-Shell Nanostructures for Efficient Plasmon-Enhanced Electrocatalysis.

Conjugating plasmonic metals with catalytically active materials with controlled configurations can harness their light energy harvesting ability in catalysis. Herein, we present a well-defined core-shell nanostructure composed of an octahedral Au nanocrystal core and a PdPt alloy shell as a bifunctional energy conversion platform for plasmon-enhanced electrocatalysis. The prepared Au@PdPt core-shell nanostructures exhibited significant enhancements in electrocatalytic activity for methanol oxidation and oxygen reduction reactions under visible-light irradiation. Our experimental and computational studies revealed that the electronic hybridization of Pd and Pt allows the alloy material to have a large imaginary dielectric function, which can efficiently induce the shell-biased distribution of plasmon energy upon illumination and, hence, its relaxation at the catalytically active region to promote electrocatalysis.

Direct strain correlations at the single-atom level in three-dimensional core-shell interface structures.

Nanomaterials with core-shell architectures are prominent examples of strain-engineered materials. The lattice mismatch between the core and shell materials can cause strong interface strain, which affects the surface structures. Therefore, surface functional properties such as catalytic activities can be designed by fine-tuning the misfit strain at the interface. To precisely control the core-shell effect, it is essential to understand how the surface and interface strains are related at the atomic scale. Here, we elucidate the surface-interface strain relations by determining the full 3D atomic structure of Pd@Pt core-shell nanoparticles at the single-atom level via atomic electron tomography. Full 3D displacement fields and strain profiles of core-shell nanoparticles were obtained, which revealed a direct correlation between the surface and interface strain. The strain distributions show a strong shape-dependent anisotropy, whose nature was further corroborated by molecular statics simulations. From the observed surface strains, the surface oxygen reduction reaction activities were predicted. These findings give a deep understanding of structure-property relationships in strain-engineerable core-shell systems, which can lead to direct control over the resulting catalytic properties.

Surface Engineering of Palladium Nanocrystals: Decoupling the Activity of Different Surface Sites on Nanocrystal Catalysts.

The existence of various surface active sites within a nanocrystal (NC) catalyst complicates understanding their respective catalytic properties and designing an optimal catalyst structure for a desired catalytic reaction. Here, we developed a novel approach that allows unequivocal investigation on the intrinsic catalytic reactivity of the edge and terrace atoms of NCs. Through the comparison of the catalytic behaviors of edge-covered Pd NCs, which were prepared by the selective deposition of catalytically inactive Au atoms onto the edge sites of rhombic dodecahedral (RD) Pd NCs, with those of the pristine RD Pd NCs toward alkyne hydrogenation and Suzuki-Miyaura coupling reactions, we could decouple the activity of the edge and {110}-plane atoms of the Pd NCs without uncertainties. We expect that this study will provide an opportunity to scrutinize the surface properties of various NC catalysts to a more precise level and devise ideal catalysts for intended catalytic reactions.

Extracellular Vesicles Generated Using Bioreactors and their Therapeutic Effect on the Acute Kidney Injury Model.

Extracellular vesicles (EVs) are nano-sized vesicles secreted by cells, having beneficial effects for various types of regenerative processes. Although EVs have shown promising effects as therapeutic agents, these effects are difficult to research due to the limitations of EV production. In this study, an EV production method based on a flat-plate bioreactor is introduced. The bioreactor produces approximately seven times more mesenchymal stem cell-derived EVs than static culture conditions. The mechanism underlying the increased production of EVs in a flat-plate bioreactor and its application to acute kidney injury is investigated. This study describes the mechanism of EV production by demonstrating the link between EV biogenesis and increased calcium ion concentration under flow conditions. EVs secreted by cells cultured in the bioreactor have therapeutic efficacy in terms of improving kidney damage, resulting in tissue regeneration in a cisplatin-induced acute kidney injury model. This method will help overcome the limitations of EV production, and the analysis of the application of EVs will increase their reliability as well as the understanding of the use of bioreactor-derived EVs as therapeutic agents.

A novel role of CRTC2 in promoting nonalcoholic fatty liver disease.

OBJECTIVE: Diet-induced obesity is often associated with nonalcoholic fatty liver disease (NAFLD), which instigates severe metabolic disorders, including cirrhosis, hepatocellular carcinoma, and type 2 diabetes. We have shown that hepatic depletion of CREB regulated transcription co-activator (CRTC) 2 protects mice from the progression of diet-induced fatty liver phenotype, although the exact mechanism by which CRTC2 modulates this process is elusive to date. Here, we investigated the role of hepatic CRTC2 in the instigation of NAFLD in mammals. METHODS: Crtc2 liver-specific knockout (Crtc2 LKO) mice and Crtc2 flox/flox (Crtc2 f/f) mice were fed a high fat diet (HFD) for 7-8 weeks. Body weight, liver weight, hepatic lipid contents, and plasma triacylglycerol (TG) levels were determined. Western blot analysis was performed to determine Sirtuin (SIRT) 1, tuberous sclerosis complex (TSC) 2, and mammalian target of rapamycin complex (mTORC) 1 activity in the liver. Effects of Crtc2 depletion on lipogenesis was determined by measuring lipogenic gene expression (western blot analysis and qRT-PCR) in the liver as well as Oil red O staining in hepatocytes. Effects of miR-34a on mTORC1 activity and hepatic lipid accumulation was assessed by AAV-miR-34a virus in mice and Ad-miR-34a virus and Ad-anti-miR-34a virus in hepatocytes. Autophagic flux was assessed by western blot analysis after leupeptin injection in mice and bafilomycin treatment in hepatocytes. Lipophagy was assessed by transmission electron microscopy and confocal microscopy. Expression of CRTC2 and p-S6K1 in livers of human NAFLD patients was assessed by immunohistochemistry. RESULTS: We found that expression of CRTC2 in the liver is highly induced upon HFD-feeding in mice. Hepatic depletion of Crtc2 ameliorated HFD-induced fatty liver disease phenotypes, with a pronounced inhibition of the mTORC1 pathway in the liver. Mechanistically, we found that expression of TSC2, a potent mTORC1 inhibitor, was enhanced in Crtc2 LKO mice due to the decreased expression of miR-34a and the subsequent increase in SIRT1-mediated deacetylation processes. We showed that ectopic expression of miR-34a led to the induction of mTORC1 pathway, leading to the hepatic lipid accumulation in part by limiting lipophagy and enhanced lipogenesis. Finally, we found a strong association of CRTC2, miR-34a and mTORC1 activity in the NAFLD patients in humans, demonstrating a conservation of signaling pathways among species. CONCLUSIONS: These data collectively suggest that diet-induced activation of CRTC2 instigates the progression of NAFLD by activating miR-34a-mediated lipid accumulation in the liver via the simultaneous induction of lipogenesis and inhibition of lipid catabolism. Therapeutic approach to specifically inhibit CRTC2 activity in the liver could be beneficial in combating NAFLD in the future.

Multifluorescence Single Extracellular Vesicle Analysis by Time-Sequential Illumination and Tracking.

We demonstrate a fluorescence-based nanoparticle tracking analysis (NTA) system for the characterization of both the size and membrane protein expression of individual extracellular vesicles (EVs). A sheet of lasers with four different wavelengths was sequentially shone onto extracellular vesicles according to a preprogrammed schedule, providing scattering images intercalated by three fluorescent images. The presence of extracellular vesicles was tracked frame by frame from scattering images. Fluorescence-labeled membrane proteins on EVs were detected by comparing scattering and fluorescent images. The tetraspanins (CD9, CD63, and CD81) of individual HEK293 EVs analyzed by both NTA and total internal reflection fluorescence microscopy showed that the proposed NTA system can contribute to the understanding of individual extracellular vesicles.

Single-vesicle imaging and co-localization analysis for tetraspanin profiling of individual extracellular vesicles.

Extracellular vesicles (EVs) are secreted nano-sized vesicles that contain cellular proteins, lipids, and nucleic acids. Although EVs are expected to be biologically diverse, current analyses cannot adequately characterize this diversity because most are ensemble methods that inevitably average out information from diverse EVs. Here we describe a single vesicle analysis, which directly visualizes marker expressions of individual EVs using a total internal-reflection microscopy and analyzes their co-localization to investigate EV subpopulations. The single-vesicle imaging and co-localization analysis successfully illustrated the diversity of EVs and revealed distinct patterns of tetraspanin expressions. Application of the analysis demonstrated similarities and dissimilarities between the EV fractions that had been acquired from different conventional EV isolation methods. The analysis method developed in this study will provide a new and reliable tool for investigating characteristics of single EVs, and the findings of the analysis might increase understanding of the characteristics of EVs.

Characterization of the Proteolytic Activity of a Halophilic Aspergillus reticulatus Strain SK1-1 Isolated from a Solar Saltern.

Salterns are hypersaline environments that are inhabited by diverse halophilic microorganisms, including fungi. In this study, we isolated a fungal strain SK1-1 from a saltern in the Republic of Korea, which was identified as Asperillus reticulatus. This is the first reported saline-environment-derived A. reticulatus that belongs to the Aspergillus penicillioides clade and encompasses xerophilic fungi. SK1-1 was halophilic, obligately requiring NaCl for growth, with a maximum radial growth of 6%-9% (w/v) NaCl. To facilitate the biotechnological application of halophilic fungi, we screened the SK1-1 strain for proteolytic activity. Proteases have widespread applications in food processing, detergents, textiles, and waste treatment, and halophilic proteases can enable protein degradation in high salt environments. We assessed the proteolytic activity of the extracellular crude enzyme of SK1-1 using azocasein as a substrate. The crude protease exhibited maximum activity at 40-50 °C, pH 9.5-10.5, and in the absence of NaCl. It was also able to retain up to 69% of its maximum activity until 7% NaCl. Protease inhibitor assays showed complete inhibition of the proteolytic activity of crude enzymes by Pefabloc® SC. Our data suggest that the halophilic A. reticulatus strain SK1-1 produces an extracellular alkaline serine protease.

Immunostaining Extracellular Vesicles Based on an Aqueous Two-Phase System: For Analysis of Tetraspanins.

Immunostaining of extracellular vesicles (EVs) has become necessary for the characterization of EV subtypes, clarification of the EV biogenesis/cellular uptake pathway, drug delivery, etc. Immunostained EVs must be in suspension for further downstream analyses or uses. However, conventional EV immunostaining methods yielding EVs in suspension lack either sufficient recovery or staining specificity because of the washing steps. In this study, we have devised and tested a method to wash immunostained EVs with successive aqueous two-phase system (ATPS) separations. The ATPS is a liquid-liquid extraction procedure that ensures a gentle separation of target molecules. The ATPS has been successfully employed to separate EVs from other impurities with high yield and high purity. Immunostained EVs were washed with the ATPS and compared with other immunostaining methods to confirm the proposed method's high EV recovery and staining accuracy. According to the result, the ATPS-based EV immunostaining method required as low as ∼1 µg without compromise of accuracy and recovery.

Role of CRTC2 in Metabolic Homeostasis: Key Regulator of Whole-Body Energy Metabolism?

Cyclic adenosine monophosphate (cAMP) signaling is critical for regulating metabolic homeostasis in mammals. In particular, transcriptional regulation by cAMP response element-binding protein (CREB) and its coactivator, CREB-regulated transcription coactivator (CRTC), is essential for controlling the expression of critical enzymes in the metabolic process, leading to more chronic changes in metabolic flux. Among the CRTC isoforms, CRTC2 is predominantly expressed in peripheral tissues and has been shown to be associated with various metabolic pathways in tissue-specific manners. While initial reports showed the physiological role of CRTC2 in regulating gluconeogenesis in the liver, recent studies have further delineated the role of this transcriptional coactivator in the regulation of glucose and lipid metabolism in various tissues, including the liver, pancreatic islets, endocrine tissues of the small intestines, and adipose tissues. In this review, we discuss recent studies that have utilized knockout mouse models to delineate the role of CRTC2 in the regulation of metabolic homeostasis.

Heterogeneous Subcellular Origin of Exosome-Mimetic Nanovesicles Engineered from Cells.

Cell-engineered nanovesicles (CNVs) are considered as an alternative to exosomes, because they can be produced efficiently on a large scale and have been successfully reported in several applied research studies. However, CNVs may originate from various organelles, i.e., some of them may cause adverse effects on recipient cells, and their origin has not yet been identified. In this study, we air-sprayed human embryonic kidney 293 (HEK293) cells into lipid-bilayer CNVs. To identify the subcellular origin of the CNVs, we prepared nine different HEK293 cell lines by transfection with organelle-specific fluorescent protein plasmids that target the plasma membrane, peroxisome, lysosome, early endosome, late endosome, nucleus, mitochondrion, Golgi apparatus, and endoplasmic reticulum. The origin of CNVs were identified by measuring fluorescence expressions for organelle-specific markers using fluorescence nanoparticle tracking analysis (NTA). In the results, we found that CNVs derived from the plasma membrane constituted the largest portion, but CNVs derived from the other organelles comprised a non-negligible portion as well. This information will be useful to guide advanced research on outer membrane vesicles and exosome-mimetic nanovesicles engineered from cells.

Pattern of adverse events induced by aflibercept and ranibizumab: A nationwide spontaneous adverse event reporting database, 2007-2016.

Data regarding the safety of anti-vascular endothelial growth factor (anti-VEGF) treatment is limited.To compare the adverse events (AEs) induced by aflibercept and ranibizumab using a spontaneous reporting system and determine the signals.We used data from the Korea Institute of Drug Safety & Risk Management-Korea Adverse Event Reporting System Database (KIDS-KD), collected between 2007 and 2016. Differences in patient demographics, report type, reporter, causality, and serious-AEs between aflibercept and ranibizumab were compared. Metrics including proportional reporting ratio (PRR), reporting odds ratio (ROR), and information component (IC), were used to compare signals with the AEs on the drug labels in the United States of America and Korea. Logistic regression analysis was performed to identify AEs that are more likely to occur with drug use.A total of 32 aflibercept and 103 ranibizumab cases of AEs were identified. The proportion of AEs that were reported voluntarily was higher with aflibercept (50.5%) use than ranibizumab (4.9%), whereas the AEs reported by post-marketing surveillance were higher with ranibizumab (46.6%) use than aflibercept (31.3%). The percentage of AEs in patients >60 years old, reports by consumers, and the ratio of SAEs to AEs associated with aflibercept (84. %, 9.4%, and 75.0%, respectively) were higher than those of ranibizumab (77.7%, 1.9%, and 19.4%, respectively). The number of newly detected AEs after aflibercept and ranibizumab treatment was 3 and 8, respectively. Among these, conjunctivitis and medicine ineffective were not included on the aflibercept and ranibizumab labels, respectively. Endophthalmitis (OR 6.96, 95% CI 2.74-17.73) was more likely to be reported in patients with aflibercept than in patients without aflibercept, whereas medicine ineffective (OR 18.49, 95% CI 2.39-143.29) and retinal disorder (OR 7.03, 95% CI 1.60-30.96) were more likely to be reported in patients with ranibizumab than in patients without ranibizumab.New signals have been identified for aflibercept and ranibizumab. Further research is necessary to evaluate the causality of AEs that were detected as signals in this study.

Noble-Metal Nanocrystals with Controlled Facets for Electrocatalysis.

Noble-metal nanocrystals (NCs) show excellent catalytic performance for many important electrocatalysis reactions. The crystallographic properties of the facets by which the NCs are bound, closely associated with the shape of the NCs, have a profound influence on the electrocatalytic function of the NCs. To develop an efficient strategy for the synthesis of NCs with controlled facets as well as compositions, understanding of the growth mechanism of the NCs and their interaction with the chemical species involved in NC synthesis is quite important. Furthermore, understanding the facet-dependent catalytic properties of noble-metal NCs and the corresponding mechanisms for various electrocatalysis reactions will allow for the rational design of robust electrocatalysts. In this review, we summarize recently developed synthesis strategies for the preparation of mono- and bimetallic noble-metal NCs by classifying them by the type of facets through which they are enclosed and discuss the electrocatalytic applications of noble-metal NCs with controlled facets, especially for reactions associated with fuel-cell applications, such as the oxygen reduction reaction and fuel (methanol, ethanol, and formic acid) oxidation reactions.