Characterization of a New Citrus Mutant Induced by Gamma Irradiation with a Unique Fruit Shape, Gwonje-Early, and Determination of Specific Selection Markers Using Allele-Specific PCR.

Gamma-ray irradiation is one of the most widely used mutagens worldwide. We previously conducted mutation breeding using gamma irradiation to develop new Citrus unshiu varieties. Among these mutants, Gwonje-early had an ovate shape, a protrusion of the upper part of the fruit, and a large fruit size compared with wild-type (WT) fruits. We investigated the external/internal morphological characteristics and fruit sugar/acid content of Gwonje-early. Additionally, we investigated genome-wide single-nucleotide polymorphisms (SNPs) and insertion/deletion (InDel) variants in Gwonje-early using whole-genome re-sequencing. Functional annotation by Gene Ontology analysis confirmed that InDels were more commonly annotated than SNPs. To identify specific molecular markers for Gwonje-early, allele-specific PCR was performed using homozygous SNPs detected via Gwonje-early genome re-sequencing. The GJ-SNP1 and GJ-SNP4 primer sets were effectively able to distinguish Gwonje-early from the WT and other commercial citrus varieties, demonstrating their use as specific molecular markers for Gwonje-early. These findings also have important implications in terms of intellectual property rights and the variety protection of Gwonje-early. Our results may provide insights into the understanding of morphological traits and the molecular breeding mechanisms of citrus species.

Supermultipotency and unpredictability in the developing superior colliculus.

A recent study by Cheung, Pauler, Koppensteiner et al. combining lineage tracing with single-cell RNA sequencing (scRNA-seq) has revealed unexpected features of the developing superior colliculus (SC). Extremely multipotent individual progenitors generate all types of SC neurons and glial cells that were found to localize in a non-predetermined pattern, demonstrating a remarkable degree of unpredictability in SC development.

Bioconversion of citrus waste into mucic acid by xylose-fermenting Saccharomyces cerevisiae.

Mucic acid holds promise as a platform chemical for bio-based nylon synthesis; however, its biological production encounters challenges including low yield and productivity. In this study, an efficient and high-yield method for mucic acid production was developed by employing genetically engineered Saccharomyces cerevisiae expressing the NAD+-dependent uronate dehydrogenase (udh) gene. To overcome the NAD+ dependency for the conversion of pectin to mucic acid, xylose was utilized as a co-substrate. Through optimization of the udh expression system, the engineered strain achieved a notable output, producing 20 g/L mucic acid with a highest reported productivity of 0.83 g/L-h and a theoretical yield of 0.18 g/g when processing pectin-containing citrus peel waste. These results suggest promising industrial applications for the biological production of mucic acid. Additionally, there is potential to establish a viable bioprocess by harnessing pectin-rich fruit waste alongside xylose-rich cellulosic biomass as raw materials.

Enhancing Precision in L-band Electron Paramagnetic Resonance Tooth Dosimetry: Incorporating Digital Image Processing and Radiation Therapy Plans for Geometric Correction.

ABSTRACT: Following unforeseen exposure to radiation, quick dose determination is essential to prioritize potential patients that require immediate medical care. L-band electron paramagnetic resonance tooth dosimetry can be efficiently used for rapid triage as this poses no harm to the human incisor, although geometric variations among human teeth may hinder accurate dose estimation. Consequently, we propose a practical geometric correction method using a mobile phone camera. Donated human incisors were irradiated with calibrated 6-MV photon beam irradiation, and dose-response curves were developed by irradiation with a predetermined dose using custom-made poly(methyl methacrylate) slab phantoms. Three radiation treatment plans for incisors were selected and altered to suit the head phantom. The mean doses on tooth structures were calculated using a commercial treatment planning system, and the electron paramagnetic resonance signals of the incisors were measured. The enamel area was computed from camera-acquired tooth images. The relative standard uncertainty was rigorously estimated both with and without geometric correction. The effects on the electron paramagnetic resonance signal caused by axial and rotational movements of tooth samples were evaluated through finite element analysis. The mean absolute deviations of mean doses both with and without geometric correction showed marginal improvement. The average relative differences without and with geometric correction significantly decreased from 21.0% to 16.8% (p = 0.01). The geometric correction method shows potential in improving dose precision measurement with minimal delay. Furthermore, our findings demonstrated the viability of using treatment planning system doses in dose estimation for L-band electron paramagnetic resonance tooth dosimetry.

Adaptive laboratory evolution and transcriptomics-guided engineering of Escherichia coli for increased isobutanol tolerance.

As a renewable energy from biomass, isobutanol is considered as a promising alternative to fossil fuels. To biotechnologically produce isobutanol, strain development using industrial microbial hosts, such as Escherichia coli, has been conducted by introducing a heterologous isobutanol synthetic pathway. However, the toxicity of produced isobutanol inhibits cell growth, thereby restricting improvements in isobutanol titer, yield, and productivity. Therefore, the development of robust microbial strains tolerant to isobutanol is required. In this study, isobutanol-tolerant mutants were isolated from two E. coli parental strains, E. coli BL21(DE3) and MG1655(DE3), through adaptive laboratory evolution (ALE) under high isobutanol concentrations. Subsequently, 16 putative genes responsible for isobutanol tolerance were identified by transcriptomic analysis. When overexpressed in E. coli, four genes (fadB, dppC, acs, and csiD) conferred isobutanol tolerance. A fermentation study with a reverse engineered isobutanol-producing E. coli JK209 strain showed that fadB or dppC overexpression improved isobutanol titers by 1.5 times, compared to the control strain. Through coupling adaptive evolution with transcriptomic analysis, new genetic targets utilizable were identified as the basis for the development of an isobutanol-tolerant strain. Thus, these new findings will be helpful not only for a fundamental understanding of microbial isobutanol tolerance but also for facilitating industrially feasible isobutanol production.

Brn3b regulates the formation of fear-related midbrain circuits and defensive responses to visual threat.

Defensive responses to visually threatening stimuli represent an essential fear-related survival instinct, widely detected across species. The neural circuitry mediating visually triggered defensive responses has been delineated in the midbrain. However, the molecular mechanisms regulating the development and function of these circuits remain unresolved. Here, we show that midbrain-specific deletion of the transcription factor Brn3b causes a loss of neurons projecting to the lateral posterior nucleus of the thalamus. Brn3b deletion also down-regulates the expression of the neuropeptide tachykinin 2 (Tac2). Furthermore, Brn3b mutant mice display impaired defensive freezing responses to visual threat precipitated by social isolation. This behavioral phenotype could be ameliorated by overexpressing Tac2, suggesting that Tac2 acts downstream of Brn3b in regulating defensive responses to threat. Together, our experiments identify specific genetic components critical for the functional organization of midbrain fear-related visual circuits. Similar mechanisms may contribute to the development and function of additional long-range brain circuits underlying fear-associated behavior.

Identification of Late Ripening Citrus Mutant, Ara-unshiu (Citrus unshiu), and Its Selectable Marker.

'Miyagawa-wase' mandarin (Citrus unshiu Marc. cv. Miyagawa-wase early) is one of the most widely cultivated citrus varieties on Jeju Island in Korea. Mutation breeding is a useful tool for the induction of genetic diversity for the rapid creation of new plant variants. We previously reported the use of gamma irradiation for the development of new citrus varieties. Here, we report a new mutant, Ara-unshiu, with a unique late fruit ripening phenotype. We investigated the fruit morphological characteristics including weight, vertical/transverse diameter, peel thickness, hardness, and color difference, as well as sugar and acid contents of the Ara-unshiu compared to wild-type controls. We then used whole genome re-sequencing and functional annotation by gene ontology to identify and characterize single nucleotide polymorphism (SNP) and insertion/deletion (InDel) variants in the Ara-unshiu, finding a greater abundance of annotated genes containing InDels compared to SNPs. Finally, we used allele-specific PCR to identify molecular markers among the homozygous SNPs detected from the Ara-unshiu genome sequencing. We report a primer set that effectively distinguishes the Ara-unshiu from the wild-type control and other citrus varieties. Our findings provide insights into the mechanisms controlling the timing of fruit ripening and tools for the molecular breeding of citrus varieties.

Characterization and structural analysis of the endo-1,4-ß-xylanase GH11 from the hemicellulose-degrading Thermoanaerobacterium saccharolyticum useful for lignocellulose saccharification.

Xylanases are important for the enzymatic breakdown of lignocellulose-based biomass to produce biofuels and other value-added products. We report functional and structural analyses of TsaGH11, an endo-1,4-ß-xylanase from the hemicellulose-degrading bacterium, Thermoanaerobacterium saccharolyticum. TsaGH11 was shown to be a thermophilic enzyme that favors acidic conditions with maximum activity at pH 5.0 and 70 °C. It decomposes xylans from beechwood and oat spelts to xylose-containing oligosaccharides with specific activities of 5622.0 and 3959.3 U mg-1, respectively. The kinetic parameters, Km and kcat towards beechwood xylan, are 12.9 mg mL-1 and 34,015.3 s-1, respectively, resulting in kcat/Km value of 2658.7 mL mg-1 s-1, higher by 102-103 orders of magnitude compared to other reported GH11s investigated with the same substrate, demonstrating its superior catalytic performance. Crystal structures of TsaGH11 revealed a ß-jelly roll fold, exhibiting open and close conformations of the substrate-binding site by distinct conformational flexibility to the thumb region of TsaGH11. In the room-temperature structure of TsaGH11 determined by serial synchrotron crystallography, the electron density map of the thumb domain of the TsaGH11 molecule, which does not affect crystal packing, is disordered, indicating that the thumb domain of TsaGH11 has high structural flexibility at room temperature, with the water molecules in the substrate-binding cleft being more disordered than those in the cryogenic structure. These results expand our knowledge of GH11 structural flexibility at room temperature and pave the way for its application in industrial biomass degradation.

Upstream processes of citrus fruit waste biorefinery for complete valorization.

Citrus fruit waste (CW) is a useful biomass and its valorization into fuels and biochemicals has received much attention. For economic feasibility, increased efficiency of the preceding extraction and enzyme saccharification processes is necessary. However, at present, there is a lack of systematic reviews addressing these two integral upstream processes in concert for CW biorefinery. Here, the state-of-the-art advancements in enzyme extraction and saccharification processes-using which relevant essential oils, flavonoids, and sugars can be obtained-are reviewed. Specifically, the extraction options for two commercially available CW-derived products, essential oils and pectin, are discussed. With respect to enzyme saccharification, the use of an undefined commercial mixture routinely results in suboptimal sugar production. In this respect, applicable strategies for enzyme mixture customization are suggested for maximizing the hydrolytic efficiency of CW. The enzyme degradation system for CW-derived carbohydrates and its extensive application for sugar production are also discussed.

Safety and Efficacy of a Large-Bore Biliary Metallic Stent for Malignant Biliary Obstruction.

Self-expandable metallic stents (SEMSs) are typically inserted in patients with unresectable malignant biliary obstruction. However, SEMSs are susceptible to occlusion. To overcome this issue, we developed a large-bore, dumbbell-shaped, fully covered SEMS (FCSEMS-L) and compared its efficacy and safety with those of a conventional FCSEMS (FCSEMS-C) in patients with malignant biliary obstruction. METHODS: Patients with unresectable distal malignant biliary obstruction were retrospectively enrolled between January 2011 and February 2021. All patients underwent endoscopic insertion of FCSEMSs. Recurrent biliary obstruction (RBO), patient survival time, complications, and prognosis were analyzed. RESULTS: RBO occurred in 31 patients (35.6%) who received an FCSEMS-L, and in 34 (45.9%) who received an FCSEMS-C. Stent occlusion occurred in 19 patients (21.8%) who received an FCSEMS-L, and in 22 (29.7%) who received an FCSEMS-C. Stent migration occurred in 12 patients (13.8%) with an FCSEMS-L and 12 (16.2%) with an FCSEMS-C. The median time to RBO (TRBO) was 301 days with an FCSEMS-L and 203 days with an FCSEMS-C. The median survival time was 479 days with an FCSEMS-L and 523 days with an FCSEMS-C. The TRBO and patient survival time did not significantly differ between the two groups. CONCLUSIONS: There were no significant differences in efficacy and complication rates between the fully covered large bore SEMSs and conventional fully covered SEMSs.

Metal-Induced Fluorescence Quenching of Photoconvertible Fluorescent Protein DendFP.

Sensitive and accurate detection of specific metal ions is important for sensor development and can advance analytical science and support environmental and human medical examinations. Fluorescent proteins (FPs) can be quenched by specific metal ions and spectroscopically show a unique fluorescence-quenching sensitivity, suggesting their potential application as FP-based metal biosensors. Since the characteristics of the fluorescence quenching are difficult to predict, spectroscopic analysis of new FPs is important for the development of FP-based biosensors. Here we reported the spectroscopic and structural analysis of metal-induced fluorescence quenching of the photoconvertible fluorescent protein DendFP. The spectroscopic analysis showed that Fe2+, Fe3+, and Cu2+ significantly reduced the fluorescence emission of DendFP. The metal titration experiments showed that the dissociation constants (Kd) of Fe2+, Fe3+, and Cu2+ for DendFP were 24.59, 41.66, and 137.18 µM, respectively. The tetrameric interface of DendFP, which the metal ions cannot bind to, was analyzed. Structural comparison of the metal-binding sites of DendFP with those of iq-mEmerald and Dronpa suggested that quenchable DendFP has a unique metal-binding site on the ß-barrel that does not utilize the histidine pair for metal binding.

The Citrus Mutant Jedae-unshiu Induced by Gamma Irradiation Exhibits a Unique Fruit Shape and Increased Flavonoid Content.

Satsuma mandarin (Citrus unshiu Marc. cv. Miyagawa-wase) is the most widely cultivated citrus variety in Korea. Although most varieties are imported from Japan, efforts have focused on developing new domestic varieties. We produced mutants by irradiating C. unshiu Marc. cv. Miyagawa-wase scions with gamma rays and grafting them onto C. unshiu Marc. cv. Miyagawa-wase branches. We compared the characteristics of these mutants with Miyagawa-wase fruit as a control. A mutant line named Jedae-unshiu with a unique fruit shape was selected for investigation in detail. The phenotype of Jedae-unshiu fruit demonstrated vertical troughs on the flavedo, smooth albedo without rough protruding fibers, and good adhesion between peel and flesh. In addition, Jedae-unshiu had thicker peels and higher fruit hardness than the control. Higher levels of hesperetin and narirutin, representative flavonoids, accumulated in the peel and flesh of Jedae-unshiu than those of the control. Cellular-level microscopic observations of the mature fruit peels demonstrated epidermal cell disruption in the control but not in Jedae-unshiu. Our results suggest that Jedae-unshiu has high possibility for development as a good storage variety containing large amounts of flavonoids, in addition to potential for ornamental value due to the unique shape of the fruit.

Low Incidence of Hepatocellular Carcinoma after Antiviral Therapy in Patients with Chronic Hepatitis C and Hemophilia.

Background: Hepatocellular carcinoma (HCC) rarely develops in patients with chronic hepatitis C (CHC) who achieve sustained virological response (SVR). We assessed the incidence of HCC in CHC patients with hemophilia after treatment with pegylated interferon plus ribavirin (PegIFN/RBV) and direct-acting antivirals (DAAs). Methods: Patients (n = 202) were enrolled between March 2007 and July 2019. A total of 139 patients were treated with PegIFN/RBV (genotype 1, n = 98; genotype 2, n = 41). Sixty-three patients were treated with DAAs (genotype 1, n = 44; genotype 2, n = 19). The cumulative incidence rates of HCC were estimated using the Kaplan−Meier method and compared using the log-rank test. Results: For genotype 1, SVR was achieved in 78.6% (77/98) and 90.9% (40/44) of patients in the PegIFN/RBV and DAAs groups, respectively. For genotype 2, SVR was achieved in 95.1% (39/41) and 94.7% (18/19) of patients in the PegIFN/RBV and DAAs groups, respectively. Six HCC cases were identified. The cumulative incidence of HCC was 4.1% at 14 years in PegIFN/RBV and 1.7% at 5 years in DAAs. The 14-year cumulative incidence of HCC was 1.9% in the SVR group and 21.7% in the no-SVR group in the PegIFN/RBV group (p < 0.001). Conclusions: Treatment with PegIFN/RBV led to stable SVR and a low incidence of HCC. Although the follow-up period was short, DAAs led to more stable SVR than PegIFN/RBV and a low incidence of HCC in CHC patients with hemophilia.

An efficient algorithm calculating common solvent accessible volume.

The solvent accessible surface area and the solvent accessible volume are measurements commonly used in implicit solvent models to include the effect of forces exerted by solvents on the protein surfaces (or the atoms on protein surfaces). The two measurements have limitations in describing interactions between proteins (or proteins' atoms) mediated/bridged by solvents. This is because describing the interactions between proteins should be able to capture the chain of protein-solvent-protein interactions while the solvent accessible surface area or the solvent accessible volume can capture only protein-solvent interactions. If we represent the solvent as a continuous medium, we can consider an atom of a protein can effectively interact with the solvent within a certain distance from its surface (or its own solvent-interacting sphere). In this case, the protein-solvent-protein interactions can be measured by the amount of solvent interacting with two proteins' atoms at the same time (or the volume shared by the two atoms' solvent-interacting spheres excluding the volumes occupied by proteins' atoms). We call the shared volume as the common solvent accessible volume (CSAV); there has been no method developed to determine the CSAV. In this work, we propose a new sweep-line-based method that efficiently calculates the common solvent accessible volume. The performance and accuracy of the proposed sweep-line-based method are compared with those of the naïve voxel-based method. The proposed method takes log-linear time to the number of atoms involved in a CSAV calculation and linear time to the resolution. Our results, tested with 52 protein structures of various sizes, show that the proposed sweep-line-based method is superior to the voxel-based method in both computational efficiency and accuracy.

α-Dioxygenases (α-DOXs): Promising Biocatalysts for the Environmentally Friendly Production of Aroma Compounds.

Fatty aldehydes (FALs) can be derived from fatty acids (FAs) and related compounds and are frequently used as flavors and fragrances. Although chemical methods have been conventionally used, their selective biotechnological production aiming at more efficient and eco-friendly synthetic routes is in demand. α-Dioxygenases (α-DOXs) are heme-dependent oxidative enzymes biologically involved in the initial step of plant FA α-oxidation during which molecular oxygen is incorporated into the Cα -position of a FA (Cn ) to generate the intermediate FA hydroperoxide, which is subsequently converted into the shortened corresponding FAL (Cn-1 ). α-DOXs are promising biocatalysts for the flavor and fragrance industries, they do not require NAD(P)H as cofactors or redox partner proteins, and they have a broad substrate scope. Here, we highlight recent advances in the biocatalytic utilization of α-DOXs with emphasis on newly discovered cyanobacterial α-DOXs as well as analytical methods to measure α-DOX activity in vitro and in vivo.

Biochemical and Structural Analysis of a Glucose-Tolerant ß-Glucosidase from the Hemicellulose-Degrading Thermoanaerobacterium saccharolyticum.

ß-Glucosidases (Bgls) convert cellobiose and other soluble cello-oligomers into glucose and play important roles in fundamental biological processes, providing energy sources in living organisms. Bgls are essential terminal enzymes of cellulose degradation systems and attractive targets for lignocellulose-based biotechnological applications. Characterization of novel Bgls is important for broadening our knowledge of this enzyme class and can provide insights into its further applications. In this study, we report the biochemical and structural analysis of a Bgl from the hemicellulose-degrading thermophilic anaerobe Thermoanaerobacterium saccharolyticum (TsaBgl). TsaBgl exhibited its maximum hydrolase activity on p-nitrophenyl-ß-d-glucopyranoside at pH 6.0 and 55 °C. The crystal structure of TsaBgl showed a single (ß/α)8 TIM-barrel fold, and a ß8-α14 loop, which is located around the substrate-binding pocket entrance, showing a unique conformation compared with other structurally known Bgls. A Tris molecule inhibited enzyme activity and was bound to the active site of TsaBgl coordinated by the catalytic residues Glu163 (proton donor) and Glu351 (nucleophile). Titration experiments showed that TsaBgl belongs to the glucose-tolerant Bgl family. The gatekeeper site of TsaBgl is similar to those of other glucose-tolerant Bgls, whereas Trp323 and Leu170, which are involved in glucose tolerance, show a unique configuration. Our results therefore improve our knowledge about the Tris-mediated inhibition and glucose tolerance of Bgl family members, which is essential for their industrial application.

Long-term survival after CCRT and HAIC followed by ALPPS for hepatocellular carcinoma with portal vein invasion: a case report.

There are various methods for treating advanced hepatocellular carcinoma with portal vein invasion, such as systemic chemotherapy, transarterial chemoembolization, transarterial radioembolization, and concurrent chemoradiotherapy. These methods have similar clinical efficacy but are designed with a palliative aim. Herein, we report a case that experienced complete remission through "associating liver partition and portal vein ligation for staged hepatectomy (ALPPS)" after concurrent chemoradiotherapy and hepatic artery infusion chemotherapy. In this patient, concurrent chemoradiotherapy and hepatic artery infusion chemotherapy induced substantial tumor shrinkage, and hypertrophy of the nontumor liver was sufficiently induced by portal vein ligation (stage 1 surgery) followed by curative resection (stage 2 surgery). Using this approach, long-term survival with no evidence of recurrence was achieved at 16 months. Therefore, the optimal use of ALPPS requires sufficient consideration in cases of significant hepatocellular carcinoma shrinkage for curative purposes.

Two novel cyanobacterial α-dioxygenases for the biosynthesis of fatty aldehydes.

α-Dioxygenases (α-DOXs) are known as plant enzymes involved in the α-oxidation of fatty acids through which fatty aldehydes, with a high commercial value as flavor and fragrance compounds, are synthesized as products. Currently, little is known about α-DOXs from non-plant organisms. The phylogenic analysis reported here identified a substantial number of α-DOX enzymes across various taxa. Here, we report the functional characterization and Escherichia coli whole-cell application of two novel α-DOXs identified from cyanobacteria: CalDOX from Calothrix parietina and LepDOX from Leptolyngbya sp. The catalytic behavior of the recombinantly expressed CalDOX and LepDOX revealed that they are heme-dependent like plant α-DOXs but exhibit activities toward medium carbon fatty acids ranging from C10 to C14 unlike plant α-DOXs. The in-depth molecular investigation of cyanobacterial α-DOXs and their application in an E. coli whole system employed in this study is useful not only for the understanding of the molecular function of α-DOXs, but also for their industrial utilization in fatty aldehyde biosynthesis.Key points• Two novel α-dioxygenases from Cyanobacteria are reported• Both enzymes prefer medium-chain fatty acids• Both enzymes are useful for fatty aldehyde biosynthesis.

Increased Production of Colanic Acid by an Engineered Escherichia coli Strain, Mediated by Genetic and Environmental Perturbations.

Colanic acid (CA) is a major exopolysaccharide synthesized by Escherichia coli that serves as a constituent of biofilm matrices. CA demonstrates potential applications in the food, cosmetics, and pharmaceutical industry. Moreover, L-fucose, a monomeric constituent of CA, exhibits various physiological activities, such as antitumor, anti-inflammatory, and skin-whitening. Here, the effects of genetic and environmental perturbations were investigated for improving CA production by E. coli. When rcsF, a positive regulator gene of CA synthesis, was expressed in E. coli ΔwaaF, a CA-producing strain constructed previously, the CA titer increased to 3051.2 mg/L as compared to 2052.8 mg/L observed with E. coli ΔwaaF. Among the environmental factors tested, namely, osmotic and oxidative stresses and pH, pH was a primary factor that significantly improved CA production. When the pH of the culture medium of E. coli ΔwaaF + rcsF was maintained at 7, the CA titer significantly increased to 4351.6 mg/L. The CA yield obtained with E. coli ΔwaaF + rcsF grown at pH 7 was 5180.4 mg CA/g dry cell weight, which is the highest yield of CA reported so far. This engineered E. coli system with optimization of environmental conditions can be employed for fast and economically-feasible production of CA.

Stable Cycling of a 4 V Class Lithium Polymer Battery Enabled by In Situ Cross-Linked Ethylene Oxide/Propylene Oxide Copolymer Electrolytes with Controlled Molecular Structures.

Commercial lithium-ion batteries are vulnerable to fire accidents, mainly due to volatile and flammable liquid electrolytes. Although solid polymer electrolytes (SPEs) are considered promising alternatives with antiflammability and processability for roll-to-roll mass production, several requirements have not yet been fulfilled for a viable lithium polymer battery. Such requirements include ionic conductivity, electrochemical stability, and interfacial resistance. In this work, the ionic conductivity of the SPEs is optimized by controlling the molecular weight and structural morphology of the plasticizers as well as introducing propylene oxide (PO) groups. Electrochemical stability is also enhanced using ethylene oxide (EO)/PO copolymer electrolytes, making the SPEs compatible with high-Ni LiNixCoyMn1-x-yO2 cathodes. The in situ cross-linking method, in which a liquid precursor first wets the electrode and is then solidified by a subsequent thermal treatment, enables the SPEs to soak into the 60 µm thick electrode with a high loading density of more than 8 mg cm-2. Thus, interfacial resistance between the SPE and the electrode is minimized. By using the in situ cross-linked EO/PO copolymer electrolytes, we successfully demonstrate a 4 V class lithium polymer battery, which performs stable cycling with a marginal capacity fading even over 100 cycles.