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The genetic compensation response (GCR) has recently been proposed as a possible explanation for the phenotypic discrepancies between gene-knockout and gene-knockdown1,2; however, the underlying molecular mechanism of the GCR remains uncharacterized. Here, using zebrafish knockdown and knockout models of the capn3a and nid1a genes, we show that mRNA bearing a premature termination codon (PTC) promptly triggers a GCR that involves Upf3a and components of the COMPASS complex. Unlike capn3a-knockdown embryos, which have small livers, and nid1a-knockdown embryos, which have short body lengths2, capn3a-null and nid1a-null mutants appear normal. These phenotypic differences have been attributed to the upregulation of other genes in the same families. By analysing six uniquely designed transgenes, we demonstrate that the GCR is dependent on both the presence of a PTC and the nucleotide sequence of the transgene mRNA, which is homologous to the compensatory endogenous genes. We show that upf3a (a member of the nonsense-mediated mRNA decay pathway) and components of the COMPASS complex including wdr5 function in GCR. Furthermore, we demonstrate that the GCR is accompanied by an enhancement of histone H3 Lys4 trimethylation (H3K4me3) at the transcription start site regions of the compensatory genes. These findings provide a potential mechanistic basis for the GCR, and may help lead to the development of therapeutic strategies that treat missense mutations associated with genetic disorders by either creating a PTC in the mutated gene or introducing a transgene containing a PTC to trigger a GCR.
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Códon sem Sentido/genética , Teste de Complementação Genética , Complexos Multiproteicos/metabolismo , RNA Mensageiro/genética , Peixe-Zebra/genética , Animais , Proteínas de Ligação ao Cálcio/deficiência , Proteínas de Ligação ao Cálcio/genética , Deleção de Genes , Células HCT116 , Histonas/metabolismo , Humanos , Complexos Multiproteicos/química , Degradação do RNAm Mediada por Códon sem Sentido , Organismos Geneticamente Modificados , Proteínas de Peixe-Zebra/deficiência , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
Amidst these growing sustainability concerns, producing NH4 + via electrochemical NO3 - reduction reaction (NO3RR) emerges as a promising alternative to the conventional Haber-Bosch process. In a pioneering approach, this study introduces Ru incorporation into Co3O4 lattices at the nanoscale and further couples it with electroreduction conditioning (ERC) treatment as a strategy to enhance metal oxide reducibility and induce oxygen vacancies, advancing NH4 + production from NO3RR. Here, supported by a suite of ex situ and in situ characterization measurements, the findings reveal that Ru enrichment promotes Co species reduction and oxygen vacancy formation. Further, as evidenced by the theoretical calculations, Ru integration lowers the energy barrier for oxygen vacancy formation, thereby facilitating a more energy-efficient NO3RR-to-NH4 + pathway. Optimal catalytic activity is realized with a Ru loading of 10 at.% (named 10Ru/Co3O4), achieving a high NH4 + production rate (98 nmol s-1 cm-2), selectivity (97.5%) and current density (≈100 mA cm-2) at -1.0 V vs RHE. The findings not only provide insights into defect engineering via the incorporation of secondary sites but also lay the groundwork for innovative catalyst design aimed at improving NH4 + yield from NO3RR. This research contributes to the ongoing efforts to develop sustainable electrochemical processes for nitrogen cycle management.
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The surface photochemical activity of goethite, which occurs widely in surface soils and sediments, plays a crucial role in the environmental transformation of various pollutants and natural organic matter. This study systemically investigated the mechanism of different types of surface hydroxyl groups on goethite in generating reactive oxygen species (ROSs) and Fe(III) reduction under sunlight irradiation. Surface hydroxyl groups were found to induce photoreductive dissolution of Fe(III) at the goethite-water interface to produce Fe2+(aq), while promoting the production of ROSs. Substitution of the surface hydroxyl groups on goethite by fluoride significantly inhibited the photochemical activity of goethite, demonstrating their important role in photochemical activation of goethite. The results showed that the surface hydroxyl groups (especially the terminating hydroxyl groups, ≡FeOH) led to the formation of Fe(III)-hydroxyl complexes via ligand-metal charge transfer on the goethite surface upon photoexcitation, facilitating the production of Fe2+(aq) and â¢OH. The bridging hydroxyl groups (≡Fe2OH) were shown to mainly catalyze the production of H2O2, leading to the subsequent light-driven Fenton reaction to produce â¢OH. These findings provide important insights into the activation of molecular oxygen on the goethite surface driven by sunlight in the environment, and the corresponding degradation of anthropogenic and natural organic compounds caused by the generated ROSs.
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Espécies Reativas de Oxigênio , Espécies Reativas de Oxigênio/química , Oxirredução , Compostos Férricos/química , Compostos de Ferro/química , Ferro/química , Minerais/química , Radical Hidroxila/química , Processos FotoquímicosRESUMO
Sophora alopecuroides has important uses in medicine, wind breaking, and sand fixation. The CHY-zinc-finger and RING-finger (CHYR) proteins are crucial for plant growth, development, and environmental adaptation; however, genetic data regarding the CHYR family remain scarce. We aimed to investigate the CHYR gene family in S. alopecuroides and its response to abiotic stress, and identified 18 new SaCHYR genes from S. alopecuroides whole-genome data, categorized into 3 subclasses through a phylogenetic analysis. Gene structure, protein domains, and conserved motifs analyses revealed an exon-intron structure and conserved domain similarities. A chromosome localization analysis showed distribution across 12 chromosomes. A promoter analysis revealed abiotic stress-, light-, and hormone-responsive elements. An RNA-sequencing expression pattern analysis revealed positive responses of SaCHYR genes to salt, alkali, and drought stress. SaCHYR4 overexpression considerably enhanced alkali and drought tolerance in Arabidopsis thaliana. These findings shed light on SaCHYR's function and the resistance mechanisms of S. alopecuroides, presenting new genetic resources for crop resistance breeding.
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Regulação da Expressão Gênica de Plantas , Família Multigênica , Filogenia , Proteínas de Plantas , Sophora , Estresse Fisiológico , Estresse Fisiológico/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Sophora/genética , Arabidopsis/genética , Genoma de Planta , Secas , Cromossomos de Plantas/genéticaRESUMO
Photonic integrated circuits (PICs) based on gallium nitride (GaN) platforms have been widely explored for various applications at C-band (1530â nmâ¼1565â nm) and visible light wavelength range. However, for O-band (1260â nmâ¼1360â nm) commonly used in short reach/cost sensitive markets, GaN-based PICs still have not been fully investigated. In this article, a microring resonator with an intrinsic Q-factor of â¼2.67 × 104 and an extinction ratio (ER) of 35.1â dB at 1319.9â nm and 1332.1â nm, is monolithically integrated with a transverse electric-polarized focusing grating coupler and a ridge waveguide on a GaN-on-sapphire platform. This shows a great potential to further exploit the optical properties of GaN materials and integrate GaN-based PICs with the mature GaN active electronic and optoelectronic devices to form a greater platform of optoelectronic-electronic integrated circuits (OEICs) for data-center and telecom applications.
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Electrochemical generation of hydrogen peroxide (H2 O2 ) is an attractive alternative to the energy-intensive anthraquinone oxidation process. Metal-free carbon-based materials such as graphene show great promise as efficient electrocatalysts in alkaline media. In particular, the graphene edges possess superior electrochemical properties than the basal plane. However, identification and enhancement of the catalytically active sites at the edges remain challenging. Furthermore, control of surface wettability to enhance gas diffusion and promote the performance in bulk electrolysis is largely unexplored. Here, a metal-free edge-rich vertical graphene catalyst is synthesized and exhibits a superior performance for H2 O2 production, with a high onset potential (0.8 V versus reversible hydrogen electrode (RHE) at 0.1 mA cm-2 ) and 100% Faradaic efficiency at various potentials. By tailoring the oxygen-containing functional groups using various techniques of electrochemical oxidation, thermal annealing and oxygen plasma post-treatment, the edge-bound in-plane ether-type (COC) groups are revealed to account for the superior catalytic performance. To manipulate the surface wettability, a simple vacuum-based method is developed to effectively induce material hydrophobicity by accelerating hydrocarbon adsorption. The increased hydrophobicity greatly enhances gas transfer without compromising the Faradaic efficiency, enabling a H2 O2 productivity of 1767 mmol gcatalyst -1 h-1 at 0.4 V versus RHE.
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MAIN CONCLUSION: Overexpression of SaAQP can improve the salt tolerance of transgenic soybean hairy roots and A. thaliana. Salt stress severely affects crop yield and food security. There is a need to improve the salt tolerance of crops, but the discovery and utilization of salt-tolerance genes remains limited. Owing to its strong stress tolerance, Sophora alopecuroides is ideal for the identification of salt-tolerance genes. Therefore, we aimed to screen and identify the salt-tolerance genes in S. alopecuroides. With a yeast expression library of seedlings, salt-tolerant genes were screened using a salt-containing medium to simulate salt stress. By combining salt-treatment screening and transcriptome sequencing, 11 candidate genes related to salt tolerance were identified, including genes for peroxidase, inositol methyltransferase, aquaporin, cysteine synthase, pectinesterase, and WRKY. The expression dynamics of candidate genes were analyzed after salt treatment of S. alopecuroides, and salt tolerance was verified in yeast BY4743. The candidate genes participated in the salt-stress response in S. alopecuroides, and their overexpression significantly improved the salt tolerance of yeast. Salt tolerance mediated by SaAQP was further verified in soybean hairy roots and Arabidopsis thaliana, and it was found that SaAQP might enhance the salt tolerance of A. thaliana by participating in a reactive oxygen species scavenging mechanism. This result provides new genetic resources in plant breeding for salt resistance.
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Tolerância ao Sal , Sophora , Regulação da Expressão Gênica de Plantas , Melhoramento Vegetal , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Tolerância ao Sal/genética , Sophora/genética , Sophora/metabolismo , Estresse FisiológicoRESUMO
In order to reduce the overpotential of hydrogen evolution reaction (HER), the ternary coating Co-W-P was deposited on the surface of the nickel foam by electrochemical deposition to obtain a highly active electrode. Based on the measured double layer capacitance (Cdl) and HER activity, there is volcanic behavior between the intrinsic activity of Co-W-P and the Co:W ratio in the electrolyte. W and P play different roles in the formation of nanoparticles, and work together to achieve the large electrochemical surface area and excellent activity. When applied to the modification of other catalysts (Ni-P and Fe-P), the higher intrinsic activity was obtained after the introduction of W.
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Salt stress seriously restricts crop yield and quality, leading to an urgent need to understand its effects on plants and the mechanism of plant responses. Although phytohormones are crucial for plant responses to salt stress, the role of phytohormone signal transduction in the salt stress responses of stress-resistant species such as Sophora alopecuroides has not been reported. Herein, we combined transcriptome and metabolome analyses to evaluate expression changes of key genes and metabolites associated with plant hormone signal transduction in S. alopecuroides roots under salt stress for 0 h to 72 h. Auxin, cytokinin, brassinosteroid, and gibberellin signals were predominantly involved in regulating S. alopecuroides growth and recovery under salt stress. Ethylene and jasmonic acid signals may negatively regulate the response of S. alopecuroides to salt stress. Abscisic acid and salicylic acid are significantly upregulated under salt stress, and their signals may positively regulate the plant response to salt stress. Additionally, salicylic acid (SA) might regulate the balance between plant growth and resistance by preventing reduction in growth-promoting hormones and maintaining high levels of abscisic acid (ABA). This study provides insight into the mechanism of salt stress response in S. alopecuroides and the corresponding role of plant hormones, which is beneficial for crop resistance breeding.
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Estresse Salino/genética , Transdução de Sinais/genética , Sophora/genética , Ácido Abscísico/metabolismo , Brassinosteroides/metabolismo , Citocininas/genética , Etilenos/metabolismo , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica de Plantas/genética , Ácidos Indolacéticos/metabolismo , Melhoramento Vegetal/métodos , Reguladores de Crescimento de Plantas/genética , Proteínas de Plantas/genética , Ácido Salicílico/metabolismo , Tolerância ao Sal/genética , Sophora/metabolismo , Estresse Fisiológico/genética , Transcriptoma/genética , Regulação para Cima/genéticaRESUMO
Salt stress is the main abiotic stress that limits crop yield and agricultural development. Therefore, it is imperative to study the effects of salt stress on plants and the mechanisms through which plants respond to salt stress. In this study, we used transcriptomics and metabolomics to explore the effects of salt stress on Sophora alopecuroides. We found that salt stress incurred significant gene expression and metabolite changes at 0, 4, 24, 48, and 72 h. The integrated transcriptomic and metabolomic analysis revealed that the differentially expressed genes (DEGs) and differential metabolites (DMs) obtained in the phenylpropanoid biosynthesis pathway were significantly correlated under salt stress. Of these, 28 DEGs and seven DMs were involved in lignin synthesis and 23 DEGs and seven DMs were involved in flavonoid synthesis. Under salt stress, the expression of genes and metabolites related to lignin and flavonoid synthesis changed significantly. Lignin and flavonoids may participate in the removal of reactive oxygen species (ROS) in the root tissue of S. alopecuroides and reduced the damage caused under salt stress. Our research provides new ideas and genetic resources to study the mechanism of plant responses to salt stress and further improve the salt tolerance of plants.
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Regulação da Expressão Gênica de Plantas , Metaboloma , Fenilpropionatos/metabolismo , Proteínas de Plantas/metabolismo , Tolerância ao Sal , Sophora/metabolismo , Transcriptoma , Perfilação da Expressão Gênica , Lignina/biossíntese , Proteínas de Plantas/genética , Sophora/genética , Sophora/crescimento & desenvolvimento , Estresse FisiológicoRESUMO
A stable MnOx @C@MnOx core-shell heterostructure consisting of vertical MnOx nanosheets grown evenly on the surface of the MnOx @carbon nanowires are obtained by simple liquid phase method combined with thermal treatment. The hierarchical MnOx @C@MnOx heterostructure electrode possesses a high specific capacitance of 350 F g-1 and an excellent cycle performance owing to the existence of the pore structure among the ultrasmall MnOx nanoparticles and the rapid transmission of electrons between the active material and carbon coating layer. Particularly, according to the in situ Raman spectra analysis, no characteristic peaks corresponding to MnOOH are found during charging/discharging, indicating that pseudocapacitive behavior of the MnOx electrode have no relevance to the intercalation/deintercalation of protons (H+ ) in the electrolyte. Further combining in situ X-ray powder diffraction analysis, the diffraction peak of α-MnO2 can be detected in the process of charging, while Mn3 O4 phase is found in discharge products. Therefore, these results demonstrate that the MnOx undergoes a reversible phase transformation reaction of Mn3 O4 âα-MnO2 . Moreover, the assembled all-solid-state asymmetric supercapacitor with a MnOx @C@MnOx electrode delivers a high energy density of 23 Wh kg-1 , an acceptable power density of 2500 W kg-1 , and an excellent cyclic stability performance of 94% after 2000 cycles, showing the potential for practical application.
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DNA origami methods enable the fabrication of various nanostructures and nanodevices, but their effective use depends on an understanding of their structural and mechanical properties and the effects of basic structural features. Frequency-modulation atomic force microscopy is introduced to directly characterize, in aqueous solution, the crossover regions of sets of 2D DNA origami based on different crossover/nick designs. Rhombic-shaped nanostructures formed under the influence of flexible crossovers placed between DNA helices are observed in DNA origami incorporating crossovers every 3, 4, or 6 DNA turns. The bending rigidity of crossovers is determined to be only one-third of that of the DNA helix, based on interhelical electrostatic forces reported elsewhere, and the measured pitches of the 3-turn crossover design rhombic-shaped nanostructures undergoing negligible bending. To evaluate the robustness of their structural integrity, they are intentionally and simultaneously stressed using force-controlled atomic force microscopy. DNA crossovers are verified to have a stabilizing effect on the structural robustness, while the nicks have an opposite effect. The structural and mechanical properties of DNA origami and the effects of crossovers and nicks revealed in this paper can provide information essential for the design of versatile DNA origami structures that exhibit specified and desirable properties.
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DNA/química , Nanoestruturas/química , Estudos Cross-Over , Microscopia de Força Atômica , Nanotecnologia/métodos , Conformação de Ácido NucleicoRESUMO
Lithium cathode materials have been considered as promising candidates for energy storage applications because of their high power/energy densities, low cost, and low toxicity. However, the Li/Ni cation mixing limits their application as practical electrode materials. The cation mixing of lithium transition-metal oxides, which was first considered only as the origin of performance degeneration, has recently been reconsidered as a way to stabilize the structure of active materials. Here we find that as the duration of the post-synthesis thermal treatment (at 500 °C) of LiNi1/3Co1/3Mn1/3O2 (NCM) was increased, the Li/Ni molar ratio in the final product was found to decrease, and this was attributed to the reduction in nickel occupying lithium sites; the cation mixing subtly changed; and those subtle variations remarkably influence their cycling performance. The cathode material with appropriate cation mixing exhibits a much slower voltage decay and capacity fade during long-term cycling. Combining X-ray diffraction, Rietveld analysis, the Fourier transform infrared technique, field-emission scanning electron microscopy, and electrochemical measurements, we demonstrate that an optimal degree of Ni2+ occupancy in the lithium layer enhances the electrochemical performance of layered NMC materials and that this occurs through a "pillaring" effect. The results provide new insights into "cation mixing" as a new concept for material design utilization of layered cathodes for lithium-ion batteries, thereby promoting their further application in lithium-ion batteries with new functions and properties.
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Raman spectroscopy gas detection technology which uses a single wavelength laser detection of gas samples without contact and loss is suitable for dissolved gas detection in oil-immersed transformers. Combined with the features of Raman spectral lines, the analysis of the detection mechanism in Raman spectroscopy technology has been made. Raman spectral lines have been presented in the expression of the convolution of Lorentz function and Gaussian function, which shows preferable Raman spectrum peak linear outline basic characteristics. In this paper, the basic features of the peak height, the peak position and the half peak width, are the main targets of peak searching, and the fundamental purpose of this paper is to obtain qualitative and quantitative analysis of the sample. Therefore, the experimental data of Raman spectroscopy is designed based on the comparison method to realize the automatic peak seeking model to achieve the detection target. Therefore, according to the automatic peak searching model based on the comparison method is designed to achieve the detection target. The simulation results of using peak searching model in the Voigt linear model show that spectrum peak height and peak position are in conformity with the theory in the test experiment. Based on the establishment of Raman spectroscopy detection platform aiming at the dissolved gas in transformer oil, the analysis of the experimental data show that the actual values of the half peak height and width are (8.7ï¼11.5)(cm-1)in the Voigt linear model with deviations. Setting the value as 10.257 cm-1 to modify the parameter, then compared with the research results, it shows that the modified Voigt linear model and the peak searching model have better adaptability and practicability. Combined with the gas detection in the experimental platform in the experimental platform of Raman spectroscopy,detection of seven kinds of transformer fault characteristic gas and analysis of peak searching have been completed effectively. In terms of methane gas, the linear relationship among the unit gas content, Raman characteristic peak intensity and the area has been obtained, which has laid a foundation for the quantitative analysis of the dissolved gas in transformer oil.
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Porous Mn3O4 hexagonal nanoplates were synthesized through annealing the hydrohausmannite precursor obtained by a one-pot hydrothermal process and by precisely controlling the concentrations of potassium hydroxide and glucose. The effect of potassium hydroxide and glucose on the growth of hexagonal nanoplates was investigated, and a growth mechanism was also proposed. Due to its abundant pores, the pure Mn3O4-based electrode exhibits excellent cycling stability with 100% capacity retention after 5000 cycles. The asymmetric supercapacitor exhibited high performance with an energy density of 17.276 W h kg(-1) at a power density of 207.3 W kg(-1) in a wide potential window of 1.5 V.
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Monodispersed LiFePO4 nanocrystals with diverse morphologies were successfully synthesized via a mild and controllable solvothermal approach with a mixture of ethylene glycol and oleic acid as the solvent. Morphology evolution of LiFePO4 nanoparticles from nanoplates to nanorods can be simply realized by varying the volume ratio of oleic acid to ethylene glycol. Moreover, the mechanism of competitive adsorption between ethylene glycol and oleic acid was proposed for the formation of different morphologies. Electrochemical measurements show that the LiFePO4/C nanorods have an initial discharge capacity of 155 mA h g(-1) at 0.5 C with a capacity retention of 80% at a high rate of 5 C, which confirms that LiFePO4/C nanorods exhibit excellent rate capability and cycling stability.
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Organic small molecules are proven to be capable of passivating the bulk/interfacial defects in inorganic perovskite solar cells. Considering the burdensome situation to screen the functional small molecules, we employ a modified machine learning (ML) strategy to guide screening suitable small molecules toward efficient solar cells through three modified ML algorithms to construct the prediction model: (i) random forest algorithm (RF), (ii) support vector machine algorithm (SVR), and (iii) XGBoost. Among them, the XGBoost algorithm displays a better overall predictive performance, whereby the R2 index reaches 0.939. Accordingly, eight small molecules are selected to modify the interface of perovskite films, and both the theoretical and experimental results certify that the difluorobenzylamine with additional fluorine atoms has a better interface modification effect among the small molecules containing functional groups, e.g., the benzene ring and amino group. The high accuracy of the modified machine learning model enables us to simplify the small-molecule screening process and form an important step for ongoing developments in perovskite solar cells and other optoelectronic devices.
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This paper presents a high-performance MEMS accelerometer with a DC/AC electrostatic stiffness tuning capability based on double-sided parallel plates (DSPPs). DC and AC electrostatic tuning enable the adjustment of the effective stiffness and the calibration of the geometric offset of the proof mass, respectively. A dynamical model of the proposed accelerometer was developed considering both DC/AC electrostatic tuning and the temperature effect. Based on the dynamical model, a self-centering closed loop is proposed for pulling the reference position of the force-to-rebalance (FTR) to the geometric center of DSPP. The self-centering accelerometer operates at the optimal reference position by eliminating the temperature drift of the readout circuit and nulling the net electrostatic tuning forces. The stiffness closed-loop is also incorporated to prevent the pull-in instability of the tuned low-stiffness accelerometer under a dramatic temperature variation. Real-time adjustments of the reference position and the DC tuning voltage are utilized to compensate for the residue temperature drift of the proposed accelerometer. As a result, a novel controlling approach composed of a self-centering closed loop, stiffness-closed loop, and temperature drift compensation is achieved for the accelerometer, realizing a temperature drift coefficient (TDC) of approximately 7 µg/°C and an Allan bias instability of less than 1 µg.
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Immune checkpoint inhibitor (ICI) therapy, which targets programmed cell death protein 1, has demonstrated enhanced survival outcomes in numerous patients with cancer. Historically, individuals with autoimmune diseases have been excluded from clinical trials involving cancer immunotherapies due to concerns about the potential worsening of their underlying autoimmune conditions. In the present case report, a patient with non-small cell lung cancer and bullous pemphigoid (BP) who underwent treatment with the ICI pembrolizumab is described. In this specific clinical case, no severe exacerbation of the underlying autoimmune disease was observed. Contrarily, the patient not only tolerated pembrolizumab well but also experienced amelioration of the BP lesions after the treatment. This case challenges the conventional exclusion criteria for ICI therapy in patients with autoimmune diseases, suggesting the potential safety and efficacy of such treatments in this specific population. However, further investigations and larger-scale studies are warranted to validate these findings and provide a more comprehensive understanding of the implications of ICI therapy in patients with autoimmune comorbidities.
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Lithium-sulfur (Li-S) batteries represent the most promising next-generation energy storage systems because of their high theoretical specific capacity and energy density. However, the severe shuttle effect and volume expansion of sulfur cathodes have impeded their commercial viability. Hence, accelerating the conversion of lithium polysulfides (LiPSs) is crucial for achieving efficient Li-S batteries. In this study, we employ a straightforward electrostatic self-assembly method to coat ultra-thin MXene nanosheets onto a S@MnO2 core-shell structure, resulting in a highly conductive three-dimensional network. This unique structure not only suppresses the diffusion of LiPSs but also accelerates electron and ion transfer, ensuring a rapid and efficient conversion of LiPSs. The CV curves of symmetrical cells and the Li2S deposition curves demonstrate a significant improvement in the catalytic performance of batteries with S@MnO2@MXene. The capacity of Li-S batteries achieved an impressive 842 mAh/g at the current density of 1C, with a minimal capacity decay of only 0.84 mAh/g per cycle within 500 cycles. Additionally, increasing the sulfur loading mass to 5.88 mg cm-2 resulted in an areal capacity of 6.33 mAh cm-2, demonstrating practical application potential.