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Although all‐solid‐state batteries are suggested as a means to tackle the safety concerns associated with current Li‐ion batteries, there is presently a lack of comprehensive understanding regarding their thermal safety. In this context, critical factors contributing to the thermal runaway of thiophosphate solid electrolytes with charged Li1‐xNi0.8Co0.1Mn0.1O2 (NCM) under thermal and mechanical abuse conditions are demonstrated, considering parameters such as heating rate under thermal abuse conditions and the hybridization of S atom in structure. In particular, the thermal behavior of various solid electrolytes, including thiophosphates, thioantimonates, and halides, is investigated to clarify critical elements in Li6PS5Cl (LPSCl) contributing to its thermal instability when combined with charged NCM. Various ex situ analyses, along with density functional theory calculations, reveal a correlation between the hybridization of S atoms and the thermal instability of solid electrolytes, suggesting that sulfur acts as a key element triggering the thermal runaway of sulfide‐based solid electrolytes. [ABSTRACT FROM AUTHOR]

The current COVID-19 mRNA vaccines were developed and applied for pandemic-emergent conditions. These vaccines use a small piece of the virus's genetic material (mRNA) to stimulate an immune response against COVID-19. However, their potential effects on individuals with chronic inflammatory conditions and vaccination routes remain questionable. Therefore, we investigated the effects of mRNA vaccines in a mouse model of chronic inflammation, focusing on their cardiac toxicity and immunogenicity dependent on the injection route. mRNA vaccine intravenous administration with or without chronic inflammation exacerbated cardiac pericarditis and myocarditis; immunization induced mild inflammation and inflammatory cytokine IL-1beta and IL-6 production in the heart. Further, IV mRNA vaccination induced cardiac damage in LPS chronic inflammation, particularly serum troponin I (TnI), which dramatically increased. IV vaccine administration may induce more cardiotoxicity in chronic inflammation. These findings highlight the need for further research to understand the underlying mechanisms of mRNA vaccines with chronic inflammatory conditions dependent on injection routes. [ABSTRACT FROM AUTHOR]

Developing new adjuvants that can effectively induce humoral and cellular immune responses while broadening the immune response is of great value. In this study, we aimed to develop single-stranded RNA adjuvants expressing (1) granulocyte monocyte colony-stimulating factor or (2) interleukin 18 based on the encephalomyocarditis virus internal ribosome entry site; we also tested their efficacy in combination with ovalbumin or inactivated influenza vaccines. Notably, cytokine-expressing RNA adjuvants increased the expression of antigen-presenting cell activation markers in mice. Specifically, when combined with ovalbumin, RNA adjuvants expressing granulocyte monocyte colony-stimulating factor increased CD4+ T-cell responses, while those expressing interleukin 18 increased CD8+ T-cell responses. Cytokine-expressing RNA adjuvants further increased the frequency of polyclonal T cells with the influenza vaccine and reduced the clinical illness scores and weight loss of mice after viral challenge. Collectively, our results suggest that cytokine-expressing RNA adjuvants can be applied to protein-based or inactivated vaccines to increase their efficacy. [ABSTRACT FROM AUTHOR]

The E6 and E7 proteins of specific subtypes of human papillomavirus (HPV), including HPV 16 and 18, are highly associated with cervical cancer as they modulate cell cycle regulation. The aim of this study was to investigate the potential antitumor effects of a messenger RNA‐HPV therapeutic vaccine (mHTV) containing nononcogenic E6 and E7 proteins. To achieve this, C57BL/6j mice were injected with the vaccine via both intramuscular and subcutaneous routes, and the resulting effects were evaluated. mHTV immunization markedly induced robust T cell‐mediated immune responses and significantly suppressed tumor growth in both subcutaneous and orthotopic tumor‐implanted mouse model, with a significant infiltration of immune cells into tumor tissues. Tumor retransplantation at day 62 postprimary vaccination completely halted progression in all mHTV‐treated mice. Furthermore, tumor expansion was significantly reduced upon TC‐1 transplantation 160 days after the last immunization. Immunization of rhesus monkeys with mHTV elicited promising immune responses. The immunogenicity of mHTV in nonhuman primates provides strong evidence for clinical application against HPV‐related cancers in humans. All data suggest that mHTV can be used as both a therapeutic and prophylactic vaccine. [ABSTRACT FROM AUTHOR]

Argyrodite‐type Li6PS5Cl (LPSCl) has attracted much attention as a solid electrolyte for all‐solid‐state batteries (ASSBs) because of its high ionic conductivity and good mechanical flexibility. LPSCl, however, has challenges of translating research into practical applications, such as irreversible electrochemical degradation at the interface between LPSCl and cathode materials. Even for Li‐ion batteries (LIBs), liquid electrolytes have the same issue as electrolyte decomposition due to interfacial instability. Nonetheless, current LIBs are successfully commercialized because functional electrolyte additives give rise to the formation of stable cathode‐electrolyte interphase (CEI) and solid‐electrolyte interphase (SEI) layers, leading to supplementing the interfacial stability between electrolyte and electrode. Herein, inspired by the role of electrolyte additives for LIBs, trimethylsilyl compounds are introduced as solid electrolyte additives for improving the interfacial stability between sulfide‐based solid electrolytes and cathode materials. 2‐(Trimethylsilyl)ethanethiol (TMS‐SH), a solid electrolyte additive, is oxidatively decomposed during charge, forming a stable CEI layer. As a result, the CEI layer derived from TMS‐SH suppresses the interfacial degradation between LPSCl and LiCoO2, thereby leading to the excellent electrochemical performance of Li | LPSCl | LiCoO2, such as superior cycle life over 2000 cycles (85.0% of capacity retention after 2000 cycles). [ABSTRACT FROM AUTHOR]

In this paper, experimental validation of high precision web handling for a two-actuator-based roll-to-roll (R2R) system is presented. To achieve this, the tension control loop is utilized to regulate the tension in the unwinder module, and the velocity loop is utilized to regulate the web speed in the rewinder module owing to the limitation of the number of actuators. Moreover, the radius estimation algorithm is applied to achieve an accurate web speed and the control sequence of the web handling in the longitudinal axis is developed to manipulate the web handling for convenience. Having these, the tension control performances are validated within ±0.79, ±1.32 and ±1.58 percent tension tracking error and 1.6, 1.53 and 1.33 percent web speed error at the speeds of 0.1 m/s, 0.2 m/s, and 0.3 m/s, respectively. The tension control performance is verified within ±0.3 N tracking error in the changes of the reference tension profile at 0.1 m/s web speed. Lastly, the air floating roller is used to minimize the friction terms and the inertia of the idle roller in the tension zone so that tension control performance can be better achieved during web transportation. [ABSTRACT FROM AUTHOR]

DNA binding fluorescent proteins are useful probes for a broad range of biological applications. Fluorescent protein (FP)-tagging allows DNA binding proteins expressed within a living cell to be directly visualised, in real-time, to study DNA binding patterns and dynamics. Moreover, FP-tagged DNA binding proteins (FP-DBP) have allowed the imaging of single proteins bound to large elongated DNA molecules with a fluorescence microscope. Although there are numerous DNA binding proteins, only a small portion of them have been exploited to construct FP-DBPs to study molecular motion in a cell or in vitro single-molecule visualisation. Therefore, it would be informative to review FP-DBP for further development. Here, we summarise the design of FP-DBPs and their brightness, photostability, pKa, maturation rate, and binding affinity (Kd) characteristics. Then, we review the applications of FP-DBP in cells to study chromosome dynamics, DNA replication, transcription factors, DNA damage, and repair. Finally, we focus on single DNA molecule visualisation using FP-DBP. [ABSTRACT FROM AUTHOR]

The recent advances in the single cell genome analysis are generating a considerable amount of novel insights into complex biological systems. However, there are still technical challenges because each cell has a single copy of DNA to be amplified in most single cell genome analytical methods. In this paper, we present a novel approach to directly visualize a genomic map on a large DNA molecule instantly stained with red and green DNA-binding fluorescent proteins without DNA amplification. For this visualization, we constructed a few types of fluorescent protein-fused DNA-binding proteins: H-NS (histone-like nucleoid-structuring protein), DNA-binding domain of BRCA1 (breast cancer 1), high mobility group-1 (HMG), and lysine tryptophan (KW) repeat motif. Because H-NS and HMG preferentially bind A/T-rich regions, we combined A/T specific binder (H-NS-mCherry and HMG-mCherry as red color) and a non-specific complementary DNA binder (BRCA1-eGFP and 2(KW)2-eGFP repeat as green color) to produce a sequence-specific two-color DNA physical map for efficient optical identification of single DNA molecules. [ABSTRACT FROM AUTHOR]

Abstract: Bioorthogonal metabolic DNA labeling with fluorochromes is a powerful strategy to visualize DNA molecules and their functions. Here, we report the development of a new DNA metabolic labeling strategy enabled by the catalyst‐free bioorthogonal ligation using vinyl thioether modified thymidine and o‐quinolinone quinone methide. With the newly designed vinyl thioether‐modified thymidine (VTdT), we added labeling tags on cellular DNA, which could further be linked to fluorochromes in cells. Therefore, we successfully visualized the DNA localization within cells as well as single DNA molecules without other staining reagents. In addition, we further characterized this bioorthogonal DNA metabolic labeling using DNase I digestion, MS characterization of VTdT as well as VTdT‐oQQF conjugate in cell nuclei or mitochondria. This technique provides a powerful strategy to study DNA in cells, which paves the way to achieve future spatiotemporal deciphering of DNA synthesis and functions. [ABSTRACT FROM AUTHOR]

DNA polymerase I offers great promise for a wide range of biotechnological applications due to its capability to add labeled nucleotides into double-stranded large DNA molecules by using both polymerase and nuclease domains. Accordingly, it is crucially important to thoroughly characterize this enzyme for further developments. Although the enzyme has been thus far characterized using mainly traditional analytical instruments, here we utilized an advanced and convenient means of mass spectrometry to elucidate enzymatic functions and mechanisms by measuring DNA oligomers generated by polymerase and nuclease reactions. Our analysis revealed several novel enzymatic features, including the observation that polymerase readily dissociates from the DNA molecules containing a wide single-stranded section. From this finding, we reasoned a serious situation of DNA break because polymerase domains cannot efficiently repair the wide single-stranded section, which is susceptible to DNA breaks. Furthermore, we deduced a plausible explanation for a paradoxical question as to why two domains of polymerase and 5′-nuclease are linked by a small and flexible polypeptide in polymerase I. The polypeptide link seems to prevent a 5′-nuclease from causing DNA breaks by locating a polymerase domain closely for immediate repair reaction. Here we present experimental evidence to prove our hypothesis via a set of mass spectrometric analyses as well as single DNA molecule observation and bacterial cell growth assay. Consequently, mass spectrometric analysis for DNA polymerase I provides a meaningful biological insight that a polypeptide link can be a molecular leash to control an aggressive domain in order to prevent unmanageable damages. [ABSTRACT FROM AUTHOR]

Large DNA molecules are a promising platform for in vitro single-molecule biochemical analysis to investigate DNA-protein interactions by fluorescence microscopy. For many studies, intercalating fluorescent dyes have been primary DNA staining reagents, but they often cause photo-induced DNA breakage as well as structural deformation. As a solution, we previously developed several fluorescent-protein DNA-binding peptides or proteins (FP-DBP) for reversibly staining DNA molecules without structural deformation or photo-induced damage. However, they cannot stain DNA in a condition similar to a physiological salt concentration that most biochemical reactions require. Given these concerns, here we developed a salt-tolerant FP-DBP: truncated transcription activator-like effector (tTALE-FP), which can stain DNA up to 100 mM NaCl. Moreover, we found an interesting phenomenon that the tTALE-FP stained DNA evenly in 1 × TE buffer but showed AT-rich specific patterns from 40 mM to 100 mM NaCl. Using an assay based on fluorescence resonance energy transfer, we demonstrated that this binding pattern is caused by a higher DNA binding affinity of tTALE-FP for AT-rich compared to GC-rich regions. Finally, we used tTALE-FP in a single molecule fluorescence assay to monitor real-time restriction enzyme digestion of single DNA molecules. Altogether, our results demonstrate that this protein can provide a useful alternative as a DNA stain over intercalators. [ABSTRACT FROM AUTHOR]

Large DNA molecules have been utilized as a model system to investigate polymer physics. However, DNA visualization via intercalating dyes has generated equivocal results due to dye-induced structural deformation, particularly unwanted unwinding of the double helix. Thus, the contour length increases and the persistence length changes so unpredictably that there has been a controversy. In this paper, we used TAMRA-polypyrrole to stain single DNA molecules. Since this staining did not change the contour length of B-form DNA, we utilized TAMRA-polypyrrole stained DNA as a tool to measure the persistence length by changing the ionic strength. Then, we investigated DNA stretching in nanochannels by varying the ionic strength from 0.06 mM to 47 mM to evaluate several polymer physics theories proposed by Odijk, de Gennes and recent papers to deal with these regimes. [ABSTRACT FROM AUTHOR]

Since the coronavirus pandemic, mRNA vaccines have revolutionized the field of vaccinology. Lipid nanoparticles (LNPs) are proposed to enhance mRNA delivery efficiency; however, their design is suboptimal. Here, a rational method for designing LNPs is explored, focusing on the ionizable lipid composition and structural optimization using machine learning (ML) techniques. A total of 213 LNPs are analyzed using random forest regression models trained with 314 features to predict the mRNA expression efficiency. The models, which predict mRNA expression levels post-administration of intradermal injection in mice, identify phenol as the dominant substructure affecting mRNA encapsulation and expression. The specific phospholipids used as components of the LNPs, as well as the N/P ratio and mass ratio, are found to affect the efficacy of mRNA delivery. Structural analysis highlights the impact of the carbon chain length on the encapsulation efficiency and LNP stability. This integrated approach offers a framework for designing advanced LNPs and has the potential to unlock the full potential of mRNA therapeutics., (© 2024 The Author(s). Small published by Wiley‐VCH GmbH.)

Large DNA molecules tethered on the functionalized glass surface have been utilized in polymer physics and biochemistry particularly for investigating interactions between DNA and its binding proteins. Here, we report a method that uses fluorescent microscopy for visualizing large DNA molecules tethered on the surface. First, glass coverslips are biotinylated and passivated by coating with biotinylated polyethylene glycol, which specifically binds biotinylated DNA via avidin protein linkers and significantly reduces undesirable binding from non-specific interactions of proteins or DNA molecules on the surface. Second, the DNA molecules are biotinylated by two different methods depending on their terminals. The blunt ended DNA is tagged with biotinylated dUTP at its 3' hydroxyl terminus, by terminal transferase, while the sticky ended DNA is hybridized with biotinylated complimentary oligonucleotides by DNA ligase. Finally, a microfluidic shear flow makes single DNA molecules stretch to their full contour lengths after being stained with fluorescent protein-DNA binding peptide (FP-DBP).

Fluorescent proteins that also bind DNA molecules are useful reagents for a broad range of biological applications because they can be optically localized and tracked within cells, or provide versatile labels for in vitro experiments. We report a novel design for a fluorescent, DNA-binding protein (FP-DBP) that completely 'paints' entire DNA molecules, whereby sequence-independent DNA binding is accomplished by linking a fluorescent protein to two small peptides (KWKWKKA) using lysine for binding to the DNA phosphates, and tryptophan for intercalating between DNA bases. Importantly, this ubiquitous binding motif enables fluorescent proteins (Kd = 14.7 μM) to confluently stain DNA molecules and such binding is reversible via pH shifts. These proteins offer useful robust advantages for single DNA molecule studies: lack of fluorophore mediated photocleavage and staining that does not perturb polymer contour lengths. Accordingly, we demonstrate confluent staining of naked DNA molecules presented within microfluidic devices, or localized within live bacterial cells., (© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.)