共找到 20 条结果
Deuterium substitution (CH3 → CD3) can enhance metabolic stability via the kinetic isotope effect and enable bioanalytical mass shifts. We report an electrochemical Kolbe heterocoupling that installs CD3 at aliphatic positions by coupling carboxylic acids with AcOD-d4. The reaction proceeds at room temperature under constant current with Pt electrodes and base. Diverse CD3-labeled building blocks are obtained in moderate to good yields, and the method avoids CD3I and deuterated solvents.
Electron exchange communication between nitroxide radical sites localized along polymer backbones creates a compelling platform for spin electronics, resistive memory, and optoelectronics. While radical site proximity, chain flexibility, and local ordering form the basis for this communication, how site-to-site spacer structure governs bulk redox charge transfer remains an open question. Herein, epoxide-cyclic anhydride ring-opening copolymerization produces TEMPO-functional radical polyesters, where strictly alternating enchainment installs a radical at every repeat unit while anhydride comonomer varies spacer structure from flexible aliphatic through alicyclic, bicyclic, and semiaromatic. SQUID magnetometry and EPR spectroscopy confirm radical contents of 86-98%; except for the thioether-containing polyester, where sulfur-specific quenching occurs. Density functional theory calculations reveal that rigid aromatic spacers position radical sites closer than flexible aliphatic ones of comparable through-bond atom counts. However, solid-state electrical conductivity measurements demonstrate that glass transition is the primary determinant of bulk charge transport, regardless of whether it is set through spacer flexibility, blending, or block copolymerization.
Thermoelectric generators (TEGs) can harvest waste heat for small electronics. However, printed planar TEGs are often limited by low thermocouple density within a given footprint. Here, we present a paper-based rolled thermoelectric generator (Rolled TEG) that autonomously transforms from a planar sheet into a cylindrical geometry via paper self-folding using a fully printed fabrication process. Silver nanoparticle ink is inkjet printed to form electrodes and interconnects, while PEDOT:PSS is screen printed to form p-type thermoelectric legs, enabling series-connected architectures on paper substrates. The self-folding transformation rearranges thermocouples onto the inner cylindrical surface, increasing thermocouple density per projected installation area while preserving leg length and electrical connectivity. By systematically examining printing conditions and electrode pattern design, we clarify how internal resistance and output characteristics are governed and demonstrate that the rolled geometry enhances footprint-normalized power generation by achieving a footprint-normalized power density of 18.9 nW cm-2, which is 28.1 times higher than that of the Planar TEG. This work establishes autonomous paper self-folding as an effective design strategy for compact, fully printed thermoelectric devices.
Single carbon-atom insertion represents a powerful molecular-editing strategy for homologation and controlled carbon-chain growth while preserving preexisting functional groups. Despite their ubiquity in bioactive chemical space, primary aliphatic amines─including amino acids─remain challenging substrates for minimal chain expansion. Here, we report a general and iterative strategy that enables formal one-carbon insertion into primary aliphatic amines. The transformation proceeds through in situ generation of triazanium intermediates from commercially available O-diphenylphosphinyl hydroxylamine, followed by rearrangement to form alkyl radical species. Selective interception of these radicals by aldoxime acceptors enables the installation of a skeletal sp2 carbon, whereas ketoxime acceptors enable the dual installation of a skeletal sp2 carbon and a peripheral functional group, furnishing synthetically versatile oxime ether products. Subsequent reductive conversion provides access to diverse sp3-carbon frameworks, including iterative one-carbon homologation. This method operates under mild conditions, exhibits broad functional-group tolerance, and is readily applicable to the late-stage molecular modification of bioactive compounds.
We present Apollo 3, a new manual genome annotation tool that integrates with the JBrowse 2 genome browser. Its functionality is inspired by existing manual genome annotation tools such as Apollo, Artemis, and Otter, but uses an updated and more scalable architecture and technology stack. It allows the simultaneous editing of multiple genomes, including the visualization of synteny to inform those annotations. Apollo 3 can be used as a standalone annotation editor, or it can be installed on a server and used collaboratively. We describe the application's design, features, and use cases.
The nitrate-nitrite-nitric oxide (NO) pathway has a wide range of effects including blood pressure (BP) regulation. Nitrate, produced endogenously, and absorbed from dietary sources (e.g., beetroot), is concentrated in/secreted by the salivary glands and reduced by lingual bacterial nitrate reductases to nitrite (the 'enterosalivary circulation'). Following systemic absorption, nitrite is further reduced to vasodilating NO intravascularly. The role of salivary pH in nitrate reduction has mainly been studied in isolated oral installation studies, rather than systemic studies employing the enterosalivary circulation. Our aim was to determine the acute relationship between salivary pH, salivary nitrite production, plasma [nitrite] and the BP-lowering effect of dietary nitrate, by altering salivary pH using sugar-containing chewing gum (vs. sugar-free chewing gum). We performed a 7-h crossover study, with 14 healthy volunteers consuming beetroot juice (Beet-It®, 70-mL, ~400-mg nitrate) and randomized to initially chew either sugar-containing acidic chewing (bubble) gum or sugar-free non-acidic chewing gum, with the alternate gum on the second visit. Sugar-containing gum decreased salivary pH by 1.4 ± 0.09 vs. sugar-free gum (p < .0001). While there was no significant effect on salivary nitrate secretion or plasma [nitrate], salivary nitrite production was increased by 45% (30.0 ± 11.0 μmol·h-1; [p < .01]), with plasma [nitrite] increasing by 25% (222.2 ± 50.3 nmol·L-1; [p = .0001]). Systolic and diastolic BP were decreased by 2.7 ± 0.5 and 1.9 ± 0.5 mmHg, respectively (both p < .0001). Acutely lowering salivary pH (with sugar-containing gum) augments salivary nitrite production, plasma [nitrite] and BP-lowering with dietary nitrate. This mechanism has potential to enhance other nitrate-nitrite-NO pathway effects, for example, exercise performance.
Out-of-hospital cardiac arrest (OHCA) poses a severe public health challenge, and improving the accessibility of automated external defibrillators (AEDs) is crucial for increasing survival rates. This study introduces a comprehensive spatial deployment strategy for AEDs in metropolitan areas, integrating current status assessment, multi-factor modeling, and phased planning, validated in Shanghai's urban region. Based on Geographic Information System (GIS) software, the current status of AED accessibility was evaluated using two indicators: geographic coverage rate and population coverage rate. Subsequently, a model was constructed by integrating elements of population distribution, medical services, and the urban built environment to determine priority zones for device deployment. 92.022% of the AEDs installed before 2021 were located within the model's recommended installation zones. Based on the model results, a three-phase device deployment plan was proposed. The completion of the first phase of the deployment plan achieved comparable service coverage to the real-world plan with a reduced number of equipment installations and about 10% more population coverage than the real-world plan. The first phase of the deployment plan expanded geographic coverage by 12.135% and increased the population served by 22.267% compared to the pre-2021 scenario. After completion of all three phases of the deployment plan, it will cover 83.653% of the geographic area and 96.486% of the population in the study area. Multi-source data-driven geographic information strategy for AED deployment strategy showed greater efficiency to better support decision makers in improving accessibility of AEDs in metropolitan areas.
ISO 14644-1 defines ISO-8 cleanrooms, often found in pharmaceutical manufacturing facilities, by maximum allowable concentrations of airborne particles at specified threshold sizes (commonly ≥0.5 μm and ≥5.0 μm) within a classification particle size range of 0.1-5 μm. For ISO Class 8, though ISO 14644- 1 lists sizes from 0.1 to 5.0 μm, classification commonly uses the operationally relevant limits of ≥0.5 μm and ≥5.0 μm. While ISO classification establishes a compliance boundary, it does not fully leverage modern optical particle counter (OPC) time series data to detect early environmental degradation driven by controllable operational factors in non-sterile areas (traffic, door cycling, material handling, and housekeeping drift). FDA's Process Analytical Technology (PAT) guidance combined with Quality by Design (QbD) principles, promotes timely measurement, enhanced process understanding, and multivariate/statistical approaches to manage variability proactively. This paper proposes a PAT aligned framework for installing and operating particle counting systems in ISO-8 non-sterile controlled areas to detect pre-limit particle size distribution (PSD) shifts. The approach integrates (i) risk-based monitoring network design, (ii) PSD-shift features (coarse to fine ratios, event rate, recovery time, persistence) that translate raw counts into actionable change signals, and (iii) statistical change detection (EWMA/CUSUM) to distinguish transient events from sustained shifts. One thing which is often overlooked is physical collection issues, especially when pulling samples through long tubes or curved lines. Particles at 5 microns or more tend to drop out under those conditions, skewing results noticeably. So this guidance covers considerations for choosing particle counters and where to place ports for truer readings. Instead of waiting until thresholds breach, the system proposed in this article acts earlier using feedback loops tied directly to control actions there by providing a robust continuous monitoring system.
The protein design field is rapidly advancing, with frequent emergence of new models and pipelines for designing de novo proteins with tailored properties and functions not found in nature. However, the current tool landscape is fragmented, tools are hard to install and deploy, and require significant computational expertise to integrate into end-to-end, scalable pipelines. A particular challenge is managing many sequences, structures, and metrics for downstream testing and retrospective analysis of input parameters. To address this need, we introduce Ovo, an open-source de novo protein design ecosystem that consolidates models, workflows, data management, and interactive visualization into a scalable, infrastructure-agnostic platform. Ovo features Nextflow-based workflow orchestration, a storage layer, and both command-line and graphical interfaces that democratize scaffold design, binder design and diversification, and validation workflows. Ovo's novel ProteinQC module computes comprehensive sequence and structure descriptors, contextualizing designs against reference sets. Ovo plugins let the community add new workflows and user interfaces to accelerate adoption of emerging methods and facilitate community-driven benchmarking. Ovo lowers engineering barriers and demystifies the design process, allowing experts and non-technical users to design proteins at scale. With community-driven development, Ovo can accelerate de novo protein design and advance discovery in therapeutics and biotechnology.
Older municipal solid waste (MSW) landfills that received organic waste over past decades struggle in aftercare with declining gas production and fluctuating gas quality, making gas extraction systems inefficient. Methane (CH4) oxidation systems, such as biowindows, provide a sustainable, cost-effective passive alternative for mitigating residual emissions. This study presents long-term monitoring of three pilot biowindows at an Austrian MSW landfill to replace gas extraction wells and reduce CH4 emissions. Over a 10-year period, CH4 emissions and their relationship with environmental factors were analyzed through periodic field campaigns during two monitoring phases (2014-2016 and 2021-2024). Monitoring included gas and temperature profiles, surface screenings, and chamber flux assessments. Statistical analyses, including Pearson correlation and decision tree modeling, identified key factors influencing CH4 emissions, which were driven by a combination of temporal trends, gas composition dynamics, and environmental factors like ambient pressure and temperature. Results revealed a 60 % reduction in median hotspot CH4 emission rates (from 162 to 70 g/m2 d) at one biowindow between the two monitoring phases, while another biowindow demonstrated high performance over the period. In contrast, the third biowindow, installed at a newer landfill section, exhibited hotspots and increased emissions (median 80-265 g/m2 d across campaigns), likely linked to formerly installed and still remaining leachate and gas collection infrastructure, highlighting the need for site-specific interventions to address localized issues and optimize performance. The study confirms that biowindows require minimal maintenance during their first decade of operation and can achieve considerable CH4 reductions under appropriate conditions and well-designed construction.
The plasma membrane and accompanying cortex serve as major hubs of signal transduction and cytoskeletal activities that collectively regulate cell physiological processes such as migration, polarity, macropinocytosis, phagocytosis, and cytokinesis. Yet, dynamically tracking membrane-cortex associated protein or lipid kinetics from live-cell image series remains challenging, primarily due to the difficulty of accurately extracting and aligning the cell boundary between consecutive frames as the cell continuously deforms and moves. Here, we present Membrane Kymograph Generator, a cross-platform software that accepts multichannel time-lapse live-cell fluorescent imaging datasets and automates boundary tracking, inter-frame alignment, and intensity sampling along the boundary. The software implements a rotational offset minimization algorithm that aligns boundaries across consecutive frames by exhaustively searching for the optimal angular shift that minimizes point-to-point distances, while handling variations in boundary point counts due to cell shape changes. The software outputs kymographs representing spatiotemporal dynamics of membrane-associated proteins or biosensors, allows users to fine-tune visualization parameters through an interactive interface, and provides built-in correlation analysis tools for multi-channel datasets. Furthermore, a native Python API enables programmatic usage for batch processing and further downstream analysis. Validation tests demonstrated that the Membrane Kymograph Generator accurately tracks, visualizes, and quantitates the spatial kinetics of a wide array of membrane proteins and lipid biosensors over extended time periods, in a variety of cell types including Dictyostelium amoeba, human neutrophils, mouse macrophages, and mammalian cancer cells. The GUI-based software is user-friendly, requires no technical expertise, and significantly reduces the manual effort required for kymograph generation and analysis while ensuring high accuracy and reproducibility. Membrane Kymograph Generator is free and open-source, licensed under GNU General Public License 3.0 or later. It can be installed on both x86-64 and AArch64/ARM64 computers running Windows, macOS, or any standard Linux distribution. The software is distributed as standalone installer files and portable executables targeting specific architectures and operating systems, requiring no dependency resolution. The source code, documentation/wiki, installers, portable binaries, and test data are freely available at https://github.com/tatsatb/membrane-kymograph-generator. The software can also be installed via PIP (package ID: membrane-kymograph, https://pypi.org/project/membrane-kymograph) and accessed programmatically via a built-in Python API. The source code is also archived on Zenodo (DOI: 10.5281/zenodo.20318834). Supplementary data are available at Bioinformatics online.
An in-package treatment combining cold plasma (CP) and ultraviolet-C light-emitting diodes (UVC[LED]) was developed and evaluated for its efficacy in microbial inactivation and its impact on the quality attributes of small king oyster mushrooms (Pleurotus eryngii). The mushrooms were packaged in air-filled nylon-coated low-density polyethylene pouches. CP was applied by attaching both ends of the pouch to a pair of electrodes, while UVC[LED] was administered by installing LED modules above and below the pouch surface. Comparison of reduction levels in indigenous mesophilic aerobic bacteria revealed that the optimal parameters were 30 W for CP and 25 V, 500 Hz, and 50% duty cycle for UVC[LED]. When each treatment was applied for 7 min, sequential applications of CP and UVC[LED] (CP → UVC[LED] or UVC[LED] → CP) resulted in the greatest reduction of Salmonella inoculated in small king oyster mushrooms, compared to individual or simultaneous treatments. During 14 days of storage at 4 °C, UVC[LED] → CP treatment reduced the number of indigenous mesophilic aerobic bacteria and Salmonella by 0.8-1.3 and 0.6-1.7 log CFU/g, respectively. Compared to untreated samples, the treated mushrooms exhibited significantly higher total phenolic content, DPPH radical scavenging activity, and ascorbic acid concentration throughout storage, without noticeable changes in color. These results suggest that UVC[LED] → CP treatment is an effective non-thermal approach to enhance the microbial safety and antioxidant properties of mushrooms, offering potential as a postharvest decontamination technology.
Marginal water sources have become increasingly important in fulfilling human and environmental needs. In situ contaminant removal is frequently used as a low-cost method of improving water quality in areas impacted by industries such as mining, which can cause complex adverse effects in surrounding watersheds. Biochar has been used as a soil amendment and considered as a sorbent for aqueous contaminants at a benchtop scale, but studies examining its use to improve surface waters at the field scale have been limited. This study observed the effects of manure-based biochar on two mining-influenced water bodies. Lead and zinc concentrations in an intermittent stream were minimally affected, and exchangeable ions from the biochar increased electrical conductivity and nutrient concentrations to undesirable levels in water downstream from the installation. Lead and zinc concentrations decreased downstream from biochar installed at a small seep in a mine waste repository, but further analysis of the solids showed that lead and zinc were sorbed to iron (oxyhydr)oxide solids precipitating from this water before encountering the biochar. When compared with optimal controlled conditions observed in benchtop studies, field conditions, such as contact time, did not allow for effective use of biochar and resulted in substantial environmental degradation of downstream waters.
Bicycle infrastructure preferences vary by rider experience and comfort, yet both bicyclists and motorists perceive separated bicycle lanes as safer than shared lane markings (sharrows). Despite widespread adoption, evidence on the real-world behavioral impacts of sharrows remains limited. This study evaluates a natural experiment in Santa Ana, California, where sharrows were installed in a downtown commercial corridor when separated bicycle lanes were not feasible. Researchers collected baseline data in September 2015 and follow-up data in February 2016 and early 2017, totaling 54 hours of direct bicycle observation across two target areas. Results revealed a mixed and cautionary pattern of change. West-to-East mean bicycle counts increased significantly from 0.986 (2015) to 1.734 (2016; p = 0.045) but declined to 1.500 by 2017. East-to-West counts showed modest, non-significant year-over-year growth. Critically, sidewalk riding increased significantly from 2015 to 2016 (p = 0.002) and remained elevated through 2017 (p = 0.009), and wrong-way riding increased significantly from 2015 to 2016 (p = 0.003). Female ridership declined from 2015 to 2016 (p = 0.036) but recovered by 2017. Ridership among adults over 65 declined post-installation without meaningful recovery. Overall, sharrow installation was not associated with lasting ridership gains or improved safe bicycling behavior. Declines among women and older adults suggest sharrows may not adequately meet the needs of all roadway users. Drawing on the present data and prior literature, policymakers should consider pairing sharrows with education programs, driver awareness campaigns, and enhanced infrastructure to support safety, equity, and sustained bicycling engagement.
The first asymmetric total synthesis of Lycopodium alkaloids bearing a 1,2-amino alcohol motif, including (-)-palhinine B, (-)-palhinine C, and (+)-palhinine B, has been achieved. The synthesis features early stage installation of a chiral C2-OH group from a tyrosine-derived 1,2-amino alcohol, which governs the overall stereochemical outcome at C7, C13, C15, and a quaternary stereogenic center at C12 of the tetracyclic palhinine skeleton. A highly diastereoselective Diels-Alder reaction of a masked ortho-benzoquinone, followed by a thiol-mediated acyl radical cyclization, enables rapid construction of the isotwistane core. DFT-guided analysis revealed the challenge of late-stage C2-OH inversion and led to a successful chemo- and diastereoselective reduction of a caged hemiaminal ketone, providing access to the C2 epimer. This work establishes a unified asymmetric strategy for complex Lycopodium alkaloids and highlights the integration of computation with synthetic design.
High-throughput genomic analyses of germline and cancer genomes facilitate the identification of causal and actionable genetic variants. The recent advances in next-generation sequencing technology generated large-scale genomic and/or multi-omics dataset. Due to huge volume of data, scientists are facing challenges in visualizing, and interpreting the data. Currently available tools to visualize genetic variants from VCF files are not very user-friendly as most of them require knowledge of command line tools or scripts to install and run those software. Therefore, graphical user interface based tools or software are needed to summarize and visualize the VCF data. We have developed a Shiny App, interactive tool using the R programming language that utilizes existing R packages like "vcfR" and "maftools" to visualize and generate quality control metrics for genetic data. Our tool is powered by Shiny, making it easier to summarize and visualize genomic data using a GUI. XVCF has been developed for the summarization and visualization of genomic variation data. The tool offers an easy and friendly interface, allowing users to perform data loading, summarization, and visualization interactively. XVCF extract useful information such as read depth, mapping quality, genotype, quality control summary, and allele frequency from unannotated data. In the second module of XVCF, the cancer genomic data is analyzed using "maftools" to produce oncoplot, lollipop plot, gene summary, etc. XVCF is available for free download from https://github.com/rashidma/XVCF. Being a shiny R package, XVCF can be installed across different operating systems and utilize different computer hardware configurations. Visualizing genomic data has always been challenging. Existing tools/software seem to be difficult to use due to lack of technical computer programming knowledge. We offer XVCF to visualize and/or summarize genomic data at a greater ease due to its graphical user interface and powerful cross-platform R shiny framework.
This paper examines emergent haptic phenomena in distributed mechanically-actuated sound installations through a structural acoustic analysis of Babbling Brook, a large-scale installation comprising forty solenoid-actuated computer keyboards (eighty solenoids in total) and an eight-channel audio keystroke layer. Most haptics research in the arts has focused on direct-contact interfaces and wearable devices designed for individual users. Environmental phenomena produced from haptics have received comparatively little attention, in part because they are difficult to measure directly in walk-through gallery settings. The installation is organized around four discrete density modes that span a perceptual continuum: at low densities listeners perceive discrete mechanical keystrokes, while at high densities the acoustic texture approaches a continuous, broadband noise-like regime reminiscent of rain and water streams. Because direct vibrometric and perceptual measurement were not possible in the gallery setting, we analyze the installation's audio as an acoustic proxy for the underlying haptic transformation, using DSP metrics associated with signal stationarity and auditory-texture perception (envelope smoothness variance, spectral flux, crest factor, waveform kurtosis, spectral spread, and low-frequency modulation). Three vintage computer keyboards with distinct switch mechanisms (rubber dome, Topre capacitive, and Alps clone) are analyzed to examine how mechanical profile shapes the resulting texture, and the same pipeline is applied to three natural water recordings as an external reference. Across modes, four of seven metrics differ with large effect sizes (Kruskal-Wallis $H > 8.6$, $p < 0.035$, $\eta ^{2} > 0.70$). Keyboard-specific profiles that are distinct at sparse densities converge at high densities as waveform statistics approach Gaussian values. Mode IV swarm statistics overlap the natural-water reference on five of seven metrics and are smoother than water on envelope and spectral-variability measures, indicating that the swarm enters the broadband stationary regime of naturalistic water sounds, but lacks water's structured macroscopic fluctuations. Together, these results provide quantitative acoustic evidence supportive of an emergent perceptual transformation from a distributed systems approach to environmental haptics in the arts. Direct vibrotactile measurement and perceptual validation are identified as future work.
Healthcare facility power-outages in low- and middle-income countries (LMICs) are common and affect patient outcomes. Decentralised solar energy has the potential to reduce power-outages in healthcare settings, yet adoption in LMIC remains modest. This study aimed to identify and prioritise the barriers, facilitators and strategies of on-site healthcare facility solar power implementation. Methodology was conducted in three phases. In phase 1, a systematic review was completed to summarise barriers and facilitators to implementation of on-site solar power in LMIC healthcare facilities. Any qualitative or mixed-methods study relating to solar power installation from 2009 to 2024 in LMICs was eligible to be included. Barriers and facilitators were extracted from the identified studies and mapped to the Consolidated Framework for Implementation Research. In phase 2, the importance of these barrier and facilitator factors was contextually prioritised through an international survey of healthcare workers on a 5-point Likert scale. In phase 3, strategies were developed from these barrier and facilitator factors to create a solar implementation tool (SOLAR-IT). In phase 1, out of a total of 3678 individual records, 28 studies were included in the systematic review, from which 19 barriers and 29 facilitators were identified. In phase 2, the global survey was completed by 260 participants from 47 LMICs. The barrier with the highest ranked importance was the upfront capital expenditure of solar panel installation, while the most important facilitator was to reduce the cost of solar panels through government intervention. In phase 3, 23 strategies were synthesised within four domains to create the SOLAR-IT. This study describes globally prioritised implementation factors and a strategy tool, which can enable teams to speed up implementation of on-site solar power in a time of extreme global need.
The demand for new energy infrastructure is increasing across the United States, but heterogenous permitting processes and embedded requirements across different local jurisdictions can cause project delays, increase "soft costs," and hinder developer expansion. This study analyzes the variability in local permitting requirements across the U.S. and develops a quantitative approach to describe their clarity and effectiveness in enabling infrastructure project development. By using an Energy Language Model (ELM), a large language model (LLM) for energy technologies, we systematically gathered permitting information from nearly 300 state-, county-, and city-level documents, creating a structured dataset of requirements and procedures on an unprecedented scale and speed. Our analysis revealed that local (city and county) permitting requirement documents are underrepresented compared to state-level guidance documents, which can impede timely and cost-effective installation of new electric infrastructure. Our validation process showed that the final database has an accuracy of approximately 95%. We, further, created a new quantitative method to score permitting requirements for clarity and efficiency, with electric vehicle supply equipment as an initial use case. The average local permitting document scored a 1.8 out of 5, which we interpret as meaning that half of the requirements developers face when installing electric infrastructure are ambiguous, increasing both cost and time. We also created a "Generalized Permit Process", highlighting common procedural steps and identifying specific opportunities for municipalities to improve their documentation. This research establishes a systematic and scalable framework for evaluating the complexities of local infrastructure permitting processes by combining LLM-powered data collection and quantitative scoring. The framework enables policymakers and developers to identify and mitigate procedural bottlenecks, with the expectation that these improvements can accelerate application review and approval, reduce project costs, and expedite connection to utility distribution grids. As a foundational approach for streamlining local project development processes, this study's methods are intended to be extended to a wide range of energy applications.
Precise and efficient replacement of large genomic DNA segments without inducing double-strand breaks (DSBs) remains a central challenge in genome engineering. Traditional homologous recombination relies on DSBs and long homologous arms, yet it remains inefficient, while recombinase or integrase systems suffer from residual sequences at integration sites. Prime editing (PE), limited by the processivity of reverse transcriptase, struggles to integrate large fragments (>100 bp). To address this challenge, we introduce Prime Editing-Microhomology-Enabled Replacement (PREMIER), a DSB-free platform by installing single-stranded microhomology arms at donor and genomic junctions via PE. In cell lines, PREMIER achieved a mean efficiency of 63.4% (median 65.2%) in diverse target sites, with peak efficiencies reaching 85.9%, exceeding homology-directed repair by 10-20-fold and reducing off-target integrations by over 100-fold compared to nonhomologous end joining. It bypasses the need for long homology arms, simplifies donor preparation, achieves targeted replacement of sequences up to 10.3 kb. In vivo, PREMIER integrates a 6.2-kb oncogene cassette into the mouse liver. Additionally, PREMIER replaces murine Trp53 with human TP53 CDS, generating functional humanized mice. Altogether, PREMIER provides a precise, high-efficiency, and DSB-free strategy for large-scale genome rewriting, offering a powerful tool for complex modeling and therapeutic genome editing.