Single Modification Research Articles

Rheological analyses of binders modified with triple combinations of Crumb-Rubber, Sasobit and Styrene-Butadiene-Styrene

The development of polymer-modified asphalt (PMA) has been a widely used technique by many researchers. Moreover, further modification of PMA binders has generated interest due to possible improvements in performance and cost. In this regard, Sasobit wax has been an alternative to reduce costs in Crumb-Rubber (CR) and Styrene-Butadiene-Styrene Copolymer (SBS) binders. However, there is little detail on the rheological behaviour of CR-SBS-Sasobit triple-modified samples for failure and damage resistance properties. Therefore, this study shows various properties of binders with triple modification of CR-SBS-Sasobit at different dosages. For this purpose, the development of single, double and triple modifications has been proposed to be tested by Multiple Stress Creep Recovery (MSCR) for permanent deformation. In the case of radial cracking and thermal cracking, it is evaluated by linear amplitude sweep test (LAS) and binder creep energy test (BYET). The results indicate that a CR-SBS combination in single or double addition achieves good thermal stability and behaviour. However, a higher elasticity is achieved in the case of a triple modification with CR-SBS-Sasobit. Specifically, Sasobit achieves 67% and 28% reduction in the cumulative deformation of MSCR for a variation of 40–80 °C, respectively. Concerning fatigue life (Nf), it is shown that CR-SBS modifications do not always acquire higher Nf than single modifications. However, adding Sasobit in triple modifications increases these values, generating bitumen with higher strength. It is also shown that adding Sasobit is a viable modifier in yield, even increasing the yield energy (Er) for samples with equal CR-SBS and reducing the percentage of SBS in the sample.

Enhanced electrochemical performance of nanoscale single crystal NMC811 modification by coating LiNbO3

Single crystallization is an important strategy to resolve intergranular cracks and unnecessary side reactions with electrolytes in layered transition metal oxide cathodes LiNi0.8Mn0.1Co0.1O2 (NMC811). Due to the limitations of high-temperature sintering and multi-step calcination, single crystal NMC811 generally shows irregular particles with a size of 2–3 µm. However, the prolonged Li-ion diffusion pathway and the stress generated by the uneven de-/intercalation sluggish Li-ion diffusion kinetics, what is more, cause structural damage such as intragranular cracks. A slow Li extraction rate or particle size reduction will ameliorate the structural damage and improve the cycling stability. As the most promising cathodes for next-generation power batteries, NMC811 required fast charge performance and cycle stability. Particle size reduction appears to be the displacement option. Nanonization is an effective strategy to mitigate intragranular cracks of single crystal NMC811. However, the serious aggregation and increased specific surface area become new challenges. In this article, we synthesized monodisperse nanoscale single crystal NMC811 by molten salt method and modified the surface by LiNbO3 coating. The electrochemical performance shows that nanoscale single crystal NMC811 has faster kinetic and higher capacity retention, so the strategy of combining nanonization and surface coating is an alternative way to prepare high specific capacity and cycle stable single crystal NMC811.

Seismic Performance of Vertical Irregular Steel Frame Structure under Mainshock-Aftershock

Abstract: The majority of the structures are involved with architectural importance and it is highly impossible to achieve uniform structural properties in all directions. Hence earthquake resistant design codes considered it as irregular frames based on relative difference in the story properties. In many cases these irregularities are responsible for structural collapse of buildings under earthquake ground motions. The seismic response of buildings with irregular distribution of stiffness along the height may be differentfrom that ofregular building. Also, past earthquakes showed that structure may be subjected to sequence of ground motions but current codes do not have guidelines for such cases. It is considered that aftershock do not cause any more damage to damaged structure by the preceding mainshock ground motion. In this study steel moment resisting frame buildings are evaluated to understand the seismic response of vertical stiffness irregular frames subjected to mainshock-aftershock ground motions. The 9-story steel moment resisting frame building situated in Los Angeles is used in this study was originally developed as part of the SAC steel project. In this study soft and stiff storey case was considered at three different locations along the height, i.e., at bottom storey, mid-height storey and top storey. The single modification factor is used for irregularity. For comparison purpose dynamic properties of regular and irregular frames are kept same. Two sets of 6 records were selected representing a seismic hazard level of 2% and 10% probability of exceedance in 50 years respectively as mainshock. Two sets of 30 mainshockaftershock ground motion are considered for the study. These ground motions were developed using randomized approach. A non-linear time history analysis of regular and irregular building is carried out separately under mainshock and mainshockaftershock. The effect of building irregularity was studied for single storey modification at bottom storey, mid-storey and top storey with comparison to regular building. The comparison of a regular and irregular building is carried out in terms of maximum roof displacement and interstorey drift ratio. Also, comparison of frames under mainshock and mainshock-aftershock is done.

Coherent pairs of measures of the second kind on the real line and Sobolev orthogonal polynomials. An application to a Jacobi case

AbstractThe aim here is to consider the orthogonal polynomials with respect to an inner product of the type , where and is a coherent pair of positive measures of the second kind on the real line (CPPM2K on the real line). Properties of and the connection formulas they satisfy with the orthogonal polynomials associated with the measure ν0 are analyzed. It is also shown that the zeros of are the eigenvalues of a matrix, which is a single line modification of the Jacobi matrix associated with the measure ν0. The paper also looks at a special example of a CPPM2K on the real line, where one of the measures is the Jacobi measure, and provides a much more detailed study of the properties of the orthogonal polynomials and the corresponding connection coefficients. In particular, the relation that these connection coefficients have with the Wilson polynomials is exposed.

A novel scoring model based on RNA modification "writers" can predict the prognosis and guide immunotherapy in gastric cancer.

Although extensive research has been carried out on the epigenetic regulation of single RNA modifications in gastric cancer, little is known regarding the crosstalk of four major RNA adenosine modifications, namely, m6A, m1A, alternative polyadenylation and adenosine-to-inosine RNA editing. By analyzing 26 RNA modification "writers" in 1750 gastric cancer samples, we creatively constructed a scoring model called the "Writers" of the RNA Modification Score (WRM_Score), which was able to quantify the RNA modification subtypes of individual patients. In addition, we explored the relationship between WRM_Score and transcriptional and posttranscriptional regulation, tumor microenvironment, clinical features and molecular subtypes. We constructed an RNA modification scoring model including two different subgroups: WRM_Score_low and WRM_Score_high. The former was associated with survival benefit and good efficacy of immune checkpoint inhibitors (ICIs) due to gene repair and immune activation, while the latter was related to poor prognosis and bad efficacy of ICIs because of stromal activation and immunosuppression. The WRM score based on immune and molecular characteristics of the RNA modification pattern is a reliable predictor of the prognosis of gastric cancer and the therapeutic efficacy of immune checkpoint inhibitors in gastric cancer.

Review of Recent Advances in Transcritical CO2 Heat Pump and Refrigeration Cycles and Their Development in the Vehicle Field

Refrigerant substitution is an urgent need in the context of reducing carbon emissions and slowing global warming. CO2 is now being proposed as a promising solution based on its excellent properties and system performance, especially in low-temperature environments. This paper presents an overview of recent advances in system configuration and operation characteristics to improve the performance of transcritical CO2 heat pump and refrigeration systems. The paper first introduces the basic research background, system cycle, and thermodynamic characteristics. Secondly, CO2 cycle improvements with single modifications and modification combinations are reviewed. Then, some important operation characteristics and control methods are discussed. Additionally, the paper provides a detailed description of the development of transcritical CO2 heat pump and refrigeration systems in the vehicle field. At the end of this review, conclusions and opportunities for future work in this field are presented.

P, K doped crystalline g-C3N4 grafted with cyano groups for efficient visible-light-driven H2O2 evolution

Graphitic carbon nitride (g-C3N4), especially in heptazine forms, has long been considered as a promising non-toxic, benign and sustainable photocatalyst for producing the important chemical and potential green energy H2O2. However, bulk g-C3N4 shows moderate activity originated from its intrinsic drawbacks such as restricted visible-light harvesting ability and sluggish charge separation. Herein, a co-modified crystalline g-C3N4 by P, K ions and cyano group was successfully synthesized via co-thermal polymerization of precursors followed by molten salt treatment. The advanced g-C3N4 exhibits an outstanding photocatalytic H2O2 generation yield of 4424 μmol g−1h−1, more than 81-fold of the pristine counterpart, also superior than that of g-C3N4 processed by single modification treatment. This superior photocatalytic H2O2 evolution efficiency is attributed to the rational design of crystalline g-C3N4, enabling super visible photons capture, more efficient charge carries separation and substantially reduced charge recombination. The current work may furnish an integratable strategy for solar fuel production.

Coordination of RNA modifications in the brain and beyond.

Gene expression regulation is a critical process throughout the body, especially in the nervous system. One mechanism by which biological systems regulate gene expression is via enzyme-mediated RNA modifications, also known as epitranscriptomic regulation. RNA modifications, which have been found on nearly all RNA species across all domains of life, are chemically diverse covalent modifications of RNA nucleotides and represent a robust and rapid mechanism for the regulation of gene expression. Although numerous studies have been conducted regarding the impact that single modifications in single RNA molecules have on gene expression, emerging evidence highlights potential crosstalk between and coordination of modifications across RNA species. These potential coordination axes of RNA modifications have emerged as a new direction in the field of epitranscriptomic research. In this review, we will highlight several examples of gene regulation via RNA modification in the nervous system, followed by a summary of the current state of the field of RNA modification coordination axes. In doing so, we aim to inspire the field to gain a deeper understanding of the roles of RNA modifications and coordination of these modifications in the nervous system.

Front Cover: Single Modification at the N‐Terminus of Norvancomycin to Combat Drug‐Resistant Gram‐Positive Bacteria (ChemMedChem 9/2023)

The Front Cover shows the single modification at the N-terminus of norvancomycin, especially lipo-sulfonium furnishment. This provides norvancomycin additional interaction with the bacterial membrane via the electrostatic interaction and hydrophobic moieties’ insertion into the membrane inner leaflet. Thus, it restores the antibacterial activity against glycopeptide-resistant bacteria. Cover design by Dongliang Guan. More information can be found in the Research Article by Dongliang Guan, Lefu Lan, Wei Huang et al.

Chemical Synthesis of Alpha-Synuclein Proteins via Solid-Phase Peptide Synthesis and Native Chemical Ligation.

Alpha-Synuclein (α-Synuclein) is a 140 amino acid protein implicated in neurodegenerative disorders known as synucleino-pathies, where it accumulates in proteinaceous inclusions in the brain. The normal physiological function of α-Synuclein remains obscure, as it exists in several non-neuronal cells in which its function has not been studied. Given the tremendous interest in studying α-Synuclein, and the existing limitations in the production of modified forms of the protein, we developed a method for the chemical synthesis of α-Synuclein by combining peptide fragment synthesis via automated microwave-assisted solid-phase peptide synthesis and ligation strategies. Our synthetic pathway enables the synthesis of protein variants of interest, carrying either mutations or posttranslational modifications, for further investigations of the effects on the structure and aggregation behavior of the protein. Ultimately, our study forms the foundation for future syntheses and studies of other custom-made α-Synuclein variants with a single or several modifications, as necessary.

Estimating human papillomavirus vaccine efficacy from a single-arm trial: proof-of-principle in the Costa Rica Vaccine Trial.

The World Health Organization recommends a 1- or 2-dose human papillomavirus (HPV) vaccination schedule for females aged 9 to 20 years. Studies confirming the efficacy of a single dose and vaccine modifications are needed, but randomized controlled trials are costly and face logistical and ethical challenges. We propose a resource-efficient single-arm trial design that uses untargeted and unaffected HPV types as controls. We estimated HPV vaccine efficacy (VE) from a single arm by comparing 2 ratios: the ratio of the rate of persistent incident infection with vaccine-targeted HPV 16 and 18 (HPV 16/18) and cross-protected types HPV 31, 33, and 45 (HPV 31/33/45) to vaccine-unaffected types HPV 35, 39, 51, 52, 56, 58, 59, and 66 (HPV 35/39/51/52/56/58/59/66) vs the ratio of prevalence of these types at the time of trial enrollment. We compare VE estimates using only data from the bivalent HPV 16/18 vaccine arm of the Costa Rica Vaccine Trial with published VE estimates that used both the vaccine and control arms. Our single-arm approach among 3727 women yielded VE estimates against persistent HPV 16/18 infections similar to published 2-arm estimates from the trial (according-to-protocol cohort: 91.0% , 95% CI = 82.9% to 95.3% [single-arm] vs 90.9% , 95% CI = 82.0% to 95.9% [2-arm]; intention-to-treat cohort: 41.7%, 95% CI = 32.4% to 49.8% [single-arm] vs 49.0% , 95% CI = 38.1% to 58.1% [2-arm]). VE estimates were also similar in analytic subgroups (number of doses received; baseline HPV serology status). We demonstrate that a single-arm design yields valid VE estimates with similar precision to a randomized controlled trial. Single-arm studies can reduce the sample size and costs of future HPV vaccine trials while avoiding concerns related to unvaccinated control groups. ClinicalTrials.gov Identifier: NCT00128661.

Integration of Bragg gratings in aerosol-jetted polymer optical waveguides for strain monitoring capabilities.

We demonstrate and discuss the integration of Bragg gratings in aerosol-jetted polymer optical waveguides, produced in the optical assembly and connection technology for component-integrated bus systems (OPTAVER) process. By using a femtosecond laser and adaptive beam shaping, an elliptical focal voxel generates different types of single pulse modification by nonlinear absorption in the waveguide material, which are arranged periodically to form Bragg gratings. Integration of a single grating structure or, alternatively, an array of Bragg grating structures in the multimode waveguide yields a pronounced reflection signal with typical multimodal properties, i.e., a number of reflection peaks with non-Gaussian shapes. However, the main wavelength of reflection, located around 1555 nm, is evaluable by means of an appropriate smoothing algorithm. When loaded by mechanical bending, a pronounced Bragg wavelength shift of this reflected peak up to 160 pm is detected. This demonstrates that the additively manufactured waveguides can be used not only for signal transmission but also as a sensor.

NSUN3-mediated mitochondrial tRNA 5-formylcytidine modification is essential for embryonic development and respiratory complexes in mice

In mammalian mitochondria, translation of the AUA codon is supported by 5-formylcytidine (f5C) modification in the mitochondrial methionine tRNA anticodon. The 5-formylation is initiated by NSUN3 methylase. Human NSUN3 mutations are associated with mitochondrial diseases. Here we show that Nsun3 is essential for embryonic development in mice with whole-body Nsun3 knockout embryos dying between E10.5 and E12.5. To determine the functions of NSUN3 in adult tissue, we generated heart-specific Nsun3 knockout (Nsun3HKO) mice. Nsun3HKO heart mitochondria were enlarged and contained fragmented cristae. Nsun3HKO resulted in enhanced heart contraction and age-associated mild heart enlargement. In the Nsun3HKO hearts, mitochondrial mRNAs that encode respiratory complex subunits were not down regulated, but the enzymatic activities of the respiratory complexes decreased, especially in older mice. Our study emphasizes that mitochondrial tRNA anticodon modification is essential for mammalian embryonic development and shows that tissue-specific loss of a single mitochondrial tRNA modification can induce tissue aberration that worsens in later adulthood.

Multi-task adaptive pooling enabled synergetic learning of RNA modification across tissue, type and species from low-resolution epitranscriptomes

Post- and co-transcriptional RNA modifications are found to play various roles in regulating essential biological processes at all stages of RNA life. Precise identification of RNA modification sites is thus crucial for understanding the related molecular functions and specific regulatory circuitry. To date, a number of computational approaches have been developed for in silico identification of RNA modification sites; however, most of them require learning from base-resolution epitranscriptome datasets, which are generally scarce and available only for a limited number of experimental conditions, and predict only a single modification, even though there are multiple inter-related RNA modification types available. In this study, we proposed AdaptRM, a multi-task computational method for synergetic learning of multi-tissue, type and species RNA modifications from both high- and low-resolution epitranscriptome datasets. By taking advantage of adaptive pooling and multi-task learning, the newly proposed AdaptRM approach outperformed the state-of-the-art computational models (WeakRM and TS-m6A-DL) and two other deep-learning architectures based on Transformer and ConvMixer in three different case studies for both high-resolution and low-resolution prediction tasks, demonstrating its effectiveness and generalization ability. In addition, by interpreting the learned models, we unveiled for the first time the potential association between different tissues in terms of epitranscriptome sequence patterns. AdaptRM is available as a user-friendly web server from http://www.rnamd.org/AdaptRM together with all the codes and data used in this project.

Single Modification at the N-Terminus of Norvancomycin to Combat Drug-Resistant Gram-Positive Bacteria.

In the arsenal of glycopeptide antibiotics, norvancomycin, which differs from vancomycin by a single methyl group, has received much less attention. Facing the risks of serious antibiotic resistance and even the collapse of last-line defenses, we designed and synthesized 40 novel norvancomycin derivatives to combat the threat. 32 compounds are single N-terminally modified derivatives generated through simple and efficient methods. Diversity at the N-terminus was greatly enriched, mainly by lipophilic attachment and strategies for the introduction of lipo-sulfonium moieties for extensive structure-activity relationship analysis. The first incorporation of a sulfonium moiety into the norvancomycin structure gave rise to compounds that exhibited 4- to 2048-fold higher activity against vancomycin-resistant bacteria VISA and VRE. This N-terminal modification for norvancomycin provides an alternatively useful and promising strategy to restore the antibacterial activity of glycopeptide antibiotics against resistant bacteria, highlighting the same importance of the N-terminal site as well as the vancosamine position, which is worth further study and development.

Cas9-induced single cut enables highly efficient and template-free repair of a muscular dystrophy causing founder mutation

With thousands of patients worldwide, CAPN3 c.550delA is the most frequent mutation causing severe, progressive, and untreatable limb girdle muscular dystrophy. We aimed to genetically correct this founder mutation in primary human muscle stem cells. We designed editing strategies providing CRISPR-Cas9 as plasmid and mRNA first in patient-derived induced pluripotent stem cells and applied this strategy then in primary human muscle stem cells from patients. Mutation-specific targeting yielded highly efficient and precise correction of CAPN3 c.550delA to wild type for both cell types. Most likely a single cut generated by SpCas9 resulted in a 5' staggered overhang of one base pair, which triggered an overhang-dependent base replication of an A:T at the mutation site. This recovered the open reading frame and the CAPN3 DNA sequence was repaired template-free to wild type, which led to CAPN3 mRNA and protein expression. Off-target analysis using amplicon sequencing of 43 in silico predicted sites demonstrates the safety of this approach. Our study extends previous usage of single cut DNA modification since our gene product has been repaired into the wild-type CAPN3 sequence with the perspective of a real cure.

The Base Pairs of Isoguanine and 8-Aza-7-deazaisoguanine with 5-Methylisocytosine as Targets for DNA Functionalization.

The isoguanine-isocytosine base pair (isoG-isoC) represents an important expansion of the DNA coding system. The base pair is more stable than the canonical adenine-thymine or guanine-cytosine pairs. However, nothing is known on the functionalization of the noncanonical isoG-isoC pair at the isoguanine site. In this work, functionalization of the isoG-isoC and the isosteric base pair that contains 8-aza-7-deazaisoguanine in place of isoguanine is studied. Short ethynyl, more space demanding octadiynyl, and dendritic tripropargylamine residues attached to the isoG-isoC base pairs were introduced to oligonucleotides. 12-mer duplexes were formed by hybridization with single base pair modification. The use of the two modified nucleobases gave us the freedom to shift nucleobase substituents within the major groove of double helical DNA. Clickable side chains at position-7 stabilize the base pair, whereas 8-substituents reduce its stability strongly. The weak isoguanine-thymine or 8-aza-7-deazaisoguanine-thymine base pairs show a similar sensitivity to the position of nucleobase functionalization as base pair matches formed with 5-methylisocytosine. CD spectra of all modified duplexes display the typical shape of a B-DNA with only marginal changes. Fluorescent pyrene labeled DNA with long, short, and branched linkers was generated using click chemistry. Pyrene click adducts with long linkers are essential to maintain or to increase base pair stability. Labeled duplexes are more fluorescent than corresponding single strands. For the dendritic linker excimer emission was observed for single strands but only monomer emission in duplexes.

Numerical investigation of flow past a circular cylinder modified with a single groove at low Reynolds number

Flow past a circular cylinder modified by grooves is one of the passive control techniques to control the drag and lift forces. The presence of grooves tends to reduce the hydrodynamic drag and lift. The aim of this work was to numerically investigate the influence of a single groove modification on a circular cylinder and how it was impacting the hydrodynamic coefficients based on its position and shape at low Reynolds numbers. Three types of grooves were considered, namely, dimple, square, and triangular, with identical width and depth which is 0.1 times the diameter. The Reynolds numbers considered were 100, 150, and 200. The computational domain was set to a blockage ratio of 0.01 after carrying out a detailed domain independence test. It was observed that the drag and lift reduction was maximum at groove location 90° with the decrements being more for the square groove shape. The viscous drag showed a decrease in its values for all the groove positions located on the flow-facing side of the cylinder; however, the pressure drag showed an increment in its values for these groove locations, and these increments were relative of larger magnitudes, thus influencing a net increase in the total mean drag when compared to the smooth case; this was evident at groove locations 120° and 150°. The vorticity patterns for the grooves located on the front side showed large irregularities in magnitude between the anticlockwise and the clockwise vortices with the groove side vortices being of larger magnitudes, and for the wake side, the irregularities were negligible. The coefficient of pressure and separation angles were also investigated. The CP vs θ plots showed discontinuity at groove locations with abrupt variations at the front face of the cylinder; for the wake side grooves, these discontinuities were low and almost coincided with the CP vs θ plots of the smooth cylinder. The grooves located at 90° showed a delay in separation and at 60° the boundary layer tends to detach earlier.

Nanopores as versatile single-molecule tools: From DNA turbines to protein sequencing.

Nanopores offer ample opportunities for studying single biomolecules. I will present some recent examples of nanopore research from my lab: (1) DNA origami turbines powered by nanoscale flow. We demonstrated driven rotary motion of a nanoscale DNA origami turbine which harnesses energy from a water/ion flow generated by a static chemical or electrical potential gradient in a solid-state nanopore. Sustained unidirectional rotary motion of up to 20 revolutions/s were observed. These artificial nano-engines operate autonomously in physiological conditions, converting energy into useful mechanical work. (2) Nanopore-based sequential reading of peptides. We demonstrated a nanopore-based single-molecule peptide reader capable of reliably detecting single amino-acid substitutions within individual peptides. A peptide is linked to a DNA molecule and sequentially pulled through a biological nanopore by a DNA helicase in single amino-acid steps. Stepping ion-current signals enable discrimination of single-amino-acid substitutions in single reads. Notably, we demonstrated the capability to ‘rewind’ peptide reads, obtaining indefinitely many independent reads of the same molecule, yielding an undetectably low read errors. Recently, we expanded this concept to discriminating single post-translational modifications within peptides of mixed charge. These proof-of-concept experiments constitute a promising basis for the development of a single-molecule protein sequencer.

Novel and efficient method for the reconstruction of adenoviruses through isothermal assembly and its potential applications.

Adenovirus based vectors are useful tools for vaccine development, gene therapy, and oncolytic virotherapy. Here we describe a novel approach for the genetic engineering of any portion of the adenovirus genome and the reconstruction of either fully replication competent or defective virions. This innovative strategy is rapid, effective and suitable for the manipulation of the entire genome broadening the spectrum of potential applications for the adenovirus system. Our strategy involved insertion of restriction enzyme recognition sequences absent in the native virus into the termini of the adenovirus genome in order to facilitate recovery. These restriction enzyme sites, together with the two inverted terminal repeats and packaging sequences, were synthesized and then subcloned into the pBR322 vector. The remaining internal portion of the adenovirus genome was separated and amplified via PCR into six segments, of which groups of two were joined together by PCR and then subcloned into pBR322 plasmids. During the PCR reaction, an overlap of 30-40 bp was added to the termini of the adjacent fragments, allowing for the subsequent isothermal assembly and correct orientation of all fragments. This approach allows for the genetic modification of each genomic fragment according to the specific research goals, (e.g., deletion, substitution, addition, etc.) To recreate the entire viral genome, the four engineered fragments (each comprised of two adenovirus genomic sections) as well as the pBR322 backbone, were reassembled into a single construct utilizing an isothermal assembly reaction. Finally, the reassembled plasmid containing the entire genome was linearized and transfected into HEK293 cells to recover the complete reconstructed adenoviral vector. Using this approach, we have successfully generated two recombinant reporter adenoviruses, one of human adenovirus serotype 14 and another of serotype 55. The E3 region was replaced by the reporter genes (GFP and Luciferase) to visualize and track the recovery process. Subsequent infection of A549 cells with these reconstructed adenovirus vectors demonstrated that they were replication competent. This method shortens the viral reconstruction process because the one-step isothermal assembly requires less than 4 days, and recombinant adenovirus recovery occurs within 10 days. This new method allows for single or multiple genetic modifications within any portion of the viral genome and can be used to construct or manipulate any adenovirus whose complete genome sequence is known.