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Recombinant DNA technology has profoundly advanced virtually every aspect of biological and medical sciences, from basic research to biotechnology. Here, I discuss conceptual connections linking fundamental discoveries that were enabled by the technology, advances in the understanding of gene regulation in both prokaryotes and eukaryotes, and the recent FDA-approved CRISPR-based gene therapy for sickle cell anemia and β-thalassemia based on transcriptional derepression., Competing Interests: Declaration of interests The author declares no competing interests., (Copyright © 2024. Published by Elsevier Inc.)

Vaccine manufacturing fosters the prevention, control, and eradication of infectious diseases. Recombinant DNA and in vitro (IVT) mRNA vaccine manufacturing technologies were enforced to combat the recent pandemic. Despite the impact of these technologies, there exists no scientific announcement that compares them. Digital Shadows are employed in this study to simulate each technology, investigating root cause deviations, technical merits, and liabilities, evaluating cost scenarios. Under this lens we provide an unbiased, advanced comparative technoeconomic study, one that determines which of these manufacturing platforms are suited for the two types of vaccines considered (monoclonal antibodies or antigens). We find recombinant DNA technology to exhibit higher Profitability Index due to lower capital and starting material requirements, pertaining to lower Minimum Selling Price per Dose values, delivering products of established quality. However, the potency of the mRNA, the streamlined and scalable synthetic processes involved and the raw material availability, facilitate faster market penetration and product flexibility, constituting these vaccines preferable whenever short product development cycles become a necessity., (© 2024. The Author(s).)

In fall 1972, Paul Berg's laboratory published articles in PNAS describing two methods for constructing recombinant DNAs in vitro. He received half of the 1980 Nobel Prize in Chemistry for this landmark accomplishment. Here, we describe how this discovery came about, revolutionizing both biological research and the pharmaceutical industry., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Covalently closed circular DNA (cccDNA) exists as a stable episomal minichromosome in the nucleus of hepatocytes and is responsible for hepatitis B virus (HBV) persistence. We recently reported a technique involving recombinant cccDNA (rcccDNA) of HBV by site-specific DNA recombination. A floxed monomeric HBV genome was engineered into a precursor plasmid (prcccDNA) which was excised via Cre/loxP-mediated DNA recombination to form a 3.3-kb rcccDNA bearing a loxP-chimeric intron. The foreign sequence was efficiently removed during RNA splicing, rendering a functionally seamless insertion. We characterized rcccDNA formation, effective viral transcription, and replication induced by rcccDNA both in vitro and in vivo. Furthermore, we closely simulated chronic hepatitis by using a replication-defective recombinant adenoviral vector to deliver rcccDNA to the transgenic mice expressing Cre recombinase, which led to prominent HBV persistence. Here, we describe a detailed protocol about how to construct and evaluate Cre/loxP-based recombinant HBV cccDNA system both in vitro and in vivo., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Advances in genetic manipulation and genome engineering techniques have enabled on-demand targeted deletion, insertion, and substitution of DNA sequences. One important step in these techniques is the design of editing sequences (e.g. primers, homologous arms) to precisely target and manipulate DNA sequences of interest. Experimental biologists can employ multiple tools in a stepwise manner to assist editing sequence design (ESD), but this requires various software involving non-standardized data exchange and input/output formats. Moreover, necessary quality control steps might be overlooked by non-expert users. This approach is low-throughput and can be error-prone, which illustrates the need for an automated ESD system. In this paper, we introduce AutoESD (https://autoesd.biodesign.ac.cn/), which designs editing sequences for all steps of genetic manipulation of many common homologous-recombination techniques based on screening-markers. Notably, multiple types of manipulations for different targets (CDS or intergenic region) can be processed in one submission. Moreover, AutoESD has an entirely cloud-based serverless architecture, offering high reliability, robustness and scalability which is capable of parallelly processing hundreds of design tasks each having thousands of targets in minutes. To our knowledge, AutoESD is the first cloud platform enabling precise, automated, and high-throughput ESD across species, at any genomic locus for all manipulation types., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Vaccinia virus is a large double-stranded DNA virus that is widely used to express foreign genes from different origins. We generated recombinant vaccinia virus that expresses a viral inhibitor to examine its effect on virus-induced necroptosis. We provide a detailed protocol to describe the generation of recombinant vaccinia virus, validation of protein expression, and determination of necroptosis using live cell imaging. This approach can be adapted to examine the effect of other cell death regulators on virus-induced cell death. For complete details on the use and execution of this protocol, please refer to Liu et al. (2021)., Competing Interests: The authors declare no competing interests., (© 2021 The Author(s).)

Recombinant MrNV capsid protein has been shown to effectively deliver plasmid DNA and dsRNA into Sf9 insect cells and shrimp tissues. To extend its application to cancer cell-targeting drug delivery, we created three different types of chimeric MrNV virus-like particles (VLPs) (R-MrNV, I-MrNV, and E-MrNV) that have specificity toward the epidermal growth factor receptor (EGFR), a cancer cell biomarker, by incorporating the EGFR-specific GE11 peptide at 3 different locations within the host cell recognition site of the capsid. All three chimeric MrNV-VLPs preserved the ability to form a mulberry-like VLP structure and to encapsulate EGFP DNA plasmid with an efficiency comparable to that previously reported for normal MrNV (N-MrNV). Compared to N-MrNV, the chimeric R-MrNV and E-MrNV carrying the exposed GE-11 peptide showed a significantly enhanced binding and internalization abilities that were specific towards EGFR expression in colorectal cancer cells (SW480). Specific targeting of chimeric MrNV to EGFR was proven by both EGFR silencing with siRNA vector and a competition with excess GE-11 peptide as well as the use of EGFR-negative colorectal cells (SW620) and breast cancer cells (MCF7). We demonstrated here that both chimeric R-MrNV and E-MrNV could be used to encapsulate cargo such as exogenous DNA and deliver it specifically to EGFR-positive cells. Our study presents the potential use of surface-modified VLPs of shrimp virus origin as nanocontainers for targeted cancer drug delivery., (© 2021. The Author(s).)

Like protein-coding genes, long noncoding RNA (lncRNA) genes are composed of introns and exons. After their transcription, lncRNAs are subject to constitutive and/or alternative splicing. Here, we describe the current knowledge on lncRNA splice variants and their functional implications in cell biology., Competing Interests: Declaration of Interests The authors declare no conflict of interest., (Crown Copyright © 2021. Published by Elsevier Ltd. All rights reserved.)

Chloroplasts are attractive platforms for synthetic biology applications since they are capable of driving very high levels of transgene expression, if mRNA production and stability are properly regulated. However, plastid transformation is a slow process and currently limited to a few plant species. The liverwort Marchantia polymorpha is a simple model plant that allows rapid transformation studies; however, its potential for protein hyperexpression has not been fully exploited. This is partially due to the fact that chloroplast post-transcriptional regulation is poorly characterized in this plant. We have mapped patterns of transcription in Marchantia chloroplasts. Furthermore, we have obtained and compared sequences from 51 bryophyte species and identified putative sites for pentatricopeptide repeat protein binding that are thought to play important roles in mRNA stabilization. Candidate binding sites were tested for their ability to confer high levels of reporter gene expression in Marchantia chloroplasts, and levels of protein production and effects on growth were measured in homoplastic transformed plants. We have produced novel DNA tools for protein hyperexpression in this facile plant system that is a test-bed for chloroplast engineering.

Dissolved oxygen (DO)-stat fed-batch culture, which allows a high cell density culture of microorganisms under constant DO conditions, was applied to anti-CRP single-chain variable fragment (scFv) production using recombinant Escherichia coli. The DO-stat fed-batch culture was successfully performed under various DO conditions for more than 50 h, resulting in increased scFv production from 0.5 to 0.8 g/L by flask and batch cultures to 2.8-3.0 g/L by the fed-batch culture under the conditions of 5-40% of DO saturation. The formation of inclusion bodies was effectively depressed during DO-stat fed-batch operation; consequently, the solubility of anti-CRP scFv was significantly improved from 36-43% by the flask and batch cultures to 96-98% by the DO-stat fed-batch culture under a wide range of DO conditions. From the kinetic analysis of fed-batch experiments, it was also found that the successful folding of anti-CRP scFv in the cytoplasm occurred when metabolic rates, such as the specific growth rate and specific glucose consumption rate, were relatively low. These results show that the fed-batch culture operated by the DO-stat feeding strategy was effective for the enhanced production of anti-CRP scFv with high solubility., (Copyright © 2021 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Efficient expression vectors for unicellular ciliate eukaryotic Tetrahymena thermophila are still needed in recombinant biology and biotechnology applications. Previously, the construction of the T. thermophila Macronuclear Artificial Chromosome 1 (TtAC1) vector revealed additional needs for structural improvements such as better in vivo stability and maintenance as a recombinant protein expression platform. In this study, we designed an efficiently maintained artificial chromosome by biomimetic of the native macronuclear rDNA minichromosome. TtAC2 was constructed by sequential cloning of subtelomeric 3'NTS region (1.8 kb), an antibiotic resistance gene cassette (2 kb neo4), a gene expression cassette (2 kb TtsfGFP), rDNA coding regions plus a dominant C3 origin sequence (10.3 kb), and telomeres (2.4 kb) in a pUC19 backbone plasmid (2.6 kb). The 21 kb TtAC2 was characterized using fluorescence microscopy, qPCR, western blot and Southern blot after its transformation to vegetative T. thermophila CU428.2 strain, which has a recessive B origin allele. All experimental data show that circular or linear forms of novel TtAC2 were maintained as free replicons in T. thermophila macronucleus with or without antibiotic treatment. Notably, TtAC2 carrying strains expressed a TtsfGFP marker protein, demonstrating the efficacy and functionality of the protein expression platform. We show that TtAC2 is functionally maintained for more than two months, and can be efficiently used in recombinant DNA, and protein production applications., (Copyright © 2021 Elsevier GmbH. All rights reserved.)

Understanding how promoters work in non-host cells is complex. Nonetheless, understanding this process is crucial while performing gene expression modulation studies. This study began with the process of constructing a shuttle vector with CMV and OpIE2 promoters in a tandem arrangement to achieve gene expression in both mammalian and insect cells, respectively. In this system, inhibitory regions in the 5' end of the OpIE2 insect viral promoter were found to be blocking the activity of the CMV promoter in mammalian cells. Initially, the OpIE2 promoter was cloned downstream of the CMV promoter and upstream of the EGFP reporter gene. After introducing the constructed shuttle vector to insect and mammalian cells, a significant drop in the CMV promoter activity in mammalian cells was observed. To enhance the CMV promoter activity, several modifications were made to the shuttle vector including site-directed mutagenesis to remove all ATG codons from the downstream promoter (OpIE2), separating the two promoters to eliminate the effect of transcription interference between them, and finally, identifying some inhibitory regions in the OpIE2 promoter sequence. When these inhibitory regions were removed, high expression levels in insect and mammalian cells were maintained. In conclusion, a shuttle vector was constructed that works efficiently in both mammalian and insect cell lines in the absence of baculovirus infection or gene expression. Moreover, the shuttle vector can be used as a platform to further study the reason for this inhibition, which may give new insights about transcription and promoters' mode of action in both insect and mammalian hosts., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Vaccinia virus (VACV)-based vectors are in extensive use as vaccines and cancer immunotherapies. VACV engineering has traditionally relied on homologous recombination between a parental viral genome and a transgene-bearing transfer plasmid, an inefficient process that necessitates the use of a selection or screening marker to isolate recombinants. Recent extensions of this approach have sought to enhance the recovery of transgene-bearing viruses through the use of CRISPR-Cas9 engineering to cleave the viral genome in infected cells. However, these methods do not completely eliminate the generation of WT viral progeny and thus continue to require multiple rounds of viral propagation and plaque purification. Here, we describe MAVERICC (marker-free vaccinia virus engineering of recombinants through in vitroCRISPR/Cas9 cleavage), a new strategy to engineer recombinant VACVs in a manner that overcomes current limitations. MAVERICC also leverages the CRISPR/Cas9 system but requires no markers and yields essentially pure preparations of the desired recombinants in a single step. We used this approach to introduce point mutations, insertions, and deletions at multiple locations in the VACV genome, both singly and in combination. The efficiency and versatility of MAVERICC make it an ideal choice for generating mutants and mutant libraries at arbitrarily selected locations in the viral genome to build complex VACV vectors, effect vector improvements, and facilitate the study of poxvirus biology., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: K.C. is a member of the scientific advisory boards of Integrum Scientific, LLC, the Pandemic Security Initiative of Celdara Medical, LLC, and Biovaxys Technology Corp., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Our understanding of folate metabolism in Leishmania has greatly benefited from studies of resistance to the inhibitor methotrexate (MTX). Folates are reduced in Leishmania by the bifunctional dihydrofolate reductase thymidylate synthase (DHFR-TS) and by pteridine reductase (PTR1). To further our understanding of folate metabolism in Leishmania, a Cos-seq genome-wide gain of function screen was performed against MTX and against the two thymidylate synthase (TS) inhibitors 5-fluorouracil and pemetrexed. The screen revealed DHFR-TS and PTR1 but also the nucleoside transporter NT1 and one hypothetical gene derived from chromosome 31. For MTX, the concentration of folate in the culture medium affected the enrichment pattern for genes retrieved by Cos-seq. We generated a L. infantum DHFR-TS null mutant that was thymidine auxotroph, a phenotype that could be rescued by the addition of thymidine or by transfection of the flavin dependent bacterial TS gene ThyX. In these DHFR-TS null mutants it was impossible to obtain a chromosomal null mutant of PTR1 except if DHFR-TS or PTR1 were provided episomally. The transfection of ThyX however did not allow the elimination of PTR1 in a DHFR-TS null mutant. Leishmania can survive without copies of either DHFR-TS or PTR1 but not without both. Provided that our results observed with the insect stage parasites are also replicated with intracellular parasites, it would suggest that antifolate therapy in Leishmania would only work if both DHFR-TS and PTR1 would be targeted simultaneously., Competing Interests: The authors have declared that no competing interests exist.

Restriction enzymes provided the foundation on which molecular cloning was built, and they remain as essential tools in current recombinant DNA technology. The three classes of restriction enzymes and their features are introduced here., (© 2021 Cold Spring Harbor Laboratory Press.)

Many Escherichia coli expression vectors make use of the lac operon. In general, the lac operator ( lacO ) is located downstream from the promoter of the target gene, so that binding of the lac repressor blocks transcription initiation until lactose or the isopropyl-β-d-thiogalactopyranoside (IPTG) analog is added. The protocol given here is intended for use with IPTG-inducible vectors. l-Arabinose-inducible systems derived from the ara operon offer an alternative to expression systems based on the lac operon; guidance for their use is also provided., (© 2021 Cold Spring Harbor Laboratory Press.)

New therapies against hepatitis B virus (HBV) require the elimination of covalently closed circular DNA (cccDNA), the episomal HBV genome. HBV plasmids containing an overlength 1.3-mer genome and bacterial backbone (pHBV1.3) are used in many different models, but do not replicate the unique features of cccDNA. Since the stable cccDNA pool is a barrier to HBV eradication in patients, we developed a recombinant circular HBV genome (rcccDNA) to mimic the cccDNA using Cre/LoxP technology. We validated four LoxP insertion sites into the HBV genome using hydrodynamic tail vein injection into murine liver, demonstrating high levels of HBV surface antigen (HBsAg) and HBV DNA expression with rcccDNA formation. HBsAg expression from rcccDNA was >30,000 ng/mL over 78 days, while HBsAg-expression from pHBV1.3 plasmid DNA declined from 2753 ng/mL to 131 ng/mL over that time in immunodeficient mice (P < 0.001), reflective of plasmid DNA silencing. We then cloned Cre-recombinase in cis on the LoxP-HBV plasmids, achieving plasmid stability in bacteria with intron insertion into Cre and demonstrating rcccDNA formation after transfection in vitro and in vivo. These cis-Cre/LoxP-HBV plasmids were then used to create HBx-mutant and GFP reporter plasmids to further probe cccDNA biology and antiviral strategies against cccDNA. Overall, we believe these auto-generating rcccDNA plasmids will be of great value to model cccDNA for testing new therapies against HBV infection., (Copyright © 2020 Elsevier B.V. All rights reserved.)

A metal-resistant engineered Pichia pastoris was developed here to fulfil the metal bioleaching in aqueous conditions. Parent and recombinant yeasts were grown in YPD medium containing different concentrations of ion metals. XRD, electron microscopy and particle size analyser were used for the characterisation and the nanoparticle analyses. The nanoparticle production kinetics were studied by ICP-OES. The cytotoxicity of nanoparticles was assayed against human cell lines. Media colours changed to a range from purplish-brown to grey during early fermentation stages. The maximum biosorption capacities were recorded 81.23 and 493.35 mg/g for gold and palladium in batch conditions, respectively. Various physical investigations proved monodispersed spherical nanoparticles around 100 nm in size. Pure palladium nanoparticles and PdCl 2 represented the least cytotoxic potency towards T47D and EPG85.257 cells. The results demonstrated that the genetically modified yeast is a cost-effective, high-throughput, robust, and facile system for metal biosorption.

K. pneumoniae BLh-1 strain was genetically modified aiming at obtaining high ethanol productivity in cultivations using residual glycerol from biodiesel synthesis as substrate. The recombinant strain K. pneumoniae Kp17 was obtained by inserting the multicopy plasmid pTOPOBL17 containing the AdhE gene, and its own promoter, from K. pneumoniae BLh-1. Influence of Fe 2+ supplementation and initial glycerol concentration on culture conditions were analyzed, both in rotatory shaker and in batch bioreactors. In the bioreactor cultures, K. pneumoniae Kp17 strain produced 4.5 g L -1 of ethanol (productivity of 0.50 g L -1  h -1 and yields of 0.15 g g -1 ) after 24-h cultivation, corresponding to an increase of approximately 40% in ethanol concentration compared to wild strain, K. pneumoniae BLh-1. Best conditions were then applied in exponential fed-batch bioreactors, with final ethanol concentration of 17.30 g L -1 (productivity of 0.59 g L -1  h -1 and yields of 0.16 g g -1 ) after 30 h of feeding, representing 11.5% of increment in titer of ethanol compared to the wild strain. Mutant cells kept 92.5% of the plasmids under batch in 24 h, and 71.9% under fed-batch after 27 h of exponential feeding. The findings in this work show the possibility of using a simple approach to genetically modify K. pneumoniae to be employed this versatile bacterium for the bioconversion of residual glycerol into ethanol.

In eukaryotic cells, the genome is packaged into chromatin and exists in different states, ranging from open euchromatic regions to highly condensed heterochromatic regions. Chromatin states are highly dynamic and are organized by an interplay of histone post-translational modifications and effector proteins, both of which are central in the regulation of gene expression. For this, chromatin effector proteins must first search the nucleus for their targets, before binding and performing their role. A key question is how chromatin effector proteins search, interact with and alter the different chromatin environments. Here we present a modular fluorescence based in vitro workflow to directly observe dynamic interactions of effector proteins with defined chromatin fibres, replicating different chromatin states. We discuss the design and creation of chromatin assemblies, the synthesis of modified histones, the fabrication of microchannels and the approach to data acquisition and analysis. All of this with the aim to better understand the complex in vivo relationship between chromatin structure and gene expression., (Copyright © 2020 Elsevier Inc. All rights reserved.)

This protocol describes procedures for cloning blunt-ended DNA fragments into linearized plasmid vectors. To obtain the maximum number of "correct" ligation products when cloning blunt-ended target fragments, the two components of DNA in the ligation reaction must be present at an appropriate ratio. If the molar ratio of plasmid vector to target DNA is too high, then the ligation reaction may generate an undesirable number of circular empty plasmids, both monomeric and polymeric; if too low, the ligation reaction may generate an excess of linear and circular homopolymers and heteropolymers of varying sizes, orientations, and compositions. For this reason, the orientation of the foreign DNA and the number of inserts in each recombinant clone must always be validated by restriction endonuclease mapping or some other means., (© 2020 Cold Spring Harbor Laboratory Press.)

This protocol describes the standard, old-fashioned but reliable procedure for cloning linear DNA fragments whose ends are incompatible with each other but are compatible with those of the linearized vector., (© 2020 Cold Spring Harbor Laboratory Press.)

Coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has become a global pandemic in the past four months and causes respiratory disease in humans of almost all ages. Although several drugs have been announced to be partially effective treatments for this disease, no approved vaccine is available. Here, we described the construction of a recombinant Lactobacillus plantarum strain expressing the SARS-CoV-2 spike protein. The results showed that the spike gene with optimized codons could be efficiently expressed on the surface of recombinant L. plantarum and exhibited high antigenicity. The highest protein yield was obtained under the following conditions: cells were induced with 50 ng/mL SppIP at 37 °C for 6-10 h. The recombinant spike (S) protein was stable under normal conditions and at 50 °C, pH = 1.5, or a high salt concentration. Recombinant L. plantarum may provide a promising food-grade oral vaccine candidate against SARS-CoV-2 infection., Competing Interests: Declaration of competing interest All authors have approved the final version of the manuscript. The authors declare no conflicts of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Allitol is a kind of rare sugar alcohol with potential application value. An engineered strain, which simultaneously expressed D-psicose-3-epimerase (DPE), ribitol dehydrogenase (RDH), and formate dehydrogenase (FDH) three enzymes, was constructed by cloning above three genes into one plasmid and transformed into the host E. coli strain, and used as the whole-cell catalysts for biotransformation of allitol from the low-cost substrate of D-fructose. The whole cell allitol biotransformation conditions were optimized. The medium, recombinant gene induction conditions, and the substrate feeding rate for cultivation of the catalytic cells were optimized. Then, the fed-batch culture was made and scaled up to 10 L fermentor. Finally, 63.44 g/L allitol was obtained from 100 g/L D-fructose after 3 h of biotransformation, and the allitol crystals of 99.9% purity were obtained by using cooling recrystallization. The allitol production method developed in this research has high product purity, and is highly efficient, easily scaled up, and suitable for large-scale production of highly purified allitol.

During mitosis chromosomes reorganise into highly compact, rod-shaped forms, thought to consist of consecutive chromatin loops around a central protein scaffold. Condensin complexes are involved in chromatin compaction, but the contribution of other chromatin proteins, DNA sequence and histone modifications is less understood. A large region of fission yeast DNA inserted into a mouse chromosome was previously observed to adopt a mitotic organisation distinct from that of surrounding mouse DNA. Here, we show that a similar distinct structure is common to a large subset of insertion events in both mouse and human cells and is coincident with the presence of high levels of heterochromatic H3 lysine nine trimethylation (H3K9me3). Hi-C and microscopy indicate that the heterochromatinised fission yeast DNA is organised into smaller chromatin loops than flanking euchromatic mouse chromatin. We conclude that heterochromatin alters chromatin loop size, thus contributing to the distinct appearance of heterochromatin on mitotic chromosomes., Competing Interests: MF, PT, AP, HO, LY, SW, RA No competing interests declared, JD Reviewing editor, eLife, (© 2020, Fitz-James et al.)

Gynogenesis is an important breeding method in aquaculture and has been widely applied to many fish species. If gynogenetic progenies are to inherit paternal partial genomic DNA, this will increase genetic variation and will provide a useful outcome for breeding. In this study, we investigated the genetic variation in homeobox (Hox) gene clusters (HoxA4a, HoxA9a, HoxA11b, HoxB1b, HoxC4a, HoxC6b, and HoxD10a) among koi carp (Cyprinus carpio haematopterus, KOC; the stimulation sperm source), grass carp (Ctenopharyngodon idellus), and gynogenetic grass carp (GGC). We found paternal DNA (a special DNA fragment and HoxC6b) derived from KOC and a recombinant gene belonging to HoxC6b in GGC. We are the first to report the recombinant HoxC6b in GGC. Our study provides further evidence for paternal DNA transmission to gynogenetic progenies, which is a finding with great significance for the genetic breeding of fish.

Recombinant DNA technology, in which artificially "cut and pasted" DNA in vitro is introduced into living cells, contributed extensively to the rapid development of molecular biology over the past 5 decades since the latter half of the 20 th century. Although the original technology required special experiences and skills, the development of polymerase chain reaction (PCR) has greatly eased in vitro genetic manipulation for various experimental methods. The current development of a simple genome-editing technique using CRISPR-Cas9 gave great impetus to molecular biology. Genome editing is a major technique for elucidating the functions of many unknown genes. Genetic manipulation technologies rely on enzymes that act on DNA. It involves artificially synthesizing, cleaving, and ligating DNA strands by making good use of DNA-related enzymes present in organisms to maintain their life activities. In this review, I focus on key enzymes involved in the development of genetic manipulation technologies.

The dehydroshikimate dehydratase (DSD) from Corynebacterium glutamicum encoded by the qsuB gene is related to the previously described QuiC1 protein (39.9% identity) from Pseudomonas putida. Both QuiC1 and QsuB are two-domain bacterial DSDs. The N-terminal domain provides dehydratase activity, while the C-terminal domain has sequence identity with 4-hydroxyphenylpyruvate dioxygenase. Here, the QsuB protein and its N-terminal domain (N-QsuB) were expressed in the T7 system, purified and characterized. QsuB was present mainly in octameric form (60%), while N-QsuB had a predominantly monomeric structure (80%) in aqueous buffer. Both proteins possessed DSD activity with one of the following cofactors (listed in the order of decreasing activity): Co2+, Mg2+, Mn2+. The Km and kcat values for the QsuB enzyme (Km ~ 1 mM, kcat ~ 61 s-1) were two and three times higher than those for N-QsuB. 3,4-DHBA inhibited QsuB (Ki ~ 0.38 mM, Ki' ~ 0.96 mM) and N-QsuB (Ki ~ 0.69 mM) enzymes via mixed and noncompetitive inhibition mechanism, respectively. E. coli MG1655ΔaroEPlac‒qsuB strain produced three times more 3,4-DHBA from glucose in test tube fermentation than the MG1655ΔaroEPlac‒n-qsuB strain. The C-terminal domain activity towards 3,4-DHBA was not established in vitro. This domain was proposed to promote protein oligomerization for maintaining structural stability of the enzyme. The dimer formation of QsuB protein was more predictable (ΔG = ‒15.8 kcal/mol) than the dimerization of its truncated version N-QsuB (ΔG = ‒0.4 kcal/mol)., Competing Interests: The authors have declared that no competing interests exist.

Infectious Bursal Disease Virus (IBDV), a member of the Birnaviridae family, causes an immunosuppressive disease in young chickens. Although several reverse genetics systems are available for IBDV, the isolation of most field-derived strains, such as very virulent IBDV (vvIBDV) and their subsequent rescue, has remained challenging due to the lack of replication of those viruses in vitro. Such rescue required either the inoculation of animals, embryonated eggs, or the introduction of mutations in the capsid protein (VP2) hypervariable region (HVR) to adapt the virus to cell culture, the latter option concomitantly altering its virulence in vivo. We describe an improved ex vivo IBDV rescue system based on the transfection of an avian cell line with RNA polymerase II-based expression vectors, combined with replication on primary chicken bursal cells, the main cell type targeted in vivo of IBDV. We validated this system by rescuing to high titers two recombinant IBDV strains: a cell-culture adapted attenuated strain and a vvIBDV. Sequencing of VP2 HVR confirmed the absence of unwanted mutations that may alter the biological properties of the recombinant viruses. Therefore, this approach is efficient, economical, time-saving, reduces animal suffering and can be used to rescue other non-cell culture adapted IBDV strains.

Fluorescence/luminescence-based techniques play an increasingly important role in the development of test systems for the characterization of future drug candidates, especially in terms of receptor binding in the field of G protein-coupled receptors (GPCRs). In this article, we present the establishment of a homogeneous live cell-based BRET binding assay for the histamine H 2 receptor with different fluorescently labeled squaramide-type compounds synthesized in the course of this study. Py-1-labeled ligand 8 (UR-KAT478) was found to be most suitable in BRET saturation binding experiments with respect to receptor affinity (pK d  = 7.35) and signal intensity. Real-time kinetic experiments showed a full association of 8 within approximately 30 min and a slow dissociation of the ligand from the receptor. Investigation of reference compounds in BRET-based competition binding with 8 yielded pK i values in agreement with radioligand binding data. This study shows that the BRET binding assay is a versatile test system for the characterization of putative new ligands at the histamine H 2 receptor and represents a valuable fluorescence-based alternative to canonical binding assays.

This study focuses on preparing the monoclonal antibody (MAb) against subgroup A of avian leukosis virus (ALV-A) and identifying its antigenic epitope. The ALV-A gp85 gene with a size of 1005bp was amplified and expressed into a recombinant protein with a size of 46KD in E.coli. The products expressed after purification were inoculated into BALB/c mice for preparing antibody-secreting splenic lymphocytes and further obtaining hybridoma cells. Finally, one new hybridoma cell (A18GH) secreting MAb against ALV-A was screened, and the MAb was able to detect ALV-A/K strains in an indirect immunofluorescence assay (IFA), but not ALV-B/J strains. A total of 14 overlapping truncated ALV-A gp85 protein segments were expressed and eight peptides containing different antigenic amino acids were artificially synthesized for analyzing the antigenic epitope of the MAb using a western blot or an ELISA, and the results indicate that the antigenic epitope consists of seven amino acids within the 146- ATRFLLR -152 region of the ALV-A gp85 protein. A biological information analysis shows that the antigenic epitope has a high antigenic index and develops a curved linear spatial structure. Further, its 7 amino acids are completely within the 17 representative ALV-A strains, 4 are within the 11 ALV-K strains, and fewer are within the ALV-B/J/E strains. This study will significantly assist in a further understanding of the protein structure and function of ALV-A, and in the establishment of specific ALV-A detection methods., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The covalently closed circular DNA (cccDNA) of hepatitis B virus (HBV) is critical for viral persistence in vivo. The lack of reliable, characterized and convenient small animal models for studying cccDNA persistence has long been a bottleneck for basic and translational research on HBV cure. A mouse model that can maintain intrahepatic cccDNA is urgently needed. Through combining the Cre/loxP-mediated recombination and adeno-associated virus (AAV) vector delivery strategy, we establish a novel recombinant cccDNA (rcccDNA) mouse model. AAV-rcccDNA mice supported long-term maintenance of intrahepatic rcccDNA which could be easily detected by Southern blotting within 30 weeks after transduction. Quantitative PCR could detect the rcccDNA signal throughout the experiment duration (>51 weeks). Furthermore, rcccDNA supported persistent serum antigenemia (>72 weeks) and intrahepatic HBsAg and HBcAg expression (>51 weeks). Flow cytometry analysis and single-cell RNA sequencing showed that AAV-rcccDNA mice displayed a compromised CD8 + T cell response. Meanwhile, minimal intrahepatic inflammation and fibrosis were observed. Furthermore, three anti-HBV compounds, AKEX0007, a post-transcriptional inhibitor, Bay 41-4109, a capsid allosteric modulator, and Entecavir were assessed in this AAV-rcccDNA mouse model. The changes of viral markers by these drugs were consistent with their mode of action although neither of them diminished the level of rcccDNA. This mouse model recapitulated the immune tolerant state of HBV infection with long term maintenance of cccDNA and antigenemia, which will provide a suitable platform for studying cccDNA persistence and developing intervention strategies that would eventually break the tolerance and clear the virus., (Copyright © 2020. Published by Elsevier B.V.)

The fast-paced field of synthetic biology is fundamentally changing the global biosecurity framework. Current biosecurity regulations and strategies are based on previous governance paradigms for pathogen-oriented security, recombinant DNA research, and broader concerns related to genetically modified organisms (GMOs). Many scholarly discussions and biosecurity practitioners are therefore concerned that synthetic biology outpaces established biosafety and biosecurity measures to prevent deliberate and malicious or inadvertent and accidental misuse of synthetic biology's processes or products. This commentary proposes three strategies to improve biosecurity: Security must be treated as an investment in the future applicability of the technology; social scientists and policy makers should be engaged early in technology development and forecasting; and coordination among global stakeholders is necessary to ensure acceptable levels of risk., (© 2020 The Authors. Published under the terms of the CC BY 4.0 license.)

In spite of the enormous potential of CRISPR/Cas in basic and applied science, the levels of undesired genomic modifications cells still remain mostly unknown and controversial. Nowadays, the efficiency and specificity of the cuts generated by CRISPR/Cas is the main concern. However, there are also other potential drawbacks when DNA donors are used for gene repair or gene knock-ins. These GE strategies should take into account not only the specificity of the nucleases, but also the fidelity of the DNA donor to carry out their function. The current methods to quantify the fidelity of DNA donor are costly and lack sensitivity to detect illegitimate DNA donor integrations. In this work, we have engineered two reporter cell lines (K562&#95;SEWAS84 and K562GWP) that efficiently quantify both the on-target and the illegitimate DNA donor integrations in a WAS -locus targeting setting. K562&#95;SEWAS84 cells allow the detection of both HDR-and HITI-based donor integration, while K562GWP cells only report HDR-based GE. To the best of our knowledge, these are the first reporter systems that allow the use of gRNAs targeting a relevant locus to measure efficacy and specificity of DNA donor-based GE strategies. By using these models, we have found that the specificity of HDR is independent of the delivery method and that the insertion of the target sequence into the DNA donor enhances efficiency but do not affect specificity. Finally, we have also shown that the higher the number of the target sites is, the higher the specificity and efficacy of GE will be.

Peste des petits ruminants (PPR) is an acute, highly infectious, and highly pathogenic disease, which mainly damages small ruminants such as goats and sheep. Hemagglutinin protein (H), the main antigenic protein of peste des petits ruminants virus (PPRV), has been a hot spot in the research of genetic engineering vaccine for PPRV. In this study, the silkworm baculovirus surface display technology is combined with the transmembrane structure of the silkworm baculovirus envelope protein GP64 and different characteristics of the promoters to display four kinds of fusion proteins, which contain P ph -H, P ph -HJ, P ie1 -H, and P ie1 -HJ. The fusion proteins displayed on baculovirus surface have been detected by western blotting, cell surface immunofluorescence, and immunogold electron microscopy. In addition, the dominant form of PPR H displayed on baculovirus surface has been determined which is fusion protein mediated by P ph containing the hemagglutinin protein and full-length GP64, P ph -H. Furthermore, by comparing the fluorescence intensity of binding of hemagglutinin protein and signaling lymphocyte activation molecules (SLAM) in Vero-SLAM cells by immunocytochemistry, P ph -H can be combined with the receptor protein of PPRV, SLAM. It provides technical support for displaying the different structure of hemagglutinin and exploring the key sites of hemagglutinin and SLAM binding. Meanwhile, it is important for exploring the pathogenesis and immune mechanism of PPRV.

Cotton leaf curl disease (CLCuD), caused by whitefly (Bemisiatabaci) transmitted single-stranded DNA viruses belonging to the Genus, Begomovirus (family, Geminiviridae) in association with satellite molecules; is responsible for major economic losses in cotton in three northwest (NW) Indian states Haryana, Punjab, and Rajasthan. Annual CLCuD incidences during 2012 to 2014 were estimated to be 37.5%, 63.6%, and 38.8% respectively. Cotton leaves were collected from symptomatic plants annually for three years and subjected to DNA isolation, followed by rolling circle amplification (RCA), cloning, and DNA sequencing of apparently full-length begomoviral genomes and associated betasatellites and alphasatellites. Among the thirteen CLCuD-begomoviral genomes recovered, eight were identified as Cotton leaf curl Multan virus-Rajasthan (CLCuMuV-Ra), one as -Pakistan (PK) and another as -Faisalabad (Fai), whereas, three were as Cotton leaf curl Kokhran virus-Burewala (CLCuKoV-Bu), indicating that CLCuMuV-Ra was the most prevalent begomovirus species. Five of the eight CLCuMuV-Ra sequences were found to be recombinants. The CLCuMuV-Ra- associated satellites consisted of Cotton leaf curl Multan betasatellite (CLCuMB), and Gossypium darwinii symptomless alphasatellite (GDarSLA), and Croton yellow vein mosaic alphasatellite (CrYVMoA). The second most abundant helper virus species, CLCuKoV-Bu, was associated with CLCuMB and GDarSLA., Competing Interests: There is no competing interest of “Sumitomo Chemical India Pvt. Ltd.” on this research. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Common variable immunodeficiency (CVID) results in defective B cell differentiation and impaired antibody production and is the most common symptomatic primary immunodeficiency. Our aim was to evaluate the correlation among B cell subgroups, κ-deleting recombination excision circle (KREC) copy numbers, and clinical and immunological data of the patients with CVID, and evaluate the patients according to classifications currently available to define the role of KREC copy numbers in the diagnosis of CVID. KREC analysis was performed using a quantitative real-time polymerase chain reaction assay, and B cell subgroups were measured by flow cytometry. The median age of the patients (n = 30) was 25 (6-69) years. Parental consanguinity ratio was 33%. The median age at diagnosis was 15 (4-59), and follow-up period was 6 (1-37) years. CD19 + and CD4 + cell counts at the time of diagnosis were low in 66.7% and 46.7% of the patients, respectively. CD19 + cell counts were positively correlated with KREC copy numbers in patients and healthy controls. CD19 + cell counts and KREC copy numbers were significantly reduced in CVID patients compared to healthy controls as expected. KRECs are quantitative markers for B cell defects. We found low CD4 + cell numbers, recent thymic emigrants, and lymphopenia in some of the patients at diagnosis, which reminds the heterogeneity of CVID's etiology. In this study, a positive correlation was shown between CD19 + cell counts and KREC copy numbers. Low KREC copy numbers indicated B cell deficiency; however, high KREC copy numbers were not sufficient to rule out CVID.

Tomato yellow leaf curl virus (TYLCV) and its related viruses are prone to recombination. It was reported that random homologous recombination between 20% diverging TYLCV related species is rarely deleterious and may be associated with a fitness advantage. Indeed, TYLCV-IS76, a recombinant between the 20% divergent TYLCV and tomato yellow leaf curl Sardinia virus (TYLCSV), exhibited a higher fitness than that of parental viruses. As this typical fitness advantage was observed with TYLCV-IS76 representatives of different pedigrees, it was thought that it is induced by beneficial intra-genomic interactions rather than by specific mutations. This hypothesis was further supported with TYLCV-IS141, a TYLCV recombinant with a short TYLCSV inherited fragment of around 141 nts, slightly longer than that of TYLCV-IS76. Indeed, the typical fitness advantage was detected irrespective of the position of the recombination breakpoint (loci 76 or 141) and the sequences of the TYLCV and TYLCSV inherited fragments., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2020. Published by Elsevier Inc.)

Herpes simplex virus 1 (HSV-1) is a representative alphaherpesvirus that can provoke a series of severe diseases to human being, but its exact pathogenesis is not perfectly understood. UL2, a uracil-DNA glycosylase involved in the process of HSV-1 DNA replication, has been shown to be predominantly targeted to the nuclei in our previous study, yet little is established regarding the subcellular localization signal or its related function of UL2 during HSV-1 propagation. Here, by creating a number of UL2 variants merged with enhanced yellow fluorescent protein, an authentic nuclear localization signal (NLS) of UL2 was, for the first time, identified and profiled to amino acids (aa) 1 to 17 (MKRACSRSPSPRRRPSS), and 12 RRR 14 was indispensable for its nuclear accumulation. Besides, the predicted nuclear export signal (aa 225 to 240) of UL2 was determined to be nonfunctional. Based on the HSV-1 bacterial artificial chromosome and homologous recombination technique, three recombinant viruses with mutations of the identified NLS, deletion and revertant of UL2 were constructed to assess the effect of UL2 nuclear targeting on HSV-1 replication. Compared to the wild type HSV-1, UL2 deletion remarkably restrained viral production, and mutation of NLS targeting UL2 to cytoplasm (pan-cellular distribution) in recombinant virus-infected cells showed a certain degree of deficiency in HSV-1 proliferation. Moreover, recombinant virus with UL2 deletion exhibited serious damages of viral DNA synthesis and mRNA expression, and these processes were partially disrupted in the recombinant virus with UL2 NLS mutation. Collectively, we had established a functional NLS in UL2 and showed that the NLS-mediated nuclear translocation of UL2 was important for efficient production of HSV-1. These data were of significance for further clarifying the biological function of UL2 during HSV-1 infection.

The manipulation of gene expression is an essential tool to study the function of genes or signaling pathways. Uniform and robust gene manipulation is crucial for successful assays. However, neuronal cells are generally difficult-to-transfect cells with conventional DNA/RNA transfection reagents. Therefore, virus-mediated gene delivery is a primary choice for the studies of gene functions in neurons. In this chapter, we will describe the methods for lentivirus-mediated gene expression or knockdown in DRG neurons.

The phenotypes conferred by recombinant plasmids upon host cells often exhibit variability between replicate populations. This statistical noise is mostly a consequence of adaptive evolution in response to fitness burdens imposed by the plasmids themselves. We developed a novel strategy, 'ribosome pegging', to exclude common unwanted mutations that benefit host cells at the expense of heterologous gene expression. Plasmids that constitutively co-expressed the fluorescent reporter tagRFP and ribosomal protein L23 (rplW) were used to transform Escherichia coli cells that lacked the essential chromosomal rplW gene. Cells within the population that expressed too little L23, or too much, were evidently inviable. Ribosome pegging obviates the need for antibiotics, thus facilitating the deployment of recombinant bacteria in uncontrolled environments. We show that ribosome-pegged E. coli carrying a plasmid that constitutively expresses L23 and an artificially evolved enzyme protects fruit flies from otherwise toxic doses of the insecticide malathion., (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Escherichia coli O157:H7 is considered as emerging foodborne pathogens that occur globally. Three major virulence protein factors; EspA(E), intimin(I), Tir(T) and Stx2 toxin have been found to be highly associated with bloody diarrhoea or, Haemolytic Uremic Syndrome. In this study, a trivalent recombinant EIT in combination with the binding domain of STX toxin were encapsulated with chitosan nanoparticles as a combination vaccine candidate. Mice were immunized either subcutaneously or orally with these antigens and challenged with E. coli O157:H7. Results of the binding inhibition assay with caco2 cell monolayer show a significant reduction in the adhesion percentage of pre-treated E. coli O157:H7 with immunized mice sera. Evaluation of neutralizing abilities of immune sera pre-incubated with CD 50 dose of STX2 by Vero cells cytotoxicity neutralization assay shows less morphological reforms in comparison with the control groups. Results of mice mortality challenge with STX2 demonstrate around 66% of survived in immunized mice. In a challenge experiment with E. coli O157:H7, all the immunized mice showed a significant decrease in bacterial colonization and shedding. The results indicate that the use of multiple recombinant proteins in combination with natural nanostructure effectively evocated strong humoral and mucosal response, increasing the protection capacity of the synthetic antigen.

β-conglycinin is one of the most allergenic proteins, and its constituent subunits α', α, and β are all potential allergens to humans. In the present study, we concentrated on the destructed antigenic sites of β subunit of β-conglycinin after high hydrostatic pressure (HHP) treatment. In this paper, the overlapping gene fragments of the β subunit of β-conglycinin were amplified by polymerase chain reaction (PCR) and cloned into T7 phage vectors. After being packaged in vitro, the recombinant T7 phage was constructed, and the overlapping fragments of the β subunit were displayed on the phage surface. The recombinant phages that expressed the overlapping fragments of the β subunit were used to react with specific antiserum by indirect ELISA to identify the HHP destructed antigenic sites. After three rounds of expression and identification, we used synthetic peptide technology to identify that the obtained fragment was a conformational epitope. We further confirmed that HHP treatment changed the conformational structure of β-conglycinin, which reduced the antigenicity of the protein., (Copyright © 2019 Elsevier B.V. All rights reserved.)

A heterologous xylose utilization pathway, either xylose reductase-xylitol dehydrogenase (XR-XDH) or xylose isomerase (XI), is usually introduced into Saccharomyces cerevisiae to construct a xylose-fermenting strain for lignocellulosic ethanol production. To investigate the molecular basis underlying the effect of different xylose utilization pathways on the xylose metabolism and ethanol fermentation, transcriptomes of flocculating industrial strains with the same genetic background harboring different xylose utilization pathways were studied. A different source of xylA did not obviously affect the change of the strains transcriptome, but compared with the XR-XDH strain, several key genes in the central carbon pathway were downregulated in the XI strains, suggesting a lower carbon flow to ethanol. The carbon starvation caused by lower xylose metabolism in XI strains further influenced the stress response and cell metabolism of amino acid, nucleobase, and vitamin. Besides, the downregulated genes mostly included those involved in mitotic cell cycle and the cell division-related process. Moreover, the transcriptomes analysis indicated that the after integrate xylA in the δ region, the DNA and chromosome stability and cell wall integrity of the strains were affected to some extent. The aim of this was to provide some reference for constructing efficient xylose-fermenting strains.

We evaluate the efficacy of recombinant DNA vaccine ABA392 against haemorrhagic septicaemia infection through intranasal administration route by targeting the mucosal immunity. The DNA vaccine was constructed and subjected to animal study using the Sprague Dawley (SD) rat. The study was divided into two major parts: (i) active and (ii) passive immunization studies, involving 30 animals for each part. Each group was then divided into five test groups: two test samples G1 and G2 with 50 and 100 µg ml -1 purified DNA vaccine; one positive control G5 with 10 6  CFU per ml formalin-killed PMB2; and two negative controls, G3 and G4 with normal saline and pVAX1 vector. Both studies were conducted for the determination of immunogenicity by total white blood cell count (TWBC), indirect ELISA and histopathological changes for the presence of the bronchus-associated lymphoid tissue (BALT). Our findings demonstrate that TWBC, IgA and IgG increased after each of the three vaccination regimes: groups G1, G2 and G5. Test samples G1 and G2 showed significant differences (P < 0·05) compared to the negative controls, G3 and G4, but no significant differences from the positive control G5. Groups G1, G2 and G5 showed more formation of BALT compared to the negative controls, G3 and G4. Our results show that intranasal inoculation of recombinant DNA vaccine ABA392 can provoke mucosal immunity which makes it a potential prophylactic against HS. SIGNIFICANCE AND IMPACT OF THE STUDY: New approach of combating haemorrhagic septicaemia disease among bovines by recombinant DNA vaccine is crucial to overcome the loss of edible products from the infected bovines. DNA vaccine can potentially serve as a better immunogen which would elicit both cellular and humoral immunity, and it is also stable for its molecular reproduction. This research report demonstrates an effective yet simple way of administering the DNA vaccine via the intranasal route in rats, to provoke the mucosal immunity through the development of immunoglobulins IgA, IgG and bronchus-associated lymphoid tissue which guard as the first-line defence at the host's mucosal lining., (© 2019 The Society for Applied Microbiology.)

Nucleic acid amplification (NAA) is a cornerstone of modern molecular and synthetic biology. Routine application by non-specialists, however, is hampered by difficulties with storing and handling the requisite labile and expensive reagents, such as deoxynucleoside triphosphates (dNTPs) and polymerases, and the complexity of protocols for their use. Here, a recombinant E. coli extract is reported that provides all the enzymes to support high-fidelity DNA amplification, and with labile dNTPs generated in situ from cheap and stable deoxynucleosides. Importantly, this is obtained from a single, engineered cell strain, through minimal processing, as a lysate capable of replacing the cold-stored commercial reagents in a typical PCR. This inexpensive preparation is highly active, as 1 L of bacterial culture is enough to supply ~10 6 NAA reactions. Lyophilized lysate can be used after a single-step reconstitution, resulting overall in a greatly simplified workflow and a promising synthetic biology tool, in particular for applications such as diagnostics.

Nontypeable Haemophilus influenzae (NTHi) is the primary cause of bacterially induced acute exacerbations of chronic obstructive pulmonary disease (COPD). NTHi adheres to and invades host respiratory epithelial cells as a means to persist in the lower airways of adults with COPD. Therefore, we mined the genomes of NTHi strains isolated from the airways of adults with COPD to identify novel proteins to investigate their role in adherence and invasion of human respiratory epithelial cells. An isogenic knockout mutant of the open reading frame NTHI1441 showed a 76.6% ± 5.5% reduction in invasion of human bronchial and alveolar epithelial cells at 1, 3, and 6 h postinfection. Decreased invasion of the NTHI1441 mutant was independent of either intracellular survival or adherence to cells. NTHI1441 is conserved among NTHi genomes. Results of whole-bacterial-cell enzyme-linked immunosorbent assay (ELISA) and flow cytometry experiments identified that NTHI1441 has epitopes expressed on the bacterial cell surface. Adults with COPD develop increased serum IgG against NTHI1441 after experiencing an exacerbation with NTHi. This study reveals NTHI1441 as a novel NTHi virulence factor expressed during infection of the COPD lower airways that contributes to invasion of host respiratory epithelial cells. The role in host cell invasion, conservation among strains, and expression of surface-exposed epitopes suggest that NTHI1441 is a potential target for preventative and therapeutic interventions for disease caused by NTHi., (Copyright © 2019 American Society for Microbiology.)