Insertion Of Sequences Research Articles

Development of a Recombinase-Mediated Cassette Exchange System for Gene Knockout and Expression of Non-Native Gene Sequences in Rickettsia

Background/Objectives: Incidence of vector-borne diseases, including rickettsioses and anaplasmosis, has been increasing in many parts of the world. The obligate intracellular nature of rickettsial pathogens has hindered the development of robust genetic tools for the study of gene function and the identification of therapeutic targets. Transposon mutagenesis has contributed to recent progress in the identification of virulence factors in this important group of pathogens. Methods: Combining the efficiency of the himar1 transposon method with a recombinase-mediated system, we aimed to develop a genetic tool enabling the exchange of the transposon with a cassette encoding non-native sequences. Results: This approach was used in Rickettsia parkeri to insert a himar1 transposon encoding fluorescent protein and antibiotic resistance genes for visualization and selection, flanked by mismatched loxP sites to enable subsequent recombinase-mediated cassette exchange (RMCE). RMCE mediated by a plasmid-encoded Cre recombinase was then employed to replace the transposon with a different cassette containing alternate fluorescent and selection markers and epitopes of Anaplasma phagocytophilum antigens. The resulting genetically modified R. parkeri was trialed as a live-attenuated vaccine against spotted fever rickettsiosis and anaplasmosis in mice. Conclusions: The use of this system provides a well-established and relatively efficient way of inserting non-native sequences into the rickettsial genome, with applications for the study of gene function and vaccine development.

Insertion of a short non-viral sequence in the 3' noncoding region of the hemagglutinin-neuraminidase increases mumps virus neurovirulence

Insertion of a short non-viral sequence in the 3' noncoding region of the hemagglutinin-neuraminidase increases mumps virus neurovirulence

RB-TnSeq barcode abundance data sets for Novosphingobium aromaticivorans grown on the β-5-linked aromatic dimer dehydrodiconiferyl alcohol.

A randomly barcoded transposon insertion sequencing (RB-TnSeq) library of Novosphingobium aromaticivorans DSM12444 was grown in media containing either glucose or the β-5-linked aromatic dimer dehydrodiconiferyl alcohol (DC-A) as the sole carbon source. The cultures were grown to saturation and then sequenced, yielding the barcode abundance data sets presented here.

Monitoring of Hepatitis E Virus Infection and Replication by Functional Tagging of the ORF2 Protein

Background and AimsHepatitis E virus (HEV) infection is a leading cause of acute hepatitis worldwide. Understanding of the mechanisms underlying productive HEV infection remains incomplete and would benefit from technological advances improving current model systems. MethodsWe exploited transposon-mediated random insertion and selection of viable clones to identify sites in the HEV ORF2 protein, corresponding to the viral capsid, allowing the insertion of reporter sequences in a functional context. ResultsShort sequence insertions (5 aa) were tolerated at four distinct sites in the C-terminal region of ORF2 protein, without significantly affecting viral capsid expression and subcellular localization as well as virus production. Full-length HEV genomes harboring larger sequence insertions such as an HA epitope tag, a highly sensitive miniaturized luciferase reporter (HiBiT) or a split GFP at these sites conserved their ability to produce infectious virus, while with about 1-log decrease in viral titers. Findings were confirmed in two different HEV genotype 3. In addition, we demonstrate that HiBiT-tagged HEV, offering rapid and several-log amplitude detection, can be used for the evaluation of antiviral drugs and neutralizing antibodies. ConclusionsWe describe a convenient, quantitative and potentially scalable system for the monitoring of HEV infection and replication in tissue culture. Impact And ImplicationsHepatitis E virus (HEV) infection is one of the most frequent causes of acute hepatitis and jaundice worldwide. As treatment options are limited and a vaccine is not universally available, the development of molecular tools to facilitate the identification of new therapeutic strategies is crucial. Based on a screening approach to identify viable insertion sites in the viral genome, we describe a versatile system for preparing recombinant viruses harboring split-reporter tags, i.e. luciferase and GFP. Proof-of-concept experiments revealed that convenient and quantitative monitoring of viral infection and replication is possible with this system, allowing to evaluate antiviral drugs and neutralizing antibodies.

Cure of Congenital Purpura Fulminans via Expression of Engineered Protein C Through Neonatal Genome Editing in Mice.

PC (protein C) is a plasma anticoagulant encoded by PROC; mutation in both PROC alleles results in neonatal purpura fulminans-a fatal systemic thrombotic disorder. In the present study, we aimed to develop a genome editing treatment to cure congenital PC deficiency. We generated an engineered APC (activated PC) to insert a furin-cleaving peptide sequence between light and heavy chains. The engineered PC was expressed in the liver of mice using an adeno-associated virus vector or CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat-associated 9)-mediated genome editing using an adeno-associated virus vector in vivo. The engineered PC could be released in its activated form and significantly prolonged the plasma coagulation time independent of the cofactor activity of PS (protein S) in vitro. The adeno-associated virus vector-mediated expression of the engineered PC, but not wild-type PC, prolonged coagulation time owing to the inhibition of activated coagulation FV (factor V) in a dose-dependent manner and abolished pathological thrombus formation in vivo in C57BL/6J mice. The insertion of EGFP (enhanced green fluorescent protein) sequence conjugated with self-cleaving peptide sequence at Alb locus via neonatal in vivo genome editing using adeno-associated virus vector resulted in the expression of EGFP in 7% of liver cells, mainly via homology-directed repair, in mice. Finally, we succeeded in improving the survival of PC-deficient mice by expressing the engineered PC via neonatal genome editing in vivo. These results suggest that the expression of engineered PC via neonatal genome editing is a potential cure for severe congenital PC deficiency.

Design of a Golden Gate Cloning Plasmid for the Generation of a Chimeric Virus-Like Particle-Based Subunit Vaccine Against Porcine Circovirus Type 2.

Porcine circovirus type 2 (PCV2) is a pervasive pathogen in the swine industry, leading to a spectrum of disorders known as porcine circovirus associated diseases (PCVAD). The PCV2 Cap protein contains critical antigenic epitopes and is the primary target for vaccine development. Current vaccines include inactivated viral particles and virus-like particles (VLPs), with experimental vaccines exploring various innovative approaches. This study introduces a novel Golden Gate assembly-based, Escherichia coli expression destination plasmid, pPRSVCP-E18, designed for the in-frame insertion of short peptide-coding DNA sequences into a highly antigenic site of Papaya ringspot virus (PRSV) coat protein (CP). We successfully cloned four PCV2 Cap peptides into this plasmid, expressed the chimeric CP proteins in E. coli under flask and bioreactor conditions, and assembled them into VLPs. These VLPs, when adjuvated and administered to BALB/c mice, elicited a specific IgG response against the PCV2 Cap peptides. Our findings demonstrate the plasmid's high efficiency for Golden Gate cloning and its potential in developing subunit vaccines against PCV2, contributing to the sustainable control of PCVAD in the swine industry.

Genome-wide identification of root colonization fitness genes in plant growth promoting Pseudomonas asiatica employing transposon-insertion sequencing

BackgroundPseudomonas spp. are well-studied plant growth promoters, particularly in the context of root colonization. However, the specific genetic factors that determine its fitness in the rhizosphere remain largely unexplored. This study breaks new ground by employing transposon insertion sequencing (Tn-Seq) to identify the genetic factors in Pseudomonas asiatica JR11 that are crucial for colonizing corn roots.ResultsWe created a transposon mutant library of P. asiatica JR11 with 91,884 insertion sites and subjected it to three consecutive enrichment cycles within the corn root system. A total of 79 genes were identified as essential for root colonization (negatively-selected), while 22 genes were found to counteract root colonization efficiency (positively-selected), with both sets being commonly present across all three cycles. These genes involve amino acid metabolism, cell wall biosynthesis, and protein functions. Additionally, we found four negatively-selected and four positively-selected hypothetical proteins that consistently influenced root colonization fitness.ConclusionsThe identification of these molecular determinants opens up exciting possibilities for further research. Understanding these pathways could lead to the development of novel strategies for enhancing the fitness of P. asiatica JR11 during corn root colonization, with potential implications for plant growth promotion and agricultural practices.

Genetic screen identifies cell wall enzyme is key for freshwater survival of Francisella tularensis.

Human infection with Francisella tularensis, a potentially lethal bacterial pathogen, typically occurs after exposure to contaminated water, soil, food, or an infected animal. While F. tularensis can persist in environmental sources over long periods of time, the genetic requirements that permit its long-term viability are not understood. To address this question, we developed a laboratory model for persistence of F. tularensis in fresh water, finding that viable cells could be recovered for 3 - 8 weeks after incubation at 4°C. Using this model, we took an unbiased, transposon insertion sequencing approach to identify genes critical for this persistence of F. tularensis cells. We found that mutants in mpl, a gene encoding murein peptide ligase, are defective for persistence in fresh water. Previous studies had identified mpl as critical for intramacrophage survival. Murein peptide ligase plays a role in peptidoglycan recycling, suggesting that F. tularensis uses this enzyme to maintain cell wall integrity during hypoosmotic and intramacrophage stress conditions. Our results highlight the importance of understanding how bacterial cell envelopes have evolved and adapted to maintain their integrity in a variety of stress conditions.

Interbrain neural correlates of self and other integration in joint statistical learning

While statistical learning is often studied individually, its collective representation through self-other integration remains unclear. This study examines dynamic self-other integration and its multi-brain mechanism using simultaneous recordings from dyads. Participants (N = 112) each repeatedly responded to half of a fixed stimulus sequence with either an active partner (joint context) or a passive observer (baseline context). Significant individual statistical learning was evident in the joint context, characterized by decreased reaction time (RT) and intra-brain neural responses, followed by a quadratic trend (i.e., first increasing and then decreasing) upon insertion of an interference sequence. More importantly, Brain-to-Brain Coupling (BtBC) in the theta band also showed learning and modulation-related trends, with its slope negatively and positively correlating with the slopes of RT and intra-brain functional connectivity, respectively. These results highlight the dynamic nature of self-other integration in joint statistical learning, with statistical regularities implicitly and spontaneously modulating this process. Notably, the BtBC serves as a key neural correlate underlying the dynamics of self-other integration.

Proposal of Patescibacterium danicum gen. Nov., sp. nov. in the ubiquitous ultrasmall bacterial phylum Patescibacteriota phyl. Nov

Abstract Candidatus Patescibacteria is a diverse bacterial phylum that is notable for members with ultrasmall cell size, reduced genomes, limited metabolic capabilities and dependence on other prokaryotic hosts. Despite the prevalence of the name Ca. Patescibacteria in the scientific literature, it is not officially recognized under the International Code of Nomenclature of Prokaryotes (ICNP) and lacks a nomenclatural type. Here, we rectify this situation by describing two closely related circular metagenome-assembled genomes (MAGs) and by proposing one of them (ABY1TS) to serve as the nomenclatural type for the species Patescibacterium danicumTS gen. Nov., sp. nov. according to the rules of the SeqCode. Rank-normalized phylogenomic inference confirmed the stable placement of P. danicumTS in the class ABY1 within Ca. Patescibacteria. Based on these results, we propose Patescibacterium gen. Nov. to serve as the type genus for associated higher taxa, including the phylum Patescibacteriota phyl. Nov. We complement our proposal with a genomic characterization, metabolic reconstruction, and biogeographical analysis of Patescibacterium. Our results confirm small genome sizes (< 1Mbp), low GC content (>36%), and the occurrence of long gene coding insertions in the 23S rRNA sequences, along with reduced metabolic potential, inferred symbiotic lifestyle, and a global distribution. In summary, this effort, focused on the fourth-largest phylum in the bacterial domain, aims to provide nomenclatural stability in the scientific literature.

InSeq analysis of defined Legionella pneumophila libraries identifies a transporter-encoding gene cluster important for intracellular replication in mammalian hosts.

Legionella pneumophila is an intracellular bacterial pathogen that replicates inside human alveolar macrophages to cause a severe pneumonia known as Legionnaires' disease. L. pneumophila requires the Dot/Icm Type IV secretion system to deliver hundreds of bacterial proteins to the host cytosol that manipulate cellular processes to establish a protected compartment for bacterial replication known as the Legionella-containing vacuole. To better understand mechanisms apart from the Dot/Icm system that support survival and replication in this vacuole, we used transposon insertion sequencing in combination with defined mutant sublibraries to identify L. pneumophila fitness determinants in primary mouse macrophages and the mouse lung. This approach validated that many previously identified genes important for intracellular replication were critical for infection of a mammalian host. Further, the screens uncovered additional genes contributing to L. pneumophila replication in mammalian infection models. This included a cluster of seven genes in which insertion mutations resulted in L. pneumophila fitness defects in mammalian hosts. Generation of isogenic deletion mutants and genetic complementation studies verified the importance of genes within this locus for infection of mammalian cells. Genes in this cluster are predicted to encode nucleotide-modifying enzymes, a protein of unknown function, and an atypical ATP-binding cassette (ABC) transporter with significant homology to multidrug efflux pumps that has been named Lit, for Legionella infectivity transporter. Overall, these data provide a comprehensive overview of the bacterial processes that support L. pneumophila replication in a mammalian host and offer insight into the unique challenges posed by the intravacuolar environment.IMPORTANCEIntracellular bacteria employ diverse mechanisms to survive and replicate inside the inhospitable environment of host cells. Legionella pneumophila is an opportunistic human pathogen and a model system for studying intracellular host-pathogen interactions. Transposon sequencing is an invaluable tool for identifying bacterial genes contributing to infection, but current animal models for L. pneumophila are suboptimal for conventional screens using saturated mutant libraries. This study employed a series of defined transposon mutant libraries to identify determinants of L. pneumophila fitness in mammalian hosts, which include a newly identified bacterial transporter called Lit. Understanding the requirements for survival and replication inside host cells informs us about the environment bacteria encounter during infection and the mechanisms they employ to make this environment habitable. Such knowledge will be key to addressing future challenges in treating infections caused by intracellular bacteria.

ReaL-MGE is a tool for enhanced multiplex genome engineering and application to malonyl-CoA anabolism

The complexities encountered in microbial metabolic engineering continue to elude prediction and design. Unravelling these complexities requires strategies that go beyond conventional genetics. Using multiplex mutagenesis with double stranded (ds) DNA, we extend the multiplex repertoire previously pioneered using single strand (ss) oligonucleotides. We present ReaL-MGE (Recombineering and Linear CRISPR/Cas9 assisted Multiplex Genome Engineering). ReaL-MGE enables precise manipulation of numerous large DNA sequences as demonstrated by the simultaneous insertion of multiple kilobase-scale sequences into E. coli, Schlegelella brevitalea and Pseudomonas putida genomes without any off-target errors. ReaL-MGE applications to enhance intracellular malonyl-CoA levels in these three genomes achieved 26-, 20-, and 13.5-fold elevations respectively, thereby promoting target polyketide yields by more than an order of magnitude. In a further round of ReaL-MGE, we adapt S. brevitalea to malonyl-CoA elevation utilizing a restricted carbon source (lignocellulose from straw) to realize production of the anti-cancer secondary metabolite, epothilone from lignocellulose. Multiplex mutagenesis with dsDNA enables the incorporation of lengthy segments that can fully encode additional functions. Additionally, the utilization of PCR to generate the dsDNAs brings flexible design advantages. ReaL-MGE presents strategic options in microbial metabolic engineering.

New-generation editing

Recent work on bacterial insertion sequences reveals that some of them use an RNA sequence (called Bridge RNA or Seek RNA) to define both donor and target DNA specificity. This opens the way to easy insertion of kilobase DNA sequences at pre-defined sites in the genome, announcing a host of new possibilities. The system still needs a lot of tweaking, as it has only been demonstrated in bacteria, but it holds great promise for genome editing and engineering.

Pseudomonas aeruginosa faces a fitness trade-off between mucosal colonization and antibiotic tolerance during airway infection.

Pseudomonas aeruginosa frequently causes antibiotic-recalcitrant pneumonia, but the mechanisms driving its adaptation during human infections remain unclear. To reveal the selective pressures and adaptation strategies at the mucosal surface, here we investigated P. aeruginosa growth and antibiotic tolerance in tissue-engineered airways by transposon insertion sequencing (Tn-seq). Metabolic modelling based on Tn-seq data revealed the nutritional requirements for P. aeruginosa growth, highlighting reliance on glucose and lactate and varying requirements for amino acid biosynthesis. Tn-seq also revealed selection against biofilm formation during mucosal growth in the absence of antibiotics. Live imaging in engineered organoids showed that biofilm-dwelling cells remained sessile while colonizing the mucosal surface, limiting nutrient foraging and reduced growth. Conversely, biofilm formation increased antibiotic tolerance at the mucosal surface. Moreover, mutants with exacerbated biofilm phenotypes protected less tolerant but more cytotoxic strains, contributing to phenotypic heterogeneity. P. aeruginosa must therefore navigate conflicting physical and biological selective pressures to establish chronic infections.

DOCK8 deficiency due to a deep intronic variant in two kindreds with hyper-IgE syndrome

Dedicator of cytokinesis 8 (DOCK8) deficiency underlies the majority of cases of patients with autosomal recessive form of the hyper-immunoglobulin E syndrome (HIES). Most DOCK8 mutations involve deletions and splice junction mutations that abrogate protein expression. However, a few patients whose presentation is reminiscent of DOCK8 deficiency have no identifiable mutations. Using Whole Exome Sequencing (WES), we identified a deep intronic homozygous DOCK8 variant located in intron 36 (c.4626 + 76 A > G) in two unrelated patients with features of HIES that resulted in an in-frame 75 base pair intronic sequence insertion in DOCK8 cDNA, resulting in a premature stop codon (p.S1542ins6Ter). This variant resulted in variable decrease in DOCK8 expression that was associated with impaired T cell receptor-triggered actin polymerization, decreased IL-6-induced STAT3 phosphorylation, reduced expression of the Th17 cell markers CCR6 and IL-17, and higher frequencies of GATA3+ T cells indicative of Th2 skewing. Our approach extends the reach of WES in identifying disease-related intronic variants. It highlights the role of non-coding mutations in immunodeficiency disorders, including DOCK8 deficiency, and emphasizes the need to explore these mutations in unexplained inborn errors of immunity.

High-throughput transposon mutagenesis in the family Enterobacteriaceae reveals core essential genes and rapid turnover of essentiality.

The Enterobacteriaceae are a scientifically and medically important clade of bacteria, containing the model organism Escherichia coli, as well as major human pathogens including Salmonella enterica and Klebsiella pneumoniae. Essential gene sets have been determined for several members of the Enterobacteriaceae, with the Keio E. coli single-gene deletion library often regarded as a gold standard. However, it remains unclear how gene essentiality varies between related strains and species. To investigate this, we have assembled a collection of 13 sequenced high-density transposon mutant libraries from five genera within the Enterobacteriaceae. We first assess several gene essentiality prediction approaches, investigate the effects of transposon density on essentiality prediction, and identify biases in transposon insertion sequencing data. Based on these investigations, we develop a new classifier for gene essentiality. Using this new classifier, we define a core essential genome in the Enterobacteriaceae of 201 universally essential genes. Despite the presence of a large cohort of variably essential genes, we find an absence of evidence for genus-specific essential genes. A clear example of this sporadic essentiality is given by the set of genes regulating the σE extracytoplasmic stress response, which appears to have independently acquired essentiality multiple times in the Enterobacteriaceae. Finally, we compare our essential gene sets to the natural experiment of gene loss in obligate insect endosymbionts that have emerged from within the Enterobacteriaceae. This isolates a remarkably small set of genes absolutely required for survival and identifies several instances of essential stress responses masked by redundancy in free-living bacteria.IMPORTANCEThe essential genome, that is the set of genes absolutely required to sustain life, is a core concept in genetics. Essential genes in bacteria serve as drug targets, put constraints on the engineering of biological chassis for technological or industrial purposes, and are key to constructing synthetic life. Despite decades of study, relatively little is known about how gene essentiality varies across related bacteria. In this study, we have collected gene essentiality data for 13 bacteria related to the model organism Escherichia coli, including several human pathogens, and investigated the conservation of essentiality. We find that approximately a third of the genes essential in any particular strain are non-essential in another related strain. Surprisingly, we do not find evidence for essential genes unique to specific genera; rather it appears a substantial fraction of the essential genome rapidly gains or loses essentiality during evolution. This suggests that essentiality is not an immutable characteristic but depends crucially on the genomic context. We illustrate this through a comparison of our essential genes in free-living bacteria to genes conserved in 34 insect endosymbionts with naturally reduced genomes, finding several cases where genes generally regarded as being important for specific stress responses appear to have become essential in endosymbionts due to a loss of functional redundancy in the genome.

Surface exclusion of IncC conjugative plasmids and their relatives.

The phenomenon of exclusion allows conjugative plasmids to selectively impede the entry of identical or related elements into their host cell to prevent the resulting instability. Entry exclusion blocks DNA translocation into the recipient cell, whereas surface exclusion destabilizes the mating pair. IncC conjugative plasmids largely contribute to the dissemination of antibiotic-resistance genes in Gammaproteobacteria. IncC plasmids are known to exert exclusion against their relatives, including IncC and IncA plasmids, yet the entry exclusion factor eexC alone does not account for the totality of the exclusion phenotype. In this study, a transposon-directed insertion sequencing approach identified sfx as necessary and sufficient for the remaining exclusion phenotype. Sfx is an exclusion factor unrelated to the ones described to date. A cell fractionation assay localized Sfx in the outer membrane. Reverse transcription PCR and beta-galactosidase experiments showed that sfx is expressed constitutively at a higher level than eexC. A search in Gammaproteobacteria genomes identified Sfx homologs encoded by IncC, IncA and related, untyped conjugative plasmids and an uncharacterized family of integrative and mobilizable elements that likely rely on IncC plasmids for their mobility. Mating assays demonstrated that sfx is not required in the donor for exclusion, ruling out Sfx as the exclusion target. Instead, complementation assays revealed that the putative adhesin TraN in the donor mediates the specificity of surface exclusion. Mating assays with TraN homologs from related untyped plasmids from Aeromonas spp. and Photobacterium damselae identified two surface exclusion groups, with each Sfx being specific of TraN homologs from the same group. Together, these results allow us to better understand the apparent incompatibility between IncA and IncC plasmids and to propose a mechanistic model for surface exclusion mediated by Sfx in IncC plasmids and related elements, with implications for the rampant dissemination of antibiotic resistance.

A Versatile Method to Create Antibody/Split‐Enzyme Complexes and Its Application to a Rapid, Homogeneous, and Universal Electrochemical Immunosensing System

AbstractIn this study, a versatile method is developed to create an antibody/split‐enzyme complex for use in electrochemical immunosensors. SpyCatchers are genetically fused at each terminus of flavin adenine dinucleotide‐dependent glucose dehydrogenase (FAD‐GDH) and a protease recognition sequence is inserted into a loop region to prepare a soluble protein and the split GDH. SpyTag‐fused single‐chain variable fragments (scFvs) are employed, which leads to the simple preparation of antibody‐split enzyme complexes (split AECs). The addition of pentameric C‐reactive protein (CRP), as a representative multimeric protein, restored the GDH activity of the split AECs, as the two split AEC fragments formed active GDH when in close proximity. CRP in human serum is electrochemically detected within 5 min without any washing steps with a clinically relevant CRP detection range (0.03–1 mg dL−1). The detection system is expanded to detect hemoglobin, SARS‐CoV‐2 spike protein, and inactivated SARS‐CoV‐2 by exchanging the scFvs. These results show that the convenient preparation method of the split GDH and the split AEC by the insertion of the protease recognition sequence and the use of SpyCatcher/SpyTag can realize a rapid, homogeneous, and universal electrochemical detection system that can be integrated into a commercially available electrochemical glucose sensor.

A novel method for the preparation of reproducible, stable, and non-infectious quality control materials for Chlamydia trachomatis nucleic acid detection.

Chlamydia trachomatis (CT) is a significant sexually transmitted pathogen known to evoke severe complications, including infertility. Nucleic acid amplification tests (NAATs) are recommended by the World Health Organization to detect CT infection. Furthermore, the establishment of methods, performance validation, internal quality control, and external quality assessment for CT NAATs necessitate the utilization of quality control materials (QCs). QCs are specimens or solutions that are analyzed for quality control purposes in a test system. In this study, we established a novel cell line that stably integrates CT amplification target sequences for producing QCs for CT NAATs. Utilizing clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 technology, we integrated the CT plasmid-mediated sequence (comprising the full length of the cryptic plasmid and the major outer membrane protein gene, 9,136 bp) into the MUC4 gene of HEK293T cells. Positive clones were screened through flow cytometric sorting, single-cell culture, and PCR-based identification, followed by the establishment of stable cell lines. These cells were then processed using optimized cell preservation procedures to prepare QCs. The sequence insertion copy number was confirmed by real-time quantitative PCR. This novel CT QCs demonstrate excellent clinical applicability, non-infectiousness, quantifiability, and stability. With an integrated sequence exceeding 9 kb in length, it offers exceptional flexibility for adapting to new kit developments. Furthermore, maintaining a well-defined copy number and stable shelf life, the QCs closely aligns with the quality control requirements of CT NAATs. This study presents an innovative method for preparing QCs for CT nucleic acid detection, making a valuable contribution to improving the performance of CT NAATs.IMPORTANCEUntreated CT infections impose significant burdens on individuals and communities, underscoring the importance of early and accurate testing via CT NAATs for disease control. QCs are instrumental in identifying testing process issues. Hence, we developed a cell line integrating CT-amplified target sequences as readily accessible non-infectious QCs. These QCs boast several advantages: the integration of over 9 kb of CT sequence allows for broad applicability, allowing flexible adaptation to the development of new kits. Confirming the CT sequence copy number provides a reliable basis for QC concentration preparation and kit detection limit evaluation. Optimized preservation protocol enhances QC stability during storage, facilitating convenient shipment to clinical laboratories at ambient temperatures. In summary, our novel CT QCs offer a powerful tool for improving CT NAAT performance and present a fresh perspective on QC preparation for detecting nucleic acids from intracellular parasitic pathogens.

The mitochondrial genome of Lavandula angustifolia Mill. (Lamiaceae) sheds light on its genome structure and gene transfer between organelles

BackgroundLavandula angustifolia holds importance as an aromatic plant with extensive applications spanning the fragrance, perfume, cosmetics, aromatherapy, and spa sectors. Beyond its aesthetic and sensory applications, this plant offers medicinal benefits as a natural herbal remedy and finds use in household cleaning products. While extensive genomic data, inclusive of plastid and nuclear genomes, are available for this species, researchers have yet to characterize its mitochondrial genome. This gap in knowledge hampers deeper understanding of the genome organization and its evolutionary significance.ResultsThrough the course of this study, we successfully assembled and annotated the mitochondrial genome of L. angustifolia, marking a first in this domain. This assembled genome encompasses 61 genes, which comprise 34 protein-coding genes, 24 transfer RNA genes, and three ribosomal RNA genes. We identified a chloroplast sequence insertion into the mitogenome, which spans a length of 10,645 bp, accounting for 2.94% of the mitogenome size. Within these inserted sequences, there are seven intact tRNA genes (trnH-GUG, trnW-CCA, trnD-GUC, trnS-GGA, trnN-GUU, trnT-GGU, trnP-UGG) and four complete protein-coding genes (psbA, rps15, petL, petG) of chloroplast derivation. Additional discoveries include 88 microsatellites, 15 tandem repeats, 74 palindromic repeats, and 87 forward long repeats. An RNA editing analysis highlighted an elevated count of editing sites in the cytochrome c oxidase genes, notably ccmB with 34 editing sites, ccmFN with 32, and ccmC with 29. All protein-coding genes showed evidence of cytidine-to-uracil conversion. A phylogenetic analysis, utilizing common protein-coding genes from 23 Lamiales species, yielded a tree with consistent topology, supported by high confidence values.ConclusionsAnalysis of the current mitogenome resource revealed its typical circular genome structure. Notably, sequences originally from the chloroplast genome were found within the mitogenome, pointing to the occurrence of horizontal gene transfer between organelles. This assembled mitogenome stands as a valuable resource for subsequent studies on mitogenome structures, their evolution, and molecular biology.