Full-length Plasmid Research Articles

Characterization and genomic analysis of the Lyme disease spirochete bacteriophage ϕBB-1.

Lyme disease is a tick-borne infection caused by the spirochete Borrelia (Borreliella) burgdorferi. Borrelia species have highly fragmented genomes composed of a linear chromosome and a constellation of linear and circular plasmids some of which are required throughout the enzootic cycle. Included in this plasmid repertoire by almost all Lyme disease spirochetes are the 32-kb circular plasmid cp32 prophages that are capable of lytic replication to produce infectious virions called ϕBB-1. While the B. burgdorferi genome contains evidence of horizontal transfer, the mechanisms of gene transfer between strains remain unclear. While we know that ϕBB-1 transduces cp32 and shuttle vector DNA during in vitro cultivation, the extent of ϕBB-1 DNA transfer is not clear. Herein, we use proteomics and long-read sequencing to further characterize ϕBB-1 virions. Our studies identified the cp32 pac region and revealed that ϕBB-1 packages linear cp32s via a headful mechanism with preferential packaging of plasmids containing the cp32 pac region. Additionally, we find ϕBB-1 packages fragments of the linear chromosome and full-length plasmids including lp54, cp26, and others. Furthermore, sequencing of ϕBB-1 packaged DNA allowed us to resolve the covalently closed hairpin telomeres for the linear B. burgdorferi chromosome and most linear plasmids in strain CA-11.2A. Collectively, our results shed light on the biology of the ubiquitous ϕBB-1 phage and further implicates ϕBB-1 in the generalized transduction of diverse genes and the maintenance of genetic diversity in Lyme disease spirochetes.

Mitochondrial Calcium Uniporter (MCU) that Modulates Mitochondrial Calcium Uptake and Facilitates Endometrial Cancer Progression through Interaction with VDAC1.

Although endometrial cancer represents a frequently diagnosed malignancy of the female reproductive tract, we know very little about the factors that control endometrial cancer. Our study was presented to investigate the function of MCU in endometrial tumorigenesis and the molecular mechanisms involved. A total of 94 endometrial cancer patients were recruited into our cohort. MCU and VDAC1 expression was examined in tumor and normal tissues via immunohistochemistry and immunofluorescence. Associations of MCU and VDAC1 expression with clinicopathological characteristics were evaluated. After transfection with shRNA targeting MCU or full-length MCU plasmids, clone formation, wound healing, transwell and MitoTracker Red staining were separately presented in Ishikawa and RL95-2 cells. Moreover, Western blotting or immunofluorescence was utilized to examine the expression of MCU, VDAC1, Na+/Ca2+/Li+ exchanger (NCLX), and β-catenin under VDAC1 knockdown and/or MCU overexpression or knockdown. MCU and VDAC1 expression were prominently up-regulated in endometrial cancer tissues and were significantly associated with histological grade, depth of myometrial invasion and lymph node status. MCU up-regulation enhanced clone formation, migration, and mitochondrial activity of endometrial cancer cells. The opposite results were investigated when MCU was silenced. MCU or VDAC1 silencing reduced the expression of MCU, VDAC1, NCLX, and β-catenin. Moreover, VDAC1 knockdown alleviated the promoting effect of MCU overexpression on the above proteins. This investigation demonstrated that MCU-induced mitochondrial calcium uptake plays a critical role in endometrial tumorigenesis through interaction with VDAC1.

Labor- and cost-effective long-read amplicon sequencing using a plasmid analysis service: Application to transposon-inserted alleles in Japanese morning glory.

The sequencing of PCR fragments amplified from specific regions of genomes is a fundamental technique in molecular genetics. Sanger sequencing is commonly used for this analysis; however, amplicon sequencing utilizing next-generation sequencing has become widespread. In addition, long-read amplicon sequencing, using Nanopore or PacBio sequencers to analyze long PCR fragments, has emerged, although it is often more expensive than Sanger sequencing. Recently, low-cost commercial services for full-length plasmid DNA sequencing using Nanopore sequencers have been launched in several countries, including Japan. This study explored the potential of these services to sequence long PCR fragments without the need for cloning into plasmid DNA, as cloning long PCR fragments or blunt-end PCR fragments into plasmid DNA is often challenging. PCR fragments of 4-11 kb, amplified from the DFR-B gene involved in the biosynthesis of anthocyanin, with or without Tpn1 transposons in Japanese morning glory (Ipomoea nil), were circularized using T4 DNA ligase and analyzed as templates. Although some inaccuracies in the length of homopolymer stretches were observed, the remaining sequences were obtained without significant errors. This method could potentially reduce the labor and costs associated with cloning, primer synthesis, and sequence assembly, thus making it a viable option for the analysis of long PCR fragment sequences. Moreover, this study reconfirmed that Tpn1 transposons are major mutagens in I. nil and demonstrated their transposition in the Violet line, a long-used standard in plant physiology.

Interaction of pAsa5 and pAsa8 Plasmids in Aeromonas salmonicida subsp. salmonicida.

The plasmid known as pAsa5 is present in Aeromonas salmonicida subsp. salmonicida, a fish pathogen. The pAsa5 plasmid carries genes that are essential for the bacterium's virulence. Recombination events are known to occur in pAsa5, resulting in the loss of certain segments or the acquisition of additional genetic elements. For example, the transposon carried by the large pAsa8 plasmid was found to be inserted into the pAsa5 plasmid in the SHY16-3432 strain, enabling the addition of antibiotic resistance genes to this plasmid, which does not normally possess any. In this study, we present the isolation of additional strains carrying pAsa8. Further analyses of these strains revealed that a fusion between pAsa5 and the complete version of pAsa8 is possible. The pAsa8 transposon insertion in pAsa5 seen in the SHY16-3432 strain appears to be an aberrant event compared to the fusion of the two full-length plasmids. A 22-nucleotide sequence, present in both plasmids, serves as the site for the fusion of the two plasmids. Moreover, it is possible to introduce pAsa8 through conjugation into naive strains of A. salmonicida subsp. salmonicida and once the plasmid is within a new strain, the fusion with pAsa5 is detectable. This study reveals a previously unexplored aspect of pAsa5 plasmid biology, highlighting an additional risk for the spread of antibiotic resistance genes in A. salmonicida subsp. salmonicida.

Transcriptome Sequencing Reveals That Intact Expression of the Chicken Endogenous Retrovirus chERV3 In Vitro Can Possibly Block the Key Innate Immune Pathway.

Endogenous retroviruses (ERVs) are viral sequences that have integrated into the genomes of vertebrates. Our preliminary transcriptome sequencing analysis revealed that chERV3 is active and is located on chromosome 1:32602284-32615631. We hypothesized that chERV3 may have a role in the host innate immune response to viral infection. In this study, using reverse genetics, we constructed the puc57-chERV3 full-length reverse cloning plasmid in vitro. We measured the p27 content in culture supernatant by enzyme-linked immunosorbent assay (ELISA). Finally, transcriptome analysis was performed to analyze the function of chERV3 in innate immunity. The results showed that chERV3 may generate p27 viral particles. We found that compared to the negative control (NC) group (transfected with pMD18T-EGFP), the chERV3 group exhibited 2538 up-regulated differentially expressed genes (DEGs) and 1828 down-regulated DEGs at 24 hours (h) and 1752 up-regulated DEGs and 1282 down-regulated DEGs at 48 h. Based on Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses, the down-regulated DEGs were enriched mainly in immune-related processes such as the inflammatory response, innate immune response, and Toll-like receptor signaling pathway. GSEA showed that the Toll-like receptor signaling pathway was suppressed by chERV3 at both time points. We hypothesized that chERV3 can influence the activation of the innate immune pathway by blocking the Toll-like receptor signaling pathway to achieve immune evasion.

Causal Variations at IRF6 Gene Identified in Van der Woude Syndrome Pedigrees.

The purpose of this study is to analyze the clinical characteristics of patients with Van der Woude syndrome (VWS) and to detect variations in each patient. Finally, the combination of genotype and phenotype can make a clear diagnosis of VWS patients with different phenotype penetrance. Five Chinese VWS pedigree were enrolled. Whole exome sequencing of the proband was performed, and the potential pathogenic variation was further verified by Sanger sequencing in the patient and their parents. The human mutant IRF6 coding sequence was generated from the human full-length IRF6 plasmid by site-directed mutagenesis and cloned into the GV658 vector, RT-qPCR and Western blot were used to detect the expression of IRF6. We found one de novo nonsense variation (p. Gln118Ter) and three novel missense variations (p. Gly301Glu, p. Gly267Ala, and p. Glu404Gly) co-segregated with VWS. RT-qPCR analysis revealed that p. Glu404Gly significantly reduced the expression level of IRF6 mRNA. Western blot of cell lysates confirmed that IRF6 p. Glu404Gly abundance levels were lower than those for IRF6 wild type. This discovery of the novel variation ( IRF6 p. Glu404Gly) expands the spectrum of known variations in VWS in Chinese humans. Genetic results combined with clinical phenotypes and differential diagnosis points from other diseases can make a definitive diagnosis and provide genetic counseling for families.

Hepatitis B virus suppresses complement C9 synthesis by limiting the availability of transcription factor USF-1 and inhibits formation of membrane attack complex: implications in disease pathogenesis

BackgroundThe complement system functions primarily as a first-line host defense against invading microbes, including viruses. However, the interaction of Hepatitis B virus (HBV) with the complement-components during chronic HBV infection remains largely unknown. We investigated the mechanism by which HBV inhibits the formation of cytolytic complement membrane-attack complex (MAC) and studied its impact on MAC-mediated microbicidal activity and disease pathogenesis.MethodsBlood/liver tissues were collected from chronically HBV-infected patients and controls. HepG2hNTCP cells were infected with HBV particles and Huh7 cells were transfected with full-length linear HBV-monomer or plasmids containing different HBV-ORFs and expression of complement components or other host genes were evaluated. Additionally, ELISA, Real-time PCR, Western blot, bioinformatics analysis, gene overexpression/knock-down, mutagenesis, chromatin immunoprecipitation, epigenetic studies, immunofluorescence, and quantification of serum HBV-DNA, bacterial-DNA and endotoxin were performed.ResultsAmong the MAC components (C5b-C9), significant reduction was noted in the expression of C9, the major constituent of MAC, in HBV-infected HepG2hNTCP cells and in Huh7 cells transfected with full-length HBV as well as HBX. C9 level was also marked low in sera/liver of chronic hepatitis B (CHB) and Immune-tolerant (IT) patients than inactive carriers and healthy controls. HBX strongly repressed C9-promoter activity in Huh7 cells but CpG-island was not detected in C9-promoter. We identified USF-1 as the key transcription factor that drives C9 expression and demonstrated that HBX-induced hypermethylation of USF-1-promoter is the leading cause of USF-1 downregulation that in turn diminished C9 transcription. Reduced MAC formation and impaired lysis of HBV-transfected Huh7 and bacterial cells were observed following incubation of these cells with C9-deficient CHB sera but was reversed upon C9 supplementation. Significant inverse correlation was noted between C9 concentration and HBV-DNA, bacterial-DNA and endotoxin content in HBV-infected patients. One-year Tenofovir therapy resulted in improvement in C9 level and decline in viral/bacterial/endotoxin load in CHB patients.ConclusionCollectively, HBX suppressed C9 transcription by restricting the availability of USF-1 through hypermethylation of USF-1-promoter and consequently hinder the formation and lytic functions of MAC. Early therapy is needed for both CHB and IT to normalize the aberrant complement profile and contain viral and bacterial infection and limit disease progression.

Demonstration of bacterium-free very rapid reverse genetics system using mumps virus.

The reverse genetics system is a very powerful tool for analyzing the molecular mechanisms of viral propagation and pathogenesis. However, full-length genome plasmid construction is highly time-consuming and laborious, and undesired mutations may be introduced by Escherichia coli. This study shows a very rapid E. coli-free method of full-genome construction using the mumps virus as an example. This method was able to reduce dramatically the time for full-genome construction, which was used very efficiently for virus rescue, from several days or more to ~2 days, with a similar accuracy and yield to the conventional method using E. coli/plasmid.

Construction of Recombinant Rabies Virus Vectors Expressing H or F Protein of Peste des Petits Ruminants Virus.

Simple SummaryPeste des petits ruminants (PPR) is one of the most contagious and fatal diseases of small ruminants. In this study, two recombinant viruses rSRV9-H and rSRV9-F, which express the envelope glycoprotein H (hemagglutinin protein) or F (fusion protein) protein, respectively, were successfully generated with a rabies virus as vector. The constructed viruses had good proliferative activity and stability and provided potential bivalent inactivated vaccine candidate strains for the prevention of PPR and livestock rabies.Peste des petits ruminants (PPR) is one of the most contagious and fatal diseases of small ruminants in the world and is classified as a category A epidemic disease. It is the target of a global eradication campaign led by the Office International des Epizooties (OIE) and Food and Agriculture Organization of the United Nations (FAO). The PPR live attenuated vaccine is currently the most widely used and approved vaccine, but the use of this vaccine interferes with the serological testing of the PPR elimination program, and there is a potential safety risk. Viral vector vaccines are one of the most promising methods to solve this dilemma. In this study, the full-length infectious clone plasmid of rabies virus (RABV), pD-SRV9-PM-LASV, was used as the backbone, and the envelope glycoprotein H (hemagglutinin protein) or F (fusion protein) gene of PPRV was inserted into the backbone plasmid to construct the infectious clones pD-SRV9-PM-PPRV-H and pD-SRV9-PM-PPRV-F, which express the PPRV H and PPRV F genes, respectively. The correct construction of these infectious clones was verified after sequencing and double digestion. The infectious clones were transfected with a helper plasmid into BSR/T7 cells, and recombinant viruses were successfully rescued by direct immunofluorescence, indirect immunofluorescence, Western blotting, and transmission electron microscopy and named rSRV9-H and rSRV9-F. The results of growth kinetics studies indicated that the inserted gene did not affect virus proliferation. Stability studies revealed that the inserted target gene was stably expressed in recombinant RABV for at least 15 generations. In this study, the recombinant viruses rSRV9-H and rSRV9-F were successfully rescued. The constructed viruses had good proliferative activity and stability and provided potential bivalent inactivated vaccine candidate strains for the prevention of PPR and livestock rabies.

GREPore-seq: A Robust Workflow to Detect Changes After Gene Editing Through Long-range PCR and Nanopore Sequencing

To achieve the enormous potential of gene-editing technology in clinical therapies, one needs to evaluate both the on-target efficiency and unintended editing consequences comprehensively. However, there is a lack of a pipelined, large-scale, and economical workflow for detecting genome editing outcomes, in particular insertion or deletion of a large fragment. Here, we describe an approach for efficient and accurate detection of multiple genetic changes after CRISPR/Cas9 editing by pooled nanopore sequencing of barcoded long-range PCR products. Recognizing the high error rates of Oxford nanopore sequencing, we developed a novel pipeline to capture the barcoded sequences by grepping reads of nanopore amplicon sequencing (GREPore-seq). GREPore-seq can assess nonhomologous end-joining (NHEJ)-mediated double-stranded oligodeoxynucleotide (dsODN) insertions with comparable accuracy to Illumina next-generation sequencing (NGS). GREPore-seq also reveals a full spectrum of homology-directed repair (HDR)-mediated large gene knock-in, correlating well with the fluorescence-activated cell sorting (FACS) analysis results. Of note, we discovered low-level fragmented and full-length plasmid backbone insertion at the CRISPR cutting site. Therefore, we have established a practical workflow to evaluate various genetic changes, including quantifying insertions of short dsODNs, knock-ins of long pieces, plasmid insertions, and large fragment deletions after CRISPR/Cas9-mediated editing. GREPore-seq is freely available at GitHub (https://github.com/lisiang/GREPore-seq) and the National Genomics Data Center (NGDC) BioCode (https://ngdc.cncb.ac.cn/biocode/tools/BT007293).

Multiplexed Assembly and Annotation of Synthetic Biology Constructs Using Long-Read Nanopore Sequencing.

Recombinant DNA is a fundamental tool in biotechnology and medicine. These DNA sequences are often built, replicated, and delivered in the form of plasmids. Validation of these plasmid sequences is a critical and time-consuming step, which has been dominated for the last 35 years by Sanger sequencing. As plasmid sequences grow more complex with new DNA synthesis and cloning techniques, we need new approaches that address the corresponding validation challenges at scale. Here we prototype a high-throughput plasmid sequencing approach using DNA transposition and Oxford Nanopore sequencing. Our method, Circuit-seq, creates robust, full-length, and accurate plasmid assemblies without prior knowledge of the underlying sequence. We demonstrate the power of Circuit-seq across a wide range of plasmid sizes and complexities, generating full-length, contiguous plasmid maps. We then leverage our long-read data to characterize epigenetic marks and estimate plasmid contamination levels. Circuit-seq scales to large numbers of samples at a lower per-sample cost than commercial Sanger sequencing, accelerating a key step in synthetic biology, while low equipment costs make it practical for individual laboratories.

Establishment of a reverse genetics system for rabies virus strain Komatsugawa.

The rabies virus strain Komatsugawa isolated from a dog in Tokyo in the 1940s retains biological properties as a field strain, providing an effective model for studying rabies pathogenesis. To facilitate molecular studies on the pathogenesis, this study aimed to establish a reverse genetics system for the Komatsugawa strain. By transfecting the full-length genome plasmid of this strain, infectious virus with artificially introduced genetic markers in its genome was rescued. The recombinant strain had biological properties similar to those of the original strain. These findings indicate that a reverse genetics system for the Komatsugawa strain has successfully been established.

T598 and T601 phosphorylation sites of canine parvovirus NS1 are crucial for viral replication and pathogenicity

Canine parvovirus-2 (CPV-2) is an important pathogen causing severe diseases in dogs and other wild carnivores. Phosphorylation of NS1 may be related to CPV-2 pathogenicity, but the exact mechanism is unclear. Here, we conducted parvovirus disease surveillance in Shaanxi Province of China and 51 fecal swabs were detected to be infected with CPV-2. The 7 CPV-2 strains were identified, all of which belonged to CPV-2c. The complete genome sequence of one of the strains (CPV-2c XY) was cloned into pKQLL plasmid to construct a full-length infectious clone plasmid pX-CPV-2c, which carried a genetic marker. The plasmid pX-CPV-2c was transfected into F81 cells for virus rescue. And the rescued virus, which was designed as X-CPV-2c, showed the similar biological property to parental CPV-2c XY in vitro and in vivo. We further constructed four NS1 phosphorylation site mutant strains (X-CPV-2cT584A, X-CPV-2cS592A, X-CPV-2cT598A/T601A and X-CPV-2cT617A) on the basis of X-CPV-2c. After the analysis and comparison of biological characteristics, the low pathogenic strain X-CPV-2cT598A/T601A was further screened out, which emphasized the importance of phosphorylation sites 598 T/601 T for the pathogenicity of CPV-2. Overall, our data indicated that T598 and T601, the C-terminal phosphorylation site of CPV-2 NS1, play important roles in viral pathogenicity and laid the foundation for the development of new attenuated live vaccine vectors.

Structural Diversity, Fitness Cost, and Stability of a BlaNDM-1-Bearing Cointegrate Plasmid in Klebsiella pneumoniae and Escherichia coli.

Cointegrate/hybrid plasmids combine the genetic elements of two or more plasmids and generally carry abundant antimicrobial resistance determinants. Hence, the spread of cointegrate plasmids will accelerate the transmission of AMR genes. To evaluate the transmission risk caused by cointegrate plasmids, we investigated the structural diversity, fitness cost, and stability of a cointegrate plasmid in Klebsiella pneumoniae YZ6 and Escherichia coli EC600. The cointegrate plasmid pSL131_IncA/C_IncX3 was from a clinical Salmonella Lomita strain. After transferring the plasmid into E. coli EC600 by conjugation, we observed plasmids with different structures, including a full-length original plasmid and two truncated versions. By contrast, DNA fragment deletion and blaCTX-M-14 gene insertion in the plasmid were detected in a transconjugant derived from K. pneumoniae YZ6. These results suggest that the structure of the plasmid was unstable during conjugation. Furthermore, both the full-length plasmid in EC600 and the structurally reorganized plasmid in YZ6 imposed a fitness cost on the bacterial host and enhanced biofilm formation ability. Serial passaging in antibiotic-free medium resulted in a rapid decline of the plasmid in YZ6. However, the stability of the structurally reorganized plasmid in YZ6 was improved via serial passaging in antibiotic-containing medium. SNP calling revealed that mutations of the outer membrane porin may play an essential role in this process. These findings indicate that structural versatility could contribute to the dissemination of cointegrate plasmids. Although the plasmid incurred a fitness cost in other Enterobacteriaceae species, positive selection could alleviate the adverse effects.

Identification of TRPV4 as a novel target in invasiveness of colorectal cancer

BackgroundEmerging evidence has indicated the critical role of TRPV4 in diverse human cancers. However, the underlying molecular mechanism of TRPV4 in colon cancer invasiveness is still unknown.MethodsImmunohistochemistry staining was used to analyze the expression of TRPV4 and ZEB1 in clinical tissues; Wound healing and transwell assays were applied to determine the cell invasiveness; Western blot was used to explore the relation between TRPV4 and ZEB1.ResultsColon cancer cells were transfected with siRNA against TRPV4 or HC067047 (a selective TRPV4 antagonist), TRPV4 full-length plasmid or siRNA against ZEB1, or both, in order to measure cell migration and invasion. And we found that TRPV4 silencing or inhibition exhibited an inhibitory role in colon cancer cell migration and invasion, coupled with compromised EMT process, and suppressed AKT activity. TRPV4 stimulated expression of ZEB1 and consequently contributed to EMT process and invasiveness. It was also revealed that overexpression of TRPV4 and ZEB1 in clinical patients with local metastasis, and positive correlation between TRPV4 and ZEB1.ConclusionsOur results uncovered the role of TRPV4 in tumor metastasis and highlighted the potential mechanism of TRPV4-ZEB1 axis in indicating EMT.

DNA Adduct Detection after Post-Labeling Technique with PCR Amplification (DNA-ADAPT-qPCR) Identifies the Pre-ribosomal RNA Gene as a Direct Target of Platinum-Acridine Anticancer Agents.

To study the DNA damage caused by a potent platinum-acridine anticancer agent (PA) in cancer cells, an assay based on biorthogonal post-labeling using a click chemistry-enabled, azide-modified derivative (APA) was developed. The method involves biotinylation, affinity capture, and bead-based enrichment of APA-modified genomic DNA. The key steps of the assay were validated and optimized in model duplexes, including full-length plasmids, restriction fragments, and a DNA ladder. Native DNA treated with APA and subsequently subjected to post-labeling with a biotin affinity tag was enzymatically digested and fragments were analyzed by in-line LC-MS and MS/MS. The monofunctional-intercalative adducts formed by APA in 5'-pyrimidine/guanine sequences in double-stranded DNA were quantitatively biotinylated by strain-promoted 1,3-dipolar cycloaddition chemistry. When applied to DNA extracted from A549 lung cancer cells, the assay in combination with qPCR amplification demonstrates that platinum-acridines form adducts in the gene sequences encoding pre-ribosomal RNA, a potential pharmacological target of these agents.

Integration of Complete Plasmids Containing Bont Genes into Chromosomes of Clostridium parabotulinum, Clostridium sporogenes, and Clostridium argentinense.

At least 40 toxin subtypes of botulinum neurotoxins (BoNTs), a heterogenous group of bacterial proteins, are produced by seven different clostridial species. A key factor that drives the diversity of neurotoxigenic clostridia is the association of bont gene clusters with various genomic locations including plasmids, phages and the chromosome. Analysis of Clostridium sporogenes BoNT/B1 strain CDC 1632, C. argentinense BoNT/G strain CDC 2741, and Clostridium parabotulinum BoNT/B1 strain DFPST0006 genomes revealed bont gene clusters within plasmid-like sequences within the chromosome or nested in large contigs, with no evidence of extrachromosomal elements. A nucleotide sequence (255,474 bp) identified in CDC 1632 shared 99.5% identity (88% coverage) with bont/B1-containing plasmid pNPD7 of C. sporogenes CDC 67071; CDC 2741 contig AYSO01000020 (1.1 MB) contained a ~140 kb region which shared 99.99% identity (100% coverage) with plasmid pRSJ17_1 of C. argentinense BoNT/G strain 89G; and DFPST0006 contig JACBDK0100002 (573 kb) contained a region that shared 100% identity (99%) coverage with the bont/B1-containing plasmid pCLD of C. parabotulinum Okra. This is the first report of full-length plasmid DNA-carrying complete neurotoxin gene clusters integrated in three distinct neurotoxigenic species: C. parabotulinum, C. sporogenes and C. argentinense.

Construction and identification of an infectious clone for CDV-3 strain of canine distemper virus

In order to establish an infectious clone for CDV-3, a commercial vaccine strain of canine distemper virus for mink, to provide reference for the studies of pathogenesis and novel vaccine development of CDV. Thirteen pairs of primers were used to amplify the full-length genome of CDV-3 strain. Five long fragments were obtained based on single restriction site analysis of the whole genome of CDV-3 by RT-PCR. Five fragments were successively inserted into the multiple clone sites in the modified eukaryotic vector of pcDNA3.2 by restriction enzymes and splicing. Meanwhile, the hammerhead ribozyme and hepatitis delta virus ribozyme sequences were added to the beginning of F1 fragment and the ending of F5 fragment, respectively. Then, the full-length cDNA recombinant plasmid of CDV-3 was obtained and named as pcDNA3.2-CDV-3. In addition, three helper plasmids, expressing the N protein, P protein and L protein of the CDV-3 strain respectively, were constructed. The 293T cells were transfected with the full-length cDNA recombinant plasmid and three helper plasmids by Lipofectamine™ 2000. At 3 days post transfection, the supernatant was added to the monolayer of Vero cells to observe the typical syncytium of CDV. Indirect immunofluorescence and artificial label identification of recombinant virus rCDV-3 were conducted after the occurrence of lesions. Finally, the growth characteristics of wtCDV-3 and rCDV-3 were compared after passaging of rCDV-3. The identification of the full-length cDNA recombinant plasmid and three helper plasmids by restriction enzyme digestion and sequencing were consistent with expected. The Vero cells infected with the recombinant rCDV-3 showed typical syncytic. The identification of indirect immunofluorescence and labeled marker, and observation under electron microscope proved that the rCDV-3 was indeed rescued from the recombinant plasmid of pcDNA3.2-CDV-3. In comparison of the virus titers of wtCDV-3, rCDV-3 replicated massively and rapidly and reached the maximize virus titer of 10⁷·⁶⁶⁷ TCID₅₀/mL within 36 h post infection (p.i.) in Vero cells, while wtCDV-3 grew gradually to 10⁶·⁶⁶⁷ TCID₅₀/mL at 72 h p.i. in Vero cells. This reverse genetic system of CDV-3 strain has been established successfully, to provide reference for the studies of pathogenesis and novel vaccine development of CDV.

The Novel Zinc Finger Protein 587B Gene, ZNF587B, Regulates Cell Proliferation and Metastasis in Ovarian Cancer Cells in vivo and in vitro.

BackgroundThe zinc finger protein 587B (ZNF587B) is a novel cisplatin-sensitive gene that was identified in our previous research by using a genome-scale CRISPR-Cas9 knockout library in ovarian cancer (OC) cell lines. ZNF587B belongs to the C2H2-type zinc finger protein (ZFP) family. Many ZFP protein could inhibit tumor development and malignancy. However, the function of ZNF587B remains unknown.MethodsQuantitative PCR (qPCR) was utilized to compare ZNF587B mRNA expression levels in OC and normal ovarian cell lines. The small interfering RNA (siRNA) and full-length ZNF587B eukaryotic expression plasmid were constructed and transfected into OC cells later. Colony formation, 5-ethynyl-2′-deoxyuridine (EdU) assay, transwell assay, and xenograft experiment were conducted to evaluate the effect of ZNF587B on OC cells.ResultsZNF587B was downregulated by approximately 43% and 17% in the OC cell lines SKOV3 and A2780, respectively, compared with that in the normal ovarian cell line IOSE80. Overexpression of ZNF587B reduced cell proliferation, colony formation, migration, and invasion, which could be reversed by knockdown of ZNF587B via siRNA. Xenograft experiments also confirmed that ZNF587B could suppress tumor growth. Survival data of OC patients in the SurvExpress database showed that with respect to overall survival, low-risk patients grouped by the prognostic index had a higher expression of ZNF587B and a better prognosis than high-risk group (HR = 1.77, 95% CI: 0.55–0.70, p = 0.023). Moreover, overexpression of ZNF587B promoted OC cells apoptosis when pretreated with cisplatin.ConclusionZNF587B is a novel potential tumor suppressor of OC and may be a therapeutic target for OC.

Evolution of satellite plasmids can prolong the maintenance of newly acquired accessory genes in bacteria

Transmissible plasmids spread genes encoding antibiotic resistance and other traits to new bacterial species. Here we report that laboratory populations of Escherichia coli with a newly acquired IncQ plasmid often evolve ‘satellite plasmids’ with deletions of accessory genes and genes required for plasmid replication. Satellite plasmids are molecular parasites: their presence reduces the copy number of the full-length plasmid on which they rely for their continued replication. Cells with satellite plasmids gain an immediate fitness advantage from reducing burdensome expression of accessory genes. Yet, they maintain copies of these genes and the complete plasmid, which potentially enables them to benefit from and transmit the traits they encode in the future. Evolution of satellite plasmids is transient. Cells that entirely lose accessory gene function or plasmid mobility dominate in the long run. Satellite plasmids also evolve in Snodgrassella alvi colonizing the honey bee gut, suggesting that this mechanism may broadly contribute to the importance of IncQ plasmids as agents of bacterial gene transfer in nature.