Heterologous DNA Research Articles

Tn1 transposition in the course of natural transformation enables horizontal antibiotic resistance spread in Acinetobacter baylyi.

Transposons are genetic elements that change their intracellular genomic position by transposition and are spread horizontally between bacteria when located on plasmids. It was recently discovered that transposition from fully heterologous DNA also occurs in the course of natural transformation. Here, we characterize the molecular details and constraints of this process using the replicative transposon Tn1 and the naturally competent bacterium Acinetobacter baylyi . We find that chromosomal insertion of Tn1 by transposition occurs at low but detectable frequencies and preferably around the A. baylyi terminus of replication. We show that Tn1 transposition is facilitated by transient expression of the transposase and resolvase encoded by the donor DNA. RecA protein is essential for the formation of a circular, double-stranded cytoplasmic intermediate from incoming donor DNA, and RecO is beneficial but not essential in this process. Absence of the recipient RecBCD nuclease stabilizes the double-stranded intermediate. Based on these results, we suggest a mechanistic model for transposition during natural transformation.

Sequential Genome Editing and Induced Excision of the Transgene in N. tabacum BY2 Cells.

While plant cells in suspension are becoming a popular platform for expressing biotherapeutic proteins, the need to pre-engineer these cells to better comply with their role as host cell lines is emerging. Heterologous DNA and selectable markers are used for transformation and genome editing designated to produce improved host cell lines for overexpression of recombinant proteins. The removal of these heterologous DNA and selectable markers, no longer needed, can be beneficial since they limit additional gene stacking in subsequent transformations and may pose excessive metabolic burden on the cell machinery. In this study we developed an innovative stepwise methodology in which the CRISPR-Cas9 is used sequentially to target genome editing, followed by its own excision. The first step included a stable insertion of a CRISPR-Cas9 cassette, targeted to knockout the β(1,2)-xylosyltranferase (XylT) and the α(1,3)-fucosyltransferase (FucT) genes in Nicotiana tabacum L. cv Bright Yellow 2 (BY2) cell suspension. The second step included the excision of the inserted cassette of 14.3 kbp by induction of specific sgRNA designed to target the T-DNA boundaries. The genome editing step and the transgene removal step are achieved in one transformation run. This mechanism enables CRISPR genome editing and subsequently eliminating the introduced transgenes thus freeing the cells from foreign DNA no longer needed.

Cross-Protection against MERS-CoV by Prime-Boost Vaccination Using Viral Spike DNA and Protein.

Middle East respiratory syndrome coronavirus (MERS-CoV) causes severe respiratory illness and has a high mortality of ∼34%. However, since its discovery in 2012, an effective vaccine has not been developed for it. To develop a vaccine against multiple strains of MERS-CoV, we targeted spike glycoprotein (S) using prime-boost vaccination with DNA and insect cell-expressed recombinant proteins for the receptor-binding domain (RBD), S1, S2, SΔTM, or SΔER. Our S subunits were generated using an S sequence derived from the MERS-CoV EMC/2012 strain. We examined humoral and cellular immune responses of various combinations with DNA plasmids and recombinant proteins in mice. Mouse sera immunized with SΔER DNA priming/SΔTM protein boosting showed cross-neutralization against 15 variants of S-pseudovirions and the wild-type KOR/KNIH/002 strain. In addition, these immunizations provided full protection against the KOR/KNIH/002 strain challenge in human DPP4 knock-in mice. These findings suggest that vaccination with the S subunits derived from one viral strain can provide cross-protection against variant MERS-CoV strains with mutations in S. DNA priming/protein boosting increased gamma interferon production, while protein-alone immunization did not. The RBD subunit alone was insufficient to induce neutralizing antibodies, suggesting the importance of structural conformation. In conclusion, heterologous DNA priming with protein boosting is an effective way to induce both neutralizing antibodies and cell-mediated immune responses for MERS-CoV vaccine development. This study suggests a strategy for selecting a suitable platform for developing vaccines against MERS-CoV or other emerging coronaviruses.IMPORTANCE Coronavirus is an RNA virus with a higher mutation rate than DNA viruses. Therefore, a mutation in S-protein, which mediates viral infection by binding to a human cellular receptor, is expected to cause difficulties in vaccine development. Given that DNA-protein vaccines promote stronger cell-mediated immune responses than protein-only vaccination, we immunized mice with various combinations of DNA priming and protein boosting using the S-subunit sequences of the MERS-CoV EMC/2012 strain. We demonstrated a cross-protective effect against wild-type KOR/KNIH/002, a strain with two mutations in the S amino acids, including one in its RBD. The vaccine also provided cross-neutralization against 15 different S-pseudotyped viruses. These suggested that a vaccine targeting one variant of S can provide cross-protection against multiple viral strains with mutations in S. The regimen of DNA priming/Protein boosting can be applied to the development of other coronavirus vaccines.

The NS1 protein of the parvovirus MVM Aids in the localization of the viral genome to cellular sites of DNA damage.

The autonomous parvovirus Minute Virus of Mice (MVM) localizes to cellular DNA damage sites to establish and sustain viral replication centers, which can be visualized by focal deposition of the essential MVM non-structural phosphoprotein NS1. How such foci are established remains unknown. Here, we show that NS1 localized to cellular sites of DNA damage independently of its ability to covalently bind the 5’ end of the viral genome, or its consensus DNA binding sequence. Many of these sites were identical to those occupied by virus during infection. However, localization of the MVM genome to DNA damage sites occurred only when wild-type NS1, but not its DNA-binding mutant was expressed. Additionally, wild-type NS1, but not its DNA binding mutant, could localize a heterologous DNA molecule containing the NS1 binding sequence to DNA damage sites. These findings suggest that NS1 may function as a bridging molecule, helping the MVM genome localize to cellular DNA damage sites to facilitate ongoing virus replication.

Chimeric Virus-Like Particles and Capsomeres Induce Similar CD8+ T Cell Responses but Differ in Capacity to Induce CD4+ T Cell Responses and Antibody Responses.

Despite extensive research, the development of an effective malaria vaccine remains elusive. The induction of robust and sustained T cell and antibody response by vaccination is an urgent unmet need. Chimeric virus-like particles (VLPs) are a promising vaccine platform. VLPs are composed of multiple subunit capsomeres which can be rapidly produced in a cost-effective manner, but the ability of capsomeres to induce antigen-specific cellular immune responses has not been thoroughly investigated. Accordingly, we have compared chimeric VLPs and their sub-unit capsomeres for capacity to induce CD8+ and CD4+ T cell and antibody responses. We produced chimeric murine polyomavirus VLPs and capsomeres each incorporating defined CD8+ T cell, CD4+ T cell or B cell repeat epitopes derived from Plasmodium yoelii CSP. VLPs and capsomeres were evaluated using both homologous or heterologous DNA prime/boost immunization regimens for T cell and antibody immunogenicity. Chimeric VLP and capsomere vaccine platforms induced robust CD8+ T cell responses at similar levels which was enhanced by a heterologous DNA prime. The capsomere platform was, however, more efficient at inducing CD4+ T cell responses and less efficient at inducing antigen-specific antibody responses. Our data suggest that capsomeres, which have significant manufacturing advantages over VLPs, should be considered for diseases where a T cell response is the desired outcome.

Lactic Acid Bacteria as Delivery Vehicle for Therapeutics Applications.

Lactic acid bacteria comprise a large group of Gram-positive organisms capable of converting sugar into lactic acid. They have been studied due to their therapeutic potential on the mucosal surface. Among the species, Lactococcus lactis is considered the model bacterium and it has been explored as an important vehicle for providing therapeutic molecules and antigens in the mucosa. They can be genetically engineered to produce a variety of molecules as well as deliver heterologous DNA and protein. DNA vaccines consist of the administration of a bacterial plasmid under the control of a eukaryotic promoter encoding the antigen of interest. The resulting proteins are capable of stimulating the immune system, becoming a promising technique for immunization against a variety of tumors and infection diseases and having several advantages compared to conventional nucleic acid delivery methods (such as bioballistic delivery, electroporation, and intramuscular administration).

Optimization and application of a high-resolution melting protocol in the characterization of avian infectious laryngotracheitis virus

A previous sequence analysis of a US5 gene fragment of infectious laryngotracheitis virus (ILTV) performed in an Argentinian epidemiological study allowed to differentiate between wild and vaccine strains. This analysis also defined five ILTV haplotypes with specific variations at positions 461, 484, 832, 878 and 894 of the US5 gene. This characterization of viral strains may also be accomplished using the High-Resolution Melting Analysis (HRMA), which has been described as an effective, fast and sensitive method to detect mutations in PCR products. In the present study, an HRM protocol was developed with the aim of characterizing the circulating ILTV strains in Argentina. The specificity of this tool was confirmed in different DNA diluents, without interference from heterologous DNA or other cellular metabolites. Additionally, the salt concentration in the elution buffer used for DNA extraction did not alter the curve profiles. Higher concentrations of DNA (Ct≅26.0) displayed well-defined curve profiles, whereas lower concentrations (Ct≅32.5) exhibited more heterogeneous curves. The HRMA showed 97.49% concordance with the reference technique, i.e., sequencing. The HRM protocol has the capability to perform DNA amplification prior to its characterization. Thus, eventually this technique may be used simultaneously as a diagnostic tool. This advantage implies a significant reduction in the time and effort involved in sample processing.

Probing the operability regime of an engineered ribocomputing unit in terms of dynamic range maintenance with extracellular changes and time

Synthetic biology aims at engineering gene regulatory circuits to end with cells (re)programmed on purpose to implement novel functions or discover natural behaviors. However, one overlooked question is whether the resulting circuits perform as intended in variety of environments or with time. Here, we considered a recently engineered genetic system that allows programming the cell to work as a minimal computer (arithmetic logic unit) in order to analyze its operability regime. This system involves transcriptional and post-transcriptional regulations. In particular, we studied the analog behavior of the system, the effect of physicochemical changes in the environment, the impact on cell growth rate of the heterologous expression, and the ability to maintain the arithmetic functioning over time. Conclusively, our results suggest 1) that there are wide input concentration ranges that the system can correctly process, the resulting outputs being predictable with a simple mathematical model, 2) that the engineered circuitry is quite sensitive to temperature effects, 3) that the expression of heterologous small RNAs is costly for the cell, not only of heterologous proteins, and 4) that a proper genetic reorganization of the system to reduce the amount of heterologous DNA in the cell can improve its evolutionary stability.

Integration of large heterologous DNA fragments into the genome of Thermococcus kodakarensis.

In this study, a transformation system enabling large-scale gene recombination was developed for the hyperthermophilic archaeon Thermococcus kodakarensis. Using the uracil auxotroph T. kodakarensis KU216 (∆pyrF) as a parent strain, we constructed multiple host strains harboring two 1-kbp DNA regions from the genomes of either the hyperthermophilic archaeon Pyrococcus furiosus or Methanocaldococcus jannaschii. The two regions were selected so that the regions between them on the respective genomes would include pyrF genes, which can potentially be used for selection. Transformation using these host strains and genomic DNA from P. furiosus or M. jannaschii were carried out. Transformants with exogenous pyrF were obtained only using host strains with regions from P. furiosus, and only when the distances between the two regions were relatively short (2-5 kbp) on the P. furiosus genome. To insert longer DNA fragments, we examined the possibilities of using P. furiosus cells to provide intact genomic DNA. A cell pellet of P. furiosus was overlaid with that of T. kodakarensis so that cells were in direct contact. As a result, we were able to isolate T. kodakarensis strains harboring DNA fragments from P. furiosus with lengths of up to 75 kbp in a single transformation step.

Cross-reactivity Reduced Dengue Virus 2 Vaccine has No cross-protection Against Heterotypic Dengue Viruses

Aim: This study assessed how prime-boost strategies influence the immunogenicity of a cross-reactivity reduced dengue virus 2 vaccine (DENV-2 RD). Materials & methods: Mice were immunized with DENV-2 RD vaccines in a heterologous DNA and virus-like particle (VLP) prime-boost. Elicited antibodies were analyzed for neutralization and protective efficacy against four DENV serotypes. Results: DENV-2 RD DNA-VLP had induced higher and broader levels of total IgG and neutralizing antibodies with statistically significant IgG titers against DENV-2 and -3. Only pups of DENV-2 RD DNA-VLP immunized female mice were fully protected against homotypic DENV challenge and partially protected (60% survival rate) against heterotypic DENV-3 lethal challenge. Conclusion: DENV-2 RD vaccine requires a multivalent format to effectively elicit a balanced and protective immunity across all four DENV serotypes.

Comparative analysis of two HIV-1 multiepitope polypeptides for stimulation of immune responses in BALB/c mice

A licensed vaccine against human immunodeficiency virus-1 (HIV-1) infection has not become available up to now. Hence, it is more rational to use immune-informatics tools for prediction of T cell epitopes (in silico study) and development of an effective epitope-driven vaccine against hypervariable pathogens. Multiepitope vaccines were considered as the next generation of an effective vaccine against HIV-1 infection. In the current study, we developed two different constructs encoding T cell epitopes derived from Nef, Vif, Vpu, Gp160 and P24 proteins in BALB/c mice. To overcome their poor immunogenicity, four different cell penetrating peptides (MPG and HR9 for DNA delivery, and CyLoP-1 and LDP-NLS for protein delivery), Montanide adjuvant, and heterologous prime-boost immunization strategy were utilized. The generation of cytokines, Granzyme B, and total IgG and its subclasses was determined using ELISA. Our data indicated that the levels of IFN-γ and Granzyme B in mice injected with Nef-Vif-Gp160-P24 multiepitope constructs were higher than those immunized with Nef-Vpu-Gp160-P24 multiepitope constructs. Moreover, the heterologous DNA priming/ multiepitope peptide boosting in both Nef-Vif-Gp160-P24 and Nef-Vpu-Gp160-P24 regimens induced significantly high antigen-specific IgG2a and IgG2b responses in comparison with other groups. There was no significant difference between MPG and HR9 as well as CyLoP-1 and LDP-NLS as a delivery system for enhancement of immune responses. Generally, the heterologous DNA prime/ multiepitope peptide boost modalities for both constructs could significantly enhance the levels of IgG2a, IgG2b, IFN-γ, and Granzyme B directed toward Th1 immune responses as compared to homologous prime/ boost with DNA or polypeptide constructs.

Engineered Recombinant Escherichia coli Probiotic Strains Integrated with F4 and F18 Fimbriae Cluster Genes in the Chromosome and Their Assessment of Immunogenic Efficacy in Vivo.

F4 (K88) and F18 fimbriaed enterotoxigenic Escherichia coli (ETEC) are the predominant causes of porcine postweaning diarrhea (PWD), and vaccines are considered the most effective preventive approach against PWD. Since heterologous DNA integrated into bacterial chromosomes could be effectively expressed with stable inheritance, we chose probiotic EcNc (E. coli Nissle 1917 prototype cured of cryptic plasmids) as a delivery vector to express the heterologous F4 or both F4 and F18 fimbriae and sequentially assessed their immune efficacy of anti-F4 and F18 fimbriae in both murine and piglet models. Employing the CRISPR-cas9 technology, yjcS, pcadA, lacZ, yieN/trkD, maeB, and nth/tppB sites in the chromosome of an EcNc strain were targeted as integration sites to integrate F4 or F18 fimbriae cluster genes under the Ptet promotor to construct two recombinant integration probiotic strains (RIPSs), i.e., nth integration strain (EcNcΔnth/tppB::PtetF4) and multiple integration strain (EcNc::PtetF18x4::PtetF4x2). Expression of F4, both F4 and F18 fimbriae on the surfaces of two RIPSs, was verified with combined methods of agglutination assay, Western blot, and immunofluorescence microscopy. The recombinant strains have improved adherence to porcine intestinal epithelial cell lines. Mice and piglets immunized with the nth integration strain and multiple integration strain through gavage developed anti-F4 and both anti-F4 and anti-F18 IgG immune responses. Moreover, the serum antibodies from the immunized mice and piglets significantly inhibited the adherence of F4+ or both F4+ and F18+ ETEC wild-type strains to porcine intestinal cell lines in vitro, indicating the potential of RIPSs as promising probiotic strains plus vaccine candidates against F4+/F18+ ETEC infection.

Design and validation of a transposon that promotes expression of genes in episomal DNA

Functional metagenomics, or the cloning and expression of DNA isolated directly from environmental samples, represents a source of novel compounds with biotechnological potential. However, attempts to identify such compounds in metagenomic libraries are generally inefficient in part due to lack of expression of heterologous DNA. In this research, the TnC_T7 transposon was developed to supply transcriptional machinery during functional analysis of metagenomic libraries. TnC_T7 contains bidirectional T7 promoters, the gene encoding the T7 RNA polymerase (T7RNAP), and a kanamycin resistance gene. The T7 RNA polymerase gene is regulated by the inducible arabinose promoter (PBAD), thereby facilitating inducible expression of genes adjacent to the randomly integrating transposon. The high processivity of T7RNAP should make this tool particularly useful for obtaining gene expression in long inserts. TnC_T7 functionality was validated by conducting in vitro transposition of pKR-C12 or fosmid pF076_GFPmut3*, carrying metagenomic DNA from soil. We identified transposon insertions that enhanced GFP expression in both vectors, including insertions in which the promoter delivered by the transposon was located as far as 8.7 kb from the GFP gene, indicating the power of the high processivity of the T7 polymerase. The results gathered in this research demonstrate the potential of TnC_T7 to enhance gene expression in functional metagenomic studies.

Heterologous prime-boost immunization co-targeting dual antigens inhibit tumor growth and relapse

ABSTRACT Therapeutic cancer vaccines aim to induce an effective immune response against cancer, and the effectiveness of these vaccines is influenced by the choice of immunogen, vaccine type, and immunization strategy. Although treatment with cancer vaccines can improve tumor burden and survival, in most animal studies, it is challenging to achieve a complete response against tumor growth and recurrence, without the use of other therapies in combination. Here, we present a novel approach where dual antigens (survivin and MUC1) are co-targeted using three DNA vaccines, followed by a single booster of a recombinant modified vaccinia Ankara (MVA) vaccine. This heterologous vaccination strategy induced higher levels of interferon (IFN)-γ-secretion and stronger antigen-specific T-cell responses than those induced individually by the DNA vaccines and the MVA vaccine in mice. This strategy also increased the number of active tumor-infiltrating T cells that efficiently inhibit tumor growth in tumor-bearing mice. Heterologous DNA prime-MVA boost immunization was capable of inducing a robust antigen-specific immune-memory, as seen from the resistance to subsequent survivin- and MUC1-expressing tumors. Moreover, the therapeutic effects of DNA prime-MVA boost and DNA prime-adenovirus boost strategies were compared. DNA prime-MVA boost immunization performed better, as indicated by the T effector ratio and the induction of Th1 immunity. This study provides the basis for the use of heterologous DNA prime-MVA boost vaccination regime targeting two antigens simultaneously as a promising immunotherapeutic strategy against cancer.

An Engineered Cas-Transposon System for Programmable and Site-Directed DNA Transpositions.

Efficient site-directed insertion of heterologous DNA into a genome remains an outstanding challenge. Recombinases that can integrate kilobase-sized DNA constructs are difficult to reprogram to user-defined loci, while genomic insertion using CRISPR-Cas methods relies on inefficient host DNA repair machinery. Here, we describe a Cas-Transposon (CasTn) system for genomic insertions that uses a Himar1 transposase fused to a catalytically dead dCas9 nuclease to mediate programmable, site-directed transposition. Using cell-free in vitro assays, we demonstrated that the Himar-dCas9 fusion protein increased the frequency of transposon insertion at a single targeted TA dinucleotide by >300-fold compared to a random transposase, and that site-directed transposition is dependent on target choice while robust to log-fold variations in protein and DNA concentrations. We also showed that Himar-dCas9 mediates directed transposition into plasmids in Escherichia coli. This work highlights CasTn as a new modality for host-independent, programmable, site-directed DNA insertions.

PADS Arsenal: a database of prokaryotic defense systems related genes.

Defense systems are vital weapons for prokaryotes to resist heterologous DNA and survive from the constant invasion of viruses, and they are widely used in biochemistry investigation and antimicrobial drug research. So far, numerous types of defense systems have been discovered, but there is no comprehensive defense systems database to organize prokaryotic defense gene datasets. To fill this gap, we unveil the prokaryotic antiviral defense system (PADS) Arsenal (https://bigd.big.ac.cn/padsarsenal), a public database dedicated to gathering, storing, analyzing and visualizing prokaryotic defense gene datasets. The initial version of PADS Arsenal integrates 18 distinctive categories of defense system with the annotation of 6 600 264 genes retrieved from 63,701 genomes across 33 390 species of archaea and bacteria. PADS Arsenal provides various ways to retrieve defense systems related genes information and visualize them with multifarious function modes. Moreover, an online analysis pipeline is integrated into PADS Arsenal to facilitate annotation and evolutionary analysis of defense genes. PADS Arsenal can also visualize the dynamic variation information of defense genes from pan-genome analysis. Overall, PADS Arsenal is a state-of-the-art open comprehensive resource to accelerate the research of prokaryotic defense systems.

The MultiBac system: a perspective.

Baculovirus expression is a time-tested technique to produce proteins in insect cells, in high quality and quantity for a range of applications. MultiBac is a baculovirus expression system we developed originally for producing multiprotein complexes comprising many subunits, for structural and mechanistic studies. First introduced in 2004, MultiBac is now in use in many laboratories worldwide, accelerating research programmes in academia and industry. We have continuously optimized our MultiBac system, providing customized reagents and standard operating protocols to facilitate its use also by non-specialists. More recently, we have generated MultiBac genomes tailored for specific purposes, for example, to produce humanized glycoproteins, high-value pharmaceutical targets including kinases, viral polymerases, and virus-like particles (VLPs) as promising vaccine candidates. By altering the host tropism of the baculovirion, we created MultiBacMam, a heterologous DNA delivery toolkit to target mammalian cells, tissues and organisms. Introducing CRISPR/Cas modalities, we set the stage for large-scale genomic engineering applications utilizing this high-capacity DNA delivery tool. Exploiting synthetic biology approaches and bottom-up design, we engage in optimizing the properties of our baculoviral genome, also to improve manufacturing at scale. Here we provide a perspective of our MultiBac system and its developments, past, present and future.

Simultaneous use of natural adjuvants and cell penetrating peptides improves HCV NS3 antigen-specific immune responses

To improve an effective hepatitis C virus (HCV) therapeutic vaccine, induction of a strong and long term HCV antigen-specific immune response is an important parameter. HCV non-structural protein 3 (NS3) has antigenic properties and plays a major role in viral clearance. In this study, DNA constructs encoding HCV NS3 and heat shock protein 27 (Hsp27)-NS3 genes, and the recombinant (r) NS3 and rHsp27-NS3 proteins complexed with HR9 and Cady-2 cell penetrating peptides (CPPs) were utilized to evaluate antibody, cytokine and Granzyme B secretion in mice. Herein, the formation of NS3 and Hsp27-NS3 DNA/ HR9 CPP complexes were revealed by gel retardation assay and protection against DNase and protease. Cady-2 peptide was used to form the nanoparticles with rNS3 and rHsp27-NS3 proteins. The size and charge of the nanoparticles were confirmed by SEM and Zetasizer instruments. Next, in vitro transfection of the nanoparticles was assessed by flow cytometry and western blotting. Finally, humoral and cellular immune responses were evaluated using different modalities in mice. Our data showed that HR9 and Cady-2 could form stable nanoparticles with DNA and proteins, respectively and enhance their delivery into HEK-293 T cells in a non-covalent approach. Furthermore, the heterologous Hsp27-NS3 DNA + HR9 prime/rHsp27-NS3+Cady-2 protein boost elicited a higher Th1 cellular immune response with a predominant IgG2a, IgG2b, IFN-γ profile and strong Granzyme B secretion than those induced by other groups. Briefly, the combination of a natural adjuvant (Hsp27) and CPPs (HR9 and Cady-2) could significantly stimulate effective immune responses as a promising approach for development of HCV therapeutic vaccines.

Gene and protein delivery using four cell penetrating peptides for HIV-1 vaccine development.

Cell penetrating peptides (CPPs) can potently transport therapeutic molecules to target cells for treatment of a variety of diseases. Thus, their use is critical to improve therapeutic vaccines. Histidine-rich nona-arginine (HR9) and primary amphipathic peptide (MPG) showed the ability to transfer DNA into the cells. Moreover, the peptide derived from the C-terminal of the tumor suppressor protein p14ARF (M918) and arginine-rich peptide (penetratin) were utilized to deliver polypeptides and proteins into the living cells. In this study, the immunostimulatory properties of HIV-1 Nef DNA and protein constructs were evaluated using small heat shock protein 20 (sHsp20) and Freund's emulsion as an adjuvant, and four CPPs (HR9, MPG, M918, and penetratin) as a gene or protein carrier in BALB/c mice. Our data indicated that the HR9/DNA, MPG/DNA, M918/protein, and penetratin/protein complexes formed the stable nanoparticles that were effectively delivered in HEK-293T cell line at certain ratios. Moreover, a heterologous Hsp20-Nef DNA + MPG prime/rHsp20-Nef protein+M918 boost regimen significantly elicited higher levels of IgG2a, IgG2b, IFN-gamma, and Granzyme B directed toward Th1 responses in a long period (3 months) after the last immunization compared to other groups. Furthermore, the effective role of Hsp20 was detected as a natural adjuvant in enhancing immune responses against HIV-1 Nef antigen. These findings demonstrated that the simultaneous use of M918 and MPG CPPs as protein and gene carriers improves HIV-1 Nef-specific B- and T-cell immune responses as a promising approach for development of HIV-1 monovalent vaccine.

Condensin-Mediated Chromosome Folding and Internal Telomeres Drive Dicentric Severing by Cytokinesis.

In Saccharomyces cerevisiae, dicentric chromosomes stemming from telomere fusions preferentially break at the fusion. This process restores a normal karyotype and protects chromosomes from the detrimental consequences of accidental fusions. Here, we address the molecular basis of this rescue pathway. We observe that tandem arrays tightly bound by the telomere factor Rap1 or a heterologous high-affinity DNA binding factor are sufficient to establish breakage hotspots, mimicking telomere fusions within dicentrics. We also show that condensins generate forces sufficient to rapidly refold dicentrics prior to breakage by cytokinesis and are essential to the preferential breakage at telomere fusions. Thus, the rescue of fused telomeres results from a condensin- and Rap1-driven chromosome folding that favors fusion entrapment where abscission takes place. Because a close spacing between the DNA-bound Rap1 molecules is essential to this process, Rap1 may act by stalling condensins.