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Molecular Therapy. Methods & Clinical... Jun 2024Recombinant adeno-associated virus (rAAV)-based gene therapy is entering clinical and commercial stages at an unprecedented pace. Triple transfection of HEK293 cells is...
Recombinant adeno-associated virus (rAAV)-based gene therapy is entering clinical and commercial stages at an unprecedented pace. Triple transfection of HEK293 cells is currently the most widely used platform for rAAV manufacturing. Here, we develop low-cis triple transfection that decreases transgene plasmid use by 10- to 100-fold and overcomes several major limitations associated with standard triple transfection. This new method improves packaging of yield-inhibiting transgenes by up to 10-fold, and generates rAAV batches with reduced plasmid backbone contamination that otherwise cannot be eliminated in downstream processing. When tested in mice and compared with rAAV produced by standard triple transfection, low-cis rAAV shows comparable or superior potency and results in diminished plasmid backbone DNA and RNA persistence in tissue. Mechanistically, low-cis triple transfection relies on the extensive replication of transgene cassette (i.e., inverted terminal repeat-flanked vector DNA) in HEK293 cells during production phase. This cost-effective method can be easily implemented and is widely applicable to producing rAAV of high quantity, purity, and potency.
PubMed: 38558570
DOI: 10.1016/j.omtm.2024.101230 -
PLoS Pathogens Apr 2024Lyme disease is a tick-borne infection caused by the spirochete Borrelia (Borreliella) burgdorferi. Borrelia species have highly fragmented genomes composed of a linear...
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.
Topics: Humans; Borrelia burgdorferi; Bacteriophages; Plasmids; Lyme Disease; Genomics; DNA
PubMed: 38558079
DOI: 10.1371/journal.ppat.1012122 -
Nucleic Acids Research Jun 2024Gametocyte development of the Plasmodium parasite is a key step for transmission of the parasite. Male and female gametocytes are produced from a subpopulation of...
Gametocyte development of the Plasmodium parasite is a key step for transmission of the parasite. Male and female gametocytes are produced from a subpopulation of asexual blood-stage parasites, but the mechanisms that regulate the differentiation of sexual stages are still under investigation. In this study, we investigated the role of PbARID, a putative subunit of a SWI/SNF chromatin remodeling complex, in transcriptional regulation during the gametocyte development of P. berghei. PbARID expression starts in early gametocytes before the manifestation of male and female-specific features, and disruption of its gene results in the complete loss of gametocytes with detectable male features and the production of abnormal female gametocytes. ChIP-seq analysis of PbARID showed that it forms a complex with gSNF2, an ATPase subunit of the SWI/SNF chromatin remodeling complex, associating with the male cis-regulatory element, TGTCT. Further ChIP-seq of PbARID in gsnf2-knockout parasites revealed an association of PbARID with another cis-regulatory element, TGCACA. RIME and DNA-binding assays suggested that HDP1 is the transcription factor that recruits PbARID to the TGCACA motif. Our results indicated that PbARID could function in two chromatin remodeling events and paly essential roles in both male and female gametocyte development.
Topics: Animals; Female; Male; Mice; Chromatin Assembly and Disassembly; Plasmodium berghei; Protozoan Proteins; Transcription Factors; Genotype; Sequence Analysis, RNA; Chromatin; Amino Acid Sequence; Sequence Analysis, Protein; Phylogeny; Transcriptome; Genome, Protozoan
PubMed: 38554111
DOI: 10.1093/nar/gkae207 -
International Journal of Molecular... Mar 2024Histones are nuclear proteins essential for packaging genomic DNA and epigenetic gene regulation. Paralogs that can substitute core histones (H2A, H2B, H3, and H4),... (Review)
Review
Histones are nuclear proteins essential for packaging genomic DNA and epigenetic gene regulation. Paralogs that can substitute core histones (H2A, H2B, H3, and H4), named histone variants, are constitutively expressed in a replication-independent manner throughout the cell cycle. With specific chaperones, they can be incorporated to chromatin to modify nucleosome stability by modulating interactions with nucleosomal DNA. This allows the regulation of essential fundamental cellular processes for instance, DNA damage repair, chromosomal segregation, and transcriptional regulation. Among all the histone families, histone H2A family has the largest number of histone variants reported to date. Each H2A variant has multiple functions apart from their primary role and some, even be further specialized to perform additional tasks in distinct lineages, such as testis specific shortH2A (sH2A). In the past decades, the discoveries of genetic alterations and mutations in genes encoding H2A variants in cancer had revealed variants' potentiality in driving carcinogenesis. In addition, there is growing evidence that H2A variants may act as novel prognostic indicators or biomarkers for both early cancer detection and therapeutic treatments. Nevertheless, no studies have ever concluded all identified variants in a single report. Here, in this review, we summarize the respective functions for all the 19 mammalian H2A variants and their roles in cancer biology whilst potentiality being used in clinical setting.
Topics: Male; Animals; Humans; Histones; Chromatin; Nucleosomes; DNA; Mammals; Neoplasms
PubMed: 38542118
DOI: 10.3390/ijms25063144 -
Vaccine Apr 2024An issue with many current vaccines is the dependency on broadly inflammatory adjuvants, such as aluminum hydroxide or aluminum salts that affect many immune- and...
An issue with many current vaccines is the dependency on broadly inflammatory adjuvants, such as aluminum hydroxide or aluminum salts that affect many immune- and non-immune cells. These adjuvants are not necessarily activating all antigen-presenting cells (APCs) that take up the antigen and most likely they also activate APCs with no antigen uptake, as well as many non-immune cells. Conjugation of antigen and adjuvant would enable the use of smaller amounts of adjuvant and avoid unnecessary tissue damage and activation of bystander cells. It would ensure that all APCs that take up the antigen would also become activated and avoid that immature and non-activated APCs present the antigen to T cells without a co-stimulatory signal, leading to tolerogenesis. We have developed a novel vaccine that co-deliver antigen and a nucleotide adjuvant to the same APC and lead to a strong activation response in dendritic cells and macrophages. The vaccine is constructed as a fusion-protein with an antigen fused to the DNA/RNA-binding domain from the Hc2 protein from Chlamydia trachomatis. We have found that the fusion protein is able to package polyinosinic:polycytidylic acid (poly(I:C)) or dsDNA into small particles. These particles were taken up by macrophages and dendritic cells and led to strong activation and maturation of these cells. Immunization of mice with the fusion protein packaged poly(I:C) led to a stronger antibody response compared to immunization with a combination of poly(I:C) and antigen without the Hc2 DNA/RNA-binding domain.
Topics: Animals; Mice; Antibody Formation; Nucleotides; Dendritic Cells; Antigens; Vaccines; Poly I-C; Adjuvants, Immunologic; DNA
PubMed: 38538405
DOI: 10.1016/j.vaccine.2024.03.058 -
The Journal of Physical Chemistry. B Apr 2024The basic packaging unit of eukaryotic chromatin is the nucleosome that contains 145-147 base pair duplex DNA wrapped around an octameric histone protein. While the DNA...
The basic packaging unit of eukaryotic chromatin is the nucleosome that contains 145-147 base pair duplex DNA wrapped around an octameric histone protein. While the DNA sequence plays a crucial role in controlling the positioning of the nucleosome, the molecular details behind the interplay between DNA sequence and nucleosome dynamics remain relatively unexplored. This study analyzes this interplay in detail by performing all-atom molecular dynamics simulations of nucleosomes, comparing the human α-satellite palindromic (ASP) and the strong positioning "Widom-601" DNA sequence at time scales of 12 μs. The simulations are performed at salt concentrations 10-20 times higher than physiological salt concentrations to screen the electrostatic interactions and promote unwrapping. These microsecond-long simulations give insight into the molecular-level sequence-dependent events that dictate the pathway of DNA unwrapping. We find that the "ASP" sequence forms a loop around SHL ± 5 for three sets of simulations. Coincident with loop formation is a cooperative increase in contacts with the neighboring N-terminal H2B tail and C-terminal H2A tail and the release of neighboring counterions. We find that the Widom-601 sequence exhibits a strong breathing motion of the nucleic acid ends. Coincident with the breathing motion is the collapse of the full N-terminal H3 tail and formation of an α-helix that interacts with the H3 histone core. We postulate that the dynamics of these histone tails and their modification with post-translational modifications (PTMs) may play a key role in governing this dynamics.
Topics: Humans; Nucleosomes; Histones; Chromatin; DNA; Molecular Dynamics Simulation
PubMed: 38530903
DOI: 10.1021/acs.jpcb.3c07363 -
BioRxiv : the Preprint Server For... Feb 2024The phylum consists of large and giant viruses that range in genome size from about 100 kilobases (kb) to more than 2.5 megabases. Here, using metagenome mining...
The phylum consists of large and giant viruses that range in genome size from about 100 kilobases (kb) to more than 2.5 megabases. Here, using metagenome mining followed by extensive phylogenomic analysis and protein structure comparison, we delineate a distinct group of viruses with double-stranded (ds) DNA genomes in the range of 35-45 kb that appear to be related to the . In phylogenetic trees of the conserved double jelly-roll major capsid proteins (MCP) and DNA packaging ATPases, these viruses do not show affinity to any particular branch of the and accordingly would comprise a class which we propose to name "" (after Ukrainian Mriya, dream). Structural comparison of the MCP suggests that, among the extant virus lineages, mriyaviruses are the closest one to the ancestor of the . In the phylogenetic trees, mriyaviruses split into two well-separated branches, the family and proposed new family "". The previously characterized members of these families, Yaravirus and Pleurochrysis sp. endemic viruses, infect amoeba and haptophytes, respectively. The genomes of the rest of the mriyaviruses were assembled from metagenomes from diverse environments, suggesting that mriyaviruses infect various unicellular eukaryotes. Mriyaviruses lack DNA polymerase, which is encoded by all other members of the , and RNA polymerase subunits encoded by all cytoplasmic viruses among the , suggesting that they replicate in the host cell nuclei. All mriyaviruses encode a HUH superfamily endonuclease that is likely to be essential for the initiation of virus DNA replication via the rolling circle mechanism.
PubMed: 38529486
DOI: 10.1101/2024.02.29.582850 -
Environment International Apr 2024Perfluoroalkyl and polyfluoroalkyl substances (PFASs) are synthetic chemicals, encompassing compounds like perfluorooctane sulfonate (PFOS), which have widespread...
Perfluoroalkyl and polyfluoroalkyl substances (PFASs) are synthetic chemicals, encompassing compounds like perfluorooctane sulfonate (PFOS), which have widespread applications across various industries, including food packaging and firefighting. In recent years, China has increasingly employed 6:2 Cl-PFESA as an alternative to PFOS. Although the association between PFAS exposure and hepatocellular carcinoma (HCC) has been demonstrated, the underlying mechanisms that promote HCC proliferation are uncleared. Therefore, we aimed to investigate the effects and differences of PFOS and 6:2 Cl-PFESA on HCC proliferation through in vivo and in vitro tumor models. Our results reveal that both PFOS and 6:2 Cl-PFESA significantly contribute to HCC proliferation in vitro and in vivo. Exposure led to reduced population doubling times, enlarged cell colony sizes, enhanced DNA synthesis efficiency, and a higher proportion of cells undergoing mitosis. Furthermore, both PFOS and 6:2 Cl-PFES) have been shown to activate the PI3K/AKT/mTOR signaling pathway and inhibit necroptosis. This action consequently enhances the proliferation of HCC cells. Our phenotypic assay findings suggest that the tumorigenic potential of 6:2 Cl-PFESA surpasses that of PFOS; in a subcutaneous tumor model using nude mice, the mean tumor weight for the 6:2 Cl-PFESA-treated cohort was 2.33 times that observed in the PFOS cohort (p < 0.01). Despite 6:2 Cl-PFESA being considered a safer substitute for PFOS, the pronounced effects of this chemical on HCC cell growth warrant a thorough assessment of hepatotoxicity risks linked to its usage.
Topics: Fluorocarbons; Carcinoma, Hepatocellular; Humans; Alkanesulfonic Acids; Liver Neoplasms; Cell Proliferation; Animals; Mice; Cell Line, Tumor; Signal Transduction; China
PubMed: 38513556
DOI: 10.1016/j.envint.2024.108582 -
PloS One 2024Chromodomain helicase DNA binding domain (CHD) proteins, including CHD7 and CHD8, remodel chromatin to enable transcriptional programs. Both proteins are important for...
Chromodomain helicase DNA binding domain (CHD) proteins, including CHD7 and CHD8, remodel chromatin to enable transcriptional programs. Both proteins are important for proper neural development as heterozygous mutations in Chd7 and Chd8 are causative for CHARGE syndrome and correlated with autism spectrum disorders, respectively. Their roles in mature neurons are poorly understood despite influencing the expression of genes required for cell adhesion, neurotransmission, and synaptic plasticity. The Drosophila homolog of CHD7 and CHD8, Kismet (Kis), promotes neurotransmission, endocytosis, and larval locomotion. Endocytosis is essential in neurons for replenishing synaptic vesicles, maintaining protein localization, and preserving the size and composition of the presynaptic membrane. Several forms of endocytosis have been identified including clathrin-mediated endocytosis, which is coupled with neural activity and is the most prevalent form of synaptic endocytosis, and activity-dependent bulk endocytosis, which occurs during periods of intense stimulation. Kis modulates the expression of gene products involved in endocytosis including promoting shaggy/GSK3β expression while restricting PI3K92E. kis mutants electrophysiologically phenocopy a liquid facets mutant in response to paradigms that induce clathrin-mediated endocytosis and activity-dependent bulk endocytosis. Further, kis mutants do not show further reductions in endocytosis when activity-dependent bulk endocytosis or clathrin-mediated endocytosis are pharmacologically inhibited. We find that Kis is important in postsynaptic muscle for proper endocytosis but the ATPase domain of Kis is dispensable for endocytosis. Collectively, our data indicate that Kis promotes both clathrin-mediated endocytosis and activity-dependent bulk endocytosis possibly by promoting transcription of several endocytic genes and maintaining the size of the synaptic vesicle pool.
Topics: Animals; Clathrin; Chromatin; Chromatin Assembly and Disassembly; Synaptic Transmission; Drosophila; Endocytosis; DNA Helicases
PubMed: 38512854
DOI: 10.1371/journal.pone.0300255 -
ELife Mar 2024To find nucleosomes, chromatin remodelers slide and hop along DNA, and their direction of approach affects the direction that nucleosomes slide in.
To find nucleosomes, chromatin remodelers slide and hop along DNA, and their direction of approach affects the direction that nucleosomes slide in.
Topics: Nucleosomes; DNA-Binding Proteins; Saccharomyces cerevisiae Proteins; Chromatin Assembly and Disassembly; Chromatin
PubMed: 38488335
DOI: 10.7554/eLife.96836