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Scientific Reports Dec 2023Efficient manufacture of recombinant adeno-associated virus (rAAV) vectors for gene therapy remains challenging. Packaging cell lines containing stable integration of...
Efficient manufacture of recombinant adeno-associated virus (rAAV) vectors for gene therapy remains challenging. Packaging cell lines containing stable integration of the AAV rep/cap genes have been explored, however rAAV production needs to be induced using wild-type adenoviruses to promote episomal amplification of the integrated rep/cap genes by mobilizing a cis-acting replication element (CARE). The adenovirus proteins responsible are not fully defined, and using adenovirus during rAAV manufacture leads to contamination of the rAAV preparation. 'TESSA' is a helper adenovirus with a self-repressing Major Late Promoter (MLP). Its helper functions enable efficient rAAV manufacture when the rep and cap genes are provided in trans but is unable to support rAAV production from stable packaging cells. Using rAAV-packaging cell line HeLaRC32, we show that expression of the adenovirus L4 22/33K unit is essential for rep/cap amplification but the proteins are titrated away by binding to replicating adenovirus genomes. siRNA-knockdown of the adenovirus DNA polymerase or the use of a thermosensitive TESSA mutant decreased adenovirus genome replication whilst maintaining MLP repression, thereby recovering rep/cap amplification and efficient rAAV manufacture. Our findings have direct implications for engineering more efficient adenovirus helpers and superior rAAV packaging/producer cells.
Topics: Humans; Transfection; HeLa Cells; Plasmids; Viral Proteins; Adenoviridae; Dependovirus; Genetic Vectors; Virus Replication
PubMed: 38066084
DOI: 10.1038/s41598-023-48901-z -
Current Opinion in Genetics &... Jun 2024The human genome is not just a simple string of DNA, it is a complex and dynamic entity intricately folded within the cell's nucleus. This three-dimensional... (Review)
Review
The human genome is not just a simple string of DNA, it is a complex and dynamic entity intricately folded within the cell's nucleus. This three-dimensional organization of chromatin, the combination of DNA and proteins in the nucleus, is crucial for many biological processes and has been prominently studied for its intricate relationship to gene expression. Indeed, the transcriptional machinery does not operate in isolation but interacts intimately with the folded chromatin structure. Techniques for chromatin conformation capture, including genome-wide sequencing approaches, have revealed key organizational features of chromatin, such as the formation of loops by CCCTC-binding factor (CTCF) and the division of loci into chromatin compartments. While much of the recent research and reviews have focused on CTCF loops, we discuss several new revelations that have emerged concerning chromatin compartments, with a particular focus on what is known about mechanistic drivers of compartmentalization. These insights challenge the traditional views of chromatin organization and reveal the complexity behind the formation and maintenance of chromatin compartments.
Topics: Humans; Chromatin; CCCTC-Binding Factor; Genome, Human; Cell Nucleus; DNA; Chromatin Assembly and Disassembly; Animals
PubMed: 38626581
DOI: 10.1016/j.gde.2024.102193 -
International Journal of Molecular... Feb 2024Fibrosis represents a process characterized by excessive deposition of extracellular matrix (ECM) proteins. It often represents the evolution of pathological conditions,... (Review)
Review
Fibrosis represents a process characterized by excessive deposition of extracellular matrix (ECM) proteins. It often represents the evolution of pathological conditions, causes organ failure, and can, in extreme cases, compromise the functionality of organs to the point of causing death. In recent years, considerable efforts have been made to understand the molecular mechanisms underlying fibrotic evolution and to identify possible therapeutic strategies. Great interest has been aroused by the discovery of a molecular association between epithelial to mesenchymal plasticity (EMP), in particular epithelial to mesenchymal transition (EMT), and fibrogenesis, which has led to the identification of complex molecular mechanisms closely interconnected with each other, which could explain EMT-dependent fibrosis. However, the result remains unsatisfactory from a therapeutic point of view. In recent years, advances in epigenetics, based on chromatin remodeling through various histone modifications or through the intervention of non-coding RNAs (ncRNAs), have provided more information on the fibrotic process, and this could represent a promising path forward for the identification of innovative therapeutic strategies for organ fibrosis. In this review, we summarize current research on epigenetic mechanisms involved in organ fibrosis, with a focus on epigenetic regulation of EMP/EMT-dependent fibrosis.
Topics: Humans; Epigenesis, Genetic; DNA Methylation; Epithelial-Mesenchymal Transition; Fibrosis; Chromatin Assembly and Disassembly
PubMed: 38474021
DOI: 10.3390/ijms25052775 -
RNA Biology Jan 2024Viruses remain a global threat to animals, plants, and humans. The type 1 human immunodeficiency virus (HIV-1) is a member of the retrovirus family and carries an RNA... (Review)
Review
Viruses remain a global threat to animals, plants, and humans. The type 1 human immunodeficiency virus (HIV-1) is a member of the retrovirus family and carries an RNA genome, which is reverse transcribed into viral DNA and further integrated into the host-cell DNA for viral replication and proliferation. The RNA structures from the HIV-1 genome provide valuable insights into the mechanisms underlying the viral replication cycle. Moreover, these structures serve as models for designing novel therapeutic approaches. Here, we review structural data on RNA from the HIV-1 genome as well as computational studies based on these structural data. The review is organized according to the type of structured RNA element which contributes to different steps in the viral replication cycle. This is followed by an overview of the HIV-1 transactivation response element (TAR) RNA as a model system for understanding dynamics and interactions in the viral RNA systems. The review concludes with a description of computational studies, highlighting the impact of biomolecular simulations in elucidating the mechanistic details of various steps in the HIV-1's replication cycle.
Topics: Animals; Humans; HIV-1; HIV Long Terminal Repeat; Virus Replication; RNA, Viral
PubMed: 38100535
DOI: 10.1080/15476286.2023.2289709 -
Biomolecules Dec 2023In this study, a previously little-studied group of viruses-virophages-was searched for and identified in the viromes of the ancient oligotrophic Lake Baikal. Virophages...
In this study, a previously little-studied group of viruses-virophages-was searched for and identified in the viromes of the ancient oligotrophic Lake Baikal. Virophages are small dsDNA viruses that parasitize giant viruses (e.g., ), which in turn affect unicellular eukaryotes. We analyzed eight viromes obtained from the deep-water areas of three basins of Lake Baikal and the shallow-water strait Maloye More in different seasons. The sequences of virophages were revealed in all viromes and were dominant after bacteriophages and algal viruses. Sixteen putative complete genomes of virophages were assembled, all of which contained four conserved genes encoding major capsid protein (MCP), minor capsid protein (mCP), maturation cysteine protease (PRO), and FtsK-HerA family DNA-packaging ATPase (ATPase). The MCP-based cluster analysis showed a sequence separation according to seasons, and a dependence on the geographical localization was not detected.
Topics: Virophages; Capsid Proteins; Lakes; Virome; Adenosine Triphosphatases; Water
PubMed: 38136644
DOI: 10.3390/biom13121773 -
Journal of Obstetrics and Gynaecology :... Dec 2023BAG3 is a co-chaperone BAG family protein that plays important roles in protein homeostasis, cell survival, cell motility, and tumour metastasis. This study aimed to...
BAG3 is a co-chaperone BAG family protein that plays important roles in protein homeostasis, cell survival, cell motility, and tumour metastasis. This study aimed to clarify the clinicopathological and prognostic implications of mRNA expression in tumours. We performed bioinformatics analysis on mRNA expression using TCGA, XIANTAO, UALCAN, and Kaplan-Meier plotter databases. mRNA expression was downregulated in breast and endometrial cancers and positively correlated with favourable PAM50 subtyping in breast cancer,clinical stage and short overall survival in ovarian cancer and negatively correlated with T stage, clinical stage, and histological grade in cervical and endometrial cancers. The top -related pathways included ligand-receptor interactions and activity, DNA packaging and nucleosomes, hormonal responses, membrane regions, microdomains and rafts, and endosomes in breast cancer; ligand-receptor interactions, transmembrane transporters and channels, cell adhesion, and keratinisation in cervical cancer; ligand-receptor interactions, anion transmembrane transporters, lipoproteins, keratinisation, cell adhesion, and protein processing in endometrial cancer; metabolism of porphyrin, chlorophyll, pentose, uronic acid, ascorbate, and alternate and cell adhesion in ovarian cancer. expression could represent a potential marker for carcinogenesis, histogenesis, aggressive behaviours, and prognosis in gynecological cancers.IMPACT STATEMENT BAG3 regulates cell activity, autophagy, and resistance to apoptosis through multiple domains and plays an important role in tumour development. BAG3 positively regulates tumour cell invasion and migration in cervical and ovarian cancers. BAG3 expression is closely associated with histogenesis, clinicopathology, and prognosis in gynecological cancers and is involved in signalling pathways associated with the control of cell proliferation, migration, invasion, and drug resistance in tumours. Abnormal BAG3 expression can be employed as a possible marker of tumour development, invasion, and prognosis, providing new ideas for treating cancer.
Topics: Female; Humans; Prognosis; Cell Line, Tumor; Adaptor Proteins, Signal Transducing; RNA, Messenger; Ligands; Apoptosis Regulatory Proteins; Ovarian Neoplasms; Computational Biology; Endometrial Neoplasms; Breast Neoplasms
PubMed: 37377218
DOI: 10.1080/01443615.2023.2228899 -
Entropy (Basel, Switzerland) Jul 2023The transformation of high-molecular DNA from a random swollen coil in a solution to a discrete nanosized particle with the ordered packaging of a rigid and highly...
The transformation of high-molecular DNA from a random swollen coil in a solution to a discrete nanosized particle with the ordered packaging of a rigid and highly charged double-stranded molecule is one of the amazing phenomena of polymer physics. DNA condensation is a well-known phenomenon in biological systems, yet its molecular mechanism is not clear. Understanding the processes occurring in vivo is necessary for the usage of DNA in the fabrication of new biologically significant nanostructures. Entropy plays a very important role in DNA condensation. DNA conjugates with metal nanoparticles are useful in various fields of nanotechnology. In particular, they can serve as a basis for creating multicomponent nanoplatforms for theranostics. DNA must be in a compact state in such constructions. In this paper, we tested the methods of DNA integration with silver, gold and palladium nanoparticles and analyzed the properties of DNA conjugates with metal nanoparticles using the methods of atomic force microscopy, spectroscopy, viscometry and dynamic light scattering. DNA size, stability and rigidity (persistence length), as well as plasmon resonance peaks in the absorption spectra of systems were studied. The methods for DNA condensation with metal nanoparticles were analyzed.
PubMed: 37509999
DOI: 10.3390/e25071052 -
Frontiers in Cellular and Infection... 2023Green synthesis of NPs has gained extensive acceptance as they are reliable, eco-friendly, sustainable, and stable. Chemically synthesized NPs cause lung inflammation,... (Review)
Review
Green synthesis of NPs has gained extensive acceptance as they are reliable, eco-friendly, sustainable, and stable. Chemically synthesized NPs cause lung inflammation, heart problems, liver dysfunction, immune suppression, organ accumulation, and altered metabolism, leading to organ-specific toxicity. NPs synthesized from plants and microbes are biologically safe and cost-effective. These microbes and plant sources can consume and accumulate inorganic metal ions from their adjacent niches, thus synthesizing extracellular and intracellular NPs. These inherent characteristics of biological cells to process and modify inorganic metal ions into NPs have helped explore an area of biochemical analysis. Biological entities or their extracts used in NPs include algae, bacteria, fungi, actinomycetes, viruses, yeasts, and plants, with varying capabilities through the bioreduction of metallic NPs. These biosynthesized NPs have a wide range of pharmaceutical applications, such as tissue engineering, detection of pathogens or proteins, antimicrobial agents, anticancer mediators, vehicles for drug delivery, formulations for functional foods, and identification of pathogens, which can contribute to translational research in medical applications. NPs have various applications in the food and drug packaging industry, agriculture, and environmental remediation.
Topics: Anti-Infective Agents; Actinobacteria; Agriculture; Drug Delivery Systems; Nanoparticles
PubMed: 37662011
DOI: 10.3389/fcimb.2023.1224778 -
Nucleic Acids Research Sep 2023DNA packaging within chromatin depends on histone chaperones and remodelers that form and position nucleosomes. Cells express multiple such chromatin regulators with...
DNA packaging within chromatin depends on histone chaperones and remodelers that form and position nucleosomes. Cells express multiple such chromatin regulators with overlapping in-vitro activities. Defining specific in-vivo activities requires monitoring histone dynamics during regulator depletion, which has been technically challenging. We have recently generated histone-exchange sensors in Saccharomyces cerevisiae, which we now use to define the contributions of 15 regulators to histone dynamics genome-wide. While replication-independent exchange in unperturbed cells maps to promoters, regulator depletions primarily affected gene bodies. Depletion of Spt6, Spt16 or Chd1 sharply increased nucleosome replacement sequentially at the beginning, middle or end of highly expressed gene bodies. They further triggered re-localization of chaperones to affected gene body regions, which compensated for nucleosome loss during transcription complex passage, but concurred with extensive TF binding in gene bodies. We provide a unified quantitative screen highlighting regulator roles in retaining nucleosome binding during transcription and preserving genomic packaging.
Topics: Chromatin; Chromatin Assembly and Disassembly; DNA; Histone Chaperones; Histones; Nucleosomes; Protein Binding; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 37493599
DOI: 10.1093/nar/gkad615 -
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