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Gene Jun 2024The adeno-associated virus (AAV) is a defective single-stranded DNA virus with the simplest structure reported to date. It constitutes a capsid protein and... (Review)
Review
The adeno-associated virus (AAV) is a defective single-stranded DNA virus with the simplest structure reported to date. It constitutes a capsid protein and single-stranded DNA. With its high transduction efficiency, low immunogenicity, and tissue specificity, it is the most widely used and promising gene therapy vector. The clustered regularly interspaced short palindromic sequence (CRISPR)/CRISPR-associated protein 9 (Cas9) gene editing system is an emerging technology that utilizes cas9 nuclease to specifically recognize and cleave target genes under the guidance of small guide RNA and realizes gene editing through homologous directional repair and non-homologous recombination repair. In recent years, an increasing number of animal experiments and clinical studies have revealed the great potential of AAV as a vector to deliver the CRISPR/cas9 system for treating genetic diseases and viral infections. However, the immunogenicity, toxicity, low transmission efficiency in brain and ear tissues, packaging size limitations of AAV, and immunogenicity and off-target effects of Cas9 protein pose several clinical challenges. This research reviews the role, challenges, and countermeasures of the AAV-CRISPR/cas9 system in gene therapy.
PubMed: 38945310
DOI: 10.1016/j.gene.2024.148733 -
Journal of Phycology Jun 2024Mazzaella, a genus with no genomic resources available, has extensive distribution in the cold waters of the Pacific, where they represent ecologically and economically...
Mazzaella, a genus with no genomic resources available, has extensive distribution in the cold waters of the Pacific, where they represent ecologically and economically important species. In this study, we aimed to sequence, assemble, and annotate the complete mitochondrial and chloroplast genomes from two Mazzaella spp. and characterize the intraspecific variation among them. We report for the first time seven whole organellar genomes (mitochondria: OR915856, OR947465, OR947466, OR947467, OR947468, OR947469, OR947470; chloroplast: OR881974, OR909680, OR909681, OR909682, OR909683, OR909684, OR909685) obtained through high-throughput sequencing for six M. laminarioides sampled from three Chilean regions and one M. membranacea. Sequenced Mazzaella mitogenomes have identical gene number, gene order, and genome structure. The same results were observed for assembled plastomes. A total of 52 genes were identified in mitogenomes, and a total of 235 genes were identified in plastomes. Although the M. membranacea plastome included a full-length pbsA gene, in all M. laminarioides samples, the pbsA gene was split in three open reading frames (ORFs). Within M. laminarioides, we observed important plastome lineage-specific variations, such as the pseudogenization of the two hypothetical protein-coding genes, ycf23 and ycf45. Nonsense mutations in the ycf23 and ycf45 genes were only detected in the northern lineage. These results are consistent with phylogenetic reconstructions and divergence time estimation using concatenated coding sequences that not only support the monophyly of M. laminarioides but also underscore that the three M. laminarioides lineages are in an advanced stage of divergence. These new results open the question of the existence of still undisclosed species in M. laminarioides.
PubMed: 38944824
DOI: 10.1111/jpy.13478 -
Insect Biochemistry and Molecular... Jun 2024The larvae of the moth Hyalophora cecropia spin silk cocoons with morphologically distinct layers. We investigated the expression of the individual silk protein...
The larvae of the moth Hyalophora cecropia spin silk cocoons with morphologically distinct layers. We investigated the expression of the individual silk protein components of these cocoons in relation to the morphology of the silk gland and its affiliation to the different layers of the cocoon. The study used transcriptomic and proteomic analyses to identify 91 proteins associated with the silk cocoons, 63 of which have a signal peptide indicating their secretory nature. We checked the specificity of their expression in different parts of the SG and the presence of the corresponding protein products in each cocoon layer. Differences were observed among less abundant proteins with unclear functions. The representation of proteins in the inner envelope and intermediate space was similar, except for a higher proportion of probable contaminating proteins, mostly originating from the gut. On the other hand, the outer envelope contains a number of putative enzymes with unclear function. However, the protein most specific to the outer layer has sequence homology to putative serine/threonine kinase-like proteins and some adhesive proteins, and its closest homolog in Bombyx mori was found in the scaffold silk. This research provides valuable insights into the silk production of the cecropia moth, highlighting both similarities and differences to other moth species.
PubMed: 38944399
DOI: 10.1016/j.ibmb.2024.104152 -
Antiviral Research Jun 2024Herpes simplex virus type 1 (HSV-1) is a neurotropic alphaherpesvirus that establishes a lifelong infection in sensory neurons of infected individuals, accompanied with...
Herpes simplex virus type 1 (HSV-1) is a neurotropic alphaherpesvirus that establishes a lifelong infection in sensory neurons of infected individuals, accompanied with intermittent reactivation of latent virus causing (a)symptomatic virus shedding. Whereas acyclovir (ACV) is a safe and highly effective antiviral to treat HSV-1 infections, long-term usage can lead to emergence of ACV resistant (ACV) HSV-1 and subsequently ACV refractory disease. Here, we isolated an HSV-1 strain from a patient with reactivated herpetic eye disease that did not respond to ACV treatment. The isolate carried a novel non-synonymous F289S mutation in the viral UL23 gene encoding the thymidine kinase (TK) protein. Because ACV needs conversion by viral TK and subsequently cellular kinases to inhibit HSV-1 replication, the UL23 gene is commonly mutated in ACV HSV-1 strains. The potential role of the F289S mutation causing ACV was investigated using CRISPR/Cas9-mediated HSV-1 genome editing. Reverting the F289S mutation in the original clinical isolate to the wild-type sequence S289F resulted in an ACV-sensitive (ACV) phenotype, and introduction of the F289S substitution in an ACV HSV-1 reference strain led to an ACV phenotype. In summary, we identified a new HSV-1 TK mutation in the eye of a patient with ACV refractory herpetic eye disease, which was identified as the causative ACV mutation with the aid of CRISPR/Cas9-mediated genome engineering technology. Direct editing of clinical HSV-1 isolates by CRISPR/Cas9 is a powerful strategy to assess whether single residue substitutions are causative to a clinical ACV phenotype.
PubMed: 38944159
DOI: 10.1016/j.antiviral.2024.105950 -
Journal of Invertebrate Pathology Jun 2024Decapod iridescent virus 1 (DIV1) stands as a significant pathogen affecting crustaceans, posing a grave threat to the shrimp industries in aquaculture dependent...
Decapod iridescent virus 1 (DIV1) stands as a significant pathogen affecting crustaceans, posing a grave threat to the shrimp industries in aquaculture dependent nations. Within the Iridoviridae family, the conserved envelope protein DIV1-168L plays a pivotal role in virion entry. Nonetheless, the host factors that interact with 168L remain unidentified. To address this gap, we established a cDNA library derived from Litopenaeus vannamei gill tissue and conducted yeast two-hybrid screening to identify host factors that interact with 168L. Additionally, we performed co-immunoprecipitation assays to verify the interaction between cuticle protein 8 (CP8) and 168L. Expression pattern analysis revealed the presence of CP8 transcripts in the gill and epidermis. Furthermore, immunohistochemistry results demonstrated the expression of CP8 in gill cells and its localization in the gill filament epithelium. Fluorescence analysis indicated that full-length CP8 colocalized with 168L in the cytoplasm of Sf9 cells. Removal of the signal peptide from the N-terminal of CP8 eliminated its concentration in the cytoplasm. Additionally, CP8 expression was significantly inhibited during DIV1 infection. Therefore, our research contributes to a better understanding of the entry mechanism of iridovirids. The GenBank accession number for the DIV1 sequence is MF197913.1.
PubMed: 38944151
DOI: 10.1016/j.jip.2024.108162 -
The Journal of Biological Chemistry Jun 2024One of seven natural CO fixation pathways, the anaerobic Wood-Ljungdahl Pathway (WLP) is unique in generating CO as a metabolic intermediate, operating through...
One of seven natural CO fixation pathways, the anaerobic Wood-Ljungdahl Pathway (WLP) is unique in generating CO as a metabolic intermediate, operating through organometallic intermediates, and in conserving (versus utilizing) net ATP. The key enzyme in the WLP is acetyl-CoA synthase (ACS), which uses an active site [2Ni-4Fe-4S] cluster (A-cluster), a CO tunnel, and an organometallic (Ni-CO, Ni-methyl, and Ni-acetyl) reaction sequence to generate acetyl-CoA. Here we reveal that an alcove, which interfaces the tunnel and the A-cluster, is essential for CO fixation and autotrophic growth by the WLP. In vitro spectroscopy, kinetics, binding, and in vivo growth experiments reveal that a Phe229A substitution at one wall of the alcove decreases CO affinity thirty-fold and abolishes autotrophic growth; however, a F229W substitution enhances CO binding 80-fold. Our results indicate the structure of the alcove is exquisitely tuned to concentrate CO near the A-cluster; protect ACS from CO loss during catalysis, provide a haven for inhibitory CO, and stabilize the tetrahedral coordination at the Ni site where CO binds. The directing, concentrating, and protective effects of the alcove explain the inability of F209A to grow autotrophically. The alcove also could help explain current controversies over whether ACS binds CO and methyl through a random or ordered mechanism. Our work redefines what we historically refer to as the metallocenter "active site". The alcove is so crucial for enzymatic function that we propose it is part of the active site. The community should now look for such alcoves in all "gas handling" metalloenzymes.
PubMed: 38944127
DOI: 10.1016/j.jbc.2024.107503 -
Computational Biology and Chemistry Jun 2024The bioinformatic analysis of cannabinoid receptors (CBRs) CB and CB reveals a detailed picture of their structure, evolution, and physiological significance within the...
The bioinformatic analysis of cannabinoid receptors (CBRs) CB and CB reveals a detailed picture of their structure, evolution, and physiological significance within the endocannabinoid system (ECS). The study highlights the evolutionary conservation of these receptors evidenced by sequence alignments across diverse species including humans, amphibians, and fish. Both CBRs share a structural hallmark of seven transmembrane (TM) helices, characteristic of class A G-protein-coupled receptors (GPCRs), which are critical for their signalling functions. The study reports a similarity of 44.58 % between both CBR sequences, which suggests that while their evolutionary paths and physiological roles may differ, there is considerable conservation in their structures. Pathway databases like KEGG, Reactome, and WikiPathways were employed to determine the involvement of the receptors in various signalling pathways. The pathway analyses integrated within this study offer a detailed view of the CBRs interactions within a complex network of cannabinoid-related signalling pathways. High-resolution crystal structures (PDB ID: 5U09 for CB and 5ZTY for CB) provided accurate structural information, showing the binding pocket volume and surface area of the receptors, essential for ligand interaction. The comparison between these receptors' natural sequences and their engineered pseudo-CBRs (p-CBRs) showed a high degree of sequence identity, confirming the validity of using p-CBRs in receptor-ligand interaction studies. This comprehensive analysis enhances the understanding of the structural and functional dynamics of cannabinoid receptors, highlighting their physiological roles and their potential as therapeutic targets within the ECS.
PubMed: 38943725
DOI: 10.1016/j.compbiolchem.2024.108138 -
Current Opinion in Structural Biology Jun 2024The intrinsically disordered, lipid-modified membrane anchor of small GTPases is emerging as a critical modulator of function through its ability to sort lipids in a... (Review)
Review
The intrinsically disordered, lipid-modified membrane anchor of small GTPases is emerging as a critical modulator of function through its ability to sort lipids in a conformation-dependent manner. We reviewed recent computational and experimental studies that have begun to shed light on the sequence-ensemble-function relationship in this unique class of lipidated intrinsically disordered regions (LIDRs).
PubMed: 38943706
DOI: 10.1016/j.sbi.2024.102869 -
Chembiochem : a European Journal of... Jun 2024While foldamers have been extensively studied as protein mimics and especially as α-helix mimics, their use as capping motif to enhance α-helix propensity remains...
While foldamers have been extensively studied as protein mimics and especially as α-helix mimics, their use as capping motif to enhance α-helix propensity remains comparatively much limited. In this study, we leverage the structural similarities between urea-based helical foldamers and α-helix to investigate the efficacy of oligoureas as N- or C-caps for reinforcing α-helical structures in water. Short oligoureas, comprising 3 to 4 residues, were strategically introduced at the N- or C-terminus of two peptide sequences (S-peptide and an Ala-rich model sequence). The impact of these foldamer insertions on peptide conformation was examined using electronic circular dichroism (ECD) and solution NMR. This research identifies specific foldamer sequences capable of promoting a-helicity when incorporated at either terminus of the peptides. Not only does this work broaden the application scope of foldamers, but it also provides valuable insights into novel strategies for modulating peptide conformation in aqueous environments. The findings presented in this study may have implications for peptide design and the development of bioactive foldamer-based peptide mimics.
PubMed: 38943628
DOI: 10.1002/cbic.202400427 -
Molekuliarnaia Biologiia 2024The mobile genetic elements IS630/Tc 1/mariner (ITm) are widespread DNA transposons that make a significant contribution to the evolution of eukaryotic genomes. With the...
The mobile genetic elements IS630/Tc 1/mariner (ITm) are widespread DNA transposons that make a significant contribution to the evolution of eukaryotic genomes. With the start of large-scale application of next-generation sequencing (NGS) technologies and the emergence of many new whole genome sequences of organisms in nucleotide sequence collections, the ITm elements have been identified in most taxa of the eukaryotic tree of life. Although ITm diversity has been studied in detail, new elements are still found, thus expanding the respective DNA transposon group and calling for review of its classification. Bivalve L31 elements were for the first time analyzed in detail to describe their structures, diversity, distribution, and phylogenetic position among the ITm elements. The L31 transposons were found to form an independent superfamily of an ancient origin within the ITm group. Rather high diversity was observed within the L31 clade; i.e., five phylogenetic clusters were identified. In mollusks, the L31 transposons have been detected only in the subclass Autobranchia and predominate in diversity and number in the infraclass Pteriomorphia. A protein encoded by open reading frame 2 (ORF2) was shown to be an integral structural component of almost all full-length L31 elements. The results provide for a better understanding of the evolution of particular ITm transposons. Further study of the L31 transposons in other taxa (cnidarians) and functional investigation of the ORF2 protein product will help to better understand the evolution of DNa transposons, the mechanisms of their horizontal transfer, and their contribution to eukaryotic biodiversity.
Topics: Animals; DNA Transposable Elements; Bivalvia; Phylogeny; Evolution, Molecular; Open Reading Frames
PubMed: 38943580
DOI: No ID Found