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Chemical Society Reviews Jul 2024Nucleic acid aptamers are oligonucleotide chains with molecular recognition properties. Compared with antibodies, aptamers show advantages given that they are readily... (Review)
Review
Nucleic acid aptamers are oligonucleotide chains with molecular recognition properties. Compared with antibodies, aptamers show advantages given that they are readily produced chemical synthesis and elicit minimal immunogenicity in biomedicine applications. Notably, aptamer-encoded nucleic acid assemblies further improve the binding affinity of aptamers with the targets due to their multivalent synergistic interactions. Specially, aptamers can be engineered with special topological arrangements in nucleic acid assemblies, which demonstrate spatial and valence matching towards antigens on viruses, thus showing potential in the detection and therapeutic applications of viruses. This review presents the recent progress on the aptamers explored for SARS-CoV-2 detection and infection treatment, wherein applications of aptamer-based assembly systems are introduced in detail. Screening methods and chemical modification strategies for aptamers are comprehensively summarized, and the types of aptamers employed against different target domains of SARS-CoV-2 are illustrated. The evolution of aptamer-based assembly systems for the detection and neutralization of SARS-CoV-2, as well as the construction principle and characteristics of aptamer-based DNA assemblies are demonstrated. The typically representative works are presented to demonstrate how to assemble aptamers rationally and elaborately for specific applications in SARS-CoV-2 diagnosis and neutralization. Finally, we provide deep insights into the current challenges and future perspectives towards aptamer-based nucleic acid assemblies for virus detection and neutralization in nanomedicine.
Topics: Aptamers, Nucleotide; SARS-CoV-2; Humans; COVID-19; COVID-19 Drug Treatment; Antiviral Agents
PubMed: 38829187
DOI: 10.1039/d3cs00774j -
Journal of Visualized Experiments : JoVE May 2024The construction of gene expression vectors is an important component of laboratory work in experimental biology. With technical advancements like Gibson Assembly,...
The construction of gene expression vectors is an important component of laboratory work in experimental biology. With technical advancements like Gibson Assembly, vector construction becomes relatively simple and efficient. However, when the full-length genome of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) cannot be easily amplified by a single polymerase chain reaction (PCR) from cDNA, or it is difficult to acquire a full-length gene expression vector by homologous recombination of multiple inserts in vitro, the current Gibson Assembly technique fails to achieve this goal. Consequently, we aimed to divide the PRRSV genome into several fragments and introduce appropriate restriction sites into the reverse primer for obtaining PCR-amplified fragments. After joining the previous DNA fragment into the vector by homologous recombination technology, the new vector acquired the restriction enzyme cleavage site. Thus, we can linearize the vector by using the newly added enzyme cleavage site and introduce the next DNA fragment downstream of the upstream DNA fragment. The introduced restriction enzyme cleavage site at the 3' end of the upstream DNA fragment will be eliminated, and a new cleavage site will be introduced into the 3' end of the downstream DNA fragment. In this way, we can join DNA fragments to the vector one by one. This method is applicable to successfully construct the PRRSV expression vector and is an effective method for assembling a large number of fragments into the expression vector.
Topics: Porcine respiratory and reproductive syndrome virus; Genetic Vectors; Cloning, Molecular; Animals; Polymerase Chain Reaction; Swine; Genome, Viral
PubMed: 38829112
DOI: 10.3791/66320 -
MLife Mar 2024O-glycosylation is an ancient yet underappreciated protein posttranslational modification, on which many bacteria and viruses heavily rely to perform critical biological... (Review)
Review
O-glycosylation is an ancient yet underappreciated protein posttranslational modification, on which many bacteria and viruses heavily rely to perform critical biological functions involved in numerous infectious diseases or even cancer. But due to the innate complexity of O-glycosylation, research techniques have been limited to study its exact role in viral attachment and entry, assembly and exit, spreading in the host cells, and the innate and adaptive immunity of the host. Recently, the advent of many newly developed methodologies (e.g., mass spectrometry, chemical biology tools, and molecular dynamics simulations) has renewed and rekindled the interest in viral-related O-glycosylation in both viral proteins and host cells, which is further fueled by the COVID-19 pandemic. In this review, we summarize recent advances in viral-related O-glycosylation, with a particular emphasis on the mucin-type O-linked α-N-acetylgalactosamine (O-GalNAc) on viral proteins and the intracellular O-linked β-N-acetylglucosamine (O-GlcNAc) modifications on host proteins. We hope to provide valuable insights into the development of antiviral reagents or vaccines for better prevention or treatment of infectious diseases.
PubMed: 38827513
DOI: 10.1002/mlf2.12105 -
Plant & Cell Physiology Jun 2024Marchantia polymorpha has become an important model system for comparative studies and synthetic biology. The systematic characterisation of genetic elements would make...
Marchantia polymorpha has become an important model system for comparative studies and synthetic biology. The systematic characterisation of genetic elements would make heterologous gene expression more predictable in this testbed for gene circuit assembly and bioproduction. Yet, the toolbox of genetic parts for Marchantia includes only a few constitutive promoters that need benchmarking to assess their utility. We compared the expression patterns of previously characterized and new constitutive promoters. We found that driving expression with the double enhancer version of the cauliflower mosaic virus 35S promoter (pro35S×2) provided the highest yield of proteins although it also inhibits the growth of transformants. In contrast, promoters derived from the Marchantia ETHYLENE RESPONSE FACTOR 1 (MpERF1) and the CLASS II HOMEODOMAIN-LEUCINE ZIPPER (MpC2HDZ) genes drove expression to higher levels across all tissues without growth penalty and can provide intermediate levels of gene expression. In addition, we showed that the cytosol is the best subcellular compartment to target heterologous proteins for higher levels of expression without a significant growth burden. To demonstrate the potential of these promoters in Marchantia, we expressed the polycistronic RUBY betalain synthesis cassette to demonstrate coordinated expression of metabolic enzymes. A heat-shock inducible promoter was used to further mitigate growth burdens associated with high amounts of betalain accumulation. We have expanded the existing toolkit for gene expression in Marchantia and provide new resources for the Marchantia research community.
PubMed: 38822700
DOI: 10.1093/pcp/pcae063 -
Chemical Science May 2024Hepatitis C virus (HCV) continues to be a significant public health challenge, affecting an estimated 71 million people globally and posing risks of severe liver...
Hepatitis C virus (HCV) continues to be a significant public health challenge, affecting an estimated 71 million people globally and posing risks of severe liver diseases. Despite advancements in treatments, diagnostic limitations hinder the global elimination efforts targeted by 2030. This study introduces an innovative diagnostic approach, integrating catalytic hairpin assembly (CHA) with plasmonic core-satellite gold nanoparticle (AuNP) assemblies, to enable sensitive and specific detection of HCV RNA. We optimized the stoichiometry of DNA hairpins to form highly stable three-way junctions (3WJs), minimizing non-specific reactions in an enzyme-free, isothermal amplification process. The resulting dual-transduction biosensor combines colorimetric and surface-enhanced Raman spectroscopy (SERS) techniques, utilizing the Raman reporter malachite green isothiocyanate (MGITC) for signal generation. Our system targets a conserved 23-nucleotide sequence within the HCV 5'-UTR, essential for RNA replication, facilitating pan-genotypic HCV detection that complements direct-acting antiviral strategies. We evaluated the biosensor's efficacy using fluorescence spectroscopy, native PAGE, AFM, and TEM. Findings indicate that the 60 nm core AuNPs surrounded by 20 nm satellite AuNPs achieved a ten-fold increase in sensitivity over the 10 nm satellites, detecting HCV RNA concentrations as low as 1.706 fM. This sensitivity is crucial, given the extremely low viral loads present during early infection stages. Our research demonstrates the promise of enzyme-free molecular biosensors for HCV, with the potential to provide cost-efficient, rapid, point-of-care testing, although further sensitivity enhancements are needed to address the challenges of early-stage detection.
PubMed: 38817589
DOI: 10.1039/d4sc00891j -
ACS Applied Bio Materials Jun 2024The ultimate vaccine against infections caused by Nipah virus should be capable of providing protection at the respiratory tract─the most probable port of entry for...
The ultimate vaccine against infections caused by Nipah virus should be capable of providing protection at the respiratory tract─the most probable port of entry for this pathogen. Intranasally delivered vaccines, which target nasal-associated lymphoid tissue and induce both systemic and mucosal immunity, are attractive candidates for enabling effective vaccination against this lethal disease. Herein, the water-soluble polyphosphazene delivery vehicle assembles into nanoscale supramolecular constructs with the soluble extracellular portion of the Hendra virus attachment glycoprotein─a promising subunit vaccine antigen against both Nipah and Hendra viruses. These supramolecular constructs signal through Toll-like receptor 7/8 and promote binding interactions with mucin─an important feature of effective mucosal adjuvants. High mass contrast of phosphorus-nitrogen backbone of the polymer enables a successful visualization of nanoconstructs in their vitrified state by cryogenic electron microscopy. Here, we characterize the self-assembly of polyphosphazene macromolecule with biologically relevant ligands by asymmetric flow field flow fractionation, dynamic light scattering, fluorescence spectrophotometry, and turbidimetric titration methods. Furthermore, a polyphosphazene-enabled intranasal Nipah vaccine candidate demonstrates the ability to induce immune responses in hamsters and shows superiority in inducing total IgG and neutralizing antibodies when benchmarked against the respective clinical stage alum adjuvanted vaccine. The results highlight the potential of polyphosphazene-enabled nanoassemblies in the development of intranasal vaccines.
Topics: Organophosphorus Compounds; Administration, Intranasal; Polymers; Nipah Virus; Animals; Viral Vaccines; Vaccines, Subunit; Particle Size; Materials Testing; Biocompatible Materials; Nanoparticles; Immunization
PubMed: 38812435
DOI: 10.1021/acsabm.4c00441 -
Vaccine May 2024Self-assembling virus-like particles (VLPs) are promising platforms for vaccine development. However, the unpredictability of the physical properties, such as...
Self-assembling virus-like particles (VLPs) are promising platforms for vaccine development. However, the unpredictability of the physical properties, such as self-assembly capability, hydrophobicity, and overall stability in engineered protein particles fused with antigens, presents substantial challenges in their downstream processing. We envision that these challenges can be addressed by combining more precise computer-aided molecular dynamics (MD) simulations with experimental studies on the modified products, with more to-date forcefield descriptions and larger models closely resembling real assemblies, realized by rapid advancement in computing technology. In this study, three chimeric designs based on the hepatitis B core (HBc) protein as model vaccine candidates were constructed to study and compare the influence of inserted epitopes as well as insertion strategy on HBc modifications. Large partial VLP models containing 17 chains for the HBc chimeric model vaccines were constructed based on the wild-type (wt) HBc assembly template. The findings from our simulation analysis have demonstrated good consistency with experimental results, pertaining to the surface hydrophobicity and overall stability of the chimeric vaccine candidates. Furthermore, the different impact of foreign antigen insertions on the HBc scaffold was investigated through simulations. It was found that separately inserting two epitopes into the HBc platform at the N-terminal and the major immunogenic regions (MIR) yields better results compared to a serial insertion at MIR in terms of protein structural stability. This study substantiates that an MD-guided design approach can facilitate vaccine development and improve its manufacturing efficiency by predicting products with extreme surface hydrophobicity or structural instability.
PubMed: 38811268
DOI: 10.1016/j.vaccine.2024.05.040 -
Veterinary Research May 2024Pseudorabies virus (PRV) is recognized as the aetiological agent responsible for Aujeszky's disease, or pseudorabies, in swine populations. Rab6, a member of the small...
Pseudorabies virus (PRV) is recognized as the aetiological agent responsible for Aujeszky's disease, or pseudorabies, in swine populations. Rab6, a member of the small GTPase family, is implicated in various membrane trafficking processes, particularly exocytosis regulation. Its involvement in PRV infection, however, has not been documented previously. In our study, we observed a significant increase in the Rab6 mRNA and protein levels in both PK-15 porcine kidney epithelial cells and porcine alveolar macrophages, as well as in the lungs and spleens of mice infected with PRV. The overexpression of wild-type Rab6 and its GTP-bound mutant facilitated PRV proliferation, whereas the GDP-bound mutant form of Rab6 had no effect on viral propagation. These findings indicated that the GTPase activity of Rab6 was crucial for the successful spread of PRV. Further investigations revealed that the reduction in Rab6 levels through knockdown significantly hampered PRV proliferation and disrupted virus assembly and egress. At the molecular level, Rab6 was found to interact with the PRV glycoproteins gB and gE, both of which are essential for viral assembly and egress. Our results collectively suggest that PRV exploits Rab6 to expedite its assembly and egress and identify Rab6 as a promising novel target for therapeutic treatment for PRV infection.
Topics: Animals; Herpesvirus 1, Suid; Swine; rab GTP-Binding Proteins; Mice; Pseudorabies; Virus Release; Virus Assembly; Swine Diseases; Cell Line
PubMed: 38807225
DOI: 10.1186/s13567-024-01328-4 -
Liver International : Official Journal... Jul 2024In 2016, the Global Health Sector Strategy, ratified by the 69th World Health Assembly, set the ambitious goal of eliminating hepatitis C virus (HCV) and hepatitis B... (Review)
Review
In 2016, the Global Health Sector Strategy, ratified by the 69th World Health Assembly, set the ambitious goal of eliminating hepatitis C virus (HCV) and hepatitis B virus infections by 2030, emphasizing the importance of national screening programmes. Achieving this goal depends on each country's ability to identify and treat 80% of chronic hepatitis C cases, a critical threshold set by the World Health Organization. Traditionally, estimates of HCV prevalence have been based on interferon era studies that focused on high-risk subgroups rather than the general population. In addition, the incomplete data available from national registries also limited the understanding of HCV prevalence. The 2016 report from the European Centre for Disease Prevention and Control highlighted that HCV rates varied across European counties, ranging from .1% to 5.9%. However, data were only available for 13 countries, making the overall picture less clear. Additionally, the epidemiological data may have underestimated the true burden of HCV due to lack of awareness among those with chronic infection. The main objective of this review is to provide a comprehensive summary of HCV epidemiology in Europe in the current era of direct-acting antivirals (DAAs). The data included in the analysis range from the end of 2013 to December 2023 and have been categorised according to the United Nations Geoscheme. The resulting synthesis underscores the noteworthy impact of DAA treatment on the epidemiological situation.
Topics: Humans; Europe; Prevalence; Antiviral Agents; Hepatitis C, Chronic; Hepacivirus
PubMed: 38804727
DOI: 10.1111/liv.15981 -
Advances in Experimental Medicine and... 2024Poxvirus assembly has been an intriguing area of research for several decades. While advancements in experimental techniques continue to yield fresh insights, many... (Review)
Review
Poxvirus assembly has been an intriguing area of research for several decades. While advancements in experimental techniques continue to yield fresh insights, many questions are still unresolved. Large genome sizes of up to 380 kbp, asymmetrical structure, an exterior lipid bilayer, and a cytoplasmic life cycle are some notable characteristics of these viruses. Inside the particle are two lateral bodies and a protein wall-bound-biconcave core containing the viral nucleocapsid. The assembly progresses through five major stages-endoplasmic reticulum (ER) membrane alteration and rupture, crescent formation, immature virion formation, genome encapsidation, virion maturation and in a subset of viruses, additional envelopment of the virion prior to its dissemination. Several large dsDNA viruses have been shown to follow a comparable sequence of events. In this chapter, we recapitulate our understanding of the poxvirus morphogenesis process while reviewing the most recent advances in the field. We also briefly discuss how virion assembly aids in our knowledge of the evolutionary links between poxviruses and other Nucleocytoplasmic Large DNA Viruses (NCLDVs).
Topics: Poxviridae; Virus Assembly; Humans; Genome, Viral; Virion; Animals; Evolution, Molecular; Endoplasmic Reticulum
PubMed: 38801570
DOI: 10.1007/978-3-031-57165-7_3