Search Results (120)

Aptamers are powerful tools for detecting and analyzing biomolecules that consist of proteins or nucleic acids. However, their application to aptamers against viroids—highly structured self-replicating RNAs—has not yet been explored. In this study, a magnetic bead- and high-throughput sequencing-based SELEX (MB-HTS-SELEX) protocol for selecting potential aptamers against potato spindle tuber viroid (PSTVd) is presented. Full-length biotinylated-PSTVd RNA was transcribed in vitro, immobilized on streptavidin-coated magnetic beads, and incubated with a library of ~3.32 × 10¹⁴ molecules of random single-stranded oligo-DNAs (oligo-ssDNAs) of 20, 30, or 40 nucleotides (L20, L30, or L40, respectively) flanked by primer binding sites for downstream PCR amplification. Simultaneous biotin labeling of the anti-aptamer strand of the resulting double-stranded DNA (dsDNA) amplicons facilitated strand separation using streptavidin-coated magnetic beads. After 10 selection rounds, high-throughput sequencing, followed by bioinformatics analysis of the generated sequences, allowed for the detection of several enriched sequences, representing putative PSTVd-binding aptamers. Subsequent pull-down assays showed that the most abundant oligo-ssDNA in L30 was docked on PSTVd molecules. This combination method may ameliorate the selection of high-affinity aptamers against PSTVd, reduce the number of selection cycles, time, and other costs of aptamer production, thereby promoting future massive and cost-effective viroid detection and characterization. Full article

Background: Self-amplifying mRNA (sa-mRNA) represents a promising platform for vaccines and gene therapies, offering sustained protein expression at low doses through self-replication. For vaccines targeting respiratory pathogens, pulmonary delivery of sa-mRNA lipid nanoparticles (LNPs) is particularly advantageous, enabling direct delivery to the infection site and induction of mucosal immunity. Objective: In this study, we evaluated the stability of sa-mRNA–LNPs under refrigerated and frozen conditions and developed a dry powder formulation suitable for inhalation, produced by freeze-drying followed by cryomilling with leucine. Methods: sa-mRNA–LNPs formulated in HEPES buffer with 20% (w/v) sucrose were stored for up to 8 weeks as liquid or freeze-dried samples at various temperatures (−80 °C, −20 °C, 4 °C, and 20 °C). Biological stability was assessed by transfection efficiency in HeLa cells, while physical stability was characterized by encapsulation efficiency, zeta potential, particle size, and polydispersity index. Results: Liquid formulations remained stable for at least 8 weeks at −80 °C and −20 °C but rapidly lost stability at 4 °C and 20 °C. Freeze-drying effectively preserved sa-mRNA–LNP functionality and structural integrity for up to 8 weeks at 4 °C, with only minor structural changes. Subsequent cryomilling in the presence of 4 wt-% leucine produced a respirable dry powder while retaining approximately 60% of the original sa-mRNA–LNP functionality. Although cryomilling induced some structural alterations, the remaining functional fraction remained stable during storage. The resulting powders displayed favorable aerosol performance for deep lung delivery, as demonstrated by cascade impaction (MMAD = 4.13 ± 0.26 µm). Conclusions: In conclusion, freeze-drying effectively preserved sa-mRNA–LNP integrity at 4 °C, whereas cryomilling with leucine produced a respirable dry powder suitable for pulmonary delivery, providing a foundation for globally accessible, needle-free sa-mRNA vaccines against respiratory diseases. Full article

The origin of life embodies two fundamental questions: how and when did life begin? It is commonly conjectured that life began on Earth around 4 billion years ago. This requires that the complex organization of RNA, DNA, triplet codon, protein, and lipid membrane (RDTPM) architecture was easy to establish between the time the Earth cooled enough for liquid water and the time when early microorganisms appeared. These bracketing events create a narrow window of time to construct a completely operational self-replicating organic system of very high complexity. Another conjecture is that life did not begin on Earth but was seeded from life-bearing space objects (e.g., asteroids, comets, space dust), commonly referred to as panspermia. The second conjecture implies that life formed somewhere else and was part of the solar nebula, originating from an earlier generation star where there was more time available for the development of life. In this paper, the goal is to provide a hypothetical perspective related to the timing for the origin of pre-biotic chemistry and life itself. Using a form of complexity growth, biological features spanning from the present day back to early life on Earth were examined for trends across time. Genome sizes, gene number, protein–protein binding sites, energy for cell construction, mass of individual cells, the rate of cell mass growth, and a molecular complexity measure all yield highly significant regressions of linearly increasing complexity when plotted over the last 4 Gyr (billion years). When extrapolated back in time, intersections with simple complexities associated with each variable yield a mean value of 8.6 Gyr before the present time. This era coincides with the peak of star and planet formation in the universe. This speculative analysis is consistent with the second conjecture for the origin of life. The major assumptions of such an analysis are presented and discussed. Full article

Alphaviruses have historically been viewed as acute, self-limiting pathogens. However, growing evidence shows that viral RNA and antigens can persist in vertebrate hosts long after the resolution of acute infection, a phenomenon known as viral persistence. Viral persistence reflects a dynamic interplay between viral replication—including shifts from lytic to non-lytic infection—and host defenses, which together establish cellular and tissue niches that enable evasion of immune-mediated clearance. Within vertebrate hosts, alphaviruses exhibit broad tissue tropism, infecting diverse cell types that may differentially support long-term persistence. Emerging evidence suggests that viral persistence arises through three interconnected processes: (i) selective infection of specific cellular niches, (ii) reprogramming of host cellular pathways, and (iii) modulation of immune responses. Yet, the extent to which viral or host determinants shape this balance, and how persistence contributes to chronic disease, remains unresolved. Here, we synthesize current in vitro and in vivo evidence of alphavirus persistence in vertebrate hosts and discuss potential mechanisms by which alphaviruses establish and maintain persistent infection beyond the acute phase. We further underscore critical gaps in current knowledge and outline future research avenues essential for elucidating the mechanisms underlying alphavirus pathogenesis. Full article

Background: Kenaf (Hibiscus cannabinus L.) is an important fiber crop belonging to the genus Hibiscus in the Malvaceae family. Research on its chloroplast genome holds significant importance for deciphering the evolutionary relationships of the Hibiscus species, developing genetic markers, and promoting kenaf (H. cannabinus) genetic breeding. Methods: Based on high-throughput sequencing technology, this study completed the sequencing and assembly of the kenaf (H. cannabinus) chloroplast genome. Results: (1) The kenaf (H. cannabinus) chloroplast genome exhibits a typical circular quadripartite structure with a total length of 163,019 bp, including a large single-copy region (LSC) of 90,467 bp, a small single-copy region (SSC) of 19,486 bp, and a pair of inverted repeat regions (IRa/IRb) of 26,533 bp each. The total GC content is 36.62%, among which, the IR region has the highest GC content (42.61%) and the SSC region the lowest (30.87%). (2) A total of 131 genes were annotated, including 85 mRNAs, 37 tRNAs, 8 rRNAs, and 1 pseudogene. Their functions cover photosynthesis (e.g., pet and atp family genes), self-replication (e.g., rpl, rps, and rpo family genes), and genes with unknown functions (e.g., ycf1 and ycf2). A codon usage bias analysis revealed that the relative synonymous codon usage (RSCU) value of the stop codon UAA is the highest (1.6329), and codons ending with A/U are preferentially used (e.g., GCU for alanine with RSCU = 1.778). (3) A repeat sequence analysis identified various interspersed repeat sequences (predominantly 30~31 bp in length, with a relatively high proportion in the 30~40 bp range, including forward and palindromic types) and simple sequence repeats (cpSSRs). Among them, single-base repeat SSRs account for the highest proportion (e.g., (A)8 and (T)9), and specific SSR primers were designed. (4) A comparative evolutionary analysis indicated that the Ka/Ks ratios (nonsynonymous substitution rate/synonymous substitution rate) of core chloroplast genes (e.g., rps2 and rpoC2) in kenaf (H. cannabinus) are all less than 1 (0.145~0.415), suggesting that they are under purifying selection. The collinearity similarity of chloroplast genomes between kenaf (H. cannabinus) and its closely related species reaches over 99.97%, and the IR region boundaries are relatively conserved. The phylogenetic tree shows that kenaf (H. cannabinus) clusters with closely related Hibiscus species with a 100% bootstrap value, indicating a close genetic relationship. Conclusions: This study provides basic data for the functional analysis of the kenaf (H. cannabinus) chloroplast genome, the phylogeny of Hibiscus, and the utilization of genetic resources. Full article

The term extracellular vesicles (EVs) includes a variety of anucleated, non-self-replicative particles released by cells, whose cargo content is compartmentalized by a lipidic bilayer membrane and includes proteins, DNA, and RNA (both coding and non-coding) molecules. MicroRNAs (miRs) are small non-coding RNA involved in gene expression regulation that functionally participate in inter-cellular communication as EV cargo. Natural Killer (NK) cells are innate immunity lymphocytes specialized in the killing of cancer cells and virally infected cells. Increasing evidence shows that NK cell-derived EVs contribute to the anti-tumoral activity of NK cells and that such effects are, at least in part, mediated by the miR cargo of these EVs. Conversely, cancer cells release EVs whose cargo includes proteins and miRs that impair NK cell function. These interactions highlight a central role for EV miRs both in the NK-mediated cytotoxicity and as a major immune-escape mechanism for cancer cells, ultimately contributing to the overall success or failure of NK cells in eliciting their anti-tumoral activity. Full article

Self-amplifying RNA (saRNA) is a promising platform for the production of vaccines, anti-tumor therapeutics, and gene therapy solutions. One of the advantages of the saRNA platform is the ability to use small doses of the therapeutic while maintaining prolonged expression of the target protein. However, the presence of auxiliary sequences encoding non-structural alphavirus proteins, which facilitate the replication of saRNA in cells, necessitates a thorough assessment of the biosafety of this platform. In our review, we focus on saRNA functions in the context of its interaction with the innate immune system. Firstly, an analysis is conducted of the side effects of candidate saRNA therapeutics, as observed in preclinical and clinical trials. Then, the mechanisms underlying the function of saRNA products derived from various alphavirus genomes in cell systems are discussed, as well as the reasons for their reactogenicity. The key approaches to optimizing the saRNA platform, which are aimed at reducing the activation of the innate immune response and cytopathic effects, are described. To summarize, this review enables us to systematize our knowledge on the advantages and disadvantages of saRNA, as well as potential approaches to improving this platform in order to develop more effective and safer therapeutics. Full article

Self-amplifying RNA-based (saRNA) technology represents the last frontier in using synthetic RNA in vaccinology. Typically, saRNA consists of positive-strand RNA molecules of viral origin (almost exclusively from alphaviruses) where the sequences of structural proteins are replaced with the open reading frame coding the antigen of interest. For in vivo delivery, they are complexed with lipid nanoparticles (LNPs), just like current COVID-19 vaccines based on synthetic messenger RNA (mRNA). Given their ability to amplify themselves inside the cell, optimal intracellular levels of the immunogenic antigen can be achieved by delivering lower amounts of saRNA molecules compared to mRNA-based vaccines. However, the excessive intracellular accumulation of saRNA may represent a relevant drawback since, as already described in alphavirus-infected cells, the recipient cell may react by incorporating excessive RNA molecules into extracellular vesicles (EVs). These EVs can shed and enter neighboring as well as distant cells, where the EV-associated saRNA can start a new replication cycle. This mechanism could lead to an unwanted and unnecessary spread of saRNA throughout the body, posing relevant safety issues. This perspective article discusses the molecular mechanisms through which saRNAs can be transmitted among different cells/tissues. In addition, a simple way to control the possible excessive saRNA intercellular propagation through the co-expression of an EV-anchored protein inhibiting the saRNA replication is proposed. Based on current knowledge, a safety improvement of saRNA-based vaccines appears to be mandatory for their usage in healthy humans. Full article

Hepatitis E, caused by the hepatitis E virus (HEV), is a zoonotic disease that extends beyond hepatocellular necrosis to replicate in multiple organs. While most infections are self-limiting, HEV infection during pregnancy is associated with severe outcomes, including acute liver failure, preterm delivery, and miscarriage, with the mechanisms underlying this high pathogenicity remaining poorly understood. This study established a pregnant tree shrew model with a late-stage HEV infection and a cellular model using zoonotic HEV genotypes GT3 and GT4 to investigate the effects of estrogen on HEV replication. Results showed that negative-strand RNA detection revealed replicative intermediates in feces and tissues during the acute phase, with peak viral loads occurring within one week and the highest titers in bile. Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels rose at 3 days post-inoculation (DPI), peaking at 7 DPI. Elevated estrogen levels post-miscarriage correlated with increased viral loads, a trend mirrored in cell culture models showing linear relationships between estrogen and viral replication. Histopathology demonstrated viral hepatitis lesions in liver tissues and abnormalities in the uterus, ovaries, and brain, including hydropic degeneration, neuronal disruption, and granulosa cell necrosis. This study developed a pregnant tree shrew model for HEV infection, providing a robust tool for exploring pathogenic mechanisms during pregnancy and genotype-specific differences in zoonotic HEV pathogenicity. These findings offer new insights into the role of estrogen in HEV replication and its contribution to adverse pregnancy outcomes. Full article

The ability of a nucleic acid molecule to self-replicate is the driving force behind the evolution of cellular life and the transition from RNA to DNA as the genetic material. Thus, the physicochemical properties of genome replication, such as the requirement for a terminal hydroxyl group for de novo DNA synthesis, are conserved in all three domains of life: eukaryotes, bacteria, and archaea. Canonical DNA replication is initiated from specific chromosomal sequences termed origins. Early bacterial models of DNA replication proposed origins as regulatory points for spatiotemporal control, with replication factors acting on a single origin on the chromosome. In eukaryotes and archaea, however, replication initiation usually involves multiple origins, with complex spatiotemporal regulation in the former. An alternative replication initiation mechanism, recombination-dependent replication, is observed in every cellular domain (and viruses); DNA synthesis is initiated instead from the 3′ end of a recombination intermediate. In the domain archaea, species including Haloferax volcanii are not only capable of initiating DNA replication without origins but grow faster without them. This raises questions about the necessity and nature of origins. Why have archaea retained such an alternative DNA replication initiation mechanism? Might recombination-dependent replication be the ancestral mode of DNA synthesis that was used during evolution from the primordial RNA world? This review provides a historical overview of major advancements in the study of DNA replication, followed by a comparative analysis of replication initiation systems in the three domains of life. Our current knowledge of origin-dependent and recombination-dependent DNA replication in archaea is summarised. Full article

Hepatitis E virus (HEV) is a positive-sense, single-stranded RNA virus that poses a significant public health risk, yet its study is hindered by the complexity of conventional RNA-based reverse genetics systems. These systems require multiple steps, including genome cloning, in vitro transcription, and capping, making them labor-intensive and susceptible to RNA degradation. In this study, we developed a single-step, plasmid-based HEV expression system that enabled direct intracellular transcription of the full-length HEV genome under a cytomegalovirus immediate-early (CMV-IE) promoter. The viral genome was flanked by hammerhead (HH) and hepatitis delta virus (HDV) ribozymes to ensure precise self-cleavage and the generation of authentic 5′ and 3′ termini. This system successfully supported HEV genome replication, viral protein expression, and progeny virion production at levels comparable to those obtained using in vitro-transcribed, capped HEV RNA. Additionally, a genetic marker introduced into the plasmid construct was stably retained in progeny virions, demonstrating the feasibility of targeted genetic modifications. However, plasmid-derived HEV exhibited delayed replication kinetics, likely due to the absence of an immediate 5′ cap. Attempts to enhance capping efficiency through co-expression of the vaccinia virus capping enzyme failed to improve HEV replication, suggesting that alternative strategies, such as optimizing the promoter design for capping, may be required. This plasmid-based HEV reverse genetics system simplifies the study of HEV replication and pathogenesis and provides a versatile platform for the genetic engineering of the HEV genome. Full article

Background: Previous evidence demonstrated DNA methylation changes in response to stress in plants, showing rapid changes within a limited time frame. Exposure to self-DNA inhibits seedling root elongation, and it was shown that it causes changes in CG DNA methylation in Lactuca sativa. We assessed cytosine methylation changes and associated gene expression patterns in roots of Arabidopsis thaliana Col-0 seedlings exposed to self-DNA for 6 and 24 h. Methods: We used whole genome bisulfite sequencing (WGBS) and RNA-seq analyses to assess genomic cytosine methylation and corresponding gene expression, respectively, on DNA and RNA extracted with commercial kits from roots exposed to self-DNA by an original setup. Fifteen hundred roots replicates, including the control in distilled water, were collected after exposure. Sequencing was performed on a NovaSeq 6000 platform and Ultralow Methyl-Seq System for RNA and DNA WGBS, respectively. Results: Gene expression in roots exposed to self-DNA differed from that of untreated controls, with a total of 305 genes differentially expressed and 87 ontologies enriched in at least one treatment vs. control comparison, and particularly after 24 h of exposure. DNA methylation, particularly in CHG and CHH contexts, was also different, with hyper- and hypomethylation prevailing in treatments vs. controls at 6 h and 24 h, respectively. Differentially expressed genes (DEGs) analysis, Gene Ontology (GO) enrichment analysis, and differentially methylated regions (DMRs) analysis, provided an integrated understanding of the changes associated with self-DNA exposure. Our results suggest differential gene expression associated with DNA methylation in response to self-DNA exposure in A. thaliana roots, enhanced after prolonged exposure. Conclusions: Main functional indications of association between DNA methylation and gene expression involved hypomethylation and downregulation of genes related to nucleotide/nucleoside metabolism (ATP synthase subunit) and cell wall structure (XyG synthase), consistent with previous observations from metabolomics and physiological studies. Further confirmation of these findings will contribute to improving our understanding of the plant molecular response to self-DNA and its implications in stress responses. Full article

Research on the birth and evolution of life are reviewed with reference to the maximum entropy production principle (MEPP). It has been shown that this principle is essential for consistent understanding of the birth and evolution of life. First, a recent work for the birth of a self-replicative system as pre-RNA life is reviewed in relation to the MEPP. A critical condition of polymer concentration in a local system is reported by a dynamical system approach, above which, an exponential increase of entropy production is guaranteed. Secondly, research works of early stage of evolutions are reviewed; experimental research for the numbers of cells necessary for forming a multi-cellular organization, and numerical research of differentiation of a model system and its relation with MEPP. It is suggested by this review article that the late stage of evolution is characterized by formation of society and external entropy production. A hypothesis on the general route of evolution is discussed from the birth to the present life which follows the MEPP. Some examples of life which happened to face poor thermodynamic condition are presented with thermodynamic discussion. It is observed through this review that MEPP is consistently useful for thermodynamic understanding of birth and evolution of life, subject to a thermodynamic condition far from equilibrium. Full article

Self-amplifying RNA is synthetic nucleic acid engineered to replicate within cells without generating viral particles. Derived from alphavirus genomes, saRNA retains the non-structural elements essential for replication while replacing the structural elements with an antigen of interest. By enabling efficient intracellular amplification, saRNA offers a promising alternative to conventional mRNA vaccines, enhancing antigen expression while requiring lower doses. However, this advantage comes with challenges. In this review, we highlight the key limitations of saRNA technology and explore potential strategies to overcome them. By identifying these challenges, we aim to provide insights that can guide the future design of saRNA-based therapeutics, extending their potential beyond vaccine applications. Full article

We hypothesize that predictable variations in environmental conditions caused by night/day cycles created opportunities and hazards that initiated information dynamics central to life’s origin. Increased daytime temperatures accelerated key chemical reactions but also caused the separation of double-stranded polynucleotides, leading to hydrolysis, particularly of single-stranded RNA. Daytime solar UV radiation promoted the synthesis of organic molecules but caused broad damage to protocell macromolecules. We hypothesize that inter-related simultaneous adaptations to these hazards produced molecular dynamics necessary to store and use information. Self-replicating RNA heritably reduced the hydrolysis of single strands after separation during warmer daytime periods by promoting sequences that formed hairpin loops, generating precursors to transfer RNA (tRNA), and initiating tRNA-directed evolutionary dynamics. Protocell survival during daytime promoted sequences in self-replicating RNA within protocells that formed RNA–peptide hybrids capable of scavenging UV-induced free radicals or catalyzing melanin synthesis from tyrosine. The RNA–peptide hybrids are precursors to ribosomes and the triplet codes for RNA-directed protein synthesis. The protective effects of melanin production persist as melanosomes are found throughout the tree of life. Similarly, adaptations mitigating UV damage led to the replacement of Na⁺ by K⁺ as the dominant mobile cytoplasmic cation to promote diel vertical migration and selected for homochirality. We conclude that information dynamics emerged in early life through adaptations to predictably fluctuating opportunities and hazards during night/day cycles, and its legacy remains observable in extant life. Full article