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Scientific Reports May 2024Our preliminary investigation has identified the potential of serum fucosylated extracellular vesicles (EVs) miR-4732-5p in the early diagnosis of lung adenocarcinoma...
Our preliminary investigation has identified the potential of serum fucosylated extracellular vesicles (EVs) miR-4732-5p in the early diagnosis of lung adenocarcinoma (LUAD) by a fucose-captured strategy utilizing lentil lectin (LCA)-magnetic beads and subsequent screening of high throughput sequencing and validation of real-time quantitative polymerase chain reaction (RT-qPCR). Considering the relatively complicated procedure, expensive equipment, and stringent laboratory condition, we have constructed an electrochemical biosensor assay for the detection of miR-4732-5p. miR-4732-5p is extremely low in serum, down to the fM level, so it needs to be detected by highly sensitive electrochemical methods based on the Mg-dependent DNAzyme splitting nucleic acid lock (NAL) cycle and hybridization chain reaction (HCR) signal amplification. In this study, signal amplification is achieved through the dual amplification reactions using NAL cycle in combination with HCR. In addition, hybridized DNA strands bind to a large number of methylene blue (MB) molecules to enhance signaling. Based on the above strategy, we further enhance our signal amplification strategies to improve detection sensitivity and accuracy. The implementation of this assay proceeded as follows: initially, miR-4732-5p was combined with NAL, and then Mg-dependent DNAzyme splitted NAL to release auxiliary DNA (S1) strands, which were subsequently captured by the immobilized capture probe DNA (C1) strands on the electrode surface. Following this, abundant quantities of DNA1 (H1) and DNA2 (H2) tandems were generated by HCR, and S1 strands then hybridized with the H1 and H2 tandems through base complementary pairing. Finally, MB was bonded to the H1 and H2 tandems through π-π stacking interaction, leading to the generation of a signal current upon the detection of a potential capable of inducing a redox change of MB by the electrode. Furthermore, we evaluated the performance of our developed electrochemical biosensor assay. The results demonstrated that our assay is a reliable approach, characterized by its high sensitivity (with a detection limit of 2.6 × 10 M), excellent specificity, good accuracy, reproducibility, and stability. Additionally, it is cost-effective, requires simple operation, and is portable, making it suitable for the detection of serum fucosylated extracellular vesicles miR-4732-5p. Ultimately, this development has the potential to enhance the diagnostic efficiency for patients with early-stage LUAD.
Topics: Humans; MicroRNAs; Biosensing Techniques; Extracellular Vesicles; Adenocarcinoma of Lung; Lung Neoplasms; Electrochemical Techniques; Biomarkers, Tumor; Early Detection of Cancer; Female; Male; Middle Aged
PubMed: 38755208
DOI: 10.1038/s41598-024-61060-z -
International Journal of Molecular... Apr 2024Three new phenanthridine peptide derivatives (, , and ) were synthesized to explore their potential as spectrophotometric probes for DNA and RNA. UV/Vis and circular...
Three new phenanthridine peptide derivatives (, , and ) were synthesized to explore their potential as spectrophotometric probes for DNA and RNA. UV/Vis and circular dichroism (CD) spectra, mass spectroscopy, and computational analysis confirmed the presence of intramolecular interactions in all three compounds. Computational analysis revealed that compounds alternate between bent and open conformations, highlighting the latter's crucial influence on successful polynucleotide recognition. Substituting one glycine with lysine in two regioisomers (, ) resulted in stronger binding interactions with DNA and RNA than for a compound containing two glycines (), thus emphasizing the importance of lysine. The regioisomer with lysine closer to the phenanthridine ring () exhibited a dual and selective fluorimetric response with non-alternating AT and ATT polynucleotides and induction of triplex formation from the AT duplex. The best binding constant (K) with a value of 2.5 × 10 M was obtained for the interaction with AT and ATT polynucleotides. Furthermore, apart from distinguishing between different types of ds-DNA and ds-RNA, the same compound could recognize GC-rich DNA through distinct induced CD signals.
Topics: Phenanthridines; Lysine; Peptides; DNA; Circular Dichroism; RNA; Nucleic Acid Conformation
PubMed: 38732083
DOI: 10.3390/ijms25094866 -
Animals : An Open Access Journal From... Apr 2024Leptospirosis is a zoonosis of great importance for One Health. In this context, the Amazonian biome may harbor numerous hosts for spp. that contribute to the...
Leptospirosis is a zoonosis of great importance for One Health. In this context, the Amazonian biome may harbor numerous hosts for spp. that contribute to the maintenance of the pathogen in the environment. Some reptiles, such as chelonians, have been little studied in terms of their involvement with leptospires. The objective of this study was to detect spp. DNA in turtles kept in captivity in a region of the Brazilian Amazon. A total of 147 samples of blood ( = 40), cloacal fluid ( = 27), cloacal lavage ( = 40), and stomach ( = 40) were collected from 40 chelonians. After DNA extraction, the samples were subjected to amplification of a 331 base pair product of the 16S rRNA gene using the Lep1 and Lep2 primers. PCR products were Sanger sequenced, assembled, and subjected to online blast search and phylogenetic analysis. Of the animals tested, 40% (16/40, 95% confidence interval [CI]: 25-55) had at least one or two samples positive for spp. Considering the total number of samples collected, 12.93% (19/147) were positive, being blood clots (27.5%; 11/40), followed by cloacal washings (10%; 4/40), cloacal fluid (11.11%; 3/27) and gastric washings (2.5%; 1/40). Of these, 11 samples were sequenced and showed 99% to 100% identity with sequences, which was confirmed by phylogenetic analysis. This is the first study to detect pathogenic DNA in chelonians in a region of the Brazilian Amazon. It has been concluded that turtles in captivity have been exposed to pathogenic .
PubMed: 38731336
DOI: 10.3390/ani14091334 -
Nature Communications May 2024Widespread manganese-sensing transcriptional riboswitches effect the dependable gene regulation needed for bacterial manganese homeostasis in changing environments....
Widespread manganese-sensing transcriptional riboswitches effect the dependable gene regulation needed for bacterial manganese homeostasis in changing environments. Riboswitches - like most structured RNAs - are believed to fold co-transcriptionally, subject to both ligand binding and transcription events; yet how these processes are orchestrated for robust regulation is poorly understood. Through a combination of single-molecule and bulk approaches, we discover how a single Mn ion and the transcribing RNA polymerase (RNAP), paused immediately downstream by a DNA template sequence, are coordinated by the bridging switch helix P1.1 in the representative Lactococcus lactis riboswitch. This coordination achieves a heretofore-overlooked semi-docked global conformation of the nascent RNA, P1.1 base pair stabilization, transcription factor NusA ejection, and RNAP pause extension, thereby enforcing transcription readthrough. Our work demonstrates how a central, adaptable RNA helix functions analogous to a molecular fulcrum of a first-class lever system to integrate disparate signals for finely balanced gene expression control.
Topics: Riboswitch; Gene Expression Regulation, Bacterial; Nucleic Acid Conformation; Transcription, Genetic; Lactococcus lactis; DNA-Directed RNA Polymerases; RNA, Bacterial; Manganese; Transcription Factors; Bacterial Proteins; Single Molecule Imaging
PubMed: 38729929
DOI: 10.1038/s41467-024-48409-8 -
Journal of Bacteriology May 2024The DNA damage response of the multi-drug-resistant nosocomial pathogen possesses multiple features that distinguish it from the commonly used LexA repression system....
The DNA damage response of the multi-drug-resistant nosocomial pathogen possesses multiple features that distinguish it from the commonly used LexA repression system. These include the absence of LexA in this genus, the evolution of a UmuD polymerase manager into the UmuDAb repressor of error-prone polymerases, the use of a corepressor unique to (DdrR), and an unusually large UmuDAb binding site. We defined cis- and trans-acting factors required for UmuDAb DNA binding and gene repression, and tested whether DdrR directly enhances its DNA binding. We used DNA binding assays to characterize UmuDAb's binding to its proposed operator present upstream of the six co-repressed or genes. UmuDAb bound tightly and cooperatively to this site with ~10-fold less affinity than LexA. DdrR enhanced the binding of both native and dimerization-deficient UmuDAb forms, but only in greater than equimolar ratios relative to UmuDAb. UmuDAb mutants unable to dimerize or effect gene repression showed impaired DNA binding, and a strain expressing the G124D dimerization mutant could not repress transcription of the UmuDAb-DdrR regulon. Competition electrophoretic mobility shift assays conducted with mutated operator probes showed that, unlike typical SOS boxes, the UmuDAb operator possessed a five-base pair central core whose sequence was more crucial for binding than the flanking palindrome. The presence of only one of the two flanking arms of the palindrome was necessary for UmuDAb binding. Overall, the data supported a model of an operator with two UmuDAb binding sites. The distinct characteristics of UmuDAb and its regulated promoters differ from the typical LexA repression model, demonstrating a novel method of repression.IMPORTANCE is a gram-negative bacterium responsible for hospital-acquired infections. Its unique DNA damage response can activate multiple error-prone polymerase genes, allowing it to gain mutations that can increase its virulence and antibiotic resistance. The emergence of infectious strains carrying multiple antibiotic resistance genes, including carbapenem resistance, lends urgency to discovering and developing ways to combat infections resistant to treatment with known antibiotics. Deciphering how the regulators UmuDAb and DdrR repress the error-prone polymerases could lead to developing complementary treatments to halt this mechanism of generating resistance.
PubMed: 38727225
DOI: 10.1128/jb.00432-23 -
RSC Chemical Biology May 2024Modified nucleosides are integral to modern drug development, serving as crucial building blocks for creating safer, more potent, and more precisely targeted therapeutic... (Review)
Review
Modified nucleosides are integral to modern drug development, serving as crucial building blocks for creating safer, more potent, and more precisely targeted therapeutic interventions. Nucleobase modifications often confer antiviral and anti-cancer activity as monomers. When incorporated into nucleic acid oligomers, they increase stability against degradation by enzymes, enhancing the drugs' lifespan within the body. Moreover, modification strategies can mitigate potential toxic effects and reduce immunogenicity, making drugs safer and better tolerated. Particularly, 1-methylpseudouridine modification improved the efficacy of the mRNA coding for spike protein of COVID-19. This became a crucial step for developing COVID-19 vaccine applied during the 2020 pandemic. This makes 1-methylpseudouridine, and its "parent" analogue pseudouridine, potent nucleotide analogues for future RNA therapy and vaccine development. This review focuses on the structure and properties of pseudouridine and 1-methylpseudouridine. RNA has a greater structural versatility, different conformation, and chemical reactivity than DNA. Watson-Crick pairing is not strictly followed by RNA that has more unusual base pairs and base-triplets. This requires detailed structural studies and structure-activity relationship analyses for RNA, also when modifications are incorporated. Recent successes in this direction are revised in this review. We describe recent successes with using pseudouridine and 1-methylpseudouridine in mRNA drug candidates. We also highlight remaining challenges that need to be solved to develop new mRNA vaccines and therapies.
PubMed: 38725905
DOI: 10.1039/d4cb00022f -
Journal of Molecular Biology May 2024During the late stage of infection, alphabaculoviruses produce many occlusion bodies (OBs) in the nuclei of the insect host's cells through the hyperexpression of...
During the late stage of infection, alphabaculoviruses produce many occlusion bodies (OBs) in the nuclei of the insect host's cells through the hyperexpression of polyhedrin (POLH), a major OB component encoded by polh. The strong polh promoter has been used to develop a baculovirus expression vector system for recombinant protein expression in cultured insect cells and larvae. However, the relationship between POLH accumulation and the polh coding sequence remains largely unelucidated. This study aimed to assess the importance of polh codon usage and/or nucleotide sequences in POLH accumulation by generating a baculovirus Bombyx mori nucleopolyhedrovirus (BmNPV) expressing mutant polh (co-polh) optimized according to the codon preference of its host insect. Although the deduced amino acid sequence of CO-POLH was the same as that of wild-type POLH, POLH accumulation was significantly lower in cells infected with the co-polh mutant. This reduction was due to decreased polh mRNA levels rather than translational repression. Analysis of mutant viruses with chimeric polh revealed that a 30 base-pair (bp) 5' proximal polh coding region was necessary for maintaining high polh mRNA levels. Sequence comparison of wild-type polh and co-polh identified five nucleotide differences in this region, indicating that these nucleotides were critical for polh hyperexpression. Furthermore, luciferase reporter assays showed that the 30 bp 5' coding region was sufficient for maintaining the polh promoter-driven high level of polh mRNA. Thus, our whole-gene scanning by codon optimization identified important hidden nucleotides for polh hyperexpression in alphabaculoviruses.
PubMed: 38724003
DOI: 10.1016/j.jmb.2024.168595 -
PloS One 2024Recessive dystrophic epidermolysis bullosa is a rare genodermatosis caused by a mutation of the Col7a1 gene. The Col7a1 gene codes for collagen type VII protein, a major...
Creation and characterization of novel rat model for recessive dystrophic epidermolysis bullosa: Frameshift mutation of the Col7a1 gene leads to severe blistered phenotype.
Recessive dystrophic epidermolysis bullosa is a rare genodermatosis caused by a mutation of the Col7a1 gene. The Col7a1 gene codes for collagen type VII protein, a major component of anchoring fibrils. Mutations of the Col7a1 gene can cause aberrant collagen type VII formation, causing an associated lack or absence of anchoring fibrils. This presents clinically as chronic blistering, scarring, and fibrosis, often leading to the development of cutaneous squamous cell carcinoma. Patients also experience persistent pain and pruritus. Pain management and supportive bandaging remain the primary treatment options. The pathology of recessive dystrophic epidermolysis bullosa was first described in the 1980s, and there has since been a multitude of encouraging treatment options developed. However, in vivo research has been hindered by inadequate models of the disease. The various mouse models in existence possess longevity and surface area constraints, or do not adequately model a normal human disease state. In this paper, we describe a novel rat model of recessive dystrophic epidermolysis bullosa that offers an alternative to previous murine models. An 8-base pair deletion was induced in the Col7a1 gene of Lewis rats, which was subsequently found to cause a premature stop codon downstream. Homozygous mutants presented with a fragile and chronically blistered phenotype postnatally. Further histological analysis revealed subepidermal clefting and the absence of anchoring fibrils. The generation of this novel model offers researchers an easily maintained organism that possesses a larger surface area for experimental topical and transfused therapies to be tested, which may provide great utility in the future study of this debilitating disease.
Topics: Collagen Type VII; Animals; Epidermolysis Bullosa Dystrophica; Disease Models, Animal; Rats; Frameshift Mutation; Phenotype; Genes, Recessive; Rats, Inbred Lew; Blister; Skin; Male
PubMed: 38722855
DOI: 10.1371/journal.pone.0302991 -
Journal of Nanobiotechnology May 2024Tumor vaccines, a crucial immunotherapy, have gained growing interest because of their unique capability to initiate precise anti-tumor immune responses and establish...
Tumor vaccines, a crucial immunotherapy, have gained growing interest because of their unique capability to initiate precise anti-tumor immune responses and establish enduring immune memory. Injected tumor vaccines passively diffuse to the adjacent draining lymph nodes, where the residing antigen-presenting cells capture and present tumor antigens to T cells. This process represents the initial phase of the immune response to the tumor vaccines and constitutes a pivotal determinant of their effectiveness. Nevertheless, the granularity paradox, arising from the different requirements between the passive targeting delivery of tumor vaccines to lymph nodes and the uptake by antigen-presenting cells, diminishes the efficacy of lymph node-targeting tumor vaccines. This study addressed this challenge by employing a vaccine formulation with a tunable, controlled particle size. Manganese dioxide (MnO) nanoparticles were synthesized, loaded with ovalbumin (OVA), and modified with A or T DNA single strands to obtain MnO/OVA/A and MnO/OVA/T, respectively. Administering the vaccines sequentially, upon reaching the lymph nodes, the two vaccines converge and simultaneously aggregate into MnO/OVA/A-T particles through base pairing. This process enhances both vaccine uptake and antigen delivery. In vitro and in vivo studies demonstrated that, the combined vaccine, comprising MnO/OVA/A and MnO/OVA/T, exhibited robust immunization effects and remarkable anti-tumor efficacy in the melanoma animal models. The strategy of controlling tumor vaccine size and consequently improving tumor antigen presentation efficiency and vaccine efficacy via the DNA base-pairing principle, provides novel concepts for the development of efficient tumor vaccines.
Topics: Animals; Cancer Vaccines; Lymph Nodes; Mice; Ovalbumin; Mice, Inbred C57BL; Oxides; Nanoparticles; Manganese Compounds; Immunity, Cellular; Female; Cell Line, Tumor; DNA; Immunotherapy; Melanoma, Experimental; Particle Size; Antigens, Neoplasm
PubMed: 38720322
DOI: 10.1186/s12951-024-02498-1 -
Molecular Pharmacology May 2024The clinical use of RNA interference (RNAi) molecular mechanisms has introduced a novel, growing class of RNA therapeutics capable of treating diseases by controlling...
The clinical use of RNA interference (RNAi) molecular mechanisms has introduced a novel, growing class of RNA therapeutics capable of treating diseases by controlling target gene expression at the posttranscriptional level. With the newly approved nedosiran (Rivfloza{trade mark, serif}), there are now six RNAi-based therapeutics approved by the United States Food and Drug Administration (FDA). Interestingly, five of the six FDA-approved small interfering RNA (siRNA) therapeutics [patisiran (Onpattro{trade mark, serif}), lumasiran (Oxlumo{trade mark, serif}), inclisiran (Leqvio{trade mark, serif}), vutrisiran (Amvuttra{trade mark, serif}), and nedosiran] were revealed to act on the 3'-untranslated regions of target mRNAs, instead of coding sequences, thereby following the common mechanistic action of genome-derived microRNAs (miRNA). Furthermore, three of the FDA-approved siRNA therapeutics [patisiran, givosiran (Givlaari{trade mark, serif}), and nedosiran] induce target mRNA degradation or cleavage via near-complete rather than complete base-pair complementarity. These features along with previous findings confound the currently held characteristics to distinguish siRNAs and miRNAs or biosimilars, of which all converge in the RNAi regulatory pathway action. Herein, we discuss the RNAi mechanism of action and current criteria for distinguishing between miRNAs and siRNAs while summarizing the common and unique chemistry and molecular pharmacology of the six FDA-approved siRNA therapeutics. The term "RNAi" therapeutics, as used previously, provides a coherently unified nomenclature for broader RNAi forms as well as the growing number of therapeutic siRNAs and miRNAs or biosimilars that best aligns with current pharmacological nomenclature by mechanism of action. The common and unique chemistry and molecular pharmacology of six FDA-approved siRNA therapeutics are summarized, in which nedosiran is newly approved. We point out rather a surprisingly mechanistic action as miRNAs for five siRNA therapeutics and discuss the differences and similarities between siRNAs and miRNAs that supports using a general and unified term "RNAi" therapeutics to align with current drug nomenclature criteria in pharmacology based on mechanism of action and embraces broader forms and growing number of novel RNAi therapeutics.
PubMed: 38719476
DOI: 10.1124/molpharm.124.000895