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Methods in Molecular Biology (Clifton,... 2024Double-stranded RNA is produced by viruses during their replicative cycle. It is a potent immune modulator and indicator of viral infection within the body....
Double-stranded RNA is produced by viruses during their replicative cycle. It is a potent immune modulator and indicator of viral infection within the body. Extracellular vesicles (EVs) are lipid-bound particles released from cells homeostatically. Recent studies have shown that a commercially available dsRNA, poly inosinic: poly cytidylic acid (poly IC), can be detected within EVs. This finding opens the door for studying EVs as (1) carriers for dsRNA and (2) indicators of viral infection. To study dsRNA-containing EVs, we must have reliable methods for producing, isolating, and detecting them. This chapter uses U937, a pro-monocytic, human myeloid leukemia cell line, as the EV producer following poly IC treatment, and an immunoblot using an anti-dsRNA antibody (J2) for detection. Two methods for isolating the EVs and two methods for isolating the RNA from these EVs are described. Together, these methods effectively produce, isolate, and detect long dsRNA from EVs.
Topics: Humans; Extracellular Vesicles; Poly I-C; U937 Cells; RNA, Double-Stranded
PubMed: 38888782
DOI: 10.1007/978-1-0716-3890-3_16 -
Environmental Geochemistry and Health Jun 2024Gold nanoparticles (AuNPs) were extensively employed for in-situ detection sulfadiazine (SDZ) residues, yet current synthesis methods suffer from complex procedures,...
A highly sensitive and selective colorimetric aptasensor for detecting sulfadiazine in river waters based on gold nanoparticles synthesized from discarded Longan seed extract.
Gold nanoparticles (AuNPs) were extensively employed for in-situ detection sulfadiazine (SDZ) residues, yet current synthesis methods suffer from complex procedures, reagent pollution of the environment, and low particle quality. This study presents a novel synthesis method using discarded longan seed extract as a reducing agent to synthesized high-quality AuNPs, and then can be used for in-situ SDZ detection. Response surface methodology (RSM) was employed to optimize synthesis parameters, which resulted in five optimal combinations that enhanced the flexibility of synthesis. These AuNPs, ranging in size from 18.26 nm to 33.8 nm with zeta potentials from - 29.5 mV to - 14.3 mV, were successfully loaded with functional groups from longan seed extract. In the detection of SDZ, the colorimetric aptasensor demonstrated excellent sensitivity and selectivity over other antibiotics with a limit of detection and quantification at 70.98 ng·mL and 236.59 ng·mL in the concentration range of 200-800 ng·mL. Recoveries of spiked SDZ samples ranged from 97.90% to 106.7%, with RSD values below 9.25%. Meanwhile, the aptasensor exhibited exceptional diagnostic efficacy (AUC: 0.976) compared to UV absorption methods in the ROC evaluation. In conclusion, this study highlights the potential of using AuNPs synthesized from longan seed extract coupled with aptamer technology as a straightforward detection method for SDZ in river water, offering promising applications in environmental monitoring.
Topics: Gold; Metal Nanoparticles; Seeds; Colorimetry; Rivers; Water Pollutants, Chemical; Plant Extracts; Sulfadiazine; Aptamers, Nucleotide; Limit of Detection; Biosensing Techniques
PubMed: 38884845
DOI: 10.1007/s10653-024-02018-y -
Applied Microbiology and Biotechnology Jun 2024Mushroom poisoning contributes significantly to global foodborne diseases and related fatalities. Amanita mushrooms frequently cause such poisonings; however,...
Mushroom poisoning contributes significantly to global foodborne diseases and related fatalities. Amanita mushrooms frequently cause such poisonings; however, identifying these toxic species is challenging due to the unavailability of fresh and intact samples. It is often necessary to analyze residues, vomitus, or stomach extracts to obtain DNA sequences for the identification of species responsible for causing food poisoning. This usually proves challenging to obtain usable DNA sequences that can be analyzed using conventional molecular biology techniques. Therefore, this study aimed to develop a DNA mini-barcoding method for the identification of Amanita species. Following the evaluation and optimization of universal primers for DNA mini-barcoding in Amanita mushrooms, we found that the internal transcribed spacer (ITS) gene sequence primer ITS-a was the most suitable DNA barcode primer for identifying Amanita species. Forty-three Amanita samples were subsequently amplified and sequenced. The sequences obtained were analyzed for intra- and inter-species genetic distances, and a phylogenetic tree was constructed. The findings indicated that the designed primers had strong universality among the Amanita samples and could accurately identify the target gene fragment with a length of 290 bp. Notably, the DNA mini-barcode accurately identified the 43 Amanita samples, demonstrating high consistency with the conventional DNA barcode. Furthermore, it effectively identified DNA from digested samples. In summary, this DNA mini-barcode is a promising tool for detecting accidental ingestion of toxic Amanita mushrooms. It may be used as an optimal barcode for species identification and traceability in events of Amanita-induced mushroom poisoning. KEY POINTS: • Development of a DNA mini-barcoding method for Amanita species identification without fresh samples. • The ITS-a primer set was optimized for robust universality in Amanita samples. • The mini-barcode is suitable for screening toxic mushroom species in mushroom poisoning cases.
Topics: Mushroom Poisoning; Amanita; DNA Barcoding, Taxonomic; Phylogeny; DNA, Fungal; DNA Primers; DNA, Ribosomal Spacer; Sequence Analysis, DNA; Humans
PubMed: 38884656
DOI: 10.1007/s00253-024-13219-x -
Experimental Biology and Medicine... 2024Rheumatoid fibroblast-like synoviocytes (RFLS) have an important role in the inflammatory pathogenesis of rheumatoid arthritis (RA). Toll-like receptor 3 (TLR3) is...
Rheumatoid fibroblast-like synoviocytes (RFLS) have an important role in the inflammatory pathogenesis of rheumatoid arthritis (RA). Toll-like receptor 3 (TLR3) is upregulated in RFLS; its activation leads to the production of interferon-β (IFN-β), a type I IFN. IFN-stimulated gene 56 (ISG56) is induced by IFN and is involved in innate immune responses; however, its role in RA remains unknown. Therefore, the purpose of this study was to investigate the role of TLR3-induced ISG56 in human RFLS. RFLS were treated with polyinosinic-polycytidylic acid (poly I:C), which served as a TLR3 ligand. ISG56, melanoma differentiation-associated gene 5 (MDA5), and C-X-C motif chemokine ligand 10 (CXCL10) expression were measured using quantitative reverse transcription-polymerase chain reaction, western blotting, and enzyme-linked immunosorbent assay. Using immunohistochemistry, we found that ISG56 was expressed in synovial tissues of patients with RA and osteoarthritis. Under poly I:C treatment, ISG56 was upregulated in RFLS. In addition, we found that the type I IFN-neutralizing antibody mixture suppressed ISG56 expression. ISG56 knockdown decreased CXCL10 expression and MDA5 knockdown decreased ISG56 expression. In addition, we found that ISG56 was strongly expressed in the synovial cells of patients with RA. TLR3 signaling induced ISG56 expression in RFLS and type I IFN was involved in ISG56 expression. ISG56 was also found to be associated with CXCL10 expression, suggesting that ISG56 may be involved in TLR3/type I IFN/CXCL10 axis, and play a role in RA synovial inflammation.
Topics: Humans; Toll-Like Receptor 3; Arthritis, Rheumatoid; Poly I-C; Signal Transduction; Synoviocytes; Chemokine CXCL10; Interferon-Induced Helicase, IFIH1; Cells, Cultured; Synovial Membrane; Adaptor Proteins, Vesicular Transport; RNA-Binding Proteins; Adaptor Proteins, Signal Transducing; Apoptosis Regulatory Proteins
PubMed: 38881847
DOI: 10.3389/ebm.2024.10122 -
Journal of Microbiology and... Jun 2024The accurate and rapid detection of methicillin-resistant (MRSA) holds significant clinical importance. This work presents a new method for detecting...
The accurate and rapid detection of methicillin-resistant (MRSA) holds significant clinical importance. This work presents a new method for detecting methicillin-resistant () in clinical samples. The method uses an aptamer-based colorimetric assay that combines a recognizing probe to identify the target and split DNAzyme to amplify the signal, resulting in a highly sensitive and direct analysis of methicillin-resistance. The identification of the PBP2a protein on the membrane of in clinical samples leads to the allosterism of the recognizing probe, and thus provides a template for the proximity ligation of split DNAzyme. The proximity ligation of split DNAzyme forms an intact DNAzyme to identify the loop section in the L probe and generates a nicking site to release the loop sequence ("3" and "4" fragments). The "3" and "4" fragments forms an intact sequence to induce the catalytic hairpin assembly, exposing the G-rich section. The released the G-rich sequence of LR probe induces the formation of G-quadruplex-hemin DNAzyme as a colorimetric signal readout. The absorption intensity demonstrated a strong linear association with the logarithm of the concentration across a wide range of 5 orders of magnitude dynamic range under the optimized experimental parameters. The limit of detection was calculated to be 23 CFU/ml and the method showed high selectivity for MRSA.
Topics: DNA, Catalytic; Colorimetry; Methicillin-Resistant Staphylococcus aureus; Aptamers, Nucleotide; Staphylococcal Infections; Humans; Biosensing Techniques; Bacterial Proteins; Staphylococcus aureus; Sensitivity and Specificity; Methicillin Resistance; Penicillin-Binding Proteins
PubMed: 38881169
DOI: 10.4014/jmb.2404.04012 -
Parasites & Vectors Jun 2024The Anopheles dirus complex plays a significant role as a malaria vector in the Greater Mekong Subregion (GMS), with varying degrees of vector competence among species....
BACKGROUND
The Anopheles dirus complex plays a significant role as a malaria vector in the Greater Mekong Subregion (GMS), with varying degrees of vector competence among species. Accurate identification of sibling species in this complex is essential for understanding malaria transmission dynamics and deploying effective vector control measures. However, the original molecular identification assay, Dirus allele-specific polymerase chain reaction (AS-PCR), targeting the ITS2 region, has pronounced nonspecific amplifications leading to ambiguous results and misidentification of the sibling species. This study investigates the underlying causes of these inconsistencies and develops new primers to accurately identify species within the Anopheles dirus complex.
METHODS
The AS-PCR reaction and thermal cycling conditions were modified to improve specificity for An. dirus member species identification. In silico analyses with Benchling and Primer-BLAST were conducted to identify problematic primers and design a new set for Dirus complex species identification PCR (DiCSIP). DiCSIP was then validated with laboratory and field samples of the An. dirus complex.
RESULTS
Despite several optimizations by reducing primer concentration, decreasing thermal cycling time, and increasing annealing temperature, the Dirus AS-PCR continued to produce inaccurate identifications for Anopheles dirus, Anopheles scanloni, and Anopheles nemophilous. Subsequently, in silico analyses pinpointed problematic primers with high Guanine-Cytosine (GC) content and multiple off-target binding sites. Through a series of in silico analyses and laboratory validation, a new set of primers for Dirus complex species identification PCR (DiCSIP) has been developed. DiCSIP primers improve specificity, operational range, and sensitivity to identify five complex member species in the GMS accurately. Validation with laboratory and field An. dirus complex specimens demonstrated that DiCSIP could correctly identify all samples while the original Dirus AS-PCR misidentified An. dirus as other species when used with different thermocyclers.
CONCLUSIONS
The DiCSIP assay offers a significant improvement in An. dirus complex identification, addressing challenges in specificity and efficiency of the previous ITS2-based assay. This new primer set provides a valuable tool for accurate entomological surveys, supporting effective vector control strategies to reduce transmission and prevent malaria re-introducing in the GMS.
Topics: Anopheles; Animals; Polymerase Chain Reaction; DNA Primers; Mosquito Vectors; Malaria; Asia, Southeastern; Sensitivity and Specificity
PubMed: 38880909
DOI: 10.1186/s13071-024-06321-6 -
Analytica Chimica Acta Aug 2024The monitoring of concentration variation of the newly developed growth differentiation factor 15 (GDF15) biomarker in human serum is of great significance for...
BACKGROUND
The monitoring of concentration variation of the newly developed growth differentiation factor 15 (GDF15) biomarker in human serum is of great significance for diagnosing cardiovascular diseases. Current methods for the detection of the GDF15 protein mainly are based on antibody-assisted immunoassays, which encounter the limitations in terms of sensitivity, complexity and costs. The development of simple and sensitive biosensors for GDF15 can therefore facilitate the diagnosis of cardiovascular diseases.
RESULTS
A new bimetallic quasi-Cu/Co-MOF nanozyme with high catalytic performance for electrochemical reduction of HO is synthesized via simple one-step precipitation and low-temperature calcination method. Such nanozymes are further employed as amplification tags and coupled with cyclic entropy-driven DNA signal enhancement strategies to construct ultrasensitive aptamer-based biosensor for detecting GDF15 in human serums. GDF15 molecules associate with two aptamers and release the ssDNA trigger sequences via target-binding induced displacement reaction. These ssDNAs subsequently initiate cyclic DNA-fueled strand displacement and catalytic hairpin assembly (CHA) reaction cascades for confining many quasi-Cu/Co-MOF nanozymes on sensor electrode, which yield drastically amplified HO reduction current for detecting GDF15 down to 0.12 pg mL with a dynamic range of 0.5 pg mL to 20 ng mL. The electrochemical aptasensor also presents good reproducibility and selectivity and exhibits the capability to detect GDF15 in diluent serums.
SIGNIFICANCE
Our aptamer-based GDF15 protein electrochemical assay clearly outperforms current existing antibody-based methods and the quasi-Cu/Co-MOF nanozyme/entropy-driven cascaded signal amplification means can be used as a universal strategy for sensitive monitoring of different biomolecular markers for diverse applications.
Topics: Aptamers, Nucleotide; Growth Differentiation Factor 15; Copper; Humans; Metal-Organic Frameworks; Electrochemical Techniques; Cobalt; Biosensing Techniques; Entropy; Hydrogen Peroxide; Limit of Detection; Nucleic Acid Amplification Techniques; DNA
PubMed: 38879214
DOI: 10.1016/j.aca.2024.342816 -
Med (New York, N.Y.) Jun 2024Reducing the synthesis of apoC-III reduces fasting triglycerides in individuals lacking lipoprotein lipase activity. Recently, Stroes et al. published a phase 3 trial...
Reducing the synthesis of apoC-III reduces fasting triglycerides in individuals lacking lipoprotein lipase activity. Recently, Stroes et al. published a phase 3 trial on the effects of olezarsen, a third-generation antisense oligonucleotide that blocks apoC-III mRNA, on triglycerides and risk of acute pancreatitis.
Topics: Humans; Apolipoprotein C-III; Hyperlipoproteinemia Type I; Oligonucleotides; Triglycerides; Oligonucleotides, Antisense; Pancreatitis; Benzimidazoles
PubMed: 38878767
DOI: 10.1016/j.medj.2024.05.004 -
Biosensors & Bioelectronics Oct 2024Sensitive monitoring of luteinizing hormone (LH), a glycoprotein that regulates the synthesis of regulatory steroid hormones, can facilitate the diagnosis of various...
Highly catalytic sulfur-doped and bimetal-coordinated CoFe(CN)NO nanoparticles coupled with PER/HCR amplification cascades for sensitive electrochemical aptamer luteinizing hormone assay.
Sensitive monitoring of luteinizing hormone (LH), a glycoprotein that regulates the synthesis of regulatory steroid hormones, can facilitate the diagnosis of various reproductive diseases. In this work, a new and highly catalytic Sulfur-doped and bimetal-coordinated CoFe(CN)NO (denoted as S-CoFe(CN)NO) nanoparticles are synthesized. Such material is further used to construct high performance sensing interface and coupled with primer exchange reaction (PER) and hybridization chain reaction (HCR) amplification cascades for sensitive electrochemical aptamer-based LH assay. Target LH molecules bind aptamer sequences in DNA duplex probes to liberate ssDNA strands, which initiate subsequent PER/HCR amplification cascades for the capture of many ferrocene (Fc)-tagged DNAs on sensing interface. S-CoFe(CN)NO subsequently leads to catalytic oxidation of these Fc tags for yielding substantially magnified currents for realizing ultrasensitive assay of LH with the detection limit of 0.69 pM in range from 5 pM to 10 nM. Owing to the high specificity of aptamer, such sensor has high selectivity and can achieve low levels of LH assay in diluted serum samples. With the successful demonstration for detecting trace LH, such sensor can be easily extended as a universal aptamer-based electrochemical sensing method for monitoring various target analytes in the biomedical and biological fields.
Topics: Aptamers, Nucleotide; Biosensing Techniques; Humans; Electrochemical Techniques; Luteinizing Hormone; Limit of Detection; Catalysis; Sulfur; Metal Nanoparticles; Cobalt; Nucleic Acid Hybridization; Nanoparticles; Ferrous Compounds
PubMed: 38878701
DOI: 10.1016/j.bios.2024.116473 -
Mikrochimica Acta Jun 2024A fluorescence biosensor for determination of aflatoxin B (AFB) based on polydiacetylene (PDA) liposomes and exonuclease III (EXO III)-assisted recycling amplification...
A fluorescence biosensor for determination of aflatoxin B (AFB) based on polydiacetylene (PDA) liposomes and exonuclease III (EXO III)-assisted recycling amplification was developed. The AFB aptamer partially hybridizes with complementary DNA (cDNA), which is released upon recognition of AFB by the aptamer. Subsequently, the cDNA hybridizes with hairpin H to form double-stranded DNA that undergoes digestion by EXO III, resulting in the cyclic release of cDNA and generation of capture DNA for further reaction. The capture DNA then hybridizes with probe modified on PDA liposomes, leading to aggregation of liposomes and subsequent fluorescence production. This strategy exhibited a limit of detection of 0.18 ng/mL within the linear range 1-100 ng/mL with a determination coefficient > 0.99. The recovery ranged from 92.81 to 106.45%, with relative standard deviations (RSD) between 1.73 and 4.26%, for corn, brown rice, peanut butter, and wheat samples. The stability, accuracy, and specificity of the method demonstrated the applicability for real sample analysis.
Topics: Polyacetylene Polymer; Liposomes; Exodeoxyribonucleases; Biosensing Techniques; Aflatoxin B1; Limit of Detection; Aptamers, Nucleotide; Nucleic Acid Amplification Techniques; Polyynes; Spectrometry, Fluorescence; Zea mays; Triticum; Oryza; Polymers; Food Contamination
PubMed: 38877314
DOI: 10.1007/s00604-024-06482-2