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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 -
Journal of Medical Virology Jun 2024Ebolavirus disease (EVD) is an often-lethal disease caused by the genus Ebolavirus (EBOV). Although vaccines are being developed and recently used, outbreak control...
Ebolavirus disease (EVD) is an often-lethal disease caused by the genus Ebolavirus (EBOV). Although vaccines are being developed and recently used, outbreak control still relies on a combination of various factors, including rapid identification of EVD cases. This allows rapid patient isolation and control measure implementation. Ebolavirus diagnosis is performed in treatment centers or reference laboratories, which usually takes a few hours to days to confirm the outbreak or deliver a clear result. A fast and field-deployable molecular detection method, such as the isothermal amplification recombinase-aided amplification (RAA), could significantly reduce sample-to-result time. In this study, a RT-RAA assay was evaluated for EBOV detection. Various primer and probe combinations were screened; analytical sensitivity and cross-specificity were tested. A total of 40 archived samples from the 2014 to 2016 Ebola outbreak in West Africa were tested with both the reference method real-time RT-PCR and the established RT-RAA assay. The assay could detect down to 22.6 molecular copies per microliter. No other pathogens were detected with the Ebolavirus RT-RAA assay. Testing 40 samples yield clinical sensitivity and specificity of 100% each. This rapid isothermal RT-RAA assay can replace the previous RT-RPA and continue to offer rapid EBOV diagnostics.
Topics: Ebolavirus; Hemorrhagic Fever, Ebola; Nucleic Acid Amplification Techniques; Humans; Sensitivity and Specificity; Recombinases; Molecular Diagnostic Techniques; Africa, Western; Disease Outbreaks; RNA, Viral; DNA Primers
PubMed: 38874258
DOI: 10.1002/jmv.29744 -
Analytical Methods : Advancing Methods... Jun 2024This study proposes a strategy using a microfluidic ratiometric electrochemical aptasensor to detect PCB77 with excellent sensitivity and specificity. This sensing...
This study proposes a strategy using a microfluidic ratiometric electrochemical aptasensor to detect PCB77 with excellent sensitivity and specificity. This sensing platform combines a microfluidic chip, a wireless integrated circuit system for aptamer-based electrochemical detection, and a mobile phone control terminal for parameter configuration, identification, observation, and wireless data transfer. The sensing method utilizes a cDNA (MB-COOH-cDNA-SH) that is labelled with the redox probe Methylene Blue (MB) at the 5' end and has a thiol group at the 3' end. Additionally, it utilizes a single strand PCB aptamer that has been modified with ferrocenes at the 3' end (aptamer-Fc). Through gold-thiol binding, the labelled probe of MB-COOH-cDNA-SH was self-assembled onto the surface of an Au/NbCT/GO modified electrode. On exposure to aptamer-Fc, it will hybridize with MB-COOH-cDNA-SH to form a stable double-stranded structure on the electrode surface. When PCB77 is present, aptamer-Fc binds specifically to the target, enabling the double-stranded DNA to unwind. Such variation caused changes in the differential pulse voltammetry (DPV) peak currents of both MB and Fc. A substantial improvement is observed in the ratio between the two DPV peaks. Under the optimum experimental conditions, this assay has a response that covers the 0.0001 to 1000 ng mL PCB77 concentration range, and the detection limit is 1.56 × 10 ng mL. The integration of a ratiometric electrochemical aptasensor with designed microfluidic and integrated devices in this work is an innovative and promising approach that offers an efficient platform for on-site applications.
Topics: Aptamers, Nucleotide; Polychlorinated Biphenyls; Electrochemical Techniques; Biosensing Techniques; Limit of Detection; Microfluidic Analytical Techniques; Gold; Methylene Blue; Ferrous Compounds; Electrodes
PubMed: 38874006
DOI: 10.1039/d4ay00830h -
Molecular Pharmaceutics Jul 2024Oligonucleotides are short nucleic acids that serve as one of the most promising classes of drug modality. However, attempts to establish a physicochemical evaluation...
Toward the Establishment of a Harmonized Physicochemical Profiling Platform for Therapeutic Oligonucleotides: A Case Study for Aptamers Where the Higher-Order Structure Influences Physical Properties.
Oligonucleotides are short nucleic acids that serve as one of the most promising classes of drug modality. However, attempts to establish a physicochemical evaluation platform of oligonucleotides for acquiring a comprehensive view of their properties have been limited. As the chemical stability and the efficacy as well as the solution properties at a high concentration should be related to their higher-order structure and intra-/intermolecular interactions, their detailed understanding enables effective formulation development. Here, the higher-order structure and the thermodynamic stability of the thrombin-binding aptamer (TBA) and four modified TBAs, which have similar sequences but were expected to have different higher-order structures, were evaluated using ultraviolet spectroscopy (UV), circular dichroism (CD), differential scanning calorimetry (DSC), and nuclear magnetic resonance (NMR). Then, the relationship between the higher-order structure and the solution properties including solubility, viscosity, and stability was investigated. The impact of the higher-order structure on the antithrombin activity was also confirmed. The higher-order structure and intra-/intermolecular interactions of the oligonucleotides were affected by types of buffers because of different potassium concentrations, which are crucial for the formation of the G-quadruplex structure. Consequently, solution properties, such as solubility and viscosity, chemical stability, and antithrombin activity, were also influenced. Each instrumental analysis had a complemental role in investigating the higher-order structure of TBA and modified TBAs. The utility of each physicochemical characterization method during the preclinical developmental stages is also discussed.
Topics: Aptamers, Nucleotide; Circular Dichroism; Oligonucleotides; Calorimetry, Differential Scanning; Viscosity; Magnetic Resonance Spectroscopy; Solubility; Thermodynamics; G-Quadruplexes; Drug Stability; Humans
PubMed: 38872243
DOI: 10.1021/acs.molpharmaceut.4c00177 -
Journal of Medical Case Reports Jun 2024Homozygous mutations in the APOA5 gene constitute a rare cause of monogenic hypertriglyceridemia, or familial chylomicronemia syndrome (FCS). We searched PubMed and...
BACKGROUND
Homozygous mutations in the APOA5 gene constitute a rare cause of monogenic hypertriglyceridemia, or familial chylomicronemia syndrome (FCS). We searched PubMed and identified 16 cases of homozygous mutations in the APOA5 gene. Severe hypertriglyceridemia related to monogenic mutations in triglyceride-regulating genes can cause recurrent acute pancreatitis. Standard therapeutic approaches for managing this condition typically include dietary interventions, fibrates, and omega-3-fatty acids. A novel therapeutic approach, antisense oligonucleotide volanesorsen is approved for use in patients with FCS.
CASE PRESENTATION
We report a case of a 25-years old Afghani male presenting with acute pancreatitis due to severe hypertriglyceridemia up to 29.8 mmol/L caused by homozygosity in APOA5 (c.427delC, p.Arg143Alafs*57). A low-fat diet enriched with medium-chain TG (MCT) oil and fibrate therapy did not prevent recurrent relapses, and volanesorsen was initiated. Volanesorsen resulted in almost normalized triglyceride levels. No further relapses of acute pancreatitis occurred. Patient reported an improve life quality due to alleviated chronic abdominal pain and headaches.
CONCLUSIONS
Our case reports a rare yet potentially life-threatening condition-monogenic hypertriglyceridemia-induced acute pancreatitis. The implementation of the antisense drug volanesorsen resulted in improved triglyceride levels, alleviated symptoms, and enhanced the quality of life.
Topics: Humans; Male; Adult; Pancreatitis; Apolipoprotein A-V; Hypertriglyceridemia; Recurrence; Homozygote; Mutation; Oligonucleotides; Hyperlipoproteinemia Type I; Diet, Fat-Restricted; Triglycerides
PubMed: 38872171
DOI: 10.1186/s13256-024-04532-0 -
Biosensors & Bioelectronics Oct 2024A new di-recognition nitrogen-doped carbon dot nanosurface aptamer molecularly imprinted polymer (CD@MIPApt) nanocatalytic di-functional probe was prepared by microwave...
A new di-recognition nitrogen-doped carbon dot nanosurface aptamer molecularly imprinted polymer (CD@MIPApt) nanocatalytic di-functional probe was prepared by microwave irradiation. The probe was utilized nitrogen-doped silver carbon dots (CD) as the matrix, glyphosate (Gly) as the template molecule, α-methyl acrylate as the monomer, ethylene glycol dimethacrylate as the cross-linker, and aptamer as the biorecognition element. It could not only recognize Gly but also exhibits catalytic amplification function. It was found that CD@MIPApt catalyzed the redox reaction of polyethylene glycol 400 (PEG400)-AgNO to generate silver nanoparticles (AgNPs). The AgNPs indicator component exhibit the effects of surface-enhanced Raman scattering (SERS), resonance Rayleigh scattering (RRS) and surface plasmon resonance absorption (Abs). In the presence of Gly, it binds to the surface imprinted site of CD@MIPApt, to reduce AgNPs generation due to the catalytic activity of CD@MIPApt decreasing. Thus, the SERS/RRS/Abs signal values decreased linearly. The linear ranges of SERS/RRS/Abs assay were 0.1-2.5 nM, 0.25-2.75 nM and 0.5-5 nM respectively. The detection limits were 0.034 nM, 0.071 nM and 0.18 nM Gly.
Topics: Glycine; Glyphosate; Silver; Molecularly Imprinted Polymers; Aptamers, Nucleotide; Metal Nanoparticles; Spectrum Analysis, Raman; Limit of Detection; Biosensing Techniques; Surface Plasmon Resonance; Herbicides; Carbon
PubMed: 38870829
DOI: 10.1016/j.bios.2024.116487