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1.
J Chromatogr B Analyt Technol Biomed Life Sci ; 1194: 123184, 2022 Apr 01.
Article in English | MEDLINE | ID: covidwho-1701949

ABSTRACT

INTRODUCTION: Ferritin is the major iron-storage glycoprotein found in all tissues. Ferritin glycosylation can be assessed by the differential affinities of ferritin glycoforms for Concanavalin A (ConA), a lectin. The fraction of serum ferritin bound to ConA is called "glycosylated ferritin" (GF). Low GF reflects macrophagic activation and is an essential biomarker used in adult-onset Still's disease (AOSD), macrophage activation syndrome (MAS) and Gaucher disease diagnosis and therapeutic management. To date, no complete assay description and method validation according to the ISO 15189 standard has been published. This study aimed to describe and validate our method used for GF measurement and describe GF values observed in patients. MATERIALS AND METHODS: Ferritin glycoforms were separated based on their affinities for ConA using commercially available TRIS-barbital buffer, Sepharose and ConA/Sepharose 4B gels. Ferritin concentrations were measured on the Siemens Dimension Vista 1500®. We analysed 16,843 GF values obtained between 2000 and 2021 from our database of patients. RESULTS: Optimal separation of ferritin glycoforms was obtained by 15-min incubation of serum with ConA/Sepharose at pH 8. The optimized volume were 0.4 mL for total serum ferritin (TSF) 30-1000 µg/L and 0.5 mL for TSF 1000-2500 µg/L. Serum with higher TSF should be pre-diluted in the TRIS-barbital buffer. Reproducibility of ferritin measurement in the TRIS-barbital buffer matrix was excellent (intra-assay CV < 1%; inter-assay CV < 4%). Reproducibility of GF assay was good (intra-assay CV < 10% for low and high ferritin samples, respectively; and inter-assay CV < 10%). Inter-operator variability was 21.6% for GF < 20%. Ferritin was stable for up to 3 days in the TRIS-barbital buffer. An inter-laboratory exchange program conducted with another French hospital showed good agreement between results. In our database, <20% GF levels were scarce, compatible with the low prevalence of Still's disease, MAS, and Gaucher disease. The 95% confidence interval for GF was [26-58]%, lower than values described in the literature for healthy individuals. CONCLUSION: Thanks to good performances, this technique can become readily available for laboratories servicing patients with AOSD, MAS (including severe COVID-19 patients) and Gaucher disease patients.


Subject(s)
Chemistry Techniques, Analytical/methods , Concanavalin A/metabolism , Ferritins/blood , Macrophage Activation Syndrome/blood , Still's Disease, Adult-Onset/blood , Biomarkers/blood , Biomarkers/metabolism , Ferritins/metabolism , Gaucher Disease/blood , Gaucher Disease/metabolism , Humans , Macrophage Activation Syndrome/metabolism , Protein Binding , Still's Disease, Adult-Onset/metabolism
2.
Mikrochim Acta ; 189(1): 34, 2021 12 23.
Article in English | MEDLINE | ID: covidwho-1633639

ABSTRACT

DNA is recognized as a powerful biomarker for clinical diagnostics because its specific sequences are closely related to the cause and development of diseases. However, achieving rapid, low-cost, and sensitive detection of short-length target DNA still remains a considerable challenge. Herein, we successfully combine the catalytic hairpin assembly (CHA) technique with capillary action to develop a new and cost-effective method, a target DNA- and pH-responsive DNA hydrogel-based capillary assay, for the naked eye detection of 24 nt short single-stranded target DNA. Upon contact of target DNA, three individual hairpin DNAs hybridize with each other to sufficiently amplify Y-shaped DNA nanostructures (Y-DNA) until they are completely consumed via CHA cycling reactions. Each arm of the resultant Y-DNA contains sticky ends with i-motif DNA structure-forming sequences that can be self-assembled in an acidic environment (pH 5.0) to form target DNA- and pH-responsive DNA hydrogels by means of i-motif DNA-driven crosslinking. When inserting a capillary tube in the resultant solution, the liquid level inside clearly reduces due to the decrease in capillary force induced by the gels. In this way, the developed assay demonstrates sensitive and quantitative detection, with a detection limit of approximately 10 pM of 24 nt short complementary DNA (cDNA) targeting SARS-CoV-2 RNA genes at room temperature within 1 h. The assay is further shown to successfully detect target cDNA in serum, and it is also applied to detect several types of target sequences. Requiring no analytic equipment, precise temperature control, or enzymatic reactions, the developed DNA hydrogel-based capillary assay has potential as a promising naked eye detection platform for target DNA in resource-limited clinical settings.


Subject(s)
Chemistry Techniques, Analytical/methods , DNA, Catalytic/chemistry , DNA, Complementary/analysis , Hydrogels/chemistry , RNA, Viral/genetics , SARS-CoV-2/chemistry , Capillary Action , Chemistry Techniques, Analytical/instrumentation , DNA, Catalytic/genetics , DNA, Complementary/genetics , Hydrogen-Ion Concentration , Inverted Repeat Sequences , Limit of Detection , Nucleic Acid Amplification Techniques , Nucleic Acid Hybridization
5.
Chembiochem ; 22(7): 1176-1189, 2021 04 06.
Article in English | MEDLINE | ID: covidwho-967966

ABSTRACT

The recent pandemic of the novel coronavirus disease 2019 (COVID-19) has caused huge worldwide disruption due to the lack of available testing locations and equipment. The use of optical techniques for viral detection has flourished in the past 15 years, providing more reliable, inexpensive, and accurate detection methods. In the current minireview, optical phenomena including fluorescence, surface plasmons, surface-enhanced Raman scattering (SERS), and colorimetry are discussed in the context of detecting virus pathogens. The sensitivity of a viral detection method can be dramatically improved by using materials that exhibit surface plasmons or SERS, but often this requires advanced instrumentation for detection. Although fluorescence and colorimetry lack high sensitivity, they show promise as point-of-care diagnostics because of their relatively less complicated instrumentation, ease of use, lower costs, and the fact that they do not require nucleic acid amplification. The advantages and disadvantages of each optical detection method are presented, and prospects for applying optical biosensors in COVID-19 detection are discussed.


Subject(s)
Biosensing Techniques/methods , COVID-19/diagnosis , Chemistry Techniques, Analytical/methods , SARS-CoV-2/isolation & purification , Animals , Humans
6.
Anal Methods ; 13(1): 34-55, 2021 01 07.
Article in English | MEDLINE | ID: covidwho-962238

ABSTRACT

RNA-based viruses likely make up the highest pandemic threat among all known pathogens in about the last 100 years, since the Spanish Flu of 1918 with 50 M deaths up to COVID-19. Nowadays, an efficient and affordable testing strategy for such viruses have become the paramount target for the fields of virology and bioanalytical chemistry. The detection of the viruses (influenza, hepatitis, HIV, Zika, SARS, Ebola, SARS-CoV-2, etc.) and human antibodies to these viruses is described and tabulated in terms of the reported methods of detection, time to results, accuracy and specificity, if they are reported. The review is focused, but not limited to publications in the last decade. Finally, the limits of detection for each representative publication are tabulated by detection methods and discussed. These methods include PCR, lateral flow immunoassays, LAMP-based methods, ELISA, electrochemical methods (e.g., amperometry, voltammetry), fluorescence spectroscopy, AFM, SPR and SERS spectroscopy, silver staining and CRISPR-Cas based methods, bio-barcode detection, and resonance light scattering. The review is likely to be interesting for various scientists, and particularly helpful with information for establishing interdisciplinary research.


Subject(s)
Chemistry Techniques, Analytical/methods , Immunoassay/methods , RNA Viruses/isolation & purification , Antibodies, Viral/analysis , Data Accuracy , Humans , Limit of Detection , SARS-CoV-2/isolation & purification , Sensitivity and Specificity
7.
Pharmacol Rev ; 72(4): 862-898, 2020 10.
Article in English | MEDLINE | ID: covidwho-767668

ABSTRACT

RNA-based therapies, including RNA molecules as drugs and RNA-targeted small molecules, offer unique opportunities to expand the range of therapeutic targets. Various forms of RNAs may be used to selectively act on proteins, transcripts, and genes that cannot be targeted by conventional small molecules or proteins. Although development of RNA drugs faces unparalleled challenges, many strategies have been developed to improve RNA metabolic stability and intracellular delivery. A number of RNA drugs have been approved for medical use, including aptamers (e.g., pegaptanib) that mechanistically act on protein target and small interfering RNAs (e.g., patisiran and givosiran) and antisense oligonucleotides (e.g., inotersen and golodirsen) that directly interfere with RNA targets. Furthermore, guide RNAs are essential components of novel gene editing modalities, and mRNA therapeutics are under development for protein replacement therapy or vaccination, including those against unprecedented severe acute respiratory syndrome coronavirus pandemic. Moreover, functional RNAs or RNA motifs are highly structured to form binding pockets or clefts that are accessible by small molecules. Many natural, semisynthetic, or synthetic antibiotics (e.g., aminoglycosides, tetracyclines, macrolides, oxazolidinones, and phenicols) can directly bind to ribosomal RNAs to achieve the inhibition of bacterial infections. Therefore, there is growing interest in developing RNA-targeted small-molecule drugs amenable to oral administration, and some (e.g., risdiplam and branaplam) have entered clinical trials. Here, we review the pharmacology of novel RNA drugs and RNA-targeted small-molecule medications, with a focus on recent progresses and strategies. Challenges in the development of novel druggable RNA entities and identification of viable RNA targets and selective small-molecule binders are discussed. SIGNIFICANCE STATEMENT: With the understanding of RNA functions and critical roles in diseases, as well as the development of RNA-related technologies, there is growing interest in developing novel RNA-based therapeutics. This comprehensive review presents pharmacology of both RNA drugs and RNA-targeted small-molecule medications, focusing on novel mechanisms of action, the most recent progress, and existing challenges.


Subject(s)
RNA/drug effects , RNA/pharmacology , Aptamers, Nucleotide/pharmacology , Aptamers, Nucleotide/therapeutic use , Betacoronavirus , COVID-19 , Chemistry Techniques, Analytical/methods , Chemistry Techniques, Analytical/standards , Clustered Regularly Interspaced Short Palindromic Repeats , Coronavirus Infections/drug therapy , Drug Delivery Systems/methods , Drug Development/organization & administration , Drug Discovery , Humans , MicroRNAs/pharmacology , MicroRNAs/therapeutic use , Oligonucleotides, Antisense/pharmacology , Oligonucleotides, Antisense/therapeutic use , Pandemics , Pneumonia, Viral/drug therapy , RNA/adverse effects , RNA, Antisense/pharmacology , RNA, Antisense/therapeutic use , RNA, Guide/pharmacology , RNA, Guide/therapeutic use , RNA, Messenger/drug effects , RNA, Messenger/pharmacology , RNA, Ribosomal/drug effects , RNA, Ribosomal/pharmacology , RNA, Small Interfering/pharmacology , RNA, Small Interfering/therapeutic use , RNA, Viral/drug effects , Ribonucleases/metabolism , Riboswitch/drug effects , SARS-CoV-2
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