scholarly journals Direct detection of circulating microRNA-122 using dynamic chemical labelling with single molecule detection overcomes stability and isomiR challenges for biomarker qualification

2019 ◽  
Author(s):  
Barbara López-Longarela ◽  
Emma E. Morrison ◽  
John D. Tranter ◽  
Lianne Chahman-Vos ◽  
Jean-François Léonard ◽  
...  
Keyword(s):  
Liver Injury ◽  
Single Molecule ◽  
Limit Of Detection ◽  
Direct Detection ◽  
Circulating Microrna ◽  
Serum Samples ◽  
Drug Induced ◽  
Patients At Risk ◽  
Area Under Roc Curve ◽  
Fit For Purpose

AbstractCirculating microRNAs are biomarkers reported to be stable and translational across species. miR-122 (miR-122-5p) is a hepatocyte-specific microRNA biomarker for drug-induced liver injury (DILI). Our objective was to develop an extraction-free and amplification-free detection method for measuring miR-122 that has translational utility in context of DILI. We developed a single molecule dynamic chemical labelling (DCL) assay based on miR-122 hybridization to an abasic peptide nucleic acid probe that contained a reactive amine instead of a nucleotide at a specific position in the sequence. The single molecule DCL assay specifically measured miR-122 directly from 10 µL of serum or plasma without any extraction steps, with a fit-for-purpose limit of detection of 1.32 pM. In 192 human serum samples, DCL accurately identified patients at risk of DILI (area under ROC curve 0.98 (95%CI 0.96-1), P<0.0001). The miR-122 assay also quantified liver injury in rats and dogs. When DCL beads were added to serum, the miR-122 signal was stabilised (no loss of signal after 14 days at room temperature). By contrast, there was substantial degradation of miR-122 in the absence of beads (≈60% lost in 1 day). RNA sequencing demonstrated the presence of multiple miR-122 isomiRs with DILI that were at low concentration or not present in healthy patient serum. Sample degradation over time produced more isomiRs, particularly rapidly with DILI. PCR was inaccurate when analysing miR-122 isomiRs, whereas the DCL assay demonstrated accurate quantification. In summary, the DCL assay can accurately measure miR-122 directly from serum and plasma to diagnose liver injury in humans and other species, and can overcome important microRNA biomarker analytical and biological challenges.

scholarly journals Simultaneous Detection of Drug-Induced Liver Injury Protein and microRNA Biomarkers Using Dynamic Chemical Labelling on a Luminex MAGPIX System

Analytica ◽  
2021 ◽  
Vol 2 (4) ◽  
pp. 130-139
Author(s):  
Antonio Marín-Romero ◽  
Mavys Tabraue-Chávez ◽  
Bárbara López-Longarela ◽  
Mario A. Fara ◽  
Rosario M. Sánchez-Martín ◽  
...  
Keyword(s):  
Liver Injury ◽  
Simultaneous Detection ◽  
Single Step ◽  
Qualitative And Quantitative Analysis ◽  
Serum Samples ◽  
Drug Induced ◽  
Drug Induced Liver Injury ◽  
Qualitative And Quantitative ◽  
Post Marketing ◽  
Mechanistic Basis

Drug-induced liver injury (DILI) is a potentially fatal adverse event and a leading cause for pre- and post-marketing drug withdrawal. Several multinational DILI initiatives have now recommended a panel of protein and microRNA (miRNA) biomarkers that can detect early liver injury and inform about mechanistic basis. This manuscript describes the development of seqCOMBO, a unique combo-multiplexed assay which combines the dynamic chemical labelling approach and an antibody-dependant method on the Luminex MAGPIX system. SeqCOMBO enables a versatile multiplexing platform to perform qualitative and quantitative analysis of proteins and miRNAs in patient serum samples simultaneously. To the best of our knowledge, this is the first method to profile protein and miRNA biomarkers to diagnose DILI in a single-step assay.

scholarly journals Spectrofluorometric Analysis of Autofluorescing Components of Crude Serum from a Rat Liver Model of Ischemia and Reperfusion

Molecules ◽  
2020 ◽  
Vol 25 (6) ◽  
pp. 1327 ◽  
Author(s):  
Anna C. Croce ◽  
Andrea Ferrigno ◽  
Clarissa Berardo ◽  
Giovanni Bottiroli ◽  
Mariapia Vairetti ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Liver Injury ◽  
Direct Detection ◽  
Ischemia Reperfusion ◽  
Cost Effective ◽  
Serum Samples ◽  
Spectral Parameters ◽  
Pure Compounds ◽  
Spectral Profiles ◽  
Response To Stress

Autofluorescence (AF) of crude serum was investigated with reference to the potential of its intrinsic AF biomarkers for the noninvasive diagnosis of liver injury. Spectral parameters of pure compounds representing retinol (vitamin A) and fluorescing free fatty acids were characterized by spectrofluorometry, to assess spectral parameters for the subsequent AF analysis of serum, collected from rats undergoing liver ischemia/reperfusion (I/R). Differences in AF spectral profiles detected between control and I/R were due to the increase in the AF components representing fatty acids in I/R serum samples. No significant changes occurred for retinol levels, consistently with the literature reporting that constant retinol levels are commonly observed in the blood, except for malnutrition or chronic severe liver disease. Conversely, fatty acids, in particular arachidonic and linoleic acid and their derivatives, act as modulating agents in inflammation, representing both a protective and damaging response to stress stimuli. The biometabolic and pathophysiological meaning of serum components and the possibility of their direct detection by AF spectrofluorometry open up interesting perspectives for the development of AF serum analysis, as a direct, cost effective, supportive tool to assess liver injury and related systemic metabolic alterations, for applications in experimental biomedicine and foreseen translation to the clinics.

scholarly journals Dynamic single-molecule sensing via nanoparticle micromanipulation for rapid and ultrasensitive biomarker detection

2021 ◽  
Author(s):  
Qiang Zeng ◽  
Xiaoyan Zhou ◽  
Yuting Yang ◽  
Yi Sun ◽  
Jingan Wang ◽  
...  
Keyword(s):  
Single Molecule ◽  
Limit Of Detection ◽  
Direct Detection ◽  
Specific Binding ◽  
Single Molecules ◽  
Amyloid Β ◽  
Molecular Complexes ◽  
Biomarker Detection ◽  
Complex Samples ◽  
Practical Applications

Abstract The ability to measure many single molecules simultaneously in larger and complex samples is critical to the translation of single-molecule sensors for practical applications in biomarker detection. The challenges lie in the limits imposed by mass transportation and thermodynamics, resulting in long assay time and/or insufficient sensitivity. Here, we report an approach called Sensing Single Molecule under MicroManipulation (SSM3) to circumvent the above limits. In SSM3, the transportation rate of analyte molecules and the kinetics of molecular interaction are fine-tuned by the nanoparticle micromanipulation. The heterogeneous lifetime of molecular complexes is quantified to discriminate specific binding from nonspecific background noise. By the highly-specific digital counting of single molecules, we demonstrate 15-minute assays for direct detection of microRNAs and amyloid-β proteins via electrical or magnetic micromanipulation, with the limit of detection at the subfemtomolar level. The presented approach could inspire more practical applications of single molecule sensors.

Circulating microRNA profiles in human drug-induced liver injury

2014 ◽  
Vol 229 ◽  
pp. S94
Author(s):  
Bastiaan Vliegenthart ◽  
Jonathan Shaffer ◽  
Laura Peeters ◽  
David Webb ◽  
Nick Bateman ◽  
...  
Keyword(s):  
Liver Injury ◽  
Circulating Microrna ◽  
Drug Induced ◽  
Drug Induced Liver Injury

scholarly journals Polymerase-free measurement of microRNA-122 with single base specificity using single molecule arrays: Detection of drug-induced liver injury

PLoS ONE ◽  
2017 ◽  
Vol 12 (7) ◽  
pp. e0179669 ◽  
Author(s):  
David M. Rissin ◽  
Barbara López-Longarela ◽  
Salvatore Pernagallo ◽  
Hugh Ilyine ◽  
A. D. Bastiaan Vliegenthart ◽  
...  
Keyword(s):  
Liver Injury ◽  
Single Molecule ◽  
Drug Induced ◽  
Single Base ◽  
Drug Induced Liver Injury ◽  
Base Specificity

scholarly journals 645. Absence of Toxemia in Clostridioides difficile infection: Results from Ultrasensitive Toxin Assay of Serum

2020 ◽  
Vol 7 (Supplement_1) ◽  
pp. S381-S382
Author(s):  
Rebecca Sprague ◽  
Karolyne Warny ◽  
Nira Pollock ◽  
Kaitlyn Daugherty ◽  
Qianyun Lin ◽  
...  
Keyword(s):  
Clinical Outcomes ◽  
Single Molecule ◽  
Limit Of Detection ◽  
High Serum ◽  
Serum Samples ◽  
Systemic Complications ◽  
Clostridioides Difficile ◽  
Number Of Patients ◽  
Wide Range ◽  
Clostridioides Difficile Infection

Abstract Background Clostridioides difficile infection (CDI) is the major cause of hospital-acquired bacterial infectious diarrheacaused by Toxin A (TcdA) and Toxin B (Tcd B), secreted from pathogenic strains of C.difficle bacteria. This infection can vary greatly in symptom severity and presentation. In fulminant CDI, these toxins lead to systemic complications such as toxemia, however, identification of toxemia in CDI patients is extremely rare. We hypothesized that this rarity of detection may be due to low concentrations of circulating toxins in the blood, below the limit of detection of commercially available assays. Methods The previously developed Single Molecule Array (Simoa) assay, capable of detecting TcdA and TcdB in stool, was modified for the detection of toxins in serum and applied to a panel of serum samples from patients with confirmed CDI. Results Our cohort included 169 patients with a median age of 68 years (IQR 54-78), most with severe CDI and many with severe clinical outcomes attributed to CDI (Table 1). We found no detectable TcdA or TcdB in the serum of our patient cohort despite a wide range of toxin concentrations in paired stool (Figure 1). The detection of toxin may be limited by the interference of anti-toxin anti-bodies circulating in serum. When serum samples were spiked with TcdA and/or TcdBvarying amounts of IgA, IgG or IgM anti-toxin, high serum anti-toxin antibody concentrations were associated with loss of Simoa signal, suggesting substantial inhibition of toxin measurements. Table 1. Demographics, Baseline Laboratory Values, and Clinical Outcomes for the cohort Figure 1. Comparison of TcdA and TcdB concentrations, as measured by Simoa, in serum and stool. Clinical cutoffs are shown: stool, 20 pg/ml for TcdA and for TcdB; serum 15.0 pg/ml for TcdA and is 26.7 pg/ml for TcdB. Signals below these cut-offs are below backgrounds and so negative. Conclusion In contrast to earlier published findings which reported on the presence of detectable toxin in the serum of a small number of patients with CDI, our work did not support this observation. Although Simoa is highly sensitive for detection of picogram quantities of TcdA or TcdB it was unable to detect either toxin in serum during CDI. This result does not support the hypothesis that toxemia develops even in severe C. difficile infection. Disclosures Alice Banz, Ph.D, BioMerieux (Employee) Kevin W. Garey, PharmD, MS, FASHP, Merck & Co. (Grant/Research Support, Scientific Research Study Investigator) Carolyn D. Alonso, MD, FIDSA, Alnylam Pharmaceuticals (Employee)Merck (Research Grant or Support) Ciarán Kelly, MD, Artugen (Consultant)Facile Therapeutics (Consultant)Finch (Consultant)First Light Biosciences (Consultant)Matrivax (Consultant)Merck (Consultant)Vedanta (Consultant)

Leberschaden unter oraler Antikoagulation, Statin- und ACE-Hemmertherapie

Praxis ◽  
2010 ◽  
Vol 99 (21) ◽  
pp. 1259-1265 ◽  
Author(s):  
Bruggisser ◽  
Terraciano ◽  
Rätz Bravo ◽  
Haschke
Keyword(s):  
Liver Injury ◽  
Wird Eine ◽  
Drug Induced ◽  
Drug Induced Liver Injury

Ein 71-jähriger Patient stellt sich mit Epistaxis und ikterischen Skleren auf der Notfallstation vor. Der Patient steht unter einer Therapie mit Phenprocoumon, Atorvastatin und Perindopril. Anamnestisch besteht ein langjähriger Alkoholabusus. Laborchemisch werden massiv erhöhte Leberwerte (ALAT, Bilirubin) gesehen. Der INR ist unter oraler Antikoagulation und bei akuter Leberinsuffizienz >12. Die weiterführenden Abklärungen schliessen eine Virushepatitis und eine Autoimmunhepatitis aus. Nachdem eine Leberbiopsie durchgeführt werden kann, wird eine medikamentös-toxische Hepatitis, ausgelöst durch die Komedikation von Atorvastatin, Phenprocoumon und Perindopril bei durch Alkohol bereits vorgeschädigter Leber diagnostiziert. Epidemiologie, Pathophysiologie und Klink der medikamentös induzierten Leberschäden (drug induced liver injury, DILI), speziell von Coumarinen, Statinen und ACE-Hemmern werden im Anschluss an den Fallbericht diskutiert.

Drug-induced liver injury network (DILIN)

Hepatology ◽  
2004 ◽  
Vol 40 (4) ◽  
pp. 773-773 ◽  
Author(s):  
Jay H. Hoofnagle
Keyword(s):  
Liver Injury ◽  
Drug Induced ◽  
Drug Induced Liver Injury

Drug induced liver injury associated with Flupirtine in 6 cases

2011 ◽  
Vol 49 (08) ◽  
Author(s):  
C Agne ◽  
K Rifai ◽  
HH Kreipe ◽  
MP Manns ◽  
F Puls
Keyword(s):  
Liver Injury ◽  
Drug Induced ◽  
Drug Induced Liver Injury
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