Advantages of highly sensitive NGS based test to detect low levels of mtDNA mutations and large deletions

Mitochondrion ◽  
2015 ◽  
Vol 24 ◽  
pp. S44-S45
Author(s):  
Hui Yu ◽  
Jing Wang ◽  
Eric Schmitt ◽  
Meagan Palculict ◽  
Michelle Miller ◽  
...  
2021 ◽  
Vol 33 (1) ◽  
Author(s):  
Johannes Haedrich ◽  
Claudia Stumpf ◽  
Michael S. Denison

Abstract Background Low maximum and action levels set by the European Union for polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs) and dioxin-like polychlorinated biphenyls (DL-PCBs) in pig meat (pork) have led to a demand for reliable and cost-effective bioanalytical screening methods implemented upstream of gas chromatography/high-resolution mass spectrometry confirmatory technology, that can detect low levels of contamination in EU-regulated foods with quick turn-around times. Results Based on the Chemically Activated LUciferase gene eXpression (CALUX) bioassay, extraction and clean-up steps were optimized for recovery and reproducibility within working ranges significantly lower than in current bioassays. A highly sensitive “3rd generation” recombinant rat hepatoma cell line (H4L7.5c2) containing 20 dioxin responsive elements was exposed to pork sample extracts, and their PCDD/Fs and DL-PCBs levels were evaluated by measuring luciferase activity. The method was validated according to the provisions of Commission Regulation (EU) 2017/644 of 5 April 2017 with spiking experiments performed selectively for PCDD/Fs and DL-PCBs and individual calibration for PCDD/Fs, DL-PCBs and the calculated sum of PCDD/Fs and DL-PCBs. The resulting performance parameters met all legal specifications as confirmed by re-calibration using authentic samples. Cut-off concentrations for assessing compliance with low maximum levels and action levels set for PCDD/Fs and DL-PCBs within a range of 0.50–1.25 pg WHO-TEQ/g fat were derived, ensuring low rates of false-compliant results (ß-error < 1%) and keeping the rate of false-noncompliant results well under control (α-error < 12%). Conclusions We present a fast and efficient bioanalytical routine method validated according to the European Union’s legal requirements on the basis of authentic samples, allowing the analyst to reliably identify pork samples and any other EU-regulated foods of animal origin suspected to be noncompliant with a high level of performance and turn-around times of 52 h. This was facilitated in particular by a quick and efficient extraction step followed by selective clean-up, use of a highly sensitive “3rd generation” H4L7.5c2 recombinant rat hepatoma cell CALUX bioassay, and optimized assay performance with improved calibrator precision and reduced lack-of-fit errors. New restrictions are proposed for the calibrator bias and the unspecific background contribution to reportable results. The procedure can utilize comparably small sample amounts and allows an annual throughput of 840–1000 samples per lab technician. The described bioanalytical method contributes to the European Commission's objective of generating accurate and reproducible analytical results according to Commission Regulation (EU) 2017/644 across the European Union.


2020 ◽  
Vol 77 (4) ◽  
pp. 1417-1430
Author(s):  
Gallen Triana-Baltzer ◽  
Kristof Van Kolen ◽  
Clara Theunis ◽  
Setareh Moughadam ◽  
Randy Slemmon ◽  
...  

Background: Early and accurate detection and staging is critical to managing Alzheimer’s disease (AD) and supporting clinical trials. Cerebrospinal fluid (CSF) biomarkers for amyloid-β peptides, tau species, and various neurodegenerative and inflammatory analytes are leading the way in this regard, yet there is room for improved sensitivity and specificity. In particular tau is known to be present in many different fragments, conformations, and post-translationally modified forms. While the exact tau species that might best reflect AD pathology is unknown, a growing body of evidence suggests that forms with high levels of phosphorylation in the mid-region may be especially enriched in AD. Objective: Develop an assay for measuring p217tau in CSF. Methods: Here we describe the development and validation of a novel sELISA for measuring CSF tau species containing phosphorylation at threonines 212 & 217, aka p217 + tau, using the PT3 antibody. Results: While the analyte is present at extremely low levels the assay is sufficiently sensitive and specific to quantitate p217 + tau with excellent precision, accuracy, and dilution linearity, allowing good differentiation between diagnostic subgroups. The p217 + tau measurements appear to track AD pathology better than the commonly used p181tau epitope, suggesting superior diagnostic and staging performance. Finally, the assay can also be configured to differentiate antibody-bound versus antibody-free tau, and therefore can be used to measure target engagement by p217 + tau-targeting immunotherapeutics. Conclusion: The assay for measuring p217 + tau described here is highly sensitive, accurate, precise, dilution linear, and shows good potential for identifying and staging AD.


2014 ◽  
Vol 220 ◽  
pp. 101-111 ◽  
Author(s):  
Sharma Rao Balakrishnan ◽  
U. Hashim ◽  
G.R. Letchumanan ◽  
M. Kashif ◽  
A.R. Ruslinda ◽  
...  

1996 ◽  
Vol 74 (2) ◽  
pp. 187-196 ◽  
Author(s):  
Jian-Ping Jin ◽  
Michael P. Walsh ◽  
Mary E. Resek ◽  
Gail A. McMartin

Calponin is a thin filament associated protein found in smooth muscle as a potential modulator of contraction. Five mouse monoclonal antibodies (mAbs CP1, CP3, CP4, CP7, and CP8) were prepared against chicken gizzard α-calponin. The CP1 epitopic structure is conserved in smooth muscles across vertebrate phyla and is highly sensitive to CNBr cleavage in contrast with the chicken-specific CP4 and the avian–mammalian-specific CP8 epitopes that are resistant to CNBr fragmentation. Using this panel of mAbs against multiple epitopes, only α-calponin was detected in adult chicken smooth muscles and throughout development of the gizzard. Western blotting showed that the calponin content varied among different smooth muscle tissues and correlated with that of h-caldesmon. In contrast with the constitutive expression of calponin in phasic smooth muscle of the digestive tract, very low levels of calponin were detected in adult avian tracheas and no calponin expression was detected in embryonic and young chick tracheas. These results provide information on the structural conservation of calponins and suggest a relationship between calponin expression and smooth muscle functional states.Key words: smooth muscle calponin, caldesmon, expression, development, chicken trachea.


1979 ◽  
Vol 57 (5) ◽  
pp. 1003-1009 ◽  
Author(s):  
D. C. Mahon ◽  
K. K. Nair ◽  
P. C. Oloffs

The effects of low-level, chronic feeding of the insecticide chlordane on the cytochemistry of the hepatocyte nuclei of male nits, with or without liver cirrhosis, were determined. Chlordane was given orally at 0.1 mg kg−1 day−1. Cirrhosis was induced by exposure of the animals to carbon tetrachloride (CCl4) vapours twice weekly for 10 weeks. The animals were killed after 20 weeks and the Feulgen-stained hepatocyte nuclei examined microspectrophotometrically for nuclear size, DNA content, and the degree of chromatin condensation. The five experimental groups were: (A) control; (B) chlordane only for 20 weeks; (C) cirrhosis only (CCl4 weeks 0–10); (D) chlordane (weeks 0–20) plus cirrhosis; (E) cirrhosis (CCl4, weeks 0–10) followed by chlordane (weeks 10–20). All treatments significantly (p < 0.01) increased nuclear size. Although DNA contents were not affected by chlordane alone, in cirrhotic animals treatment with chlordane caused significantly higher DNA contents than that in cirrhotics not receiving chlordane. This suggests that hepatic dysfunction increases the liver's sensitivity to respond to chlordane. Chromatin condensation was decreased by chlordane in both healthy and cirrhotic animals; animals with cirrhosis, but not receiving chlordane, had more condensed chromatin than the controls.The results of this study show that computer analysis of images of Feulgen-stained nuclei is a highly sensitive technique to detect the effects of chemicals on the degree of chromatin condensation even at dosage levels below the no-effect levels previously reported.


2016 ◽  
Author(s):  
Héloïse Rouzé ◽  
Gaël J Lecellier ◽  
Denis Saulnier ◽  
Serge Planes ◽  
Yannick Gueguen ◽  
...  

The adaptative bleaching hypothesis (ABH) states that depending on the symbiotic flexibility of coral hosts (i.e., the ability of corals to “switch” or “shuffle” their algal symbionts), coral bleaching can lead to a change in the composition of their associated Symbiodinium community, and, thus, contribute to the coral’s overall survival. In order to determine the flexibility of corals, molecular tools are required to provide accurate species delineations, and to detect low levels of coral-associated Symbiodinium. Here, we used highly sensitive quantitative (real-time) PCR (qPCR) technology to analyse five common coral species from Moorea (French Polynesia), previously screened using only traditional conventional molecular methods, to assess the presence of low-abundance (background) Symbiodinium. Similar to other studies, each coral species exhibited a strong specificity to a particular clade, irrespective of the environment. In addition, however, each of the five species harboured at least one additional Symbiodinium clade, among clades A-D, at background levels. Unexpectedly, and for the first time in French Polynesia, clade B was detected as a coral symbiont. These results increase the number of known coral-Symbiodinium associations from corals found in French Polynesia, and likely indicate an underestimation of the ability of the corals in this region to associate with and/or “shuffle” different Symbiodinium clades. Altogether our data suggest that corals from French Polynesia may manage a trade-off between optimizing symbioses with a specific Symbiodinium clade(s), and maintaining associations with particular background clades that may play a role in the ability of corals to respond to environmental change.


Author(s):  
Robert L. Shuler ◽  
Theodore Koukouvitis ◽  
Dyske Suematsu

Objectives: We study partial unlock or reopening interaction with seasonal effects in a managed epidemic to quantify overshoot effects on small and large unlock steps and discover robust strategies for reducing overshoot. Methods: We simulate partial unlock of social distancing for epidemics over a range of replication factor, immunity duration and seasonality factor for strategies targeting immunity thresholds using overshoot optimization. Results: Seasonality change must be taken into account as one of the steps in an easing sequence, and a two step unlock, including seasonal effects, minimizes overshoot and deaths. It may cause undershoot, which causes rebounds and assists survival of the pathogen. Conclusions: Partial easing levels, even low levels for economic relief while waiting on a vaccine, have population immunity thresholds based on the reduced replication rates and may experience overshoot as well. We further find a two step strategy remains highly sensitive to variations in case ratio, replication factor, seasonality and timing. We demonstrate a three or more step strategy is more robust, and conclude that the best possible approach minimizes deaths under a range of likely actual conditions which include public response.


Blood ◽  
2000 ◽  
Vol 95 (4) ◽  
pp. 1336-1341 ◽  
Author(s):  
Stefano Duga ◽  
Rosanna Asselta ◽  
Elena Santagostino ◽  
Sirous Zeinali ◽  
Tatjana Simonic ◽  
...  

Congenital afibrinogenemia is a rare autosomal recessive disorder characterized by bleeding that varies from mild to severe and by complete absence or extremely low levels of plasma and platelet fibrinogen. Although several mutations in the fibrinogen genes associated with dysfibrinogenemia and hypofibrinogenemia have been described, the genetic defects of congenital afibrinogenemia are largely unknown, except for a recently reported 11-kb deletion of the fibrinogen A-chain gene. Nevertheless, mutation mechanisms other than the deletion of a fibrinogen gene are likely to exist because patients with afibrinogenemia showing no gross alteration within the fibrinogen cluster have been reported. We tested this hypothesis by studying the affected members of two families, one Italian and one Iranian, who had no evidence of large deletions in the fibrinogen genes. Sequencing of the fibrinogen genes in the 2 probands detected 2 different homozygous missense mutations in exons 7 and 8 of the Bβ-chain gene, leading to amino acid substitutions Leu353Arg and Gly400Asp, respectively. Transient transfection experiments with plasmids expressing wild-type and mutant fibrinogens demonstrated that the presence of either mutation was sufficient to abolish fibrinogen secretion. These findings demonstrated that missense mutations in the Bβ fibrinogen gene could cause congenital afibrinogenemia by impairing fibrinogen secretion.


Science ◽  
2013 ◽  
Vol 341 (6141) ◽  
pp. 1237758 ◽  
Author(s):  
Annapurna Poduri ◽  
Gilad D. Evrony ◽  
Xuyu Cai ◽  
Christopher A. Walsh

Genetic mutations causing human disease are conventionally thought to be inherited through the germ line from one’s parents and present in all somatic (body) cells, except for most cancer mutations, which arise somatically. Increasingly, somatic mutations are being identified in diseases other than cancer, including neurodevelopmental diseases. Somatic mutations can arise during the course of prenatal brain development and cause neurological disease—even when present at low levels of mosaicism, for example—resulting in brain malformations associated with epilepsy and intellectual disability. Novel, highly sensitive technologies will allow more accurate evaluation of somatic mutations in neurodevelopmental disorders and during normal brain development.


2012 ◽  
Vol 19 (4) ◽  
pp. R131-R147 ◽  
Author(s):  
Valdemar Máximo ◽  
Jorge Lima ◽  
Hugo Prazeres ◽  
Paula Soares ◽  
Manuel Sobrinho-Simões

The biology and the genetics of Hürthle cell tumors are reviewed starting from the characterization and differential diagnosis of the numerous benign and malignant, neoplastic and nonneoplastic lesions of the thyroid in which Hürthle cell transformation is frequently observed. The clinicopathologic and molecular evidence obtained from the comparative study of the aforementioned conditions indicate that Hürthle cell appearance represents a phenotype that is superimposed on the genotypic and conventional histopathologic features of the tumors. Hürthle cell tumors differ from their non-Hürthle counterparts regarding the prevalence of large deletions of mitochondrial DNA (mtDNA), mutations of mtDNA genes coding for oxidative phosphorylation (OXPHOS) proteins (namely mutations of complex I subunit genes) and mutations of nuclear genes coding also for mitochondrial OXPHOS proteins. Such mitochondrial alterations lead to energy production defects in Hürthle cell tumors; the increased proliferation of mitochondria may reflect a compensatory mechanism for such defects and is associated with the overexpression of factors involved in mitochondrial biogenesis. The mitochondrial abnormalities are also thought to play a major role in the predisposition for necrosis instead of apoptosis which seems to be blocked in most Hürthle cell tumors. Finally, the results obtained in experimental models using cybrid cell lines and the data obtained from histopathologic and molecular studies of familial Hürthle cell tumors are used, together with the aforementioned genetic and epigenetic alterations, to progress in the understanding of the mechanisms through which mitochondrial abnormalities may be involved in the different steps of thyroid carcinogenesis, from tumor initiation to metastization.


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