scholarly journals Multicenter Evaluation of Independent High-Throughput and RT-qPCR Technologies for the Development of Analytical Workflows for Circulating miRNA Analysis

Cancers ◽  
2020 ◽  
Vol 12 (5) ◽  
pp. 1166 ◽  
Author(s):  
Anna Babayan ◽  
Martin H. D. Neumann ◽  
Andrei Herdean ◽  
Jonathan M. Shaffer ◽  
Melanie Janning ◽  
...  
Keyword(s):  
High Throughput ◽  
Extracellular Vesicle ◽  
Small Rna Sequencing ◽  
Test Results ◽  
Healthy Individuals ◽  
Or Practice ◽  
Healthy Control ◽  
Qpcr Platform ◽  
Nsclc Patients ◽  
The Eu

Background: Among emerging circulating biomarkers, miRNA has the potential to detect lung cancer and follow the course of the disease. However, miRNA analysis deserves further standardization before implementation into clinical trials or practice. Here, we performed international ring experiments to explore (pre)-analytical factors relevant to the outcome of miRNA blood tests in the context of the EU network CANCER-ID. Methods: Cell-free (cfmiRNA) and extracellular vesicle-derived miRNA (EVmiRNA) were extracted using the miRNeasy Serum/Plasma Advanced, and the ExoRNeasy Maxi kit, respectively, in a plasma cohort of 27 NSCLC patients and 20 healthy individuals. Extracted miRNA was investigated using small RNA sequencing and hybridization platforms. Validation of the identified miRNA candidates was performed using quantitative PCR. Results: We demonstrate the highest read counts in healthy individuals and NSCLC patients using QIAseq. Moreover, QIAseq showed 15.9% and 162.9% more cfmiRNA and EVmiRNA miRNA counts, respectively, in NSCLC patients compared to healthy control samples. However, a systematic comparison of selected miRNAs revealed little agreement between high-throughput platforms, thus some miRNAs are detected with one technology, but not with the other. Adding to this, 35% (9 of 26) of selected miRNAs in the cfmiRNA and 42% (11 of 26) in the EVmiRNA fraction were differentially expressed by at least one qPCR platform; about half of the miRNAs (54%) were concordant for both platforms. Conclusions: Changing of (pre)-analytical methods of miRNA analysis has a significant impact on blood test results and is therefore a major confounding factor. In addition, to confirm miRNA biomarker candidates screening studies should be followed by targeted validation using an independent platform or technology.

Abstract 46: High-Density Lipoproteins Control Monocyte Differentiation Through microRNA Intercellular Communication

2013 ◽  
Vol 33 (suppl_1) ◽  
Author(s):  
Kasey C Vickers ◽  
Michael G Levin ◽  
Michael P Anderson ◽  
Qing Xu ◽  
Joshua Anzinger ◽  
...  
Keyword(s):  
Gene Expression ◽  
Rna Sequencing ◽  
High Throughput ◽  
Small Rna ◽  
Cellular Response ◽  
Culture Media ◽  
Recipient Cell ◽  
Inflammatory Cells ◽  
High Density Lipoproteins ◽  
Small Rna Sequencing

Many HDL-microRNAs (miRNA) are well-characterized post-transcriptional regulators of inflammation, and are significantly increased on HDL with hypercholesterolemia and atherosclerosis in humans and mice. Therefore, we hypothesize that inflammatory cells uniquely control their own gene expression through cellular miRNA export to HDL and then regulate recipient cell gene expression through HDL-mediated miRNA delivery. To test this hypothesis, we used high-throughput proteomics, Open Arrays, small RNA sequencing, and gene expression microarrays. Human monocytes (plasma elutriation) were differentiated into dendritic cells and multiple macrophage phenotypes. Each cell-type was incubated with pure reconstituted HDL (rHDL), which was then purified from culture media by apolipoprotein A-I immunoprecipitation after 24 h, and both cellular and HDL-miRNAs were profiled using TaqMan Open Arrays. Macrophages were found to export high levels of miRNAs to HDL that inhibit monocyte/macrophage differentiation (miR-146a, miR-223); however, monocytes were also found to export many miRNAs associated with differentiation, including miR-92a, miR-222, miR-17, miR-20a, miR106a, and miR-21. Furthermore, many miRNAs were found to be transcribed in inflammatory cells, but completely exported to HDL and not retained in the cell. Most interestingly, HDL treatment was found to induce miR-223 transcription in monocytes, as determined by primary miR-223 transcript levels; however, intracellular levels of the mature form (miR-223) did not change. These results suggest that HDL induces the export of miRNAs it transports. PAR-CLIP with high-throughput small RNA sequencing was used to demonstrate that miRNAs are transferred from macrophages to endothelial cells and loaded onto cellular Argonaute 2-continaining RNA-induced silencing complexes. To demonstrate this in mice, human HDL, containing endogenous levels of miR-223, were injected into miR-223-null mice and inflammation-associated miRNA delivery was mapped in vivo. In summary, we found profound differences in the cellular response to HDL treatment and HDL-miRNA communication amongst inflammatory cell phenotypes that are physiologically relevant to cardiovascular disease.

A Simple, Rapid and Non-Radiolabeled Immune Assay to Detect Anti-AChR Antibodies in Myasthenia Gravis

2018 ◽  
Vol 50 (3) ◽  
pp. 229-235 ◽  
Author(s):  
Suresh Bokoliya ◽  
Shripad Patil ◽  
Madhu Nagappa ◽  
Arun Taly
Keyword(s):  
Myasthenia Gravis ◽  
Case Control Study ◽  
Enzyme Linked Immunosorbent Assay ◽  
Test Results ◽  
Dot Blot ◽  
Dot Blot Assay ◽  
Healthy Control ◽  
Neurological Illnesses ◽  
Control Study ◽  
Immune Assay

AbstractObjectiveTo assess the practicality of dot-blot testing for rapid and sensitive detection of the antiacetylcholine receptor (anti-AChR) antibodies in myasthenia gravis (MG).MethodsIn this case-control study, we tested serum specimens of 85 patients with MG, 85 healthy control individuals, and 85 patients without MG who have other autoimmune and neurological illnesses. All the serum specimens were tested for anti-AChR antibodies using 3 assays: in-house enzyme-linked immunosorbent assay (ELISA), the dot-blot assay, and commercial ELISA.ResultsIn-house ELISA, commercial ELISA, and dot-blot test results were positive for anti-AChR antibodies in 65 (76.5%) patients with MG. The results of all 3 tests were negative for anti-AChR antibodies in healthy controls and patients without MG. We observed perfect concordance (K = 1, P <.001) between all 3 tests. In-house ELISA correlated significantly (r = 0.873, P <.001) with commercial ELISA. In-house ELISA and the dot-blot test demonstrated similar diagnostic performance in detecting anti-AChR antibodies.ConclusionsThe dot-blot assay is a simple, nonradioactive immune assay for rapid detection of anti-AChR antibodies in MG.

scholarly journals Elabela levels in patients with type 2 diabetes: can it be a marker for diabetic nephropathy?

2020 ◽  
Vol 20 (2) ◽  
pp. 833-840
Author(s):  
Erhan Onalan ◽  
Yusuf Doğan ◽  
Ebru Onalan ◽  
Nevzat Gozel ◽  
Ilay Buran ◽  
...  
Keyword(s):  
Type 2 Diabetes ◽  
Diabetic Nephropathy ◽  
High Serum ◽  
Control Group ◽  
Diabetic Patients ◽  
Healthy Individuals ◽  
Urine Albumin ◽  
Healthy Control ◽  
Group 2

Backround: Elabela (ELA) is a hormone that is secreted at high levels in the kidneys of a healthy adult. This study aims to investigate whether serum ELA levels of patients with Type 2 Diabetes vary with the severity of renal damage. Methods: Our study included 50 healthy control subjects and 100 diabetic patients, who were categorized into groups based on urine albumin/creatinine ratios (ACR). Patients included in the study were assigned to four groups: Group 1 (healthy control), Group 2 (ACR<29mg/g), Group 3 (ACR=30-299 mg/g), and Group 4 (ACR>300 mg/g normal or high serum creatinine). Physical examination findings, demographic characteristics of the study group were recorded, and serum ELA levels and other laboratory parameters were assessed using appropriate methods. Results: The results of the study indicated that ELA levels determined in healthy individuals gradually decreased through stages of normal albuminuria, microalbuminuria, and macroalbuminuria. Moreover, ELA had a significant negative corre- lation with LDL-C (r=-0.201, p=0.014), glucose (r=-0.437, P<0.001), retinopathy (r=-0.222, P=0.006), serum BUN (r=- 0.161, P=0.049), and a positive correlation with eGFR (r=0.250, P=0.002). Conclusions: The fact that ELA levels are higher in healthy individuals compared to diabetic patients without microalbu- minuria, and higher in diabetic patients without microalbuminuria compared to patients with advanced albuminuria and kidney damage, suggests that the ELA level can be an important clinical prognostic variable and even a promising agent for the treatment of diabetic nephropathy patients. Keywords: Elabela, diabetes, diabetic kidney disease, albuminuria.

scholarly journals EVQuant; high-throughput quantification and characterization of extracellular vesicle (sub)populations

2020 ◽  
Author(s):  
T.A. Hartjes ◽  
J.A. Slotman ◽  
M.S. Vredenbregt ◽  
N. Dits ◽  
R. Van der Meel ◽  
...  
Keyword(s):  
Protein Content ◽  
Size Distribution ◽  
High Throughput ◽  
Liquid Biopsy ◽  
Ideal Liquid ◽  
Extracellular Vesicle ◽  
Cell Of Origin ◽  
Clinical Implementation ◽  
High Throughput Assay

AbstractExtracellular vesicles (EVs) reflect the cell of origin in terms of nucleic acids and protein content. They are found in biofluids and represent an ideal liquid biopsy biomarker source for many diseases. Unfortunately, clinical implementation is limited by available technologies for EV analysis. We have developed a simple, robust and sensitive microscopy-based high-throughput assay (EVQuant) to overcome these limitations and allow widespread use in the EV community. The EVQuant assay can detect individual immobilized EVs as small as 35 nm and determine their concentration in biofluids without extensive EV isolation or purification procedures. It can also identify specific EV subpopulations based on combinations of biomarkers and is used here to identify prostate-derived urinary EVs as CD9-/CD63+. Moreover, characterization of individual EVs allows analysis of their size distribution. The ability to identify, quantify and characterize EV (sub-)populations in high-throughput substantially extents the applicability of the EVQuant assay over most current EV quantification assays.

scholarly journals Testing the Force Absorption of Composite Materials to Select the Best for Making a Helmet

2021 ◽  
Vol 15 (4) ◽  
pp. 581-584
Author(s):  
Božo Bujanić ◽  
Matija Košak
Keyword(s):  
Carbon Fibers ◽  
Material Selection ◽  
Glass Fibers ◽  
Test Procedure ◽  
Test Results ◽  
Aramid Fibers ◽  
Absorption Properties ◽  
Eu Project ◽  
Impact Absorption ◽  
The Eu

The paper presents and describes the procedure of testing the materials that were available for the production of a multifunctional protective helmet. The procedure was carried out at the company Šestan-Busch d.o.o. as part of the EU project for the development and production of a multifunctional protective helmet. The test results showed that carbon fibers polymers as a composite material have the best impact absorption properties which was a key criterion for material selection. Other materials; glass fibers polymers, aramid fibers polymers and combinations in the test procedure showed worse results compared to the selected criterion.

Abstract PO-063: Extracellular vesicle miRNAs and autophagic CTCs: Predictive and prognostic biomarkers in radiotherapy treated NSCLC patients

2021 ◽  
Author(s):  
Diego de Miguel Perez ◽  
Rosario Guerrero Tejada ◽  
Alessandro Russo ◽  
Francisco Gabriel Ortega ◽  
Antonio Martínez-Única ◽  
...  
Keyword(s):  
Extracellular Vesicle ◽  
Prognostic Biomarkers ◽  
Nsclc Patients

scholarly journals Altered diversity and composition of gut microbiota in Wilson's disease

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Xiangsheng Cai ◽  
Lin Deng ◽  
Xiaogui Ma ◽  
Yusheng Guo ◽  
Zhiting Feng ◽  
...  
Keyword(s):  
Gut Microbiota ◽  
Gut Microbiome ◽  
Wilson’S Disease ◽  
Wilson's Disease ◽  
Healthy Individuals ◽  
Lower Abundance ◽  
Rrna Sequencing ◽  
Healthy Control ◽  
The Impact ◽  
Bacterial Groups

AbstractWilson’s disease (WD) is an autosomal recessive inherited disorder of chronic copper toxicosis with high mortality and disability. Recent evidence suggests a correlation between dysbiosis in gut microbiome and multiple diseases such as genetic and metabolic disease. However, the impact of intestinal microbiota polymorphism in WD have not been fully elaborated and need to be explore for seeking some microbiota benefit for WD patients. In this study, the 16S rRNA sequencing was performed on fecal samples from 14 patients with WD and was compared to the results from 16 healthy individuals. The diversity and composition of the gut microbiome in the WD group were significantly lower than those in healthy individuals. The WD group presented unique richness of Gemellaceae, Pseudomonadaceae and Spirochaetaceae at family level, which were hardly detected in healthy controls. The WD group had a markedly lower abundance of Actinobacteria, Firmicutes and Verrucomicrobia, and a higher abundance of Bacteroidetes, Proteobacteria, Cyanobacteria and Fusobacteria than that in healthy individuals. The Firmicutes to Bacteroidetes ratio in the WD group was significantly lower than that of healthy control. In addition, the functional profile of the gut microbiome from WD patients showed a lower abundance of bacterial groups involved in the host immune and metabolism associated systems pathways such as transcription factors and ABC-type transporters, compared to healthy individuals. These results implied dysbiosis of gut microbiota may be influenced by the host metabolic disorders of WD, which may provide a new understanding of the pathogenesis and new possible therapeutic targets for WD.

scholarly journals Alterations in the Ocular Surface Fungal Microbiome in Fungal Keratitis Patients

2019 ◽  
Vol 7 (9) ◽  
pp. 309 ◽  
Author(s):  
Prashanthi ◽  
Jayasudha ◽  
Chakravarthy ◽  
Padakandla ◽  
SaiAbhilash ◽  
...  
Keyword(s):  
Ocular Surface ◽  
Alpha Diversity ◽  
Illumina Miseq ◽  
Diversity Indices ◽  
Fungal Keratitis ◽  
Its2 Region ◽  
Healthy Individuals ◽  
Healthy Control ◽  
Fungal Microbiome ◽  
Map Analysis

Keratitis, an inflammatory disease of the eye, when neglected could lead to sight-threatening complications and ultimately blindness. Globally, over a million people are affected by keratitis annually. Keratitis has a microbial etiology and is caused by bacteria, fungi, viruses, etc. The present study compared the ocular surface fungal microbiome of healthy individuals and individuals with fungal keratitis. Fungal microbiomes from the conjunctival swabs of healthy individuals and from conjunctival swabs and corneal scrapings of individuals with fungal keratitis were generated using ITS2 region amplicons. Microbiomes were sequenced using Illumina MiSeq 2 × 250 base pair chemistry with a paired-end protocol. Based on Alpha diversity indices, phylum and genera level diversity, abundance differences, and heat map analysis, the fungal microbiomes of conjunctival swabs and corneal scrapings of individuals with fungal keratitis exhibited dysbiosis (alterations in the diversity and abundance) compared to the ocular surface microbiome of the healthy control individuals. This is the first report indicating dysbiosis in the fungal microbiome of conjunctival swabs and corneal scrapings in individuals with fungal keratitis. A total of 11 genera present in the majority of the eyes constituted the variable core ocular microbiome.

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