scholarly journals Determination of Exosome Mitochondrial DNA as a Biomarker of Renal Cancer Aggressiveness

Cancers ◽  
2021 ◽  
Vol 14 (1) ◽  
pp. 199
Author(s):  
Elena Arance ◽  
Viviana Ramírez ◽  
Alejandro Rubio-Roldan ◽  
Francisco M. Ocaña-Peinado ◽  
Catalina Romero-Cachinero ◽  
...  
Keyword(s):  
Mitochondrial Dna ◽  
Liquid Biopsy ◽  
Digital Pcr ◽  
Mtdna Copy Number ◽  
Non Invasive ◽  
Cancer Aggressiveness ◽  
Next Generation Sequencing Ngs ◽  
First Time

Here, the role of non-invasive biomarkers in liquid biopsy was evaluated, mainly in exosomes and mitochondrial DNA (mtDNA) as promising, novel, and stable biomarkers for renal cell carcinoma (RCC). A total of 140 fractions (named from B to F) obtained by ultracentrifugations of whole blood samples from 28 individuals (13 patients and 15 controls) were included. Nanoparticle Tracking Analysis (NTA) was conducted to characterized exosomal fraction. Subsequently, an analysis of digital PCR (dPCR) using the QuantStudio™ 3D Digital PCR platform was performed and the quantification of mtDNA copy number by QuantStudioTM 12K Flex Real-Time PCR System (qPCR) was developed. Moreover, Next Generation Sequencing (NGS) analyses were included using MiSeq system (Illumina, San Diego, CA, USA). An F fraction, which contains all exosome data and all mitochondrial markers, was identified in dPCR and qPCR with statistically significant power (adjusted p values ≤ 0.03) when comparing cases and controls. Moreover, present analysis in mtDNA showed a relevant significance in RCC aggressiveness. To sum up, this is the first time a relation between exosomal mtDNA markers and clinical management of RCC is analyzed. We suggest a promising strategy for future liquid biopsy RCC analysis, although more analysis should be performed prior to application in routine clinical practice.

scholarly journals Determination of Exosome Mitochondrial DNA as a Biomarker of Renal Cancer Aggressiveness

2021 ◽  
Author(s):  
Elena Arance ◽  
Viviana Ramírez ◽  
Alejandro Rubio-Roldan ◽  
Francisco M. Ocaña-Peinado ◽  
Catalina Romero-Cachinero ◽  
...  
Keyword(s):  
Mitochondrial Dna ◽  
Hypervariable Region ◽  
Digital Pcr ◽  
Complex Iii ◽  
Mtdna Copy Number ◽  
Non Invasive ◽  
Apocytochrome B ◽  
Cancer Aggressiveness ◽  
Next Generation Sequencing Ngs ◽  
First Time

Abstract Background: Components of liquid biopsy are promising non-invasive biomarkers for monitoring the renal cell carcinoma (RCC) status. Present study tries to evaluate the role of exosomes and mitochondrial DNA (mtDNA) as promise, novel and stable biomarkers that could improve current ones in RCC. Methods: A total of 140 fractions obtained by ultracentrifugations of whole blood samples stored in EDTA anticoagulant from 28 (13 patients and 15 controls) were included in present study. An exosome collection protocol and exhaustive analysis of all phases obtained by ultracentrifugations (named from B to F) was performed. Subsequently, an analysis of dPCR (digital PCR) using the QuantStudio™ 3D Digital PCR platform and the quantification of mtDNA copy number using by QuantStudioTM 12K Flex Real-Time PCR System (qPCR) was developed. Moreover, Next Generation Sequencing (NGS) analyses were included using MiSeq system (Illumina, CA, USA). Results: F fraction, which contains all exosomes´ data and all mitochondrial markers (hypervariable region 1 (HV1) and apocytochrome b of complex III (MT-CYB)) were statistically identified in dPCR and qPCR (p values < 0.05) when a comparison between cases and controls was performed. Moreover, the studied mitochondrial genes shown a relevant significance in RCC aggressiveness analyses (metastasis and stages 4).Conclusions: To sum up, this is the first time a relation between mtDNA genetic markers in exosome and clinical management of RCC is suggested.

scholarly journals Exploring Secondary Metabolite Profiles of Stachybotrys spp. by LC-MS/MS

Toxins ◽  
2019 ◽  
Vol 11 (3) ◽  
pp. 133 ◽  
Author(s):  
Annika Jagels ◽  
Viktoria Lindemann ◽  
Sebastian Ulrich ◽  
Christoph Gottschalk ◽  
Benedikt Cramer ◽  
...  
Keyword(s):  
Secondary Metabolites ◽  
Future Research ◽  
Structural Modifications ◽  
Metabolite Profiles ◽  
The Individual ◽  
First Time ◽  
Analytical Standards ◽  
Respective Species

The genus Stachybotrys produces a broad diversity of secondary metabolites, including macrocyclic trichothecenes, atranones, and phenylspirodrimanes. Although the class of the phenylspirodrimanes is the major one and consists of a multitude of metabolites bearing various structural modifications, few investigations have been carried out. Thus, the presented study deals with the quantitative determination of several secondary metabolites produced by distinct Stachybotrys species for comparison of their metabolite profiles. For that purpose, 15 of the primarily produced secondary metabolites were isolated from fungal cultures and structurally characterized in order to be used as analytical standards for the development of an LC-MS/MS multimethod. The developed method was applied to the analysis of micro-scale extracts from 5 different Stachybotrys strains, which were cultured on different media. In that process, spontaneous dialdehyde/lactone isomerization was observed for some of the isolated secondary metabolites, and novel stachybotrychromenes were quantitatively investigated for the first time. The metabolite profiles of Stachybotrys species are considerably influenced by time of growth and substrate availability, as well as the individual biosynthetic potential of the respective species. Regarding the reported adverse effects associated with Stachybotrys growth in building environments, combinatory effects of the investigated secondary metabolites should be addressed and the role of the phenylspirodrimanes re-evaluated in future research.

scholarly journals Skin Electrical Resistance Measurement of Oxygen-Containing Terpenes as Penetration Enhancers: Role of Stratum Corneum Lipids

Molecules ◽  
2019 ◽  
Vol 24 (3) ◽  
pp. 523 ◽  
Author(s):  
Xue-min Zhu ◽  
Yu Li ◽  
Fei Xu ◽  
Wei Gu ◽  
Guo-jun Yan ◽  
...  
Keyword(s):  
Stratum Corneum ◽  
Electrical Resistance ◽  
Attenuated Total Reflection ◽  
Penetration Enhancers ◽  
Rapid Screening ◽  
Factors Affecting ◽  
Skin Cell ◽  
First Time

The measurement of skin electrical resistance (SER) has drawn a great deal of attention for the rapid screening of transdermal penetration enhancers (PEs). However, the mechanisms underlying the SER measurement are still unclear. This study was to investigate the effects and mechanisms of seven oxygen-containing terpenes on the SER kinetics. Stratum corneum (SC) lipids were proved to play a key role in SER measurement. Then, the factors affecting the SER measurement were optimized. By the determination of SER kinetics, cyclic terpenes (1,8-cineole, terpinen-4-ol, menthol and α-terpineol) were demonstrated to possess higher enhancement ratio (ER) values compared with linear terpenes (linalool, geraniol and citral). For the first time, the linear correlation was found between ER of terpenes and the interaction energy of terpene–ceramide complexes revealed by molecular simulation. The attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR) analysis revealed that the effect of cyclic terpenes on SC lipid arrangement was obviously stronger than that of linear terpenes. In addition, by evaluating HaCaT skin cell viability, little difference was found between the toxicities of cyclic and linear terpenes. In conclusion, measurement of SER could be a feasible approach for the efficient evaluation of the PEs that mainly act on SC lipids.

Frequent LOH of CYP2D6 in ER+ breast cancer determined by next-generation sequencing (NGS).

2013 ◽  
Vol 31 (15_suppl) ◽  
pp. 534-534
Author(s):  
Mark J. Ratain ◽  
James Sun ◽  
Yusuke Nakamura ◽  
Nancy J Cox ◽  
Tarek Sahmoud ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Sequence Data ◽  
Biological Significance ◽  
Gene Copy Number ◽  
Gene Copy ◽  
Full Cohort ◽  
Next Generation Sequencing Ngs ◽  
Apparent Association

534 Background: The role of CYP2D6 genetic variation in predicting response to tamoxifen in ER+ breast cancer is a subject of ongoing debate. There has been great variability in approaches to both genotyping and phenotyping, and in particular many investigators have extracted DNA from breast cancer samples rather than peripheral blood. We hypothesized that CYP2D6 gene copy number alterations are common in ER+ breast cancer, affecting genotype results, and used NGS to characterize CYP2D6 in patients with ER+ disease. Methods: CYP2D6 sequencing was performed as part of a comprehensive NGS profile of cancer-related genes for 261 predominantly relapsed and metastatic ER+ breast cancer FFPE specimens. Sequence data were resolved into genotypes according to the * allele nomenclature. Tumor LOH was determined at CYP2D6, and its error impact on genotyping methods was estimated. To assess biological significance, the prevalence of CYP2D6 alleles and LOH in ER+ disease was compared against a control set of 99 ER- tumors. Results: CYP2D6 allele frequencies in our full cohort (ER+, 261; ER-, 99) were consistent with prior studies; 64.4%, 16.8%, 9.0% vs. 63.1%, 17.2%, 7.0% for *1/*2, *4, and *41 respectively, and 1%-2% for the rarer alleles *9, *10, and *5. The rate of CYP2D6 LOH was higher in ER+ disease (41% vs. 26%, p<0.01), with all excess arising from copy-loss (as opposed to copy-neutral) changes (22% vs. 7%, p<0.002). The estimated impact of LOH on germline genotype assessment from tumor was considerable; an assay sensitive at >20% minor allele frequency (e.g., Sanger sequencing) can misclassify >10% of heterozygotes, leading to significant Hardy-Weinberg disequilibrium (e.g., p=8.3x10-8 for *4). Interestingly, an enrichment of reduced or non-functional CYP2D6 alleles in ER+ samples was observed (61% vs. 47%, p<0.03). Conclusions: Our results demonstrate the distorting effect of extensive LOH on genotype assessment of CYP2D6 in breast cancer. Therefore, tumor DNA should not be routinely used for determination of germline 2D6 genotype, although it appears possible to use NGS. The apparent association between reduced function CYP2D6 alleles and ER+ breast cancer in our dataset requires further investigation.

Cerebrospinal fluid mitochondrial DNA levels in patients with multiple sclerosis

2018 ◽  
Vol 25 (11) ◽  
pp. 1535-1538 ◽  
Author(s):  
Nicolas Fissolo ◽  
Laura Cervera-Carles ◽  
Luisa María Villar Guimerans ◽  
Alberto Lleó ◽  
Jordi Clarimón ◽  
...  
Keyword(s):  
Multiple Sclerosis ◽  
Cerebrospinal Fluid ◽  
Mitochondrial Dna ◽  
Clinically Isolated Syndrome ◽  
Chain Reaction ◽  
Pathological Conditions ◽  
Polymerase Chain ◽  
Digital Polymerase Chain Reaction

The role of cerebrospinal fluid (CSF) mitochondrial DNA (mtDNA) levels as biomarker in multiple sclerosis (MS) is unknown. We determined CSF mtDNA levels in a cohort of 237 individuals, including patients with MS and clinically isolated syndrome (CIS), inflammatory and non-inflammatory neurological controls, and cognitively healthy controls (HC). mtDNA concentration was measured by droplet digital polymerase chain reaction. CSF mtDNA levels were increased in all pathological conditions compared with HC, though no differences were observed between relapse-onset and progressive MS clinical forms, CIS patients and neurological controls. These findings do not support the determination of CSF mtDNA levels as a useful biomarker in MS clinical practice.

scholarly journals Droplet Digital PCR Analysis of Liquid Biopsy Samples Unveils the Diagnostic Role of hsa-miR-133a-3p and hsa-miR-375-3p in Oral Cancer

Biology ◽  
2020 ◽  
Vol 9 (11) ◽  
pp. 379
Author(s):  
Salvatore Crimi ◽  
Luca Falzone ◽  
Giuseppe Gattuso ◽  
Caterina Maria Grillo ◽  
Saverio Candido ◽  
...  
Keyword(s):  
Oral Cancer ◽  
Cancer Patients ◽  
Liquid Biopsy ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Pcr Analysis ◽  
Screening Programs ◽  
Diagnostic Potential ◽  
Oral Cancer Patients

Despite the availability of screening programs, oral cancer deaths are increasing due to the lack of diagnostic biomarkers leading to late diagnosis and a poor prognosis. Therefore, there is an urgent need to discover novel effective biomarkers for this tumor. On these bases, the aim of this study was to validate the diagnostic potential of microRNAs (miRNAs) through the analysis of liquid biopsy samples obtained from ten oral cancer patients and ten healthy controls. The expression of four selected miRNAs was evaluated by using droplet digital PCR (ddPCR) in a pilot cohort of ten oral cancer patients and ten healthy donors. Bioinformatics analyses were performed to assess the functional role of these miRNAs. The expression levels of the predicted down-regulated hsa-miR-133a-3p and hsa-miR-375-3p were significantly reduced in oral cancer patients compared to normal individuals while no significant results were obtained for the up-regulated hsa-miR-503-5p and hsa-miR-196a-5p. ROC analysis confirmed the high sensitivity and specificity of hsa-miR-375-3p and hsa-miR-133a-3p. Therefore, both miRNAs are significantly down-regulated in cancer patients and can be used as biomarkers for the early diagnosis of oral cancer. The analysis of circulating miRNAs in a larger series of patients is mandatory to confirm the results obtained in this pilot study.

scholarly journals Features of a lullaby as a type of polycode text

2020 ◽  
pp. 158-168
Author(s):  
Ariuka I. Gelyaeva ◽  
Dzhamiliyat Dzh. Khuchinaeva
Keyword(s):  
Main Method ◽  
Linguistic Understanding ◽  
Nature Of Information ◽  
The Difference ◽  
First Time ◽  
Communicative Space ◽  
Empirical Material ◽  
Main Strategy

The article is devoted to identification of the features of the Karachai-Balkarian lullaby as a text of a complex semiotic nature and determination of the role of verbal and non-verbal components in the organization of an integral communicative space. It is postulated that the difference in sign is characteristic not only of modern texts, born of scientific and technological progress, which changed the sign nature of information, but also of a lullaby. It is noted that in linguistics, the study of a lullaby as a polycode text is not one of the intensively developed ones, and in the Karachai-Balkarian language its linguistic understanding and description as a multilayered text is carried out for the first time, which determines the relevance of the work. The main method for analyzing empirical material is the semiotic method of studying the text as an integral system of interdependent signs and symbols. Elements of discourse and phonosemantic analysis are also used. The article separately examines various semiotic layers and components syncretically presented in the text of the lullaby. The article analyzes the units of the lexical-semantic and grammatical levels of the language, which represent the verbal layer, as well as non-verbal means used in addition to the language code that accompany the verbal text of the song. Refrain “ballyau-ballyau”, “bellyay-bellyay” and the dominant sounds of the text «l-l’», «б-б’» according to their acoustic and physical characteristics, they are subordinated to the main strategy of a lullaby - calming and lulling a child. Particular attention is paid to determining the role of verbal and non-verbal signs in the implementation of synsemantics of the text of a lullaby. The novelty of the research lies in the identification of the specificity of the lullaby as a text of a different-sign nature, which manifests itself in the syncretism of various semiotic components, which in the text space are in complementarity relations. The multidimensionality of vectors of interaction of verbal, paraverbal, kinetic, symbolic components in a lullaby is revealed. It has been established that the verbal layer mainly performs an informative function, non-verbal means - expressive and pictorial functions.

BIOM-12. DEVELOPMENT OF 5-ALA BASED LIQUID BIOPSY FOR THE NON-INVASIVE DIAGNOSIS OF GLIOBLASTOMA

2021 ◽  
Vol 23 (Supplement_6) ◽  
pp. vi12-vi12
Author(s):  
Anudeep Yekula ◽  
Tiffaney Hsia ◽  
Leonora Balaj ◽  
Bob Carter
Keyword(s):  
Liquid Biopsy ◽  
Aminolevulinic Acid ◽  
Expression Patterns ◽  
Protoporphyrin Ix ◽  
Digital Pcr ◽  
Sequencing Analysis ◽  
Guided Surgery ◽  
Non Invasive ◽  
Fluorescence Guided Surgery ◽  
Healthy Control

Abstract INTRODUCTION Tumor specificity of 5-aminolevulinic acid (5-ALA) induced protoporphyrin IX (PpIX) fluorescence is widely applied for fluorescence-guided surgery (FGS) in gliomas. We recently showed the feasibility of detecting tumour-specific fluorescent PpIX extracellular vesicles (EVs) derived from the plasma of glioblastoma (GBM) patients undergoing 5-ALA based fluorescence-guided surgery. Here, we further develop methods to characterize, sort and study fluorescent PpIX EVs in plasma of patients with glioma. METHODS We used imaging flow cytometry and Astrios EQ nanoFACS to characterize and sort PpIX EVs, respectively. Downstream RNA analysis utilized transcriptome sequencing analysis and droplet digital PCR (EGFRvIII mRNA). RESULTS All GBM cell lines (Gli36vIII, U87, Gli36 WT) dosed with 5-ALA demonstrated PpIX fluorescence, and released PpIX positive EVs. There was a high correlation between fluorescence in cells and the number of PpIX EVs released (r2=0.9). We sorted 100,000 PpIX EVs from Gli36vIII cells dosed with 5-ALA and detected 65 copies and 24 copies of mutant EGFRvIII mRNA and wildtype EGFR mRNA per 100,000 EVs, respectively. RNAseq analysis of the sorted PpIX EVs showed expression patterns reflective of parent cells. Furthermore, 100,000 sorted PpIX EVs from the plasma of a patient with EGFRvIII glioma yielded 22 copies of EGFRvIII mRNA while &lt; 5 copies were detected in 1ml of plasma and healthy control plasma, demonstrating the tumor-specific nature of PpIX EVs. Finally, we performed transcriptome analysis on 250,000 PpIX EVs each from 8 patients undergoing 5-ALA based FGS. We identified several mRNAs including Gli3, STAG2, ELF3, PHLPP1 which play an important role in cancer. CONCLUSION The ability to sort and characterize tumor specific PpIX EVs following 5-ALA administration opens new avenues for liquid biopsy-based glioma diagnosis.

scholarly journals Liquid biopsy for patients with IBD-associated neoplasia

BMC Cancer ◽  
2020 ◽  
Vol 20 (1) ◽  
Author(s):  
Hideaki Kinugasa ◽  
Sakiko Hiraoka ◽  
Kazuhiro Nouso ◽  
Shumpei Yamamoto ◽  
Mami Hirai ◽  
...  
Keyword(s):  
Liquid Biopsy ◽  
Target Genes ◽  
Tumor Tissue ◽  
Digital Pcr ◽  
Circulating Tumor Dna ◽  
Tissue Samples ◽  
Non Invasive ◽  
Tumor Tissues ◽  
Inflammatory Bowel

Abstract Background It is often difficult to diagnose inflammatory bowel disease (IBD)-associated neoplasia endoscopically due to background inflammation. In addition, due to the absence of sensitive tumor biomarkers, countermeasures against IBD-associated neoplasia are crucial. The purpose of this study is to develop a new diagnostic method through the application of liquid biopsy. Methods Ten patients with IBD-associated cancers and high-grade dysplasia (HGD) with preserved tumor tissue and blood were included. Tumor and non-tumor tissues were analyzed for 48 cancer-related genes using next-generation sequencing. Simultaneously, circulating tumor DNA (ctDNA) was analyzed for mutations in the target genes using digital PCR. Results Out of 10 patients, seven had IBD-related cancer and three had IBD-related HGD. Two patients had carcinoma in situ; moreover, three had stageII and two had stage III. To avoid false positives, the mutation rate cutoff was set at 5% based on the control results; seven of 10 (70%) tumor tissue samples were mutation-positive. Mutation frequencies for each gene were as follows: TP53 (20.9%; R136H), TP53 (25.0%; C110W), TP53 (8.5%; H140Q), TP53 (31.1%; R150W), TP53 (12.8%; R141H), KRAS (40.0%; G12V), and PIK3CA (34.1%; R 88Q). The same mutations were detected in the blood of these seven patients. However, no mutations were detected in the blood of the remaining three patients with no tumor tissue mutations. The concordance rate between tumor tissue DNA and blood ctDNA was 100%. Conclusion Blood liquid biopsy has the potential to be a new method for non-invasive diagnosis of IBD-associated neoplasia.

The effect of age on the unfolding of elastin lamellae and collagen fibers with stretch in human carotid arteries

1981 ◽  
Vol 59 (10) ◽  
pp. 1050-1057 ◽  
Author(s):  
Zenon J. Samila ◽  
Stefan A. Carter
Keyword(s):  
Elastic Properties ◽  
Carotid Arteries ◽  
Collagen Fibers ◽  
Low Degrees ◽  
First Time ◽  
The Relationship ◽  
Effect Of Age ◽  
Human Carotid

We measured folding of elastin lamellae and collagen fibers in human carotid arteries and correlated the results with the elastic properties of the vessels. Specimens cut into circumferential strips were stretched to various degrees, fixed, and stained for elastin and collagen. Folding was measured on photographic projections. Elastin lamellae unfolded quickly with initial stretch. In old vessels they did not straighten as much as in the young suggesting that the content of the interlamellar space may interfere with the unfolding. Collagen fibers straightened more during stretch in stiffer older vessels than in the young, already at low degrees of stretch. Young's modulus at extension of 5% appeared to correlate with unfolding of elastin lamellae in young extensible vessels. The modulus correlated significantly with unfolding of collagen fibers at extensions of 15% and greater, and the slope of the regression of the modulus on folding increased with stretch. Our findings provide for the first time morphologic evidence for the role of elastin lamellae in the determination of the elastic properties at low extensions, for the importance of collagen fibers in increasing stiffness with further stretch, and for the relationship between increased stiffness with age and the earlier recruitment of the collagen fibers.

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