scholarly journals Fast and specific enrichment of cancer-related exosomes by DNA-nanoweight-assisted centrifugation

2021 ◽  
Author(s):  
zhangwei lu ◽  
ma yuxyan ◽  
ye shi ◽  
bin jia ◽  
xintong li ◽  
...  
Keyword(s):  
Membrane Vesicles ◽  
Clinical Samples ◽  
Hybridization Chain Reaction ◽  
Conventional Elisa ◽  
Dna Tetrahedron ◽  
Diagnosis Of Cancer ◽  
Tumor Exosomes ◽  
Biological Environment ◽  
Higher Sensitivity ◽  
Mcf 7

Exosomes are nanoscale membrane vesicles actively released by cells and play an important role in the diagnosis of cancer-related diseases. However, it is challenging to efficiently enrich exosomes from extracellular fluids. In this work, we used DNA-tetrahedron as a nanoweight during centrifugation to precisely enrich tumor exosomes from a complex biological environment. Two different DNA tetrahedral nanostructures (DTAs), each carrying a specific aptamer for exosome biomarker recognition, were incubated with clinical samples simultaneously. One DTA triggered the cross-linking of multiple target exosomes, and therefore enabled low-speed and fast centrifugation for enrichment. The other DTA further narrowed down the target exosome subtype and initiated a hybridization chain reaction (HCR) for sensitive signal amplification. The method enabled the detection of 180 MCF-7-derived exosomes per microliter and 560 HepG2-derived exosomes per microliter, with 1000-fold higher sensitivity than conventional ELISA. This easy-to-operate method can enrich exosomes with excellent specificity and therefore will be appealing in biomedical research and clinical diagnosis.

Integrated Quantitative Analysis of the Phosphoproteome and Transcriptome in Tamoxifen-resistant Breast Cancer*

2020 ◽  
Author(s):  
Lungwani Muungo
Keyword(s):  
Breast Cancer ◽  
High Rate ◽  
System Level ◽  
Clinical Samples ◽  
Transcriptome Data ◽  
Data Set ◽  
Reporter Assays ◽  
Treated Breast ◽  
Mcf 7 ◽  
Resistant Cells

Quantitative phosphoproteome and transcriptome analysisof ligand-stimulated MCF-7 human breast cancer cells wasperformed to understand the mechanisms of tamoxifen resistanceat a system level. Phosphoproteome data revealed thatWT cells were more enriched with phospho-proteins thantamoxifen-resistant cells after stimulation with ligands.Surprisingly, decreased phosphorylation after ligand perturbationwas more common than increased phosphorylation.In particular, 17?-estradiol induced down-regulation inWT cells at a very high rate. 17?-Estradiol and the ErbBligand heregulin induced almost equal numbers of up-regulatedphospho-proteins in WT cells. Pathway and motifactivity analyses using transcriptome data additionallysuggested that deregulated activation of GSK3? (glycogensynthasekinase 3?) and MAPK1/3 signaling might be associatedwith altered activation of cAMP-responsive elementbindingprotein and AP-1 transcription factors intamoxifen-resistant cells, and this hypothesis was validatedby reporter assays. An examination of clinical samples revealedthat inhibitory phosphorylation of GSK3? at serine 9was significantly lower in tamoxifen-treated breast cancerpatients that eventually had relapses, implying that activationof GSK3? may be associated with the tamoxifen-resistantphenotype. Thus, the combined phosphoproteomeand transcriptome data set analyses revealed distinct signal

Ultrasensitive ratiometric detection of Pb2+ using DNA tetrahedron-mediated hyperbranched hybridization chain reaction

2021 ◽  
Vol 1147 ◽  
pp. 170-177
Author(s):  
Pingping Ji ◽  
Guimei Han ◽  
Yan Huang ◽  
Hongxin Jiang ◽  
Qiwen Zhou ◽  
...  
Keyword(s):  
Hybridization Chain Reaction ◽  
Chain Reaction ◽  
Ratiometric Detection ◽  
Dna Tetrahedron

scholarly journals CSMD: a computational subtraction-based microbiome discovery pipeline for species-level characterization of clinical metagenomic samples

2019 ◽  
Author(s):  
Yu Liu ◽  
Paul W Bible ◽  
Bin Zou ◽  
Qiaoxing Liang ◽  
Cong Dong ◽  
...  
Keyword(s):  
Species Level ◽  
Supplementary Information ◽  
Clinical Samples ◽  
Plasma Dna ◽  
Bioinformatics Pipeline ◽  
Microbial Identification ◽  
Reference Databases ◽  
Microbial Dna ◽  
Higher Sensitivity

Abstract Motivation Microbiome analyses of clinical samples with low microbial biomass are challenging because of the very small quantities of microbial DNA relative to the human host, ubiquitous contaminating DNA in sequencing experiments and the large and rapidly growing microbial reference databases. Results We present computational subtraction-based microbiome discovery (CSMD), a bioinformatics pipeline specifically developed to generate accurate species-level microbiome profiles for clinical samples with low microbial loads. CSMD applies strategies for the maximal elimination of host sequences with minimal loss of microbial signal and effectively detects microorganisms present in the sample with minimal false positives using a stepwise convergent solution. CSMD was benchmarked in a comparative evaluation with other classic tools on previously published well-characterized datasets. It showed higher sensitivity and specificity in host sequence removal and higher specificity in microbial identification, which led to more accurate abundance estimation. All these features are integrated into a free and easy-to-use tool. Additionally, CSMD applied to cell-free plasma DNA showed that microbial diversity within these samples is substantially broader than previously believed. Availability and implementation CSMD is freely available at https://github.com/liuyu8721/csmd. Supplementary information Supplementary data are available at Bioinformatics online.

scholarly journals Isolation of Mucorales from biological environment and identification of Rhizopus among the isolates using PCRRFLP

2019 ◽  
Vol 21 (2) ◽  
pp. 98-103
Author(s):  
Mahboobeh Madani ◽  
Mohammadali Zia
Keyword(s):  
Reliable Method ◽  
Enzymatic Digestion ◽  
Accurate Diagnosis ◽  
Molecular Methods ◽  
Clinical Samples ◽  
Molecular Approaches ◽  
Pcr Products ◽  
Microscopic Studies ◽  
Biological Environment

Background and aims: Mucorales are fungi belonging to the category of Zygomycetes, found much in nature. Culture-based methods for clinical samples are often negative, difficult and time-consuming and mainly identify isolates to the genus level, and sometimes only as Mucorales. Therefore, applying fast and accurate diagnosis methods such as molecular approaches seems necessary. This study aims at isolating Mucorales for determination of Rhizopus genus between the isolates using molecular methods. Methods: In this descriptive observational study, a total of 500 samples were collected from air and different surfaces and inoculated on Sabouraud Dextrose Agar supplemented with chloramphenicol. Then, the fungi belonging to Mucorales were identified and their pure culture was provided. DNA extraction was done using extraction kit and the chloroform method. After amplification, the samples belonging to Mucorales were identified by observing 830 bp bands. For enzymatic digestion, enzyme BmgB1 was applied for identification of Rhizopus species by formation of 593 and 235 bp segments. Results: One hundred pure colonies belonging to Mucorales were identified using molecular methods and after enzymatic digestion, 21 isolates were determined as Rhizopus species. The sequencing of PCR products and macroscopic and microscopic studies confirmed the existence of R. stolonifera, R. oryzae and R. caespitosus in the samples. Conclusion: Generally, developing a reliable method for determining Zygomycete species can be a useful tool for better understanding of the epidemiology of mucoromycosis.

scholarly journals Evaluation of the AllplexTM Gastrointestinal Panel—Parasite Assay for Protozoa Detection in Stool Samples: A Retrospective and Prospective Study

2020 ◽  
Vol 8 (4) ◽  
pp. 569 ◽  
Author(s):  
Brice Autier ◽  
Jean-Pierre Gangneux ◽  
Florence Robert-Gangneux
Keyword(s):  
Prospective Study ◽  
Multiplex Pcr ◽  
Clinical Samples ◽  
Molecular Assay ◽  
Pcr Assay ◽  
Cryptosporidium Spp ◽  
Multiplex Pcr Assay ◽  
Parasite Detection ◽  
Stool Samples ◽  
Higher Sensitivity

This study aims at evaluating the performances of the multiplex PCR AllplexTM Gastrointestinal Panel-Parasite Assay (GIPPA), which detects G. duodenalis, Cryptosporidium spp., E. histolytica, D. fragilis, B. hominis, and C. cayetanensis, by comparison to microscopy. A retrospective evaluation was conducted on a series of positive clinical samples (n = 99) stored at −80 °C or at +4 °C. A five-month prospective study was then conducted on all samples sent to our lab for parasite detection (n = 586). In the retrospective cohort, sensitivity was 81% for both G. duodenalis (26/32) and D. fragilis (21/26) and 100% for Cryptosporidium spp. (26/26, including 6 different species), B. hominis (26/26), and C. cayetanensis (4/4). During the prospective study, 95 samples were positive by microscopy and 207 by multiplex PCR assay. The molecular assay showed a significantly higher sensitivity of PCR, especially for G. duodenalis (100% vs. 60.7%, p < 0.01), D. fragilis (97.2% vs. 14.1%, p < 0.001), and B. hominis (99.4% vs. 44.2%, p < 0.001) but also for E. histolytica (100% vs. 50.0%). The sensitivity of the AllplexTM GIPPA on the first stool sample was equivalent to the sensitivity of microscopy on multiple stool samples but inferior to multiplex PCR on multiple stool samples. Taken together, the AllplexTM GIPPA is suitable for the routine detection of protozoa in fecal samples.

Preliminary study of three tumor markers for hepatocellular carcinoma: AFP, AFP-L3, and Glypican-3

2007 ◽  
Vol 25 (18_suppl) ◽  
pp. 15014-15014
Author(s):  
H. Li ◽  
K. Z. Qu ◽  
R. M. Ner ◽  
A. D. Sferruzza ◽  
R. A. Bender
Keyword(s):  
Reference Range ◽  
Hepatocellular Cancer ◽  
Clinical Samples ◽  
Heparin Sulfate Proteoglycan ◽  
Heparin Sulfate ◽  
Increased Risk ◽  
Preliminary Study ◽  
Malignant Liver ◽  
Higher Sensitivity

15014 Background: a-Fetoprotein (AFP) is the most widely used biochemical marker for detection of hepatocellular cancer (HCC). AFP is also elevated in acute and chronic liver disease, limiting its utility as an early indicator of HCC. Recently, AFP-L3 and glypican 3 (GPC3) have been reported for early detection of HCC. AFP-L3 is a glycoform of AFP produced by malignant liver cells. Measurement of AFP-L3 as a percentage of total AFP helps distinguish non-malignant hepatic disease from HCC. GPC3 is a heparin sulfate proteoglycan anchored to the plasma membrane. The level of GPC3 in the serum of HCC patients is higher than in the serum of healthy adults or patients with non-malignant hepatopathy. We tested the concordance of AFP, AFP, AFP-L3%, and GPC3and GPC3 to determine if their simultaneous measurement may increase the sensitivity of identifying individuals at risk for HCC. Method: De-identified clinical samples (n = 176) submitted for AFP-L3% measurement were tested. AFP and AFP-L3 levels were measured using the LiBASys automated analyzer (Wako Chemicals USA Inc, Richmond, VA). GPC3 levels were measured with a commercial ELISA kit (BioMosaics, Inc, Burlington, VT). To assess concordance we categorized results according to accepted cut-off values for AFP and AFP-L3, and a clinically established reference range for GPC3. Results: Fourteen of the 176 samples had elevated GPC3 (=180pg/mL), 6 (43%) of which had normal AFP values (<20ng/mL). Forty- seven of the 176 samples had elevated AFP-L3% (L3 =10%), 6 (12.8%) of which had normal AFP levels. Conclusion: These data suggest that GPC3 and AFP-L3% may be used as supplemental markers, and the simultaneous determination of AFP, AFP-L3, and GPC3 may provide a higher sensitivity for identifying individuals with increased risk of HCC. [Table: see text] No significant financial relationships to disclose.

The enrichment of CD44 in exosomes from breast cancer cells treated with doxorubicin promotes chemoresistance

2020 ◽  
Author(s):  
xiaohong wang ◽  
kai cheng ◽  
guoqiang zhang ◽  
yue yu ◽  
song liu ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cancer Cells ◽  
Breast Cancer Cells ◽  
Chemotherapy Resistance ◽  
Clinical Samples ◽  
Cd44 Expression ◽  
Candidate Target ◽  
And Function ◽  
Cancer Chemoresistance ◽  
Mcf 7

Abstract Background: Exosomes have been shown to be associated with chemotherapy resistance transmission between cancer cells. However, the cargo and function of exosomes changed in response to doxorubicin remains unclear. Methods: We compared proteome profiles of exosomes extracted from the supernatant of MCF-7(S/Exo) and MCF-7/ADR(A/Exo) cells. We confirmed the differential expression of the candidate target-exosomic-CD44 by immune gold staining and western blot. We further studied the changes of chemosensitivity and CD44 expression in MCF-7 cells co-incubated with A/Exo. We analyzed the levels of exosomal CD44 from patient plasma, and compared the sensitivity and specificity of exosomic CD44 and plasma CD44 on diagnosis of chemoresistance. We modified the MCF-7-derived exosomes loaded with siRNA against CD44 to observe the effects of targeting reduced CD44 expression in lumimal A breast cancer cells. Results: DOX increased the exosomes release from MCF-7/ADR cells and the exosomes mediated proteins intercellular transfer in breast cancer chemoresistance regulation. The candidate target of CD44 in A/Exo was much higher than in S/Exo and the increase levels of exosomic CD44 (21.65-fold) was much higher than cellular CD44 (6.55-fold). The same results were obtained in clinical samples. Exosome-siRNA targeted CD44 (Exos-siCD44) could efficiently targeted to silence its expression. When co-cultured on Exos-siCD44, breast cancer cells exhibited reduced cell proliferation and enhanced susceptibility to DOX and the same phenomenon was observed in mice. Conclusion：Drug-resistant breast cancer cells spread resistance capacity to sensitive ones by releasing exosomes to transfer proteins in intercellular.

Multiplexed Immunosensing Platform Coupled to Hybridization Chain Reaction for Electrochemical Determination of MicroRNAs in Clinical Samples

2018 ◽  
Vol 31 (2) ◽  
pp. 293-302 ◽  
Author(s):  
Ludmila Jirakova ◽  
Roman Hrstka ◽  
Susana Campuzano ◽  
Jose M. Pingarrón ◽  
Martin Bartosik
Keyword(s):  
Electrochemical Determination ◽  
Clinical Samples ◽  
Hybridization Chain Reaction ◽  
Chain Reaction

scholarly journals Detection and Quantification of eDNA-Associated Bacterial Membrane Vesicles by Flow Cytometry

2019 ◽  
Vol 20 (21) ◽  
pp. 5307 ◽  
Author(s):  
Valentina Puca ◽  
Eva Ercolino ◽  
Christian Celia ◽  
Giuseppina Bologna ◽  
Luisa Di Marzio ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Fluorescence Microscopy ◽  
Biofilm Formation ◽  
Lactobacillus Reuteri ◽  
Bacterial Species ◽  
Cell Communication ◽  
Membrane Vesicles ◽  
Extracellular Dna ◽  
Clinical Samples ◽  
H Pylori

Bacteria generate membrane vesicles, which are structures known as extracellular vesicles (EVs), reported to be involved in different pathogenic mechanisms, as it has been demonstrated that EVs participate in biofilm formation, cell-to-cell communication, bacteria–host interactions, and nutrients supply. EVs deliver nucleic acids, proteins, and polysaccharides. It has been reported that Helicobacter pylori (H. pylori) and Lactobacillus reuteri (L. reuteri), of both planktonic and biofilm phenotypes, produce EVs carrying extracellular DNA (eDNA). Here, we used polychromatic flow cytometry (PFC) to identify, enumerate, and characterize EVs as well as the eDNA-delivering EV compartment in the biofilm and planktonic phenotypes of H.pylori ATCC 43629 and L. reuteri DSM 17938. Biofilm formation was demonstrated and analyzed by fluorescence microscopy, using a classical live/dead staining protocol. The enumeration of EVs and the detection of eDNA-associated EVs were performed by PFC, analyzing both whole samples (cells plus vesicles) and EVs isolated by ultracentrifugation confirm EVs isolated by ultracentrifugation. PFC analysis was performed relying on a known-size beaded system and a mix of three different fluorescent tracers. In detail, the whole EV compartment was stained by a lipophilic cationic dye (LCD), which was combined to PKH26 and PicoGreen that selectively stain lipids and DNA, respectively. Fluorescence microscopy results displayed that both H. pylori and L. reuteri produced well-structured biofilms. PFC data highlighted that, in both detected bacterial species, biofilms produced higher EVs counts when paralleled to the related planktonic phenotypes. Furthermore, the staining with PicoGreen showed that most of the generated vesicles were associated with eDNA. These data suggest that the use of PFC, set according to the parameters here described, allows for the study of the production of eDNA-associated EVs in different microbial species in the same or several phases of growth, thus opening new perspectives in the study of microbial derived EVs in clinical samples.

scholarly journals Extracellular vesicle (EV)-polyphenol nanoaggregates for microRNA-based cancer diagnosis

2019 ◽  
Vol 11 (1) ◽  
Author(s):  
Minjeong Jang ◽  
Giwoong Choi ◽  
Yoon Young Choi ◽  
Jae Eun Lee ◽  
Jik-Han Jung ◽  
...  
Keyword(s):  
Molecular Beacon ◽  
Diagnostic System ◽  
Extracellular Vesicle ◽  
Diagnostic Techniques ◽  
Clinical Samples ◽  
Human Blood Plasma ◽  
Strongly Correlated ◽  
Efficient Resource ◽  
Diagnosis Of Cancer ◽  
Geo Database

AbstractSmall extracellular vesicles (EVs), including exosomes, in body fluids have important applications in the noninvasive liquid biopsy-based diagnosis of cancer. Current EV-based diagnostic techniques still face practical challenges, such as inefficient EV isolation. Here, we report an efficient, resource-free pre-enrichment approach using (–)-epigallocatechin-3-gallate (EGCG), a polyphenolic biomolecule, to isolate and detect exosomal microRNAs (miRNAs) in human blood plasma samples. Our system comprises three steps: (1) EGCG-mediated EV aggregation, (2) filter-based EV isolation, and (3) molecular beacon-based detection of target miRNA in EVs. Using blood samples from cancer patients with gastric cancer or hepatocellular carcinoma, we constructed an EGCG-assisted miRNA diagnostic system. For both cancers, the levels of target miRNAs (miR-21, -27a, and -375) in EVs were strongly correlated with those in the publicly available GEO database. Our approach, an easy-to-use method for efficient EV isolation and the detection of miRNA in clinical samples, is applicable for molecular diagnostics in precision medicine.

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