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

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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Evaluation of Various Diagnostic Techniques for the Diagnosis of Pulmonary and Extra Pulmonary Tuberculosis at a Tertiary Care Center in North India

Infectious Disorders - Drug Targets ◽

10.2174/1871526519666191011165702 ◽

2020 ◽

Vol 20 (4) ◽

pp. 433-439

Author(s):

Monika Rajani ◽

Molay Banerjee

Keyword(s):

Care Center ◽

Tertiary Care ◽

Diagnostic System ◽

Diagnostic Techniques ◽

Automated System ◽

North India ◽

Mortality And Morbidity ◽

Indicator Tube ◽

Extra Pulmonary ◽

Uv Lamp

Introduction: Tuberculosis (TB) is a one of the main causes of mortality and morbidity worldwide. Bactec MGIT (Mycobacteria Growth Indicator Tube) system is a rapid, reliable automated system for early diagnosis of pulmonary and extra pulmonary TB in setups where purchase of expensive instruments is not possible. The present study was thus carried out to evaluate AFB microscopy, culture on Lowenstein Jensen media and micro MGIT system for early and accurate diagnosis of Tuberculosis. Methods: A total of 280 samples were processed for direct AFB smear examination, and culture on micro MGIT and LJ media. The identification of Mycobacterium tuberculosis complex in positive cultures was done by MPT64 Ag card test (BD MGIT TBC Identification Test). Results: Out of the processed samples, (47.1%) 132/280 were positive for Mycobacterium spp by Micro MGIT, (35%) 98/280 on LJ medium and (25.7%) 72/280 by AFB smear. A total of (48.5%) 136 samples were positive by a combination of Micro MGIT and LJ medium. Among the total positive samples (136/280), Micro MGIT was found to be positive in 97% (132/136) of samples, LJ was positive in 72% (98/136), while 52.9% (72/136) were positive by AFB smear. Conclusion: Manual MGIT System is a simple and efficient, safe to use the diagnostic system. It does not require any expensive/special instrumentation other than the UV lamp for the detection of fluorescence. In areas with limited resources where the purchase of expensive instruments such as the MGIT 960 is out of scope, the use of manual MGIT for rapid susceptibility testing for MDR-TB could be an option. We would recommend testing MGIT 960 using first and secondline drugs to determine DST.

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Raman Spectral Signatures of Serum-Derived Extracellular Vesicle-Enriched Isolates May Support the Diagnosis of CNS Tumors

Cancers ◽

10.3390/cancers13061407 ◽

2021 ◽

Vol 13 (6) ◽

pp. 1407

Author(s):

Matyas Bukva ◽

Gabriella Dobra ◽

Juan Gomez-Perez ◽

Krisztian Koos ◽

Maria Harmati ◽

...

Keyword(s):

Lumbar Disc ◽

Area Under The Curve ◽

Principal Component ◽

Extracellular Vesicle ◽

Classification Performance ◽

Cns Tumors ◽

Clinical Samples ◽

Support Vector ◽

Serum Samples ◽

Raman Spectroscopic

Investigating the molecular composition of small extracellular vesicles (sEVs) for tumor diagnostic purposes is becoming increasingly popular, especially for diseases for which diagnosis is challenging, such as central nervous system (CNS) malignancies. Thorough examination of the molecular content of sEVs by Raman spectroscopy is a promising but hitherto barely explored approach for these tumor types. We attempt to reveal the potential role of serum-derived sEVs in diagnosing CNS tumors through Raman spectroscopic analyses using a relevant number of clinical samples. A total of 138 serum samples were obtained from four patient groups (glioblastoma multiforme, non-small-cell lung cancer brain metastasis, meningioma and lumbar disc herniation as control). After isolation, characterization and Raman spectroscopic assessment of sEVs, the Principal Component Analysis–Support Vector Machine (PCA–SVM) algorithm was performed on the Raman spectra for pairwise classifications. Classification accuracy (CA), sensitivity, specificity and the Area Under the Curve (AUC) value derived from Receiver Operating Characteristic (ROC) analyses were used to evaluate the performance of classification. The groups compared were distinguishable with 82.9–92.5% CA, 80–95% sensitivity and 80–90% specificity. AUC scores in the range of 0.82–0.9 suggest excellent and outstanding classification performance. Our results support that Raman spectroscopic analysis of sEV-enriched isolates from serum is a promising method that could be further developed in order to be applicable in the diagnosis of CNS tumors.

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Development and Validation of a Novel M6A-related LncRNA Prognostic Signature Associated with Immune Microenvironment for Ovarian Serous Cystadenocarcinoma

10.21203/rs.3.rs-1120501/v1 ◽

2021 ◽

Author(s):

Rui Geng ◽

Tian Chen ◽

Zihang Zhong ◽

Senmiao Ni ◽

Jianling Bai ◽

...

Keyword(s):

Immune Modulation ◽

Cox Regression ◽

Great Influence ◽

Clinical Samples ◽

Consensus Clustering ◽

Prognostic Signature ◽

Cancer Stemness ◽

Gynecological Malignancy ◽

Pan Cancer ◽

Geo Database

Abstract Background: OV is the most lethal gynecological malignancy. M6A and lncRNAs have great influence on OV development and patients' immunotherapy response. Here, we decided to establish a reliable signature in the light of mRLs. Method: The lncRNAs associated with m6A in OV were analyzed and obtained by co-expression analysis in the light of TCGA-OV database. Univariate, LASSO and multivariate Cox regression analyses were employed to establish the model in the light of the mRLs. K-M analysis, PCA, GSEA, and nomogram based on the TCGA-OV and GEO database were conducted to prove the predictive value and independence of the model. The underlying relationship between the model and TME and cancer stemness properties were further investigated through immune features comparison, consensus clustering analysis, and Pan-cancer analysis.Results: A prognostic signature comprising four mRLs: WAC-AS1, LINC00997, DNM3OS, and FOXN3-AS1, was constructed and verified for OV according to TCGA and GEO database. The expressions of the four mRLs were confirmed by qRT-PCR in clinical samples. Applying this signature, people can identify patients more effectively. All the sample were assigned into two clusters, and the clusters had different overall survival, clinical features, and tumor microenvironment. Finally, Pan-cancer analysis further demonstrated the four mRLs significantly related to immune infiltration, TME and cancer stemness properties in various cancer types. Conclusion: This study provided an accurate prognostic signature for patients with OV and elucidated the potential mechanism of the mRLs in immune modulation and treatment response, giving new insights into identifying new therapeutic targets.

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Studies on Diagnosis of Bovine Brucellosis by Immunocyto-chemistry and Immunohistochemistry

Indian Journal of Animal Research ◽

10.18805/ijar.b-4203 ◽

2021 ◽

Author(s):

K.S. Lakshmikanth ◽

N.S. Sharma ◽

D. Pathak ◽

Paviter Kaur

Keyword(s):

Biochemical Analysis ◽

Placental Tissue ◽

Diagnostic Techniques ◽

Clinical Samples ◽

Bovine Brucellosis ◽

Reproductive Disorders ◽

Chain Reaction ◽

Tissue Samples ◽

Polymerase Chain ◽

Positive Results

Background: Brucellosis is a major threat to livestock economy and an important zoonotic disease. A rapid and accurate diagnosis is a necessity to curb the spread and progress of the disease. The current study aimed to evaluate sensitivity of Immunocytochemistry and Immunohistochemistry methods for detection of Brucella spp.Methods: A total of 50 samples comprising of fetal stomach content, vaginal discharges and placenta were collected from cattle and buffaloes suffering from abortions and other reproductive disorders in and around Ludhiana, Punjab during the period 2017-2018. All the samples were processed for isolation and confirmed with biochemical analysis and Polymerase chain reaction (PCR). The isolates obtained and 43 clinical samples excluding placental samples were subjected to Immunocytochemistry (ICC). Immunohistochemistry (ICH) was performed on placental samples.Result: A total of four isolates were recovered from the screened samples. The four isolates also yielded positive results in Immunocytochemistry. Among the 43 clinical samples screened by Immunocytochemistry, five were positive, however only 3 isolates were recovered on isolation. A total of seven placental tissue samples were processed and subjected to immunohistochemistry. Of the three placental samples positive by immunohistochemistry, only one sample was isolated on culture. The results suggest that both immunocytochemistry and immunohistochemistry are sensitive diagnostic techniques in comparison to isolation.

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Clinical applications of spectroscopic techniques in conjunction with multivariate analysis in virus diagnosis

Biomedical Spectroscopy and Imaging ◽

10.3233/bsi-210213 ◽

2021 ◽

pp. 1-27

Author(s):

Marfran C. D. Santos ◽

João V. M. Mariz ◽

Raissa V. O. Silva ◽

Camilo L. M. Morais ◽

Kássio M. G. Lima

Keyword(s):

Multivariate Analysis ◽

Near Infrared ◽

Low Cost ◽

Diagnostic Techniques ◽

Clinical Samples ◽

Spectroscopic Techniques ◽

Global Pandemic ◽

Molecular Fluorescence ◽

Viral Diagnosis ◽

Near Future

In view of the global pandemic that started in 2020, caused by COVID-19, the importance of the existence of fast, reliable, cheap diagnostic techniques capable of detecting the virus even in the first days of infection became evident. This review discusses studies involving the use of spectroscopic techniques in the detection of viruses in clinical samples. Techniques based on mid-infrared, near-infrared, Raman, and molecular fluorescence are explained and it was demonstrated how they can be used in conjunction with computational tools of multivariate analysis to build models capable of detecting viruses. Studies that used real clinical samples from 2011 to 2021 were analyzed. The results demonstrate the potential of the techniques in detecting viruses. Spectroscopic techniques, as well as chemometric techniques, were also explained. Viral diagnosis based on spectroscopy has interesting advantages compared to standard techniques such as: fast results, no need for reagents, non-destructiveness for the sample, no need for sample preparation, relatively low cost, among others. Several studies have corroborated the real possibility that, in the near future, we may have spectroscopic tools being successfully applied in viral diagnosis.

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Diagnosis of melioidosis: the role of molecular techniques

Future Microbiology ◽

10.2217/fmb-2020-0202 ◽

2021 ◽

Vol 16 (4) ◽

pp. 271-288

Author(s):

Ian Gassiep ◽

Delaney Burnard ◽

Michelle J Bauer ◽

Robert E Norton ◽

Patrick N Harris

Keyword(s):

Laboratory Diagnosis ◽

Diagnostic Techniques ◽

Molecular Techniques ◽

Molecular Diagnostic ◽

Clinical Samples ◽

Molecular Diagnostic Techniques ◽

Detection And Identification ◽

Life Years ◽

Culture Independent ◽

Future Utility

Melioidosis is an emerging infectious disease with an estimated global burden of 4.64 million disability-adjusted life years per year. A major determinant related to poor disease outcomes is delay to diagnosis due to the fact that identification of the causative agent Burkholderia pseudomallei may be challenging. Over the last 25 years, advances in molecular diagnostic techniques have resulted in the potential for rapid and accurate organism detection and identification direct from clinical samples. While these methods are not yet routine in clinical practice, laboratory diagnosis of infectious diseases is transitioning to culture-independent techniques. This review article aims to evaluate molecular methods for melioidosis diagnosis direct from clinical samples and discuss current and future utility and limitations.

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Diagnostic techniques in the assessment of haematological malignancies

Oxford Textbook of Medicine ◽

10.1093/med/9780198746690.003.0512 ◽

2020 ◽

pp. 5181-5188

Author(s):

Wendy N. Erber

Keyword(s):

Bone Marrow ◽

Blood Count ◽

Molecular Genetic ◽

Diagnostic Techniques ◽

Blood Film ◽

Clinical Samples ◽

Clinical Relapse ◽

Haematological Malignancies ◽

Prognostic Prediction ◽

Detection Of Mutations

The diagnosis of haematological malignancies requires an understanding of the diseases and the uses and limitations of the range of available investigations. The relative importance of different investigations varies by disease entity. The blood count is one of the most widely used tests in all of medicine and often the first indication of an underlying haematological malignancy. Some blood count features are ‘diagnostic’ and others may give an indication of a bone marrow defect. Morphological assessment of a stained blood film adds value to an abnormal blood count. It may identify abnormal morphology of red cells, leucocytes, or platelets which may be specific and diagnostic, or give clues suggesting a diagnosis. Bone marrow aspirate (liquid sample) gives cytological detail, and trephine biopsy provides information about marrow cellularity, architecture, cellular distribution, and extent of fibrosis. Immunophenotyping detects cellular antigens in clinical samples and is essential in the diagnosis and classification of haematological malignancies. It is also used for disease staging and monitoring, to detect surrogate markers of genetic aberrations, identify potential immunotherapeutic targets, and to aid prognostic prediction. Cytogenetics assesses the number and structure of whole chromosomes and chromosomal regions in neoplastic cells and is performed to diagnose and classify some haematological malignancies. Molecular genetic methods facilitate the detection of mutations, rearrangements, or translocations in genes. Applications in malignant haematology include confirming clonality, detecting disease-associated genotypes, determining prognosis, disease monitoring following therapy, predicting imminent clinical relapse, and identifying patients who are likely (or not) to respond to new targeted inhibitor therapies.

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A facile deoxyuridine/biotin-modified molecular beacon for simultaneous detection of proteins and nucleic acids via a label-free and background-eliminated fluorescence assay

The Analyst ◽

10.1039/c9an01016e ◽

2019 ◽

Vol 144 (18) ◽

pp. 5504-5510 ◽

Cited By ~ 2

Author(s):

Fei Yin ◽

Liqi Liu ◽

Xia Sun ◽

Laiyong Hou ◽

Yu Lu ◽

...

Keyword(s):

Early Diagnosis ◽

Nucleic Acids ◽

Molecular Beacon ◽

Simultaneous Detection ◽

Cancer Biomarkers ◽

Label Free ◽

Early Diagnosis Of Cancer ◽

Different Types ◽

Diagnosis Of Cancer ◽

Modified Molecular Beacon

Simultaneous detection of different types of cancer biomarkers (nucleic acids and proteins) could facilitate early diagnosis of cancer and clinical treatment.

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Isolation of extracellular vesicles improves the detection of mutant DNA from plasma of metastatic melanoma patients

Scientific Reports ◽

10.1038/s41598-020-72834-6 ◽

2020 ◽

Vol 10 (1) ◽

Cited By ~ 3

Author(s):

Davide Zocco ◽

Simona Bernardi ◽

Mauro Novelli ◽

Chiara Astrua ◽

Paolo Fava ◽

...

Keyword(s):

Metastatic Melanoma ◽

Braf Inhibitor ◽

Extracellular Vesicle ◽

Therapeutic Monitoring ◽

Clinical Samples ◽

Outer Side ◽

Alternative Source ◽

Gene Copies ◽

Melanoma Patients ◽

Promising Source

Abstract Detection of BRAFV600E within cell free tumor DNA (ctDNA) is emerging as a promising means to improve patients’ stratification or enable BRAF inhibitor (BRAFi) therapeutic monitoring in a minimally invasive manner. Here, we investigated whether extracellular vesicle-(EV)-associated-DNA (EV-DNA) has value as an alternative source of circulating BRAFV600E. To do so, we identified a clinical practice-compatible protocol for the isolation of EV-DNA and assessed BRAF gene status on plasma samples from metastatic melanoma patients at the beginning and during BRAFi therapy. This protocol uses a peptide with high affinity for EVs and it has been found to recover more mutant DNA from plasma than standard ultracentrifugation. Molecular analyses revealed that mutant DNA is largely unprotected from nuclease digestion, interacting with the outer side of the EV membrane or directly with the peptide. When used on clinical samples, we found that the protocol improves the detection of BRAFV600E gene copies in comparison to the reference protocol for ctDNA isolation. Taken together, these findings indicate that EVs are a promising source of mutant DNA and should be considered for the development of next-generation liquid biopsy approaches.

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