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Keywords = EV-associated DNA (EV-DNA)

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30 pages, 883 KiB  
Review
Approaches and Challenges in Characterizing the Molecular Content of Extracellular Vesicles for Biomarker Discovery
by Suman Kumari, Christopher Lausted, Kelsey Scherler, Alphonsus H. C. Ng, Yue Lu, Inyoul Lee, Leroy Hood and Kai Wang
Biomolecules 2024, 14(12), 1599; https://doi.org/10.3390/biom14121599 - 14 Dec 2024
Viewed by 871
Abstract
Extracellular vesicles (EVs) are lipid bilayer nanoparticles released from all known cells and are involved in cell-to-cell communication via their molecular content. EVs have been found in all tissues and body fluids, carrying a variety of biomolecules, including DNA, RNA, proteins, metabolites, and [...] Read more.
Extracellular vesicles (EVs) are lipid bilayer nanoparticles released from all known cells and are involved in cell-to-cell communication via their molecular content. EVs have been found in all tissues and body fluids, carrying a variety of biomolecules, including DNA, RNA, proteins, metabolites, and lipids, offering insights into cellular and pathophysiological conditions. Despite the emergence of EVs and their molecular contents as important biological indicators, it remains difficult to explore EV-mediated biological processes due to their small size and heterogeneity and the technical challenges in characterizing their molecular content. EV-associated small RNAs, especially microRNAs, have been extensively studied. However, other less characterized RNAs, including protein-coding mRNAs, long noncoding RNAs, circular RNAs, and tRNAs, have also been found in EVs. Furthermore, the EV-associated proteins can be used to distinguish different types of EVs. The spectrum of EV-associated RNAs, as well as proteins, may be associated with different pathophysiological conditions. Therefore, the ability to comprehensively characterize EVs’ molecular content is critical for understanding their biological function and potential applications in disease diagnosis. Here, we set out to provide an overview of EV-associated RNAs and proteins as well as approaches currently being used to characterize them. Full article
(This article belongs to the Special Issue Extracellular Vesicles as Biomarkers of Diseases)
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<p>Depiction of EV subtypes and their biogenesis. Exosomes, a type of small EVs, are formed from the invagination of the endosomal membrane. Small ectosomes, another type of sEVs, are generated by the outward budding of the membrane. Microvesicles, indicated as large EVs, are produced through the direct budding of the plasma membrane. Apoptotic vesicles, a type of lEV, are produced by blebbing and fragmentation during apoptosis.</p>
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16 pages, 2534 KiB  
Article
Molecular Profiling of Endocrine Resistance in HR+/HER2-Metastatic Breast Cancer: Insights from Extracellular Vesicles-Derived DNA and ctDNA in Liquid Biopsies
by Ana Martínez-Rodríguez, Jesús Fuentes-Antrás, Víctor Lorca, Alfonso López de Sá, Pedro Pérez-Segura, Fernando Moreno, Jose Angel García-Sáenz and Vanesa García-Barberán
Int. J. Mol. Sci. 2024, 25(23), 13045; https://doi.org/10.3390/ijms252313045 - 4 Dec 2024
Viewed by 696
Abstract
Standard treatments in hormone receptor-positive (HR+)/HER2-metastatic breast cancer (mBC) typically involve endocrine therapy (ET) combined with CDK4/6 inhibitors, yet resistance to ET remains a persistent challenge in advanced cases. A deeper knowledge of the use of liquid biopsy is crucial for the implementation [...] Read more.
Standard treatments in hormone receptor-positive (HR+)/HER2-metastatic breast cancer (mBC) typically involve endocrine therapy (ET) combined with CDK4/6 inhibitors, yet resistance to ET remains a persistent challenge in advanced cases. A deeper knowledge of the use of liquid biopsy is crucial for the implementation of precision medicine in mBC with real-time treatment guidance. Our study assesses the prognostic value of PIK3CA and ESR1 mutations in DNA derived from extracellular vesicles (EV-DNA) in longitudinal plasma from 59 HR+/HER2-mBC patients previously exposed to aromatase inhibitors, with a comparative analysis against circulating tumor DNA (ctDNA). Mutations were evaluated by digital PCR. PIK3CA and ESR1 mutations were found in 22 and 25% of patients. Baseline ESR1 mutations in EV-DNA were associated with shorter progression-free survival (PFS) across the cohort, with the Y537S mutation showing a particularly strong impact on the outcome of fulvestrant-treated patients. In contrast, PIK3CA mutations in EV-DNA did not significantly correlate with PFS, whereas in ctDNA, they were linked to poor outcomes. Altogether, this study positions EV-DNA as a valuable biomarker alongside ctDNA, enriching the understanding of different analytes in liquid biopsy and supporting strategies for HR+/HER2-mBC in precision oncology. Full article
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<p>Distribution and dynamics of <span class="html-italic">ESR1</span> and <span class="html-italic">PIK3CA</span> mutations. Frequency of gene mutations at T0, T1, and T2 (<b>A</b>). Frequency of <span class="html-italic">ESR1</span> mutations for each codon according to study treatment (<b>B</b>). VAF mean and SD detected in <span class="html-italic">ESR1</span> at T1 according to study treatment (<b>C</b>). Frequency of specific <span class="html-italic">PIK3CA</span> mutations according to study treatment (<b>D</b>). Frequency of gene mutations at T0, T1, and T2 according to study treatment (<b>E</b>). * <span class="html-italic">p</span> &lt; 0.05.</p>
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<p>Concordance and correlation between EV-DNA and ctDNA. (<b>A</b>) Concordance of all digital PCR assessments for <span class="html-italic">PIK3CA</span> and <span class="html-italic">ESR1</span> genes. PPA, positive percentage agreement; NPA, negative percentage agreement; OPA, overall percentage agreement; Kappa, Cohen’s kappa coefficient. Correlation of VAFs between EV-DNA and ctDNA for each <span class="html-italic">PIK3CA</span> mutation (<b>B</b>–<b>D</b>). Correlation of VAFs between EV-DNA and ctDNA for each <span class="html-italic">ESR1</span> mutation (<b>E</b>,<b>F</b>). Comparison of VAFs between ctDNA and EV-DNA in mutated cases (<b>G</b>). *** <span class="html-italic">p</span> &lt; 0.001.</p>
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<p>Comparison of concordant and discordant results between EV-DNA and ctDNA. VAFs and cop/µL distribution in concordant samples compared to discordant samples (<b>A</b>,<b>B</b>). Differences in cop/µL based on discordancy type and concordance (<b>C</b>,<b>D</b>). Percentage of total concentration for each size range in: EV-DNA and cfDNA (<b>E</b>), EV-DNA in concordant and discordant samples (<b>F</b>), cfDNA in concordant and discordant samples (<b>G</b>). * <span class="html-italic">p</span> &lt; 0.05, *** <span class="html-italic">p</span> &lt; 0.001, ns = not significant.</p>
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<p>Prognostic value of <span class="html-italic">ESR1</span> and <span class="html-italic">PIK3CA</span> mutations at baseline. Prognostic value of <span class="html-italic">ESR1</span> mutations in EV-DNA in the entire cohort (<b>A</b>), in patients receiving AI-based therapy (<b>B</b>), and in patients receiving fulvestrant-based therapy (<b>C</b>). Prognostic value of Y537S mutation in EV-DNA in patients receiving fulvestrant-based therapy (<b>D</b>) and fulvestrant monotherapy (<b>E</b>). Prognostic value in the entire cohort of <span class="html-italic">ESR1</span> mutations in ctDNA (<b>F</b>) and combining EV-DNA and ctDNA (<b>G</b>). Prognostic value of <span class="html-italic">PIK3CA</span> mutations in EV-DNA (<b>H</b>) and ctDNA (<b>I</b>) in the entire cohort. ** <span class="html-italic">p</span> &lt; 0.01, ns = not significant.</p>
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15 pages, 2466 KiB  
Article
Profiling Plasma Extracellular Vesicle Metabotypes and miRNAs: An Unobserved Clue for Predicting Relapse in Patients with Early-Stage NSCLC
by Vivi Bafiti, Eleni Thanou, Sotiris Ouzounis, Athanasios Kotsakis, Vasilis Georgoulias, Evi Lianidou, Theodora Katsila and Athina Markou
Cancers 2024, 16(22), 3729; https://doi.org/10.3390/cancers16223729 - 5 Nov 2024
Viewed by 980
Abstract
Background and Objective: Lung cancer, the second most prevalent cancer globally, poses significant challenges in early detection and prognostic assessment. Despite advancements in targeted therapies and immunotherapy, the timely identification of relapse remains elusive. Blood-based liquid biopsy biomarkers, including circulating tumor cells (CTCs), [...] Read more.
Background and Objective: Lung cancer, the second most prevalent cancer globally, poses significant challenges in early detection and prognostic assessment. Despite advancements in targeted therapies and immunotherapy, the timely identification of relapse remains elusive. Blood-based liquid biopsy biomarkers, including circulating tumor cells (CTCs), cell-free DNA (cfDNA), circulating tumor DNA (ctDNA), circulating-free RNAs (cfRNAs), and extracellular vesicles (EVs)/exosomes, offer promise for non-invasive monitoring. Methods: We employ a comprehensive approach integrating miRNA/lncRNA/metabolomic datasets, following a mixed-methods content analysis, to identify candidate biomarkers in NSCLC. NSCLC-associated miRNA/gene/lncRNA associations were linked to in silico-derived molecular pathways. Results: For data validation, mass spectrometry-based untargeted metabolomics of plasma EVs highlighted miRNA/lncRNA/metabotypes, linking “glycerophospholipid metabolism” to lncRNA H19 and “alanine, aspartate and glutamate metabolism” to miR-29a-3p. Prognostic significance was established for miR-29a-3p, showing lower expression in NSCLC patients with disease progression compared to stable disease (p = 0.004). Kaplan–Meier survival analysis indicated that patients with miR-29a-3p under-expression had significantly shorter overall survival (OS) (p = 0.038). Despite the expression of lncRNA H19 in plasma EVs being undetected, its expression in plasma cfRNAs correlated significantly with disease progression (p = 0.035). Conclusions: Herein, we showcase the potential of plasma EV-derived miR-29a-3p as a prognostic biomarker and underscore the intricate interplay of miRNAs, lncRNAs, and metabolites in NSCLC biology. Our findings offer new insights and avenues for further exploration, contributing to the ongoing quest for effective biomarkers in early-stage NSCLC. Full article
(This article belongs to the Special Issue RNA in Non-Small-Cell Lung Cancer)
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<p>A graphical representation of the in silico pipeline designed and employed to identify miRNAs and lncRNAs that can serve as candidate biomarkers for NSCLC. Transcriptomics datasets were retrieved from the GEO database and the DGE analysis was performed with the GEO2R tool. NSCLC lncRNAs were mined through LncTarD2.0 and then cross-linked with miRNAs-DEGs. Pathway analysis was conducted based on the identified lncRNAs and miRNAs. Next, cross-omics data integration between RNA and EV metabotypes was implemented to reveal candidate biomarkers in NSCLC.</p>
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<p>Network representation of the NSCLC-related gene, miRNA, and lncRNA associations. (<b>A</b>) miRNA–gene associations (a directed graph); red nodes: up-regulated genes; blue nodes: down-regulated genes; purple nodes: miRNAs. The nodes are interconnected with arrowed edges indicating the direction of the association. (<b>B</b>) miRNA/gene/lncRNA associations; two types of nodes are depicted, also in different shapes. Rectangular nodes: target elements; circular nodes: regulatory elements; blue nodes: down-regulated lncRNAs; red notes: up-regulated lncRNAs; yellow nodes: transcription factors; purple nodes: miRNAs; green nodes: protein-coding genes. Three edge types indicate the relationships among the nodes in question; solid edges: regulatory relationships; dotted edges: binding or interaction; double-dashed edges: associations; edge colors: regulation direction; black edges: an increase in expression (positively E); purple edges: a decrease in expression (negatively E); orange edges: a decrease in function (negatively F); blue edges: a positive function (positively F); and red edges: an interaction between nodes (interact). Nodes with bold outlines: candidate biomarkers.</p>
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<p>Untargeted metabolomics in plasma EVs from NSCLC patients reveals key perturbed metabolic pathways. Metabolite Set Enrichment Analysis (MSEA) was performed using Metaboanalyst v.6. The enriched pathways were ranked by significance, as indicated by the color scale (top ten pathways with <span class="html-italic">p</span>-values &lt; 0.05).</p>
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<p>(<b>A</b>) Relative-fold change (2<sup>−ΔΔCq</sup>) of <span class="html-italic">miR-29a-3p</span> in EVs from early-stage NSCLC patient samples in terms of relapse and survival, (<b>B</b>) Kaplan–Meier estimates of OS for NSCLC patients with respect to <span class="html-italic">miR-29a-3p</span> expression. ** <span class="html-italic">p</span> ≤ 0.01.</p>
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14 pages, 1987 KiB  
Communication
Phenotypic Biomarkers of Aqueous Extracellular Vesicles from Retinoblastoma Eyes
by Anne Amacker, Chen-Ching Peng, Nan Jiang, Shreya Sirivolu, Nikki Higa, Kevin Stachelek, Bibiana Reiser, Peter Kuhn, David Cobrinik, Paolo Neviani, Jesse L. Berry, Tijana Jovanovic-Talisman and Liya Xu
Int. J. Mol. Sci. 2024, 25(21), 11660; https://doi.org/10.3390/ijms252111660 - 30 Oct 2024
Viewed by 858
Abstract
Recent advancements in aqueous humor (AH) cell-free DNA (cfDNA) genomics have opened new avenues for ex vivo molecular profiling of retinoblastoma (RB), the most common pediatric intraocular malignancy, where biopsy is typically prohibited. While these insights offer a genetic blueprint of the tumor, [...] Read more.
Recent advancements in aqueous humor (AH) cell-free DNA (cfDNA) genomics have opened new avenues for ex vivo molecular profiling of retinoblastoma (RB), the most common pediatric intraocular malignancy, where biopsy is typically prohibited. While these insights offer a genetic blueprint of the tumor, they lack multi-omic molecular phenotyping, which is essential for understanding the functional state. Extracellular vesicles (EVs), naturally present in AH, are promising by offering time-resolved phenotypic information. We employed multiplex bead-based flow cytometry and Single Extracellular Vesicle Nanoscopy (SEVEN) to analyze EV phenotypes in AH from a cohort of five RB, with three uveal melanoma (UM) and two age-matched glaucoma (GLC) samples serving as controls. The studies identified CD133-enriched EVs uniquely in RB AH, absent in both GLC and UM AH. This was corroborated by further analysis of five RB cell lines, including two commercial (Y79, Weri) and three in-house developed lines, confirming CD133 enrichment and supporting its role as an RB-specific EV marker. Single-vesicle analysis demonstrated a strong association of CD133 with CD81 and CD63, with minimal CD9 presence. These results, validated through complementary techniques, position CD133 as a critical marker in RB-derived EVs, paving the way for enhanced multi-omic RB characterization and potential advancements in clinical diagnostics. Full article
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<p>Surface marker profiles of EV/EPs in aqueous humor from RB, GLC, and UM cases were analyzed using multiplex bead-based flow cytometry. (<b>A</b>) Workflow design of the AH MACSPlex experiment. (<b>B</b>) Mean Fluorescence Intensity bar plots for two GLC aqueous humor samples, scaled to 5 μL. (<b>C</b>) Mean Fluorescence Intensity bar plots for three Uveal Melanoma samples collected prior to treatment. (<b>D</b>) Mean Fluorescence Intensity bar plots for five RB aqueous humor samples collected at the time of primary enucleation.</p>
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<p>The ratio of surface marker mean fluorescence intensity (MFI) to mean tetraspanin MFI from RB, GLC, and UM cases using MACSPlex analysis. (<b>A</b>) Heatmap depicting the MFI for each surface marker compared to the mean CD9/63/81 MFI for each GLC AH sample, UM AH sample, RB AH sample, and RB cell line. (<b>B</b>) The ratio of CD133 tetraspanin signal to mean tetraspanin MFI for all AH samples and RB cell lines included in the analysis.</p>
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<p>Concordance of MACSPlex Individual Channel Results and SP-IRIS Analysis in Aqueous Humor Sample Case 79. (<b>A</b>) Single and triple-channel MACSPlex experiments were conducted on the AH sample Case 79, where APC-conjugated CD9, CD63, or CD81 antibodies were individually added and processed through flow cytometry. Mean Fluorescence Intensity (MFI) analysis of CD9, CD63, CD81, and CD133 of an RB aqueous humor sample (Case 79) is presented for the antibody mix, CD9-only, CD63-only, and CD81-only experiments. (<b>B</b>) The same Case 79 AH underwent SP-IRIS analysis using the ExoviewR100 system for tetraspanin expression profiling. Representative fluorescent images were captured by fluorescent-conjugated antibodies (red = CD63-AF647, green = CD81-AF555, and blue = CD9-AF488). (<b>C</b>) Subpopulation breakdown of single and double positive EV populations using MACSPlex results compared to Exoview on Case 79.</p>
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<p>Multiparametric characterization of single EVs from AH using SEVENi. (<b>A</b>) Representative raw SMLM images of zoomed-in regions; sample dilutions are indicated in the methods section. (<b>B</b>) Number of detected EVs per region of interest normalized for 1 μL of AH; Error bars, SEM. (<b>C</b>) Distribution sizes, molecular contents, and circularity for detected EVs. Boxes denote interquartile ranges, center lines denote medians, crosses denote means, and the dots denote EVs beyond 1.5 times the interquartile range. <span class="html-italic">n</span> = 3 technical replicates (15 ROIs) for CD63/CD81 capture and CD133 capture, <span class="html-italic">n</span> = 4 technical replicates (20 ROIs) for CD9 capture and IgG capture; ** <span class="html-italic">p</span> &lt; 0.01; **** <span class="html-italic">p</span> &lt; 0.0001; ns: not significant. Values and statistics are included in <a href="#app1-ijms-25-11660" class="html-app">Tables S1 and S2</a>.</p>
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18 pages, 3677 KiB  
Article
Differential Protein Expression in Extracellular Vesicles Defines Treatment Responders and Non-Responders in Multiple Sclerosis
by Gabriel Torres Iglesias, MariPaz López-Molina, Lucía Botella, Fernando Laso-García, Beatriz Chamorro, Mireya Fernández-Fournier, Inmaculada Puertas, Susana B. Bravo, Elisa Alonso-López, Exuperio Díez-Tejedor, María Gutiérrez-Fernández and Laura Otero-Ortega
Int. J. Mol. Sci. 2024, 25(19), 10761; https://doi.org/10.3390/ijms251910761 - 6 Oct 2024
Viewed by 1489
Abstract
Multiple sclerosis (MS) remains the leading cause of neurological disability among young adults worldwide, underscoring the urgent need to define the best therapeutic strategy. Recent advances in proteomics have deepened our understanding of treatment mechanisms and revealed promising biomarkers for predicting therapeutic outcomes. [...] Read more.
Multiple sclerosis (MS) remains the leading cause of neurological disability among young adults worldwide, underscoring the urgent need to define the best therapeutic strategy. Recent advances in proteomics have deepened our understanding of treatment mechanisms and revealed promising biomarkers for predicting therapeutic outcomes. This study focuses on the identification of a protein profile of circulating extracellular vesicles (EVs) derived from neurons, oligodendrocytes, and B and T cells able to differentiate treatment responders and non-responders in 80 patients with MS. In the patients who responded to treatment, T cell-derived EVs were enriched in LV151, a protein involved in the promotion of anti-inflammatory cytokines, whereas Bcell-derived EVs showed elevated PSMD6 and PTPRC, related to immunoproteasome function. Oligodendrocyte- and neuron-derived EVs showed upregulated CO6A1 and COEA1, involved in extracellular matrix reorganisation, as well as LAMA5, NonO, SPNT, and NCAM, which are critical for brain repair. In contrast, non-responders showed higher levels of PSMD7 and PRS10 from B cell-derived EVs, associated with DNA damage, and increased levels of PERM and PERL from T cell-derived EVs, linked to nuclear factor kappa B activation and drug-resistant proteins such as HS90A and RASK. These findings highlight a distinct panel of proteins in EVs that could serve as an early indicator of treatment efficacy in MS. Full article
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<p>Flow diagram of the study illustrating the procedures applied to patients with MS and their distributions into groups. The blue figures represent responders, while the gray figure indicates non-responders. Abbreviations: EDSS, expanded disability status scale; MRI, magnetic resonance imaging; NEDA, no evidence of disease activity; EV, extracellular vesicles; MS, multiple sclerosis.</p>
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<p>(<b>A</b>) Electron microscope image of EVs smaller than 200 nm. (<b>B</b>) Size dispersion and concentration of EV samples analysed by NTA. (<b>C</b>) Western blot image demonstrating the positivity of specific markers CD9, CD81, and CD63 in the EV membrane. Negative control samples are from plasma. The gel image was cropped. Abbreviations: EV, extracellular vesicles; kDa, Kilodalton.</p>
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<p>The differentially expressed proteins analysed by volcano plots between responders and non-responders in PRE and POST in (<b>A</b>) T cells, (<b>B</b>) B cells, (<b>C</b>) neurons, and (<b>D</b>) oligodendrocytes. The red dots represent proteins common to both groups, while the black dots represent differentially expressed proteins.</p>
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<p>(<b>A</b>) Differentially upregulated proteins identified in responders. (<b>B</b>) Most differentially overexpressed proteins in non-responders. The blue color represents responder patients, while the green color indicates non-responder patients. * <span class="html-italic">p</span> &lt; 0.05.</p>
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<p>(<b>A</b>) Venn diagram showing the differentially expressed and shared proteins in responders and non-responders. (<b>B</b>) Pathway enrichment analysis using reactome databases (accessed on 16 August 2023) of 133 differentially unregulated proteins from responders (in blue) and 137 proteins from non-responders (in green). (<b>C</b>) Functional enrichment analysis using FunRich tool of the upregulated proteins in T cells, B cells, neurons, and oligodendrocytes in EVs in PRE- and POST-treatment of responders and non-responders. The blue color represents functions present in responder patients, the green color indicates functions in non-responder patients, and the yellow color represents those common to both groups.</p>
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<p>(<b>A</b>) Protein–protein interaction network for proteins described in responders and non-responders. (<b>B</b>) Heatmap analysis of differentially expressed proteins in responders vs. non-responders in T cells, B cells, neurons, and oligodendrocytes. In the colour bar, red represents high expression, and grey represents low expression.</p>
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13 pages, 2152 KiB  
Article
Association of Serum Extracellular Vesicle miRNAs with Cognitive Functioning and Quality of Life in Parkinson’s Disease
by Paulina Vaitkienė, Aistė Pranckevičienė, Andrius Radžiūnas, Augustina Mišeikaitė, Giedrė Miniotaitė, Violeta Belickienė, Ovidijus Laucius and Vytenis Deltuva
Biomolecules 2024, 14(8), 1000; https://doi.org/10.3390/biom14081000 - 13 Aug 2024
Viewed by 1217
Abstract
The identification of mechanisms associated with Parkinson disease (PD) development in cognitive functioning would be of great usefulness to clarify PD pathogenesis and to develop preventive and therapeutic strategies. In this study, blood serum extracellular vesicle (EV) levels of the candidate microRNAs (small [...] Read more.
The identification of mechanisms associated with Parkinson disease (PD) development in cognitive functioning would be of great usefulness to clarify PD pathogenesis and to develop preventive and therapeutic strategies. In this study, blood serum extracellular vesicle (EV) levels of the candidate microRNAs (small noncoding RNAs that play a role in gene expression regulation):,miR-7, miR-21, miR-153, miR-155, miR-200a and miR-214, have been investigated for association with PD in a group of 93 patients with cognitive parameters, PD symptoms, affected quality of life and some clinical characteristics. MiRNA was extracted from patients’ blood serum EVs, transcribed into cDNA and their expression was evaluated using RT-PCR. The miR-153 and miR-200a showed the most plausible correlations with cognitive functioning parameters such as general intellectual functioning, psychomotor speed, mental flexibility, and nonverbal executive functions. Moreover, lower levels of miR-153 were associated with attention span, working memory and psychomotor speed with learning. Increased levels of miR-200a, miR-7, miR-214, and miR-155 were also linked with neurological functioning, such as bradykinesia, tremor, balance and others. Despite the fact that due to small sample size, our results should be considered as preliminary, our study suggests that miRNA expression in EVs could be associated with symptom severity, cognitive impairment and quality of life in PD. Full article
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<p>MiRNA expression and intensity of bradykinesia, balance difficulty, ON/OFF phenomena occurrence, severity of dyskinesia and intensity of tremor. Dots mark individual miRNA expression; whiskers mark minimum and maximum miRNA expression levels; and the black line in the middle marks the median of values. Numbers below mark patients grouped by intensity of symptoms from 0 to 4: 0—no symptoms, 1—low, 2—mild, 3—strong, and 4—severe. Expression of miRNA was normalized with cel-miR-39-3p; Student’s <span class="html-italic">t</span> test *: (<span class="html-italic">p</span> ≤ 0.05), **: (<span class="html-italic">p</span> ≤ 0.01), and ***: (<span class="html-italic">p</span> ≤ 0.001).</p>
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<p>Relationship between Parkinson’s Disease symptoms and miRNAs expression in patients’ blood serum EVs. Comprehensive illustration of the modulatory role of miRNAs in impairment in PD shows the relationship (positive and negative dependence, and strong tendency) between miRNAs and clinical functions, PD symptoms, cognitive functions, and quality of life. PD symptoms and clinical parameters were assessed as they worsened, while quality of life and cognitive parameters were evaluated as they improved.</p>
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25 pages, 5542 KiB  
Review
Extracellular Vesicles in Ovarian Cancer: From Chemoresistance Mediators to Therapeutic Vectors
by Barathan Muttiah, Nur Dina Muhammad Fuad, Faizul Jaafar and Nur Atiqah Haizum Abdullah
Biomedicines 2024, 12(8), 1806; https://doi.org/10.3390/biomedicines12081806 - 9 Aug 2024
Cited by 1 | Viewed by 1567
Abstract
Ovarian cancer (OC) remains the deadliest gynecological malignancy, with alarming projections indicating a 42% increase in new cases and a 51% rise in mortality by 2040. This review explores the challenges in OC treatment, focusing on chemoresistance mechanisms and the potential of extracellular [...] Read more.
Ovarian cancer (OC) remains the deadliest gynecological malignancy, with alarming projections indicating a 42% increase in new cases and a 51% rise in mortality by 2040. This review explores the challenges in OC treatment, focusing on chemoresistance mechanisms and the potential of extracellular vesicles (EVs) as drug delivery agents. Despite advancements in treatment strategies, including cytoreductive surgery, platinum-based chemotherapy, and targeted therapies, the high recurrence rate underscores the need for innovative approaches. Key resistance mechanisms include drug efflux, apoptosis disruption, enhanced DNA repair, cancer stem cells, immune evasion, and the complex tumor microenvironment. Cancer-associated fibroblasts and extracellular vesicles play crucial roles in modulating the tumor microenvironment and facilitating chemoresistance. EVs, naturally occurring nanovesicles, emerge as promising drug carriers due to their low toxicity, high biocompatibility, and inherent targeting capabilities. They have shown potential in delivering chemotherapeutics like doxorubicin, cisplatin, and paclitaxel, as well as natural compounds such as curcumin and berry anthocyanidins, enhancing therapeutic efficacy while reducing systemic toxicity in OC models. However, challenges such as low production yields, heterogeneity, rapid clearance, and inefficient drug loading methods need to be addressed for clinical application. Ongoing research aims to optimize EV production, loading efficiency, and targeting, paving the way for novel and more effective therapeutic strategies in OC treatment. Overcoming these obstacles is crucial to unlocking the full potential of EV-based therapies and improving outcomes for OC patients. Full article
(This article belongs to the Section Cancer Biology and Oncology)
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<p>Advanced strategies in OC treatment. Initial Treatment for OC involves cytoreductive surgery and platinum-based chemotherapy to remove tumor tissue and target residual cancer cells. Recurrent OC management varies based on platinum sensitivity, utilizing combination chemotherapy or alternative agents like topotecan. Targeted Therapies include antiangiogenic agents, PARP inhibitors, and immune checkpoint inhibitors for extended progression-free survival. Hormone Therapy explores estrogen and progesterone dependencies with aromatase inhibitors and anti-estrogen treatments for OC treatment options.</p>
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<p>The chemoresistance mechanisms in OC focusing on EV and their interactions with the TME. Cancer cells and CAFs release EVs into the extracellular space. EV contents, including miRNAs (e.g., miR-21, miR-200c), proteins (e.g., HSP90), ABC transporters, and other cargo (e.g., DNA fragments, lipid droplets), are taken up by neighboring cancer cells and distant metastatic sites. The EV cargo promotes DNA repair and inhibits apoptosis, contributing to chemoresistance. TME-mediated communication has interactions involving the ECM and EVs, leading to invasion and metastasis.</p>
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<p>Advancements highlight the growing versatility and potential of EVs as drug delivery vehicles, offering promising avenues for targeted and effective therapies across various medical fields.</p>
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67 pages, 2808 KiB  
Review
Circulating Liquid Biopsy Biomarkers in Glioblastoma: Advances and Challenges
by Attila A. Seyhan
Int. J. Mol. Sci. 2024, 25(14), 7974; https://doi.org/10.3390/ijms25147974 - 21 Jul 2024
Cited by 2 | Viewed by 2963
Abstract
Gliomas, particularly glioblastoma (GBM), represent the most prevalent and aggressive tumors of the central nervous system (CNS). Despite recent treatment advancements, patient survival rates remain low. The diagnosis of GBM traditionally relies on neuroimaging methods such as magnetic resonance imaging (MRI) or computed [...] Read more.
Gliomas, particularly glioblastoma (GBM), represent the most prevalent and aggressive tumors of the central nervous system (CNS). Despite recent treatment advancements, patient survival rates remain low. The diagnosis of GBM traditionally relies on neuroimaging methods such as magnetic resonance imaging (MRI) or computed tomography (CT) scans and postoperative confirmation via histopathological and molecular analysis. Imaging techniques struggle to differentiate between tumor progression and treatment-related changes, leading to potential misinterpretation and treatment delays. Similarly, tissue biopsies, while informative, are invasive and not suitable for monitoring ongoing treatments. These challenges have led to the emergence of liquid biopsy, particularly through blood samples, as a promising alternative for GBM diagnosis and monitoring. Presently, blood and cerebrospinal fluid (CSF) sampling offers a minimally invasive means of obtaining tumor-related information to guide therapy. The idea that blood or any biofluid tests can be used to screen many cancer types has huge potential. Tumors release various components into the bloodstream or other biofluids, including cell-free nucleic acids such as microRNAs (miRNAs), circulating tumor DNA (ctDNA), circulating tumor cells (CTCs), proteins, extracellular vesicles (EVs) or exosomes, metabolites, and other factors. These factors have been shown to cross the blood-brain barrier (BBB), presenting an opportunity for the minimally invasive monitoring of GBM as well as for the real-time assessment of distinct genetic, epigenetic, transcriptomic, proteomic, and metabolomic changes associated with brain tumors. Despite their potential, the clinical utility of liquid biopsy-based circulating biomarkers is somewhat constrained by limitations such as the absence of standardized methodologies for blood or CSF collection, analyte extraction, analysis methods, and small cohort sizes. Additionally, tissue biopsies offer more precise insights into tumor morphology and the microenvironment. Therefore, the objective of a liquid biopsy should be to complement and enhance the diagnostic accuracy and monitoring of GBM patients by providing additional information alongside traditional tissue biopsies. Moreover, utilizing a combination of diverse biomarker types may enhance clinical effectiveness compared to solely relying on one biomarker category, potentially improving diagnostic sensitivity and specificity and addressing some of the existing limitations associated with liquid biomarkers for GBM. This review presents an overview of the latest research on circulating biomarkers found in GBM blood or CSF samples, discusses their potential as diagnostic, predictive, and prognostic indicators, and discusses associated challenges and future perspectives. Full article
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<p>Updated WHO classification of tumors of the CNS. The 2021 WHO classification of CNS tumors introduced significant changes, such as limiting the diagnosis of GBM to only <span class="html-italic">IDH</span> wild type tumors, reclassifying previously diagnosed <span class="html-italic">IDH</span>-mutated GBMs as astrocytomas, <span class="html-italic">IDH</span>-mutated, and grade 4, and requiring the presence of <span class="html-italic">IDH</span> mutations for tumors to be classified as astrocytomas or oligodendrogliomas. For the abbreviations, go to the abbreviations list at the end of the text. Created with <a href="http://BioRender.com" target="_blank">BioRender.com</a> (accessed on 6 March 2023).</p>
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<p>Examples of biomarkers measured in circulation, along with the advantages and disadvantages of liquid biopsy versus tumor tissue biopsy, are shown. This schematic representation illustrates circulating biomarkers released from the tumor into the bloodstream through the partially disrupted BBB. These biomarkers may also be directly secreted into the CSF. In patients with GBM, a compromised BBB allows circulating biomarkers such as ctDNAs, miRNAs, EVs, CTCs, proteins, and metabolites to enter the bloodstream or CSF. These biomarkers can be collected through blood or CSF draws and subsequently analyzed. The illustration provides a breakdown of tumoral components within the circulatory system. Various analytical methods, including PCR, qRT-PCR, NGS, WGS, immunoaffinity capture, ELISA, mass spectrometry, chemiluminescent immunoassay, and density gradient centrifugation, have been used to detect circulating analytes. Each circulating analyte can be assessed for tumor-specific changes such as various types of mutations, epigenetic modifications, DNA fragmentation patterns, nucleosome patterning, chromosomal aberrations, and the presence, absence, or changes in levels of ctDNAs, miRNAs (and other noncoding RNAs as well as mRNAs), CTCs, proteins, cytokines, metabolites, EVs, or exosomes, along with post-translational modifications. Each type of biomarker detection method, whether blood- or CSF-based or tissue-based, has unique advantages and disadvantages in diagnosing and monitoring GBM patients. Abbreviations: BBB, blood–brain barrier; CSF, cerebrospinal fluid; CTCs, circulating tumor cells; ctDNA, circulating tumor DNA; DNA, deoxyribonucleic acid; EVs, extracellular vesicles; NGS, next-generation sequencing; PCR, polymerase chain reaction; RNA, ribonucleic acid. Created with <a href="http://BioRender.com" target="_blank">BioRender.com</a> (accessed on 11 July 2024).</p>
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<p>Comparison of examples of liquid biopsy techniques, highlighting their capabilities, shortcomings, and available technologies. Abbreviations: CNA, copy number alterations; CTC, circulating tumor cells; ctDNA, circulating tumor DNA; DNA, deoxyribonucleic acid; ddPCR, droplet digital PCR; miRNA, microRNA; NGS, next-generation sequencing; PCR, polymerase chain reaction; RNA, ribonucleic acid. Created with <a href="http://BioRender.com" target="_blank">BioRender.com</a> (accessed on 11 July 2024).</p>
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22 pages, 741 KiB  
Review
Tumor-Derived Extracellular Vesicles as Liquid Biopsy for Diagnosis and Prognosis of Solid Tumors: Their Clinical Utility and Reliability as Tumor Biomarkers
by Prerna Dabral, Nobel Bhasin, Manish Ranjan, Maysoon M. Makhlouf and Zakaria Y. Abd Elmageed
Cancers 2024, 16(13), 2462; https://doi.org/10.3390/cancers16132462 - 5 Jul 2024
Cited by 2 | Viewed by 1575
Abstract
Early cancer detection and accurate monitoring are crucial to ensure increased patient survival. Recent research has focused on developing non-invasive biomarkers to diagnose cancer early and monitor disease progression at low cost and risk. Extracellular vesicles (EVs), nanosized particles secreted into extracellular spaces [...] Read more.
Early cancer detection and accurate monitoring are crucial to ensure increased patient survival. Recent research has focused on developing non-invasive biomarkers to diagnose cancer early and monitor disease progression at low cost and risk. Extracellular vesicles (EVs), nanosized particles secreted into extracellular spaces by most cell types, are gaining immense popularity as novel biomarker candidates for liquid cancer biopsy, as they can transport bioactive cargo to distant sites and facilitate intercellular communications. A literature search was conducted to discuss the current approaches for EV isolation and the advances in using EV-associated proteins, miRNA, mRNA, DNA, and lipids as liquid biopsies. We discussed the advantages and challenges of using these vesicles in clinical applications. Moreover, recent advancements in machine learning as a novel tool for tumor marker discovery are also highlighted. Full article
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<p>A representative chart showing different methods for isolation of extracellular vesicles (EVs) from body fluids.</p>
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22 pages, 2908 KiB  
Review
Extracellular Vesicular miRNA in Pancreatic Cancer: From Lab to Therapy
by Prashant Kumar Tiwari, Poojhaa Shanmugam, Vamika Karn, Saurabh Gupta, Richa Mishra, Sarvesh Rustagi, Mandeep Chouhan, Devvret Verma, Niraj Kumar Jha and Sanjay Kumar
Cancers 2024, 16(12), 2179; https://doi.org/10.3390/cancers16122179 - 8 Jun 2024
Cited by 2 | Viewed by 1813
Abstract
Pancreatic cancer is a prevalent lethal gastrointestinal cancer that generally does not show any symptoms until it reaches advanced stages, resulting in a high mortality rate. People at high risk, such as those with a family history or chronic pancreatitis, do not have [...] Read more.
Pancreatic cancer is a prevalent lethal gastrointestinal cancer that generally does not show any symptoms until it reaches advanced stages, resulting in a high mortality rate. People at high risk, such as those with a family history or chronic pancreatitis, do not have a universally accepted screening protocol. Chemotherapy and radiotherapy demonstrate limited effectiveness in the management of pancreatic cancer, emphasizing the urgent need for innovative therapeutic strategies. Recent studies indicated that the complex interaction among pancreatic cancer cells within the dynamic microenvironment, comprising the extracellular matrix, cancer-associated cells, and diverse immune cells, intricately regulates the biological characteristics of the disease. Additionally, mounting evidence suggests that EVs play a crucial role as mediators in intercellular communication by the transportation of different biomolecules, such as miRNA, proteins, DNA, mRNA, and lipids, between heterogeneous cell subpopulations. This communication mediated by EVs significantly impacts multiple aspects of pancreatic cancer pathogenesis, including proliferation, angiogenesis, metastasis, and resistance to therapy. In this review, we delve into the pivotal role of EV-associated miRNAs in the progression, metastasis, and development of drug resistance in pancreatic cancer as well as their therapeutic potential as biomarkers and drug-delivery mechanisms for the management of pancreatic cancer. Full article
(This article belongs to the Special Issue Oncogenesis of Pancreatic Cancer: Where Are We)
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<p>Exosomes arise from intraluminal vesicles (ILVs) within multivesicular bodies (MVBs), a process facilitated by the ESCRT complex or alternative ESCRT-independent pathway. Upon ILV formation, MVBs undergo trafficking toward either the plasma membrane or the lysosome. At the plasma membrane, fusion of MVBs is mediated by the SNARE complex, culminating in exosome release. On the contrary, microvesicles emerge by budding directly from the plasma membrane. Apoptotic bodies, exclusive to apoptotic cells, are shed from the cell surface. These extracellular vesicles are lipid bilayer-enclosed and contain various biomolecules including nucleic acids (DNA and RNA), diverse proteins, receptors, lipids, and metabolites.</p>
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<p>(<b>A</b>) Schematic representation depicting the miRNA-10a-5p induces the expression of transcriptional factors Vimentin, Slug, and Snail while suppressing E-cadherin and Claudin1, ultimately facilitating cell migration and metastasis. (<b>B</b>) miRNA-501-3p targets TGFBR3, leading to the activation of TGF-β signaling and phosphorylation of P27, thereby inducing AKT signaling and promoting invasion. (<b>C</b>) miRNA-301 in metastasis through JAK/STAT and PTEN/PI3K pathways. (<b>D</b>) miRNA-125-5p promotes migration and metastasis by inducing MEK2/ERK2 and inhibiting STARD13, whereas miRNA-21 induces metalloprotease activity, thereby promoting cell migration. (The direction of arrow shown in the box, ↑—increased expression; ↓—decreased expression; ⊣—inhibition).</p>
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<p>Exosome-associated miRNAs are involved in various roles such as (<b>1</b>) invasion, proliferation, and metastasis; (<b>2</b>) drug resistance; (<b>3</b>) PDAC treatment; and (<b>4</b>) overcoming drug resistance. The direction of arrow shown in the box, ↑—increased expression.</p>
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18 pages, 5816 KiB  
Article
Proteomic Profiling of Plasma- and Gut-Derived Extracellular Vesicles in Obesity
by Pedro Baptista Pereira, Estefania Torrejón, Inês Ferreira, Ana Sofia Carvalho, Akiko Teshima, Inês Sousa-Lima, Hans Christian Beck, Bruno Costa-Silva, Rune Matthiesen, Maria Paula Macedo and Rita Machado de Oliveira
Nutrients 2024, 16(5), 736; https://doi.org/10.3390/nu16050736 - 4 Mar 2024
Cited by 1 | Viewed by 2465
Abstract
Obesity entails metabolic alterations across multiple organs, highlighting the role of inter-organ communication in its pathogenesis. Extracellular vesicles (EVs) are communication agents in physiological and pathological conditions, and although they have been associated with obesity comorbidities, their protein cargo in this context remains [...] Read more.
Obesity entails metabolic alterations across multiple organs, highlighting the role of inter-organ communication in its pathogenesis. Extracellular vesicles (EVs) are communication agents in physiological and pathological conditions, and although they have been associated with obesity comorbidities, their protein cargo in this context remains largely unknown. To decipher the messages encapsulated in EVs, we isolated plasma-derived EVs from a diet-induced obese murine model. Obese plasma EVs exhibited a decline in protein diversity while control EVs revealed significant enrichment in protein-folding functions, highlighting the importance of proper folding in maintaining metabolic homeostasis. Previously, we revealed that gut-derived EVs’ proteome holds particular significance in obesity. Here, we compared plasma and gut EVs and identified four proteins exclusively present in the control state of both EVs, revealing the potential for a non-invasive assessment of gut health by analyzing blood-derived EVs. Given the relevance of post-translational modifications (PTMs), we observed a shift in chromatin-related proteins from glycation to acetylation in obese gut EVs, suggesting a regulatory mechanism targeting DNA transcription during obesity. This study provides valuable insights into novel roles of EVs and protein PTMs in the intricate mechanisms underlying obesity, shedding light on potential biomarkers and pathways for future research. Full article
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<p>Diet-induced obesity increases the number of plasma extracellular vesicles. (<b>a</b>) Analysis of the number of particles per mL of sample from control (NCD) and diet-induced obese mice (HFD). (<b>b</b>) Protein quantification in plasma EVs in mg/mL, obtained by BCA. (<b>c</b>) Protein content per EV, represented in mg of protein per particle. All statistical analysis were performed using unpaired <span class="html-italic">t</span>-test with Welch’s correction. All data are presented as mean ± standard error of the mean. * <span class="html-italic">p</span>-value &lt; 0.05. Each <span class="html-italic">n</span> represents a sample of 20 animals, <span class="html-italic">n</span> = 3 for both groups, with blue dots representing NCD samples and orange triangles representing HFD samples.</p>
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<p>Comparative protein profile in plasma-derived extracellular vesicles from NCD and HFD-fed mice. (<b>a</b>) Schematic representation illustrating the experimental workflow for the analysis of plasma EVs. Each sample, obtained from mice fed either an NCD or an HFD, underwent two rounds of analysis using liquid chromatography-tandem mass spectrometry (LC-MS/MS). (<b>b</b>) Venn diagram representing the overlap of proteins identified in NCD and HFD plasma EVs with a false discovery rate (FDR) &lt; 0.01. (<b>c</b>) List of proteins exclusively identified in HFD plasma EVs. (<b>d</b>) Volcano plot representing the fold change and <span class="html-italic">p</span>-value of regulated proteins shared between NCD and HFD plasma EVs. Dotted horizontal line indicates the threshold for a <span class="html-italic">p</span>-value &lt; 0.05. A positive log<sub>2</sub> (fold change) value indicates higher protein levels in diet-induced obese mice when compared to control mice. Down-regulated proteins are represented in light blue, while up-regulated proteins are indicated in light red. (<b>e</b>) List of significantly regulated proteins (<span class="html-italic">p</span>-value &lt; 0.05) in plasma EVs from HFD mice compared to NCD mice.</p>
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<p>Gene Ontology over-representation analysis of plasma EVs proteins identified exclusively in NCD mice. (<b>a</b>) Bar plot and (<b>b</b>) table of enriched molecular functions and their related identified genes. Black rectangles indicate the association between the identified gene and the enriched term.</p>
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<p>Obesity-associated changes in proteins shared between plasma and gut EVs. (<b>a</b>) Venn diagram displaying the intersection of proteins identified in HFD mice and NCD mice, from all proteins shared between PDEV and GDEV. All proteins presented an FDR &lt; 0.01. (<b>b</b>) List of four proteins shared between PDEV and GDEV, present exclusively in NCD mice.</p>
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<p>Analysis of protein acetylation and glycation in plasma and gut EVs. (<b>a</b>) Venn diagram representing the intersection of acetylated proteins identified in NCD and HFD plasma EVs. (<b>b</b>) Venn diagram representing the intersection of glycated proteins identified in NCD and HFD plasma EVs. (<b>c</b>) Venn diagram representing the intersection of acetylated proteins identified in NCD and HFD gut EVs. (<b>d</b>) Venn diagram representing the intersection of glycated proteins identified in NCD and HFD gut EVs. All proteins presented an FDR &lt; 0.01. The identification of acetylated and glycated proteins was only conducted on proteins previously identified in both NCD and HFD samples (223 proteins for plasma EVs and 1804 proteins for gut EVs).</p>
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<p>Over-representation analysis of gut EVs proteins featuring acetylation and glycation. (<b>a</b>) GO enrichment analysis according to cellular components. (<b>b</b>) GO enrichment analysis according to molecular functions. (<b>c</b>) KEGG enrichment analysis for KEGG pathways. Dot plots represent the five most significantly enriched terms in each group.</p>
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15 pages, 669 KiB  
Review
Extracellular Vesicles in the Pathogenesis, Clinical Characterization, and Management of Dermatomyositis: A Narrative Review
by Cristina Ricco, Ahmed Eldaboush, Ming-Lin Liu and Victoria P. Werth
Int. J. Mol. Sci. 2024, 25(4), 1967; https://doi.org/10.3390/ijms25041967 - 6 Feb 2024
Cited by 1 | Viewed by 2003
Abstract
Extracellular vesicles (EVs) are lipid-bilayer particles secreted from cells that primarily assist in cell-to-cell communication through the content of their cargo, such as proteins and RNA. EVs have been implicated in the pathogenesis of various autoimmune diseases, including dermatomyositis (DM), an inflammatory autoimmune [...] Read more.
Extracellular vesicles (EVs) are lipid-bilayer particles secreted from cells that primarily assist in cell-to-cell communication through the content of their cargo, such as proteins and RNA. EVs have been implicated in the pathogenesis of various autoimmune diseases, including dermatomyositis (DM), an inflammatory autoimmune disease characterized by distinct cutaneous manifestations, myopathy, and lung disease. We sought to review the role of EVs in DM and understand how they contribute to the pathogenesis and clinical characterization of the disease. We summarized the research progress on EVs in dermatomyositis based on recent publications. EV cargoes, such as double-stranded DNA, microRNA, and proteins, contribute to DM pathogenesis and mediate the proinflammatory response and cytokine release through signaling pathways such as the stimulator of interferon genes (STING) pathway. These nucleic acids and proteins have been proposed as disease-specific, stable biomarkers to monitor disease activity and responses to therapy. They also correlate with clinical parameters, inflammatory markers, and disease severity scores. Furthermore, some markers show an association with morbidities of DM, such as muscle weakness and interstitial lung disease. The continued study of EVs will help us to further elucidate our understanding of dermatomyositis. Full article
(This article belongs to the Special Issue Exosomes 2.0)
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<p>EVs in the type 1 IFN pathway. (<b>A</b>) Through an association with mitochondrial antiviral signaling protein (MAVS), which is depicted as an EV surface protein marker, studies have shown an upregulation of the type 1 IFN pathway. (<b>B</b>) Through direct endocytosis of EV cargo, the type 1 IFN pathway can be stimulated. (<b>C</b>) EVs released from infected cells can activate pDCs via interaction with Toll-like receptor 7 (TLR7) and produce type 1 IFNs [<a href="#B34-ijms-25-01967" class="html-bibr">34</a>,<a href="#B35-ijms-25-01967" class="html-bibr">35</a>].</p>
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17 pages, 1492 KiB  
Article
A Method for Using Cell-Penetrating Peptides for Loading Plasmid DNA into Secreted Extracellular Vesicles
by Jekaterina Nebogatova, Heleri Heike Härk, Anett Puskar, Ly Porosk, Paolo Guazzi, Moataz Dowaidar, Ülo Langel and Kaido Kurrikoff
Biomolecules 2023, 13(12), 1751; https://doi.org/10.3390/biom13121751 - 6 Dec 2023
Cited by 1 | Viewed by 1942
Abstract
The low bioavailability and high toxicity of plasmid DNA (pDNA)-based therapeutics pose challenges for their in vivo application. Extracellular vesicles (EVs) have great potential to overcome these limitations, as they are biocompatible native cargo carriers. Various methods for loading pDNA into EVs, including [...] Read more.
The low bioavailability and high toxicity of plasmid DNA (pDNA)-based therapeutics pose challenges for their in vivo application. Extracellular vesicles (EVs) have great potential to overcome these limitations, as they are biocompatible native cargo carriers. Various methods for loading pDNA into EVs, including electroporation, sonication, and co-incubation, have been previously investigated, but their success has been questionable. In this study, we report a unique method for loading EVs with pDNA through transient transfection using cell-penetrating peptides (CPPs). With this method, we found a 104-fold increase in the expression levels of the luciferase reporter protein in recipient cells compared to the untreated cells. These data point to the high transfection efficacy and bioavailability of the delivered encapsulated nucleic acid. Furthermore, the in vivo experimental data indicate that the use of pDNA-loaded EVs as native delivery vehicles reduces the toxic effects associated with traditional nucleic acid (NA) delivery and treatment. Full article
(This article belongs to the Special Issue Functional Peptides and Their Interactions)
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<p>Quantification of Cy5-labeled pDNA in exosomes purified from donor cells’ conditioned media assessed via flow cytometry: (<b>a</b>) schematic representation of experimental setup of quantification of Cy5-labeled pDNA in total EV population (immuno-labeling protocol); (<b>b</b>) schematic representation of experimental setup of quantification of Cy5-labeled pDNA in total EV population (double transfection protocol); (<b>c</b>) flow cytometry assay. pDNA was labeled with Cy5 dye, and exosomes were either labeled with exosome-specific CD63 mAb or expressed CD63-GFP protein. pDNA containing EV population was gated if Cy5 and CD63 signal were present (Cy5+/GFP+). Transfection was performed with CPP PF14 in CHO-K1 or N2a cell lines. N = 2. Results are shown in % of the total EV population. Panel (<b>a</b>,<b>b</b>) were created with BioRender.com, acessed on 20 May 2023.</p>
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<p>Quantification of radio-labeled pDNA in EVs purified from donor cells’ conditioned media: (<b>a</b>) schematic representation of experimental setup of quantification radio-labeled pDNA in total isolated EV population; (<b>b</b>) quantification of radio-labeled pDNA detected in EVs assessed by measuring [α32P]dCTP sample signals. CPP PF14 was used for complex formulation. The CHO K1 cell line was used as a donor cell line for loaded EV production. N = 3. Results are shown as % of the total sample input of labeled pDNA. Panel (<b>a</b>) was created with BioRender.com acessed on 20 May 2023.</p>
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<p>The signal level of luciferase reporter expressed in adherent recipient CHO-K1 cells after transfecting with pLuc-loaded EVs, extracted from either (<b>a</b>) an adherent donor CHO-K1 cell line, (<b>b</b>) or a suspension HEK293FT donor cell line. On the <span class="html-italic">x</span>-axis, the dosageof pDNA in pDNA/CPP complex per well used for transection is shown. N = 6. For statistical analyses, multiple unpaired <span class="html-italic">t</span>-tests were performed. Results are shown in RLU/mg and normalized to total protein in the lysate.</p>
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<p>Comparison of EV isolation methods. Conditioned media were purified via polymer precipitation, tangential flow filtration, or size exclusion chromatography. Inside the bar % of sample measurements from conditioned media input are shown: (<b>a</b>) double-sandwich ELISA assay for quantitative and qualitative analyses of exosomes. Results are shown in ng/µL; (<b>b</b>) NTA for the determination of a size distribution profile of small particles with a specific diameter in liquid suspension. N = 1. Results are shown in particle/mL.</p>
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<p>Cell transfection with isolated exosomes in the luciferase reporter assay. A549 and HEK293FT cell lines were used as recipient cell lines. The signal from the luciferase reporter was measured in recipient and donor cells. N = 6. For statistical analyses, multiple unpaired <span class="html-italic">t</span>-test was performed. Results are shown in RLU/mg, normalized to total protein in the lysate.</p>
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<p>In vivo transfection. Eight-week-old female BALB/c mice were administered intravenously via the tail vein: (<b>a</b>) results of postmortem ex vivo tissue analysis are shown as the fold over pDNA control. Tissues were harvested 24 h after administration; (<b>b</b>) levels of ASAT and ALAT serum marker enzymes in whole blood were measured 24 h after administration. N = 1.</p>
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21 pages, 7244 KiB  
Article
In Doxorubicin-Adapted Hodgkin Lymphoma Cells, Acquiring Multidrug Resistance and Improved Immunosuppressive Abilities, Doxorubicin Activity Was Enhanced by Chloroquine and GW4869
by Naike Casagrande, Cinzia Borghese, Michele Avanzo and Donatella Aldinucci
Cells 2023, 12(23), 2732; https://doi.org/10.3390/cells12232732 - 29 Nov 2023
Cited by 2 | Viewed by 1852
Abstract
Classical Hodgkin lymphoma (cHL) is a highly curable disease (70–80%), even though long-term toxicities, drug resistance, and predicting clinical responses to therapy are major challenges in cHL treatment. To solve these problems, we characterized two cHL cell lines with acquired resistance to doxorubicin, [...] Read more.
Classical Hodgkin lymphoma (cHL) is a highly curable disease (70–80%), even though long-term toxicities, drug resistance, and predicting clinical responses to therapy are major challenges in cHL treatment. To solve these problems, we characterized two cHL cell lines with acquired resistance to doxorubicin, KM-H2dx and HDLM-2dx (HRSdx), generated from KM-H2 and HDLM-2 cells, respectively. HRSdx cells developed cross-resistance to vinblastine, bendamustin, cisplatin, dacarbazine, gemcitabine, brentuximab vedotin (BV), and γ-radiation. Both HDLM-2 and HDLM-2dx cells had intrinsic resistance to BV but not to the drug MMAE. HDLM-2dx acquired cross-resistance to caelyx. HRSdx cells had in common decreased CD71, CD80, CD54, cyt-ROS, HLA-DR, DDR1, and CD44; increased Bcl-2, CD58, COX2, CD26, CCR5, and invasive capability; increased CCL5, TARC, PGE2, and TGF-β; and the capability of hijacking monocytes. In HRSdx cells less sensitive to DNA damage and oxidative stress, the efflux drug transporters MDR1 and MRP1 were not up-regulated, and doxorubicin accumulated in the cytoplasm rather than in the nucleus. Both the autophagy inhibitor chloroquine and extracellular vesicle (EV) release inhibitor GW4869 enhanced doxorubicin activity and counteracted doxorubicin resistance. In conclusion, this study identifies common modulated antigens in HRSdx cells, the associated cross-resistance patterns, and new potential therapeutic options to enhance doxorubicin activity and overcome resistance. Full article
(This article belongs to the Special Issue Molecular and Cellular Mechanisms of Lymphomas)
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<p>Characteristics of HRS and HRSdx cells. (<b>A</b>) Phase-contrast photo-micrographs of HRS and HRSdx cells. (<b>B</b>) Morphological images of HRS (upper panels) and HRSdx cells (red, lower panels) obtained after May–Grünwald–Giemsa staining (MGG). (Magnification, ×20; scale bar, 10 µm.) (<b>C</b>) Growth curves of HRS and HRSdx cells. The number of viable cells was evaluated via trypan blue dye exclusion assays. The calculated doubling times (DTs, in days) for each cell line were reported in the figure. (<b>D</b>) Clonogenic growth. Cells were seeded in medium containing 0.8% methylcellulose. After 14 days, aggregates with ≥40 cells were scored as colonies. Values (total number of colonies) are the mean ± SD of eight replicates of three independent experiments. (<b>E</b>) HRS and HRSdx cells were lysed, and NF-kB p65 transcription factor activity was analyzed in nuclear extracts using the Transcription Factor Kit (p65). Results are represented as the percent of control (activity HRSdx respect to HRS parental cells) and are the mean ± SD of three independent experiments. Chemotaxis assays in Boyden chambers. (<b>F</b>) Migration of HRS and HRSdx cells through fibronectin-coated (20 µg/mL) chambers towards 20% complete medium. Data are the percentages of cells that migrated from the serum-free upper chamber to the lower complete medium chamber. (<b>G</b>) Invasion. Migration of HRS and HRSdx cells through Matrigel-coated (50 ug/mL) chambers towards 20% complete medium. Data are the percentages of cells that migrated from the Matrigel-coated upper chamber to the lower complete medium chamber. (<b>H</b>) Western blot analysis for ROCK, RhoA, and vinculin in HRS and HRSdx cells. Images were acquired using a ChemiDoc XRS system (Bio-Rad). (<b>I</b>) Flow cytometry expression of CCR5 and CXCR4 in HRS and HRSdx cells. Red arrows indicate up-regulated antigens. Mean fluorescence intensities are reported in the boxes. * <span class="html-italic">p</span> &lt; 0.05 HRSdx vs. HRS cells.</p>
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<p>Phenotypes of HRS and HRSdx cells. Flow cytometry assay of molecules expressed by HRS and HRSdx cells. Representative flow cytometry histograms showing the expression of (<b>A</b>) survival factors and antiapoptotic molecules, (<b>B</b>) markers of the putative cancer stem cells, and (<b>C</b>) molecules involved in the interactions with collagen and stromal cells (<b>D</b>) or with white blood cells (lymphocytes, monocytes, eosinophils, and mast cells). Mean fluorescence intensities are reported in the boxes. Red arrows indicate up-regulated antigens and black arrows down-modulated antigens in doxorubicin resistant HRSdx cells with respect to parental HRS cells. (<b>E</b>) Venn diagrams showing the molecules modulated in both HRSdx cells and those specifically * up-regulated (red) or * down-regulated (blue) in KM-H2dx and HDLM-2dx.</p>
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<p>Tumor cell expression and secretion of immunosuppressive molecules and monocyte immunosuppressive education by tumor cell conditioned medium (CM). (<b>A</b>) Flow cytometry assay of immunosuppressive molecules expressed by HRS and HRSdx cells. Red arrows indicate up-regulated antigens and black arrows down-modulated antigens in HRSdx cells with respect to HRS cells. (<b>B</b>) Cytokines secreted by HRS and HRSdx cells cultured for 72 h in complete medium. Their concentrations were evaluated by an ELISA assay and reported as pg × 10<sup>6</sup> cells, excluding L-lactate (*, ng/10<sup>6</sup> cells). Values for KM-H2 and HDLM-2 are shown in the respective insert. Bar charts report the fold-increase in the concentration of each chemokine secreted by HRSdx cells with respect to HRS cells. (<b>C</b>) Monocytic THP-1 cells were cultured with HRS-CM and HRSdx-CM, and then, CD206, PDL-1, and IDO expression was evaluated via flow cytometry. Mean fluorescence intensities are reported in the boxes. Red arrows indicate antigens up-regulated by HRSdx-CM with respect to HRS-CM. (<b>D</b>) Immunosuppression (IS). Schematic representation of common HRSdx modifications in cells leading to immunosuppression (antigens, cytokines, monocytes, tumor education). The red arrow indicates up-regulated antigens and the black arrow down-modulated antigens (HRSdx respect to HRS cells).</p>
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<p>Cross-resistance pattern and γ-radiation activity in HRS and HRSdx cells. (<b>A</b>) The resistance factor (RF) value is the ratio of the HRSdx IC<sub>50</sub> (KM-H2dx and HDLM-2dx) over the HRS IC<sub>50</sub> (KM-H2 and HDLM-2). RFs are reported in ascending order. RF &lt; 1 indicates cross-sensitivity (CS), and RF ≥1 indicates cross-resistance (CR). An RF ranging from 1 to 2 indicates low CR, an RF from 2 to 5 indicates moderate CR, and an RF ≥ 5 indicates high CR. (<b>B</b>–<b>D</b>) Tumor cells were treated with γ-radiation (0–12 Gy). Then, cell viability, clonogenic growth, and cell cycle distribution were evaluated. (<b>B</b>) Cells were double-stained with Annexin-V-FITC and 7AAD and analyzed via flow cytometry. Bar charts show the percentage of viable cells (Annexin-V and 7AAD negative cells). (<b>C</b>) Clonogenic growth assay. Untreated and γ-radiation-treated cells were seeded in medium containing 0.8% methylcellulose and cultured for 14 days; aggregates with ≥40 cells were scored as colonies. Values (total number of colonies) are the mean ± SD of eight replicates. (<b>D</b>) Bar charts show the percentage of cells in each cell cycle phase, evaluated after propidium iodide staining and flow cytometry analysis. (<b>E</b>) Representative cytofluorimetric histograms of the cell cycle progression after γ-radiation treatment. Results are the mean ± SD of three independent experiments. * <span class="html-italic">p</span> &lt; 0.05 HRSdx vs. parental HRS cells.</p>
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<p>Expression of drug transporters, uptake, distribution, and DNA damage via doxorubicin in HRS and HRSdx cells. (<b>A</b>) Relative mRNA expression of MDR1/ABCB1 and MRP1/ABCC1 in HRS and HRSdx cells using GAPDH gene expression as internal control. (<b>B</b>) Western blot for MDR1, MRP1, and α-tubulin expression. (<b>C</b>) Flow cytometry-based doxorubicin accumulation assay. HRS and HRSdx cells were incubated with doxorubicin (0–200 ng/mL) for 2 h. Then, the percentage of red fluorescence-positive cells was evaluated via flow cytometry. (<b>D</b>) Cells were incubated with doxorubicin (DOX, 1 µg/mL). After 2 h, doxorubicin internalization and distribution were evaluated via confocal microscopy. (<b>E</b>) HRS and HRSdx cells were incubated for 24 h with doxorubicin (KM-H2 IC<sub>90</sub> = 100 ng/mL and HDLM-2 IC<sub>90</sub> = 175 ng/mL). Then, γ-H2AX expression was evaluated via flow cytometry. (<b>F</b>) KM-H2dx and HDLM-2dx cells were incubated for 24 h with doxorubicin (KM-H2dx IC<sub>90</sub> = 300 ng/mL and HDLM-2dx IC<sub>90</sub> = 450 ng/mL). γ-H2AX expression was evaluated via flow cytometry. Results are the mean ± SD of three independent experiments. * <span class="html-italic">p</span> &lt; 0.05 HRSdx vs. parental HRS cells.</p>
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<p>Sensitivity of HRS and HRSdx cells to oxidative stress. HRS and HRSdx cells were treated with H<sub>2</sub>O<sub>2</sub> (0–0.5 mM). (<b>A</b>) After 1 h, cell viability was evaluated with a trypan blue dye exclusion assay. (<b>B</b>) Alternatively, cells were double-stained with Annexin-V-FITC and 7AAD and analyzed via flow cytometry. Bar charts show the percentage of viable cells (Annexin-V- and 7AAD-negative cells). (<b>C</b>) HRS and HRSdx cells were treated for 24 h with H<sub>2</sub>O<sub>2</sub> (0.25 mM). After 24 h, cell viability was evaluated with a trypan blue dye exclusion assay. (<b>D</b>) TrxR enzymatic activity was evaluated using a TrxR assay kit and expressed as U/mg of protein. Results are the mean ± SD of three independent experiments. * <span class="html-italic">p</span> &lt; 0.05 HRSdx vs. parental HRS cells.</p>
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<p>Chloroquine and GW4869 enhance cell death induced by doxorubicin. HRS and HRSdx cells were cultured with doxorubicin (DOX) in the presence or not in the presence of (<b>A</b>) a non-toxic concentration of chloroquine (CQ) (2.5 µM) or (<b>B</b>) GW4869 (2 µM). After 72 h, cell viability was evaluated via a trypan blue dye exclusion assay. Results are the mean ± SD of three independent experiments. * <span class="html-italic">p</span> &lt; 0.05 DOX vs. DOX plus CQ or DOX plus GW4869. Arrows indicate the IC<sub>50</sub> of doxorubicin, in the presence or not in the presence of CQ or GW4869. The difference in the IC<sub>50</sub> is shown by the horizontal black double-headed arrow.</p>
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Article
Genotypic Frequencies of Mutations Associated with Alpha-1 Antitrypsin Deficiency in Unrelated Bone Marrow Donors from the Murcia Region Donor Registry in the Southeast of Spain
by Irene Cuenca, Carmen Botella, María Rosa Moya-Quiles, Víctor Jimenez-Coll, José Antonio Galian, Helios Martinez-Banaclocha, Manuel Muro-Pérez, Alfredo Minguela, Isabel Legaz and Manuel Muro
Diagnostics 2023, 13(17), 2845; https://doi.org/10.3390/diagnostics13172845 - 2 Sep 2023
Viewed by 1369
Abstract
Alpha-1 antitrypsin (AAT1) deficiency (AAT1D) is an inherited disease with an increased risk of chronic obstructive pulmonary disease (COPD), liver disease, and skin and blood vessel problems. AAT1D is caused by mutations in the SERPINE1 gene (Serine Protease Inhibitor, group A, member 1). [...] Read more.
Alpha-1 antitrypsin (AAT1) deficiency (AAT1D) is an inherited disease with an increased risk of chronic obstructive pulmonary disease (COPD), liver disease, and skin and blood vessel problems. AAT1D is caused by mutations in the SERPINE1 gene (Serine Protease Inhibitor, group A, member 1). Numerous variants of this gene, the Pi system, have been identified. The most frequent allelic variants are Pi*M, Pi*S, and Pi*Z. The development of COPD requires both a genetic predisposition and the contribution of an environmental factor, smoking being the most important. Studies on this deficiency worldwide are very scarce, and it is currently considered a rare disease because it is underdiagnosed. The aim of this study was to analyze the genotypic frequencies of mutations associated with AAT1 deficiency in unrelated bone marrow donors from the donor registry of the Region of Murcia in southeastern Spain due to the high risk of presenting with different pathologies and underdiagnosis in the population. A total of 112 DNA-healthy voluntary unrelated bone marrow donors from different parts of the Region of Murcia were analyzed retrospectively. AAT1 deficiency patient testing involved an automated biochemical screening routine. The three main variants, Pi*M, Pi*Z, and Pi*S, were analyzed in the SERPINE1 gene. Our results showed a frequency of 3.12% of the Pi*Z (K342) mutation in over 224 alleles tested in the healthy population. The frequency of Pi*S (V264) was 11.1%. The frequency of the haplotype with the most dangerous mutation, EK342 EE264, was 4.46%, and the frequency of EK342 EV264 was 1.78% in the healthy population. Frequencies of other EE342 EV264-mutated haplotypes accounted for 18.7%. As for the EE342 VV264 haplotype, 0.89% of the total healthy population presented heterozygous for the EV264 mutation and one individual presented homozygous for the VV264 mutation. In conclusion, the frequencies of Pi mutations in the healthy population of the Region of Murcia were not remarkably different from the few studies reported in Spain. The genotype and haplotype frequencies followed the usual pattern. Health authorities should be aware of this high prevalence of the Pi*S allelic variant and pathological genotypes such as Pi*MZ and Pi*SZ in the healthy population if they consider screening the smoking population. Full article
(This article belongs to the Special Issue Genetics and Cytopathology Testing in Disease Diagnosis)
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Figure 1

Figure 1
<p>Map of the Region of Murcia showing the origin and number of study subjects from different municipalities (Source: Laboratory Information System (SIL) of the Immunology Service of the HCUVA, Modulab of the Werfen company, Barcelona, Spain).</p>
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<p>The most representative results for Pi* genotypes were obtained for the following mutations: MM, MZ, MS, SS, or SZ.</p>
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