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Micromachines May 2024Laboratory automation effectively increases the throughput in sample analysis, reduces human errors in sample processing, as well as simplifies and accelerates the...
Laboratory automation effectively increases the throughput in sample analysis, reduces human errors in sample processing, as well as simplifies and accelerates the overall logistics. Automating diagnostic testing workflows in peripheral laboratories and also in near-patient settings -like hospitals, clinics and epidemic control checkpoints- is advantageous for the simultaneous processing of multiple samples to provide rapid results to patients, minimize the possibility of contamination or error during sample handling or transport, and increase efficiency. However, most automation platforms are expensive and are not easily adaptable to new protocols. Here, we address the need for a versatile, easy-to-use, rapid and reliable diagnostic testing workflow by combining open-source modular automation (Opentrons) and automation-compatible molecular biology protocols, easily adaptable to a workflow for infectious diseases diagnosis by detection on paper-based diagnostics. We demonstrated the feasibility of automation of the method with a low-cost diagnostic test that utilizes magnetic beads for pathogen DNA isolation, isothermal amplification, and detection on a paper-based microarray. In summary, we integrated open-source modular automation with adaptable molecular biology protocols, which was also faster and cheaper to perform in an automated than in a manual way. This enables a versatile diagnostic workflow for infectious diseases and we demonstrated this through a low-cost test on paper-based microarrays.
PubMed: 38930678
DOI: 10.3390/mi15060708 -
International Journal of Molecular... Jun 2024This study aimed to assess the expression profile of messenger RNA (mRNA) and microRNA (miRNA) related to the dopaminergic system in five types of breast cancer in...
This study aimed to assess the expression profile of messenger RNA (mRNA) and microRNA (miRNA) related to the dopaminergic system in five types of breast cancer in Polish women. Patients with five breast cancer subtypes were included in the study: luminal A ( = 130), luminal B ( = 196, including HER2-, = 100; HER2+, = 96), HER2+ ( = 36), and TNBC ( = 43); they underwent surgery, during which tumor tissue was removed along with a margin of healthy tissue (control material). The molecular analysis included a microarray profile of mRNAs and miRNAs associated with the dopaminergic system, a real-time polymerase chain reaction preceded by reverse transcription for selected genes, and determinations of their concentration using enzyme-linked immunosorbent assay (ELISA). The conducted statistical analysis showed that five mRNAs statistically significantly differentiated breast cancer sections regardless of subtype compared to control samples; these were dopamine receptor 2 (), dopamine receptor 3 (), dopamine receptor 25 (), transforming growth factor beta 2 (), and caveolin 2 (). The predicted analysis showed that hsa-miR-141-3p can regulate the expression of and , whereas hsa-miR-4441 is potentially engaged in the expression regulation of and . In addition, the expression pattern of mRNA can also be regulated by has-miR-16-5p. The overexpression of DRD2 and DRD3, with concomitant silencing of DRD5 expression, confirms the presence of dopaminergic abnormalities in breast cancer patients. Moreover, these abnormalities may be the result of miR-141-3P, miR-16-5p, and miR-4441 activity, regulating proliferation or metastasis.
Topics: Humans; Female; MicroRNAs; Breast Neoplasms; Gene Expression Regulation, Neoplastic; Middle Aged; Dopamine; Adult; Gene Expression Profiling; Aged; RNA, Messenger; Receptors, Dopamine D3; Receptors, Dopamine D2; Transforming Growth Factor beta2
PubMed: 38928253
DOI: 10.3390/ijms25126546 -
International Journal of Molecular... Jun 2024Breast cancer poses a global health challenge, yet the influence of ethnicity on the tumor microenvironment (TME) remains understudied. In this investigation, we...
Breast cancer poses a global health challenge, yet the influence of ethnicity on the tumor microenvironment (TME) remains understudied. In this investigation, we examined immune cell infiltration in 230 breast cancer samples, emphasizing diverse ethnic populations. Leveraging tissue microarrays (TMAs) and core samples, we applied multiplex immunofluorescence (mIF) to dissect immune cell subtypes across TME regions. Our analysis revealed distinct immune cell distribution patterns, particularly enriched in aggressive molecular subtypes triple-negative and HER2-positive tumors. We observed significant correlations between immune cell abundance and key clinicopathological parameters, including tumor size, lymph node involvement, and patient overall survival. Notably, immune cell location within different TME regions showed varying correlations with clinicopathologic parameters. Additionally, ethnicities exhibited diverse distributions of cells, with certain ethnicities showing higher abundance compared to others. In TMA samples, patients of Chinese and Caribbean origin displayed significantly lower numbers of B cells, TAMs, and FOXP3-positive cells. These findings highlight the intricate interplay between immune cells and breast cancer progression, with implications for personalized treatment strategies. Moving forward, integrating advanced imaging techniques, and exploring immune cell heterogeneity in diverse ethnic cohorts can uncover novel immune signatures and guide tailored immunotherapeutic interventions, ultimately improving breast cancer management.
Topics: Humans; Tumor Microenvironment; Female; Breast Neoplasms; Tissue Array Analysis; Middle Aged; Fluorescent Antibody Technique; Adult; Aged; Ethnicity; Biomarkers, Tumor
PubMed: 38928207
DOI: 10.3390/ijms25126501 -
International Journal of Molecular... Jun 2024Bladder cancer (BC) is the 12th most commonly diagnosed cancer worldwide. Although there are several well-established molecular and immunological classifications,...
Bladder cancer (BC) is the 12th most commonly diagnosed cancer worldwide. Although there are several well-established molecular and immunological classifications, prognostic and predictive markers for tumor cells and immune cells are still needed. Using a tissue microarray, we analyzed the expression of the chemokine CC motif ligand 5 (CCL5) by immunohistochemistry (IHC) in 175 muscle-invasive BC samples. The application of a single cutoff for the staining status of tumor cells (TCs; positive vs. negative) and immune cells (ICs; positive vs. negative) revealed 75 patients (42.9%) and 123 patients (70.3%) with CCL5-positive TCs or ICs, respectively. IHC results were associated with prognostic and predictive data. Multivariate Cox regression analysis revealed that positive CCL5 staining in TCs was associated with significantly shorter disease-specific survival (DSS; RR = 1.51; = 0.047), but CCL5-negative ICs were associated with significantly shorter overall survival (OS; RR = 1.66; = 0.005), DSS (RR = 2.02; = 0.001) and recurrence-free survival (RFS; RR = 1.94; = 0.002). Adjuvant chemotherapy was favorable for patients with CCL5-negative ICs for OS (RR = 0.30; = 0.006), DSS (RR = 0.36; = 0.022) and RFS (RR = 0.41; = 0.046) but not for patients with CCL5-positive ICs, except in the subgroup of N1 + N2 patients, where it was associated with better OS. We suggest that CCL5 expression can be a prognostic and predictive marker for muscle-invasive bladder cancer patients.
Topics: Humans; Urinary Bladder Neoplasms; Chemokine CCL5; Male; Female; Aged; Prognosis; Middle Aged; Biomarkers, Tumor; Neoplasm Invasiveness; Aged, 80 and over; Adult; Immunohistochemistry
PubMed: 38928033
DOI: 10.3390/ijms25126325 -
Genes Jun 2024Uterine pathologies pose a challenge to women's health on a global scale. Despite extensive research, the causes and origin of some of these common disorders are not... (Comparative Study)
Comparative Study
Uterine pathologies pose a challenge to women's health on a global scale. Despite extensive research, the causes and origin of some of these common disorders are not well defined yet. This study presents a comprehensive analysis of transcriptome data from diverse datasets encompassing relevant uterine pathologies such as endometriosis, endometrial cancer and uterine leiomyomas. Leveraging the Comparative Analysis of Shapley values (CASh) technique, we demonstrate its efficacy in improving the outcomes of the classical differential expression analysis on transcriptomic data derived from microarray experiments. CASh integrates the microarray game algorithm with Bootstrap resampling, offering a robust statistical framework to mitigate the impact of potential outliers in the expression data. Our findings unveil novel insights into the molecular signatures underlying these gynecological disorders, highlighting CASh as a valuable tool for enhancing the precision of transcriptomics analyses in complex biological contexts. This research contributes to a deeper understanding of gene expression patterns and potential biomarkers associated with these pathologies, offering implications for future diagnostic and therapeutic strategies.
Topics: Female; Humans; Transcriptome; Endometriosis; Leiomyoma; Gene Expression Profiling; Endometrial Neoplasms; Uterine Neoplasms; Uterine Diseases; Algorithms
PubMed: 38927658
DOI: 10.3390/genes15060723 -
Genes May 2024Given the crucial role of the personalized management and treatment of hearing loss (HL), etiological investigations are performed early on, and genetic analysis... (Review)
Review
Given the crucial role of the personalized management and treatment of hearing loss (HL), etiological investigations are performed early on, and genetic analysis significantly contributes to the determination of most syndromic and nonsyndromic HL cases. Knowing hundreds of syndromic associations with HL, little comprehensive data about HL in genomic disorders due to microdeletion or microduplications of contiguous genes is available. Together with the description of a new patient with a novel 3.7 Mb deletion of the Xq21 critical locus, we propose an unreported literature review about clinical findings in patients and their family members with Xq21 deletion syndrome. We finally propose a comprehensive review of HL in contiguous gene syndromes in order to confirm the role of cytogenomic microarray analysis to investigate the etiology of unexplained HL.
Topics: Humans; Chromosome Deletion; Chromosomes, Human, X; Hearing Loss; Male; Syndrome; Female; Pedigree
PubMed: 38927613
DOI: 10.3390/genes15060677 -
Biomedicines Jun 2024Breast cancer (BC) poses a challenge in establishing new treatment strategies and identifying new prognostic and predictive markers due to the extensive genetic...
UNLABELLED
Breast cancer (BC) poses a challenge in establishing new treatment strategies and identifying new prognostic and predictive markers due to the extensive genetic heterogeneity of BC. Very few studies have investigated the impact of mRNA expression of these genes on the survival of BC patients.
METHODS
We examined the impact of the mRNA expression of breast cancer gene type 1 (), breast cancer gene type 2 (), and partner and localizer of () on the metastasis-free survival (MFS) of patients with early BC using microarray gene expression analysis.
RESULTS
The study was performed in a cohort of 461 patients with a median age of 62 years at initial diagnosis. The median follow-up time was 147 months. We could show that the lower expression of and is significantly associated with longer MFS ( < 0.050). On the contrary, the lower expression of PALB2 was correlated with a shorter MFS ( = 0.049). Subgroup survival analysis identified the prognostic influence of mRNA expression for among patients with luminal-B-like BC and for and in the subset of patients with luminal-A-like BC ( < 0.050).
CONCLUSIONS
According to our observations, , , and expression might become valuable biomarkers of disease progression.
PubMed: 38927568
DOI: 10.3390/biomedicines12061361 -
Biology May 2024Gynecological diseases are triggered by aberrant molecular pathways that alter gene expression, hormonal balance, and cellular signaling pathways, which may lead to...
Gynecological diseases are triggered by aberrant molecular pathways that alter gene expression, hormonal balance, and cellular signaling pathways, which may lead to long-term physiological consequences. This study was able to identify highly preserved modules and key hub genes that are mainly associated with gynecological diseases, represented by endometriosis (EM), ovarian cancer (OC), cervical cancer (CC), and endometrial cancer (EC), through the weighted gene co-expression network analysis (WGCNA) of microarray datasets sourced from the Gene Expression Omnibus (GEO) database. Five highly preserved modules were observed across the EM (GSE51981), OC (GSE63885), CC (GSE63514), and EC (GSE17025) datasets. The functional annotation and pathway enrichment analysis revealed that the highly preserved modules were heavily involved in several inflammatory pathways that are associated with transcription dysregulation, such as NF-kB signaling, JAK-STAT signaling, MAPK-ERK signaling, and mTOR signaling pathways. Furthermore, the results also include pathways that are relevant in gynecological disease prognosis through viral infections. Mutations in the gene that encodes for ERα, which were shown to also affect signaling pathways involved in inflammation, further indicate its importance in gynecological disease prognosis. Potential drugs were screened through the Drug Repurposing Encyclopedia (DRE) based on the up-and downregulated hub genes, wherein a bacterial ribosomal subunit inhibitor and a benzodiazepine receptor agonist were the top candidates. Other drug candidates include a dihydrofolate reductase inhibitor, glucocorticoid receptor agonists, cholinergic receptor agonists, selective serotonin reuptake inhibitors, sterol demethylase inhibitors, a bacterial antifolate, and serotonin receptor antagonist drugs which have known anti-inflammatory effects, demonstrating that the gene network highlights specific inflammatory pathways as a therapeutic avenue in designing drug candidates for gynecological diseases.
PubMed: 38927277
DOI: 10.3390/biology13060397 -
Anticancer Research Jul 2024Clinical diagnostic value of circ-ARHGER28 in breast cancer (BC), and the biological functions of circ-ARHGER28 on the proliferation and apoptosis of MCF-7 cells were...
BACKGROUND/AIM
Clinical diagnostic value of circ-ARHGER28 in breast cancer (BC), and the biological functions of circ-ARHGER28 on the proliferation and apoptosis of MCF-7 cells were investigated.
MATERIALS AND METHODS
Human circRNA microarray was performed to analyze the expression of circRNAs in BC patients. RT-qPCR combined with bioinformatics analysis was applied to verify the candidate circRNAs in BC tissues and peripheral blood samples. Circ-ARHGER28 was chosen as the candidate gene for further research. The clinical diagnostic value and biological functions of circ-ARHGER28 were analyzed. The overexpression and negative control vector of circ-ARHGER28 were constructed and transfected to MCF-7 cells. The CCK 8 assay and clone formation experiments were applied to detect the cell proliferative and migratory abilities. Flow cytometry was used to analyze cell apoptosis and cell cycle distribution. RT-qPCR and Western blot were performed to detect apoptosis and expression of PI3K/AKT/mTOR-associated genes and proteins.
RESULTS
Overexpression of circ-ARHGER28 inhibited the proliferation, colony formation and migration of MCF-7 cells, while increasing the population of the cells in the G/M phase and the apoptotic rate. Apoptosis associated genes and proteins were significantly increased, whereas gene and protein expression of PI3K, AKT and mTOR were decreased in the cells.
CONCLUSION
Circular RNA ARHGER28 exhibits promising diagnostic value for BC. Circ-ARHGER28 inhibited MCF-7 cell proliferation and increased the apoptotic rate. The function of circ-ARHGER28 was associated with the PI3K/AKT/mTOR signaling pathway. Circ-ARHGER28 could be an ideal biomarker for BC diagnosis and a novel target for BC therapy.
Topics: Humans; Breast Neoplasms; Cell Proliferation; Female; Apoptosis; RNA, Circular; MCF-7 Cells; Proto-Oncogene Proteins c-akt; Gene Expression Regulation, Neoplastic; TOR Serine-Threonine Kinases; Biomarkers, Tumor; Signal Transduction; Phosphatidylinositol 3-Kinases; Cell Movement; Middle Aged
PubMed: 38925846
DOI: 10.21873/anticanres.17100 -
American Journal of Medical Genetics.... Jun 2024Low-pass whole genome sequencing (LP-WGS) has been applied as alternative method to detect copy number variants (CNVs) in the clinical setting. Compared with chromosomal...
Low-pass whole genome sequencing (LP-WGS) has been applied as alternative method to detect copy number variants (CNVs) in the clinical setting. Compared with chromosomal microarray analysis (CMA), the sequencing-based approach provides a similar resolution of CNV detection at a lower cost. In this study, we assessed the efficiency and reliability of LP-WGS as a more affordable alternative to CMA. A total of 1363 patients with unexplained neurodevelopmental delay/intellectual disability, autism spectrum disorders, and/or multiple congenital anomalies were enrolled. Those patients were referred from 15 nonprofit organizations and university centers located in different states in Brazil. The analysis of LP-WGS at 1x coverage (>50kb) revealed a positive testing result in 22% of the cases (304/1363), in which 219 and 85 correspond to pathogenic/likely pathogenic (P/LP) CNVs and variants of uncertain significance (VUS), respectively. The 16% (219/1363) diagnostic yield observed in our cohort is comparable to the 15%-20% reported for CMA in the literature. The use of commercial software, as demonstrated in this study, simplifies the implementation of the test in clinical settings. Particularly for countries like Brazil, where the cost of CMA presents a substantial barrier to most of the population, LP-WGS emerges as a cost-effective alternative for investigating copy number changes in cytogenetics.
PubMed: 38924610
DOI: 10.1002/ajmg.a.63802