Pub Date : 2025-02-01DOI: 10.1016/j.mcp.2025.102009
Gabriella Páll , Tamás Pándics , Erzsébet Pintér , Mária Kun , Anna Karoliny , Lajos A. Réthy
{"title":"Uncovering molecular sensitization patterns for peanut in East-Central European children: The dominance of Ara h 6","authors":"Gabriella Páll , Tamás Pándics , Erzsébet Pintér , Mária Kun , Anna Karoliny , Lajos A. Réthy","doi":"10.1016/j.mcp.2025.102009","DOIUrl":"10.1016/j.mcp.2025.102009","url":null,"abstract":"","PeriodicalId":49799,"journal":{"name":"Molecular and Cellular Probes","volume":"79 ","pages":"Article 102009"},"PeriodicalIF":2.3,"publicationDate":"2025-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142980505","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-02-01DOI: 10.1016/j.mcp.2024.102004
Xiuqin Chen , Min Zheng , Su Lin , Meiqing Huang , Shaoying Chen , Shilong Chen
High-throughput genetic screening serves as an indispensable approach for deciphering gene functions and the intricate relationships between phenotypes and genotypes. The CRISPR/Cas9 system, with its ability to precisely edit genomes on a large scale, has revolutionized the field by enabling the construction of comprehensive genomic libraries. This technology has become a cornerstone for genome-wide screenings in disease research. This review offers a comprehensive examination of how CRISPR/Cas9-based genetic screening has been leveraged to uncover genes that play a role in disease mechanisms, focusing on areas such as cancer development and viral replication processes. The insights presented in this review hold promise for the development of novel therapeutic strategies and precision medicine approaches.
{"title":"The application of CRISPR/Cas9–based genome-wide screening to disease research","authors":"Xiuqin Chen , Min Zheng , Su Lin , Meiqing Huang , Shaoying Chen , Shilong Chen","doi":"10.1016/j.mcp.2024.102004","DOIUrl":"10.1016/j.mcp.2024.102004","url":null,"abstract":"<div><div>High-throughput genetic screening serves as an indispensable approach for deciphering gene functions and the intricate relationships between phenotypes and genotypes. The CRISPR/Cas9 system, with its ability to precisely edit genomes on a large scale, has revolutionized the field by enabling the construction of comprehensive genomic libraries. This technology has become a cornerstone for genome-wide screenings in disease research. This review offers a comprehensive examination of how CRISPR/Cas9-based genetic screening has been leveraged to uncover genes that play a role in disease mechanisms, focusing on areas such as cancer development and viral replication processes. The insights presented in this review hold promise for the development of novel therapeutic strategies and precision medicine approaches.</div></div>","PeriodicalId":49799,"journal":{"name":"Molecular and Cellular Probes","volume":"79 ","pages":"Article 102004"},"PeriodicalIF":2.3,"publicationDate":"2025-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142873326","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-02-01DOI: 10.1016/j.mcp.2025.102012
Pritam Bhagwan Bhosale , Abuyaseer Abusaliya , Hun Hwan Kim , Vetrivel Preethi , Se Hyo Jeong , Min Yeong Park , Chung Kil Won , Jeong Doo Heo , Meejung Ahn , Je Kyung Seong , Gon Sup Kim
Despite numerous attempts to understand the molecular mechanisms behind the development of liver cancer, it continues to pose a significant worldwide health challenge. Transcriptome sequencing, a powerful tool in molecular biology, has played a pivotal role in uncovering the intricate gene expression profiles underlying hepatocellular carcinoma (HCC). In the present study, we identified a total of 808 differentially expressed genes (DEGs), with 584 exhibiting downregulation, and 224 showing upregulation following apigetrin treatment. We utilized a combination of bioinformatics tools and platforms, including Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment and mapping, Protein-Protein Interaction (PPI), and GEPIA. We found that DEGs were related to the apoptotic cell death process and identified hub genes, namely CASP8, RB1, and TGFBR2. These genes were further validated through both GEPIA analysis and western blot experiments. Our findings collectively demonstrate that apigetrin has the potential to modulate genes related to liver cancer and trigger molecular pathways that lead to apoptotic cell death in liver cancer cells. This study underscores the potential of apigetrin as an innovative treatment strategy for HCC, emphasizing the need for additional research to elucidate its mechanisms of action and evaluate its clinical efficacy.
{"title":"Comprehensive analysis of Apigetrin's effects on liver cancer cells: Insights from bioinformatics, in vitro studies, and next-generation transcriptome sequencing","authors":"Pritam Bhagwan Bhosale , Abuyaseer Abusaliya , Hun Hwan Kim , Vetrivel Preethi , Se Hyo Jeong , Min Yeong Park , Chung Kil Won , Jeong Doo Heo , Meejung Ahn , Je Kyung Seong , Gon Sup Kim","doi":"10.1016/j.mcp.2025.102012","DOIUrl":"10.1016/j.mcp.2025.102012","url":null,"abstract":"<div><div>Despite numerous attempts to understand the molecular mechanisms behind the development of liver cancer, it continues to pose a significant worldwide health challenge. Transcriptome sequencing, a powerful tool in molecular biology, has played a pivotal role in uncovering the intricate gene expression profiles underlying hepatocellular carcinoma (HCC). In the present study, we identified a total of 808 differentially expressed genes (DEGs), with 584 exhibiting downregulation, and 224 showing upregulation following apigetrin treatment. We utilized a combination of bioinformatics tools and platforms, including Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment and mapping, Protein-Protein Interaction (PPI), and GEPIA. We found that DEGs were related to the apoptotic cell death process and identified hub genes, namely CASP8, RB1, and TGFBR2. These genes were further validated through both GEPIA analysis and western blot experiments. Our findings collectively demonstrate that apigetrin has the potential to modulate genes related to liver cancer and trigger molecular pathways that lead to apoptotic cell death in liver cancer cells. This study underscores the potential of apigetrin as an innovative treatment strategy for HCC, emphasizing the need for additional research to elucidate its mechanisms of action and evaluate its clinical efficacy.</div></div>","PeriodicalId":49799,"journal":{"name":"Molecular and Cellular Probes","volume":"79 ","pages":"Article 102012"},"PeriodicalIF":2.3,"publicationDate":"2025-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143048647","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-02-01DOI: 10.1016/j.mcp.2025.102014
Akbar Amirfiroozy , Maryam Naghinejad , Azim Rezamand , Hamid Farhangi , Zahra Golchehre , Hossein Jalali , Mohammad Taheri , Mohammad Keramatipour
Osteopetrosis is a group of genetically and clinically diverse inherited disorders characterized by an increase in bone density. The main known cause is an abnormality in the development or function of osteoclasts. Hence, the process of bone resorption is impaired, resulting in: 1- a reduction in bone marrow volume and, subsequently, a decrement in the hematopoietic capacity of bone marrow, which leads to anemia and compromised immunological function; 2- improper bone development, which leads to pressure on peripheral nerves, causing auditory, visual, and movement impairments; and 3- disturbance in the formation of bone microstructure that leads to susceptibility to bone fracture. This study aimed to evaluate the clinical symptoms and genetic causes of 30 patients (probands) who suffered from malignant infantile osteopetrosis, a subtype of this disorder. The Sanger sequencing technique was used to sequence four common genes (TCIRG1, CLCN7, SNX10, and OSTM1) in osteopetrosis. Subsequently, the selected variants were subjected to segregation analysis between the probands and their parents. Consequently, the sequencing of these four genes in probands revealed 16 pathogenic and likely pathogenic mutations, five of which had never been reported before. The TCIRG1 gene has three novel splice site variations and one frameshift variant. The CLCN7 gene had a novel missense variant. Also, a total of five variants of uncertain significance (VUSs) were identified in the analyzed sequences, of which three haven't been reported to date, and two were observed in osteopetrosis patients. Therefore, by documenting these novel likely pathogenic variants and VUS in known genes associated with this disease in patients, specialists can conduct more accurate genetic analysis and counseling when encountering these variants. Additionally, this documentation will facilitate the reclassification of these variants.
{"title":"A comprehensive report of the clinical and mutational profiles of 30 Iranian malignant infantile osteopetrosis patients","authors":"Akbar Amirfiroozy , Maryam Naghinejad , Azim Rezamand , Hamid Farhangi , Zahra Golchehre , Hossein Jalali , Mohammad Taheri , Mohammad Keramatipour","doi":"10.1016/j.mcp.2025.102014","DOIUrl":"10.1016/j.mcp.2025.102014","url":null,"abstract":"<div><div>Osteopetrosis is a group of genetically and clinically diverse inherited disorders characterized by an increase in bone density. The main known cause is an abnormality in the development or function of osteoclasts. Hence, the process of bone resorption is impaired, resulting in: 1- a reduction in bone marrow volume and, subsequently, a decrement in the hematopoietic capacity of bone marrow, which leads to anemia and compromised immunological function; 2- improper bone development, which leads to pressure on peripheral nerves, causing auditory, visual, and movement impairments; and 3- disturbance in the formation of bone microstructure that leads to susceptibility to bone fracture. This study aimed to evaluate the clinical symptoms and genetic causes of 30 patients (probands) who suffered from malignant infantile osteopetrosis, a subtype of this disorder. The Sanger sequencing technique was used to sequence four common genes (<em>TCIRG1</em>, <em>CLCN7, SNX10</em>, and <em>OSTM1</em>) in osteopetrosis. Subsequently, the selected variants were subjected to segregation analysis between the probands and their parents. Consequently, the sequencing of these four genes in probands revealed 16 pathogenic and likely pathogenic mutations, five of which had never been reported before. The <em>TCIRG1</em> gene has three novel splice site variations and one frameshift variant. The <em>CLCN7</em> gene had a novel missense variant. Also, a total of five variants of uncertain significance (VUSs) were identified in the analyzed sequences, of which three haven't been reported to date, and two were observed in osteopetrosis patients. Therefore, by documenting these novel likely pathogenic variants and VUS in known genes associated with this disease in patients, specialists can conduct more accurate genetic analysis and counseling when encountering these variants. Additionally, this documentation will facilitate the reclassification of these variants.</div></div>","PeriodicalId":49799,"journal":{"name":"Molecular and Cellular Probes","volume":"79 ","pages":"Article 102014"},"PeriodicalIF":2.3,"publicationDate":"2025-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143061452","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-02-01DOI: 10.1016/j.mcp.2024.102000
Andrew Peifer, Anna Kidney, Geetha Nattanmai, Kate Wahl, Sherly Jose, Elizabeth Owuor, Linnell Randall, Erin Klingbeil, Kimberlee A. Musser, Kara Mitchell
blaROB-1 is the only widely found β-lactamase in Neisseria meningitidis, and its presence is on the rise. To enhance our bacterial meningitis testing procedure, we clinically validated a real-time PCR assay to rapidly detect the blaROB gene and predict drug resistance in Neisseria meningitidis. A screen of 101 clinical isolates and 37 clinical specimens of blood and cerebrospinal fluid received between January 2018 and June 2024 found 8 isolates and 2 cerebrospinal fluid specimens that were positive for blaROB.
{"title":"Rapid diagnostic testing method to detect ROB β-lactamase gene in Neisseria meningitidis","authors":"Andrew Peifer, Anna Kidney, Geetha Nattanmai, Kate Wahl, Sherly Jose, Elizabeth Owuor, Linnell Randall, Erin Klingbeil, Kimberlee A. Musser, Kara Mitchell","doi":"10.1016/j.mcp.2024.102000","DOIUrl":"10.1016/j.mcp.2024.102000","url":null,"abstract":"<div><div>bla<sub>ROB-1</sub> is the only widely found β-lactamase in <em>Neisseria meningitidis</em>, and its presence is on the rise. To enhance our bacterial meningitis testing procedure, we clinically validated a real-time PCR assay to rapidly detect the <em>bla</em><sub>ROB</sub> gene and predict drug resistance in <em>Neisseria meningitidis</em>. A screen of 101 clinical isolates and 37 clinical specimens of blood and cerebrospinal fluid received between January 2018 and June 2024 found 8 isolates and 2 cerebrospinal fluid specimens that were positive for <em>bla</em><sub>ROB</sub>.</div></div>","PeriodicalId":49799,"journal":{"name":"Molecular and Cellular Probes","volume":"79 ","pages":"Article 102000"},"PeriodicalIF":2.3,"publicationDate":"2025-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142824533","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-02-01DOI: 10.1016/j.mcp.2024.101997
Le Zhang , Jiahong Li , Qiwen Wan , Chaozhi Bu , Weilai Jin , Fuqiang Yuan , Wenhao Zhou
The therapeutic potential of intestinal stem cell-derived extracellular vesicles (ISCs-EVs) in necrotizing enterocolitis (NEC) remains largely unexplored. This research aims to investigate the therapeutic effects of ISCs-EVs on NEC. Lgr5-positive ISCs were screened from the small intestine of mice by flow cytometry, and ISCs-EVs were isolated by density gradient centrifugation. Subsequently, ISCs-EVs were identified through transmission electron microscopy, nanoparticle tracking analysis, and western blotting. Subsequently, we evaluated the efficacy of ISCs-EVs in a mouse model of NEC and found that they enhanced survival (more than 20 %), reduced intestinal damage (restore the number of intestinal crypts and decrease the expression of MPO and cleaved-caspase 3 in intestinal tissues), promoted angiogenesis (the mRNA expression of VEGF was increased by approximately 35 %), and mitigated inflammation (decreased the level of MUC1, p-NF-κB, IL-6 and TNF-α). Furthermore, in vitro assessments demonstrated that ISCs-EVs reduced apoptosis (P < 0.01) and stimulated proliferation (P < 0.05) of IEC-6 cells, while enhancing mucin secretion in LS174T cells. In summary, our study provides a comprehensive assessment of the therapeutic effects of ISCs-EVs on NEC, using both animal and cell models. This highlights their potential for use in NEC treatment.
{"title":"Intestinal stem cell-derived extracellular vesicles ameliorate necrotizing enterocolitis injury","authors":"Le Zhang , Jiahong Li , Qiwen Wan , Chaozhi Bu , Weilai Jin , Fuqiang Yuan , Wenhao Zhou","doi":"10.1016/j.mcp.2024.101997","DOIUrl":"10.1016/j.mcp.2024.101997","url":null,"abstract":"<div><div>The therapeutic potential of intestinal stem cell-derived extracellular vesicles (ISCs-EVs) in necrotizing enterocolitis (NEC) remains largely unexplored. This research aims to investigate the therapeutic effects of ISCs-EVs on NEC. Lgr5-positive ISCs were screened from the small intestine of mice by flow cytometry, and ISCs-EVs were isolated by density gradient centrifugation. Subsequently, ISCs-EVs were identified through transmission electron microscopy, nanoparticle tracking analysis, and western blotting. Subsequently, we evaluated the efficacy of ISCs-EVs in a mouse model of NEC and found that they enhanced survival (more than 20 %), reduced intestinal damage (restore the number of intestinal crypts and decrease the expression of MPO and cleaved-caspase 3 in intestinal tissues), promoted angiogenesis (the mRNA expression of VEGF was increased by approximately 35 %), and mitigated inflammation (decreased the level of MUC1, p-NF-κB, IL-6 and TNF-α). Furthermore, in vitro assessments demonstrated that ISCs-EVs reduced apoptosis (P < 0.01) and stimulated proliferation (P < 0.05) of IEC-6 cells, while enhancing mucin secretion in LS174T cells. In summary, our study provides a comprehensive assessment of the therapeutic effects of ISCs-EVs on NEC, using both animal and cell models. This highlights their potential for use in NEC treatment.</div></div>","PeriodicalId":49799,"journal":{"name":"Molecular and Cellular Probes","volume":"79 ","pages":"Article 101997"},"PeriodicalIF":2.3,"publicationDate":"2025-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142792408","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-02-01DOI: 10.1016/j.mcp.2024.101998
Fangyue Guo , Jianghao Li , Peizhi Ma , Mengying Liu , Jing Wu , Hai Qu , Yehuan Zheng , Mengying Wang , Seyed Sepehr Marashi , Zhijian Zhang , Shanfeng Zhang , Guangyu Fu , Pei Li
Ciprofloxacin (CIP) is a broad-spectrum fluoroquinolone antibiotic, and its excessive residues in food and water sources pose potential risks to human health. Therefore, there is a need for a rapid and convenient method for its accurate quantification. The clustered regularly interspaced short palindromic repeat (CRISPR)/Cas12a system has gained extensive application in signal detection and amplification due to the trans-cleavage activity of Cas12a. In this study, we devised a novel magnetic bead-based dual sandwich aptamer coupled with a CRISPR/Cas12a system for the precise quantification of CIP in milk, river water, and honey. Through the incorporation of a magnetic bead-based dual aptamer sandwich approach, the concentration of CIP in the samples was pre-enriched. Additionally, by optimizing the Fluorescence-Quencher (F-Q) probe concentration, detection aptamer (APTd) concentration, and assay duration, the limit of blank (LOB) of the system was determined as 362 nM, while the limit of detection (LOD) was determined as 403 nM. This enabled the accurate quantification of CIP within the linear range of 0.5 μM to 0.2 mM with high specificity. Moreover, the performance of this detection method was comparable to that of high-performance liquid chromatography (HPLC) in river water, milk, and honey samples.
{"title":"A magnetic bead-based dual-aptamer sandwich assay for quantitative detection of ciprofloxacin using CRISPR/Cas12a","authors":"Fangyue Guo , Jianghao Li , Peizhi Ma , Mengying Liu , Jing Wu , Hai Qu , Yehuan Zheng , Mengying Wang , Seyed Sepehr Marashi , Zhijian Zhang , Shanfeng Zhang , Guangyu Fu , Pei Li","doi":"10.1016/j.mcp.2024.101998","DOIUrl":"10.1016/j.mcp.2024.101998","url":null,"abstract":"<div><div>Ciprofloxacin (CIP) is a broad-spectrum fluoroquinolone antibiotic, and its excessive residues in food and water sources pose potential risks to human health. Therefore, there is a need for a rapid and convenient method for its accurate quantification. The clustered regularly interspaced short palindromic repeat (CRISPR)/Cas12a system has gained extensive application in signal detection and amplification due to the trans-cleavage activity of Cas12a. In this study, we devised a novel magnetic bead-based dual sandwich aptamer coupled with a CRISPR/Cas12a system for the precise quantification of CIP in milk, river water, and honey. Through the incorporation of a magnetic bead-based dual aptamer sandwich approach, the concentration of CIP in the samples was pre-enriched. Additionally, by optimizing the Fluorescence-Quencher (F-Q) probe concentration, detection aptamer (APTd) concentration, and assay duration, the limit of blank (LOB) of the system was determined as 362 nM, while the limit of detection (LOD) was determined as 403 nM. This enabled the accurate quantification of CIP within the linear range of 0.5 μM to 0.2 mM with high specificity. Moreover, the performance of this detection method was comparable to that of high-performance liquid chromatography (HPLC) in river water, milk, and honey samples.</div></div>","PeriodicalId":49799,"journal":{"name":"Molecular and Cellular Probes","volume":"79 ","pages":"Article 101998"},"PeriodicalIF":2.3,"publicationDate":"2025-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142814709","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-02-01DOI: 10.1016/j.mcp.2024.102005
Ingrid Lojova , Marcel Kucharik , Zuzana Pös , Andrej Balaz , Andrea Zatkova , Eva Tothova Tarova , Jaroslav Budis , Ludevit Kadasi , Tomas Szemes , Jan Radvanszky
Myotonic dystrophy type 1 (DM1) is a serious multisystem disorder caused by GCA repeat expansions in the DMPK gene. Early and accurate diagnosis, often requiring reliable DNA-diagnostic techniques, is critical for preventing life-threatening cardiac complications. Clinically, two main diagnostic challenges exist. Firstly, because of overlapping symptomatology with other conditions, conventional DNA-testing methods focusing on DM1 expansion detection ensure diagnostic results only in a small subset of patients, and frequently, further DNA-testing in remaining cases is necessary. Secondly, because of variable symptomatology and age of onset, not all DM1 patients are referred for DM1 genetic testing, leading to unrecognized but at-risk cases. When using conventional methods, the main technical problems are expanded-allele sizing and sensitivity to the presence of sequence interruptions. On a set of 50 individual genomes, including ten DM1 patients, we tested the performance of short-read whole-genome sequencing (WGS), one of the most up-to-date molecular testing methods. We identified all expansion-range DM1 alleles and characterized sequence interruptions in seven expansion-range/premutation-range alleles. Although neither the tested conventional methods, nor WGS allowed expanded-allele sizing, conventional methods provided higher sizing limits for normal-range alleles. Genotyping concordance rate was found to be 95–99 %. WGS was found to be superior in elucidating the sequence structure of the motifs, even if they fall outside the sizing limit (from partial reads). In addition, WGS enables the identification of genetic modifiers in other genes and the detection of alternative diagnoses in DM1-negative patients by extension of the bioinformatic evaluation of the generated data.
{"title":"Advancing molecular diagnostics of myotonic dystrophy type 1 using short-read whole genome sequencing","authors":"Ingrid Lojova , Marcel Kucharik , Zuzana Pös , Andrej Balaz , Andrea Zatkova , Eva Tothova Tarova , Jaroslav Budis , Ludevit Kadasi , Tomas Szemes , Jan Radvanszky","doi":"10.1016/j.mcp.2024.102005","DOIUrl":"10.1016/j.mcp.2024.102005","url":null,"abstract":"<div><div>Myotonic dystrophy type 1 (DM1) is a serious multisystem disorder caused by GCA repeat expansions in the <em>DMPK</em> gene. Early and accurate diagnosis, often requiring reliable DNA-diagnostic techniques, is critical for preventing life-threatening cardiac complications. Clinically, two main diagnostic challenges exist. Firstly, because of overlapping symptomatology with other conditions, conventional DNA-testing methods focusing on DM1 expansion detection ensure diagnostic results only in a small subset of patients, and frequently, further DNA-testing in remaining cases is necessary. Secondly, because of variable symptomatology and age of onset, not all DM1 patients are referred for DM1 genetic testing, leading to unrecognized but at-risk cases. When using conventional methods, the main technical problems are expanded-allele sizing and sensitivity to the presence of sequence interruptions. On a set of 50 individual genomes, including ten DM1 patients, we tested the performance of short-read whole-genome sequencing (WGS), one of the most up-to-date molecular testing methods. We identified all expansion-range DM1 alleles and characterized sequence interruptions in seven expansion-range/premutation-range alleles. Although neither the tested conventional methods, nor WGS allowed expanded-allele sizing, conventional methods provided higher sizing limits for normal-range alleles. Genotyping concordance rate was found to be 95–99 %. WGS was found to be superior in elucidating the sequence structure of the motifs, even if they fall outside the sizing limit (from partial reads). In addition, WGS enables the identification of genetic modifiers in other genes and the detection of alternative diagnoses in DM1-negative patients by extension of the bioinformatic evaluation of the generated data.</div></div>","PeriodicalId":49799,"journal":{"name":"Molecular and Cellular Probes","volume":"79 ","pages":"Article 102005"},"PeriodicalIF":2.3,"publicationDate":"2025-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142878315","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-02-01DOI: 10.1016/j.mcp.2025.102008
Viktória Szabó , Balázs Varsányi , Mirella Barboni , Ágnes Takács , Krisztina Knézy , Mária Judit Molnár , Zoltán Zsolt Nagy , Bence György , Carlo Rivolta
The rapid advancements in the field of genetics have significantly propelled the development of gene therapies, paving the way for innovative treatments of various hereditary disorders. This review focuses on the genetics of ophthalmologic conditions, highlighting the currently approved ophthalmic gene therapy and exploring emerging therapeutic strategies under development. Inherited retinal dystrophies represent a heterogeneous group of genetic disorders that manifest across a broad spectrum from infancy to late middle age. Key clinical features include nyctalopia (night blindness), constriction of the visual field, impairments in color perception, reduced central visual acuity, and rapid eye movements. Recent technological advancements, such as multimodal imaging, psychophysical assessments, and electrophysiological testing, have greatly enhanced our ability to understand disease progression and establish genotype-phenotype correlations.
Additionally, the integration of molecular diagnostics into clinical practice is revolutionizing patient stratification and the design of targeted interventions, underscoring the transformative potential of personalized medicine in ophthalmology. The review also covers the challenges and opportunities in developing gene therapies for other ophthalmic conditions, such as age-related macular degeneration and optic neuropathies. We discuss the viral and non-viral vector systems used in ocular gene therapy, highlighting their advantages and limitations. Additionally, we explore the potential of emerging technologies like CRISPR/Cas9 in treating genetic eye diseases. We briefly address the regulatory landscape, concerns, challenges, and future directions of gene therapy in ophthalmology. We emphasize the need for long-term safety and efficacy data as these innovative treatments move from bench to bedside.
{"title":"Insights into eye genetics and recent advances in ocular gene therapy","authors":"Viktória Szabó , Balázs Varsányi , Mirella Barboni , Ágnes Takács , Krisztina Knézy , Mária Judit Molnár , Zoltán Zsolt Nagy , Bence György , Carlo Rivolta","doi":"10.1016/j.mcp.2025.102008","DOIUrl":"10.1016/j.mcp.2025.102008","url":null,"abstract":"<div><div>The rapid advancements in the field of genetics have significantly propelled the development of gene therapies, paving the way for innovative treatments of various hereditary disorders. This review focuses on the genetics of ophthalmologic conditions, highlighting the currently approved ophthalmic gene therapy and exploring emerging therapeutic strategies under development. Inherited retinal dystrophies represent a heterogeneous group of genetic disorders that manifest across a broad spectrum from infancy to late middle age. Key clinical features include nyctalopia (night blindness), constriction of the visual field, impairments in color perception, reduced central visual acuity, and rapid eye movements. Recent technological advancements, such as multimodal imaging, psychophysical assessments, and electrophysiological testing, have greatly enhanced our ability to understand disease progression and establish genotype-phenotype correlations.</div><div>Additionally, the integration of molecular diagnostics into clinical practice is revolutionizing patient stratification and the design of targeted interventions, underscoring the transformative potential of personalized medicine in ophthalmology. The review also covers the challenges and opportunities in developing gene therapies for other ophthalmic conditions, such as age-related macular degeneration and optic neuropathies. We discuss the viral and non-viral vector systems used in ocular gene therapy, highlighting their advantages and limitations. Additionally, we explore the potential of emerging technologies like CRISPR/Cas9 in treating genetic eye diseases. We briefly address the regulatory landscape, concerns, challenges, and future directions of gene therapy in ophthalmology. We emphasize the need for long-term safety and efficacy data as these innovative treatments move from bench to bedside.</div></div>","PeriodicalId":49799,"journal":{"name":"Molecular and Cellular Probes","volume":"79 ","pages":"Article 102008"},"PeriodicalIF":2.3,"publicationDate":"2025-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142980504","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
breast cancer-associated fibroblast (CAF) is linked to metastasis and is poor for breast cancer prognosis. Since Clostridium Toxin A (Botox-A) had represented a cytotoxic effect on fibroblasts, this study aims to assess Botox-A cytotoxicity in both normal fibroblasts and exosome-induced CAFs.
Material and method
the serum exosomes of 40 BC patients and 30 healthy individuals were isolated and lncRNA H19 (lnch19) levels were assessed by qRT-PCR method. After that, Breast Cancer (BC) exosomes co-cultured with Human foreskin fibroblasts (HFF) and qRT-PCR were applied to evaluate α-SMA, Vimentin, BCL-2, and BAX expression. Both Normal and malignant HFFs co-cultured with Botox-A, and Botox-A loaded exosome for 24 and 48 h and their apoptosis, Cell proliferation, and viability were monitored by MTT assay, Annexin V-FITC and PI staining and qRT-PCR for BCL-2, BAX, and cyclin D1 mRNAs.
Results
Serum exosomes of BC patients had significantly higher levels of lncRNA H19 than healthy individuals. MTT assay results showed Botox-A decreased vital Human foreskin fibroblasts in a dose-dependent manner. BC exosomes significantly increased α-SMA, Vimentin, and BCL-2 mRNA levels in Human foreskin fibroblasts, on the other hand, BAX decreased meaningfully. Co-culture of exosome-treated HFF cells with both Botox-A and Botox-A loaded exosomes significantly boosted BCL-2 mRNA levels, completely contrary to BAX and cyclid d1 expression. Meanwhile, flow cytometry results confirmed a high rate of apoptosis in malignant Human foreskin fibroblasts treated with Botox-A loaded exosome.
Conclusion
The findings of this study indicate that exosomal lncRNA H19 could be a diagnostic marker for Breast Cancer and these Breast cancer exosomes can induce malignant phenotype in fibroblasts and turn them into CAFs. Botox-A could be toxic for both normal fibroblasts and CAFs, inducing apoptosis and suppressing cell proliferation among them.
{"title":"Botox-A induced apoptosis and suppressed cell proliferation in fibroblasts pre-treated with breast cancer exosomes","authors":"Hossein Sayaf , Niloufar Salimian , Mahnaz Mohammadi , Parisa Ahmadi , Amir Gholamzad , Sadegh Babashah , Maliheh Entezari , Najma Farahani , Maryam Montazeri , Mehrdad Hashemi","doi":"10.1016/j.mcp.2024.102007","DOIUrl":"10.1016/j.mcp.2024.102007","url":null,"abstract":"<div><h3>Background</h3><div>breast cancer-associated fibroblast (CAF) is linked to metastasis and is poor for breast cancer prognosis. Since Clostridium Toxin A (Botox-A) had represented a cytotoxic effect on fibroblasts, this study aims to assess Botox-A cytotoxicity in both normal fibroblasts and exosome-induced CAFs.</div></div><div><h3>Material and method</h3><div>the serum exosomes of 40 BC patients and 30 healthy individuals were isolated and lncRNA H19 (lnch19) levels were assessed by qRT-PCR method. After that, Breast Cancer (BC) exosomes co-cultured with Human foreskin fibroblasts (HFF) and qRT-PCR were applied to evaluate α-SMA, Vimentin, BCL-2, and BAX expression. Both Normal and malignant HFFs co-cultured with Botox-A, and Botox-A loaded exosome for 24 and 48 h and their apoptosis, Cell proliferation, and viability were monitored by MTT assay, Annexin V-FITC and PI staining and qRT-PCR for BCL-2, BAX, and cyclin D1 mRNAs.</div></div><div><h3>Results</h3><div>Serum exosomes of BC patients had significantly higher levels of lncRNA H19 than healthy individuals. MTT assay results showed Botox-A decreased vital Human foreskin fibroblasts in a dose-dependent manner. BC exosomes significantly increased α-SMA, Vimentin, and BCL-2 mRNA levels in Human foreskin fibroblasts, on the other hand, BAX decreased meaningfully. Co-culture of exosome-treated HFF cells with both Botox-A and Botox-A loaded exosomes significantly boosted BCL-2 mRNA levels, completely contrary to BAX and cyclid d1 expression. Meanwhile, flow cytometry results confirmed a high rate of apoptosis in malignant Human foreskin fibroblasts treated with Botox-A loaded exosome.</div></div><div><h3>Conclusion</h3><div>The findings of this study indicate that exosomal lncRNA H19 could be a diagnostic marker for Breast Cancer and these Breast cancer exosomes can induce malignant phenotype in fibroblasts and turn them into CAFs. Botox-A could be toxic for both normal fibroblasts and CAFs, inducing apoptosis and suppressing cell proliferation among them.</div></div>","PeriodicalId":49799,"journal":{"name":"Molecular and Cellular Probes","volume":"79 ","pages":"Article 102007"},"PeriodicalIF":2.3,"publicationDate":"2025-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142899949","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
扫码关注我们
求助内容：
应助结果提醒方式：
京公网安备 11010802042870号