Biology subjects

Shiddiky, M. J. A.

Publications and source records attributed to Shiddiky, M. J. A..

3 recordsLinked to original sources

bioRxiv2026

Exo-LAMP: A Rapid Extraction-Free Isothermal Assay for Exosomal mRNA Detection

Preeclampsia arises from early placental dysfunction that precedes clinical presentation, creating a need for molecular readouts that enable timely risk assessment. Placenta derived exosomes carry nucleic acid biomarkers with diagnostic potential, yet current analytical workflows rely on multistep isolation and extraction procedures that are slow and resource intensive. Here, we report Exo-LAMP, an extraction-free isothermal assay for detecting placental exosomal KISS1 mRNA, representing the first isothermal approach for exosomal mRNA detection. Placental alkaline phosphatase (PLAP) positive syncytiotrophoblast derived exosomes are selectively captured from biofluids using PLAP antibody conjugated magnetic beads within 20 minutes. Captured exosomes are treated with RNase inhibitor and lysed at 90C for 5 minutes, enabling direct RNA release while preserving transcript integrity and eliminating conventional extraction steps. The released RNA is analyzed in the same vessel by colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP), providing semi-quantitative readout within 25 to 30 minutes. The assay quantifies KISS1 mRNA across 103 to 108 exosomes per reaction, with an estimated limit of detection of about 2 exosomes and a limit of quantification of 103 exosomes. Stoichiometric analysis indicates approximately one KISS1 transcript per 27 exosomes, compared with one per 38 exosomes measured by reverse transcription quantitative polymerase chain reaction (RT-qPCR). In both the exosome-depleted serum spike-in and the plasma spike-in samples containing mixed vesicle populations, the assay detects target derived transcripts at 1% abundance and retains measurable signal at the quantification limit in the presence of up to 108 non-target exosomes. These results establish Exo-LAMP as a rapid and simplified platform for exosomal mRNA analysis that integrates selective capture, lysis, and amplification in a single workflow completed in less than 1 hour. The method provides a general framework for semi-quantitative detection of exosomal transcripts and can be adapted to other coding and non-coding RNA targets for liquid biopsy applications.

bioengineering↗

bioRxiv2026

Global dissemination of FC-ϵ RI and p53 signalling perturbation contribute to Dasatinib resistance in Pancreatic Cancer Cell-lines

Acquired Dasatinib Resistance (ADR) hinders efficacious treatment of Pancreatic Cancer (PC), often mediated by dynamic signalling reprogramming due to prolonged drug intake. With a novel signalling cross-talk network modelling, this study analyses transcriptomics data of dasatinib-resistant and dasatinib-sensitive pancreatic cancer cell lines and prioritizes key signalling molecules via systemic coordination of their magnitude of dysregulation and the degree of signalling cross-talk among enriched pathways. Results found the p53 and FC-{epsilon} RI signalling pathways demonstrating significant perturbation enrichment, complementarily orchestrating a total of 87% of the global perturbation map in dasatinib resistance. Further statistical characterization of the cross-talk network identified 10 key resistant biomarkers, including THBS1, CDKN1A, and BCL2L1 within p53 signalling, and RAC2 and MAPK13 within FC epsilon RI signalling. Validation with TCGA transcriptomics, CPTAC relative proteomics, and StringDB protein-protein interaction data for their potential prognostics revealed BCL2L1 as pivotal for global perturbation dissemination and, thereby, a novel therapeutic target.

bioinformatics↗

bioRxiv2025

Mutation profiling of KRAS and BRAF in primary tumors and circulating tumor cells of colorectal cancer patients using PNA-LNA molecular switch

Identifying KRAS and BRAF mutation status is essential for guiding targeted therapies and enhancing treatment outcomes in colorectal cancer (CRC). This study employs the "PNA-LNA molecular switch" to detect mutations in KRAS codon 12 (c.35G>T/G12V) and BRAF codon 600 (c.1799T>A/V600E) from primary tumours and circulating tumour cells (CTCs) in CRC patients, correlating mutation status with clinicopathological parameters. DNA was isolated from 71 primary tumours and 37 CTC samples. Mutation profiles were generated using the PNA-LNA molecular switch. KRAS mutations were detected in 26 primary tumours (36.6%) and 13 CTCs (26.8%), while BRAF mutations were observed in 19 primary tumours (26.8%) and 7 CTCs (19%). No significant correlation was observed between mutation status and clinicopathological parameters in primary tumours. However, KRAS G12V mutations in CTCs significantly correlated with lymph node metastasis (p=0.002), overall pathological stage (p=0.005), and lymphovascular invasion (p=0.034). BRAF V600E mutation status showed no significant clinicopathological associations. Validation of the PNA-LNA molecular switch against Next-Generation Sequencing (NGS) showed 89% concordance with p-values < 0.001 for both genes. This method is highly comparable to NGS for detecting KRAS and BRAF mutations and shows promise as a point-of-care diagnostic tool. Larger patient cohorts are required to confirm its clinical utility.

cancer biology↗