Lanthanide-Complex-Enhanced Bioorthogonal Branched DNA Amplification.

Fluorescence in situ hybridization (FISH) is a widely used technique for detecting intracellular nucleic acids. However, its effectiveness in detecting low-copy nucleic acids is limited due to its low fluorescence intensity and background autofluorescence. To address these challenges, we present here an approach of lanthanide-complex-enhanced bioorthogonal-branched DNA amplification (LEBODA) with high sensitivity for in situ nuclear acid detection in single cells. The approach capitalizes on two levels of signal amplification. First, it utilizes click chemistry to directly link a substantial number of bridge probes to target-recognizing probes, providing an initial boost in signal intensity. Second, it incorporates high-density lanthanide complexes into each bridge probe, enabling secondary amplifications. Compared to the traditional "double Z" probes used in the RNAscope method, LEBODA exhibits 4 times the single enhancement for RNA detection signal with the click chemistry approach. Using SARS-CoV-2 pseudovirus-infected HeLa cells, we demonstrate the superiority in the detection of viral-infected cells in rare populations as low as 20% infectious rate. More encouragingly, the LEBODA approach can be adapted for DNA-FISH and single-molecule RNA-FISH, as well as other hybridization-based signal amplification methods. This adaptability broadens the potential applications of LEBODA in the sensitive detection of biomolecules, indicating promising prospects for future research and practical use.

Lanthanide Complex for Single-Molecule Fluorescent in Situ Hybridization and Background-Free Imaging.

Traditional single-molecule fluorescence in situ hybridization (smFISH) methods for RNA detection often face sensitivity challenges due to the low fluorescence intensity of the probe. Also, short-lived autofluorescence complicates obtaining clear signals from tissue sections. In response, we have developed an smFISH probe using highly grafted lanthanide complexes to address both concentration quenching and autofluorescence background. Our approach involves an oligo PCR incorporating azide-dUTP, enabling conjugation with lanthanide complexes. This method has proven to be stable, convenient, and cost-effective. Notably, for the mRNA detection in SKBR3 cells, the lanthanide probe group exhibited 2.5 times higher luminescence intensity and detected 3 times more signal points in cells compared with the Cy3 group. Furthermore, we successfully applied the probe to image HER2 mRNA molecules in breast cancer FFPE tissue sections, achieving a 2.7-fold improvement in sensitivity compared to Cy3-based probes. These results emphasize the potential of time-resolved smFISH as a highly sensitive method for nucleic acid detection, free of background fluorescence interference.

EGFR/TKIs induce excessive apoptosis of bladder carcinoma cells by arresting cell cycle and promoting mitochondrial peroxidation damage.

In recent years, bladder carcinoma (BC) has shown an increasing incidence, with poor patient outcomes. In clinical practice, BC is still mainly treated by surgery combined with chemoradiotherapy. However, as chemotherapy resistance of tumor cells becomes more and more obvious, it is urgent to find more effective BC treatment regimes. With the increasing application and growing attention paid to epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) in various neoplastic diseases, EGFR-TKIs have been considered as a new treatment direction in the future. In this study, the research team used AG1478, an EGFR-TKI, to intervene with the BC cell line T24. It was found that the cell activity was statistically decreased, the apoptosis was enhanced, and the cells were dominantly arrested in the G0/G1 phase, confirming the future therapeutic potential of EGFR-TKIs in BC. Besides, the research team further observed that AG1478 also promoted pyroptosis in T24 cells, and its mechanism is related to the induction of mitochondrial oxidative stress damage. The findings lay a more reliable foundation for the future application of EGFR-TKIs in BC.

Diagnostic, prognostic, and therapeutic potential of exosomal microRNAs in renal cancer.

Renal cell carcinoma (RCC) arises from the tubular epithelial cells of the nephron. It has the highest mortality rate among urological cancers. There are no effective therapeutic approaches and no non-invasive biomarkers for diagnosis and follow-up. Thus, suitable novel biomarkers and therapeutic targets are essential for improving RCC diagnosis/prognosis and treatment. Circulating exosomes such as exosomal microRNAs (Exo-miRs) provide non-invasive prognostic/diagnostic biomarkers and valuable therapeutic targets, as they can be easily isolated and quantified and show high sensitivity and specificity. Exosomes secreted by an RCC can exhibit alterations in the miRs' profile that may reflect the cellular origin and (patho)physiological state, as a ''signature'' or ''fingerprint'' of the donor cell. It has been shown that the transportation of renal-specific miRs in exosomes can be rapidly detected and measured, holding great potential as biomarkers in RCC. The present review highlights the studies reporting tumor microenvironment-derived Exo-miRs with therapeutic potential as well as circulating Exo-miRs as potential diagnostic/prognostic biomarkers in patients with RCC.