Biology subjects

Shafiei, N.

Publications and source records attributed to Shafiei, N..

2 recordsLinked to original sources

bioRxiv2024

A versatile correlative light and electron microscopy protocol for human brain and other biological models

Correlative light and electron microscopy (CLEM) combines light microscopy, for identifying a target via genetic labels, dyes, antibodies, and morphological features, with electron microscopy, for analyzing high-resolution subcellular ultrastructures. Here, we describe the step-by-step instructions to perform a CLEM experiment, optimized for the investigation of ultrastructural features in human brain tissue. The procedure is carried out at room-temperature and can be also adapted to other human and animal tissue samples. The procedure requires 8-days to complete and includes the stages of sample fixation for optimal ultrastructural preservation, immunofluorescence staining, image acquisition, multi-modal image correlation, and is executable within standard EM laboratories. Serving as a critical tool for characterizing human tissue and disease models, room-temperature CLEM facilitates the identification and quantification of subcellular morphological features across brain regions. Key pointsO_LIThe protocol for correlative light and electron microscopy (CLEM) is optimized for analyzing chemically fixed human brain tissues. It focuses on maintaining the integrity of ultrastructural features, thereby minimizing artifacts and structural alterations. C_LIO_LIExamining brain tissues at the ultrastructural level can provide an unprecedented amount of detail which may help advance our understanding of the mechanisms underlying neurodegenerative disorders. C_LI

neuroscience↗

bioRxiv2023

Nucleosome reorganisation in breast cancer tissues

Nucleosome repositioning in cancer is believed to cause many changes in genome organisation and gene expression. Understanding these changes is important to elucidate fundamental aspects of cancer. It is also important for medical diagnostics based on cell-free DNA (cfDNA), which originates from genomic DNA regions protected from digestion by nucleosomes. Here we have generated high resolution nucleosome maps in paired tumour and normal tissues from the same breast cancer patients using MNase-assisted histone H3 ChIP-seq and compared them with the corresponding cfDNA from blood plasma. This analysis has detected single-nucleosome repositioning at key regulatory regions in a patient-specific manner and common cancer-specific patterns across patients. The nucleosomes gained in tumour versus normal tissue were particularly informative of cancer pathways, with [~]20-fold enrichment at CpG islands, a large fraction of which marked promoters of genes encoding DNA-binding proteins. In addition, tumour tissues were characterised by a 5-10 bp decrease in the average distance between nucleosomes (nucleosome repeat length, NRL), which is qualitatively similar to the differences between pluripotent and differentiated cells. These effects were correlated with gene activity, DNA sequence repeats abundance, differential DNA methylation and binding of linker histone variants H1.4 and H1X. Our findings provide a new mechanistic understanding of nucleosome repositioning in tumour tissues that can be valuable for patient stratification and monitoring using liquid biopsies.

genomics↗