Biology subjects

Raman, N.

Publications and source records attributed to Raman, N..

5 recordsLinked to original sources

bioRxiv2024

Characterization of immune cell populations in the tumor microenvironment of colorectal cancer using high definition spatial profiling

Colorectal cancer (CRC) is the second-deadliest cancer in the world, yet a deeper understanding of spatial patterns of gene expression in the tumor microenvironment (TME) remains elusive. Here, we introduce the Visium HD platform (10x Genomics) and use it to investigate human CRC and normal adjacent mucosal tissues from formalin fixed paraffin embedded (FFPE) samples. The first assay available on Visium HD is a probe-based spatial transcriptomics workflow that was developed to enable whole transcriptome single cell scale analysis. We demonstrate highly refined unsupervised spatial clustering in Visium HD data that aligns with the hallmarks of colon tissue morphology and is notably improved over earlier Visium assays. Using serial sections from the same FFPE blocks we generate a single cell atlas of our samples, then we integrate the data to comprehensively characterize the immune cell types present in the TME, specifically at the tumor periphery. We observed enrichment of two pro-tumor macrophage subpopulations with differential gene expression profiles that were localized within distinct tumor regions. Further characterization of the T cells present in one of the samples revealed a clonal expansion that we were able to localize in the tissue using in situ gene expression analysis. In situ analysis also allowed us to perform in-depth characterization of the microenvironment of the clonally expanded T cell population and we identified a third macrophage subpopulation with gene expression profiles consistent with an anti-tumor response. Our study provides a comprehensive map of the cellular composition of the CRC TME and identifies phenotypically and spatially distinct immune cell populations within it. We show that the single cell-scale resolution afforded by Visium HD and the whole transcriptome nature of the assay allows investigations into cellular function and interaction at the tumor periphery in FFPE tissues, which has not been previously possible.

cancer biology↗

bioRxiv2024

Tumour specimen cold ischemia time impacts molecular cancer drug target discovery

Tumour tissue collections are used to uncover pathways associated with disease outcomes that can also serve as targets for cancer treatment, ideally by comparing the molecular properties of cancer tissues to matching normal tissues. The quality of such collections determines the value of the data and information generated from their analyses including expression and modifications of nucleic acids and proteins. These biomolecules are dysregulated upon ischemia and decomposed once the living cells start to decay into inanimate matter. Therefore, ischemia time before final tissue preservation is the most important determinant of the quality of a tissue collection. Here we show the impact of ischemia time on tumour and matching adjacent normal tissue samples for mRNAs in 1,664, proteins in 1,818 and phosphoproteins in 1,800 cases (tumour and matching normal samples) of four solid tumour types (CRC, HCC, LUAD and LUSC NSCLC subtypes). In CRC, ischemia times exceeding 15 minutes impacted 12.5% (mRNA), 25% (protein) and 50% (phosphosites) of differentially expressed molecules in tumour versus normal tissues. This hypoxia- and decay-induced dysregulation increased with longer ischemia times and was observed across tumour types. Interestingly, the proteomics analysis revealed that specimen ischemia time above 15 minutes is mostly associated with a dysregulation of proteins in the immune response pathway and less so with metabolic processes. We conclude that ischemia time is a crucial quality parameter for tissue collections used for target discovery and validation in prognostic cancer research.

cancer biology↗

bioRxiv2022

Functional antibodies exhibit light chain coherence

The vertebrate adaptive immune system modifies the genome of individual B cells to encode antibodies binding particular antigens1. In most mammals, antibodies are composed of a heavy and a light chain which are sequentially generated by recombination of V, D (for heavy chains), J, and C gene segments. Each chain contains three complementarity-determining regions (CDR1-3), contributing to antigen specificity. Certain heavy and light chains are preferred for particular antigens2-21. We considered pairs of B cells sharing the same heavy chain V gene and CDRH3 amino acid sequence and isolated from different donors, also known as public clonotypes22,23. We show that for naive antibodies (not yet adapted to antigens), the probability that they use the same light chain V gene is [~]10%, whereas for memory (functional) antibodies it is [~]80%. This property of functional antibodies is a phenomenon we call light chain coherence. We also observe it when similar heavy chains recur within a donor. Thus, though naive antibodies appear to recur by chance, the recurrence of functional antibodies reveals surprising constraint and determinism in the processes of V(D)J recombination and immune selection. For most functional antibodies, the heavy chain determines the light chain.

immunology↗

bioRxiv2022

enclone: precision clonotyping and analysis of immune receptors

Half a billion years of evolutionary battle forged the vertebrate adaptive immune system, an astonishingly versatile factory for molecules that can adapt to arbitrary attacks. The history of an individual encounter is chronicled within a clonotype: the descendants of a single fully rearranged adaptive immune cell. For B cells, reading this immune history for an individual remains a fundamental challenge of modern immunology. Identification of such clonotypes is a magnificently challenging problem for three reasons: O_LIThe cell history is inferred rather than directly observed: the only available data are the sequences of V(D)J molecules occurring in a sample of cells. C_LIO_LIEach immune receptor is a pair of V(D)J molecules. Identifying these pairs at scale is a technological challenge and cannot be done with perfect accuracy--real samples are mixtures of cells and fragments thereof. C_LIO_LIThese molecules can be intensely mutated during the optimization of the response to particular antigens, blurring distinctions between kindred molecules. C_LI It is thus impossible to determine clonotypes exactly. All solutions to this problem make a trade-off between sensitivity and specificity; useful solutions must address actual artifacts found in real data. We present enclone1, a system for computing approximate clonotypes from single cell data, and demonstrate its use and value with the 10x Genomics Immune Profiling Solution. To test it, we generate data for 1.6 million individual B cells, from four humans, including deliberately enriched memory cells, to tax the algorithm and provide a resource for the community. We analytically determine the specificity of enclones clonotyping algorithm, showing that on this dataset the probability of co-clonotyping two unrelated B cells is around 10-9. We prove that using only heavy chains increases the error rate by two orders of magnitude. enclone comprises a comprehensive toolkit for the analysis and display of immune receptor data. It is ultra-fast, easy to install, has public source code, comes with public data, and is documented at bit.ly/enclone. It has three "flavors" of use: (1) as a command-line tool run from a terminal window, that yields visual output; (2) as a command-line tool that yields parseable output that can be fed to other programs; and (3) as a graphical version (GUI).

immunology↗

bioRxiv2018

14-helical β-peptides Elicit Toxicity against C. albicans by Forming Pores in the Cell Membrane and Subsequently Disrupting Intracellular Organelles

Synthetic peptidomimetics of antimicrobial peptides are promising as antimicrobial drug candidates because they promote membrane disruption and exhibit greater structural and proteolytic stability. We previously reported selective antifungal 14-helical {beta}-peptides, but the mechanism of antifungal toxicity of {beta}-peptides remains unknown. To provide insight into the mechanism, we studied antifungal {beta}-peptide binding to artificial membranes and living Candida albicans cells. We investigated the ability of {beta}-peptides to interact with and permeate small unilamellar vesicle models of fungal and bacterial membranes. The partition coefficient supported a pore-mediated mechanism characterized by the existence of a critical {beta}-peptide concentration separating low and high partition coefficient regimes. Live cell intracellular tracking of {beta}-peptides showed that {beta}-peptides translocated into the cytoplasm, and then disrupted the nucleus and vacuole sequentially, leading to cell death. This understanding of the mechanisms of antifungal activity will facilitate design and development of peptidomimetic AMPs, including 14-helical {beta}-peptides, for antifungal applications.\n\nGraphical Abstract\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=46 SRC=\"FIGDIR/small/430850_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (14K):\norg.highwire.dtl.DTLVardef@10ee335org.highwire.dtl.DTLVardef@66da40org.highwire.dtl.DTLVardef@82d132org.highwire.dtl.DTLVardef@7429e1_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗