Biology subjects

Marks, K.

Publications and source records attributed to Marks, K..

6 recordsLinked to original sources

bioRxiv2025

Somatic mutations distinguish melanocyte subpopulations in human skin

To better understand the homeostatic mechanisms governing melanocytes, we performed deep phenotyping of clonal expansions of single melanocytes from human skin. In total, we interrogated the mutational landscapes, gene expression profiles, and morphological features of 297 melanocytes from 31 donors. To our surprise, a population of melanocytes with low mutation burden was maintained in sun damaged skin. These melanocytes were more stem-like, smaller, less dendritic and displayed distinct gene expression profiles compared to their counterparts with high mutation burdens. We used single-cell spatial transcriptomics (10X Xenium) to reveal the spatial distribution of melanocytes inferred to have low and high mutation burdens (LowMut and HighMut cells), based on their gene expression profiles. LowMut melanocytes were found in hair follicles as well as in the interfollicular epidermis, whereas HighMut melanocytes resided almost exclusively in the interfollicular epidermis. We propose that melanocytes in the hair follicle occupy a privileged niche, protected from UV radiation, but periodically migrate out of the hair follicle to replenish the interfollicular epidermis after waves of photodamage. More broadly, our study illustrates the advantages of a cell atlas that includes mutational information, as cells can change their cellular states and positional coordinates over time, but mutations are like scars, providing a historical record of the homeostatic processes that were operative on each cell.

cell biology↗

bioRxiv2025

A CD57+ cytotoxic CD8 T cell subset associated with fibrotic lung disease in systemic sclerosis

Interstitial lung disease (ILD) is a major cause of morbidity and mortality in systemic sclerosis (SSc); however, the immunopathologic mechanisms driving lung disease in SSc are unclear. T cells have been implicated as a likely driver of lung injury in SSc. Here, we have evaluated T cells in blood and lungs of patients with SSc-ILD and identified a specific population of cytotoxic CD8 T cells that is expanded in SSc-ILD patients. Cytotoxic effector memory CD8 T cells marked by CD57 expression are preferentially expanded in SSc-ILD patients compared to SSc patients without ILD and controls and show prominent clonal expansion. These CD57+ T effector memory (TEM) cells differ from T effector memory cells re-expressing CD45RA (TEMRA) transcriptomically and functionally, with cytotoxic function that is enhanced by CD155 engagement of the costimulatory receptor CD226. Analyses of cells from ILD lungs indicate endothelial cells as a likely source of CD155 to activate CD57+ cytotoxic T cells. Together, the results implicate a CD57+ cytotoxic CD8 T cell population as a potential mediator of lung injury in SSc-ILD.

immunology↗

bioRxiv2024

A minimally invasive thrombotic stroke model to study circadian rhythm in awake mice

Experimental stroke models in rodents are essential for mechanistic studies and therapeutic development. However, these models have several limitations negatively impacting their translational relevance. Here we aimed to develop a minimally invasive thrombotic stroke model through magnetic particle delivery that does not require craniotomy, is amenable to reperfusion therapy, can be combined with in vivo imaging modalities, and can be performed in awake mice. We found that the model results in reproducible cortical infarcts within the middle cerebral artery (MCA) with cytologic and immune changes similar to that observed with more invasive distal MCA occlusion models. Importantly, the injury produced by the model was ameliorated by tissue plasminogen activator (tPA) administration. We also show that MCA occlusion in awake animals results in bigger ischemic lesions independent of day/night cycle. Magnetic particle delivery had no overt effects on physiologic parameters and systemic immune biomarkers. In conclusion, we developed a novel stroke model in mice that fulfills many requirements for modeling human stroke.

neuroscience↗

bioRxiv2024

PTEN and ARID1A haploinsufficiency equip colonic epithelium for oncogenic transformation

Normal aged tissues are thought to exist as a patchwork of mutant clones. However, the relevance of driver mutations in normal tissue in terms of cancer initiation has not been well described. Here, we sought a quantitative understanding of how different cancer drivers achieve an age-related mutational footprint in the human colonic epithelium and to relate the clonal behaviours they generate to cancer risk. Metanalysis of contemporary multiregional sampling studies of colorectal tumours revealed many of the weak or moderate cancer drivers are trunk mutations present in the last common ancestor from which cancers arise. To study the processes by which such driver mutations could contribute to cancer predisposition, immunohistochemistry was used to detect PTEN, SMAD4 and ARID1A deficient clones in normal colon FFPE surgical resection samples (N=182 patients). Age-related changes in clone size and frequency identified positive biases in clone dynamics that acted to increase the mutational footprint for ARID1A and PTEN but not SMAD4. In vitro engineered monoallelic loss of PTEN and ARID1A implicated specific altered downstream pathways and acquired pro-oncogenic cellular fates corresponding to haploinsufficiency for these genes. In situ analysis confirmed enhanced proliferation in both PTEN and ARID1A deficient clones and creation of an immune exclusive microenvironment associated with ARID1A deficiency. The behaviours resulting from haploinsufficiency of PTEN and ARID1A exemplify how priming of the tissue through somatic mosaicism could contribute alternative combinations of genetic events leading to transformation.

cancer biology↗

bioRxiv2019

EM-seq: Detection of DNA Methylation at Single Base Resolution from Picograms of DNA

Bisulfite sequencing is widely used to detect 5mC and 5hmC at single base resolution. However, bisulfite treatment damages DNA resulting in fragmentation, loss of DNA and biased sequencing data. To overcome this, we developed Enzymatic Methyl-seq (EM-seq), an enzymatic based approach that uses as little as 100 pg of DNA. EM-seq outperformed bisulfite converted libraries in all metrics examined including coverage, duplication, sensitivity and nucleotide composition. EM-seq libraries displayed even GC distribution, improved correlation across input amounts as well as increased representation of genomic features. These data indicate that EM-seq is more accurate and reliable than whole genome bisulfite sequencing (WGBS).

genomics↗

bioRxiv2019

In vitro characterization of the yeast DcpS, a scavenger mRNA decapping enzyme

Eukaryotic mRNAs are modified at their 5 end early during transcription by the addition of N7-methylguanosine (m7G), which forms the \"cap\" on the first 5 nucleotide. Identification of the 5 nucleotide on mRNA is necessary for determination of the Transcription Start Site (TSS). We explored the effect of various reaction conditions on the activity of the yeast scavenger mRNA decapping enzyme DcpS (yDcpS) and examined decapping of 30 chemically distinct cap structures varying the state of methylation, sugar, phosphate linkage, and base composition on 25mer RNA oligonucleotides. Contrary to the generally accepted belief that DcpS enzymes only decap short oligonucleotides, we found that yDcpS efficiently decaps RNA transcripts as long as 1400 nucleotides. Further, we validated the application of yDcpS for enriching capped RNA using a strategy of specifically tagging the 5 end of capped RNA by first decapping and then recapping it with an affinity-tagged guanosine nucleotide.

molecular biology↗