Biology subjects

Nefzger, C.

Publications and source records attributed to Nefzger, C..

3 recordsLinked to original sources

bioRxiv2025

Spatial Transcriptomic Signature of Progressive Fibrosis in Human MASLD: Role of Senescence and Metabolic Reprogramming.

Granular detail about the location and nature of liver cell interactions and the metabolic, inflammatory and fibrogenic pathways driving progressive fibrosis in metabolic dysfunction-associated steatotic liver disease (MASLD) is needed to deliver novel therapeutic targets. Here we used spatial transcriptomic data from human MASLD liver biopsies to identify the major cell types and their potential interconnected activities within specific tissue regions across the spectrum of MASLD. Gene expression data were generated using 10X Genomics Visium technology from 33 formalin-fixed paraffin-embedded liver biopsy samples and overlaid with annotated anatomical regions. Differential gene expression (DEG) and pathway analyses, cellular deconvolution and ligand-receptor interactions were conducted for each annotated anatomical category, with specific protein expression validated using CODEX spatial proteomics and immunohistochemistry staining. Unsupervised gene expression data grouped the annotated spots into 2 main clusters enriched for early/intermediate vs late fibrosis and transcriptome-based cellular deconvolution was well aligned with annotated histopathological features. In addition to extracellular matrix/receptor interactions and immune cell recruitment and trafficking, several genes encoding immunoglobulins were highly upregulated in late-stage fibrosis and were spatially associated with a senescence signature. Upregulated DEGs for early/intermediate-stage fibrosis were significantly enriched for metabolic pathways, oxidative phosphorylation and fatty acid metabolism. In contrast glycolysis genes were strongly co-expressed with late stage fibrosis. MASLD progression is accompanied by a decline in normal liver metabolic function and significant reprogramming of metabolic fuel utilisation. The spatial association of a senescence signature with expression of genes encoding immunoglobulins and complement has been linked to aging and is associated with progressive fibrosis. This work provides a valuable discovery dataset spanning different stages of human liver fibrosis and highlights the complex crosstalk between metabolic perturbations and inflammation underpinning fibrosis progression.

genomics↗

bioRxiv2023

Substrate stiffness facilitates improved induced pluripotent stem cell production through modulation of both early and late phases of cell reprogramming

Cell reprogramming involves time-intensive, costly processes that ultimately produce low numbers of reprogrammed cells of variable quality. By screening a range of polyacrylamide hydrogels (pAAm gels) of varying stiffness (1 kPA - 1.3 MPa) we found that a gel of medium stiffness significantly increases the overall number of reprogrammed cells by up to ten-fold with accelerated reprogramming kinetics, as compared to the standard Tissue Culture PolyStyrene (TCPS)-based protocol. We observe that though the gel improves both early and late phases of reprogramming, improvement in the late (reprogramming prone population maturation) phase is more pronounced and produces iPSCs having different characteristics and lower remnant transgene expression than those produced on TCPS. Comparative RNA-Seq analyses coupled with experimental validation reveals that modulation of Bone Morphogenic Protein (BMP) signalling by a novel reprogramming regulator, Phactr3, upregulated in the gel at an earliest time-point without the influence of transcription factors used for reprogramming, plays a crucial role in the improvement in the early reprogramming kinetics and overall reprogramming outcomes. This study provides new insights into the mechanism via which substrate stiffness modulates reprogramming kinetics and iPSC quality outcomes, opening new avenues for producing higher numbers of quality iPSCs or other reprogrammed cells at shorter timescales.

bioengineering↗

bioRxiv2018

A modular dCas9-SunTag DNMT3A epigenome editing system overcomes pervasive off-target activity of direct fusion dCas9-DNMT3A constructs

AbstractDNA methylation is a covalent modification of the genome that plays important roles in genome regulation and vertebrate development. Although detection of this modification in the genome has been possible for several decades, the ability to deliberately and specifically manipulate local DNA methylation states in the genome has been extremely limited. Consequently, this has impeded the direct determination of the consequence of DNA methylation on transcriptional regulation and transcription factor binding in the native chromatin context. Thus, highly specific targeted epigenome editing tools are needed to address this outstanding question. Recent adaptations of genome editing technologies, such as the fusion of the DNMT3A methyltransferase catalytic domain to catalytically inactive Cas9 (dC9-D3A), have aimed to provide new tools for altering DNA methylation at desired loci. Here, we performed a deeper analysis of the performance of these tools, revealing consistent off-target binding events and DNA methylation deposition in the genome, limiting the capacity of these tools to unambiguously assess the functional consequences of DNA methylation. To address this, we developed a modular dCas9-SunTag (dC9Sun-D3A) system that can recruit multiple DNMT3A catalytic domains to a target site for editing DNA-methylation. dC9Sun-D3A is tunable, specific and exhibits much higher induction of DNA methylation at target sites than the dC9-D3A direct fusion protein. Importantly, genome-wide characterization of dC9Sun-D3A binding sites and DNA methylation revealed minimal off-target protein binding and induction of DNA methylation with dC9Sun-D3A, compared to pervasive off-target binding and methylation by the dC9-D3A direct fusion construct. Furthermore, we used dC9Sun-D3A to test the impact of DNA methylation upon the DNA binding of CTCF and NRF1 upon targeted methylation of their core binding sites, demonstrating the binding sensitivity of these proteins to DNA methylation in situ. Overall, this modular dC9Sun-D3A system enables precise DNA methylation deposition with the lowest amount of off-target DNA methylation reported to date, allowing accurate functional determination of the role of DNA methylation at single loci.

molecular biology↗