Biology subjects

Forster, J.

Publications and source records attributed to Forster, J..

9 recordsLinked to original sources

bioRxiv2025

Loss of NRMT1 allows expression of multiple differentiation pathways and alters transcription of secreted proteins in C2C12 myoblasts

Muscle stem cells (satellite cells) retain their identity and function through expression of the paired homeobox transcription factor PAX7. PAX7 is able to both stimulate satellite cell proliferation and activate target genes involved in establishing myogenic identity, including myogenic factor 5 (MYF5) and the other myogenic regulatory factors (MRFs). Upregulation of the MRFs promotes commitment to the muscle lineage by initiating withdrawal from the cell cycle, upregulating expression of muscle-specific transcripts, and directing myoblast fusion. We have previously shown that knockout of the N-terminal methyltransferase NRMT1 in C2C12 mouse myoblasts results in significantly decreased Pax7 expression, an inability of the cells to differentiate into myotubes, and abnormal upregulation of osteogenic markers. Here, we use RNA-sequencing to more comprehensively determine how loss of NRMT1 affects the transcriptional profile of proliferating and differentiating C2C12 myoblasts. We see that upon inducing differentiation, NRMT1 knockout cells can downregulate cell cycle, DNA replication, and histone gene expression. Though they also have significantly downregulated Pax7 and Myf5 expression, other muscle-specific transcripts are significantly increased over wild type, indicating the muscle transcriptional program is not completely inhibited. However, signaling pathways involved in the differentiation of other types of mesenchymal and hematopoietic lineages are also increased with NRMT1 loss and expression of chemotactic genes is downregulated. Together, these data indicate that NRMT1 knockout cells can upregulate genes needed for cell cycle withdrawal and muscle specification but fail to suppress markers of other lineages and activate normal chemotactic signaling, which may lead to the observed differentiation phenotypes.

genomics↗

bioRxiv2025

Redirecting resistance evolution in BRAFV600 melanoma by inhibition of the peroxiredoxin-thioredoxin system

Drug-tolerant persister cells (DTPs) exhibit remarkable cell state heterogeneity and phenotypic evolvability. However, the central question of how DTPs epigenetically coordinate their metabolic flexibility to adapt early to therapeutic stress remains unanswered. We have recently shown that the histone demethylase KDM5B, which is intrinsically expressed in differentiated melanoma DTPs, reprograms the metabolic cell landscape. However, the exact mechanism by which KDM5B affects underlying metabolic enzymes, and whether this reveals new druggable vulnerabilities, remain unknown. By transcriptional and epigenetic profiling of BRAFV600 melanoma cells following KDM5B gene silencing, we discovered a direct molecular axis between the epigenetic regulator KDM5B and the PRDX/TXN ROS detoxification system. This metabolic axis is regulated independently of KDM5Bs demethylase activity. Furthermore, RNAi approaches and the pharmacological inhibition of the PRDX/TXN system led to ROS-induced cell death in differentiated melanoma DTPs and a delay of resistance development to MAPK inhibition. This process was independent of lipid-ROS-driven ferroptosis. Additionally, single-cell transcriptome analyses from pre-clinical melanoma models under continuous MAPK inhibitory treatment demonstrated altered cellular differentiation dynamics, with a reduction in the early evolution into the mesenchymal DTP state under concomitant PRDX inhibition. Interestingly, the degree of melanoma cell state differentiation at the onset of treatment was a major determinant for the transition towards the neural crest-like DTP state. Our study identified a high degree of epigenetic-metabolic connectivity and flexibility within the melanoma DTP pool and urges caution with single redox pathway-targeted strategies for tumor elimination in the future. Prospectively, our results point towards a new resistance targeting strategy for BRAFV600 melanoma patients based on pharmacological re-direction of the evolution of melanoma cell states already at therapy onset. HighlightsO_LITranscriptional and epigenetic profiling identified KDM5B as a regulator of the PRDX/TXN-ROS detoxification system C_LIO_LIPRDX inhibition increases the vulnerability of KDM5Bhigh DTPs to ROS, independent of ferroptosis C_LIO_LIPRDX inhibition delays resistance to MAPK inhibition in BRAFV600 melanoma cells C_LIO_LILongitudinal single-cell transcriptome analysis reveals that PRDX inhibition re-directs early DTP evolution C_LIO_LIPRDX/TXN gene expression is predictive for melanoma patient survival C_LI

cancer biology↗

bioRxiv2023

Methods for the analysis of skin microbiomes: a comparison of sampling processes and 16S rRNA hypervariable regions.

BackgroundThe skin microbiome is increasingly recognised as a critical component of the innate skin immune response and is central to the pathogenesis of many inflammatory skin disorders including atopic dermatitis. Previous studies have not looked in detail at the impact of changing both sampling methodology and hypervariable region sequencing for skin microbiome analysis. ObjectivesWe set out to undertake a detailed analysis comparing microbe population diversity and resolution at the single species level by swab, tape, scrape and scrape then swab sampling assayed by hypervariable region sequencing. MethodsIn triplicate samples from the antecubital fossa were taken from healthy volunteers for 16s RNA analysis by primer amplification for hypervariable regions 1-3, and 3-4. ResultsAlpha (phylogenetic) diversity was the greatest with tape sampling and V1-3 analysis, whereas for V4, tape and swab were equivalent. Scrape sampling showed lower alpha diversity at both V1-3 and V4. All measures of beta diversity showed the scrape methodology yielded a lesser diversity than the others. Phyla composition was similar across all sampling methodologies. Minor differences in composition were noted between V1-3 and V4 sequencing, but V1-3 was optimal for identification of firmicutes (including staphylococci). ConclusionsIn the methodological planning of skin microbiome analysis skin scientists need to consider the microbes of interest to choose the optimal hypervariable region to sequence, and harmonisation of methodological approaches would be beneficial to the field. For detection of staphylococci on flexural skin, we would recommend tape sampling with analysis of V1-3. Whats already known about this topic?O_LIPrevious microbiome studies have utilised many different methodologies, but a detailed comparison of their utility in skin research has not been undertaken C_LIO_LIMethodological choices in skin microbiome analysis affect both sensitivity to detect diversity and sensitivity to identify at a species level C_LIO_LIOptimisation to detect diversity and species identification can require different approaches C_LI What does this study add?O_LIOur study compared a matrix the four most widely utilised skin sampling methodologies and the two principle means for hypervariable gene analysis. C_LIO_LIWe show that alpha diversity is optimal with tape sampling C_LIO_LIAdditionally, in contrast to gut microbiome analyses, in the skin hypervariable region V1-3 analysis is superior to V4 when the species of interest are staphylococci. C_LI

microbiology↗

bioRxiv2022

Epigenetic plasticity via adaptive DNA hypermethylation and clonal expansion underlie resistance to oncogenic pathway inhibition in pancreatic cancer

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer with poor prognosis. Drug resistance is the major cause for therapeutic failure in PDAC patients with progressive disease. The mechanisms underlying resistance formation are complex and remain poorly understood. To gain insights into molecular changes during the formation of resistance to oncogenic MAPK pathway inhibition we utilized short-term passaged primary tumor cells from ten PDACs of genetically engineered mice. We followed gain and loss of resistance upon MEKi exposure and withdrawal by longitudinal integrative analysis of whole genome sequencing, whole genome bisulfite sequencing, RNA-sequencing and mass spectrometry data. We found that resistant cell populations under increasing MEKi treatment evolved by the expansion of a single clone but were not a direct consequence of known resistance-conferring mutations. Rather, resistant cells showed adaptive DNA hypermethylation of 209 and hypomethylation of 8 genomic sites, most of which overlap with regulatory elements known to be active in murine PDAC cells. Both DNA methylation changes and MEKi resistance were transient and reversible upon drug withdrawal. The effector caspase CASP3 is one of the 114 genes for which transcriptional downregulation inversely correlated with the methylation status of the associated DNA region. CASP3 inactivation in resistant cells led to attenuation of drug-induced apoptosis which could be reversed by DNA methyltransferase inhibition with remarkable sensitivity exclusively in the resistant cells. Overall, our data provide a context for characterization and targeting of epigenetically mediated resistance mechanisms in PDAC.

cancer biology↗

bioRxiv2021

Microphaser - a small-scale phasing approach for improved personalized neopeptidome creation

MotivationHaplotype phasing approaches have been shown to improve accuracy of the search space of neoantigen prediction by determining if adjacent variants are located on the same chromosomal copy. However, the aneuploid nature of cancer cells as well as the admixture of different clones in tumor bulk sequencing data are challenging the current diploid based phasing algorithms. We present microphaser, a small-scale phasing approach to improve haplotyping variants in cancer samples. Microphaser aims to create a more accurate neopeptidome for downstream neoantigen prediction. ResultsMicrophaser achieves large concordance with state-of-the-art phasing-aware neoantigen prediction pipeline neoepiscope, with differences in edge cases and an improved filtering step. AvailabilityMicrophaser is written in the Rust programming language. It is made available via Github (https://github.com/koesterlab/microphaser)and Bioconda. The corresponding prediction pipeline (https://github.com/snakemake-workflows/dna-seq-neoantigen-prediction) has been written within the Snakemake workflow management system and can be deployed as part of the snakemake-workflows project.

bioinformatics↗

bioRxiv2021

FIND-IT: Ultrafast mining of genome diversity

Novel crop improvement methodologies, including the exploitation of natural genetic variation, are urgently required to feed our rapidly growing human population in the context of global climate change. Here we describe a Fast Identification of Nucleotide variants by DigITal PCR (FIND-IT) method for the rapid identification of pre-targeted genetic variants or rare alleles in large genomic populations. Libraries of 500,000 individuals can be screened and desired variants isolated within two weeks. FIND-IT is widely applicable for mining valuable diversity in any genomic population, including elite breeding and wild germplasm collections. The method provides single nucleotide resolution that has been validated by identifying and isolating knockout lines, non-synonymous codon changes and variants of miRNA and transcription factor binding sites in the agronomically important crop barley. In contrast to existing methods, FIND-IT does not require transformation, cloning or enzymatic steps, and is exempt from GMO regulations. Thus, FIND-IT can be applied immediately to elite crop cultivars and can be tailored to minimize or eliminate time-consuming backcrossing requirements.

genetics↗

bioRxiv2020

A Novel Efficient L-Lysine Exporter Identified by Functional Metagenomics

Lack of active export system often limits the industrial bio-based production processes accumulating the intracellular product and hence complexing the purification steps. L-lysine, an essential amino acid, is produced biologically in quantities exceeding two million tons per year; yet, L-lysine production is challenged by efficient export system at high titres during fermentation. To address this issue, new exporter candidates for efficient efflux of L-lysine are needed. Using metagenomic functional selection, we identified 58 genes encoded on 28 unique metagenomic fragments from cow gut microbiome library that improved L-lysine tolerance. These genes include a novel putative L-lysine transporter, belonging to a previously uncharacterized EamA superfamily. Characterization using Xenopus oocyte expression system as well as an Escherichia coli host demonstrates activity as a L-lysine transporter. This novel exporter improved L-lysine tolerance in E. coli by 40% and enhanced the specific productivity of L-lysine in an industrial Corynebacterium glutamicum strain by 12%. Our approach allows the sequence-independent discovery of novel exporters and can be deployed to increase titres and productivity of toxicity-limited bioprocesses.

molecular biology↗

bioRxiv2018

Tinnitus development is associated with synaptopathy of inner hair cells in Mongolian gerbils

Human hearing loss (HL) is often accompanied by comorbidities like tinnitus which is affecting up to 15% of the adult population. Rodent animal studies could show that tinnitus may not only be a result of apparent HL due to cochlear hair cell damage but can also be a consequence of synaptopathy at the inner hair cells (IHC) already induced by moderate sound traumata. Here we investigate synaptopathy previously shown in mice in our animal model, the Mongolian gerbil, and relate it to behavioral signs of tinnitus. Tinnitus was induced by a mild monaural acoustic trauma leading to monaural noise induced HL in the animals, quantified by auditory brainstem response (ABR) audiometry. Behavioral signs of tinnitus percepts were detected by measurement of prepulse inhibition of the acoustic startle response in a gap-noise paradigm. 14 days after trauma, the cochleae of both ears were isolated and IHC synapses were counted within several spectral regions of the cochlea. Behavioral signs of tinnitus were only found in animals with IHC synaptopathy, independent of type of HL. On the other hand, animals with apparent HL but without behavioral signs of tinnitus showed a reduction in amplitudes of ABR waves I&II but no significant changes in the number of synapses at the IHC. We conclude - in line with the literature - that HL is caused by damage to the IHC or by other reasons but that the development of tinnitus, at least in our animal model, is closely linked to synaptopathy at the IHC.

neuroscience↗

bioRxiv2017

Genetics of the Research Domain Criteria (RDoC): genome-wide association study of delay discounting

Delay discounting (DD), which is the tendency to discount the value of delayed versus current rewards, is elevated in a constellation of diseases and behavioral conditions. We performed a genome-wide association study of DD using 23,127 research participants of European ancestry. The most significantly associated SNP was rs6528024 (P = 2.40 x 10-8), which is located in an intron of the gene GPM6B. We also showed that 12% of the variance in DD was accounted for by genotype, and that the genetic signature of DD overlapped with attention-deficit/hyperactivity disorder, schizophrenia, major depression, smoking, personality, cognition, and body weight.

genetics↗