Biology subjects

Furth, P. A.

Publications and source records attributed to Furth, P. A..

9 recordsLinked to original sources

bioRxiv2026

Detection of Candidate Circular RNAs to Monitor Anti-Hormonal Response in the Mammary Gland

Anti-hormonal therapies such as selective estrogen receptor modulators like tamoxifen or aromatase inhibitors like letrozole represent a cornerstone for breast cancer prevention and therapy of estrogen receptor-positive breast cancer. Therapeutic monitoring can include blood tests and imaging; however, genetically-based approaches are not yet in practice. Ideally, a test would be able to detect a positive molecular response across different estrogen pathway-suppressive approaches. Circular RNAs are a species of non-coding RNAs detectable in plasma that have been proposed as non-invasive therapeutic biomarkers. To determine whether a set of specific circular RNAs is altered across estrogen-suppressive pathway approaches, we analyzed mammary gland-specific total RNA sequencing data from two individual genetically engineered mouse models (GEMMs) of estrogen pathway-induced breast cancer, with or without exposure to tamoxifen or letrozole. The nf-core/circrna pipeline was used to identify circRNAs that were differentially expressed in response to either tamoxifen or letrozole. We then screened for circRNAs that were differentially regulated by both anti-hormonals. Four up-regulated and 31 down-regulated circRNAs with host genes known to be expressed in human breast epithelial cells were identified as showing reproducible differential regulation in response to anti-hormonal treatment.

cancer biology↗

bioRxiv2026

Common Pitfalls in CircRNA Detection and Quantification

1Circular RNAs have garnered considerable interest, as they have been implicated in numerous biological processes and diseases. Through their stability, they are often considered promising biomarker candidates or therapeutic targets. Due to the lack of a poly(A) tail, circRNAs are best detected in total RNA-seq data after depleting ribosomal RNA. However, we observe that the application of circRNA detection in the vastly more ubiquitous poly(A)-enriched RNA-seq data still occurs. In this study, we systematically compare the detection of circRNAs in two matched poly(A) and ribosomal RNA-depleted data sets. Our results indicate that the comparably few circRNAs detected in poly(A) data are likely false positives. In addition, we demonstrate that the quality of sample processing, as measured by the fraction of ribosomal reads, significantly affects the sensitivity of circRNA detection, leading to a bias in downstream analysis. Our findings establish best practices for circRNA research: total RNA sequencing with effective rRNA depletion is the preferred approach for accurate circRNA profiling, whereas poly(A)-enriched data are unsuitable for comprehensive detection. Employing multiple circRNA detection tools and prioritizing back-splice junctions identified by several algorithms enhances confidence in the selection of candidates. These recommendations, validated across diverse datasets and tissue types, provide generalizable principles for robust circRNA analysis. Key PointsO_LIRibosomal RNA contamination substantially impairs the accuracy of circRNA detection. This technical confounding factor has thus far received limited attention in the field. C_LIO_LITool agreement for circRNA calls is moderate in total RNA-seq but essentially absent in poly(A)-enriched RNA-seq data, underscoring the importance of using multiple tools for circRNA detection. C_LIO_LIBack-splice junctions detected in poly(A)-enriched RNA-seq data are predominantly tool-specific artifacts rather than genuine circRNAs, challenging the validity of circRNA identification in poly(A)-enriched datasets. C_LI

bioinformatics↗

bioRxiv2025

Heritability and transcriptional impact of JAK3, STAT5A and STAT6 variants in a Tyrolean family

The Janus Kinase (JAK) and Signal Transducers and Activators of Transcription (STAT) pathway regulate a range of biological processes, including immune response and hematopoiesis. While a major research focus has been on somatic human mutations in disease, less is known about the heritability of germline variants and their physiological impact. Here we identify the rare JAK3P151R, JAK3R925S, STAT5AV494L and STAT6Q633H variants in an extended family spanning three generations and integrate in silico analyses, AlphaFold 3 structural predictions and investigate the immune transcriptomes in probands carrying one or more variants. All four variants are inherited through the germline without any evident clinical or physiological manifestations in the carriers. As individual variants, not all persons carrying a specific variant showed the same immune transcriptome. The presence of activated basal transcriptomes was limited to some, but not all, individuals carrying the above variants above. A next step in understanding the role of germline variants will be to understand how and why other factors including both other germline variants as well as environmental and developmental factors influence the likelihood of expression of an activated basal transcriptome.

genomics↗

bioRxiv2025

Disease-associated mutations in the STAT5B SH2 domain reprogram hepatic cholesterol and lipid metabolism

Growth hormone (GH) signaling through STAT5B is a central regulator of hepatic metabolism, yet the functional consequences of disease-associated STAT5B variants remain poorly understood. Here, we analyzed mice carrying STAT5BY665F (gain-of-function) and STAT5BY665H (loss-of-function) variants and dissect their impact on metabolic regulation. STAT5BY665F mice developed hepatic lipid accumulation, hypercholesterolemia, and enhanced insulin sensitivity, whereas STAT5BY665H mice displayed reduced body weight and impaired insulin responsiveness. Transcriptomic analyses revealed that STAT5BY665F activated lipid, cholesterol, and immune transcriptional programs, while STAT5BY665H failed to induce these pathways. Notably, STAT5BY665F substantially feminized male liver gene expression, inducing 77% of female-biased genes while repressing 51% of male-biased genes, thereby mimicking the persistent STAT5B activation characteristic of female livers. ChIP-seq demonstrated extensive STAT5BY665F enhancer occupancy at metabolic and immune loci, contrasting with the minimal chromatin engagement of STAT5BY665H. Beyond the liver, STAT5BY665F broadly reprogrammed adipose tissue gene expression, activating lipid metabolism and immune regulatory networks, whereas STAT5BY665H exerted more restricted effects. Together, these findings illustrate how alterations in STAT5B activity affect enhancer activation and can lead to changes in metabolic function and hepatic sexual dimorphism.

genomics↗

bioRxiv2024

STAT5B leukemic mutations, altering SH2 tyrosine 665, have opposing impacts on immune gene programs

STAT5B is a vital transcription factor for lymphocytes. Here, function of two STAT5B mutations from human T cell leukemias: one substituting tyrosine 665 with phenylalanine (STAT5BY665F), the other with histidine (STAT5BY665H) was interrogated. In silico modeling predicted divergent energetic effects on homodimerization with a range of pathogenicity. In primary T cells in vitro STAT5BY665F showed gain-of-function while STAT5BY665H demonstrated loss-of-function. Introducing the mutation into the mouse genome illustrated that the gain-of-function Stat5bY665F mutation resulted in accumulation of CD8+ effector and memory and CD4+ regulatory T-cells, altering CD8+/CD4+ ratios. In contrast, STAT5BY665H knock-in mice showed diminished CD8+ effector and memory and CD4+ regulatory T cells. In contrast to wild-type STAT5, the STAT5BY665F variant displayed greater STAT5 phosphorylation, DNA binding and transcriptional activity following cytokine activation while the STAT5BY665H variant resembled a null. The work exemplifies how joining in silico and in vivo studies of single nucleotides deepens our understanding of disease-associated variants, revealing structural determinants of altered function, defining mechanistic roles, and, specifically here, identifying a gain-of function variant that does not directly induce hematopoietic malignancy.

genetics↗

bioRxiv2024

RNA sequencing depth guidelines for the study of alternative splicing

A key parameter in the experimental design of RNA-seq projects is the choice of sequencing depth. Considering a limited budget, one needs to find a tradeoff between the number of samples and the sensitivity of the analysis, particularly concerning lowly expressed genes. While previous studies have proposed a lower bound for the comprehensive analysis of differential gene expression, for the analysis of alternative splicing, it has only been proposed for human adipose tissue. However, alternative splicing differs across tissues and conditions. We analyzed publicly available and newly generated deep-sequenced paired-end RNA-seq samples (between 150 and >500 million reads, read length 50-150 bp) from human buffy coat cells and diverse sets of tissues, including gluteal subcutaneous fat, heart, and hypothalamus. Our results show that the sequencing depth typically used in published cohorts is not sufficient to comprehensively capture the landscape of alternative splicing. This motivates the use of deeper sequencing or long-read technologies in future studies. Toward this goal, we offer guidelines for choosing a suitable sequencing depth. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=177 SRC="FIGDIR/small/617406v2_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@1aecd9org.highwire.dtl.DTLVardef@1b3d0fborg.highwire.dtl.DTLVardef@5d15c4org.highwire.dtl.DTLVardef@140035b_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioinformatics↗

bioRxiv2023

Blood transcriptomics analysis offers insights into variant-specific immune response to SARS-CoV-2

Bulk RNA sequencing (RNA-seq) of blood is typically used for gene expression analysis in biomedical research but is still rarely used in clinical practice. In this study, we argue that RNA-seq should be considered a routine diagnostic tool, as it offers not only insights into aberrant gene expression and splicing but also delivers additional readouts on immune cell type composition as well as B-cell and T-cell receptor (BCR/TCR) repertoires. We demonstrate that RNA-seq offers vital insights into a patients immune status via integrative analysis of RNA-seq data from patients infected with various SARS-CoV-2 variants (in total 240 samples with up to 200 million reads sequencing depth). We compare the results of computational cell-type deconvolution methods (e.g., MCP-counter, xCell, EPIC, quanTIseq) to complete blood count data, the current gold standard in clinical practice. We observe varying levels of lymphocyte depletion and significant differences in neutrophil levels between SARS-CoV-2 variants. Additionally, we identify B and T cell receptor (BCR/TCR) sequences using the tools MiXCR and TRUST4 to show that - combined with sequence alignments and pBLAST - they could be used to classify a patients disease. Finally, we investigated the sequencing depth required for such analyses and concluded that 10 million reads per sample is sufficient. In conclusion, our study reveals that computational cell-type deconvolution and BCR/TCR methods using bulk RNA-seq analyses can supplement missing CBC data and offer insights into immune responses, disease severity, and pathogen-specific immunity, all achievable with a sequencing depth of 10 million reads per sample. Key PointsO_LIComputational deconvolution of transcriptomes can estimate immune cell abundances in SARS-CoV-2 patients, supplementing missing CBC data. C_LIO_LI10 million RNA sequencing reads per sample suffice for analyzing immune responses and disease severity, including BCR/TCR identification. C_LI

bioinformatics↗

bioRxiv2023

circRNA-sponging: a pipeline for extensive analysis of circRNA expression and their role in miRNA sponging

MotivationCircular RNAs (circRNAs) are long non-coding RNAs (lncRNAs) often associated with diseases and considered potential biomarkers for diagnosis and treatment. Among other functions, circRNAs have been shown to act as microRNA (miRNA) sponges, preventing the role of miRNAs that repress their targets. However, there is no pipeline to systematically assess the sponging potential of circRNAs. ResultsWe developed circRNA-sponging, a nextflow pipeline that (1) identifies circRNAs via backsplicing junctions detected in RNA-seq data, (2) quantifies their expression values in relation to their linear counterparts spliced from the same gene, (3) performs differential expression analysis, (4) identifies and quantifies miRNA expression from miRNA-sequencing (miRNA-seq) data, (5) predicts miRNA binding sites on circRNAs, (6) systematically investigates potential circRNA-miRNA sponging events, (7) creates a network of competing endogenous RNAs, and (8) identifies potential circRNA biomarkers. We showed the functionality of the circRNA-sponging pipeline using RNA sequencing data from brain tissues, where we identified two distinct types of circRNAs characterized by a specific ratio of the number of the binding site to the length of the transcript. The circRNA-sponging pipeline is the first end-to-end pipeline to identify circRNAs and their sponging systematically with raw total RNA-seq and miRNA-seq files, allowing us to better indicate the functional impact of circRNAs as a routine aspect in transcriptomic research. Availabilityhttps://github.com/biomedbigdata/circRNA-sponging Contactmarkus.daniel.hoffmann@tum.de; markus.list@tum.de Supplementary MaterialSupplementary data are available at Bioinformatic Advances online.

bioinformatics↗

bioRxiv2017

Expanding primary cells from mucoepidermoid and other salivary gland neoplasms for genetic and chemosensitivity testing

Restricted availability of cell and animal models is a rate-limiting step for investigation of salivary gland neoplasm pathophysiology and therapeutic response. Conditionally reprogrammed cell (CRC) technology enables establishment of primary epithelial cell cultures from patient material. This study tested a translational workflow for acquisition, expansion and testing of CRC-derived primary cultures of salivary gland neoplasms from patients presenting to an academic surgical practice. Results showed cultured cells were sufficient for epithelial cell-specific transcriptome characterization to detect candidate therapeutic pathways and fusion genes in addition to screening for cancer-risk-associated single nucleotide polymorphisms (SNPs) and driver gene mutations through exome sequencing. Focused study of primary cultures of a low-grade mucoepidermoid carcinoma demonstrated Amphiregulin-Mechanistic Target of Rapamycin-AKT/Protein kinase B (AKT) pathway activation, identified through bioinformatics and subsequently confirmed as present in primary tissue and preserved through different secondary 2D and 3D culture media and xenografts. Candidate therapeutic testing showed that the allosteric AKT inhibitor MK2206 reproducibly inhibited cell survival across different culture formats. In contrast, the cells appeared resistant to the adenosine triphosphate competitive AKT inhibitor GSK690693. Procedures employed here illustrate an approach for reproducibly obtaining material for pathophysiological studies of salivary gland neoplasms, and other less common epithelial cancer types, that can be executed without compromising pathological examination of patient specimens. The approach permits combined genetic and cell-based physiological and therapeutic investigations in addition to more traditional pathologic studies and can be used to build sustainable bio-banks for future inquiries.

cancer biology↗