Biology subjects

Prochazka, J.

Publications and source records attributed to Prochazka, J..

At least 19 recordsLinked to original sources

bioRxiv2026

Automated Retinal Dysplasia Segmentation in Mouse Optical Coherence Tomography Scans Using a UNet-Based model

PurposeOptical coherence tomography (OCT) is the state-of-the-art non-invasive imaging technique for preclinical retinopathy studies. However, manual pathology annotations in mouse OCT scans are labour-intensive and susceptible to inter-rater variability. To alleviate these issues, we developed a neural network-based model for automated annotation of retinal dysplasia in mouse OCT scans. MethodsOur model was trained on 205 expert-annotated OCT stacks and validated on 40 unseen stacks with additional expert annotation comparisons in a subset of them. ResultsThe model detects pathologies with high accuracy (F1-score > 0.95) and consistency with experts (median Dice score > 0.8). We integrated the model into a cross-platform app ("OCTOPUS") that includes batch processing, automated annotation, manual annotation editing, and quantitative longitudinal tracking with pathological area estimation on the fundus image, and exportable results in CSV and SVG formats. ConclusionsOur open-source tool aims to facilitate and standardise OCT assessments between laboratories for efficient preclinical screening and high-throughput phenotyping in mouse models of retinal disease.

bioinformatics↗

bioRxiv2026

Rhomboid protease RHBDL2 is a calcium-activated suppressor of EGFR signalling in keratinocytes.

Signalling via the epidermal growth factor receptor (EGFR) is indispensable for morphogenesis and tissue homeostasis. It is activated by extracellular ligands, typically released from transmembrane precursors by proteolysis. Ligand shedding activity is provided by the conserved rhomboid intramembrane serine proteases in Drosophila, but by the unrelated ADAM family metalloproteases in mammals, leaving the functions of mammalian non-mitochondrial rhomboids underexplored. Using quantitative proteomics, we show that EGFR is the main endogenous substrate of the human rhomboid protease RHBDL2 in keratinocytes. By shedding the EGFR ectodomain, thus producing a decoy receptor, RHBDL2 suppresses EGFR signalling, limiting cell migration and invasion. Conspicuously, RHBDL2 activity is upregulated by elevated intracellular calcium concentration, a condition typical for keratinocyte differentiation. These effects are recapitulated in primary human keratinocytes, and human skin equivalents deficient in RHBDL2 display incomplete differentiation and are morphologically disordered compared to wild type cells. We propose that context-specific fine-tuning of EGFR signalling and sensitivity to cross-talk from other signalling pathways could be important and hitherto overlooked roles of rhomboid proteases in mammals.

cell biology↗

bioRxiv2025

Post-transcriptional control of enamel matrix mineralization by TENT5A-mediated cytoplasmic polyadenylation

Tooth enamel formation, or amelogenesis, is a complex biomineralization process regulated by ameloblasts cells. These cells secrete enamel matrix proteins (EMPs), including Amelogenin (AMELX) and Ameloblastin (AMBN), which are essential for hydroxyapatite crystal deposition during enamel mineralization. Precise regulation of this process ensures the mechanical and chemical strength of dental enamel. Using Tent5a knock-out (KO) mouse, we demonstrated that TENT5A, a non-canonical poly(A) polymerase is crucial for the stability, translation, and secretion of EMP mRNAs, particularly AMELX, during amelogenesis. TENT5A-deficient mice exhibited Amelogenesis imperfecta, characterized by enamel hypomineralization, reduced thickness, and disrupted ultrastructure, as revealed by micro-computed tomography. Through nanopore direct mRNA sequencing, we identified that TENT5A polyadenylates Amelx and other mRNAs encoding secreted proteins, enhancing their stability and translation in the endoplasmic reticulum. Moreover, loss of TENT5A altered AMELX secretion and extracellular self-assembly, impairing matrix organization and hydroxyapatite deposition. This study highlights the role of TENT5A in post-transcriptional regulation during enamel formation, demonstrating its importance in enamel homeostasis. TeaserTENT5A adds poly(A) tails to specific mRNAs in enamel, enhancing their expression, which is crucial for dental mineralization.

developmental biology↗

bioRxiv2025

Phenotype of mice carrying an NMDA receptor GluN2B protein-truncating variant associated with intellectual disability

Pathogenic variants in GRIN2B, encoding the NMDA receptor (NMDAR) GluN2B subunit, are linked to intellectual disability (ID) and related neurodevelopmental disorders. While most disease-associated variants are missense, protein-truncating variants (PTVs) may cause haploinsufficiency with less severe phenotypes. Here, we characterize a knock-in mouse model carrying the GluN2B-L825Ffs*15 PTV (Grin2b+/{Delta}). Proteomic analysis revealed markedly reduced full-length GluN2B protein and no detectable truncated GluN2B, accompanied by a compensatory increase in GluN2A. Electrophysiology in hippocampal neurons demonstrated reduced NMDA-induced currents, diminished ifenprodil sensitivity, and accelerated NMDAR-mediated EPSC deactivation, consistent with a shift toward GluN2A-containing receptors. AMPAR-mEPSC amplitudes were increased, indicating altered excitatory synaptic function. Behaviorally, Grin2b+/{Delta} mice exhibited hypoactivity, increased anxiety in males, and impaired sensorimotor gating in both sexes, while learning, memory, and social behaviors remained largely intact. These results demonstrate that a monoallelic GluN2B PTV alters NMDAR subunit composition and function, producing moderate behavioral effects, and provide insight into mechanisms underlying GRIN2B-associated ID.

neuroscience↗

bioRxiv2025

IFNG-producing self-reactive CD4+ T cells drive autoimmune adrenalitis in a mouse model of Addison's disease

Autoimmune Addisons disease (AD) is a rare but life-threatening disorder caused by immune-mediated destruction of the adrenal cortex, and progress in therapy has been limited by insufficient mechanistic insight. Here, we establish a model of Experimental Autoimmune Adrenalitis (EAA) that recapitulates key features of AD and reveals sex-dependent differences in disease manifestation within the model. Immunization with peptides derived from the adrenal self-antigen CYP11A1 induces corticosterone insufficiency. We show that autoimmune adrenalitis is driven by IFNG produced by self-reactive CD4 T cells, promoting granulomatous inflammation in the adrenal cortex. Together, these findings identify IFNG as a central effector of autoimmune adrenalitis and suggest that targeting the IFNG pathway may represent a potential therapeutic strategy for AD. SignificanceAddisons disease (AD) is a rare autoimmune disorder that destroys the adrenal cortex, yet its underlying mechanisms remain unknown. We developed a mouse model of Experimental Autoimmune Adrenalitis (EAA) that mirrors the hormonal and immunological features of AD. Our study reveals that IFNG-producing CD4 T cells drive adrenal inflammation and dysfunction, identifying IFNG as a key pathogenic factor and a potential therapeutic target.

immunology↗

bioRxiv2025

FAM83H regulates postnatal T cell development through thymic stroma organization

Family of Sequence Similarity 83H (FAM83H/ SACK1H) is primarily expressed in epithelial cells, where it interacts with casein kinase 1 (CK1) and keratins to regulate cytoskeletal organization, cell proliferation, and vesicular trafficking. Mutations in FAM83H are known to cause amelogenesis imperfecta, highlighting its critical role in enamel formation. We generated Fam83h-deficient mice (Fam83hem2(IMPC)Ccpcz, Fam83h-/-) and mice lacking a part of the N-terminal CK1-binding domain (Fam83h{Delta}87/{Delta}87). Consistent with other Fam83h-deficient models, these mice are subviable, smaller in size, and exhibit a sparse, scruffy coat, scaly skin, general weakness, and hypoactivity. Notably, both strains show impaired lymphoid cell development in early postnatal life. In the thymus, Fam83h expression is confined to thymic epithelial cells (TECs), and its deficiency in stromal cells results in disrupted thymic architecture and severe block in the expansion of DN3 (double-negative stage 3) T cells, ultimately leading to insufficient T cell production. Single-cell transcriptomic analysis reveals that Fam83h-/- cortical TECs (cTECs) express reduced levels of the TEC master regulator Foxn1, and its multiple downstream target genes, suggesting a critical role for FAM83H likely in coordination with CK1in cTEC maturation. HighlightsFam83h-deficient mice exhibit multiple epithelial defects, but no obvious enamel defects Fam83h deficiency disrupts lymphocyte development Fam83h deficiency impairs thymic epithelial cell maturation and thymus function

immunology↗

bioRxiv2025

Establishing Comprehensive Transthoracic Echocardiography Reference Ranges for Mouse Models: Insights into the Impact of Anesthesia, Sex, and Age

Mouse models play a critical role in cardiology research, offering valuable insights into the molecular mechanisms, genetics, and potential treatments for cardiovascular diseases. However, the ability to transfer findings in mice between studies is limited by the absence of standardized protocols and valid reference values for the assessment of normal cardiac function in mice. This study aims to establish comprehensive transthoracic echocardiography (TTE) reference ranges for mice, particularly focusing on C57BL/6N wildtype controls. The study, which includes data from over 15,000 mice through the International Mouse Phenotyping Consortium (IMPC), highlights how variables such as sex, age, body weight, and anesthesia affect TTE parameters. The findings showed that anesthesia is the primary predictor of variability in cardiac function. Isoflurane and tribromoethanol anesthetized mice presented with modified cardiac function compared to conscious mice. Additionally, we observed minimal sex differences in cardiac morphology and function, except for small variations influenced by anesthesia. The effects of aging on cardiac function were modest, characterized by a decrease in heart rate and subtle changes in ventricular dimensions without evidence of pathological remodeling, likely attributable to disease-free cardiovascular aging. Validation of the reference ranges across multiple mouse strains showed that these values provide a reliable baseline for experiments involving cardiac function in mice. The data underscore the importance of using anesthesia-specific reference values when interpreting TTE results, ensuring robust comparisons in genetic and pharmacological studies. These reference ranges serve as quality assurance tools for future cardiac studies in mice, offering insights into typical TTE parameter values, supporting the detection of experimental perturbations, and contributing to more effective translation of findings from mouse to human.

genetics↗

bioRxiv2024

TACSTD2 expression marks the early transition to colon adenomas

This study aimed to address new molecular events occurring in precancerous stages of colorectal cancer (CRC) by integrated analysis of gene expression data and DNA methylation profiles. Whole-transcriptome sequencing analysis was performed on 16 fresh frozen colorectal adenoma and matched mucosa specimens along with validation of candidates in an independent cohort of 20 fresh frozen paired adenoma and adjacent mucosa tissues as well as eight independent public datasets (335 cases). Genome-wide methylation profiles were determined for 5 adenoma pairs and confirmed by pyrosequencing on 20 tissue pairs used for validation. Functional analysis was performed in vitro and in vivo using the inflammation-associated azoxymethane/dextran sodium sulfate (AOM/DSS) and the sporadic colorectal carcinogenesis (six AOM injections) mouse models as well as ApcMin/+ mice. Candidates were investigated by immunohistochemical staining of human adenomas and early-stage (pT1) CRC tumors. A total of 1,917 differentially expressed genes and 148,191 differentially methylated CpG sites were detected in adenomas compared with adjacent mucosa samples. Based on the transcriptome data and relevance to CRC, TACSTD2, MMP7, MMP1, CLDN2, CLDN1, and ETV4, were selected for further validation. TACSTD2 promoter hypomethylation in adenoma tissues was validated in 20 additional tissue pairs and corresponded with increased TACSTD2 expression. TACSTD2 was also overexpressed in an in vitro transformation model of human colonic epithelial cells. The TACSTD2 protein TROP2 was elevated in human adenomas, pT1 tumors, and in murine adenomas, while it was absent in the unaffected adjacent mucosa. TROP2 overexpression might trigger the development of precancerous lesions and could help identify early transformation foci in colon biopsies. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=110 SRC="FIGDIR/small/620817v1_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@1f23753org.highwire.dtl.DTLVardef@10e6abeorg.highwire.dtl.DTLVardef@181f500org.highwire.dtl.DTLVardef@fc0d38_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2023

Genetic activation of canonical RNA interference in mice

Canonical RNA interference (RNAi) is sequence-specific mRNA degradation guided by small interfering RNAs (siRNAs) made from double-stranded RNA (dsRNA) by RNase III Dicer. RNAi has different roles including gene regulation, antiviral immunity or defense against transposable elements. In mammals, RNAi is constrained by Dicer, which is adapted to produce microRNAs, another class of small RNAs. However, RNAi exists in mouse oocytes, which employs a truncated Dicer variant. A homozygous mutation to express only the truncated variant ({Delta}HEL1) causes dysregulation of microRNAs and perinatal lethality in mice. Here, we report the phenotype and RNAi activity in Dicer{Delta}HEL1/wtmice, which are viable, show minimal miRNome changes but their endogenous siRNA levels are increased by an order of magnitude. We show that siRNA abundance is limited by available dsRNA but not by PKR, a dsRNA sensor of innate immunity. Expressing dsRNA from a transgene, functional RNAi in vivo was induced in heart. Dicer{Delta}HEL1/wt mice thus represent a new model for researching mammalian canonical RNAi in vivo and offer an unprecedented platform for addressing claims about its biological roles.

molecular biology↗

bioRxiv2023

ABIN1 is a negative regulator of effector functions in cytotoxic T cells

T cells are pivotal in the adaptive immune defense, necessitating a delicate balance between robust response against infections and self-tolerance. Their activation involves intricate cross-talk among signaling pathways triggered by the T-cell antigen receptors (TCR) and co-stimulatory or inhibitory receptors. The molecular regulation of these complex signaling networks is still incompletely understood. We identified an adaptor protein, ABIN1 as a component of the signaling complexes of GITR and OX40 co-stimulation receptors. T cells lacking ABIN1 are hyper-responsive ex vivo, and exhibit enhanced responses to cognate infections, and superior ability to induce experimental autoimmune diabetes in mice. We observed that ABIN1 negatively regulates NF-{kappa}B and p38 pathways. The latter was at least partially responsible for the upregulation of key effector proteins, IFNG and GZMB in ABIN1-deficient T cells after TCR stimulation. Our findings reveal the intricate role of ABIN1 in T-cell regulation and its potential as a target for therapeutic fine-tuning of T-cell responses.

immunology↗

bioRxiv2023

Aberrant activation of the innate immune sensor PKR by self dsRNA is prevented by direct interaction with ADAR1

Loss of dsRNA editing by Adar1 leads to aberrant interferon induction in Adar null mouse embryos. Adar Mavs mutants, in which this interferon induction is prevented, die within two weeks of birth. We show here that early death of pups is reduced in Adar Mavs EIF2k2 (Pkr) mutants, identifying PKR as the second aberrantly activated dsRNA sensor in Adar mutant mice. In intestines of Adar Mavs pups transit amplifying progenitor cells in intestinal crypts die and intestinal villi are lost in pups before death; intestinal defects are prevented in Adar Mavs Eifak2. In human A549 lung cancer cells ADAR1 forms a complex with PKR. AlphaFold modelling predicts a direct inhibitory interaction of ADAR1 dsRBDIII with the PKR near the kinase active site and a new mode for ADAR1 dsRNA-binding. Mutations at residues required for ADAR1 dsRNA binding or for predicted PKR contacts, prevent interaction with PKR.

immunology↗

bioRxiv2023

Early embryogenesis in CHDFIDD mouse model reveals facial clefts and altered craniofacial neurogenesis

Congenital heart defects, facial dysmorphism and intellectual development disorder (CHDFIDD) is associated with mutations in CDK13 gene which encodes a transcription regulating Cyclin-dependent kinase 13 (CDK13). Here we analyzed early embryonic stages of CHDFIDD mouse models with hypomorphic mutation in Cdk13 gene with very similar phenotypic manifestations plus cleft lip/palate and knockout of Cdk13 which exhibits robust phenotype with midfacial cleft. Cdk13 is strongly expressed in the mouse embryonic craniofacial structures, namely in the forebrain, nasal epithelium and maxillary mesenchyme. In vitro, CDK13 protein is located not only in nuclear region but also in the cellular protrusions in cultured mesenchymal cells and cells isolated from dorsal root ganglia. In Cdk13-deficient embryos, we found hypoplastic branches of the trigeminal nerve including maxillary branch and additionally we detected significant gene expression changes of molecules involved in neurogenesis (Mef2c, Pou4f1, Sod1, Cdk5rap2, Nrcam) within the developing palatal shelves. Key palate-associated molecules (Msx1 and Meis2) were downregulated during early craniofacial development in mutant embryos. These results demonstrate the role of CDK13 in regulation of facial morphogenesis and also growth of craniofacial peripheral nerves.

developmental biology↗

bioRxiv2022

Human neutralizing antibodies to cold linear epitopes and to subdomain 1 of SARS-CoV-2

Emergence of SARS-CoV-2 variants diminishes the efficacy of vaccines and antiviral monoclonal antibodies. Continued development of immunotherapies and vaccine immunogens resilient to viral evolution is therefore necessary. Using coldspot-guided antibody discovery, a screening approach that focuses on portions of the virus spike that are both functionally relevant and averse to change, we identified human neutralizing antibodies to highly conserved viral epitopes. Antibody fp.006 binds the fusion peptide and cross-reacts against coronaviruses of the four genera, including the nine human coronaviruses, through recognition of a conserved motif that includes the S2 site of proteolytic cleavage. Antibody hr2.016 targets the stem helix and neutralizes SARS-CoV-2 variants. Antibody sd1.040 binds to subdomain 1, synergizes with antibody rbd.042 for neutralization and, like fp.006 and hr2.016, protects mice when present as bispecific antibody. Thus, coldspot-guided antibody discovery reveals donor-derived neutralizing antibodies that are cross-reactive with Orthocoronavirinae, including SARS-CoV-2 variants. One sentence summaryBroadly cross-reactive antibodies that protect from SARS-CoV-2 variants are revealed by virus coldspot-driven discovery.

immunology↗

bioRxiv2022

Retinitis pigmentosa associated mutations in mouse Prpf8 cause misexpression of circRNAs and degeneration of cerebellar granule neurons

A subset of patients suffering from a familial retinitis pigmentosa (RP) carry mutations in several spliceosomal components including PRPF8 protein. Here, we established two novel alleles of murine Prpf8 that genocopy or mimic aberrant PRPF8 found in RP patients - the substitution p.Tyr2334Asn and an extended protein variant p.Glu2331ValfsX15. Homozygous mice expressing either of the aberrant Prpf8 variants developed within first 2 months progressive atrophy of the cerebellum due to extensive granule neuron loss. Comparison of transcriptome from pre-degenerative and degenerative tissues revealed a subset of circRNAs that were deregulated in all tissues and both Prpf8-RP mouse strains. To identify potential risk factors that sensitize cerebellum for Prpf8 mutations we monitored expression of several splicing proteins during first eight weeks. We observed downregulation of all selected splicing proteins in wild-type cerebellum, which coincided with neurodegeneration onset. The decrease in splicing protein expression was further pronounced in mouse strains expressing mutated Prpf8. Collectively, we propose a model where physiological reduction of spliceosomal components during postnatal tissue maturation sensitizes cells to expression of aberrant Prpf8 and the subsequent deregulation of circRNAs triggers neuron death.

neuroscience↗

bioRxiv2022

Molecular basis of indispensable accuracy of mammalian miRNA biogenesis

Mammalian Dicer is the gatekeeper into the essential gene-regulating miRNA pathway. What is committing mammalian Dicer to the miRNA pathway remains unknown. We report that Dicers highly conserved DExD/H helicase domain is the key structural element supporting accurate miRNA biogenesis. While ATPase activity of the domain is non-essential, its loss is lethal in mice. It is required during canonical miRNA biogenesis for efficient recognition, high-fidelity cleavage, and strand selection. Structure of Dicer-miRNA precursor complexes showed that the DExD/H domain acquired helicase-unrelated function defining Dicer conformations, which affect substrate loading and facilitate pre-selection of miRNA precursors. Dicer lacking the DExD/H domain favors conformations enabling reduced substrate selectivity and supporting RNA interference, a different small RNA pathway. Therefore, Dicers DExD/H domain ensures indispensable high-fidelity precursor processing of mammalian miRNAs, which constitutes a structural "mold" for adaptive miRNA evolution.

molecular biology↗

bioRxiv2021

Comprehensive transcriptional profiling and mouse phenotyping reveals dispensable role for adipose tissue selective long noncoding RNA Gm15551

1Cold and nutrient activated brown adipose tissue (BAT) is capable of increasing systemic energy expenditure via uncoupled respiration and secretion of endocrine factors thereby protecting mice against diet-induced obesity and improving insulin response and glucose tolerance in men. Long non-coding RNAs (lncRNAs) have recently been identified as fine tuning regulators of cellular function. While certain lncRNAs have been functionally characterised in adipose tissue, their overall contribution in the activation of BAT remains elusive. We identified lncRNAs correlating to inter-scapular brown adipose tissue (iBAT) function in high fat diet (HFD) and cold stressed mice. We focused on Gm15551 which has an adipose tissue specific expression profile, is highly upregulated during adipogenesis and downregulated by {beta}-adrenergic activation in mature adipocytes. Albeit we performed comprehensive transcriptional and adipocyte physiology profiling in vitro and in vivo, we could not detect an effect of gain or loss of function of Gm15551.

molecular biology↗

bioRxiv2020

DDI2 protease activity controls embryonic development and inflammation via TCF11/NRF1

DDI2 is an aspartic protease that cleaves polyubiquitinated substrates. Upon proteotoxic stress, DDI2 activates the ER-bound transcription factor TCF11/NRF1 (NFE2L1), a master regulator of proteostasis maintenance in mammalian cells, and ensures the expression of rescue factors including proteasome subunits. Here we describe the consequences of DDI2 ablation both in vivo and in cells. Knock-out of DDI2 in mice resulted in embryonic lethality at E12.5 with severe developmental failure. Molecular characterization of the embryos and surrogate DDI2 knock-out cell lines showed insufficient proteasome expression with proteotoxic stress, accumulation of high molecular weight ubiquitin conjugates, and induction of the unfolded protein and integrated stress responses. We also show that DDI2 KO-induced proteotoxic stress causes the cell-autonomous innate immune system to induce a type I interferon signature. These results indicate an important role for DDI2 in the proteostasis network of cells and tissues and in the maintenance of a balanced immune response. HighlightsO_LIDDI2-deficiency in mice causes severe developmental failure and embryonic lethality at mid-late gestation C_LIO_LIDDI2-deficiency causes severe proteotoxic stress and proteasome impairment C_LIO_LIDDI2-deficiency induces the UPR and ISR signaling pathways C_LIO_LIDDI2-deficient cells survive via STAT3-dependent interferon signaling C_LI

cell biology↗

bioRxiv2020

Plectin Ensures Intestinal Epithelial Integrity and Protects Colon Against Colitis

Plectin, a highly versatile cytolinker protein, provides tissues with mechanical stability through the integration of intermediate filaments (IFs) with cell junctions. Here, we hypothesize that plectin-controlled cytoarchitecture is a critical determinant of the intestinal barrier function and homeostasis. Mice lacking plectin in intestinal epithelial cells (IEC; Ple{Delta}IEC) spontaneously developed colitis characterized by extensive detachment of IECs from the basement membrane (BM), increased intestinal permeability, and inflammatory lesions. Moreover, plectin expression was reduced in colons of ulcerative colitis (UC) patients and negatively correlated with the severity of colitis. Mechanistically, plectin deficiency in IECs led to aberrant keratin filament (KF) network organization and formation of dysfunctional hemidesmosomes (HDs) and intercellular junctions. In addition, the hemidesmosomal 6{beta}4 integrin (Itg) receptor showed attenuated association with KFs, and protein profiling revealed prominent downregulation of junctional constituents. Consistent with effects of plectin loss in the intestinal epithelium, plectin-deficient IECs exhibited remarkably reduced mechanical stability and limited adhesion capacity in vitro. Feeding mice with a low-residue liquid diet that reduced mechanical stress and antibiotic treatment successfully mitigated epithelial damage in the Ple{Delta}IEC colon.

molecular biology↗