Biology subjects

Tureckova, J.

Publications and source records attributed to Tureckova, J..

6 recordsLinked to original sources

bioRxiv2025

PU.1-driven enrichment enables microglia profiling from frozen brain tissue using the high-throughput Smart-seq3xpress method

Single-cell transcriptomics has revealed the central role of microglia in brain development, homeostasis, and disease, particularly in the context of neuroinflammation. While single-cell RNA-sequencing enables targeted microglial analysis from fresh tissue, studying these cells in cryopreserved or archival samples remains challenging due to the lack of protocols for their specific enrichment. We introduce a method for the selective isolation of microglial nuclei from fresh-frozen brain tissue using the transcription factor PU.1 as a nuclear marker. To stabilize PU.1 for reliable detection, a brief formaldehyde fixation step is applied. The protocol is fully compatible with Smart-seq3xpress, a high-sensitivity, full-length transcriptomic method offering isoform- and allele-level resolution, making the workflow scalable and cost-efficient. We benchmarked the method in a mouse model of ischemic stroke, evaluating both technical performance and its ability to capture biologically meaningful microglial states. Compared to standard single-nucleus protocols, our approach yielded higher gene and UMI counts and a greater proportion of coding reads. Transcriptomic profiles closely matched those from whole-cell RNA-sequencing including the detection of activation markers and diverse microglial subpopulations. This approach addresses key limitations of single-nucleus RNA - sequencing and opens new possibilities for studying microglial states in cryopreserved and archival brain tissue, broadening access to cellular insights in both basic and translational research.

neuroscience↗

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↗

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

Astrocyte-like subpopulation of NG2 glia in the adult mouse cortex exhibits characteristics of neural progenitor cells and is capable of forming neuron-like cells after ischemic injury

Glia cells expressing neuron-glial antigen 2 (NG2) play a critical role as oligodendrocyte precursor cells (OPCs) in the healthy brain; however, their differentiation potential after ischemic injury remains an unresolved question. Here, we aimed to elucidate the heterogeneity and role of NG2 glia in the ischemic brain. We used transgenic mice to label NG2-expressing cells and their progeny with red fluorescent protein tdTomato in the healthy brains and those after focal cerebral ischemia (FCI). Based on single-cell RNA sequencing, the labeled glial cells were divided into five distinct subpopulations. The identity of these subpopulations was determined based on gene expression patterns. In addition, membrane properties were further analyzed using the patch-clamp technique. Three of the observed subpopulations represented OPCs, whereas the fourth group exhibited characteristics of cells destined for oligodendrocyte fate. The fifth subpopulation of NG2 glia carried astrocytic markers. Importantly, we detected features of neural progenitors in these cells. This subpopulation was present in both healthy and post-ischemic tissue; however, its gene expression changed after ischemia, with genes related to neurogenesis being more abundant. Neurogenic gene expression was monitored over time and complemented by immunohistochemical staining, which showed increased numbers of Purkinje cell protein 4-positive NG2 cells at the edge of the ischemic lesion 12 days after FCI, and NeuN-positive NG2 cells 28 days after injury, indicating the existence of neuron-like cells that develop from NG2 glia in the ischemic tissue. Our results provide further insight into the differentiation plasticity and neurogenic potential of NG2 glia after stroke. Main PointsO_LIDifferent subpopulations of NG2 glia in the healthy and ischemic adult cortex were identified based on their gene expression and membrane properties. C_LIO_LIAstrocyte-like NG2 glia exhibit neurogenic gene expression and are more abundant in post-ischemic tissue. C_LIO_LIProgeny of NG2-positive cells carrying neuronal marker NeuN was observed at the edge of the ischemic lesion. C_LI

neuroscience↗

bioRxiv2021

Bispecific antibody prevents SARS-CoV-2 escape and protects mice from disease

Neutralizing antibodies targeting the receptor binding domain (RBD) of the SARS-CoV-2 Spike (S) are among the most promising approaches against coronavirus disease 2019 (COVID-19)1,2. We developed a bispecific, IgG1-like molecule (CoV-X2) based on two antibodies derived from COVID-19 convalescent donors, C121 and C1353. CoV-X2 simultaneously binds two independent sites on the RBD and, unlike its parental antibodies, prevents detectable S binding to Angiotensin-Converting Enzyme 2 (ACE2), the virus cellular receptor. Furthermore, CoV-X2 neutralizes SARS-CoV-2 and its variants of concern, as well as the escape mutants generated by the parental monoclonals. In a novel animal model of SARS-CoV-2 infection with lung inflammation, CoV-X2 protects mice from disease and suppresses viral escape. Thus, simultaneous targeting of non-overlapping RBD epitopes by IgG-like bispecific antibodies is feasible and effective, combining into a single molecule the advantages of antibody cocktails.

immunology↗

bioRxiv2019

Decoding the transcriptional response to ischemic stroke in young and aged mouse brain

Ischemic stroke is one of the leading causes of mortality and major healthcare and economic burden. It is a well-recognized disease of aging, yet it is unclear how the age-dependent vulnerability occurs and what are the underlying mechanisms. To address these issues, we performed a comprehensive RNA-Seq analysis of aging, ischemic stroke and their interaction using a model of permanent middle cerebral artery occlusion (MCAO) in 3 and 18 month old female mice. We assessed differential gene expression across injury status and age, estimated cell type proportion changes, assayed the results against a range of transcriptional signatures from the literature and performed unsupervised co-expression analysis, identifying modules of genes with varying response to injury. We uncovered selective vulnerability of neuronal populations and increased activation of type-I interferon (IFN-I) signaling and several other inflammatory pathways in aged mice. We extended these findings via targeted expression analysis in tissue as well as acutely purified cellular populations to show differential temporal dynamics of IFN-I signaling between age groups and contribution of individual cell types. Together, these results paint a picture of ischemic stroke as a complex age-related disease and provide insights into interaction of aging and stroke on cellular and molecular level.\n\nGraphical summary\n\nO_FIG O_LINKSMALLFIG WIDTH=156 HEIGHT=200 SRC=\"FIGDIR/small/769331v1_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (46K):\norg.highwire.dtl.DTLVardef@1590c10org.highwire.dtl.DTLVardef@126fc76org.highwire.dtl.DTLVardef@88e20aorg.highwire.dtl.DTLVardef@b000d9_HPS_FORMAT_FIGEXP M_FIG C_FIG

genomics↗