Biology subjects

Abaffy, P.

Publications and source records attributed to Abaffy, P..

8 recordsLinked to original sources

bioRxiv2026

Stimulation of rodent and human beta-cell proliferation using synthetic modified mRNAs encoding cell cycle regulators

Pancreatic beta cells exhibit marked resistance to proliferation, posing a barrier to therapeutic strategies aimed at restoring beta-cell mass in diabetes. Here, we present a transient, non-integrative approach to stimulate beta-cell proliferation using in vitro transcribed (IVT) mRNAs encoding cell cycle regulators. In rodent beta cells and human-beta cell derived EndoC-BH5 cells, chemically modified IVT mRNAs activated cell cycle entry and subsequent mitosis. A single dose of cyclin D1 and CDK4 IVT mRNAs nearly doubled the number of rat beta cells. However, achieving cell division in human beta cells required co-delivery of MYC IVT mRNA. The mitogenic response of beta cells peaked within 36-60 hours, and declined thereafter, reflecting the transient nature of IVT mRNA. Transcriptomic profiling revealed temporary activation of proliferative pathways and reversible downregulation of beta-cell maturation markers. Importantly, we detected no evidence of sustained proliferation. Our findings demonstrate that mRNA-based delivery of cell cycle regulators can overcome the intrinsic cell cycle block in beta cells and may provide a controllable approach for beta-cell regeneration.

bioengineering↗

bioRxiv2025

Single-nucleus transcriptome analysis provides new insights into B chromosome elimination in sorghum

B chromosomes (Bs) are supernumerary entities found in many plant species, with some exhibiting tissue-specific elimination. In Sorghum purpureosericeum, extensive level of the B chromosome elimination occurs during embryogenesis. It progresses quickly and affects most of the embryonic organs, leaving the B chromosome maintained mainly in limited regions of meristems. Dynamic of the process and rarity of the transcripts associated with elimination make its capturing challenging. To address this, we performed single-nucleus RNA sequencing (snRNA-seq) on embryos actively undergoing B chromosome elimination. The snRNA-seq approach enabled the detection of a greater number of B-linked transcripts compared to previous method. Among all, we identified the nuclei with B-specific transcripts, which prevalently clustered together forming cluster 10 and contributing to cluster G2/M. The detailed analysis of cluster 10 revealed two distinct subpopulations of B-containing nuclei with divergent transcriptional profiles. The genes expressed in one subpopulation (sCL10-1) indicated that elimination of the B chromosome might be a factor for the split of the two subpopulations; namely that nuclei in sCL10-1 prepare for / undergo elimination, while the other subpopulation is expected to exhibit regular segregation of the B chromosome. Our analysis shows a direction so far missing in current studies and highlights a clear benefit of the single-cell approaches for studying specific behaviour of the B chromosomes.

plant biology↗

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↗

bioRxiv2023

Characterization of regeneration initiating cells during Xenopus laevis tail regeneration

Embryos are regeneration and wound healing masters. They not only rapidly close their wounds, remodel injured tissue without a scar, but also regenerate body parts. Many animal models with variable regenerative capabilities have already been studied. Additionally, with the introduction of high throughput techniques, novel regeneration mechanisms including genes and signaling pathways, and specialized cell types required for regeneration control in spatial and temporal aspects have been identified. Until now our knowledge has been limited to primarily the late phases of regeneration (> 1 day post injury). In this paper, we reveal the critical steps for regeneration initiation. We have discovered Regeneration Initiating Cells (RICs) using single cell and spatial transcriptomic analyses during tail regeneration in Xenopus laevis. RICs are formed transiently from the basal epidermal cells and are critical for the modification of the surrounding extracellular matrix to allow for migration of other cell types such as regeneration organizing cells that further promote regeneration. Absence or deregulation of RICs leads to excessive extracellular matrix deposition and regeneration defects.

developmental biology↗

bioRxiv2023

Spatiotemporal transcriptomic map of ischemic brain injury

The role of non-neuronal cells in the resolution of cerebral ischemia remains to be fully understood. To decode key cellular processes that occur after ischemia, we performed spatial and single-cell transcriptomic profiling of mouse brain tissue during the first week of injury. Cortical gene expression was severely disrupted, being defined by inflammation and cell death in the lesion core, and glial scar formation on the periphery. For each of the three major glial populations, an inflammatory-responsive state, resembling the reactive states observed in neurodegenerative contexts, was documented. The recovered spectrum of ischemia-induced oligodendrocyte states supports the emerging hypothesis that oligodendrocytes actively respond to and modulate the neuroinflammatory stimulus. Thus, we present a landmark transcriptomic dataset that provides a comprehensive view of spatiotemporal organization of processes in the post-ischemic brain and documents the conservation of glial response in CNS pathology.

neuroscience↗

bioRxiv2021

Evolutionary conservation of maternal RNA localization in fishes and amphibians revealed by TOMO-Seq

Asymmetrical localization of biomolecules inside the egg, results in uneven cell division and establishment of many biological processes, cell types and the body plan. However, our knowledge about evolutionary conservation of localized mRNAs is still limited to a few candidates. Our goal was to compare localization profiles along the animal-vegetal axis of mature eggs from four models, Xenopus laevis, Danio rerio, Ambystoma mexicanum and Acipenser ruthenus using the spatial expression method called TOMO-Seq. We revealed that RNAs of many known important genes such as germ layer determinants, germ plasm factors and members of key signalling pathways, are localized in completely different profiles among the models. There was also a poor correlation between vegetally localized genes but a relatively good correlation between animally localized genes. These findings indicate that the regulation of embryonic development within the animal kingdom is highly diverse and cannot be deduced based on a single model.

developmental biology↗

bioRxiv2019

Preparation of single-cell suspension from mouse breast cancer focusing on preservation of original cell state information and cell type composition

Single-cell analysis of gene expression has become a very popular method during the last decade. Unfortunately, appropriate standardization and workflow optimization remain elusive. The first step of the single cell analysis requires that the solid tissue be disassociated into a suspension of individual cells. However, during this step several technical bias can arise which can later result in the misinterpretation of the data. The goal of this study was to identify and quantify the effect of these technical factors on the quality of the single-cell suspension and the subsequent interpretation of the produced expression data. We tested the effects of various enzymes used for dissociation, several centrifugation forces, dissociation temperatures and the addition of Actinomycin D, a gene expression inhibitor. RT-qPCR was used to assess the effect from each parameter alteration, while a single-cell RNA sequencing experiment was used to confirm the optimized factors. Our concluding results provide a complete protocol for the tissue dissociation of mouse mammary tumour from 4T1 cells that preserves the original cell state and is suitable for any single-cell RNA sequencing analysis. Furthermore, our workflow may serve as a guide for the optimization of the dissociation procedure of any other tissue of interest, which would ultimately improve the reproducibility of the reported data.

cancer biology↗

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↗