bims-ginsta Biomed News
on Genome instability
Issue of 2024–07–14
thirty-one papers selected by
Jinrong Hu, National University of Singapore
  1. A non-canonical role for a small nucleolar RNA in ribosome biogenesis and senescence.
  2. A human pluripotent stem cell-based somitogenesis model using microfluidics.
  3. Cell-intrinsic mechanical regulation of plasma membrane accumulation at the cytokinetic furrow.
  4. A single-cell atlas of chromatin accessibility in mouse organogenesis.
  5. Emergence of large-scale cell death through ferroptotic trigger waves.
  6. FAM110A promotes mitotic spindle formation by linking microtubules with actin cytoskeleton.
  7. Cell-type-specific loops linked to RNA polymerase II elongation in human neural differentiation.
  8. Activation of fetal-like molecular programs during regeneration in the intestine and beyond.
  9. Resolving chaperone-assisted protein folding on the ribosome at the peptide level.
  10. Mechanical forces remodel the cardiac extracellular matrix during zebrafish development.
  11. Control of epiblast cell fate by mechanical cues.
  12. Inter-cellular mRNA Transfer Alters Human Pluripotent Stem Cell State.
  13. Simple aneuploidy evades p53 surveillance and promotes niche factor-independent growth in human intestinal organoids.
  14. Alveolar fibroblast lineage orchestrates lung inflammation and fibrosis.
  15. Endonucleosis mediates internalization of cytoplasm into the nucleus.
  16. Human DDX6 regulates translation and decay of inefficiently translated mRNAs.
  17. Senescent cell heterogeneity and responses to senolytic treatment are related to cell cycle status during cell growth arrest.
  18. Pioneer factors: Emerging rules of engagement for transcription factors on chromatinized DNA.
  19. Polymerization of ZBTB transcription factors regulates chromatin occupancy.
  20. Replicative senescence is ATM driven, reversible, and accelerated by hyperactivation of ATM at normoxia.
  21. Aging limits stemness and tumorigenesis in the lung by reprogramming iron homeostasis.
  22. Molecular Regulation of Cardiac Conduction System Development.
  23. Mechanical stress during confined migration causes aberrant mitoses and c-MYC amplification.
  24. Overview of chromatin regulatory processes during surface ectodermal development and homeostasis.
  25. PUFFFIN: an ultra-bright, customisable, single-plasmid system for labelling cell neighbourhoods.
  26. Pressure sensing of lysosomes enables control of TFEB responses in macrophages.
  27. Human promoter directionality is determined by transcriptional initiation and the opposing activities of INTS11 and CDK9.
  28. Mapping the chromatin accessibility landscape of zebrafish embryogenesis at single-cell resolution by SPATAC-seq.
  29. On the evolutionary developmental biology of the cell.
  30. A roadmap for ribosome assembly in human mitochondria.
  31. Keep calm and reboot - how cells restart transcription after DNA damage and DNA repair.
  1. Cell. 2024 Jun 28. pii: S0092-8674(24)00693-7. [Epub ahead of print]
    A non-canonical role for a small nucleolar RNA in ribosome biogenesis and senescence.
    Yujing Cheng, Siwen Wang, He Zhang, Jong-Sun Lee, Chunyang Ni, Jason Guo, Eric Chen, Shenming Wang, Asha Acharya, Tsung-Cheng Chang, Michael Buszczak, Hao Zhu, Joshua T Mendell.
      Cellular senescence is an irreversible state of cell-cycle arrest induced by various stresses, including aberrant oncogene activation, telomere shortening, and DNA damage. Through a genome-wide screen, we discovered a conserved small nucleolar RNA (snoRNA), SNORA13, that is required for multiple forms of senescence in human cells and mice. Although SNORA13 guides the pseudouridylation of a conserved nucleotide in the ribosomal decoding center, loss of this snoRNA minimally impacts translation. Instead, we found that SNORA13 negatively regulates ribosome biogenesis. Senescence-inducing stress perturbs ribosome biogenesis, resulting in the accumulation of free ribosomal proteins (RPs) that trigger p53 activation. SNORA13 interacts directly with RPL23, decreasing its incorporation into maturing 60S subunits and, consequently, increasing the pool of free RPs, thereby promoting p53-mediated senescence. Thus, SNORA13 regulates ribosome biogenesis and the p53 pathway through a non-canonical mechanism distinct from its role in guiding RNA modification. These findings expand our understanding of snoRNA functions and their roles in cellular signaling.
    Keywords:  RPL23; SNORA13; nucleolar stress; p53; ribosome biogenesis; senescence; snoRNA
    DOI:  https://doi.org/10.1016/j.cell.2024.06.019
  2. Cell Stem Cell. 2024 Jun 28. pii: S1934-5909(24)00212-1. [Epub ahead of print]
    A human pluripotent stem cell-based somitogenesis model using microfluidics.
    Yue Liu, Yung Su Kim, Xufeng Xue, Yuchuan Miao, Norio Kobayashi, Shiyu Sun, Robin Zhexuan Yan, Qiong Yang, Olivier Pourquié, Jianping Fu.
      Emerging human pluripotent stem cell (hPSC)-based embryo models are useful for studying human embryogenesis. Particularly, there are hPSC-based somitogenesis models using free-floating culture that recapitulate somite formation. Somitogenesis in vivo involves intricately orchestrated biochemical and biomechanical events. However, none of the current somitogenesis models controls biochemical gradients or biomechanical signals in the culture, limiting their applicability to untangle complex biochemical-biomechanical interactions that drive somitogenesis. Herein, we develop a human somitogenesis model by confining hPSC-derived presomitic mesoderm (PSM) tissues in microfabricated trenches. Exogenous microfluidic morphogen gradients imposed on the PSM tissues cause axial patterning and trigger spontaneous rostral-to-caudal somite formation. A mechanical theory is developed to explain the size dependency between somites and the PSM. The microfluidic somitogenesis model is further exploited to reveal regulatory roles of cellular and tissue biomechanics in somite formation. This study presents a useful microengineered, hPSC-based model for understanding the biochemical and biomechanical events that guide somite formation.
    Keywords:  biomechanics; embryo model; human development; human pluripotent stem cells; in vitro modeling; microfluidics; somitogenesis
    DOI:  https://doi.org/10.1016/j.stem.2024.06.004
  3. Proc Natl Acad Sci U S A. 2024 Jul 16. 121(29): e2320769121
    Cell-intrinsic mechanical regulation of plasma membrane accumulation at the cytokinetic furrow.
    Roberto Alonso-Matilla, Alice R Lam, Teemu P Miettinen.
      Cytokinesis is the process where the mother cell's cytoplasm separates into daughter cells. This is driven by an actomyosin contractile ring that produces cortical contractility and drives cleavage furrow ingression, resulting in the formation of a thin intercellular bridge. While cytoskeletal reorganization during cytokinesis has been extensively studied, less is known about the spatiotemporal dynamics of the plasma membrane. Here, we image and model plasma membrane lipid and protein dynamics on the cell surface during leukemia cell cytokinesis. We reveal an extensive accumulation and folding of the plasma membrane at the cleavage furrow and the intercellular bridge, accompanied by a depletion and unfolding of the plasma membrane at the cell poles. These membrane dynamics are caused by two actomyosin-driven biophysical mechanisms: the radial constriction of the cleavage furrow causes local compression of the apparent cell surface area and accumulation of the plasma membrane at the furrow, while actomyosin cortical flows drag the plasma membrane toward the cell division plane as the furrow ingresses. The magnitude of these effects depends on the plasma membrane fluidity, cortex adhesion, and cortical contractility. Overall, our work reveals cell-intrinsic mechanical regulation of plasma membrane accumulation at the cleavage furrow that is likely to generate localized differences in membrane tension across the cytokinetic cell. This may locally alter endocytosis, exocytosis, and mechanotransduction, while also serving as a self-protecting mechanism against cytokinesis failures that arise from high membrane tension at the intercellular bridge.
    Keywords:  actomyosin; biophysical model; cytokinesis; membrane tension; plasma membrane
    DOI:  https://doi.org/10.1073/pnas.2320769121
  4. Nat Cell Biol. 2024 Jul 08.
    A single-cell atlas of chromatin accessibility in mouse organogenesis.
    Keyong Sun, Xin Liu, Xun Lan.
      Organogenesis is a highly complex and precisely regulated process. Here we profiled the chromatin accessibility in >350,000 cells derived from 13 mouse embryos at four developmental stages from embryonic day (E) 10.5 to E13.5 by SPATAC-seq in a single experiment. The resulting atlas revealed the status of 830,873 candidate cis-regulatory elements in 43 major cell types. By integrating the chromatin accessibility atlas with the previous transcriptomic dataset, we characterized cis-regulatory sequences and transcription factors associated with cell fate commitment, such as Nr5a2 in the development of gastrointestinal tract, which was preliminarily supported by the in vivo experiment in zebrafish. Finally, we integrated this atlas with the previous single-cell chromatin accessibility dataset from 13 adult mouse tissues to delineate the developmental stage-specific gene regulatory programmes within and across different cell types and identify potential molecular switches throughout lineage development. This comprehensive dataset provides a foundation for exploring transcriptional regulation in organogenesis.
    DOI:  https://doi.org/10.1038/s41556-024-01435-6
  5. Nature. 2024 Jul 10.
    Emergence of large-scale cell death through ferroptotic trigger waves.
    Hannah K C Co, Chia-Chou Wu, Yi-Chen Lee, Sheng-Hong Chen.
      Large-scale cell death is commonly observed during organismal development and in human pathologies1-5. These cell death events extend over great distances to eliminate large populations of cells, raising the question of how cell death can be coordinated in space and time. One mechanism that enables long-range signal transmission is trigger waves6, but how this mechanism might be used for death events in cell populations remains unclear. Here we demonstrate that ferroptosis, an iron- and lipid-peroxidation-dependent form of cell death, can propagate across human cells over long distances (≥5 mm) at constant speeds (around 5.5 μm min-1) through trigger waves of reactive oxygen species (ROS). Chemical and genetic perturbations indicate a primary role of ROS feedback loops (Fenton reaction, NADPH oxidase signalling and glutathione synthesis) in controlling the progression of ferroptotic trigger waves. We show that introducing ferroptotic stress through suppression of cystine uptake activates these ROS feedback loops, converting cellular redox systems from being monostable to being bistable and thereby priming cell populations to become bistable media over which ROS propagate. Furthermore, we demonstrate that ferroptosis and its propagation accompany the massive, yet spatially restricted, cell death events during muscle remodelling of the embryonic avian limb, substantiating its use as a tissue-sculpting strategy during embryogenesis. Our findings highlight the role of ferroptosis in coordinating global cell death events, providing a paradigm for investigating large-scale cell death in embryonic development and human pathologies.
    DOI:  https://doi.org/10.1038/s41586-024-07623-6
  6. Proc Natl Acad Sci U S A. 2024 Jul 16. 121(29): e2321647121
    FAM110A promotes mitotic spindle formation by linking microtubules with actin cytoskeleton.
    Cecilia Aquino-Perez, Mahira Safaralizade, Roman Podhajecky, Hong Wang, Zdenek Lansky, Robert Grosse, Libor Macurek.
      Precise segregation of chromosomes during mitosis requires assembly of a bipolar mitotic spindle followed by correct attachment of microtubules to the kinetochores. This highly spatiotemporally organized process is controlled by various mitotic kinases and molecular motors. We have recently shown that Casein Kinase 1 (CK1) promotes timely progression through mitosis by phosphorylating FAM110A leading to its enrichment at spindle poles. However, the mechanism by which FAM110A exerts its function in mitosis is unknown. Using structure prediction and a set of deletion mutants, we mapped here the interaction of the N- and C-terminal domains of FAM110A with actin and tubulin, respectively. Next, we found that the FAM110A-Δ40-61 mutant deficient in actin binding failed to rescue defects in chromosomal alignment caused by depletion of endogenous FAM110A. Depletion of FAM110A impaired assembly of F-actin in the proximity of spindle poles and was rescued by expression of the wild-type FAM110A, but not the FAM110A-Δ40-61 mutant. Purified FAM110A promoted binding of F-actin to microtubules as well as bundling of actin filaments in vitro. Finally, we found that the inhibition of CK1 impaired spindle actin formation and delayed progression through mitosis. We propose that CK1 and FAM110A promote timely progression through mitosis by mediating the interaction between spindle microtubules and filamentous actin to ensure proper mitotic spindle formation.
    Keywords:  actin; microtubules; mitosis; mitotic spindle; protein kinase
    DOI:  https://doi.org/10.1073/pnas.2321647121
  7. Cell Genom. 2024 Jul 09. pii: S2666-979X(24)00200-3. [Epub ahead of print] 100606
    Cell-type-specific loops linked to RNA polymerase II elongation in human neural differentiation.
    Katelyn R Titus, Zoltan Simandi, Harshini Chandrashekar, Dominik Paquet, Jennifer E Phillips-Cremins.
      DNA is folded into higher-order structures that shape and are shaped by genome function. The role of long-range loops in the establishment of new gene expression patterns during cell fate transitions remains poorly understood. Here, we investigate the link between cell-specific loops and RNA polymerase II (RNA Pol II) during neural lineage commitment. We find thousands of loops decommissioned or gained de novo upon differentiation of human induced pluripotent stem cells (hiPSCs) to neural progenitor cells (NPCs) and post-mitotic neurons. During hiPSC-to-NPC and NPC-to-neuron transitions, genes changing from RNA Pol II initiation to elongation are >4-fold more likely to anchor cell-specific loops than repressed genes. Elongated genes exhibit significant mRNA upregulation when connected in cell-specific promoter-enhancer loops but not invariant promoter-enhancer loops or promoter-promoter loops or when unlooped. Genes transitioning from repression to RNA Pol II initiation exhibit a slight mRNA increase independent of loop status. Our data link cell-specific loops and robust RNA Pol II-mediated elongation during neural cell fate transitions.
    DOI:  https://doi.org/10.1016/j.xgen.2024.100606
  8. Cell Stem Cell. 2024 Jul 05. pii: S1934-5909(24)00186-3. [Epub ahead of print]31(7): 949-960
    Activation of fetal-like molecular programs during regeneration in the intestine and beyond.
    Sara Viragova, Dong Li, Ophir D Klein.
      Tissue regeneration after damage is generally thought to involve the mobilization of adult stem cells that divide and differentiate into progressively specialized progeny. However, recent studies indicate that tissue regeneration can be accompanied by reversion to a fetal-like state. During this process, cells at the injury site reactivate programs that operate during fetal development but are typically absent in adult homeostasis. Here, we summarize our current understanding of the molecular signals and epigenetic mediators that orchestrate "fetal-like reversion" during intestinal regeneration. We also explore evidence for this phenomenon in other organs and species and highlight open questions that merit future examination.
    Keywords:  Hippo; YAP; colon; dedifferentiation; developmental reprogramming; epithelium; fetal-like reversion; intestinal epithelium; intestine; liver; paligenosis; plasticity; regeneration; stem cells; stomach
    DOI:  https://doi.org/10.1016/j.stem.2024.05.009
  9. Nat Struct Mol Biol. 2024 Jul 10.
    Resolving chaperone-assisted protein folding on the ribosome at the peptide level.
    Thomas E Wales, Aleksandra Pajak, Alžběta Roeselová, Santosh Shivakumaraswamy, Steven Howell, Svend Kjær, F Ulrich Hartl, John R Engen, David Balchin.
      Protein folding in vivo begins during synthesis on the ribosome and is modulated by molecular chaperones that engage the nascent polypeptide. How these features of protein biogenesis influence the maturation pathway of nascent proteins is incompletely understood. Here, we use hydrogen-deuterium exchange mass spectrometry to define, at peptide resolution, the cotranslational chaperone-assisted folding pathway of Escherichia coli dihydrofolate reductase. The nascent polypeptide folds along an unanticipated pathway through structured intermediates not populated during refolding from denaturant. Association with the ribosome allows these intermediates to form, as otherwise destabilizing carboxy-terminal sequences remain confined in the ribosome exit tunnel. Trigger factor binds partially folded states without disrupting their structure, and the nascent chain is poised to complete folding immediately upon emergence of the C terminus from the exit tunnel. By mapping interactions between the nascent chain and ribosomal proteins, we trace the path of the emerging polypeptide during synthesis. Our work reveals new mechanisms by which cellular factors shape the conformational search for the native state.
    DOI:  https://doi.org/10.1038/s41594-024-01355-x
  10. Development. 2024 Jul 01. pii: dev202310. [Epub ahead of print]151(13):
    Mechanical forces remodel the cardiac extracellular matrix during zebrafish development.
    Alessandra Gentile, Marga Albu, Yanli Xu, Newsha Mortazavi, Agatha Ribeiro da Silva, Didier Y R Stainier, Felix Gunawan.
      The cardiac extracellular matrix (cECM) is fundamental for organ morphogenesis and maturation, during which time it undergoes remodeling, yet little is known about whether mechanical forces generated by the heartbeat regulate this remodeling process. Using zebrafish as a model and focusing on stages when cardiac valves and trabeculae form, we found that altering cardiac contraction impairs cECM remodeling. Longitudinal volumetric quantifications in wild-type animals revealed region-specific dynamics: cECM volume decreases in the atrium but not in the ventricle or atrioventricular canal. Reducing cardiac contraction resulted in opposite effects on the ventricular and atrial ECM, whereas increasing the heart rate affected the ventricular ECM but had no effect on the atrial ECM, together indicating that mechanical forces regulate the cECM in a chamber-specific manner. Among the ECM remodelers highly expressed during cardiac morphogenesis, we found one that was upregulated in non-contractile hearts, namely tissue inhibitor of matrix metalloproteinase 2 (timp2). Loss- and gain-of-function analyses of timp2 revealed its crucial role in cECM remodeling. Altogether, our results indicate that mechanical forces control cECM remodeling in part through timp2 downregulation.
    Keywords:  Biomechanical forces; Cardiac ECM remodeling; Cardiac development; Zebrafish cardiogenesis
    DOI:  https://doi.org/10.1242/dev.202310
  11. bioRxiv. 2024 Jun 25. pii: 2024.06.24.600402. [Epub ahead of print]
    Control of epiblast cell fate by mechanical cues.
    Charlène Guillot, Yannis Djeffal, Mattia Serra, Olivier Pourquié.
      In amniotes, embryonic tissues originate from multipotent epiblast cells, arranged in a mosaic of presumptive territories. How these domains fated to specific lineages become segregated during body formation remains poorly understood. Using single cell RNA sequencing analysis and lineage tracing in the chicken embryo, we identify epiblast cells contributing descendants to the neural tube, somites and lateral plate after completion of gastrulation. We show that intercalation after cell division generates important movements of epiblast cells which lead to their relocation to different presumptive territories, explaining this broad spectrum of fates. This tissue remodeling phase is transient, being soon followed by the establishment of boundaries restricting cell movements therefore defining the presumptive territories of the epiblast. Finally, we find that the epiblast faces distinct mechanical constraints along the antero-posterior axis, leading to cell fate alterations when challenged. Together, we demonstrate the critical role of mechanical cues in epiblast fate determination.
    DOI:  https://doi.org/10.1101/2024.06.24.600402
  12. bioRxiv. 2024 Jun 27. pii: 2024.06.27.600209. [Epub ahead of print]
    Inter-cellular mRNA Transfer Alters Human Pluripotent Stem Cell State.
    Yosuke Yoneyama, Ran-Ran Zhang, Masaki Kimura, Yuqi Cai, Mike Adam, Sreeja Parameswaran, Hideki Masaki, Naoaki Mizuno, Joydeep Bhadury, So Maezawa, Hiroshi Ochiai, Hiromitsu Nakauchi, S Steven Potter, Matthew T Weirauch, Takanori Takebe.
      Inter-cellular transmission of mRNA is being explored in mammalian species using immortal cell lines (1-3). Here, we uncover an inter-cellular mRNA transfer phenomenon that allows for the adaptation and reprogramming of human primed pluripotent stem cells (hPSCs). This process is induced by the direct cell contact-mediated coculture with mouse embryonic stem cells (mESCs) under the condition impermissible for human primed PSC culture. Mouse-derived mRNA contents are transmitted into adapted hPSCs only in the coculture. Transfer-specific mRNA analysis show the enrichment for divergent biological pathways involving transcription/translational machinery and stress-coping mechanisms, wherein such transfer is diminished when direct cell contacts are lost. After 5 days of mESC culture, surface marker analysis, and global gene profiling confirmed that mRNA transfer-prone hPSC efficiently gains a naïve-like state. Furthermore, transfer-specific knockdown experiments targeting mouse-specific transcription factor-coding mRNAs in hPSC show that mouse-derived Tfcp2l1 , Tfap2c, and Klf4 are indispensable for human naïve-like conversion. Thus, inter-species mRNA transfer triggers cellular reprogramming in mammalian cells. Our results support that episodic mRNA transfer can occur in cell cooperative and competitive processes(4), which provides a fresh perspective on understanding the roles of mRNA mobility for intra- and inter-species cellular communications.
    DOI:  https://doi.org/10.1101/2024.06.27.600209
  13. Mol Biol Cell. 2024 Jul 10. mbcE24040166
    Simple aneuploidy evades p53 surveillance and promotes niche factor-independent growth in human intestinal organoids.
    Blake A Johnson, Albert Z Liu, Tianhao Bi, Yi Dong, Taibo Li, Dingjingyu Zhou, Akshay Narkar, Yufei Wu, Sean X Sun, Tatianna C Larman, Jin Zhu, Rong Li.
      Aneuploidy is nearly ubiquitous in tumor genomes, but the role of aneuploidy in the early stages of cancer evolution remains unclear. Here, by inducing heterogeneous aneuploidy in non-transformed human colon organoids (colonoids), we investigated how the effects of aneuploidy on cell growth and differentiation may promote malignant transformation. Previous work implicated p53 activation as a downstream response to aneuploidy induction. We found that simple aneuploidy, characterized by 1-3 gained or lost chromosomes, resulted in little or modest p53 activation and cell cycle arrest when compared with more complex aneuploid cells. Single-cell RNA sequencing analysis revealed that the degree of p53 activation was strongly correlated with karyotype complexity. Single-cell tracking showed that cells could continue to divide despite the observation of one to a few lagging chromosomes. Unexpectedly, colonoids with simple aneuploidy exhibited impaired differentiation after niche factor withdrawal. These findings demonstrate that simple aneuploid cells can escape p53 surveillance and may contribute to niche factor-independent growth of cancer-initiating colon stem cells. [Media: see text].
    DOI:  https://doi.org/10.1091/mbc.E24-04-0166
  14. Nature. 2024 Jul 10.
    Alveolar fibroblast lineage orchestrates lung inflammation and fibrosis.
    Tatsuya Tsukui, Paul J Wolters, Dean Sheppard.
      Fibroblasts are present throughout the body and function to maintain tissue homeostasis. Recent studies have identified diverse fibroblast subsets in healthy and injured tissues1,2, but the origins and functional roles of injury-induced fibroblast lineages remain unclear. Here we show that lung-specialized alveolar fibroblasts take on multiple molecular states with distinct roles in facilitating responses to fibrotic lung injury. We generate a genetic tool that uniquely targets alveolar fibroblasts to demonstrate their role in providing niches for alveolar stem cells in homeostasis and show that loss of this niche leads to exaggerated responses to acute lung injury. Lineage tracing identifies alveolar fibroblasts as the dominant origin for multiple emergent fibroblast subsets sequentially driven by inflammatory and pro-fibrotic signals after injury. We identify similar, but not completely identical, fibroblast lineages in human pulmonary fibrosis. TGFβ negatively regulates an inflammatory fibroblast subset that emerges early after injury and stimulates the differentiation into fibrotic fibroblasts to elicit intra-alveolar fibrosis. Blocking the induction of fibrotic fibroblasts in the alveolar fibroblast lineage abrogates fibrosis but exacerbates lung inflammation. These results demonstrate the multifaceted roles of the alveolar fibroblast lineage in maintaining normal alveolar homeostasis and orchestrating sequential responses to lung injury.
    DOI:  https://doi.org/10.1038/s41586-024-07660-1
  15. Nat Commun. 2024 Jul 11. 15(1): 5843
    Endonucleosis mediates internalization of cytoplasm into the nucleus.
    Ourania Galanopoulou, Evangelia C Tachmatzidi, Elena Deligianni, Dimitris Botskaris, Kostas C Nikolaou, Sofia Gargani, Yannis Dalezios, Georges Chalepakis, Iannis Talianidis.
      Setd8 regulates transcription elongation, mitotic DNA condensation, DNA damage response and replication licensing. Here we show that, in mitogen-stimulated liver-specific Setd8-KO mice, most of the hepatocytes are eliminated by necrosis but a significant number of them survive via entering a stage exhibiting several senescence-related features. Setd8-deficient hepatocytes had enlarged nuclei, chromosomal hyperploidy and nuclear engulfments progressing to the formation of intranuclear vesicles surrounded by nuclear lamina. These vesicles contain glycogen, cytoplasmic proteins and even entire organelles. We term this process "endonucleosis". Intranuclear vesicles are absent in hepatocytes of Setd8/Atg5 knockout mice, suggesting that the process requires the function of the canonical autophagy machinery. Endonucleosis and hyperploidization are temporary, early events in the surviving Setd8-deficient cells. Larger vesicles break down into microvesicles over time and are eventually eliminated. The results reveal sequential events in cells with extensive DNA damage, which function as part of survival mechanisms to prevent necrotic death.
    DOI:  https://doi.org/10.1038/s41467-024-50259-3
  16. Elife. 2024 Jul 11. pii: RP92426. [Epub ahead of print]13
    Human DDX6 regulates translation and decay of inefficiently translated mRNAs.
    Ramona Weber, Chung-Te Chang.
      Recent findings indicate that the translation elongation rate influences mRNA stability. One of the factors that has been implicated in this link between mRNA decay and translation speed is the yeast DEAD-box helicase Dhh1p. Here, we demonstrated that the human ortholog of Dhh1p, DDX6, triggers the deadenylation-dependent decay of inefficiently translated mRNAs in human cells. DDX6 interacts with the ribosome through the Phe-Asp-Phe (FDF) motif in its RecA2 domain. Furthermore, RecA2-mediated interactions and ATPase activity are both required for DDX6 to destabilize inefficiently translated mRNAs. Using ribosome profiling and RNA sequencing, we identified two classes of endogenous mRNAs that are regulated in a DDX6-dependent manner. The identified targets are either translationally regulated or regulated at the steady-state-level and either exhibit signatures of poor overall translation or of locally reduced ribosome translocation rates. Transferring the identified sequence stretches into a reporter mRNA caused translation- and DDX6-dependent degradation of the reporter mRNA. In summary, these results identify DDX6 as a crucial regulator of mRNA translation and decay triggered by slow ribosome movement and provide insights into the mechanism by which DDX6 destabilizes inefficiently translated mRNAs.
    Keywords:  CCR4-NOT complex; DDX6; biochemistry; chemical biology; chromosomes; codon optimization; gene expression; human; mRNA decay; ribosome stalling
    DOI:  https://doi.org/10.7554/eLife.92426
  17. bioRxiv. 2024 Jun 26. pii: 2024.06.22.600200. [Epub ahead of print]
    Senescent cell heterogeneity and responses to senolytic treatment are related to cell cycle status during cell growth arrest.
    Francesco Neri, Shuyuan Zheng, Mark Watson, Pierre-Yves Desprez, Akos A Gerencser, Judith Campisi, Denis Wirtz, Pei-Hsun Wu, Birgit Schilling.
      Cellular senescence has been strongly linked to aging and age-related diseases. It is well established that the phenotype of senescent cells is highly heterogeneous and influenced by their cell type and senescence-inducing stimulus. Recent single-cell RNA-sequencing studies identified heterogeneity within senescent cell populations. However, proof of functional differences between such subpopulations is lacking. To identify functionally distinct senescent cell subpopulations, we employed high-content image analysis to measure senescence marker expression in primary human endothelial cells and fibroblasts. We found that G2-arrested senescent cells feature higher senescence marker expression than G1-arrested senescent cells. To investigate functional differences, we compared IL-6 secretion and response to ABT263 senolytic treatment in G1 and G2 senescent cells. We determined that G2-arrested senescent cells secrete more IL-6 and are more sensitive to ABT263 than G1-arrested cells. We hypothesize that cell cycle dependent DNA content is a key contributor to the heterogeneity within senescent cell populations. This study demonstrates the existence of functionally distinct senescent subpopulations even in culture. This data provides the first evidence of selective cell response to senolytic treatment among senescent cell subpopulations. Overall, this study emphasizes the importance of considering the senescent cell heterogeneity in the development of future senolytic therapies.
    DOI:  https://doi.org/10.1101/2024.06.22.600200
  18. Curr Opin Struct Biol. 2024 Jul 10. pii: S0959-440X(24)00102-7. [Epub ahead of print]88 102875
    Pioneer factors: Emerging rules of engagement for transcription factors on chromatinized DNA.
    Manuel Carminati, Luca Vecchia, Lisa Stoos, Nicolas H Thomä.
      Pioneering transcription factors (TFs) can drive cell fate changes by binding their DNA motifs in a repressive chromatin environment. Recent structures illustrate emerging rules for nucleosome engagement: TFs distort the nucleosomal DNA to gain access or employ alternative DNA-binding modes with smaller footprints, they preferentially access solvent-exposed motifs near the entry/exit sites, and frequently interact with histones. The extent of TF-histone interactions, in turn, depends on the motif location on the nucleosome, the type of DNA-binding fold, and adjacent domains present. TF-histone interactions can phase TF motifs relative to nucleosomes, and we discuss how these complex and surprisingly diverse interactions between nucleosomes and TFs contribute to function.
    DOI:  https://doi.org/10.1016/j.sbi.2024.102875
  19. Mol Cell. 2024 Jul 11. pii: S1097-2765(24)00513-6. [Epub ahead of print]84(13): 2511-2524.e8
    Polymerization of ZBTB transcription factors regulates chromatin occupancy.
    Paul M C Park, Jiho Park, Jared Brown, Moritz Hunkeler, Shourya S Roy Burman, Katherine A Donovan, Hojong Yoon, Radosław P Nowak, Mikołaj Słabicki, Benjamin L Ebert, Eric S Fischer.
      BCL6, an oncogenic transcription factor (TF), forms polymers in the presence of a small-molecule molecular glue that stabilizes a complementary interface between homodimers of BCL6's broad-complex, tramtrack, and bric-à-brac (BTB) domain. The BTB domains of other proteins, including a large class of TFs, have similar architectures and symmetries, raising the possibility that additional BTB proteins self-assemble into higher-order structures. Here, we surveyed 189 human BTB proteins with a cellular fluorescent reporter assay and identified 18 ZBTB TFs that show evidence of polymerization. Through biochemical and cryoelectron microscopy (cryo-EM) studies, we demonstrate that these ZBTB TFs polymerize into filaments. We found that BTB-domain-mediated polymerization of ZBTB TFs enhances chromatin occupancy within regions containing homotypic clusters of TF binding sites, leading to repression of target genes. Our results reveal a role of higher-order structures in regulating ZBTB TFs and suggest an underappreciated role for TF polymerization in modulating gene expression.
    Keywords:  BTB domain; ZBTB proteins; higher-order assembly; homotypic clusters; protein polymerization; transcription factors
    DOI:  https://doi.org/10.1016/j.molcel.2024.06.010
  20. bioRxiv. 2024 Jun 26. pii: 2024.06.24.600514. [Epub ahead of print]
    Replicative senescence is ATM driven, reversible, and accelerated by hyperactivation of ATM at normoxia.
    Alexander Stuart, Titia de Lange.
      Programmed telomere shortening limits tumorigenesis through the induction of replicative senescence. Here we address three long-standing questions concerning senescence. First, we show that the ATM kinase is solely responsible for the induction of replicative senescence. Senescence was delayed by ATM inhibition (ATMi) or overexpression of TRF2, the shelterin subunit dedicated to ATM repression. In contrast, there was no evidence for ATR signaling contributing to replicative senescence even when ATMi was combined with ATR inhibition. Second, we show ATMi can induce apparently normal cell divisions in a subset of senescent cells, indicating that senescence can be reversed. Third, we show that the extended replicative life span at low (physiological) oxygen is due to diminished ATM activity. At low oxygen, cells show a decreased ATM response to dysfunctional telomeres and genome-wide DSBs compared to 20% oxygen. As this effect could be reversed by NAC, the attenuated response of ATM to critically short telomeres and the resulting extended life span at low oxygen is likely due to ROS-induced formation of cysteine disulfide-bridges that crosslink ATM dimers into a form that is not activated by DSBs. These findings show how primary human cells detect shortened telomeres and reveal the molecular mechanism underlying the telomere tumor suppressor pathway.
    DOI:  https://doi.org/10.1101/2024.06.24.600514
  21. bioRxiv. 2024 Jun 28. pii: 2024.06.23.600305. [Epub ahead of print]
    Aging limits stemness and tumorigenesis in the lung by reprogramming iron homeostasis.
    Xueqian Zhuang, Qing Wang, Simon Joost, Alexander Ferrena, David T Humphreys, Zhuxuan Li, Melissa Blum, Klavdija Bastl, Selena Ding, Yuna Landais, Yingqian Zhan, Yang Zhao, Ronan Chaligne, Joo-Hyeon Lee, Sebastian E Carrasco, Umeshkumar K Bhanot, Richard P Koche, Matthew J Bott, Pekka Katajisto, Yadira M Soto-Feliciano, Thomas Pisanic, Tiffany Thomas, Deyou Zheng, Emily S Wong, Tuomas Tammela.
      Aging is associated with a decline in the number and fitness of adult stem cells 1-4 . Aging-associated loss of stemness is posited to suppress tumorigenesis 5,6 , but this hypothesis has not been tested in vivo . Here, using physiologically aged autochthonous genetically engineered mouse models and primary cells 7,8 , we demonstrate aging suppresses lung cancer initiation and progression by degrading stemness of the alveolar cell of origin. This phenotype is underpinned by aging-associated induction of the transcription factor NUPR1 and its downstream target lipocalin-2 in the cell of origin in mice and humans, leading to a functional iron insufficiency in the aged cells. Genetic inactivation of the NUPR1-lipocalin-2 axis or iron supplementation rescue stemness and promote tumorigenic potential of aged alveolar cells. Conversely, targeting the NUPR1- lipocalin-2 axis is detrimental to young alveolar cells via induction of ferroptosis. We find that aging-associated DNA hypomethylation at specific enhancer sites associates with elevated NUPR1 expression, which is recapitulated in young alveolar cells by inhibition of DNA methylation. We uncover that aging drives a functional iron insufficiency, which leads to loss of stemness and tumorigenesis, but promotes resistance to ferroptosis. These findings have significant implications for the therapeutic modulation of cellular iron homeostasis in regenerative medicine and in cancer prevention. Furthermore, our findings are consistent with a model whereby most human cancers initiate in young individuals, revealing a critical window for such cancer prevention efforts.
    DOI:  https://doi.org/10.1101/2024.06.23.600305
  22. Curr Cardiol Rep. 2024 Jul 11.
    Molecular Regulation of Cardiac Conduction System Development.
    Lucie Boulgakoff, Gaetano D'Amato, Lucile Miquerol.
       PURPOSE OF REVIEW: The cardiac conduction system, composed of pacemaker cells and conducting cardiomyocytes, orchestrates the propagation of electrical activity to synchronize heartbeats. The conduction system plays a crucial role in the development of cardiac arrhythmias. In the embryo, the cells of the conduction system derive from the same cardiac progenitors as the contractile cardiomyocytes and and the key question is how this choice is made during development.
    RECENT FINDINGS: This review focuses on recent advances in developmental biology using the mouse as animal model to better understand the cellular origin and molecular regulations that control morphogenesis of the cardiac conduction system, including the latest findings in single-cell transcriptomics. The conducting cell fate is acquired during development starting with pacemaking activity and last with the formation of a complex fast-conducting network. Cardiac conduction system morphogenesis is controlled by complex transcriptional and gene regulatory networks that differ in the components of the cardiac conduction system.
    Keywords:  Cardiac Conduction; Embryonic Development; Molecular Control; Morphogenesis; Transcription Factors
    DOI:  https://doi.org/10.1007/s11886-024-02094-7
  23. Proc Natl Acad Sci U S A. 2024 Jul 16. 121(29): e2404551121
    Mechanical stress during confined migration causes aberrant mitoses and c-MYC amplification.
    Giulia Bastianello, Gururaj Rao Kidiyoor, Conor Lowndes, Qingsen Li, Raoul Bonnal, Jeffrey Godwin, Fabio Iannelli, Lorenzo Drufuca, Ramona Bason, Fabrizio Orsenigo, Dario Parazzoli, Mattia Pavani, Valeria Cancila, Stefano Piccolo, Giorgio Scita, Andrea Ciliberto, Claudio Tripodo, Massimiliano Pagani, Marco Foiani.
      Confined cell migration hampers genome integrity and activates the ATR and ATM mechano-transduction pathways. We investigated whether the mechanical stress generated by metastatic interstitial migration contributes to the enhanced chromosomal instability observed in metastatic tumor cells. We employed live cell imaging, micro-fluidic approaches, and scRNA-seq to follow the fate of tumor cells experiencing confined migration. We found that, despite functional ATR, ATM, and spindle assembly checkpoint (SAC) pathways, tumor cells dividing across constriction frequently exhibited altered spindle pole organization, chromosome mis-segregations, micronuclei formation, chromosome fragility, high gene copy number variation, and transcriptional de-regulation and up-regulation of c-MYC oncogenic transcriptional signature via c-MYC locus amplifications. In vivo tumor settings showed that malignant cells populating metastatic foci or infiltrating the interstitial stroma gave rise to cells expressing high levels of c-MYC. Altogether, our data suggest that mechanical stress during metastatic migration contributes to override the checkpoint controls and boosts genotoxic and oncogenic events. Our findings may explain why cancer aneuploidy often does not correlate with mutations in SAC genes and why c-MYC amplification is strongly linked to metastatic tumors.
    Keywords:  aneuploidy; chromosome segregation; mechanical stress; mitosis; mitotic spindle
    DOI:  https://doi.org/10.1073/pnas.2404551121
  24. Dev Biol. 2024 Jul 04. pii: S0012-1606(24)00173-8. [Epub ahead of print]515 30-45
    Overview of chromatin regulatory processes during surface ectodermal development and homeostasis.
    Meagan C Branch, Madison Weber, Meng-Yen Li, Pooja Flora, Elena Ezhkova.
      The ectoderm is the outermost of the three germ layers of the early embryo that arise during gastrulation. Once the germ layers are established, the complex interplay of cellular proliferation, differentiation, and migration results in organogenesis. The ectoderm is the progenitor of both the surface ectoderm and the neural ectoderm. Notably, the surface ectoderm develops into the epidermis and its associated appendages, nails, external exocrine glands, olfactory epithelium, and the anterior pituitary. Specification, development, and homeostasis of these organs demand a tightly orchestrated gene expression program that is often dictated by epigenetic regulation. In this review, we discuss the recent discoveries that have highlighted the importance of chromatin regulatory mechanisms mediated by transcription factors, histone and DNA modifications that aid in the development of surface ectodermal organs and maintain their homeostasis post-development.
    Keywords:  Chromatin regulation; Development; Epigenetics; Homeostasis; Surface ectoderm
    DOI:  https://doi.org/10.1016/j.ydbio.2024.07.001
  25. EMBO J. 2024 Jul 12.
    PUFFFIN: an ultra-bright, customisable, single-plasmid system for labelling cell neighbourhoods.
    Tamina Lebek, Mattias Malaguti, Giulia Lm Boezio, Lida Zoupi, James Briscoe, Alistair Elfick, Sally Lowell.
      Cell communication coordinates developmental processes, maintains homeostasis, and contributes to disease. Therefore, understanding the relationship between cells in a shared environment is crucial. Here we introduce Positive Ultra-bright Fluorescent Fusion For Identifying Neighbours (PUFFFIN), a cell neighbour-labelling system based upon secretion and uptake of positively supercharged fluorescent protein s36GFP. We fused s36GFP to mNeonGreen or to a HaloTag, facilitating ultra-bright, sensitive, colour-of-choice labelling. Secretor cells transfer PUFFFIN to neighbours while retaining nuclear mCherry, making identification, isolation, and investigation of live neighbours straightforward. PUFFFIN can be delivered to cells, tissues, or embryos on a customisable single-plasmid construct composed of interchangeable components with the option to incorporate any transgene. This versatility enables the manipulation of cell properties, while simultaneously labelling surrounding cells, in cell culture or in vivo. We use PUFFFIN to ask whether pluripotent cells adjust the pace of differentiation to synchronise with their neighbours during exit from naïve pluripotency. PUFFFIN offers a simple, sensitive, customisable approach to profile non-cell-autonomous responses to natural or induced changes in cell identity or behaviour.
    Keywords:  HaloTag; Neighbour Labelling; PUFFFIN; Pluripotency; Synthetic Signalling
    DOI:  https://doi.org/10.1038/s44318-024-00154-w
  26. Nat Cell Biol. 2024 Jul 12.
    Pressure sensing of lysosomes enables control of TFEB responses in macrophages.
    Ruiqi Cai, Ori Scott, Gang Ye, Trieu Le, Ekambir Saran, Whijin Kwon, Subothan Inpanathan, Blayne A Sayed, Roberto J Botelho, Amra Saric, Stefan Uderhardt, Spencer A Freeman.
      Polymers are endocytosed and hydrolysed by lysosomal enzymes to generate transportable solutes. While the transport of diverse organic solutes across the plasma membrane is well studied, their necessary ongoing efflux from the endocytic fluid into the cytosol is poorly appreciated by comparison. Myeloid cells that employ specialized types of endocytosis, that is, phagocytosis and macropinocytosis, are highly dependent on such transport pathways to prevent the build-up of hydrostatic pressure that otherwise offsets lysosomal dynamics including vesiculation, tubulation and fission. Without undergoing rupture, we found that lysosomes incurring this pressure owing to defects in solute efflux, are unable to retain luminal Na+, which collapses its gradient with the cytosol. This cation 'leak' is mediated by pressure-sensitive channels resident to lysosomes and leads to the inhibition of mTORC1, which is normally activated by Na+-coupled amino acid transporters driven by the Na+ gradient. As a consequence, the transcription factors TFEB/TFE3 are made active in macrophages with distended lysosomes. In addition to their role in lysosomal biogenesis, TFEB/TFE3 activation causes the release of MCP-1/CCL2. In catabolically stressed tissues, defects in efflux of solutes from the endocytic pathway leads to increased monocyte recruitment. Here we propose that macrophages respond to a pressure-sensing pathway on lysosomes to orchestrate lysosomal biogenesis as well as myeloid cell recruitment.
    DOI:  https://doi.org/10.1038/s41556-024-01459-y
  27. Elife. 2024 Jul 08. pii: RP92764. [Epub ahead of print]13
    Human promoter directionality is determined by transcriptional initiation and the opposing activities of INTS11 and CDK9.
    Joshua D Eaton, Jessica Board, Lee Davidson, Chris Estell, Steven West.
      RNA polymerase II (RNAPII) transcription initiates bidirectionally at many human protein-coding genes. Sense transcription usually dominates and leads to messenger RNA production, whereas antisense transcription rapidly terminates. The basis for this directionality is not fully understood. Here, we show that sense transcriptional initiation is more efficient than in the antisense direction, which establishes initial promoter directionality. After transcription begins, the opposing functions of the endonucleolytic subunit of Integrator, INTS11, and cyclin-dependent kinase 9 (CDK9) maintain directionality. Specifically, INTS11 terminates antisense transcription, whereas sense transcription is protected from INTS11-dependent attenuation by CDK9 activity. Strikingly, INTS11 attenuates transcription in both directions upon CDK9 inhibition, and the engineered recruitment of CDK9 desensitises transcription to INTS11. Therefore, the preferential initiation of sense transcription and the opposing activities of CDK9 and INTS11 explain mammalian promoter directionality.
    Keywords:  CDK9; INTS11; Integrator; RNA polymerase II; chromosomes; cleavage and polyadenylation; gene expression; human; promoter; transcription; transcription initiation; transcription termination
    DOI:  https://doi.org/10.7554/eLife.92764
  28. Nat Cell Biol. 2024 Jul 08.
    Mapping the chromatin accessibility landscape of zebrafish embryogenesis at single-cell resolution by SPATAC-seq.
    Keyong Sun, Xin Liu, Runda Xu, Chang Liu, Anming Meng, Xun Lan.
      Currently, the dynamic accessible elements that determine regulatory programs responsible for the unique identity and function of each cell type during zebrafish embryogenesis lack detailed study. Here we present SPATAC-seq: a split-pool ligation-based assay for transposase-accessible chromatin using sequencing. Using SPATAC-seq, we profiled chromatin accessibility in more than 800,000 individual nuclei across 20 developmental stages spanning the sphere stage to the early larval protruding mouth stage. Using this chromatin accessibility map, we identified 604 cell states and inferred their developmental relationships. We also identified 959,040 candidate cis-regulatory elements (cCREs) and delineated development-specific cCREs, as well as transcription factors defining diverse cell identities. Importantly, enhancer reporter assays confirmed that the majority of tested cCREs exhibited robust enhanced green fluorescent protein expression in restricted cell types or tissues. Finally, we explored gene regulatory programs that drive pigment and notochord cell differentiation. Our work provides a valuable open resource for exploring driver regulators of cell fate decisions in zebrafish embryogenesis.
    DOI:  https://doi.org/10.1038/s41556-024-01449-0
  29. Trends Genet. 2024 Jul 05. pii: S0168-9525(24)00150-1. [Epub ahead of print]
    On the evolutionary developmental biology of the cell.
    Leslie S Babonis.
      Organisms are complex assemblages of cells, cells that produce light, shoot harpoons, and secrete glue. Therefore, identifying the mechanisms that generate novelty at the level of the individual cell is essential for understanding how multicellular life evolves. For decades, the field of evolutionary developmental biology (Evo-Devo) has been developing a framework for connecting genetic variation that arises during embryonic development to the emergence of diverse adult forms. With increasing access to new single cell 'omics technologies and an array of techniques for manipulating gene expression, we can now extend these inquiries inward to the level of the individual cell. In this opinion, I argue that applying an Evo-Devo framework to single cells makes it possible to explore the natural history of cells, where this was once only possible at the organismal level.
    Keywords:  atavism; cell type biodiversity; heterochrony; homeosis; novelty; plasticity
    DOI:  https://doi.org/10.1016/j.tig.2024.06.003
  30. Nat Struct Mol Biol. 2024 Jul 11.
    A roadmap for ribosome assembly in human mitochondria.
    Elena Lavdovskaia, Elisa Hanitsch, Andreas Linden, Martin Pašen, Venkatapathi Challa, Yehor Horokhovskyi, Hanna P Roetschke, Franziska Nadler, Luisa Welp, Emely Steube, Marleen Heinrichs, Mandy Mong-Quyen Mai, Henning Urlaub, Juliane Liepe, Ricarda Richter-Dennerlein.
      Mitochondria contain dedicated ribosomes (mitoribosomes), which synthesize the mitochondrial-encoded core components of the oxidative phosphorylation complexes. The RNA and protein components of mitoribosomes are encoded on two different genomes (mitochondrial and nuclear) and are assembled into functional complexes with the help of dedicated factors inside the organelle. Defects in mitoribosome biogenesis are associated with severe human diseases, yet the molecular pathway of mitoribosome assembly remains poorly understood. Here, we applied a multidisciplinary approach combining biochemical isolation and analysis of native mitoribosomal assembly complexes with quantitative mass spectrometry and mathematical modeling to reconstitute the entire assembly pathway of the human mitoribosome. We show that, in contrast to its bacterial and cytosolic counterparts, human mitoribosome biogenesis involves the formation of ribosomal protein-only modules, which then assemble on the appropriate ribosomal RNA moiety in a coordinated fashion. The presence of excess protein-only modules primed for assembly rationalizes how mitochondria cope with the challenge of forming a protein-rich ribonucleoprotein complex of dual genetic origin. This study provides a comprehensive roadmap of mitoribosome biogenesis, from very early to late maturation steps, and highlights the evolutionary divergence from its bacterial ancestor.
    DOI:  https://doi.org/10.1038/s41594-024-01356-w
  31. FEBS Lett. 2024 Jul 11.
    Keep calm and reboot - how cells restart transcription after DNA damage and DNA repair.
    Lise-Marie Donnio, Giuseppina Giglia-Mari.
      The effects of genotoxic agents on DNA and the processes involved in their removal have been thoroughly studied; however, very little is known about the mechanisms governing the reinstatement of cellular activities after DNA repair, despite restoration of the damage-induced block of transcription being essential for cell survival. In addition to impeding transcription, DNA lesions have the potential to disrupt the precise positioning of chromatin domains within the nucleus and alter the meticulously organized architecture of the nucleolus. Alongside the necessity of resuming transcription mediated by RNA polymerase 1 and 2 transcription, it is crucial to restore the structure of the nucleolus to facilitate optimal ribosome biogenesis and ensure efficient and error-free translation. Here, we examine the current understanding of how transcriptional activity from RNA polymerase 2 is reinstated following DNA repair completion and explore the mechanisms involved in reassembling the nucleolus to safeguard the correct progression of cellular functions. Given the lack of information on this vital function, this Review seeks to inspire researchers to explore deeper into this specific subject and offers essential suggestions on how to investigate this complex and nearly unexplored process further.
    Keywords:  DNA repair; RNA polymerase 1; RNA polymerase 2; UV lesions; chromatin; homeostasis; nucleolus
    DOI:  https://doi.org/10.1002/1873-3468.14964
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