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Autographa californica multiple nucleopolyhedrovirus (AcMNPV) orf106 ( ac106 ) is highly conserved in baculoviruses. Previous studies have shown that ac106 is required for the production of infectious budded virions (BVs). However, the functional role of ac106 in virion morphogenesis remains unknown. In this report, an ac106 knockout virus and an ac106 repair virus were constructed. The effect of ac106 deletion on virion morphogenesis was investigated, and the expression and subcellular localization of the Ac106 protein were characterized. Our data indicated that ac106 is required for the nuclear egress of nucleocapsids and intranuclear microvesicle formation, as well as subsequent BV and occlusion-derived virion (ODV) production and the embedding of ODVs into polyhedra. Ac106 is a baculovirus late protein that is concentrated in discrete foci of virus-induced membrane structures in the intranuclear ring zone of virus-infected cells. Further studies on the relationship between Ac106 and four other proteins that are also required for intranuclear microvesicle formation, Ac75, Ac76, Ac93, and P48 (Ac103), revealed that Ac106 is associated with Ac75, Ac76, Ac93, P48, and itself. Ac106 is required for Ac75, Ac93, and P48 accumulation in foci of virus-induced intranuclear membrane structures and the intranuclear transport of Ac76. Analysis of the subcellular localization of ODV integral envelope proteins upon deletion of the genes required for intranuclear microvesicle formation indicated that intranuclear microvesicle formation may be essential for ODV integral envelope protein transport into the nucleus, supporting the hypothesis that intranuclear microvesicles originate from the nuclear membrane.IMPORTANCEBaculovirus occlusion-derived virions (ODVs) are known to acquire their envelopes from virus-induced intranuclear microvesicles within the nucleoplasm, and this strategy of intranuclear envelopment of nucleocapsids to form virions is unique among viruses. However, the mechanism of ODV morphogenesis, particularly intranuclear microvesicle formation, remains unclear. In this study, we identified ac106 as the fifth gene, in addition to ac75 , ac76 , ac93 , and p48 ( ac103 ), which are required for intranuclear microvesicle formation. Further studies on the relationship between ac106 and the other four genes, as well as the effect of ac106 or ac75 deletion on the localization of ODV integral envelope proteins, indicated that intranuclear microvesicle formation may be essential for the transport of ODV integral envelope proteins into the nucleus, which strongly supports the hypothesis that intranuclear microvesicles originate from the nuclear membrane. These findings greatly enhance our understanding of the molecular mechanism of baculovirus ODV morphogenesis., Competing Interests: The authors declare no conflict of interest.

Microautophagy degrades cargos in the vacuole by direct engulfment of the vacuolar membrane. Micronucleophagy selectively degrades a portion of the nucleus in budding yeast. The vacuole contacts the nucleus via the nucleus-vacuole junction (NVJ), and in micronucleophagy a portion of the nucleus containing nucleolar proteins is made to protrude into the vacuole at the NVJ, followed by abscission and degradation. Microautophagy and micronucleophagy are induced by inactivation of target of rapamycin complex 1 (TORC1) protein kinase after nutrient starvation. Here, we show that the VAMP-associated proteins (VAPs) Scs2 and its paralog Scs22 are required for NVJ integrity and micronucleophagic degradation of nucleolar proteins. On the other hand, nucleolar dynamics prerequisite for micronucleophagy were not impaired in VAP mutant cells. Finally, yeast VAPs were critical for viability during prolonged nutrient starvation. This study sheds light on the emerging role of VAP in adaptation in responses to nutrient starvation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Gastric cancer (GC) is a common digestive system cancer, characterized by a significant mortality rate. Mitochondria is an indispensable organelle in eukaryotic cells. It was previously revealed that a series of nucleus-encoded mitochondrial genes (NMG) mutations and dysfunctions potentially contribute to the initiation and progression of GC. However, the correlation between NMG mutations and survival outcomes for GC patients is still unclear. In this study, NMG expression profile and clinical information in GC samples were collected from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases. Through consistent clustering and functional enrichment analysis, we have identified three NMG clusters and three gene clusters that are associated with patterns of immune cell infiltration. Prognostic genes were identified through Univariate Cox regression analysis. The principal component analysis was conducted to set up a scoring system. Subsequently, the Single‑cell RNA sequencing (scRNA-seq) data of GC patients and cancer cell drug sensitivity data were retrieved from the GEO database. Patients with high NMG scores exhibited increased microsatellite instability status and a heightened tumor mutation rate compared to those with low NMG scores. Survival analysis revealed that GC samples with high NMG scores could achieve a better prognosis. Additionally, These patients were observed to be more responsive to immunotherapy. Moreover, we delved into prognostic genes at the level of single cells, revealing that MRPL4 and MRPL37 exhibit high expression in epithelial cells, while TPM1 demonstrates high expression in tissue stem cells. Utilizing cancer cell drug sensitivity data from the Drug Sensitivity in Cancer (GDSC) database, we noted a heightened sensitivity to chemotherapy in the high NMG group. Furthermore, we discovered a significant enrichment of cuproptosis-related genes in clusters with high NMG scores. Consequently, employing the scoring system could facilitate the prediction of GC patients' sensitivity to cuproptosis-induced therapy. Our study confirmed the potency of this scoring system as a therapeutic response biomarker for gastric cancer, potentially informing clinical treatment strategies., Competing Interests: Declarations Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Cell migration through small constrictions during cancer metastasis requires significant deformation of the nucleus, with associated mechanical stress on the nuclear lamina and chromatin. However, how mechanical deformation impacts various subnuclear structures, including protein and nucleic acid-rich biomolecular condensates, is largely unknown. Here, we find that cell migration through confined spaces gives rise to mechanical deformations of the chromatin network, which cause embedded nuclear condensates, including nucleoli and nuclear speckles, to deform and coalesce. Chromatin deformations exhibit differential behavior in the advancing vs. trailing region of the nucleus, with the trailing half being more permissive for de novo condensate formation. We show that this results from increased chromatin heterogeneity, which gives rise to a shift in the binodal phase boundary. Taken together, our findings show how chromatin deformation impacts condensate assembly and properties, which can potentially contribute to cellular mechanosensing., Competing Interests: Competing interests C.P.B. is a founder of and consultant for Nereid Therapeutics. The remaining authors declare no competing interests., (© 2024. The Author(s).)

A nucleus is crucial for both sonodynamic therapy (SDT) and antitumor immunity. However, how to burst ROS generation in situ , accurately damage a nucleus, and meanwhile activate a cGAS-STING pathway-induced innate immune response are still a great challenge. Here, we present TBzT-CPi, a small molecule with a D-A-π-A1 structure that simultaneously amplifies nucleus-targeted SDT and cGAS-STING pathway-dependent immune stimulation. TBzT-CPi could accumulate in the nucleus upon ultrasound irradiation and generate ROS in situ , which damages DNA and simultaneously triggers immunogenic cell death (ICD). Stirringly, nucleus-targeting SDT not only efficiently induces apoptosis in tumor cells but also modifies the immunosuppressive tumor microenvironment by activating cytotoxic T lymphocytes, maturing dendritic cells, and secreting cytokines. These findings pave the way for developing nucleus-targeting sonosensitizers for sonodynamic immunotherapy of cancer.

Canonically PD-L1 functions as the inhibitory immune checkpoint on cell surface. Recent studies have observed PD-L1 expression in the nucleus of cancer cells. But the biological function of nuclear PD-L1 (nPD-L1) in tumor growth and antitumor immunity is unclear. Here we enforced nPD-L1 expression and established stable cells. nPD-L1 suppressed tumorigenesis and aggressiveness in vitro and in vivo. Compared with PD-L1 deletion, nPD-L1 expression repressed tumor growth and improved survival more markedly in immunocompetent mice. Phosphorylated AMPKα (p-AMPKα) facilitated nuclear PD-L1 compartmentalization and then cooperated with it to directly phosphorylate S146 of histone variant macroH2A1 (mH2A1) to epigenetically activate expression of genes of cellular senescence, JAK/STAT, and Hippo signaling pathways. Lipoic acid (LA) that induced nuclear PD-L1 translocation suppressed tumorigenesis and boosted antitumor immunity. Importantly, LA treatment synergized with PD-1 antibody and overcame immune checkpoint blockade (ICB) resistance, which likely resulted from nPD-L1-increased MHC-I expression and sensitivity of tumor cells to interferon-γ. These findings offer a conceptual advance for PD-L1 function and suggest LA as a promising therapeutic option for overcoming ICB resistance.

Loss of cytosolic actin filaments upon TORC2 inhibition triggers chromosome fragmentation in yeast, which results from altered base excision repair of Zeocin-induced lesions. To find the link between TORC2 kinase and this yeast chromosome shattering (YCS) we performed phosphoproteomics. YCS-relevant phospho-targets included plasma membrane-associated regulators of actin polymerization, such as Las17, the yeast Wiscott-Aldrich Syndrome protein. Induced degradation of Las17 was sufficient to trigger YCS in presence of Zeocin, bypassing TORC2 inhibition. In yeast, Las17 does not act directly at damage, but instead its loss, like TORC2 inhibition, raises nuclear actin levels. Nuclear actin, in complex with Arp4, forms an essential subunit of several nucleosome remodeler complexes, including INO80C, which facilitates DNA polymerase elongation. Here we show that the genetic ablation of INO80C activity leads to partial YCS resistance, suggesting that elevated levels of nuclear G-actin may stimulate INO80C to increase DNA polymerase processivity and convert single-strand lesions into double-strand breaks., Competing Interests: Competing interests The authors declare that no competing interests., (© 2024. The Author(s).)

Brain-derived neurotrophic factor (BDNF), a member of the neurotrophin family, is deeply involved in the development and higher function of the nervous system, including learning and memory. By contrast, a reduction in BDNF levels is associated with various neurological disorders such as dementia and depression. Therefore, the inducers of Bdnf expression might be valuable in ameliorating or protecting against a decline in brain functions. We previously reported that, through high-throughput screening to identify inducers of Bdnf expression in Bdnf-luciferase transgenic mice, several herbal extracts induced Bdnf expression in cortical neurons. In the present study, we found that Panax notoginseng root extract (PNRE) potently induced Bdnf expression in primary cultured cortical neurons primarily via the L-type voltage-dependent Ca 2+ channel (L-VDCC) and calcineurin. PNRE promoted nuclear translocation of cAMP-responsive element-binding protein-regulated transcription coactivator 1 (CRTC1). These findings suggest that PNRE activates the L-VDCC/calcineurin/CRTC1 axis, which is the primary signaling pathway involved in the neuronal activity-dependent expression of Bdnf. Moreover, we demonstrated that PNRE increased the dendritic complexity of cortical neurons in vitro. Thus, by upregulating Bdnf expression, PNRE is a potential candidate for improving cognitive impairment seen in several kinds of dementia.

Background: Osteoarthritis (OA) is a progressive condition affecting the joints that lacking effective therapy. However, the underlying molecular mechanism has not been fully clarified., Methods: A model of OA was established in Sprague-Dawley (SD) rats through intra-articularly injected with monoiodoacetate (MIA). Western blot analysis was used to identify the levels of UBE2I and hnRNPA2B1 in articular cartilage. Overexpression and siRNA vectors for UBE2I were constructed and transfected into rat chondrocytes. CCK-8, TUNEL and transwell assay were utilized to assess the cell viability, apoptosis and migration ability. Western blot analysis was used to determine the levels of chondrogenic-specific genes including SOX9, COL2A1, Aggrecan, and PRG4. Then, molecular interactions were confirmed by immunoprecipitation., Results: We observed significant upregulation of UBE2I and hnRNPA2B1 expression in articular cartilage samples of OA. The Pearson correlation analysis revealed positive correlation between UBE2I and hnRNPA2B1 levels. Functional experiments showed that increased UBE2I expression significantly suppressed cell growth, migration, and reduced the expression of chondrogenic-specific genes, while decreasing UBE2I levels had the opposite effects. Molecular interactions between UBE2I and hnRNPA2B1were determined via co-localization and immunoprecipitation. SUMO1 and SUMO3 proteins were enriched by immunoprecipitation using hnRNPA2B1 antibodies. Rescue experiments were performed using SUMOylation inhibitor (2-D08) and SUMOylation activator (N106). Overexpression of UBE2I increased the expression of hnRNPA2B1 in the cytoplasm and decreased the level in the nucleus, which was reversed by the treatment of 2-D08. Conversely, UBE2I knockdown and N106 treatment had the opposite effect., Conclusions: UBE2I modulated the nuclear translocation of hnRNPA2B1 by promoting SUMOylation in OA., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Pyruvate kinase M2 (PKM2) is a key metabolic enzyme. Yet, its role in cerebral ischemia injury remains unclear. In this study we demonstrated that PKM2 expression was increased in the microglia after mouse cerebral ischemia-reperfusion (I/R) injury. We found that microglial polarization-mediated pro-inflammatory effect was mediated by PKM2 after cerebral I/R. Mechanistically, our results revealed that nuclear PKM2 mediated ischemia-induced microglial polarization through association with acetyl-H3K9. Hif-1α mediated the effect of nuclear PKM2/histone H3 on microglial polarization. PKM2-dependent Histone H3/Hif-1α modifications contributed the expression of CCL2 and induced up-regulation of microglial polarization in peri-infarct, resulting in neuroinflammation. Inhibiting nuclear translocation of microglial PKM2 reduced ischemia-induced pro-inflammation and promoted neuronal survival. Together, this study identifies nucleus PKM2 as a crucial mediator for regulating ischemia-induced neuroinflammation, suggesting PKM2 as a potential therapeutic target in ischemic stroke., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Photosynthesis requires chloroplasts, in which most proteins are nucleus-encoded and produced via cytoplasmic translation. The translation initiation factor eIF5B gates the transition from initiation (I) to elongation (E), and the Kozak motif is associated with translation efficiency, but their relationship is previously unknown. Here, with ribosome profiling, we determined the genome-wide I-E transition efficiencies. We discovered that the most prevalent Kozak motif is associated with high I-E transition efficiency in Arabidopsis, rice, and wheat, thus implicating the potential of the Kozak motif in facilitating the I-E transition. Indeed, the effects of Kozak motifs in promoting translation depend on HOT3/eIF5B1 in Arabidopsis. HOT3 preferentially promotes the translation of photosynthesis-associated nuclear genes in a Kozak motif-dependent manner, which explains the chloroplast defects and reduced photosynthesis activity of hot3 mutants. Our study linked the Kozak motif to eIF5B-mediated I-E transition during translation and uncovered the function of HOT3 in the cytoplasmic translational control of chloroplast biogenesis and photosynthesis., Competing Interests: Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Subcellular compartmentalization of metabolic enzymes establishes a unique metabolic environment that elicits specific cellular functions. Indeed, the nuclear translocation of certain metabolic enzymes is required for epigenetic regulation and gene expression control. Here, we show that the nuclear localization of the mitochondrial enzyme methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) ensures mitosis progression. Nuclear MTHFD2 interacts with proteins involved in mitosis regulation and centromere stability, including the methyltransferases KMT5A and DNMT3B. Loss of MTHFD2 induces severe methylation defects and impedes correct mitosis completion. MTHFD2 deficient cells display chromosome congression and segregation defects and accumulate chromosomal aberrations. Blocking the catalytic nuclear function of MTHFD2 recapitulates the phenotype observed in MTHFD2 deficient cells, whereas restricting MTHFD2 to the nucleus is sufficient to ensure correct mitotic progression. Our discovery uncovers a nuclear role for MTHFD2, supporting the notion that translocation of metabolic enzymes to the nucleus is required to meet precise chromatin needs., Competing Interests: Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Nuclear metabolism and DNA damage response are intertwined processes, but the precise molecular links remain elusive. Here, we explore this crosstalk using triple-negative breast cancer (TNBC) as a model, a subtype often prone to DNA damage accumulation. We show that the de novo purine synthesis enzyme IMPDH2 is enriched on chromatin in TNBC compared to other subtypes. IMPDH2 chromatin localization is DNA damage dependent, and IMPDH2 repression leads to DNA damage accumulation. On chromatin, IMPDH2 interacts with and modulates PARP1 activity by controlling the nuclear availability of NAD + to fine-tune the DNA damage response. However, when IMPDH2 is restricted to the nucleus, it depletes nuclear NAD + , leading to PARP1 cleavage and cell death. Our study identifies a non-canonical nuclear role for IMPDH2, acting as a convergence point of nuclear metabolism and DNA damage response., Competing Interests: Competing interests The author declares no competing interests., (© 2024. The Author(s).)

Background: Circular RNAs (circRNAs) are differentially expressed in various cardiovascular diseases, including myocardial infarction (MI) injury. However, their functional role in necroptosis-induced loss of cardiomyocytes remains unclear. We identified a cardiac necroptosis-associated circRNA transcribed from the Cacna1c gene (circCacna1c) to investigate the involvement of circRNAs in cardiomyocyte necroptosis., Methods: To investigate the role of circCacna1c during oxidative stress, H9c2 cells and neonatal rat cardiomyocytes were treated with hydrogen peroxide (H 2 O 2 ) to induce reactive oxygen species (ROS)-induced cardiomyocyte death. The N 6 -methyladenosine (m 6 A) modification level of circCacna1c was determined by methylated RNA immunoprecipitation quantitative polymerase chain reaction (MeRIP-qPCR) analysis. Additionally, an RNA pull-down assay was performed to identify interacting proteins of circCacna1c in cardiomyocytes, and the regulatory role of circCacna1c in target protein expression was tested using a western blotting assay. Furthermore, the MI mouse model was constructed to analyze the effect of circCacna1c on heart function and cardiomyocyte necroptosis., Results: The expression of circCacna1c was found to be reduced in cardiomyocytes exposed to oxidative stress and in mouse hearts injured by MI. Overexpression of circCacna1c inhibited necroptosis of cardiomyocytes induced by hydrogen peroxide and MI injury, resulting in a significant reduction in myocardial infarction size and improved cardiac function. Mechanistically, circCacna1c directly interacts with heterogeneous nuclear ribonucleoprotein F (Hnrnpf) in the cytoplasm, preventing its nuclear translocation and leading to reduced Hnrnpf levels within the nucleus. This subsequently suppresses Hnrnpf-dependent receptor-interacting protein kinase 1 (RIPK1) expression. Furthermore, fat mass and obesity-associated protein (FTO) mediates demethylation of m 6 A modification on circCacna1c during necrosis and facilitates degradation of circCacna1c., Conclusion: Our study demonstrates that circCacna1c can improve cardiac function following MI-induced heart injury by inhibiting the Hnrnpf/RIPK1-mediated cardiomyocyte necroptosis. Therefore, the FTO/circCacna1c/Hnrnpf/RIPK1 axis holds great potential as an effective target for attenuating cardiac injury caused by necroptosis in ischemic heart disease., Competing Interests: Declarations Ethics approval and consent to participate All animal protocols in this study were performed in accordance with the Basel Declaration and the approval of the Ethics Committee of Medical College of the Qingdao University (Number: QDU-AEC-2022329, Date: July 4th, 2022). Consent for publication Not applicable. Competing interests The authors have declared that no competing interest exists., (© 2024. The Author(s).)

Over the course of evolution, land plant mitochondrial genomes have lost many transfer RNA (tRNA) genes and the import of nucleus-encoded tRNAs is essential for mitochondrial protein synthesis. By contrast, plastidial genomes of photosynthetic land plants generally possess a complete set of tRNA genes and the existence of plastidial tRNA import remains a long-standing question. The early vascular plants of the Selaginella genus show an extensive loss of plastidial tRNA genes while retaining photosynthetic capacity, and represent an ideal model for answering this question. Using purification, northern blot hybridization, and high-throughput tRNA sequencing, a global analysis of total and plastidial tRNA populations was undertaken in Selaginella kraussiana. We confirmed the expression of all plastidial tRNA genes and, conversely, observed that nucleus-encoded tRNAs corresponding to these plastidial tRNAs were generally excluded from the chloroplasts. We then demonstrated a selective and differential plastidial import of around forty nucleus-encoded tRNA species, likely compensating for the insufficient coding capacity of plastidial-encoded tRNAs. In-depth analysis revealed differential import of tRNA isodecoders, leading to the identification of specific situations. This includes the expression and import of nucleus-encoded tRNAs expressed from plastidial or bacterial-like genes inserted into the nuclear genome. Overall, our results confirm the existence of molecular processes that enable tRNAs to be selectively imported not only into mitochondria, as previously described, but also into chloroplasts, when necessary., Competing Interests: Competing interests statement:The authors declare no competing interest.

Pseudo-Response Regulator (PRR) proteins constitute a fundamental set of circadian clock components in plants. PRRs have an amino acid sequence stretch with similarity to the receiver (REC) domain of response regulators (RRs) in the Multi-Step Phosphorelay (MSP). However, it has never been elucidated whether PRRs interact with Histidine-containing Phosphotransfer (HPt) proteins, which transfer a phosphate to RRs. Here, we studied whether PRRs interact with HPts in the moss Physcomitrium patens by the Yeast Two-Hybrid system and Bimolecular Fluorescence Complementation. P. patens PRR1/2/3 interacted with HPt1/2 in the nucleus, but not with HPt3, suggesting that P. patens PRRs function as authentic RRs. We discuss these results in relation to the evolution and diversity of the plant circadian clocks., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Setsuyuki Aoki reports financial support was provided by Japan Society for the Promotion of Science. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Plant pathogens secrete nuclear effectors into the host nuclei to modulate the host immune system. Although several nuclear effectors of fungal pathogens have been recently reported, the molecular mechanism of NLS-associated transport vehicles of nuclear effectors and the roles of NLS in transcriptional reprogramming of host immunity genes remain enigmatic. We previously reported the MoHTR1, a nuclear effector of the rice blast fungus, Magnaporthe oryzae. MoHTR1 is translocated to rice nuclei but not in fungal nuclei. Here, we identify the core NLS (RxKK) responsible for MoHTR1's nuclear localization. MoHTR1 is translocated in the host nucleus through interaction with rice importin α. MoHTR1 NLS empowers it to translocate the cytoplasmic effectors of M. oryzae into rice nuclei. Furthermore, other nuclear effector candidates of the blast pathogen and rice proteins which have RxKK also exhibit nuclear localization, highlighting the crucial role of RxKK in this process. We also unveil the importance of SUMOylation in the stability of MoHTR1 and translocation of MoHTR1 to host nuclei. Moreover, MoHTR1 NLS is essential for the pathogenicity of M. oryzae by reprogramming immunity-associated genes in the host. Our findings provide insights into the significance of plant-specific NLS on fungal nuclear effectors and its role in plant-pathogen interactions., Competing Interests: Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Background: Fibroepithelial lesions (FELs) of the breast encompass a broad spectrum of lesions, ranging from commonly encountered fibroadenomas (FAs) to rare phyllodes tumors (PTs). Accurately diagnosing and grading these lesions is crucial for making management decisions, but it can be challenging due to their overlapping features and the subjective nature of histological assessment. Here, we evaluated the role of digital nuclear morphometric analysis in FEL diagnosis and prognosis., Methods: A digital nuclear morphometric analysis was conducted on 241 PTs and 59 FAs. Immunohistochemical staining for cytokeratin and Leukocyte common antigen (LCA) was used to exclude non-stromal components, and nuclear area, perimeters, calipers, circularity, and eccentricity in the stromal cells were quantified with QuPath software. The correlations of these features with FEL diagnosis and prognosis was assessed., Results: All nuclear features, including area, perimeter, circularity, maximum caliper, minimum caliper and eccentricity, showed significant differences between FAs and benign PTs (p ≤ 0.002). Only nuclear area, perimeter, minimum caliper and eccentricity correlated significantly with PT grading (p ≤ 0.022). For differentiation of FAs from benign PTs, the model integrating all differential nuclear features demonstrated a specificity of 90% and sensitivity of 70%. For PT grading, the nuclear morphometric score showed a specificity of 78% and sensitivity of 96% for distinguishing benign/borderline from malignant PTs. In addition, a relationship of nuclear circularity was found with PT recurrence. The Kaplan-meier analysis, using the best cutoff determined by ROC curve, showed shorter event free survival in benign PTs with high circularity (chi-square = 4.650, p = 0.031)., Conclusions: Our data suggested the digital nuclear morphometric analysis could have potentials to objectively differentiate different FELs and predict PT outcome. These findings could provide the evidence-based data to support the development of deep-learning based algorithm on nuclear morphometrics in FEL diagnosis., Competing Interests: Declarations Ethical approval The study was approved by Joint Chinese University of Hong Kong—New Territories East Cluster clinical research ethics committee. Tissue from patients was acquired with informed consent following the local institutional review and the Declaration of Helsinki. Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

A subset of oropharyngeal squamous cell carcinoma (OPSCC) is caused by the high-risk human papilloma virus (HPV), which expresses p16 INK4a immunoreactivity. Dual-color immunofluorescence (IF) analysis of TP53 binding protein-1 (53BP1) and a proliferative indicator, Ki-67, to elucidate genomic instability (GIN) in tumor tissues revealed that abnormal 53BP1 expression is closely associated with carcinogenesis in diverse organs. We have previously demonstrated that the number of 53BP1 nuclear foci (NF) in cervical cells increases with cancer progression. The distribution of 53BP1 NF was similar to that of punctate HPV signals, as determined by in situ hybridization, and the pattern of p16 INK4a overexpression. The present study aimed to confirm the type of 53BP1 expression using dual-color IF as an indicator of GIN in oropharyngeal squamous epithelial lesions, including HPV-dependent and -independent OPSCC. This study identified significant differences in the nuclear expression of 53BP1 between benign oropharyngeal epithelial lesions and OPSCC, and between HPV-dependent and HPV-independent OPSCC. We concluded that the incidence of abnormal 53BP1 expression in OPSCC is significantly associated with stage classification and overall survival. Therefore, double IF analysis of 53BP1 and Ki-67 expression may be a useful tool for estimating the malignant potential and prognosis of OPSCC., Competing Interests: Declarations Competing interests The authors declare no competing interests. Ethics committee approval and informed consent This study was performed retrospectively in accordance with the tenets of the Declaration of Helsinki. The Ethics Committee of Nagasaki university hospital approved the study (approval date: November 20, 2018; #18111921) and waived the need for informed consent. All methods were carried out in accordance with relevant guidelines and regulations. Patient profiles were anonymized by coding and collectively summarized with the obtained data as the final dataset. Patients were able to opt out of the study by following the instructions provided on the institute website., (© 2024. The Author(s).)

To ensure the integrity of our genetic code, a coordinated network of signalling and repair proteins, known as the DNA damage response (DDR), detects and repairs DNA insults, the most toxic being double-strand breaks (DSBs). Tudor interacting repair regulator (TIRR) is a key factor in DSB repair, acting through its interaction with p53 binding protein 1 (53BP1). TIRR is also an RNA binding protein, yet its role in RNA regulation during the DDR remains elusive. Here, we show that TIRR selectively binds to a subset of messenger RNAs (mRNAs) in response to DNA damage. Upon DNA damage, TIRR interacts with the nuclear export protein Exportin-1 through a nuclear export signal. Furthermore, TIRR plays a crucial role in the modulation of RNA processing bodies (PBs). TIRR itself and TIRR-bound RNA co-localize with PBs, and TIRR depletion results in nuclear RNA retention and impaired PB formation. We also suggest a potential link between TIRR-regulated RNA export and efficient DDR. This work reveals intricate involvement of TIRR in orchestrating mRNA nuclear export and storage within PBs, emphasizing its significance in the regulation of RNA-mediated DDR., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Poly (ADP-ribose) glycohydrolase (PARG) inhibitors are currently under clinical development for the treatment of DNA repair-deficient cancers; however, their precise mechanism of action is still unclear. Here, we report that PARG inhibition leads to excessive PARylated poly (ADP-ribose) polymerase 1 (PARP1) reducing the ability of PARP1 to properly localize to sites of DNA damage. Strikingly, the mis-localized PARP1 accumulates as aggregates throughout the nucleus. Abrogation of the catalytic activity of PARP1 prevents aggregate formation, indicating that PAR chains play a key role in this process. Finally, we find that PARP1 nuclear aggregates were highly persistent and were associated with cleaved cytoplasmic PARP1, ultimately leading to cell death. Overall, our data uncover an unexpected mechanism of PARG inhibitor cytotoxicity, which will shed light on the use of these drugs as anti-cancer therapeutics., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The differentiation and maturation of osteoblasts are essential for bone formation. Zn 2+ plays a crucial role in cell differentiation and is involved in osteogenic differentiation. The concentration and distribution of Zn 2+ in the nucleus and cytoplasm indicate the differentiation states of osteoblasts. However, there is an absence of a real-time method for monitoring the dynamic fluctuations of endogenous Zn 2+ within the nucleus. Here, a novel Zn 2+ fluorescent probe ( NTAD-N1 ) with nuclear membrane permeability was designed and developed, allowing for distribution throughout the entire cell, including the nucleus. The NTAD-N1 probe successfully showed the dynamic distribution and concentration changes of Zn 2+ in the nucleus and cytoplasm of preosteoblast MC3T3-E1 during the 21-day differentiation period. The results showed that free Zn 2+ increased significantly during differentiation of osteoblasts (2-21 days). Importantly, after 4 days of differentiation, osteoblasts are mainly distributed in the nucleus, which is confirmed by metallothionein expression. Subsequently, the level of free Zn 2+ in the cytoplasm remained at a high level, which promoted the increase in alkaline phosphatase activity and inhibited the activity of cis-aconitase in the tricarboxylic acid cycle, resulting in the accumulation of citric acid. This series of events promotes the formation of mineralized nodules. In the process of osteoblast differentiation, the detection time of Zn 2+ (≤7 days) is ahead of the late marker of alkaline phosphatase (14 days) and mineralized nodules (14-21 days). This indicates that Zn 2+ can be used as a biomarker and an intervention point for early differentiation of osteoblasts.

Pulmonary hypertension (PH) is a serious pulmonary vascular disease characterized by vascular remodeling. Circular RNAs (CircRNAs) play important roles in pulmonary hypertension, but the mechanism of PH is not fully understood, particularly the roles of circRNAs located in the nucleus. Circ-calmodulin 4 (circ-calm4) is expressed in both the cytoplasm and the nucleus of pulmonary arterial smooth muscle cells (PASMCs). This study aimed to investigate the role of endonuclear circ-calm4 in PH and elucidate its underlying signaling pathway in ferroptosis. Immunoblotting, quantitative real-time polymerase chain reaction (PCR), malondialdehyde (MDA) assay, immunofluorescence, iron assay, dot blot, and chromatin immunoprecipitation (ChIP) were performed to investigate the role of endonuclear circ-calm4 in PASMC ferroptosis. Increased endonuclear circ-calm4 facilitated ferroptosis in PASMCs under hypoxic conditions. We further identified the cartilage oligomeric matrix protein (COMP) as a downstream effector of circ-calm4 that contributed to the occurrence of hypoxia-induced ferroptosis in PASMCs. Importantly, we confirmed that endonuclear circ-calm4 formed circR-loops with the promoter region of the COMP gene and negatively regulated its expression. Inhibition of COMP restored the phenotypes related to ferroptosis under hypoxia stimulation combined with antisense oligonucleotide (ASO)-circ-calm4 treatment. We conclude that the circ-calm4/COMP axis contributed to hypoxia-induced ferroptosis in PASMCs and that circ-calm4 formed circR-loops with the COMP promoter in the nucleus and negatively regulated its expression. The circ-calm4/COMP axis may be useful for the design of therapeutic strategies for protecting cellular functionality against ferroptosis and pulmonary hypertension., Competing Interests: Declaration of competing interest All authors have read and approved the submission of the manuscript. The manuscript has not been published and is not being considered for publication elsewhere, in whole or in part, in any language., (Copyright © 2024 Elsevier B.V. All rights reserved.)

TGF-β stimulates CCN2 expression which in turn amplifies TGF-β signaling. This process promotes extracellular matrix production and accelerates the pathological progression of fibrotic diseases. Alternative splicing plays an important role in multiple disease development, while U2 small nuclear RNA auxiliary factor 2 (U2AF2) is an essential factor in the early steps of pre-mRNA splicing. However, the molecular mechanism underlying abnormal CCN2 expression upon TGF-β stimulation remains unclear. This study elucidates that SIRT4 acts as a master regulator for CCN2 expression in response to TGF-β by modulating U2AF2-mediated alternative splicing. Analyses of renal biopsy specimens from patients with CKD and mouse fibrotic kidney tissues revealed marked nuclear accumulation of SIRT4. The tubulointerstitial fibrosis was alleviated by global deletion or tubular epithelial cell (TEC)-specific knockout of Sirt4 , and aggravated by adeno-associated virus-mediated SIRT4 overexpression in TECs. Furthermore, SIRT4 was found to translocate from the mitochondria to the cytoplasm through the BAX/BAK pore under TGF-β stimulation. In the cytoplasm, TGF-β activated the ERK pathway and induced the phosphorylation of SIRT4 at Ser36, which further promoted its interaction with importin α1 and subsequent nuclear translocation. In the nucleus, SIRT4 was found to deacetylate U2AF2 at K413, facilitating the splicing of CCN2 pre-mRNA to promote CCN2 protein expression. Importantly, exosomes containing anti-SIRT4 antibodies were found to effectively mitigate the UUO-induced kidney fibrosis in mice. Collectively, these findings indicated that SIRT4 plays a role in kidney fibrosis by regulating CCN2 expression via the pre-mRNA splicing., Competing Interests: GY, JX, XY, XL, YL, LL, LK, ZL, SY No competing interests declared, (© 2024, Yang, Xiang, Yang et al.)

Nuclear migration is critical for the proper positioning of neurons in the developing brain. It is known that bidirectional microtubule motors are required for nuclear transport, yet the mechanism of the coordination of opposing motors is still under debate. Using mouse cerebellar granule cells, we demonstrate that Nesprin-2 serves as a nucleus-motor adaptor, coordinating the interplay of kinesin-1 and dynein. Nesprin-2 recruits dynein-dynactin-BicD2 independently of the nearby kinesin-binding LEWD motif. Both motor binding sites are required to rescue nuclear migration defects caused by the loss of function of Nesprin-2. In an intracellular cargo transport assay, the Nesprin-2 fragment encompassing the motor binding sites generates persistent movements toward both microtubule minus and plus ends. Nesprin-2 drives bidirectional cargo movements over a prolonged period along perinuclear microtubules, which advance during the migration of neurons. We propose that Nesprin-2 keeps the nucleus mobile by coordinating opposing motors, enabling continuous nuclear transport along advancing microtubules in migrating cells., (© 2024 Zhou et al.)

Huntington's disease (HD) is caused by a polyglutamine expansion of the huntingtin protein, resulting in the formation of polyglutamine aggregates. The mechanisms of toxicity that result in the complex HD pathology remain only partially understood. Here, we show that nuclear polyglutamine aggregates induce nuclear envelope (NE) blebbing and ruptures that are often repaired incompletely. These ruptures coincide with disruptions of the nuclear lamina and lead to lamina scar formation. Expansion microscopy enabled resolving the ultrastructure of nuclear aggregates and revealed polyglutamine fibrils sticking into the cytosol at rupture sites, suggesting a mechanism for incomplete repair. Furthermore, we found that NE repair factors often accumulated near nuclear aggregates, consistent with stalled repair. These findings implicate nuclear polyQ aggregate-induced loss of NE integrity as a potential contributing factor to Huntington's disease and other polyglutamine diseases., (© 2024 Korsten et al.)

Glucocorticoid receptor (GR) activation may promote metastasis in oestrogen receptor-negative and triple-negative breast cancer (TNBC). However, the role of the GRβ isoform, which has opposing effects to the main isoform, has not been studied in clinical samples. We aimed to analyse the intracellular localisation of total GR and GRβ in vitro using plasmid constructs and fluorescent immunocytochemistry. Additionally, our goal was to perform immunostaining for total GR and GRβ on two cohorts: (i) on 194 clinical breast cancer samples to compare the expression in different molecular subtypes, and (ii) on 161 TNBC samples to analyse the association of GR with survival. We supplemented our analysis with RNA data from 1097 TNBC cases. We found that in the absence of the ligand, GR resided in the cytoplasm of breast cancer cells, while upon ligand activation, it translocated to the nucleus. A negative correlation was found between cytoplasmic GRtotal and Ki67 in luminal A tumours, while the opposite trend was observed in TNBC samples. Tumours with strong lymphoid infiltration showed higher cytoplasmic GRtotal staining compared to those with weaker infiltration. Patients with high nuclear GRtotal staining had shorter progression-free survival in univariate analysis. High cytoplasmic GRβ was a marker for better overall survival in multivariate analysis (10-year overall survival HR [95% CI]: 0.46 [0.22-0.95], p = 0.036). As a conclusions, this study is the first to investigate GRβ expression in breast tumours. Different expression and cellular localisation of GRtotal and GRβ were observed in the context of molecular subtypes, underscoring the complex role of GR in breast cancer. An inverse association between cytoplasmic GRtotal and the Ki67 proliferation index was observed in luminal A and TNBC. Regarding the impact of GR on outcomes in TNBC patients, while cytoplasmic GRβ was associated with a better prognosis, patients with nuclear GRtotal staining may be at a higher risk of disease progression, as it negatively affects survival. Caution should be exercised when using glucocorticoids in patients with nuclear GR staining, as it may negatively impact survival.

Skeletal myofibers are syncytia made from the fusion of dozens or hundreds of mononuclear progenitor cells. Along myogenesis, the arriving nuclei from the progenitor cells have a long journey before being positioned at the periphery of a mature myofiber. Once at the periphery, nuclei are regularly spaced and each nucleus is transcriptionally responsible for its surrounding proportion of cytoplasm, known as the myonuclear domain. Disruption of these domains can be observed in various myopathies, suggesting their importance for skeletal muscle functionality. However, little is known about mechanisms regulating the myonuclear domain stability and organization. Here we take the example of MACF1, a microtubule-associated protein, as an essential actor in myonuclear domain organization, to highlight the potential role of microtubules and their associated proteome network for the stability of these domains and hence for proper myofiber functionality., (© 2024 médecine/sciences – Inserm.)

Myocardial injury induced by ischemia-reperfusion (I/R) remains a difficult clinical problem. However, the exact mechanisms underlying I/R-induced have yet to be clarified. CARD9 is an important cytoplasmic-binding protein. In this study, an immunocoprecipitation assay showed that SUMOylation of the CARD9 protein promoted the binding of CARD9 to HOXB5, but hindered the O-GlcNAc glycosylation of HOXB5, a predicted transcription factor of Parkin and a key factor in mitophagy. O-GlcNAc glycosylation is an important signal for translocation of proteins from the cytoplasm to the nucleus. CARD9 protein SUMOylation is regulated by PIAS3, which is related to I/R-induced myocardial injury. Therefore, we propose that knockdown of PIAS3 inhibits SUMOylation of the CARD9 protein, facilitates the dissociation of CARD9 and HOXB5, which increases the O-GlcNAc-mediated glycosylation of HOXB5, while the resulting HOXB5 nuclear translocation promotes Parkin-induced mitophagy and alleviates myocardial I/R injury., (© 2024 The Author(s). Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

Mitochondria serve as the cellular powerhouse, and their distinct DNA makes them a prospective target for gene editing to treat genetic disorders. However, the impact of genome editing on mitochondrial DNA (mtDNA) stability remains a mystery. Our study reveals previously unknown risks of genome editing that both nuclear and mitochondrial editing cause discernible transfer of mitochondrial DNA segments into the nuclear genome in various cell types including human cell lines, primary T cells, and mouse embryos. Furthermore, drug-induced mitochondrial stresses and mtDNA breaks exacerbate this transfer of mtDNA into the nuclear genome. Notably, we observe that mitochondrial editors, including mitoTALEN and recently developed base editor DdCBE, can also enhance crosstalk between mtDNA and the nuclear genome. Moreover, we provide a practical solution by co-expressing TREX1 or TREX2 exonucleases during DdCBE editing. These findings imply genome instability of mitochondria during induced DNA breaks and explain the origins of mitochondrial-nuclear DNA segments., (© 2024. The Author(s).)

Regulated start-codon selection has the potential to reshape the proteome through the differential production of upstream open reading frames, canonical proteins, and alternative translational isoforms 1-3 . However, conditions under which start codon selection is altered remain poorly defined. Here, using transcriptome-wide translation-initiation-site profiling 4 , we reveal a global increase in the stringency of start-codon selection during mammalian mitosis. Low-efficiency initiation sites are preferentially repressed in mitosis, resulting in pervasive changes in the translation of thousands of start sites and their corresponding protein products. This enhanced stringency of start-codon selection during mitosis results from increased association between the 40S ribosome and the key regulator of start-codon selection, eIF1. We find that increased eIF1-40S ribosome interaction during mitosis is mediated by the release of a nuclear pool of eIF1 upon nuclear envelope breakdown. Selectively depleting the nuclear pool of eIF1 eliminates the change to translational stringency during mitosis, resulting in altered synthesis of thousands of protein isoforms. In addition, preventing mitotic translational rewiring results in substantially increased cell death and decreased mitotic slippage in cells that experience a mitotic delay induced by anti-mitotic chemotherapies. Thus, cells globally control stringency of translation initiation, which has critical roles during the mammalian cell cycle in preserving mitotic cell physiology., Competing Interests: Competing interests D.P.B. has equity in Alnylam Pharmaceuticals, where he is a co-founder and advisor. The other authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Among flowering plants, genome size varies remarkably, by >2200-fold, and this variation depends on the loss and gain of noncoding DNA sequences that form distinct heterochromatin complexes during interphase. In plants with giant genomes, most chromatin remains condensed during interphase, forming a dense network of heterochromatin threads called interphase chromonemata. Using super-resolution light and electron microscopy, we studied the ultrastructure of chromonemata during and after replication in root meristem nuclei of Nigella damascena L. During S-phase, heterochromatin undergoes transient decondensation locally at DNA replication sites. Due to the abundance of heterochromatin, the replication leads to a robust disassembly of the chromonema meshwork and a general reorganization of the nuclear morphology visible even by conventional light microscopy. After replication, heterochromatin recondenses, restoring the chromonema structure. Thus, we show that heterochromatin replication in interphase nuclei of giant-genome plants induces a global nuclear reorganization., (© 2024 Society for Experimental Biology and John Wiley & Sons Ltd.)

Motivation: Heart failure (HF), a major cause of morbidity and mortality, necessitates precise diagnostic and prognostic methods., Results: This study presents a novel deep learning approach, Transformer-based Analysis of Images of Tissue for Effective Remedy (TRAITER), for HF diagnosis and prognosis. Using image segmentation techniques and a Vision Transformer, TRAITER predicts HF likelihood from cardiac tissue cell nuclear morphology images and the potential for left ventricular reverse remodeling (LVRR) from dual-stained images with cell nuclei and DNA damage markers. In HF prediction using 31 158 images from 9 patients, TRAITER achieved 83.1% accuracy. For LVRR prediction with 231 840 images from 46 patients, TRAITER attained 84.2% accuracy for individual images and 92.9% for individual patients. TRAITER outperformed other neural network models in terms of receiver operating characteristics, and precision-recall curves. Our method promises to advance personalized HF medicine decision-making., Availability and Implementation: The source code and data are available at the following link: https://github.com/HamanoLaboratory/predict-of-HF-and-LVRR., (© The Author(s) 2024. Published by Oxford University Press.)

Defining the time of action for morphogens requires tools capable of temporally controlled perturbations. To study how the transcription factor Dorsal affects patterning of the Drosophila embryonic dorsal-ventral axis, we used two light-inducible tags that trigger either nuclear export or degradation of Dorsal under blue light. Nuclear export of Dorsal leads to loss of the high-threshold, ventrally expressed target gene snail (sna), while the low-threshold, laterally expressed target gene short-gastrulation (sog) is retained. In contrast, degradation of Dorsal results in retention of sna, loss of sog, and lower nuclear levels compared to when Dorsal is exported from the nucleus. To understand why nuclear export causes loss of sna but degradation does not, we investigated Dorsal kinetics using photobleaching and found that it rapidly re-enters the nucleus even under blue-light conditions favoring export. The associated kinetics of Dorsal being rapidly imported and exported continuously are likely responsible for loss of sna but, alternatively, can support sog. Collectively, our results indicate that this dynamic patterning process is influenced by both Dorsal concentration and nuclear retention., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

The conservative post-transcriptional modification in mammals and Drosophila is adenosine-to-inosine (A-to-I) deamination in double-stranded RNA, catalyzed by RNA-editing enzymes known as adenosine deaminases acting on RNA (ADARs). The traditional nuclear import pathway for ADARs involves the recognition of a putative classical nuclear localization sequence (NLS) by importin α4 and α5. In our previous research, ADAR in silkworm, Bombyx mori (BmADARa) was confirmed predominantly located in the nucleus. However, the location of the NLS in BmADARa and its impact on nuclear import and self-dimerization remained unclear. Utilizing NLS prediction software, we predicted the presence of a bipartite NLS within the amino-terminal, 85 amino acids of BmADARa (N85). This prediction was validated through point mutation, which demonstrated that the bipartite NLS could directly mediate nuclear import of BmADARa. Co-immunoprecipitation analysis revealed that BmADARa is mainly dependent on BmKaryopherin α3 (homologous to mammalian importin α4) for nuclear import, although both BmKaryopherin α3 and BmImportin α5 could recognize bipartite NLS. The N-terminal truncated mutants and the bipartite NLS mutants of BmADARa suggest that the bipartite NLS is the major nuclear import site and a crucial structure for self-dimerization of BmADARa. In conclusion, the N-terminal bipartite NLS of BmADARa is recognized by BmKaryopherin α3 and BmImportin α5, facilitating its nuclear import. This promotes BmADARa self-dimerization and maintains the stability of dimerization, thereby enhancing its editing efficiency on target substrates. The results of this research demonstrate the role of bipartite NLS in BmADARa editing and laying a foundation for further research on the regulation of BmADARa in the growth and development in B. mori., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Intramuscular adipogenesis plays an important role in muscle development, which determines the quality of goat meat. However, its underlying cellular and molecular mechanisms remain poorly understood. In this study, we provided detailed cellular atlases of goat longissimus dorsi during muscle development at single-nucleus resolution, and identified the subpopulations of fibroblasts/fibro-adipogenic progenitors (FAPs) and muscle satellite cell (MuSC), as well as the differentiation trajectory of FAPs subpopulations. Cellular ligand-receptor interaction analysis revealed enriched BMP and IGF pathways implicated in within-tissue crosstalk centered around FAPs. Through single-nucleus gene regulatory network analysis and in vitro interference verification, we found that TCF7L2 was a critical transcriptional factor (TF) in early adipogenesis in skeletal muscle. Overall, our work reveals the cellular intricacies and diversity of goat longissimus dorsi during muscle development, implementing insights into the critical roles of BMP, IGF pathways and TCF7L2 TF in intramuscular adipogenesis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Mutations have underpinned research into gene characterization across all domains of life. This includes the discovery of the genes involved in the development of asexual spores in filamentous fungi. Mutants in the ascomycete Paecilomyces variotii were isolated with impaired biosynthesis of the characteristic yellow pigment produced by this filamentous fungus. The affected genes were identified as pvpP, encoding the polyketide synthase that is required for synthesis of the pigment YWA1, and abaA and wetA that are two genes that encode components of the AbaA-BrlA-WetA module required for the development of asexual spores in species in the Eurotiales order. WetA was further characterized. A strain expressing a functional WetA-GFP fusion was created and used to find that WetA is expressed primarily in spores and concentrated in their nuclei, providing evidence that this conserved protein likely functions as a regulator of transcription in conidia. Analysis of the phenotypes of the P. variotii wetA mutant suggests that how this three-protein module impacts fungal biology will vary from species-to-species, despite being conserved amongst filamentous Ascomycete species., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Nicholas Chong reports financial support was provided by Australasian Mycological Society. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Inflammasomes play important roles in resisting infections caused by various pathogens. HSV-1 is a highly contagious virus among humans. The process by which HSV-1 particles bud from the nucleus is unique to herpes viruses, but the specific mechanism is still unclear. Here, we screened genes involved in HSV-1 replication. We found that TET3 plays an essential role in HSV-1 infection. TET3 recognizes the UL proteins of HSV-1 and, upon activation, can directly bind to caspase-1 to activate an ASC-independent inflammasome in the nucleus. The subsequent cleavage of GSDMD in the nucleus is crucial for the budding of HSV-1 particles from the nucleus. Inhibiting the perforation ability of GSDMD on the nuclear membrane can significantly reduce the maturation and spread of HSV-1. Our results may provide a new approach for the treatment of HSV-1 in the future., (© 2024. The Author(s), under exclusive licence to CSI and USTC.)

Cisplatin resistance is one of the major causes of treatment failure in head and neck squamous cell carcinoma (HNSCC). There is an urgent need to uncover the underlying mechanism for developing effective treatment strategies. A quantitative proteomics assay was used to identify differential proteins in cisplatin-resistant cells. Mitochondrial topoisomerase I (TOP1MT) localization was determined using laser confocal microscopy and nucleocytoplasmic separation assay. Chromatin immunoprecipitation sequencing, dual-luciferase reporter assay, and RNA immunoprecipitation were used to identify the interaction between pseudogenes, miRNAs, and real genes. In vivo experiments verified the interaction between TOP1MT and pseudogenes on cisplatin resistance. TOP1MT was identified as a driving factor of cisplatin resistance in vitro, in vivo, and in HNSCC patients. Moreover, TOP1MT exceptionally translocated to the nucleus in cisplatin-resistant HNSCC cells in a signal peptide-dependent manner. Nuclear TOP1MT (nTOP1MT) transcriptionally regulated the mitochondrial functional pseudogene MTATP6P1, which bound to miR-137 and miR-491-5p as a competing endogenous RNA (ceRNA) and promoted the expression of MTATP6. An increase in MTATP6 enhanced mitochondrial oxidative phosphorylation (OXPHOS), which conferred cisplatin resistance in HNSCC. Our findings revealed that nTOP1MT transcriptionally activated MTAPT6P1 and increased MTATP6 expression via ceRNA, which facilitated OXPHOS and cisplatin resistance. These results provide novel insight for overcoming cisplatin resistance in HNSCC., Competing Interests: Declaration of Conflicting InterestsThe authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

For investigations into fate specification and morphogenesis in time-lapse images of preimplantation embryos, automated 3D instance segmentation and tracking of nuclei are invaluable. Low signal-to-noise ratio, high voxel anisotropy, high nuclear density, and variable nuclear shapes can limit the performance of segmentation methods, while tracking is complicated by cell divisions, low frame rates, and sample movements. Supervised machine learning approaches can radically improve segmentation accuracy and enable easier tracking, but they often require large amounts of annotated 3D data. Here, we first report a previously unreported mouse line expressing near-infrared nuclear reporter H2B-miRFP720. We then generate a dataset (termed BlastoSPIM) of 3D images of H2B-miRFP720-expressing embryos with ground truth for nuclear instances. Using BlastoSPIM, we benchmark seven convolutional neural networks and identify Stardist-3D as the most accurate instance segmentation method. With our BlastoSPIM-trained Stardist-3D models, we construct a complete pipeline for nuclear instance segmentation and lineage tracking from the eight-cell stage to the end of preimplantation development (>100 nuclei). Finally, we demonstrate the usefulness of BlastoSPIM as pre-train data for related problems, both for a different imaging modality and for different model systems., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Phosphorylation of histone H3 at threonine 11 (H3T11ph) affects transcription and chromosome stability. However, the enzymes responsible for depositing H3T11ph and the functions of H3T11ph in plants remain unknown. Here we report that in Arabidopsis thaliana, PYRUVATE KINASE 6 (PK6), PK7 and PK8 enter the nucleus under conditions of sufficient glucose and light exposure, where they interact with SWI2/SNF2-RELATED 1 COMPLEX 4 (SWC4) and phosphorylate H3 at threonine 11. Mutations in these kinases or knockdown of SWC4 resulted in FLC-dependent early flowering, short hypocotyls and short pedicels. Genome-wide, H3T11ph is highly enriched at transcription start sites and transcription termination sites, and positively correlated with gene transcript levels. PK6 and SWC4 targeted FLC, MYB73, PRE1, TCP4 and TCP10, depositing H3T11ph at these loci and promoting their transcription, and PK6 occupancy at these loci requires SWC4. Together, our results reveal that nuclear-localized PK6, PK7 and PK8 modulate H3T11ph and plant growth., Competing Interests: Competing interests The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

This study aimed to evaluate the predictive values of phosphoglucomutase-1 (PGM1) expression for prognosis in patients with hepatocellular carcinoma (HCC). PGM1 expression was assessed by immunohistochemistry in tissue microarrays. The relationship of PGM1 expression level with pathologic parameters and prognosis values was respectively analyzed by χ 2 test and Cox regression. The accuracy of independent risk factors in predicting prognosis was calculated by receiver operating characteristic curve. HCC patient-derived xenograft models were performed to evaluate the nuclear PGM1 antitumor effect. The results showed that PGM1 expression was low in HCC tissues. Nuclear PGM1 was an independent prognostic factor for overall survival and time to recurrence. Cox regression showed that nuclear PGM1, serum α-fetoprotein, liver cirrhosis, and TNM staging stage were independent risk predictors for HCC. Receiver operating characteristic curve demonstrated that combination of independent predictors had better prognostic value than TNM staging alone. Moreover, patient-derived xenograft models showed antitumor effect of nuclear PGM1. We found that low expression of nuclear PGM1 was detected in HCC tissues and associated with poor prognostic. Nuclear PGM1 was an independent prognostic factor in patients with HCC. Furthermore, nuclear PGM1 combining other independent risk factors showed a better prognostic value. Nuclear PGM1 was a useful prognostic biomarker for patients with HCC., Competing Interests: The authors declare no conflict of interest., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

A new fate of cell surface receptors, cleaved activation in the nucleus, is summarized. The intracellular domain (ICD) of cell surface receptors, cleaved by enzymes like γ-secretase, translocates to the nucleus to form transcriptional complexes participating in the onset and development of tumors. The fate is clinically significant, as inhibitors of cleavage enzymes have shown effectiveness in treating advanced tumors by reducing tumorigenic ICDs. Additionally, the construction of synthetic receptors also conforms with the fate mechanism. This review details each step of cleaved activation in the nucleus, elucidates tumorigenic mechanisms, explores application in antitumor therapy, and scrutinizes possible limitations., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

RNA-binding proteins (RBPs) can undergo phase separation and form condensates, processes that, in turn, can be critical for their functionality. In a recent study, Huang, Ellis, and colleagues show that cellular stress can trigger transient alterations in nuclear TAR DNA-binding protein 43 (TDP-43), leading to changes crucial for proper neuronal function. These findings have implications for understanding neurological TDP-43 proteinopathies., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The nucleoskeleton is essential for nuclear architecture as well as genome integrity and gene expression. In addition to lamins, titin or spectrins, dynamic actin filament polymerization has emerged as a potential intranuclear structural element but its functions are less well explored. Here we found that calcium elevations trigger rapid nuclear actin assembly requiring the nuclear membrane protein SUN2 independently of its function as a component of the LINC complex. Instead, SUN2 colocalized and associated with the formin and actin nucleator INF2 in the nuclear envelope in a calcium-regulated manner. Moreover, SUN2 is required for active RNA polymerase II (RNA Pol II) clustering in response to calcium elevations. Thus, our data uncover a SUN2-formin module linking the nuclear envelope to intranuclear actin assembly to promote signal-dependent spatial reorganization of active RNA Pol II., (© 2024. The Author(s).)

Nuclear pore complexes (NPCs) mediate all traffic between the nucleus and the cytoplasm and are among the most stable protein assemblies in cells. Budding yeast cells carry two variants of NPCs which differ in the presence or absence of the nuclear basket proteins Mlp1, Mlp2, and Pml39. The binding of these basket proteins occurs very late in NPC assembly and Mlp-positive NPCs are excluded from the region of the nuclear envelope that borders the nucleolus. Here, we use recombination-induced tag exchange to investigate the stability of all the NPC subcomplexes within individual NPCs. We show that the nuclear basket proteins Mlp1, Mlp2, and Pml39 remain stably associated with NPCs through multiple cell-division cycles, and that Mlp1/2 are responsible for the exclusion of NPCs from the nucleolar territory. In addition, we demonstrate that binding of the FG-nucleoporins Nup1 and Nup2 depletes also Mlp-negative NPCs from this region by an independent pathway. We develop a method for single NPC tracking in budding yeast and observe that NPCs exhibit increased mobility in the absence of nuclear basket components. Our data suggest that the distribution of NPCs on the nucleus is governed by multiple interaction of nuclear basket proteins with the nuclear interior., Competing Interests: Conflicts of interest: The authors declare no financial conflict of interest.

Nuclear Bcl-xL is found to promote cancer metastasis independently of its mitochondria-based anti-apoptotic activity. How Bcl-xL is translocated into the nucleus and how nuclear Bcl-xL regulates histone H3 trimethyl Lys4 (H3K4me3) modification have yet to be understood. Here, we report that C-terminal Binding Protein 2 (CtBP2) binds to Bcl-xL via its N-terminus and translocates Bcl-xL into the nucleus. Knockdown of CtBP2 by shRNA decreases the nuclear portion of Bcl-xL and reverses Bcl-xL-induced invasion and metastasis in mouse models. Furthermore, knockout of CtBP2 not only reduces the nuclear portion of Bcl-xL but also suppresses Bcl-xL transcription. The binding between Bcl-xL and CtBP2 is required for their interaction with MLL1, a histone H3K4 methyltransferase. Pharmacologic inhibition of the MLL1 enzymatic activity reverses Bcl-xL-induced H3K4me3 and TGFβ mRNA upregulation, as well as invasion. Moreover, the cleavage under targets and release using nuclease (CUT&RUN) assay coupled with next-generation sequencing reveals that H3K4me3 modifications are particularly enriched in the promotor regions of genes encoding TGFβ and its signaling pathway members in cancer cells overexpressing Bcl-xL. Altogether, the metastatic function of Bcl-xL is mediated by its interaction with CtBP2 and MLL1 and this study offers new therapeutic strategies to treat Bcl-xL-overexpressing cancer., Competing Interests: Declaration of competing interest The authors declare no competing financial interests., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Lysosomes are involved in a myriad of cellular functions, such as degradation of macromolecules, endocytosis and exocytosis, modulation of several signaling pathways, and regulation of cell metabolism. To fulfill these diverse functions, lysosomes can undergo several dynamic changes in their content, size, pH, and location within cells. Here, we studied some of these parameters during embryonic chick skeletal muscle cells. We used an anti-lysosome-associated membrane protein 2 (LAMP2) antibody to specifically determine the intracellular localization of lysosomes in these cells. Our data shows that lysosomes are highly enriched in the perinuclear region of chick embryonic muscle cells. We also showed that the wingless signaling pathway (Wnt)/β-catenin signaling pathway can modulate the location of LAMP2 in chick myogenic cells. Our results highlight the role of lysosomes during muscle differentiation and particularly the presence of a subcellular population of lysosomes that are concentrated in the perinuclear region of muscle cells., (© 2024 International Federation for Cell Biology.)

A limited number of bacteria are able to colonize the nuclei of eukaryotes. 'Candidatus Endonucleobacter' infects the nuclei of deep-sea mussels, where it replicates to ≥80,000 bacteria per nucleus and causes nuclei to swell to 50 times their original size. How these parasites are able to replicate and avoid apoptosis is not known. Dual RNA-sequencing transcriptomes of infected nuclei isolated using laser-capture microdissection revealed that 'Candidatus Endonucleobacter' does not obtain most of its nutrition from nuclear DNA or RNA. Instead, 'Candidatus Endonucleobacter' upregulates genes for importing and digesting sugars, lipids, amino acids and possibly mucin from its host. It likely prevents apoptosis of host cells by upregulating 7-13 inhibitors of apoptosis, proteins not previously seen in bacteria. Comparative phylogenetic analyses revealed that 'Ca. Endonucleobacter' acquired inhibitors of apoptosis through horizontal gene transfer from their hosts. Horizontal gene transfer from eukaryotes to bacteria is assumed to be rare, but may be more common than currently recognized., (© 2024. The Author(s).)

Generation of mammalian red blood cells requires the expulsion of polarized nuclei late in terminal erythroid differentiation. However, the mechanisms by which spherical erythroblasts determine the direction of nuclear polarization and maintain asymmetry during nuclear expulsion are poorly understood. Given the analogy of erythroblast enucleation to asymmetric cell division and the key role of Aurora kinases in mitosis, we sought to investigate the function of Aurora kinases in erythroblast enucleation. We found that AURKA (Aurora kinase A) is abundantly expressed in orthochromatic erythroblasts. Intriguingly, high-resolution confocal microscopy analyses revealed that AURKA co-localized with the centrosome on the side of the nucleus opposite its membrane contact point during polarization and subsequently translocated to the anterior end of the protrusive nucleus upon nuclear exit. Mechanistically, AURKA regulated centrosome maturation and localization via interaction with γ-tubulin to provide polarization orientation for the nucleus. Furthermore, we identified ECT2 (epithelial cell transforming 2), a guanine nucleotide exchange factor, as a new interacting protein and ubiquitination substrate of AURKA. After forming the nuclear protrusion, AURKA translocated to the anterior end of the protrusive nucleus to directly degrade ECT2, which is partly dependent on kinase activity of AURKA. Moreover, knockdown of ECT2 rescued impaired enucleation caused by AURKA inhibition. Our findings have uncovered a previously unrecognized role of Aurora kinases in the establishment of nuclear polarization and eventual nuclear extrusion and provide new mechanistic insights into erythroblast enucleation.