Nuclear Protein Research Articles

Cancer epithelial cells participate in the self-organization of lung tumor spheroids. A morphological approach.

The tumor microenvironment is known to play an important role in tumor progression. However, the specific mechanisms underlying this process are still not known in detail and more research is needed on the elements that control tumor progression in lung cancer. In this work, we aimed to investigate the involvement of epithelial and stromal cancer cells in growth, cell migration and epithelial-to-mesenchymal transition (EMT) in a 3D in vitro model consisting of cell spheroids cultured in a type I collagen scaffold. Spheroids were manufactured using different combinations of epithelial cells, particularly H460 and H1792 cell lines, with cancer-associated fibroblasts (CAFs) and normal fibroblasts (NFs), both isolated from adenocarcinoma patients. We evaluated the morphology of the spheroids by analysis of F-actin and pankeratin with confocal microscopy. We determined the ultrastructure of cells in the spheroids by transmission electron microscopy and the expression of CDH1, CDH2 and VIM by RT-PCR. We observed that, on the one hand, the type of epithelial cell influences the morphology of spheroids. Stromal cells stimulated spheroid growth and cell dissemination through the collagen matrix, either alone or organized in branches with a nucleus of epithelial cells preceded by fibroblast cells. They also induced the appearance of new cell groups in the scaffold and the presence of EMT markers. The results presented here indicate the participation of both epithelial and stromal cells in the control of spheroid self-organization. The experimental model proposed here, although preliminary, is useful for the study of some aspects related to tumor progression in lung cancer.

Nuclear TOP1MT Confers Cisplatin Resistance via Pseudogene in HNSCC.

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.

Wētā Aotearoa—Polyphyly of the New Zealand Anostostomatidae (Insecta: Orthoptera)

The Anostostomatidae of Aotearoa New Zealand are well-characterized at the genus and species level, but the higher-level systematics of the family as a whole remain poorly resolved. We tested the hypothesis that the New Zealand anaostostomatid fauna consists of a single monophyletic group consistent with a single common ancestor. For phylogenetic analysis, we sampled the genera in Aotearoa New Zealand as well as representatives of the family from Australia and New Caledonia. Maximum likelihood analyses including topological comparison statistics with a DNA alignment of thirteen mitochondrial and four nuclear protein coding genes rejected the monophyly of lineages in New Zealand. We found phylogenetic support for four separate New Zealand lineages; three with their closest relatives in Australia and one in New Caledonia. The New Zealand genus Hemiandrus is paraphyletic and the establishment of a morphologically distinct genus is justified. We determined that six of the valid species previously placed in Hemiandrus form a distinct clade that we designated here as Anderus gen. nov. The putative Hemiandrus that we sampled from Australia was sister to neither of the New Zealand lineages.

Poly(ADP-Ribose) Polymerase (PARP) Inhibitors for Cancer Therapy: Advances, Challenges, and Future Directions

Poly(ADP-ribose) polymerases (PARPs) are crucial nuclear proteins that play important roles in various cellular processes, including DNA repair, gene transcription, and cell death. Among the 17 identified PARP family members, PARP1 is the most abundant enzyme, with approximately 1–2 million molecules per cell, acting primarily as a DNA damage sensor. It has become a promising biological target for anticancer drug studies. Enhanced PARP expression is present in several types of tumors, such as melanomas, lung cancers, and breast tumors, correlating with low survival outcomes and resistance to treatment. PARP inhibitors, especially newly developed third-generation inhibitors currently undergoing Phase II clinical trials, have shown efficacy as anticancer agents both as single drugs and as sensitizers for chemo- and radiotherapy. This review explores the properties, characteristics, and challenges of PARP inhibitors, discussing their development from first-generation to third-generation compounds, more sustainable synthesis methods for discovery of new anti-cancer agents, their mechanisms of therapeutic action, and their potential for targeting additional biological targets beyond the catalytic active site of PARP proteins. Perspectives on green chemistry methods in the synthesis of new anticancer agents are also discussed.

Single-cell sequencing analysis revealed that NEAT1 was a potential biomarker and therapeutic target of prostate cancer.

Prostate cancer (PCa) usually manifests atypical symptoms in the early stage, and once symptoms appear, most PCa patients have developed to the advanced stage, failing to undergo radical surgery. In this study, PCa occurrence-related biomarkers were explored based on single-cell RNA sequencing (scRNA-seq) data. scRNA-seq data of prostate normal (Normal), benign prostatic hyperplasia (BPH), and PCa (Tumor) samples were acquired from the Gene Expression Omnibus (GEO). Cellular subsets associated with PCa occurrence were obtained using cell annotation. Additionally, the mRNA expression of nuclear enriched abundant transcript 1 (NEAT1) was detected by quantitative real-time PCR (qRT-PCR). The effects of NEAT1 on cell proliferation and apoptosis were analyzed by 5-ethynyl-2-deoxyuridine (EdU) and flow cytometry. Subsequently, cell-derived xenograft (CDX) models were constructed and divided into the LV-NC and LV-shNEAT1 groups. After the tumor tissues of CDX model mice in each group were extracted, the cell growth and Ki67 expression were observed separately using hematoxylin-eosin (H&E) staining and immunohistochemistry (IHC). Ten cellular subsets were obtained via cell annotation, and significantly differential changes were observed between Basal intermediate and Luminal during the course of BPH to PCa. NEAT1-Luminal was highly recruited in the Tumor group with low stemness and high malignancy scores. Matrix metallopeptidase 7 (MMP7)- keratin 17 (KRT17)-Basal intermediate had high ratios in the Tumor group with low stemness and high malignancy scores. The results of pseudotime analysis revealed that NEAT1-Luminal in the Tumor group were consistently distributed with tumor stage cells. In vitro assays showed that NEAT1 expression was elevated in PCa cells, and NEAT1 knockdown could inhibit cell proliferation and induce apoptosis. CDX assays indicated that silencing NEAT1 could reduce the growth rate of PCa tumor volume in CDX model mice. H&E staining results showed that nuclei of tumor cells were reduced and exhibited lighter color in the LV-shNEAT1 group compared with the LV-NC group. IHC results showed that Ki67 positivity was significantly lower in the LV-shNEAT1 group than in the LV-NC group. NEAT1 expression is increased in PCa, and NEAT1 can be a potential biomarker and therapeutic target for PCa.

MCM proteins are up-regulated in placentas of women with reduced insulin sensitivity.

In the first trimester of pregnancy the human placenta grows rapidly, making it sensitive to changes in the intrauterine environment. To test whether exposure to an environment in utero often associated with obesity modifies placental proteome and function, we performed untargeted proteomics (LC-MS/MS) in placentas from 19 women (gestational age 35-48 days, i.e. 5+0-6+6 weeks). Maternal clinical traits (body mass index, leptin, glucose, C-peptide and insulin sensitivity) and gestational age were recorded. DNA replication and cell cycle pathways were enriched in the proteome of placentas of women with low maternal insulin sensitivity. Driving these pathways were the minichromosome maintenance (MCM) proteins MCM2, MCM3, MCM4, MCM5, MCM6 and MCM7 (MCM-complex). These proteins are part of the pre-replicative complex and participate in DNA damage repair. Indeed, MCM6 and γH2AX (DNA-damage marker) protein levels correlated in first trimester placental tissue (r = 0.514, P<0.01). MCM6 and γH2AX co-localized to nuclei of villous cytotrophoblast cells, the proliferative cell type of the placenta, suggesting increased DNA damage in this cell type. To mimic key features of the intrauterine obesogenic environment, a first trimester trophoblast cell line, i.e., ACH-3P, was exposed to high insulin (10 nM) or low oxygen tension (2.5% O2). There was a significant correlation between MCM6 and γH2AX protein levels, but these were independent of insulin or oxygen exposure. These findings show that chronic exposure in utero to reduced maternal insulin sensitivity during early pregnancy induces changes in the early first trimester placental proteome. Pathways related to DNA replication, cell cycle and DNA damage repair appear especially sensitive to such an in utero environment.

Quantitation of the DNA-dependent protein kinase inhibitor peposertib (M3814) and metabolite in human plasma by LC-MS/MS.

The DNA-dependent protein kinase (DNA-PK) is an abundant nuclear protein that mediates DNA double-strand break repair by nonhomologous end joining (NHEJ). As such, DNA-PK is critical for V(D)J recombination in lymphocytes and for survival in cells exposed to ionizing radiation and clastogens. Peposertib (M3814) is a small molecule DNA-PK inhibitor currently in preclinical and clinical development for cancer treatment. We have developed a high-performance liquid chromatography-mass spectrometry method for quantitating peposertib and its metabolite in 0.1 mL human plasma. After MTBE liquid-liquid extraction, chromatographic separation was achieved with a Phenomenex Synergi polar reverse phase (4μm, 2× 50 mm) column and a gradient of 0.1% formic acid in acetonitrile and water over an 8min run time. Mass spectrometric detection was performed on an ABI SCIEX 4000 with electrospray, positive-mode ionization. The assay was linear from 10 to 3000 ng/mL for peposertib and 1-300 ng/mL for the metabolite and proved to be both accurate (97.3%-103.7%) and precise (<8.9%CV) fulfilling criteria from the Food and Drug Administration (FDA) guidance on bioanalytical method validation. This liquid chromatography-tandem mass spectroscopy (LC-MS/MS) assay will support several ongoing clinical studies by defining peposertib pharmacokinetics.

Early detection of cervical tumor cell of origin through Oncogene-induced senescent HPV-positive cells.

Human Papilloma Virus (HPV) is an oncogenic virus and is the most common cause of cervical cancer. HPV has been shown to induce senescence. Cellular senescence is involved in cancer progression and tumorigenesis. Identification and isolation of cells of tumor origin before tumorigeneses is an important step in cancer prevention and treatment. This study aimed to investigate the early cervical atypical senescent cytological preneoplastic change in non-menopausal women. Cervical smears of 121 patients were randomly selected and included in the study which cytopathologically diagnosed as atypical squamous cells of undetermined significance (AS-CUS) in correlation to HPV status, parakeratosis (PK), p16 immunostaining, enlarged Squamous cells nuclei (ES) and inflammatory cells infiltration (ICI). Results revealed that out of the total 121 patients, 32 cases (26%) were positive for high-risk HPV (HR-HPV), 26 cases (22%) were positive for low-risk HPV (LR-HPV) and 63 (52%) were negative for HPV. HPV infections were significantly associated with age groups (p<0.026), PK (p = 0.043), p16 (p = 0.001), ES (p = 0.002) and ICI (p = 0.049). The positive immunostaining expression of p16 was only noticed in two HR-HPV patients. ES cells were found in 9.5% of HPV-negative cases, 27% of LR-HPV cases and 40.5% of HR-HPV cases. High PK cell positivity was seen only in HR-HPV. High ICI scores were seen in 40.6% of HR-HPV patients, 26.9 % of LR-HPV and 17.4 % of negative HPV patients. It was concluded that high PK positivity, high ICI score, positive p16 immunostaining and ES were correlated with HR-HPV in non-menopausal women. These findings could provide potential diagnostic clues for HPV-harboring senescent cells as a strategy for reducing HPV risk of cervical cancer development and identifying the cell of tumor origin, which could be beneficial for improving the utility of senolytic agents and immunotherapy in clinical practice.

Spatiotemporal dynamics of nucleocytoplasmic transport

Nucleocytoplasmic transport is essential for cellular function, presenting a canonical example of rapid molecular sorting inside cells. It consists of a coordinated interplay between import/export of molecules in/out the cell nucleus. Here, we investigate the role of spatiotemporal dynamics of the nucleocytoplasmic transport and its regulation. We develop a biophysical model that captures the main features of the nucleocytoplasmic transport, in particular, its regulation through the Ran cycle. Our model yields steady-state profiles for the molecular components of the Ran cycle, their relaxation times, as well as the nuclear-to-cytoplasmic molecule ratio. We show that these quantities are affected by their spatial dynamics and heterogeneity within the nucleus. Specifically, we find that the spatial nonuniformity of Ran guanine exchange factor (RanGEF)—particularly its proximity to the nuclear envelope—increases the Ran content in the nucleus. We further show that RanGEF's accumulation near the nuclear envelope results from its intrinsic dynamics as a nuclear cargo, transported by the Ran cycle itself. Overall, our work highlights the critical role of molecular spatial dynamics in cellular processes and proposes new avenues for theoretical and experimental inquiries into the nucleocytoplasmic transport. Published by the American Physical Society 2024

Mutations of the Cx43 Gene in Non-Small Cell Lung Cancer: Association with Aberrant Localization of Cx43 Protein Expression and Tumor Progression

Background and Objectives: The Connexin43 (Cx43) gene is a suspected tumor suppressor gene, as re-expressed wild-type Cx43 genes reduce the malignancy potential of tumor cells. However, the role of Cx43 gene expression in human lung tumorigenesis remains unclear. Materials and Methods: Tumor tissues from 165 primary lung cancer patients were collected to study Cx43 protein expression and gene mutations using immunohistochemistry and direct DNA sequencing. In addition, Cx43 genes with or without mutations were transfected to CL-3 human lung cancer cells to confirm the function of these mutant forms of the Cx43 gene. Results: Aberrant localization of Cx43 protein in the nucleus and cytoplasm of tumor cells was detected in 14 out of 165 non-small cell lung cancer (NSCLC) patients. Mutations in the Cx43 gene were also found in patients with aberrant Cx43 localization, and transfection of these mutant genes into lung cancer cells enhanced their proliferation. Conclusions: To our knowledge, this is the first study to demonstrate Cx43 gene mutations in human lung neoplasm, supporting the hypothesis that Cx43 may function as a tumor suppressor in some lung cancer patients. Additionally, the findings suggest an association between aberrant localization of Cx43 protein expression and tumor progression.

Multi-stage electroporation dynamics induced by ionization wave–substrate interaction in cold plasma jet

The reactive species-independent nature of cold plasma's electric field is pivotal in biomedical applications. This work proposes to connect the plasma fluid model and the asymptotic Smoluchowski model for electroporation, providing a unified framework to investigate the evolution of the electric field in the biological substrate and the multi-stage electroporation response of the human cell. Two common substrates with distinct dielectric properties, namely, the cultivation medium and epidermis, are selected to report three stages of ionization wave (IW)–substrate interaction. The three-stage streamer discharge dynamics (restrike, axial-radial transition, and radial expansion of IW) induce three-stage cell electroporation dynamics (slow charging, fast charging, and electroporation), though the two processes are asynchronous. Specifically, the inner membrane covered the cell nucleus with ultra-short charging time that undergoes only the first two discharge stages in both substrates. Whether the cell membrane is exposed to the third stage of discharge depends on the permittivity of the substrate. The asynchrony can be attributed to the difference in the charging time of the cell membranes and substrates affected by the substrate permittivity. The presented model can provide quantitative insights into the cell electroporation induced by the IW–substrate interaction and theoretical guidance for plasma biomedical applications.

Repeated FRAP of the actin-binding protein CapG in the cell nucleus—a functional assay for EGF signaling in the single live breast cancer cell

Compartmentalization and differential distribution of proteins within a cell maintain cellular function and viability. CapG is a gelsolin-related actin-binding protein that distributes in steady state diffusively throughout cytoplasm and cell nucleus. To detect changes in CapG’s nucleocytoplasmic shuttling in response to external stimuli on the single cell level, we established repeated FRAP experiments of one and the same breast cancer cell. With this experimental set up, we found that ATP-depletion reversibly decreased CapG’s shuttling into the cell nucleus. The addition of epidermal growth factor (EGF) increased CapG’s nuclear shuttling within minutes. Serum-starvation doubled the number of breast cancer cells from 40 to 80% displaying increased CapG shuttling in response to EGF. Testing five different potential CapG phosphorylation sites, we found that serine 70 mediates the increase in CapG’s nuclear shuttling triggered by EGF. Thus, repeated FRAP of CapG in the cell nucleus can be used as functional readout of signaling cascades in the same single live breast cancer cell.

7449 A Genome-wide CRISPR Screen Identifies Drivers of Impaired Glucose Uptake in Human Insulin Resistance

Abstract Disclosure: N. Haider: None. Y. Lee: None. P. Yi: None. C.R. Kahn: None. Insulin resistance is a major risk factor in the development of type 2 diabetes (T2D), and metabolic syndrome. Although 25% of people within the general non-diabetic population are insulin resistant, the primary underlying cause of insulin resistance remains elusive. In this study, we have used induced pluripotent stem cells (iPSC) derived from non-diabetic humans at both ends of the insulin sensitivity spectrum, i.e., the top 20% of insulin resistance vs. the top 20% of insulin sensitive, as well as individuals with T2D to model human insulin resistance. We find that iPSCs differentiated into myoblasts (iMyos) from T2D and non-diabetic individuals in the highest quintile of insulin resistance (I-Res) show impaired insulin signaling and defective insulin-stimulated glucose uptake, as compared to iMyos from the insulin sensitive individuals (I-Sen), indicating that these cells in vitro mirror the alterations seen in vivo in a cell autonomous manner. To uncover the primary upstream drivers that contribute to the impaired glucose uptake associated with insulin resistance, we conducted a genome-wide loss-of-function screen using a CRISPR-based approach to identify genes that positively or negatively regulated glucose uptake in I-Res and T2D iMyos. Knockdown of ∼525 genes show effect to rescue the impaired glucose uptake in both I-Res and T2D iMyos, i.e., were negative regulators of glucose uptake. We hypothesized that important negative regulators of glucose uptake might be over-expressed at the protein level in I-Res and T2D iMyos and crossing the iMyos proteomics data with the CRISPR screen revealed 8 top candidates that are increased at the protein level and whose knockdown rescued glucose uptake in I-Res and T2D iMyos. These included candidates associated with cytoskeleton changes, protein kinases, transmembrane transport, mitochondrial and nuclear proteins. In summary, iPSC derived myoblasts from patients with insulin resistance and T2D mirror defects in glucose uptake observed in vivo. Combining CRISPR screening and global proteomics, we have identified important upstream regulators of insulin resistance in humans. Presentation: 6/2/2024

Microtubule forces drive nuclear damage in LMNA cardiomyopathy.

Nuclear homeostasis requires a balance of forces between the cytoskeleton and nucleus. Mutations in the LMNA gene, which encodes the nuclear envelope proteins lamin A/C, disrupt this balance by weakening the nuclear lamina. This results in nuclear damage in contractile tissues and ultimately muscle disease. Intriguingly, disrupting the LINC complex that connects the cytoskeleton to the nucleus has emerged as a promising strategy to ameliorate LMNA-associated cardiomyopathy. Yet how LINC complex disruption protects the cardiomyocyte nucleus remains unclear. To address this, we developed an assay to quantify the coupling of cardiomyocyte contraction to nuclear deformation and interrogated its dependence on the nuclear lamina and LINC complex. We found that, surprisingly, the LINC complex was mostly dispensable for transferring contractile strain to the nucleus, and that increased nuclear strain in lamin A/C-deficient cardiomyocytes was not rescued by LINC complex disruption. Instead, LINC complex disruption eliminated the cage of microtubules encircling the nucleus. Disrupting microtubules was sufficient to prevent nuclear damage and rescue cardiac function induced by lamin A/C deficiency. We computationally simulated the stress fields surrounding cardiomyocyte nuclei and show how microtubule forces generate local vulnerabilities that damage lamin A/C-deficient nuclei. Our work pinpoints localized, microtubule-dependent force transmission through the LINC complex as a pathological driver and therapeutic target for LMNA-cardiomyopathy.

12363 A Unique Case Of hüRthle Cell Carcinoma of the Thyroid Gland and Meningioma

Abstract Disclosure: I. Elsayed: None. R. Khan: None. H. Dhaliwal: None. Introduction: MEN1 gene is a tumor suppressor gene on chromosome 11q13 that encodes the nuclear protein menin, which regulates cell growth and cycle. When menin protein is deficient, MEN1 develops. MEN1 mutation usually involves tumors within the parathyroid glands, pancreas, or pituitary. Case Description: A 66-year-old male with past medical history of hypertension, hyperlipidemia, and coronary artery disease presented with sudden onset dizziness, nausea and vomiting for 1 hour. He had similar symptoms on and off for several months, however on the day of presentation, these symptoms were much more acute and severe. Stroke work-up was done including CT and CTA head which revealed a 1.7cm enhancing lesion to R temporal lobe with surrounding edema with an incidental 3 x 3 cm heterogeneous mass in the large portion of the right thyroid gland. Steroids initiated. MRI brain imaging revealed findings consistent with meningioma with local mass effect and pronounced parenchymal edema. Patient underwent successful resection of the mass and pathology confirmed diagnosis of Meningioma, WHO Grade 1. Ear Nose Throat specialist was consulted for incidental thyroid mass. Ultrasound guided Fine needle aspiration biopsy was, Pathology report returned suspicious for follicular neoplasm, Hürthle cell-type. Patient initially underwent right substernal semi-thyroidectomy. Pathology confirmed diagnosis of Oncocytic (Hürthle cell) carcinoma, minimally invasive, 4.3 cm with a single focus of capsular invasion, negative for lymphovascular invasion Discussion: This case presents a 66-year-old male with a rare coexistence of two distinct tumors: a meningioma and a Hürthle cell carcinoma of the thyroid gland. While meningiomas and Hürthle cell carcinoma are not commonly associated with MEN1 syndrome, there have been reports of atypical cases with unusual tumor associations. MEN1 is usually characterized by abnormalities in at least two affected endocrine glands, but variations can involve other endocrine and non-endocrine tumors as well. 25% of thyroid disease can be seen in MEN 1 syndrome; typically papillary thyroid cancer. Only one case in literature found Hürthle cell carcinoma and meningioma in the setting of MEN-1 phenotype. A second case unique case was Hürthle cell carcinoma with metastases to the meninges. The discovery of both a meningioma and a Hürthle cell carcinoma in this patient emphasizes the importance of clinicians being vigilant for the possibility of multiple neoplasms. This case highlights that there may be a relation between role of MEN1 with atypical neoplasms not commonly seen. Presentation: 6/2/2024

Mechanism of Alzheimer type II astrocyte development in hepatic encephalopathy

Type C hepatic encephalopathy (Type C HE) is a major and complex neurological condition that occurs following chronic liver failure. The molecular basis of Type C HE remains elusive. Type C HE is characterized by mental confusion, cognitive and motor disturbances. The presence of Alzheimer type II astrocytes (AT2A) is the key histopathological finding observed in Type C HE. However, nothing is currently known regarding AT2A development and its involvement in cognitive, and motor deficits in Type C HE. We, therefore, examined in rats the mechanisms by which liver failure contributes to the progression of AT2A, and its role in the development of cognitive and motor deficits in thioacetamide (TAA) model of Type C HE. We and others earlier reported increased oxidative/nitrosative stress (ONS), JNK1/2, and cMyc activation in ammonia-treated astrocyte cultures, as well as in brains from chronic liver failure. We now found increased levels of astrocytic glia maturation factor (GMF, a factor strongly implicated in neuroinflammation), as well as various inflammatory factors (IL-1β, TNF-α, IL-6, MMP-3, COX2, CXCL1, and PGE2), and reduced levels of GFAP and increased levels of aggregated nuclear protein Lamin A/C in rat brain cortex post-chronic liver failure. We also found increased levels of GMF and inflammatory factors (MMP-3, COX2, CXCL1, and PGE2) in astrocytes post-ammonia treatment in vitro. Additionally, pharmacological inhibition of upstream signaling of GMF (ONS, JNK1/2, and cMyc) or GMF inhibitors W-7 and trifluoperazine significantly reduced the levels of inflammatory factors, the number of AT2A cells, as well as the cognitive and motor deficits in TAA-treated rats. Increased levels of GMF were also identified in human post-mortem brain sections. These findings strongly suggest that increased levels of astrocytic GMF due to elevated levels of ONS, JNK1/2, and cMyc and the subsequent inflammation contribute to the development of AT2A and the consequent cognitive, and motor deficits in chronic liver failure.

A human progeria-associated BAF-1 mutation modulates gene expression and accelerates aging in C. elegans.

Alterations in the nuclear envelope are linked to a variety of rare diseases termed laminopathies. A single amino acid substitution at position 12 (A12T) of the human nuclear envelope protein BAF (Barrier to Autointegration Factor) causes Néstor-Guillermo Progeria Syndrome (NGPS). This premature ageing condition leads to growth retardation and severe skeletal defects, but the underlying mechanisms are unknown. Here, we have generated a novel in vivo model for NGPS by modifying the baf-1 locus in C. elegans to mimic the human NGPS mutation. These baf-1(G12T) mutant worms displayed multiple phenotypes related to fertility, lifespan, and stress resistance. Importantly, nuclear morphology deteriorated faster during aging in baf-1(G12T) compared to wild-type animals, recapitulating an important hallmark of cells from progeria patients. Although localization of BAF-1(G12T) was similar to wild-type BAF-1, lamin accumulation at the nuclear envelope was reduced in mutant worms. Tissue-specific chromatin binding andtranscriptome analyses showed reduced BAF-1 association in most genes deregulated by the baf-1(G12T) mutation, suggesting that altered BAF chromatin association induces NGPS phenotypes via altered gene expression.

Inhibition of O-GlcNAc transferase activates type I interferon-dependent antitumor immunity by bridging cGAS-STING pathway.

The O-GlcNAc transferase (OGT) is an essential enzyme that mediates protein O-GlcNAcylation, a unique form of posttranslational modification of many nuclear and cytosolic proteins. Recent studies observed increased OGT and O-GlcNAcylation levels in a broad range of human cancer tissues compared to adjacent normal tissues, indicating a universal effect of OGT in promoting tumorigenesis. Here, we show that OGT is essential for tumor growth in immunocompetent mice by repressing the cyclic GMP-AMP synthase (cGAS)-dependent DNA sensing pathway. We found that deletion of OGT (Ogt-/-) caused a marked reduction in tumor growth in both syngeneic mice tumor models and a genetic mice colorectal cancer (CRC) model induced by mutation of the Apc gene (Apcmin). Pharmacological inhibition or genetic deletion of OGT induced a robust genomic instability (GIN), leading to cGAS-dependent production of the type I interferon (IFN-I) and IFN-stimulated genes (ISGs). As a result, deletion of Cgas or Sting from Ogt-/- cancer cells restored tumor growth, and this correlated with impaired CD8+ T-cell-mediated antitumor immunity. Mechanistically, we found that OGT-dependent cleavage of host cell factor C1 (HCF-1) is required for the avoidance of GIN and IFN-I production in tumors. In summary, our results identify OGT-mediated genomic stability and activate cGAS-STING pathway as an important tumor-cell-intrinsic mechanism to repress antitumor immunity.

Super-resolved analysis of colocalization between replication and transcription along the cell cycle in a model of oncogene activation

To understand how oncogenes affect genome organization, it is essential to visualize fundamental processes such as DNA replication and transcription at high resolution in intact cells. At the same time, it is important to determine the progression of the cell along the cell cycle, as cell cycle regulation is crucial for the control of cell proliferation and oncogenesis. Here, we present a super-resolution imaging-based method to analyze single cell nuclei sorted according to specific phases of the cell cycle. The sorting is based on the evaluation of the number and the intensity of pixels in the replication foci image and the colocalization analysis is based on image cross-correlation spectroscopy (ICCS). We evaluate the colocalization between replication and transcription, at different cell cycle phases, in a model of PML-RARα oncogene activation. We find that colocalization between replication and transcription is higher in cells in early S phase compared to cells in middle and late S phase. When we turn on the PML-RARα oncogene, this colocalization pattern is preserved but we detect an increase of colocalization between replication and transcription in the early S phase which points to an effect of the PML-RARα oncogene on the coordination between replication and transcription.

Isolation and focal treatment of brain aneurysms using interfacial fluid trapping.

Current approaches for localized intravascular treatments rely on using solid implants, such as metallic coils for embolizing aneurysms, or on direct injection of a therapeutic agent that can disperse from the required site of action. Here, we present a fluid-based strategy for localizing intravascular therapeutics that leverages surface tension and immiscible fluid interactions, to allow confined and focal treatment at brain aneurysm sites. We first show, computationally and experimentally, that an immiscible phase can be robustly positioned at the neck of human aneurysm models to seal and isolate the aneurysm's cavity for further treatment, including in wide-neck aneurysms. We then demonstrate localized delivery and confined treatment, by selective staining of cell nuclei within the aneurysm cavity as well as by hydrogel-based embolization in patient-specific aneurysm models. Altogether, our interfacial flow-driven strategy offers a potential approach for intravascular localized treatment of cardiovascular and other diseases.