The IMPDH cytoophidium couples metabolism and fetal development in mice.

The cytoophidium is an evolutionarily conserved subcellular structure formed by filamentous polymers of metabolic enzymes. In vertebrates, inosine monophosphate dehydrogenase (IMPDH), which catalyses the rate-limiting step in guanosine triphosphate (GTP) biosynthesis, is one of the best-known cytoophidium-forming enzymes. Formation of the cytoophidium has been proposed to alleviate the inhibition of IMPDH, thereby facilitating GTP production to support the rapid proliferation of certain cell types such as lymphocytes, cancer cells and pluripotent stem cells (PSCs). However, past studies lacked appropriate models to elucidate the significance of IMPDH cytoophidium under normal physiological conditions. In this study, we demonstrate that the presence of IMPDH cytoophidium in mouse PSCs correlates with their metabolic status rather than pluripotency. By introducing IMPDH2 Y12C point mutation through genome editing, we established mouse embryonic stem cell (ESC) lines incapable of forming IMPDH polymers and the cytoophidium. Our data indicate an important role of IMPDH cytoophidium in sustaining a positive feedback loop that couples nucleotide biosynthesis with upstream metabolic pathways. Additionally, we find that IMPDH2 Y12C mutation leads to decreased cell proliferation and increased DNA damage in teratomas, as well as impaired embryo development following blastocoel injection. Further analysis shows that IMPDH cytoophidium assembly in mouse embryonic development begins after implantation and gradually increases throughout fetal development. These findings provide insights into the regulation of IMPDH polymerisation in embryogenesis and its significance in coordinating cell metabolism and development.

Clinical Characteristics and Treatment Outcomes of Oral Cancers Using Transoral Robotic Surgery in an Endemic Region.

Oral cancer poses a major health challenge in Taiwan, consistently ranking among the highest globally in both incidence and cancer-related mortality. Transoral robotic surgery (TORS) has potential advantages over open surgery, but its long-term oncologic outcomes are not well established. In this study, we sought to elucidate the role of TORS in improving treatment outcomes among oral cancer patients. A case-control study with propensity score matching was conducted in a single teaching hospital in Taiwan. It included 72 oral cancer patients in each group to analyze and compare survival outcomes between the surgical approaches. The TORS group demonstrated a higher negative resection margin rate, a lower mortality risk and better overall survival than the open-surgery group. Multivariate Cox regression analysis confirmed TORS's association with a reduced risk of death. Kaplan-Meier survival analysis and log-rank tests indicated significantly better survival outcomes for the TORS group across all cancer stages. Moreover, the TORS group exhibited improved overall survival rates for stage III and IV patients compared to the conventional open-surgery group. In conclusion, this study suggests that TORS may offer better overall survival rates and potential advantages over conventional surgery for oral cancer treatment.

Cancer survivorship and risk of pregnancy complications, adverse obstetric outcomes, and maternal morbidities in female adolescents and young adults: a nationwide population-based study from Taiwan.

BACKGROUND: Cancer treatment in female adolescent and young adult (AYA) cancer survivors (i.e., those diagnosed between 15 and 39 years of age) may adversely affect multiple bodily functions, including the reproductive system. METHODS: We initially assembled a retrospective, nationwide population-based cohort study by linking data from two nationwide Taiwanese data sets. We subsequently identified first pregnancies and singleton births to AYA cancer survivors (2004-2018) and select AYA without a previous cancer diagnosis matched to AYA cancer survivors for maternal age and infant birth year. RESULTS: The study cohort consisted of 5151 and 51,503 births to AYA cancer survivors and matched AYA without a previous cancer diagnosis, respectively. The odds for overall pregnancy complications (odds ratio [OR], 1.09; 95% confidence interval [CI], 1.01-1.18) and overall adverse obstetric outcomes (OR, 1.07; 95% CI, 1.01-1.13) were significantly increased in survivors compared with matched AYA without a previous cancer diagnosis. Specifically, cancer survivorship was associated with an increased risk of preterm labour, labour induction, and threatened abortion or threatened labour requiring hospitalisation. CONCLUSIONS: AYA cancer survivors are at increased risk for pregnancy complications and adverse obstetric outcomes. Efforts to integrate individualised care into clinical guidelines for preconception and prenatal care should be thoroughly explored.

Structural basis of human PRPS2 filaments.

BACKGROUND: PRPP synthase (PRPS) transfers the pyrophosphate groups from ATP to ribose-5-phosphate to produce 5-phosphate ribose-1-pyrophosphate (PRPP), a key intermediate in the biosynthesis of several metabolites including nucleotides, dinucleotides and some amino acids. There are three PRPS isoforms encoded in human genome. While human PRPS1 (hPRPS1) and human PRPS2 (hPRPS2) are expressed in most tissues, human PRPS3 (hPRPS3) is exclusively expressed in testis. Although hPRPS1 and hPRPS2 share 95% sequence identity, hPRPS2 has been shown to be less sensitive to allosteric inhibition and specifically upregulated in certain cancers in the translational level. Recent studies demonstrate that PRPS can form a subcellular compartment termed the cytoophidium in multiple organisms across prokaryotes and eukaryotes. Forming filaments and cytoophidia is considered as a distinctive mechanism involving the polymerization of the protein. Previously we solved the filament structures of Escherichia coli PRPS (ecPRPS) using cryo-electron microscopy (cryo-EM) 1. RESULTS: Order to investigate the function and molecular mechanism of hPRPS2 polymerization, here we solve the polymer structure of hPRPS2 at 3.08 Å resolution. hPRPS2 hexamers stack into polymers in the conditions with the allosteric/competitive inhibitor ADP. The binding modes of ADP at the canonical allosteric site and at the catalytic active site are clearly determined. A point mutation disrupting the inter-hexamer interaction prevents hPRPS2 polymerization and results in significantly reduced catalytic activity. CONCLUSION: Findings suggest that the regulation of hPRPS2 polymer is distinct from ecPRPS polymer and provide new insights to the regulation of hPRPS2 with structural basis.

Structural basis for ligand binding modes of CTP synthase.

Cytidine triphosphate synthase (CTPS), which comprises an ammonia ligase domain and a glutamine amidotransferase domain, catalyzes the final step of de novo CTP biosynthesis. The activity of CTPS is regulated by the binding of four nucleotides and glutamine. While glutamine serves as an ammonia donor for the ATP-dependent conversion of UTP to CTP, the fourth nucleotide GTP acts as an allosteric activator. Models have been proposed to explain the mechanisms of action at the active site of the ammonia ligase domain and the conformational changes derived by GTP binding. However, actual GTP/ATP/UTP binding modes and relevant conformational changes have not been revealed fully. Here, we report the discovery of binding modes of four nucleotides and a glutamine analog 6-diazo-5-oxo-L-norleucine in Drosophila CTPS by cryo-electron microscopy with near-atomic resolution. Interactions between GTP and surrounding residues indicate that GTP acts to coordinate reactions at both domains by directly blocking ammonia leakage and stabilizing the ammonia tunnel. Additionally, we observe the ATP-dependent UTP phosphorylation intermediate and determine interacting residues at the ammonia ligase. A noncanonical CTP binding at the ATP binding site suggests another layer of feedback inhibition. Our findings not only delineate the structure of CTPS in the presence of all substrates but also complete our understanding of the underlying mechanisms of the allosteric regulation and CTP synthesis.

CTPS and IMPDH form cytoophidia in developmental thymocytes.

The cytoophidium, a filamentous structure formed by metabolic enzymes, has emerged as a novel regulatory machinery for certain proteins. The rate-limiting enzymes of de novo CTP and GTP synthesis, cytidine triphosphate synthase (CTPS) and inosine monophosphate dehydrogenase (IMPDH), are the most characterized cytoophidium-forming enzymes in mammalian models. Although the assembly of CTPS cytoophidia has been demonstrated in various organisms including multiple human cancers, a systemic survey for the presence of CTPS cytoophidia in mammalian tissues in normal physiological conditions has not yet been reported. Herein, we examine major organs of adult mouse and observe that CTPS cytoophidia are displayed by a specific thymocyte population ranging between DN3 to early DP stages. Most of these cytoophidium-presenting cells have both CTPS and IMPDH cytoophidia and undergo rapid cell proliferation. In addition, we show that cytoophidium formation is associated with active glycolytic metabolism as the cytoophidium-presenting cells exhibit higher levels of c-Myc, phospho-Akt and PFK. Inhibition of glycolysis with 2DG, however, disrupts most of cytoophidium structures and impairs cell proliferation. Our findings not only indicate that the regulation of CTPS and IMPDH cytoophidia are correlated with the metabolic switch triggered by pre-TCR signaling, but also suggest physiological roles of the cytoophidium in thymocyte development.

Histone acetyltransferase p300 mediates the upregulation of CTEN induced by the activation of EGFR signaling in cancer cells.

Upregulation of C-terminal tensin-like (CTEN) is induced by the activation of epidermal growth factor receptor (EGFR) signaling and mainly contributes to cancer cell migration and invasion. CTEN is known as a downstream target of the EGFR-RAF-MEK-ERK pathway but the regulatory mechanism underlying EGFR signaling regulates the increased expression of CTEN is still incompletely understood. In this study, we investigated the epigenetic regulation of CTEN gene transcription upon EGFR activation. Analyses of chromatin accessibility revealed that the structure of CTEN promoter became more loosed and the acetylation state of the histone tails within the core promoter region was increased after EGF treatment. Moreover, activation of EGFR signaling facilitates histone acetyltransferase p300 to be recruited to CTEN promoter through MEK-ERK pathway. MEK-ERK activation also induces the phosphorylation of p300, thereby enhancing the levels of histone acetylation within CTEN promoter, which in turn upregulates CTEN gene expression. Our work provides new insights into the actions of EGFR signaling to upregulate CTEN, which may lead to the rational design of novel therapeutic approaches.

Histidine-Dependent Protein Methylation Is Required for Compartmentalization of CTP Synthase.

CTP synthase (CTPS) forms compartmentalized filaments in response to substrate availability and environmental nutrient status. However, the physiological role of filaments and mechanisms for filament assembly are not well understood. Here, we provide evidence that CTPS forms filaments in response to histidine influx during glutamine starvation. Tetramer conformation-based filament formation restricts CTPS enzymatic activity during nutrient deprivation. CTPS protein levels remain stable in the presence of histidine during nutrient deprivation, followed by rapid cell growth after stress relief. We demonstrate that filament formation is controlled by methylation and that histidine promotes re-methylation of homocysteine by donating one-carbon intermediates to the cytosolic folate cycle. Furthermore, we find that starvation stress and glutamine deficiency activate the GCN2/ATF4/MTHFD2 axis, which coordinates CTPS filament formation. CTPS filament formation induced by histidine-mediated methylation may be a strategy used by cancer cells to maintain homeostasis and ensure a growth advantage in adverse environments.

CTP synthase forms the cytoophidium in human hepatocellular carcinoma.

CTP synthase (CTPS) can aggregate into an intracellular macrostructure, the cytoophidium, in various organisms including human cells. Previous studies have shown that assembly of human CTPS cytoophidia may be correlated with the cellular metabolic status, and is able to promote the activity of CTPS. A correlation between the cytoophidium and cancer metabolism has been proposed but not yet been revealed. In the current study we provide clear evidence of the presence of CTPS cytoophidia in various human cancers and some non-cancerous tissues. Moreover, among 203 tissue samples of hepatocellular carcinoma, 56 (28%) samples exhibited many cytoophidia, whereas no cytoophidia were detected in adjacent non-cancerous hepatocytes for all samples. Our findings suggest that the CTPS cytoophidium may participate in the adaptive metabolism of human hepatocellular carcinoma.

Highly Sensitive Aluminum-Based Biosensors using Tailorable Fano Resonances in Capped Nanostructures.

Metallic nanostructure-based surface plasmon sensors are capable of real-time, label-free, and multiplexed detections for chemical and biomedical applications. Recently, the studies of aluminum-based biosensors have attracted a large attention because aluminum is a more cost-effective metal and relatively stable. However, the intrinsic properties of aluminum, having a large imaginary part of the dielectric function and a longer evanescent length, limit its sensing capability. Here we show that capped aluminum nanoslits fabricated on plastic films using hot embossing lithography can provide tailorable Fano resonances. Changing height of nanostructures and deposited metal film thickness modulated the transmission spectrum, which varied from Wood's anomaly-dominant resonance, asymmetric Fano profile to surface plasmon-dominant resonance. For biolayer detections, the maximum surface sensitivity occurred at the dip of asymmetric Fano profile. The optimal Fano factor was close to -1.3. The wavelength and intensity sensitivities for surface thickness were up to 2.58 nm/nm and 90%/nm, respectively. The limit of detection (LOD) of thickness reached 0.018 nm. We attributed the enhanced surface sensitivity for capped aluminum nanoslits to a reduced evanescent length and sharp slope of the asymmetric Fano profile. The protein-protein interaction experiments verified the high sensitivity of capped nanostructures. The LOD was down to 236 fg/mL.

Cytoophidium assembly reflects upregulation of IMPDH activity.

Cytidine triphosphate synthase (CTPS) and inosine monophosphate dehydrogenase (IMPDH) (both of which have two isoforms) can form fiber-like subcellular structures termed 'cytoophidia' under certain circumstances in mammalian cells. Although it has been shown that filamentation of CTPS downregulates its activity by disturbing conformational changes, the activity of IMPDH within cytoophidia is still unclear. Most previous IMPDH cytoophidium studies were performed under conditions involving inhibitors that impair GTP synthesis. Here, we show that IMPDH forms cytoophidia without inhibition of GTP synthesis. First, we find that an elevated intracellular CTP concentration or treatment with 3'-deazauridine, a CTPS inhibitor, promotes IMPDH cytoophidium formation and increases the intracellular GTP pool size. Moreover, restriction of cell growth triggers the disassembly of IMPDH cytoophidia, implying that their presence is correlated with active cell metabolism. Finally, we show that the presence of IMPDH cytoophidia in mouse pancreatic islet cells might correlate with nutrient uptake in the animal. Collectively, our findings reveal that formation of IMPDH cytoophidia reflects upregulation of purine nucleotide synthesis, suggesting that the IMPDH cytoophidium plays a role distinct from that of the CTPS cytoophidium in controlling intracellular nucleotide homeostasis.

SMN is required for the maintenance of embryonic stem cells and neuronal differentiation in mice.

Survival motor neuron (SMN) is the determining factor in spinal muscular atrophy, the most common genetic cause of childhood mortality. We have previously found that SMN regulates stem cell division, proliferation and differentiation in Drosophila. However, it is unknown whether a similar effect exists in vertebrates. Here, we show that SMN is enriched in highly proliferative embryonic stem cells (ESCs) in mice and reduction of SMN impairs the pluripotency of ESCs. Moreover, we find that SMN reduction activates ERK signaling and affects neuronal differentiation in vitro. Teratomas with reduced SMN grow more slowly and show weaker signals of neuronal differentiation than those with a normal level of SMN. Finally, we show that over-expression of SMN is protective for ESCs from retinoic acid-induced differentiation. Taken together, our results suggest that SMN plays a role in the maintenance of pluripotent ESCs and neuronal differentiation in mice.

Telomere elongation and naive pluripotent stem cells achieved from telomerase haplo-insufficient cells by somatic cell nuclear transfer.

Haplo-insufficiency of telomerase genes in humans leads to telomere syndromes such as dyskeratosis congenital and idiopathic pulmonary fibrosis. Generation of pluripotent stem cells from telomerase haplo-insufficient donor cells would provide unique opportunities toward the realization of patient-specific stem cell therapies. Recently, pluripotent human embryonic stem cells (ntESCs) have been efficiently achieved by somatic cell nuclear transfer (SCNT). We tested the hypothesis that SCNT could effectively elongate shortening telomeres of telomerase haplo-insufficient cells in the ntESCs with relevant mouse models. Indeed, telomeres of telomerase haplo-insufficient (Terc(+/-)) mouse cells are elongated in ntESCs. Moreover, ntESCs derived from Terc(+/-) cells exhibit naive pluripotency as evidenced by generation of Terc(+/-) ntESC clone pups by tetraploid embryo complementation, the most stringent test of naive pluripotency. These data suggest that SCNT could offer a powerful tool to reprogram telomeres and to discover the factors for robust restoration of telomeres and pluripotency of telomerase haplo-insufficient somatic cells.

Amniotic fluid stem cells prevent follicle atresia and rescue fertility of mice with premature ovarian failure induced by chemotherapy.

Chemotherapy used to treat cancer may cause irreversible premature ovarian failure (POF). Of late, amniotic fluid stem cells (AFSCs) provide a novel source for regenerative medicine because of their primitive stage, low immunogenicity, and easy accessibility. In this study, we isolated AFSCs from transgenic mice that ubiquitously express enhanced green fluorescence protein (EGFP). These AFSCs exhibited morphologies, immunophenotypes, and mesoderm trilineage differentiation potentials similar to mesenchymal stem cells (MSCs). Further, AFSCs proliferated faster than MSCs and expressed OCT4, a marker for pluripotency. To investigate their potential in recovering fertility in POF model, AFSCs were transplanted into the ovaries of mice with POF six weeks post induction using chemotherapeutic drugs, busulfan and cyclophosphamide. AFSCs could rescue the reproductive ability of mice with POF by preventing follicle atresia and sustaining the healthy follicles. Notably, the transplanted AFSCs did not differentiate into granulosa and germline cells in vivo. After one month, the decreased numbers of transplanted AFSCs accompanied with the reduced beneficial effects indicated that the therapeutic efficacy were directly from AFSCs. These findings demonstrated the therapeutic effects of AFSCs and suggested the promise of AFSCs for treating infertility and POF caused by chemotherapy.

Isolation and characterization of novel murine epiphysis derived mesenchymal stem cells.

BACKGROUND: While bone marrow (BM) is a rich source of mesenchymal stem cells (MSCs), previous studies have shown that MSCs derived from mouse BM (BMMSCs) were difficult to manipulate as compared to MSCs derived from other species. The objective of this study was to find an alternative murine MSCs source that could provide sufficient MSCs. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we described a novel type of MSCs that migrates directly from the mouse epiphysis in culture. Epiphysis-derived MSCs (EMSCs) could be extensively expanded in plastic adherent culture, and they had a greater ability for clonogenic formation and cell proliferation than BMMSCs. Under specific induction conditions, EMSCs demonstrated multipotency through their ability to differentiate into adipocytes, osteocytes and chondrocytes. Immunophenotypic analysis demonstrated that EMSCs were positive for CD29, CD44, CD73, CD105, CD166, Sca-1 and SSEA-4, while negative for CD11b, CD31, CD34 and CD45. Notably, EMSCs did not express major histocompatibility complex class I (MHC I) or MHC II under our culture system. EMSCs also successfully suppressed the proliferation of splenocytes triggered by concanavalin A (Con A) or allogeneic splenocytes, and decreased the expression of IL-1, IL-6 and TNF-α in Con A-stimulated splenocytes suggesting their anti-inflammatory properties. Moreover, EMSCs enhanced fracture repair, ameliorated necrosis in ischemic skin flap, and improved blood perfusion in hindlimb ischemia in the in vivo experiments. CONCLUSIONS/SIGNIFICANCES: These results indicate that EMSCs, a new type of MSCs established by our simple isolation method, are a preferable alternative for mice MSCs due to their better growth and differentiation potentialities.