Publications by authors named "Chia-Chun Chang"

24 Publications

  • Page 1 of 1

Structural basis for ligand binding modes of CTP synthase.

Proc Natl Acad Sci U S A 2021 Jul;118(30)

School of Life Science and Technology, ShanghaiTech University, Shanghai, 201210, China;

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 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.
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http://dx.doi.org/10.1073/pnas.2026621118DOI Listing
July 2021

CTPS forms the cytoophidium in zebrafish.

Exp Cell Res 2021 Jun 12;405(2):112684. Epub 2021 Jun 12.

School of Life Science and Technology, ShanghaiTech University, Shanghai, 201210, China; Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, OX1 3PT, United Kingdom. Electronic address:

Cytidine triphosphate synthase (CTPS) catalyzes the rate-limiting step of de novo CTP biosynthesis. An intracellular structure of CTPS, the cytoophidium, has been found in many organisms including prokaryotes and eukaryotes. Formation of the cytoophidium has been suggested to regulate the activity and stability of CTPS and may participate in certain physiological events. Herein, we demonstrate that both CTPS1a and CTPS1b in zebrafish are able to form the cytoophidium in cultured cells. A point mutation, H355A, abrogates cytoophidium assembly of zebrafish CTPS1a and CTPS1b. In addition, we show the presence of CTPS cytoophidia in multiple tissues of larval and adult fish under normal conditions, while treatment with a CTPS inhibitor 6-diazo-5-oxo-l-norleucine (DON) can induce more cytoophidia in some tissues. Our findings reveal that forming the CTPS cytoophidium is a natural phenomenon of zebrafish and provide valuable information for future research on the physiological importance of this intracellular structure in vertebrates.
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http://dx.doi.org/10.1016/j.yexcr.2021.112684DOI Listing
June 2021

IMPDH forms the cytoophidium in zebrafish.

Dev Biol 2021 May 25;478:89-101. Epub 2021 May 25.

School of Life Science and Technology, ShanghaiTech University, Shanghai, 201210, China. Electronic address:

Inosine monophosphate dehydrogenase (IMPDH) catalyzes the rate-limiting step in de novo guanine nucleotide biosynthesis. Its activity is negatively regulated by the binding of GTP. IMPDH can form a membraneless subcellular structure termed the cytoophidium in response to certain changes in the metabolic status of the cell. The polymeric form of IMPDH, which is the subunit of the cytoophidium, has been shown to be more resistant to the inhibition by GTP at physiological concentrations, implying a functional correlation between cytoophidium formation and the upregulation of GTP biosynthesis. Herein we demonstrate that zebrafish IMPDH1b and IMPDH2 isoforms can assemble abundant cytoophidium in most of cultured cells under stimuli, while zebrafish IMPDH1a shows distinctive properties of forming the cytoophidium in different cell types. Point mutations that disrupt cytoophidium structure in mammalian models also prevent the aggregation of zebrafish IMPDHs. In addition, we discover the presence of the IMPDH cytoophidium in various tissues of larval and adult fish under normal growth conditions. Our results reveal that polymerization and cytoophidium assembly of IMPDH can be a regulatory machinery conserved among vertebrates, and with specific physiological purposes.
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http://dx.doi.org/10.1016/j.ydbio.2021.05.017DOI Listing
May 2021

CTPS and IMPDH form cytoophidia in developmental thymocytes.

Exp Cell Res 2021 Aug 20;405(1):112662. Epub 2021 May 20.

Institute of Biotechnology, National Taiwan University, Taipei, 106, Taiwan; Agricultural Biotechnology Research Center, Academia Sinica, Taipei, 115, Taiwan; Animal Resource Center, National Taiwan University, Taipei, 106, Taiwan. Electronic address:

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.
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http://dx.doi.org/10.1016/j.yexcr.2021.112662DOI Listing
August 2021

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

Biochem Biophys Res Commun 2021 01 10;534:53-58. Epub 2020 Dec 10.

Department of Biochemical Science and Technology, National Taiwan University, Taipei, Taiwan. Electronic address:

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.
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http://dx.doi.org/10.1016/j.bbrc.2020.12.009DOI Listing
January 2021

Detection and phylogenetic analysis of porcine circovirus type 3 in Taiwan.

Arch Virol 2021 Jan 9;166(1):259-263. Epub 2020 Nov 9.

Graduate Institute of Microbiology and Public Health, College of Veterinary Medicine, National Chung Hsing University, 145 Xingda Road, Taichung, 40227, Taiwan, Republic of China.

Porcine circovirus type 3 (PCV3) is a newly emerging porcine circovirus that infects pig populations worldwide. In this study, we investigated the prevalence of PCV3 in Taiwan and analyzed the phylogenetic relationships between the Taiwanese PCV3 strains and those from other countries. A total of 463 clinical specimens from sick pigs were collected in 2016-2019 and analyzed for PCV3 by PCR. The positivity rate for PCV3 was 10.6% in 2016, increasing markedly to 34.78% in 2019. A phylogenetic analysis based on full-length genomic sequences of PCV3 divided the PCV3 strains into three clades, with the Taiwanese strains in clade 1.
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http://dx.doi.org/10.1007/s00705-020-04870-6DOI Listing
January 2021

Drosophila CTP synthase can form distinct substrate- and product-bound filaments.

J Genet Genomics 2019 11 29;46(11):537-545. Epub 2019 Nov 29.

School of Life Science and Technology, ShanghaiTech University, Shanghai, 201210, China. Electronic address:

Intracellular compartmentation is a key strategy for the functioning of a cell. In 2010, several studies revealed that the metabolic enzyme CTP synthase (CTPS) can form filamentous structures termed cytoophidia in prokaryotic and eukaryotic cells. However, recent structural studies showed that CTPS only forms inactive product-bound filaments in bacteria while forming active substrate-bound filaments in eukaryotic cells. In this study, using negative staining and cryo-electron microscopy, we demonstrate that Drosophila CTPS, whether in substrate-bound or product-bound form, can form filaments. Our results challenge the previous model and indicate that substrate-bound and product-bound filaments can coexist in the same species. We speculate that the ability to switch between active and inactive cytoophidia in the same cells provides an additional layer of metabolic regulation.
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http://dx.doi.org/10.1016/j.jgg.2019.11.006DOI Listing
November 2019

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

Cell Rep 2018 09;24(10):2733-2745.e7

Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, Taoyuan 33302, Taiwan; Department of Biochemistry and Molecular Biology, College of Medicine, Chang Gung University, Taoyuan 33302, Taiwan; Molecular Medicine Research Center, Chang Gung University, Taoyuan 33302, Taiwan; Liver Research Center, Chang Gung Memorial Hospital, Linkou, Taiwan. Electronic address:

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.
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http://dx.doi.org/10.1016/j.celrep.2018.08.007DOI Listing
September 2018

Interfilament interaction between IMPDH and CTPS cytoophidia.

FEBS J 2018 10 31;285(20):3753-3768. Epub 2018 Aug 31.

Department of Physiology, Anatomy and Genetics, University of Oxford, UK.

Inosine monophosphate dehydrogenase (IMPDH) and cytidine triphosphate synthase (CTPS) are two metabolic enzymes that perform rate-limiting steps in the de novo synthesis of purine and pyrimidine nucleotides, respectively. It has been shown that IMPDH and CTPS can comprise a filamentous macrostructure termed the cytoophidium, which may play a role in regulation of their catalytic activity. Although these two proteins may colocalise in the same cytoophidium, how they associate with one another is still elusive. As reported herein, we established a model HeLa cell line coexpressing OFP-tagged IMPDH2 and GFP-tagged CTPS1 and recorded the assembly, disassembly and movement of the cytoophidium in live cells. Moreover, by using super-resolution confocal imaging, we demonstrate how IMPDH- and CTPS-based filaments are aligned or intertwined in the mixed cytoophidium. Collectively, our findings provide a panorama of cytoophidium dynamics and suggest that IMPDH and CTPS cytoophidia may coordinate by interfilament interaction.
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http://dx.doi.org/10.1111/febs.14624DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6220823PMC
October 2018

Enhancing Surface Sensing Sensitivity of Metallic Nanostructures using Blue-Shifted Surface Plasmon Mode and Fano Resonance.

Sci Rep 2018 Jun 27;8(1):9762. Epub 2018 Jun 27.

Research Center for Applied Sciences, Academia Sinica, 128, section 2, Academia Road, Nangkang, Taipei, 11529, Taiwan.

Improving surface sensitivities of nanostructure-based plasmonic sensors is an important issue to be addressed. Among the SPR measurements, the wavelength interrogation is commonly utilized. We proposed using blue-shifted surface plasmon mode and Fano resonance, caused by the coupling of a cavity mode (angle-independent) and the surface plasmon mode (angle-dependent) in a long-periodicity silver nanoslit array, to increase surface (wavelength) sensitivities of metallic nanostructures. It results in an improvement by at least a factor of 4 in the spectral shift as compared to sensors operated under normal incidence. The improved surface sensitivity was attributed to a high refractive index sensitivity and the decrease of plasmonic evanescent field caused by two effects, the Fano coupling and the blue-shifted resonance. These concepts can enhance the sensing capability and be applicable to various metallic nanostructures with periodicities.
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http://dx.doi.org/10.1038/s41598-018-28122-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6021451PMC
June 2018

IMP/GTP balance modulates cytoophidium assembly and IMPDH activity.

Cell Div 2018 15;13. Epub 2018 Jun 15.

1Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, OX1 3PT UK.

Background: Inosine monophosphate dehydrogenase (IMPDH), the rate-limiting enzyme in de novo GTP biosynthesis, plays an important role in cell metabolism and proliferation. It has been demonstrated that IMPDH can aggregate into a macrostructure, termed the cytoophidium, in mammalian cells under a variety of conditions. However, the regulation and function of the cytoophidium are still elusive.

Results: In this study, we report that spontaneous filamentation of IMPDH is correlated with rapid cell proliferation. Intracellular IMP accumulation promoted cytoophidium assembly, whereas elevated GTP level triggered disassociation of aggregates. By using IMPDH2 CBS domain mutant cell models, which are unable to form the cytoophidium, we have determined that the cytoophidium is of the utmost importance for maintaining the GTP pool and normal cell proliferation in the condition that higher IMPDH activity is required.

Conclusions: Together, our results suggest a novel mechanism whereby cytoophidium assembly upregulates IMPDH activity and mediates guanine nucleotide homeostasis.
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http://dx.doi.org/10.1186/s13008-018-0038-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6004095PMC
June 2018

CTP synthase forms the cytoophidium in human hepatocellular carcinoma.

Exp Cell Res 2017 12 31;361(2):292-299. Epub 2017 Oct 31.

Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford OX1 3PT, United Kingdom; School of Life Science and Technology, ShanghaiTech University, Shanghai 201210, China. Electronic address:

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.
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http://dx.doi.org/10.1016/j.yexcr.2017.10.030DOI Listing
December 2017

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

Sci Rep 2017 03 8;7:44104. Epub 2017 Mar 8.

Research Center for Applied Sciences, Academia Sinica, 128, section 2, Academia Road, Nangkang, Taipei 11529, Taiwan.

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.
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http://dx.doi.org/10.1038/srep44104DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5341018PMC
March 2017

Enhancing the Surface Sensitivity of Metallic Nanostructures Using Oblique-Angle-Induced Fano Resonances.

Sci Rep 2016 09 9;6:33126. Epub 2016 Sep 9.

Research Center for Applied Sciences, Academia Sinica, 128, section 2, Academia Road, Nangkang, Taipei 11529, Taiwan.

Surface sensitivity is an important factor that determines the minimum amount of biomolecules detected by surface plasmon resonance (SPR) sensors. We propose the use of oblique-angle-induced Fano resonances caused by two-mode coupling or three-mode coupling between the localized SPR mode and long-range surface plasmon polariton modes to increase the surface sensitivities of silver capped nanoslits. The results indicate that the coupled resonance between the split SPR (-kSPR) and cavity modes (two-mode coupling) has a high wavelength sensitivity for small-angle incidence (2°) due to its short decay length. Additionally, three-mode coupling between the split SPR (-kSPR), substrate (+kSub) and cavity modes has a high intensity sensitivity for large-angle incidence due to its short decay length, large resonance slope and enhanced transmission intensity. Compared to the wavelength measurement, the intensity measurement has a lower detectable (surface) concentration below 1 ng/ml (0.14 pg/mm(2)) and is reduced by at least 3 orders of magnitude. In addition, based on the calibration curve and current system noise, a theoretical detection limit of 2.73 pg/ml (0.38 fg/mm(2)) can be achieved. Such a surface concentration is close to that of prism-based SPR with phase measurement (0.1-0.2 fg/mm(2) under a phase shift of 5 mdeg).
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http://dx.doi.org/10.1038/srep33126DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5016831PMC
September 2016

Production of Live Offspring from Vitrified-Warmed Oocytes Collected at Metaphase I Stage.

PLoS One 2016 22;11(6):e0157785. Epub 2016 Jun 22.

Institute of Biotechnology, National Taiwan University, Taipei, Taiwan.

Vitrification of matured oocytes is widely adopted in human clinics and animal research laboratories. Cryopreservation of immature oocytes, particularly those at metaphase I (MI), remains a challenge. In the present work, mouse MI oocytes denuded of cumulus cells were vitrified and warmed (V/W) either prior to (V/W-BEFORE-IVM, n = 562) or after (V/W-AFTER-IVM, n = 664) in vitro maturation (IVM). Derivative metaphase II (MII) oocytes were then used for intracytoplasmic sperm injection (ICSI). In the control groups, in vivo matured MII oocytes were used freshly (FRESH-MII, n = 517) or after V/W (MII-V/W, n = 617). In vitro and in vivo developmental competencies were compared among groups. Satisfactory blastocyst rates were achieved in V/W-BEFORE-IVM (27.5%) and V/W-AFTER-IVM (32.4%) groups, albeit as expected still lower than those from fresh-MII (56.1%) or MII-V/W (45.6%) oocytes. Similarly, the term development rates from V/W-BEFORE-IVM and V/W-AFTER-IVM were 12.4% and 16.7% respectively, acceptable but lower than those of the fresh-MII (41.2%) and MII-V/W (23.3%) groups. These data demonstrate that oocytes collected at MI stage are amenable to V/W, which can be performed before or after IVM with acceptable development rates including production of healthy pups. These findings provide useful knowledge to researchers and clinical practitioners for preservation and use of the otherwise discarded MI oocytes.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0157785PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4917218PMC
July 2017

Cytoophidium assembly reflects upregulation of IMPDH activity.

J Cell Sci 2015 Oct 24;128(19):3550-5. Epub 2015 Aug 24.

Institute of Biotechnology, National Taiwan University, Taipei 106, Taiwan, Republic of China Agricultural Biotechnology Research Center, Academia Sinica, Taipei 115, Taiwan, Republic of China

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.
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http://dx.doi.org/10.1242/jcs.175265DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4610212PMC
October 2015

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

Cell Rep 2014 Dec 20;9(5):1603-1609. Epub 2014 Nov 20.

Center for Advanced Models for Translational Sciences and Therapeutics, University of Michigan Medical Center, Ann Arbor, MI 48109, USA. Electronic address:

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.
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http://dx.doi.org/10.1016/j.celrep.2014.10.052DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4268138PMC
December 2014

Nucleotide synthesis is regulated by cytoophidium formation during neurodevelopment and adaptive metabolism.

Biol Open 2014 Oct 17;3(11):1045-56. Epub 2014 Oct 17.

Medical Research Council Functional Genomics Unit, Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford OX1 3PT, United Kingdom

The essential metabolic enzyme CTP synthase (CTPsyn) can be compartmentalised to form an evolutionarily-conserved intracellular structure termed the cytoophidium. Recently, it has been demonstrated that the enzymatic activity of CTPsyn is attenuated by incorporation into cytoophidia in bacteria and yeast cells. Here we demonstrate that CTPsyn is regulated in a similar manner in Drosophila tissues in vivo. We show that cytoophidium formation occurs during nutrient deprivation in cultured cells, as well as in quiescent and starved neuroblasts of the Drosophila larval central nervous system. We also show that cytoophidia formation is reversible during neurogenesis, indicating that filament formation regulates pyrimidine synthesis in a normal developmental context. Furthermore, our global metabolic profiling demonstrates that CTPsyn overexpression does not significantly alter CTPsyn-related enzymatic activity, suggesting that cytoophidium formation facilitates metabolic stabilisation. In addition, we show that overexpression of CTPsyn only results in moderate increase of CTP pool in human stable cell lines. Together, our study provides experimental evidence, and a mathematical model, for the hypothesis that inactive CTPsyn is incorporated into cytoophidia.
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http://dx.doi.org/10.1242/bio.201410165DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4232762PMC
October 2014

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

PLoS One 2014 8;9(9):e106538. Epub 2014 Sep 8.

Institute of Biotechnology, National Taiwan University, Taipei, Taiwan; Department of Animal Science and Technology, National Taiwan University, Taipei, Taiwan.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0106538PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4157795PMC
May 2015

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

Brain Struct Funct 2015 17;220(3):1539-53. Epub 2014 Mar 17.

Institute of Biotechnology, National Taiwan University, No. 81, Chang-Xiang St., Da-an District, Taipei, 106, Taiwan, ROC.

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.
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http://dx.doi.org/10.1007/s00429-014-0743-7DOI Listing
January 2016

CTP synthase forms cytoophidia in the cytoplasm and nucleus.

Exp Cell Res 2014 Apr 3;323(1):242-253. Epub 2014 Feb 3.

MRC Functional Genomics Unit, Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford OX1 3PT, UK. Electronic address:

CTP synthase is an essential metabolic enzyme responsible for the de novo synthesis of CTP. Multiple studies have recently showed that CTP synthase protein molecules form filamentous structures termed cytoophidia or CTP synthase filaments in the cytoplasm of eukaryotic cells, as well as in bacteria. Here we report that CTP synthase can form cytoophidia not only in the cytoplasm, but also in the nucleus of eukaryotic cells. Both glutamine deprivation and glutamine analog treatment promote formation of cytoplasmic cytoophidia (C-cytoophidia) and nuclear cytoophidia (N-cytoophidia). N-cytoophidia are generally shorter and thinner than their cytoplasmic counterparts. In mammalian cells, both CTP synthase 1 and CTP synthase 2 can form cytoophidia. Using live imaging, we have observed that both C-cytoophidia and N-cytoophidia undergo multiple rounds of fusion upon glutamine analog treatment. Our study reveals the coexistence of cytoophidia in the cytoplasm and nucleus, therefore providing a good opportunity to investigate the intracellular compartmentation of CTP synthase.
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http://dx.doi.org/10.1016/j.yexcr.2014.01.029DOI Listing
April 2014

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

PLoS One 2012 27;7(4):e36085. Epub 2012 Apr 27.

Institute of Biotechnology, National Taiwan University, Taipei, Taiwan.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0036085PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3338631PMC
September 2012

Physicochemical characterization and gene transfection efficiency of lipid emulsions with various co-emulsifiers.

Int J Pharm 2005 Jan 19;289(1-2):197-208. Epub 2004 Dec 19.

School of Medicine, Fu Jen Catholic University, Taipei, Taiwan.

Transfection systems based on complexes of DNA and lipid emulsions were evaluated with respect to their effectiveness, toxicity, physicochemical characteristics, and cell-type dependence. The potential of a series of co-emulsifiers to serve as vectors was investigated. The co-emulsifiers examined included 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), Tween, cholesterol, stearylamine, and polyethylenimine (PEI). Squalane and 1,2-dioleoyl-sn-glycero-3-trimethylammonium-propane (DOTAP), respectively, were the main oil phase and cationic lipid added to the lipid emulsions. Cell viability was reduced after inclusion of either of the two cationic components of stearylamine and PEI. DOPE and cholesterol showed both higher transfection activity and cell viability as compared to the other co-emulsifiers. The incorporation of DOPE and cholesterol also prevented droplet aggregation of the emulsions after long-term storage. Results of the transfection of COS-1, A549, or HaCat cell lines with lipid emulsions indicated differences in transfection activities of each formulation for the different cell lines. It is concluded that DOPE and cholesterol as co-emulsifiers for DOTAP were preferable for stability and DNA transfection of emulsions.
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http://dx.doi.org/10.1016/j.ijpharm.2004.11.008DOI Listing
January 2005

Lipid nano/submicron emulsions as vehicles for topical flurbiprofen delivery.

Drug Deliv 2004 Mar-Apr;11(2):97-105

Graduate Institute of Natural Products, Chang Gung University, Tao-Yuan, Taiwan.

The application of lipid nano/submicron emulsions as topical drug carrier systems for the percutaneous absorption of flurbiprofen was investigated. The lipid emulsions were made up of isopropyl myristate (IPM), soybean oil, or coconut oil as the oil phase, egg lecithin as the predominant emulsifier, and double-distilled water as the external phase. Stearylamine (SA) and deoxycholic acid (DA) also were used to produce positively and negatively charged emulsions. To evaluate the physicochemical properties of the lipid emulsions, particle size by laser light scattering, the image of atomic force microscopy, and relaxation time values by Nuclear Magnetic Resonance (NMR) were determined. The in vitro permeation data showed that incorporation of SA significantly reduced the topical delivery of flurbiprofen. On the other hand, incorporation of DA exhibited no or a negligible effect on drug permeation. Enhancement of drug absorption was observed when adding oleic acid as part of the oil phase. The in vivo topical application of flurbiprofen from selected lipid emulsions showed a similar trend to the in vitro status. Furthermore, the intersubject variability was considerably reduced by lipid emulsions than by aqueous suspensions in both the in vitro and in vivo experiments. The irritant profiles of lipid emulsions showed that IPM elicited higher irritation than soybean oil. The incorporation of oleic acid also produced skin disruption. The results in the present study suggest the feasibility of lipid emulsions for the topical delivery of flurbiprofen.
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http://dx.doi.org/10.1080/10717540490280697DOI Listing
July 2004
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