Drosophila CTP synthase regulates collective cell migration by controlling the polarized endocytic cycle.

Phosphatidylinositol (PI) 4,5-bisphosphate (PIP2) is involved in many biological functions. However, the mechanisms of PIP2 in collective cell migration remain elusive. This study highlights the regulatory role of cytidine triphosphate synthase (CTPsyn) in collective border cell migration through regulating the asymmetrical distribution of PIP2. We demonstrated that border cell clusters containing mutant CTPsyn cells suppressed migration. CTPsyn was co-enriched with Actin at the leading edge of the Drosophila border cell cluster where PIP2 was enriched, and this enrichment depended on the CTPsyn activity. Genetic interactions of border cell migration were found between CTPsyn mutant and genes in PI biosynthesis. The CTPsyn reduction resulted in loss of the asymmetric activity of endocytosis recycling. Also, genetic interactions were revealed between components of the exocyst complex and CTPsyn mutant, indicating that CTPsyn activity regulates the PIP2-related asymmetrical exocytosis activity. Furthermore, CTPsyn activity is essential for RTK-polarized distribution in the border cell cluster. We propose a model in which CTPsyn activity is required for the asymmetrical generation of PIP2 to enrich RTK signaling through endocytic recycling in collective cell migration.

Case Report: First Case of Non-restrictive Ventricular Septal Defect With Congestive Heart Failure in a Chinese Han Male Infant Carrying a Class II Chromosome 17p13.3 Microduplication.

Chromosome 17p13.3 microduplication syndrome is considered a multisystem disorder that results in a wide variety of clinical manifestations including dysmorphic facial characteristics, brain structural malformations, developmental restriction, growth restriction, and neurocognitive disorders. The two major classes of chromosome 17p13.3 microduplication, which have different clinical presentations, are associated with specific genetic regions. Among the various known phenotypes, scattered cases with congenital heart disease (CHD) have been reported for both classes of chromosome 17p13.3 microduplication syndrome. Unfortunately, there is insufficient understanding of the correlation between chromosome anomaly induced alterations in gene expression and aberrant cardiac development, and thus early diagnosis of CHD among patients with chromosome 17p13.3 microduplication is difficult without routine prenatal cardiac assessment. One such congenital heart anomalies known to affect a substantial number of newborns worldwide is ventricular septal defect (VSD), which has been found in 17p13.3 microduplication carriers, and seems to sometimes undergo spontaneous closure. We report an unprecedented case of moderate sized perimembranous-outlet VSD and congestive heart failure (CHF) in a Chinese Han male infant with a class II chromosome 17p13.3 microduplication. Despite the fact that cytogenic testing and fetal echocardiography confirmed a 249-Kb chromosome duplication within 17p13.3 that encompassed the PAFAH1B1 gene and showed the presence of VSD during prenatal period, this patient still developed a range of symptoms including sustained prolonged feeding, dyspnea, diaphoresis and retarded growth. A physical examination indicated hepatomegaly and a grade III/VI pan-systolic murmur along the left upper sternal border. Laboratory testing showed a high serum pro-B-type natriuretic peptide (pro-BNP). Imaging studies revealed cardiomegaly and a persistent VSD with related pulmonary stenosis. Since the clinical findings were compatible with CHF, we provided mainline treatment with digoxin, captopril, and furosemide, as well as fluid restriction. Despite sustained poor weight gain, the feeding behavior and the respiratory conditions of the patient improved gradually. This case report and literature review suggest that patients carrying chromosome 17p13.3 microduplication who have VSD may have an increased risk of developing CHF as young infants and hence a comprehensive cardiac evaluation is warranted to allow the early diagnosis and management of any severe heart anomalies.

Cyclic Strain Mitigates Nanoparticle Internalization by Vascular Smooth Muscle Cells.

Background: Intravascular delivery of nanoparticles for theranostic application permits direct interaction of nanoparticles and vascular cells. Since vascular smooth muscle cells (VSMCs), the major components of the vascular wall, are constantly subjected to mechanical stimulation from hemodynamic influence, we asked whether cyclic strain may modulate internalization of magnetic nanoparticles (MNPs) by cultured VSMCs. Methods: Cyclic strain (1 Hz and 10%) was applied with Flexcell system in cultured VSMCs from rats, with cell-associated MNPs (MNPcell) determined by a colorimetric iron assay. Transmission and scanning electron microscopy were used for morphology studies. Confocal microscopy was used to demonstrate distribution of actin assembly in VSMCs. Results: Incubation of poly(acrylic acid) (PAA)-coated MNPs with VSMCs for 4 h induced microvilli formation and MNP internalization. Application of cyclic strain for 4-12 h significantly reduced MNPcell by up to 65% (p < 0.05), which was associated with blunted microvilli and reduced vesicle size/cell, but not vesicle numbers/cell. Confocal microscopy demonstrated that both cyclic strain and fibronectin coating of the culture plate reduced internalized MNPs, which were co-localized with vinculin. Furthermore, cytochalasin D reduced MNPcell, suggesting a role of actin polymerization in MNP uptake by VSMCs; however, a myosin II ATPase inhibitor, blebbistatin, exhibited no effect. Cyclic strain also attenuated uptake of PAA-MNPs by LN-229 cells and uptake of poly-L-lysine-coated MNPs by VSMCs. Conclusion: In such a dynamic milieu, cyclic strain may impede cellular internalization of nanocarriers, which spares the nanocarriers and augments their delivery to the target site in the lumen of vessels or outside of the circulatory system.

New insights into precocious puberty and ADHD: a nationwide cohort study.

BACKGROUND: Attention deficit-hyperactivity disorder (ADHD) is one of the most common neurodevelopmental disorders in children; however, studies delineating the association between ADHD and central precocious puberty are limited. This study aimed to understand whether children with ADHD are at a higher risk of central precocious puberty. METHODS: This population-based retrospective cohort study was conducted using the National Health Insurance Research Database of Taiwan to investigate the association between ADHD and the incidence of central precocious puberty between 2000-2015. We identified ADHD individuals treated with methylphenidate, atomoxetine or not. The control cohort consisted of individuals without ADHD. The outcome measure was central precocious puberty diagnosis. RESULTS: Among 290,148 children (mean age: 5.83 years), central precocious puberty incidence was 4.24 and 1.95 per 105 person-years in the ADHD and control groups, respectively. Children with ADHD treated with medication had a higher risk than those without ADHD. However, medication use did not affect the incidence of central precocious puberty among children with ADHD. CONCLUSION: This study showed an association between ADHD and a higher risk of central precocious puberty. Early referral of children with ADHD to a pediatric endocrinologist for evaluation may facilitate correct diagnoses and early interventions. IMPACT: ADHD is associated with a higher risk of central precocious puberty. This study provides relevant findings, as it is the first nationwide, population-based cohort study to investigate the association between ADHD and the risk of central precocious puberty with a 15-year follow-up. Early referral of children with ADHD to a pediatric endocrinologist for the evaluation of suspected precocious puberty could facilitate correct diagnosis. Early intervention treatment with gonadotropin-releasing hormone agonist might improve final height in children with central precocious puberty.

Differentiation of primary central nervous system lymphoma from glioblastoma using optical coherence tomography based on attention ResNet.

Significance: Differentiation of primary central nervous system lymphoma from glioblastoma is clinically crucial to minimize the risk of treatments, but current imaging modalities often misclassify glioblastoma and lymphoma. Therefore, there is a need for methods to achieve high differentiation power intraoperatively. Aim: The aim is to develop and corroborate a method of classifying normal brain tissue, glioblastoma, and lymphoma using optical coherence tomography with deep learning algorithm in an ex vivo experimental design. Approach: We collected tumor specimens from ordinal surgical operations and measured them with optical coherence tomography. An attention ResNet deep learning model was utilized to differentiate glioblastoma and lymphoma from normal brain tissues. Results: Our model demonstrated a robust classification power of detecting tumoral tissues from normal tissues and moderate discrimination between lymphoma and glioblastoma. Moreover, our results showed good consistency with the previous histological findings in the pathological manifestation of lymphoma, and this could be important from the aspect of future clinical practice. Conclusion: We proposed and demonstrated a quantitative approach to distinguish different brain tumor types. Using our method, both neoplasms can be identified and classified with high accuracy. Hopefully, the proposed method can finally assist surgeons with decision-making intraoperatively.

Sialylation of CD55 by ST3GAL1 Facilitates Immune Evasion in Cancer.

Altered glycosylations, which are associated with expression and activities of glycosyltransferases, can dramatically affect the function of glycoproteins and modify the behavior of tumor cells. ST3GAL1 is a sialyltransferase that adds sialic acid to core 1 glycans, thereby terminating glycan chain extension. In breast carcinomas, overexpression of ST3GAL1 promotes tumorigenesis and correlates with increased tumor grade. In pursuing the role of ST3GAL1 in breast cancer using ST3GAL1-siRNA to knockdown ST3GAL1, we identified CD55 to be one of the potential target proteins of ST3GAL1. CD55 is an important complement regulatory protein, preventing cells from complement-mediated cytotoxicity. CD55 had one N-linked glycosylation site in addition to a Ser/Thr-rich domain, which was expected to be heavily O-glycosylated. Detailed analyses of N- and O-linked oligosaccharides of CD55 released from scramble or ST3GAL1 siRNA-treated breast cancer cells by tandem mass spectrometry revealed that the N-glycan profile was not affected by ST3GAL1 silencing. The O-glycan profile of CD55 demonstrated a shift in abundance to nonsialylated core 1 and monosialylated core 2 at the expense of the disialylated core 2 structure after ST3GAL1 silencing. We also demonstrated that O-linked desialylation of CD55 by ST3GAL1 silencing resulted in increased C3 deposition and complement-mediated lysis of breast cancer cells and enhanced sensitivity to antibody-dependent cell-mediated cytotoxicity. These data demonstrated that ST3GAL1-mediated O-linked sialylation of CD55 acts like an immune checkpoint molecule for cancer cells to evade immune attack and that inhibition of ST3GAL1 is a potential strategy to block CD55-mediated immune evasion.

SNAP29 mediates the assembly of histidine-induced CTP synthase filaments in proximity to the cytokeratin network.

Under metabolic stress, cellular components can assemble into distinct membraneless organelles for adaptation. One such example is cytidine 5'-triphosphate synthase (CTPS, for which there are CTPS1 and CTPS2 forms in mammals), which forms filamentous structures under glutamine deprivation. We have previously demonstrated that histidine (His)-mediated methylation regulates the formation of CTPS filaments to suppress enzymatic activity and preserve the CTPS protein under glutamine deprivation, which promotes cancer cell growth after stress alleviation. However, it remains unclear where and how these enigmatic structures are assembled. Using CTPS-APEX2-mediated in vivo proximity labeling, we found that synaptosome-associated protein 29 (SNAP29) regulates the spatiotemporal filament assembly of CTPS along the cytokeratin network in a keratin 8 (KRT8)-dependent manner. Knockdown of SNAP29 interfered with assembly and relaxed the filament-induced suppression of CTPS enzymatic activity. Furthermore, APEX2 proximity labeling of keratin 18 (KRT18) revealed a spatiotemporal association of SNAP29 with cytokeratin in response to stress. Super-resolution imaging suggests that during CTPS filament formation, SNAP29 interacts with CTPS along the cytokeratin network. This study links the cytokeratin network to the regulation of metabolism by compartmentalization of metabolic enzymes during nutrient deprivation.

Fine-Tuning of Type I Interferon Response by STAT3.

Type I interferon (IFN-I) is induced during innate immune response and is required for initiating antiviral activity, growth inhibition, and immunomodulation. STAT1, STAT2, and STAT3 are activated in response to IFN-I stimulation. STAT1, STAT2, and IRF9 form ISGF3 complex which transactivates downstream IFN-stimulated genes and mediates antiviral response. However, the role of STAT3 remains to be characterized. Here, we review the multiple actions of STAT3 on suppressing IFN-I responses, including blocking IFN-I signaling, downregulating the expression of ISGF3 components, and antagonizing the transcriptional activity of ISGF3. Finally, we discuss the evolution of the suppressive activity of STAT3 and the therapeutic potential of STAT3 inhibitors in host defense against viral infections and IFN-I-associated diseases.

The methionine salvage pathway-involving ADI1 inhibits hepatoma growth by epigenetically altering genes expression via elevating S-adenosylmethionine.

The 5'-methylthioadenosine (MTA) cycle-participating human acireductone dioxygenase 1 (ADI1) has been implicated as a tumor suppressor in prostate cancer, yet its role remains unclear in hepatocellular carcinoma (HCC). Here, we demonstrated a significant reduction of ADI1, either in protein or mRNA level, in HCC tissues. Additionally, higher ADI1 levels were associated with favorable postoperative recurrence-free survival in HCC patients. By altering ADI1 expression in HCC cells, a negative correlation between ADI1 and cell proliferation was observed. Cell-based and xenograft experiments were performed by using cells overexpressing ADI1 mutants carrying mutations at the metal-binding sites (E94A and H133A, respectively), which selectively disrupted differential catalytic steps, resulting in staying or leaving the MTA cycle. The results showed that the growth suppression effect was mediated by accelerating the MTA cycle. A cDNA microarray analysis followed by verification experiments identified that caveolin-1 (CAV1), a growth-promoting protein in HCC, was markedly decreased upon ADI1 overexpression. Suppression of CAV1 expression was mediated by an increase of S-adenosylmethionine (SAMe) level. The methylation status of CAV1 promoter was significantly altered upon ADI1 overexpression. Finally, a genome-wide methylation analysis revealed that ADI1 overexpression altered promoter methylation profiles in a set of cancer-related genes, including CAV1 and genes encoding antisense non-coding RNAs, long non-coding RNAs, and microRNAs, resulting in significant changes of their expression levels. In conclusion, ADI1 expression promoted MTA cycle to increase SAMe levels, which altered genome-wide promoter methylation profiles, resulting in altered gene expression and HCC growth suppression.

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.

IMP/GTP balance modulates cytoophidium assembly and IMPDH activity.

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.

LIMCH1 regulates nonmuscle myosin-II activity and suppresses cell migration.

Nonmuscle myosin II (NM-II) is an important motor protein involved in cell migration. Incorporation of NM-II into actin stress fiber provides a traction force to promote actin retrograde flow and focal adhesion assembly. However, the components involved in regulation of NM-II activity are not well understood. Here we identified a novel actin stress fiber-associated protein, LIM and calponin-homology domains 1 (LIMCH1), which regulates NM-II activity. The recruitment of LIMCH1 into contractile stress fibers revealed its localization complementary to actinin-1. LIMCH1 interacted with NM-IIA, but not NM-IIB, independent of the inhibition of myosin ATPase activity with blebbistatin. Moreover, the N-terminus of LIMCH1 binds to the head region of NM-IIA. Depletion of LIMCH1 attenuated myosin regulatory light chain (MRLC) diphosphorylation in HeLa cells, which was restored by reexpression of small interfering RNA-resistant LIMCH1. In addition, LIMCH1-depleted HeLa cells exhibited a decrease in the number of actin stress fibers and focal adhesions, leading to enhanced cell migration. Collectively, our data suggest that LIMCH1 plays a positive role in regulation of NM-II activity through effects on MRLC during cell migration.

Ubiquitination and filamentous structure of cytidine triphosphate synthase.

Living organisms respond to nutrient availability by regulating the activity of metabolic enzymes. Therefore, the reversible post-translational modification of an enzyme is a common regulatory mechanism for energy conservation. Recently, cytidine-5'-triphosphate (CTP) synthase was discovered to form a filamentous structure that is evolutionarily conserved from flies to humans. Interestingly, induction of the formation of CTP synthase filament is responsive to starvation or glutamine depletion. However, the biological roles of this structure remain elusive. We have recently shown that ubiquitination regulates CTP synthase activity by promoting filament formation in Drosophila ovaries during endocycles. Intriguingly, although the ubiquitination process was required for filament formation induced by glutamine depletion, CTP synthase ubiquitination was found to be inversely correlated with filament formation in Drosophila and human cell lines. In this article, we discuss the putative dual roles of ubiquitination, as well as its physiological implications, in the regulation of CTP synthase structure.

In Vitro Generation of Murine Plasmacytoid Dendritic Cells from Common Lymphoid Progenitors using the AC-6 Feeder System.

Plasmacytoid dendritic cells (pDCs) are powerful type I interferon (IFN-I) producing cells that are activated in response to infection or during inflammatory responses. Unfortunately, study of pDC function is hindered by their low frequency in lymphoid organs, and existing methods for in vitro DC generation predominantly favor the production of cDCs over pDCs. Here we present a unique approach to efficiently generate pDCs from common lymphoid progenitors (CLPs) in vitro. Specifically, the protocol described details how to purify CLPs from bone marrow and generate pDCs by coculturing with γ-irradiated AC-6 feeder cells in the presence of Flt3 ligand. A unique characteristic of this culture system is that the CLPs migrate underneath the AC-6 cells and become cobblestone area-forming cells, a critical step for expanding pDCs. Morphologically distinct DCs, namely pDCs and cDCs, were generated after approximately 2 weeks with a composition of 70-90% pDCs under optimal conditions. Typically, the number of pDCs generated by this method is roughly 100-fold of the number of CLPs seeded. Therefore, this is a novel system with which to robustly generate the large numbers of pDCs required to facilitate further studies into the development and function of these cells.

Regulation of CTP Synthase Filament Formation During DNA Endoreplication in Drosophila.

CTP synthase (CTPsyn) plays an essential role in DNA, RNA, and lipid synthesis. Recent studies in bacteria, yeast, and Drosophila all reveal a polymeric CTPsyn structure, which dynamically regulates its enzymatic activity. However, the molecular mechanism underlying the formation of CTPsyn polymers is not completely understood. In this study, we found that reversible ubiquitination regulates the dynamic assembly of the filamentous structures of Drosophila CTPsyn. We further determined that the proto-oncogene Cbl, an E3 ubiquitin ligase, controls CTPsyn filament formation in endocycles. While the E3 ligase activity of Cbl is required for CTPsyn filament formation, Cbl does not affect the protein levels of CTPsyn. It remains unclear whether the regulation of CTPsyn filaments by Cbl is through direct ubiquitination of CTPsyn. In the absence of Cbl or with knockdown of CTPsyn, the progression of the endocycle-associated S phase was impaired. Furthermore, overexpression of wild-type, but not enzymatically inactive CTPsyn, rescued the endocycle defect in Cbl mutant cells. Together, these results suggest that Cbl influences the nucleotide pool balance and controls CTPsyn filament formation in endocycles. This study links Cbl-mediated ubiquitination to the polymerization of a metabolic enzyme and reveals a role for Cbl in endocycles during Drosophila development.

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.

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

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.

ADI1, a methionine salvage pathway enzyme, is required for Drosophila fecundity.

BACKGROUND: Methionine, an essential amino acid, is required for protein synthesis and normal cell metabolism. The transmethylation pathway and methionine salvage pathway (MTA cycle) are two major pathways regulating methionine metabolism. Recently, methionine has been reported to play a key role in Drosophila fecundity. RESULTS: Here, we revealed that the MTA cycle plays a crucial role in Drosophila fecundity using the mutant of aci-reductone dioxygenase 1 (DADI1), an enzyme in the MTA cycle. In dietary restriction condition, the egg production of adi1 mutant flies was reduced compared to that of control flies. This fecundity defect in mutant flies was rescued by reintroduction of Dadi1 gene. Moreover, a functional homolog of human ADI1 also recovered the reproduction defect, in which the enzymatic activity of human ADI1 is required for normal fecundity. Importantly, methionine supply rescued the fecundity defect in Dadi1 mutant flies. The detailed analysis of Dadi1 mutant ovaries revealed a dramatic change in the levels of methionine metabolism. In addition, we found that three compounds namely, methionine, SAM and Methionine sulfoxide, respectively, may be required for normal fecundity. CONCLUSIONS: In summary, these results suggest that ADI1, an MTA cycle enzyme, affects fly fecundity through the regulation of methionine metabolism.