HMGB2 Promotes De Novo Lipogenesis to Accelerate Hepatocyte Proliferation During Liver Regeneration.

Liver regeneration is a well-orchestrated compensatory process that is regulated by multiple factors. We recently reported the importance of the chromatin protein, a high-mobility group box 2 (HMGB2) in mouse liver regeneration. However, the molecular mechanism remains unclear. In this study, we aimed to study how HMGB2 regulates hepatocyte proliferation during liver regeneration. Seventy-percent partial hepatectomy (PHx) was performed in wild-type (WT) and HMGB2-knockout (KO) mice, and the liver tissues were used for microarray, immunohistochemistry, quantitative polymerase chain reaction (qPCR), and Western blotting analyses. In the WT mice, HMGB2-positive hepatocytes colocalized with cell proliferation markers. In the HMGB2-KO mice, hepatocyte proliferation was significantly decreased. Oil Red O staining revealed the transient accumulation of lipid droplets at 12-24 hr after PHx in the WT mouse livers. In contrast, decreased amount of lipid droplets were found in HMGB2-KO mouse livers, and it was preserved until 36 hr. The microarray, immunohistochemistry, and qPCR results demonstrated that the expression of lipid metabolism-related genes was significantly decreased in the HMGB2-KO mouse livers. The in vitro experiments demonstrated that a decrease in the amount of lipid droplets correlated with decreased cell proliferation activity in HMGB2-knockdown cells. HMGB2 promotes de novo lipogenesis to accelerate hepatocyte proliferation during liver regeneration.

The crucial role of SETDB1 in structural and functional transformation of epithelial cells during regeneration after intestinal ischemia reperfusion injury.

Su (var) 3-9, enhancer of seste, trithorax (SET)-domain bifurcated histone lysine methyltransferase (SETDB1) plays a crucial role in maintaining intestinal stem cell homeostasis; however, its physiological function in epithelial injury is largely unknown. In this study, we investigated the role of SETDB1 in epithelial regeneration using an intestinal ischemia/reperfusion injury (IRI) mouse model. Jejunum tissues were sampled after 75 min of ischemia followed by 3, 24, and 48 h of reperfusion. Morphological evaluations were performed using light microscopy and electron microscopy, and the involvement of SETDB1 in epithelial remodeling was investigated by immunohistochemistry. Expression of SETDB1 was increased following 24 h of reperfusion and localized in not only the crypt bottom but also in the transit amplifying zone and part of the villi. Changes in cell lineage, repression of cell adhesion molecule expression, and decreased histone H3 methylation status were detected in the crypts at the same time. Electron microscopy also revealed aberrant alignment of crypt nuclei and fusion of adjacent villi. Furthermore, increased SETDB1 expression and epithelial remodeling were confirmed with loss of stem cells, suggesting SETDB1 affects epithelial cell plasticity. In addition, crypt elongation and increased numbers of Ki-67 positive cells indicated active cell proliferation after IRI; however, the expression of PCNA was decreased compared to sham mouse jejunum. These morphological changes and the aberrant expression of proliferation markers were prevented by sinefungin, a histone methyltransferase inhibitor. In summary, SETDB1 plays a crucial role in changes in the epithelial structure after IRI-induced stem cell loss.

Protective role of estrogen through G-protein coupled receptor 30 in a colitis mouse model.

Estrogen and its receptors are involved in the pathogenesis of gastrointestinal diseases such as colitis. However, the role of the membrane estrogen receptor G-protein-coupled receptor 30 (GPR30) in colitis is poorly understood. We therefore investigated the effect of estrogen in dextran sulfate sodium (DSS)-induced colitis. Male C57BL/6 mice were administered 1.5% DSS for 5 days and treated with 17ß-estradiol (E2), GPR30 agonist (G1), or GPR30 antagonist (G15) for 8 days. Inflammation grade was evaluated by disease activity index (DAI) and histomorphological score. Colon tissues were immunohistochemically analyzed and revealed high expression of membrane GPR30, histone 3 lysine 36 dimethylation, and lysine 79 trimethylation in normal mouse colon epithelial cells but significantly decreased expression in DSS-treated mice, whereas the expression was partially preserved after treatment with E2 or G1. Colon shortening and DAI were significantly lower in E2- and G1-treated mice compared to DSS-treated mice. Caudal type homeobox 2 (CDX2) expression and cell proliferation differed in normal colon epithelial cells but overlapped in those of DSS-treated mice. Administration of E2 and G1 reduced CDX2 expression and cell proliferation. Altered expression of claudin-2 and occludin were observed in the colonic epithelium of DSS-treated mice, and these changes were significantly lower in the colon of E2- and G1-treated mice. These results indicate that estrogen regulates histone modification, cell proliferation, and CDX2 expression through GPR30, which affects intestinal epithelial barrier function. We conclude that estrogen protects against intestinal epithelial damage through GPR30 by enhancing intestinal epithelial barrier function in DSS-induced colitis in mice.

Regulation of Multidrug Efflux Pumps by TetR Family Transcriptional Repressor Negatively Affects Secondary Metabolism in Streptomyces coelicolor A3(2).

Streptomyces spp. are well-known producers of bioactive secondary metabolites (SMs) that serve as pharmaceutical agents. In addition to their ability to produce SMs, Streptomyces spp. have evolved diverse membrane transport systems to protect cells against antibiotics produced by itself or other microorganisms. We previously screened mutants of Streptomyces coelicolor that show a phenotype of reduced undecylprodigiosin (RED) production in a combined-culture with Tsukamurella pulmonis. Here, we identified a point mutation, which reduced RED production, by performing genome resequencing and genetic complementation. We found that inactivation of the sco1718 gene encoding the TetR family transcriptional regulator (TFR) produced a deficient phenotype for several SMs in Streptomyces coelicolor A3(2). In the genome of S. coelicolor A3(2), two other sets of TFR and two-component ATP-binding cassette (ABC) transporter genes (sco4358-4360 and sco5384-5382) were found which had similar effects on the phenotype for both secondary metabolism and antibiotic resistance. An electrophoretic mobility shift assay and quantitative reverse transcription-PCR experiments demonstrated that TFRs repressed the expression of each adjacent two-component ABC transporter genes by binding to the operator sequence. Notably, the Δsco1718 mutant showed increased resistance to several antibiotics of other actinomycete origin. Our results imply the switching of cell metabolism to direct offense (antibiotic production) or defense (efflux pump activation) using costly and limited quantities of cell energy sources (e.g., ATP) in the soil ecosystem. IMPORTANCE The bacterial metabolic potential to synthesize diverse secondary metabolites in the environment has been revealed by recent (meta)genomics of both unculturable and culturable bacteria. These studies imply that bacteria are continuously exposed to harmful chemical compounds in the environment. Streptomyces spp. contain antibiotic efflux pumps and SM biosynthetic gene clusters. However, the mechanism by which soil bacteria, including Streptomyces, survive against toxic compounds in the environment remains unclear. Here, we identified three sets of TFR-ABC transporter genes in Streptomyces coelicolor A3(2). We found that each TFR controlled the expression of respective ABC transporter, and the expression of all ABC transporters negatively impacted SM production and increased antibiotic resistance. Notably, bioinformatic analysis indicated that these TFR-ABC transporter gene sets are highly conserved and widely distributed in the genome of Streptomyces species, indicating the importance of systematic regulation that directs antibiotic production and xenobiotic excretion.

Pivotal role of High-Mobility Group Box 2 in ovarian folliculogenesis and fertility.

BACKGROUND: High-Mobility Group Box 1 (HMGB1) and HMGB2 are chromatin-associated proteins that belong to the HMG protein family, and are involved in the regulation of DNA transcription during cell differentiation, proliferation and regeneration in various tissues. However, the role of HMGB2 in ovarian folliculogenesis is largely unknown. METHODS: We investigated the functional role of HMGB1 and HMGB2 in ovarian folliculogenesis and fertilization using C57BL/6 wild type (WT) and HMGB2-knockout (KO) mice. Ovarian tissues were obtained from WT and HMGB2-KO mice at postnatal days 0, 3, 7, and 2, 6 months of age, then performed immunohistochemistry, qPCR and Western blotting analyses. Oocyte fertilization capability was examined by natural breeding and in vitro fertilization experiments. RESULTS: In HMGB2-KO mice, ovary weight was decreased due to reduced numbers of oocytes and follicles. Natural breeding and in vitro fertilization results indicated that HMGB2-KO mice are subfertile, but not sterile. Immunohistochemistry showed that oocytes expressed HMGB2, but not HMGB1, in neonatal and adult WT ovaries. Interestingly, in HMGB2-KO ovaries, a compensatory increase in HMGB1 was found in oocyte nuclei of neonatal and 2-month-old mice; however, this was lost at 6 months of age. CONCLUSIONS: The depletion of HMGB2 led to alterations in ovarian morphology and function, suggesting that HMGB2 plays an essential role in ovarian development, folliculogenesis and fertilization.

An Advanced Detection System for In Situ Hybridization Using a Fluorescence Resonance Energy Transfer-based Molecular Beacon Probe.

In situ hybridization (ISH) is a powerful method for detecting specific RNAs at the cellular level. Although conventional ISH using hapten-labeled probes are useful for detecting multiple RNAs, the detection procedures are still complex and required longer time. Therefore, we introduced a new application of fluorescence resonance energy transfer (FRET)-based molecular beacon (MB) probes for ISH. MCF-7 cells and C57BL/6J mouse uterus were used for ISH. MB probes for ERα mRNA and 28S rRNA were labeled with Cy3/BHQ-2 and 6-FAM/DABCYL, and conventional probes were labeled with digoxigenin. Fluorescence measurements revealed that of more-rapid hybridization kinetics compared to conventional probes. In MCF-7 cells, 28S rRNA was detected in nucleolus and cytoplasm of all cells, whereas ERα mRNA was detected in some nucleolus. In the uterus, 28S rRNA was clearly detected using complementary MB probe, but there were no signals in control slides. Moreover, 28S rRNA was detected in all cells, whereas ERα mRNA was detected mainly in the epithelium. Fluorescence intensity of 28S rRNA was decreased significantly in 1 or 2 base-mismatched sequences, that indicates highly specific detection of target RNAs. In conclusion, the FRET-based MB probes are very useful for ISH, providing rapid hybridization, high sensitivity and specificity.

Click chemistry for fluorescence imaging via combination of a BODIPY-based 'turn-on' probe and a norbornene glucosamine.

In the present study, we synthesized a novel near-infrared turn-on BODIPY probe and a new norbornene-modified glucosamine derivative. The probe exhibits a significant NIR fluorescence emission with a turn-on response and can perform tumour-specific imaging in tumour-bearing mice. The non-natural glucosamine provides metabolic glycoengineering labelling. It can be expressed on cells as chemical tags and further reacted with fluorescence dyes for cell labelling. The combination of the two derivatives enables quick and sensitive cell imaging in vitro and in vivo using the iEDDA reaction.

Crucial role of high-mobility group box 2 in mouse ovarian follicular development through estrogen receptor beta.

High-mobility group box 2 (HMGB2) is a chromatin-associated protein that is an important regulator of gene transcription, recombination, and repair processes. The functional importance of HMGB2 has been reported in various organs, including the testis, heart, and cartilage. However, its role in the ovary is largely unknown. In this study, ovary tissues from wild-type (WT) and HMGB2-knock-out (KO) mice were examined by histopathological staining and immunohistochemistry. The ovary size and weight were significantly lower in HMGB2-KO mice than in age-matched WT littermates. Histopathological analysis revealed ovarian atrophy and progressive fibrosis in 10-month-old HMGB2-KO mouse ovaries. Compared to age-matched WT mice, the numbers of oocytes and developing follicles were significantly decreased at 2 months of age and were completely depleted at 10 months of age in HMGB2-KO mice. Immunohistochemistry revealed the expression of HMGB2 in the granulosa cells of developing follicles, oocytes, some corpora lutea, and stromal cells. Importantly, HMGB2-positive cells were co-localized with estrogen receptor beta (ERß), but not ERα. Estrogen response element-binding activity was demonstrated by southwestern histochemistry, and it was decreased in HMGB2-KO mouse ovaries. Cell proliferation activity was also decreased in HMGB2-KO mouse ovaries in parallel with the decreased folliculogenesis. These results indicated that the depletion of HMGB2 induced ovarian atrophy that was characterized by a decreased ovarian size and weight, progressive fibrosis, as well as decreased oocytes and folliculogenesis. In conclusion, we demonstrated the crucial role of HMGB2 in mouse ovarian folliculogenesis through ERß expression.

Systematic Approach to DNA Aptamer Design Using Amino Acid-Nucleic Acid Hybrids (ANHs) Targeting Thrombin.

Chemical modifications of innate DNA/RNA aptamers facilitate the improvement of their function. Herein, we report our modular strategy to manipulate a thrombin-binding DNA aptamer (TBA) to improve its anticoagulation activity and binding affinity. A set of amino acid conjugates, termed amino acid-nucleic acid hybrids or ANHs, was synthesized and incorporated into a TBA loop sequences. We found that substitutions with hydrophobic amino acids in the loop region possessed significantly enhanced antithrombin activity, up to 3-fold higher than the native TBA. We investigated the correlations between thrombin-binding affinity and the features of our amino-acid conjugates using experimental techniques including circular dichroism spectroscopy, surface plasmon resonance assay, and molecular modeling. The present study demonstrates a systematic approach to aptamer design based on amino-acid characteristics, allowing the development of advanced aptamers.

Improving Thermodynamic Stability and Anticoagulant Activity of a Thrombin Binding Aptamer by Incorporation of 8-trifluoromethyl-2'-deoxyguanosine.

In this study, we incorporated 8-trifluoromethyl-2'-deoxyguanosine (FG) into a thrombin binding aptamer (TBA). Circular dichroism, nuclear magnetic resonance (NMR), electrophoresis, and prothrombin time (PT) assay were performed to investigate the structure, thermodynamic stability, biological stability, and anticoagulant activity of the FG-modified TBA sequences. We found that the replacement of FG into TBA sequences led to a remarkable improvement in the melting temperature up to 30 °C compared with the native sequence. The trifluoromethyl group allowed us to investigate the TBA G-quadruplex structure by 19F NMR spectroscopy. Furthermore, PT assays showed that the modified sequences can significantly improve the anticoagulant activity in comparison with the native TBA. Finally, we demonstrated that the trifluoromethyl-modified TBA sequence could function as an anticoagulant reagent in live rats. Our results strongly suggested that FG is a powerful nucleoside derivative to increase the thermodynamic stability and anticoagulant activity of TBA.

Influenza Virus Z-RNAs Induce ZBP1-Mediated Necroptosis.

Influenza A virus (IAV) is a lytic RNA virus that triggers receptor-interacting serine/threonine-protein kinase 3 (RIPK3)-mediated pathways of apoptosis and mixed lineage kinase domain-like pseudokinase (MLKL)-dependent necroptosis in infected cells. ZBP1 initiates RIPK3-driven cell death by sensing IAV RNA and activating RIPK3. Here, we show that replicating IAV generates Z-RNAs, which activate ZBP1 in the nucleus of infected cells. ZBP1 then initiates RIPK3-mediated MLKL activation in the nucleus, resulting in nuclear envelope disruption, leakage of DNA into the cytosol, and eventual necroptosis. Cell death induced by nuclear MLKL was a potent activator of neutrophils, a cell type known to drive inflammatory pathology in virulent IAV disease. Consequently, MLKL-deficient mice manifest reduced nuclear disruption of lung epithelia, decreased neutrophil recruitment into infected lungs, and increased survival following a lethal dose of IAV. These results implicate Z-RNA as a new pathogen-associated molecular pattern and describe a ZBP1-initiated nucleus-to-plasma membrane "inside-out" death pathway with potentially pathogenic consequences in severe cases of influenza.

Linear consecutive hexaoxazoles as G4 ligands inducing chair-type anti-parallel topology of a telomeric G-quadruplex.

G-quadruplex structures (G4s) in guanine-rich regions of DNA play critical roles in various biological phenomena, including replication, translation, and gene expression. There are three types of G4 topology, i.e., parallel, anti-parallel, and hybrid, and ligands that selectively interact with or stabilize a specific topology have been extensively explored to enable studies of topology-related functions. Here, we describe the synthesis of a new series of G4 ligands based on 6LCOs (6-linear consecutive oxazoles), i.e., L2H2-2M2EA-6LCO (2), L2A2-2M2EAc-6LCO (3), and L2G2-2M2EG-6LCO (4), which bear four aminoalkyl, acetamidealkyl, and guanidinylalkyl side chains, respectively. Among them, ligand 2 stabilized telomeric G4 and induced anti-parallel topology independently of the presence of cations. The anti-parallel topology induced by 2 was identified as chair-type by means of 19F NMR spectroscopy and fluorescence experiments with 2-aminopurine-labeled DNA.

A Nucleoside Derivative 5-Vinyluridine (VrU) for Imaging RNA in Cells and Animals.

In the present study, we used a nucleoside derivative 5-vinyluridine (VrU) for labeling during cell division and for tumor imaging in living mice. We demonstrated that the functional nucleoside bearing a 5-vinyl group is metabolically incorporated into cellular RNA and can be used to image RNA using a Diels-Alder reaction. The reagent allows for simultaneous and clear imaging of DNA and RNA in mammalian cells at single-cell resolution. We extended this approach to observe DNA and RNA behaviors in several basic stages of cell division. We further demonstrated that the derivative can be used for fluorescence imaging of tumor in live mice.

A disintegrin and metalloproteinase with thrombospondin motifs 2 cleaves and inactivates Reelin in the postnatal cerebral cortex and hippocampus, but not in the cerebellum.

Reelin plays important roles in regulating neuronal development, modulating synaptic function, and counteracting amyloid ß toxicity. A specific proteolytic cleavage (N-t cleavage) of Reelin abolishes its biological activity. We recently identified ADAMTS-3 (a disintegrin and metalloproteinase with thrombospondin motifs 3) as the major N-t cleavage enzyme in the embryonic and early postnatal brain. The contribution of other proteases, particularly in the postnatal brain, has not been demonstrated in vivo. ADAMTS-2, -3 and -14 share similar domain structures and substrate specificity, raising the possibility that ADAMTS-2 and -14 may cleave Reelin. We found that recombinant ADAMTS-2 protein expressed in cultured cell lines cleaves Reelin at the N-t site as efficiently as ADAMTS-3 while recombinant ADAMTS-14 hardly cleaves Reelin. The disintegrin domain is necessary for the Reelin-cleaving activity of ADAMTS-2 and -3. ADAMTS-2 is expressed in the adult brain at approximately the same level as ADAMTS-3. We generated ADAMTS-2 knockout (KO) mice and found that ADAMTS-2 significantly contributes to the N-t cleavage and inactivation of Reelin in the postnatal cerebral cortex and hippocampus, but much less in the cerebellum. Therefore, it was suggested that ADAMTS-2 can be a therapeutic target for adult brain disorders such as schizophrenia and Alzheimer's disease.

19F NMR Spectroscopy for the Analysis of DNA G-Quadruplex Structures Using 19F-Labeled Nucleobase.

G-quadruplex structures have been suggested to be biologically important in processes such as transcription and translation, gene expression and regulation in human cancer cells, and regulation of telomere length. Investigation of G-quadruplex structures associated with biological events is therefore essential to understanding the functions of these molecules. We developed the 19F-labeled nucleobases and introduced them into DNA sequences for the 19F NMR spectroscopy analysis. We present the 19F NMR methodology used in our research group for the study of G-quadruplex structures in vitro and in living cells.

Stability and properties of Z-DNA containing artificial nucleobase 2'-O-methyl-8-methyl guanosine.

We synthesized several DNA oligonucleotides containing one or several 2'-O-methyl-8-methyl guanosine (m8Gm) and demonstrated that these oligonucleotides not only stabilize the Z-DNA with a wide range of sequences under low salt conditions but also possess high thermal stability. Using artificial nucleobase-containing oligonucleotides, we studied the interaction of the Zα domain with Z-DNA. Furthermore, we showed that the m8Gm-contained oligonucleotides allow to study the photochemical reaction of Z-DNA.

2'-O-Methyl-8-methylguanosine as a Z-Form RNA Stabilizer for Structural and Functional Study of Z-RNA.

In contrast to Z-DNA that was stabilized and well-studied for its structure by chemical approaches, the stabilization and structural study of Z-RNA remains a challenge. In this study, we developed a Z-form RNA stabilizer m8Gm, and demonstrated that incorporation of m8Gm into RNA can markedly stabilize the Z-RNA at low salt conditions. Using the m8Gm-contained Z-RNA, we determined the structure of Z-RNA and investigated the interaction of protein and Z-RNA.

A multi-functional guanine derivative for studying the DNA G-quadruplex structure.

In the present study, we developed a multi-functional guanine derivative, 8FG, as a G-quadruplex stabilizer, a fluorescent probe for the detection of G-quadruplex formation, and a 19F sensor for the observation of the G-quadruplex. We demonstrate that the functional nucleoside bearing a 3,5-bis(trifluoromethyl)benzene group at the 8-position of guanine stabilizes the DNA G-quadruplex structure and fluoresces following the G-quadruplex formation. Furthermore, we show that the functional sensor can be used to directly observe DNA G-quadruplexes by 19F-NMR in living cells. To our knowledge, this is the first study showing that the nucleoside derivative simultaneously allows for three kinds of functions at a single G-quadruplex DNA. Our results suggest that the multi-functional nucleoside derivative can be broadly used for studying the G-quadruplex structure and serves as a powerful tool for examining the molecular basis of G-quadruplex formation in vitro and in living cells.

Antiparallel RNA G-quadruplex Formed by Human Telomere RNA Containing 8-Bromoguanosine.

In this study, by combining nuclear magnetic resonance (NMR), circular dichroism (CD), liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS), and gel electrophoresis, we report an unusual topological structure of the RNA G-quadruplex motif formed by human telomere RNA r(UAGGGU) containing 8-bromoguanosine. Results showed that the RNA sequence formed an antiparallel tetramolecular G-quadruplex, in which each pair of diagonal strands run in opposite directions. Furthermore, guanosines were observed both in syn- and anti-conformations. In addition, two of these G-quadruplex subunits were found to be stacking on top of each other, forming a dimeric RNA G-quadruplex. Our findings provide a new insight into the behavior of RNA G-quadruplex structures.