Published literature

Development (31) Products

  Mouse&Rat miRNA OneArray  
 Cellular and Molecular Biology Letters. DOI: 10.1515/cmble-2015-0034.
 Mechanical Strain Affects Some Microrna Profiles in Pre-Oeteoblasts 
 
  Abstract
MicroRNAs (miRNAs) are important regulators of cell proliferation, differentiation and function. Mechanical strain is an essential factor for osteoblast proliferation and differentiation. A previous study revealed that a physiological mechanical tensile strain of 2500 microstrain (弮庰) at 0.5 Hz applied once a day for 1 h over 3 consecutive days promoted osteoblast differentiation. However, the mechanoresponsive miRNAs of these osteoblasts were not identified. In this study, we applied the same mechanical tensile strain to in vitro cultivated mouse MC3T3-E1 pre-osteoblasts and identified the mechanoresponsive miRNAs. Using miRNA microarray and qRT-PCR assays, the expression patterns of miRNAs were evaluated and 5 of them were found to be significantly different between the mechanical loading group and the control group: miR-3077-5p, 3090-5p and 3103-5p were significantly upregulated and miR-466i-3p and 466h-3p were downregulated. Bioinformatics analysis revealed possible target genes for these differentially expressed miRNAs. Some target genes correlated with osteoblast differentiation. These findings indicated that the mechanical strain changed the expression levels of these miRNAs. This might be a potential regulator of osteoblast differentiation and responses to mechanical strain.
   

  Mouse OneArray  
 Science Signaling. 2015, 8(375):ra41. doi: 10.1126/scisignal.2005781.
 Actin cytoskeletal remodeling with protrusion formation is essential for heart regeneration in Hippo-deficient mice
 
 
 Yuka Morikawa, Min Zhang, Todd Heallen, John Leach, Ge Tao, Yang Xiao, Yan Bai, Wei Li, James T. Willerson, James F. Martin
  Abstract
The mammalian heart regenerates poorly, and damage commonly leads to heart failure. Hippo signaling is an evolutionarily conserved kinase cascade that regulates organ size during development and prevents adult mammalian cardiomyocyte regeneration by inhibiting the transcriptional coactivator Yap, which also responds to mechanical signaling in cultured cells to promote cell proliferation. To identify Yap target genes that are activated during cardiomyocyte renewal and regeneration, we performed Yap chromatin immunoprecipitation sequencing (ChIP-Seq) and mRNA expression profiling in Hippo signalingdeficient mouse hearts. We found that Yap directly regulated genes encoding cell cycle progression proteins, as well as genes encoding proteins that promote F-actin polymerization and that link the actin cytoskeleton to the extracellular matrix. Included in the latter group were components of the dystrophin glycoprotein complex, a large molecular complex that, when defective, results in muscular dystrophy in humans. Cardiomyocytes near the scar tissue of injured Hippo signalingdeficient mouse hearts showed cellular protrusions suggestive of cytoskeletal remodeling. The hearts of mdx mutant mice, which lack functional dystrophin and are a model for muscular dystrophy, showed impaired regeneration and cytoskeleton remodeling, but normal cardiomyocyte proliferation, after injury. Our data showed that, in addition to genes encoding cell cycle progression proteins, Yap regulated genes that enhance cytoskeletal remodeling. Thus, blocking the Hippo pathway input to Yap may tip the balance so that Yap responds to mechanical changes associated with heart injury to promote repair.
   

  Mouse OneArray  
 PLoS One. 2015, 10(3):e0118832. doi: 10.1371/journal.pone.0118832. eCollection 2015.
 Behavior Training Reverses Asymmetry in Hippocampal Transcriptome of the Cav3.2 Knockout Mice
 
 
 Ni-Chun Chung, Ying-Hsueh Huang, Chuan-Hsiung Chang, James C. Liao, Chih-Hsien Yang, Chien-Chang Chen, Ingrid Y. Liu
  Abstract
.Homozygous Cav3.2 knockout mice, which are defective in the pore-forming subunit of a low voltage activated T-type calcium channel, have been documented to show impaired maintenance of late-phase long-term potentiation (L-LTP) and defective retrieval of context-associated fear memory. To investigate the role of Cav3.2 in global gene expression, we performed a microarray transcriptome study on the hippocampi of the Cav3.2-/- mice and their wild-type littermates, either naïve (untrained) or trace fear conditioned. We found a significant left-right asymmetric effect on the hippocampal transcriptome caused by the Cav3.2 knockout. Between the naive Cav3.2-/- and the naive wild-type mice, 3522 differentially expressed genes (DEGs) were found in the left hippocampus, but only 4 DEGs were found in the right hippocampus. Remarkably, the effect of Cav3.2 knockout was partially reversed by trace fear conditioning. The number of DEGs in the left hippocampus was reduced to 6 in the Cav3.2 knockout mice after trace fear conditioning, compared with the wild-type naïve mice. To our knowledge, these results demonstrate for the first time the asymmetric effects of the Cav3.2 and its partial reversal by behavior training on the hippocampal transcriptome.
   

  Mouse OneArray  
 PLoS One. 2015, 10(3):e0121765. doi: 10.1371/journal.pone.0121765. eCollection 2015.
 Smyd1 Facilitates Heart Development by Antagonizing Oxidative and ER Stress Responses
 
 
 Tara L. Rasmussen, Yanlin Ma, Chong Yon Park, June Harriss, Stephanie A. Pierce, Joseph D. Dekker, Nicolas Valenzuela, Deepak Srivastava, Robert J. Schwartz, M. David Stewart, Haley O. Tucker
  Abstract
Smyd1/Bop is an evolutionary conserved histone methyltransferase previously shown by conventional knockout to be critical for embryonic heart development. To further explore the mechanism(s) in a cell autonomous context, we conditionally ablated Smyd1 in the first and second heart fields of mice using a knock-in (KI) Nkx2.5-cre driver. Robust deletion of floxed-Smyd1 in cardiomyocytes and the outflow tract (OFT) resulted in embryonic lethality at E9.5, truncation of the OFT and right ventricle, and additional defects consistent with impaired expansion and proliferation of the second heart field (SHF). Using a transgenic (Tg) Nkx2.5-cre driver previously shown to not delete in the SHF and OFT, early embryonic lethality was bypassed and both ventricular chambers were formed; however, reduced cardiomyocyte proliferation and other heart defects resulted in later embryonic death at E11.5-12.5. Proliferative impairment prior to both early and mid-gestational lethality was accompanied by dysregulation of transcripts critical for endoplasmic reticulum (ER) stress. Mid-gestational death was also associated with impairment of oxidative stress defense-a phenotype highly similar to the previously characterized knockout of the Smyd1-interacting transcription factor, skNAC. We describe a potential feedback mechanism in which the stress response factor Tribbles3/TRB3, when directly methylated by Smyd1, acts as a co-repressor of Smyd1-mediated transcription. Our findings suggest that Smyd1 is required for maintaining cardiomyocyte proliferation at minimally two different embryonic heart developmental stages, and its loss leads to linked stress responses that signal ensuing lethality.
   

  Mouse OneArray  
 The International Journal of Biochemistry & Cell Biology. 2015, 60:99-111. doi: 10.1016/j.biocel.2014.12.024.
 Identification of stage-specific markers during differentiation of hair cells from mouse inner ear stem cells or progenitor cells in vitro
 
 
 Quanwen Liu, JiarongChen, XiangliGao, JieDing, ZihuaTang, CuiZhang, Jianling Chen, Liang Li, PingChen, JinfuWang
  Abstract
The induction of inner ear hair cells from stem cells or progenitor cells in the inner ear proceeds through a committed inner ear sensory progenitor cell stage prior to hair cell differentiation. To increase the efficacy of inducing inner ear hair cell differentiation from the stem cells or progenitor cells, it is essential to identify comprehensive markers for the stem cells/progenitor cells from the inner ear, the committed inner ear sensory progenitor cells and the differentiating hair cells to optimize induction conditions. Here, we report that we efficiently isolated and expanded the stem cells or progenitor cells from postnatal mouse cochleae, and induced the generation of inner ear progenitor cells and subsequent differentiation of hair cells. We profiled the gene expression of the stem cells or progenitor cells, the inner ear progenitor cells, and hair cells using aRNA microarray analysis. The pathway and gene ontology (GO) analysis of differentially expressed genes was performed. Analysis of genes exclusively detected in one particular cellular population revealed 30, 38, and 31 genes specific for inner ear stem cells, inner ear progenitor cells, and hair cells, respectively. We further examined the expression of these genes in vivo and determined that Gdf10+Ccdc121, Tmprss9+Orm1, and Chrna9+Espnl are marker genes specific for inner ear stem cells, inner ear progenitor cells, and differentiating hair cells, respectively. The identification of these marker genes will likely help the effort to increase the efficacy of hair cell induction from the stem cells or progenitor cells.
   

  Human OneArray  
 PLoS One. 2014 December 5. doi: 10.1371/journal.pone.0114781.
 Functional Study of One Nucleotide Mutation in Pri-MiR-125a Coding Region which Related to Recurrent Pregnancy Loss
 
 
 Yi Hu, Zheng-Hao Huo, Chun-Mei Liu, Shi-Guo Liu, Ning Zhang, Kun-Lun Yin, Xu Ma, Hong-Fei Xia
  Abstract
MicroRNAs (miRNAs) are short non-coding RNAs which modulate gene expression by binding to complementary segments present in the 3涸TR of the mRNAs of protein coding genes. MiRNAs play very important roles in maintaining normal human body physiology conditions, meanwhile, abnormal miRNA expressions have been found related to many human diseases spanning from psychiatric disorders to malignant cancers. Recently, emerging reports have indicated that disturbed miRNAs expression contributed to the pathogenesis of recurrent pregnancy loss (RPL). In this study, we identified a new mutation site (+29A>G, position relative to pre-miR-125a) by scanning pri-miR-125a coding region in 389 Chinese Han RPL patients. This site was co-existed with two polymorphisms (rs12976445 and rs41275794) in patients heterogeneously and changed the predicted secondary structures of pri-miR-125a. Subsequent in vitro analysis indicated that the A>G mutation reduced mature miR-125a expression, and further led to less efficient inhibition of verified target genes. Functional analysis showed that mutant pri-mir-125a can enhance endometrial stromal cells (ESCs) invasive capacity and increase the sensitivity of ESCs cells to mifepristone. Moreover, we further analyzed the possible molecular mechanism by RIP-chip assay and found that mutant pri-mir-125a disturbed the expression of miR-125a targetome, the functions of which includes embryonic development, cell proliferation, migration and invasion. These data suggest that A>G mutation in pri-miR-125a coding region contributes to the genetic predisposition to RPL by disordering the production of miR-125a, which consequently meddled in gene regulatory network between mir-125a and mRNA.
   

  Array technology and applications  
 Pediatrics & Neonatology. 2014 Nov 4. doi:10.1016/j.pedneo.2014.08.001.
 Genetic Evaluation of Children with Global Developmental DelayCurrent Status of Network Systems in Taiwan
 
 
 Shio-Jean Lin, Yong-Lin Foo, Julie Chi Chow, Ming-Chi Lai, Wen-Hui Tsai, Li-Chen Tung, Mei-Chin Kuo
  Abstract
This review article aims to introduce the screening and referral network of genetic evaluation for children with developmental delay in Taiwan. For these children, integrated systems provide services from the medical, educational, and social welfare sectors. All cities and counties in Taiwan have established a network for screening, detection, referral, evaluation, and intervention services. Increased awareness improves early detection and intervention. There remains a gap between supply and demand, especially with regard to financial resources and professional manpower. Genetic etiology has a major role in prenatal causes of developmental delay. A summary of reports on some related genetic disorders in the Taiwanese population is included in this review. Genetic diagnosis allows counseling with regard to recurrence risk and prevention. Networking with neonatal screening, laboratory diagnosis, genetic counseling, and orphan drugs logistics systems can provide effective treatment for patients. In Taiwan, several laboratories provide genetic tests for clinical diagnosis. Accessibility to advanced expensive tests such as gene chips or whole exome sequencing is limited because of funding problems; however, the service system in Taiwan can still operate in a relatively cost-effective manner. This experience in Taiwan may serve as a reference for other countries.
   

  Human OneArray  
 BMC Genomics. 2014, 15:P48. doi:10.1186/1471-2164-15-S2-P48.
 Analysis of correlations between zona pellucida birefringence and molecular markers of oocyte developmental competence
 
 
 Markus Montag, Marc-André Sirard, Mourad Assidi
  Abstract
Human infertility is the incapacity of a couple to conceive after one year of unprotected sexual intercourse. Selection of the best gametes for subsequent steps of fertilization and embryo transfer was shown to be the crucial step in infertility treatment procedure. Oocyte selection using morphological criteria has been the gold standard method in assisted reproductive technologies (ART) clinics. Zona Pellucida (ZP) , a filamentous matrix of glycosylated glycoproteins surrounding the oocyte, is one of these morphological criteria of oocyte selection. In fact, ZP thickness and birefringence was reported to be positively correlated with higher ability of the oocyte to achieve successful pregnancy, but this selection approach has limitations in terms of accuracy, objectivity and constancy. Recent studies using OMICs approaches have identified key molecular markers in somatic cells (cumulus and/or granulosa cells) and follicular fluid that quantitatively and non-invasively predict the oocyte quality for better selection, higher pregnancy rates and efficient infertility treatment. These biomarkers could be a valuable reinforcement of the morphological selection criteria widely used in IVF clinics. In this context, this study was designed to study the relationship between some molecular predictors of oocyte quality found by our group and the conventional morphological parameters of oocyte quality. We expect to find a positive correlation between the ZP birefringence and molecular markers of oocyte competence. Such integrative strategy should lead to a powerful combined approach that will precisely predict the oocyte developmental potential, allowing therefore efficient infertility treatment and elective single embryo transfer (eSET).
   

  Mouse OneArray  
 European Journal of Oral Sciences. 2013, 110. doi: 10.1111/eos.12056.
 Expression of Clu and Tgfb1 during murine tooth development: effects of in-vivo transfection with anti-miR-214
 
 
 Amer Sehic, Cuong Khuu, Steinar Risnes, Harald Osmundsen, Qalb-E-Saleem Khan
  Abstract
Expression of clusterin (Clu) in the murine first molar tooth germ was markedly increased at postnatal developmental stages. The time-course of expression of this gene paralleled those of other genes encoding proteins involved during the secretory phase of odontogenesis, as described previously. Immunohistochemical studies of clusterin in murine molar tooth germs suggested this protein to be located in outer enamel epithelium, regressing enamel organ, secretory ameloblasts, and the dental epithelium connecting the tooth to the oral epithelium at an early eruptive stage. Immunolabelling of transforming growth factor beta-1 (TGF-b1) revealed it to be located close to clusterin. The levels of expression of Clu and Tgfb1 were markedly decreased following in-vivo transfection with anti-miR-214. In contrast, the expression of several genes associated with regulation of growth and development were increased by this treatment. We suggest that clusterin has functions during secretory odontogenesis and the early eruptive phase. Bioinformatic analysis after treatment with anti-miR-214 suggested that, whilst cellular activities associated with tooth mineralization and eruption were inhibited, activities associated with an alternative developmental activity (i.e. biosynthesis of contractile proteins) appeared to be stimulated. These changes probably occur through regulation mediated by a common cluster of transcription factors and support suggestions that microRNAs (miRNAs) are highly significant as regulators of differentiation during odontogenesis.
   

  Mouse OneArray  
 Methods in Molecular Biology. 2012, 887:95-110. doi: 10.1007/978-1-61779-860-3_10.
 Deoxyoligonucleotide Microarrays for Gene Expression Profiling in Murine Tooth Germs
 
 
 Anne-Marthe Jevnaker, Maria A. Landin, Harald Osmundsen
  Abstract
The use of deoxyoligonucleotide microarrays facilitates rapid expression profiling of gene expression using samples of about 1 弮g of total RNA. Here are described practical aspects of the procedures involved, including essential reagents. Analysis of results is discussed from a practical, experimental, point of view together with software required to carry out the required statistical analysis to isolate populations of differentially expressed genes.
   

  Human OneArray  
 Biology of Reproduction. 2012, 86(5):1-10. doi: 10.1095/biolreprod.111.097295.
 Mono-(2-Ethylhexyl) Phthalate (MEHP) Promotes Invasion and Migration of Human Testicular Embryonal Carcinoma Cells
 
 
 Yi-Chen Lin, Pei-Li Yao, John H. Richburg
  Abstract
Testicular dysgenesis syndrome refers to a collection of diseases in men, including testicular cancer, that arise as a result of abnormal testicular development. Phthalates are a class of chemicals used widely in the production of plastic products and other consumer goods. Unfortunately, phthalate exposure has been linked to reproductive dysfunction and has been shown to adversely affect normal germ cell development. In this study, we show that mono-(2-ethylhexyl) phthalate (MEHP) induces matrix metalloproteinase 2 (MMP2) expression in testicular embryonal carcinoma NT2/D1 cells but has no significant effect on MMP9 expression. NT2/D1 cells also have higher levels of MYC expression following MEHP treatment. It is widely recognized that activation of MMP2 and MYC is tightly associated with tumor metastasis and tumor progression. Gelatin zymographic analysis indicates that MEHP strongly activates MMP2 in NT2/D1 cells. Addition of the MMP2-specific inhibitor SB-3CT inhibited MEHP-enhanced cell invasion and migration, demonstrating that MMP2 plays a functional role in promoting testicular embryonal carcinoma progression in response to MEHP exposure. Furthermore, we investigated genome-wide gene expression profiles of NT2/D1 cells following MEHP exposure at 0, 3, and 24 h. Microarray analysis and semiquantitative RT-PCR revealed that MEHP exposure primarily influenced genes in cell adhesion and transcription in NT2/D1 cells. Gap junction protein-alpha 1, vinculin, and inhibitor of DNAbinding protein-1 were significantly down-regulated by MEHP treatment, while claudin-6 and beta 1-catenin expression levels were up-regulated. This study provides insight into mechanisms that may account for modulating testicular cancer progression following phthalate exposure.
   

  Mouse OneArray  
 Journal of Molecular Neuroscience. 2012, 47(3):604-618. doi: 10.1007/s12031-011-9690-4.
 The Effects of Unilateral Naris Occlusion on Gene Expression Profiles in Mouse Olfactory Mucosa
 
 
 Christopher T. Waggener, David M. Coppola
  Abstract
Unilateral naris occlusion has been the method of choice for effecting stimulus deprivation in studies of olfactory plasticity. Early experiments emphasized the deleterious effects of this technique on the developing olfactory system while more recent studies have pointed to several apparently compensatory responses. However, the evidence for deprivation-induced compensatory processes in olfaction remains fragmentary. High-throughput methods such as microarray analysis can help fill the deficits in our understanding of naris occlusion as a mode of stimulus deprivation. Here we report for young adult mice the effects of early postnatal naris occlusion on the olfactory mucosal transcriptome using microarray analysis with RTPCR confirmation. The transcripts of key genes involved in olfactory reception, transduction, and transmission were up-regulated in deprived-side olfactory mucosa, with opposite effects in non-deprived-side mucosa, compared to controls. Results support the hypothesis that odor environment triggers a previously unknown homeostatic control mechanism in olfactory receptor neurons designed to maximize information transfer.
   

  Mouse OneArray  
 PLoS ONE. 2012, 7(2):e31005. doi: 10.1371/journal.pone.0031005.
 Conditional Ablation of Ezh2 in Murine Hearts Reveals Its Essential Roles in Endocardial Cushion Formation, Cardiomyocyte Proliferation and Survival
 
 
 Li Chen, Yanlin Ma, Eun Young Kim, Wei Yu, Robert J. Schwartz, Ling Qian, Jun Wang
  Abstract
Ezh2 is a histone trimethyltransferase that silences genes mainly via catalyzing trimethylation of histone 3 lysine 27 (H3K27Me3). The role of Ezh2 as a regulator of gene silencing and cell proliferation in cancer development has been extensively investigated; however, its function in heart development during embryonic cardiogenesis has not been well studied. In the present study, we used a genetically modified mouse system in which Ezh2 was specifically ablated in the mouse heart. We identified a wide spectrum of cardiovascular malformations in the Ezh2 mutant mice, which collectively led to perinatal death. In the Ezh2 mutant heart, the endocardial cushions (ECs) were hypoplastic and the endothelial-to-mesenchymal transition (EMT) process was impaired. The hearts of Ezh2 mutant mice also exhibited decreased cardiomyocyte proliferation and increased apoptosis. We further identified that the Hey2 gene, which is important for cardiomyocyte proliferation and cardiac morphogenesis, is a downstream target of Ezh2. The regulation of Hey2 expression by Ezh2 may be independent of Notch signaling activity. Our work defines an indispensible role of the chromatin remodeling factor Ezh2 in normal cardiovascular development.
   

  Mouse OneArray  
 Development. 2012, 139(4):709-19. doi: 10.1242/dev.073197.
 Bmp signaling regulates a dose-dependent transcriptional program to control facial skeletal development
 
 
 Margarita Bonilla-Claudio, Jun Wang, Yan Bai, Elzbieta Klysik, Jennifer Selever, James F. Martin
  Abstract
We performed an in depth analysis of Bmp4, a critical regulator of development, disease, and evolution, in cranial neural crest (CNC). Conditional Bmp4 overexpression, using a tetracycline-regulated Bmp4 gain-of-function allele, resulted in facial skeletal changes that were most dramatic after an E10.5 Bmp4 induction. Expression profiling uncovered a signature of Bmp4-induced genes (BIG) composed predominantly of transcriptional regulators that control self-renewal, osteoblast differentiation and negative Bmp autoregulation. The complimentary experiment, CNC inactivation of Bmp2, Bmp4 and Bmp7, resulted in complete or partial loss of multiple CNC-derived skeletal elements, revealing a crucial requirement for Bmp signaling in membranous bone and cartilage development. Importantly, the BIG signature was reduced in Bmp loss-of-function mutants, indicating Bmp-regulated target genes are modulated by Bmp dose. Chromatin immunoprecipitation (ChIP) revealed a subset of the BIG signature, including Satb2, Smad6, Hand1, Gadd45帠 and Gata3, that was bound by Smad1/5 in the developing mandible, revealing direct Smad-mediated regulation. These data support the hypothesis that Bmp signaling regulates craniofacial skeletal development by balancing self-renewal and differentiation pathways in CNC progenitors.
   

  Data Analysis  
 PLoS ONE. 2011, 6(4):e18628. doi: 10.1371/journal.pone.0018628.
 MicroRNAs Dynamically Remodel Gastrointestinal Smooth Muscle Cells
 
 
 Chanjae Park, Wei Yan, Sean M. Ward, Sung Jin Hwang, Qiuxia Wu, William J. Hatton, Jong Kun Park, Kenton M. Sanders, Seungil Ro
  Abstract
Smooth muscle cells (SMCs) express a unique set of microRNAs (miRNAs) which regulate and maintain the differentiation state of SMCs. The goal of this study was to investigate the role of miRNAs during the development of gastrointestinal (GI) SMCs in a transgenic animal model. We generated SMC-specific Dicer null animals that express the reporter, green fluorescence protein, in a SMC-specific manner. SMC-specific knockout of Dicer prevented SMC miRNA biogenesis, causing dramatic changes in phenotype, function, and global gene expression in SMCs: the mutant mice developed severe dilation of the intestinal tract associated with the thinning and destruction of the smooth muscle (SM) layers; contractile motility in the mutant intestine was dramatically decreased; and SM contractile genes and transcriptional regulators were extensively down-regulated in the mutant SMCs. Profiling and bioinformatic analyses showed that SMC phenotype is regulated by a complex network of positive and negative feedback by SMC miRNAs, serum response factor (SRF), and other transcriptional factors. Taken together, our data suggest that SMC miRNAs are required for the development and survival of SMCs in the GI tract.
   

  Human OneArray  
 CLINICAL IMMUNOLOGY. 2011 Sep 16. doi: 10.1111/j.1365-3083.2011.02637.x.
 Healthy first degree relatives of patients with type 1 diabetes exhibit significant differences in basal gene expression pattern of immunocompetent cells compared to controls: expression pattern as predeterminant of autoimmune diabetes
 
 
 Michal Kolar, Radek Blatny, Zbynek Halbhuber, Jana Vcelakova, Miluse Hubackova, Lenka Petruzelkova, Zdenek Sumnik, Barbora Obermannova, Pavlina Pithova, Vendula Stavikova, Maria Krivjanska, Ales Neuwirth, Stanislava Kolouskova, Dominik Filipp, Katerina Stechova
  Abstract
Introduction:? Expression features of genetic landscape which predispose an individual to the type 1 diabetes are poorly understood. We addressed this question by comparing gene expression profile of freshly isolated peripheral blood mononuclear cells isolated from either patients with type 1 diabetes, or their first degree relatives or healthy controls. Our aim was to establish whether a distinct type of "prodiabetogenic" gene expression pattern in the group of relatives of T1D patients could be identified. Methods:? Whole-genome expression profile of nine T1D patients, their ten first-degree relatives and ten healthy controls were analyzed using the human high density expression microarray chip. Functional aspects of candidate genes were assessed using the MetaCore software. Results:? The highest number of differentially expressed genes (547) was found between the autoantibody negative healthy relatives and the healthy controls. Some of them represent genes critically involved in the regulation of innate immune responses such as TLR signalling and CCR3 signalling in eosinophiles, humoral immune reactions such as BCR pathway, costimulation and cytokine responses mediated by CD137, CD40 and CD28 signalling and IL-1 proinflammatory pathway. Conclusion:? Our data demonstrate that expression profile of healthy relatives of patients with T1D is clearly distinct from the pattern found in the healthy controls. That especially concerns differential activation status of genes and signalling pathways involved in proinflammatory processes and those of innate immunity and humoral reactivity. Thus, we posit that the study of the healthy relative's gene expression pattern is instrumental for identification of novel markers associated with the development of diabetes.
   

  Mouse OneArray  
 PLoS One. 2011, 6(8):e23682. doi: 10.1371/journal.pone.0023682.
 Comprehensive Assessment of Host Responses to Ionizing Radiation by Nuclear Factor-庥B Bioluminescence Imaging-Guided Transcriptomic Analysis
 
 
 Q Chang CT, Lin H, Ho TY, Li CC, Lo HY, Wu SL, Huang YF, Liang JA, Hsiang CY.
  Abstract
The aim of this study was to analyze the host responses to ionizing radiation by nuclear factor-庥B (NF-庥B) bioluminescence imaging-guided transcriptomic tool. Transgenic mice carrying the NF-庥B-driven luciferase gene were exposed to a single dose of 8.5 Gy total-body irradiation. In vivo imaging showed that a maximal NF-庥B-dependent bioluminescent intensity was observed at 3 h after irradiation and ex vivo imaging showed that liver, intestine, and brain displayed strong NF-庥B activations. Microarray analysis of these organs showed that irradiation altered gene expression signatures in an organ-specific manner and several pathways associated with metabolism and immune system were significantly altered. Additionally, the upregulation of fatty acid binding protein 4, serum amyloid A2, and serum amyloid A3 genes, which participate in both inflammation and lipid metabolism, suggested that irradiation might affect the cross pathways of metabolism and inflammation. Moreover, the alteration of chemokine (CC-motif) ligand 5, chemokine (CC-motif) ligand 20, and Jagged 1 genes, which are involved in the inflammation and enterocyte proliferation, suggested that these genes might be involved in the radiation enteropathy. In conclusion, this report describes the comprehensive evaluation of host responses to ionizing radiation. Our findings provide the fundamental information about the in vivo NF-庥B activity and transcriptomic pattern after irradiation. Moreover, novel targets involved in radiation injury are also suggested.
   

  Mouse OneArray  
 Acta Histochem. 2012, 114(4):379-85. doi: 10.1016/j.acthis.2011.07.008.
 Expression of insulin-like growth factor binding protein-4 (IGFBP-4) in acute pancreatitis induced by l-arginine in mice
 
 
 Jia Qing Shen, Jie Shen, Xing Peng Wang.
  Abstract
The mechanisms of injury and regeneration after acute pancreatitis are still incompletely understood. Insulin-like growth factor binding proteins (IGFBPs) have been reported to play roles in various pancreatic diseases, but the involvement of insulin-like growth factor binding protein-4 (IGFBP-4) in acute pancreatitis is unknown. The aim of the study was to examine the expression of IGFBP-4 in mice with acute pancreatitis induced by two doses of L-arginine. IGFBP-4 expression was assayed by microarray test, real-time RT-PCR, Western blotting, ELISA and by an immunohistochemical assay. Microarray test of pancreatic mRNA showed that IGFBP-4 mRNA increased significantly after L-arginine treatment and the increase was confirmed by real-time RT-PCR. Western blotting and ELISA assay showed similar patterns of increase of IGFBP-4 in pancreatic tissues and serum. In the control pancreas, IGFBP-4 was mainly immunolocalized in the pancreatic islets. In the pancreatic tissues of mice with pancreatitis induced by L-arginine, the immunolocalization of IGFBP-4 was detected in both acinar cells and pancreatic islets. In conclusion, our results suggest that IGFBP-4 may play a potential role in pancreatic injury and regeneration in a murine model of acute pancreatitis induced by L-arginine.
   

  Mouse OneArray, Mouse&Rat miRNA OneArray  
 Physiol Genomics. 2011, 43(9):488-98. doi: 10.1152/physiolgenomics.00248.2010.
 Effects of in vivo transfection with anti-miR-214 on gene expression in murine molar tooth germ
 
 
 Sehic A, Risnes S, Khuu C, Khan QE, Osmundsen H.
  Abstract
MicroRNAs (miRNAs) are an abundant class of noncoding RNAs that are believed to be important in many biological processes through regulation of gene expression. Little is known of their function in tooth morphogenesis and differentiation. MicroRNA-214 (miR-214), encoded by the polycistronic Dnm30os gene, is highly expressed during development of molar tooth germ and was selected as a target for silencing with anti-miR-214. Mandibular injection of 1-100 pmol of anti-miR-214 close to the developing first molar in newborn mice resulted in significant decrease in expression of miR-214, miR-466h, and miR-574-5p in the tooth germ. Furthermore, levels of miR-199a-3p, miR-199a-5p, miR-690, miR-720, and miR-1224 were significantly increased. Additionally, the expression of 863 genes was significantly increased and the expression of 305 genes was significantly decreased. Among the genes with increased expression was Twist-1 and Ezh2, suggested to regulate expression of miR-214. Microarray results were validated using real-time RT-PCR and Western blotting. Among genes with decreased expression were Amelx, Calb1, Enam, and Prnp; these changes also being reflected in levels of corresponding encoded proteins in the tooth germ. In the anti-miR-214-treated molars the enamel exhibited evidence of hypomineralization with remnants of organic material and reduced surface roughness after acid etching, possibly due to the transiently decreased expression of Amelx and Enam. In contrast, several genes encoding contractile proteins exhibited significantly increased expression. mRNAs involved in amelogenesis (Ambn, Amelx, Enam) were not found among targets of miRNAs that were differentially expressed following treatment with anti-miR-214. It is therefore suggested that effects of miR-214 on amelogenesis are indirect, perhaps mediated by the observed miR-214-dependent changes in levels of expression of numerous transcription factors.
   

  Experimental Accessories  
 Mol Endocrinol. 2010, 24(3):540-51. doi: 10.1210/me.2009-0432.
 MicroRNA-224 Is Involved in Transforming Growth Factor-帣-Mediated Mouse Granulosa Cell Proliferation and Granulosa Cell Function by Targeting Smad4
 
 
 Guidong Yao, Mianmian Yin, Jie Lian, Hui Tian, Lin Liu, Xin Li, Fei Sun
  Abstract
"Many members of the TGF- superfamily are indicated to play important roles in ovarian follicular development, such as affecting granulosa cell function and oocyte maturation. Abnormalities associated with TGF- 1 signaling transduction could result in female infertility. MicroRNAs (miRNAs), as small noncoding RNAs, were recently found to regulate gene expression at posttranscriptional levels. However, little is known about the role of miRNAs in TGF- -mediated granulosa cell proliferation and granulosa cell function. In this study, the miRNA expression profiling was identified from TGF- 1-treated mouse preantral granulosa cells (GCs), and three miRNAs were found to be significantly up-regulated and 13 miRNAs were down-regulated. Among up-regulated miRNAs, miR-224 was the second most significantly elevated miRNA. This up-regulation was attenuated by treatment of GCs with SB431542 (an inhibitor of TGF superfamily type I receptors, thus blocking phosphorylation of the downstream effectors Smad2/3), indicating that miR-224 expression was regulated by TGF- 1/Smads pathway. The ectopic expression of miR-224 can enhance TGF- 1-induced GC proliferation through targeting Smad4. Inhibition of endogenous miR- 224 partially suppressed GC proliferation induced by TGF- 1. In addition, both miR-224 and TGF- 1 can promote estradiol release from GC, at least in part, through increasing CYP19A1 mRNA levels. This is the first demonstration that miRNAs can control reproductive functions resulting in promoting TGF- 1-induced GC proliferation and ovarian estrogen release. Such miRNA-mediated effects could be potentially used for regulation of reproductive processes or for treatment of reproductive disorders."
   

  Human OneArray  
 J Assist Reprod Genet. 2011, 28(2):173-88. doi: 10.1007/s10815-010-9491-7.
 Biomarkers of human oocyte developmental competence expressed in cumulus cells before ICSI: a preliminary study.
 
 
 Assidi M, Montag M, Van Der Ven K, Sirard MA.
  Abstract
To identify reliable genomic biomarkers expressed in cumulus cells that accurately and non-invasively predict the oocyte developmental competence and reinforce the already used morphological criteria. Eight consenting patients were selected for ovarian stimulation and ICSI procedures. Cumulus-oocyte complexes were transvaginally punctured and individually selected based on both good morphological criteria and high zona pellucida birefringence. Following ICSI, two 3-day embryos per patient were transferred. Pregnancy outcome was recorded and proven implantation was thereafter confirmed. Differential gene expression was assessed using two microarray platforms. Further real-time PCR validation, Ingenuity pathways analysis and intra-patient analysis were performed on 17 selected candidates. Seven genes were differentially (p???0.05) associated to successful pregnancy and implantation. These biomarkers could be used to predict the oocyte developmental competence. These genomic markers are a powerful reinforcement of morphological approaches of oocyte selection. Their large-scale validation could increase pregnancy outcome and single embryo transfer efficiency.
   

  Human OneArray  
 Systems Biology in Reproductive Medicine. 2010, 56(4):318-26. doi: 10.3109/19396361003646109.
 Expression Profiling of Protein Tyrosine Kinases and Their Ligand Activators in Leiomyoma Uteri.
 
 
 Yong Jiang, Guangli Suo, Anil Sadarangani, Bryan Cowan, Jean Y. J. Wang.
  Abstract
The aim of this study is to compare the expression patterns of tyrosine kinases and their ligand activators between matched myometrium and leiomyoma tissues. Total RNA extracted from 42 pairs of matched leiomyomal and adjacent myometrial tissues were hybridized to a set of 840 customized oligonucleotide microarrays to compare the expression profiles of 244 selected human genes including 90 tyrosine kinases and 103 ligands. Among the 244 genes surveyed, 38 were found to exhibit differential expression between pairs of myometrium and leiomyoma tissues. Clustering analysis of the expression ratios of these 38 genes from 42 pairs of samples led to the subdivision of fibroid samples into three groups, based in principle on the expression ratios of two peptide ligands, CYR61 and EFNA4. Real-time quantitative RT-PCR measurements of an additional 32 pairs of samples further confirmed the three subgroups. The altered expression of ligand activators between myometrium and leiomyoma suggest that tyrosine kinases regulated by CYR61 and EFNA4 may be exploited as therapeutic targets to develop non-surgical treatments of symptomatic leiomyomas.
   

  Human OneArray  
 NATURE. 2010, 466(7305):503-7. doi: 10.1038/nature09261.
 Histone H4K20/H3K9 demethylase PHF8 regulates zebrafish brain and craniofacial development.
 
 
 Hank H. Qi, Madathia Sarkissian, Gang-Qing Hu, Zhibin Wang, Arindam Bhattacharjee, D. Benjamin Gordon, Michelle Gonzales, Fei Lan, Pat P. Ongusaha, Maite Huarte, Nasser K. Yaghi, Huijun Lim, Benjamin A. Garcia, Leonardo Brizuela, Keji Zhao, Thomas M. Roberts, Yang Shi.
  Abstract
X-linked mental retardation (XLMR) is a complex human disease that causes intellectual disability. Causal mutations have been found in approximately 90 X-linked genes; however, molecular and biological functions of many of these genetically defined XLMR genes remain unknown. PHF8 (PHD (plant homeo domain) finger protein 8) is a JmjC domain-containing protein and its mutations have been found in patients with XLMR and craniofacial deformities. Here we provide multiple lines of evidence establishing PHF8 as the first mono-methyl histone H4 lysine 20 (H4K20me1) demethylase, with additional activities towards histone H3K9me1 and me2. PHF8 is located around the transcription start sites (TSS) of approximately 7,000 RefSeq genes and in gene bodies and intergenic regions (non-TSS). PHF8 depletion resulted in upregulation of H4K20me1 and H3K9me1 at the TSS and H3K9me2 in the non-TSS sites, respectively, demonstrating differential substrate specificities at different target locations. PHF8 positively regulates gene expression, which is dependent on its H3K4me3-binding PHD and catalytic domains. Importantly, patient mutations significantly compromised PHF8 catalytic function. PHF8 regulates cell survival in the zebrafish brain and jaw development, thus providing a potentially relevant biological context for understanding the clinical symptoms associated with PHF8 patients. Lastly, genetic and molecular evidence supports a model whereby PHF8 regulates zebrafish neuronal cell survival and jaw development in part by directly regulating the expression of the homeodomain transcription factor MSX1/MSXB, which functions downstream of multiple signalling and developmental pathways. Our findings indicate that an imbalance of histone methylation dynamics has a critical role in XLMR.
   

  Human OneArray  
 European Journal of Cell Biology. 2010, 89(7):537-46. doi: 10.1016/j.ejcb.2010.01.005.
 Expression of the homeobox gene, HOPX, is modulated by cell differentiation in human keratinocytes and is involved in the expression of differentiation markers.
 
 
 Jun-Mo Yang, Seon Mi Sim, Hyo-Yeon Kim, Geon Tae Park.
  Abstract
Homeodomain only protein X (HOPX), an unusual homeodomain protein, was originally identified as a key regulator of cardiac development. We first demonstrated that the expression of HOPX was dependent on the differentiation of human keratinocytes and has an effect on the expression of differentiation markers. HOPX was suppressed in proliferating human keratinocytes and was gradually induced by calcium-triggered differentiation of human keratinocytes. In the epidermis, HOPX is highly expressed in the terminally differentiated suprabasal layers. Among the transcript variants of HOPX, the variant 3 driven by promoter A was the main transcript and it was regulated by cell differentiation in human keratinocytes. The expression of HOPX was induced through the phorbol-12-myristate-13-acetate (PMA)-dependent protein kinase C (PKC) signaling pathway, and not by the demethylating agent, 5-aza-dC (5-aza-2'-deoxycitidine) suggesting the suppression of HOPX is not associated with DNA methylation in human keratinocytes. The RNA interference (RNAi) silencing experiment showed that the knockdown of HOPX expression resulted in the increase of such differentiation markers as involucrin and loricrin. Exogenous expression of HOPX down-regulated the expression of differentiation marker genes in immortalized human keratinocytes (HaCaT). Collectively, HOPX is modulated by cell differentiation in human keratinocytes and this might contribute to homeostasis of keratinocytes by controlling differentiation-dependent genes.
   

  Human OneArray  
 Basic & Clinical Medicine. 2010, 30:5.
 Expression and regulation of HES1 in sperms with low motility from asthenospermia patients.
 
 
 LI Yan,WEN Liu,CHEN De-yu.
  Abstract
To analyze the expression and regulation of HES1 in sperms with low motility.MethodsThirty semen samples from asthenospermia patients and 20 semen samples from healthy and fertile adults were collected,total RNAs were extracted to produce cDNAs probes.Hybridization with Phalanx OneArrayTM containing 30 968 probes was carried out after the labeled cDNAs were purified by PCR product purification kit.Realtime RT-PCR was used to analyze the expression of hsa-miR-487a and hsa-miR-193b;the expression of the target genes of hsa-miR-487a and hsa-miR-193b were searched from gene-expression profiles in asthenospermia patients' sperms.Results The expression level of HES1 in low motility sperms was up-regulated.The expression level of hsa-miR-193b in low motility sperms was 2.19 times higher than that in high motility sperms,hsa-miR-487a was 0.43% of that in high motility sperms.Conclusion The expression level of HES1 in low motility sperms was up-regulated.Hsa-miR-487a and hsa-miR-193b may affect the expression of HES1 and so regulate sperm motility.
   

  Mouse OneArray  
 PNAS. 2010, 107(21):9753-8. doi: 10.1073/pnas.0912585107.
 Pitx2 prevents susceptibility to atrial arrhythmias by inhibiting left-sided pacemaker specification.
 
 
 Wang J, Klysik E, Sood S, Johnson RL, Wehrens XH, Martin JF
  Abstract
Atrial fibrillation (AF), the most prevalent sustained cardiac arrhythmia, often coexists with the related arrhythmia atrial flutter (AFL). Limitations in effectiveness and safety of current therapies make an understanding of the molecular mechanism underlying AF more urgent. Genome-wide association studies implicated a region of human chromosome 4q25 in familial AF and AFL, approximately 150 kb distal to the Pitx2 homeobox gene, a developmental left-right asymmetry (LRA) gene. To investigate the significance of the 4q25 variants, we used mouse models to investigate Pitx2 in atrial arrhythmogenesis directly. When challenged by programmed stimulation, Pitx2(null+/-) adult mice had atrial arrhythmias, including AFL and atrial tachycardia, indicating that Pitx2 haploinsufficiency predisposes to atrial arrhythmias. Microarray and in situ studies indicated that Pitx2 suppresses sinoatrial node (SAN)-specific gene expression, including Shox2, in the left atrium of embryos and young adults. In vivo ChIP and transfection experiments indicated that Pitx2 directly bound Shox2 in vivo, supporting the notion that Pitx2 directly inhibits the SAN-specific genetic program in left atrium. Our findings implicate Pitx2 and Pitx2-mediated LRA-signaling pathways in prevention of atrial arrhythmias.
   

  Mouse OneArray  
 Eur J Oral Sci. 2010, 118(2):118-30. doi: 10.1111/j.1600-0722.2010.00722.x.
 Gene expression and dental enamel structure in developing mouse incisor.
 
 
 Risnes S, Khan QE, Khuu C, Osmundsen H, Amer Sehic
  Abstract
At the mouse incisor tip the initially differentiated ameloblasts produce a thin, prism-free enamel, while further apically, in the immediate adjacent segment, the enamel thickness increases and the four-layered enamel of mouse incisor is formed. Comparative gene-expression profiling was carried out on RNA isolated from these two segments of incisor tooth germs at embryonic day (E)17.5 and at postnatal days (P)0, 1, 2, and 10 using microarrays to measure messenger RNA (mRNA) and microRNA (miRNA) species present in the segments. Validation of expression data was achieved using real-time reverse transcription-polymerase chain reaction (RT-PCR) and western blotting. Bioinformatic data suggested enhanced cellular apoptosis in the incisal tip segment, which, together with diminished expression of the Amelx and Enam genes, may contribute to the production of the thin enamel seen in this tooth segment. For genes exhibiting higher levels of expression in the adjacent segment where complex enamel is being formed, bioinformatic analysis suggested significant associations with cellular functions involving the actin cytoskeleton, cellular development, morphology, and movement. This is suggested to reflect that ameloblasts with Tomes' process are being organized in transverse rows, facilitating the transverse movement that results in prism decussation in the inner enamel of the adjacent segment. Bioinformatic analysis of miRNA expression data lends support to these suggestions.
   

  Experimental Accessories  
 Reprod Biol Endocrinol. 2009, 11;7:13. doi: 10.1186/1477-7827-7-13.
 Altered microRNA expression in patients with non-obstructive azoospermia
 
 
 Jie Lian, Xiansheng Zhang, Hui Tian, Ning Liang, Yong Wang, Chaozhao Liang, Xin Li, Fei Sun
  Abstract
MicroRNAs (miRNAs), a class of small non-coding RNA molecules, are indicated to play essential roles in spermatogenesis. However, little is known about the expression patterns or function of miRNAs in human testes involved in infertility. In this study, the miRNA expression profiles of testes of patients with non-obstructive azoospermia (NOA) and normal controls were performed by using microarray technologies. Altered microRNA expression in NOA patients was found, with 154 differentially down-regulated and 19 up-regulated miRNAs. These findings have been confirmed by real-time reverse transcription-polymerase chain reaction (RT-PCR) assays on select miRNAs, including miR-302a, miR-491-3p, miR-520d-3p and miR-383. Several down-regulated miRNA clusters in patients with NOA were identified, such as the oncogenic potential of the mir-17-92 cluster and mir-371,2,3 cluster. This is the first report that the expression of miRNAs is altered in testicular tissues of patients with NOA, suggesting a role of miRNAs in regulating spermatogenesis in human males.
   

  Human OneArray, Mouse OneArray  
 BLOOD. 2009, 114(15):3181-90. doi: 10.1182/blood-2009-02-205708.
 Characterization of Gaucher disease bone marrow mesenchymal stromal cells reveals an altered in?ammatory secretome.
 
 
 Philippe M. Campeau, Moutih Rafei, Marie-No ?le Boivin, Ying Sun, Gregory A. Grabowski, Jacques Galipeau
  Abstract
Gaucher disease causes pathologic skeletal changes that are not fully explained. Considering the important role of mesenchymal stromal cells (MSCs) in bone structural development and maintenance, we analyzed the cellular biochemistry of MSCs from an adult patient with Gaucher disease type 1 (N370S/L444P mutations). Gaucher MSCs possessed a low glucocerebrosidase activity and consequently had a 3-fold increase in cellular glucosylceramide. Gaucher MSCs have a typical MSC marker phenotype, normal osteocytic and adipocytic differentiation, growth, exogenous lactosylceramide trafficking, cholesterol content, lysosomal morphology, and total lysosomal content, and a marked increase in COX-2, prostaglandin E2, interleukin-8, and CCL2 production compared with normal controls. Transcriptome analysis on normal MSCs treated with the glucocerebrosidase inhibitor conduritol B epoxide showed an up-regulation of an array of inflammatory mediators, including CCL2, and other differentially regulated pathways. These cells also showed a decrease in sphingosine-1-phosphate. In conclusion, Gaucher disease MSCs display an altered secretome that could contribute to skeletal disease and immune disease manifestations in a manner distinct and additive to Gaucher macrophages themselves.
   

  Human OneArray  
 Biochemical and Biophysical Research Communications. 2009, 387(2):239-44. doi: 10.1016/j.bbrc.2009.06.128.
 Dual knockdown of N-ras and epiregulin synergistically suppressed the growth of human hepatoma cells.
 
 
 Meng Zhao, Hong-wei He, Huan-xing Sun, Kai-huan Ren, Rong-guang Shao
  Abstract
Hepatocellular carcinoma (HCC) is a major challenge because of its resistance to conventional cytotoxic chemotherapy and radiotherapy. Multi-targeted therapy might be a new option for HCC treatment. Our previous study showed that N-ras gene was activated in HCC and was inhibited by RNA interference. In the present study, we investigated the alternation of gene expression by microarray in N-Ras-siRNA-treated HepG2 cells. The results revealed that the EREG gene, encoding epiregulin, was dramatically up-regulated in response to silence of N-ras. We speculated that the up-regulation of epiregulin was involved in the compensatory mechanism of N-ras knockdown for cell growth. Therefore, we evaluated whether dual silence of N-ras and epiregulin display a greater suppression of cell growth. The results confirmed that dual knockdown of N-ras and epiregulin synergistically inhibited cell growth. Our results also showed that dual knockdown of N-ras and epiregulin significantly induced cell arrest at G0/G1 phase. Furthermore, Western blot assay showed that dual knockdown of N-ras and epiregulin markedly reduced the phosphorylations of ERK1/2, Akt and Rb, and inhibited the expression of cyclin D1. Our findings imply that multi-targeted silence of oncogenes might be an effective treatment for HCC.
   

  Human OneArray  
 Zhonghua Xin Xue Guan Bing Za Zhi. 2009, 37(2):120-125.
 Identification of differentially expressed genes in myocardium of patients with heart failure by human whole genomic oligonucleotide microarray-assisted pathways analysis.
 
 
 Wu XX, Wan T, Wu HJ, Zhi G, Xiao CS, Gao CQ, WU Jia-jin
  Abstract
To identify the differentially expressed gene profiles in myocardium of patients with heart failure using human whole genomic oligonucleotide microarray-assisted pathway analysis. Phalanx whole genomic oligonucleotide microarrays were used to detect the gene expression profiles of myocardium in four patients died of heart failure and 4 brain died patients without heart diseases. The microarray findings were confirmed by real-time quantitative reverse transcriptase-polymerase chain reaction. The genes with a threshold of 1.2 times fold-change were selected and BioCarta Pathway and KEGG (Kyoto Encyclopaedia of Genes and Genomes) pathway databases were used to identify functionallyrelated gene pathways. A total of 2806 genes with differentially expression were detected between the failing and non-failing heart samples,expression changes of 399 genes were more than 2-folds. Eleven pathways were identified by BioCarta pathway database and sixteen pathways were identified by KEGG PATHWAY Database. Genomic microarray-assisted pathway analysis could help to identify gene expression profiles in failing heart.