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1.  Regulation of Active DNA Demethylation by an α-Crystallin Domain Protein in Arabidopsis 
Molecular cell  2014;55(3):361-371.
DNA methylation patterns are dynamically controlled by DNA methylation and active DNA demethylation, but the mechanisms of regulation of active DNA demethylation are not well understood. Through forward genetic screens for Arabidopsis mutants showing DNA hypermethylation at specific loci and increased silencing of reporter genes, we identified IDM2 (increased DNA methylation 2) as a regulator of DNA demethylation and gene silencing. IDM2 dysfunction causes DNA hypermethylation and silencing of reporter genes and some endogenous genes. These effects of idm2 mutations are similar to those of mutations in IDM1, a regulator of active DNA demethylation. IDM2 encodes an α-crystallin domain protein in the nucleus. IDM2 and IDM1 interact physically and partially colocalize at discrete subnuclear foci. IDM2 is required for the full activity of H3K18 acetylation but not H3K23 acetylation of IDM1 in planta. Our results suggest that IDM2 functions in active DNA demethylation and in antisilencing by regulating IDM1.
PMCID: PMC4302764  PMID: 25002145
2.  An AP Endonuclease Functions in Active DNA Dimethylation and Gene Imprinting in Arabidopsis 
PLoS Genetics  2015;11(1):e1004905.
Active DNA demethylation in plants occurs through base excision repair, beginning with removal of methylated cytosine by the ROS1/DME subfamily of 5-methylcytosine DNA glycosylases. Active DNA demethylation in animals requires the DNA glycosylase TDG or MBD4, which functions after oxidation or deamination of 5-methylcytosine, respectively. However, little is known about the steps following DNA glycosylase action in the active DNA demethylation pathways in plants and animals. We show here that the Arabidopsis APE1L protein has apurinic/apyrimidinic endonuclease activities and functions downstream of ROS1 and DME. APE1L and ROS1 interact in vitro and co-localize in vivo. Whole genome bisulfite sequencing of ape1l mutant plants revealed widespread alterations in DNA methylation. We show that the ape1l/zdp double mutant displays embryonic lethality. Notably, the ape1l+/−zdp−/− mutant shows a maternal-effect lethality phenotype. APE1L and the DNA phosphatase ZDP are required for FWA and MEA gene imprinting in the endosperm and are important for seed development. Thus, APE1L is a new component of the active DNA demethylation pathway and, together with ZDP, regulates gene imprinting in Arabidopsis.
Author Summary
DNA cytosine methylation (5-methylcytosine, 5-meC) is an important epigenetic mark, and methylation patterns are coordinately controlled by methylation and demethylation reactions during development and reproduction. In plants, REPRESSOR OF SILENCING (ROS1) is one of the well characterized 5-meC DNA glycosylases that initiate active DNA demethylation by 5-meC excision. Our previous work showed that a 3′-DNA phosphatase, ZDP, functions downstream of ROS1 during active DNA demethylation in Arabidopsis. Here we found that the apurinic/apyrimidinic endonuclease APE1L functions downstream of ROS1 in a ZDP-independent branch of the active DNA demethylation pathway in Arabidopsis. In plants, gene imprinting requires the 5-meC DNA glycosylase Demeter (DME) that has been proposed to initiate a base excision repair pathway for active DNA demethylation in the central cell in female gametophyte. However, besides DME, no other base excision repair enzymes have been found to be important for gene imprinting. Our results show that APE1L and ZDP act jointly downstream of DME to regulate gene imprinting in plants, and suggest that DME-initiated active DNA demethylation in the central cell and endosperm uses both APE- and ZDP-dependent mechanisms.
PMCID: PMC4287435  PMID: 25569774
3.  Local Application of Sodium Salicylate Enhances Auditory Responses in the Rat’s Dorsal Cortex of the Inferior Colliculus 
Sodium salicylate (SS) is a widely used medication with side effects on hearing. In order to understand these side effects, we recorded sound-driven local-field potentials in a neural structure, the dorsal cortex of the inferior colliculus (ICd). Using a microiontophoretic technique, we applied SS at sites of recording and studied how auditory responses were affected by the drug. Furthermore, we studied how the responses were affected by combined local application of SS and an agonists/antagonist of the type-A or type-B γ-aminobutyric acid receptor (GABAA or GABAB receptor). Results revealed that SS applied alone enhanced auditory responses in the ICd, indicating that the drug had local targets in the structure. Simultaneous application of the drug and a GABAergic receptor antagonist synergistically enhanced amplitudes of responses. The synergistic interaction between SS and a GABAA receptor antagonist had a relatively early start in reference to the onset of acoustic stimulation and the duration of this interaction was independent of sound intensity. The interaction between SS and a GABAB receptor antagonist had a relatively late start, and the duration of this interaction was dependent on sound intensity. Simultaneous application of the drug and a GABAergic receptor agonist produced an effect different from the sum of effects produced by the two drugs released individually. These differences between simultaneous and individual drug applications suggest that SS modified GABAergic inhibition in the ICd. Our results indicate that SS can affect sound-driven activity in the ICd by modulating local GABAergic inhibition.
PMCID: PMC4231951  PMID: 25452744
auditory system; GABAA receptor; GABAB receptor; hearing; midbrain; tinnitus; microiontophoresis
4.  Protocol: a beginner’s guide to the analysis of RNA-directed DNA methylation in plants 
Plant Methods  2014;10:18.
DNA methylation is a conserved epigenetic mark that controls genome stability, development and environmental responses in many eukaryotes. DNA methylation can be guided by non-coding RNAs that include small interfering RNAs and scaffold RNAs. Although measurement of DNA methylation and regulatory non-coding RNAs is desirable for many biologists who are interested in exploring epigenetic regulation in their areas, conventional methods have limitations and are technically challenging. For instance, traditional siRNA detection through RNA hybridization requires relatively large amount of small RNAs and involves radioactive isotopes. An alternative approach is RT-qPCR that employs stem loop primers during reverse transcription; however, it requires a prerequisite that the exact sequences of siRNAs should be known.
By using the model organism Arabidopsis thaliana, we developed an easy-to-follow, integrative procedure for time-efficient, quantitative measurement of DNA methylation, small interfering RNAs, and scaffold RNAs. Starting with simplified nucleic acid manipulation, we examined DNA methylation levels by using Chop PCR (methylation-sensitive enzyme digestion followed by PCR), which allowed for fast screening for DNA methylation mutants without the need of transgenic reporters. We deployed a simple bioinformatics method for mining published small RNA databases, in order to obtain the nucleotide (nt) sequences of individual 24nt siRNAs within the regions of interest. The protocol of commercial TaqMan Small RNA Assay was subsequently optimized for reliable quantitative detection of individual siRNAs. We used nested qPCR to quantify scaffold RNAs that are of low abundance and without Poly-A tails. In addition, nuclei fraction enables separation of chromatin-associated scaffold RNAs from their cognate non-scaffold transcripts that have been released from chromatin.
We have developed a procedure for quantitative investigations on nucleic acids that are core components of RNA-directed DNA methylation. Our results not only demonstrated the efficacy of this procedure, but also provide lists of methylation-sensitive restriction enzymes, novel DNA methylation marker loci, and related siRNA sequences, all of which can be valuable for future epigenetic studies. Importantly, step-by-step protocols are provided in details such that the approaches can be easily followed by biologists with little experience in epigenetics.
PMCID: PMC4065543  PMID: 24955108
Epigenetics; RNA-directed DNA methylation; siRNA; Scaffold RNA; Pol IV; Pol V; Nuclei fractionation
5.  Mechanisms of Small RNA Generation from Cis-NATs in Response to Environmental and Developmental Cues 
Molecular Plant  2013;6(3):704-715.
This review summarizes the current findings on the occurrence and characteristics of cis-natural antisense transcripts (NATs) and nat-siRNAs; discusses the biogenesis, regulations and functions of nat-siRNAs; and highlights the advantages and limitations of new technologies on cis-NATs detection.
A large proportion of eukaryotic genomes is transcribed from both positive and negative strands of DNA and thus may generate overlapping sense and antisense transcripts. Some of these so-called natural antisense transcripts (NATs) are possibly co-regulated. When the overlapping sense and antisense transcripts are expressed at the same time in the same cell in response to various developmental and environmental cues; they may form double-stranded RNAs, which could be recognized by the small RNA biogenesis machinery and processed into small interfering RNAs (siRNAs). cis-NAT-derived siRNAs (nat-siRNAs) are present in plants, animals, and fungi. In plants, the presence of nat-siRNAs is supported not only by Northern blot and genetic analyses, but also by the fact that there is an overall sixfold enrichment of siRNAs in the overlapping regions of cis-NATs and 19%–29% of the siRNA-generating cis-NATs in plants give rise to siRNAs only in their overlapping regions. Silencing mediated by nat-siRNAs is one of the mechanisms for regulating the expression of the cis-NATs. This review focuses on challenging issues related to the biogenesis mechanisms as well as regulation and detection of nat-siRNAs. The advantages and limitations of new technologies for detecting cis-NATs, including direct RNA sequencing and strand-specific RNA sequencing, are also discussed.
PMCID: PMC3660955  PMID: 23505223
gene expression; gene regulation; gene silencing.
6.  Chemical probes in plant epigenetics studies 
Plant Signaling & Behavior  2013;8(9):e25364.
Transcription potential is determined by the accessibility of DNA sequences within the context of chromatin, which is coordinately controlled by various epigenetic modifications. Chemical inhibition of epigenetic regulators provides a quick and effective approach to investigate the roles of epigenetic modifications in controlling many biological processes, especially for species in which genetic information is limited. This mini-review provides a brief overview of epigenetic regulators in the model organism Arabidopsis thaliana and summarizes compounds that have been applied in plant epigenetics studies, with highlights in the applications of these chemical probes in mechanistic and functional investigations.
PMCID: PMC4002629  PMID: 23838953
plant epigenetics; chemical inhibitors; DNA methylation; histone modification; methyltransferase inhibitors
7.  The Quantitative Imaging Network: NCI's Historical Perspective and Planned Goals 
Translational Oncology  2014;7(1):1-4.
The purpose of this editorial is to provide a brief history of National Institutes of Health National Cancer Institute (NCI) workshops as related to quantitative imaging within the oncology setting. The editorial will then focus on the recently supported NCI initiatives, including the Quantitative Imaging Network (QIN) initiative and its organizational structure, including planned research goals and deliverables. The publications in this issue of Translational Oncology come from many of the current members of this QIN research network.
PMCID: PMC3998696  PMID: 24772201
8.  Dynamic Chemical Communication between Plants and Bacteria through Airborne Signals: Induced Resistance by Bacterial Volatiles 
Journal of Chemical Ecology  2013;39(7):1007-1018.
Certain plant growth-promoting rhizobacteria (PGPR) elicit induced systemic resistance (ISR) and plant growth promotion in the absence of physical contact with plants via volatile organic compound (VOC) emissions. In this article, we review the recent progess made by research into the interactions between PGPR VOCs and plants, focusing on VOC emission by PGPR strains in plants. Particular attention is given to the mechanisms by which these bacterial VOCs elicit ISR. We provide an overview of recent progress in the elucidation of PGPR VOC interactions from studies utilizing transcriptome, metabolome, and proteome analyses. By monitoring defense gene expression patterns, performing 2-dimensional electrophoresis, and studying defense signaling null mutants, salicylic acid and ethylene have been found to be key players in plant signaling pathways involved in the ISR response. Bacterial VOCs also confer induced systemic tolerance to abiotic stresses, such as drought and heavy metals. A review of current analytical approaches for PGPR volatile profiling is also provided with needed future developments emphasized. To assess potential utilization of PGPR VOCs for crop plants, volatile suspensions have been applied to pepper and cucumber roots and found to be effective at protecting plants against plant pathogens and insect pests in the field. Taken together, these studies provide further insight into the biological and ecological potential of PGPR VOCs for enhancing plant self-immunity and/or adaptation to biotic and abiotic stresses in modern agriculture.
PMCID: PMC3738840  PMID: 23881442
PGPR; ISR; IST; Volatile organic compounds; Headspace
9.  Effect of advanced glycation end products on the expression of hypoxia-inducible factor-1α and vascular endothelial growth factor proteins in RF/6A cells 
The aim of this study was to investigate the effect of advanced glycation end products (AGEs) on the expression of hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF) proteins in RF/6A cells cultured in vitro, and to investigate the association between the expression of HIF-1α and VEGF proteins. RF/6A cells were cultured in vitro and treated with AGEs and non-glycated albumin control at various concentrations (0, 50, 100, 200, 400 and 800 mg/l) for 24 h. The expression of the VEGF protein was detected by ELISA, and western blot analysis was used to determine the levels of HIF-1α protein. The expression of HIF-1α and VEGF proteins was significantly higher in the AGE group compared with the non-glycated control group (all P<0.05). With the increase in concentration of AGEs, the expression levels of HIF-1α and VEGF protein increased and reached a maximum at 200 mg/l AGE, then decreased at 400 and 800 mg/l. However this effect was not observed in the non-glycated control groups. There was a positive correlation between the expression of HIF-1α and VEGF (P<0.05). AGEs induced the expression of HIF-1α and VEGF proteins in RF/6A cells in a concentration-dependent manner. AGEs may upregulate the expression of VEGF protein by increasing the levels of HIF-1α protein, demonstrating the potential role of HIF-1α-targeted therapy in neovascularization.
PMCID: PMC3671897  PMID: 23737911
hypoxia-inducible factor-1α; vascular endothelial growth factor; advanced glycation end products; RF/6A cells
10.  Molecular Mimicry Regulates ABA Signaling by SnRK2 Kinases and PP2C Phosphatases 
Science (New York, N.Y.)  2011;335(6064):85-88.
Abscisic acid (ABA) is an essential hormone for plants to survive environmental stresses. At the center of the ABA signaling network is a subfamily of type 2C protein phosphatases (PP2Cs), which form exclusive interactions with ABA receptors and subfamily 2 Snfl-related kinase (SnRK2s). Here, we report a SnRK2-PP2C complex structure, which reveals marked similarity in PP2C recognition by SnRK2 and ABA receptors. In the complex, the kinase activation loop docks into the active site of PP2C, while the conserved ABA-sensing tryptophan of PP2C inserts into the kinase catalytic cleft, thus mimicking receptor-PP2C interactions. These structural results provide a simple mechanism that directly couples ABA binding to SnRK2 kinase activation and highlight a new paradigm of kinase-phosphatase regulation through mutual packing of their catalytic sites.
PMCID: PMC3584687  PMID: 22116026
11.  Positive and Negative Relationship between Anxiety and Depression of Patients in Pain: A Bifactor Model Analysis 
PLoS ONE  2012;7(10):e47577.
The relationship between anxiety and depression in pain patients has not been clarified comprehensively. Previous research has identified a common factor in anxiety and depression, which may explain why depression and anxiety are strongly correlated. However, the specific clinical features of anxiety and depression seem to pull in opposite directions.
The purpose of this study is to develop a statistical model of depression and anxiety, based on data from pain patients using Hospital Anxiety and Depression Scale (HADS). This model should account for the positive correlation between depression and anxiety in terms of a general factor and also demonstrate a latent negative correlation between the specific factors underlying depression and anxiety.
The anxiety and depression symptoms of pain patients were evaluated using the HADS and the severity of their pain was assessed with the visual analogue scale (VAS). We developed a hierarchical model of the data using an IRT method called bifactor analysis. In addition, we tested this hierarchical model with model fit comparisons with unidimensional, bidimensional, and tridimensional models. The correlations among anxiety, depression, and pain severity were compared, based on both the bidimensional model and our hierarchical model.
The bidimensional model analysis found that there was a large positive correlation between anxiety and depression (r = 0.638), and both scores were significantly positively correlated with pain severity. After extracting general factor of distress using bifactor analysis, the specific factors underlying anxiety and depression were weakly but significantly negatively correlated (r = −0.245) and only the general factor was significantly correlated with pain severity. Compared with the three first-order models, the bifactor hierarchical model had the best model fit.
Our results support the hypothesis that apart from distress, anxiety and depression are inversely correlated. This finding has not been convincingly demonstrated in previous research.
PMCID: PMC3475698  PMID: 23094064
12.  RNA-directed DNA Methylation 
Current opinion in plant biology  2011;14(2):142-147.
DNA methylation is an important epigenetic mechanism for silencing transposons and other repetitive elements, and for stable repression of specific transgenes and endogenous genes. Plants can utilize small interfering RNAs (siRNAs) to guide de novo DNA methyltransferases for the establishment of sequence-specific DNA methylation. Genetic and biochemical approaches have identified many components involved in RNA-directed DNA methylation (RdDM). These components function in one or more of the following three aspects: biogenesis of siRNAs, production of scaffold RNAs, and the assembly of an effector complex that involves the complementary pairing between the guide siRNAs and nascent scaffold RNAs and which recruits the DNA methyltransferases. Recent studies not only unveiled new molecular players and novel interactions, but also suggested spatial and temporal segregation of the RdDM process within the nucleus.
PMCID: PMC3096526  PMID: 21420348
13.  Genome-wide analysis of plant nat-siRNAs reveals insights into their distribution, biogenesis and function 
Genome Biology  2012;13(3):R20.
Many eukaryotic genomes encode cis-natural antisense transcripts (cis-NATs). Sense and antisense transcripts may form double-stranded RNAs that are processed by the RNA interference machinery into small interfering RNAs (siRNAs). A few so-called nat-siRNAs have been reported in plants, mammals, Drosophila, and yeasts. However, many questions remain regarding the features and biogenesis of nat-siRNAs.
Through deep sequencing, we identified more than 17,000 unique siRNAs corresponding to cis-NATs from biotic and abiotic stress-challenged Arabidopsis thaliana and 56,000 from abiotic stress-treated rice. These siRNAs were enriched in the overlapping regions of NATs and exhibited either site-specific or distributed patterns, often with strand bias. Out of 1,439 and 767 cis-NAT pairs identified in Arabidopsis and rice, respectively, 84 and 119 could generate at least 10 siRNAs per million reads from the overlapping regions. Among them, 16 cis-NAT pairs from Arabidopsis and 34 from rice gave rise to nat-siRNAs exclusively in the overlap regions. Genetic analysis showed that the overlapping double-stranded RNAs could be processed by Dicer-like 1 (DCL1) and/or DCL3. The DCL3-dependent nat-siRNAs were also dependent on RNA-dependent RNA polymerase 2 (RDR2) and plant-specific RNA polymerase IV (PolIV), whereas only a fraction of DCL1-dependent nat-siRNAs was RDR- and PolIV-dependent. Furthermore, the levels of some nat-siRNAs were regulated by specific biotic or abiotic stress conditions in Arabidopsis and rice.
Our results suggest that nat-siRNAs display distinct distribution patterns and are generated by DCL1 and/or DCL3. Our analysis further supported the existence of nat-siRNAs in plants and advanced our understanding of their characteristics.
PMCID: PMC3439971  PMID: 22439910
14.  The level and distribution of the GABABR1 and GABABR2 receptor subunits in the rat's inferior colliculus 
The type B γ-aminobutyric acid receptor (GABAB receptor) is an important neurotransmitter receptor in the midbrain auditory structure, the inferior colliculus (IC). A functional GABAB receptor is a heterodimer consisting of two subunits, GABABR1 and GABABR2. Western blotting and immunohistochemical experiments were conducted to examine the expression of the two subunits over the IC including its central nucleus, dorsal cortex, and external cortex (ICc, ICd, and ICx). Results revealed that the two subunits existed in both cell bodies and the neuropil throughout the IC. The two subunits had similar regional distributions over the IC. The combined level of cell body and neuropil labeling was higher in the ICd than the other two subdivisions. Labeling in the ICc and ICx was stronger in the dorsal than the ventral regions. In spite of regional differences, no defined boundaries were formed between different areas. For both subunits, the regional distribution of immunoreactivity in the neuropil was parallel to that of combined immunoreactivity in the neuropil and cell bodies. The density of labeled cell bodies tended to be higher but sizes of cell bodies tended to be smaller in the ICd than in the other subdivisions. No systematic regional changes were found in the level of cell body immunoreactivity, except that GABABR2-immunoreactive cell bodies in the ICd had slightly higher optic density (OD) than in other regions. Elongated cell bodies existed throughout the IC. Many labeled cell bodies along the outline of the IC were oriented in parallel to the outline. No strong tendency of orientation was found in labeled cell bodies in ICc. Regional distributions of the subunits in ICc correlated well with inputs to this subdivision. Our finding regarding the contrast in the level of neuropil immunoreactivity among different subdivisions is consistent with the fact that the GABAB receptor has different pre- and postsynaptic functions in different IC regions.
PMCID: PMC3506002  PMID: 23189044
hearing; auditory system; auditory midbrain; GABA; GABAB receptor; GABABR1 subunit; GABABR2 subunit; inhibition
15.  Potent in vitro and in vivo anticancer activities of des-methyl, des-amino pateamine A, a synthetic analogue of marine natural product pateamine A 
Molecular cancer therapeutics  2009;8(5):1250-1260.
We report here that des-methyl, des-amino pateamine A (DMDA-PatA), a structurally simplified analogue of the marine natural product pateamine A, has potent antiproliferative activity against a wide variety of human cancer cell lines while showing relatively low cytotoxicity against nonproliferating, quiescent human fibroblasts. DMDA-PatA retains almost full in vitro potency in P-glycoprotein-overexpressing MES-SA/D×5-R×1 human uterine sarcoma cells that are significantly resistant to paclitaxel, suggesting that DMDA-PatA is not a substrate for P-glycoprotein-mediated drug efflux. Treatment of proliferating cells with DMDA-PatA leads to rapid shutdown of DNA synthesis in the S phase of the cell cycle. Cell-free studies show that DMDA-PatA directly inhibits DNA polymerases α and γ in vitro albeit at concentrations considerably higher than those that inhibit cell proliferation. DMDA-PatA shows potent anticancer activity in several human cancer xenograft models in nude mice, including significant regressions observed in the LOX and MDA-MB-435 melanoma models. DMDA-PatA thus represents a promising natural product-based anticancer agent that warrants further investigation.
PMCID: PMC3026899  PMID: 19417157
16.  Sustained growth promotion in arabidopsis with long-term exposure to the beneficial soil bacterium Bacillus subtilis (GB03) 
Plant Signaling & Behavior  2009;4(10):948-953.
Volatile emissions from the commercial growth promoting soil bacterium Bacillus subtilis (GB03) are effective in augmenting short-term growth, photosynthetic capacity and salt tolerance in Petri-dish grown arabidopsis seedlings. In contrast, the impact sustained GB03 volatile exposure on plant growth and development has yet to be examined. here is provided physical and physiological data establishing that bacterial volatiles induce long-term growth promotion, elevated photosynthetic capacity and iron accumulation, as well as delayed albeit higher seed count compared with water-treated control plants. Plants were grown unrestricted in double Magenta boxes containing solid MS media for up to twelve weeks with GB03 volatiles introduced in separate containers within the chamber so that plant bacterial interactions were only by air-borne transmission. These results establish that GB03 volatiles induce sustained beneficial effects on Arabidopsis growth including robust and extended vegetative growth followed by elevated seed set.
PMCID: PMC2801358  PMID: 19826235
Bacillus subtilis (GB03); photosynthetic efficiency; plant growth promoting rhizobacteria (PGPR); seed set; volatile organic compound (VOCs)
17.  B7 Blockade Alters the Balance between Regulatory T cells and Tumor-reactive T Cells for Immunotherapy of Cancer 
In prostate cancer bearing host, regulatory T cells restrain activity of tumor antigen specific T cells. Since B7:CD28 interactions are needed for both function of CD4+CD25+ Treg cells and the CD8+ effective T cells, targeting this pathway may help to overcome the immunotherapy barriers.
Experimental design
The anti-B7−1/B7−2 mAbs were administrated to a transgenic mouse model of prostate cancer (TRAMP) ectopically expressing SV40 large T antigen (TAg) in different tumor development stages for prevention and therapy of prostate cancer. The treatment was also tested in treating transplanted MC38 colon adenocarcinoma in mice.
Here we showed that short-term administration of anti-B7−1/B7−2 mAbs in TRAMP mice leads to significant inhibited primary tumor growth and the size of metastatic lesions. The treatment is effective to inhibit MC38 colon cancer growth. Correspondingly, this treatment results in a transient reduction of Treg in both thymus and the periphery. In vivo cytotoxicity assay revealed TAg-specific CTL effectors in anti-B7 treated, but not control IgG-treated TRAMP mice.
Transient blockade of B7−1/2 alters the balance between Treg and cancer-reactive T cells to enhance cancer immunotherapy.
PMCID: PMC2693886  PMID: 19188167
regulatory T cells; costimulatory molecule; prostate cancer
19.  Fibroblast growth factor receptor signaling is essential for lens fiber cell differentiation 
Developmental biology  2008;318(2):276-288.
The vertebrate lens provides an excellent model to study the mechanisms that regulate terminal differentiation. Although fibroblast growth factors (FGFs) are thought to be important for lens cell differentiation, it is unclear which FGF receptors mediate these processes during different stages of lens development. Deletion of three FGF receptors (Fgfr1-3) early in lens development demonstrated that expression of only a single allele of Fgfr2 or Fgfr3 was sufficient for grossly normal lens development, while mice possessing only a single Fgfr1 allele developed cataracts and microphthalmia. Profound defects were observed in lenses lacking all three Fgfrs. These included lack of fiber cell elongation, abnormal proliferation in prospective lens fiber cells, reduced expression of the cell cycle inhibitors p27kip1 and p57kip2, increased apoptosis and aberrant or reduced expression of Prox1, Pax6, c-Maf, E-cadherin and α-, β- and γ-crystallins. Therefore, while signaling by FGF receptors is essential for lens fiber differentiation, different FGF receptors function redundantly.
PMCID: PMC2574794  PMID: 18455718
apoptosis; cell cycle; FGF receptor; lens development; lens fiber differentiation; redundancy; conditional knockout
20.  Modulation of NKT Cell Development by B7-CD28 Interaction: An Expanding Horizon for Costimulation 
PLoS ONE  2008;3(7):e2703.
It has been demonstrated that the development of NKT cells requires CD1d. The contribution of costimulatory molecules in this process has not been studied. Here we show that in mice with targeted mutations of B7-1/2 and CD28, the TCRβ+α-Galcer/CD1d + (iVα14 NKT) subset is significantly reduced in the thymus, spleen and liver. This is mainly due to decreased cell proliferation; although increased cell death in the thymi of CD28-deficient mice was also observed. Moreover, in the B7-1/2- and CD28-deficient mice, we found a decreased percentage of the CD4−NK1.1+ subset and a correspondingly increased portion of the CD4+NK1.1− subset. In addition, the mice with a targeted mutation of either B7 or CD28 had a reduced susceptibility to Con A induced hepatitis, which is known to be mediated by NKT cells. Our results demonstrate that the development, maturation and function of NKT cell are modulated by the costimulatory pathway and thus expand the horizon of costimulation into NKT, which is widely viewed as a bridge between innate and adaptive immunity. As such, costimulation may modulate all major branches of cell-mediated immunity, including T cells, NK cells and NKT cells.
PMCID: PMC2442875  PMID: 18628995
21.  FOXP3 is an X-linked breast cancer suppressor gene and an important repressor of the HER-2/ErbB2 oncogene 
Cell  2007;129(7):1275-1286.
The X-linked Foxp3 is a member of the forkhead/winged helix transcription factor family. Germ-line mutations cause lethal autoimmune diseases in males. Serendipitously, we observed that Foxp3sf/+ heterozygous mice developed cancer at a high rate. The majority of the cancers were mammary carcinomas in which the wild-type Foxp3 allele was inactivated and ErbB2 was over-expressed. Foxp3 bound and repressed the ErbB2 promoter. Deletion, functionally significant somatic mutations and down-regulation of the FOXP3 gene were commonly found in human breast cancer samples and correlated significantly with HER-2 over-expression, regardless of the status of HER-2 amplification. In toto, the data demonstrate that FOXP3 is an X-linked breast cancer suppressor gene and an important regulator of the HER-2/ErbB2 oncogene.
PMCID: PMC1974845  PMID: 17570480
22.  Water diffusion-exchange effect on the paramagnetic relaxation enhancement in off-resonance rotating frame 
The off-resonance rotating frame technique based on the spin relaxation properties of off-resonance T1ρ can significantly increase the sensitivity of detecting paramagnetic labeling at high magnetic fields. However, the in vivo detectable dimension for labeled cell clusters/tissues in T1ρ -weighted images is limited by the water diffusion-exchange between mesoscopic scale compartments. An experimental investigation of the effect of water diffusion-exchange between compartments on the paramagnetic relaxation enhancement of paramagnetic agent compartment is presented for in vitro/in vivo models. In these models, the size of paramagnetic agent compartment is comparable to the mean diffusion displacement of water molecules during the long RF pulses that are used to generate the off-resonance rotating frame. The three main objectives of this study were: (1) to qualitatively correlate the effect of water diffusion-exchange with the RF parameters of the long pulse and the rates of water diffusion, (2) to explore the effect of water diffusion-exchange on the paramagnetic relaxation enhancement in vitro, and (3) to demonstrate the paramagnetic relaxation enhancement in vivo. The in vitro models include the water permeable dialysis tubes or water permeable hollow fibers embedded in cross-linked proteins gels. The MWCO of the dialysis tubes was chosen from 0.1 to 15 kDa to control the water diffusion rate. Thin hollow fibers were chosen to provide sub-millimeter scale compartments for the paramagnetic agents. The in vivo model utilized the rat cerebral vasculatures as a paramagnetic agent compartment, and intravascular agents (Gd-DTPA)30-BSA were administrated into the compartment via bolus injections. Both in vitro and in vivo results demonstrate that the paramagnetic relaxation enhancement is predominant in the T1ρ -weighted imaging in the presence of water diffusion exchange. The T1ρ contrast has substantially higher sensitivity than the conventional T1 contrast in detecting paramagnetic agents, especially at low paramagnetic agent volumetric fractions, low paramagnetic agent concentrations, and low RF amplitudes. Short pulse duration, short pulse recycle delay and efficient paramagnetic relaxation can reduce the influence of water diffusion-exchange on the paramagnetic enhancement. This study paves the way for the design of off-resonance rotating experiments to detect labeled cell clusters/tissue compartments in vivo at a sub-millimeter scale.
PMCID: PMC2041893  PMID: 17412624
off-resonance rotating frame; paramagnetic relaxation enhancement; diffusion-exchange effect; volumetric coefficient; imaging contrast; rat brain images; gadolinium chelates
23.  Dynamics of paramagnetic agents by off-resonance rotating frame technique in the presence of magnetization transfer effect 
The simple method for measuring the rotational correlation time of paramagnetic ion chelates via off-resonance rotating frame technique is challenged in vivo by the magnetization transfer effect. A theoretical model for the spin relaxation of water protons in the presence of paramagnetic ion chelates and magnetization transfer effect is described. This model considers the competitive relaxations of water protons by the paramagnetic relaxation pathway and the magnetization transfer pathway. The influence of magnetization transfer to the total residual z-magnetization has been quantitatively evaluated in the context of magnetization map and various difference magnetization profiles for macromolecule conjugated Gd-DTPA in cross-linked protein gels. The numerical simulations and experimental validations confirm that the rotational correlation time for paramagnetic ion chelates can be measured even in the presence of strong magnetization transfer. This spin relaxation model also provides novel approaches to enhance the detection sensitivity for paramagnetic labeling by suppressing spin relaxations caused by magnetization transfer. The inclusion of the magnetization transfer effect allows us to use the magnetization map as a simulation tool to design efficient paramagnetic labeling targeting at specific tissues, to design experiments running at low RF power depositions, and to optimize the sensitivity for detecting paramagnetic labeling. Thus, the presented method will be a very useful tool for in vivo applications such as molecular imaging via paramagnetic labeling.
PMCID: PMC1941718  PMID: 17123851
off-resonance rotating frame; paramagnetic relaxation enhancement; magnetization transfer effect; magnetization map; magnetization profiles; difference magnetization profiles; dynamic of paramagnetic ion chelates
24.  FOXP3 is a novel transcriptional repressor for the breast cancer oncogene SKP2  
The Journal of Clinical Investigation  2007;117(12):3765-3773.
S-phase kinase-associated protein 2 (SKP2) is a component of the E3 ubiquitin ligase SKP1-Cul1-Fbox complex. Overexpression of SKP2 results in cell cycle dysregulation and carcinogenesis; however, the genetic lesions that cause this upregulation are poorly understood. We recently demonstrated that forkhead box P3 (FOXP3) is an X-linked breast cancer suppressor and an important repressor of the oncogene ERBB2/HER2. Since FOXP3 suppresses tumor growth regardless of whether the tumors overexpress ERBB2/HER2, additional FOXP3 targets may be involved in its tumor suppressor activity. Here, we show that mammary carcinomas from mice heterozygous for a Foxp3 mutation exhibited increased Skp2 expression. Ectopic expression of FOXP3 in mouse mammary cancer cells repressed SKP2 expression with a corresponding increase in p27 and polyploidy. Conversely, siRNA silencing of the FOXP3 gene in human mammary epithelial cells increased SKP2 expression. We also show that Foxp3 directly interacted with and repressed the Skp2 promoter. Moreover, the analysis of over 200 primary breast cancer samples revealed an inverse correlation between FOXP3 and SKP2 levels. Finally, we demonstrated that downregulation of SKP2 was critical for FOXP3-mediated growth inhibition in breast cancer cells that do not overexpress ERBB2/HER2. Our data provide genetic, biochemical, and functional evidence that FOXP3 is a novel transcriptional repressor for the oncogene SKP2.
PMCID: PMC2075479  PMID: 18008005
25.  Massive and destructive T cell response to homeostatic cue in CD24-deficient lymphopenic hosts 
The Journal of Experimental Medicine  2006;203(7):1713-1720.
In response to a lymphopenic cue, T lymphocytes undergo a slow-paced homeostatic proliferation in an attempt to restore T cell cellularity. The molecular interaction that maintains the pace of homeostatic proliferation is unknown. In this study, we report that in lymphopenic CD24-deficient mice, T cells launch a massive proliferation that results in the rapid death of the recipient mice. The dividing T cells have phenotypes similar to those activated by cognate antigens. The rapid homeostatic proliferation is caused by a lack of CD24 on dendritic cells (DCs). Interestingly, although CD24 expression in T cells is required for optimal homeostatic proliferation in the wild-type (WT) host, mice lacking CD24 on all cell types still mount higher homeostatic proliferation than the WT mice. Thus, a lack of CD24 in the non–T host cells bypassed the requirement for T cell expression of CD24 in homeostatic proliferation in the WT host. Our data demonstrate that CD24 expressed on the DCs limits T cell response to homeostatic cue and prevents fatal damage associated with uncontrolled homeostatic proliferation.
PMCID: PMC2118348  PMID: 16769998

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