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1.  Genome-wide alteration of 5-hydroxymethylcytosine in a mouse model of fragile X-associated tremor/ataxia syndrome 
Human Molecular Genetics  2013;23(4):1095-1107.
Fragile X-associated tremor/ataxia syndrome (FXTAS) is a late-onset neurodegenerative disorder in which patients carry premutation alleles of 55–200 CGG repeats in the FMR1 gene. To date, whether alterations in epigenetic regulation modulate FXTAS has gone unexplored. 5-Hydroxymethylcytosine (5hmC) converted from 5-methylcytosine (5mC) by the ten-eleven translocation (TET) family of proteins has been found recently to play key roles in neuronal functions. Here, we undertook genome-wide profiling of cerebellar 5hmC in a FXTAS mouse model (rCGG mice) and found that rCGG mice at 16 weeks showed overall reduced 5hmC levels genome-wide compared with age-matched wild-type littermates. However, we also observed gain-of-5hmC regions in repetitive elements, as well as in cerebellum-specific enhancers, but not in general enhancers. Genomic annotation and motif prediction of wild-type- and rCGG-specific differential 5-hydroxymethylated regions (DhMRs) revealed their high correlation with genes and transcription factors that are important in neuronal developmental and functional pathways. DhMR-associated genes partially overlapped with genes that were differentially associated with ribosomes in CGG mice identified by bacTRAP ribosomal profiling. Taken together, our data strongly indicate a functional role for 5hmC-mediated epigenetic modulation in the etiology of FXTAS, possibly through the regulation of transcription.
doi:10.1093/hmg/ddt504
PMCID: PMC3900112  PMID: 24108107
2.  AGO3 Slicer activity regulates mitochondria–nuage localization of Armitage and piRNA amplification 
The Journal of Cell Biology  2014;206(2):217-230.
The endonuclease AGO3 and mitochondria-associated protein Zucchini together control the dynamic subcellular localization of Armitage between mitochondria and germline granules to regulate secondary piRNA amplification.
In Drosophila melanogaster the reciprocal “Ping-Pong” cycle of PIWI-interacting RNA (piRNA)–directed RNA cleavage catalyzed by the endonuclease (or “Slicer”) activities of the PIWI proteins Aubergine (Aub) and Argonaute3 (AGO3) has been proposed to expand the secondary piRNA population. However, the role of AGO3/Aub Slicer activity in piRNA amplification remains to be explored. We show that AGO3 Slicer activity is essential for piRNA amplification and that AGO3 inhibits the homotypic Aub:Aub Ping-Pong process in a Slicer-independent manner. We also find that expression of an AGO3 Slicer mutant causes ectopic accumulation of Armitage, a key component in the primary piRNA pathway, in the Drosophila melanogaster germline granules known as nuage. AGO3 also coexists and interacts with Armitage in the mitochondrial fraction. Furthermore, AGO3 acts in conjunction with the mitochondria-associated protein Zucchini to control the dynamic subcellular localization of Armitage between mitochondria and nuage in a Slicer-dependent fashion. Collectively, our findings uncover a new mechanism that couples mitochondria with nuage to regulate secondary piRNA amplification.
doi:10.1083/jcb.201401002
PMCID: PMC4107788  PMID: 25049272
3.  Genome-wide antagonism between 5-hydroxymethylcytosine and DNA methylation in the adult mouse brain 
Frontiers in biology  2014;9(1):66-74.
Mounting evidence points to critical roles for DNA modifications, including 5-methylcytosine (5mC) and its oxidized forms, in the development, plasticity and disorders of the mammalian nervous system. The novel DNA base 5-hydroxymethylcytosine (5hmC) is known to be capable of initiating passive or active DNA demethylation, but whether and how extensively 5hmC functions in shaping the post-mitotic neuronal DNA methylome is unclear. Here we report the genome-wide distribution of 5hmC in dentate granule neurons from adult mouse hippocampus in vivo. 5hmC in the neuronal genome is highly enriched in gene bodies, especially in exons, and correlates with gene expression. Direct genome-wide comparison of 5hmC distribution between embryonic stem cells and neurons reveals extensive differences, reflecting the functional disparity between these two cell types. Importantly, integrative analysis of 5hmC, overall DNA methylation and gene expression profiles of dentate granule neurons in vivo reveals the genome-wide antagonism between these two states of cytosine modifications, supporting a role for 5hmC in shaping the neuronal DNA methylome by promoting active DNA demethylation.
doi:10.1007/s11515-014-1295-1
PMCID: PMC4284063  PMID: 25568643
dentate granule neuron; active DNA demethylation; TET; methylome
4.  Molecular mechanism of ERK dephosphorylation by striatal-enriched protein tyrosine phosphatase (STEP) 
Journal of neurochemistry  2013;128(2):315-329.
Striatal-enriched tyrosine phosphatase (STEP) is an important regulator of neuronal synaptic plasticity, and its abnormal level or activity contributes to cognitive disorders. One crucial downstream effector and direct substrate of STEP is extracellular signal-regulated protein kinase (ERK), which has important functions in spine stabilisation and action potential transmission. The inhibition of STEP activity toward phospho-ERK has the potential to treat neuronal diseases, but the detailed mechanism underlying the dephosphorylation of phospho-ERK by STEP is not known. Therefore, we examined STEP activity toward pNPP, phospho-tyrosine-containing peptides, and the full-length phospho-ERK protein using STEP mutants with different structural features. STEP was found to be a highly efficient ERK tyrosine phosphatase that required both its N-terminal regulatory region and key residues in its active site. Specifically, both KIM and KIS of STEP were required for ERK interaction. In addition to the N-terminal KIS region, S245, hydrophobic residues L249/L251, and basic residues R242/R243 located in the KIM region were important in controlling STEP activity toward phospho-ERK. Further kinetic experiments revealed subtle structural differences between STEP and HePTP that affected the interactions of their KIMs with ERK. Moreover, STEP recognised specific positions of a phospho-ERK peptide sequence through its active site, and the contact of STEP F311 with phospho-ERK V205 and T207 were crucial interactions. Taken together, our results not only provide the information for interactions between ERK and STEP, but will also help in the development of specific strategies to target STEP-ERK recognition, which could serve as a potential therapy for neurological disorders.
doi:10.1111/jnc.12463
PMCID: PMC3947313  PMID: 24117863
ERK; phosphorylation; phosphatase; synaptic plasticity; Striatal enriched tyrosine phosphatases (STEP); neurological disorders
5.  Incorporating DNA Methylation Dynamics Into Epigenetic Codes 
Summary
Genomic function is dictated by a combination of DNA sequence and the molecular mechanisms controlling access to genetic information. Access to DNA can be determined by the interpretation of covalent modifications that influence the packaging of DNA into chromatin, including DNA methylation and histone modifications. These modifications are believed to be forms of “epigenetic codes” that exist in discernable combinations that reflect cellular phenotype. Although DNA methylation is known to play important roles in gene regulation and genomic function, its contribution to the encoding of epigenetic information is just beginning to emerge. Here we discuss paradigms associated with the various components of DNA methylation/demethylation and recent advances in the understanding of its dynamic regulation in the genome, integrating these mechanisms into a framework to explain how DNA methylation could contribute to epigenetic codes.
doi:10.1002/bies.201300090
PMCID: PMC4278575  PMID: 24242211
epigenetics; chromatin; DNA methylation; DNA demethylation
6.  Understanding CELSRs - Cadherin EGF LAG seven-pass G-type receptors 
Journal of neurochemistry  2014;131(6):699-711.
The cadherin EGF LAG seven-pass G-type receptors (CELSRs) are a special subgroup of adhesion G protein-coupled receptors (GPCRs), which are pivotal regulators of many biological processes such as neuronal/endocrine cell differentiation, vessel valve formation and the control of planar cell polarity during embryonic development. All three members of the CELSR family (CELSR1-3) have large ecto-domains that form homophilic interactions and encompass more than 2,000 amino acids. Mutations in the ecto-domain or other gene locations of CELSRs are associated with neural tube defects (NTDs) and other diseases in humans. Celsr knockout (KO) animals have many developmental defects. Therefore, specific agonists or antagonists of CELSR members may have therapeutic potential. Although significant progress has been made regarding the functions and biochemical properties of CELSRs, our knowledge of these receptors is still lacking, especially considering that they are broadly distributed but have few characterized functions in a limited number of tissues. The dynamic activation and inactivation of CELSRs and the presence of endogenous ligands beyond homophilic interactions remain elusive, as do the regulatory mechanisms and downstream signaling of these receptors. Given this motivation, future studies with more advanced cell biology or biochemical tools, such as conditional KO mice, may provide further insights into the mechanisms underlying CELSR function, laying the foundation for the design of new CELSR-targeted therapeutic reagents.
doi:10.1111/jnc.12955
PMCID: PMC4261025  PMID: 25280249
G Protein-Coupled Receptor (GPCR); Adhesion; CELSR; Development; Planar Cell Polarity (PCP)
7.  Bacillus cereus AR156-Induced Resistance to Colletotrichum acutatum Is Associated with Priming of Defense Responses in Loquat Fruit 
PLoS ONE  2014;9(11):e112494.
The effectiveness of a biocontrol agent Bacillus cereus AR156 for control of anthracnose rot caused by Colletotrichum acutatum in harvested loquat fruit and the possible mechanisms of its action have been investigated. Treatment of fruit with B. cereus AR156 resulted in lower disease incidence and smaller lesion diameters compared with that of untreated fruit. The treatment enhanced activities of defense-related enzymes including chitinase, β-1, 3-glucanase, phenylalanine ammonia-lyase, peroxidase and polyphenoloxidase, and promoted accumulation of H2O2. Total phenolic content and 2,2-diphenyl-1-picrylhydrazyl radical scavenging activity were also increased by treatment. Transcripts of three defense-related genes were enhanced only in fruit undergoing both B. cereus AR156 treatment and C. acutatum inoculation compared with those receiving either intervention alone. These results suggest that the disease resistance against C. acutatum in loquat fruit is enhanced by B. cereus AR156 and that the induced resistance is associated with induction and priming of defense responses in the fruit.
doi:10.1371/journal.pone.0112494
PMCID: PMC4227702  PMID: 25386680
8.  A study on the mechanical characteristics of the EBM-printed Ti-6Al-4V LCP plates in vitro 
Purpose
The electron beam melting (EBM) Ti-6Al-4V material technology has been developed over a short time period. It was introduced through a research to develop Ti-6Al-4V implants for patients, but EBM printed locking compression plates have not been used for clinical implants. The main purpose of this study is to find whether the EBM Ti-6Al-4V plate suit for clinical implants.
Methods
First, we scanned an AO-locking compression plate (LCP) and printed LCP samples using EBM. Next, we evaluated the EBM plate surface roughness through optical microscopy as well as the LCP and EBM plates’ mechanical characteristics using the ASTM standard, which is commonly used to test the mechanical properties of bone plates subject to bending. Each sample was examined using a single-cycle four-point bending test and hardness testing to acquire data on bending stiffness, bending strength, bending structural stiffness, and hardness.
Results
The results show significant differences in bending stiffness, bending strength, bending structural stiffness, and hardness between the samples using EBM and the original LCP plates. The EBM-printed samples’ surface roughness was 0.49 ± 0.02 μm. The mean hardness of the LCP sample was 266.67 HV10 ± 5.8, and the EBM-printed sample mean hardness was 341.1 HV10 ± 1.93. The EBM samples’ bending stiffness was 87.67%, which is greater than using the LCP plates’; and the bending strength was 190.7% greater, the bending structural stiffness was 73.2% greater, and the hardness was 27.9% greater.
Conclusions
The results show that the EBM plates’ general mechanical strength was significantly greater than the LCP plates. An EBM plate is advantageous for clinical implants because it can be customized with great potential for improvement.
doi:10.1186/s13018-014-0106-3
PMCID: PMC4221727  PMID: 25370215
Ti-6Al-4V; EBM; Mechanical properties; Bone plate
9.  REGIOSELECTIVE MULTICOMPONENT DOMINO REACTIONS PROVIDING RAPID AND EFFICIENT ROUTES TO FUSED ACRIDINES 
Heterocycles  2013;88(2):1065-1077.
Regioselective three-component reactions of aromatic aldehydes with indazol-5-amine and 2-hydroxy-1,4-naphthoquinone in HOAc under microwave irradiation have been developed. In this one-pot reaction, a series of new pyrazole-fused benzo[h]acridine derivatives with 1,2-diketone unit were synthesized with high chemical yields. The resulting pyrazole-fused acridines were employed to further react with aldehydes and ammonium acetate to give polycyclic oxazole-fused pyrazolo[3,4-j]acridines. The present green synthesis shows several advantages including operational simplicity and fast reaction rates, which makes it a useful and attractive process of library generation for drug discovery.
doi:10.3987/COM-13-S(S)65
PMCID: PMC4214857  PMID: 25364095
10.  The Complete Mitochondrial Genomes of Six Species of Tetranychus Provide Insights into the Phylogeny and Evolution of Spider Mites 
PLoS ONE  2014;9(10):e110625.
Many spider mites belonging to the genus Tetranychus are of agronomical importance. With limited morphological characters, Tetranychus mites are usually identified by a combination of morphological characteristics and molecular diagnostics. To clarify their molecular evolution and phylogeny, the mitochondrial genomes of the green and red forms of Tetranychus urticae as well as T. kanzawai, T. ludeni, T. malaysiensis, T. phaselus, T. pueraricola were sequenced and compared. The seven mitochondrial genomes are typical circular molecules of about 13,000 bp encoding and they are composed of the complete set of 37 genes that are usually found in metazoans. The order of the mitochondrial (mt) genes is the same as that in the mt genomes of Panonychus citri and P. ulmi, but very different from that in other Acari. The J-strands of the mitochondrial genomes have high (∼84%) A+T contents, negative GC-skews and positive AT-skews. The nucleotide sequence of the cox1 gene, which is commonly used as a taxon barcode and molecular marker, is more highly conserved than the nucleotide sequences of other mitochondrial genes in these seven species. Most tRNA genes in the seven genomes lose the D-arm and/or the T-arm. The functions of these tRNAs need to be evaluated. The mitochondrial genome of T. malaysiensis differs from the other six genomes in having a slightly smaller genome size, a slight difference in codon usage, and a variable loop in place of the T-arm of some tRNAs by a variable loop. A phylogenic analysis shows that T. malaysiensis first split from other Tetranychus species and that the clade of the family Tetranychoidea occupies a basal position in the Trombidiformes. The mt genomes of the green and red forms of T. urticae have limited divergence and short evolutionary distance.
doi:10.1371/journal.pone.0110625
PMCID: PMC4199730  PMID: 25329165
11.  Probing the microRNA pathway with small molecules 
Bioorganic & medicinal chemistry  2013;21(20):6119-6123.
MicroRNA (miRNA)/RNA interference (RNAi) is recognized as one of the most important mechanisms regulating gene expression at the posttranscriptional level in eukaryotic cells. The main components within the miRNA/RNAi pathway are now known and well characterized, but studies on the molecular mechanisms that regulate the activity of the miRNA/RNAi pathway are just beginning to emerge. High-throughput reporter assays have been developed to monitor the activity of the miRNA/RNAi pathway and applied in a proof-of-concept pilot screening, which has led to the identification of some inhibitors and activators that either generally or specifically regulate the activity of the miRNA/RNAi pathway. In addition, combined with multidisciplinary approaches like proteomics, biochemistry, and genetics, some protein co-factors were found to play important roles in the regulation of the miRNA/RNAi pathway. Herein we highlight the high-throughput reporter assays developed in recent years and the resulting discovery of miRNA/RNAi enhancers and inhibitors.
doi:10.1016/j.bmc.2013.05.030
PMCID: PMC3789859  PMID: 23791866
microRNA/RNAi pathway; reporter system; high-throughput screening; small molecule; chemical biology approach; enhancer; inhibitor
12.  Evidence of Type-II Band Alignment in III-nitride Semiconductors: Experimental and theoretical investigation for In0.17Al0.83N/GaN heterostructures 
Scientific Reports  2014;4:6521.
Type-II band alignment structure is coveted in the design of photovoltaic devices and detectors, since it is beneficial for the transport of photogenerated carriers. Regrettably, for group-III-nitride wide bandgap semiconductors, all existing devices are limited to type-I heterostructures, owing to the unavailable of type-II ones. This seriously restricts the designing flexibility for optoelectronic devices and consequently the relevant performance of this material system. Here we show a brandnew type-II band alignment of the lattice-matched In0.17Al0.83N/GaN heterostructure from the perspective of both experimental observations and first-principle theoretical calculations. The band discontinuity is dominated by the conduction band offset ΔEC, with a small contribution from the valence band offset ΔEV which equals 0.1 eV (with being above). Our work may open up new prospects to realize high-performance III-Nitrides optoelectronic devices based on type-II energy band engineering.
doi:10.1038/srep06521
PMCID: PMC4185379  PMID: 25283334
13.  The Protective Effect of MicroRNA-320 on Left Ventricular Remodeling after Myocardial Ischemia-Reperfusion Injury in the Rat Model 
The primary objective of this study investigated the role of microRNA-320 (miR-320) on left ventricular remodeling in the rat model of myocardial ischemia-reperfusion (I/R) injury, and we intended to explore the myocardial mechanism of miR-320-mediated myocardium protection. We collected 120 male Wistar rats (240–280 g) in this study and then randomly divided them into three groups: (1) sham surgery group (sham group: n = 40); (2) ischemia-reperfusion model group (I/R group: n = 40); and (3) I/R model with antagomir-320 group (I/R + antagomir-320 group: n = 40). Value changes of heart function in transesophageal echocardiography were recorded at various time points (day 1, day 3, day 7, day 15 and day 30) after surgery in each group. Myocardial sections were stained with hematoxylin and eosin (H&E) and examined with optical microscope. The degree of myocardial fibrosis was assessed by Sirius Red staining. Terminal dUTP nick end-labeling (TUNEL) and qRT-PCR methods were used to measure the apoptosis rate and to determine the miR-320 expression levels in myocardial tissues. Transesophageal echocardiography showed that the values of left ventricular ejection fraction (LVEF), left ventricular fractional shortening (LVFS), left ventricular systolic pressure (LVSP) and ±dp/dtmax in the I/R group were obviously lower than those in the sham group, while the left ventricular end-diastolic pressure (LVEDP) value was higher than that in the sham group. The values of LVEF, LVFS, LVSP and ±dp/dtmax showed a gradual decrease in the I/R group, while the LVEDP value showed an up tendency along with the extension of reperfusion time. The H&E staining revealed that rat myocardial tissue in the I/R group presented extensive myocardial damage; for the I/R + antagomir-320 group, however, the degree of damage in myocardial cells was obviously better than that of the I/R group. The Sirius Red staining results showed that the degree of myocardial fibrosis in the I/R group was more severe along with the extension of the time of reperfusion. For the I/R + antagomir-320 group, the degree of myocardial fibrosis was less severe than that in the I/R group. Tissues samples in both the sham and I/R + antagomir-320 groups showed a lower apoptosis rate compared to I/R group. The qRT-PCR results indicated that miR-320 expression in the I/R group was significantly higher than that in both the sham and I/R + antagomir-320 groups. The expression level of miR-320 is significantly up-regulated in the rat model of myocardial I/R injury, and it may be implicated in the prevention of myocardial I/R injury-triggered left ventricular remodeling.
doi:10.3390/ijms151017442
PMCID: PMC4227171  PMID: 25268616
microRNA-320; myocardial ischemia-reperfusion injury; left ventricular remodeling
14.  The catalytic region and PEST domain of PTPN18 distinctly regulate the HER2 phosphorylation and ubiquitination barcodes 
Cell Research  2014;24(9):1067-1090.
The tyrosine phosphorylation barcode encoded in C-terminus of HER2 and its ubiquitination regulate diverse HER2 functions. PTPN18 was reported as a HER2 phosphatase; however, the exact mechanism by which it defines HER2 signaling is not fully understood. Here, we demonstrate that PTPN18 regulates HER2-mediated cellular functions through defining both its phosphorylation and ubiquitination barcodes. Enzymologic characterization and three crystal structures of PTPN18 in complex with HER2 phospho-peptides revealed the molecular basis for the recognition between PTPN18 and specific HER2 phosphorylation sites, which assumes two distinct conformations. Unique structural properties of PTPN18 contribute to the regulation of sub-cellular phosphorylation networks downstream of HER2, which are required for inhibition of HER2-mediated cell growth and migration. Whereas the catalytic domain of PTPN18 blocks lysosomal routing and delays the degradation of HER2 by dephosphorylation of HER2 on pY1112, the PEST domain of PTPN18 promotes K48-linked HER2 ubiquitination and its rapid destruction via the proteasome pathway and an HER2 negative feedback loop. In agreement with the negative regulatory role of PTPN18 in HER2 signaling, the HER2/PTPN18 ratio was correlated with breast cancer stage. Taken together, our study presents a structural basis for selective HER2 dephosphorylation, a previously uncharacterized mechanism for HER2 degradation and a novel function for the PTPN18 PEST domain. The new regulatory role of the PEST domain in the ubiquitination pathway will broaden our understanding of the functions of other important PEST domain-containing phosphatases, such as LYP and PTPN12.
doi:10.1038/cr.2014.99
PMCID: PMC4152746  PMID: 25081058
phosphatase; receptor; ubiquitination; HER2; PTPN18; EGFR
15.  Lack of association between LEPR Lys656Asn or Ser343Ser polymorphisms and cancer susceptibility: A meta-analysis 
Biomedical Reports  2014;2(6):849-854.
Previous studies have shown conflicting results between the association of leptin receptor (LEPR) genetic polymorphisms and cancer risk. The frequent LEPR Lys656Asn or Ser343Ser genetic polymorphism has been demonstrated to be functional and may promote genetic susceptibility to cancers. However, the association between the LEPR Lys656Asn or Ser343Ser genetic polymorphism and cancer risk remains to be determined. To improve the understanding of the LEPR Lys656Asn or Ser343Ser genetic polymorphism role in global cancer, a comprehensive meta-analysis was conducted that comprised 2,480 cases and 3,162 controls. The LEPR Lys656Asn or Ser343Ser genetic polymorphism did not significantly affect the cancer risk. In the stratified analysis, there was no significant association of the LEPR Lys656Asn or Ser343Ser variants with any type of cancer under any model. In addition, significantly increased risks were found in the Asian population in heterozygous codominant [odds ratio (OR), 1.24 (1.01–1.53)] and dominant [OR, 1.24 (1.02–1.50)] genetic models. A significantly increased susceptibility to cancer was not found when stratified by study design. There were no significant differences found in genotype method and sample size in cases among the genotypes. These findings indicated a lack of association between LEPR Lys656Asn or Ser343Ser polymorphisms and cancer susceptibility, however, these polymorphisms may increase the cancer susceptibility among the Asian population, particularly in the dominant genetic model. The single-nucleotide polymorphism is also suggested to function as a dominant mutation, which requires verification or association with functional studies.
doi:10.3892/br.2014.326
PMCID: PMC4179704  PMID: 25279158
leptin receptor; Lys656Asn; Ser343Ser; cancer; genetic polymorphism; meta-analysis
16.  Emulsifiers and thickeners on extrusion-cooked instant rice product 
Extrusion-cooked instant rice was prepared by optimizing the formulation with emulsifiers, glycerol monostearate (GMS), soybean lecithin (LC), and sodiumstearoyl lactylate (SSL), and thickeners, gum Arabic (GA), sodium alginate (SA), and sticky rice (SR). The emulsifiers addition caused increase of degree of gelatinization (DG), and decrease of water soluble carbohydrate (WSC), α-amylase sensitivity, water soluble index (WAI) and adhesive for extrudates, while the thickeners addition increased extrudates DG, bulk density (BD), WSC, α-amylase sensitivity, WAI, hydration rate (HR) and adhesiveness. Based on the data generated by a single additive at various levels, optimum formulation was obtained employing orthogonal matrix system with combination of the selected additives for extrusion cooking. Extrudates were evaluated for optimum hydration time followed by drying to prepare the finished product. Texture profile analysis and sensory evaluation indicate that quality of the finished product is equivalent to that of the round shaped rice and superior to a commercial instant rice product. This study also demonstrates possibility of value-added and versatile instant rice product development using broken rice.
doi:10.1007/s13197-011-0400-6
PMCID: PMC3671062  PMID: 24425967
Extrusion cooking; Instant rice; Emulsifier; Thickener; Broken rice; Orthogonal matrix
17.  Chemical Modification-Assisted Bisulfite Sequencing (CAB-Seq) for 5-Carboxylcytosine Detection in DNA 
5-Methylcytosine (5mC) in DNA can be oxidized stepwise to 5-hydroxymethylcytosine (5hmC), 5- formylcytosine (5fC), and 5-carboxylcytosine (5caC) by the TET family proteins. Thymine DNA glycosylase can further remove 5fC and 5caC, connecting 5mC oxidation with active DNA demethylation. Here we present a chemical modification-assisted bisulfite sequencing (CAB-Seq) that can detect 5caC with single-base resolution in DNA. We optimized 1-ethyl-3- [3-dimethylaminopropyl]carbodiimide hydrochloride (EDC)- catalyzed amide bond formation between the carboxyl group of 5caC and a primary amine group. We found that the modified 5caC can survive the bisulfite treatment without deamination. Therefore, this chemical labeling coupled with bisulfite treatment provides a base-resolution detection and sequencing method for 5caC.
doi:10.1021/ja4044856
PMCID: PMC3727251  PMID: 23758547
18.  Genome-wide profiling of 5-formylcytosine reveals its roles in epigenetic priming 
Cell  2013;153(3):678-691.
SUMMARY
TET proteins oxidize 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). 5fC and 5caC are excised by mammalian DNA glycosylase TDG, implicating 5mC oxidation in DNA demethylation. Here we show that the genomic locations of 5fC can be determined by coupling chemical reduction with biotin tagging. Genome-wide mapping of 5fC in mouse embryonic stem cells (mESCs) reveals that 5fC preferentially occurs at poised enhancers among other gene regulatory elements. Application to Tdg null mESCs further suggests that 5fC production coordinates with p300 in remodeling epigenetic states of enhancers. This process, which is not influenced by 5hmC, appears to be associated with further oxidation of 5hmC and commitment to demethylation through 5fC. Finally, we resolved 5fC at base-resolution by hydroxylamine-based protection from bisulfite-mediated deamination, thereby confirming sites of 5fC accumulation. Our results reveal roles of active 5mC/5hmC oxidation and TDG-mediated demethylation in epigenetic tuning at regulatory elements.
doi:10.1016/j.cell.2013.04.001
PMCID: PMC3657391  PMID: 23602153
19.  Subtelomeric hotspots of aberrant 5-hydroxymethylcytosine-mediated epigenetic modifications during reprogramming to pluripotency 
Nature cell biology  2013;15(6):700-711.
Mammalian somatic cells can be directly reprogrammed into induced pluripotent stem cells (iPSCs) by introducing defined sets of transcription factors. Somatic cell reprogramming involves epigenomic reconfiguration, conferring iPSCs with characteristics similar to embryonic stem cells (ESCs). Human ES cells contain 5-hydroxymethylcytosine (5hmC), which is generated through the oxidation of 5-methylcytosine by the TET enzyme family. Here we show that 5hmC levels increase significantly during reprogramming to human iPSCs mainly due to TET1 activation, and this hydroxymethylation change is critical for optimal epigenetic reprogramming, but does not compromise primed pluripotency. Compared with hES cells, we find iPS cells tend to form large-scale (100 kb-1.3 Mb) aberrant reprogramming hotspots in subtelomeric regions, most of which display incomplete hydroxymethylation on CG sites. Strikingly, these 5hmC aberrant hotspots largely coincide (~80%) with aberrant iPS-ES non-CG methylation regions. Our results suggest that TET1-mediated 5hmC modification could contribute the epigenetic variation of iPSCs and iPSC-hESC differences.
doi:10.1038/ncb2748
PMCID: PMC3998089  PMID: 23685628
20.  A Novel Method for Fast Change-Point Detection on Simulated Time Series and Electrocardiogram Data 
PLoS ONE  2014;9(4):e93365.
Although Kolmogorov-Smirnov (KS) statistic is a widely used method, some weaknesses exist in investigating abrupt Change Point (CP) problems, e.g. it is time-consuming and invalid sometimes. To detect abrupt change from time series fast, a novel method is proposed based on Haar Wavelet (HW) and KS statistic (HWKS). First, the two Binary Search Trees (BSTs), termed TcA and TcD, are constructed by multi-level HW from a diagnosed time series; the framework of HWKS method is implemented by introducing a modified KS statistic and two search rules based on the two BSTs; and then fast CP detection is implemented by two HWKS-based algorithms. Second, the performance of HWKS is evaluated by simulated time series dataset. The simulations show that HWKS is faster, more sensitive and efficient than KS, HW, and T methods. Last, HWKS is applied to analyze the electrocardiogram (ECG) time series, the experiment results show that the proposed method can find abrupt change from ECG segment with maximal data fluctuation more quickly and efficiently, and it is very helpful to inspect and diagnose the different state of health from a patient's ECG signal.
doi:10.1371/journal.pone.0093365
PMCID: PMC3972110  PMID: 24690633
21.  High-yield novel leech hyaluronidase to expedite the preparation of specific hyaluronan oligomers 
Scientific Reports  2014;4:4471.
Hyaluronidases (HAases), particularly leech HAases, have attracted intense attention due to their broad applications in medical treatments and great potential for the enzymatic production of hyaluronan oligosaccharides. However, little is known about this third interesting family of HAases. Here, we applied the random amplification of cDNA ends polymerase chain reaction (RACE-PCR) approach to identify the first leech HAase-encoding gene. By combining protein engineering and high-density culture, we achieved high-level production (8.42 × 105 U ml−1) in the yeast Pichia pastoris secretory expression system. Compared with the commercial bovine testicular HAase, the recombinant leech HAase exhibited superior enzymatic properties. Furthermore, analysis of the hydrolytic process suggested that this novel enzyme adopts a nonprocessive endolytic mode, yielding a narrow-spectrum of specific HA oligosaccharides with different incubation times. Large-scale production of this novel leech HAase will not only greatly promote medical applications but also facilitate the enzymatic production of specific HA oligosaccharides.
doi:10.1038/srep04471
PMCID: PMC3966032  PMID: 24667183
23.  Coordination of Engineered Factors with TET1/2 Promotes Early-Stage Epigenetic Modification during Somatic Cell Reprogramming 
Stem Cell Reports  2014;2(3):253-261.
Summary
Somatic cell reprogramming toward induced pluripotent stem cells (iPSCs) holds great promise in future regenerative medicine. However, the reprogramming process mediated by the traditional defined factors (OSMK) is slow and extremely inefficient. Here, we develop a combination of modified reprogramming factors (OySyNyK) in which the transactivation domain of the Yes-associated protein is fused to defined factors and establish a highly efficient and rapid reprogramming system. We show that the efficiency of OySyNyK-induced iPSCs is up to 100-fold higher than the OSNK and the reprogramming by OySyNyK is very rapid and is initiated in 24 hr. We find that OySyNyK factors significantly increase Tet1 expression at the early stage and interact with Tet1/2 to promote reprogramming. Our studies not only establish a rapid and highly efficient iPSC reprogramming system but also uncover a mechanism by which engineered factors coordinate with TETs to regulate 5hmC-mediated epigenetic control.
Graphical Abstract
Highlights
•A combination of modified reprogramming factors (OySyNyK) is developed•A highly efficient and rapid reprogramming system is established•TET1/2 proteins are involved in rapid iPSC induction by OySyNyK•OySyNyK factors coordinate with TET proteins to promote rapid reprogramming
The reprogramming process mediated by the traditional defined factors (OSMK) is slow and inefficient. Sun, Jin, Chen, and colleagues have developed a combination of modified reprogramming factors (OySyNyK) in which the transactivation domain of the Yes-associated protein is fused to defined factors and establish a highly efficient and rapid reprogramming system.
doi:10.1016/j.stemcr.2014.01.012
PMCID: PMC3964280  PMID: 24672749
24.  Identifying Bipolar Disorder Susceptibility Loci in a Densely Affected Pedigree 
Molecular psychiatry  2012;18(12):1245-1246.
doi:10.1038/mp.2012.176
PMCID: PMC3899577  PMID: 23247078
Bipolar disorder; genetics; genome-wide association; copy number variation; whole genome sequencing; exome sequencing; rare variation
25.  Use of model systems to understand the etiology of fragile X-associated primary ovarian insufficiency (FXPOI) 
Fragile X-associated primary ovarian insufficiency (FXPOI) is among the family of disorders caused by the expansion of a CGG repeat sequence in the 5' untranslated region of the X-linked gene FMR1. About 20% of women who carry the premutation allele (55 to 200 unmethylated CGG repeats) develop hypergonadotropic hypogonadism and cease menstruating before age 40. Some proportion of those who are still cycling show hormonal profiles indicative of ovarian dysfunction. FXPOI leads to subfertility and an increased risk of medical conditions associated with early estrogen deficiency. Little progress has been made in understanding the etiology of this clinically significant disorder. Understanding the molecular mechanisms of FXPOI requires a detailed knowledge of ovarian FMR1 mRNA and FMRP’s function. In humans, non-invasive methods to discriminate the mechanisms of the premutation on ovarian function are not available, thus necessitating the development of model systems. Vertebrate (mouse and rat) and invertebrate (Drosophila melanogaster) animal studies for the FMR1 premutation and ovarian function exist and have been instrumental in advancing our understanding of the disease phenotype. For example, rodent models have shown that FMRP is highly expressed in oocytes where it is important for folliculogenesis. The two premutation mouse models studied to date show evidence of ovarian dysfunction and, together, suggest that the long repeat in the transcript itself may have some pathological effect quite apart from any effect of the toxic protein. Further, ovarian morphology in young animals appears normal and the primordial follicle pool size does not differ from that of wild-type animals. However, there is a progressive premature decline in the levels of most follicle classes. Observations also include granulosa cell abnormalities and altered gene expression patterns. Further comparisons of these models are now needed to gain insight into the etiology of the ovarian dysfunction. Premutation model systems in non-human primates and those based on induced pluripotent stem cells show particular promise and will complement current models. Here, we review the characterization of the current models and describe the development and potential of the new models. Finally, we will discuss some of the molecular mechanisms that might be responsible for FXPOI.
doi:10.1186/1866-1955-6-26
PMCID: PMC4139715  PMID: 25147583
Primary ovarian insufficiency; Premature ovarian failure; Fragile X syndrome; Fertility; Repeat expansion disorder; CGG repeat

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