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1.  Directed evolution combined with synthetic biology strategies expedite semi-rational engineering of genes and genomes 
Bioengineered  2015;6(3):136-140.
Owing to our limited understanding of the relationship between sequence and function and the interaction between intracellular pathways and regulatory systems, the rational design of enzyme-coding genes and de novo assembly of a brand-new artificial genome for a desired functionality or phenotype are difficult to achieve. As an alternative approach, directed evolution has been widely used to engineer genomes and enzyme-coding genes. In particular, significant developments toward DNA synthesis, DNA assembly (in vitro or in vivo), recombination-mediated genetic engineering, and high-throughput screening techniques in the field of synthetic biology have been matured and widely adopted, enabling rapid semi-rational genome engineering to generate variants with desired properties. In this commentary, these novel tools and their corresponding applications in the directed evolution of genomes and enzymes are discussed. Moreover, the strategies for genome engineering and rapid in vitro enzyme evolution are also proposed.
doi:10.1080/21655979.2015.1011029
PMCID: PMC4601291  PMID: 25621864
directed evolution; DNA assembly; enzyme; genome engineering; metabolic engineering; recombineering; synthetic biology
2.  The Drosophila Helicase MLE Targets Hairpin Structures in Genomic Transcripts 
PLoS Genetics  2016;12(1):e1005761.
RNA hairpins are a common type of secondary structures that play a role in every aspect of RNA biochemistry including RNA editing, mRNA stability, localization and translation of transcripts, and in the activation of the RNA interference (RNAi) and microRNA (miRNA) pathways. Participation in these functions often requires restructuring the RNA molecules by the association of single-strand (ss) RNA-binding proteins or by the action of helicases. The Drosophila MLE helicase has long been identified as a member of the MSL complex responsible for dosage compensation. The complex includes one of two long non-coding RNAs and MLE was shown to remodel the roX RNA hairpin structures in order to initiate assembly of the complex. Here we report that this function of MLE may apply to the hairpins present in the primary RNA transcripts that generate the small molecules responsible for RNA interference. Using stocks from the Transgenic RNAi Project and the Vienna Drosophila Research Center, we show that MLE specifically targets hairpin RNAs at their site of transcription. The association of MLE at these sites is independent of sequence and chromosome location. We use two functional assays to test the biological relevance of this association and determine that MLE participates in the RNAi pathway.
Author Summary
In virtually all RNA molecules, single stranded regions undergo complementary base-pairing with neighboring regions to form double-stranded structures called stem-loops or hairpins. During the fundamental processes of transcription and translation, these RNA structures are reshaped by helicases—enzymes that separate paired regions of nucleic acids. In addition, small non-coding RNA molecules involved in regulating gene expression rely on sequences that allow them to form hairpin structures. In this paper we present evidence that the Drosophila helicase MLE, well known for its role in dosage compensation, participates in the processing of hairpin RNAs in the pathway that leads to induced RNA interference.
doi:10.1371/journal.pgen.1005761
PMCID: PMC4710571  PMID: 26752049
3.  Gossypol Acetic Acid Prevents Oxidative Stress-Induced Retinal Pigment Epithelial Necrosis by Regulating the FoxO3/Sestrin2 Pathway 
Molecular and Cellular Biology  2015;35(11):1952-1963.
The late stage of dry age-related macular degeneration (AMD), or geographic atrophy (GA), is characterized by extensive retinal pigment epithelial (RPE) cell death, and a cure is not available currently. We have recently demonstrated that RPE cells die from necrosis in response to oxidative stress, providing a potential novel mechanism for RPE death in AMD. In this study, we screened U.S. Food and Drug Administration-approved natural compounds and identified gossypol acetic acid (GAA) as a potent inhibitor of oxidative stress-induced RPE cell death. GAA induces antioxidative response and inhibits accumulation of excessive reactive oxygen species in cells, through which it prevents the activation of intrinsic necrotic pathway in response to oxidative stress. Sestrin2 (SESN2) is found to mediate GAA function in antioxidative response and RPE survival upon oxidative stress. Moreover, Forkhead box O3 transcription factor (FoxO3) is further found to be required for GAA-mediated SESN2 expression and RPE survival. Mechanistically, GAA promotes FoxO3 nuclear translocation and binding to the SESN2 enhancer, which in turn increases its transcriptional activity. Taken together, we have identified GAA as a potent inhibitor of oxidative stress-induced RPE necrosis by regulating the FoxO3/SESN2 pathway. This study may have significant implications in the therapeutics of age-related diseases, especially GA.
doi:10.1128/MCB.00178-15
PMCID: PMC4420921  PMID: 25802279
4.  CGG repeats in RNA modulate expression of TDP-43 in mouse and fly models of fragile X tremor ataxia syndrome 
Human Molecular Genetics  2014;23(22):5906-5915.
Determining the molecular mechanism(s) leading to Purkinje neuron loss in the neurodegenerative disorder fragile X-associated tremor/ataxia syndrome (FXTAS) is limited by the complex morphology of this cell type. Purkinje neurons are notoriously difficult to isolate and maintain in culture presenting considerable difficultly to identify molecular changes in response to expanded CGG repeat (rCGG)-containing mRNA that induces neurotoxicity in FXTAS. Several studies have uncovered a number of RNA-binding proteins involved in translation that aberrantly interact with the CGG-containing RNA; however, whether these interactions alter the translational profile of cells has not been investigated. Here we employ bacTRAP translational profiling to demonstrate that Purkinje neurons ectopically expressing 90 CGG repeats exhibit a dramatic change in their translational profile even prior to the onset of rCGG-induced phenotypes. This approach identified ∼500 transcripts that are differentially associated with ribosomes in r(CGG)90-expressing mice. Functional annotation cluster analysis revealed broad ontologies enriched in the r(CGG)90 list, including RNA binding and response to stress. Intriguingly, a transcript for the Tardbp gene, implicated in a number of other neurodegenerative disorders, exhibits altered association with ribosomes in the presence of r(CGG)90 repeats. We therefore tested and showed that reduced association of Tardbp mRNA with the ribosomes results in a loss of TDP-43 protein expression in r(CGG)90-expressing Purkinje neurons. Furthermore, we showed that TDP-43 could modulate the rCGG repeat-mediated toxicity in a Drosophila model that we developed previously. These findings together suggest that translational dysregulation may be an underlying mechanism of rCGG-induced neurotoxicity in FXTAS.
doi:10.1093/hmg/ddu314
PMCID: PMC4204772  PMID: 24986919
5.  Ocean acidification increases the accumulation of toxic phenolic compounds across trophic levels 
Nature Communications  2015;6:8714.
Increasing atmospheric CO2 concentrations are causing ocean acidification (OA), altering carbonate chemistry with consequences for marine organisms. Here we show that OA increases by 46–212% the production of phenolic compounds in phytoplankton grown under the elevated CO2 concentrations projected for the end of this century, compared with the ambient CO2 level. At the same time, mitochondrial respiration rate is enhanced under elevated CO2 concentrations by 130–160% in a single species or mixed phytoplankton assemblage. When fed with phytoplankton cells grown under OA, zooplankton assemblages have significantly higher phenolic compound content, by about 28–48%. The functional consequences of the increased accumulation of toxic phenolic compounds in primary and secondary producers have the potential to have profound consequences for marine ecosystem and seafood quality, with the possibility that fishery industries could be influenced as a result of progressive ocean changes.
Increasing atmospheric CO2 concentrations causes ocean acidification, which alters marine chemical environments with unknown consequences for marine ecosystems. Here, Gao et al. show that ocean acidification increases levels of phenolic compounds in phytoplankton and zooplankton, implying a food chain impact.
doi:10.1038/ncomms9714
PMCID: PMC4640080  PMID: 26503801
6.  Identification of susceptibility genes in non-syndromic cleft lip with or without cleft palate using whole-exome sequencing 
Background
Non-syndromic cleft lip with or without cleft palate (NSCL/P) is among the most common congenital malformations. The etiology of NSCL/P remains poorly characterized owing to its complex genetic heterogeneity. The objective of this study was to identify genetic variants that increase susceptibility to NSCL/P.
Material and Methods
Whole-exome sequencing (WES) was performed in 8 fetuses with NSCL/P in China. Bioinformatics analysis was performed using commercially available software. Variants detected by WES were validated by Sanger sequencing.
Results
By filtering out synonymous variants in exons, we identified average 8575 nonsynonymous single nucleotide variants (SNVs). We subsequently compared the SNVs against public databases including NCBI dbSNP build 135 and 1000 Genomes Project and obtained an average of 203 SNVs. Total 12 reported candidate genes were verified by Sanger sequencing. Sanger sequencing also confirmed 16 novel SNVs shared by two or more samples.
Conclusions
We have found and confirmed 16 susceptibility genes responsible for NSCL/P, which may play important role in the etiology of NSCL/P. The susceptibility genes identified in this study will not only be useful in revealing the etiology of NSCL/P but also in diagnosis and treatment of the patients with NSCL/P.
Key words:Non-syndromic cleft lip with or without cleft palate, whole-exome sequencing, sanger sequencing, susceptibility gene, single nucleotide variants (SNVs).
doi:10.4317/medoral.20758
PMCID: PMC4670259  PMID: 26449438
7.  Environmental enrichment modulates 5-hydroxymethylcytosine dynamics in hippocampus 
Genomics  2014;104(5):376-382.
Gene-environment interactions mediated at the epigenetic level may provide an initial step in delivering an appropriate response to environmental changes. 5-hydroxymethylcytosine (5hmC), a DNA base derived from 5-methylcytosine (5mC), accounts for ~40% of modified cytosine in brain and has been implicated in DNA methylation-related plasticity. To identify the role of 5hmC in gene-environment interactions, we exposed both young (6-week-old) and aged (18-month-old) mice to both an enriched environment and a standard environment. Exposure to EE significantly improves learning and memory in aged mice and reduces 5hmC abundance in mouse hippocampus. Furthermore, we mapped the genome-wide distribution of 5hmC and found that the alteration of 5hmC modification occurred mainly at gene bodies. In particular, genes involved in axon guidance are enriched among the genes with altered 5hmC modification. These results together suggest that environmental enrichment could modulate the dynamics of 5hmC in hippocampus, which could potentially contribute to improved learning and memory in aged animals.
doi:10.1016/j.ygeno.2014.08.019
PMCID: PMC4252786  PMID: 25205305
8.  Propofol sedation versus no sedation in detection of pharyngeal and upper gastrointestinal superficial squamous cell carcinoma using endoscopic narrow band imaging: a multicenter prospective trial 
Intravenous propofol can provide a superior quality of sedation compared to standard sedation for upper gastrointestinal endoscopy. However, the utility of propofol sedation for the endoscopic early detection of superficial pharyngeal and esophageal squamous cell carcinoma has not been investigated. In a multicenter, prospective trial, 255 patients with esophageal squamous cell carcinomas (ESCCs) were assigned to receive propofol sedation or no sedation according to their own willingness. The primary aim was to compare the detection rates of superficial cancer in the pharyngeal region and the esophagus between two groups. The secondary aim was to evaluate factors associated with technical adequacy. The detection rate was higher in the propofol sedation vs. no sedation group for H&N region (6.06% vs. 2.40%), but not significantly (P=0.22). However, the small lesion (less than 10 mm in diameter) detection rate was higher in sedation vs. no sedation group for H&N region (88.89% vs. 33.33%; P=0.048). The median time for pharyngeal observation in the sedation group was faster than in the no sedation group (20.6 s vs. 44.3 s; P<0.001). Ninety-five percent of H&N region evaluations were totally complete in sedation compared with sixty percent in the no sedation group (P<0.001). The overall p value indicated that only smoking habit was associated with incomplete pharyngeal observation (P<0.05), and it was more difficult to accomplish a complete pharyngeal observation in patients who smoked more than 10 packs per day. Intravenous propofol sedation compared to no intravenous sedation during conventional upper gastrointestinal endoscopy can facilitate a more complete pharyngeal examination and increase the detection rate of superficial H&N squamous cell carcinoma in high risk patients.
PMCID: PMC4694378  PMID: 26770478
Esophageal squamous cell carcinoma; head and neck cancer; superficial cancer; high-risk patients; propofol sedation
9.  A feed-forward mechanism involving Drosophila fragile X mental retardation protein triggers a replication stress-induced DNA damage response 
Human Molecular Genetics  2014;23(19):5188-5196.
Fragile X syndrome, a common form of inherited mental retardation, is caused by loss of the fragile X mental retardation protein (FMRP). As a selective RNA-binding protein, FMRP is localized predominately in cytoplasm, where it regulates translational control. However, there is a small portion of FMRP present in the nucleus, and its function there has been elusive. Here, we show that Drosophila dFMR1 in nucleus is required for replication stress-induced H2Av phosphorylation in the DNA damage response (DDR). Replication stress could induce the expression of dFmr1 and promote the nuclear accumulation of dFMR1. We show that, upon the stimulation of replication stress, dFMR1 is associated with chromatin in a domain-specific manner, which is essential for its ability to induce the phosphorylation of H2Av. These results together reveal an unexpected nuclear role of FMRP in DDR and uncover a feed-forward mechanism by which dFmr1 and early DDR induced by replication stress reciprocally regulate each other, thereby synergistically triggering activity of the DDR signaling cascade.
doi:10.1093/hmg/ddu241
PMCID: PMC4159158  PMID: 24833720
10.  Phospho-selective mechanisms of arrestin conformations and functions revealed by unnatural amino acid incorporation and 19F-NMR 
Nature Communications  2015;6:8202.
Specific arrestin conformations are coupled to distinct downstream effectors, which underlie the functions of many G-protein-coupled receptors (GPCRs). Here, using unnatural amino acid incorporation and fluorine-19 nuclear magnetic resonance (19F-NMR) spectroscopy, we demonstrate that distinct receptor phospho-barcodes are translated to specific β-arrestin-1 conformations and direct selective signalling. With its phosphate-binding concave surface, β-arrestin-1 ‘reads' the message in the receptor phospho-C-tails and distinct phospho-interaction patterns are revealed by 19F-NMR. Whereas all functional phosphopeptides interact with a common phosphate binding site and induce the movements of finger and middle loops, different phospho-interaction patterns induce distinct structural states of β-arrestin-1 that are coupled to distinct arrestin functions. Only clathrin recognizes and stabilizes GRK2-specific β-arrestin-1 conformations. The identified receptor-phospho-selective mechanism for arrestin conformation and the spacing of the multiple phosphate-binding sites in the arrestin enable arrestin to recognize plethora phosphorylation states of numerous GPCRs, contributing to the functional diversity of receptors.
G-protein-coupled receptors (GPCRs) signal via G proteins or arrestin-mediated pathways; the plasticity of arrestin proteins is thought to underlie their function. Here, the authors use NMR to examine how β-arrestin-1 recognizes different GPCR phospho-barcodes, and how this triggers structural rearrangements to fulfill selective functions.
doi:10.1038/ncomms9202
PMCID: PMC4569848  PMID: 26347956
11.  Factors for Endoscopic Submucosal Dissection in Early Colorectal Neoplasms: A Single Center Clinical Experience in China 
Clinical Endoscopy  2015;48(5):405-410.
Background/Aims
Early colorectal (CR) neoplasm can be cured by endoscopic submucosal dissection (ESD), but clinical experience and factors associated with complications from ESD for CR neoplasms in China have not been reported.
Methods
Seventy-eight cases of early CR neoplasm treated with endoscopic resection performed between December 2012 and December 2013 at Beijing Military General Hospital were included. Factors associated with ESD complications and procedure times were evaluated.
Results
The en bloc resection rate was 88.5% (69/78), tumor size was 32.1±10.7 mm, and procedure time was 71.8±49.5 minutes. The major complication was perforation, which occurred in 8.97% of the ESD procedures. Multivariate logistic regression analysis indicated that only tumor size (p=0.022) was associated with ESD perforation. Tumor size (p<0.001) and the non-lifting sign (p=0.017) were independent factors for procedure time, and procedure time (p=0.016) was a key factor for en bloc resection. After a median 10 months (range, 4 to 16) of follow-up, no patients had local recurrence.
Conclusions
This study indicated that ESD is an applicable method for large early CR neoplasm in the colon and rectum. Tumor size and the non-lifting sign might be considerable factors for increased complication rate and procedural time of ESD.
doi:10.5946/ce.2015.48.5.405
PMCID: PMC4604279  PMID: 26473124
Endoscopic submucosal dissection; Early colorectal neoplasms; Complications; Procedure time
12.  Cascade bicyclizations of o-alkynyl aldehydes with thiazolium salt: A new access toward poly-functionalized indeno[2,1-b]pyrroles 
A new cascade bicyclization of o-alkynyl aldehydes with thiazolium salt is described, in which 25 examples of densely functionalized indeno[2,1-b]pyrroles are achieved in a functional-group-compatible manner. Thiazole carbenes generated in situ from thiazolium salt play dual roles as a reaction partner and as a NHC catalyst. The synthetic utility of these bicyclization reactions results in subsequent C–C bond-forming events to rapidly build up molecular complexity.
Graphical abstract
doi:10.1039/c5cc04306a
PMCID: PMC4547933  PMID: 26178784
13.  Human Leukocyte Antigen DQB1 (HLA-DQB1) Polymorphisms and the Risk for Guillain-Barré Syndrome: A Systematic Review and Meta-Analysis 
PLoS ONE  2015;10(7):e0131374.
Guillain-Barré syndrome (GBS) is an autoimmune disorder of the peripheral nervous system. There is no consensus regarding reported associations between human leukocyte antigen DQB1 (HLA-DQB1) polymorphisms and the risk for developing GBS. Here, we evaluated possible associations between HLA-DQB1 polymorphisms and the risk for GBS using a meta-analysis. We searched PubMed for case-control genetic association studies for HLA-DQB1 polymorphisms (*020x, *030x, *040x, *050x, and *060x) and the risk for GBS. Fixed-effect meta-analytical methods were used for the outcome measure and subgroup analyses. Estimated odds ratios (ORs) and 95% confidence intervals (CIs) were used to investigate the associations between HLA-DQB1 polymorphisms and the risk for GBS. Nine case-control studies involving 780 cases of GBS and 1353 controls were identified in the current study. The meta-analysis demonstrated no significant associations between HLA-DQB1 polymorphisms and the risk for GBS in Asian and Caucasian populations. There were two associations that approached significance: HLA-DQB1*030x in Asian patients (P = 0.07; OR: 0.76, 95% CI: 0.57–1.03) and HLA-DQB1*060x in all patients (P = 0.08; OR: 1.48, 95% CI: 0.96–2.29). Additional studies with larger sample sizes are required to establish a definitive assessment of the contribution of HLA-DQB1 polymorphisms to GBS risk.
doi:10.1371/journal.pone.0131374
PMCID: PMC4512729  PMID: 26204120
14.  Detection of differentially methylated regions from whole-genome bisulfite sequencing data without replicates 
Nucleic Acids Research  2015;43(21):e141.
DNA methylation is an important epigenetic modification involved in many biological processes and diseases. Recent developments in whole genome bisulfite sequencing (WGBS) technology have enabled genome-wide measurements of DNA methylation at single base pair resolution. Many experiments have been conducted to compare DNA methylation profiles under different biological contexts, with the goal of identifying differentially methylated regions (DMRs). Due to the high cost of WGBS experiments, many studies are still conducted without biological replicates. Methods and tools available for analyzing such data are very limited.
We develop a statistical method, DSS-single, for detecting DMRs from WGBS data without replicates. We characterize the count data using a rigorous model that accounts for the spatial correlation of methylation levels, sequence depth and biological variation. We demonstrate that using information from neighboring CG sites, biological variation can be estimated accurately even without replicates. DMR detection is then carried out via a Wald test procedure. Simulations demonstrate that DSS-single has greater sensitivity and accuracy than existing methods, and an analysis of H1 versus IMR90 cell lines suggests that it also yields the most biologically meaningful results. DSS-single is implemented in the Bioconductor package DSS.
doi:10.1093/nar/gkv715
PMCID: PMC4666378  PMID: 26184873
15.  Fat mass and obesity-associated (FTO) protein interacts with CaMKII and modulates the activity of CREB signaling pathway 
Human Molecular Genetics  2014;23(12):3299-3306.
Polymorphisms in the fat mass and obesity-associated (FTO) gene have been associated with obesity in humans. FTO is a nuclear protein and its physiological function remains largely unknown, but alterations in its expression in mice influence energy expenditure, food intake and, ultimately, body weight. To understand the molecular functions of FTO, we performed a yeast two-hybrid screen to identify the protein(s) that could directly interact with human FTO protein. Using multiple assays, we demonstrate that FTO interacts with three isoforms of calcium/calmodulin-dependent protein kinase II: α, β and γ, which are protein kinases that phosphorylate a broad range of substrates. This interaction is functional; overexpression of FTO delays the dephosphorylation of cAMP response element-binding protein (CREB) in human neuroblastoma (SK-N-SH) cells, which in turn leads to a dramatic increase in the expression of the CREB targets neuropeptide receptor 1 (NPY1R) and brain-derived neurotrophic factor (BDNF), which already are known to regulate food intake and energy homeostasis. Thus, our results suggest that FTO could modulate obesity by regulating the activity of the CREB signaling pathway.
doi:10.1093/hmg/ddu043
PMCID: PMC4030783  PMID: 24488767
16.  -374T/A polymorphism of the receptor for advanced glycation end products is associated with decreased risk of breast cancer in a Chinese population 
Purpose: we aimed to investigate the receptor for advanced glycation end products (RAGE) -374T/A polymorphism and breast cancer risk in a Chinese population. Methods: The study subjects included 188 women with histologically confirmed breast cancer and 210 controls. The RAGE genotypes were determined using Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) assay. Pearson’s χ2 test was used to test the association between cases and controls and genotype frequencies. The association between the polymorphism and risk of breast cancer was estimated by odds ratio (OR) and 95% confidence interval (95% CI). Results: The AA genotype was significantly higher in breast cancer patients than in controls (37.77% vs. 28.10%, P = 0.002). Furthermore, the A allele frequency was significantly higher in the case group than in the control group (55.32% vs. 42.14%, P < 0.001). With the TT genotype as reference, the adjusted OR for AA homozygous carriers reached to 0.36 (95% CI: 0.17-0.88; P = 0.03). Under the dominant model of inheritance, the TA+AA genotype was associated with significantly decreased risk for breast cancer (adjusted OR = 0.38, 95% CI = 0.27-0.87; P = 0.02). The A allele carriage also presented a lower risk for breast cancer (adjusted OR = 0.42; 95% CI, 0.33-0.91; P = 0.04). Conclusion: Our findings suggest that the polymorphic variants of RAGE-374T/A may have an influence on breast cancer risk among Chinese women.
PMCID: PMC4538026  PMID: 26309709
Receptor for advanced glycation end products; breast cancer; polymorphisms; risk
17.  Cell cycle-linked MeCP2 phosphorylation modulates adult neurogenesis involving the Notch signaling pathway 
Nature communications  2014;5:5601.
Neuronal activity regulates the phosphorylation states at multiple sites on MeCP2 in postmitotic neurons. The precise control of the phosphorylation status of MeCP2 in neurons is critical for the normal development and function of the mammalian brain. However, it is unknown whether phosphorylation at any of the previously identified sites on MeCP2 can be induced by signals other than neuronal activity in other cell types, and what functions MeCP2 phosphorylation may have in those contexts. Here we show that, in neural progenitor cells isolated from the adult mouse hippocampus, cell cycle-linked phosphorylation at serine 421 on MeCP2 is directly regulated by aurora kinase B, and modulates the balance between proliferation and neural differentiation through the Notch signaling pathway. Our findings suggest MeCP2 S421 phosphorylation may function as a general epigenetic switch accessible by different extracellular stimuli through different signaling pathways for regulating diverse biological functions in different cell types.
doi:10.1038/ncomms6601
PMCID: PMC4288926  PMID: 25420914
18.  Candidate colorectal cancer predisposing gene variants in Chinese early-onset and familial cases 
AIM: To investigate whether whole-exome sequencing may serve as an efficient method to identify known or novel colorectal cancer (CRC) predisposing genes in early-onset or familial CRC cases.
METHODS: We performed whole-exome sequencing in 23 Chinese patients from 21 families with non-polyposis CRC diagnosed at ≤ 40 years of age, or from multiple affected CRC families with at least 1 first-degree relative diagnosed with CRC at ≤ 55 years of age. Genomic DNA from blood was enriched for exome sequences using the SureSelect Human All Exon Kit, version 2 (Agilent Technologies) and sequencing was performed on an Illumina HiSeq 2000 platform. Data were processed through an analytical pipeline to search for rare germline variants in known or novel CRC predisposing genes.
RESULTS: In total, 32 germline variants in 23 genes were identified and confirmed by Sanger sequencing. In 6 of the 21 families (29%), we identified 7 mutations in 3 known CRC predisposing genes including MLH1 (5 patients), MSH2 (1 patient), and MUTYH (biallelic, 1 patient), five of which were reported as pathogenic. In the remaining 15 families, we identified 20 rare and novel potentially deleterious variants in 19 genes, six of which were truncating mutations. One previously unreported variant identified in a conserved region of EIF2AK4 (p.Glu738_Asp739insArgArg) was found to represent a local Chinese variant, which was significantly enriched in our early-onset CRC patient cohort compared to a control cohort of 100 healthy Chinese individuals scored negative by colonoscopy (33.3% vs 7%, P < 0.001).
CONCLUSION: Whole-exome sequencing of early-onset or familial CRC cases serves as an efficient method to identify known and potential pathogenic variants in established and novel candidate CRC predisposing genes.
doi:10.3748/wjg.v21.i14.4136
PMCID: PMC4394074  PMID: 25892863
Colorectal cancer; Cancer predisposition; Early-onset; Germline variants; Exome sequencing
19.  MicroRNA-135b regulates apoptosis and chemoresistance in colorectal cancer by targeting large tumor suppressor kinase 2 
American Journal of Cancer Research  2015;5(4):1382-1395.
Colorectal cancer remains the third most common cause of death from cancer worldwide. MicroRNA emerges as a good area of research for current cancer therapy. Here, we identified miR-135b to be a contributor to anti-apoptosis and chemoresistance in colorectal cancer. We observed high levels of miR-135b in colorectal cancer cell lines and clinical tissues, compared to colorectal epithelium cell line and noncancerous tissues. Furthermore, enforced expression of miR-135b attenuated doxorubicin-induced apoptosis in colorectal cells. (Doxorubicin alone can trigger significant apoptosis). In elucidating the molecular mechanism by which miR-135b participate in the regulation of apoptosis and chemoresistance in colorectal cancer, we discovered that large tumor suppressor kinase 2 (LATS2) is a direct target of miR-135b. The role of miR-135b was confirmed in colorectal tumor xenograft models. The growth of established tumors was suppressed by an inhibition of miR-135b expression and enhanced apoptosis was further assessed by TUNEL assay. Taken together, our results reveal that miR-135b and LATS2 axis may be a novel therapeutic target for colorectal cancer.
PMCID: PMC4473317  PMID: 26101704
miR-135b; chemoresistance; colorectal cancer; LATS2
20.  Study of novel coating strategy for coronary stents: simutaneous coating of VEGF and anti- CD34 antibody 
Introduction
Intravascular coronary stenting has been used in the treatment of coronary artery disease (CAD), with a major limitation of in-stent restenosis (ISR). The 316 stainless steel has been widely used for coronary stents. In this study, we developed a novel coating method to reduce ISR by simultaneously coating vascular endothelial growth factor (VEGF) and anti-CD34 antibody on 316L stainless steel.
Methods
Round 316L stainless steel sheets in the D-H group were polymerized with compounds generated from condensation reaction of dopamine and heparin using N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS). Sixteen sheets from the D-H group were further immersed into 1ug/ml VEGF165 and 3mg/ml heparin sodium one after another for 10 times, and named as the D-(H-V)10 group. Eight sheets from the D-(H-V)10 group were coated with anti-CD34 antibody and termed as the D-(H-V)10-A group. Immunofluorescence assay and ELISA were used to evaluate whether the 316L stainless steel disks were successfully coated with VEGF and anti-CD34 antibody.
Results
The results of immunofluorescence assay and ELISA showed that VEGF could be detected in the D-(H-V)10 and D-(H-V)10-A group, suggesting the steel sheets were successfully covered with VEGF. Anti-CD34 antibody could only be observed in the D-(H-V)10-A group, which was the only group coated with CD34 antibody. Both results suggested that the 316L stainless steel sheets were successfully coated with VEGF and anti-CD34 antibody.
Conclusion
Our study developed a method to simultaneously coat VEGF and anti-CD34 antibody to stainless metal steel. This research serves as a fundamental role for a novel coating strategy.
Descriptors: Coronary Artery Disease. Drug-Eluting Stents. Coronary Restenosis. Vascular Endothelial Growth Factor. Antigens, CD34.
doi:10.5935/1678-9741.20150016
PMCID: PMC4462960  PMID: 26107446
Coronary Artery Disease; Drug-Eluting Stents; Coronary Restenosis; Vascular Endothelial Growth Factor; Antigens, CD34
21.  Crystallographic orientation-dependent pattern replication in direct imprint of aluminum nanostructures 
In the present work, we perform molecular dynamics simulations corroborated by experimental validations to elucidate the underlying deformation mechanisms of single-crystalline aluminum under direct imprint using a rigid silicon master. We investigate the influence of crystallographic orientation on the microscopic deformation behavior of the substrate materials and its correlation with the macroscopic pattern replications. Furthermore, the surface mechanical properties of the patterned structures are qualitatively characterized by nanoindentation tests. Our results reveal that dislocation slip and deformation twinning are two primary plastic deformation modes of single-crystalline aluminum under the direct imprint. However, both the competition between the individual deformation mechanisms and the geometry between activated dislocation slip systems and imprinted surface vary with surface orientation, which in turn leads to a strong crystallographic orientation dependence of the pattern replications. It is found that the (010) orientation leads to a better quality of pattern replication of single-crystalline aluminum than the (111) orientation.
doi:10.1186/s11671-015-0788-4
PMCID: PMC4385152  PMID: 25852392
Direct imprint; Single-crystalline aluminum; Crystallographic orientation; Nanoindentation; Molecular dynamics
22.  Base-resolution methylation patterns accurately predict transcription factor bindings in vivo 
Nucleic Acids Research  2015;43(5):2757-2766.
Detecting in vivo transcription factor (TF) binding is important for understanding gene regulatory circuitries. ChIP-seq is a powerful technique to empirically define TF binding in vivo. However, the multitude of distinct TFs makes genome-wide profiling for them all labor-intensive and costly. Algorithms for in silico prediction of TF binding have been developed, based mostly on histone modification or DNase I hypersensitivity data in conjunction with DNA motif and other genomic features. However, technical limitations of these methods prevent them from being applied broadly, especially in clinical settings. We conducted a comprehensive survey involving multiple cell lines, TFs, and methylation types and found that there are intimate relationships between TF binding and methylation level changes around the binding sites. Exploiting the connection between DNA methylation and TF binding, we proposed a novel supervised learning approach to predict TF–DNA interaction using data from base-resolution whole-genome methylation sequencing experiments. We devised beta-binomial models to characterize methylation data around TF binding sites and the background. Along with other static genomic features, we adopted a random forest framework to predict TF–DNA interaction. After conducting comprehensive tests, we saw that the proposed method accurately predicts TF binding and performs favorably versus competing methods.
doi:10.1093/nar/gkv151
PMCID: PMC4357735  PMID: 25722376
23.  Organic Fluorescent Dyes Supported on Activated Boron Nitride: A Promising Blue Light Excited Phosphors for High-Performance White Light-Emitting Diodes 
Scientific Reports  2015;5:8492.
We report an effective and rare-earth free light conversion material synthesized via a facile fabrication route, in which organic fluorescent dyes, i.e. Rhodamine B (RhB) and fluorescein isothiocyanate (FITC) are embedded into activated boron nitride (αBN) to form a composite phosphor. The composite phosphor shows highly efficient Förster resonance energy transfer and greatly improved thermal stability, and can emit at broad visible wavelengths of 500–650 nm under the 466 nm blue-light excitation. By packaging of the composite phosphors and a blue light-emitting diode (LED) chip with transparent epoxy resin, white LED with excellent thermal conductivity, current stability and optical performance can be realized, i.e. a thermal conductivity of 0.36 W/mk, a Commission Internationale de 1'Eclairage color coordinates of (0.32, 0.34), and a luminous efficiency of 21.6 lm·W−1. Our research opens the door toward to the practical long-life organic fluorescent dyes-based white LEDs.
doi:10.1038/srep08492
PMCID: PMC4329563  PMID: 25682730
24.  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
25.  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

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