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1.  Parkin deficiency contributes to pancreatic tumorigenesis by inducing spindle multipolarity and misorientation 
Cell Cycle  2013;12(7):1133-1141.
Parkin, an E3 ubiquitin ligase well known for its role in the pathogenesis of juvenile Parkinson disease, has been considered as a candidate tumor suppressor in certain types of cancer. It remains unknown whether parkin is involved in the development of pancreatic cancer, the fourth leading cause of cancer-related deaths worldwide. Herein, we demonstrate the downregulation and copy number loss of the parkin gene in human pancreatic cancer specimens. The expression of parkin negatively correlates with clinicopathological parameters indicating the malignancy of pancreatic cancer. In addition, knockdown of parkin expression promotes the proliferation and tumorigenic properties of pancreatic cancer cells both in vitro and in mice. We further find that parkin deficiency increases the proportion of cells with spindle multipolarity and multinucleation. Parkin-depleted cells also show a significant increase in spindle misorientation. These findings indicate crucial involvement of parkin deficiency in the pathogenesis of pancreatic cancer.
doi:10.4161/cc.24215
PMCID: PMC3646869  PMID: 23470638
parkin; pancreatic cancer; cell proliferation; spindle multipolarity; spindle misorientation
2.  Mice deficient in Epg5 exhibit selective neuronal vulnerability to degeneration 
The Journal of Cell Biology  2013;200(6):731-741.
Cortical layer 5 pyramidal neurons and spinal cord motor neurons are selectively vulnerable to degeneration after loss of the autophagy gene Epg5.
The molecular mechanism underlying the selective vulnerability of certain neuronal populations associated with neurodegenerative diseases remains poorly understood. Basal autophagy is important for maintaining axonal homeostasis and preventing neurodegeneration. In this paper, we demonstrate that mice deficient in the metazoan-specific autophagy gene Epg5/epg-5 exhibit selective damage of cortical layer 5 pyramidal neurons and spinal cord motor neurons. Pathologically, Epg5 knockout mice suffered muscle denervation, myofiber atrophy, late-onset progressive hindquarter paralysis, and dramatically reduced survival, recapitulating key features of amyotrophic lateral sclerosis (ALS). Epg5 deficiency impaired autophagic flux by blocking the maturation of autophagosomes into degradative autolysosomes, leading to accumulation of p62 aggregates and ubiquitin-positive inclusions in neurons and glial cells. Epg5 knockdown also impaired endocytic trafficking. Our study establishes Epg5-deficient mice as a model for investigating the pathogenesis of ALS and indicates that dysfunction of the autophagic–endolysosomal system causes selective damage of neurons associated with neurodegenerative diseases.
doi:10.1083/jcb.201211014
PMCID: PMC3601354  PMID: 23479740
3.  Comparisons of Contact Chemoreception and Food Acceptance by Larvae of Polyphagous Helicoverpa armigera and Oligophagous Bombyx mori 
Journal of Chemical Ecology  2013;39:1070-1080.
We compared food choice and the initial response to deterrent treated diet between fifth instars of Helicoverpa armigera, a polyphagous generalist pest, and Bombyx mori, an oligophagous specialist beneficial. Bombyx mori was more behaviorally sensitive to salicin than to caffeine. The relative sensitivities were reversed for H. armigera, which was tolerant to the highest levels of salicin found in natural sources but sensitive to caffeine. A single gustatory receptor neuron (GRN) in the medial styloconic sensillum of B. mori was highly sensitive to salicin and caffeine. The styloconic sensilla of H. armigera did not respond consistently to either of the bitter compounds. Phagostimulants also were tested. Myo-inositol and sucrose were detected specifically by two GRNs located in B. mori lateral styloconic sensillum, whereas, in H. armigera, sucrose was sensed by a GRN in the lateral sensillum, and myo-inositol by a GRN in the medial sensillum. Myo-inositol responsiveness in both species occurred at or below 10−3 mM, which is far below the naturally occurring concentration of 1 mM in plants. Larval responses to specific plant secondary compounds appear to have complex determinants that may include host range, metabolic capacity, and gustatory repertoire.
Electronic supplementary material
The online version of this article (doi:10.1007/s10886-013-0303-2) contains supplementary material, which is available to authorized users.
doi:10.1007/s10886-013-0303-2
PMCID: PMC3764315  PMID: 23779267
Food preference; Gustation; Styloconic sensillum; Gustatory receptor neuron; Sugar response; Bitter response
4.  Identification of a novel family of BRAFV600E inhibitors 
Journal of Medicinal Chemistry  2012;55(11):5220-5230.
The BRAF oncoprotein is mutated in about half of malignant melanomas and other cancers, and a kinase activating single valine to glutamate substitution at residue 600 (BRAFV600E) accounts for over 90% of BRAF-mediated cancers. Several BRAFV600E inhibitors have been developed, although they harbor some liabilities, thus motivating the development of other BRAFV600E inhibitor options. We report here the use of an ELISA based high-throughput screen to identify a family of related quinolol/naphthol compounds that preferentially inhibit BRAFV600E over BRAFWT and other kinases. We also report the X-ray crystal structure of a BRAF/quinolol complex revealing the mode of inhibition, employ structure-based medicinal chemistry efforts to prepare naphthol analogs that inhibit BRAFV600E in vitro with IC50 values in the 80–200 nM under saturating ATP concentrations, and demonstrate that these compounds inhibit MAPK signaling in melanoma cells. Prospects for improving the potency and selectivity of these inhibitors are discussed.
doi:10.1021/jm3004416
PMCID: PMC3383862  PMID: 22537109
5.  Osteopontin, a possible modulator of cancer stem cells and their malignant niche 
Oncoimmunology  2013;2(5):e24169.
The reciprocal interaction between cancer stem cells and their niche is important for oncogenesis and tumor progression, but the factors involved in this interrelationship remain largely unknown. We have recently demonstrated that osteopontin plays a crucial role in the crosstalk between CD44+ colorectal cancer stem cells and tumor-associated macrophages.
doi:10.4161/onci.24169
PMCID: PMC3667903  PMID: 23762797
cancer stem cells; CD44; niche; osteopontin; tumor-associated macrophages
6.  Appoptosin is a Novel Proapoptotic Protein and Mediates Cell Death in Neurodegeneration 
Apoptosis is an essential cellular process in multiple diseases and a major pathway for neuronal death in neurodegeneration. The detailed signaling events/pathways leading to apoptosis, especially in neurons, require further elucidation. Here we identify a β-amyloid precursor protein (APP)-interacting protein, designated as appoptosin, whose levels are upregulated in brain samples from Alzheimer’s disease and infarct patients, and in rodent stroke models, as well as in neurons treated with β-amyloid (Aβ) and glutamate. We further demonstrate that appoptosin induces reactive oxygen species release and intrinsic caspase-dependent apoptosis. The physiological function of appoptosin is to transport/exchange glycine/5-amino-levulinic acid across the mitochondrial membrane for heme synthesis. Downregulation of appoptosin prevents cell death and caspase activation caused by glutamate or Aβ insults. APP modulates appoptosin-mediated apoptosis through interaction with appoptosin. Our study identifies appoptosin as a crucial player in apoptosis and a novel proapoptotic protein involved in neuronal cell death, providing a possible new therapeutic target for neurodegenerative disorders and cancers.
doi:10.1523/JNEUROSCI.3668-12.2012
PMCID: PMC3509748  PMID: 23115192
7.  A unified approach for allele frequency estimation, SNP detection and association studies based on pooled sequencing data using EM algorithms 
BMC Genomics  2013;14(Suppl 1):S1.
Background
Genome-wide association studies (GWAS) have identified many common polymorphisms associated with complex traits. However, these associated common variants explain only a small fraction of the phenotypic variances, leaving a substantial portion of genetic heritability unexplained. As a result, searches for "missing" heritability are drawing increasing attention, particularly for rare variant studies that often require a large sample size and, thus, extensive sequencing effort. Although the development of next generation sequencing (NGS) technologies has made it possible to sequence a large number of reads economically and efficiently, it is still often cost prohibitive to sequence thousands of individuals that are generally required for association studies. A more efficient and cost-effective design would involve pooling the genetic materials of multiple individuals together and then sequencing the pools, instead of the individuals. This pooled sequencing approach has improved the plausibility of association studies for rare variants, while, at the same time, posed a great challenge to the pooled sequencing data analysis, essentially because individual sample identity is lost, and NGS sequencing errors could be hard to distinguish from low frequency alleles.
Results
A unified approach for estimating minor allele frequency, SNP calling and association studies based on pooled sequencing data using an expectation maximization (EM) algorithm is developed in this paper. This approach makes it possible to study the effects of minor allele frequency, sequencing error rate, number of pools, number of individuals in each pool, and the sequencing depth on the estimation accuracy of minor allele frequencies. We show that the naive method of estimating minor allele frequencies by taking the fraction of observed minor alleles can be significantly biased, especially for rare variants. In contrast, our EM approach can give an unbiased estimate of the minor allele frequency under all scenarios studied in this paper. A SNP calling approach, EM-SNP, for pooled sequencing data based on the EM algorithm is then developed and compared with another recent SNP calling method, SNVer. We show that EM-SNP outperforms SNVer in terms of the fraction of db-SNPs among the called SNPs, as well as transition/transversion (Ti/Tv) ratio. Finally, the EM approach is used to study the association between variants and type I diabetes.
Conclusions
The EM-based approach for the analysis of pooled sequencing data can accurately estimate minor allele frequencies, call SNPs, and find associations between variants and complex traits. This approach is especially useful for studies involving rare variants.
doi:10.1186/1471-2164-14-S1-S1
PMCID: PMC3549804  PMID: 23369070
8.  Caspase cleavage of cytochrome c1 disrupts mitochondrial function and enhances cytochrome c release 
Cell Research  2011;22(1):127-141.
Mitochondrial catastrophe can be the cause or consequence of apoptosis and is associated with a number of pathophysiological conditions. The exact relationship between mitochondrial catastrophe and caspase activation is not completely understood. Here we addressed the underlying mechanism, explaining how activated caspase could feedback to attack mitochondria to amplify further cytochrome c (cyto.c) release. We discovered that cytochrome c1 (cyto.c1) in the bc1 complex of the mitochondrial respiration chain was a novel substrate of caspase 3 (casp.3). We found that cyto.c1 was cleaved at the site of D106, which is critical for binding with cyto.c, following apoptotic stresses or targeted expression of casp.3 into the mitochondrial intermembrane space. We demonstrated that this cleavage was closely linked with further cyto.c release and mitochondrial catastrophe. These mitochondrial events could be effectively blocked by expressing non-cleavable cyto.c1 (D106A) or by caspase inhibitor z-VAD-fmk. Our results demonstrate that the cleavage of cyto.c1 represents a critical step for the feedback amplification of cyto.c release by caspases and subsequent mitochondrial catastrophe.
doi:10.1038/cr.2011.82
PMCID: PMC3351920  PMID: 21577235
cytochrome c1 cleavage; mitochondrial catastrophe; caspase; apoptosis
9.  Depression and Associated Factors in Internal Migrant Workers in China 
Journal of affective disorders  2011;134(1-3):198-207.
Background
Internal migrant workers are a large population in China. Current health related studies among this population mainly focused on infectious disease, maternal health and occupational diseases and injuries. However, very limited studies were paid attention to mental health of migrant workers though it is an important public health issue.
Aims
The current study aims to understand prevalence of depression symptoms and factors associated with depression among Chinese migrant workers using novel methods to develop a comprehensive sample.
Methods
Respondent-driven sampling (RDS) was employed to recruit the target population, who are required 1) not to hold a hukou indicative of living in central areas or near suburbs of Chengdu city; 2) to be 16 years or older; 3) not to be a student. The Center for Epidemiologic Depression Scale (CES-D) was used to measure depression symptoms of migrant workers. And then Structural Equation Model (SEM) was applied to explore factors associated with depression among Chinese migrant workers.
Results
Among 1,180 migrant workers, 23.7% of them had clinically relevant depression symptoms (CES-D score >= 16), and 12.8% were consistent with a clinical diagnosis of depression (CES-D score >= 21). Self-rated economic status, city adaptation status, and self-rated health had negative effects on depression. Social economic status (SES) affected depression, and was mediated by self-rated economic status and self-rated health. City adaptation status was affected by length of residence in the city, satisfaction with one’s job, and the social support that one could obtain while living in the city.
Conclusions
The findings indicated a higher prevalence of depression symptoms among migrant workers comparing to general population reported by previous studies, identified possible factors associated with depression symptoms, and also explored relationships between these factors. Our study provides a model to understand mental health of Chinese internal migrant workers and to generate important research questions for the future.
doi:10.1016/j.jad.2011.05.043
PMCID: PMC3189449  PMID: 21705089
China; migrant worker; depression; factor; respondent-driven sampling; RDS
10.  Fine mapping of a region of chromosome 11q13 reveals multiple independent loci associated with risk of prostate cancer 
Human Molecular Genetics  2011;20(14):2869-2878.
Genome-wide association studies have identified prostate cancer susceptibility alleles on chromosome 11q13. As part of the Cancer Genetic Markers of Susceptibility (CGEMS) Initiative, the region flanking the most significant marker, rs10896449, was fine mapped in 10 272 cases and 9123 controls of European origin (10 studies) using 120 common single nucleotide polymorphisms (SNPs) selected by a two-staged tagging strategy using HapMap SNPs. Single-locus analysis identified 18 SNPs below genome-wide significance (P< 10−8) with rs10896449 the most significant (P= 7.94 × 10−19). Multi-locus models that included significant SNPs sequentially identified a second association at rs12793759 [odds ratio (OR) = 1.14, P= 4.76 × 10−5, adjusted P= 0.004] that is independent of rs10896449 and remained significant after adjustment for multiple testing within the region. rs10896438, a proxy of previously reported rs12418451 (r2= 0.96), independent of both rs10896449 and rs12793759 was detected (OR = 1.07, P= 5.92 × 10−3, adjusted P= 0.054). Our observation of a recombination hotspot that separates rs10896438 from rs10896449 and rs12793759, and low linkage disequilibrium (rs10896449–rs12793759, r2= 0.17; rs10896449–rs10896438, r2= 0.10; rs12793759–rs10896438, r2= 0.12) corroborate our finding of three independent signals. By analysis of tagged SNPs across ∼123 kb using next generation sequencing of 63 controls of European origin, 1000 Genome and HapMap data, we observed multiple surrogates for the three independent signals marked by rs10896449 (n= 31), rs10896438 (n= 24) and rs12793759 (n= 8). Our results indicate that a complex architecture underlying the common variants contributing to prostate cancer risk at 11q13. We estimate that at least 63 common variants should be considered in future studies designed to investigate the biological basis of the multiple association signals.
doi:10.1093/hmg/ddr189
PMCID: PMC3118760  PMID: 21531787
11.  Multi-Patterned Dynamics of Mitochondrial Fission and Fusion in a Living Cell 
PLoS ONE  2012;7(5):e19879.
Mitochondria are highly-dynamic organelles, but it is challenging to monitor quantitatively their dynamics in a living cell. Here we developed a novel approach to determine the global occurrence of mitochondrial fission and fusion events in living human epithelial cells (Hela) and mouse embryonic fibroblast cells (MEF). Distinct patterns of sequential events including fusion followed by fission (Fu-Fi), the so-called “kiss and run” model previously described, fission followed by fusion (Fi-Fu), fusion followed by fusion (Fu-Fu), and fission followed by fission (Fi-Fi) were observed concurrently. The paired events appeared in high frequencies with short lifetimes and large sizes of individual mitochondria, as compared to those for unpaired events. The high frequencies of paired events were found to be biologically significant. The presence of membrane uncoupler CCCP enhanced the frequency of paired events (from both Fu-Fi and Fi-Fu patterns) with a reduced mitochondrial size. Knock-out of mitofusin protein Mfn1 increased the frequency of fission with increased lifetime of unpaired events whereas deletion of both Mfn1 and Mfn2 resulted in an instable dynamics. These results indicated that the paired events were dominant but unpaired events were not negligible, which provided a new insight into mitochondrial dynamics. In addition to kiss and run model of action, our data suggest that, from a global visualization over an entire cell, multiple patterns of action appeared in mitochondrial fusion and fission.
doi:10.1371/journal.pone.0019879
PMCID: PMC3359327  PMID: 22649485
12.  Production in Escherichia coli of Poly(3-Hydroxybutyrate-co-3-Hydroxyvalerate) with Differing Monomer Compositions from Unrelated Carbon Sources ▿  
Applied and Environmental Microbiology  2011;77(14):4886-4893.
The industrial production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) has been hindered by high cost and a complex control strategy caused by the addition of propionate. In this study, based on analysis of the PHBV biosynthesis process, we developed a PHBV biosynthetic pathway from a single unrelated carbon source via threonine biosynthesis in Escherichia coli. To accomplish this, we (i) overexpressed threonine deaminase, which is the key factor for providing propionyl-coenzyme A (propionyl-CoA), from different host bacteria, (ii) removed the feedback inhibition of threonine by mutating and overexpressing the thrABC operon in E. coli, and (iii) knocked out the competitive pathways of catalytic conversion of propionyl-CoA to 3-hydroxyvaleryl-CoA. Finally, we constructed a series of strains and mutants which were able to produce the PHBV copolymer with differing monomer compositions in a modified M9 medium supplemented with 20 g/liter xylose. The largest 3-hydroxyvalerate fraction obtained in the copolymer was 17.5 mol%.
doi:10.1128/AEM.00091-11
PMCID: PMC3147381  PMID: 21652742
13.  Interactions between Genetic Variants in the Adiponectin, Adiponectin Receptor 1 and Environmental Factors on the Risk of Colorectal Cancer 
PLoS ONE  2011;6(11):e27301.
Background
Metabolic syndrome traits play an important role in the development of colorectal cancer. Adipokines, key metabolic syndrome cellular mediators, when abnormal, may induce carcinogenesis.
Methodology/Principal Findings
To investigate whether polymorphisms of important adipokines, adiponectin (ADIPOQ) and its receptors, either alone or in combination with environmental factors, are implicated in colorectal cancer, a two-stage case-control study was conducted. In the first stage, we evaluated 24 tag single nucleotide polymorphisms (tag SNPs) across ADIPOQ ligand and two ADIPOQ receptors (ADIPOR1 and ADIPOR2) among 470 cases and 458 controls. One SNP with promising association was then analyzed in stage 2 among 314 cases and 355 controls. In our study, ADIPOQ rs1063538 was consistently associated with increased colorectal cancer risk, with an odds ratio (OR) of 1.94 (95%CI: 1.48–2.54) for CC genotype compared with TT genotype. In two-factor gene-environment interaction analyses, rs1063538 presented significant interactions with smoking status, family history of cancer and alcohol use, with ORs of 4.52 (95%CI: 2.78–7.34), 3.18 (95%CI: 1.73–5.82) and 1.97 (95%CI: 1.27–3.04) for smokers, individuals with family history of cancer or drinkers with CC genotype compared with non-smokers, individuals without family history of cancer or non-drinkers with TT genotype, respectively. Multifactor gene-environment interactions analysis revealed significant interactions between ADIPOQ rs1063538, ADIPOR1 rs1539355, smoking status and BMI. Individuals carrying one, two and at least three risk factors presented 1.18–fold (95%CI:0.89–fold to 1.58–fold), 1.87–fold (95%CI: 1.38–fold to2.54–fold) and 4.39–fold (95%CI: 2.75–fold to 7.01–fold) increased colorectal cancer risk compared with those who without risk factor, respectively (P trend <0.0001).
Conclusions/Significance
Our results suggest that variants in ADIPOQ may contribute to increased colorectal cancer risk in Chinese and this contribution may be modified by environmental factors, such as smoking status, family history of cancer and BMI.
doi:10.1371/journal.pone.0027301
PMCID: PMC3210156  PMID: 22087284
14.  RNF185, a Novel Mitochondrial Ubiquitin E3 Ligase, Regulates Autophagy through Interaction with BNIP1 
PLoS ONE  2011;6(9):e24367.
Autophagy is an evolutionarily conserved catabolic process that allows recycling of cytoplasmic organelles, such as mitochondria, to offer a bioenergetically efficient pathway for cell survival. Considerable progress has been made in characterizing mitochondrial autophagy. However, the dedicated ubiquitin E3 ligases targeting mitochondria for autophagy have not been revealed. Here we show that human RNF185 is a mitochondrial ubiquitin E3 ligase that regulates selective mitochondrial autophagy in cultured cells. The two C-terminal transmembrane domains of human RNF185 mediate its localization to mitochondrial outer membrane. RNF185 stimulates LC3II accumulation and the formation of autophagolysosomes in human cell lines. We further identified the Bcl-2 family protein BNIP1 as one of the substrates for RNF185. Human BNIP1 colocalizes with RNF185 at mitochondria and is polyubiquitinated by RNF185 through K63-based ubiquitin linkage in vivo. The polyubiquitinated BNIP1 is capable of recruiting autophagy receptor p62, which simultaneously binds both ubiquitin and LC3 to link ubiquitination and autophagy. Our study might reveal a novel RNF185-mediated mechanism for modulating mitochondrial homeostasis through autophagy.
doi:10.1371/journal.pone.0024367
PMCID: PMC3170314  PMID: 21931693
15.  Multiple gene dysfunctions lead to high cancer-susceptibility: evidences from a whole-exome sequencing study 
A total of $275 million has been launched to The Cancer Genome Atlas Project for genomic mapping of more than 20 types of cancers. The major challenge is to develop high throughput and cost-effective techniques for human genome sequencing. We developed a targeted exome sequencing technology to routinely determine human exome sequence. As a proof-of-concept, we chose a unique patient, who underwent three high mortalities cancers, i.e., breast, gallbladder and lung cancers, to reveal the genetic cause of high-cancer-susceptibility. Total 24,545 SNPs were detected. 10,868 (44.27%) SNPs were within coding regions, and 1,077 (4.38%) located in the UTRs. 3367 genes were hit by 4480 non-sysnonymous mutations in CDS with truncation of 30 proteins; and 10 mutations occurred at the splice sites that would generate different protein isoforms. Substitutions or premature terminations occurred in 132 proteins encoded by cancer-associated genes. CARD8 was completely loss; ANAPC1 was pre-translationally terminated from the transcripts of one allele. On the Ras-MAPK pathway, 18 genes were homozygously mutated. 15 growth factors/cytokines and their receptors, 9 transcription factors, 6 proteins on WNT signaling pathway, and 16 cell surface and extracellular proteins may be dysfunctioned. Exome sequencing made it possible for individualized cancer therapy.
PMCID: PMC3186053  PMID: 21984973
whole-exome sequencing; cancer; genetic variations; high-cancer-susceptibility
16.  The DNA Methylome of Human Peripheral Blood Mononuclear Cells 
PLoS Biology  2010;8(11):e1000533.
Analysis across the genome of patterns of DNA methylation reveals a rich landscape of allele-specific epigenetic modification and consequent effects on allele-specific gene expression.
DNA methylation plays an important role in biological processes in human health and disease. Recent technological advances allow unbiased whole-genome DNA methylation (methylome) analysis to be carried out on human cells. Using whole-genome bisulfite sequencing at 24.7-fold coverage (12.3-fold per strand), we report a comprehensive (92.62%) methylome and analysis of the unique sequences in human peripheral blood mononuclear cells (PBMC) from the same Asian individual whose genome was deciphered in the YH project. PBMC constitute an important source for clinical blood tests world-wide. We found that 68.4% of CpG sites and <0.2% of non-CpG sites were methylated, demonstrating that non-CpG cytosine methylation is minor in human PBMC. Analysis of the PBMC methylome revealed a rich epigenomic landscape for 20 distinct genomic features, including regulatory, protein-coding, non-coding, RNA-coding, and repeat sequences. Integration of our methylome data with the YH genome sequence enabled a first comprehensive assessment of allele-specific methylation (ASM) between the two haploid methylomes of any individual and allowed the identification of 599 haploid differentially methylated regions (hDMRs) covering 287 genes. Of these, 76 genes had hDMRs within 2 kb of their transcriptional start sites of which >80% displayed allele-specific expression (ASE). These data demonstrate that ASM is a recurrent phenomenon and is highly correlated with ASE in human PBMCs. Together with recently reported similar studies, our study provides a comprehensive resource for future epigenomic research and confirms new sequencing technology as a paradigm for large-scale epigenomics studies.
Author Summary
Epigenetic modifications such as addition of methyl groups to cytosine in DNA play a role in regulating gene expression. To better understand these processes, knowledge of the methylation status of all cytosine bases in the genome (the methylome) is required. DNA methylation can differ between the two gene copies (alleles) in each cell. Such allele-specific methylation (ASM) can be due to parental origin of the alleles (imprinting), X chromosome inactivation in females, and other as yet unknown mechanisms. This may significantly alter the expression profile arising from different allele combinations in different individuals. Using advanced sequencing technology, we have determined the methylome of human peripheral blood mononuclear cells (PBMC). Importantly, the PBMC were obtained from the same male Han Chinese individual whose complete genome had previously been determined. This allowed us, for the first time, to study genome-wide differences in ASM. Our analysis shows that ASM in PBMC is higher than can be accounted for by regions known to undergo parent-of-origin imprinting and frequently (>80%) correlates with allele-specific expression (ASE) of the corresponding gene. In addition, our data reveal a rich landscape of epigenomic variation for 20 genomic features, including regulatory, coding, and non-coding sequences, and provide a valuable resource for future studies. Our work further establishes whole-genome sequencing as an efficient method for methylome analysis.
doi:10.1371/journal.pbio.1000533
PMCID: PMC2976721  PMID: 21085693
17.  Essential role of the voltage-dependent anion channel (VDAC) in mitochondrial permeability transition pore opening and cytochrome c release induced by arsenic trioxide 
Oncogene  2004;23(6):1239-1247.
The precise molecular mechanism underlying arsenic trioxide (As2O3)-induced apoptosis is a subject of extensive study. Here, we show that clinically relevant doses of As2O3 can induce typical apoptosis in IM-9, a multiple myeloma cell line, in a Bcl-2 inhibitable manner. We confirmed that As2O3 directly induced cytochrome c (cyto c) release from isolated mouse liver mitochondria via the mitochondrial permeability transition pore, and we further identified the voltage-dependent anion channel (VDAC) as a biological target of As2O3 responsible for eliciting cyto c release in apoptosis. First, pretreatment of the isolated mitochondria with an anti-VDAC antibody specifically prevented As2O3-induced cyto c release. Second, in proteoliposome experiments, VDAC by itself was sufficient to mediate As2O3-induced cyto c release, which could be specifically inhibited by Bcl-XL. Third, As2O3 induced mitochondria membrane potential (ΔΨm) reduction and cyto c release only in the VDAC-expressing, but not in the VDAC-deficient yeast strain. Finally, we found that As2O3 induced the increased expression and homodimerization of VDAC in IM-9 cells, but not in Bcl-2 overexpressing cells, suggesting that VDAC homodimerization could potentially determine its gating capacity to cyto c, and Bcl-2 blockage of VDAC homodimerization represents a novel mechanism for its inhibition of apoptosis.
doi:10.1038/sj.onc.1207205
PMCID: PMC2913247  PMID: 14647451
apoptosis; arsenic trioxide; Bcl-2 proteins; cytochrome c; VDAC
18.  Treatment planning to improve delivery accuracy and patient throughput in helical tomotherapy 
Purpose
To investigate delivery quality assurance (DQA) discrepancies observed for a subset of helical tomotherapy patients.
Methods and Materials
Six tomotherapy patient plans were selected for analysis. Three had passing DQA ion chamber (IC) measurements while three had measurements deviating from the expected dose by more than 3.0%. All plans utilized similar parameters, including: 2.5 cm field-width, 15 s gantry period, and pitch values ranging from 0.143–0.215. Preliminary analysis suggested discrepancies were associated with plans having predominantly small leaf open times (LOTs). To test this, patients with failing DQA measurements were replanned using an increased pitch of 0.287. New DQA plans were generated and IC measurements performed. Exit fluence data was also collected during DQA delivery for dose reconstruction purposes.
Results
Sinogram analysis showed increases in mean LOTs ranging from 29.8–83.1% for the increased pitch replans. IC measurements for these plans showed a reduction in dose discrepancies, bringing all measurements within ±3.0%. The replans were also more efficient to deliver, resulting in reduced treatment times. Dose reconstruction results were in excellent agreement with IC measurements, illustrating the impact of leaf-timing inaccuracies on plans having predominantly small LOTs.
Conclusions
The impact of leaf-timing inaccuracies on plans with small mean LOTs can be considerable. These inaccuracies result from deviations in MLC leaf latency from the linear approximation used by the treatment planning system and can be important for plans having a 15 s gantry period. The ability to reduce this effect while improving delivery efficiency by increasing the pitch is demonstrated.
doi:10.1016/j.ijrobp.2009.02.004
PMCID: PMC2768470  PMID: 19394157
tomotherapy; treatment planning; delivery accuracy; patient throughput; leaf latency
19.  Cytotoxic Activities of New Jadomycin Derivatives 
The Journal of antibiotics  2005;58(6):405-408.
Cytotoxic activities of jadomycin B and five new jadomycin derivatives against four cancer cell lines (HepG2, IM-9, IM-9/Bcl-2 and H460) were evaluated. Jadomycin S was most potent against HepG2, IM-9 and IM-9/Bcl-2 while jadomycin F was most potent against H460. Their potencies correlated with the degrees of apoptosis induced. Structure-activity-relationship analyses clearly demonstrate that the side chains of the oxazolone ring derived from the incorporated amino acids make a significant impact on biological activity. Therefore, jadomycin offers an ideal scaffold to manipulate structure and could be exploited to make many novel bioactive compounds with altered activities.
doi:10.1038/ja.2005.51
PMCID: PMC2881663  PMID: 16156517
jadomycin; derivative; Streptomyces venezuelae; cytotoxic
20.  Site-Specific Molecular Signatures Predict Aggressive Disease in HNSCC 
Head and Neck Pathology  2008;2(4):243-256.
It is known that head and neck squamous cell carcinomas (HNSCC) originating from different anatomic locations can exhibit varying behavior that is not predictable by histopathology of the primary tumor. Using a microarray containing 27,323 cDNA clones, we generated sets of gene expression profiles for 36 HNSCC primary tumors (12 oral cavity, 12 oropharynx, and 12 larynx/hypopharynx). From these datasets, we ranked genes according to their ability to differentiate between patients whose disease progressed within a 24 month period (aggressive phenotype) and those that did not (non-aggressive phenotype) based on levels of gene expression. A merging of datasets from the three sites revealed that only a fraction of identified genes were shared between any two sites. This contrasted greatly with the significant overlap (approximately 50%) in down-regulated genes identified in tumor/normal comparisons using cases both from oropharynx and larynx/hypopharynx. From these data, we conclude that HNSCC tumors originating from different anatomic sites share consistent changes in gene expression when comparing primary tumors to normal adjacent mucosa; these common changes most likely reflect alterations required for tumor development. In contrast, once a tumor has developed, tumor-host interactions at the different anatomic sites are likely responsible for the site-specific signatures associated with aggressive versus non-aggressive disease. Predictions of outcome based on gene expression profiling are therefore heavily influenced by the anatomic site of the primary tumor.
doi:10.1007/s12105-008-0071-4
PMCID: PMC2807580  PMID: 20614290
Microarray; Squamous cell carcinoma; Head and neck cancer; Prognosis
21.  Spatial-angular compounding for elastography using beam steering on linear array transducers 
Medical physics  2006;33(3):618-626.
Spatial-angular compounding is a new technique that enables the reduction of noise artifacts in ultrasound elastography. Under this method, compounded elastograms are obtained from a spatially weighted average of local strain estimated from radio frequency (rf) echo signals acquired at different insonification angles. In previous work, the acquisition of the rf signals was performed through the lateral translation of a phased-array transducer. Clinical applications of angular compounding would, however, require the utilization of beam steering on linear-array transducers to obtain angular data sets, which is more efficient than translating phased-array transducers. In this article, we investigate the performance of angular compounding for elastography by using beam steering on a linear-array transducer. Quantitative experimental results demonstrate that spatial angular compounding provides significant improvement in both the elastographic signal-to-noise ratio and the contrast-to-noise ratio. For the linear array transducer used in this study, the optimum angular increment is around 1.5°–3.75°, and the maximum angle that can be used in angular compounding should not exceed 10°.
doi:10.1118/1.2168429
PMCID: PMC1815397  PMID: 16878565
angular compounding; angled beams; compounding; elastography; elastogram; elasticity; elasticity imaging; strain; stiffness; signal-to-noise; ultrasound
22.  Correlation of RF Signals During Angular Compounding 
A theoretical analysis of the correlation between radio-frequency (RF) echo signal data acquired from the same location but at different angles is presented. The accuracy of the theoretical results is verified with computer simulations. Refinements to previous analyses of the correlation of RF signals originating from the same spatial location at different angular positions are made. We extend the analysis to study correlation of RF signals coming from different spatial locations and eventually correlation of RF signal segments that intersect at the same spatial location. The theory predicts a faster decorrelation with a change in the insonification angle for longer RF echo signal segments. As the RF signal segment becomes shorter, the decorrelation rate with angle is slower and approaches the limit corresponding to the correlation of RF signals originating from the same spatial location. Theoretical results provide a clear understanding of angular compounding techniques used to improve the signal-to-noise ratio in ultrasonic parametric imaging and in elastography.
PMCID: PMC1479311  PMID: 16118977
23.  Redox Regulation of Apoptosis before and after Cytochrome C Release 
Programmed cell death, or apoptosis, is one of the most studied areas of modern biology. Apoptosis is a genetically regulated process, which plays an essential role in the development and homeostasis of higher organisms. Mitochondria, known to play a central role in regulating cellular metabolism, was found to be critical for regulating apoptosis induced under both physiological and pathological conditions. Mitochondria are a major source of reactive oxygen species (ROS) but they can also serve as its target during the apoptosis process. Release of apoptogenic factors from mitochondria, the best known of which is cytochrome c, leads to assembly of a large apoptosis-inducing complex called the apoptosome. Cysteine proteases (called caspases) are recruited to this complex and, following their activation by proteolytic cleavage, activate other caspases, which in turn target for specific cleavage a large number of cellular proteins. The redox regulation of apoptosis during and after cytochrome c release is an area of intense investigation. This review summarizes what is known about the biological role of ROS and its targets in apoptosis with an emphasis on its intricate connections to mitochondria and the basic components of cell death.
PMCID: PMC1343461  PMID: 16467897
Reactive oxygen species; Mitochondria; Cytochrome c; Caspase
24.  Role of Apo2L/TRAIL and Bcl-2-family Proteins in Apoptosis of Multiple Myeloma* 
Leukemia & lymphoma  2003;44(7):1209-1214.
Apo2 Ligand or Tumour Necrosis Factor (TNF)-Related Apoptosis-Inducing Ligand (Apo2L/TRAIL) is a member of the TNF gene superfamily that selectively induces apoptosis in tumor cells of diverse origins through engagement of death receptors. We have recently demonstrated that Type I interferons (IFN-α and β) induce apoptosis in multiple myeloma (MM) cell lines and in plasma cells from MM patients. Moreover, Apo2L selectively induces apoptosis of patient MM tumor cells while sparing non-malignant cells. Apo2L induction is one of the earliest events following IFN administration in these cells. IFNs activate Caspases and the mitochondrial-dependent apoptotic pathway mediated by Apo2L production. Cell death induced by IFNs and Apo2L can be blocked by a dominant-negative Apo2L receptor, DR5, and is regulated by members of the Bcl-2 family of proteins. This review is focused on the apoptotic signaling pathways regulated by Apo2L and Bcl-2-family proteins and summarizes what is known about their clinical role.
doi:10.1080/1042819031000068052
PMCID: PMC1199086  PMID: 12916874
Multiple myeloma; Apoptosis; Apo2L/TRAIL; Bcl-2 family proteins; Interferon
25.  Identification of genes regulated by Wnt/β-catenin pathway and involved in apoptosis via microarray analysis 
BMC Cancer  2006;6:221.
Background
Wnt/β-catenin pathway has critical roles in development and oncogenesis. Although significant progress has been made in understanding the downstream signaling cascade of this pathway, little is known regarding Wnt/β-catenin pathway modification of the cellular apoptosis.
Methods
To identify potential genes regulated by Wnt/β-catenin pathway and involved in apoptosis, we used a stably integrated, inducible RNA interference (RNAi) vector to specific inhibit the expression and the transcriptional activity of β-catenin in HeLa cells. Meanwhile, we designed an oligonucleotide microarray covering 1384 apoptosis-related genes. Using oligonucleotide microarrays, a series of differential expression of genes was identified and further confirmed by RT-PCR.
Results
Stably integrated inducible RNAi vector could effectively suppress β-catenin expression and the transcriptional activity of β-catenin/TCF. Meanwhile, depletion of β-catenin in this manner made the cells more sensitive to apoptosis. 130 genes involved in some important cell-apoptotic pathways, such as PTEN-PI3K-AKT pathway, NF-κB pathway and p53 pathway, showed significant alteration in their expression level after the knockdown of β-catenin.
Conclusion
Coupling RNAi knockdown with microarray and RT-PCR analyses proves to be a versatile strategy for identifying genes regulated by Wnt/β-catenin pathway and for a better understanding the role of this pathway in apoptosis. Some of the identified β-catenin/TCF directed or indirected target genes may represent excellent targets to limit tumor growth.
doi:10.1186/1471-2407-6-221
PMCID: PMC1574340  PMID: 16959035

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