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1.  Potential of Computer-aided Diagnosis of High Spectral and Spatial (HiSS) MRI in the Classification of Breast Lesions 
To compare the performance of CADx analysis of pre-contrast HiSS MRI to that of clinical DCE-MRI in the diagnostic classification of breast lesions.
Materials and Methods:
Thirty-four malignant and seven benign lesions were scanned using 2D HiSS and clinical 4D DCE-MRI protocols. Lesions were automatically segmented. Morphological features were calculated for HiSS whereas both morphological and kinetic features were calculated for DCE-MRI. After stepwise feature selection, Bayesian artificial neural networks merged selected features, and ROC analysis evaluated the performance with leave-one-lesion-out validation.
AUC values of 0.92 ± 0.06 and 0.90 ± 0.05 were obtained using CADx on HiSS and DCE-MRI, respectively, in the task of classifying benign and malignant lesions. While we failed to show that the higher HiSS performance was significantly better than DCE-MRI, non-inferiority testing confirmed that HiSS was not worse than DCE-MRI.
CADx of HiSS (without contrast) performed similarly to CADx on clinical DCE-MRI; thus, computerized analysis of HiSS may provide sufficient information for diagnostic classification. The results are clinically important for patients in whom contrast agent is contra-indicated. Even in the limited acquisition mode of 2D single slice HiSS, by using quantitative image analysis to extract characteristics from the HiSS images, similar performance levels were obtained as compared to those from current clinical 4D DCE-MRI. As HiSS acquisitions become possible in 3D, CADx methods can also be applied. Since HiSS and DCE-MRI are based on different contrast mechanisms, the use of the two protocols in combination may increase diagnostic accuracy.
PMCID: PMC4143529  PMID: 24023011
High spectral and spatial resolution (HiSS) MRI; computer-aided diagnosis (CADx); breast cancer; dynamic contrast enhanced MRI (DCE-MRI); contrast-agent induced nephrotoxicity
2.  Etomidate-Remifentanil is more Suitable for Monitored Anesthesia Care during Gastroscopy in Older Patients than Propofol-Remifentanil 
This prospective and randomized study was designed to compare safety, potential complications, and patient and examiner satisfaction of 2 anesthetic combinations – etomidate-remifentanil and propofol-remifentanil – in elderly patients undergoing diagnostic gastroscopy.
A group of 720 patients, aged 60–80 years, scheduled for diagnostic gastroscopy under sedation were prospectively randomized. After 0.4–0.6 μg kg−1 of remifentanil was infused, etomidate or propofol was administered. Patients in the etomidate group received doses of etomidate at 0.1–0.15 mg kg−1 followed by 4–6 mg. Patients in the propofol group received doses of propofol at 1–2 mg kg−1 followed by 20–40 mg. Physiological indexes were evaluated for the 715 of 720 patients that completed the treatment. The onset time, duration time, and discharge time were recorded. Physicians, anesthetists, and patients were surveyed to assess their satisfaction.
Systolic pressure and diastolic pressure decreased significantly after the procedure in the propofol group (P<0.001). The average heart rate was significantly lower in the propofol group (P<0.05). No periods of desaturation (SpO2 <95%) were observed in either group. The onset time was earlier in the etomidate group (P=0.00). All adverse events, with the exception of myoclonus, were greater in the propofol group, and physician and patient satisfaction in both groups was similar.
Etomidate-remifentanil administration for sedation and analgesia during gastroscopy resulted in more stable hemodynamic responses and less adverse events in older patients.
PMCID: PMC4288392  PMID: 25553506
Etomidate; Gastroscopy; Propofol
3.  Combined Use of T2-weighted MRI and T1-weighted DCE-MRI in the Automated Analysis of Breast Lesions 
A multi-parametric computer-aided diagnosis (CADx) scheme that combines information from T1-weighted DCE-MRI and T2-weighted MRI was investigated using a database of 110 malignant and 86 benign breast lesions. Automatic lesion segmentation was performed, and three categories of lesion features (geometric, T1-weighted DCE, and T2-weighted) were automatically extracted. Stepwise feature selection was performed considering only geometric features, only T1-weighted DCE features, only T2-weighted features, and all features. Features were merged with Bayesian artificial neural networks, and diagnostic performance was evaluated by ROC analysis. With leave-one-lesion-out cross-validation, an AUC value of 0.77 ± 0.03 was achieved with T2-weighted-only features, indicating high diagnostic value of information in T2-weighted images. AUC values of 0.79 ± 0.03 and 0.80 ± 0.03 were obtained for geometric-only features and T1-weighted DCE-only features, respectively. When all features were considered, an AUC value of 0.85 ± 0.03 was achieved. We observed p-values of 0.0006, 0.023, and 0.0014 between the {geometric-only, T1-weighted DCE-only, and T2-weighted-only features} and all features conditions, respectively. When ranked, the p-values satisfied the Holm-Bonferroni multiple-comparison test; thus, the improvement of multi-parametric CADx was statistically significant. A CADx scheme that combines information from T1-weighted DCE and T2-weighted MRI may be advantageous over conventional T1-weighted DCE-MRI CADx.
PMCID: PMC4156840  PMID: 21523818
breast cancer; computer-aided diagnosis; T1-weighted dynamic contrast enhanced magnetic resonance imaging; T2-weighted magnetic resonance imaging
4.  Relationships between computer-extracted mammographic texture pattern features and BRCA1/2 mutation status: a cross-sectional study 
Mammographic density is similar among women at risk of either sporadic or BRCA1/2-related breast cancer. It has been suggested that digitized mammographic images contain computer-extractable information within the parenchymal pattern, which may contribute to distinguishing between BRCA1/2 mutation carriers and non-carriers.
We compared mammographic texture pattern features in digitized mammograms from women with deleterious BRCA1/2 mutations (n = 137) versus non-carriers (n = 100). Subjects were stratified into training (107 carriers, 70 non-carriers) and testing (30 carriers, 30 non-carriers) datasets. Masked to mutation status, texture features were extracted from a retro-areolar region-of-interest in each subject’s digitized mammogram. Stepwise linear regression analysis of the training dataset identified variables to be included in a radiographic texture analysis (RTA) classifier model aimed at distinguishing BRCA1/2 carriers from non-carriers. The selected features were combined using a Bayesian Artificial Neural Network (BANN) algorithm, which produced a probability score rating the likelihood of each subject’s belonging to the mutation-positive group. These probability scores were evaluated in the independent testing dataset to determine whether their distribution differed between BRCA1/2 mutation carriers and non-carriers. A receiver operating characteristic analysis was performed to estimate the model’s discriminatory capacity.
In the testing dataset, a one standard deviation (SD) increase in the probability score from the BANN-trained classifier was associated with a two-fold increase in the odds of predicting BRCA1/2 mutation status: unadjusted odds ratio (OR) = 2.00, 95% confidence interval (CI): 1.59, 2.51, P = 0.02; age-adjusted OR = 1.93, 95% CI: 1.53, 2.42, P = 0.03. Additional adjustment for percent mammographic density did little to change the OR. The area under the curve for the BANN-trained classifier to distinguish between BRCA1/2 mutation carriers and non-carriers was 0.68 for features alone and 0.72 for the features plus percent mammographic density.
Our findings suggest that, unlike percent mammographic density, computer-extracted mammographic texture pattern features are associated with carrying BRCA1/2 mutations. Although still at an early stage, our novel RTA classifier has potential for improving mammographic image interpretation by permitting real-time risk stratification among women undergoing screening mammography.
Electronic supplementary material
The online version of this article (doi:10.1186/s13058-014-0424-8) contains supplementary material, which is available to authorized users.
PMCID: PMC4268674  PMID: 25159706
6.  A Chemical Probe Targets DNA 5-Formylcytosine Sites and Inhibits TDG Excision, Polymerases Bypass, and Gene Expression 
Dynamic regulation and faithful maintenance of proper DNA methylation patterns are essential for many cellular functions. 5-Formylcytosine (5fC), a newly discovered oxidized form of methylcytosine (mC) is involved in active DNA demethylation process. The latest progresses suggest exciting novel functional roles of this residue. Chemical tools are desired to further elucidate the functional roles of 5fC and to modulate dynamics of DNA demethylation and downstream biological processes. Here we designed and constructed a chemical probe, consisting of an aldehyde targeting group and an intercalation group. This molecule can selectively react with 5fC and subsequently inhibit base excision by thymine DNA glycosylase (TDG) and cause significant pausing for both DNA and RNA polymerase elongation. Further investigation using a GFP reporter system in living cells revealed that the ligand modification in 5fC sites at 5′-UTR of the GFP gene greatly inhibited the GFP expression level. These results altogether confirmed our successful design and established a new approach for generating functional ligands that target the formylcytosine sites and modulate 5fC-related biological processes.
PMCID: PMC4038700  PMID: 24883182
7.  Ubiquitin-Specific Protease 5 Is Required for the Efficient Repair of DNA Double-Strand Breaks 
PLoS ONE  2014;9(1):e84899.
During the DNA damage response (DDR), ubiquitination plays an important role in the recruitment and regulation of repair proteins. However, little is known about elimination of the ubiquitination signal after repair is completed. Here we show that the ubiquitin-specific protease 5 (USP5), a deubiquitinating enzyme, is involved in the elimination of the ubiquitin signal from damaged sites and is required for efficient DNA double-strand break (DSB) repair. Depletion of USP5 sensitizes cells to DNA damaging agents, produces DSBs, causes delayed disappearance of γH2AX foci after Bleocin treatment, and influences DSB repair efficiency in the homologous recombination pathway but not in the non-homologous end joining pathway. USP5 co-localizes to DSBs induced by laser micro-irradiation in a RAD18-dependent manner. Importantly, polyubiquitin chains at sites of DNA damage remained for longer periods in USP5-depleted cells. Our results show that disassembly of polyubiquitin chains by USP5 at sites of damage is important for efficient DSB repair.
PMCID: PMC3891734  PMID: 24454762
8.  Correlation between social factors and anxiety-depression in function dyspepsia: do relationships exist? 
Przegla̜d Gastroenterologiczny  2014;9(6):348-353.
Function dyspepsia (FD) may cause patients to suffer from anxiety and depression, and psychosocial disorders would have a significant effect on FD symptoms.
To examine the prevalence of anxiety and depression among function dyspepsia (FD) patients and to identify social factors of anxiety-depression among FD patients.
Material and methods
Patients with FD, who fulfilled the Rome III criteria, were enrolled. All patients were administered a validated Chinese version of the self-rating scale (SDS) and self-rating anxiety scale (SAS), and investigated regarding the patients’ social factors.
A total of 907 patients were enrolled, including 516 (56.89%) FD patients within anxiety-depression status; SDS mean scores were 51.57 ±8.22; SAS mean scores were 51.04 ±7.53; 52.28% were male and 64.25% were female (χ2 = 262.54, p < 0.01); 56.16% were aged 18–29 years, 54.15% were aged 30–39 years, 54.77% were aged 40–49 years, 62.02% were aged 50–59 years, 69.23% were aged above 60 years (χ2 = 18.14, p < 0.01); 67.44% were the retirees; 63.31% were manual workers; 55.10% were soldiers; 43.57% were mental workers; 38.89% were students (χ2 = 716.53, p < 0.01); 64.20% had junior high school degree or below; 57.36% had high school degrees; 42.03% had college degrees; 44.44% had master's or above degrees (χ2 = 27.21, p < 0.05); 38.10% were in good health condition; 61.90% were in poor health condition (χ2 = 7.94, p < 0.01); 20.31% had correlative family history; and 79.69% had no correlative family history (χ2 = 2.23, p > 0.05).
The FD patients have higher rates of anxiety and depression. Gender, age, occupation, education level, and health condition have a significant effect on anxiety and depression status. Female gender, advanced age, high-stress occupation, lower education level, and poor health condition all are risk factors. Family history has no relationship with anxiety and depression among FD patients.
PMCID: PMC4300350  PMID: 25653730
digestion; function dyspepsia; anxiety; depression; socioeconomic factors
9.  Novel method for site-specific induction of oxidative DNA damage reveals differences in recruitment of repair proteins to heterochromatin and euchromatin 
Nucleic Acids Research  2013;42(4):2330-2345.
Reactive oxygen species (ROS)-induced DNA damage is repaired by the base excision repair pathway. However, the effect of chromatin structure on BER protein recruitment to DNA damage sites in living cells is poorly understood. To address this problem, we developed a method to specifically produce ROS-induced DNA damage by fusing KillerRed (KR), a light-stimulated ROS-inducer, to a tet-repressor (tetR-KR) or a transcription activator (TA-KR). TetR-KR or TA-KR, bound to a TRE cassette (∼90 kb) integrated at a defined genomic locus in U2OS cells, was used to induce ROS damage in hetero- or euchromatin, respectively. We found that DNA glycosylases were efficiently recruited to DNA damage in heterochromatin, as well as in euchromatin. PARP1 was recruited to DNA damage within condensed chromatin more efficiently than in active chromatin. In contrast, recruitment of FEN1 was highly enriched at sites of DNA damage within active chromatin in a PCNA- and transcription activation-dependent manner. These results indicate that oxidative DNA damage is differentially processed within hetero or euchromatin.
PMCID: PMC3936713  PMID: 24293652
10.  Damage-dependent regulation of MUS81-EME1 by Fanconi anemia complementation group A protein 
Nucleic Acids Research  2013;42(3):1671-1683.
MUS81-EME1 is a DNA endonuclease involved in replication-coupled repair of DNA interstrand cross-links (ICLs). A prevalent hypothetical role of MUS81-EME1 in ICL repair is to unhook the damage by incising the leading strand at the 3′ side of an ICL lesion. In this study, we report that purified MUS81-EME1 incises DNA at the 5′ side of a psoralen ICL residing in fork structures. Intriguingly, ICL repair protein, Fanconi anemia complementation group A protein (FANCA), greatly enhances MUS81-EME1-mediated ICL incision. On the contrary, FANCA exhibits a two-phase incision regulation when DNA is undamaged or the damage affects only one DNA strand. Studies using truncated FANCA proteins indicate that both the N- and C-moieties of the protein are required for the incision regulation. Using laser-induced psoralen ICL formation in cells, we find that FANCA interacts with and recruits MUS81 to ICL lesions. This report clarifies the incision specificity of MUS81-EME1 on ICL damage and establishes that FANCA regulates the incision activity of MUS81-EME1 in a damage-dependent manner.
PMCID: PMC3919598  PMID: 24170812
11.  Computerized Analysis of Mammographic Parenchymal Patterns on a Large Clinical Dataset of Full-Field Digital Mammograms: Robustness Study with Two High-Risk Datasets 
Journal of Digital Imaging  2012;25(5):591-598.
The purpose of this study was to demonstrate the robustness of our prior computerized texture analysis method for breast cancer risk assessment, which was developed initially on a limited dataset of screen-film mammograms. This current study investigated the robustness by (1) evaluating on a large clinical dataset, (2) using full-field digital mammograms (FFDM) as opposed to screen-film mammography, and (3) incorporating analyses over two types of high-risk patient sets, as well as patients at low risk for breast cancer. The evaluation included the analyses on the parenchymal patterns of women at high risk of developing of breast cancer, including both BRCA1/2 gene mutation carriers and unilateral cancer patients, and of women at low risk of developing breast cancer. A total of 456 cases, including 53 women with BRCA1/2 gene mutations, 75 women with unilateral cancer, and 328 low-risk women, were retrospectively collected under an institutional review board approved protocol. Regions-of-interest (ROIs), were manually selected from the central breast region immediately behind the nipple. These ROIs were subsequently used in computerized feature extraction to characterize the mammographic parenchymal patterns in the images. Receiver operating characteristic analysis was used to assess the performance of the computerized texture features in the task of distinguishing between high-risk and low-risk subjects. In a round robin evaluation on the FFDM dataset with Bayesian artificial neural network analysis, AUC values of 0.82 (95% confidence interval [0.75, 0.88]) and 0.73 (95% confidence interval [0.67, 0.78]) were obtained between BRCA1/2 gene mutation carriers and low-risk women, and between unilateral cancer and low-risk women, respectively. These results from computerized texture analysis on digital mammograms demonstrated that high-risk and low-risk women have different mammographic parenchymal patterns. On this large clinical dataset, we validated our methods for quantitative analyses of mammographic patterns on FFDM, statistically demonstrating again that women at high risk tend to have dense breasts with coarse and low-contrast texture patterns.
PMCID: PMC3447101  PMID: 22246204
Computerized texture analysis; Breast cancer risk assessment; Mammographic parenchymal patterns; Full-field digital mammograms; Quantitative imaging analysis
12.  Damage response of XRCC1 at sites of DNA single strand breaks is regulated by phosphorylation and ubiquitylation after degradation of poly(ADP-ribose) 
Journal of Cell Science  2013;126(19):4414-4423.
Single-strand breaks (SSBs) are the most common type of oxidative DNA damage and they are related to aging and many genetic diseases. The scaffold protein for repair of SSBs, XRCC1, accumulates at sites of poly(ADP-ribose) (pAR) synthesized by PARP, but it is retained at sites of SSBs after pAR degradation. How XRCC1 responds to SSBs after pAR degradation and how this affects repair progression are not well understood. We found that XRCC1 dissociates from pAR and is translocated to sites of SSBs dependent on its BRCTII domain and the function of PARG. In addition, phosphorylation of XRCC1 is also required for the proper dissociation kinetics of XRCC1 because (1) phosphorylation sites mutated in XRCC1 (X1 pm) cause retention of XRCC1 at sites of SSB for a longer time compared to wild type XRCC1; and (2) phosphorylation of XRCC1 is required for efficient polyubiquitylation of XRCC1. Interestingly, a mutant of XRCC1, LL360/361DD, which abolishes pAR binding, shows significant upregulation of ubiquitylation, indicating that pARylation of XRCC1 prevents the poly-ubiquitylation. We also found that the dynamics of the repair proteins DNA polymerase beta, PNK, APTX, PCNA and ligase I are regulated by domains of XRCC1. In summary, the dynamic damage response of XRCC1 is regulated in a manner that depends on modifications of polyADP-ribosylation, phosphorylation and ubiquitylation in live cells.
PMCID: PMC3784821  PMID: 23868975
Single strand breaks; XRCC1; Damage response; Phosphorylation; PolyADP-ribosylation; Ubiquitylation
13.  Caspase-1 activation and mature interleukin-1β release are uncoupled events in monocytes 
AIM: To investigate whether caspase-1 activation/intracellular processing of pro-interleukin-1β (pro-IL-1β) and extracellular release of mature IL-1β from activated monocytes are separable events.
METHODS: All experiments were performed on fresh or overnight cultured human peripheral blood monocytes (PBMCs) that were isolated from healthy donors. PBMCs were activated by lipopolysaccharide (LPS) stimulation before being treated with Adenosine triphosphate (ATP, 1 mmol/L), human α-defensin-5 (HD-5, 50 μg/mL), and/or nigericin (Nig, 30 μmol/L). For each experiment, the culture supernatants were collected separately from the cells. Cell lysates and supernatants were both subject to immunoprecipitation with anti-IL-1β antibodies followed by western blot analysis with anti-caspase-1 and anti-IL-1β antibodies.
RESULTS: We found that pro-IL-1β was processed to mature IL-1β in LPS-activated fresh and overnight cultured human monocytes in response to ATP stimulation. In the presence of HD-5, this release of IL-1β, but not the processing of pro-IL-1β to IL-1β, was completely inhibited. Similarly, in the presence of HD-5, the release of IL-1β, but not the processing of IL-1β, was significantly inhibited from LPS-activated monocytes stimulated with Nig. Finally, we treated LPS-activated monocytes with ATP and Nig and collected the supernatants. We found that both ATP and Nig stimulation could activate and release cleaved caspase-1 from the monocytes. Interestingly, and contrary to IL-1β processing and release, caspase-1 cleavage and release was not blocked by HD-5. All images are representative of three independent experiments.
CONCLUSION: These data suggest that caspase-1 activation/processing of pro-IL-1β by caspase-1 and the release of mature IL-1β from human monocytes are distinct and separable events.
PMCID: PMC3652645  PMID: 23710297
Caspase-1; Human defensin; Monocytes; Interleukin-1β processing and release; Inflammasome
14.  Application of a nanotechnology antimicrobial spray to prevent lower urinary tract infection: a multicenter urology trial 
Journal of Translational Medicine  2012;10(Suppl 1):S14.
Catheter-associated urinary tract infection (CAUTI) is a common nosocomial device-associated infection. It is now recognized that the high infection rates were caused by the formation of biofilm on the surface of the catheters that decreases the susceptibility to antibiotics and results in anti-microbial resistance.
In this study, we performed an in vitro test to explore the mechanism of biofilm formation and subsequently conducted a multi-center clinical trial to investigate the efficacy of CAUTI prevention with the application of JUC, a nanotechnology antimicrobial spray.
Siliconized latex urinary catheters were cut into fragments and sterilized by autoclaving. The sterilized sample fragments were randomly divided into the therapy and control group, whereby they were sprayed with JUC and distilled water respectively and dried before use.
The experimental standard strains of Escherichia coli (E. coli) were isolated from the urine samples of patients. At 16 hours and 7 days of incubation, the samples were extracted for confocal laser scanning microscopy.
A total of 1,150 patients were accrued in the clinical study. Patients were randomized according to the order of surgical treatment. The odd array of patients was assigned as the therapy group (JUC), and the even array of patients was assigned as the control group (normal saline).
After 16 hours of culture, bacterial biofilm formed on the surface of sample fragments from the control group. In the therapy group, no bacterial biofilm formation was observed on the sample fragments. No significant increase in bacterial colony count was observed in the therapy group after 7 days of incubation.
On the 7th day of catheterization, urine samples were collected for bacterial culture before extubation. Significant difference was observed in the incidence of bacteriuria between the therapy group and control group (4.52% vs. 13.04%, p < 0.001).
In this study, the effectiveness of JUC in preventing CAUTI in a hospital setting was demonstrated in both in vitro and clinical studies.
PMCID: PMC3445864  PMID: 23046566
15.  Relationships among Trust in Messages, Risk Perception, and Risk Reduction Preferences Based upon Avian Influenza in Taiwan  
Improvements in communications technology enable consumers to receive information through diverse channels. In the case of avian influenza, information repeated by the mass media socially amplifies the consumer awareness of risks. Facing indeterminate risks, consumers may feel anxious and increase their risk perception. When consumers trust the information published by the media, their uncertainty toward avian influenza may decrease. Consumers might take some actions to reduce risk. Therefore, this study focuses on relationships among trust in messages, risk perception and risk reduction preferences. This study administered 525 random samples and consumer survey questionnaires in different city of Taiwan in 2007. Through statistical analysis, the results demonstrate: (1) the higher the trust consumers have in messages about avian influenza, the lower their risk perceptions are; (2) the higher the consumers’ risk perceptions are and, therefore, the higher their desired level of risk reductive, the more likely they are to accept risk reduction strategies; (3) consumer attributes such as age, education level, and marital status correlate with significant differences in risk perception and risk reduction preferences acceptance. Gender has significant differences only in risk reduction preferences and not in risk perception.
PMCID: PMC3447584  PMID: 23066394
avian influenza; trust in message; risk perception; risk reduction preference
16.  Monoubiquitinated Histone H2A Destabilizes Photolesion-containing Nucleosomes with Concomitant Release of UV-damaged DNA-binding Protein E3 Ligase* 
The Journal of Biological Chemistry  2012;287(15):12036-12049.
Background: The compaction of DNA into nucleosomes interferes with DNA repair.
Results: Monoubiquitination of core histone H2A destabilizes nucleosomes containing UV-damaged DNA.
Conclusion: Destabilized nucleosomes enable the release of the DNA damage-binding complex DDB1-CUL4BDDB2, which assists in histone ubiquitination.
Significance: This mechanism explains how the ubiquitination of histone H2A, in addition to chromatin remodeling, promotes repair and facilitates genome stability.
How the nucleotide excision repair (NER) machinery gains access to damaged chromatinized DNA templates and how the chromatin structure is modified to promote efficient repair of the non-transcribed genome remain poorly understood. The UV-damaged DNA-binding protein complex (UV-DDB, consisting of DDB1 and DDB2, the latter of which is mutated in xeroderma pigmentosum group E patients, is a substrate-recruiting module of the cullin 4B-based E3 ligase complex, DDB1-CUL4BDDB2. We previously reported that the deficiency of UV-DDB E3 ligases in ubiquitinating histone H2A at UV-damaged DNA sites in the xeroderma pigmentosum group E cells contributes to the faulty NER in these skin cancer-prone patients. Here, we reveal the mechanism by which monoubiquitination of specific H2A lysine residues alters nucleosomal dynamics and subsequently initiates NER. We show that DDB1-CUL4BDDB2 E3 ligase specifically binds to mononucleosomes assembled with human recombinant histone octamers and nucleosome-positioning DNA containing cyclobutane pyrimidine dimers or 6-4 photoproducts photolesions. We demonstrate functionally that ubiquitination of H2A Lys-119/Lys-120 is necessary for destabilization of nucleosomes and concomitant release of DDB1-CUL4BDDB2 from photolesion-containing DNA. Nucleosomes in which these lysines are replaced with arginines are resistant to such structural changes, and arginine mutants prevent the eviction of H2A and dissociation of polyubiquitinated DDB2 from UV-damaged nucleosomes. The partial eviction of H3 from the nucleosomes is dependent on ubiquitinated H2A Lys-119/Lys-120. Our results provide mechanistic insight into how post-translational modification of H2A at the site of a photolesion initiates the repair process and directly affects the stability of the human genome.
PMCID: PMC3320950  PMID: 22334663
Cancer; Chromatin Histone Modification; DNA-binding Protein; DNA Damage; E3 Ubiquitin Ligase; DDB1-CUL4BDDB2; H2A Lys-119/Lys-120; NER; Nucleosome; Ubiquitination
17.  Computerized assessment of breast lesion malignancy using DCE-MRI: robustness study on two independent clinical datasets from two manufacturers 
Academic radiology  2010;17(7):822-829.
Rationale and Objectives
To conduct a pre-clinical evaluation of the robustness of our computerized system for breast lesion characterization on two breast magnetic resonance imaging (MRI) databases that were acquired using scanners from two different manufacturers.
Materials and Methods
Two clinical breast MRI databases were acquired from a Siemens scanner and a GE scanner, which shared similar imaging protocols and retrospectively collected under an IRB-approved protocol. In our computerized analysis system, once a breast lesion is identified by the radiologist, the computer performs automatic lesion segmentation and feature extraction, and outputs an estimated probability of malignancy. We used a Bayesian neural network with automatic relevance determination for joint feature selection and classification. To evaluate the robustness of our classification system, we first used Database 1 for feature selection and classifier training, and Database 2 to test the trained classifier. Then, we exchanged the two datasets and repeated the process. Area under the ROC curve (AUC) was used as a performance figure of merit in the task of distinguishing between malignant and benign lesions.
We obtained an AUC of 0.85 (approximate 95% confidence interval (CI): [0.79, 0.91]) for (a) feature selection and classifier training using Database 1 and testing on Database 2; and an AUC of 0.90 (approximate 95% CI: [0.84, 0.96]) for (b) feature selection and classifier training using Database2 and testing on Database1. We failed to observe statistical significance for the difference AUC of 0.05 between the two database-conditions (P=0.24; 95% confidence interval [− 0.03, 0.1]).
These results demonstrate the robustness of our computerized classification system in the task of distinguishing between malignant and benign breast lesions on DCE-MRI images from two manufacturers. Our study showed the feasibility of developing a computerized classification system that is robust across different scanners.
PMCID: PMC2907891  PMID: 20540907
computer-aided diagnosis; breast MRI; robustness
19.  Evaluation of Computer-aided Diagnosis on a Large Clinical Full-Field Digital Mammographic Dataset 
Academic radiology  2008;15(11):1437-1445.
Rationale and Objectives:
To convert and optimize our previously developed computerized analysis methods for use with images from full-field digital mammography (FFDM) for breast mass classification in order to aid in the diagnosis of breast cancer.
Materials and Methods:
An institutional review board approved protocol was obtained, with waiver of consent for retrospective use of mammograms and pathology data. Seven hundreds and thirty-nine full-field digital mammographic images, which contained 287 biopsy-proven breast mass lesions, of which 148 lesions were malignant and 139 lesions were benign, were retrospectively collected. Lesion margins were delineated by an expert breast radiologist and were used as the truth for lesion-segmentation evaluation. Our computerized image analysis method consisted of several steps: 1) identified lesions were automatically extracted from the parenchymal background using computerized segmentation methods; 2) a set of image characteristics (mathematical descriptors) were automatically extracted from image data of the lesions and surrounding tissues; and 3) selected features were merged into an estimate of the probability of malignancy using a Bayesian artificial neural network classifier. Performance of the analyses was evaluated at various stages of the conversion using receiver operating characteristic (ROC) analysis.
An AUC value of 0.81 was obtained in the task of distinguishing between malignant and benign mass lesions in a round-robin by case evaluation on the entire FFDM dataset. We failed to show a statistically significant difference (P value=0.83) as compared with results from our previous study in which the computerized classification was performed on digitized screen-film mammograms (SFMD).
Our computerized analysis methods developed on digitized screen-film mammography can be converted for use with FFDM. Results show that the computerized analysis methods for the diagnosis of breast mass lesions on FFDM are promising, and can potentially be used to aid clinicians in the diagnostic interpretation of FFDM.
PMCID: PMC2597106  PMID: 18995194
Computer-aided diagnosis; Full-field digital mammography; Breast mass classification
20.  Rapid Recruitment of BRCA1 to DNA Double-Strand Breaks Is Dependent on Its Association with Ku80▿ † 
Molecular and Cellular Biology  2008;28(24):7380-7393.
BRCA1 is the first susceptibility gene to be linked to breast and ovarian cancers. Although mounting evidence has indicated that BRCA1 participates in DNA double-strand break (DSB) repair pathways, its precise mechanism is still unclear. Here, we analyzed the in situ response of BRCA1 at DSBs produced by laser microirradiation. The amino (N)- and carboxyl (C)-terminal fragments of BRCA1 accumulated independently at DSBs with distinct kinetics. The N-terminal BRCA1 fragment accumulated immediately after laser irradiation at DSBs and dissociated rapidly. In contrast, the C-terminal fragment of BRCA1 accumulated more slowly at DSBs but remained at the sites. Interestingly, rapid accumulation of the BRCA1 N terminus, but not the C terminus, at DSBs depended on Ku80, which functions in the nonhomologous end-joining (NHEJ) pathway, independently of BARD1, which binds to the N terminus of BRCA1. Two small regions in the N terminus of BRCA1 independently accumulated at DSBs and interacted with Ku80. Missense mutations found within the N terminus of BRCA1 in cancers significantly changed the kinetics of its accumulation at DSBs. A P142H mutant failed to associate with Ku80 and restore resistance to irradiation in BRCA1-deficient cells. These might provide a molecular basis of the involvement of BRCA1 in the NHEJ pathway of the DSB repair process.
PMCID: PMC2593434  PMID: 18936166
21.  A polycomb group protein, PHF1, is involved in the response to DNA double-strand breaks in human cell 
Nucleic Acids Research  2008;36(9):2939-2947.
DNA double-strand breaks (DSBs) represent the most toxic DNA damage arisen from endogenous and exogenous genotoxic stresses and are known to be repaired by either homologous recombination or nonhomologous end-joining processes. Although many proteins have been identified to participate in either of the processes, the whole processes still remain elusive. Polycomb group (PcG) proteins are epigenetic chromatin modifiers involved in gene silencing, cancer development and the maintenance of embryonic and adult stem cells. By screening proteins responding to DNA damage using laser micro-irradiation, we found that PHF1, a human homolog of Drosophila polycomb-like, Pcl, protein, was recruited to DSBs immediately after irradiation and dissociated within 10 min. The accumulation at DSBs is Ku70/Ku80-dependent, and knockdown of PHF1 leads to X-ray sensitivity and increases the frequency of homologous recombination in HeLa cell. We found that PHF1 interacts physically with Ku70/Ku80, suggesting that PHF1 promotes nonhomologous end-joining processes. Furthermore, we found that PHF1 interacts with a number of proteins involved in DNA damage responses, RAD50, SMC1, DHX9 and p53, further suggesting that PHF1, besides the function in PcG, is involved in genome maintenance processes.
PMCID: PMC2396414  PMID: 18385154
22.  Vertebrate POLQ and POLβ Cooperate in Base Excision Repair of Oxidative DNA Damage 
Molecular cell  2006;24(1):115-125.
Base excision repair (BER) plays an essential role in protecting cells from mutagenic base damage caused by oxidative stress, hydrolysis, and environmental factors. POLQ is a DNA polymerase, which appears to be involved in translesion DNA synthesis (TLS) past base damage. We disrupted POLQ, and its homologs HEL308 and POLN in chicken DT40 cells, and also created polq/hel308 and polq/poln double mutants. We found that POLQ-deficient mutants exhibit hypersensitivity to oxidative base damage induced by H2O2, but not to UV or cisplatin. Surprisingly, this phenotype was synergistically increased by concomitant deletion of the major BER polymerase, POLβ. Moreover, extracts from a polq null mutant cell line show reduced BER activity, and POLQ, like POLβ, accumulated rapidly at sites of base damage. Accordingly, POLQ and POLβ share an overlapping function in the repair of oxidative base damage. Taken together, these results suggest a role for vertebrate POLQ in BER.
PMCID: PMC1868411  PMID: 17018297
23.  RAD18 and Poly(ADP-Ribose) Polymerase Independently Suppress the Access of Nonhomologous End Joining to Double-Strand Breaks and Facilitate Homologous Recombination-Mediated Repair▿ †  
Molecular and Cellular Biology  2007;27(7):2562-2571.
The Saccharomyces cerevisiae RAD18 gene is essential for postreplication repair but is not required for homologous recombination (HR), which is the major double-strand break (DSB) repair pathway in yeast. Accordingly, yeast rad18 mutants are tolerant of camptothecin (CPT), a topoisomerase I inhibitor, which induces DSBs by blocking replication. Surprisingly, mammalian cells and chicken DT40 cells deficient in Rad18 display reduced HR-dependent repair and are hypersensitive to CPT. Deletion of nonhomologous end joining (NHEJ), a major DSB repair pathway in vertebrates, in rad18-deficient DT40 cells completely restored HR-mediated DSB repair, suggesting that vertebrate Rad18 regulates the balance between NHEJ and HR. We previously reported that loss of NHEJ normalized the CPT sensitivity of cells deficient in poly(ADP-ribose) polymerase 1 (PARP1). Concomitant deletion of Rad18 and PARP1 synergistically increased CPT sensitivity, and additional inactivation of NHEJ normalized this hypersensitivity, indicating their parallel actions. In conclusion, higher-eukaryotic cells separately employ PARP1 and Rad18 to suppress the toxic effects of NHEJ during the HR reaction at stalled replication forks.
PMCID: PMC1899888  PMID: 17242200
24.  Flavopiridol downregulates hypoxia-mediated hypoxia-inducible factor-1α expression in human glioma cells by a proteasome-independent pathway: Implications for in vivo therapy1 
Neuro-Oncology  2005;7(3):225-235.
Angiogenesis is a critical step required for sustained tumor growth and tumor progression. The stimulation of endothelial cells by cytokines secreted by tumor cells such as vascular endothelial growth factor (VEGF) induces their proliferation and migration. This is a prominent feature of high-grade gliomas. The secretion of VEGF is greatly upregulated under conditions of hypoxia because of the transcription factor hypoxia-inducible factor (HIF)-1α, which controls the expression of many genes, allowing rapid adaptation of cells to their hypoxic microenvironment. Flavopiridol, a novel cyclin-dependent kinase inhibitor, has been attributed with antiangiogenic properties in some cancer cell lines by its ability to inhibit VEGF production. Here, we show that flavopiridol treatment of human U87MG and T98G glioma cell lines decreases hypoxia-mediated HIF-1α expression, VEGF secretion, and tumor cell migration. These in vitro results correlate with reduced vascularity of intracranial syngeneic GL261 gliomas from animals treated with flavopiridol. In addition, we show that flavopiridol downregulates HIF-1α expression in the presence of a proteasome inhibitor, an agent that normally results in the accumulation and overexpression of HIF-1α. The potential to downregulate HIF-1α expression with flavopiridol treatment in combination with a proteasome inhibitor makes this an extremely attractive anticancer treatment strategy for tumors with high angiogenic activity, such as gliomas.
PMCID: PMC1871916  PMID: 16053697
flavopiridol; proteasome inhibitor; hypoxia; HIF-1α; VEGF; glioma
25.  MSH2–MSH6 stimulates DNA polymerase η, suggesting a role for A:T mutations in antibody genes 
Activation-induced cytidine deaminase deaminates cytosine to uracil (dU) in DNA, which leads to mutations at C:G basepairs in immunoglobulin genes during somatic hypermutation. The mechanism that generates mutations at A:T basepairs, however, remains unclear. It appears to require the MSH2–MSH6 mismatch repair heterodimer and DNA polymerase (pol) η, as mutations of A:T are decreased in mice and humans lacking these proteins. Here, we demonstrate that these proteins interact physically and functionally. First, we show that MSH2–MSH6 binds to a U:G mismatch but not to other DNA intermediates produced during base excision repair of dUs, including an abasic site and a deoxyribose phosphate group. Second, MSH2 binds to pol η in solution, and endogenous MSH2 associates with the pol in cell extracts. Third, MSH2–MSH6 stimulates the catalytic activity of pol η in vitro. These observations suggest that the interaction between MSH2–MSH6 and DNA pol η stimulates synthesis of mutations at bases located downstream of the initial dU lesion, including A:T pairs.
PMCID: PMC2213055  PMID: 15710654

Results 1-25 (28)