Clinical Validation of a Targeted Next-Generation Sequencing Panel for Lymphoid Malignancies.

Lymphoid malignancies are a heterogeneous group of hematological disorders characterized by a diverse range of morphologic, immunophenotypic, and clinical features. Next-generation sequencing (NGS) is increasingly being applied to delineate the complex nature of these malignancies and identify high-value biomarkers with diagnostic, prognostic, or therapeutic benefit. However, there are various challenges in using NGS routinely to characterize lymphoid malignancies, including pre-analytic issues, such as sequencing DNA from formalin-fixed, paraffin-embedded tissue, and optimizing the bioinformatic workflow for accurate variant calling and filtering. This study reports the clinical validation of a custom capture-based NGS panel to test for molecular markers in a range of lymphoproliferative diseases and histiocytic neoplasms. The fully validated clinical assay represents an accurate and sensitive tool for detection of single-nucleotide variants and small insertion/deletion events to facilitate the characterization and management of patients with hematologic cancers specifically of lymphoid origin.

Associations of sleeping patterns and isotemporal substitution of other behavior with the prevalence of CKD in Chinese adults.

Studies have found a U-shaped relationship between sleep duration and chronic kidney disease (CKD) risk, but limited research evaluated the association of reallocating excessive sleep to other behavior with CKD. We included 104 538 participants from the nationwide cohort of the Risk Evaluation of Cancers in Chinese Diabetic Individuals: A Longitudinal Study, with self-reported time of daily-life behavior. Using isotemporal substitution models, we found that substituting 1 h of sleeping with sitting, walking, or moderate-to-vigorous physical activity was associated with a lower CKD prevalence. Leisure-time physical activity displacement was associated with a greater prevalence reduction than occupational physical activity in working population. In stratified analysis, a lower CKD prevalence related to substitution toward physical activity was found in long sleepers. More pronounced correlations were observed in long sleepers with diabetes than in those with prediabetes, and they benefited from other behavior substitutions toward a more active way. The U-shaped association between sleep duration and CKD prevalence implied the potential effects of insufficient and excessive sleep on the kidneys, in which the pernicious link with oversleep could be reversed by time reallocation to physical activity. The divergence in the predicted effect on CKD following time reallocation to behavior of different domains and intensities and in subpopulations with diverse metabolic statuses underlined the importance of optimizing sleeping patterns and adjusting integral behavioral composition.

Colonization of multidrug-resistant Gram-negative bacteria increases risk of surgical site infection after hemorrhoidectomy: a cross-sectional study of two centers in southern China.

PURPOSE: The present study aims to determine the rectoanal colonization rate and risk factors for the colonization of present multidrug-resistant bacteria (MDRBs). In addition, the relationship between MDRB colonization and surgical site infection (SSI) following hemorrhoidectomy was explored. METHODS: A cross-sectional study was conducted in the Department of Colorectal Surgery of two hospitals. Patients with hemorrhoid disease, who underwent hemorrhoidectomy, were included. The pre-surgical screening of multidrug-resistant Gram-negative bacteria (MDR-GNB) colonization was performed using rectal swabs on the day of admission. Then, the MDRB colonization rate was determined through the rectal swab. Logistic regression models were established to determine the risk factors for MDRB colonization and SSI after hemorrhoidectomy. A p-value of < 0.05 was considered statistically significant. RESULTS: A total of 432 patients met the inclusion criteria, and the MDRB colonization prevalence was 21.06% (91/432). The independent risk factors for MDRB colonization were as follows: patients who received ≥ 2 categories of antibiotic treatment within 3 months (odds ratio (OR): 3.714, 95% confidence interval (CI): 1.436-9.605, p = 0.007), patients with inflammatory bowel disease (IBD; OR: 6.746, 95% CI: 2.361-19.608, p < 0.001), and patients with high serum uric acid (OR: 1.006, 95% CI: 1.001-1.010, p = 0.017). Furthermore, 41.57% (37/89) of MDRB carriers and 1.81% (6/332) of non-carriers developed SSIs, with a total incidence of 10.21% (43/421). Based on the multivariable model, the rectoanal colonization of MDRBs (OR: 32.087, 95% CI: 12.052-85.424, p < 0.001) and hemoglobin < 100 g/L (OR: 4.130, 95% CI: 1.556-10.960, p = 0.004) were independently associated with SSI after hemorrhoidectomy. CONCLUSION: The rectoanal colonization rate of MDRBs in hemorrhoid patients is high, and this was identified as an independent risk factor for SSI after hemorrhoidectomy.

Epidemiological characteristics and risk factors for cystic and alveolar echinococcosis in China: an analysis of a national population-based field survey.

BACKGROUND: Human cystic and alveolar echinococcosis are neglected tropical diseases that WHO has prioritized for control in recent years. Both diseases impose substantial burdens on public health and the socio-economy in China. In this study, which is based on the national echinococcosis survey from 2012 to 2016, we aim to describe the spatial prevalence and demographic characteristics of cystic and alveolar echinococcosis infections in humans and assess the impact of environmental, biological and social factors on both types of the disease. METHODS: We computed the sex-, age group-, occupation- and education level-specific prevalences of cystic and alveolar echinococcosis at national and sub-national levels. We mapped the geographical distribution of echinococcosis prevalence at the province, city and county levels. Finally, by analyzing the county-level echinococcosis cases combined with a range of associated environmental, biological and social factors, we identified and quantified the potential risk factors for echinococcosis using a generalized linear model. RESULTS: A total of 1,150,723 residents were selected and included in the national echinococcosis survey between 2012 and 2016, of whom 4161 and 1055 tested positive for cystic and alveolar echinococcosis, respectively. Female gender, older age, occupation at herdsman, occupation as religious worker and illiteracy were identified as risk factors for both types of echinococcosis. The prevalence of echinococcosis was found to vary geographically, with areas of high endemicity observed in the Tibetan Plateau region. Cystic echinococcosis prevalence was positively correlated with cattle density, cattle prevalence, dog density, dog prevalence, number of livestock slaughtered, elevation and grass area, and negatively associated with temperature and gross domestic product (GDP). Alveolar echinococcosis prevalence was positively correlated with precipitation, level of awareness, elevation, rodent density and rodent prevalence, and negatively correlated with forest area, temperature and GDP. Our results also implied that drinking water sources are significantly associated with both diseases. CONCLUSIONS: The results of this study provide a comprehensive understanding of geographical patterns, demographic characteristics and risk factors of cystic and alveolar echinococcosis in China. This important information will contribute towards developing targeted prevention measures and controlling diseases from the public health perspective.

MICon Contamination Detection Workflow for Next-Generation Sequencing Laboratories Using Microhaplotype Loci and Supervised Learning.

Innovation in sequencing instrumentation is increasing the per-batch data volumes and decreasing the per-base costs. Multiplexed chemistry protocols after the addition of index tags have further contributed to efficient and cost-effective sequencer utilization. With these pooled processing strategies, however, comes an increased risk of sample contamination. Sample contamination poses a risk of missing critical variants in a patient sample or wrongly reporting variants derived from the contaminant, which are particularly relevant issues in oncology specimen testing in which low variant allele frequencies have clinical relevance. Small custom-targeted next-generation sequencing (NGS) panels yield limited variants and pose challenges in delineating true somatic variants versus contamination calls. A number of popular contamination identification tools have the ability to perform well in whole-genome/exome sequencing data; however, in smaller gene panels, there are fewer variant candidates for the tools to perform accurately. To prevent clinical reporting of potentially contaminated samples in small next-generation sequencing panels, we have developed MICon (Microhaplotype Contamination detection), a novel contamination detection model that uses microhaplotype site variant allele frequencies. In a heterogeneous hold-out test cohort of 210 samples, the model displayed state-of-the-art performance with an area under the receiver-operating characteristic curve of 0.995.

Sepsis Due to Bloodstream Ralstonia mannitolilytica Infection After Surgical Treatment of Perianal Abscess: A Case Report.

BACKGROUND: Ralstonia mannitolilytica can cause opportunistic infections. Reports on this pathogen identified in the bloodstream are rare worldwide, especially in China. CASE DESCRIPTION: We describe a 48-year-old man who developed sepsis due to bloodstream Ralstonia mannitolilytica infection after surgery for a perianal abscess. His condition deteriorated into multiple organ dysfunction syndromes until susceptible antibiotics (ceftriaxone and levofloxacin) were administrated based on the drug sensitivity test results. The patient had a satisfactory recovery with no complications during a 6-month follow-up period. CONCLUSION: Ralstonia mannitolilytica blood-borne infection in patients evolves rapidly. The inconsistent sensitivity to antibiotics makes timely treatment difficult and can lead to serious complications. We report the clinical presentations and treatment outcomes for this patient here to remind clinicians about this rare opportunistic pathogen and to highlight the importance of bacterial culture, especially for immunocompromised patients.

Evaluation of the quality of COVID-19 prevention and control by a novel comprehensive evaluation model in a tertiary general hospital: a prospective observational study.

BACKGROUND: Prevention and control (P&C) of Corona Virus Disease 2019 (COVID-19) is still a critical task in most countries and regions. However, there are many single evaluation indexes to assess the quality of COVID-19 P&C. It is necessary to synthesize the single evaluation indexes reasonably to obtain the overall evaluation results. METHODS: This study was divided into three steps. Step 1: In February 2020, the improved Delphi method was used to establish the quality evaluation indexes system for COVID-19 P&C. Step 2: in March 2020, the CRITIC method was used to adjust the Order Relation Analysis (G1) method to obtain the subjective and objective (S&O) combination weights. The comprehensive evaluation value was obtained using the weighted Efficacy Coefficient (EC) method, weighted TOPSIS method, weighted rank-sum ratio (RSR) method, and weighted Grey Relationship Analysis (GRA) method. Finally, the linear normalization method was used to synthesize the evaluation values of different evaluation methods. Step 3: From April 2020 to May 2021, this evaluation method was used to monitor and assess COVID-19 P&C quality in critical departments prospectively. The results were reported to the departments monthly. RESULT: A quality evaluation indexes system for COVID-19 P&C was established. Kendall's consistency test shows that the four evaluation method had good consistency (χ2 = 43.429, P<0.001, Kendall's consistency coefficient = 0.835). The Spearman correlation test showed that the correlation between the combined evaluation results and the original method was statistically significant(P < 0.001). According to the Mann-Kendall test, from March 2020 to May 2021, the mean value of COVID-19 P&C quality in all critical departments showed an upward trend (P < 0.01). CONCLUSIONS: The combined comprehensive evaluation method based on the S&O combined weight was more scientific and comprehensive than the single weighting and evaluation methods. In addition, monitoring and feedback of COVID-19 P&C quality were helpful for the improvement of P&C quality.

Towards to understanding the preliminary loss and absorption of nitrogen and phosphorus under different treatments in cotton drip- irrigation in northwest Xinjiang.

Drip irrigation under plastic mulch is widely used in Xinjiang, Northwest China. It can not only save water, but also reduce nutrient loss and improve fertilizer utilization. However, it is not clear whether the leaching occurs or not, what is the leaching amount? What is the relationship among fertilization, irrigation regimes, loss, cotton absorption, and cotton field under different fertilization and irrigation management under drip irrigation? Studying these issues not only provides reference for the formulation of fertilization and irrigation systems, but also is of great significance for reducing non-point source pollution. A long-term positioning experiment was conducted from 2009 to 2012 in Baotou Lake farm in Korla City, Xinjiang, with drip-irrigated cotton (Gossypium hirsutum L.) under different N fertilizer and irrigation amounts. The treatments were designed comprising Control (CK,0 N, 0 P, and 0 K with an irrigation of 480 mm) and the following three other treatments: (1) Conventional fertilize and irrigation (CON, 357 kg N hm-2, 90 kg P hm-2, 0 kg K hm-2, and irrigation of 480 mm); (2) Conventional fertilization and Optimizing irrigation (OPT, 357 kg N hm-2, 90 kg P hm-2, 62 kg K hm-2, and irrigation of 420 mm); and (3) Optimizing fertilization and irrigation (OPTN, 240 kg N hm-2, 65 kg P hm-2, 62 kg K hm-2, and irrigation of 420 mm). The results found that the leaching would occur in arid area under drip irrigation. The loss of total N, NH4+, P, N and P loss coefficient was higher under conventional fertilize and irrigation treatment while the loss of NO3- was higher under conventional fertilization and optimizing irrigation treatment. The correlations among N, P absorption by cotton, loss of NH4+ and total phosphorus were quadratic function. The total nitrogen loss and cumulative nitrogen application was lineally correlated. The loss of NO3- and cumulative nitrogen application was exponential. The nitrogen and phosphorus absorption by cotton under conventional fertilization and optimizing irrigation treatment was 24.53% and 35.86% higher than that in conventional fertilize and irrigation treatment, respectively. The cotton yield under conventional fertilization and optimizing irrigation treatment obtained higher than that in other three treatments. Therefore, the conventional fertilization and optimizing irrigation treatment was the optimal management of water and fertilizer in our study. These results demonstrate that reasonable water, nitrogen and phosphorus fertilize could not only effectively promote the absorption of nitrogen and phosphorus, but also reduce nitrogen and phosphorus losses under drip fertigation and plastic mulching.

AmpR Increases the Virulence of Carbapenem-Resistant Klebsiella pneumoniae by Regulating the Initial Step of Capsule Synthesis.

BACKGROUND: Non-hypermucoviscous carbapenem-resistant Klebsiella pneumoniae with enhanced virulence lacking hvKP-specific virulence factors is uncommon, and the virulence mechanisms of this organism are not understood. METHODS: Following a retrospective study of carbapenem-resistant K. pneumoniae based on core genome multilocus sequence typing (cgMLST), isolates that caused high mortality were investigated with a genome-wide association study (GWAS), proteome analysis and an animal model. RESULTS: The subclone of sequence type 11 (ST11) K. pneumoniae, which belongs to complex type 3176 (CT3176) and K-locus 47 (KL47), was highlighted due to the high mortality of infected patients. GWAS analysis showed that transcriptional regulatory gene ampR was associated with the CT3176 isolates. In a mouse model, the mortality, bacterial load and pathological changes of mice infected with ampR-carrying isolates were distinct from those infected with ampR-null isolates. The ampR gene that enhances the virulence of the non-hypermucoviscous KL47 strain was unable to enhance the virulence of hypermucoviscous KL1 strain. Proteome analysis showed that the expression of WcaJ in the ampR + isolates was significantly higher than that in the ampR - isolates. Quantification of capsular polysaccharide confirmed that more capsule polysaccharide was produced by ampR+ and ampR-complementary strains compared to ampR- strains. It is suggested that the enhancement of the initial stage of capsule synthesis may be the cause of the enhanced virulence of these non-hypermucoviscous ST11 carbapenem-resistant K. pneumoniae isolates. CONCLUSION: Non-hypermucoviscous ST11 carbapenem-resistant K. pneumoniae with enhanced virulence warrants continued surveillance and investigation.

National Alveolar Echinococcosis Distribution - China, 2012-2016.

What is already known about this topic? Both alveolar echinococcosis (AE) and cystic echinococcosis are endemic in China, among which alveolar echinococcosis has a very high mortality rate. What is added by this report? The survey results showed the prevalence and scope of AE in China and identified high-risk groups including children, monks, herdsmen and illiterate people. At the same time, all the cases found in the survey (more than 90% of the patients did not go to the hospital for diagnosis and treatment before survey) were promptly diagnosed and treated. What are the implications for public health practice? This study provides information for the development of a plan for AE prevention and control and for the implementation of interventions targeted to high-risk populations.

Characterization of group B Streptococcus isolated from sterile and non-sterile specimens in China.

Group B streptococcus (GBS) is a leading cause of invasive neonatal infections and has increasingly been associated with invasive diseases in non-pregnant adults. We collected 113 GBS isolates recovered from sterile and non-sterile specimens from seven tertiary hospitals in China between October 2014 and September 2016. Medical records were retrospectively reviewed and the sequence types, serotypes, virulence, and antimicrobial resistance profiles of the isolates were characterized and correlated. Significantly higher C-reactive protein and procalcitonin levels and absolute neutrophil counts were observed in patients with invasive infections than in those with non-invasive infections (P < 0.05). The 113 isolates were grouped into 24 sequence types, 5 clonal complexes, and 6 serotypes. multivariate analysis revealed that clonal complex 17 isolates characterized by serotype iii, the surface protein gene rib, and the pilus island pi-2b were independently correlated with invasive infection (or: 6.79; 95% ci: 2.31-19.94, P < 0.001). These results suggest alternative molecular biomarkers for diagnosis and prognosis of GBS infections.

Development and Verification of an RNA Sequencing (RNA-Seq) Assay for the Detection of Gene Fusions in Tumors.

We assessed the performance characteristics of an RNA sequencing (RNA-Seq) assay designed to detect gene fusions in 571 genes to help manage patients with cancer. Polyadenylated RNA was converted to cDNA, which was then used to prepare next-generation sequencing libraries that were sequenced on an Illumina HiSeq 2500 instrument and analyzed with an in-house developed bioinformatic pipeline. The assay identified 38 of 41 gene fusions detected by another method, such as fluorescence in situ hybridization or RT-PCR, for a sensitivity of 93%. No false-positive gene fusions were identified in 15 normal tissue specimens and 10 tumor specimens that were negative for fusions by RNA sequencing or Mate Pair NGS (100% specificity). The assay also identified 22 fusions in 17 tumor specimens that had not been detected by other methods. Eighteen of the 22 fusions had not previously been described. Good intra-assay and interassay reproducibility was observed with complete concordance for the presence or absence of gene fusions in replicates. The analytical sensitivity of the assay was tested by diluting RNA isolated from gene fusion-positive cases with fusion-negative RNA. Gene fusions were generally detectable down to 12.5% dilutions for most fusions and as little as 3% for some fusions. This assay can help identify fusions in patients with cancer; these patients may in turn benefit from both US Food and Drug Administration-approved and investigational targeted therapies.

Impact of RNA degradation on fusion detection by RNA-seq.

BACKGROUND: RNA-seq is a well-established method for studying the transcriptome. Popular methods for library preparation in RNA-seq such as Illumina TruSeq® RNA v2 kit use a poly-A pulldown strategy. Such methods can cause loss of coverage at the 5' end of genes, impacting the ability to detect fusions when used on degraded samples. The goal of this study was to quantify the effects RNA degradation has on fusion detection when using poly-A selected mRNA and to identify the variables involved in this process. RESULTS: Using both artificially and naturally degraded samples, we found that there is a reduced ability to detect fusions as the distance of the breakpoint from the 3' end of the gene increases. The median transcript coverage decreases exponentially as a function of the distance from the 3' end and there is a linear relationship between the coverage decay rate and the RNA integrity number (RIN). Based on these findings we developed plots that show the probability of detecting a gene fusion ("sensitivity") as a function of the distance of the fusion breakpoint from the 3' end. CONCLUSIONS: This study developed a strategy to assess the impact that RNA degradation has on the ability to detect gene fusions by RNA-seq.

[Seroepidemiological Survey on Echinococcosis among Children in Ningxia Hui Autonomous Region in 2012].

According to the population structure, a stratified cluster sampling was carried out in 22 counties/ cities/disticts of Ningxia Hui Autonomous Region from June to August 2012. Serum anti-echinococcus IgG was detected by ELISA. Among 22995 sampled children from 91 primary schools, the sero-positive rate was 2.9%. The rate in males and females was 2.8%(333/11 840) and 3.0%(337/11 155), respectively (χ2 = 0.88, P > 0.05). Higher serum positive rate occurred in Yuanzhou District (10.6%, 169/1602), Yanchi County (9.1%, 74/810), and Zhongning County (7.1%, 96/1350) (χ2 = 1826.51, P < 0.05). The rate in rural schools (3.1%, 371/11 963) was higher than that of urban ones (2.7%, 368/13,834) (χ2 = 4.30, P < 0.05), and higher in Hui nationality (3.3%, 302/9,127) than that of Han nationality (2.7%, 368/13,834) (χ2 = 8.17, P < 0.05). The highest positive rate was found in the group of 7 to 9 years (3.2%, 180/5 662) and of 12 years (3.3%, 254/7,694) (χ2 = 4.11, P < 0.05).

ERß1: characterization, prognosis, and evaluation of treatment strategies in ERα-positive and -negative breast cancer.

BACKGROUND: The role and clinical value of ERß1 expression is controversial and recent data demonstrates that many ERß antibodies are insensitive and/or non-specific. Therefore, we sought to comprehensively characterize ERß1 expression across all sub-types of breast cancer using a validated antibody and determine the roles of this receptor in mediating response to multiple forms of endocrine therapy both in the presence and absence of ERα expression. METHODS: Nuclear and cytoplasmic expression patterns of ERß1 were analyzed in three patient cohorts, including a retrospective analysis of a prospective adjuvant tamoxifen study and a triple negative breast cancer cohort. To investigate the utility of therapeutically targeting ERß1, we generated multiple ERß1 expressing cell model systems and determined their proliferative responses following anti-estrogenic or ERß-specific agonist exposure. RESULTS: Nuclear ERß1 was shown to be expressed across all major sub-types of breast cancer, including 25% of triple negative breast cancers and 33% of ER-positive tumors, and was associated with significantly improved outcomes in ERα-positive tamoxifen-treated patients. In agreement with these observations, ERß1 expression sensitized ERα-positive breast cancer cells to the anti-cancer effects of selective estrogen receptor modulators (SERMs). However, in the absence of ERα expression, ERß-specific agonists potently inhibited cell proliferation rates while anti-estrogenic therapies were ineffective. CONCLUSIONS: Using a validated antibody, we have confirmed that nuclear ERß1 expression is commonly present in breast cancer and is prognostic in tamoxifen-treated patients. Using multiple breast cancer cell lines, ERß appears to be a novel therapeutic target. However, the efficacy of SERMs and ERß-specific agonists differ as a function of ERα expression.

Endoxifen's molecular mechanisms of action are concentration dependent and different than that of other anti-estrogens.

Endoxifen, a cytochrome P450 mediated tamoxifen metabolite, is being developed as a drug for the treatment of estrogen receptor (ER) positive breast cancer. Endoxifen is known to be a potent anti-estrogen and its mechanisms of action are still being elucidated. Here, we demonstrate that endoxifen-mediated recruitment of ERα to known target genes differs from that of 4-hydroxy-tamoxifen (4HT) and ICI-182,780 (ICI). Global gene expression profiling of MCF7 cells revealed substantial differences in the transcriptome following treatment with 4HT, endoxifen and ICI, both in the presence and absence of estrogen. Alterations in endoxifen concentrations also dramatically altered the gene expression profiles of MCF7 cells, even in the presence of clinically relevant concentrations of tamoxifen and its metabolites, 4HT and N-desmethyl-tamoxifen (NDT). Pathway analysis of differentially regulated genes revealed substantial differences related to endoxifen concentrations including significant induction of cell cycle arrest and markers of apoptosis following treatment with high, but not low, concentrations of endoxifen. Taken together, these data demonstrate that endoxifen's mechanism of action is different from that of 4HT and ICI and provide mechanistic insight into the potential importance of endoxifen in the suppression of breast cancer growth and progression.

Development, characterization, and applications of a novel estrogen receptor beta monoclonal antibody.

The role of estrogen receptor alpha (ERα) in breast cancer has been studied extensively, and its protein expression is prognostic and a primary determinant of endocrine sensitivity. However, much less is known about the role of ERß and its relevance remains unclear due to the publication of conflicting reports. Here, we provide evidence that much of this controversy may be explained by variability in antibody sensitivity and specificity and describe the development, characterization, and potential applications of a novel monoclonal antibody targeting full-length human ERß and its splice variant forms. Specifically, we demonstrate that a number of commercially available ERß antibodies are insensitive for ERß and exhibit significant cross-reaction with ERα. However, our newly developed MC10 ERß antibody is shown to be highly specific and sensitive for detection of full-length ERß and its variant forms. Strong and variable staining patterns for endogenous levels of ERß protein were detected in normal human tissues and breast tumors using the MC10 antibody. Importantly, ERß was shown to be expressed in a limited cohort of both ERα positive and ERα negative breast tumors. Taken together, these data demonstrate that the use of poorly validated ERß antibodies is likely to explain much of the controversy in the field with regard to the biological relevance of ERß in breast cancer. The use of the MC10 antibody, in combination with highly specific antibodies targeting only full-length ERß, is likely to provide additional discriminatory features in breast cancers that may be useful in predicting response to therapy.

Estrogen receptor-beta sensitizes breast cancer cells to the anti-estrogenic actions of endoxifen.

INTRODUCTION: We have previously demonstrated that endoxifen is the most important tamoxifen metabolite responsible for eliciting the anti-estrogenic effects of this drug in breast cancer cells expressing estrogen receptor-alpha (ERα). However, the relevance of ERß in mediating endoxifen action has yet to be explored. Here, we characterize the molecular actions of endoxifen in breast cancer cells expressing ERß and examine its effectiveness as an anti-estrogenic agent in these cell lines. METHODS: MCF7, Hs578T and U2OS cells were stably transfected with full-length ERß. ERß protein stability, dimer formation with ERα and expression of known ER target genes were characterized following endoxifen exposure. The ability of various endoxifen concentrations to block estrogen-induced proliferation of MCF7 parental and ERß-expressing cells was determined. The global gene expression profiles of these two cell lines was monitored following estrogen and endoxifen exposure and biological pathway analysis of these data sets was conducted to identify altered cellular processes. RESULTS: Our data demonstrate that endoxifen stabilizes ERß protein, unlike its targeted degradation of ERα, and induces ERα/ERß heterodimerization in a concentration dependent manner. Endoxifen is also shown to be a more potent inhibitor of estrogen target genes when ERß is expressed. Additionally, low concentrations of endoxifen observed in tamoxifen treated patients with deficient CYP2D6 activity (20 to 40 nM) markedly inhibit estrogen-induced cell proliferation rates in the presence of ERß, whereas much higher endoxifen concentrations are needed when ERß is absent. Microarray analyses reveal substantial differences in the global gene expression profiles induced by endoxifen at low concentrations (40 nM) when comparing MCF7 cells which express ERß to those that do not. These profiles implicate pathways related to cell proliferation and apoptosis in mediating endoxifen effectiveness at these lower concentrations. CONCLUSIONS: Taken together, these data demonstrate that the presence of ERß enhances the sensitivity of breast cancer cells to the anti-estrogenic effects of endoxifen likely through the molecular actions of ERα/ß heterodimers. These findings underscore the need to further elucidate the role of ERß in the biology and treatment of breast cancer and suggest that the importance of pharmacologic variation in endoxifen concentrations may differ according to ERß expression.

Microcephalin regulates BRCA2 and Rad51-associated DNA double-strand break repair.

Microcephalin (MCPH1) is a BRCA1 COOH terminal (BRCT) domain containing protein involved in the cellular response to DNA damage that has been implicated in autosomal recessive primary microcephaly. MCPH1 is recruited to sites of DNA double-strand breaks by phosphorylated histone H2AX (gammaH2AX), but the mechanism by which MCPH1 contributes to the repair process remains to be determined. Here, we show that MCPH1 binds to BRCA2 and regulates the localization of BRCA2 and Rad51 at sites of DNA damage. The interaction occurs through the NH(2) terminus of BRCA2 and the COOH terminal BRCT domains of MCPH1. Disruption of the interaction between MCPH1 and BRCA2 has no effect on the ability of BRCA2 to form a complex with Rad51 but is associated with substantially reduced levels of both BRCA2 and Rad51 at sites of DNA double-strand breaks. Uncoupling of MCPH1 from BRCA2 also interferes with Rad51-dependent and BRCA2-dependent homologous recombination repair activity. These results suggest that the role of MCPH1 in the DNA damage response is in part associated with the ability to localize BRCA2 to sites of DNA double-stand breaks.

Image segmentation based on GrabCut framework integrating multiscale nonlinear structure tensor.

In this paper, we propose an interactive color natural image segmentation method. The method integrates color feature with multiscale nonlinear structure tensor texture (MSNST) feature and then uses GrabCut method to obtain the segmentations. The MSNST feature is used to describe the texture feature of an image and integrated into GrabCut framework to overcome the problem of the scale difference of textured images. In addition, we extend the Gaussian Mixture Model (GMM) to MSNST feature and GMM based on MSNST is constructed to describe the energy function so that the texture feature can be suitably integrated into GrabCut framework and fused with the color feature to achieve the more superior image segmentation performance than the original GrabCut method. For easier implementation and more efficient computation, the symmetric KL divergence is chosen to produce the estimates of the tensor statistics instead of the Riemannian structure of the space of tensor. The Conjugate norm was employed using Locality Preserving Projections (LPP) technique as the distance measure in the color space for more discriminating power. An adaptive fusing strategy is presented to effectively adjust the mixing factor so that the color and MSNST texture features are efficiently integrated to achieve more robust segmentation performance. Last, an iteration convergence criterion is proposed to reduce the time of the iteration of GrabCut algorithm dramatically with satisfied segmentation accuracy. Experiments using synthesis texture images and real natural scene images demonstrate the superior performance of our proposed method.