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PDF file, 25K, Characteristics of 51 breast cancer patients in the third cohort used to determine the clinical correlation of Slit2 mRNA expression (Fig. 6D).

PDF file, 44K, Robo2 knockdown did not promote the soft agar colony forming ability of MDA-MB-231 cells when co-cultured with fibroblasts.

PDF file, 25K, Characteristics of 162 breast cancer patients in the second cohort used to determine the clinical correlation of Robo1 protein expression (Fig. 6C).

PDF file, 60K, High expression of Robo1 in cancer cells or Slit2 in stromal fibroblasts prevents B-catenin nuclear translocation in breast cancer specimen.

PDF file, 26K, Characteristics of 122 breast cancer patients in the first cohort used to determine the clinical correlation of Robo1 mRNA expression (Fig. 6A).

PDF file, 26K, Univariate and multivariate proportional hazards analysis of mortality of breast cancer patients of the second cohort.

PDF file, 34K, Activated Robo1 by rSlit2 interacted with p85 in BT20 cells.

PDF file, 73K, Robo1 protein expression in tumors derived from MDA-MB-231 and HCC1937 fat pad xenografts.

PDF file, 26K, Co-staining of Slit2 and (alpha)-SMA expression in fibroblasts of breast cancer specimen.

PDF file, 23K, Comparable total B-catenin protein expressions in shCtrl and shRobo1 MDA-MB-231 cells.

PDF file, 22K, List of primer sequences in the study.

Tumor microenvironment plays a critical role in regulating tumor progression by secreting factors that mediate cancer cell growth. Stromal fibroblasts can promote tumor growth through paracrine factors; however, restraint of malignant carcinoma progression by the microenvironment also has been observed. The mechanisms that underlie this paradox remain unknown. Here, we report that the tumorigenic potential of breast cancer cells is determined by an interaction between the Robo1 receptor and its ligand Slit2, which is secreted by stromal fibroblasts. The presence of an active Slit2/Robo1 signal blocks the translocation of β-catenin into nucleus, leading to downregulation of c-myc and cyclin D1 via the phosphoinositide 3-kinase (PI3K)/Akt pathway. Clinically, high Robo1 expression in the breast cancer cells correlates with increased survival in patients with breast cancer, and low Slit2 expression in the stromal fibroblasts is associated with lymph node metastasis. Together, our findings explain how a specific tumor microenvironment can restrain a given type of cancer cell from progression and show that both stromal fibroblasts and tumor cell heterogeneity affect breast cancer outcomes. Cancer Res; 72(18); 4652–61. ©2012 AACR.

PDF file, 90K.

DNA double-strand break (DSB) repair by homologous recombination (HR) is thought to be restricted to the S- and G2- phases of the cell cycle in part due to 53BP1 antagonizing DNA end resection in G1-phase and non-cycling quiescent (G0) cells. Here, we show that LIN37, a component of the DREAM transcriptional repressor, functions in a 53BP1-independent manner to prevent DNA end resection and HR in G0 cells. Loss of LIN37 leads to the expression of HR proteins, including BRCA1, BRCA2, PALB2, and RAD51, and promotes DNA end resection in G0 cells even in the presence of 53BP1. In contrast to 53BP1-deficiency, DNA end resection in LIN37-deficient G0 cells depends on BRCA1 and leads to RAD51 filament formation and HR. LIN37 is not required to protect DNA ends in cycling cells at G1-phase. Thus, LIN37 regulates a novel 53BP1-independent cell phase-specific DNA end protection pathway that functions uniquely in quiescent cells.

SUMMARY The immunoglobulin heavy chain (Igh) locus features a dynamic chromatin landscape to promote class switch recombination (CSR), yet the mechanisms that regulate this landscape remain poorly understood. CHD4, a component of the chromatin remodeling NuRD complex, directly binds H3K9me3, an epigenetic mark present at the Igh locus during CSR. We find that CHD4 is essential for early B cell development but is dispensable for the homeostatic maintenance of mature, naive B cells. However, loss of CHD4 in mature B cells impairs CSR because of suboptimal targeting of AID to the Igh locus. Additionally, we find that CHD4 represses p53 expression to promote B cell proliferation. This work reveals distinct roles for CHD4 in B cell development and CSR and links the H3K9me3 epigenetic mark with AID recruitment to the Igh locus., Graphical Abstract, In Brief Yen et al. demonstrate that CHD4, a component of the NuRD remodeling complex, is essential for early B cell development, represses p53 expression in mature B cells, and influences the recruitment of AID to DNA during class switch recombination.

We show that the Sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) family proteins SERCA2 and SERCA3 have redundant functions that are required for RAG gene expression and DNA breaks during V(D)J recombination. Loss of SERCA activity leads to B lymphopenia in mice and humans., A whole-genome CRISPR/Cas9 screen identified ATP2A2, the gene encoding the Sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) 2 protein, as being important for V(D)J recombination. SERCAs are ER transmembrane proteins that pump Ca2+ from the cytosol into the ER lumen to maintain the ER Ca2+ reservoir and regulate cytosolic Ca2+-dependent processes. In preB cells, loss of SERCA2 leads to reduced V(D)J recombination kinetics due to diminished RAG-mediated DNA cleavage. SERCA2 deficiency in B cells leads to increased expression of SERCA3, and combined loss of SERCA2 and SERCA3 results in decreased ER Ca2+ levels, increased cytosolic Ca2+ levels, reduction in RAG1 and RAG2 gene expression, and a profound block in V(D)J recombination. Mice with B cells deficient in SERCA2 and humans with Darier disease, caused by heterozygous ATP2A2 mutations, have reduced numbers of mature B cells. We conclude that SERCA proteins modulate intracellular Ca2+ levels to regulate RAG1 and RAG2 gene expression and V(D)J recombination and that defects in SERCA functions cause lymphopenia.

DNA double strand break (DSB) repair by homologous recombination (HR) is thought to be restricted to the S- and G2- phases of the cell cycle in part due to 53BP1 antagonizing DNA end resection in G1-phase and non-cycling quiescent (G0) cells. Here, we show that LIN37, a component of the DREAM transcriptional repressor, functions in a 53BP1-independent manner to prevent DNA end resection and HR in G0 cells. Loss of LIN37 leads to expression of HR proteins, including BRCA1, BRCA2, PALB2 and RAD51, and DNA end resection in G0 cells even in the presence of 53BP1. In contrast to 53BP1-deficiency, DNA end resection in LIN37-deficient G0 cells depends on BRCA1 and leads to RAD51 filament formation and HR. LIN37 is not required to protect DNA ends in cycling cells at G1-phase. Thus, LIN37 regulates a novel 53BP1-independent cell phase-specific DNA end protection pathway that functions uniquely in quiescent cells.

DNA double-strand breaks (DSBs) are known to be powerful inducers of homologous recombination (HR), but single-strand breaks (nicks) have also been shown to trigger HR. Both DSB- and nick-induced HR (nickHR) are exploited in advanced genome-engineering approaches based on the bacterial RNA-guided nuclease Cas9. However, the mechanisms of nickHR are largely unexplored. Here, we applied Cas9 nickases to study nickHR in mammalian cells. We find that nickHR is unaffected by inhibition of major damage signaling kinases and that it is not suppressed by nonhomologous end-joining (NHEJ) components, arguing that nick processing does not require a DSB intermediate to trigger HR. Relative to a single nick, nicking both strands enhances HR, consistent with a DSB intermediate, even when nicks are induced up to ∼1kb apart. Accordingly, HR and NHEJ compete for repair of these paired nicks, but, surprisingly, only when 5' overhangs or blunt ends can be generated. Our study advances the understanding of molecular mechanisms driving nick and paired-nick repair in mammalian cells and clarify phenomena associated with Cas9-mediated genome editing.

DNA double-strand breaks (DSBs) can be repaired through several mechanisms, including homologous recombination (HR). While HR between identical sequences is robust in mammalian cells, HR between diverged sequences is suppressed by DNA mismatch-repair (MMR) components such as MSH2. Exonuclease I (EXO1) interacts with the MMR machinery and has been proposed to act downstream of the mismatch recognition proteins in mismatch correction. EXO1 has also been shown to participate in extensive DSB end resection, an initial step in the HR pathway. To assess the contribution of EXO1 to HR in mammalian cells, DSB-inducible reporters were introduced into Exo1-/- mouse embryonic stem cells, including a novel GFP reporter containing several silent polymorphisms to monitor HR between diverged sequences. Compared to HR between identical sequences which was not clearly affected, HR between diverged sequences was substantially increased in Exo1-/- cells although to a lesser extent than seen in Msh2-/- cells. Thus, like canonical MMR proteins, EXO1 can restrain aberrant HR events between diverged sequence elements in the genome.

BRCA1 is essential for homology-directed repair (HDR) of DNA double-strand breaks in part through antagonism of the nonhomologous end-joining factor 53BP1. The ATM kinase is involved in various aspects of DNA damage signaling and repair, but how ATM participates in HDR and genetically interacts with BRCA1 in this process is unclear. To investigate this question, we used the Brca1S1598F mouse model carrying a mutation in the BRCA1 C-terminal domain of BRCA1. Whereas ATM loss leads to a mild HDR defect in adult somatic cells, we find that ATM inhibition leads to severely reduced HDR in Brca1S1598F cells. Consistent with a critical role for ATM in HDR in this background, loss of ATM leads to synthetic lethality of Brca1S1598F mice. Whereas both ATM and BRCA1 promote end resection, which can be regulated by 53BP1, 53bp1 deletion does not rescue the HDR defects of Atm mutant cells, in contrast to Brca1 mutant cells. These results demonstrate that ATM has a role in HDR independent of the BRCA1-53BP1 antagonism and that its HDR function can become critical in certain contexts.

This paper explores the starting point of VR (virtual reality) game experience by 5 “W” of care (What, Why, Who, Where and When), uses STEEP method to analyze the problems in different aspects of the user experience, and invites two senior people to evaluate 11 important factors. Then, the factor relationship hierarchy analysis graph is obtained with the ISM theoretical operation, and the VR game users’ problems and goals that urgently need to change are obtained. The main issue in the research is that the quality of the game story determines the experience of the users, and the side issues are the sound effect, art effect, and special effect.

By reviewing literature on short video apps and interviewing experts, this study determined three major aspects of Tik Tok user experience, emotionality, interactivity and usability, and identified nine key factors to construct the hierarchical structure of analytic hierarchy process (AHP). Based on questionnaires from experts, the Analytic Hierarchy Process (AHP) was used to determine each dimension and factor weight. Considering the defects of AHP, we introduced the fuzzy theory and fuzzy analytic hierarchy process (FAHP) to solve related problems. Finally, through the FAHP software developed by MATLAB 2015a GUI, the fuzzy weight of charm factors involved in the Tik Tok user experience is concluded as follows: emotional factors account for 48.77%, interactive factors for 31.23%, and usability factors for 20.00%. This study provides certain reference for design and update of short video apps.

C K-edge X-ray absorption near-edge structure (XANES) and valence-band photoemission spectroscopy (PES) measurements were taken for hydrogenated amorphous carbon films before and after ion beam irradiation. The C K-edge XANES and valence-band PES spectra indicate that irradiation breaks C–C bonds to form C C bonds and has a similar effect on the electronic structures of the films, irrespective of the hydrogen content. The valence-band PES measurements reveal that ion beam irradiation markedly enhances the density of carbon π states in the vicinity of the Fermi level.

Carcinoid tumors are a group of heterogeneous tumors with neuroendocrine differentiation and are mainly located in the gastrointestinal tract. A high frequency of cytoplasmic accumulation and/or nuclear translocation of beta-catenin with frequent mutations of exon 3 of beta-catenin gene in gastrointestinal carcinoid tumor has been previously described, but the role of Wnt/beta-catenin/APC pathway in the genesis of carcinoid tumor remains largely unknown.To further characterize the role of Wnt/beta-catenin/APC pathway, we investigated 91 gastrointestinal carcinoid tumors and, for comparison, 26 extragastrointestinal carcinoid tumors by immunohistochemical detection of beta-catenin protein and direct sequencing of exon 3 of the beta-catenin gene and exon 15 of the APC gene.Cytoplasmic accumulation and/or nuclear translocation of beta-catenin were found in 27 gastrointestinal carcinoid tumors (29.7%) but not in any extragastrointestinal carcinoid tumors. Interestingly, neither beta-catenin nor APC gene mutation was detected in all of the cases with nuclear expression of beta-catenin.Our results indicate that the role beta-catenin plays in the genesis of gastrointestinal and extragastrointestinal carcinoid tumors is different. Nuclear expression of beta-catenin does not occur in extragastrointestinal carcinoid tumors, and mutation of exon 3 of beta-catenin gene and exon 15 of APC gene does not contribute to the activation of Wnt/beta-catenin/APC pathway in gastrointestinal carcinoid tumors.

Diamond-like carbon (DLC) nanocomposite films, containing copper (Cu) nanocrystallites, were synthesized and studied. Cu bonds very weakly with carbon, and does not form a carbide phase. Therefore, Cu nanoparticles can be easily formed in a DLC matrix by depositing Cu and carbon together. The mechanical properties of DLC films that contain Cu nanoparticles are interesting since the film toughness may be increased by grain–matrix interface sliding. Hard, tough and stress-free DLC/Cu films were prepared by a sputtering Cu target in an argon/acetylene atmosphere while biasing the substrate with a radio frequency power supply. The residual stress of the film, calculated by Stoney's equation, was as low as 0.7 GPa. The reduced stress and the increased film toughness increased the critical load from 66 N for a conventional DLC film to 80 N for the DLC/Cu film, as measured in a scratch test. However, the DLC/Cu films were slightly less hard than the DLC films.

The corrosion protection afforded by an aluminum arc-spray coating and a post-treatment consisted of hot pressing and anodizing on an AZ31 magnesium alloy has been studied by using polarization and electrochemical impedance techniques. The corrosion current densities of the Al arc-sprayed AZ31 specimens in their spray-coated condition, and after hot pressed under 500 and 1600 MPa conditions, were found to be 2.4×10−4, 2.1×10−5 and 8.1×10−7 A cm−2, respectively. Apparently, the corrosion resistance of the Al arc-sprayed AZ31 specimens was significantly improved by the densification of the Al-sprayed layer. By adopting an anodizing treatment the corrosion current density of the AZ31 Mg alloy with dense Al coating was further reduced to 3.7×10−8 A cm−2, showing an improvement of the corrosion resistance of the Al-coated AZ31 alloy by the anodizing treatment. The overall electrochemical impedances ranging from 0.01 Hz to 10 kHz of the Al arc-sprayed+hot pressed+anodized (AHA) specimen were approximately 2 orders of magnitude higher than those of the AZ 31 Mg plate, indicating that through the Al arc-spraying and a post-hot pressing and anodizing treatment, the corrosion resistance in AZ31 Mg alloy can be significantly improved. This was achieved, according to the X-ray photoelectron spectroscopy (XPS) analysis, due to the formation of a protective Al2O3 layer on top of the Al-coated AZ31 Mg alloy.

Homology-directed repair (HDR) is a critical pathway for the repair of DNA double-strand breaks (DSBs) in mammalian cells. Efficient HDR is thought to be crucial for maintenance of genomic integrity during organismal development and tumor suppression. However, most mammalian HDR studies have focused on transformed and immortalized cell lines. We report here the generation of a Direct Repeat (DR)-GFP reporter-based mouse model to study HDR in primary cell types derived from diverse lineages. Embryonic and adult fibroblasts from these mice as well as cells derived from mammary epithelium, ovary, and neonatal brain were observed to undergo HDR at I-SceI endonuclease-induced DSBs at similar frequencies. When the DR-GFP reporter was crossed into mice carrying a hypomorphic mutation in the breast cancer susceptibility gene Brca1 , a significant reduction in HDR was detected, showing that BRCA1 is critical for HDR in somatic cell types. Consistent with an HDR defect, Brca1 mutant mice are highly sensitive to the cross-linking agent mitomycin C. By contrast, loss of the DSB signaling ataxia telangiectasia-mutated (ATM) kinase did not significantly alter HDR levels, indicating that ATM is dispensable for HDR. Notably, chemical inhibition of ATM interfered with HDR. The DR-GFP mouse provides a powerful tool for dissecting the genetic requirements of HDR in a diverse array of somatic cell types in a normal, nontransformed cellular milieu.

Tumor microenvironment plays a critical role in regulating tumor progression by secreting factors that mediate cancer cell growth. Stromal fibroblasts can promote tumor growth through paracrine factors; however, restraint of malignant carcinoma progression by the microenvironment also has been observed. The mechanisms that underlie this paradox remain unknown. Here, we report that the tumorigenic potential of breast cancer cells is determined by an interaction between the Robo1 receptor and its ligand Slit2, which is secreted by stromal fibroblasts. The presence of an active Slit2/Robo1 signal blocks the translocation of β-catenin into nucleus, leading to downregulation of c-myc and cyclin D1 via the phosphoinositide 3-kinase (PI3K)/Akt pathway. Clinically, high Robo1 expression in the breast cancer cells correlates with increased survival in patients with breast cancer, and low Slit2 expression in the stromal fibroblasts is associated with lymph node metastasis. Together, our findings explain how a specific tumor microenvironment can restrain a given type of cancer cell from progression and show that both stromal fibroblasts and tumor cell heterogeneity affect breast cancer outcomes. Cancer Res; 72(18); 4652–61. ©2012 AACR.

Microenvironment plays an important role in cancer development. We have reported that the cancer-associated stromal cells exhibit phenotypic and functional changes compared to stromal cells neighboring to normal tissues. However, the molecular mechanisms as well as the maintenance of these changes remain elusive. Here we showed that through co-culture with breast cancer cells for at least three to four passages, breast normal tissue-associated fibroblasts (NAFs) gained persistent activity for promoting cancer cell invasion, partly via up-regulating ADAM metallopeptidase with thrombospondin type 1 motif, 1 (ADAMTS1). Furthermore, we demonstrated that the DNA methylation pattern in the ADAMTS1 promoter has no alteration. Instead, the loss of EZH2 binding to the ADAMTS1 promoter and the resulting decrease of promoter-associated histone H3K27 methylation may account for the up-regulation of ADAMTS1. Importantly, the lack of EZH2 binding and the H3K27 methylation on the ADAMTS1 promoter were sustained in cancer cell-precocultured NAFs after removal of cancer cells. These results suggest that cancer cells are capable of inducing stromal fibroblasts to secrete ADAMTS1 persistently for their invasion and the effect is epigenetically inheritable.

Power consumption has been becoming one of the most important criteria for portable embedded devices. During application program development phase to estimate the power consumption is of great significance. In this paper, we propose a power evaluation methodology for PACDSP instruction set simulator. Programmers can easily observe the power consumption during the application development stage. The power modeling is compared with Synopsys PrimeTime PX, and the maximum deviation is less than 4% by executing the BDTI benchmarks. Furthermore, we also discuss the power consumption of the PAC Duo multimedia platform.

The p53 tumor suppressor exerts its function mainly as a transcriptional activator. Here we show that the Ras-related small GTPase Rad, an inhibitor of Rho kinase, is a direct transcriptional target of p53. Expression of Rad messenger RNA (mRNA) and protein was induced by DNA damage in a p53-dependent manner. The −2934/−2905-bp Rad promoter region, to which p53 bound, was required for p53-mediated Rad gene activation. Treatment by DNA damaging agents increased p53 occupancy and histone acetylation in the region of Rad promoter containing the p53-binding site. Expression of Rad diminished the inhibitory phosphorylation at Ser3 of cofilin, a regulator of actin dynamics, and suppressed migration and invasiveness of cancer cells. Knockdown of Rad promoted cell migration and alleviated the p53-mediated migration suppression. Frequent loss of Rad mRNA and protein expression was observed in non-small cell lung carcinoma tissues. Together our results reveal a mechanism that p53 may inhibit cell migration by disrupting actin dynamics via Rad activation and implicate a tumor suppressor role of Rad in lung cancer.

Large gastric bezoars are difficult to remove endoscopically. A 78-year-old man presenting with abdominal pain and loss of appetite for 4 months was admitted and evaluated. Gastroscopy disclosed two large phytobezoars within the stomach. Laparoscopic removal was undertaken. The bezoars were removed via a gastrotomy using the three-trocar technique. They were successfully retrieved from the abdominal cavity using an improvised "endobag" made from a simple surgical glove. Such an endobag presents several advantages; they are easy to make, sterile, economical, readily available, disposable, there is ample space to manipulate the specimen within, and there is minimal risk of contamination throughout the procedure. The authors recommend this approach for the treatment of patients with large gastric bezoars in whom laparotomy is indicated.

A commercially available polyimide and two forms of diamond-like carbon films being ion beam exposure were used to figure out their effects on the electrical characteristics of the liquid crystal cell.

A four-pole elliptic function microstrip filter at 2.4 GHz with fractional bandwidth of 4% is realized using compact miniaturized resonators. The configuration of this filter consists of two back-to-back arrays of resonators on two dielectric layers. The common ground plane is located between the two layers. The signal in the upper dielectric surface can be coupled to the opposite side of the surface through the aperture on the common ground plane. The measured results show a good agreement with the simulation.