Experimental and Numerical Study of Al2219 Powders Deposition on Al2219-T6 Substrate by Cold Spray: Effects of Spray Angle, Traverse Speed, and Standoff Distance.

Cold spray (CS) is an emerging technology for repairing and 3D additive manufacturing of a variety of metallic components using deformable metal powders. In CS deposition, gas type, gas pressure, gas temperature, and powder feed rate are the four key process parameters that have been intensively studied. Spray angle, spray gun traverse speed, and standoff distance (SoD) are the other three process parameters that have been less investigated but are also important, especially when depositing on uneven substrates or building up 3D freeform structures. Herein, the effects of spray angle, traverse speed, and SoD during CS deposition have been investigated holistically on a single material system (i.e., Al2219 powders on Al2219-T6 substrate). The coatings' mass gain, thickness, porosity, and residual stress have been characterized, and the results show that spray angle and traverse speed exercise much more effects than SoD in determining coatings' buildup. Finite element method (FEM) modeling and computational fluid dynamic (CFD) simulation have been carried out to understand the effects of these three parameters for implementing CS as repairing and additive manufacturing using aluminum-based alloy powders.

Triptolide interrupts rRNA synthesis and induces the RPL23­MDM2­p53 pathway to repress lung cancer cells.

Lung cancer has one of the highest mortalities of any cancer worldwide. Triptolide (TP) is a promising tumor suppressor extracted from the Chinese herb Tripterygium wilfordii. Our previous proteomics analysis revealed that TP significantly interfered with the ribosome biogenesis pathway; however, the underlying molecular mechanism remains poorly understood. The aim of the present study was to determine the molecular mechanism of TP's anticancer effect by investigating the association between ribosomal stress and p53 activation. It was found that TP induces nucleolar disintegration together with RNA polymerase I (Pol I) and upstream binding factor (UBF) translocation. TP interrupted ribosomal (r)RNA synthesis through inhibition of RNA Pol I and UBF transcriptional activation. TP treatment increased the binding of ribosomal protein L23 (RPL23) to mouse double minute 2 protein (MDM2), resulting in p53 being released from MDM2 and stabilized. Activation of p53 induced apoptosis and cell cycle arrest by enhancing the activation of p53 upregulated modulator of apoptosis, caspase 9 and caspase 3, and suppressing BCL2. In vivo experiments showed that TP significantly reduced xenograft tumor size and increased mouse body weight. Immunohistochemical assays confirmed that TP significantly increased the p53 level and induced nucleolus disintegration, during which nucleolin distribution moved from the nucleolus to the nucleoplasm, and RPL23 clustered at the edge of the cell membrane. Therefore, it was proposed that TP induces ribosomal stress, which leads to nucleolus disintegration, and inhibition of rRNA transcription and synthesis, resulting in increased binding of RPL23 with MDM2. Consequently, p53 is activated, which induces apoptosis and cell cycle arrest.

Instabilities in dielectric elastomers: buckling, wrinkling, and crumpling.

Instabilities in a thin sheet are ubiquitous and can be induced by various stimuli, such as a uniaxial force, liquid-vapor surface tension, etc. This paper investigates voltage-induced instabilities in a membrane of a dielectric elastomer. Instabilities including buckling, wrinkling, and crumpling are observed in the experiments. The prestretches of the dielectric elastomer are found to play a significant role in determining its instability mode. When the prestretch is small, intermediate, or large, the membrane may undergo buckling, wrinkling, or crumpling, respectively. Finite element analysis is conducted to study these instability modes, and the simulations are well consistent with the experimental observations. We hope that this investigation of mechanical and physical properties of dielectric elastomers can enhance their extensive and significant applications in soft devices and soft robots.

[Metabolic Characteristics of Leucocyte-deplated and Suspended RBC Produced by Using Lipid Whole Blood During Routine Storage].

OBJECTIVE: To investigate the differences of metabolic pathways of leucocyte-deplated RBCs prepared by using lipid whole blood and nomal blood during routine storage so as to provide some reference for clinical blood use. METHODS: Twenty U whole blood from 20 donors, including 10 U lipid blood and 10 U normal whole blood, were selected for preparing leukodepleted red blood cells, red blood cells were taken from storage bags on day 0, 14 and 35, respectively. Metabolites in the red blood cells were analyzed, red blood cell metabolic extracts were detected by UPLC-MS/MS. The metabolite data of RBC from 2 groups were analyzed by SIMCA-P 13.0 software using OPLS-DA and by SPSS 19.0 using Mann-Whitney U test. Difference of metabolic pathways was described according to different metabolites. RESULTS: The glucose, adenine, pyruvic acid, GSH, GSSG and niacinamide levels on day 0 in lipid RBCs were higher than those in the control group(P<0.05). The glucose, pyruvic acid and GSH levels on day 14 in lipid RBCs were lower than those in the control group (P<0.05), and the levels of adenine, GSSG and niacinamide were higher than that in the control group (P<0.05). The glucose level on day 0 was lower than that in the control group (P<0.05), and the levels of adenine and niacinamide were higher than those in the control group (P<0.05). but the pyruvic acid, GSH and GSSG levels were not significantly different between 2 groups (P>0.05). CONCLUSION: Compared with the normal red blood cells, the energy metabolism pathway decreases in lipid red blood cells within the storage period and pentose phosphate pathway increases.

Suppression of Epstein-Barr virus DNA load in latently infected nasopharyngeal carcinoma cells by CRISPR/Cas9.

Epstein-Barr virus (EBV) infects more than 90% of the world's adult population. Once established, latent infection of nasopharyngeal epithelial cells with EBV is difficult to eradicate and might lead to the development of nasopharyngeal carcinoma (NPC) in a small subset of individuals. In this study we explored the anti-EBV potential of CRISPR/Cas9 targeting of EBV genome in infected NPC cells. We designed gRNAs to target different regions of the EBV genome and transfected them into C666-1 cells. The levels of EBV DNA in transfected cells were decreased by about 50%. The suppressive effect on EBV DNA load lasted for weeks but could not be further enhanced by re-transfection of gRNA. Suppression of EBV by CRISPR/Cas9 did not affect survival of C666-1 cells but sensitized them to chemotherapeutic killing by cisplatin and 5-fluorouracil. Our work provides the proof-of-principle for suppressing EBV DNA load with CRISPR/Cas9 and a potential new strategy to sensitize EBV-infected NPC cells to chemotherapy.

Dynamic pattern of wrinkles in a dielectric elastomer.

A membrane of a dielectric elastomer may undergo electromechanical phase transition from the flat to wrinkled state, when the applied voltage reaches a critical value. The wrinkled region is observed to expand at the expense of the flat region during the phase transition. In this paper, we report on a dynamic pattern of wrinkles in a circular membrane of a dielectric elastomer. During phase transition, both the flat and wrinkled regions move interchangeably in the membrane. The radial prestretch is found to significantly affect electromechanical phase transition. For example, a membrane with a small prestretch can exhibit a dynamic pattern of wrinkles, which is essentially related to snap-through instability. However, a membrane with a large prestretch undergoes continuous phase transition, without exhibiting a dynamic pattern. An analytical model is developed to interpret these experimental phenomena. Finite element simulations are performed to predict the wrinkle morphology, especially the coexistence of flat and wrinkled regions. Both the theoretical calculations and finite element simulations are qualitatively consistent with the experiments. Additionally, we observe another type of electromechanical behavior involving a dynamic pattern of wrinkles with different wavelengths. The membrane first undergoes continuous transition from the flat to wrinkled state, followed by discontinuous transition from one wrinkled state to another. These results may inspire new applications for dielectric elastomers such as on-demand patterning of wrinkles for microfluidics and stretchable electronics.

Pharmacodynamic study on insomnia-curing effects of Shuangxia Decoction in Drosophila melanogaster.

The present study aimed to establish a pharmacodynamic method using the pySolo software to explore the influence of freeze-dried powders of Shuangxia Decoction (SXD) on the sleep of normal Drosophila melanogaster and the Drosophila melanogaster whose sleep was divested by light. The dose-effect and the time-effect relationships of SXD on sleep were examined. The effect-onset concentration of SXD was 0.25%, the plateau appeared at the concentration of 2.5% and the total sleep time showed a downtrend when the concentration was greater than 2.5%. The sleep time was the longest on the fourth day after SXD was given. The fruit fly sleep deprivation model was repeated by light stimulation at night. The middle dosage group (2.5%) had the best insomnia-curing effect. In conclusion, using the pySolo software, an approach for the pharmacodynamics study was established with Drosophila melanogaster as a model organism to determine the insomnia-curing effects of the traditional Chinese medicine (TCM). Our results demonstrated the reliability of this method. The freeze-dried powders of SXD could effectively improve the sleep quality of Drosophila melanogaster.

Ethylene/propylene copolymerization catalyzed by vanadium complexes containing N-heterocyclic carbenes.

Various vanadium complexes containing N-heterocyclic carbenes, VOCl3[1,3-R2(NCH=)2C:] (V1, R = 2,6-Me2C6H3; V2, R = 2,6-Et2C6H3; V3, R = 2,6-(i)Pr2C6H3; V4, R = 2,4,6-Me3C6H2), have been synthesized and employed as catalyst precursors for ethylene/propylene copolymerization after activation by Et3Al2Cl3. Complex V4 showed higher catalytic activity of ca. 38 kg copolymer per (mol of V) per h and an ethylene/propylene copolymer with random monomer distribution could be prepared. Complex V3 consumed more cocatalyst than its analogues to reach higher catalytic activity. The obtained copolymers exhibit relatively narrow polydispersity and contain more randomly distributed monomer units than that the copolymers prepared by using the traditional vanadium catalytic system.

A smoothed finite element method for analysis of anisotropic large deformation of passive rabbit ventricles in diastole.

The smoothed FEM (S-FEM) is firstly extended to explore the behavior of 3D anisotropic large deformation of rabbit ventricles during the passive filling process in diastole. Because of the incompressibility of myocardium, a special method called selective face-based/node-based S-FEM using four-node tetrahedral elements (FS/NS-FEM-TET4) is adopted in order to avoid volumetric locking. To validate the proposed algorithms of FS/NS-FEM-TET4, the 3D Lame problem is implemented. The performance contest results show that our FS/NS-FEM-TET4 is accurate, volumetric locking-free and insensitive to mesh distortion than standard linear FEM because of absence of isoparametric mapping. Actually, the efficiency of FS/NS-FEM-TET4 is comparable with higher-order FEM, such as 10-node tetrahedral elements. The proposed method for Holzapfel myocardium hyperelastic strain energy is also validated by simple shear tests through the comparison outcomes reported in available references. Finally, the FS/NS-FEM-TET4 is applied in the example of the passive filling of MRI-based rabbit ventricles with fiber architecture derived from rule-based algorithm to demonstrate its efficiency. Hence, we conclude that FS/NS-FEM-TET4 is a promising alternative other than FEM in passive cardiac mechanics.

PBX3 promotes migration and invasion of colorectal cancer cells via activation of MAPK/ERK signaling pathway.

AIM: To investigate the role of pre-B-cell leukemia homeobox (PBX)3 in migration and invasion of colorectal cancer (CRC) cells. METHODS: We detected PBX3 expression in five cell lines and surgical specimens from 111 patients with CRC using real-time reverse transcription-polymerase chain reaction. We forced expression of PBX3 in low metastatic HT-29 and SW480 cells and knocked down expression of PBX3 in highly metastatic LOVO and HCT-8 cells. Wound healing and Boyden chamber assays were used to detect cell migration and invasion after altered expression of PBX3. Western blot was performed to detect the change of signaling molecule ERK1/2 following PBX3 overexpression. RESULTS: High level of PBX3 expression was correlated with the invasive potential of CRC cells, and significantly associated with lymph node invasion (P = 0.02), distant metastasis (P = 0.04), advanced TNM stage (P = 0.03) and poor overall survival of patients (P < 0.05). Ectopic expression of PBX3 in low metastatic cells was shown to promote migration and invasion, while inhibited PBX3 expression in highly metastatic cells suppressed migration and invasion. Furthermore, upregulation of phosphorylated extracellular signal-regulated kinase (ERK)1/2 was found to be one of the targeted molecules responsible for PBX3-induced CRC cell migration and invasion. CONCLUSION: PBX3 induces invasion and metastasis of CRC cells partially through activation of the MAPK/ERK signaling pathway.

Let-7c functions as a metastasis suppressor by targeting MMP11 and PBX3 in colorectal cancer.

Accumulating evidence shows that microRNAs, functioning as either oncogenes or tumour suppressors by negatively regulating downstream target genes that are actively involved in tumour initiation and progression, may be promising biomarkers and therapy targets. Data mining through a microRNA chip database indicated that let-7c may be associated with tumour metastasis. Here, we confirmed that down-regulation of let-7c in primary cancer tissues was significantly associated with metastases, advanced TNM stages and poor survival of colorectal cancer patients. Moreover, ectopic expression of let-7c in a highly metastatic Lovo cell line remarkably suppressed cell migration and invasion in vitro by the down-regulation of K-RAS, MMP11 and PBX3, as well as tumour growth and metastases in vivo, whereas inhibition of let-7c in low-metastatic HT29 cells increased cell motility and invasion by the enhanced gene expression of K-RAS, MMP11 and PBX3. Interestingly, the luciferase reporters' activities with the 3'-UTRs of K-RAS, MMP11 and PBX3 were inhibited significantly by let-7c. Importantly, rescue experiments involving the over-expression of these genes without their 3'-UTRs completely reversed the effects of let-7c on tumour metastasis, both in vitro and in vivo. Finally, the levels of let-7c were inversely correlated with those of MMP11 and PBX3, but not with those of K-RAS. Taken together, these results demonstrate that let-7c, apart from its tumour growth suppression role, also functions as a tumour metastasis suppressor in colorectal cancer by directly destabilizing the mRNAs of MMP11 and PBX3 at least.

Suppression of lung cancer cell invasion and metastasis by connexin43 involves the secretion of follistatin-like 1 mediated via histone acetylation.

Although connexin has been recognized as a tumor suppressor in many types of cancer, the underlying mechanisms are poorly understood. We have previously shown that transfection of connexin43 (Cx43) cDNA retarded the growth of a highly metastatic human pulmonary giant cell carcinoma cell line, PG, both in vitro and in vivo. Here, we further demonstrate that the metastasis and invasion, but not the migration, of PG cells are also inhibited following Cx43 transfection. The diminishment of metastasis and invasion is associated with down-regulation of genes including MMP-2, S100A, LAMA4, and HDAC10, as well as up-regulation of genes such as MTSS1 and FSTL1 as revealed by gene chip analysis. Interestingly, the suppression effects of Cx43 are related to secreted factor(s), which are blocked by FSTL1 antibody treatment in a dose-dependent manner. Furthermore, the FSTL1 promoter was shown to be associated with acetylated histones H3 and H4 upon Cx43 transfection. These data suggest that Cx43 inhibits the invasion and metastasis of PG cells by modulating the secretion of FSTL1, which is regulated by histone acetylation. Cx43 may act as a "histone deacetylase inhibitor" to modulate gene expression and subsequent cellular functions in PG cells.

The properties of tumor-initiating cells from a hepatocellular carcinoma patient's primary and recurrent tumor.

Hepatocellular carcinoma (HCC) is associated with a high morbidity and mortality due to its high rate of recurrence. However, little is known about the biological characteristics of recurrent HCC cells. A single patient's primary and recurrent HCC-derived cell lines, Hep-11 and Hep-12, respectively, were established by primary culture. These two cell lines have the same hepatitis B virus integration site and share many common amplifications and deletions, which suggest that they have the same clonal origin. While Hep-11 cells were non-tumorigenic at 16 weeks following injection of up to 10 000 cells, injection of only 100 Hep-12 cells was sufficient to initiate tumor growth, and all single Hep-12 clones were tumorigenic in immunodeficient mice. Compared with Hep-11, Hep-12 cells expressed the oval cell markers AFP, NCAM/CD56, c-kit/CD117, as well as multiple stem cell markers such as Nanog, OCT4 and SOX2. In addition, >90% of Hep-12 cells were aldehyde dehydrogenase positive. They were also less resistant to paclitaxel, but more resistant to doxorubicin, cisplatin and hydroxycamptothecin (HCPT), which had been administrated to the patient. Furthermore, Hep-12 cells expressed higher levels of poly (adenosine diphosphate-ribose) polymerase-1 (PARP-1) than Hep-11, and PARP-1 inhibition potentiated the sensitivity to HCPT in Hep-12 cells but not in Hep-11 cells. These results indicate that a large population of the recurrent HCC-derived Hep-12 cells were tumor-initiating cells and that elevated expression of PARP-1 was related to their resistance to HCPT.

Sarcomeric-alpha-actinin defective in vinculin-binding causes Z-line expansion and nemaline-like body formation in cultured chick myotubes.

The Z-line in each striated muscle has a precisely defined width that corresponds to muscle fiber type, and it can enlarge several fold in nemaline myopathy. To explore the mechanism(s) underlying Z-line width and structure maintenance, a series of sarcomeric-alpha-actinin mutants tagged with myc-epitope was transfected into cultured chick myotubes. By double-staining transfected myotubes with myc and myofibrillar protein antibodies, we found that alpha-actinin mutants with deletion of the region from the beginning of the fourth spectrin repeat to the start of the EF-hands resulted in expansion of Z-line width, often displayed a doublet staining pattern, and resulted in formation of nemaline-like bodies in older myotubes under fluorescence microscope. Yeast-two hybridization analysis demonstrated that this region was involved in vinculin binding, and for vinculin to bind alpha-actinin, residues 1-116 and 258-323 were required. Hence, we have defined a critical region of s-alpha-actinin that affects the width and integrity of the Z-line. This region is at least involved in the interaction with vinculin.

High level expression and purification of recombinant PEX protein in cultured skeletal muscle cell expression system.

Large quantities of recombinant proteins are needed for specific therapeutic and diagnostic applications. To achieve high-level expression in eukaryotic cells, the choice of cell line as well as the expression vector is critical. In this report, we demonstrate that a combination of the skeletal muscle cell line, QM7 and a cytomegalovirus promoter-based expression vector can achieve high-level expression of secretory recombinant proteins in eukaryotic cells. We also screened a serum-free medium containing 3 microg/ml insulin suitable for QM7 differentiation and identified a very potent signal peptide from MMP9, which effectively directs secretion of heterologous proteins. The C-terminal hemopexin-like domain of MMP-2, PEX, a powerful candidate for the treatment of diseases associated with neovascularization was expressed in QM7 cells with bioactivity. This skeletal muscle cell-based system may be employed for the production of human proteins of special interests, such as those for structural determination or therapeutical development.

Conversion of cadherin isoforms in cultured human gastric carcinoma cells.

AIM: To explore the expression of cadherin isoforms in cultured human gastric carcinoma cells and its regulation. METHODS: The expressions of cell adhesion molecules (including E-cadherin, N-cadherin, alpha-catenin, beta-catenin) and cadherin transcription factors including snail, slug and twist were determined by reverse transcriptase-polymerase chain reaction(RT-PCR), immunoblotting and immunofluorescence in SV40-immortalized human gastric cell line Ges-1 and human gastric cancer cell lines MGC-803, BGC-823 and SGC-7901. RESULTS: All cell lines expressed N-cadherin, but not E-cadherin. N-cadherin immunofluorescence was detected at cell membranous adherents junctions where co-localization with immunofluorescent staining of inner surface adhesion proteins alpha- and beta-catenins was observed. The transformed Ges-1 and gastric cancer cell lines all expressed transcription factors (snail, slug and twist) which inhibited the expression of E-cadherin and triggered epithelial-mesenchymal transformation. CONCLUSION: Cadherin isoforms can change from E-cadherin to N-cadherin in transformed human gastric cancer cells, which is associated with intracellular events of stomach carcinogenesis and high expression of corresponding transcription factors.

[Selective heterologous communication among human lung epithelial cells, fibroblasts, and lung cancer PG cells].

OBJECTIVE: To further understand the roles of gap junctional intercellular communication in carcinogenesis and progression of human lung cancer. METHODS: The heterologous communication was characterized among human lung epithelial cells HLEC, human lung fibroblasts HLF, human lung giant carcinoma PG cells and its Cx43 transfectants PG/C4 by using a preloading assay. Cx43 immunofluorescent staining and Northern blot were performed to examine Cx43 gene expression. RESULTS: Although both human lung fibroblasts HLF and epithelial cells HLEC expressed Cx43 and had very strong homologous communication, HLEC cells were unable to communicate with HLF cells. The human lung carcinoma cell line PG was defective of both homologous communication, and heterologous communication with its epithelial origin HLEC. Transfection of Cx43 into PG cells, which rescued PG cells' homologous communication and enhanced heterologous communication with HLF cells, could restore heterologous coupling with HLEC cells. CONCLUSION: Selective heterologous communication exists among different kinds of human lung cells, when human lung carcinoma cells lost coupling with its epithelial origin, Cx43 gene expression is not enough to establish heterologous communication between them.

Cisplatin-induced premature senescence with concomitant reduction of gap junctions in human fibroblasts.

To examine the role of gap junctions in cell senescence, the changes of gap junctions in cisplatin-induced premature senescence of primary cultured fibroblasts were studied and compared with the replicative senescent human fibroblasts. Dye transfer assay for gap junction function and immunofluorescent staining for connexin 43 protein distribution were done respectively. Furthermore, cytofluorimetry and DAPI fluorescence staining were performed for cell cycle and apoptosis analysis. p53 gene expression level was detected with indirect immunofluorescence. We found that cisplatin (10 mM) treatment could block cell growth cycle at G1 and induced premature senescence. The premature senescence changes included high frequency of apoptosis, elevation of p53 expression, loss of membranous gap junctions and reduction of dye-transfer capacity. These changes were comparable to the changes of replicative senescence of human fibroblasts. It was also concluded that cisplatin could induce premature senescence concomitant with inhibition of gap junctions in the fibroblasts. Loss of functional gap junctions from the cell membrane may account for the reduced intercellular communication in the premature senescent fibroblasts. The cell system we used may provide a model useful for the study of the gap junction thus promoting agents against premature senescence.

Effective asymmetry in gap junctional intercellular communication between populations of human normal lung fibroblasts and lung carcinoma cells.

The dysfunction of homologous and/or heterologous gap junctional intercellular communication (GJIC) has been implicated in tumorigenesis of many kinds of cells. Here we have characterized GJIC and the expression of connexins in six human lung carcinoma cell lines and normal lung fibroblasts (HLF). Compared with HLF, all the carcinoma cells showed reduced or little homologous GJIC. They expressed remarkably reduced connexin(Cx)43 mRNA and variable levels of Cx45 mRNA, but neither Cx43 nor Cx45 protein could be detected. However, using a preloading assay, transfer of calcein was observed between donor HLF cells and first order neighboring recipient tumor cells (recipient cells in 1000-fold excess). Transfer from tumor to HLF cells under the same conditions was not seen, although increasing the ratio of donor tumor cells to recipient HLF cells and plating the cells at low density did reveal weak transfer from tumor cells to HLF. Transfection of Cx43 into giant cell carcinoma PG cells increased homologous communication and eliminated the rectifying behavior of heterologous communication. This indicates that the apparent rectification of dye transfer between normal and tumor cells was a product of low rates of heterologous transfer linked to (i) rapid dilution of the dye to below detectable limits through a very well coupled cell population (tumor to HLF) and (ii) concentration of dye in immediate neighbors in a poorly coupled cell population (HLF to tumor cells). These results suggest that the coupling levels may need to exceed a certain threshold to allow propagation of signals over a sufficient distance to affect behavior of a cell population. We propose that the relative rates of heterologous and homologous coupling of cell populations and the 'pool size' of shared metabolites in tumor cells and the surrounding normal tissue are likely to be very important in the regulation of their growth.

[Expression of matrix metalloproteinase-9 and its complex in the urine of breast cancer patients].

OBJECTIVE: To investigate the expression and clinical significance of matrix metalloproteinase-9 and its complex in the urine of the patient with breast cancer. METHODS: Using substract gel electrophoresis and western-blot analysis, expressions of MMP-9 and MMP-9/NGAL complex in breast cancer (n = 97), breast benign (n = 41) and normal (n = 60) were observed. RESULTS: There MMP-9 and MMP-9/NGAL complex expressions were 76.29% and 64.95% in breast cancer, 46.34% and 43.90% in breast benign, and 23.33% in normal respectively. The MMP-9 and MMP-9/NGAL complex expressions were higher in breast cancer than those in breast benign and in normal (chi(2) = 7.456, P < 0.01). MMP-9 and MMP-9/NGAL complex expressions in urine of breast cancer had not any relationship with tumor size, TNM stage, patient age, menopause status as well as ER status, but was correlated to lymphatic node status (chi(2) = 5.206, P < 0.05). CONCLUSIONS: MMP-9 and MMP-9/NGAL complex expressions in urine are significant in estimating lymphatic node metastasis in breast cancer and a valuable early prognostic factors and screening in breast cancer.