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EGFR and cMET cross-talk is involved in breast cancer (BC) progression and resistance to different targeted therapies, however little is known about the co-expression patterns of EGFR and cMET or its prognostic significance in BC. Protein levels of EGFR, cMET and their phosphorylated proteins were measured in 825 BC samples using reverse phase protein array (RPPA). Given unimodal distribution of proteins, the median was selected as a cut-off after sensitivity analyses. Kaplan-Meier survival curves were used to estimate relapse-free (RFS) and overall survival (OS). Cox-proportional hazards models were utilized to determine associations between EGFR and cMET with outcomes. Mean age was 58 years with 457 (55%) hormone receptor (HR) positive, 211 (26%) triple-negative (TN) and 148 (18%) HER2 positive tumors (HER2+). HER2+ was associated with higher EGFR expression and phosphorylation, compared to HR and TN (p

The new natural product 4]-hydroxy-deacetyl-18-deoxycytochalasin H (1), together with the known deacetyl-18-deoxycytochalasin H (2) and 18-deoxycytochalasin H (3) were obtained from the endophytic fungus Trichoderma harzianum isolated from leaves of Cola nitida. The structure of the new compound was unambiguously determined by 1D and 2D NMR spectroscopy, and by HRESIMS measurements, as well as by comparison with the literature. Compounds 1-3 showed potent cytotoxic activity against the murine lymphoma (L5178Y) cell line and against human ovarian cancer (A2780 sens and A2780 CisR) cell lines (IC50 0.19-6.97 µM). The A2780 cell lines included cisplatin-sensitive (sens) and -resistant (R) cells.

We consider the existence of at least two positive solutions for a system of Caputo fractional difference equations $\Delta _{{\mathrm{C}}}^{\nu _{j}}y_{j}(t)=-\lambda_{j}f_{j}(y_{1}(t+\nu_{1}-1), \ldots,y_{n}(t+\nu_{n}-1))$ , subject to boundary conditions $y_{j}(\nu_{j}-3)=\Delta y_{j}(\nu_{j}+b)=\Delta ^{2} y_{j}(\nu_{j}-3)=0$ , where $2

We consider the problem of synthesizing multiple valued logic (MVL) functions by neural networks. A differential evolution algorithm is proposed to train the learnable multiple valued logic network. The optimum window and biasing parameters to be chosen for convergence are derived. Experiments performed on benchmark problems demonstrate the convergence and robustness of the network. Preliminary results indicate that differential evolution is suitable to train MVL networks for synthesizing MVL functions.

BACKGROUND:There is preclinical synergism between taxanes and MK-2206. We aim to determine the maximum tolerated dose, safety, and activity of combining MK-2206 and paclitaxel in metastatic cancer. METHODS:Patients received weekly doses of paclitaxel at 80mg/m2 on day 1, followed by MK-2206 orally on day 2 escalated at 90mg, 135mg, and 200mg. Treatment continued until progression, excessive toxicity, or patient request. Blood and tissue were collected for pharmacokinetic and pharmacodynamics markers. A cycle consisted of three weeks of therapy. Dose-limiting toxicity (DLT) was defined as unacceptable toxicity during the first cycle. All statistical tests were two-sided. RESULTS:Twenty-two patients were treated, nine in dose escalation and 13 in dose expansion. Median age was 55 years. Median number of cycles was four. Dose escalation was completed with no DLT. CTCAE Grade 3 or higher adverse events were fatigue (n = 2), rash (n = 2), hyperglycemia (n = 1), and neutropenia (n = 7). Four patients in the expansion phase required MK-2206 dose reduction. Phase II recommended dose was established as paclitaxel 80mg/m2 weekly on day 1, and MK-2206 135mg weekly on day 2. Paclitaxel systemic exposure was similar in the presence or absence of MK-2206. Plasma MK-2206 concentrations were similar to data from previous phase I monotherapy. There was a statistically significant decrease in expression of pAKT S473 (P = .01) and pAKT T308 (P = .002) after therapy. PI3K/AKT/mTOR downregulation in tumor tissues and circulating markers did not correlate with tumor response or clinical benefit. There were five objective responses, and nine patients had stable disease. CONCLUSION:MK-2206 was well tolerated with paclitaxel. Preliminary antitumor activity was documented.

Background Breast cancer patients who are resistant to neoadjuvant chemotherapy (NeoCT) have a poor prognosis. There is a pressing need to develop in vivo models of chemo resistant tumors to test novel therapeutics. We hypothesized that patient-derived breast cancer xenografts (BCXs) from chemo- naive and chemotherapy-exposed tumors can provide high fidelity in vivo models for chemoresistant breast cancers. Methods Patient tumors and BCXs were characterized with short tandem repeat DNA fingerprinting, reverse phase protein arrays, molecular inversion probe arrays, and next generation sequencing. Results Forty-eight breast cancers (24 post-chemotherapy, 24 chemo-naive) were implanted and 13 BCXs were established (27%). BCX engraftment was higher in TNBC compared to hormone-receptor positive cancer (53.8% vs. 15.6%, p = 0.02), in tumors from patients who received NeoCT (41.7% vs. 8.3%, p = 0.02), and in patients who had progressive disease on NeoCT (85.7% vs. 29.4%, p = 0.02). Twelve patients developed metastases after surgery; in five, BCXs developed before distant relapse. Patients whose tumors developed BCXs had a lower recurrence-free survival (p = 0.015) and overall survival (p

We investigate the existence of solutions for a Caputo fractional difference equation boundary value problem. We use Schauder fixed point theorem to deduce the existence of solutions. The proofs are based upon the theory of discrete fractional calculus. We also provide some examples to illustrate our main results.

Deregulation and activation of the phosphoinositide 3-kinase (PI3K)/Akt/mammalian (or mechanistic) target of rapamycin (mTOR) pathway have a major role in proliferation and cell survival in breast cancer. However, as single agents, mTOR inhibitors have had modest antitumor efficacy. In this study, we evaluated the effects of vertical inhibition of mTOR and Akt in breast cancer cell lines and xenografts. We assessed the effects of mTOR inhibitor rapamycin and Akt inhibitor MK-2206, given as single drugs or in combination, on cell signaling, cell proliferation and apoptosis in a panel of cancer cell lines in vitro. The antitumor efficacy was tested in vivo. We demonstrated that MK-2206 inhibited Akt phosphorylation, cell proliferation and apoptosis in a dose-dependent manner in breast cancer cell lines. Rapamycin inhibited S6 phosphorylation and cell proliferation, and resulted in lower levels of apoptosis induction. Furthermore, the combination treatment inhibited phosphorylation of Akt and S6, synergistically inhibited proliferation and induced apoptosis with a higher efficacy. In vivo combination inhibited tumor growth more than either agent alone. Our data suggest that a combination of Akt and mTOR inhibitors have greater antitumor activity in breast cancer cells, which may be a viable approach to treat patients.

Introduction: Molecular alterations in the PI3K/Akt/mTOR pathway is implicated in the pathogenesis of ER positive breast cancer with high frequency and therefore is the major focus of drug development. Activating mutations of this signaling pathway occur in more than 70% of breast tumors. The genetic alterations affecting major components of PI3K/Akt/mTOR include genes encoding the PI3K catalytic subunits p110α (PIK3CA) and the PI3K effector AKT1. Despite the efficacy of the available small molecule inhibitors for targeting different components of PI3K pathway, the contribution of PIK3CA and AKT1 genetic alterations to targeted therapy is not well understood. We speculated that ER positive breast tumors with PIK3CA and AKT1 alterations are more sensitive to PI3K and Akt inhibitors due to differential downstream pathway effectors. Methods: We used a panel of isogenic MCF7 cell lines with oncogenic mutations of PIK3CA and AKT1 established through somatic cell gene targeting. A series of pharmacological compounds currently approved for breast cancer therapy or in clinical trials in targeting PI3K/Akt/mTOR pathway were evaluated using viability, clonogenic and cell cycle analysis assays. We performed functional proteomic profiling using reverse phase proteomic analysis (RPPA) to investigate differential protein expression in response to PI3K pathway inhibition in PIK3CA and AKT1 mutants MCF7 cell lines. In vivo experiment was done to evaluate the efficacy of the inhibitors in suppressing tumor growth. Results: We demonstrated that PIK3CA (E545K) and AKT1 (E17K) sensitized cells to the inhibitory effects of BYL719 (p110α catalytic subunit inhibitor) and AZD5363 (pan-AKT kinase inhibitor) on survival, cell cycle progression and colony formation ability. The presence of PIK3CA and AKT1 mutations conferred growth advantage and exhibited increased proliferation in vitro and in vivo. These alterations dramatically increased Akt phosphorylation and induced activation of PI3K downstream effectors. RPPA analysis revealed several proteins differentially expressed in PIK3CA and AKT1 mutant cells compared with wild type (p-value < 0.05). Conclusion: PIK3CA and AKT1 mutations showed distinct effects on sensitivity to targeted pathway inhibitors in an isogenic luminal breast cancer model. The presence of these genetic alterations in estrogen receptor (ER) positive breast cancer provided proliferative advantage and enhanced their sensitivity to targeted PI3K inhibition. These results will contribute to identify single agent and combination therapies targeting PI3K pathway with maximal activity at tolerated dose and selection for individuals most likely to be responsive based on their genomic alterations. Citation Format: Maryam Shariati, Kurt W. Evans, Stephen M. Scott, Huiqin Chen, Funda Meric-Bernstam. Targeted therapy of hormone receptor-positive breast cancer harboring PIK3CA and AKT1 genetic aberrations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 135. doi:10.1158/1538-7445.AM2017-135

Background: There is growing evidence that ADAM (a disintegrin and metalloproteinase) family proteins are involved in multiple types of cancer, including breast cancer. More than 50 members of the ADAM family have been identified thus far. Previous studies have focused on one or two ADAMs but comparative research among the members of the family is lacking. This study was undertaken to identify the most important ADAMs associated with breast cancer. Methods: We analyzed mRNA expression of ADAMs in tumor bed stroma and paired normal adjacent stroma from 31 breast cancer patients. The original raw data was adapted from a breast dataset (Finak et al, 2008). We next analyzed RNA expression of 35 ADAMs in different subtypes of breast cancers [dataset from Affymetrix U133A arrays using methods available in the R statistical software package (http://cran.r-project.org)]. We further evaluated protein expression of ADAMs and related signaling molecules in 187 breast cancers, including 110 untreated primary tumors and 77 residual tumors after neoadjuvant chemotherapy, and 167 matched ‘normal’ mammary tissues using reverse phase protein arrays (RPPA). The association between ADAMs expression and survival was analyzed using Kaplan–Meier analysis. Results: The microarray analysis of tumor bed stroma revealed significantly elevated mRNA levels of 12 ADAMs, including ADAM8, ADAM 12, ADAM 19, ADAM 21, ADAM 17, ADAM 10, ADAM 28, ADAM 7, ADAM 33, ADAM 22, ADAM 6 and ADAMTS20, compared to their normal adjacent stroma. We evaluated the expression of 35 ADAMs in total 369 breast cancers, including 201 hormone receptor positive (HR+), 60 HER2 amplified (HER2+) and 108 triple negative (TN) patients using RNA array and demonstrated that 24 of 35 ADAMs were significantly upregulated in TNBC compared to that in HR+ and HER2+ subtypes. We further analyzed protein expression of ADAMs in 189 breast cancers and 172 matched “normal” breast tissues with RPPA. Compared to “normal” breast tissues, ADAM17, ADAM10, ADAM15, ADAM8 and ADAM9 were significantly increased in tumors (P < 0.0001, respectively), while ADAM20, ADAM23, ADAM29, and ADAM30 exhibited lower level. Kaplan–Meier analysis showed that 13-year overall survival rate in high expression of ADAM 17, ADAM 15 or ADAM10 group was significantly decreased compared to that in the low expression group (40% vs. 12%, P = 0.0026; 50% vs. 11%, P = 0.0104; 53% vs. 9%, P < 0.0001, respectively), suggesting that upregulation of ADAM17, ADAM15, or ADAM10 was significantly associated with poor outcome. ADAM17, ADAM9 or ADAM10 expression was tightly associated with elevation of anti-apoptotic molecule Bcl-X, cyclins (E1, E2, B1), PTPN12, and phosphorylation of 4-EBP1. Conclusion: Several ADAM proteins have elevated expression in breast cancer and are associated with decreased patient survival. ADAMs may, therefore, serve as potential biomarkers for predicting outcomes and oncotargets in breast cancer. Citation Format: Shuying Liu, Michael Z. Gilcrease, Huiqin Chen, Wenbin Liu, Fan Zhang, Kim-Anh Do, Gordon B. Mills, William F. Symmans, Naoto T. Ueno, Ana M. Gonzales-Angulo, Gabriel N. Hortobagyi, Debasish Tripathy. ADAMs: potential biomarkers and oncotargets in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4736. doi:10.1158/1538-7445.AM2017-4736

Wheels are important safety components in the vehicle driving system. Automobile lightweight is the direction of the modern automobile development. In this article, steel wheel lightweight was studied. The equal strength design of rim was used to reduce the weight of the wheel. Stress analysis of the wheel was studied using UG/Nastran. A professional software WheelStrength was used to predict the radial and cornering fatigue lives of the wheel. Sheet stamping process was set up to analyze the interference fit between the disk and the rim. The assembly was simulated by axisymmetric finite element method. After calculation and analysis, the stress distributions and fatigue lives for rim under different load cases have been found. The thicknesses of wheel rim bead and the interface between rim and disk cannot be reduced. Stress and fatigue simulation results were compared using different thicknesses of the optimized region. It was found that the best thickness of the optimized region was 1.5 mm. Spinning was used to form the flared preform. The thickness of the rim after spinning and rolling forming agreed well with the simulation. The results of fatigue tests indicated that the lightweight wheel met the design requirement. The weight of the rim was reduced by about 14%.

DNA polymerase d is not only the major replicative enzyme in eukaryotic chromosomal DNA synthesis but is also the primary polymerase for most DNA repair pathways. However, the subunit composition of polymerase d varies in different organisms. While polymerase d in many eukaryotic species has all 4 subunits (POLD1, 2, 3, and 4), many other organisms do not possess POLD4. Whether POLD4 is indispensable and why these differences exist are unknown. In the present study, we identified the POLD4 protein sequences of 218 eukaryotic species and determined the POLD1, 2, and 3 protein sequences of 55 species representing various taxonomic groups. No insect and nematode species examined possessed POLD4. Approximately 80% of protozoan species did not contain POLD4. Nearly 50% of fungal species did not contain POLD4. Other animal and plant species are expected to contain POLD4. Phylogenetic analyses of POLD1, 2, 3, and 4 sequences revealed that most animal and plant species inherited DNA polymerase d from protozoa, whereas some other animal and plant species may have inherited polymerase d directly from fungi. Because a large number of protozoan and fungal species do not possess POLD4, current insect and nematode species lacking POLD4 may have evolved from ancestor protozoan species lacking POLD4; thus, other protozoan and animal species lacking POLD4 may share a similar evolutionary history. Future studies should examine the origin and indispensability of POLD4 in various organisms.

// Ashley M. Holder 1, 2 , Argun Akcakanat 3 , Farrell Adkins 4 , Kurt Evans 3 , Huiqin Chen 5 , Caimiao Wei 5 , Denai R. Milton 5 , Yisheng Li 5 , Kim-Anh Do 5 , Filip Janku 3 , Funda Meric-Bernstam 1, 3 1 Department of Surgical Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA 2 Department of Surgery, Washington University in St. Louis, St. Louis, MO, USA 3 Department of Investigational Cancer Therapeutics, The University of Texas MD Anderson Cancer Center, Houston, TX, USA 4 Department of Colorectal Surgery, Cleveland Clinic Florida, Weston, FL, USA 5 Department of Biostatistics, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Correspondence to: Funda Meric-Bernstam, e-mail: fmeric@mdanderson.org Keywords: epithelial-to-mesenchymal transition (EMT), rapamycin, trametinib, biomarker, E-cadherin Received: September 02, 2014 Accepted: March 25, 2015 Published: April 13, 2015 ABSTRACT Rapamycin analogues have antitumor efficacy in several tumor types, however few patients demonstrate tumor regression. Thus, there is a pressing need for markers of intrinsic response/resistance and rational combination therapies. We hypothesized that epithelial-to-mesenchymal transition (EMT) confers rapamycin resistance. We found that the epithelial marker E-cadherin protein is higher in rapamycin sensitive (RS) cells and mesenchymal breast cancer cell lines selected by transcriptional EMT signatures are less sensitive to rapamycin. MCF7 cells, transfected with constitutively active mutant Snail, had increased rapamycin resistance (RR) compared to cells transfected with wild-type Snail. Conversely, we transfected two RR mesenchymal cell lines—ACHN and MDA-MB-231—with miR-200b/c or ZEB1 siRNA to promote mesenchymal-to-epithelial transition. This induced E-cadherin expression in both cell lines, and ACHN demonstrated a significant increase in RS. Treatment of ACHN and MDA-MB-231 with trametinib modulated EMT in ACHN cells in vitro . Treatment of MDA-MB-231 and ACHN xenografts with trametinib in combination with rapamycin resulted in significant growth inhibition in both but without an apparent effect on EMT. Future studies are needed to determine whether EMT status is predictive of sensitivity to rapalogs and to determine whether combination therapy with EMT modulating agents can enhance antitumor effects of PI3K/mTOR inhibitors.

In this paper, we will use the Krasnosel’skii fixed point theorem to investigate a discrete fractional boundary value problem of the form , , , where , , is a continuous function, , are given functionals, where Ψ, Φ are linear functionals, and λ is a positive parameter. MSC:26A33, 39A05, 39A12.

The following fractional difference boundary value problems▵νyt=-ft+ν-1,yt+ν-1,y(ν-2)=y(ν+b+1)=0are considered, where1<ν≤2is a real number and▵νy(t)is the standard Riemann-Liouville fractional difference. Based on the Krasnosel’skiǐ theorem and the Schauder fixed point theorem, we establish some conditions onfwhich are able to guarantee that this FBVP has at least two positive solutions and one solution, respectively. Our results significantly improve and generalize those in the literature. A number of examples are given to illustrate our main results.

A white-emitting phosphor Sr2SiO4: Pr3+ was synthesized through a solid-state reaction, and characterized by XRD, scanning electron microscopy (SEM), fluorescence spectrophotometer and thermo luminescence (TL) meter. Its emission spectra is composed of bluish purple (peaking at 390 nm), green (peaking at 535 nm) and red (peaking at 604 nm) light emission. They originate from the transitions of 4f → 5d, 3P0 → 3H5 and 1D2 → 3H4 of Pr3+. The afterglow emission spectrum is similar to the emission spectra. And the afterglow can last over 40 min in darkness. The TL curve shows that there is only one thermo luminescence band peak at about 376.480 K, which is responsible for the long-lasting emission.

We use the Krasnosel’skii fixed point theorem to obtain the sufficient conditions of the existence of two positive solutions for the boundary value problem of fractional difference equations depending on parameters.

It is the basis for the new casting-rolling compound forming technology to investigate the rules of rolling deformation and microstructure evolution of casting ring blank. The initial rolling temperature of casting ring blank is one of the key factors influencing deformation and dynamic recrystallization behavior. In this paper, dynamic recrystallization models of as-cast 42CrMo steel were derived from thermo-simulation experimental results. Then, under DEFORM-3D software environment, deformation and dynamic recrystallization of 42CrMo casting ring blank were simulated at different initial rolling temperature by coupled thermo-mechanical finite element method. According to the simulation results, the effects of initial rolling temperature on strain fields distribution and dynamic recrystallization of 42CrMo casting ring blank were discussed. The results show that: (1) increased initial rolling temperature makes plastic deformation expand from ring’s outer and inner-layer to middle and distribute ever more evenly; (2) increased initial rolling temperature can lead to increased dynamic recrystallization fraction, but grain size of ring’s inner and outer-layer becomes smaller and smaller, while that of mid-layer coarser and coarser.Copyright © 2013 by ASME

Senior-Løken syndrome is a rare syndromic form of nephronophthisis that is associated with retinal dystrophy. Presently, seven genes (NPHP1-6 and NPHP10) have been associated with Senior-Løken syndrome. NPHP5 mutations are known to cause classical Senior-Løken syndrome. Here, we report two sisters (II-4, II-5) from a Chinese Han ethnic family who presented with classical Senior-Løken syndrome. Both affected sisters exhibited Leber's congenital amaurosis and juvenile nephronophthisis that progressed to end-stage renal disease by the age of 16 years and 9 months in patient II-4 and 12 years and 9 months in patient II-5. Sequence analysis showed a homozygous truncated mutation in NPHP5, c.1090CT (p.R364X), in the patient II-4. This mutation is predicted to introduce a new open reading frame that results in the truncation of the C-terminal 235 amino acids of nephrocystin-5 and its consequent loss of function. Both parents carried a single heterozygous mutation in the same position, and no homozygous deletion of NPHP1 was found in this pedigree.

Background: Triple-negative breast cancer (TNBC) is an aggressive subtype lacking estrogen receptor, progesterone receptor, and HER2 overexpression. Patients with TNBC have a generally poor prognosis due to metastasis, high rates of recurrence, and lack of FDA-approved targeted therapies. We previously showed using functional proteomics that patients with high-ERK2-expressing TNBC tumors had a higher risk of death than those with low-ERK2-expressing tumors. Moreover, ERK2 but not ERK1 plays an important role in epithelial-mesenchymal transition (EMT) and is required for acquisition of stem cell-like characteristics. Compared to other breast cancer subtypes, TNBC has a higher proportion of cancer stem cells (CSCs) and is linked to EMT, two critical features associated with breast cancer progression, metastasis, and recurrence in patients. The MAPK signaling pathway is activated in TNBC, but the roles of ERK isoforms in tumor progression and metastasis are not well defined. We hypothesized that ERK2 but not ERK1 promotes EMT, the CSC phenotype, and metastasis in TNBC. Methods and Results: Knockdown of ERK2 in SUM149 and BT549 TNBC cells significantly inhibited anchorage-independent colony formation (p Conclusions and Future Directions: Our findings support our hypothesis, indicating that ERK2 promotes EMT and the CSC phenotype through EGR1 and mediates metastasis in TNBC. Future studies will determine ERK activity and pathway engagement using a novel peptide sensor based on the Sox fluorophore. We will pursue a therapeutic approach using siRNA against ERK2 incorporated in a DOTAP:cholesterol liposome. Citation Format: Mary Kathryn Pitner, Hitomi Saso, Richard Larson, Rachel M. Sammons, Huiqin Chen, Caimiao Wei, Gaurav Chauhan, Kimie Kondo, Naoto T. Ueno, Kevin Dalby, Bisrat G. Debeb, Chandra Bartholomeusz. Silencing of ERK2 reverses EMT and suppresses the CSC phenotype, inhibiting lung metastasis in triple-negative breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1624.

The objective of this research was to study the performance of young novice male drivers in a driving simulator after they were administered different doses of alcohol (placebo: 0 g/kg; medium dose: 0.75 g/kg; high dose: 1 g/kg) during the blood alcohol concentration (BAC) ascending and descending periods. The high dose of alcohol produced an average peak BAC of 74±5.477 mg/100 ml at 30 min after administration, and the medium dose produced an average peak BAC of 47.714±17.68 mg/100 ml at 10 min after administration. Compared with the placebo, the drivers' performance under the high dose of alcohol was characterised by more abrupt steering manoeuvres, a greater average speed, and a greater offset from the lane centre. The drivers were more timid under the medium dose administration. The study on driver behaviour plays an important role on constructing the early warning model, so as to put forward the corresponding intervention measures of unsafe driving behaviour and improve vehicle safety in reducing accidents due to drinking and/or drunk driving on public roads.

Introduction Translation initiation is activated in cancer through increase in eukaryotic initiation factor 4E (eIF4E), eIF4G, phosphorylated eIF4E-binding protein (p4E-BP1) and phosphorylated ribosomal protein S6 (pS6), and decreased programmed cell death protein 4 (pdcd4), a translational inhibitor. Further, translation elongation is deregulated though alterations in eukaryotic elongation factor 2 (eEF2) and eEF2 kinase (eEF2K). We sought to determine the association of these translational aberrations with clinical-pathologic factors and survival outcomes in hormone receptor-positive breast cancer. Methods Primary tumors were collected from 190 patients with Stage I to III hormone receptor-positive breast cancer. Expression of eIF4E, eIF4G, 4E-BP1, p4E-BP1 T37/46, p4E-BP1 S65, p4E-BP1 T70, S6, pS6 S235/236, pS6 S240/244, pdcd4, eEF2 and eEF2K was assessed by reverse phase protein arrays. Univariable and multivariable analyses for recurrence-free survival (RFS) and overall survival (OS) were performed. Results High eEF2, S6, pS6 S240/244, p4E-BP1 T70, and low pdcd4 were significantly associated with node positivity. Median follow-up for living patients was 96 months. High p4E-BP1 T36/47, p4E-BP1 S65, p4E-BP1 T70 and 4E-BP1 were associated with worse RFS. High p4E-BP1 T70 and pS6 S235/236, and low pdcd4, were associated with worse OS. In multivariable analysis, in addition to positive nodes, p4E-BP1 S65 remained a significant predictor of RFS (HR = 1.62, 95% CI = 1.13-2.31; P = 0.008). In addition to age, pS6 S235/236 (HR = 1.73, 95% CI = 1.03-2.90, P = 0.039), eEF2K (HR = 2.19, 95% CI = 1.35-3.56, P = 0.002) and pdcd4 (HR = 0.42, 95% CI = 0.25-0.70, P = 0.001) were associated with OS. Conclusions Increased pS6, p4E-BP1, eEF2K and decreased pdcd4 are associated with poor prognosis in hormone receptor-positive breast cancer, suggesting their role as prognostic markers and therapeutic targets.

The objective of this research was to study the risk factors that significantly influence the severity of intersection crashes for vehicle occupants, as well as for pedestrians and other vulnerable road users.Logistic regression was applied as the method in this study to analyze a data set of intersection crashes involving casualties in Victoria, Australia, for the period January 2000 to December 2009.Seven risk factors obtained were found to be significantly associated with the severity of intersection crashes, including driver age and gender, speed zone, traffic control type, time of day, crash type, and seat belt usage.This study found that male drivers as well as older drivers (age 65 and above) had higher odds of being involved in fatal intersection crashes. Intersection crashes occurring between midnight and early morning (12:00 a.m. to 5:59 a.m.), in 100 km/h speed zones, or with no traffic control had a higher odds of a fatal outcome than their counterpart categories. Furthermore, intersection crashes involving pedestrians or a non-seat belt-wearing driver were more likely to lead to a fatal outcome. In general, identification of risk factors and the discussion of the odds ratio between levels on the impact of the intersection crash severity would be beneficial for road safety stakeholders to develop initiatives to reduce the severity of intersection crashes.

Purpose: We sought to determine whether phosphoinositide 3-kinase (PI3K) pathway mutation or activation state and rapamycin-induced feedback loop activation of Akt is associated with rapamycin sensitivity or resistance. Experimental Design: Cancer cell lines were tested for rapamycin sensitivity, Akt phosphorylation, and mTOR target inhibition. Mice injected with breast or neuroendocrine cancer cells and patients with neuroendocrine tumor (NET) were treated with rapalogs and Akt phosphorylation was assessed. Results: Thirty-one cell lines were rapamycin sensitive (RS) and 12 were relatively rapamycin resistant (RR; IC50 > 100 nmol/L). Cells with PIK3CA and/or PTEN mutations were more likely to be RS (P = 0.0123). Akt phosphorylation (S473 and T308) was significantly higher in RS cells (P < 0.0001). Rapamycin led to a significantly greater pathway inhibition and greater increase in p-Akt T308 (P < 0.0001) and p-Akt S473 (P = 0.0009) in RS cells. Rapamycin and everolimus significantly increased Akt phosphorylation but inhibited growth in an in vivo NET model (BON). In patients with NETs treated with everolimus and octreotide, progression-free survival correlated with p-Akt T308 in pretreatment (R = 0.4762, P = 0.0533) and on-treatment tumor biopsies (R = 0.6041, P = 0.0102). Patients who had a documented partial response were more likely to have an increase in p-Akt T308 with treatment compared with nonresponders (P = 0.0146). Conclusion: PIK3CA/PTEN genomic aberrations and high p-Akt levels are associated with rapamycin sensitivity in vitro. Rapamycin-mediated Akt activation is greater in RS cells, with a similar observation in patients with clinical responses on exploratory biomarker analysis; thus feedback loop activation of Akt is not a marker of resistance but rather may function as an indicator of rapamycin activity. Clin Cancer Res; 18(6); 1777–89. ©2012 AACR.

Purpose: To examine gene expression differences between pre- and post-neoadjuvant systemic therapy (NST) specimens of breast cancers and identify biologic changers that may lead to new therapeutic insights. Methods: Gene expression data from prechemotherapy fine needle aspiration specimens were compared with resected residual cancers in 21 patients after 4 to 6 months of NST. We removed stroma-associated genes to minimize confounding effects. PAM50 was used to assign molecular class. Paired t test and gene set analysis were used to identify differentially expressed genes and pathways. Results: The ER and HER2 status based on mRNA expression remained stable in all but two cases, and there were no changes in proliferation metrics (Ki67 and proliferating cell nuclear antigen expression). Molecular class changed in 8 cases (33.3%), usually to normal-like class, which was associated with low residual cancer cell cellularity. The expression of 200 to 600 probe sets changed between baseline and post-NST samples. In basal-like cancers, pathways driven by increased expression of phosphoinositide 3-kinase, small G proteins, and calmodulin-dependent protein kinase II and energy metabolism were enriched, whereas immune cell–derived and the sonic hedgehog pathways were depleted in residual cancer. In non–basal-like breast cancers, notch signaling and energy metabolism (e.g., fatty acid synthesis) were enriched and sonic hedgehog signaling and immune-related pathways were depleted in residual cancer. There was no increase in epithelial–mesenchymal transition or cancer stem cell signatures. Conclusions: Our data indicate that energy metabolism related processes are upregulated and immune-related signals are depleted in residual cancers. Targeting these biologic processes may represent promising adjuvant treatment strategies for patients with residual cancer. Clin Cancer Res; 18(4); 1109–19. ©2012 AACR.

BACKGROUND: The current study was conducted to determine the frequency and association between recurrence-free survival (RFS) and MET and catalytic subunit of phosphoinositide-3-kinase (PIK3CA) copy number elevations in patients with early stage breast cancer. METHODS: Tumor DNA was extracted from 971 formalin-fixed, paraffin-embedded early breast cancers for molecular inversion probes arrays. Data were segmented using the single-nucleotide polymorphism (SNP)-FASST2 segmentation algorithm. Copy number gains were called when the copy number of each segment was greater than 2.3 or 1.7, respectively. RFS was estimated by the Kaplan-Meier method. Cox proportional hazards models were fit to determine independent associations between copy number and RFS. RESULTS: Of the 971 tumors studied, 82 (8.44%) and 134 (13.8%) had an elevation of the MET or PIK3CA copy number, respectively, and 25.6% of tumors with a MET copy number elevation had a PIK3CA copy number elevation. Patients with either a MET or PI3KCA high copy number tended to have poorer prognostic features (larger tumor size, higher tumor grade, and hormone receptor negativity). Both MET and PIK3CA high copy numbers were more likely to occur in patients with triple receptor-negative disease (P = .019 and P < .001, respectively). At a median follow-up of 7.4 years, there were 252 cases of disease recurrence. The 5-year RFS rates were 63.5% and 83.1% for MET high copy number and MET normal/low copy number, respectively (P = .06) and 73.1%, and 82.3% for PIK3CA high copy number and PIK3CA normal/low copy number, respectively (P = .15). A high copy number for either gene was not found to be an independent predictor of RFS. CONCLUSIONS: A high copy number of MET or PIK3CA was found to be associated with poorer prognostic features and triple receptor-negative disease. Coamplification was frequent. Patients with tumors with high MET copy numbers tended to have a worse RFS. Cancer 2013. © 2012 American Cancer Society.

The purpose of this work was to determine whether there are differences in PIK3CA mutation status and PTEN protein expression between primary and matched metastatic breast tumors as this could influence patient management. Paraffin sections of 50 μm were used for DNA extraction and slides of 3 μm for immunohistochemistry (IHC) and FISH. Estrogen receptor, progesterone receptor, and HER2 IHC were repeated in a central laboratory for both primary tumors and metastases. PTEN levels were assessed by IHC and phosphoinositide 3-kinase (PI3K) pathway mutations were detected by a mass spectroscopy–based approach. Median age was 48 years (range: 30–83 years). Tumor subtype included 72% hormone receptor positive/HER2 negative, 20% HER2-positive, and less than 7.8% triple receptor negative. Tissues were available for PTEN IHC in 46 primary tumors and 52 metastases. PTEN was lost in 14 (30%) primary tumors and 13 (25%) metastases. There were five cases of PTEN loss and eight cases of PTEN gain from primary tumors to metastases (26% discordance). Adequate DNA was obtained from 46 primary tumors and from 50 metastases for PIK3CA analysis. PIK3CA mutations were detected in 19 (40%) of primary tumors and 21 (42%) of metastases. There were five cases of PIK3CA mutation loss and four cases of mutation gain (18% discordance). There was an increase of the level of PIK3CA mutations in four cases and decrease in one case from primary tumors to metastases. There is a high level of discordance in PTEN level, PIK3CA mutations, and receptor status between primary tumors and metastases that may influence patient selection and response to PI3K-targeted therapies. Mol Cancer Ther; 10(6); 1093–101. ©2011 AACR.

Aim This article evaluates the risk of recurrence for patients who have small node-negative breast cancer by age and tumor subtype. Methods One thousand twelve patients with a T1a,bN0 breast cancer diagnosed between 1990 and 2002 who did not receive chemotherapy or trastuzumab were included. Patients and tumor characteristics were compared using the χ 2 or Wilcoxon's rank sum tests. Survival outcomes were estimated with the Kaplan-Meier method and compared using the log-rank statistic. Cox proportional hazards models were used to determine association of breast cancer subtypes and age at diagnosis with other covariates. Results Median age was 51.5 years. There were 771 hormone receptor (HR)-positive, 98 HER2-positive, and 143 triple-negative breast cancers (TNBC). Six hundred ninety-three patients were > 50 years, and 33 patients were ≤ 35 years. For 5-year survival estimates, there were 118 deaths and overall survival was 94.6% (95% confidence interval [CI] = 93.2%, 96.1%). After adjusting for breast cancer subtype and other tumor characteristics, patients ≤ 35 had 2.51 (95% CI=1.21-5.22) times greater risk of worse recurrence-free survival (RFS), and 2.60 (95% CI=1.05-6.46) times greater risk of worse distant RFS (DRFS) compared to patients > 50 years old. Compared to patients with HR-positive disease, patients with HER2-positive breast cancer had 4.98 (95% CI=2.91-8.53) times the risk of worse RFS and 4.70 (95% CI=2.51-8.79) times greater risk of worse DRFS, and patients with TNBC had 2.71 (95% CI=1.59-4.59) times greater risk of worse RFS and 2.08 (95% CI=1.04-4.17) times greater risk of worse DRFS. Conclusions In this cohort, patients with T1a,bN0 breast cancer, young age and breast cancer subtype were significantly associated with RFS and DRFS.

Purpose: To investigate the incidence of germline and somatic BRCA1/2 mutations in unselected patients with triple-negative breast cancer (TNBC) and determine the prognostic significance of carrying a mutation. Methods: DNA was obtained from 77 TNBC and normal tissues. BRCA1/2 exons/flanking regions were sequenced from tumor and patients classified as mutant or wild type (WT). Sequencing was repeated from normal tissue to identify germline and somatic mutations. Patient characteristics were compared with chi-square. Survival was estimated by Kaplan–Meier method and compared with log-rank. Cox proportional hazards models were fit to determine the independent association of mutation status with outcome. Results: Median age was 51 years (27–83 years). Fifteen patients (19.5%) had BRCA mutations: 12 (15.6%) in BRCA1 (one somatic), and 3 (3.9%) in BRCA2. Patients with BRCA mutations tended to be younger than WT, (P = 0.005). Grade, histology, and stage were not associated with mutation status. At a median follow-up of 43 months (7–214 months), there were 33 (42.9%) recurrences and 35 (45.5%) deaths. Five-year recurrence-free survival estimates were 51.7% for WT versus 86.2% for patients with mutations, (P = 0.031); and 5-year overall survival estimates were 52.8% for WT versus 73.3% for patients with mutations (P = 0.225). After adjustment, patients with BRCA mutations had a significantly better RFS (HR: 0.19, 95% CI: 0.045–0.79, P = 0.016) compared with WT. Conclusions: In this unselected cohort of TNBC, we found a 19.5% incidence of BRCA mutations. Genetic testing should be discussed with patients with TNBC. Patients with TNBC with BRCA mutations had a significantly lower risk of relapse. Clin Cancer Res; 17(5); 1082–9. ©2011 AACR.

Food borne illnesses from the ingestion of S. typhimurium have been of primary concern due to their common occurrence in food products of daily consumption. In this paper, micron size, magnetoelastic (ME) biosensors for the detection of S. typhimurium were fabricated and tested in liquid solutions containing known concentrations of S. typhimurium cells. The biosensors are comprised of a ME sensor platform and immobilized bio-molecular recognition element (E2 phage) that has been engineered to bind the S. typhimurium multi-valently. The micron size ME sensor platforms were manufactured using microelectronics fabrication techniques. Phage was engineered at Auburn University and immobilized onto all surfaces of the sensor. The ME biosensor oscillates with a characteristic resonance frequency when subjected to a time varying magnetic field. Binding between the phage and bacteria, adds mass to the sensor that causes a shift in the sensor's resonance frequency. Sensors with the dimension of 500x100x4 μm were exposed to S. typhimurium with increasing known concentrations ranging from 5 x10 1 to 5 x 10 7 cfu/ml. The ME biosensor exhibited high sensitivity and a detection limit better than 50 cfu/ml.

Current bacterial detection methods require the collection of samples followed by preparation and analysis in the laboratory, both time and labour consuming steps. More importantly, because of cost, only a limited number of samples can be taken and analyzed. This paper presents the results of an investigation to directly detect Salmonella typhimurium on fresh tomato surfaces using phage-based magnetoelastic (ME) biosensors. The biosensor is composed of a ME resonator platform coated with filamentous E2 phage, engineered to bind with S. typhimurium . The ME biosensors are wireless sensors, whose resonance oscillation and resonance frequency are actuated and detected through magnetic fields. The sensors used in this study were 0.028 mm × 0.2 mm × 1 mm in size. In this study, the tomato surface was spiked with S. typhimurium suspensions with concentrations ranging from 5 × 10 1 to 5 × 10 8 CFU/ml and then allowed to dry in air. The detection was conducted by directly placing ME measurement biosensors and control sensors on the spiked surface for 30 min in a humid environment. The control sensors were identical to the measurement biosensors, but without phage. Both measurement and control sensors were blocked with BSA to reduce non-specific binding. The resonance frequencies of both measurement and control sensors were measured prior to and after the placement of the sensors on the tomato. Shifts in the resonance frequency of the measurement biosensors were observed, while the control sensors showed negligible change. Scanning electron microscopy (SEM) was used to verify the specific binding of S. typhimurium to the biosensor. Results of multiple biosensor detection and corresponding analyzes showed statistically different responses between the measurement and control sensors for tomatoes spiked with S. typhimurium suspensions with concentrations of 5 × 10 2 CFU/ml and greater. This study demonstrates the direct detection of food-borne bacteria on fresh produce.

Dihydrolipoamide dehydrogenase (DLDH), a flavin-dependent oxidoreductase is essential for energy metabolism. As an oxidoreductase it catalyzes the NAD + -dependent oxidation of dihydrolipoamide. In this study, a putative Bombyx mori dihydrolipoamide dehydrogenase (BmDLDH) gene was cloned, expressed, purified and characterized for the first time. The BmDLDH gene was amplified from a pool of silkworm cDNAs by PCR and cloned into Escherichia coli expression vector pET-28a(+). The recombinant His-tagged BmDLDH protein was expressed in E. coli BL21 (DE3) and purified by metal chelating affinity chromatography. The amino acid sequence of recombinant protein was confirmed by mass spectroscopic analysis. Furthermore, the oxidoreductase activity in the reverse reaction indicated that the soluble recombinant BmDLDH produced at lower growth temperature was able to catalyze the lipoamide-dependent oxidation of NADH.

Magnetoelastic (ME) sensors provide a fast, sensitive method to detect bacteria with smaller sensors have higher mass sensitivity for detecting lower concentrations of bacteria. However, signals from smaller sensors are weaker and have more noise due to manufacturing defects. In this paper, we present a biosensor system for the detection of Salmonella typhimurium using multiple magnetoelastic sensors, each with the size of 2000 × 400 × 30 μm. The sensors are immobilized with E2 phage, which specifically binds with S. typhimurium. Unlike traditional methods, our system uses a step pulse to “shock” the sensor, causing it to vibrate at its natural resonance frequency and produce a signal in the pickup coil due to reverse magnetostriction. A Fast Fourier Transform (FFT) was used to determine the resonance frequency. As the biosensor captures S. typhimurium cells, its mass increases with a corresponding decrease of its resonance frequency.The detection system was composed of one coil with a reference sensor to monitor stability, and another coil with three measurement sensors separated in three tubes for simultaneous detection of bacteria. With multi-sensors the effect of a manufacturing defect is decreased and we get the benefit of averaging for more accurate and reliable results. Stability tests show that the variance of frequency detection is less than 122 ppm of its resonance frequency. SEM pictures of the sensor surface show a uniform binding of S. typhimurium cells. Cells were counted and the mass change calculated. The measured frequency change corresponds well to the theoretical change. The results show that the multiple phage based ME biosensors are able to simultaneously detect S. typhimurium and offer good sensitivity and reliability of detection.

Serpins are a broadly distributed family of protease inhibitors. In this study, the gene encoding Bombyx mori serpin-2 (Bmserpin-2) was cloned and expressed in E. coli. The Bmserpin-2 cDNA contains a 1125 bp open reading frame (ORF). The deduced protein has 374 amino-acid residues, contains a conserved SERPIN domain and shares extensive homology with other invertebrate serpins. RT-PCR analysis showed that Bmserpin-2 was expressed in all developmental stages of B. mori larvae and various larval tissues. Subcellular localization analysis indicated that Bmserpin-2 protein was located in the cytoplasm. Interestingly, real-time quantitative PCR revealed that the expression of Bmserpin-2 in the midgut of susceptible B. mori strain 306 significantly increased at 72 hours post inoculation (hpi) when infected with BmNPV. However, there was no significant increase of the Bmserpin-2 expression in resistant strain NB infected with BmNPV. Thus, our data indicates that Bmserpin-2 may be involved in B. mori antiviral response.

Purpose: To evaluate HER2 status in residual tumor identified at the time of surgery in patients not achieving a pathologic complete response (pCR) and to determine the effect of alterations in HER2 status on recurrence-free survival (RFS). Experimental Design: Clinicopathologic data for patients with HER2-overexpressing breast cancer receiving neoadjuvant therapy with a taxane, anthracycline, and concomitant trastuzumab between 2004 and 2007 were reviewed. Surgical specimens for patients achieving less than a pCR were assessed to determine if there was enough residual tissue to evaluate posttreatment HER2 status. RFS was determined using the Kaplan-Meier method and compared by the log-rank statistic. Results: A pCR was achieved in 72 of the 142 (50.7%) patients. Residual tumor was sufficient to assess posttreatment HER2 status in 25 patients. Fluorescence in situ hybridization done on pretreatment specimens confirmed HER2 amplification before beginning therapy. Eight (32.0%) posttreatment tumors were found to be HER2-negative by fluorescence in situ hybridization. At a median follow-up of 37 months (range, 8-56 months), the RFS was significantly better for patients with tumors that retained HER2 amplification (87.5% versus 50%, P = 0.04). Conclusion: High pCR rates are achieved in patients with HER2-positive breast cancer treated with neoadjuvant trastuzumab in combination with anthracyclines and taxanes. One third of patients with significant residual disease loses HER2 amplification, and this change is associated with poor RFS. Residual tumor identified at the time of surgery should be reassessed for HER2 status, and novel adjuvant therapy strategies need to be studied in this population. (Clin Cancer Res 2009;15(23):7381–8)

In this study, we report the cloning and characteristics of an adiponectin-like receptor gene from Bombyx mori (BmAdipoR) with highly conserved deduced amino-acid sequences and similar structure to the human adiponectin receptor (AdipoR). Structural analysis of the translated cDNA suggested it encoded a membrane protein with seven transmembrane domains. BmAdipoR was found to be expressed in multiple tissues and highly expressed in Malpighian tubules, fat body and testis. BmNPV (Bombyx mori nucleopolyhedrovirus) bacmid system combined with confocal microscopy revealed that BmAdipoR was targeted to the cell membrane. We also found that infection with BmNPV did not have an effect on BmAdipoR mRNA quantity in the midgut of susceptible Bombyx mori strain (306) at 48 h, but BmAdipoR mRNA quantity increased significantly at 72 h. We concluded that BmAdipoR gene was a membrane protein ubiquitously expressed in Bombyx mori tissues and that its expression was altered by treating with BmNPV.

1524 Background: Identifying the clinical impact of mutations in cancer-related genes can help define their role in cancer. Here, we aim to classify frequent hotspot mutations in metastatic breast ...