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The tumor microenvironment and wound healing after injury, both contain extremely high concentrations of the extracellular signaling molecule, adenosine triphosphate (ATP) compared to normal tissue. P2Y2 receptor, an ATP-activated purinergic receptor, is typically associated with pulmonary, endothelial, and neurological cell signaling. Here we report its role and importance in breast epithelial cell signaling and how it’s altered in metastatic breast cancer. In response to ATP activation, P2Y2 receptor signaling causes an increase of intracellular Ca2+in non-tumorigenic breast epithelial cells, while their tumorigenic and metastatic counterparts have significantly reduced Ca2+responses. The non-tumorigenic cells respond to increased Ca2+with actin polymerization and localization to cellular junctions, while the metastatic cells remained unaffected. The increase in intracellular Ca2+after ATP stimulation could be blunted using a P2Y2 antagonist, which also prevented actin mobilization in non-tumorigenic breast epithelial cells. Furthermore, the lack of Ca2+concentration changes and actin mobilization in the metastatic breast cancer cells could be due to reduced P2Y2 expression, which correlates with poorer overall survival in breast cancer patients. This study elucidates rapid changes that occur after elevated intracellular Ca2+in breast epithelial cells and how metastatic cancer cells have adapted to evade this cellular response.STATEMENT OF SIGNIFICANCEThis work shows non-tumorigenic breast epithelial cells increase intracellular Ca2+after ATP-P2Y2 signaling and re-localize actin, while metastatic cells lack this response, due to decreased P2Y2 expression, which correlates with poorer survival.

Significance In breast cancer, the chronically stiffening mechanical microenvironment promotes tumor growth/invasion. However, the molecular details and integration of cancer mechanotransduction signaling pathways are not well understood. We find that nontumorigenic breast epithelial cells are mechanically sensitive and respond to acute mechanical stimuli with the generation of ROS-stimulated calcium signaling in a microtubule-dependent manner. These results establish that epithelial cells conserve a mechanotransduction pathway from muscle and bone (X-ROS). Using common breast cancer genetic mutations, we further report that oncogene activation reduces X-ROS pathway mechanoresponsiveness at the level of ROS sensitivity. This definition of how oncogene activation disrupts highly conserved mechanotransduction signaling mechanisms could improve the understanding of altered tumor cell responses to the mechanical microenvironment and reveal new therapeutic opportunities., Changes in the mechanical microenvironment and mechanical signals are observed during tumor progression, malignant transformation, and metastasis. In this context, understanding the molecular details of mechanotransduction signaling may provide unique therapeutic targets. Here, we report that normal breast epithelial cells are mechanically sensitive, responding to transient mechanical stimuli through a two-part calcium signaling mechanism. We observed an immediate, robust rise in intracellular calcium (within seconds) followed by a persistent extracellular calcium influx (up to 30 min). This persistent calcium was sustained via microtubule-dependent mechanoactivation of NADPH oxidase 2 (NOX2)-generated reactive oxygen species (ROS), which acted on transient receptor potential cation channel subfamily M member 8 (TRPM8) channels to prolong calcium signaling. In contrast, the introduction of a constitutively active oncogenic KRas mutation inhibited the magnitude of initial calcium signaling and severely blunted persistent calcium influx. The identification that oncogenic KRas suppresses mechanically-induced calcium at the level of ROS provides a mechanism for how KRas could alter cell responses to tumor microenvironment mechanics and may reveal chemotherapeutic targets for cancer. Moreover, we find that expression changes in both NOX2 and TRPM8 mRNA predict poor clinical outcome in estrogen receptor (ER)-negative breast cancer patients, a population with limited available treatment options. The clinical and mechanistic data demonstrating disruption of this mechanically-activated calcium pathway in breast cancer patients and by KRas activation reveal signaling alterations that could influence cancer cell responses to the tumor mechanical microenvironment and impact patient survival.

9 pages, 5 figures, 1 table, Modern small-molecule drug discovery relies on the selective targeting of biological macromolecules by low-molecular weight compounds. Therefore, the binding affinities of candidate drugs to their targets are key for pharmacological activity and clinical use. For drug discovery methods where multiple drug candidates can simultaneously bind to the same target, a competition is established, and the resulting equilibrium depends on the dissociation constants and concentration of all the species present. Such coupling between all equilibrium-governing parameters complicates analysis and development of improved mixture-based, high-throughput drug discovery techniques. In this work, we present an iterative computational algorithm to solve coupled equilibria between an arbitrary number of ligands and a biomolecular target that is efficient and robust. The algorithm does not require the estimation of initial values to rapidly converge to the solution of interest. We explored binding equilibria under ligand/receptor conditions used in mixture-based library screening by affinity selection–mass spectrometry (AS–MS). Our studies support a facile method for affinity-ranking hits. The ranking method involves varying the receptor-to-ligand concentration ratio in a pool of candidate ligands in two sequential AS–MS analyses. The ranking is based on the relative change in bound ligand concentration. The method proposed does not require a known reference ligand and produces a ranking that is insensitive to variations in the concentration of individual compounds, thereby enabling the use of unpurified compounds generated by mixture-based combinatorial synthesis techniques

Post-translational modifications (PTMs) of the microtubule network impart differential functions across normal cell types and their cancerous counterparts. The removal of the C-terminal tyrosine of α-tubulin (deTyr-Tub) as performed by the tubulin carboxypeptidase (TCP) is of particular interest in breast epithelial and breast cancer cells. The recent discovery of the genetic identity of the TCP to be a vasohibin (VASH1/2) coupled with a small vasohibin-binding protein (SVBP) allows for the functional effect of this tubulin PTM to be directly tested for the first time. Our studies revealed the immortalized breast epithelial cell line MCF10A undergoes apoptosis following transfection with TCP constructs, but the addition of oncogenic KRas or Bcl-2/Bcl-xL overexpression prevents subsequent apoptotic induction in the MCF10A background. Functionally, an increase in deTyr-Tub via TCP transfection in MDA-MB-231 and Hs578t breast cancer cells leads to enhanced focal gelatin degradation. Given the elevated deTyr-Tub at invasive tumor fronts and the correlation with poor breast cancer survival, these new discoveries help clarify how the TCP synergizes with oncogene activation, increases focal gelatin degradation, and may correspond to increased tumor cell invasion. These connections could inform more specific microtubule-directed therapies to target deTyr-tubulin.

Cytoskeletal remodeling in circulating tumor cells (CTCs) facilitates metastatic spread. Previous oncology studies examine sustained aberrant calcium (Ca2+) signaling and cytoskeletal remodeling scrutinizing long-term phenotypes such as tumorigenesis and metastasis. The significance of acute Ca2+ signaling in tumor cells that occur within seconds to minutes is overlooked. This study investigates rapid cytoplasmic Ca2+ elevation in suspended cells on actin and tubulin cytoskeletal rearrangements and the metastatic microtentacle (McTN) phenotype. The compounds Ionomycin and Thapsigargin acutely increase cytoplasmic Ca2+, suppressing McTNs in the metastatic breast cancer cell lines MDA-MB-231 and MDA-MB-436. Functional decreases in McTN-mediated reattachment and cell clustering during the first 24 h of treatment are not attributed to cytotoxicity. Rapid cytoplasmic Ca2+ elevation was correlated to Ca2+-induced actin cortex contraction and rearrangement via myosin light chain 2 and cofilin activity, while the inhibition of actin polymerization with Latrunculin A reversed Ca2+-mediated McTN suppression. Preclinical and phase 1 and 2 clinical trial data have established Thapsigargin derivatives as cytotoxic anticancer agents. The results from this study suggest an alternative molecular mechanism by which these compounds act, and proof-of-principle Ca2+-modulating compounds can rapidly induce morphological changes in free-floating tumor cells to reduce metastatic phenotypes.

Alström syndrome is an autosomal recessive obesity ciliopathy caused by loss-of-function mutations in the ALMS1 gene. In addition to multi-organ dysfunction, such as cardiomyopathy, retinal degeneration, and renal dysfunction, the disorder is characterized by high rates of obesity, insulin resistance and early onset type 2 diabetes mellitus (T2DM). We previously published data supporting the alms1 loss-of-function zebrafish mutant as a monogenic model for mechanistic interrogation of T2DM phenotypes. Both adult and larval alms1-/- animals exhibit reduced insulin+ areas alongside defective peripheral glucose uptake and concomitant hyperinsulinemia in larval alms1-/- animals. Gene expression changes in beta-cells isolated from alms1-/- mutants revealed changes consistent with insulin hyperproduction, insulin hypersecretion and glucose sensing failure. Therefore, we used this model, in which hyperinsulinemia is the primary and causative defect underlying generation of T2DM, in a high-throughput screen to examine small molecule compounds effecting pancreatic mass in vivo. The external development, simple growing conditions, and large number of progeny in the zebrafish allow for rapid screening with parallel elimination of lethal compounds. We examined the Microsource Spectrum Collection 2000, a library of 2,560 compounds, for those which increase beta-cell mass in wildtype, alms1-/- animals or both. These identified compounds are indicated in future studies for beta-cell proliferation pathways, T2DM diabetes-specific interactions, and potential targets for future therapeutic development. Disclosure J. Dunleavey: None. D.A. Annis: None. T.L. Hostelley: None. N.A. Zaghloul: None. Funding National Institute of Diabetes and Digestive and Kidney Diseases

This article examines estimation of the one-step-ahead transition probabilities in a reversible Markov chain on a countable state space. A symmetrized moment estimator is proposed that exploits the reversible structure. Examples are given where the symmetrized estimator has superior asymptotic properties to those of a naive estimator, implying that knowledge of reversibility can sometimes improve estimation. The asymptotic mean and variance of the estimators are quantified. The results are proven using only elementary results such as the law of large numbers and the central limit theorem.

In this paper, we provide a stochastic ordering of the Studentized range statistics under a balanced one-way ANOVA model. Based on this result we show that, when restricted to the multiple comparisons with the best, the Newman-Keuls (NK) procedure strongly controls experimentwise error rate for a sequence of null hypotheses regarding the number of largest treatment means. In other words, the NK procedure provides an upper confidence bound for the number of best treatments.

The ranking and selection problem has been well-studied in the case of continuous responses. In this paper, we address the situation in which continuous responses are replaced by discrete orderings. When individuals in the population provide exhaustive rank-orderings of the alternatives, two common decision rules are the majority rule and the rank-sum method. In the former case, the alternative receiving the most first-place votes is declared superior, while in the latter, the alternative with the smallest rank-sum is deemed the best. Both the Pitman efficiencies and the lower bounds on Bahadur efficiencies of the majority rule relative to the rank-sum method are derived, assuming that the rank data are generated from either the Plackett–Luce or the translative strengths models. In addition, finite sample properties of the two methods are compared with the maximum likelihood approach through simulation studies. Our results suggest two things. First, when it is substantially more difficult to obtain a complete rank-ordering than simply the top choice, the majority rule performs adequately and efforts would be better spent asking many voters to provide top choice rather than fewer voters to provide complete orderings. Second, the rank-sum rule compares favorably to, and is substantially more robust than, the maximum likelihood approach.

Maximum likelihood estimation for exponential families depends exclusively on the first two moments of the data. Recognizing this, Wedderburn [1974. Quasi-likelihood functions, generalized linear models, and the Gauss–Newton method. Biometrika 61, 439–447] proposed estimating regression parameters based on a quasi-likelihood function requiring only the relationship between the mean and variance. We extend quasi-likelihood to situations in which there exists vague prior information on the mean parameters. It is shown when data are exponential family with quadratic variance functions, maximum a posteriori inference under a conjugate prior relies solely on two moments of the data and the prior distribution. This result suggests a Bayesian analog of quasi-likelihood for which only two moments of the data and two moments of the prior need be specified.

This article discusses the properties of various knockout tournament designs and presents theoretical results. Potential playoff schemes for Division I-A football are examined via simulation studies. Several metrics are used to assess the relative merits of playoff scenarios, which differ in number, selection, and seeding of playoff teams. Most suggest that college football would benefit from a limited playoff system. Interestingly, for the class of playoff systems examined, the number of teams influences the performance far more than does the seeding procedure.

In the minimum inhibitory concentration (MIC) test literature, discussion concerning the effect of laboratory-to-laboratory variation is lacking. We present 2 sets of drug dilution test quality control data that illustrate considerable laboratory differences in measured MIC. In both isolates (Escherichia coli, ATCC 25922; Staphylococcus aureus, ATCC 29213) the laboratory-to-laboratory variability accounts for approximately half of the total variability. We illustrate the impact of this variability on the probability of correctly classifying the susceptibility level of an isolate and on the estimation of resistance prevalence. For example, we show that laboratory differences in the probability of correctly classifying the isolate (specifically near the lower breakpoint) can vary up to 80%.

Box's paper helicopter has been used to teach experimental design for more than a decade. It is simple, inexpensive, and provides real data for an involved, multifactor experiment. Unfortunately it can also further an all-too-common practice that Professor Box himself has repeatedly cautioned against, namely ignoring the fundamental science while rushing to solve problems that may not be sufficiently understood. Often this slighting of the science so as to get on with the statistics is justified by referring to Box's oft-quoted maxim that “All models are wrong, however some are useful.” Nevertheless, what is equally true, to paraphrase both Professor Box and George Orwell, is that “All models are wrong, but some are more wrong than others.” To experiment effectively it is necessary to understand the relevant science so as to distinguish between what is usefully wrong, and what is dangerously wrong.This article presents an improved analysis of Box's helicopter problem relying on statistical and engineering...

The American Philosophical Association suggests that studying philosophy can have an important impact on one’s creative thinking ability. This paper abstracts from the empirical research on creativity a reasonable model of creative thinking, and discusses what the account implies about fostering creative thinking in philosophy courses. Given the empirical research on creativity and the nature of philosophy, studying philosophy can have an important impact on one’s creative thinking ability, but faculty need to focus on it in their courses.

In this paper, we propose a team rating system that accounts for differences in strength of schedule by computing an additive correction factor which adjusts team winning percentage. This approach permits teams to be ranked appropriately despite disparate schedules. Our method is easy to compute as it only requires a simple matrix inversion, and we prove that the needed inverse exists for all reasonable classes of schedules. We also prove some mathematical properties of the ratings system and apply our method to data from the 2007 Major League Baseball season.

Rating -- and subsequently ranking -- college football teams requires making sense of sometimes conflicting pair-wise comparisons. Classical statistical techniques fall into one of two classes: win/loss models, which focus on binary outcomes, and point-scoring models, which consider the distribution of component scores. Annis and Craig (2005) illustrate deficiencies of both, and propose a hybrid method that considers both sources of data. Their method, while providing satisfactory results in many circumstances, can be difficult to implement numerically. This paper presents a refinement of their hybrid rating algorithm which preserves their original intent but simplifies greatly its implementation. Like its predecessor, the new model enjoys robustness to model misspecification, while offering substantial simplification.

An Article Submitted to Journal of Quantitative Analysis in Sports, Manuscript 1030 When faced with protecting a three-point lead in the waning seconds of a basketball game, which is a preferable strategy: playing defense or fouling the offense before they can attempt a game-tying shot? Gonzaga University head coach, Mark Few, was faced with such a decision against Michigan State in the semi-finals of the Maui Invitational (November 22, 2005) and elected to play defense. The strategy backfired, as Michigan State’s Maurice Ager made a three-point basket at the buzzer to force overtime. (Gonzaga eventually won in triple overtime.) Was this failure to hold the lead at the end of regulation bad luck or bad strategy? Put another way, which strategy (conventional defense or intentionally fouling) maximizes the defensive team’s chances of winning the game? Drawing on the Gonzaga/Michigan State game for inspiration, this paper addresses this question and concludes that, contrary to popular belief, intentionally fouling is preferable to playing tight defense.

Existing paired comparison models used for ranking football teams primarily focus on either wins and losses or points scored (either via each team's total or a margin of victory). While reasonable, each approach fails to produce satisfactory rankings in frequently arising situations due to its ignorance of additional data. We propose a new, hybrid model incorporating both wins and constituent scores and show that it outperforms its competitors and is robust against model mis-specification based on a series of simulation studies. We conclude by illustrating the method using the 2003-04 and 2004-05 college football seasons.

SUMMARY A common method for measuring the drug-specic minimum inhibitory concentration (MIC) of an antibacterial agent is via a two-fold broth dilution test known as the MIC test. Because this procedure implicitly rounds data upward, inference based on unadjusted measurements is biased and overestimates bacterial resistance to a drug. We detail this test procedure and its associated bias, which, in many cases, has an expected value of approximately 0.5 on the log 2 scale. In addition, new bias-corrected estimates of resistance are proposed. A numeric example is used to illustrate the extent to which the traditional resistance estimate can overestimate the true proportion of resistant strains, a phenomenon which is remedied by using the proposed estimates. Published in 2005 by John Wiley & Sons, Ltd.

The objective of this study was to investigate the influence of photoperiod and 0, 1, 5, or 10 applications at weekly intervals of GA3 foliar sprays at 500 mg·liter–1 on growth and flowering of Craspedia globosa `Drumstick' Benth. Long days (LD) hastened flowering and increased the number of flowers per plant. Short days (SD) increased foliage height and foliage fresh and dry weights. Foliage and total plant heights increased and days to bud and secondary inflorescence width decreased linearly as GA3 application frequency increased. Chemical name used: (1α,2β,4aα,4bβ,10β)-2,4a,7-trihydroxy-1-methyl-8-methylen egibb-3-ene-1,10-dicarboxylic acid 1,4a-lactone (gibberellic acid, GA3).

(2008). Bayes Linear Statistics: Theory and Methods. Journal of the American Statistical Association: Vol. 103, No. 483, pp. 1319-1319.

(2006). Kendall's Advanced Theory of Statistics, Vol. 1: Distribution Theory, Kendall's Advanced Theory of Statistics, Vol. 2A: Classical Inference and the Linear Model. Journal of the American Statistical Association: Vol. 101, No. 476, pp. 1721-1721.

(2006). Baseball's All-Time Best Hitters: How Statistics Can Level the Playing Field and Baseball's All-Time Best Sluggers: Adjusted Batting Performance from Strikeouts to Home Runs. The American Statistician: Vol. 60, No. 1, pp. 97-97.

(2005). Stereology for Statisticians. Journal of the American Statistical Association: Vol. 100, No. 472, pp. 1462-1462.

Flowers of Craspedi a uniflora (Billy Buttons), a New Zealand annual plant, have been introduced recently into the U.S.A. florist market. Craspedia is relatively unknown in the U.S.A., but is beginning to be utilized by florists as a cut flower and has potential for specialty cut flower production. Craspedia uniflora cut flowers were grown from seed in an inflated, double-layer, polyethylene greenhouse. Short day (SD) and long day (LD) photoperiod treatments were applied by daylength reduction via black cloth and night interruption lighting, respectively. Treatments began 10 weeks after sowing (15 leaf stage) and continued until harvest. Gibberellic acid was applied as a single foliar spray to runoff at 0 and 500 mg·liter-1 at initiation of photoperiod treatment. Long day treatment reduced days to anthesis, increased flower number, and decreased stem length and plant fresh weight. Gibberellic acid (500 mg·liter-1) increased foliage height under both photo period treatments and increased plant dry weight under LD but reduced dry weight under SD treatment. Flower fresh weight, diameter and bud number were not affected by treatment.

There is no data in the literature regarding the study techniques actually used by students in various grades. The absence of this data may in part explain the conflicting results in the literature on study technique effectiveness. This normative study investigated the study technique preferences of 914 students in grades 6–8, 10–12, and at the college level in order to determine the study techniques actually used by students at various ages. The results indicate that with increasing grade level there is an increase in the total number of study techniques described by students, and also that with increasing grade level Read Only decreases in popularity and is replaced by the study techniques of Underlining and/or Note Taking. Possible explanations for these results include the more active involvement and encoding required by Underlining and Note Taking and the increasing influence of individual differences.

College students enrolled in various kinds of philosophy courses and in a nonphilosophy control group were tested at the beginning and end of the term with different forms of the Watson-Glaser Critical Thinking Appraisal in order to determine the effect of course content on growth in critical thinking. It was found that Logic had a consistent impact on certain aspects of critical thinking, while other philosophy courses when compared to the control group did not have this consistent effect. It was also found that student sex interacts with number of previous philosophy courses for at least one aspect of critical thinking, with females scoring lower than males when both have no previous courses in philosophy and higher than males when both sexes have had previous philosophy courses. Implications of these findings for increasing the growth of critical thinking in other disciplines are discussed.