Rote

Scottish poetry sectio

Genome-wide association studies and the genetics of entrepreneurship

Contains fulltext : 88506.pdf (publisher's version ) (Closed access

Organizational innovation in the multinational enterprise: internalization theory and business history

This article engages in a methodological experiment by using historical evidence to challenge a common misperception about internalization theory. The theory has often been criticized for maintaining that it assumes a hierarchically organized MNE based on knowledge flowing from the home country. This is not an accurate description of how global firms operate in recent decades, but this article shows it has never been true historically. Using longitudinal data on individual firms from the nineteenth century onwards, it reveals evidence of how entrepreneurs and firms with multinational activity faced with market imperfections changed the design of their headquarters and their organizational structures

Phosphoflow-Based Evaluation of Mek Inhibitors as Small-Molecule Therapeutics for B-Cell Precursor Acute Lymphoblastic Leukemia.

Upstream mutations that lead to constitutive activation of Erk in B-cell precursor acute lymphoblastic leukemia (BCP-ALL) are relatively common. In the era of personalized medicine, flow cytometry could be used as a rapid method for selection of optimal therapies, which may include drugs that target the Erk pathway. Here, we evaluated the utility of phospho-flow, compared to Western blotting, to monitor Erk pathway activation and its inhibition by targeted Mek kinase inhibitors in human BCP ALL. Because the Erk pathway is not only activated endogenously, by mutations, but also by normal extracellular stimulation through stromal contact and serum growth factors, we compared Erk activation ex vivo in ALL cells in the presence and absence of stroma and serum. Phospho-flow was able to readily detect changes in the pool of pErk1/2 that had been generated by normal microenvironmental stimuli in patient-derived BCP-ALL cells passaged in NSG mice, in viably frozen primary patient samples, and in fresh patient samples. Treatment with the Mek1/2 inhibitor selumetinib resulted in a rapid, complete and persistent reduction of microenvironment-generated pErk1/2. Imaging flow cytometry confirmed reduction of nuclear pErk1/2 upon selumetinib treatment. An ALL relapsing with an activating KRasG12V mutation contained higher endogenous as well as serum/stromal-stimulated levels of pErk1/2 than the matched diagnosis sample which lacked the mutation, but selumetinib treatment reduced pErk1/2 to the same level in both samples. Selumetinib and trametinib as Mek inhibitors were mainly cytostatic, but combined treatment with the PI3K∂ inhibitor CAL101 increased cytotoxicity. Thus phospho-flow cytometry could be used as a platform for rapid, individualized in vitro drug sensitivity assessment for leukemia patients at the time of diagnosis

Phospho-flow cytometry detects Mek pathway activation and inhibition.

<p>(A) PBMC from normal donors were stimulated with 40 nM PMA for 15 minutes, then analyzed by phospho-flow. Left, pMek, right pErk. Error bars, mean ± SD of 3 independent samples. *p<0.05, Student's t-test. (B, C) Imaging cytometry analysis on PBMC not stimulated (B top) or stimulated with 40 nM PMA (B bottom) or LAX56 cells treated with DMSO (C, top) or 10 μM selumetinib (C, bottom) for 10 minutes. (B, C) left, representative images; right, summary plot. Analysis of 3500 to 20,000 events per condition; numbers in the brightfield images represent the number designation of the individual event shown. (D) Histograms of pErk1/2 phospho-flow analysis on TXL2 ALL cells cultured without stroma or FBS (left panel), or on OP9 stroma + 20% FBS (right panel), for 24 hours. Cells were treated for 4 hours with solvent DMSO (red), with 100 μM sodium pervanadate (purple), with 10 μM selumetinib (blue), or with 100 μM sodium pervanadate, plus 10 μM selumetinib (green) as indicated. Grey histograms, unstained controls; black line, secondary antibody only. Single analysis.</p

Western blot analysis shows pErk reduction through selumetinib treatment and by removal of stromal support.

<p>ICNO6 and TXL2 ALL cells were cultured for 4 hrs in αMEM + 20% FBS on OP9 stroma or in αMEM + 1% BSA without OP9 stroma, and then treated in the same media for an additional 4 hours with 10 μM selumetinib. Western blots were incubated with the antibodies indicated in the panel. Gapdh, loading control. The membrane was sequentially stripped and re-probed with antibodies.</p

Viability and proliferation of ALL cells treated with trametinib and CAL101.

<p>US7 or TXL2 cells (10⁶ cells) as indicated were treated for 72 hours with 10 μM trametinib, 10 μM CAL101, or a combination of the two. Viability and cell counts were determined by Trypan blue exclusion. Cells were either exposed to drug while on stroma (A) or without stroma (B) in complete medium with 20% FBS. Error bars, mean ±SEM of triplicate values. *p<0.05; **p<0.01, one-way ANOVA.</p

Evaluation of selumetinib and trametinib Mek1/2 inhibitors as mono-treatment on BCP-ALL cell viability in the presence and absence of stroma.

<p>The indicated ALLs were treated with different μM concentrations of selumetinib or trametinib for 72 hours in the presence (A) or absence (B) of irradiated OP9 stromal support. Note: “0” values were determined on one sample set but are shown twice, for selumetinib and trametinib, for clarity. (C) 72-hour treatment of primary LAX56 with selumetinib as indicated. Viability (left panels) and cell counts (right panels) were determined by Trypan Blue exclusion. Error bars, SD of values measured in triplicate wells. One of two experiments for US7 and TXL2 with similar results. *p<0.05 **p<0.01, one-way ANOVA.</p

Mek pathway inhibition in combination-treated BCP-ALL cells by phospho-flow and Western blot.

<p>(A) Phospho-flow on US7 cells using pErk1/2 (CST) or pMek (BD) antibodies in the left and right panels respectively, as indicated, in the presence or absence of trametinib, CAL101 or both. All samples were starved overnight in X-Vivo15 medium (t 0 samples), then treated from t 0 onward for 4 hours with αMEM + 20% FBS and OP9 stroma (‘stimulated’), or with αMEM + 1% BSA in the absence of stroma (‘starved’). At t 0 solvent DMSO or 10 μM of the indicated drugs was also added to the samples. Error bars, mean +/- SEM of three independent experiments. *p<0.05 **p<0.01, Student's t-test. (B) Western blot analysis (single analysis) of US7 cells grown for 24 hours in αMEM + 20% FBS and OP9 stroma (‘OP9’ samples, left panel), or in αMEM + 1% BSA in the absence of stroma (‘no OP9’ samples, right panel). Cells were then additionally treated for 4 hours with solvent DMSO (control) or 10 μM of the indicated drugs. Gapdh, loading control. Western blot membrane was sequentially stripped and reprobed with antibodies.</p