What we talk about when we talk about "global mindset": managerial cognition in multinational corporations

Recent developments in the global economy and in multinational corporations have placed significant emphasis on the cognitive orientations of managers, giving rise to a number of concepts such as “global mindset” that are presumed to be associated with the effective management of multinational corporations (MNCs). This paper reviews the literature on global mindset and clarifies some of the conceptual confusion surrounding the construct. We identify common themes across writers, suggesting that the majority of studies fall into one of three research perspectives: cultural, strategic, and multidimensional. We also identify two constructs from the social sciences that underlie the perspectives found in the literature: cosmopolitanism and cognitive complexity and use these two constructs to develop an integrative theoretical framework of global mindset. We then provide a critical assessment of the field of global mindset and suggest directions for future theoretical and empirical research

Designing stem-cell-based dopamine cell replacement trials for Parkinson's disease

Clinical studies of Parkinson’s disease (PD) using a dopamine cell replacment strategy have been tried for more than 30 years. The outcomes following transplantation of human fetal ventral mesencephalic tissue (hfVM) have been variable, with some patients coming off their anti-PD treatment for many years and others not responding and/or developing significant side effects, including graft-induced dyskinesia. This led to a re-appraisal of the best way to do such trials, which resulted in a new European-Union-funded allograft trial with fetal dopamine cells across several centers in Europe. This new trial, TRANSEURO (NCT01898390), is an open-label study in which some individuals in a large observational cohort of patients with mild PD who were undergoing identical assessments were randomly selected to receive transplants of hfVM. The TRANSEURO trial is currently ongoing as researchers have completed both recruitment into a large multicenter observational study of younger onset early-stage PD and transplantation of hfVM in 11 patients. While completion of TRANSEURO is not expected until 2021, we feel that sharing the rationale for the design of TRANSEURO, along with the lessons we have learned along the way, can help inform researchers and facilitate planning of transplants of dopamine-producing cells derived from human pluripotent stem cells for future clinical trials

The growth and aggressive behavior of human osteosarcoma is regulated by a CaMKII-controlled autocrine VEGF signaling mechanism.

Osteosarcoma (OS) is a hyperproliferative malignant tumor that requires a high vascular density to maintain its large volume. Vascular Endothelial Growth Factor (VEGF) plays a crucial role in angiogenesis and acts as a paracrine and autocrine agent affecting both endothelial and tumor cells. The alpha-Ca2+/Calmodulin kinase two (α-CaMKII) protein is an important regulator of OS growth. Here, we investigate the role of α-CaMKII-induced VEGF in the growth and tumorigenicity of OS. We show that the pharmacologic and genetic inhibition of α-CaMKII results in decreases in VEGF gene expression (50%) and protein secretion (55%), while α- CaMKII overexpression increases VEGF gene expression (250%) and protein secretion (1,200%). We show that aggressive OS cells (143B) express high levels of VEGF receptor 2 (VEGFR-2) and respond to exogenous VEGF (100nm) by increasing intracellular calcium (30%). This response is ameliorated by the VEGFR inhibitor CBO-P11, suggesting that secreted VEGF results in autocrine stimulated α-CaMKII activation. Furthermore, we show that VEGF and α-CaMKII inhibition decreases the transactivation of the HIF-1α and AP-1 reporter constructs. Additionally, chromatin immunoprecipitation assay shows significantly decreased binding of HIF-1α and AP-1 to their responsive elements in the VEGF promoter. These data suggest that α-CaMKII regulates VEGF transcription by controlling HIF-1α and AP-1 transcriptional activities. Finally, CBO-P11, KN-93 (CaMKII inhibitor) and combination therapy significantly reduced tumor burden in vivo. Our results suggest that VEGF-induced OS tumor growth is controlled by CaMKII and dual therapy by CaMKII and VEGF inhibitors could be a promising therapy against this devastating adolescent disease

VEGF expression is positively regulated by CaMKII in human OS.

<p>143B cells were transduced with lentiviruses expressing either scrambled (shCtrl) or α-CaMKII- targeting shRNAs (shCaMKIIα) or treated with the CaMKII inhibitor KN-93. HOS cells were transduced with retroviruses expressing either GFP (GFP-Ctrl) or CaMKIIα (GFP- CaMKIIα). <b>A.</b> Real-time PCR was performed using primers specific for VEGF or β-Actin. Values were obtained from three separate experiments each repeated in triplicate and represent the mean ±S.D. *p<0.01. <b>B.</b> α-CaMKII-inhibited 143B (shCaMKIIα or KN-93) and α-CaMKII overexpressing HOS (GFP-CaMKIIα) cells were seeded at 1 x 10⁵ cells per well of a 6-well plate. Twenty-four hours later, aliquots of supernatant were examined for human VEGF by ELISA. Values were obtained from three separate experiments each repeated in triplicate and represent the mean ±S.D. *p<0.01. <b>C.</b> α-CaMKII inhibited 143B (shCaMKIIα or KN-93) and α- CaMKII overexpressing HOS (GFP-CaMKIIα) cells were seeded at 1 x 10⁵ cells per well of a 6- well plate. Twenty-four hours later, aliquots of conditioned media were removed and added to 12-well plates seeded with 1 x 10⁵ HUVEC cells. Endothelial cell tube-like formation was measured at 12 h. Negative controls of non-conditioned culture media (DMEM + 10% FBS) and conditioned media with VEGF immunoprecipitated out (VEGF IP) were also included. Representative photomicrographs were taken at 50x magnification from 3 independent experiments, each repeated in triplicate. Scale bar = 50 μm <b>D.</b> Capillary tube-like length was quantified using the ImageJ software. Values were obtained from three separate experiments each repeated in triplicate and represent the mean ±S.D. *p<0.01.</p

Inhibition of VEGF and CaMKII in OS cells dramatically decreases tumor growth <i>in vivo</i>.

<p>143B cells were transduced with lentiviruses expressing firefly luciferase, injected intratibially and allowed to grow for 1 week. ALZET micro-osmotic pumps were subcutaneously implanted into mice delivering saline (vehicle), CBO-P11 (2 mg/kg/day), KN-93 (1 mg/kg/day), or both CBO-P11 and KN-93. <b>A.</b> Luciferase imaging was performed before treatment (W0) and 2 weeks after treatment (W2) (n = 8). <b>B.</b> Tumor size was measured based on the luminescence intensity and graphed. Values were obtained from 8 mice from each group and represent the mean ± SD. *P < 0.01. <b>C.</b> Mice were euthanized and tibiae were collected. Tibiae were scanned by μ-CT, with arrows indicating areas of bone erosion. Hematoxylin and eosin staining was performed on paraffin embedded tumors. Broken lines indicate the boundary of the tumors and separating it from normal bone microenvironment. Images were taken at 40x magnification. IHC staining using specific antibodies directed against CD31 and Ki-67 (brown) were performed. Images were taken at 200x magnification and are representative of 8 different mice. Scale bar = 50 μm.</p

VEGF expression and secretion are increased in the highly aggressive OS cell lines in comparison with non-aggressive OS cell lines.

<p><b>A.</b> Real-time PCR was performed using primers specific for VEGF or β-Actin in the human OS cell lines HOS, MG-63, MNNG/HOS and 143B. Values were obtained from three separate experiments each repeated in triplicate and represent the mean ±S.D. *p<0.01. <b>B.</b> HOS, MG-63, MNNG/HOS and 143B human OS cells were seeded at 1 x 10⁵ cells per well on a 6-well plate. Twenty-four hours later, conditioned media were examined for the presence of human VEGF by ELISA. Values were obtained from three separate experiments each replicated in triplicate and represent the mean ±S.D. *p<0.01. <b>C.</b> HOS, MG-63, MNNG/HOS and 143B human OS cells were seeded at 1 x 10⁵ cells per well on a 6-well plate. Twenty-four hours later, aliquots of supernatant were removed from dishes and added to 12-well plates seeded with 1 x 10⁵ HUVEC cells. Endothelial cell tube-like formation was examined at 12 h. Negative controls of non-conditioned culture media (DMEM + 10% FBS) and conditioned media depleted from VEGF were also included. Representative photomicrographs were taken at 50x magnification from 3 independent experiments, each repeated in triplicate. Scale bar = 50 μm <b>D.</b> Capillary tube-like length was quantified using the ImageJ software. Values were obtained from three separate experiments each repeated in triplicate and represent the mean ±S.D. *p<0.01.</p

Exogenous VEGF partially rescues normal cell phenotype in α-CaMKII silenced 143B OS cells.

<p>143B cells were transduced with lentiviruses expressing either scrambled (shCtrl) or α-CaMKII- targeting shRNAs (shCaMKIIα) <b>A.</b> MTT assay (left panel) was performed at days 1, 2, 3, 4 and 5 to determine the number of viable cells. 5x10³ 143B cells were seeded in a 96-well plate. At 24 hours after seeding, cells were treated with 100nM VEGF. Values were obtained from three separate experiments, each repeated in triplicate and represent the mean ± S.D. *p<0.01. Trypan blue exclusion assay (right panel) was performed at day 5 to determine the live cell number. At 24 hours after seeding, cells were treated with 100nM VEGF every 24 hours. Values were obtained from three separate experiments, each repeated in triplicate and represent the mean ± S.D. *p<0.01. <b>B.</b> Scratch/wound healing assay was performed on cells cultured for 12 hours. The width between the scratched areas at hour 0 was set to 100%. Representative photomicrographs were taken at 50x magnification from 3 independent experiments, each repeated in triplicate. Values represent the mean ± S.D. *p<0.01. <b>C</b>. Trans-well invasion assay allowing cells to invade for 24 hours. Representative photomicrographs were taken at 100x magnifications from 3 independent experiments, each repeated in duplicate. Values represent the mean ± S.D. *p<0.01.</p

CaMKII controls VEGF gene expression by regulating TRE and HRE.

<p><b>A.</b> Schematic illustration of the VEGF promoter showing the binding sites for the AP-1 and HIF-1α transcription factors <b>B.</b> Reporter activation assay. Cells were transfected with TRE and HRE luciferase constructs and treated with 10 μM KN-93 and/or 1 μM CBO-P11 for 24 hours and harvested 24 hours later for luciferase activity measurement. Data are expressed relative to total protein, and values represent the mean ± SD of 3 separate experiments each repeated in triplicate; *p≤ 0.01. <b>C.</b> Quantification of western blot analyses. 143B cells were treated with 10 μM KN-93 and/or 1 μM CBO-P11 for 24 hours. Immunoblots were developed using specific antibodies directed against p-c-Jun, c-Fos, HIF-1α, Lamin B1 or β-Actin. Band density was measured using imageJ software and normalized to β-Actin. <b>D.</b> ChIP assay. DNA-protein complexes were immunoprecipitated with antibodies against AP-1 (c-Fos), HIF-1α and normal rabbit IgG as a control. Immunoprecipitated DNA fragments were amplified by PCR using primers encompassing the TRE and HRE binding sites on the VEGF promoter. Three independent experiments were performed. <b>E.</b> Quantitative analyses of the endogenous VEGF Expression. Real-time PCR was performed using primers specific for VEGF or β-Actin in human OS cell lines treated with 10 μM KN-93 and/or 1μM CBO-P11 for 24 hours. Values were obtained from three separate experiments each replicated in triplicate and represent the mean ±S.D. *p<0.01.</p

VEGF acts on 143B human OS cells in an autocrine manner.

<p><b>A.</b> VEGF receptors were identified by western blot. Immunoblots were developed using specific antibodies directed against VEGFR-2, VEGFR-1 or β-Actin. The autoradiograph is representative of three experiments. <b>B.</b> Detection of [Ca²⁺]_i. 143B cells were incubated with 5μM of Fluo-4-AM and then treated with 100nM VEGF (upper panel) or 1μM CBO-P11 (lower panel). BSA was used as control. Relative fluorescence intensities at four regions-of-interests were measured, with the average fluorescent intensity plotted versus time. Values were obtained from three separate experiments each repeated in triplicate and represent the mean ±S.D. *p<0.01. <b>C.</b> Activation of the CaMKII signaling. 143B Cells were treated with 10 μM KN-93 and/or 1 μM CBO-P11 for 24 hours. Immunoblots were developed using specific antibodies directed against p-α-CaMKII, α-CaMKII, p-CREB or CREB. The autoradiographs are representative of three independent experiments.</p