Going beyond defining: Preschool educators' use of knowledge in their pedagogical reasoning about vocabulary instruction.

Previous research investigating both the knowledge of early childhood educators and the support for vocabulary development present in early childhood settings has indicated that both educator knowledge and enacted practice are less than optimal, which has grave implications for children's early vocabulary learning and later reading achievement. Further, the nature of the relationship between educators' knowledge and practice is unclear, making it difficult to discern the best path towards improved knowledge, practice, and children's vocabulary outcomes. The purpose of the present study was to add to the existing literature by using stimulated recall interviews and a grounded approach to examine how 10 preschool educators used their knowledge to made decisions about their moment-to-moment instruction in support of children's vocabulary development. Results indicate that educators were thinking in highly context-specific ways about their goals and strategies for supporting vocabulary learning, taking into account important knowledge of their instructional history with children and of the children themselves to inform their decision making in the moment. In addition, they reported thinking about research-based goals and strategies for supporting vocabulary learning that went beyond simply defining words for children. Implications for research and professional development are discussed.

A novel synthetic analogue of ω-3 17,18-epoxyeicosatetraenoic acid activates TNF receptor-1/ASK1/JNK signaling to promote apoptosis in human breast cancer cells.

A saturated analog of the cytochrome P450-mediated ω-3-17,18-epoxide of ω-3-eicosapentaenoic acid (C20E) activated apoptosis in human triple-negative MDA-MB-231 breast cancer cells. This study evaluated the apoptotic mechanism of C20E. Increased cytosolic cytochrome c expression and altered expression of pro- and antiapoptotic B-cell lymphoma-2 proteins indicated activation of the mitochondrial pathway. Caspase-3 activation by C20E was prevented by pharmacological inhibition and silencing of the JNK and p38 MAP kinases (MAPK), upstream MAPK kinases MKK4 and MKK7, and the upstream MAPK kinase kinase apoptosis signal-regulating kinase 1 (ASK1). Silencing of the death receptor TNF receptor 1 (TNFR1), but not Fas, DR4, or DR5, and the adapters TRADD and TNF receptor-associated factor 2, but not Fas-associated death domain, prevented C20E-mediated apoptosis. B-cell lymphoma-2 homology 3-interacting domain death agonist (Bid) cleavage by JNK/p38 MAPK linked the extrinsic and mitochondrial pathways of apoptosis. In further studies, an antibody against the extracellular domain of TNFR1 prevented apoptosis by TNF-α but not C20E. These findings suggest that C20E acts intracellularly at TNFR1 to activate ASK1-MKK4/7-JNK/p38 MAPK signaling and to promote Bid-dependent mitochondrial disruption and apoptosis. In in vivo studies, tumors isolated from C20E-treated nu/nu mice carrying MDA-MB-231 xenografts showed increased TUNEL staining and decreased Ki67 staining, reflecting increased apoptosis and decreased proliferation, respectively. ω-3-Epoxy fatty acids like C20E could be incorporated into treatments for triple-negative breast cancers.-Dyari, H. R. E., Rawling, T., Chen, Y., Sudarmana, W., Bourget, K., Dwyer, J. M., Allison, S. E., Murray, M. A novel synthetic analogue of ω-3 17,18-epoxyeicosatetraenoic acid activates TNF receptor-1/ASK1/JNK signaling to promote apoptosis in human breast cancer cells.

The guanine exchange factor SWAP70 mediates vGPCR-induced endothelial plasticity.

BACKGROUND: The viral G protein-coupled receptor (vGPCR) is proposed to act as one of the predominant mediators of Kaposi's sarcoma (KS), a human herpes virus 8 (HHV8)-elicited disease. The actions of vGPCR manifest pathogenesis, in part, through increased permeability of endothelial cells. Endothelial cell-cell junctions have indeed emerged as an instrumental target involved in the vasculature defects observed within the tumor microenvironment. The pathway leading to adherens junction destabilization has been shown to involve the activation of the small GTPase Rac, in the context of either latent infection or the sole expression of vGPCR. However, the precise molecular mechanisms governed by vGPCR in vascular leakage require further elucidation. FINDINGS: Guanine exchange factors (GEFs) function as critical molecular switches that control the activation of small GTPases. We therefore screened the effects of 80 siRNAs targeting GEFs on vGPCR-driven endothelial permeability and identified switch-associated protein 70 (SWAP70) as necessary for its elevating effects. Pull-down experiments further showed that Rac activation by vGPCR was dependent on SWAP70. Examination of tissues and cells from HHV8-positive patients revealed that SWAP70 was ubiquitously expressed. Furthermore, SWAP70 was found to be crucial for vGPCR-driven endothelial tube formation and endothelial sprouting in vitro. CONCLUSIONS: SWAP70 appears to act as a molecular intermediate between vGPCR and endothelial activation. Because of the important role of vGPCR-mediated endothelial plasticity in KS pathogenesis, inhibition of SWAP70 function could be of interest for blocking vGPCR-driven activities in HHV8-defined diseases.

Control of CXCR2 activity through its ubiquitination on K327 residue.

BACKGROUND: The interleukin-8 chemokine (IL-8) G-protein coupled receptor CXCR2 governs pro-inflammatory and pro-angiogenic responses in leukocytes and endothelial cells. At a molecular standpoint, CXCR2 is widely reported to operate through calcium flux, phosphoinoisitide 3 kinase (PI3K) and mitogen-activated protein kinase (MAPK). While CXCR2 trafficking is suspected to be intertwined with its signaling, the exact mechanism is not fully elucidated. RESULTS: Here, we identified the lysine 327 within the CXCR2 C-terminal tail as a key residue for ubiquitination, internalization, and signaling. First, the substitution to an arginine of K327 mutation was associated with a reduction in CXCR2 poly-ubiquitination. While WT CXCR2 was rapidly internalized following IL-8 administration, K327R mutant remained at the plasma membrane. Finally, K327R mutant failed to promote the recruitment of ß-arrestin2, as estimated by imagery and bioluminescence resonance transfer. As a consequence, the activation of intracellular signaling, including both early events such as ERK phosphorylation and the increase in calcium flux, and the latter activation of the AP1 and NF-κB transcription factors, was blunted. CONCLUSIONS: Overall, our results demonstrate that CXCR2 ubiquitination on K327 residue modulates agonist-activated CXCR2 cell sorting and intracellular signaling. Thus, the inhibition of K327 ubiquitination might emerge as an effective mean to curb exacerbated CXCR2 signaling in several pathological conditions, such as inflammatory diseases and cancer.

The endoplasmic reticulum acts as a platform for ubiquitylated components of nuclear factor κB signaling.

The innate and adaptive immune responses involve the stimulation of nuclear factor κB (NF-κB) transcription factors through the Lys(63) (K(63))-linked ubiquitylation of specific components of NF-κB signaling pathways. We found that ubiquitylated components of the NF-κB pathway accumulated on the cytosolic leaflet of the endoplasmic reticulum (ER) membrane after the engagement of cell-surface, proinflammatory cytokine receptors or antigen receptors. Through mass spectrometric analysis, we found that the ER-anchored protein metadherin (MTDH) was a partner for these ubiquitylated activators of NF-κB and that it directly bound to K(63)-linked polyubiquitin chains. Knockdown of MTDH inhibited the accumulation of ubiquitylated NF-κB signaling components at the ER, reduced the extent of NF-κB activation, and decreased the amount of proinflammatory cytokines produced. Our observations highlight an unexpected facet of the ER as a key subcellular gateway for NF-κB activation.

Glioblastoma cell-secreted interleukin-8 induces brain endothelial cell permeability via CXCR2.

Glioblastoma constitutes the most aggressive and deadly of brain tumors. As yet, both conventional and molecular-based therapies have met with limited success in treatment of this cancer. Among other explanations, the heterogeneity of glioblastoma and the associated microenvironment contribute to its development, as well as resistance and recurrence in response to treatments. Increased vascularity suggests that tumor angiogenesis plays an important role in glioblastoma progression. However, the molecular crosstalk between endothelial and glioblastoma cells requires further investigation. To examine the effects of glioblastoma-derived signals on endothelial homeostasis, glioblastoma cell secretions were collected and used to treat brain endothelial cells. Here, we present evidence that the glioblastoma secretome provides pro-angiogenic signals sufficient to disrupt VE-cadherin-mediated cell-cell junctions and promote endothelial permeability in brain microvascular endothelial cells. An unbiased angiogenesis-specific antibody array screen identified the chemokine, interleukin-8, which was further demonstrated to function as a key factor involved in glioblastoma-induced permeability, mediated through its receptor CXCR2 on brain endothelia. This underappreciated interface between glioblastoma cells and associated endothelium may inspire the development of novel therapeutic strategies to induce tumor regression by preventing vascular permeability and inhibiting angiogenesis.

Proteomes of umbilical vein and microvascular endothelial cells reflect distinct biological properties and influence immune recognition.

Human umbilical vein endothelial cells (HUVEC) are widely used as a source of endothelial cells (EC). However, HUVEC characteristics cannot be extrapolated to other types of EC, particularly microvascular ECs. Our objective was to compare the proteomes of microvascular ECs and HUVEC. Proteomes of HUVEC and human microvascular pulmonary EC (HMVEC-P) and dermal EC (HMVEC-D) from healthy Caucasian donors were compared by 2D DIGE and MS. Fatty acid binding proteins 4 and 5 were among the 159 and 30 proteins spots found to have at least twofold change in expression between HUVEC and HMVEC-D and between HUVEC and HMVEC-P samples, respectively. Eight protein spots showed twofold changed expression between HMVEC-D and HMVEC-P samples. Ingenuity® analysis revealed that proteins differentially expressed between HUVEC and HMVEC-D samples interact with retinoic acid. In vitro tubulogenesis assays showed a differential effect of retinoic acid between HUVEC and HMVEC. Moreover, serum IgG from patients with a rare vascular disease, systemic sclerosis, showed distinct reactivity profiles in HUVEC and HMVEC-D protein extracts. The proteome profiles of HUVEC and microvascular EC differ noticeably, which reflects distinct biological properties and influence immune recognition.

Semaphorin 3A elevates endothelial cell permeability through PP2A inactivation.

VE-cadherin-mediated cell-cell junction weakening increases paracellular permeability in response to both angiogenic and inflammatory stimuli. Although Semaphorin 3A has emerged as one of the few known anti-angiogenic factors to exhibit pro-permeability activity, little is known about how it triggers vascular leakage. Here we report that Semaphorin 3A induced VE-cadherin serine phosphorylation and internalisation, cell-cell junction destabilisation, and loss of barrier integrity in brain endothelial cells. In addition, high-grade glioma-isolated tumour-initiating cells were found to secrete Semaphorin 3A, which promoted brain endothelial monolayer permeability. From a mechanistic standpoint, Semaphorin 3A impinged upon the basal activity of the serine phosphatase PP2A and disrupted PP2A interaction with VE-cadherin, leading to cell-cell junction disorganization and increased permeability. Accordingly, both pharmacological inhibition and siRNA-based knockdown of PP2A mimicked Semaphorin 3A effects on VE-cadherin. Hence, local Semaphorin 3A production impacts on the PP2A/VE-cadherin equilibrium and contributes to elevated vascular permeability.

Jumping the barrier: VE-cadherin, VEGF and other angiogenic modifiers in cancer.

The endothelial barrier controls the passage of fluids, nutrients and cells through the vascular wall. This physiological function is closely related to developmental and adult angiogenesis, blood pressure control, as well as immune responses. Moreover, cancer progression is frequently characterized by disorganized and leaky blood vessels. In this context, vascular permeability drives tumour-induced angiogenesis, blood flow disturbances, inflammatory cell infiltration and tumour cell extravasation. Although various molecules have been implicated, the vascular endothelial adhesion molecule, VE-cadherin (vascular endothelial cadherin), has emerged as a critical player involved in maintaining endothelial barrier integrity and homoeostasis. Indeed, VE-cadherin coordinates the endothelial cell-cell junctions through its adhesive and signalling properties. Of note, many angiogenic and inflammatory mediators released into the tumour microenvironment influence VE-cadherin behaviour. Therefore restoring VE-cadherin function could be one very promising target for vascular normalization in cancer therapies. In this review, we will mainly focus on recent discoveries concerning the molecular mechanisms involved in modulating VE-cadherin plasticity in cancer.

Secreted factors from brain endothelial cells maintain glioblastoma stem-like cell expansion through the mTOR pathway.

Glioma stem-cells are associated with the brain vasculature. However, the way in which this vascular niche regulates stem-cell renewal and fate remains unclear. Here, we show that factors emanating from brain endothelial cells positively control the expansion of long-term glioblastoma stem-like cells. We find that both pharmacological inhibition of and RNA interference with the mammalian target of rapamycin (mTOR) pathway reduce their spheroid growth. Conversely, the endothelial secretome is sufficient to promote this mTOR-dependent survival. Thus, interfering with endothelial signals might present opportunities to identify treatments that selectively target malignant stem-cell niches.

Ets2 transcription factor, telomerase activity and breast cancer.

1. The enzyme telomerase maintains telomeres (ends of chromosomes) by synthesizing telomeric DNA at each end of the chromosomes. Its association with telomeres has implicated telomerase in cell immortalization. 2. Numerous studies have shown significant levels of telomerase activity in 85% of various types of cancer. Transcriptional control of the catalytic subunit, telomerase reverse transcriptase (TERT), dominates regulation of telomerase. Although several major factors have been identified in regulating TERT, they cannot explain all the transcriptional activity of the hTERT gene. 3. The Ets transcription factor (TF) family is becoming a regular feature in tumourigenesis, particularly in breast cancer. However, the roles and mechanisms of different Ets TFs are largely unknown. 4. The present minireview discusses the research that identified Ets as a regulator of telomerase required for breast cancer cell survival and proliferation, highlighting the discoveries central to understanding the molecular acts used by Ets TFs to mediate TERT gene transcription.

Ets2 maintains hTERT gene expression and breast cancer cell proliferation by interacting with c-Myc.

Human telomerase reverse transcriptase (hTERT) underlies cancer cell immortalization, and the expression of hTERT is regulated strictly at the gene transcription. Here, we report that transcription factor Ets2 is required for hTERT gene expression and breast cancer cell proliferation. Silencing Ets2 induces a decrease of hTERT gene expression and increase in human breast cancer cell death. Reconstitution with recombinant hTERT rescues the apoptosis induced by Ets2 depression. In vitro and in vivo analyses show that Ets2 binds to the EtsA and EtsB DNA motifs on the hTERT gene promoter. Mutation of either Ets2 binding site reduces the hTERT promoter transcriptional activity. Moreover, Ets2 forms a complex with c-Myc as demonstrated by co-immunoprecipitation and glutathione S-transferase pulldown assays. Immunological depletion of Ets2, or mutation of the EtsA DNA motif, disables c-Myc binding to the E-box, whereas removal of c-Myc or mutation of the E-box also compromises Ets2 binding to EtsA. Thus, hTERT gene expression is maintained by a mechanism involving Ets2 interactions with the c-Myc transcription factor and the hTERT gene promoter, a protein-DNA complex critical for hTERT gene expression and breast cancer cell proliferation.

Transcriptional regulation of telomerase activity: roles of the the Ets transcription factor family.

Telomerase maintains telomeres to preclude cell senescence. It remains elusive how telomerase activity is repressed in differentiated cells, but retained at high levels in stem cells and cancer. Recent studies suggest that the Ets transcription factor family, downstream of the mitogen signaling pathways of MAP kinase, regulates telomerase activity at the gene transcription level of human telomerase reverse transcriptase (hTERT). Several Ets transcription factors are involved in regulating hTERT gene expression, both directly and indirectly through the proto-oncogene c-myc. ER81 may mediate telomerase activation in telomerase-negative fibroblasts stimulated by oncogenes Her2/Neu, Ras, and Raf. Ets2 may also play an important role in regulating the hTERT gene; but further studies are required to decipher the mechanisms in the regulation of telomerase activity.