The effect of case nodes in problem-based learning on the length and quality of discussion: a 2x2 factorial study.

Background: Problem-based learning (PBL) relies heavily on case structure for their success. To make more meaningful cases, faculty introduced a "case node" that requires students to make a group decision on the action they will take at a given point in the case. The purpose of this study was to determine whether case nodes enhance PBL discussions. Methods: Two PBL cases were designed with and without a node. In 2011, 2012, and 2015, first-year medical students were assigned one PBL case with a node and one without a node. In total, 26 groups processed cases with a node while 27 groups processed the same cases without the node. All sessions were audio recorded and analyzed to determine the length and quality of discussions. Results: Groups with a node, regardless of case (M = 25.62, SD = 12.25) spent significantly more time in discussion on the node topic than those without a node (M = 16.54, SD = 10.33, p=.005, d = .80). Groups with a node, regardless of case (M = 14.38, SD = 8.04) expressed an opinion significantly more frequently than those without a node (M = 6.07, SD = 5.80, p < .001, d = 1.19). Conclusions: Case nodes increased both the length and depth of discussion on a topic and may be an effective way to enhance case-based instruction.

Contexte: Le succès de l'apprentissage par problèmes (APP) repose en grande partie sur la structure des cas. Pour rendre les cas plus significatifs, les membres du corps professoral ont introduit dans les scénarios un «nœud¼, ou un point nodal, marquant un moment où les étudiants doivent prendre une décision de groupe quant à l'action à entreprendre. L'objectif de cette étude était de déterminer si les cas comportant de tels points nodaux amélioraient la discussion dans le cadre de l'APP. Méthodes: On a conçu deux cas d'APP en deux versions, l'une comportant un nœud, l'autre non. En 2011, 2012 et 2015, on a soumis à des étudiants en première année de médecine un cas d'APP avec un nœud et un cas sans nœud. Au total, 26 groupes ont travaillé sur le cas avec un nœud et 27 groupes sur le cas sans nœud. Toutes les séances ont été enregistrées et analysées afin de déterminer la durée et la qualité des discussions. Résultats: Les groupes qui ont travaillé sur un cas comportant un nœud, quel que soit le cas (M = 25.62, SD = 12.25), ont consacré significativement plus de temps à la discussion que ceux qui avaient un cas sans nœud (M = 16.54, SD =1 0.33, p = .005, d = .80). Les premiers ont également exprimé des opinions significativement plus fréquemment, quel que soit le cas (M = 14.38, SD = 8.04), que les seconds (M = 6.07, SD = 5.80, p < .001, d = 1.19). Conclusions: Les nœuds introduits dans les cas ont entraîné des discussions à la fois plus longues et plus approfondies sur le sujet abordé. Par conséquent, ils constitueraient un moyen efficace d'améliorer l'enseignement fondé sur l'étude de cas.

The role of lysyl oxidase family members in the stabilization of abdominal aortic aneurysms.

Abdominal aortic aneurysms (AAAs) are a major cause of morbidity and mortality in the United States today. We employed a model for AAA development using apolipoprotein E knock out mice fed a high-fat diet and treated with ANG II and ß-aminopropionitrile (ß-APN) for 4 wk. ANG II induces hypertension and atherosclerotic disease, whereas ß-APN inhibits the activity of the lysyl oxidase/ lysyl oxidase-like protein (LOX/LOXL) family members. LOX/LOXL family members crosslink collagen and elastin in the extracellular matrix and therefore contribute to the integrity and stabilization of a healthy vessel wall. In this model, cotreatment with ANG II and ß-APN caused a 90% AAA incidence and increased atherosclerotic lesion formation from less than 5% to greater than 25% after 4 wk. In more atheroprotected mouse strains (C57BL/6 and BalbC), cotreatment with ANG II and ß-APN caused 50% and 40% AAA incidence, respectively. These data demonstrate the importance of LOX/LOXL to the stability of the vessel wall. Therapeutic strategies to overexpress LOX/LOXL enzymes or to support the crosslinking of soluble matrix proteins in a polymeric scaffold are a promising opportunity to achieve stabilization of AAAs.

Matrix crosslinking forces tumor progression by enhancing integrin signaling.

Tumors are characterized by extracellular matrix (ECM) remodeling and stiffening. The importance of ECM remodeling to cancer is appreciated; the relevance of stiffening is less clear. We found that breast tumorigenesis is accompanied by collagen crosslinking, ECM stiffening, and increased focal adhesions. Induction of collagen crosslinking stiffened the ECM, promoted focal adhesions, enhanced PI3 kinase (PI3K) activity, and induced the invasion of an oncogene-initiated epithelium. Inhibition of integrin signaling repressed the invasion of a premalignant epithelium into a stiffened, crosslinked ECM and forced integrin clustering promoted focal adhesions, enhanced PI3K signaling, and induced the invasion of a premalignant epithelium. Consistently, reduction of lysyl oxidase-mediated collagen crosslinking prevented MMTV-Neu-induced fibrosis, decreased focal adhesions and PI3K activity, impeded malignancy, and lowered tumor incidence. These data show how collagen crosslinking can modulate tissue fibrosis and stiffness to force focal adhesions, growth factor signaling and breast malignancy.

Lysyl oxidase-like 2 promotes migration in noninvasive breast cancer cells but not in normal breast epithelial cells.

A growing number of studies indicate the importance of the lysyl oxidase family in the promotion of epithelial neoplasms towards their more aggressive forms. However, the role of individual family members in carcinoma progression has yet to be ascertained. In this study, we analyzed LOXL2 expression in malignantly transformed MCF-7 and normal MCF-10A mammary epithelial cell line clones stably transduced with LOXL2 in vitro, and in normal and cancerous breast tissue samples in vivo. We found LOXL2 to be catalytically active in both MCF-7 and MCF-10 clones. LOXL2 overexpression promoted a more mesenchymal morphology in both cell types, but LOXL2-induced increase in migratory ability could only be established in MCF-7 clones. We demonstrated altered localization of the LOXL2 protein in breast cancer tissue compared to normal mammary tissue, and altered localization and processing of LOXL2 protein in breast cancer cell lines compared to normal cell lines, which may allow LOXL2 to interact with different intra and extracellular components during tumor progression. Results support the role of LOXL2 in selectively promoting a metastatic phenotype in breast tumor cells. Additional data suggest epigenetic molecular mechanisms in tumor specific regulation of LOXL2 expression that could be explored as a molecular target in the prevention of breast cancer progression.

Lysyl oxidase-like 2 expression is increased in colon and esophageal tumors and associated with less differentiated colon tumors.

Lysyl oxidase-like 2 (LOXL2) belongs to an amine oxidase family whose members have been implicated in crosslink formation in stromal collagens and elastin, cell motility, and tumor development and progression. We previously demonstrated the association between increased LOXL2 expression and invasive/metastatic behavior in human breast cancer cells and mouse squamous and spindle cell carcinomas, interaction between LOXL2 and SNAIL in epithelial-mesenchymal transition, and localization of the LOXL2 gene to 8p21.2-21.3, within a minimally deleted region in several cancers, including colon and esophagus. In the present study, we analyzed LOXL2 expression in colon and esophageal tumors, and explored methylation as a regulator of LOXL2 expression. Immunohistochemistry using normal tissues demonstrated intracellular localization of LOXL2 in colonic enteroendocrine cells and esophageal squamous cells at the luminal surface, but not in mitotically active cells. Tissue array analysis of 52 colon adenocarcinomas and 50 esophageal squamous cell carcinomas revealed presence of LOXL2 expression in 83 and 92% of the samples, respectively, and a significant association between increased number of LOXL2-expressing cells and less-differentiated colon carcinomas. We determined that the methylation status of the 1150 bp 5' CpG island may contribute to the regulation of the gene. Loss of heterozygosity studies, using a microsatellite within intron 4 of the LOXL2 gene, revealed that loss of LOXL2 was unlikely to play a major role in either colon or esophageal tumors. These results suggest that increased LOXL2 expression in colon and esophageal cancer may contribute to tumor progression.

Lysyl oxidase regulates breast cancer cell migration and adhesion through a hydrogen peroxide-mediated mechanism.

We have previously shown that lysyl oxidase (LOX) mRNA is up-regulated in invasive breast cancer cells and that catalytically active LOX facilitates in vitro cell invasion. Here we validate our in vitro studies by showing that LOX expression is up-regulated in distant metastatic breast cancer tissues compared with primary cancer tissues. To elucidate the mechanism by which LOX facilitates cell invasion, we show that catalytically active LOX regulates in vitro motility/migration and cell-matrix adhesion formation. Treatment of the invasive breast cancer cell lines, Hs578T and MDA-MB-231, with beta-aminopropionitrile (betaAPN), an irreversible inhibitor of LOX catalytic activity, leads to a significant decrease in cell motility/migration and adhesion formation. Conversely, poorly invasive MCF-7 cells expressing LOX (MCF-7/LOX32-His) showed an increase in migration and adhesion that was reversible with the addition of betaAPN. Moreover, a decrease in activated focal adhesion kinase (FAK) and Src kinase, key proteins involved in adhesion complex turnover, was observed when invasive breast cancer cells were treated with betaAPN. Additionally, FAK and Src activation was increased in MCF-7/LOX32-His cells, which was reversible on betaAPN treatment. Hydrogen peroxide was produced as a by-product of LOX activity and the removal of hydrogen peroxide by catalase treatment in invasive breast cancer cells led to a dose-dependent loss in Src activation. These results suggest that LOX facilitates migration and cell-matrix adhesion formation in invasive breast cancer cells through a hydrogen peroxide-mediated mechanism involving the FAK/Src signaling pathway. These data show the need to target LOX for treatment of aggressive breast cancer.

Drosophila lysyl oxidases Dmloxl-1 and Dmloxl-2 are differentially expressed and the active DmLOXL-1 influences gene expression and development.

Mammalian lysyl oxidase (LOX) is essential for the catalysis of lysyl-derived cross-links in fibrillar collagens and elastin in the extracellular matrix and has also been implicated in cell motility, differentiation, and tumor cell invasion. The active LOX has been shown to translocate to the nuclei of smooth muscle cells and regulate chromatin structure and transcription. It is difficult to interpret the role of the LOX protein as it is co-expressed with other members of the LOX amine oxidase family in most mammalian cells. To investigate the function of the LOX proteins, we have characterized the Drosophila lysyl oxidases Dmloxl-1 and Dmloxl-2. We present the gene, domain structure, and expression pattern of Dmloxl-1 and Dmloxl-2 during development. In early development, only Dmloxl-1 was expressed, which allowed functional studies. We have expressed Dmloxl-1 in S2 cells and determined that it is a catalytically active enzyme, inhibited by beta-amino-proprionitrile (BAPN), a specific LOX inhibitor. We localized DmLOXL-1 in the nuclei in embryos and in adult salivary gland cells in the nuclei, cytoplasm, and cell surface, using immunostaining and a DmLOXL-1 antibody. To address the biological function of Dmloxl-1, we raised larvae under BAPN inhibitory conditions and over-expressed Dmloxl-1 in transgenic Drosophila. DmLOXL-1 inhibition resulted in developmental delay and a shift in sex ratio; over-expression in the w(m4) variegating strain increased drosopterin production, demonstrating euchromatinization. Our previous data on the transcriptional down-regulation of seven ribosomal genes and the glue gene under inhibitory conditions and the current results collectively support a nuclear role for Dmloxl-1 in euchromatinization and gene regulation.

A molecular role for lysyl oxidase in breast cancer invasion.

We identified previously an up-regulation in lysyl oxidase (LOX) expression,an extracellular matrix remodeling enzyme, in a highly invasive/metastatic human breast cancer cell line, MDA-MB-231, compared with MCF-7, a poorly invasive/nonmetastatic breast cancer cell line. In this study, we demonstrate that the mRNA expression of LOX and other LOX family members [lysyl oxidase-like (LOXL), LOXL2, LOXL3, and LOXL4] was observed only in breast cancer cells with a highly invasive/metastatic phenotype but not in poorly invasive/nonmetastatic breast cancer cells. LOX and LOXL2 showed the strongest association with invasive potential in both highly invasive/metastatic breast cancer cell lines tested (MDA-MB-231 and Hs578T). To determine whether LOX is directly involved in breast cancer invasion, LOX antisense oligonucleotides were transfected into MDA-MB-231 and Hs578T cells, and found to inhibit invasion through a collagen IV/laminin/gelatin matrix in vitro compared with LOX sense oligonucleotide-treated and untreated controls. In addition, treatment of MDA-MB-231 and Hs578T cells with beta-aminopropionitrile (an irreversible inhibitor of LOX enzymatic activity) decreased invasive activity. Conversely, MCF-7 cells transfected with the murine LOX gene demonstrated a 2-fold increase in invasiveness that was reversible by the addition of beta-aminopropionitrile in a dose-dependent manner. In addition, endogenous LOX mRNA expression was induced when MCF-7 cells were cultured in the presence of fibroblast conditioned medium or conditioned matrix, suggesting a role for stromal fibroblasts in LOX regulation in breast cancer cells. Moreover, the correlation of LOX up-regulation and invasive/metastatic potential was additionally demonstrated in rat prostatic tumor cell lines, and human cutaneous and uveal melanoma cell lines. These results provide substantial new evidence that LOX is involved in cancer cell invasion.