Developing a Cell-like Substrate to Investigate the Mechanosensitivity of Cell-to-Cell Junctions

Indiana University-Purdue University Indianapolis (IUPUI)The role of mechanical forces in the fate and function of adherent cells has been revealed to be a pivotal factor in understanding cell biology. Cells require certain physical cues to be present in their microenvironment or the cell will begin apoptosis. Mechanical signals from the environment are interpreted at the cellular level and biochemical responses are made due to the information from outside the cell, this process is known as mechanotransduction. Misinterpretation of physical cues has been indicated in many disease states, including heart disease and asthma. When a cell is bound to the ECM, proteins such as integrins are engaged at static and stable adhesion sites. These tight and static anchoring points found at the ECM exist in stark contrast to the dynamic conditions seen at intercellular junctions. Intercellular junctions, such as gap and adherens junctions, are formed between cells to act as a mechanism to relay information and exchange material. Due to the important role intercellular junctions play in processes of wound healing, epithelial-mesenchymal transition and cancer metastasis developing more sophisticated levels of understanding of these mechanisms would provide valuable insight. Complex biological processes, including immune cell signaling and cellular ECM adhesions, have been effectively replicated in model systems. These model systems have included the use of solid supported lipid bilayers and polymeric hydrogels that display cell adhesion molecules. Studies of cellular mechanotransduction at ECM adhesion sites has also been completed with covalently functionalized polymeric substrates of adjustable elasticity. However, developing model systems that allow the accurate reproduction of properties seen at intercellular junctions, while also allowing the investigation of cellular mechanosensitivity has proven to be a difficult task. Previous work has shown that polymer-tethered lipid bilayers (PTLBs) are a viable material to allow the replication of the dynamics and adhesion seen at intercellular junctions. Although efforts have been made to produce PTLBs with different mechanical properties, there is currently not a material with sufficient tunable elastic properties for the study of cellular mechanotransduction. To establish a system that allows the study of stiffness effects across a biologically relevant range (~0.50 – 40 kPa) while maintaining the dynamic properties seen at cell-to-cell junctions, polymer gel-tethered bilayers (PGTBs) were developed. A fabrication strategy was established to allow the incorporation of a hydrogel support with easily tunable stiffness and a tethered lipid bilayer coating, which produced a powerful platform to study the effects of stiffness at intercellular junctions. Careful attention was given to maintain the beneficial properties of membrane diffusion, and it was shown that on different linking architectures lipid bilayers could be established and diffusion was preserved. Microscopy-based FCS and FRAP methodology were utilized to measure lipid diffusion in these systems, while confocal microscopy was used to analyze cell spreading and adhesion. Three distinct architectures to link the lipid membrane to the underlying polyacrylamide hydrogel were pursued in this work, a non-covalent biotin-streptavidin system, a covalently linked design with fibronectin, and a direct covalent linkage utilizing crosslinker chemistry. In this work, it was shown that cells were able to spread and adhere on these substrates, with cell adhesion zones visualized under plated cells that demonstrate the capability of the cell to rearrange the presented linkers, while maintaining a stable material. Also confirmed is the tunability of the polymer hydrogel across a wide range of stiffness, this was shown by quantitative changes in cell spreading area in response to polymer properties

Homenaje al P. Silvio Broseghini

Las expresiones de Silvio ponen de manifiesto cuán trabajosamente elaboró el tema del encuentro de la teolo gía cristiana con el pensamiento indígena y cómo le preocupó enfocar la presencia misionera

Political culture on the northern plains : North Dakota and the Nonpartisan experience

This thesis investigates the history of North Dakota during the era of the Nonpartisan League (roughly 1915 through 1921). A significant body of research on the League exists, but no study has yet specifically addressed itself to exploring the political culture of the League and of its opponents in such a way that the full nature of the Nonpartisan revolt is made clear. The League was indeed the result of farmers’ perception of economic exploitation at the hands of big business, yet it was more basically a proactive struggle for inclusion. While employing elements of agrarian ideology, the League was also the product of a world dominated by the urban-based values of business. Leaguers hoped, through occupationally-based, collective political action to gain power, dignity, and material success within that world. Although this consciousness was a departure from the received political culture of the 1910s, in other ways the Leaguers\u27 political culture was fairly mainstream. The League\u27s program of political inclusion, for example, kept well within traditional gendered boundaries. Nor was the league vision of material progress revolutionary. Being a historical inquiry, the preparation of this thesis is the result of standard historical research methodologies. In terms of theory, however, this project has been informed by post-structuralist theories of “language. Thus, particular attention has been paid to the process of language building especially during the early years of the Nonpartisan era. How Leaguers and their opponents defined themselves and their world, inclusively and exclusively, provides a more subtle understanding of the Nonpartisan revolt

Pleistocene History and Glacio-tectonic Features in the Lack Megantic Region, Quebec

Guidebook for field trips in the Rangeley Lakes - Dead River Basin region, western Maine: 62nd annual meeting October 2, 3, and 4, 1970: Trip E-

Tunable cell-surface mimetics as engineered cell substrates

Most recent breakthroughs in understanding cell adhesion, cell migration, and cellular mechanosensitivity have been made possible by the development of engineered cell substrates of well-defined surface properties. Traditionally, these substrates mimic the extracellular matrix (ECM) environment by the use of ligand-functionalized polymeric gels of adjustable stiffness. However, such ECM mimetics are limited in their ability to replicate the rich dynamics found at cell-cell contacts. This review focuses on the application of cell surface mimetics, which are better suited for the analysis of cell adhesion, cell migration, and cellular mechanosensitivity across cell-cell interfaces. Functionalized supported lipid bilayer systems were first introduced as biomembrane-mimicking substrates to study processes of adhesion maturation during adhesion of functionalized vesicles (cell-free assay) and plated cells. However, while able to capture adhesion processes, the fluid lipid bilayer of such a relatively simple planar model membrane prevents adhering cells from transducing contractile forces to the underlying solid, making studies of cell migration and cellular mechanosensitivity largely impractical. Therefore, the main focus of this review is on polymer-tethered lipid bilayer architectures as biomembrane-mimicking cell substrate. Unlike supported lipid bilayers, these polymer-lipid composite materials enable the free assembly of linkers into linker clusters at cellular contacts without hindering cell spreading and migration and allow the controlled regulation of mechanical properties, enabling studies of cellular mechanosensitivity. The various polymer-tethered lipid bilayer architectures and their complementary properties as cell substrates are discussed

A Goal Setting Intervention Positively Impacts Adolescents’ Dietary Behaviors and Physical Activity Self-Efficacy

The efficacy of a youth development intervention on improving eating and physical activity(PA) self-efficacy, goal attainment scaling, goal effort, and behaviors was examined in a repeated measures, quasi-experimental field trial. Ethnically diverse students (n=64) from a low-income middle school participated in the 10-session intervention driven by the Social Cognitive Theory with a Goal Setting Theory emphasis. Participants, 13-14 years old, made significant changes in dietary behaviors (P=0.03) and PA self-efficacy (P=0.02) after receiving the intervention. Self-efficacy did not mediate dietary behavior change but did mediate the small changes made in PA. Goal effort was not a mediator of behavior change. After the intervention, more participants rated themselves as making one lasting improvement in eating (

Guided Goal Setting: A Feasible Obesity Prevention Behavior Change Strategy for Low-Income Parents with Young Children

To reduce the risk of pediatric obesity, behaviorally-focused parental education is needed. This study examined the feasibility of guided goal setting in a pediatric obesity prevention intervention for low-income parents of young children. Parents from Head Start participated in a six-week nutrition, activity, and parenting intervention that included guided goal setting (N = 47). At each session, data were collected on goal selection, effort, and attainment. Pre- and postintervention data were collected on nutrition, activity, and child feeding behaviors (n = 24). A subset of parents completed in-depth interviews (n = 20). Parents reported a high level of goal effort (88%) and achievement (79%) and a preference for goal personalization and a list of goals from which to choose. In general, parents did not find the goal contract or weekly tracking as motivating as goal personalization. Pre- and postintervention assessment showed significant changes in the dietary energy density (p = .008) and vegetable behavior (p = .04) subscales with a marginal change in the snacking subscale (p = .08). Guided goal setting was a feasible behavioral strategy for the parents in this study since parents engaged in the process and demonstrated changes in serving more vegetables and fewer energy-dense foods

A Goal Setting Intervention Positively Impacts Adolescents’ Dietary Behaviors and Physical Activity Self-Efficacy

The efficacy of a youth development intervention on improving eating and physical activity(PA) self-efficacy, goal attainment scaling, goal effort, and behaviors was examined in a repeated measures, quasi-experimental field trial. Ethnically diverse students (n=64) from a low-income middle school participated in the 10-session intervention driven by the Social Cognitive Theory with a Goal Setting Theory emphasis. Participants, 13-14 years old, made significant changes in dietary behaviors (P=0.03) and PA self-efficacy (P=0.02) after receiving the intervention. Self-efficacy did not mediate dietary behavior change but did mediate the small changes made in PA. Goal effort was not a mediator of behavior change. After the intervention, more participants rated themselves as making one lasting improvement in eating (

No evidence for basigin/CD147 as a direct SARS-CoV-2 spike binding receptor.

The spike protein of SARS-CoV-2 is known to enable viral invasion into human cells through direct binding to host receptors including ACE2. An alternate entry receptor for the virus was recently proposed to be basigin/CD147. These early studies have already prompted a clinical trial and multiple published hypotheses speculating on the role of this host receptor in viral infection and pathogenesis. Here, we report that we are unable to find evidence supporting the role of basigin as a putative spike binding receptor. Recombinant forms of the SARS-CoV-2 spike do not interact with basigin expressed on the surface of human cells, and by using specialized assays tailored to detect receptor interactions as weak or weaker than the proposed basigin-spike binding, we report no evidence for a direct interaction between the viral spike protein to either of the two common isoforms of basigin. Finally, removing basigin from the surface of human lung epithelial cells by CRISPR/Cas9 results in no change in their susceptibility to SARS-CoV-2 infection. Given the pressing need for clarity on which viral targets may lead to promising therapeutics, we present these findings to allow more informed decisions about the translational relevance of this putative mechanism in the race to understand and treat COVID-19

A Viral Protein Mediates Superinfection Exclusion at the Whole-Organism Level but Is Not Required for Exclusion at the Cellular Level

Superinfection exclusion (SIE), the ability of an established virus infection to interfere with a secondary infection by the same or a closely related virus, has been described for different viruses, including important pathogens of humans, animals, and plants. Citrus tristeza virus (CTV), a positive-sense RNA virus, represents a valuable model system for studying SIE due to the existence of several phylogenetically distinct strains. Furthermore, CTV allows SIE to be examined at the whole-organism level. Previously, we demonstrated that SIE by CTV is a virus-controlled function that requires the viral protein p33. In this study, we show that p33 mediates SIE at the whole-organism level, while it is not required for exclusion at the cellular level. Primary infection of a host with a fluorescent protein-tagged CTV variant lacking p33 did not interfere with the establishment of a secondary infection by the same virus labeled with a different fluorescent protein. However, cellular coinfection by both viruses was rare. The obtained observations, along with estimates of the cellular multiplicity of infection (MOI) and MOI model selection, suggested that low levels of cellular coinfection appear to be best explained by exclusion at the cellular level. Based on these results, we propose that SIE by CTV is operated at two levels-the cellular and the whole-organism levels-by two distinct mechanisms that could function independently. This novel aspect of viral SIE highlights the intriguing complexity of this phenomenon, further understanding of which may open up new avenues to manage virus diseases.Peer reviewe