Chitosan-Graft-Branched Polyethylenimine Copolymers: Influence of Degree of Grafting on Transfection Behavior

BACKGROUND: Successful non-viral gene delivery currently requires compromises to achieve useful transfection levels while minimizing toxicity. Despite high molecular weight (MW) branched polyethylenimine (bPEI) is considered the gold standard polymeric transfectant, it suffers from high cytotoxicity. Inversely, its low MW counterpart is less toxic and effective in transfection. Moreover, chitosan is a highly biocompatible and biodegradable polymer but characterized by very low transfection efficiency. In this scenario, a straightforward approach widely exploited to develop effective transfectants relies on the synthesis of chitosan-graft-low MW bPEIs (Chi-g-bPEI(x)) but, despite the vast amount of work that has been done in developing promising polymeric assemblies, the possible influence of the degree of grafting on the overall behavior of copolymers for gene delivery has been largely overlooked. METHODOLOGY/PRINCIPAL FINDINGS: With the aim of providing a comprehensive evaluation of the pivotal role of the degree of grafting in modulating the overall transfection effectiveness of copolymeric vectors, we have synthesized seven Chi-g-bPEI(x) derivatives with a variable amount of bPEI grafts (minimum: 0.6%; maximum: 8.8%). Along the Chi-g-bPEI(x) series, the higher the degree of grafting, the greater the ζ-potential and the cytotoxicity of the resulting polyplexes. Most important, in all cell lines tested the intermediate degree of grafting of 2.7% conferred low cytotoxicity and higher transfection efficiency compared to other Chi-g-bPEI(x) copolymers. We emphasize that, in transfection experiments carried out in primary articular chondrocytes, Chi-g-bPEI(2.7%) was as effective as and less cytotoxic than the gold standard 25 kDa bPEI. CONCLUSIONS/SIGNIFICANCE: This work underlines for the first time the pivotal role of the degree of grafting in modulating the overall transfection effectiveness of Chi-g-bPEI(x) copolymers. Crucially, we have demonstrated that, along the copolymer series, the fine tuning of the degree of grafting directly affected the overall charge of polyplexes and, altogether, had a direct effect on cytotoxicity

Motivations of non-Buddhists visiting Buddhist temples

The current study employs the leisure motivation scale to examine motivations of non- Buddhists visiting Buddhist temples. Specifically, this investigation builds on tourism literature to explore the motivations of non-Buddhists visiting Buddhist temples in Los Angeles, California. Motivations to Buddhist temples are of particular interest given the increasing popularity in the West of Eastern spiritual activities, such as yoga and meditation, as well as the exponential growth of Buddhist-themed tourism campaigns. The findings provide insights for tourism officials responsible for promoting ways to attract tourists to Buddhist temples within their respective destinations

Access to parks and recreational facilities, physical activity, and health care costs for older adults: Evidence from U.S. counties

© 2019, © 2019 National Recreation and Park Association. Although research has documented the health benefits of physical activity and use of park and recreational facilities, the relationships of these factors to healthcare costs remain unexplored at the population level. Building upon a social-ecological model, we analyzed county-level data to examine the extent to which physical activity rate and access to parks and recreational facilities were related to the healthcare costs for older adults (i.e., those 65 years and above) in U.S. counties. The results revealed that older adults’ physical activity rate in a county was negatively associated with the county’s healthcare costs of these adults. Also, access to parks and recreational facilities was negatively associated with older adults’ healthcare costs through the physical activity rate. These findings indicate that access to parks and recreational facilities correlates with increased physical activity levels among older adults, which might reduce their healthcare costs in communities

Peeling back the layers: Deconstructing information literacy discourse in higher education

The discourses of information literacy practice create epistemological assumptions about how the practice should happen, who should be responsible and under what conditions instruction should be given. Analysis of a wide range of documents and texts emerging from the Higher Education (HE) sector suggest that information literacy (IL) is shaped by two competing and incongruent narratives. The outward facing narrative of information literacy (located in information literacy standards and guidelines) positions information literacy as an empowering practice that arms students with the knowledge and skills to battle the complexity of the modern information world. In contrast, the inward facing narrative (located in information literacy texts) positions students as lacking appropriate knowledge, skills and agency. This deficit perception, which has the capacity to influence pedagogical practice, is at odds with constructivist and action-oriented views that are espoused within information literacy instructional pedagogy. This presentation represents the first paper in a research programme that interrogates the epistemological premises and discourses of information literacy within HE

Exploring the constraint profile of winter sports resort tourist segments

Many studies have confirmed the importance of market segmentation both theoretically and empirically. Surprisingly though, no study has so far addressed the issue from the perspective of leisure constraints. Since different consumers face different barriers, we look at participation in leisure activities as an outcome of the negotiation process that winter sports resort tourists go through, to balance between related motives and constraints. This empirical study reports the findings on the applicability of constraining factors in segmenting the tourists who visit winter sports resorts. Utilizing data from 1,391 tourists of winter sports resorts in Greece, five segments were formed based on their constraint, demographic and behavioral profile. Our findings indicate that such segmentation sheds light on factors that could potentially limit the full utilization of the market. To maximize utilization, we suggest customizing marketing to the profile of each distinct winter sports resort tourist segment that emerge

A Novel Mechanism Is Involved in Cationic Lipid-Mediated Functional siRNA Delivery

A key challenge for therapeutic application of RNA interference is to efficiently deliver synthetic small interfering RNAs (siRNAs) into target cells that will lead to the knockdown of the target transcript (functional siRNA delivery). To facilitate rational development of nonviral carriers, we have investigated by imaging, pharmacological and genetic approaches the mechanisms by which a cationic lipid carrier mediates siRNA delivery into mammalian cells. We show that 95% of siRNA lipoplexes enter the cells through endocytosis and persist in endolysosomes for a prolonged period of time. However, inhibition of clathrin-, caveolin-, or lipid-raft-mediated endocytosis or macropinocytosis fails to inhibit the knockdown of the target transcript. In contrast, depletion of cholesterol from the plasma membrane has little effect on the cellular uptake of siRNA lipoplexes, but it abolishes the target transcript knockdown. Furthermore, functional siRNA delivery occurs within a few hours and is gradually inhibited by lowering temperatures. These results demonstrate that although endocytosis is responsible for the majority of cellular uptake of siRNA lipoplexes, a minor pathway, probably mediated by fusion between siRNA lipoplexes and the plasma membrane, is responsible for the functional siRNA delivery. Our findings suggest possible directions for improving functional siRNA delivery by cationic lipids.National Institutes of Health (U.S.) (NIH Grant AI56267)National Institutes of Health (U.S.) (NIH Grant CA112967)National Institutes of Health (U.S.) (NIH Grant CA119349)Natural Sciences and Engineering Research Council of Canada (NSERC) (Post-doctoral fellowship

The Adaptor Function of TRAPPC2 in Mammalian TRAPPs Explains TRAPPC2-Associated SEDT and TRAPPC9-Associated Congenital Intellectual Disability

Background: The TRAPP (Transport protein particle) complex is a conserved protein complex functioning at various steps in vesicle transport. Although yeast has three functionally and structurally distinct forms, TRAPPI, II and III, emerging evidence suggests that mammalian TRAPP complex may be different. Mutations in the TRAPP complex subunit 2 (TRAPPC2) cause X-linked spondyloepiphyseal dysplasia tarda, while mutations in the TRAPP complex subunit 9 (TRAPPC9) cause postnatal mental retardation with microcephaly. The structural interplay between these subunits found in mammalian equivalent of TRAPPI and those specific to TRAPPII and TRAPPIII remains largely unknown and we undertook the present study to examine the interaction between these subunits. Here, we reveal that the mammalian equivalent of the TRAPPII complex is structurally distinct from the yeast counterpart thus leading to insight into mechanism of disease. Principal Findings: We analyzed how TRAPPII- or TRAPPIII- specific subunits interact with the six-subunit core complex of TRAPP by co-immunoprecipitation in mammalian cells. TRAPPC2 binds to TRAPPII-specific subunit TRAPPC9, which in turn binds to TRAPPC10. Unexpectedly, TRAPPC2 can also bind to the putative TRAPPIII-specific subunit, TRAPPC8. Endogenous TRAPPC9-positive TRAPPII complex does not contain TRAPPC8, suggesting that TRAPPC2 binds to either TRAPPC9 or TRAPPC8 during the formation of the mammalian equivalents of TRAPPII or TRAPPIII, respectively. Therefore, TRAPPC2 serves as an adaptor for the formation of these complexes. A disease-causing mutation of TRAPPC2, D47Y, failed to interact with either TRAPPC9 or TRAPPC8, suggesting that aspartate 47 in TRAPPC2 is at or near the site of interaction with TRAPPC9 or TRAPPC8, mediating the formation of TRAPPII and/or TRAPPIII. Furthermore, disease-causing deletional mutants of TRAPPC9 all failed to interact with TRAPPC2 and TRAPPC10. Conclusions: TRAPPC2 serves as an adaptor for the formation of TRAPPII or TRAPPIII in mammalian cells. The mammalian equivalent of TRAPPII is likely different from the yeast TRAPPII structurally. © 2011 Zong et al.published_or_final_versio