Optimizing siRNA Efficacy through Alteration in the Target Cell-Adhesion Substrate Interaction

Short interfering RNA (siRNA) is a class of nucleotide drugs with a profound potential to improve patient health through its ability to silence the expression of specific genes at the post-transcriptional level. However, the clinical application of siRNA therapeutics remains hindered by a lack of efficient delivery systems that deposit siRNA into the cytoplasm of cells, a step necessary for siRNA’s silencing effect. Much research has focused on the development of siRNA delivery agents to overcome this challenge. There are no standard pre-clinical models for testing of siRNA delivery agents, and investigators have chosen to evaluate efficacy in a variety of systems including in vitro tissue culture and animal models. These systems have vastly different cellular microenvironments which may modulate cellular behavior and affect the response of cells to siRNA, thus altering the apparent efficacy of siRNA delivery agents. The substrate on which cells adhere is one aspect of the microenvironment that has been previously shown to alter cellular behavior. In this work, we tested the hypothesis that changing the properties of cellular adhesion substrates can change the apparent efficacy of a siRNA delivery agent. Specifically, we used a commonly employed in vitro cationic lipid siRNA delivery vector and evaluated siRNA silencing efficacy in U251 cells seeded on alginate hydrogel surfaces. These surfaces were synthesized to have systematic variation in integrin ligand arginine-glycine-aspartate (RGD) density and elastic modulus. We found that an eightfold increase in RGD content of the alginate grown substrate increased siRNA knockdown efficacy from 25 ± 12% to 52 ± 10%, with constant concentrations of siRNA and delivery agent. We found no difference in siRNA mediated knockdown efficacy over the elastic modulus range tested (53-133 kPa). These results indicate that the cell-adhesion substrate interaction can modulate siRNA protein silencing efficacy, a finding important for evaluation of siRNA therapeutics in the in vitro setting

The role of hydrophobic amino acid grafts in the enhancement of membrane-disruptive activity of pH-responsive pseudo-peptides

pH-responsive polymers have been synthesised by grafting l-valine (PV-75), l-leucine (PL-75) and l-phenylalanine (PP-75) onto the pendant carboxylic acid moieties of a pseudo-peptide, poly(l-lysine iso-phthalamide), at a stoichiometric degree of substitution of 75 mol%. The effect of such modification on the pH-, concentration- and time-dependent cell membrane-disruptive activity of the grafted polymers has been investigated using a haemolysis model. At 0.025 mg mL(−1), the grafted polymers were almost non-haemolytic at pH 7.4, but mediated considerable membrane lysis after 60 min in the pH range characteristic of early endosomes, which ranked in the order: PP-75 > PL-75 > PV-75 > poly(l-lysine iso-phthalamide). PP-75 was 35-fold more lytic on a molar basis than the membrane-lytic peptide melittin. With increasing concentration, the grafted polymers showed an increased ability to lyse cell membranes and caused noticeable membrane disruption at physiological pH. The mechanism of the polymer-mediated membrane destabilisation has been investigated. The in-vitro cytotoxicity of the grafted polymers has been assessed using a propidium iodide fluorescence assay. It has been demonstrated by confocal microscopy that the grafted polymers can induce a significant release of endocytosed materials into the cytoplasm of HeLa cells, which is a feature critical for drug delivery applications

The relation between metaphor and impoliteness in the context of interpersonal media gossip

The present study aims at examining the possible relation between metaphor and impoliteness in the context of interpersonal media gossip. To this end, we collected data related to 20 celebrities (10 actors and 10 actresses) regarding 60 linguistic situations that occurred from August 2018 to April 2020. Words under examination were 51449. According to the findings of the study, human conceptualization based on source domains such as shit/ animal waste matter, useless things, cartoon/ fictional characters, useless money, dirty substance, imaginary/ metaphysical beings, mushroom, bread, weekdays, fat, and disease/blight were accompanied by impoliteness. These findings justify the role of impoliteness in confirming the direct relation between metaphor and impoliteness to the extent that human is considered as one of the above domains. Interpersonal media gossip prepares the grounds for the use of 345 metaphors along with impoliteness which has its roots in special features of this type of gossip. Among all source domains, concepts such as shit/ animal waste matter are of the highest frequencies. Among impoliteness strategies related to naming, one must point to the negative impoliteness sub-strategy. Language users attempt to hurt the positive face of people from themselves by using positive impoliteness

Endocytosis Mechanism of Nano Metal-Organic Frameworks for Drug Delivery.

The pathway of internalization and final fate of a specific metal-organic framework (MOF) in cells has been investigated for the first time. This study is based on two calcein-loaded UiO-66 samples with particle sizes of 150 and 260 nm (i.e., cal@150 UiO-66 and cal@260 UiO-66, respectively), and shows that the active trafficking of cal@150 UiO-66 is done almost exclusively through clathrin-mediated endocytosis, whereas the uptake of cal@260 UiO-66 is a combination of both clathrin and caveolae-mediated endocytosis. Colocalization studies with a lysosomal marker showed that cal@150 UiO-66 is located mostly in lysosomes for further degradation, whereas cal@260 UiO-66 seems to avoid the lysosomal degradation and potentially deliver the cargo molecules in the cytosol, allowing their distribution to different cellular organelles. This study reveals the importance of the internalization processes of MOFs, particularly the relevance of their particle size, and also the critical significance of their final fate to become an efficient drug delivery system. Based on these results, it is possible that extremely small particle-sized MOFs are not the most efficient carriers and instead relatively medium-sized particles are required.C.A.O. and S.M. thank Becas Chile and the Cambridge Trust for funding. D.F.-J. thanks the Royal Society (UK) for funding through a University Research Fellowship.This is the author accepted manuscript. It is currently under an indefinite embargo pending publication by Wiley

Alum Activates the Bovine NLRP3 Inflammasome

peer-reviewedThere has been a move away from vaccines composed of whole or inactivated antigens toward subunit-based vaccines, which although safe, provide less immunological protection. As a result, the use of adjuvants to enhance and direct adaptive immune responses has become the focus of much targeted bovine vaccine research. However, the mechanisms by which adjuvants work to enhance immunological protection in many cases remains unclear, although this knowledge is critical to the rational design of effective next generation vaccines. This study aimed to investigate the mechanisms by which alum, a commonly used adjuvant in bovine vaccines, enhances IL-1β secretion in bovine peripheral blood mononuclear cells (PBMCs). Unlike the case with human PBMCs, alum promoted IL-1β secretion in a subset of bovine PBMCs without priming with a toll-like receptor agonist. This suggests that PBMCs from some cattle are primed to produce this potent inflammatory cytokine and western blotting confirmed the presence of preexisting pro-IL-1β in PBMCs from a subset of 8-month-old cattle. To address the mechanism underlying alum-induced IL-1β secretion, specific inhibitors identified that alum mediates lysosomal disruption which subsequently activates the assembly of an NLRP3, ASC, caspase-1, and potentially caspase-8 containing complex. These components form an inflammasome, which mediates alum-induced IL-1β secretion in bovine PBMCs. Given the demonstrated role of the NLRP3 inflammasome in regulating adaptive immunity in murine systems, these results will inform further targeted research into the potential of inflammasome activation for rational vaccine design in cattle

A Critical Discourse Analysis of Radi’s Dramas From behind the Windows and Hamlet with Season Salad Based on Van Leeuwen’s Framework "Representing Social Actions"

Different representations of social actions create distinct types of discourses. Applying van Leeuwen’s 'Social Actions' framework (2008), the present study critically analyzes the power relations between the main characters of Radi’s dramas From behind the Windows and Hamlet with Season Salad. The objective of our study is to account for the differences between the discourse of the dominant and the discourse of the dominated. In order to elucidate such differences we count and analyze the characters’ social (re)actions and, in turn, identify four types of contrasts: cognitive vs. affective and perceptive reactions; material vs. semiotic actions; transactive vs. non-transactive actions; interactive vs. instrumental actions. Two opposing discourses emerge from these contrasts. On the one hand, the dominant characters mostly react cognitively and their actions are often semiotic, transactive, and interactive. On the other hand, the dominated characters’ reactions are often affective and perceptive, while most of their actions are material, non-transactive, and instrumental. As the results show, the author’s linguistic choices underscore the power relations between the dominant and the dominated characters. Building upon the fact that our analysis sheds light on the underlying ideologies and intentions of the author, we tentatively conclude that despite its being predominantly employed in the analysis of political discourses, van Leeuwen’s framework also proves effective in the critical analysis of literary works

A Study of Discourse Anaphora in Persian Language based on Gundel, Hedberg and Zacharski’s Givenness Hierarchy

One of the main themes of discourse analysis is information structure. The impact on the syntactic, phonological and morphological form of the sentence of information structure is observable. Regarding its impact on the morphological form, there exists an area of research called ‘discourse anaphora’. The present study analyzed discourse anaphora in the novel ‘School’s Principal’ and the book ‘Language and Thought’ based on Gundel, Hedberg and Zacharski's (1993) Givenness Hierarchy. The model consists of six cognitive statuses. It is believed that the choice of the form of referring expressions depends on the cognitive status of the referent in the mind of hearer (reader). In the present study, twelve referring forms were chosen, and different cognitive statuses were identified and presented in a separate table for each form based on the collected examples. Finally, based on the numbers and statistics taken from the tables, the degree of compatibility of the predictions of the Givenness Hierarchy with Persian data was observed

Solyndra Project Report

This paper analyzes the founding and bankruptcy of Solyndra, LLC of Fremont, California. In 2005, Solyndra was founded by a technologist, Dr. Chris Gronet, based on semiconductor deposition method developed by Miguel Contreras at the National Renewable Energy Laboratory.Consequently, it was taken over by professional management with strong financial connection, access to approximately $3 billion including about half a billion dollars backed by the De partment of Energy’s loan guarantee. Solyndra’s competitive advantage was a large portfolio of patent protecting its Solar rod design which could be installed similar to florescent tubes used in lighting. Further during the period, Solyndra had a price advantage due to the high cost of polysilicon

Hierarchy Decision Model for Managing Innovative R&D Product Development Projects

Technological innovation is critical to any organization’s ability to survive and grow in today’s business environment. It is widely accepted that companies’ ability to effectively manage its Research and Development (R&D) investments and portfolio in order to deliver value through technological innovation is a key determinant of its market success and competitive advantage [13]. With globalization, Companies’ have a broader access to information and markets which have resulted in amplifying the need for technological innovation in order to succeed over the long term [11]. Although companies have access to and use tools to measure quantitative aspect of the business especially on the input side of innovation, it has been proven much more challenge to measure the qualitative aspect of innovation. Technology managers and leaders need methodological assessment tools in order to decide among the active technologically innovative projects in their organization. This paper focuses on technologically innovative projects where R&D managers are not able to directly leverage knowledge from past project successes and failures. The objective of this project is to develop a decision model based on Mission, Objectives, Goals and Activities (MOGSA) model developed by Dr. Kocaoglu and his team [18] in order to enable the management to relatively rank various projects in their R&D Portfolio. Using the relative rank from the decision model, R&D managers are able to determine appropriate funding allocation; in some cases resulting in termination of one or more projects with lower future potential in order to provide additional funding for more promising projects