Mechanisms of enhanced non-viral gene delivery to human mesenchymal stem cells induced by glucocorticoid priming

Background: Because of unique roles in wound healing, trophic tissue support, immunomodulation, differentiation ability, and immune privileged status, human mesenchymal stem cells (hMSCs), which can be easily derived from many adult tissues (e.g. bone marrow (BMSCs) and adipose tissue (AMSCs)), are under intense study for the applications of cell and gene therapeutics, as well as tissue engineering and regenerative medicine1. Genetic modification of hMSCs could allow for targeted delivery of transgenic therapeutic factors or genetically-guided differentiation. Non-viral gene delivery (i.e. cationic polymer- and lipid-mediated) is safer and more flexible than immunogenic and mutagenic viral vectors2, but it is less effective, especially in hMSCs (i.e. maximum 10-30% transfection)3. As part of an approach to understand molecular mechanisms of non-viral gene delivery4 and ‘prime’ cells to be more receptive to transfection5, our lab recently demonstrated that transgene expression from lipofected hMSCs can be increased about 10-fold by priming cells, 30 mins before plasmid DNA (pDNA) transfection, with 100 nM dexamethasone (DEX), a glucocorticoid (Gc) drug, relative to EtOH vehicle control (VC)6. This work investigates the mechanisms by which Gc priming enhances non-viral gene delivery, which are currently unknown. Studies provide insights into the biological processes of Gc priming and transfection to inform future gene delivery technologies, and characterize a simple protocol to significantly enhance non-viral gene delivery of therapeutic transgenes for future clinical applications. Please click Additional Files below to see the full abstract

Shuttle orbiter Ku-band radar/communications system design evaluation

Tasks performed in an examination and critique of a Ku-band radar communications system for the shuttle orbiter are reported. Topics cover: (1) Ku-band high gain antenna/widebeam horn design evaluation; (2) evaluation of the Ku-band SPA and EA-1 LRU software; (3) system test evaluation; (4) critical design review and development test evaluation; (5) Ku-band bent pipe channel performance evaluation; (6) Ku-band LRU interchangeability analysis; and (7) deliverable test equipment evaluation. Where discrepancies were found, modifications and improvements to the Ku-band system and the associated test procedures are suggested

Acetylcholine modulates gamma frequency oscillations in the hippocampus by activation of muscarinic M1 receptors

This is the author accepted manuscript. The final version is available from the publisher via the DOI in this record.This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/ejn.13582 This article is protected by copyright. All rights reserved.Modulation of gamma oscillations is important for the processing of information and the disruption of gamma oscillations is a prominent feature of schizophrenia and Alzheimer’s disease. Gamma oscillations are generated by the interaction of excitatory and inhibitory neurons where their precise frequency and amplitude are controlled by the balance of Accepted Article This article is protected by copyright. All rights reserved. excitation and inhibition. Acetylcholine enhances the intrinsic excitability of pyramidal neurons and supresses both excitatory and inhibitory synaptic transmission but the net modulatory effect on gamma oscillations is not known. Here, we find that the power, but not frequency, of optogenetically -induced gamma oscillations in the CA3 region of mouse hippocampal slices is enhanced by low concentrations of the broad spectrum cholinergic agonist carbachol but reduced at higher concentrations. This bidirectional modulation of gamma oscillations is replicated within a mathematical model by neuronal depolarization, but not by reducing synaptic conductances, mimicking the effects of muscarinic M1 receptor activation. The predicted role for M1 receptors was supported experimentally; bidirectional modulation of gamma oscillations by acetylcholine was replicated by a selective M1 receptor agonist and prevented by genetic deletion of M1 receptors. These results reveal that acetylcholine release in CA3 of the hippocampus modulates gamma oscillation power but not frequency in a bidirectional and dose -dependent manner by acting primarily through muscarinic M1 receptorsThis work was supported by the Wellcome Trust Neural Dynamics PhD programme (RTB) and the Wellcome Trust (JRM). We thank Eli Lilly and Co. for gifts of GSK -5 and M1 receptor knockout mice. We thank members of the Mellor lab for helpful discussions and J. Brown for comments on previous versions of the manuscript. The authors declare no competing financial interests

Glucocorticoid Priming of Nonviral Gene Delivery to hMSCs Increases Transfection by Reducing Induced Stresses

Human mesenchymal stem cells (hMSCs) are under study for cell and gene therapeutics because of their immunomodulatory and regenerative properties. Safe and efficient gene delivery could increase hMSC clinical potential by enabling expression of transgenes for control over factor production, behavior, and differentiation. Viral delivery is efficient but suffers from safety issues, while nonviral methods are safe but highly inefficient, especially in hMSCs. We previously demonstrated that priming cells with glucocorticoids (Gcs) before delivery of DNA complexes significantly increases hMSC transfection, which correlates with a rescue of transfection-induced metabolic and protein synthesis decline, and apoptosis. In this work, we show that transgene expression enhancement is mediated by transcriptional activation of endogenous hMSC genes by the cytosolic glucocorticoid receptor (cGR) and that transfection enhancement can be potentiated with a GR transcription-activation synergist. We demonstrate that the Gc-activated cGR modulates endogenous hMSC gene expression to ameliorate transfection-induced endoplasmic reticulum (ER) and oxidative stresses, apoptosis, and inflammatory responses to prevent hMSC metabolic and protein synthesis decline, resulting in enhanced transgene expression after nonviral gene delivery to hMSCs. These results provide insights important for rational design of more efficient nonviral gene delivery and priming techniques that could be utilized for clinical hMSC applications

Fitness for purpose? project-based, collaborative learning in engineering undergraduate education

In response to evolving work practices in engineering (Royal Academy of Engineering 2010) and the ever-changing nature of vocational and professional knowledge (Broad, 2016), attention has rightly turned to the development of innovative pedagogies to facilitate the entry of graduates to the employment market. Project-based Learning (PjBL) is an example of such an innovative pedagogy and has been developed in some HE contexts to provide students with authentic learning experiences which are designed to embed team working and collaboration; problem solving and solution-finding alongside the development of technical knowledge and skills (Mills and Treagust, 2003). Yet despite the introduction of collaborative inquiry-based curricula in engineering, comprehensive understanding of the pedagogical practices that these innovative practices require has not yet emerged (Damşa & Nerland, 2016). At UCL Engineering, PjBL has been an integral feature of the Integrated Engineering Programme (IEP) undergraduate curriculum since 2014. Drawing upon an on-going collaborative Institute of Education/UCL ‘seed corn’ funded project, this paper will present data collected through observation of project-based learning scenarios and discussion with engineering undergraduates in situ. It will identify and discuss the nature of student learning and engagement in project-based learning activities, with particular attention to disciplinary issues and the development of student disciplinary knowledge. In so doing, it will assess whether PjBL is, indeed, ‘fit for purpose’

A Discrete Version of the Inverse Scattering Problem and the J-matrix Method

The problem of the Hamiltonian matrix in the oscillator and Laguerre basis construction from the S-matrix is treated in the context of the algebraic analogue of the Marchenko method.Comment: 11 pages. The Laguerre basis case is adde

Highlighting the learning in project-based undergraduate engineering education: pedagogical and methodological considerations

This paper presents a discussion of findings in relation to the pedagogy of Project Based Learning (PjBL) from a collaborative learning and teaching research project at UCL Engineering. It highlights the importance of adopting a) an understanding learning as a social practice in PjBL, and b) developing valid methodological approaches when capturing student learning experiences

Where do graduates Develop their Enterprise Skills? The Value of the Contribution of Higher Education Institutions’ Context

This study investigates the value of the contribution of HEIs’ context in developing graduates enterprise skills. HEIs are under pressure to develop more enterprising graduates, particularly with the increasing numbers of graduates seeking employment and the growing dissatisfaction of employers. This study explores where graduates develop enterprise skills through investigating the impact of HE and employment contexts on their development. The paper draws on a qualitative study in the social constructionist paradigm within the pharmacy context, where interviews were conducted with pharmacy academics and employers. Results show that ability to demonstrate skills in one context does not necessarily mean ability to demonstrate them in another since the development and demonstration of enterprise skills is impacted by the contexts in which they are developed and demonstrated. The study adds value by highlighting the significant role of both HE and employment contexts in developing enterprise skills, while emphasising that these skills become more transferable through exposure to more contexts