The role of the helper lipid on the DNA transfection efficiency of lipopolyplex formulations.

Multifunctional, lipopolyplex formulations comprising a mixture of cationic liposomes and cationic, receptor-targeting peptides have potential use in gene therapy applications. Lipopolyplex formulations described here are typically far more efficient transfection agents than binary lipoplex or polyplex formulations. It has been shown previously that the peptide component mediates both DNA packaging and targeting of the nanoparticle while in this report we investigate the contribution of the lipid component. We hypothesised that the lipid components synergise with the peptides in the transfection process by promoting endosomal escape after lipid bilayer fusion. Lipopolyplexes were prepared with cationic liposomes comprising DOTAP with either neutral lipid DOPE or DOPC. DOPE promotes fusogenic, inverted hexagonal lipid structures while DOPC promotes more stable laminar structures. Lipopolyplexes containing DOPE showed substantially higher transfection efficiency than those formulated with DOPC, both in vitro and in vivo. DOPE-containing lipopolyplexes showed rapid endosomal trafficking and nuclear accumulation of DNA while DOPC-containing formulations remained within the late endo-lysosomal compartments. These findings are consistent with previous finding for the role of DOPE in lipoplexes and support the hypothesis regarding the function of the lipid components in lipopolyplexes. These findings will help to inform future lipopolyplex design, strategies and clinical development processes

The Enteropathogenic E. coli (EPEC) Tir Effector Inhibits NF-κB Activity by Targeting TNFα Receptor-Associated Factors

Enteropathogenic Escherichia coli (EPEC) disease depends on the transfer of effector proteins into epithelia lining the human small intestine. EPEC E2348/69 has at least 20 effector genes of which six are located with the effector-delivery system genes on the Locus of Enterocyte Effacement (LEE) Pathogenicity Island. Our previous work implied that non-LEE-encoded (Nle) effectors possess functions that inhibit epithelial anti-microbial and inflammation-inducing responses by blocking NF-κB transcription factor activity. Indeed, screens by us and others have identified novel inhibitory mechanisms for NleC and NleH, with key co-operative functions for NleB1 and NleE1. Here, we demonstrate that the LEE-encoded Translocated-intimin receptor (Tir) effector has a potent and specific ability to inhibit NF-κB activation. Indeed, biochemical, imaging and immunoprecipitation studies reveal a novel inhibitory mechanism whereby Tir interaction with cytoplasm-located TNFα receptor-associated factor (TRAF) adaptor proteins induces their proteasomal-independent degradation. Infection studies support this Tir-TRAF relationship but reveal that Tir, like NleC and NleH, has a non-essential contribution in EPEC's NF-κB inhibitory capacity linked to Tir's activity being suppressed by undefined EPEC factors. Infections in a disease-relevant intestinal model confirm key NF-κB inhibitory roles for the NleB1/NleE1 effectors, with other studies providing insights on host targets. The work not only reveals a second Intimin-independent property for Tir and a novel EPEC effector-mediated NF-κB inhibitory mechanism but also lends itself to speculations on the evolution of EPEC's capacity to inhibit NF-κB function

Multifunctional receptor-targeted nanocomplexes for magnetic resonance imaging and transfection of tumours.

The efficient targeted delivery of nucleic acids in vivo provides some of the greatest challenges to the development of genetic therapies. We aim to develop nanocomplex formulations that achieve targeted transfection of neuroblastoma tumours that can be monitored simultaneously by MRI. Here, we have compared nanocomplexes comprising self-assembling mixtures of liposomes, plasmid DNA and one of three different peptide ligands derived from ApoE, neurotensin and tetanus toxin for targeted transfection in vitro and in vivo. Neurotensin-targeted nanocomplexes produced the highest levels of transfection and showed a 4.7-fold increase in transfected luciferase expression over non-targeted nanocomplexes in Neuro-2A cells. Transfection of subcutaneous Neuro-2A tumours in vivo with neurotensin-targeted nanocomplexes produced a 9.3-fold increase in gene expression over non-targeted controls. Confocal microscopy analysis elucidated the time course of DNA delivery with fluorescently labelled nanocomplex formulations in cells. It was confirmed that addition of a gadolinium lipid conjugate contrast agent allowed real time in vivo monitoring of nanocomplex localisation in tumours by MRI, which was maintained for at least 24 h. The peptide-targeted nanocomplexes developed here allow for the specific enhancement of targeted gene therapy both in vitro and in vivo, whilst allowing real time monitoring of delivery with MRI

A cross-national study on the antecedents of work–life balance from the fit and balance perspective

Drawing on the perceived work–family fit and balance perspective, this study investigates demands and resources as antecedents of work–life balance (WLB) across four countries (New Zealand, France, Italy and Spain), so as to provide empirical cross-national evidence. Using structural equation modelling analysis on a sample of 870 full time employees, we found that work demands, hours worked and family demands were negatively related to WLB, while job autonomy and supervisor support were positively related to WLB. We also found evidence that resources (job autonomy and supervisor support) moderated the relationships between demands and work–life balance, with high resources consistently buffering any detrimental influence of demands on WLB. Furthermore, our study identified additional predictors of WLB that were unique to some national contexts. For example, in France and Italy, overtime hours worked were negatively associated with WLB, while parental status was positively associated with WLB. Overall, the implications for theory and practice are discussed.Peer ReviewedPostprint (author's final draft

Translating shared decision-making into health care clinical practices: Proof of concepts

Background: There is considerable interest today in shared decision-making (SDM), defined as a decision-making process jointly shared by patients and their health care provider. However, the data show that SDM has not been broadly adopted yet. Consequently, the main goal of this proposal is to bring together the resources and the expertise needed to develop an interdisciplinary and international research team on the implementation of SDM in clinical practice using a theory-based dyadic perspective. Methods: Participants include researchers from Canada, US, UK, and Netherlands, representing medicine, nursing, psychology, community health and epidemiology. In order to develop a collaborative research network that takes advantage of the expertise of the team members, the following research activities are planned: 1) establish networking and on-going communication through internet-based forum, conference calls, and a bi-weekly e-bulletin; 2) hold a two-day workshop with two key experts (one in theoretical underpinnings of behavioral change, and a second in dyadic data analysis), and invite all investigators to present their views on the challenges related to the implementation of SDM in clinical practices; 3) conduct a secondary analyses of existing dyadic datasets to ensure that discussion among team members is grounded in empirical data; 4) build capacity with involvement of graduate students in the workshop and online forum; and 5) elaborate a position paper and an international multi-site study protocol. Discussion: This study protocol aims to inform researchers, educators, and clinicians interested in improving their understanding of effective strategies to implement shared decision-making in clinical practice using a theory-based dyadic perspective

Lipid peptide nanocomplexes for gene delivery and magnetic resonance imaging in the brain.

Gadolinium-labelled nanocomplexes offer prospects for the development of real-time, non-invasive imaging strategies to visualise the location of gene delivery by MRI. In this study, targeted nanoparticle formulations were prepared comprising a cationic liposome (L) containing a Gd-chelated lipid at 10, 15 and 20% by weight of total lipid, a receptor-targeted, DNA-binding peptide (P) and plasmid DNA (D), which electrostatically self-assembled into LPD nanocomplexes. The LPD formulation containing the liposome with 15% Gd-chelated lipid displayed optimal peptide-targeted, transfection efficiency. MRI conspicuity peaked at 4h after incubation of the nanocomplexes with cells, suggesting enhancement by cellular uptake and trafficking. This was supported by time course confocal microscopy analysis of transfections with fluorescently-labelled LPD nanocomplexes. Gd-LPD nanocomplexes delivered to rat brains by convection-enhanced delivery were visible by MRI at 6 h, 24 h and 48 h after administration. Histological brain sections analysed by laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) confirmed that the MRI signal was associated with the distribution of Gd(3+) moieties and differentiated MRI signals due to haemorrhage. The transfected brain cells near the injection site appeared to be mostly microglial. This study shows the potential of Gd-LPD nanocomplexes for simultaneous delivery of contrast agents and genes for real-time monitoring of gene therapy in the brain

Gonad shielding in paediatric pelvic radiography: disadvantages prevail over benefit

Objective To re-evaluate gonad shielding in paediatric pelvic radiography in terms of attainable radiation risk reduction and associated loss of diagnostic information. Methods A study on patient dose and the quality of gonad shielding was performed retrospectively using 500 pelvic radiographs of children from 0 to 15 years old. In a subsequent study, 195 radiographs without gonad shielding were included. Patient doses and detriment adjusted risks for heritable disease and cancer were calculated with and without gonad shielding. Results For girls, gonad shields were placed incorrectly in 91% of the radiographs; for boys, in 66%. Without gonad shielding, the hereditary detriment adjusted risk for girls ranged between 0.1?×?10?6 and 1.3?×?10?6 and for boys between 0.3?×?10?6 and 3.9?×?10?6, dependent on age. With shielding, the reduction in hereditary risk for girls was on average 6?±?3% of the total risk of the radiograph, for boys 24?±?6%. Without gonad shielding, the effective dose ranged from 0.008 to 0.098 mSv. Conclusions With modern optimised X-ray systems, the reduction of the detriment adjusted risk by gonad shielding is negligibly small. Given the potential consequences of loss of diagnostic information, of retakes, and of shielding of automatic exposure-control chambers, gonad shielding might better be discontinued.Support TNWApplied Science

Keystone symposium: The role of microenvironment in tumor induction and progression, Banff, Canada, 5–10 February 2005

The first Keystone symposium on the role of microenvironment in tumor induction and progression attracted 274 delegates from 13 countries to Banff in the heart of the Canadian Rockies. The meeting was organized by Mina Bissell, Ronald DePinho and Luis Parada, and was held concurrently with the Keystone symposium on cancer and development, chaired by Matthew Scott and Roeland Nusse. The 30 oral presentations and over 130 posters provided an excellent forum for discussing emerging data in this rapidly advancing field

Does advancing male age influence the expression levels and localisation patterns of phospholipase C zeta (PLCζ) in human sperm?

Socio-economic factors have led to an increasing trend for couples to delay parenthood. However, advancing age exerts detrimental effects upon gametes which can have serious consequences upon embryo viability. While such effects are well documented for the oocyte, relatively little is known with regard to the sperm. One fundamental role of sperm is to activate the oocyte at fertilisation, a process initiated by phospholipase C zeta (PLCζ), a sperm-specific protein. While PLCζ deficiency can lead to oocyte activation deficiency and infertility, it is currently unknown whether the expression or function of PLCζ is compromised by advancing male age. Here, we evaluate sperm motility and the proportion of sperm expressing PLCζ in 71 males (22–54 years; 44 fertile controls and 27 infertile patients), along with total levels and localisation patterns of PLCζ within the sperm head. Three different statistical approaches were deployed with male age considered both as a categorical and a continuous factor. While progressive motility was negatively correlated with male age, all three statistical models concurred that no PLCζ–related parameter was associated with male age, suggesting that advancing male age is unlikely to cause problems in terms of the sperm’s fundamental ability to activate an oocyt