The Evaluation of Star-Shaped Polypeptides for Gene Delivery in Tissue Engineering

The field of tissue engineering (TE) is increasingly using biomaterial scaffolds which are augmented with therapeutics to facilitate enhanced tissue regeneration. The formation of a “gene activated” scaffold, an advanced construct containing gene therapeutics within a 3D scaffold is recognised as a safe method to provide improved spatiotemporal control of growth factor release at a defect site via the in-situ transfection of host cells. However, a versatile and biocompatible gene delivery vector which is capable of functionalising 3D scaffolds for the efficient in vivo delivery of nucleic acids is currently lacking. The primary objective of this thesis was to create a next generation gene activated scaffold which could be applied to multiple TE applications via the incorporation of innovative, bio-inspired gene delivery vectors in the form of star-shaped poly(L-lysine) polypeptides (star-PLLs). Herein, we systematically evaluate three star-PLL vectors for plasmid DNA (pDNA) delivery to Mesenchymal Stem Cells (MSCs) which encompassed structural variations to the dendrimer core (G3, G4 or G5), the number of poly(L-lysine) arms (16 arms, 32 arms or 64 arms) and the associated number of L-lysine subunits per arm (5 subunits or 40 subunits). These included 1: G3(16)PLL40 (16-star-PLL) 2: G4(32)PLL40 (32-star-PLL) & 3: G5(64)PLL5 (64-star-PLL). Numerous physicochemical and biophysical techniques were employed throughout this thesis to identify star-PLL-pDNA formulations which were non-toxic, non-immunogenic and of suitable size and charge to efficiently deliver pDNA to MSCs both in 2D monolayer culture and in 3D on collagen based scaffolds. The safety and functionality of lead star-PLL-pDNA gene activated scaffolds were then evaluated in vivo using a rodent subcutaneous implant model and a cranial bone defect model. Results from this thesis demonstrate for the first time that all three star-PLL vectors can self-assemble with pDNA to form stable, non-toxic, non-immunogenic, nano-sized complexes which can efficiently transfect MSCs, a difficult to transfect primary cell type. We highlight the 64-star-PLL structure as a particularly efficient vector, which can facilitate comparable transgene expression in MSCs to commonly used vectors such as polyethyleneimine (PEI) at a lower pDNA dose. Following delivery of the therapeutic transgenes bone morphogenetic protein-2 (pBMP-2) and vascular endothelial growth factor (pVEGF) by the 64-star-PLL vector, we demonstrate enhanced MSC mediated osteogenesis compared to using the PEI vector in both 2D culture and 3D on collagen based scaffolds. Using a rodent subcutaneous implant model, we demonstrate that star-PLL-pDNA gene activated scaffolds are biocompatible in vivo and can facilitate autologous host cell transfection. Finally, using a rodent cranial bone defect model we show that star-PLL-pDNA gene activated scaffolds containing a pBMP-2 and pVEGF (pDual) cargo can facilitate superior bone tissue regeneration compared to a gene free scaffold at just four weeks post implantation. Collectively, this thesis describes the development of a first in class, biocompatible star-PLL-pDNA gene activated scaffold platform with proven functionality in vivo for TE applications. We have identified a lead star-PLL vector which is non-toxic, non-immunogenic and can efficiently deliver a therapeutic pDNA cargo to cells in 2D monolayer culture, in 3D culture on collagen scaffolds and in vivo. This cell-free gene activated scaffold confers enhanced spatiotemporal control of growth factor release at a tissue defect leading to a transient protein expression profile which is highly desirable for TE applications. We highlight the translational reality of these star-PLL-pDNA gene activated scaffolds via demonstrating the significant regeneration of bone tissue in a rodent calvarial defect model at just four weeks post implantation compared to a gene free scaffold.</p

Towards a framework for interviewing suspects of fraud

Whereas much of the limited research concerning the actual investigative interviewing of suspects has involved police interviews, almost no research has involved other agencies who increasingly are undertaking criminal investigations. The first study therefore examined 142 actual benefit fraud interviews, revealing that, whilst investigators generally displayed ethical interviewing standards, few interviews were skilled. Shortcomings were particularly found in terms of rapport development, summarising, and how investigators closed interviews. The second study (using the same sample as with the first study) examined whether there was any association between skilled interviewing and interview outcomes, finding that better interviewing conducted under the recommended interviewing framework, tended to be associated with better outcomes, whereas less skilled interviewing generally led to interviews resulting in untested denials or just partial admissions. Powerful effect sizes were regularly reported in the study. A third study focussed on the effects of rapport upon outcomes in investigative interviews. Rapport has been frequently cited in previous studies as a feature that improves the quality of interview performance of investigators and is likely to yield much further information from suspects. The third study examined whether the particular task of rapport, built and then maintained throughout the interview, had any association with interview outcomes. The study found that there was an association between better displays of rapport building and maintenance and those preferred interview outcomes (i.e., the obtaining of a comprehensive account from suspects or full and frank admission of wrongdoing). Effect sizes were frequently found to be strong. A further (fourth) study was then conducted to establish why the interviewing performance was so mediocre. 115 investigation personnel completed a questionnaire. This study found that, whilst there appeared to be an understanding of the essential components of interviewing, there was a considerable gap between perception and reality that was believed to be caused by inconsistent approaches to evaluation by senior officers and similar problems concerning the vital task of self-evaluation.The fifth and final study, recognising that organisations (such as those studied in this thesis) necessarily tend to be more risk averse when making decisions to prosecute, examined the skill levels of prescribed tactics used, and attitude displayed, by interviewers and the extent of their presence in 85 interviews. The study found that there appeared to be an association both between the higher rated skill levels and the larger extent of the presence of these tactics and attitudes, when examined against increased shifts towards confessions. Effects sizes were found to be consistently strong in this final study. Nevertheless, as only one offence type was analysed, further research of interview performance in investigation organisations (both police and nonpolice) is vital.</div

Additional file 1: of Factors predicting pain and early discontinuation of tumour necrosis factor-Îą-inhibitors in people with rheumatoid arthritis: results from the British society for rheumatology biologics register

Univariate associations with discontinuation of TNFι-inhibitor. (DOCX 54 kb

Additional file 1: of General and disease-specific pain trajectories as predictors of social and political outcomes in arthritis and cancer

Table S1. Baseline health and socio-demographic characteristics of the sample. Comparisons are between the cancer and arthritis subsamples. Table S2. Binary logistic regression models modelling the relationship between pain and dropout or death in the Wave 1 ELSA respondents and the cancer and arthritis subsamples. Table S3. Model characteristics of each identified trajectory. Table S4. Uncorrected post hoc 2 × 2 χ2 tests on dropout by Wave 7 between classes. Table S5. Growth models for social and civic engagement variables (unstandardised). Table S6. Regression coefficients for age and trajectory on the intercept and slope for growth models for each index of social and civic engagement for respondents in the whole sample. Table S7. Regression coefficients for age and trajectory on the intercept and slope for growth models for each index of social and civic engagement for respondents with arthritis. Table S8. Regression coefficients for age and trajectory on the intercept and slope for growth models for each index of social and civic engagement for respondents with cancer. Figure S1. Histogram of the chronic pain measurement at Wave 1 for all Wave 1 ELSA respondents. Figure S2. Histogram of the chronic pain measurement at Wave 1 for respondents included in the arthritis subsample. Figure S3. Histogram of the chronic pain measurement at Wave 1 for respondents included in the cancer subsample. (DOCX 3796 kb

Percentage responses and means and SDs, in rank order for knowledge of psychologically coercive behaviours (N = 682).

<p>Percentage responses and means and SDs, in rank order for knowledge of psychologically coercive behaviours (N = 682).</p

ベイコク カシュウ キョウイクキョク ケンテイ ニホンゴ ドクホン

訂正[版

Additional file 1: of Characterization of multinucleated giant cells in synovium and subchondral bone in knee osteoarthritis and rheumatoid arthritis

Morphological MGC foam subtype and CD68 positive cells surrounding fat cells in OA and in RA. A and B. MGCs displaying a foam-like subtype were identified near to and surrounding fat cells in inflamed synovia from patients with either OA (A) or RA (B). Haematoxylin and eosin staining. Scale bar = 20 μm. Open arrows indicate foam-like MGC and A = adipocyte. C and D. Multiple mononuclear CD68 positive cells were found in a crown-like structure encircling adipocyte cells in both OA (C) and RA (D). Immunohistochemistry for CD68, using eosin contrast. Scale bar = 100 μm. (TIFF 3238 kb

Why Do Males in Scotland Die Younger than Those in England? Evidence from Three Prospective Cohort Studies

<div><h3>Background</h3><p>To examine explanations for the higher rates of male mortality in two Scottish cohorts compared with a cohort in south-east England for which similar data were collected.</p> <h3>Methodology/Principal Findings</h3><p>We compared three cohort studies which recruited participants in the late 1960s and early 1970s. A total of 13,884 men aged 45–64 years at recruitment in the Whitehall occupational cohort (south-east England), 3,956 men in the Collaborative occupational cohort and 6,813 men in the Renfrew & Paisley population-based study (both central Scotland) were included in analyses of all-cause and cause-specific mortality. All-cause mortality was 25% (age-adjusted hazard ratio 1.25, 95% confidence interval (CI)1.21 to 1.30) and 41% (hazard ratio 1.41 (95% CI 1.36 to 1.45) higher in the Collaborative and Renfrew & Paisley cohorts respectively compared to the Whitehall cohort. The higher mortality rates were substantially attenuated by social class (to 8% and 17% higher respectively), and were effectively eliminated upon the further addition of the other baseline risk factors, such as smoking habit, lung function and pre-existing self-reported morbidity. Despite this, coronary heart disease mortality remained 11% and 16% higher, stroke mortality 45% and 37% higher, mortality from accidents and suicide 51% and 70% higher, and alcohol-related mortality 46% and 73% higher in the Collaborative and Renfrew & Paisley cohorts respectively compared with the Whitehall cohort in the fully adjusted model.</p> <h3>Conclusions/Significance</h3><p>The higher all-cause, respiratory, and lung cancer male mortality in the Scottish cohorts was almost entirely explained by social class differences and higher prevalence of known risk factors, but reasons for the excess mortality from stroke, alcohol-related causes, accidents and suicide remained unknown.</p> </div

Hazard ratios for mortality in the Scottish cohorts compared to the Whitehall Study⁺ (reference) with successive adjustment for potential explanatory factors.

<p>+Analyses for all-cause mortality are based on 13,884, 3,956 and 6,813 men in the Whitehall, Collaborative and Renfrew & Paisley studies respectively. For cause-specific mortality the number of men in the analyses are 13,850, 3,948 and 6,792 respectively. The number of deaths is given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038860#pone-0038860-t002" target="_blank">Table 2</a>.</p>*<p>Angina, ECG abnormality, respiratory symptoms or breathlessness.</p>±<p>Multiply adjusted for :- age, smoking, FEV₁, cardio-respiratory symptoms or history, height, systolic blood pressure, cholesterol, body mass index.</p

Proportions and means⁺ for established risk factors by study.

+<p>Prevalences and means are adjusted for age (age is unadjusted). All measures show significant (p<0.001) heterogeneity between the studies.</p>*<p>FEV ratio is the ratio of measured FEV divided by predicted FEV calculated from subjects’ age and height based on an equation derived in those men free of respiratory symptoms in the Renfrew & Paisley study.</p