Lentivirus Display: Stable Expression of Human Antibodies on the Surface of Human Cells and Virus Particles

Background: Isolation of human antibodies using current display technologies can be limited by constraints on protein expression, folding and post-translational modifications. Here we describe a discovery platform that utilizes self-inactivating (SIN) lentiviral vectors for the surface display of high-affinity single-chain variable region (scFv) antibody fragments on human cells and lentivirus particles. Methodology/Principal Findings: Bivalent scFvFc human antibodies were fused in frame with different transmembrane (TM) anchoring moieties to allow efficient high-level expression on human cells and the optimal TM was identified. The addition of an eight amino acid HIV-1 gp41 envelope incorporation motif further increased scFvFc expression on human cells and incorporation into lentiviral particles. Both antibody-displaying human cells and virus particles bound antigen specifically. Sulfation of CDR tyrosine residues, a property recently shown to broaden antibody binding affinity and antigen recognition was also demonstrated. High level scFvFc expression and stable integration was achieved in human cells following transduction with IRES containing bicistronic SIN lentivectors encoding ZsGreen when scFvFc fusion proteins were expressed from the first cassette. Up to 10[super]6-fold enrichment of antibody expressing cells was achieved with one round of antigen coupled magnetic bead pre-selection followed by FACS sorting. Finally, the scFvFc displaying human cells could be used directly in functional biological screens with remarkable sensitivity. Conclusions/Significance: This antibody display platform will complement existing technologies by virtue of providing properties unique to lentiviruses and antibody expression in human cells, which, in turn, may aid the discovery of novel therapeutic human mAbs

Gyrase inhibitors induce an oxidative damage cellular death pathway

Modulation of bacterial chromosomal supercoiling is a function of DNA gyrase-catalyzed strand breakage and rejoining. This reaction is exploited by both antibiotic and proteic gyrase inhibitors, which trap the gyrase molecule at the DNA cleavage stage. Owing to this interaction, doublestranded DNA breaks are introduced and replication machinery is arrested at blocked replication forks. This immediately results in bacteriostasis and ultimately induces cell death. Here we demonstrate, through a series of phenotypic and gene expression analyses, that superoxide and hydroxyl radical oxidative species are generated following gyrase poisoning and play an important role in cell killing by gyrase inhibitors. We show that superoxide-mediated oxidation of iron-sulfur clusters promotes a breakdown of iron regulatory dynamics; in turn, iron misregulation drives the generation of highly destructive hydroxyl radicals via the Fenton reaction. Importantly, our data reveal that blockage of hydroxyl radical formation increases the survival of gyrase-poisoned cells. Together, this series of biochemical reactions appears to compose a maladaptive response, that serves to amplify the primary effect of gyrase inhibition by oxidatively damaging DNA, proteins and lipids

Collision-Induced Decay of Metastable Baby Skyrmions

Many extensions of the standard model predict heavy metastable particles which may be modeled as solitons (skyrmions of the Higgs field), relating their particle number to a winding number. Previous work has shown that the electroweak interactions admit processes in which these solitons decay, violating standard model baryon number. We motivate the hypothesis that baryon-number-violating decay is a generic outcome of collisions between these heavy particles. We do so by exploring a 2+1 dimensional theory which also possesses metastable skyrmions. We use relaxation techniques to determine the size, shape and energy of static solitons in their ground state. These solitons could decay by quantum mechanical tunneling. Classically, they are metastable: only a finite excitation energy is required to induce their decay. We attempt to induce soliton decay in a classical simulation by colliding pairs of solitons. We analyze the collision of solitons with varying inherent stabilities and varying incident velocities and orientations. Our results suggest that winding-number violating decay is a generic outcome of collisions. All that is required is sufficient (not necessarily very large) incident velocity; no fine-tuning of initial conditions is required.Comment: 24 pages, 7 figures, latex. Very small changes onl

Gyrase inhibitors induce an oxidative damage cellular death pathway in Escherichia coli

Modulation of bacterial chromosomal supercoiling is a function of DNA gyrase-catalyzed strand breakage and rejoining. This reaction is exploited by both antibiotic and proteic gyrase inhibitors, which trap the gyrase molecule at the DNA cleavage stage. Owing to this interaction, double-stranded DNA breaks are introduced and replication machinery is arrested at blocked replication forks. This immediately results in bacteriostasis and ultimately induces cell death. Here we demonstrate, through a series of phenotypic and gene expression analyses, that superoxide and hydroxyl radical oxidative species are generated following gyrase poisoning and play an important role in cell killing by gyrase inhibitors. We show that superoxide-mediated oxidation of iron–sulfur clusters promotes a breakdown of iron regulatory dynamics; in turn, iron misregulation drives the generation of highly destructive hydroxyl radicals via the Fenton reaction. Importantly, our data reveal that blockage of hydroxyl radical formation increases the survival of gyrase-poisoned cells. Together, this series of biochemical reactions appears to compose a maladaptive response, that serves to amplify the primary effect of gyrase inhibition by oxidatively damaging DNA, proteins and lipids

Football in the community schemes: Exploring the effectiveness of an intervention in promoting healthful behaviour change

This study aims to examine the effectiveness of a Premier League football club’s Football in the Community (FitC) schemes intervention in promoting positive healthful behaviour change in children. Specifically, exploring the effectiveness of this intervention from the perspectives of the participants involved (i.e. the researcher, teachers, children and coaches). A range of data collection techniques were utilized including the principles of ethnography (i.e. immersion, engagement and observations), alongside conducting focus groups with the children. The results allude to the intervention merely ‘keeping active children active’ via (mostly) fun, football sessions. Results highlight the important contribution the ‘coach’ plays in the effectiveness of the intervention. Results relating to working practice (i.e. coaching practice and coach recruitment) are discussed and highlighted as areas to be addressed. FitC schemes appear to require a process of positive organizational change to increase their effectiveness in strategically attending to the health agenda

An Approach to Mapping Forest Growth Stages in Queensland, Australia through Integration of ALOS PALSAR and Landsat Sensor Data

Whilst extensive clearance of forests in the eastern Australian Brigalow Belt Bioregion (BBB) has occurred since European settlement, appropriate management of those that are regenerating can facilitate restoration of biomass (carbon) and biodiversity to levels typical of relatively undisturbed or remnant formations. However, maps of forests are different stages of regeneration are needed to facilitate restoration planning, including prevention of further re-clearing. Focusing on the Tara Downs subregion of the BBB and on forests with brigalow (Acacia harpophylla) as a component, this research establishes a method for differentiating and mapping early, intermediate and remnant growth stages from Japan Aerospace Exploration Agency (JAXA) Advanced Land Observing Satellite (ALOS) Phased-Array L-band Synthetic Aperture Radar (PALSAR) Fine Beam Dual (FBD) L-band HH- and HV-polarisation backscatter and Landsat-derived Foliage Projective Cover (FPC). Using inventory data collected from 74 plots, located in the Tara Downs subregion, forests were assigned to one of three regrowth stages based on their height and cover relative to that of undisturbed stands. The image data were then segmented into objects with each assigned to a growth stage by comparing the distributions of L-band HV and HH polarisation backscatter and FPC to that of reference distributions using a z-test. Comparison with independent assessments of growth stage, based on time-series analysis of aerial photography and SPOT images, established an overall accuracy of > 70%, with this increasing to 90% when intermediate regrowth was excluded and only early-stage regrowth and remnant classes were considered. The proposed method can be adapted to respond to amendments to user-definitions of growth stage and, as regional mosaics of ALOS PALSAR and Landsat FPC are available for Queensland, has application across the state

A Randomized Phase II Trial of Pioglitazone for Lung Cancer Chemoprevention in High Risk Current and Former Smokers

Lung cancer chemoprevention, especially in high-risk former smokers, has great potential to reduce lung cancer incidence and mortality. Thiazolidinediones prevent lung cancer in preclinical studies, and diabetics receiving thiazolidinediones have lower lung cancer rates which led to our double-blind, randomized, phase II placebo-controlled trial of oral pioglitazone in high risk current or former smokers with sputum cytologic atypia or known endobronchial dysplasia. Bronchoscopy was performed at study entry and after completing of six months of treatment. Biopsies were histologically scored, and primary endpoint analysis tested worst biopsy scores (Max) between groups; Dysplasia index (DI) and average score (Avg) changes were secondary endpoints. Biopsies also received an inflammation score. The trial accrued 92 subjects (47 pioglitazone, 45 placebo), and 76 completed both bronchoscopies (39 pioglitazone, 37 placebo). Baseline dysplasia was significantly worse for current smokers, and 64% of subjects had mild or greater dysplasia at study entry. Subjects receiving pioglitazone did not exhibit improvement in bronchial dysplasia. Former smokers treated with pioglitazone exhibited a slight improvement in Max, while current smokers exhibited slight worsening. While statistically significant changes in Avg and DI were not observed in the treatment group, former smokers exhibited a slight decrease in both Avg and DI. Negligible Avg and DI changes occurred in current smokers. A trend towards decreased Ki-67 labeling index occurred in former smokers with baseline dysplasia receiving pioglitazone. While pioglitazone did not improve endobronchial histology in this high-risk cohort, specific lesions showed histologic improvement and further study is needed to better characterize responsive dysplasia

Public opinion on energy crops in the landscape: considerations for the expansion of renewable energy from biomass

Public attitudes were assessed towards two dedicated biomass crops – Miscanthus and Short Rotation Coppice (SRC), particularly regarding their visual impacts in the landscape. Results are based on responses to photographic and computer-generated images as the crops are still relatively scarce in the landscape. A questionnaire survey indicated little public concern about potential landscape aesthetics but more concern about attendant built infrastructure. Focus group meetings and interviews indicated support for biomass end uses that bring direct benefits to local communities. Questions arise as to how well the imagery used was able to portray the true nature of these tall, dense, perennial plants but based on the responses obtained and given the caveat that there was limited personal experience of the crops, it appears unlikely that wide-scale planting of biomass crops will give rise to substantial public concern in relation to their visual impact in the landscape

Heavy Drinking Is Associated with Poor Blood Pressure Control in the REasons for Geographic and Racial Differences in Stroke (REGARDS) Study

Alcohol intake has been shown to have a J-shaped association with blood pressure (BP). However, this association has not been examined in mixed race populations or in people with diabetes where tighter blood pressure control is recommended. Participants in the REGARDS study who were 45 years or older (n = 30,239) were included. Medical history (including self-reported alcohol intake) was collected by telephone while blood collection and clinical measurements were done during an in-home visit. We defined diabetes as use of medications and/or fasting glucose ≥ 126 mg/dL and hypertension as use of blood pressure lowering medications and/or BP ≥ 140/90 mmHg or BP ≥ 130/80 mmHg in people with diabetes. After adjustment for confounders, heavy drinking was associated with an increased odds of hypertension (OR = 1.59; 95% CI = 1.37, 1.87). Diabetes and gender significantly modified (interaction P < 0.05 for both) the association between alcohol use and hypertension, although heavy drinking remained associated with increased odds of hypertension in sub-group analyses. We did not observe the previously described J-shaped relationship in any sub-group except white females. These data suggest heavy alcohol consumption is associated with poor BP control and that heavy drinkers may want to consider limiting alcohol intake in order to manage hypertension