Protein coexpression using FMDV 2A : effect of “linker” residues

This article was made open access through BIS OA funding. The research was supported by the MRC.Many biomedical applications absolutely require, or are substantially enhanced by, coexpression of multiple proteins from a single vector. Foot-and-mouth disease virus 2A (F2A) and “2A-like” sequences (e.g., Thosea asigna virus 2A; T2A) are used widely for this purpose since multiple proteins can be coexpressed by linking open reading frames (ORFs) to form a single cistron. The activity of F2A “cleavage” may, however, be compromised by both the use of shorter versions of F2A and the sequences (derived from multiple-purpose cloning sites) used to link F2A to the upstream protein. To characterise these effects, different lengths of F2A and T2A were inserted between green and cherry fluorescent proteins. Mutations were introduced in the linker region immediately upstream of both F2A- and T2A-based constructs and activities determined using both cell-free translation systems and transfected cells. In shorter versions of F2A, activity may be affected by both the C-terminal sequence of the protein upstream and, equally strikingly, the residues immediately upstream introduced during cloning. Mutations significantly improved activity for shorter versions of F2A but could decrease activity in the case of T2A. These data will aid the design of cloning strategies for the co-expression of multiple proteins in biomedical/biotechnological applications.Peer reviewe

Acceptability and Feasibility of Virtual Reality to Promote Health Literacy in Primary Care from the Health Professional’s view: A Qualitative Study

ObjectiveThe development of health literacy is important in the management of chronic pain and virtual reality may be an effective medium for its development. This study aims to understand the usability and acceptability of a virtual reality-based pain education system for the facilitation of health literacy.MethodsSemi-structured interviews were conducted with health professionals who had used a VR-based pain education system within their clinical practice, to explore perceptions of feasibility. Data collection and analyses were informed by the Unified Theory of Acceptance and Use of Technology and the Integrated Model of Health Literacy.ResultsFrom 10 participants, the VR-based system was considered feasible in providing immersive experiential learning which addressed patient understanding and health-related communication.ConclusionVR appears to be perceived as an acceptable and feasible technology to support the development of health literacy in people with chronic pain. Its largest perceived benefit was its capacity to provide an immersive and entertaining alternative to conventional methods of pain education.Practice ImplicationsVirtual reality is considered as a feasible method of facilitating patient understanding and health-related communication related to chronic pain. Feasibility of such a tool relies clinically on time available, social expectations of VR, and the role of immersive and experiential learning within the management of chronic pain

Structure and stereochemistry of the base excision repair glycosylase MutY reveal a mechanism similar to retaining glycosidases.

MutY adenine glycosylases prevent DNA mutations by excising adenine from promutagenic 8-oxo-7,8-dihydroguanine (OG):A mismatches. Here, we describe structural features of the MutY active site bound to an azaribose transition state analog which indicate a catalytic role for Tyr126 and approach of the water nucleophile on the same side as the departing adenine base. The idea that Tyr126 participates in catalysis, recently predicted by modeling calculations, is strongly supported by mutagenesis and by seeing close contact between the hydroxyl group of this residue and the azaribose moiety of the transition state analog. NMR analysis of MutY methanolysis products corroborates a mechanism for adenine removal with retention of stereochemistry. Based on these results, we propose a revised mechanism for MutY that involves two nucleophilic displacement steps akin to the mechanisms accepted for 'retaining' O-glycosidases. This new-for-MutY yet familiar mechanism may also be operative in related base excision repair glycosylases and provides a critical framework for analysis of human MutY (MUTYH) variants associated with inherited colorectal cancer

Syenite-hosted gold mineralization and hydrothermal alteration at the Young-Davidson deposit, Matachewan, Ontario

The syenite-hosted Young-Davidson (YD) gold deposit is located at the western extension of the Cadillac-Larder-Lake deformation zone (CLLDZ), southwest of Kirkland Lake, in the southern Abitibi greenstone belt, Canada. Gold is predominantly hosted by syenite, and to a lesser extent by Timiskaming sedimentary rocks and mafic volcanic rocks in veins and/or small pervasively altered shear zones related to the CLLDZ. Historical gold production was mainly from quartz veins and disseminated pyrite hosted in altered syenite. Recent drilling by AuRico Gold Corporation (formerly Northgate Minerals Co.) has expanded the underground resource to 14.6 million tonnes (measured and indicated) at an average grade of 2.03 g/t Au and an underground reserve of 39.1 million tonnes (proven and probable) at an average grade of 2.79 g/t Au, where most of the gold is hosted in the syenite. Three generations of veins have been identified based on crosscutting relationships mapped at an underground ore crosscut: V1 boudinaged iron-carbonate veins, V2 folded quartz-pyrite veinlets and V3 planar quartz-carbonate veins. Structural characterization of the vein sets indicates that V2 and V3 extensional vein arrays developed during the D2 deformation, which generated the steeply dipping east-west striking penetrative S2 foliation that is found elsewhere along the CLLDZ. Petrographic analysis of mineralized syenite shows that native gold grains are most abundant in zones of intense potassic-hematite-pyrite alteration as inclusions and along cracks in vein-related and disseminated pyrite. Volcanic-hosted gold is mainly located in quartz-iron-carbonate veins hosted in narrow sheared and pervasively albite-iron-carbonate-pyrite altered volcanic rocks. Whole rock lithogeochemical analyses and mass balance calculations show that mineralized syenite is characterized by enrichments in K, S, Na, Ba and W whereas mineralized volcanic rocks are characterized by strong enrichments in Na, Al, Si, K, Fe, S, Ba and W. Geochemical contouring of 279 analyses of drill core along two N-S sections through the YD syenite show broad cross section scale correlation of gold mineralization with K2O, S and Ba. Sulfur is the most strongly correlated with gold mineralization; however, the most intense sulfidation is generally coincident with zones containing coarse altered feldspars and a fine-grained groundmass of iron-carbonate, Na- and K-feldspar and minor quartz. Bulk sulfur isotopic analyses of syenite-hosted pyrite grains indicate that the dominant source of sulfur is magmatic but there is also a component that has interacted with the reduced Archean atmosphere. Pyrite grains in syenite and volcanic rocks contain up to 15,000 ppm Co, 4,000 ppm Ni and 4,200 ppm As. Chemical mapping of Co, Ni and As from different vein associated pyrite grains show: 1) Arsenic in pyrite is low, < 1,000 ppm; 2) Pyrite shows Co-Ni zoning and gold in V1 vein pyrite is associated with Co-Ni rich pyrite overgrowths of earlier corroded pyrite grains; 3) gold along fractures in V2 vein pyrite is also associated with Co-Ni enrichment; and 4) gold in V3 veins crosscuts Co-Ni patterns in pyrite, which suggests that the V3 style of mineralization has precipitated by different mechanisms. Pyrite mapping combined with alteration assemblages, mineralization characteristics and mineral chemistry suggests that the syenite-hosted mineralization has formed from interaction between multiple fluids with distinct physicochemical conditions. The proposed formational model for the Young-Davidson deposit is of fluid mixing between magmatic and metamorphic fluids during the deformation events that generated the extensive veining systems at Young-Davidson

FMDV replicons encoding green fluorescent protein are replication competent

The study of replication of viruses that require high bio-secure facilities can be accomplished with less stringent containment using non-infectious 'replicon' systems. The FMDV replicon system (pT7rep) reported by Mclnerney et al. (2000) was modified by the replacement of sequences encoding chloramphenicol acetyl-transferase (CAT) with those encoding a functional L proteinase (Lpro) linked to a bi-functional fluorescent/antibiotic resistance fusion protein (green fluorescent protein/puromycin resistance, [GFP-PAC]). Cells were transfected with replicon-derived transcript RNA and GFP fluorescence quantified. Replication of transcript RNAs was readily detected by fluorescence, whilst the signal from replication-incompetent forms of the genome was >2-fold lower. Surprisingly, a form of the replicon lacking the Lpro showed a significantly stronger fluorescence signal, but appeared with slightly delayed kinetics. Replication can, therefore, be quantified simply by live-cell imaging and image analyses, providing a rapid and facile alternative to RT-qPCR or CAT assays

Evaluation of the accuracy of serum MMP-9 as a test for colorectal cancer in a primary care population

Background Bowel cancer is common and is a major cause of death. Meta-analysis of randomised controlled trials estimates that screening for colorectal cancer using faecal occult blood (FOB) test reduces mortality from colorectal cancer by 16%. However, FOB testing has a low positive predictive value, with associated unnecessary cost, risk and anxiety from subsequent investigation, and is unacceptable to a proportion of the target population. Increased levels of an enzyme called matrix metalloproteinase 9 (MMP-9) have been found to be associated with colorectal cancer, and this can be measured from a blood sample. Serum MMP-9 is potentially an accurate, low risk and cost-effective population screening tool. This study aims to evaluate the accuracy of serum MMP-9 as a test for colorectal cancer in a primary care population. Methods/Design People aged 50 to 69 years, who registered in participating general practices in the West Midlands Region, will be asked to complete a questionnaire that asks about symptoms. Respondents who describe any colorectal symptoms (except only abdominal bloating and/or anal symptoms) and are prepared to provide a blood sample for MMP9 estimation and undergo a colonoscopy (current gold standard investigation) will be recruited at GP based clinics by a research nurse. Those unfit for colonoscopy will be excluded. Colonoscopies will be undertaken in dedicated research clinics. The accuracy of MMP-9 will be assessed by comparing the MMP-9 level with the colonoscopy findings, and the combination of factors (e.g. symptoms and MMP-9 level) that best predict a diagnosis of malignancy (invasive disease or polyps) will be determined. Discussion Colorectal cancer is a major cause of morbidity and mortality. Most colorectal cancers arise from adenomas and there is a period for early detection by screening, but available tests have risks, are unacceptable to many, have high false positive rates or are expensive. This study will establish the potential of serum MMP-9 as a screening test for colorectal cancer. If it is confirmed as accurate and acceptable, this serum marker has the potential to assist with reducing the morbidity and mortality from colorectal cancer

Barchan-Parabolic Dune Pattern Transition From Vegetation Stability Threshold

Many dune fields exhibit a downwind transition from forward-pointing barchan dunes to stabilized, backward-pointing parabolic dunes, accompanied by an increase in vegetation. A recent model predicts this pattern transition occurs when dune surface erosion/deposition rates decrease below a threshold of half the vegetation growth rate. We provide a direct test using a unique data set of repeat topographic surveys across White Sands Dune Field and find strong quantitative support for the model threshold. We also show the threshold hypothesis applied to a barchan dune results naturally in its curvature inversion, as the point of threshold crossing progresses from the horns to the crest. This simple, general threshold framework can be an extremely useful tool for predicting the response of dune landscapes to changes in wind speed, sediment supply, or vegetation growth rate. Near the threshold, a small environmental change could result in a drastic change in dune pattern and activity

FIREBALL: Instrument pointing and aspect reconstruction

The Faint Intergalactic Redshifted Emission Balloon (FIREBALL) had its first scientific flight in June 2009. The instrument is a 1 meter class balloon-borne telescope equipped with a vacuum-ultraviolet integral field spectrograph intended to detect emission from the inter-galactic medium at redshifts 0.3 < z < 1.0. The scientific goals and the challenging environment place strict constraints on the pointing and tracking systems of the gondola. In this manuscript we briefly review our pointing requirements, discuss the methods and solutions used to meet those requirements, and present the aspect reconstruction results from the first successful scientific flight

The kinematics of ionized gas in lyman-break analogs at z ~ 0.2

We present results for 19 “Lyman-break analogs” observed with Keck/OSIRIS with an adaptive-optics-assisted spatial resolution of less than 200 pc. We detect satellites/companions, diffuse emission, and velocity shear, all with high signal-to-noise ratios. These galaxies present remarkably high velocity dispersion along the line of sight (~70 km s^(−1)), much higher than standard star-forming spirals in the low-redshift universe. We artificially redshift our data to z ~ 2.2 to allow for a direct comparison with observations of high-z Lyman-break galaxies and find striking similarities between both samples. This suggests that either similar physical processes are responsible for their observed properties, or, alternatively, that it is very difficult to distinguish between different mechanisms operating in the low- versus high-redshift starburst galaxies based on the available data. The comparison between morphologies in the UV/optical continuum and our kinemetry analysis often shows that neither is by itself sufficient to confirm or completely rule out the contribution from recent merger events. We find a correlation between the kinematic properties and stellar mass, in that more massive galaxies show stronger evidence for a disk-like structure. This suggests a co-evolutionary process between the stellar mass buildup and the formation of morphological and dynamical substructure within the galaxy