Lentiviral vectors for treatment of haemophilia

Haemophilia A and B are X‐linked recessive disorders caused by defects in coagulation factors (F) VIII and IX, respectively. Severe cases of haemophilia are characterised by episodes of spontaneous bleeding, predominantly into the joints and muscles, and can result in permanent disability and even mortality if left untreated. The haemophilias are compelling candidates for treatment with gene therapy as therapeutic benefit only requires a modest increase in the endogenous coagulation factor level, response to treatment can be easily monitored, and factor expression can be mediated by many cell types in vivo. Integration deficient lentiviral vectors (IDLVs) offer marked advantages over currently used integrating lentiviral vectors (ILVs) as side effects caused by insertional mutagenesis are potentially minimised. Previous work has shown that efficient and sustained transgene expression in non‐dividing cells, such as brain and muscle tissue, using IDLVs can be achieved. ILVs have previously been used to mediate long term expression of coagulation factors in vivo. In this study, we investigated the use of IDLVs as treatment for haemophilia with muscle and liver tissue (primarily nondividing hepatocytes) as principal targets. Transduction efficiency and relative transgene expression in vivo from ILVs and IDLVs were assessed in both tissues, and a number of strategies, including pseudotyping and tissue specific promoters, were utilised to improve targeted expression. Overall, despite achieving sustained transgene expression from IDLVs, in comparison to ILVs, the levels obtained were significantly lower and IDLVs were unable to mediate expression of human FIX at therapeutic levels in liver. Finally, the expression of bioengineered forms of human factor VIII (hFVIII) was assessed using ILVs in vivo after neonatal delivery. Long‐term expression was achieved and a 20‐fold increase in expression was observed after codon optimisation of the hFVIII cDNA sequence. In conclusion, IDLVs can mediate sustained transgene expression in vivo, however, vectors may need to be further optimised for increased expression to achieve clinical benefit for haemophilia patients

Identification of T. gondii myosin light chain-1 as a direct target of TachypleginA-2, a small-molecule inhibitor of parasite motility and invasion

This work was supported by US Public Health Service grant AI054961 (GEW/NJW), a University Research Fellowship from the Royal Society (NJW) and funding for the mass spectrometry analysis was provided by the Vermont Genetics Network/NIH Grant 8P20GM103449 from the INBRE program of the NIGMS.Motility of the protozoan parasite Toxoplasma gondii plays an important role in the parasite's life cycle and virulence within animal and human hosts. Motility is driven by a myosin motor complex that is highly conserved across the Phylum Apicomplexa. Two key components of this complex are the class XIV unconventional myosin, TgMyoA, and its associated light chain, TgMLC1. We previously showed that treatment of parasites with a small-molecule inhibitor of T. gondii invasion and motility, tachypleginA, induces an electrophoretic mobility shift of TgMLC1 that is associated with decreased myosin motor activity. However, the direct target(s) of tachypleginA and the molecular basis of the compound-induced TgMLC1 modification were unknown. We show here by ''click'' chemistry labelling that TgMLC1 is a direct and covalent target of an alkyne-derivatized analogue of tachypleginA. We also show that this analogue can covalently bind to model thiol substrates. The electrophoretic mobility shift induced by another structural analogue, tachypleginA-2, was associated with the formation of a 225.118 Da adduct on S57 and/or C58, and treatment with deuterated tachypleginA-2 confirmed that the adduct was derived from the compound itself. Recombinant TgMLC1 containing a C58S mutation (but not S57A) was refractory to click labelling and no longer exhibited a mobility shift in response to compound treatment, identifying C58 as the site of compound binding on TgMLC1. Finally, a knock-in parasite line expressing the C58S mutation showed decreased sensitivity to compound treatment in a quantitative 3D motility assay. These data strongly support a model in which tachypleginA and its analogues inhibit the motility of T. gondii by binding directly and covalently to C58 of TgMLC1, thereby causing a decrease in the activity of the parasite's myosin motor. Publisher PDFPeer reviewe

Psychophysiological measures of driver distraction and workload while intoxicated

The crash risk associated with cell phone use while driving is a contentious issue. Many states are introducing Advanced Traveler Information Systems (ATIS) that may be accessed with cell phones while driving (e.g., 511 Traveler Information Services). In these contexts, there is a need for relevant research to determine the risk of cell phone use. This study compared driver performance while conversing on a hands-free cell phone to conditions of operating common in-vehicle controls (e.g., radio, fan, air conditioning) and alcohol intoxication (BAC 0.08). In addition, the study examined the combined effects of being distracted and being intoxicated given that there may be a higher risk of a crash if the driver engages in a combination of risk factors. During simulated traffic scenarios, resource allocation was assessed through an eventrelated potential (ERP) novelty oddball paradigm. Intoxicated drivers were less attentive to all stimuli and drivers engaged in secondary tasks had weaker responses to unexpected novel sounds in brain regions associated with evaluative processing. Drivers conversing on the cell phone and in-vehicle tasks while sober had lower accuracy during the target tone task than intoxicated drivers not completing any secondary task

Grassland biodiversity restoration increases resistance of carbon fluxes to drought

Evidence suggests that the restoration of plant diversity in grasslands not only brings benefits for biodiversity conservation, but also the delivery of ecosystem services. While biodiversity-function experiments show that greater plant diversity increases resistance of plant productivity to climate extremes, it is not known whether real-world management options for grassland restoration likewise stabilize ecosystem responses to extreme climate events. We used a long-term (23 year) field experiment in northern England to test the hypothesis that management aimed at biodiversity restoration increases the resistance and recovery of ecosystem carbon (C) fluxes to short-term summer drought. This was tested by measuring plant, soil and microbial responses to a simulated drought in experimental grassland plots where fertilizer application and seed addition have been managed to enhance plant species diversity. The cessation of fertilizer application brought about small increases in plant species richness. Additionally, cessation of fertilizer application reduced overall plant productivity and promoted hemi-parasitic plants at the expense of grasses and forbs. Resistance of CO 2 fluxes to drought, measured as ecosystem respiration, was greater in non-fertilized plots, as lower plant biomass reduced water demand, likely aided by proportionally more hemi-parasitic plants further reducing plant biomass. Additionally, legumes increased under drought, thereby contributing to overall resistance of plant productivity. Recovery of soil microbial C and nitrogen was more rapid after rewetting than soil microbial community composition, irrespective of restoration treatment, suggesting high resilience of soil microbial communities to drought. Synthesis and applications. This study shows that while grassland diversity restoration management increases the resistance of carbon fluxes to drought, it also reduces agricultural yields, revealing a trade-off for land managers. Furthermore legumes, promoted through long-term restoration treatments, can help to maintain plant community productivity under drought by increasing their biomass. As such, grassland management strategies not only have consequences for ecosystem processes, but also the capacity to withstand extreme weather events

Toric anti-self-dual Einstein metrics via complex geometry

Using the twistor correspondence, we give a classification of toric anti-self-dual Einstein metrics: each such metric is essentially determined by an odd holomorphic function. This explains how the Einstein metrics fit into the classification of general toric anti-self-dual metrics given in an earlier paper (math.DG/0602423). The results complement the work of Calderbank-Pedersen (math.DG/0105263), who describe where the Einstein metrics appear amongst the Joyce spaces, leading to a different classification. Taking the twistor transform of our result gives a new proof of their theorem.Comment: v2. Published version. Additional references. 14 page

Identifying the mechanisms underpinning recognition of structured sequences of action

© 2012 The Experimental Psychology SocietyWe present three experiments to identify the specific information sources that skilled participants use to make recognition judgements when presented with dynamic, structured stimuli. A group of less skilled participants acted as controls. In all experiments, participants were presented with filmed stimuli containing structured action sequences. In a subsequent recognition phase, participants were presented with new and previously seen stimuli and were required to make judgements as to whether or not each sequence had been presented earlier (or were edited versions of earlier sequences). In Experiment 1, skilled participants demonstrated superior sensitivity in recognition when viewing dynamic clips compared with static images and clips where the frames were presented in a nonsequential, randomized manner, implicating the importance of motion information when identifying familiar or unfamiliar sequences. In Experiment 2, we presented normal and mirror-reversed sequences in order to distort access to absolute motion information. Skilled participants demonstrated superior recognition sensitivity, but no significant differences were observed across viewing conditions, leading to the suggestion that skilled participants are more likely to extract relative rather than absolute motion when making such judgements. In Experiment 3, we manipulated relative motion information by occluding several display features for the duration of each film sequence. A significant decrement in performance was reported when centrally located features were occluded compared to those located in more peripheral positions. Findings indicate that skilled participants are particularly sensitive to relative motion information when attempting to identify familiarity in dynamic, visual displays involving interaction between numerous features

Developing a biotic index for Colorado stream quality

November 1994.Also listed online under Open file reports list as no. 8.Includes bibliographical references (pages 114-118).Financed in part by the U.S. Dept. of the Interior, Geological Survey, grant no. 14-08-0001-G2008/3 11

A highly discriminatory RNA strand-specific assay to facilitate analysis of the role of cis-acting elements in foot-and-mouth disease virus replication

Foot-and-mouth-disease virus (FMDV), the aetiological agent responsible for foot-and-mouth disease (FMD), is a member of the genus Aphthovirus within the family Picornavirus. In common with all picornaviruses, replication of the single-stranded positive-sense RNA genome involves synthesis of a negative-sense complementary strand that serves as a template for the synthesis of multiple positive-sense progeny strands. We have previously employed FMDV replicons to examine viral RNA and protein elements essential to replication, but the factors affecting differential strand production remain unknown. Replicon-based systems require transfection of high levels of RNA, which can overload sensitive techniques such as quantitative PCR, preventing discrimination of specific strands. Here, we describe a method in which replicating RNA is labelled in vivo with 5-ethynyl uridine. The modified base is then linked to a biotin tag using click chemistry, facilitating purification of newly synthesised viral genomes or anti-genomes from input RNA. This selected RNA can then be amplified by strand-specific quantitative PCR, thus enabling investigation of the consequences of defined mutations on the relative synthesis of negative-sense intermediate and positive-strand progeny RNAs. We apply this new approach to investigate the consequence of mutation of viral cis-acting replication elements and provide direct evidence for their roles in negative-strand synthesis

Acute bone response to whole body vibration in healthy pre-pubertal boys

The skeleton responds to mechanical stimulation. We wished to ascertain the magnitude and speed of the growing skeleton’s response to a standardised form of mechanical stimulation, vibration. 36 prepubertal boys stood for 10 minutes in total on one of two vibrating platforms (high (>2 g) or low (<1 g) magnitude vibration) on either 1, 3 or 5 successive days (n=12 for each duration); 15 control subjects stood on an inactive platform. Blood samples were taken at intervals before and after vibration to measure bone formation (P1NP, osteocalcin) and resorption (CTx) markers as well as osteoprotegerin and sclerostin. There were no significant differences between platform and control groups in bone turnover markers immediately after vibration on days 1, 3 and 5. Combining platform groups, at day 8 P1NP increased by 25.1% (CI 12.3 to 38.0; paired t-test p=0.005) and bone resorption increased by 10.9% (CI 3.6 to 18.2; paired t-test p=0.009) compared to baseline. Osteocalcin, osteoprotogerin and sclerostin did not change significantly. The growing skeleton can respond quickly to vibration of either high or low magnitude. Further work is needed to determine the utility of such “stimulation-testing” in clinical practice