Expression of enzymatically active reverse transcriptase in Escherichia coli

Reverse transcriptase of murine retroviruses is a monomeric protein of approximately 80,000 daltons, which is encoded by the central portion of the viral pol gene. To prepare large quantities of the enzyme, we have constructed gene fusions between the trpE gene and portions of the pol gene of Moloney murine leukemia virus. The inserted pol gene sequences include the entire coding region for the mature enzyme and various amounts of additional coding sequences. Many of these constructs express high levels of reverse transcriptase activity even though the NH2 and COOH termini of the protein product only approximate the correct termini of the authentic protein

Navier-Stokes Analysis of a High Wing Transport High-Lift Configuration with Externally Blown Flaps

Insights and lessons learned from the aerodynamic analysis of the High Wing Transport (HWT) high-lift configuration are presented. Three-dimensional Navier-Stokes CFD simulations using the OVERFLOW flow solver are compared with high Reynolds test data obtained in the NASA Ames 12 Foot Pressure Wind Tunnel (PWT) facility. Computational analysis of the baseline HWT high-lift configuration with and without Externally Blown Flap (EBF) jet effects is highlighted. Several additional aerodynamic investigations, such as nacelle strake effectiveness and wake vortex studies, are presented. Technical capabilities and shortcomings of the computational method are discussed and summarized

Effects of DNA nonhomologous end-joining factors on telomere length and chromosomal stability in mammalian cells

AbstractDNA repair by nonhomologous end-joining (NHEJ) relies on the Ku70:Ku80 heterodimer in species ranging from yeast to man. In Saccharomyces cerevisiae and Schizosaccharomyces pombe, Ku also controls telomere functions. Here, we show that Ku70, Ku80, and DNA-PKcs, with which Ku interacts, associate in vivo with telomeric DNA in several human cell types, and we show that these associations are not significantly affected by DNA-damaging agents. We also demonstrate that inactivation of Ku80 or Ku70 in the mouse yields telomeric shortening in various primary cell types at different developmental stages. By contrast, telomere length is not altered in cells impaired in XRCC4 or DNA ligase IV, two other NHEJ components. We also observe higher genomic instability in Ku-deficient cells than in XRCC4-null cells. This suggests that chromosomal instability of Ku-deficient cells results from a combination of compromised telomere stability and defective NHEJ

Mechanism of membrane fusion induced by vesicular stomatitis virus G protein

The glycoproteins (G proteins) of vesicular stomatitis virus (VSV) and related rhabdoviruses (e.g., rabies virus) mediate both cell attachment and membrane fusion. The reversibility of their fusogenic conformational transitions differentiates them from many other low-pH-induced viral fusion proteins. We report single-virion fusion experiments, using methods developed in previous publications to probe fusion of influenza and West Nile viruses. We show that a three-stage model fits VSV single-particle fusion kinetics: (i) reversible, pH-dependent, G-protein conformational change from the known prefusion conformation to an extended, monomeric intermediate; (ii) reversible trimerization and clustering of the G-protein fusion loops, leading to an extended intermediate that inserts the fusion loops into the target-cell membrane; and (iii) folding back of a cluster of extended trimers into their postfusion conformations, bringing together the viral and cellular membranes. From simulations of the kinetic data, we conclude that the critical number of G-protein trimers required to overcome membrane resistance is 3 to 5, within a contact zone between the virus and the target membrane of 30 to 50 trimers. This sequence of conformational events is similar to those shown to describe fusion by influenza virus hemagglutinin (a “class I” fusogen) and West Nile virus envelope protein (“class II”). Our study of VSV now extends this description to “class III” viral fusion proteins, showing that reversibility of the low-pH-induced transition and architectural differences in the fusion proteins themselves do not change the basic mechanism by which they catalyze membrane fusion

Exercise, APOE, and Working Memory: MEG and Behavioral Evidence for Benefit of Exercise in Epsilon4 Carriers

Performance on the Sternberg working memory task, and MEG cortical response on a variation of the Sternberg task were examined in middle-aged carriers and non-carriers of the APOE ε4 allele. Physical activity was also assessed to examine whether exercise level modifies the relationship between APOE genotype and neurocognitive function. Regression revealed that high physical activity was associated with faster RT in the six- and eight-letter conditions of the Sternberg in ε4 carriers, but not in the non-carriers after controlling for age, gender, and education (N = 54). Furthermore, the MEG analysis revealed that sedentary ε4 carriers exhibited lower right temporal lobe activation on matching probe trials relative to high-active ε4 carriers, while physical activity did not distinguish non-carriers (N = 23). The M170 peak was identified as a potential marker for pre-clinical decline as ε4 carriers exhibited longer M170 latency, and highly physically active participants exhibited greater M170 amplitude to matching probe trials

In-street wind direction variability in the vicinity of a busy intersection in central London

We present results from fast-response wind measurements within and above a busy intersection between two street canyons (Marylebone Road and Gloucester Place) in Westminster, London taken as part of the DAPPLE (Dispersion of Air Pollution and Penetration into the Local Environment; www.dapple.org.uk) 2007 field campaign. The data reported here were collected using ultrasonic anemometers on the roof-top of a building adjacent to the intersection and at two heights on a pair of lamp-posts on opposite sides of the intersection. Site characteristics, data analysis and the variation of intersection flow with the above-roof wind direction (θref) are discussed. Evidence of both flow channelling and recirculation was identified within the canyon, only a few metres from the intersection for along-street and across-street roof-top winds respectively. Results also indicate that for oblique rooftop flows, the intersection flow is a complex combination of bifurcated channelled flows, recirculation and corner vortices. Asymmetries in local building geometry around the intersection and small changes in the background wind direction (changes in 15-min mean θref of 5–10 degrees) were also observed to have profound influences on the behaviour of intersection flow patterns. Consequently, short time-scale variability in the background flow direction can lead to highly scattered in-street mean flow angles masking the true multi-modal features of the flow and thus further complicating modelling challenges

Reprogramming human T cell function and specificity with non-viral genome targeting.

Decades of work have aimed to genetically reprogram T cells for therapeutic purposes1,2 using recombinant viral vectors, which do not target transgenes to specific genomic sites3,4. The need for viral vectors has slowed down research and clinical use as their manufacturing and testing is lengthy and expensive. Genome editing brought the promise of specific and efficient insertion of large transgenes into target cells using homology-directed repair5,6. Here we developed a CRISPR-Cas9 genome-targeting system that does not require viral vectors, allowing rapid and efficient insertion of large DNA sequences (greater than one kilobase) at specific sites in the genomes of primary human T cells, while preserving cell viability and function. This permits individual or multiplexed modification of endogenous genes. First, we applied this strategy to correct a pathogenic IL2RA mutation in cells from patients with monogenic autoimmune disease, and demonstrate improved signalling function. Second, we replaced the endogenous T cell receptor (TCR) locus with a new TCR that redirected T cells to a cancer antigen. The resulting TCR-engineered T cells specifically recognized tumour antigens and mounted productive anti-tumour cell responses in vitro and in vivo. Together, these studies provide preclinical evidence that non-viral genome targeting can enable rapid and flexible experimental manipulation and therapeutic engineering of primary human immune cells

Risk factors for Brucella spp. and Coxiella burnetii infection among small ruminants in Eastern India

Small ruminants are the main reservoirs for brucellosis and coxiellosis, two zoonotic diseases affecting livestock production, and posing a public health threat in India. Understanding disease prevalence and risk factors associated with small ruminant infection can help mitigate disease transmission. We report a cross-sectional survey in the states of Assam and Odisha in Eastern India. We interviewed 244 farmers to assess knowledge, attitude and practices relevant to brucellosis and coxiellosis infection. Serum samples from 411 goats and 21 sheep were analysed using enzyme-linked immunosorbent assay and Rose-Bengal Brucella agglutination plate test. Higher Brucella and Coxiella burnetii seroprevalence were found in Odisha (22% and 11.5%, respectively) than Assam (9.8% and 1.6%, respectively), and certain districts in Odisha were at higher risk. No association was found between seropositive animals and clinical signs, a challenge when attempting to identify seropositive animals in the herd. None of the farmers interviewed were aware of brucellosis, its aetiology, clinical form, or zoonotic risk. This study acts as a first indication of the extent of these diseases among small ruminants in these Indian states, highlighting how farming practices are associated with increased risk of infection. More research is urgently needed to mitigate zoonoses transmission in this region

Interacting mindreaders

Could interacting mindreaders be in a position to know things which they would be unable to know if they were manifestly passive observers? This paper argues that they could. Mindreading is sometimes reciprocal: the mindreader's target reciprocates by taking the mindreader as a target for mindreading. The paper explains how such reciprocity can significantly narrow the range of possible interpretations of behaviour where mindreaders are, or appear to be, in a position to interact. A consequence is that revisions and extensions are needed to standard theories of the evidential basis of mindreading. The view also has consequences for understanding how abilities to interact combined with comparatively simple forms of mindreading may explain the emergence, in evolution or development, of sophisticated forms of social cognition