Mechanism of Chromophore Assisted Laser Inactivation Employing Fluorescent Proteins

Chromophore Assisted Laser Inactivation (CALI) is a technique that uses irradiation of chromophores proximate to a target protein to inactivate function. Previously, EGFP mediated CALI has been used to inactivate EGFP-fusion proteins in a spatio-temporally defined manner within cells but the mechanism of inactivation is unknown 1, 2. To help elucidate the mechanism of protein inactivation mediated by fluorescent protein CALI ([FP]-CALI), the activities of purified Glutathione-S-transferase-FP (GST-EXFP) fusions were measured after laser irradiation in vitro. Singlet oxygen and free radical quenchers as well as the removal of oxygen inhibited CALI, indicating the involvement of a reactive oxygen species (ROS). At higher concentrations of protein, turbidity after CALI increased significantly indicating cross-linking of proximate fusion proteins suggesting that damage of residues on the surface the protein, distant from the active site, results in inactivation. Control experiments removed sample heating as a possible cause of these effects. Different FP mutants fused to GST vary in their CALI efficiency in the order EGFP>EYFP>ECFP, while a GST construct that binds FlAsH results in significantly higher CALI efficiency than any of the XFPs tested. It is likely that the hierarchy of XFP effectiveness reflects the balance between ROS that are trapped within the XFP structure and cause fluorophore and chromophore bleaching and those that escape to effect CALI of proximate proteins

Germplasm enhancement for resistance to Pyrenophra tritici-repentis in wheat

Yellow spot (syn. tan spot), caused by Pyrenophora tritici-repentis , is an important foliar disease of wheat in Australia that causes losses exceeding 50 % when conditions are favourable for disease development. Although good progress has been made internationally to understand yellow spot resistance, relatively few resistance genes have been identifi ed and mapped in Australian germplasm and only one (tsn1 on chromosome 5BL) is in general and known use in Australian breeding programs. Although tsn1 is an important yellow spot resistance gene, it doesn’t explain the full spectrum of resistance and there is a significant opportunity to enhance expression of yellow spot resistance through identification of resistance factors other than tsn1 . Six doubled haploid (DH) mapping populations (five of which were fixed for tsn1) were screened for yellow spot resistance at the seedling/tillering and adult plant stages at the Department of Agriculture and Food, Western Australia (DAFWA) and the Department of Environment and Primary Industries Victoria (DEPIVic) from 2009 to 2012. Four of the above populations were screened at the Department of Agriculture, Fisheries and Forestry Queensland (DAFFQ). Frequency distribution of individuals within each population for various levels of yellow spot resistance was continuous indicating that resistance is conditioned by several genes with partial effects. A few lines within each population consistently showed high levels of resistance probably resulting from a combination of several genes with additive effects. Nine new loci for yellow spot resistance were mapped

Design and development of MOSFIRE: the Multi-Object Spectrometer For Infra-Red Exploration at the Keck Observatory

MOSFIRE is a unique multi-object spectrometer and imager for the Cassegrain focus of the 10 m Keck 1 telescope. A refractive optical design provides near-IR (0.97 to 2.45 μm) multi-object spectroscopy over a 6.14' x 6.14' field of view with a resolving power of R~3,270 for a 0.7" slit width (2.9 pixels in the dispersion direction), or imaging over a field of view of 6.8' diameter with 0.18" per pixel sampling. A single diffraction grating can be set at two fixed angles, and order-sorting filters provide spectra that cover the K, H, J or Y bands by selecting 3rd, 4th, 5th or 6th order respectively. A folding flat following the field lens is equipped with piezo transducers to provide tip/tilt control for flexure compensation at the 0.1 pixel level. A special feature of MOSFIRE is that its multiplex advantage of up to 46 slits is achieved using a cryogenic Configurable Slit Unit or CSU developed in collaboration with the Swiss Centre for Electronics and Micro Technology (CSEM). The CSU is reconfigurable under remote control in less than 5 minutes without any thermal cycling of the instrument. Slits are formed by moving opposable bars from both sides of the focal plane. An individual slit has a length of 7.1" but bar positions can be aligned to make longer slits. When masking bars are removed to their full extent and the grating is changed to a mirror, MOSFIRE becomes a wide-field imager. Using a single, ASIC-driven, 2K x 2K H2-RG HgCdTe array from Teledyne Imaging Sensors with exceptionally low dark current and low noise, MOSFIRE will be extremely sensitive and ideal for a wide range of science applications. This paper describes the design and testing of the instrument prior to delivery later in 2010

West Nile Virus Range Expansion into British Columbia

Elevated temperatures and mosquito abundance may contribute

Inhaled Nanoparticles Accumulate at Sites of Vascular Disease

The development of engineered nanomaterials is growing exponentially, despite concerns over their potential similarities to environmental nanoparticles that are associated with significant cardiorespiratory morbidity and mortality. The mechanisms through which inhalation of nanoparticles could trigger acute cardiovascular events are emerging, but a fundamental unanswered question remains: Do inhaled nanoparticles translocate from the lung in man and directly contribute to the pathogenesis of cardiovascular disease? In complementary clinical and experimental studies, we used gold nanoparticles to evaluate particle translocation, permitting detection by high-resolution inductively coupled mass spectrometry and Raman microscopy. Healthy volunteers were exposed to nanoparticles by acute inhalation, followed by repeated sampling of blood and urine. Gold was detected in the blood and urine within 15 min to 24 h after exposure, and was still present 3 months after exposure. Levels were greater following inhalation of 5 nm (primary diameter) particles compared to 30 nm particles. Studies in mice demonstrated the accumulation in the blood and liver following pulmonary exposure to a broader size range of gold nanoparticles (2-200 nm primary diameter), with translocation markedly greater for particles <10 nm diameter. Gold nanoparticles preferentially accumulated in inflammation-rich vascular lesions of fat-fed apolipoproteinE-deficient mice. Furthermore, following inhalation, gold particles could be detected in surgical specimens of carotid artery disease from patients at risk of stroke. Translocation of inhaled nanoparticles into the systemic circulation and accumulation at sites of vascular inflammation provides a direct mechanism that can explain the link between environmental nanoparticles and cardiovascular disease and has major implications for risk management in the use of engineered nanomaterials

Diesel exhaust particulate increases the size and complexity of lesions in atherosclerotic mice

Abstract Objective Diesel exhaust particulate (DEP), a major component of urban air pollution, has been linked to atherogenesis and precipitation of myocardial infarction. We hypothesized that DEP exposure would increase and destabilise atherosclerotic lesions in apolipoprotein E deficient (ApoE−/−) mice. Methods ApoE−/− mice were fed a ‘Western diet’ (8 weeks) to induce ‘complex’ atherosclerotic plaques, with parallel experiments in normal chow fed wild-type mice. During the last 4 weeks of feeding, mice received twice weekly instillation (oropharyngeal aspiration) of 35 μL DEP (1 mg/mL, SRM-2975) or vehicle (saline). Atherosclerotic burden was assessed by en-face staining of the thoracic aorta and histological examination of the brachiocephalic artery. Results Brachiocephalic atherosclerotic plaques were larger in ApoE−/− mice treated with DEP (59±10%) than in controls (32±7%; P = 0.017). In addition, DEP-treated mice had more plaques per section of artery (2.4±0.2 vs 1.8±0.2; P = 0.048) and buried fibrous layers (1.2±0.2 vs 0.4±0.1; P = 0.028). These changes were associated with lung inflammation and increased antioxidant gene expression in the liver, but not with changes in endothelial function, plasma lipids or systemic inflammation. Conclusions Increased atherosclerosis is caused by the particulate component of diesel exhaust producing advanced plaques with a potentially more vulnerable phenotype. These results are consistent with the suggestion that removal of the particulate component would reduce the adverse cardiovascular effects of diesel exhaust

What Every Business Student Needs to Know About Information Systems

Whether Information Systems should or should not be part of the core business school curriculum is a recurring discussion in many universities. In this article, a task force of 40 prominent information systems scholars address the issue. They conclude that information systems is absolutely an essential body of knowledge for business school students to acquire as well as a key element of the business school\u27s long-run strategic positioning within the university. Originally prepared in response to draft accreditation guidelines prepared by AACSB International, the article includes a compilation of the concepts that the authors believe to be the core information systems knowledge that all business school students should be familiar with

Mutations involving the SRY-related gene SOX8 are associated with a spectrum of human reproductive anomalies.

© The Author(s) 2018. Published by Oxford University Press. All rights reserved. SOX8 is an HMG-box transcription factor closely related to SRY and SOX9. Deletion of the gene encoding Sox8 in mice causes reproductive dysfunction but the role of SOX8 in humans is unknown. Here, we show that SOX8 is expressed in the somatic cells of the early developing gonad in the human and influences human sex determination. We identified two individuals with 46, XY disorders/differences in sex development (DSD) and chromosomal rearrangements encompassing the SOX8 locus and a third individual with 46, XY DSD and a missense mutation in the HMG-box of SOX8. In vitro functional assays indicate that this mutation alters the biological activity of the protein. As an emerging body of evidence suggests that DSDs and infertility can have common etiologies, we also analysed SOX8 in a cohort of infertile men (n=274) and two independent cohorts of women with primary ovarian insufficiency (POI; n=153 and n=104). SOX8 mutations were found at increased frequency in oligozoospermic men (3.5%; P < 0.05) and POI (5.06%; P=4.5×10 -5 ) as compared with fertile/normospermic control populations (0.74%). The mutant proteins identified altered SOX8 biological activity as compared with the wild-type protein. These data demonstrate that SOX8 plays an important role in human reproduction and SOX8 mutations contribute to a spectrum of phenotypes including 46, XY DSD, male infertility and 46, XX POI.Link_to_subscribed_fulltex