Classical Swine Fever Virus p7 Protein Interacts with Host Protein CAMLG and Regulates Calcium Permeability at the Endoplasmic Reticulum

We have previously shown that Classical Swine Fever Virus (CSFV) p7 is an essential nonstructural protein with a viroporin activity, a critical function in the progression of virus infection. We also identified p7 domains and amino acid residues critical for pore formation. Here, we describe how p7 specifically interacts with host protein CAMLG, an integral ER transmembrane protein involved in intracellular calcium release regulation and signal response generation. Detection of interaction as well as the identification of p7 areas mediating interaction with CAMLG was performed by yeast two-hybrid. p7-CAMLG interaction was further confirmed by confocal microscopy in eukaryotic cells, co-expressing both proteins. Mutant forms of p7 having substituted native residues identified as mediating interaction with CAMLG showed a decreased co-localization compared with the native forms of p7. Furthermore, it is shown that native p7, but not the mutated forms of p7 that fail to interact with CAMLG, efficiently mediates calcium permeability in the ER. Interestingly, viruses harboring some of those mutated forms of p7 have been previously shown to have a significantly decreased virulence in swine.ARS/USDA-University of Connecticut SCA# 58-1940-1-190 and ARS/USDA-University of the Basque Country NACA#8064-32000-056-18S

Molecular Characterization of the Viroporin Function of Foot-and-Mouth Disease Virus Nonstructural Protein 2B

Nonstructural protein 2B of foot-and-mouth disease (FMD) virus (FMDV) is comprised of a small, hydrophobic, 154-amino-acid protein. Structure-function analyses demonstrated that FMDV 2B is an ion channel-forming protein. Infrared spectroscopy measurements using partially overlapping peptides that spanned regions between amino acids 28 and 147 demonstrated the adoption of helical conformations in two putative transmembrane regions between residues 60 and 78 and between residues 119 and 147 and a third transmembrane region between residues 79 and 106, adopting a mainly extended structure. Using synthetic peptides, ion channel activity measurements in planar lipid bilayers and imaging of single giant unilamellar vesicles (GUVs) revealed the existence of two sequences endowed with membrane-porating activity: one spanning FMDV 2B residues 55 to 82 and the other spanning the C-terminal region of 2B from residues 99 to 147. Mapping the latter sequence identified residues 119 to 147 as being responsible for the activity. Experiments to assess the degree of insertion of the synthetic peptides in bilayers and the inclination angle adopted by each peptide regarding the membrane plane normal confirm that residues 55 to 82 and 119 to 147 of 2B actively insert as transmembrane helices. Using reverse genetics, a panel of 13 FMD recombinant mutant viruses was designed, which harbored nonconservative as well as alanine substitutions in critical amino acid residues in the area between amino acid residues 28 and 147. Alterations to any of these structures interfered with pore channel activity and the capacity of the protein to permeabilize the endoplasmic reticulum (ER) to calcium and were lethal for virus replication. Thus, FMDV 2B emerges as the first member of the viroporin family containing two distinct pore domains

Nanofabrication by magnetic focusing of supersonic beams

We present a new method for nanoscale atom lithography. We propose the use of a supersonic atomic beam, which provides an extremely high-brightness and cold source of fast atoms. The atoms are to be focused onto a substrate using a thin magnetic film, into which apertures with widths on the order of 100 nm have been etched. Focused spot sizes near or below 10 nm, with focal lengths on the order of 10 microns, are predicted. This scheme is applicable both to precision patterning of surfaces with metastable atomic beams and to direct deposition of material.Comment: 4 pages, 3 figure

High resolution amplitude and phase gratings in atom optics

An atom-field geometry is chosen in which an atomic beam traverses a field interaction zone consisting of three fields, one having frequency $\Omega =c/\lambda$ propagating in the $\hat{z}$ direction and the other two having frequencies $\Omega +\delta_{1}$ and $\Omega +\delta_{2}$ propagating in the -$\hat{z}$ direction. For $n_{1}\delta_{1}+n_{2}\delta_{2}=0$ and $|\delta_{1}| T,|\delta_{2}| T\gg 1$, where $n_{1}$ and $n_{2}$ are positive integers and $T$ is the pulse duration in the atomic rest frame, the atom-field interaction results in the creation of atom amplitude and phase gratings having period $\lambda /[2(n_{1}+n_{2})]$. In this manner, one can use optical fields having wavelength $\lambda$ to produce atom gratings having periodicity much less than $\lambda$.Comment: 11 pages, 14 figure

Evaluation of the effect of patient education on rates of falls in older hospital patients: Description of a randomised controlled trial

Background. Accidental falls by older patients in hospital are one of the most commonly reported adverse events. Falls after discharge are also common. These falls have enormous physical, psychological and social consequences for older patients, including serious physical injury and reduced quality of life, and are also a source of substantial cost to health systems worldwide. There have been a limited number of randomised controlled trials, mainly using multifactorial interventions, aiming to prevent older people falling whilst inpatients. Trials to date have produced conflicting results and recent meta-analyses highlight that there is still insufficient evidence to clearly identify which interventions may reduce the rate of falls, and falls related injuries, in this population. Methods and design. A prospective randomised controlled trial (n = 1206) is being conducted at two hospitals in Australia. Patients are eligible to be included in the trial if they are over 60 years of age and they, or their family or guardian, give written consent. Participants are randomised into three groups. The control group continues to receive usual care. Both intervention groups receive a specifically designed patient education intervention on minimising falls in addition to usual care. The education is delivered by Digital Video Disc (DVD) and written workbook and aims to promote falls prevention activities by participants. One of the intervention groups also receives follow up education training visits by a health professional. Blinded assessors conduct baseline and discharge assessments and follow up participants for 6 months after discharge. The primary outcome measure is falls by participants in hospital. Secondary outcome measures include falls at home after discharge, knowledge of falls prevention strategies and motivation to engage in falls prevention activities after discharge. All analyses will be based on intention to treat principle. Discussion. This trial will examine the effect of a single intervention (specifically designed patient education) on rates of falls in older patients in hospital and after discharge. The results will provide robust recommendations for clinicians and researchers about the role of patient education in this population. The study has the potential to identify a new intervention that may reduce rates of falls in older hospital patients and could be readily duplicated and applied in a wide range of clinical settings. Trial Registration. ACTRN12608000015347

Effects of ocean sprawl on ecological connectivity: impacts and solutions

The growing number of artificial structures in estuarine, coastal and marine environments is causing “ocean sprawl”. Artificial structures do not only modify marine and coastal ecosystems at the sites of their placement, but may also produce larger-scale impacts through their alteration of ecological connectivity - the movement of organisms, materials and energy between habitat units within seascapes. Despite the growing awareness of the capacity of ocean sprawl to influence ecological connectivity, we lack a comprehensive understanding of how artificial structures modify ecological connectivity in near- and off-shore environments, and when and where their effects on connectivity are greatest. We review the mechanisms by which ocean sprawl may modify ecological connectivity, including trophic connectivity associated with the flow of nutrients and resources. We also review demonstrated, inferred and likely ecological impacts of such changes to connectivity, at scales from genes to ecosystems, and potential strategies of management for mitigating these effects. Ocean sprawl may alter connectivity by: (1) creating barriers to the movement of some organisms and resources - by adding physical barriers or by modifying and fragmenting habitats; (2) introducing new structural material that acts as a conduit for the movement of other organisms or resources across the landscape; and (3) altering trophic connectivity. Changes to connectivity may, in turn, influence the genetic structure and size of populations, the distribution of species, and community structure and ecological functioning. Two main approaches to the assessment of ecological connectivity have been taken: (1) measurement of structural connectivity - the configuration of the landscape and habitat patches and their dynamics; and (2) measurement of functional connectivity - the response of organisms or particles to the landscape. Our review reveals the paucity of studies directly addressing the effects of artificial structures on ecological connectivity in the marine environment, particularly at large spatial and temporal scales. With the ongoing development of estuarine and marine environments, there is a pressing need for additional studies that quantify the effects of ocean sprawl on ecological connectivity. Understanding the mechanisms by which structures modify connectivity is essential if marine spatial planning and eco-engineering are to be effectively utilised to minimise impacts

Cetacean rapid assessment : an approach to fill knowledge gaps and target conservation across large data deficient areas

The work was funded by the Pew Marine Fellows Program and WCS.1. Many species and populations of marine megafauna are undergoing substantial declines, while many are also very poorly understood. Even basic information on species presence is unknown for tens of thousands of kilometres of coastline, particularly in the developing world, which is a major hurdle to their conservation. 2. Rapid ecological assessment is a valuable tool used to identify and prioritize areas for conservation; however, this approach has never been clearly applied to marine cetaceans. Here a rapid assessment protocol is outlined that will generate broad‐scale, quantitative, baseline data on cetacean communities and potential threats, that can be conducted rapidly and cost-effectively across whole countries, or regions. 3. The rapid assessment was conducted in Tanzania, East Africa, and integrated collection of data on cetaceans from visual, acoustic, and interview surveys with existing information from multiple sources, to provide low resolution data on cetacean community relative abundance, diversity, and threats. Four principal threats were evaluated and compared spatially using a qualitative scale: cetacean mortality in fishing gear (particularly gillnets); cetacean hunting, consumption or use by humans; shipping related collision risk and noise disturbance; and dynamite fishing. 4. Ninety‐one groups of 11 species of marine mammal were detected during field surveys. Potentially the most important area for cetaceans was the Pemba Channel, a deep, high‐current waterway between Pemba Island and mainland Africa, where by far the highest relative cetacean diversity and high relative abundance were recorded, but which is also subject to threats from fishing. 5. A rapid assessment approach can be applied in data deficient areas to quickly provide information on cetaceans that can be used by governments and managers for marine spatial planning, management of developments, and to target research activities into the most important locations.PostprintPeer reviewe