Development of strategies for conditional RNA interference

BACKGROUND: RNA interference (RNAi) represents a powerful tool with which to undertake sequence-dependent suppression of gene expression. Synthesized double-stranded RNA (dsRNA) or dsRNA generated endogenously from plasmid or viral vectors can be used for RNAi. For the latter, polymerase III promoters which drive ubiquitous expression in all tissues have typically been adopted. Given that dsRNA molecules must contain few 5' and 3' over-hanging bases to maintain potency, employing polymerase II promoters to drive tissue-specific expression of RNAi may be problematic due to potential inclusion of nucleotides 5' and 3' of siRNA sequences. METHODS: To circumvent this, polymerase II promoters in combination with cis-acting hammerhead ribozymes and short-hairpin RNA sequences have been explored as a means to generate potent dsRNA molecules in tissues defined by the promoter in use. RESULTS: The novel constructs evaluated in this study produced functional siRNA which suppressed the enhanced green fluorescent protein (eGFP) both in vitro and in vivo (in mice). Additionally, the constructs did not appear to elicit a significant type-1 interferon response compared to traditional H1-transcribed shRNA. CONCLUSIONS: Given the potential 'off-target' effects of dsRNAs, it would be preferable in many cases to limit expression of dsRNA to the tissue of interest and moreover would significantly augment the resolution of RNAi technologies. Notably, the system under evaluation in this study could readily be adapted to achieve this objective.     

Mapping scrub vegetation cover from photogrammetric point clouds: A useful tool in reserve management

Scrub vegetation is a valuable habitat and resource for wildlife, but if unmanaged can encroach and dominate adjacent habitats, reducing biodiversity value. A primary task in the management of terrestrial nature reserves in the UK is monitoring and controlling scrub. The methods used to monitor and assess scrub cover are often basic, relying on qualitative assessment. Inaccurate assessments may fail to inform appropriate management of the habitats and lead to loss or degradation of important ecological features. Scrub can be monitored using UAV or satellite‐derived imagery, but it can be difficult to distinguish between other vegetation types without using high‐cost hyperspectral sensors. An alternative method using high‐resolution surface models from photogrammetric point clouds enables the isolation of vegetation types based on height. Scrub can be isolated from woodland, hedgerows, and tall ground vegetation. In this study, we calculate scrub cover using a photogrammetric point cloud modeling approach using UAVs.We illustrate the method with two case studies from the UK. The scrub cover at Daneway Banks, a calcareous grassland site in Gloucestershire, was calculated at 21.8% of the site. The scrub cover at Flat Holm Island, a maritime grassland in the Severn Estuary, was calculated at 7%. This approach enabled the scrub layer to be readily measured and if required, modeled to provide a visual guide of what a projected management objective would look like. This approach provides a new tool in reserve management, enabling habitat management strategies to be informed, and progress toward objectives monitored.     

Clinical Characteristics and Outcomes in Patients with Pulmonary Blastomycosis

Background  Blastomycosis is an uncommon granulomatous infection caused by the thermally dimorphic fungus Blastomyces dermatitidis. The most frequent clinical infections involve the lung, skin, and bone. Pulmonary manifestations range from asymptomatic self-limited infection to severe diffuse pneumonia causing respiratory failure. Objectives  To establish the clinical characteristics and outcomes of patients with pulmonary blastomycosis diagnosed at hospitals in Manitoba and northwestern Ontario, Canada. Methods  A retrospective review of medical records was done for 318 patients with blastomycosis in these regions. Results  The majority of patients were Caucasian (198 (62.5%) patients), male (193 (61%) patients), and residents of Ontario (209 (65.7%) patients). Most patients were treated in an inpatient hospital ward (266 (84%) patients) and survived (294 (92%) patients). Pulmonary involvement, either alone or associated with other sites, was present in 296 (93%) of the 318 patients; 22 (7%) patients had no evidence of pulmonary blastomycosis. The majority of patients had localized lung disease (1–3 quadrants on chest radiograph involved; 225 (82%) patients). Of 294 (92%) patients requiring hospitalization, 266 (90%) patients received all inpatient care on a general medical ward; 28 (10%) patients received some care in the intensive care unit (ICU). Factors associated with ICU admission included diffuse pulmonary disease (four quadrants involved on chest radiograph), diabetes, and prior use of antimicrobial therapy. Twenty-four (8%) patients died, and multivariate analysis showed that older age and Aboriginal ethnicity were the significant risk factors for death from blastomycosis. Conclusion  Blastomycosis is a cause of serious, potentially life-threatening pulmonary infection in this geographic region. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Contribution of A1 subunit residue Q316 in thrombin-activated factor VIII to A2 subunit dissociation

Blood coagulation factor VIII (fVIII) is activated by thrombin to form an A1/A2/A3-C1-C2 heterotrimer, which functions as a cofactor for factor IXa during intrinsic pathway factor X activation. Human thrombin-activated fVIII (fVIIIa) decays rapidly because of first-order dissociation of the A2 subunit, which may function to regulate the coagulation mechanism. The three fVIII A domains each consist of two cupredoxin-like subdomains. Substitution of the COOH-terminal A1 subdomain of porcine fVIIIa, which decays more slowly than human fVIIIa, reduces the dissociation rate constant for fVIIIa decay. Examination of a human fVIII A1-A2-A3 homology model [Pemberton, S., et al. (1997) Blood 89, 2413-2421) revealed a possible interaction between Q316 in the FG helix of the COOH-terminal A1 subdomain and M539 in the FG helix of the NH2-terminal A2 subdomain, which are sites where human and porcine fVIII differ. Decays of purified recombinant human and porcine fVIIIa and the human fVIIIa mutants Q316H, M539L and Q316H/M539L were compared at 23 and 37 degrees C. The decay rates of the Q316H and Q316H/M539L mutants, but not the M539L mutant, were significantly slower than human fVIIIa. These results indicate that the FG helix of the COOH-terminal A1 cupredoxin-like subdomain of fVIII may be under selective pressure by the requirements of hemostatic balance.     

C-8 Modifications of 3-alkyl-1,8-dibenzylxanthines as inhibitors of human cytosolic phosphoenolpyruvate carboxykinase

New modifications on the C-8 4-aminobenzyl unit of the previously reported 3-alkyl-1,8-dibenzylxanthine inhibitors of cPEPCK are presented. The most active compound reported here is the 5-chloro-1,3-dimethyl-1H-pyrazole-4-sulfonic acid amide derivative 2 with an IC(50) of 0.29+/-0.08 microM. An X-ray analysis of a heteroaromatic sulfonamide is presented showing a new pi-pi interaction.     

Structural colour: elusive iridescence strategies brought to light

Understanding structural colours in nature requires the right set of optical experiments: this is illustrated by a new study on iridescent bird of paradise feathers, which suggests the potential behavioural importance of dynamic colour changes.     

Structure of a Blinkin-BUBR1 complex reveals an interaction crucial for kinetochore-mitotic checkpoint regulation via an unanticipated binding Site

The maintenance of genomic stability relies on the spindle assembly checkpoint (SAC), which ensures accurate chromosome segregation by delaying the onset of anaphase until all chromosomes are properly bioriented and attached to the mitotic spindle. BUB1 and BUBR1 kinases are central for this process and by interacting with Blinkin, link the SAC with the kinetochore, the macromolecular assembly that connects microtubules with centromeric DNA. Here, we identify the Blinkin motif critical for interaction with BUBR1, define the stoichiometry and affinity of the interaction, and present a 2.2 ? resolution crystal structure of the complex. The structure defines an unanticipated BUBR1 region responsible for the interaction and reveals a novel Blinkin motif that undergoes a disorder-to-order transition upon ligand binding. We also show that substitution of several BUBR1 residues engaged in binding Blinkin leads to defects in the SAC, thus providing the first molecular details of the recognition mechanism underlying kinetochore-SAC signaling.     

Ferruginous pygmy-owl (Glaucidium brasilianum) and eastern screech-owl (Megascopes asio): new hosts for Philornis mimicola (Diptera: Muscidae) and Ornithodoros concanensis (Acari: Argasidae)

While banding ferruginous pygmy-owls (Glaucidium brasilianum) and Eastern screech-owls (Megascops asio) in south Texas during 2004, we recorded Philornis mimicola (Diptera: Muscidae) and Ornithodoros concanensis (Acari: Argasidae) parasitizing nestlings. Inspection of nestlings revealed 54 P. mimicola and one O. concanensis. Inspection of nest material revealed 111 P. mimicola, including 57 puparia. The effect (e.g., blood loss, anemia) of these hematophagous parasites might have contributed to the demise of at least one Eastern screech-owl nestling. This is the first record of P. mimicola and O. concanensis parasitizing ferruginous pygmy-owls and Eastern screech-owls.