Adaptive Optimisation of Illumination Beam Profiles in Fluorescence Microscopy

Wide-field fluorescence microscope techniques such as single/selective plane illumination microscope (SPIM) are typically configured to image large regions of a sample at once. Here the illumination beam provides uniform excitation of several biological features across the region, `sliced' to a thickness of between 5-10 microns. In this paper we propose a simple alteration to the optical configuration of a SPIM by switching the light-sheet- forming cylindrical lens with a spatial light modulator. This has the potential to adaptively reconfigure the light sheet geometry to improve the optical sectioning of specific biological features, rather than the thicker sectioning of several features at once across a larger observation field-of-view. We present a prototype version of such a system, referred to as an Adaptive-SPIM (A-SPIM) system. We then suggest that the direct recording of illumination beam shapes within the working microscope system can better facilitate the analysis and subsequent re-configuration of the illumination beam to a specific geometry, and summarise the design and operation of a device that we have developed specifically for this purpose. We finally present reconstructed quantitative three dimensional flux maps of illumination beams from three microscope configurations taken using this miniature high-dynamic range beam profiling device, comparing the beam geometry of a regular SPIM system with our prototype A-SPIM system, and suggesting future improvements

Biodiversity of nematode assemblages from the region of the Clarion-Clipperton Fracture Zone, an area of commercial mining interest

BACKGROUND: The possibility for commercial mining of deep-sea manganese nodules is currently under exploration in the abyssal Clarion-Clipperton Fracture Zone. Nematodes have potential for biomonitoring of the impact of commercial activity but the natural biodiversity is unknown. We investigate the feasibility of nematodes as biomonitoring organisms and give information about their natural biodiversity. RESULTS: The taxonomic composition (at family to genus level) of the nematode fauna in the abyssal Pacific is similar, but not identical to, the North Atlantic. Given the immature state of marine nematode taxonomy, it is not possible to comment on the commonality or otherwise of species between oceans. The between basin differences do not appear to be directly linked to current ecological factors. The abyssal Pacific region (including the Fracture Zone) could be divided into two biodiversity subregions that conform to variations in the linked factors of flux to the benthos and of sedimentary characteristics. Richer biodiversity is associated with areas of known phytodetritus input and higher organic-carbon flux. Despite high reported sample diversity, estimated regional diversity is less than 400 species. CONCLUSION: The estimated regional diversity of the CCFZ is a tractable figure for biomonitoring of commercial activities in this region using marine nematodes, despite the immature taxonomy (i.e. most marine species have not been described) of the group. However, nematode ecology is in dire need of further study

Ribosomal RNA Genes Challenge the Monophyly of the Hyalospheniidae (Amoebozoa: Arcellinida)

To date only five partial and two complete SSU rRNA gene sequences are available for the lobose testate amoebae (Arcellinida). Consequently, the phylogenetic relationships among taxa and the definition of species are still largely dependant on morphological characters of uncertain value, which causes confusion in the phylogeny, taxonomy and the debate on cosmopolitanism of free-living protists. Here we present a SSU rRNA-based phylogeny of the Hyalospheniidae including the most common species. Similar to the filose testate amoebae of the order Euglyphida the most basal clades have a terminal aperture; the ventral position of the pseudostome appears to be a derived character. Family Hyalospheniidae appears paraphyletic and is separated into three clades: (1) Heleopera sphagni, (2) Heleopera rosea and Argynnia dentistoma and (3) the rest of the species from genera Apodera, Hyalosphenia, Porosia and Nebela. Our data support the validity of morphological characters used to define species among the Hyalospheniidae and even suggest that taxa described as varieties may deserve the rank of species (e.g. N. penardiana var. minor). Finally our results suggest that the genera Hyalosphenia and Nebela are paraphyletic, and that Porosia bigibbosa branches inside the main Nebela clade

A consensus building exercise to determine research priorities for silver trauma

Background Emergency care research into ‘Silver Trauma’, which is simply defined as major trauma consequent upon relatively minor injury mechanisms, is facing many challenges including that at present, there is no clear prioritisation of the issues. This study aimed to determine the top research priorities to guide future research. Methods This consensus-based prioritization exercise used a three-stage modified Delphi technique. The study consisted of an idea generating (divergent) first round, a ranking evaluation in the second round, and a (convergent) consensus meeting in the third round. Results A total of 20 research questions advanced to the final round of this study. After discussing the importance and clinical significance of each research question, five research questions were prioritised by the experts; the top three research priorities were: (1). What are older people’s preferred goals of trauma care? (2). Beyond the Emergency Department (ED), what is the appropriate combined geriatric and trauma care? (3). Do older adults benefit from access to trauma centres? If so, do older trauma patients have equitable access to trauma centre compared to younger adults? Conclusion The results of this study will assist clinicians, researchers, and organisations that are interested in silver trauma in guiding their future efforts and funding toward addressing the identified research priorities

The putative proteinase maturation protein A of Streptococcus pneumoniae is a conserved surface protein with potential to elicit protective immune responses

Surface-exposed proteins often play an important role in the interaction between pathogenic bacteria and their host. We isolated a pool of hydrophobic, surface-associated proteins of Streptococcus pneumoniae. The opsonophagocytic activity of hyperimmune serum raised against this protein fraction was high and species specific. Moreover, the opsonophagocytic activity was independent of the capsular type and chromosomal genotype of the pneumococcus. Since the opsonophagocytic activity is presumed to correlate with in vivo protection, these data indicate that the protein fraction has the potential to elicit species-specific immune protection with cross-protection against various pneumococcal strains. Individual proteins in the extract were purified by two-dimensional gel electrophoresis. Antibodies raised against three distinct proteins contributed to the opsonophagocytic activity of the serum. The proteins were identified by mass spectrometry and N-terminal amino acid sequencing. Two proteins were the previously characterized pneumococcal surface protein A and oligopeptide-binding lipoprotein AmiA. The third protein was the recently identified putative proteinase maturation protein A (PpmA), which showed homology to members of the family of peptidyl-prolyl cis/trans isomerases. Immunoelectron microscopy demonstrated that PpmA was associated with the pneumococcal surface. In addition, PpmA was shown to elicit species-specific opsonophagocytic antibodies that were cross-reactive with various pneumococcal strains. This antibody cross-reactivity was in line with the limited sequence variation of ppmA. The importance of PpmA in pneumococcal pathogenesis was demonstrated in a mouse pneumonia model. Pneumococcal ppmA-deficient mutants showed reduced virulence. The properties of PpmA reported here indicate its potential for inclusion in multicomponent protein vaccines

Effect of model root exudate on denitrifier community dynamics and activity at different water-filled pore space levels in a fertilised soil

Although a “rhizosphere effect” on denitrification rates has been established, a clear understanding of the effects of exudate addition on denitrifier community dynamics remains elusive. A microcosm experiment was designed to explore the interaction between exudate addition and soil moisture on community dynamics and denitrification rates. Artificial root exudate at 5 different carbon concentrations was added daily to soil microcosms at contrasting target WFPS (50, 70 and 90%). After a 7-day period, total denitrification and N2O emission rates were measured and community dynamics assessed using molecular methods. The response of denitrifier genes to exudate addition was different, with nirS and nosZ-I showing a stronger effect than nirK and nosZ-II. Distinct community structures were observed for nirS and nosZ-I at 90% target WFPS when compared to 50% and 70%. NirS denitrifier population size showed a ca. 5-fold increase in gene copy number at 90% WFPS when exudate was added at the highest C input. Significant total denitrification and N2O emission rates were observed only at 90% WFPS, which increased with C input. Our study improves the understanding of the complex interaction between microbial communities, the abiotic environment and process rates which can inform management practices aimed at increasing complete denitrification and controlling greenhouse gas production from agriculture

An eco-climatic framework for evaluating the resilience of vegetation to water deficit

The surge in global efforts to understand the causes and consequences of drought on forest ecosystems has tended to focus on specific impacts such as mortality. We propose an ecoclimatic framework that takes a broader view of the ecological relevance of water deficits, linking elements of exposure and resilience to cumulative impacts on a range of ecosystem processes. This ecoclimatic framework is underpinned by two hypotheses: (i) exposure to water deficit can be represented probabilistically and used to estimate exposure thresholds across different vegetation types or ecosystems; and (ii) the cumulative impact of a series of water deficit events is defined by attributes governing the resistance and recovery of the affected processes. We present case studies comprising Pinus edulis and Eucalyptus globulus, tree species with contrasting ecological strategies, which demonstrate how links between exposure and resilience can be examined within our proposed framework. These examples reveal how climatic thresholds can be defined along a continuum of vegetation functional responses to water deficit regimes. The strength of this framework lies in identifying climatic thresholds on vegetation function in the absence of more complete mechanistic understanding, thereby guiding the formulation, application and benchmarking of more detailed modelling

Phagosomal acidification Is required to kill Streptococcus pneumoniae in a Zebrafish model

Streptococcus pneumoniae (the pneumococcus) is a major human pathogen causing invasive disease, including community-acquired bacteraemia, and remains a leading cause of global mortality. Understanding the role of phagocytes in killing bacteria is still limited, especially in vivo. In this study, we established a zebrafish model to study the interaction between intravenously administered pneumococci and professional phagocytes such as macrophages and neutrophils, to unravel bacterial killing mechanisms employed by these immune cells. Our model confirmed the key role of polysaccharide capsule in promoting pneumococcal virulence through inhibition of phagocytosis. Conversely, we show pneumococci lacking a capsule are rapidly internalised by macrophages. Low doses of encapsulated S. pneumoniae cause near 100% mortality within 48 hours postinfection (hpi), while 50 times higher doses of unencapsulated pneumococci are easily cleared. Time course analysis of in vivo bacterial numbers reveals that while encapsulated pneumococcus proliferates to levels exceeding 105 CFU at the time of host death, unencapsulated bacteria are unable to grow and are cleared within 20 hpi. Using genetically induced macrophage depletion, we confirmed an essential role for macrophages in bacterial clearance. Additionally, we show that upon phagocytosis by macrophages, phagosomes undergo rapid acidification. Genetic and chemical inhibition of vacuolar ATPase (v-ATPase) prevents intracellular bacterial killing and induces host death indicating a key role of phagosomal acidification in immunity to invading pneumococci. We also show that our model can be used to study the efficacy of antimicrobials against pneumococci in vivo. Collectively, our data confirm that larval zebrafish can be used to dissect killing mechanisms during pneumococcal infection in vivo and highlight key roles for phagosomal acidification in macrophages for pathogen clearance

The James Clerk Maxwell Telescope Spectral Legacy Survey

Original article can be found at: http://www.journals.uchicago.edu/loi/pasp Copyright University of Chicago Press / AAS. DOI: 10.1086/511161Stars form in the densest, coldest, most quiescent regions of molecular clouds. Molecules provide the only probes that can reveal the dynamics, physics, chemistry, and evolution of these regions, but our understanding of the molecular inventory of sources and how this is related to their physical state and evolution is rudimentary and incomplete. The Spectral Legacy Survey (SLS) is one of seven surveys recently approved by the James Clerk Maxwell Telescope (JCMT) Board of Directors. Beginning in 2007, the SLS will produce a spectral imaging survey of the content and distribution of all the molecules detected in the 345 GHz atmospheric window (between 332 and 373 GHz) toward a sample of five sources. Our intended targets are a low-mass core (NGC 1333 IRAS 4), three high-mass cores spanning a range of star-forming environments and evolutionary states (W49, AFGL 2591, and IRAS 20126), and a photodissociation region (the Orion Bar). The SLS will use the unique spectral imaging capabilities of HARP-B/ACSIS (Heterodyne Array Receiver Programme B/Auto- Correlation Spectrometer and Imaging System) to study the molecular inventory and the physical structure of these objects, which span different evolutionary stages and physical environments and to probe their evolution during the star formation process. As its name suggests, the SLS will provide a lasting data legacy from the JCMT that is intended to benefit the entire astronomical community. As such, the entire data set (including calibrated spectral data cubes, maps of molecular emission, line identifications, and calculations of the gas temperature and column density) will be publicly available.Peer reviewe