Imaging intracellular protein dynamics by spinning disk confocal microscopy

The palette of fluorescent proteins (FPs) has grown exponentially over the past decade, and as a result, live imaging of cells expressing fluorescently tagged proteins is becoming more and more mainstream. Spinning disk confocal (SDC) microscopy is a high-speed optical sectioning technique and a method of choice to observe and analyze intracellular FP dynamics at high spatial and temporal resolution. In an SDC system, a rapidly rotating pinhole disk generates thousands of points of light that scan the specimen simultaneously, which allows direct capture of the confocal image with low-noise scientific grade-cooled charge-coupled device cameras, and can achieve frame rates of up to 1000 frames per second. In this chapter, we describe important components of a state-of-the-art spinning disk system optimized for live cell microscopy and provide a rationale for specific design choices. We also give guidelines of how other imaging techniques such as total internal reflection microscopy or spatially controlled photoactivation can be coupled with SDC imaging and provide a short protocol on how to generate cell lines stably expressing fluorescently tagged proteins by lentivirus-mediated transduction.

Not drowning, (hand)waving? Molecular phylogenetics, biogeography and evolutionary tempo of the ‘Gondwanan’ midge Stictocladius Edwards (Diptera: Chironomidae)

Many insect clades, especially within the Diptera (true flies), have been considered classically ‘Gondwanan’, with an inference that distributions derive from vicariance of the southern continents. Assessing the role that vicariance has played in the evolution of austral taxa requires testing the location and tempo of diversification and speciation against the well-established predictions of fragmentation of the ancient super-continent. Several early (anecdotal) hypotheses that current austral distributions originate from the breakup of Gondwana derive from studies of taxa within the family Chironomidae (non-biting midges). With the advent of molecular phylogenetics and biogeographic analytical software, these studies have been revisited and expanded to test such conclusions better. Here we studied the midge genus Stictocladius Edwards, from the subfamily Orthocladiinae, which contains austral-distributed clades that match vicariance-based expectations. We resolve several issues of systematic relationships among morphological species and reveal cryptic diversity within many taxa. Time-calibrated phylogenetic relationships among taxa accorded partially with the predicted tempo from geology. For these apparently vagile insects, vicariance-dated patterns persist for South America and Australia. However, as often found, divergence time estimates for New Zealand at c. 50 mya post-date separation of Zealandia from Antarctica and the remainder of Gondwana, but predate the proposed Oligocene ‘drowning’ of these islands. We detail other such ‘anomalous’ dates and suggest a single common explanation rather than stochastic processes. This could involve synchronous establishment following recovery from ‘drowning’ and/or deleteriously warming associated with the mid-Eocene climatic optimum (hence ‘waving’, which refers to cycles of drowning events) plus new availability of topography providing of cool running waters, or all these factors in combination. Alternatively a vicariance explanation remains available, given the uncertain duration of connectivity of Zealandia to Australia–Antarctic–South America via the Lord Howe and Norfolk ridges into the Eocene.

Synonymization of key pest species within the Bactrocera dorsalis species complex (Diptera: Tephritidae): Taxonomic changes based on a review of 20 years of integrative morphological, molecular, cytogenetic, behavioural and chemoecological data

Bactrocera papayae Drew & Hancock, Bactrocera philippinensis Drew & Hancock, Bactrocera carambolae Drew & Hancock, and Bactrocera invadens Drew, Tsuruta & White are four horticultural pest tephritid fruit fly species that are highly similar, morphologically and genetically, to the destructive pest, the Oriental fruit fly, Bactrocera dorsalis (Hendel) (Diptera: Tephritidae). This similarity has rendered the discovery of reliable diagnostic characters problematic, which, in view of the economic importance of these taxa and the international trade implications, has resulted in ongoing difficulties for many areas of plant protection and food security. Consequently, a major international collaborative and integrated multidisciplinary research effort was initiated in 2009 to build upon existing literature with the specific aim of resolving biological species limits among B. papayae, B. philippinensis, B. carambolae, B. invadens and B. dorsalis to overcome constraints to pest management and international trade. Bactrocera philippinensis has recently been synonymized with B. papayae as a result of this initiative and this review corroborates that finding; however, the other names remain in use. While consistent characters have been found to reliably distinguish B. carambolae from B. dorsalis, B. invadens and B. papayae, no such characters have been found to differentiate the latter three putative species. We conclude that B. carambolae is a valid species and that the remaining taxa, B. dorsalis, B. invadens and B. papayae, represent the same species. Thus, we consider B. dorsalis (Hendel) as the senior synonym of B. papayae Drew and Hancock syn.n. and B. invadens Drew, Tsuruta & White syn.n. A redescription of B. dorsalis is provided. Given the agricultural importance of B. dorsalis, this taxonomic decision will have significant global plant biosecurity implications, affecting pest management, quarantine, international trade, postharvest treatment and basic research. Throughout the paper, we emphasize the value of independent and multidisciplinary tools in delimiting species, particularly in complicated cases involving morphologically cryptic taxa.

Molecular camouflage : making use of protecting groups to control the self-assembly of inorganic janus particles onto metal–chalcogenide nanotubes by Pearson Hardness

Hard and soft: Binding of inorganic Pt@Fe3O4 Janus particles to WS2 nanotubes through their Pt or Fe3O4 domains is governed by the difference in Pearson hardness: the soft Pt block has a higher sulfur affinity than the harder magnetite face; thus the binding proceeds preferentially through the Pt face. This binding preference can be reversed by masking the Pt face with an organic protecting group.

Influences of molecular structure on co-crystallization of polypyridyl metal complexes.

Heteroleptic complexes of the type \[RuL2L′](PF6)2 (L, L′ = combinations of 1,10-phenanthroline (phen) and 2,2′-bipyridine (bipy)) were found to cocrystallize with \[Ni(phen)3](PF6)2 to produce cocrystals of \[Ni(phen)3]x\[RuL2L′]1–x(PF6)2. In this report we show that the ability of the complexes to cocrystallize is influenced by the number of common ligands between complexes in solution. Supramolecular selection is a phenomenon caused by molecular recognition through which cocrystals can grow from the same solution but contain different ratios of the molecular components. It was found that systems where L = phen displayed less supramolecular selection than systems where L = bipy. With increasing supramolecular selection, the composition of cocrystals was found to vary significantly from the initial relative concentration in the cocrystallizing solution, and therefore it was increasingly difficult to control the final composition of the resultant cocrystals. Consequently, modulation of concentration-dependent properties such as phase was also found to be less predictable with increasing supramolecular selection. Notwithstanding the complication afforded by the presence of supramolecular selection, our results reaffirm the robustness of the \[M(phen)3](PF6)2 structure because it was maintained even when ca. 90% of the complexes in the cocrystals were \[Ru(phen)(bipy)2](PF6)2, which in its pure form is not isomorphous with \[M(phen)3](PF6)2. Experiments between complexes without common ligands, i.e., \[Ru(bipy)3](PF6)2 cocrystallized with \[Ni(phen)3](PF6)2, were found to approach the limit to which molecular recognition processes can be confused into cocrystallizing different molecules to form single cocrystals. For these systems the result was the formation of block-shaped crystals skewered by a needle-shaped crystals.

Review-evaluating the molecular assays for measuring the oxidative potential of particulate matter

Several cell-free assays are currently used to quantify and detect the Reactive Oxygen Species (ROS). All of them have certain limitations, do not provide direct comparison of results and, to date, none of these assays have been acknowledged as the most suitable acellular assay and none has yet been adopted for investigation of potential PM toxicity. These assays include DTT, ascorbic acid, DCFHDA and PFN assays which have been used in measurements of the particles generated from various combustion sources such as diesel engine, wood smoke (or biomass burning) and cigarette smoke, as well as for outdoor measurements. All the probes use different units for expressing redox properties of PM. Also, their reactivity is being triggered by different types of ROS. This limits the direct comparison of the results that are reporting the toxicity of the same aerosol type measured with various probes. This study is evaluating and comparing the various assays in order to develop deeper understanding of their capabilities, selectivity as well as improve understanding of the underlying chemical mechanisms. Keywords: DTT, DCFH-DA, PFN, BPEA-nit, Ascorbic acid, oxidative potential

A comparison of classification algorithms within the Classifynder pollen imaging system

We describe an investigation into how Massey University's Pollen Classifynder can accelerate the understanding of pollen and its role in nature. The Classifynder is an imaging microscopy system that can locate, image and classify slide based pollen samples. Given the laboriousness of purely manual image acquisition and identification it is vital to exploit assistive technologies like the Classifynder to enable acquisition and analysis of pollen samples. It is also vital that we understand the strengths and limitations of automated systems so that they can be used (and improved) to compliment the strengths and weaknesses of human analysts to the greatest extent possible. This article reviews some of our experiences with the Classifynder system and our exploration of alternative classifier models to enhance both accuracy and interpretability. Our experiments in the pollen analysis problem domain have been based on samples from the Australian National University's pollen reference collection (2890 grains, 15 species) and images bundled with the Classifynder system (400 grains, 4 species). These samples have been represented using the Classifynder image feature set. In addition to the Classifynder's native neural network classifier, we have evaluated linear discriminant, support vector machine, decision tree and random forest classifiers on these data with encouraging results. Our hope is that our findings will help enhance the performance of future releases of the Classifynder and other systems for accelerating the acquisition and analysis of pollen samples. © 2013 AIP Publishing LLC.

Molecular profiling of exudate from chronic ulcerated wounds

Non-healing wounds represent a significant burden to healthcare systems and societies worldwide. Current best practice treatments of chronic wounds can require patients to undergo extensive periods of therapy without any positive outcome. This consumes substantial healthcare resources and severely impacts patient quality of life. At present, there are no measures to predict a patient's response to best practice care. The hypothesis of this thesis was that biochemical markers could be found within the wound fluid of chronic ulcers and these markers could predict the healing outcome of an ulcer undergoing best practice care. Discovery phase proteomic and mass spectrometry techniques were utilised to determine novel proteins that correlated with the healing outcome of ulcers. These candidate biomarkers could be developed into simple dip-stick tools for use in clinical practice. This would aid clinicians in the choice of effective wound management strategies to address hard-to-heal wounds.

Effect of partial H2O-D2O replacement on the anisotropy of transverse proton spin relaxation in bovine articular cartilage

Anisotropy of transverse proton spin relaxation in collagen-rich tissues like cartilage and tendon is a well-known phenomenon that manifests itself as the "magic-angle" effect in magnetic resonance images of these tissues. It is usually attributed to the non-zero averaging of intra-molecular dipolar interactions in water molecules bound to oriented collagen fibers. One way to manipulate the contributions of these interactions to spin relaxation is by partially replacing the water in the cartilage sample with deuterium oxide. It is known that dipolar interactions in deuterated solutions are weaker, resulting in a decrease in proton relaxation rates. In this work, we investigate the effects of deuteration on the longitudinal and the isotropic and anisotropic contributions to transverse relaxation of water protons in bovine articular cartilage. We demonstrate that the anisotropy of transverse proton spin relaxation in articular cartilage is independent of the degree of deuteration, bringing into question some of the assumptions currently held over the origins of relaxation anisotropy in oriented tissues.

Calibration of treatment time intensity modulated EPID images for anatomical imaging

Intensity Modulated Radiotherapy (IMRT) is a well established technique for delivering highly conformal radiation dose distributions. The complexity of the delivery techniques and high dose gradients around the target volume make verification of the patient treatment crucial to the success of the treatment. Conventional treatment protocols involve imaging the patient prior to treatment, comparing the patient set-up to the planned set-up and then making any necessary shifts in the patient position to ensure target volume coverage. This paper presents a method for calibrating electronic portal imaging device (EPID) images acquired during IMRT delivery so that they can be used for verifying the patient set-up.

Subsurface imaging of vegetation, climate, and root-zone moisture interactions

Changes in global climate and land use affect important prolesses from evapotranspiration and groundwater recharge to carbon storage and biochemical cycling. Near surface soil moisture is pivotal to understand the consequences of these changes. However, the dynamic interactions between vegetation and soil moisture remain largely unresolved because it is difficult to monitor and quantify subsurface hydrologic fluxes at relevant scales. Here we use electrical resistivity to monitor the influence of climate and vegetation on root-zone moisture, bridging the gap between remotely-sensed and in-situ point measurements. Our research quantifies large seasonal differences in root-zone moisture dynamics for a forest-grassland ecotone. We found large differences in effective rooting depth and moisture distributions for the two vegetation types. Our results highlight the likely impacts of land transformations on groun ter recharge, streamflow, and land-atmosphere exchanges.

Primary and secondary neural networks of auditory prepulse inhibition: a functional magnetic resonance imaging study of sensorimotor gating of the human acoustic startle response

Feedforward inhibition deficits have been consistently demonstrated in a range of neuropsychiatric conditions using prepulse inhibition (PPI) of the acoustic startle eye-blink reflex when assessing sensorimotor gating. While PPI can be recorded in acutely decerebrated rats, behavioural, pharmacological and psychophysiological studies suggest the involvement of a complex neural network extending from brainstem nuclei to higher order cortical areas. The current functional magnetic resonance imaging study investigated the neural network underlying PPI and its association with electromyographically (EMG) recorded PPI of the acoustic startle eye-blink reflex in 16 healthy volunteers. A sparse imaging design was employed to model signal changes in blood oxygenation level-dependent (BOLD) responses to acoustic startle probes that were preceded by a prepulse at 120 ms or 480 ms stimulus onset asynchrony or without prepulse. Sensorimotor gating was EMG confirmed for the 120-ms prepulse condition, while startle responses in the 480-ms prepulse condition did not differ from startle alone. Multiple regression analysis of BOLD contrasts identified activation in pons, thalamus, caudate nuclei, left angular gyrus and bilaterally in anterior cingulate, associated with EMGrecorded sensorimotor gating. Planned contrasts confirmed increased pons activation for startle alone vs 120-ms prepulse condition, while increased anterior superior frontal gyrus activation was confirmed for the reverse contrast. Our findings are consistent with a primary pontine circuitry of sensorimotor gating that interconnects with inferior parietal, superior temporal, frontal and prefrontal cortices via thalamus and striatum. PPI processes in the prefrontal, frontal and superior temporal cortex were functionally distinct from sensorimotor gating.

Mapping Hydrophobicity on the Protein Molecular Surface at Atom-Level Resolution

A precise representation of the spatial distribution of hydrophobicity, hydrophilicity and charges on the molecular surface of proteins is critical for the understanding of the interaction with small molecules and larger systems. The representation of hydrophobicity is rarely done at atom-level, as this property is generally assigned to residues. A new methodology for the derivation of atomic hydrophobicity from any amino acid-based hydrophobicity scale was used to derive 8 sets of atomic hydrophobicities, one of which was used to generate the molecular surfaces for 35 proteins with convex structures, 5 of which, i.e., lysozyme, ribonuclease, hemoglobin, albumin and IgG, have been analyzed in more detail. Sets of the molecular surfaces of the model proteins have been constructed using spherical probes with increasingly large radii, from 1.4 to 20 A˚, followed by the quantification of (i) the surface hydrophobicity; (ii) their respective molecular surface areas, i.e., total, hydrophilic and hydrophobic area; and (iii) their relative densities, i.e., divided by the total molecular area; or specific densities, i.e., divided by property-specific area. Compared with the amino acid-based formalism, the atom-level description reveals molecular surfaces which (i) present an approximately two times more hydrophilic areas; with (ii) less extended, but between 2 to 5 times more intense hydrophilic patches; and (iii) 3 to 20 times more extended hydrophobic areas. The hydrophobic areas are also approximately 2 times more hydrophobicity-intense. This, more pronounced "leopard skin"-like, design of the protein molecular surface has been confirmed by comparing the results for a restricted set of homologous proteins, i.e., hemoglobins diverging by only one residue (Trp37). These results suggest that the representation of hydrophobicity on the protein molecular surfaces at atom-level resolution, coupled with the probing of the molecular surface at different geometric resolutions, can capture processes that are otherwise obscured to the amino acid-based formalism.

Models of protein linear molecular motors for dynamic nanodevices

Protein molecular motors are natural nano-machines that convert the chemical energy from the hydrolysis of adenosine triphosphate into mechanical work. These efficient machines are central to many biological processes, including cellular motion, muscle contraction and cell division. The remarkable energetic efficiency of the protein molecular motors coupled with their nano-scale has prompted an increasing number of studies focusing on their integration in hybrid micro- and nanodevices, in particular using linear molecular motors. The translation of these tentative devices into technologically and economically feasible ones requires an engineering, design-orientated approach based on a structured formalism, preferably mathematical. This contribution reviews the present state of the art in the modelling of protein linear molecular motors, as relevant to the future design-orientated development of hybrid dynamic nanodevices. © 2009 The Royal Society of Chemistry.