Similar Size Range Research Articles

Whole-body gene expression pattern registration in Platynereis larvae

BackgroundDigital anatomical atlases are increasingly used in order to depict different gene expression patterns and neuronal morphologies within a standardized reference template. In evo-devo, a discipline in which the comparison of gene expression patterns is a widely used approach, such standardized anatomical atlases would allow a more rigorous assessment of the conservation of and changes in gene expression patterns during micro- and macroevolutionary time scales. Due to its small size and invariant early development, the annelid Platynereis dumerilii is particularly well suited for such studies. Recently a reference template with registered gene expression patterns has been generated for the anterior part (episphere) of the Platynereis trochophore larva and used for the detailed study of neuronal development.ResultsHere we introduce and evaluate a method for whole-body gene expression pattern registration for Platynereis trochophore and nectochaete larvae based on whole-mount in situ hybridization, confocal microscopy, and image registration. We achieved high-resolution whole-body scanning using the mounting medium 2,2’-thiodiethanol (TDE), which allows the matching of the refractive index of the sample to that of glass and immersion oil thereby reducing spherical aberration and improving depth penetration. This approach allowed us to scan entire whole-mount larvae stained with nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (NBT/BCIP) in situ hybridization and counterstained fluorescently with an acetylated-tubulin antibody and the nuclear stain 4’6-diamidino-2-phenylindole (DAPI). Due to the submicron isotropic voxel size whole-mount larvae could be scanned in any orientation. Based on the whole-body scans, we generated four different reference templates by the iterative registration and averaging of 40 individual image stacks using either the acetylated-tubulin or the nuclear-stain signal for each developmental stage. We then registered to these templates the expression patterns of cell-type specific genes. In order to evaluate the gene expression pattern registration, we analyzed the absolute deviation of cell-center positions. Both the acetylated-tubulin- and the nuclear-stain-based templates allowed near-cellular-resolution gene expression registration. Nuclear-stain-based templates often performed significantly better than acetylated-tubulin-based templates. We provide detailed guidelines and scripts for the use and further expansion of the Platynereis gene expression atlas.ConclusionsWe established whole-body reference templates for the generation of gene expression atlases for Platynereis trochophore and nectochaete larvae. We anticipate that nuclear-staining-based image registration will be applicable for whole-body alignment of the embryonic and larval stages of other organisms in a similar size range.

Comparative study of liposomes, transfersomes, ethosomes and cubosomes for transcutaneous immunisation: characterisation and in vitro skin penetration

Lipid colloidal vaccines, including liposomes, transfersomes, ethosomes and cubosomes, were formulated, characterised and investigated for their ability to enhance penetration of a peptide vaccine through stillborn piglet skin in vitro. Liposomes and transfersomes were formulated using a film-hydration method, ethosomes using a modified reverse phase method and cubosomes using a lipid precursor method. The size, zeta potential, peptide loading and interfacial behaviour of the formulations were characterised. Skin penetration studies were performed using Franz diffusion cells with piglet skin as the membrane. The localization of peptide in the skin was examined using confocal laser scanning microscopy. The various formulations contained negatively charged particles of similar size (range: 134-200 nm). Addition of the saponin adjuvant Quil A to the formulations destabilised the monolayers and reduced peptide loading. Cubosomes and ethosomes showed superior skin retention compared with the other systems. Confocal laser scanning microscopy showed greater peptide penetration and accumulation in the skin treated with cubosomes and ethosomes. With the other systems peptide was only located in the vicinity of the hair follicles and within the hair shaft. We conclude from the in-vitro studies that cubosomes and ethosomes are promising lipid carriers for transcutaneous immunisation.

Impact of two particle measurement techniques on the determination of N95 class respirator filtration performance against ultrafine particles

The purpose of this experimental study was to compare two different particle measurement devices; an Electrical Low Pressure Impactor (ELPI) and a Scanning Mobility Particle Sizer (SMPS), to measure the number concentration and the size distribution of NaCl salt aerosols to determine the collection efficiency of filtering respirators against poly disperse aerosols. Tests were performed on NIOSH approved N95 filtering face-piece respirators (FFR), sealed on a manikin head. Ultrafine particles found in the aerosols were also collected and observed by transmission electron microscopy (TEM). According to the results, there is a systematic difference for the particle size distribution measured by the SMPS and the ELPI. It is largely attributed to the difference in the measurement techniques. However, in spite of these discrepancies, reasonably similar trends were found for the number concentration with both measuring instruments. The particle penetration, calculated based on mobility and aerodynamic diameters, never exceeded 5% for any size range measured at constant flow rate of 85L/min. Also, the most penetrating particle size (MPPS), with the lowest filtration efficiency, would occur at a similar ultrafine size range <100nm. With the ELPI, the MPPS was at 70nm aerodynamic diameter, whereas it occurred at 40nm mobility diameter with the SMPS.

Sabretoothed Carnivores and the Killing of Large Prey

Sabre-like canines clearly have the potential to inflict grievous wounds leading to massive blood loss and rapid death. Hypotheses concerning sabretooth killing modes include attack to soft parts such as the belly or throat, where biting deep is essential to generate strikes reaching major blood vessels. Sabretoothed carnivorans are widely interpreted as hunters of larger and more powerful prey than that of their present-day nonsabretoothed relatives. However, the precise functional advantage of the sabretooth bite, particularly in relation to prey size, is unknown. Here, we present a new point-to-point bite model and show that, for sabretooths, depth of the killing bite decreases dramatically with increasing prey size. The extended gape of sabretooths only results in considerable increase in bite depth when biting into prey with a radius of less than ∼10 cm. For sabretooths, this size-reversed functional advantage suggests predation on species within a similar size range to those attacked by present-day carnivorans, rather than “megaherbivores” as previously believed. The development of the sabretooth condition appears to represent a shift in function and killing behaviour, rather than one in predator-prey relations. Furthermore, our results demonstrate how sabretoothed carnivorans are likely to have evolved along a functionally continuous trajectory: beginning as an extension of a jaw-powered killing bite, as adopted by present-day pantherine cats, followed by neck-powered biting and thereafter shifting to neck-powered shear-biting. We anticipate this new insight to be a starting point for detailed study of the evolution of pathways that encompass extreme specialisation, for example, understanding how neck-powered biting shifts into shear-biting and its significance for predator-prey interactions. We also expect that our model for point-to-point biting and bite depth estimations will yield new insights into the behaviours of a broad range of extinct predators including therocephalians (gorgonopsian + cynodont, sabretoothed mammal-like reptiles), sauropterygians (marine reptiles) and theropod dinosaurs.

EFFECTS OF PARTICLE SIZE ON MIXTURES DURING NONELECTROSTATIC AND ELECTROSTATIC POWDER COATING

ABSTRACT Two mixtures, 44 and 256µm NaCl and 64 and 191 µm starch, were nonelectrostatically or electrostatically coated onto an aluminum target. The coating processes cause the proportion of each powder in the mixture to be different on the target than in the initial mixture. First, some loss of powder occurs during the targeting step; thus, not all of the powder land on the target. This targeting loss is caused by the individual powder characteristics and interactions that occur between powders in the mixture. Also, an uneven distribution is produced on the target due to the way the powder is dispersed. Finally, adhesion loss occurs on the target, also due to individual and mixture characteristics. During nonelectrostatic coating, the largest cause of the change in proportion was targeting losses. During electrostatic coating, the largest cause was adhesion loss. Interactions in the mixture decreased the change in proportion, except for nonelectrostatically coated NaCl.PRACTICAL APPLICATIONSFood coatings consist of mixtures of powders of different sizes. It is desirable to maintain the same proportions of the powder components on each food sample as in the applied coating. Mixtures change proportions due to differences in particle size, so all of the powders in the mixture should have a similar size range to reduce changes during coating. During nonelectrostatic coating, the differences in targeting losses are the biggest cause of this change, so coating systems need to be designed to minimize the differences in targeting losses. Electrostatic coating reduced targeting loss by a large amount; therefore, it is one method to reduce the change in proportions caused by targeting loss. However, electrostatic coating increased the differences in mixture adhesion loss, so products need to be designed to minimize the adhesion loss, such as by adding oil on the product surface.

Observations of Saharan dust microphysical and optical properties from the Eastern Atlantic during NAMMA airborne field campaign

Abstract. As part of the international project entitled "African Monsoon Multidisciplinary Analysis (AMMA)", NAMMA (NASA AMMA) aimed to gain a better understanding of the relationship between the African Easterly Waves (AEWs), the Sahara Air Layer (SAL), and tropical cyclogenesis. The NAMMA airborne field campaign was based out of the Cape Verde Islands during the peak of the hurricane season, i.e., August and September 2006. Multiple Sahara dust layers were sampled during 62 encounters in the eastern portion of the hurricane main development region, covering both the eastern North Atlantic Ocean and the western Saharan desert (i.e., 5–22° N and 10–35° W). The centers of these layers were located at altitudes between 1.5 and 3.3 km and the layer thickness ranged from 0.5 to 3 km. Detailed dust microphysical and optical properties were characterized using a suite of in-situ instruments aboard the NASA DC-8 that included a particle counter, an Ultra-High Sensitivity Aerosol Spectrometer, an Aerodynamic Particle Sizer, a nephelometer, and a Particle Soot Absorption Photometer. The NAAMA sampling inlet has a size cut (i.e., 50% transmission efficiency size) of approximately 4 μm in diameter for dust particles, which limits the representativeness of the NAMMA observational findings. The NAMMA dust observations showed relatively low particle number densities, ranging from 268 to 461 cm−3, but highly elevated volume density with an average at 45 μm3 cm−3. NAMMA dust particle size distributions can be well represented by tri-modal lognormal regressions. The estimated volume median diameter (VMD) is averaged at 2.1 μm with a small range of variation regardless of the vertical and geographical sampling locations. The Ångström Exponent assessments exhibited strong wavelength dependence for absorption but a weak one for scattering. The single scattering albedo was estimated at 0.97 ± 0.02. The imaginary part of the refractive index for Sahara dust was estimated at 0.0022, with a range from 0.0015 to 0.0044. Closure analysis showed that observed scattering coefficients are highly correlated with those calculated from spherical Mie-Theory and observed dust particle size distributions. These values are generally consistent with literature values reported from studies with similar particle sampling size range.

Carbon Nanotubes Activate Store Operated Calcium Entry (SOCE) In Human Platelets Manifested by STIM1 Capping

AbstractAbstract 3190Carbon nanotubes (CNTs) are a promising new class of materials with many potential biomedical applications. CNTs have profound impact on the development of diagnostic biosensors, drug delivery nanosystems, imaging nanoprobes for intravascular use or other devices that come in contact with blood. Therefore, the biocompatibility/toxicity of CNTs in blood is a critical safety issue. We have previously shown that agglomerates of structurally different CNTs but not fullerene (nC60), activate human platelets by inducing extracellular Ca2+ influx (Semberova, J. et al.: Nano Lett., 2009). In addition, we demonstrated that CNT-induced Ca2+ entry in platelets could be inhibited by calcium channel blockers SKF 96365 and 2-APB. Here we investigate a mechanism of CNT-induced platelet activation. Since both unmodified CNTs and nC60 are hydrophobic and insoluble in aqueous solutions, characterization of the dispersed nanomaterial agglomerates in plasma is critical for evaluation of effects on platelets. Flow particle image analysis showed that both multiwalled carbon nanotubes (MWCNTs) M60 (>95% purity; outer diameter 60–100 nm, SES) and nC60 (99.9%; MER) form agglomerates of a similar size range in plasma: median particle size 1.0 μm (10th-90th%: 0.4–2.9) μm for M60, and 0.7 μm (0.4-1.5) μm for nC60. However, roughness and surface area are markedly higher for CNT M60 (128±28 nm; 122 m2/g) compared to fullerene nC60 (4.4±3.9 nm; 0.9 m2/g). Both Field-Emission Scanning Electron Microscopy (FESEM) and Transmission Electron Microscopy (TEM) demonstrated that MWCNTs self-assemble to fiber networks which leads in platelet rich plasma to platelet entrapment, formation of pseudopodia, aggregation, platelet plasma membrane budding and release of membrane microparticles. To test whether a surface modification of CNTs leading to increased solubility would affect activity on platelets, we prepared carboxy-MWCNTs (M60(COOH)) by refluxing pristine M60 in a concentrated 3:1 (v/v) H2SO4 and HNO3 mixture at 70°C for 2 hours; followed by washing and dialysis in water. Interestingly, we observed that M60(COOH), which dispersed well in PBS, activate human blood platelets similarly like their pristine counterparts. FESEM showed that both pristine and carboxy-modified M60 self-assemble in plasma forming rope-like structures. Numerous protuberances existed on the top surface of the M60 and M60(COOH) clusters, which were formed by the relatively long, dense net, highly tangled CNTs without specific orientation. In contrast, nC60 particles are largely devoid of these surface features and minimal platelet activation was observed. In addition, TEM analysis with DAB stained platelet dense tubular system (DTS) showed nanopenetration of CNTs through platelet membranes likely causing DTS injury. Changes in calcium concentration in the intracellular compartments were studied in Fura FF loaded platelets in the presence of streptolysin and EGTA (100μM). We demonstrated a marked decrease of calcium in the intracellular stores induced by M60 or M60(COOH), as well as by SERCA inhibitor thapsigargin (TPG). Under the same conditions, nC60 did not induce depletion of calcium from the stores, similarly to the control untreated platelets. Moreover, Laser Scanning Confocal Microscopy (LSCM) using anti STIM1 (clone 44/GOK) monoclonal antibody showed that M60 caused capping of STIM1 molecules in platelets, which indicates activation of the store operated calcium entry (SOCE). Western blotting of platelet lysates showed that the anti STIM1 antibody 44/GOK detected a band at the expected MW of about 85 kDa. This was also confirmed using a polyclonal antibody against carboxy-terminal of STIM1 and corresponding blocking peptide (ProSci, Inc.). LSCM further showed a colocalization of the STIM1 and ORAI1 molecules in platelets. Similar picture of STIM1 capping in platelets was observed after treatment with TPG. In contrast, no STIM1 capping was observed in platelets treated with fullerene nC60. In conclusion, our results showed that the MWCNTs nanopenetrate membranes of DTS and activate SOCE in human platelets. The findings and conclusions in this study have not been formally disseminated by the Food and Drug Administration and should not be construed to represent any Agency determination or policy. Disclosures:No relevant conflicts of interest to declare.

Mesoporous high surface area Ce0.9Gd0.1O1.95 synthesized by spray drying

Mesoporous gadolinium doped cerium dioxide with high surface area was produced by spray drying using Pluronic 123 as surfactant. The powder, when calcined at 400°C, had a BET surface area of 136m2g−1 and was polycrystalline as confirmed by XRD and TEM. XEDS confirmed Ce, Gd and O, as the only elements present in the powders. Nitrogen adsorption measurements showed that the material was mesoporous with pore diameters of approximately 10nm, which was later confirmed by TEM studies. TEM–EELS were used to confirm that Ce was in the +4 oxidation state. SEM studies showed particles of 10nm diameter, corresponding to the crystallite size calculated from XRD data. The similar size range of the mesopores and the observed crystallite size indicates that the porosity is partly formed from intergranular mesoporosity. Using the spray drying method of a surfactant assisted liquid precursor solution it can be possible to manufacture mesoporous gadolinium doped ceria in large quantities in a continuous production.

Characterisation of Sarcoptes scabiei antigens

In pig herds, the status of Sarcoptes scabiei infections is routinely monitored by serodiagnosis. Crude antigen for ELISA is usually prepared from S. scabiei var. canis or other variations and may lead to variations in the outcome of different tests, making assay standardisation difficult. This study was performed to investigate the antigen profiles of S. scabiei, including differences between hydrophilic and more hydrophobic protein fractions, by Western blotting with sera from pigs with defined infection status. Potential cross-reactivity among S. scabiei (var. canis, suis and bovis), Dermatophagoides farinae and Tyrophagus putrescentiae was also analysed. Hydrophobic S. scabiei antigens were detectable in the range of 40-50 kDa, whilst the hydrophilic fraction showed no specific antigenicity. In the hydrophobic fractions of D. farinae and T. putrescentiae, two major protein fractions in a similar size range could be identified, but no cross-reactivity with Sarcoptes-positive sera was detectable. However, examination of the hydrophilic fractions revealed cross-reactivity between Sarcoptes-positive sera and both the house dust mite and the storage mite in the range of 115 and 28/38 kDa. Specific bands in the same range (42 and 48 kDa) could be detected in blots from hydrophobic fractions of all three tested variations of S. scabiei (var. canis, bovis and suis). These results show that there are considerable differences in mange antibody reactivity, including reactions with proteins from free-living mites, which may interfere with tests based on hydrophilic antigens. Further refinement of antigen and the use of specific hydrophobic proteins could improve ELISA performance and standardisation.

Synthesis of pyramidal, cubical and truncated octahedral magnetite nanocrystals by controlling reaction heating rate

Pyramidal, cubical and truncated octahedral magnetite nanocrystals have been synthesized by thermal de-composition of iron (III) acetylacetone (Fe(acac)3) in the presence of oleic acid under various reaction rate controlled by heating rate. The magnetite nanocrystals were characterized by transmission electron microscopy (TEM) and X-ray diffraction (XRD). High-resolution transmission electron microscopy (HRTEM) was applied to reveal the structural information of single magnetite (Fe3O4) nanocrystals. Magnetization curves of the three types of magnetite nanocrystals show that the pyramidal crystals exhibit a slight hysteresis compared to the other two despite of the similar size range. The results suggest that in addition to the surfactant selective capping and varying reaction temperature, the reaction rate variation is also an effective means for controlling the morphology and functions of the magnetite nanocrystals.

Abstract LB-242: Oncosomes: Large bioactive particles secreted by prostate cancer cells

Abstract Purpose of the study: Human prostate tumor cells were recently shown by our group to secrete large (0.5-10 m diameter) bioactive microvesicles capable of activating signal transduction and inducing proliferation and migration in cells in the tumor microenvironment (Di Vizio, et al. Cancer Res. 2009 69: 5601-9). These secreted particles, termed oncosomes, were shown to be vehicles for numerous signaling proteins involved in cell growth and motility. They also contain the membrane protein caveolin-1 (Cav-1), a mediator of prostate cancer (PCa) metastasis and a circulating PCa biomarker. The purpose of this study was to develop the tools to identify, isolate, sort and characterize PCa-derived oncosomes. Experimental procedures: Secreted particles from PCa cells were ultracentrifuged at 100,000xg and the pelleted material processed on a Moflo High-Speed Cell Sorter (Beckman-Coulter) and analyzed using Summit V4.3 software (Dako Colorado, Inc.). 1, 3 and 10 m bead standards (Spherotech Inc.) were used to set size gates. To avoid capturing aggregated material, forward scatter and pulse width were set at a linear scale and only single events were gated and analyzed. Sorted particles were visualized by confocal and electron microscopy. Results: Using the above procedure, we were able to retrieve intact particles of 1-10 m in diameter secreted by LNCaP cells overexpressing the oncoprotein MyrAkt1. The nontumorigenic prostate stromal cell line WPMY-1 secreted &amp;gt; 10-fold fewer particles in this size range. We confirmed that these were membrane-derived particles by showing that they contained MyrAkt1, which localizes to the plasma membrane. Particles in a similar size range were also sorted after purification from LNCaP cells stably expressing a Cav-1/GFP fusion protein. Sorted particles visualized microscopically appeared to be surrounded by a lipid bilayer and to have intrinsic structure that made them resistant to collapse. Particles isolated by these methods evoked degradation of FITC-labeled collagen and contained active MMP9 and MMP2, two key proteases involved in tumor cell invasion. Conclusions: The specific type of large bioactive particle we have isolated has not been previously described in any tumor system. Studies are ongoing to detect these particles in animal models of cancer and in human patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-242.

In vivo evaluation of the biodistribution and safety of PLGA nanoparticles as drug delivery systems

The remarkable physicochemical properties of particles in the nanometer range have been proven to address many challenges in the field of science. However, the possible toxic effects of these particles have raised some concerns. The aim of this article is to evaluate the effects of poly(lactide-co-glycolide) (PLGA) nanoparticles in vitro and in vivo compared to industrial nanoparticles of a similar size range such as zinc oxide, ferrous oxide, and fumed silica. An in vitro cytotoxicity study was conducted to assess the cell viability following exposure to PLGA nanoparticles. Viability was determined by means of a WST assay, wherein cell viability of greater than 75% was observed for both PLGA and amorphous fumed silica particles and ferrous oxide, but was significantly reduced for zinc oxide particles. In vivo toxicity assays were performed via histopathological evaluation, and no specific anatomical pathological changes or tissue damage was observed in the tissues of Balb/C mice. The extent of tissue distribution and retention following oral administration of PLGA particles was analyzed for 7 days. After 7 days, the particles remained detectable in the brain, heart, kidney, liver, lungs, and spleen. The results show that a mean percentage (40.04%) of the particles were localized in the liver, 25.97% in the kidney, and 12.86% in the brain. The lowest percentage was observed in the spleen. Thus, based on these assays, it can be concluded that the toxic effects observed with various industrial nanoparticles will not be observed with particles made of synthetic polymers such as PLGA when applied in the field of nanomedicine. Furthermore, the biodistribution of the particles warrants surface modification of the particles to avoid higher particle localization in the liver. From the Clinical Editor The aim of this study was to evaluate the effects of poly(lactide-co-glycolide) (PLGA) nanoparticles in vitro and in vivo compared to industrial nanoparticles including zinc oxide, ferrous oxide, and fumed silica. The authors concluded that the toxic effects observed with various industrial nanoparticles is unlikely to be observed with particles made of PLGA. The biodistribution of these particles warrants surface modification to avoid particle accumulation in the liver.

CO Oxidation of Au–Pt Nanostructures: Enhancement of Catalytic Activity of Pt Nanoparticles by Au

The CO oxidation activity of Pt deposited on Ta2O5/Ta was studied with various amounts of Au post-deposited on Pt/Ta2O5/Ta. For Pt nanoparticles with a mean size of 2–4 nm, an enhancement in the CO oxidation activity with increasing amount of post-deposited Au was found. The mixed Au–Pt nanoparticles with sizes in the range of 2–4 nm exhibited higher stability than the bare Au nanoparticles with a similar size range. In contrast to the results obtained with the Pt nanoparticles, the catalytic activity of a thicker Pt film gradually decreased with increasing amount of Au deposited. Based on the CO desorption experiments, it is suggested that the surface of the catalytically active Au–Pt bimetallic structures consists of both Au and Pt sites.

Effect of particle shape on dry particle inhalation: study of flowability, aerosolization, and deposition properties.

The shape effects of dry particles on flowability, aerosolization, and deposition properties in inhalation drug delivery are studied. The properties are compared with similar size range particles of different shapes such as sphere, needle, cube, plate, and pollen. Flowability of the particles is characterized by Carr's compressibility index and angle of slide (theta) method. The aerosolization and deposition properties of the particles are studied in vitro using an eight-stage Anderson cascade impactor with a Rotahaler. Pollen-shaped particles are found to exhibit better flowability, higher emitted dose, and higher fine particle fraction than particles of other shapes in similar size range. They showed minimum theta of 35 degrees and maximum emitted dose of 87% and fine particle fraction of 16%. The use of pollen-shaped particles can be a potential improvement in dry particle inhalation.

Characterization and purification of polydisperse reconstituted lipoproteins and nanolipoprotein particles.

Heterogeneity is a fact that plagues the characterization and application of many self-assembled biological constructs. The importance of obtaining particle homogeneity in biological assemblies is a critical goal, as bulk analysis tools often require identical species for reliable interpretation of the results—indeed, important tools of analysis such as x-ray diffraction typically require over 90% purity for effectiveness. This issue bears particular importance in the case of lipoproteins. Lipid-binding proteins known as apolipoproteins can self assemble with liposomes to form reconstituted high density lipoproteins (rHDLs) or nanolipoprotein particles (NLPs) when used for biotechnology applications such as the solubilization of membrane proteins. Typically, the apolipoprotein and phospholipids reactants are self assembled and even with careful assembly protocols the product often contains heterogeneous particles. In fact, size polydispersity in rHDLs and NLPs published in the literature are frequently observed, which may confound the accurate use of analytical methods. In this article, we demonstrate a procedure for producing a pure, monodisperse NLP subpopulation from a polydisperse self-assembly using size exclusion chromatography (SEC) coupled with high resolution particle imaging by atomic force microscopy (AFM). In addition, NLPs have been shown to self assemble both in the presence and absence of detergents such as cholate, yet the effects of cholate on NLP polydispersity and separation has not been systematically examined. Therefore, we examined the separation properties of NLPs assembled in both the absence and presence of cholate using SEC and native gel electrophoresis. From this analysis, NLPs prepared with and without cholate showed particles with well defined diameters spanning a similar size range. However, cholate was shown to have a dramatic affect on NLP separation by SEC and native gel electrophoresis. Furthermore, under conditions where different sized NLPs were not sufficiently separated or purified by SEC, AFM was used to deconvolute the elution pattern of different sized NLPs. From this analysis we were able to purify an NLP subpopulation to 90% size homogeneity by taking extremely fine elutions from the SEC. With this purity, we generate high quality NLP crystals that were over 100 μm in size with little precipitate, which could not be obtained utilizing the traditional size exclusion techniques. This purification procedure and the methods for validation are broadly applicable to other lipoprotein particles.

Strain-dependent viral dynamics and virus-cell interactions in a novel in vitro system supporting the life cycle of blood-borne hepatitis C virus #

We developed an in vitro system that can be used for the study of the life cycle of a wide variety of blood-borne hepatitis C viruses (HCV) from various patients using a three-dimensional hollow fiber culture system and an immortalized primary human hepatocyte (HuS-E/2) cell line. Unlike the conventional two-dimensional culture, this system not only enhanced the infectivity of blood-borne HCV but also supported its long-term proliferation and the production of infectious virus particles. Both sucrose gradient fractionation and electron microscopy examination showed that the produced virus-like particles are within a similar fraction and size range to those previously reported. Infection with different HCV strains showed strain-dependent different patterns of HCV proliferation and particle production. Fluctuation of virus proliferation and particle production was found during prolonged culture and was found to be associated with change in the major replicating virus strain. Induction of cellular apoptosis was only found when strains of HCV-2a genotype were used for infection. Interferon-alpha stimulation also varied among different strains of HCV-1b genotypes tested in this study. These results suggest that this in vitro infection system can reproduce strain-dependent events reflecting viral dynamics and virus-cell interactions at the early phase of blood-borne HCV infection, and that this system can allow the development of new anti-HCV strategies specific to various HCV strains.

Itokawa's cratering record as observed by Hayabusa: Implications for its age and collisional history

In this paper, we study cratering and crater erasure processes and provide an age estimate for the near-Earth Asteroid (25143) Itokawa, the target of the mission Hayabusa, based on its crater history since the time when it was formed in the main belt by catastrophic disruption or experienced a global resetting event. Using a model which was applied to the study of the crater history of Gaspra, Ida, Mathilde and Eros [O'Brien, D.P., Greenberg, R., Richardson, J.E., 2006. Icarus 183, 79–92], we calculate the time needed to accumulate the craters on Itokawa's surface, taking into account several processes which can affect crater formation and crater erasure on such a low-gravity object, such as seismic shaking. We use two models of the projectile population and two scaling laws to relate crater diameter to projectile size. Both models of the projectile population provide similar results, and depending on the scaling law used, we find that the time necessary to accumulate Itokawa's craters was at least ∼ 75 Myr , and maybe as long as 1 Gyr. Moreover, using the same model and similar parameters (scaled accordingly), we provide a good match not only to Itokawa's craters, but also to those of Eros, which has also been imaged at high enough resolution to give crater counts in a similar size range to those on Itokawa. We show that, as for Eros, the lack of small craters on Itokawa is consistent with erasure by seismic shaking, although for Itokawa, the pronounced deficiency of the smallest craters ( < 10 m in diameter) requires another process or event in addition to just seismic shaking. A small body such as Itokawa is highly sensitive to specific events that may occur during its history. For example, the two parts of Itokawa, called head and body, may well have joined each other by a low-velocity impact within the last hundred thousand years [Scheeres, D.J., Abe, M., Yoshikawa, M., Nakamura, R., Gaskell, R.W., Abell, P.A., 2007. Icarus 188, 425–429]. In addition to providing an erasure mechanism for small craters, the proposed timescale of that event is consistent with the timescale necessary in our model to form the current, depleted population of just a few small ( < 10 m ) craters on Itokawa, suggesting that it may be the explanation for the discrepancy between Itokawa's cratering record and that obtained from our equilibrium seismic shaking model. Other explanations for the depletion of the smallest craters on Itokawa, such as armoring by boulders lying on the surface, cannot be ruled out.

Evaluation of Equine Laminar Vein Function: Harvesting, dissection and the use of functional methods to distinguish between veins and arteries

Introduction Pharmacological evaluation of the unique equine laminar microvasculature is crucial to understanding its role in health and in diseases such as laminitis. However, separating the distinctive characteristics of arterial versus venous components of this complex vascular network has previously proved to be extremely difficult. Encased in a hard hoof capsule, isolation of individual blood vessels presents a considerable challenge. Exacerbating this difficulty, the laminar venous network is adapted to sustain high intravascular pressures and consequently has thickened walls, making the normally straightforward visual distinction between arteries and veins problematic. Here we describe a novel harvesting and dissection method coupled with a functional analysis procedure that facilitates distinction of arteries and veins. Methods Laminar tissue was recovered from the hoof of euthanized, clinically normal horses by dissection at the coronary band and stored in cold Krebs–Henseleit physiological salt solution prior to further dissection in the laboratory to remove 2 mm segments of vessels 100–500 µm in diameter. Active length tension measurements were made to evaluate optimal conditions for experimentation, and based on the differences in contractility and appearance, an experimental protocol was set up to allow a) initial distinction between arteries and veins and b) in vitro pharmacological evaluation. Results Active length tension studies clearly revealed the presence of two populations of vessels distinguished by either a large or a lower maximal contraction that subsequent histological evaluation confirmed to be arteries and veins respectively. Functional distinction using relative contractility to 60 mM potassium salt solution then demonstrated equine laminar veins to have increased sensitivity to the agonist endothelin 1 (ET-1) compared to arteries. Discussion In vitro evaluation of laminar vessels is possible despite anatomical obstacles. Furthermore, a clear distinction can be made between laminar veins and arteries using functional characteristics providing vessels of a similar size range are selected. Utilising these novel procedures, investigators can unambiguously analyse the pharmacological characteristics of equine laminar veins and arteries to decipher the physiological mechanisms responsible for the control of laminar blood flow.

Comparison of predation signatures left by Atlantic oyster drills ( Urosalpinx cinerea Say, Muricidae) and veined rapa whelks ( Rapana venosa Valenciennes, Muricidae) in bivalve prey

Predation signatures left by Atlantic oyster drills ( Urosalpinx cinerea, 8.8 to 29.8 mm shell length, SL) and veined rapa whelks ( Rapana venosa, 3.0 to 79.9 mm SL) in oyster and mussel prey were quantitatively described and compared for a similar size range of predators (8–33 mm SL). Rapa whelks and Atlantic oyster drills leave boreholes in the valves of mussels and oysters when predator shell lengths are less than 34 mm. Rapa whelks with shell lengths greater than 34 mm consumed their oyster and mussel prey without drilling or edge boring. The absence of a predation signature of any kind on prey was first observed at rapa whelk shell lengths greater than 10 mm. In rapa whelks, the shell length range of 10 to 34 mm appears to parallel a transition in predation strategy between evident signatures (drilling) and no signatures (non-drilling). The zones over adductor muscle scars were the most frequent site for drilling by both predators. Borehole dimensions scale with size of the predator for Urosalpinx and Rapana. Rapa whelk outer borehole diameters were significantly larger than outer borehole diameters made by similarly sized Atlantic oyster drills. Rapa whelks leave a borehole that has a more steep-sided vertical profile than Atlantic oyster drills. Observed differences in borehole morphology between species may provide a valuable tool to identify predators in field collected valves of prey species. The ability to identify the presence of rapa whelks < 34 mm shell length in a habitat would offer an earlier warning for the presence of this invader than is currently offered by collections of rapa whelks as bycatch in commercial fishing gear at shell lengths greater than 70 mm. Differences in predation strategies between native and introduced gastropods may result in functional trophic differences that have broad scale ecological consequences.

Spawning and Movement Behavior of Migratory Coastal Cutthroat Trout on the Western Copper River Delta, Alaska

AbstractWe studied the movement patterns of migratory coastal cutthroat trout Oncorhynchus clarkii clarkii in the western Copper River delta, Alaska, near the northern extent of the subspecies' distribution. Life history information for coastal cutthroat trout is scarce within this region. Movement of coastal cutthroat trout was monitored from 1994 to 1996 with radiotelemetry and tag–recapture of fish at a two‐way fishweir. Radio‐tracking of 20 adults was conducted for a period ranging from 5 to 343 d. We observed anadromous and potamodromous migrations by fish that were morphologically indistinguishable and spanned a similar size range (mean fork length = 364 mm). Within these migratory groups, fish exhibited variation in movement patterns, including the timing of entry to spawning streams and postspawning movements. Anadromous and potamodromous fish entered study streams either during fall to overwinter and then spawn or during the spring just before spawning. Spawning was most concentrated in the upper portions of the study streams and occurred upstream to the highest available areas. Postspawning movements of potamodromous fish consisted of small‐scale (&lt;2 km) movement within the spawning stream or longer movements to larger rivers or lakes. Anadromous fish migrated to estuarine or marine habitat after spawning. Both anadromous and potamodromous fish moved to lentic freshwater habitat to overwinter. Our results provide empirical evidence for an array of life histories displayed by coastal cutthroat trout in south‐central Alaska. We attribute the variety of movement patterns, at least in part, to the diverse assemblage of available habitat and seasonally abundant food resources provided by Pacific salmon Oncorhynchus spp. Future management should maintain the quality of and connectivity between reproductive, trophic, and refuge habitats distributed throughout the western Copper River delta.