Darkfield-Confocal Microscopy detection of nanoscale particle internalization by human lung cells

<p>Abstract</p> <p>Background</p> <p>Concerns over the health effects of nanomaterials in the environment have created a need for microscopy methods capable of examining the biological interactions of nanoparticles (NP). Unfortunately, NP are beyond the diffraction limit of resolution for conventional light microscopy (~200 nm). Fluorescence and electron microscopy techniques commonly used to examine NP interactions with biological substrates have drawbacks that limit their usefulness in toxicological investigation of NP. EM is labor intensive and slow, while fluorescence carries the risk of photobleaching the sample and has size resolution limits. In addition, many relevant particles lack intrinsic fluorescence and therefore can not be detected in this manner. To surmount these limitations, we evaluated the potential of a novel combination of darkfield and confocal laser scanning microscopy (DF-CLSM) for the efficient 3D detection of NP in human lung cells. The DF-CLSM approach utilizes the contrast enhancements of darkfield microscopy to detect objects below the diffraction limit of 200 nm based on their light scattering properties and interfaces it with the power of confocal microscopy to resolve objects in the z-plane.</p> <p>Results</p> <p>Validation of the DF-CLSM method using fluorescent polystyrene beads demonstrated spatial colocalization of particle fluorescence (Confocal) and scattered transmitted light (Darkfield) along the X, Y, and Z axes. DF-CLSM imaging was able to detect and provide reasonable spatial locations of 27 nm TiO₂particles in relation to the stained nuclei of exposed BEAS 2B cells. Statistical analysis of particle proximity to cellular nuclei determined a significant difference between 5 min and 2 hr particle exposures suggesting a time-dependant internalization process.</p> <p>Conclusions</p> <p>DF-CLSM microscopy is an alternative to current conventional light and electron microscopy methods that does not rely on particle fluorescence or contrast in electron density. DF-CLSM is especially well suited to the task of establishing the spatial localization of nanoparticles within cells, a critical topic in nanotoxicology. This technique has advantages to 2D darkfield microscopy as it visualizes nanoparticles in 3D using confocal microscopy. Use of this technique should aid toxicological studies related to observation of NP interactions with biological endpoints at cellular and subcellular levels.</p

Teaching molecular genetics: chapter 4—positional cloning of genetic disorders

Positional cloning is the approach of choice for the identification of genetic mutations underlying the pathological development of diseases with simple Mendelian inheritance. It consists of different consecutive steps, starting with recruitment of patients and DNA collection, that are critical to the overall process. A genetic analysis of the enrolled patients and their families is performed, based on genetic recombination frequencies generated by meiotic cross-overs and on genome-wide molecular studies, to define a critical DNA region of interest. This analysis culminates in a statistical estimate of the probability that disease features may segregate in the families independently or in association with specific molecular markers located in known regions. In this latter case, a marker can be defined as being linked to the disease manifestations. The genetic markers define an interval that is a function of their recombination frequencies with the disease, in which the disease gene is localised. The identification and characterisation of chromosome abnormalities as translocations, deletions and duplications by classical cytogenetic methods or by the newly developed microarray-based comparative genomic hybridisation (array CGH) technique may define extensions and borders of the genomic regions involved. The step following the definition of a critical genomic region is the identification of candidate genes that is based on the analysis of available databases from genome browsers. Positional cloning culminates in the identification of the causative gene mutation, and the definition of its functional role in the pathogenesis of the disorder, by the use of cell-based or animal-based experiments. More often, positional cloning ends with the generation of mice with homologous mutations reproducing the human clinical phenotype. Altogether, positional cloning has represented a fundamental step in the research on genetic renal disorders, leading to the definition of several disease mechanisms and allowing a proper diagnostic approach to many conditions

A Comprehensive Workflow for General-Purpose Neural Modeling with Highly Configurable Neuromorphic Hardware Systems

In this paper we present a methodological framework that meets novel requirements emerging from upcoming types of accelerated and highly configurable neuromorphic hardware systems. We describe in detail a device with 45 million programmable and dynamic synapses that is currently under development, and we sketch the conceptual challenges that arise from taking this platform into operation. More specifically, we aim at the establishment of this neuromorphic system as a flexible and neuroscientifically valuable modeling tool that can be used by non-hardware-experts. We consider various functional aspects to be crucial for this purpose, and we introduce a consistent workflow with detailed descriptions of all involved modules that implement the suggested steps: The integration of the hardware interface into the simulator-independent model description language PyNN; a fully automated translation between the PyNN domain and appropriate hardware configurations; an executable specification of the future neuromorphic system that can be seamlessly integrated into this biology-to-hardware mapping process as a test bench for all software layers and possible hardware design modifications; an evaluation scheme that deploys models from a dedicated benchmark library, compares the results generated by virtual or prototype hardware devices with reference software simulations and analyzes the differences. The integration of these components into one hardware-software workflow provides an ecosystem for ongoing preparative studies that support the hardware design process and represents the basis for the maturity of the model-to-hardware mapping software. The functionality and flexibility of the latter is proven with a variety of experimental results

Recognition, Perceptions and Treatment Practices for Severe Malaria in Rural Tanzania: Implications for Accessing Rectal Artesunate as a Pre-Referral

OBJECTIVES: Preparatory to a community trial investigating how best to deliver rectal artesunate as pre-referral treatment for severe malaria; local understanding, perceptions of signs/symptoms of severe malaria and treatment-seeking patterns for and barriers to seeking biomedical treatment were investigated. METHODOLOGY/PRINCIPAL FINDINGS: 19 key informant interviews, 12 in-depth interviews and 14 focus group discussions targeting care-givers, opinion leaders, and formal and informal health care providers were conducted. Monthly fever episodes and danger signs or symptoms associated with severe malaria among under-fives were recorded. Respondents recognized convulsions, altered consciousness and coma, and were aware of their risks if not treated. But, these symptoms were perceived to be caused by supernatural forces, and traditional healers were identified as primary care providers. With some delay, mothers eventually visited a health facility when convulsions were part of the illness, despite pressures against this. Although vomiting and failure to eat/suck/drink were associated with malaria, they were not considered as indicators of danger signs unless combined with another more severe symptom. Study communities were familiar with rectal application of medicines. CONCLUSIONS/SIGNIFICANCE: Communities' recognition and awareness of major symptoms of severe malaria could encourage action, but perceptions of their causes and poor discrimination of other danger signs – vomiting and failure to feed – might impede early treatment. An effective health education targeting parents/guardians, decision-makers/advisors, and formal and informal care providers might be a prerequisite for successful introduction of rectal artemisinins as an emergency treatment. Role of traditional healers in delivering such medication to the community should be explored

Identification of globular cluster stars in RAVE data - I. Application to stellar parameter calibration

We present the identification of potential members of nearby Galactic globular clusters using radial velocities from the RAdial Velocity Experiment Data Release 4 (RAVE-DR4) survey data base. Our identifications are based on three globular clusters – NGC 3201, NGC 5139 (ω Cen) and NGC 362 – all of which are shown to have ∣RV∣ > 100 km s−1. The high radial velocity of cluster members compared to the bulk of surrounding disc stars enables us to identify members using their measured radial velocities, supplemented by proper motion information and location relative to the tidal radius of each cluster. The identification of globular cluster stars in RAVE DR4 data offers a unique opportunity to test the precision and accuracy of the stellar parameters determined with the currently available Stellar Parameter Pipelines used in the survey, as globular clusters are ideal test-beds for the validation of stellar atmospheric parameters, abundances, distances and ages. For both NGC 3201 and ω Cen, there is compelling evidence for numerous members (>10) in the RAVE data base; in the case of NGC 362 the evidence is more ambiguous, and there may be significant foreground and/or background contamination in our kinematically selected sample. A comparison of the RAVE-derived stellar parameters and abundances with published values for each cluster and with BASTI isochrones for ages and metallicities from the literature reveals overall good agreement, with the exception of the apparent underestimation of surface gravities for giants, in particular for the most metal-poor stars. Moreover, if the selected members are part of the main body of each cluster our results would also suggest that the distances from Binney et al., where only isochrones more metal rich than −0.9 dex were used, are typically underestimated by ∼40 per cent with respect to the published distances for the clusters, while the distances from Zwitter et al. show stars ranging from 1 to ∼6.5 kpc – with indications of a trend towards higher distances at lower metallicities – for the three clusters analysed in this study

An affordable, quality-assured community-based system for high-resolution entomological surveillance of vector mosquitoes that reflects human malaria infection risk patterns.

ABSTRACT: BACKGROUND: More sensitive and scalable entomological surveillance tools are required to monitor low levels of transmission that are increasingly common across the tropics, particularly where vector control has been successful. A large-scale larviciding programme in urban Dar es Salaam, Tanzania is supported by a community-based (CB) system for trapping adult mosquito densities to monitor programme performance. Methodology An intensive and extensive CB system for routine, longitudinal, programmatic surveillance of malaria vectors and other mosquitoes using the Ifakara Tent Trap (ITT-C) was developed in Urban Dar es Salaam, Tanzania, and validated by comparison with quality assurance (QA) surveys using either ITT-C or human landing catches (HLC), as well as a cross-sectional survey of malaria parasite prevalence in the same housing compounds. RESULTS: Community-based ITT-C had much lower sensitivity per person-night of sampling than HLC (Relative Rate (RR) [95% Confidence Interval (CI)] = 0.079 [0.051, 0.121], P < 0.001 for Anopheles gambiae s.l. and 0.153 [0.137, 0.171], P < 0.001 for Culicines) but only moderately differed from QA surveys with the same trap (0.536 [0.406,0.617], P = 0.001 and 0.747 [0.677,0.824], P < 0.001, for An. gambiae or Culex respectively). Despite the poor sensitivity of the ITT per night of sampling, when CB-ITT was compared with QA-HLC, it proved at least comparably sensitive in absolute terms (171 versus 169 primary vectors caught) and cost-effective (153US$versus 187US$ per An. gambiae caught) because it allowed more spatially extensive and temporally intensive sampling (4284 versus 335 trap nights distributed over 615 versus 240 locations with a mean number of samples per year of 143 versus 141). Despite the very low vectors densities (Annual estimate of about 170 An gambiae s.l bites per person per year), CB-ITT was the only entomological predictor of parasite infection risk (Odds Ratio [95% CI] = 4.43[3.027,7. 454] per An. gambiae or Anopheles funestus caught per night, P =0.0373). Discussion and conclusion CB trapping approaches could be improved with more sensitive traps, but already offer a practical, safe and affordable system for routine programmatic mosquito surveillance and clusters could be distributed across entire countries by adapting the sample submission and quality assurance procedures accordingly

Accurate masses and radii of normal stars: modern results and applications

This paper presents and discusses a critical compilation of accurate, fundamental determinations of stellar masses and radii. We have identified 95 detached binary systems containing 190 stars (94 eclipsing systems, and alpha Centauri) that satisfy our criterion that the mass and radius of both stars be known to 3% or better. To these we add interstellar reddening, effective temperature, metal abundance, rotational velocity and apsidal motion determinations when available, and we compute a number of other physical parameters, notably luminosity and distance. We discuss the use of this information for testing models of stellar evolution. The amount and quality of the data also allow us to analyse the tidal evolution of the systems in considerable depth, testing prescriptions of rotational synchronisation and orbital circularisation in greater detail than possible before. The new data also enable us to derive empirical calibrations of M and R for single (post-) main-sequence stars above 0.6 M(Sun). Simple, polynomial functions of T(eff), log g and [Fe/H] yield M and R with errors of 6% and 3%, respectively. Excellent agreement is found with independent determinations for host stars of transiting extrasolar planets, and good agreement with determinations of M and R from stellar models as constrained by trigonometric parallaxes and spectroscopic values of T(eff) and [Fe/H]. Finally, we list a set of 23 interferometric binaries with masses known to better than 3%, but without fundamental radius determinations (except alpha Aur). We discuss the prospects for improving these and other stellar parameters in the near future.Comment: 56 pages including figures and tables. To appear in The Astronomy and Astrophysics Review. Ascii versions of the tables will appear in the online version of the articl

Axons Amplify Somatic Incomplete Spikes into Uniform Amplitudes in Mouse Cortical Pyramidal Neurons

BACKGROUND: Action potentials are the essential unit of neuronal encoding. Somatic sequential spikes in the central nervous system appear various in amplitudes. To be effective neuronal codes, these spikes should be propagated to axonal terminals where they activate the synapses and drive postsynaptic neurons. It remains unclear whether these effective neuronal codes are based on spike timing orders and/or amplitudes. METHODOLOGY/PRINCIPAL FINDINGS: We investigated this fundamental issue by simultaneously recording the axon versus soma of identical neurons and presynaptic vs. postsynaptic neurons in the cortical slices. The axons enable somatic spikes in low amplitude be enlarged, which activate synaptic transmission in consistent patterns. This facilitation in the propagation of sequential spikes through the axons is mechanistically founded by the short refractory periods, large currents and high opening probability of axonal voltage-gated sodium channels. CONCLUSION/SIGNIFICANCE: An amplification of somatic incomplete spikes into axonal complete ones makes sequential spikes to activate consistent synaptic transmission. Therefore, neuronal encoding is likely based on spike timing order, instead of graded analogues

The Role of Growth Retardation in Lasting Effects of Neonatal Dexamethasone Treatment on Hippocampal Synaptic Function

BACKGROUND: Dexamethasone (DEX), a synthetic glucocorticoid, is commonly used to prevent or lessen the morbidity of chronic lung disease in preterm infants. However, evidence is now increasing that this clinical practice negatively affects somatic growth and may result in long-lasting neurodevelopmental deficits. We therefore hypothesized that supporting normal somatic growth may overcome the lasting adverse effects of neonatal DEX treatment on hippocampal function. METHODOLOGY/PRINCIPAL FINDINGS: To test this hypothesis, we developed a rat model using a schedule of tapering doses of DEX similar to that used in premature infants and examined whether the lasting influence of neonatal DEX treatment on hippocampal synaptic plasticity and memory performance are correlated with the deficits in somatic growth. We confirmed that neonatal DEX treatment switched the direction of synaptic plasticity in hippocampal CA1 region, favoring low-frequency stimulation- and group I metabotropic glutamate receptor agonist (S)-3,5,-dihydroxyphenylglycine-induced long-term depression (LTD), and opposing the induction of long-term potentiation (LTP) by high-frequency stimulation in the adolescent period. The effects of DEX on LTP and LTD were correlated with an increase in the autophosphorylation of Ca(2+)/calmodulin-dependent protein kinase II at threonine-286 and a decrease in the protein phosphatase 1 expression. Neonatal DEX treatment resulted in a disruption of memory retention subjected to object recognition task and passive avoidance learning. The adverse effects of neonatal DEX treatment on hippocampal synaptic plasticity and memory performance of the animals from litters culled to 4 pups were significantly less than those for the 8-pup litters. However, there was no significant difference in maternal care between groups. CONCLUSION/SIGNIFICANCE: Our results demonstrate that growth retardation plays a crucial role in DEX-induced long-lasting influence of hippocampal function. Our findings suggest that therapeutic strategies designed to support normal development and somatic growth may exert beneficial effects to reduce lasting adverse effects following neonatal DEX treatment