Correlative light and electron microscopy in parasite research.

The interaction of a parasite and a host cell is a complex process, which involves several steps: (1) attachment to the plasma membrane, (2) entry inside the host cell, and (3) hijacking of the metabolism of the host. In biochemical experiments, only an event averaged over the whole cell population can be analyzed. The power of microscopy, however, is to investigate individual events in individual cells. Therefore, parasitologists frequently perform experiments with fluorescence microscopy using different dyes to label structures of the parasite or the host cell. Though the resolution of light microscopy has greatly improved, it is not sufficient to reveal interactions at the ultrastructural level. Furthermore, only specifically labeled structures can be seen and related to each other. Here, we want to demonstrate the additional value of electron microscopy in this area of research. Investigation of the different steps of parasite-host cell interaction by electron microscopy, however, is often hampered by the fact that there are only a few cells infected, and therefore it is difficult to find enough cells to study. A solution is to profit from low magnification, hence large overview, and specific location of the players by fluorescence labels in a light microscope with the high power resolution and structural information provided by an electron microscope, in short by correlative light and electron microscopy.

Assessment of speed of human locomotion using a differential satellite global positioning system.

PURPOSE: The objective was to explore whether a satellite-based navigation system, global positioning system used in differential mode (DGPS), could accurately assess the speed of running in humans. METHODS: A subject was equipped with a portable GPS receptor coupled to a receiver for differential corrections, while running outdoors on a straight asphalt road at 27 different speeds. Actual speed (reference method) was assessed by chronometry. RESULTS: The accuracy of speed prediction had a standard deviation (SD) of 0.08 km x h(-1) for walking, 0.11 km x h(-1) for running, yielding a coefficient of variation (SD/mean) of 1.38% and 0.82%, respectively. There was a highly significant linear relationship between actual and DGPS speed assessment (r2 = 0.999) with little bias in the prediction equation, because the slope of the regression line was close to unity (0.997). CONCLUSION: the DGPS technique appears to be a valid and inconspicuous tool for "on line" monitoring of the speed of displacement of individuals located on any field on earth, for prolonged periods of time and unlimited distance, but only in specific environmental conditions ("open sky"). Furthermore, the accuracy of speed assessment using the differential GPS mode was improved by a factor of 10 as compared to non-differential GPS.

Time of injury in the light of acute and hazardous usual alcohol consumption - A study among emergency department patients.

Purpose: To investigate how prior-to-injury and usual alcohol consumption relate to time of injury. Patients and methods: The associations between injury time of day and day of week and prior-to-injury (labeled as "acute") alcohol intake and hazardous usual alcohol consumption (considered from the point of view of both heavy episodic drinking [HED] and risky volumes of consumption) are assessed using interview data from a randomized sample of 486 injured patients treated in a Swiss emergency department (ED; Lausanne University Hospital). Results: Acute consumption was associated with both injury time of day and day of week, HED with day of week only, and risky volume with none. Conclusions: Acute consumption and HED, but not risky volume of consumption, show specific time distributions for injuries. These findings highlight the potential importance of considering the time dimension of an injury when providing emergency care and have additional implications for interventions aimed at influencing the alcohol consumption of injured patients presenting to the ED.

Analysis of acute brain slices by electron microscopy: A correlative light-electron microscopy workflow based on Tokuyasu cryo-sectioning.

Acute brain slices are slices of brain tissue that are kept vital in vitro for further recordings and analyses. This tool is of major importance in neurobiology and allows the study of brain cells such as microglia, astrocytes, neurons and their inter/intracellular communications via ion channels or transporters. In combination with light/fluorescence microscopies, acute brain slices enable the ex vivo analysis of specific cells or groups of cells inside the slice, e.g. astrocytes. To bridge ex vivo knowledge of a cell with its ultrastructure, we developed a correlative microscopy approach for acute brain slices. The workflow begins with sampling of the tissue and precise trimming of a region of interest, which contains GFP-tagged astrocytes that can be visualised by fluorescence microscopy of ultrathin sections. The astrocytes and their surroundings are then analysed by high resolution scanning transmission electron microscopy (STEM). An important aspect of this workflow is the modification of a commercial cryo-ultramicrotome to observe the fluorescent GFP signal during the trimming process. It ensured that sections contained at least one GFP astrocyte. After cryo-sectioning, a map of the GFP-expressing astrocytes is established and transferred to correlation software installed on a focused ion beam scanning electron microscope equipped with a STEM detector. Next, the areas displaying fluorescence are selected for high resolution STEM imaging. An overview area (e.g. a whole mesh of the grid) is imaged with an automated tiling and stitching process. In the final stitched image, the local organisation of the brain tissue can be surveyed or areas of interest can be magnified to observe fine details, e.g. vesicles or gold labels on specific proteins. The robustness of this workflow is contingent on the quality of sample preparation, based on Tokuyasu's protocol. This method results in a reasonable compromise between preservation of morphology and maintenance of antigenicity. Finally, an important feature of this approach is that the fluorescence of the GFP signal is preserved throughout the entire preparation process until the last step before electron microscopy.

Food's visually perceived fat content affects discrimination speed in an orthogonal spatial task.

Choosing what to eat is a complex activity for humans. Determining a food's pleasantness requires us to combine information about what is available at a given time with knowledge of the food's palatability, texture, fat content, and other nutritional information. It has been suggested that humans may have an implicit knowledge of a food's fat content based on its appearance; Toepel et al. (Neuroimage 44:967-974, 2009) reported visual-evoked potential modulations after participants viewed images of high-energy, high-fat food (HF), as compared to viewing low-fat food (LF). In the present study, we investigated whether there are any immediate behavioural consequences of these modulations for human performance. HF, LF, or non-food (NF) images were used to exogenously direct participants' attention to either the left or the right. Next, participants made speeded elevation discrimination responses (up vs. down) to visual targets presented either above or below the midline (and at one of three stimulus onset asynchronies: 150, 300, or 450 ms). Participants responded significantly more rapidly following the presentation of a HF image than following the presentation of either LF or NF images, despite the fact that the identity of the images was entirely task-irrelevant. Similar results were found when comparing response speeds following images of high-carbohydrate (HC) food items to low-carbohydrate (LC) food items. These results support the view that people rapidly process (i.e. within a few hundred milliseconds) the fat/carbohydrate/energy value or, perhaps more generally, the pleasantness of food. Potentially as a result of HF/HC food items being more pleasant and thus having a higher incentive value, it seems as though seeing these foods results in a response readiness, or an overall alerting effect, in the human brain.

Phytochrome interacting factors 4 and 5 redundantly limit seedling de-etiolation in continuous far-red light.

Phytochromes are red/far-red photosensors that regulate numerous developmental programs in plants. Among them, phytochrome A (phyA) is essential to enable seedling de-etiolation under continuous far-red (FR) light, a condition that mimics the environment under a dense canopy. The ecological relevance of this response is demonstrated by the high mortality rate of phyA mutant plants that germinate in deep vegetational shade. phyA signaling involves direct interaction of the photoreceptor with phytochrome-interacting factors PIF1 and PIF3, members of the bHLH transcription factor family. Here we investigated the involvement of PIF4 and PIF5 in phyA signaling, and found that they redundantly control de-etiolation in FR light. The pif4 pif5 double mutant is hypersensitive to low fluence rates of FR light. This phenotype is dependent on FR light perception by phyA, but does not rely on alterations in the phyA level. Our microarray analysis shows that PIF4 and PIF5 are part of an inhibitory mechanism that represses the expression of some light-responsive genes in the dark, and that they are also needed for full expression of several growth-related genes in the light. Unlike PIF1 and PIF3, PIF4 and PIF5 are not degraded in response to FR light, indicating that they are light-regulated by a different mechanism. Our genetic analysis suggests that this is achieved through sequestration of these PIFs by the closely related bHLH transcription factor HFR1 (long hypocotyl in FR light).

Shedding light on proteolytic cleavage of CD44: the responsible sheddase and functional significance of shedding.

CD44 is the major cell-surface receptor for hyaluronan, which is implicated in cell-cell and cell-matrix adhesion, cell migration, and signaling. Studies have shown that CD44-dependent migration requires CD44 to be shed from the cell surface and that matrix metalloproteinase-mediated cleavage may provide an underlying mechanism. However, the full spectrum of proteases that may participate in CD44 shedding has yet to be defined. In this issue, Anderegg et al. demonstrate that ADAM10, but not ADAM17 or MMP14, mediates constitutive shedding of CD44 in human melanoma cells and that knockdown of ADAM10 blocks the antiproliferative activity of the soluble proteolytic cleavage product of CD44.

The prediction of speed and incline in outdoor running in humans using accelerometry.

PURPOSE: To explore whether triaxial accelerometric measurements can be utilized to accurately assess speed and incline of running in free-living conditions. METHODS: Body accelerations during running were recorded at the lower back and at the heel by a portable data logger in 20 human subjects, 10 men, and 10 women. After parameterizing body accelerations, two neural networks were designed to recognize each running pattern and calculate speed and incline. Each subject ran 18 times on outdoor roads at various speeds and inclines; 12 runs were used to calibrate the neural networks whereas the 6 other runs were used to validate the model. RESULTS: A small difference between the estimated and the actual values was observed: the square root of the mean square error (RMSE) was 0.12 m x s(-1) for speed and 0.014 radiant (rad) (or 1.4% in absolute value) for incline. Multiple regression analysis allowed accurate prediction of speed (RMSE = 0.14 m x s(-1)) but not of incline (RMSE = 0.026 rad or 2.6% slope). CONCLUSION: Triaxial accelerometric measurements allows an accurate estimation of speed of running and incline of terrain (the latter with more uncertainty). This will permit the validation of the energetic results generated on the treadmill as applied to more physiological unconstrained running conditions.

Biases of the ordinary least squares and instrumental variables estimators of the intergenerational earnings elasticity : revisited in the light of panel data

The OLS estimator of the intergenerational earnings correlation is biased towards zero, while the instrumental variables estimator is biased upwards. The first of these results arises because of measurement error, while the latter rests on the presumption that the education of the parent family is an invalid instrument. We propose a panel data framework for quantifying the asymptotic biases of these estimators, as well as a mis-specification test for the IV estimator. [Author]

Localized amyloid light-chain amyloidosis and extramedullary plasmacytoma of the mitral valve.

An unusual case of localized amyloid light-chain (AL) amyloidosis and extramedullary plasmacytoma of the mitral valve is described. The worsening of a mitral regurgitation led to investigations and surgery. The valve presented marked distortion and thickening by type AL amyloid associated with a monotypic CD138+ immunoglobulin lambda plasma cell proliferation. Systemic staging showed a normal bone marrow and no evidence of amyloid deposition in other localizations. The patient's outcome after mitral valve replacement was excellent. To our knowledge, this is the first description of a localized AL amyloidosis as well as of a primary extramedullary plasmacytoma of the mitral valve.

How to assign a likelihood ratio in a footwear mark case: an analysis and discussion in the light of R v T

This article analyses and discusses issues that pertain to the choice of relevant databases for assigning values to the components of evaluative likelihood ratio procedures at source level. Although several formal likelihood ratio developments currently exist, both case practitioners and recipients of expert information (such as judiciary) may be reluctant to consider them as a framework for evaluating scientific evidence in context. The recent ruling R v T and ensuing discussions in many forums provide illustrative examples for this. In particular, it is often felt that likelihood ratio-based reasoning amounts to an application that requires extensive quantitative information along with means for dealing with technicalities related to the algebraic formulation of these approaches. With regard to this objection, this article proposes two distinct discussions. In a first part, it is argued that, from a methodological point of view, there are additional levels of qualitative evaluation that are worth considering prior to focusing on particular numerical probability assignments. Analyses will be proposed that intend to show that, under certain assumptions, relative numerical values, as opposed to absolute values, may be sufficient to characterize a likelihood ratio for practical and pragmatic purposes. The feasibility of such qualitative considerations points out that the availability of hard numerical data is not a necessary requirement for implementing a likelihood ratio approach in practice. It is further argued that, even if numerical evaluations can be made, qualitative considerations may be valuable because they can further the understanding of the logical underpinnings of an assessment. In a second part, the article will draw a parallel to R v T by concentrating on a practical footwear mark case received at the authors' institute. This case will serve the purpose of exemplifying the possible usage of data from various sources in casework and help to discuss the difficulty associated with reconciling the depth of theoretical likelihood ratio developments and limitations in the degree to which these developments can actually be applied in practice.

Are the light-harvesting I complexes from Rhodospirillum rubrum arranged around the reaction centre in a square geometry?

The basic photosynthetic unit containing the reaction centre and the light-harvesting I complex (RC-LHI) of the purple non-sulphur bacterium Rhodospirillum rubrum was purified and reconstituted into two-dimensional (2D) membrane crystals. Transmission electron microscopy using conventional techniques and cryoelectron microscopy of the purified single particles and of 2D crystals yielded a projection of the RC-LHI complex at a resolution of at least 1.6 nm. In this projection the LHI ring appears to have a square symmetry and packs in a square crystal lattice. The square geometry of the LHI ring was observed also in images of single isolated particles of the RC-LHI complex. However, although the LHI units are packed identically within the crystal lattice, a new rotational analysis developed here showed that the reaction centres take up one of four possible orientations within the ring. This fourfold disorder supports our interpretation of a square ring symmetry and suggests that a hitherto undetected component may be present within the photosynthetic unit.

Incline, speed, and distance assessment during unconstrained walking.

Body accelerations during human walking were recorded by a portable measuring device. A new method for parameterizing body accelerations and finding the pattern of walking is outlined. Two neural networks were designed to recognize each pattern and estimate the speed and incline of walking. Six subjects performed treadmill walking followed by self-paced walking on an outdoor test circuit involving roads of various inclines. The neural networks were first "trained" by known patterns of treadmill walking. Then the inclines, the speeds, and the distance covered during overground walking (outdoor circuit) were estimated. The results show a good agreement between actual and predicted variables. The standard deviation of estimated incline was less than 2.6% and the maximum of the coefficient of variation of speed estimation is 6%. To the best of our knowledge, these results constitute the first assessment of speed, incline and distance covered during level and slope walking and offer investigators a new tool for assessing levels of outdoor physical activity.