High resolution imaging of the cortex isolated from sea urchin eggs and embryos.

The cellular cortex-consisting of the plasma membrane and the adjacent outer few microns of the cytoplasm-is a critically important, dynamic and complex region in the sea urchin egg and embryo. Some 40 years ago it was discovered that isolated cortices could be obtained from eggs adhered to glass coverslips and since that time this preparation has been used in a wide range of studies, including seminal research on fertilization, exocytosis, the cytoskeleton, and cytokinesis. In this chapter, we discuss methods for isolating cortices from eggs and embryos, including those undergoing cell division. We also provide protocols for analyzing cortical architecture and dynamics using specific localization methods combined with super-resolution Structured Illumination and Stimulated Emission Depletion light microscopy and platinum replica transmission electron microscopy.

An Exploratory Study to Detect Ménière's Disease in Conventional MRI Scans Using Radiomics.

OBJECTIVE: The purpose of this exploratory study was to investigate whether a quantitative image analysis of the labyrinth in conventional magnetic resonance imaging (MRI) scans using a radiomics approach showed differences between patients with Ménière's disease (MD) and the control group. MATERIALS AND METHODS: In this retrospective study, MRI scans of the affected labyrinths of 24 patients with MD were compared to the MRI scans of labyrinths of 29 patients with an idiopathic asymmetrical sensorineural hearing loss. The 1.5- and 3-T MRI scans had been previously made in a clinical setting between 2008 and 2015. 3D Slicer 4.4 was used to extract several substructures of the labyrinth. A quantitative analysis of the normalized radiomic image features was performed in Mathematica 10. The image features of the two groups were statistically compared. RESULTS: For numerous image features, there was a statistically significant difference (p-value <0.05) between the MD group and the control group. The statistically significant differences in image features were localized in all the substructures of the labyrinth: 43 in the anterior semicircular canal, 10 in the vestibule, 22 in the cochlea, 12 in the posterior semicircular canal, 24 in the horizontal semicircular canal, 11 in the common crus, and 44 in the volume containing the reuniting duct. Furthermore, some figures contain vertical or horizontal bands (three or more statistically significant image features in the same image feature). Several bands were seen: 9 bands in the anterior semicircular canal, 1 band in the vestibule, 3 bands in the cochlea, 0 bands in the posterior semicircular canal, 5 bands in the horizontal semicircular canal, 3 bands in the common crus, and 10 bands in the volume containing the reuniting duct. CONCLUSION: In this exploratory study, several differences were found in image features between the MD group and the control group by using a quantitative radiomics approach on high resolution T2-weighted MRI scans of the labyrinth. Further research should be aimed at validating these results and translating them in a potential clinical diagnostic method to detect MD in MRI scans.

Subcortical, modality-specific pathways contribute to multisensory processing in humans.

Oftentimes, we perceive our environment by integrating information across multiple senses. Recent studies suggest that such integration occurs at much earlier processing stages than once thought possible, including in thalamic nuclei and putatively unisensory cortical brain regions. Here, we used diffusion tensor imaging (DTI) and an audiovisual integration task to test the hypothesis that anatomical connections between sensory-related subcortical structures and sensory cortical areas govern multisensory processing in humans. Twenty-five subjects (mean age 22 years, 22 females) participated in the study. In line with our hypothesis, we show that estimated strength of white-matter connections between the first relay station in the auditory processing stream (the cochlear nucleus), the auditory thalamus, and primary auditory cortex predicted one's ability to combine auditory and visual information in a visual search task. This finding supports a growing body of work that indicates that subcortical sensory pathways do not only feed forward unisensory information to the cortex, and suggests that anatomical brain connectivity contributes to multisensory processing ability in humans.

Drosophila social clustering is disrupted by anesthetics and in narrow abdomen ion channel mutants.

Members of many species tend to congregate, a behavioral strategy known as local enhancement. Selective advantages of local enhancement range from efficient use of resources to defense from predators. While previous studies have examined many types of social behavior in fruit flies, few have specifically investigated local enhancement. Resource-independent local enhancement (RILE) has recently been described in the fruit fly using a measure called social space index (SSI), although the neural mechanisms remain unknown. Here, we analyze RILE of Drosophila under conditions that allow us to elucidate its neural mechanisms. We have investigated the effects of general volatile anesthetics, compounds that compromise higher order functioning of the type typically required for responding to social cues. We exposed Canton-S flies to non-immobilizing concentrations of halothane and found that flies had a significantly decreased SSI compared with flies tested in air. Narrow abdomen (na) mutants, which display altered responses to anesthetics in numerous behavioral assays, also have a significantly reduced SSI, an effect that was fully reversed by restoring expression of na by driving a UAS-NA rescue construct with NA-GAL4. We found that na expression in cholinergic neurons fully rescued the behavioral defect, whereas expression of na in glutamatergic neurons did so only partially. Our results also suggest a role for na expression in the mushroom bodies (MBs), as suppressing na expression in the MBs of NA-GAL4 rescue flies diminishes SSI. Our data indicate that RILE, a simple behavioral strategy, requires complex neural processing.

Essential role of IL-6 in protection against H1N1 influenza virus by promoting neutrophil survival in the lung.

Influenza virus infection is considered a major worldwide public health problem. Seasonal infections with the most common influenza virus strains (e.g., H1N1) can usually be resolved, but they still cause a high rate of mortality. The factors that influence the outcome of the infection remain unclear. Here, we show that deficiency of interleukin (IL)-6 or IL-6 receptor is sufficient for normally sublethal doses of H1N1 influenza A virus to cause death in mice. IL-6 is necessary for resolution of influenza infection by protecting neutrophils from virus-induced death in the lung and by promoting neutrophil-mediated viral clearance. Loss of IL-6 results in persistence of the influenza virus in the lung leading to pronounced lung damage and, ultimately, death. Thus, we demonstrate that IL-6 is a vital innate immune cytokine in providing protection against influenza A infection. Genetic or environmental factors that impair IL-6 production or signaling could increase mortality to influenza virus infection.

Mesenchymal stem cells in cartilage repair: state of the art and methods to monitor cell growth, differentiation and cartilage regeneration.

Degenerative joint diseases caused by rheumatism, joint dysplasia or traumata are particularly widespread in countries with high life expectation. Although there is no absolutely convincing cure available so far, hyaline cartilage and bone defects resulting from joint destruction can be treated today by appropriate transplantations. Recently, procedures were developed based on autologous chondrocytes from intact joint areas. The chondrocytes are expanded in cell culture and subsequently transplanted into the defect areas of the affected joints. However, these autologous chondrocytes are characterized by low expansion capacity and the synthesis of extracellular matrix of poor functionality and quality. An alternative approach is the use of adult mesenchymal stem cells (MSCs). These cells effectively expand in 2D culture and have the potential to differentiate into various cell types, including chondrocytes. Furthermore, they have the ability to synthesize extracellular matrix with properties mimicking closely the healthy hyaline joint cartilage. Beside a more general survey of the architecture of hyaline cartilage, its composition and the pathological processes of joint diseases, we will describe here which advances were achieved recently regarding the development of closed, aseptic bioreactors for the production of autologous grafts for cartilage regeneration based on MSCs. Additionally, a novel mathematical model will be presented that supports the understanding of the growth and differentiation of MSCs. It will be particularly emphasized that such models are helpful to explain the well-known fact that MSCs exhibit improved growth properties under reduced oxygen pressure and limited supply with nutrients. Finally, it will be comprehensively shown how different analytical methods can be used to characterize MSCs on different levels. Besides discussing methods for non-invasive monitoring and tracking of the cells and the determination of their elastic properties, mass spectrometric methods to evaluate the lipid compositions of cells will be highlighted.

[National external quality assessment in auto-immunity: antineutrophil cytoplasmic antibodies]. / Contrôle national de qualité en auto-immunité : anticorps anticytoplasme des polynucléaires neutrophiles.

The French Health Products Safety Agency organized in 2007, for the scheme of the national external quality assessment, a survey on antineutrophil cytoplasmic antibodies (ANCA) including detection and identification of the antibodies. This survey allowed to assess the quality of the different methods of these assays. The detection of ANCA by the indirect immunofluorescence technique was satisfactory. However, the methods of identification gave a high rate of false negative results for the anti-myeloperoxidase antibodies (anti-MPO), especially with the immunodot technique. Concerning the titer of anti-MPO antibodies obtained by ELISA, the broad dispersion of results between reagents pointed out a lack of standardisation of the detection of these antibodies.

Dendritic backpropagation and synaptic plasticity in the mormyrid electrosensory lobe.

This study is concerned with the origin of backpropagating action potentials in GABAergic, medium ganglionic layer neurones (MG-cells) of the mormyrid electrosensory lobe (ELL). The characteristically broad action potentials of these neurones are required for the expression of spike timing dependent plasticity (STDP) at afferent parallel fibre synapses. It has been suggested that this involves active conductances in MG-cell apical dendrites, which constitute a major component of the ELL molecular layer. Immunohistochemistry showed dense labelling of voltage gated sodium channels (VGSC) throughout the molecular layer, as well as in the ganglionic layer containing MG somata, and in the plexiform and upper granule cell layers of ELL. Potassium channel labelling was sparse, being most abundant in the deep fibre layer and the nucleus of the electrosensory lobe. Intracellular recordings from MG-cells in vitro, made in conjunction with voltage sensitive dye measurements, confirmed that dendritic backpropagation is active over at least the inner half of the molecular layer. Focal TTX applications demonstrated that in most case the origin of the backpropagating action potentials is in the proximal dendrites, whereas the small narrow spikes also seen in these neurones most likely originate in the axon. It had been speculated that the slow time course of membrane repolarisation following the broad action potentials was due to a poor expression of potassium channels in the dendritic compartments, or to their voltage- or calcium-sensitive inactivation. However application of TEA and 4AP confirmed that both A-type and delayed rectifying potassium channels normally contribute to membrane repolarisation following dendritic and axonal spikes. An alternative explanation for the shape of MG action potentials is that they represent the summation of active events occurring more or less synchronously in distal dendrites. Coincidence of backpropagating action potentials with parallel fibre input produces a strong local depolarisation that could be sufficient to cause local secretion of GABA, which might then cause plastic change through an action on presynaptic GABA(B) receptors. However, STP depression remained robust in the presence of GABAB receptor antagonists.

Potassium channels in the regulation of pulmonary artery smooth muscle cell proliferation and apoptosis: pharmacotherapeutic implications.

Maintaining the proper balance between cell apoptosis and proliferation is required for normal tissue homeostasis; when this balance is disrupted, disease such as pulmonary arterial hypertension (PAH) can result. Activity of K(+) channels plays a major role in regulating the pulmonary artery smooth muscle cell (PASMC) population in the pulmonary vasculature, as they are involved in cell apoptosis, survival and proliferation. PASMCs from PAH patients demonstrate many cellular abnormalities linked to K(+) channels, including decreased K(+) current, downregulated expression of various K(+) channels, and inhibited apoptosis. K(+) is the major intracellular cation, and the K(+) current is a major determinant of cell volume. Apoptotic volume decrease (AVD), an early hallmark and prerequisite of programmed cell death, is characterized by K(+) and Cl(-) efflux. In addition to its role in AVD, cytosolic K(+) can be inhibitory toward endogenous caspases and nucleases and can suppress mitochondrial cytochrome c release. In PASMC, K(+) channel activation accelerates AVD and enhances apoptosis, while K(+) channel inhibition decelerates AVD and inhibits apoptosis. Finally, inhibition of K(+) channels, by increasing cytosolic [Ca(2+)] as a result of membrane depolarization-mediated opening of voltage-dependent Ca(2+) channels, leads to PASMC contraction and proliferation. The goals of this review are twofold: (1) to elucidate the role of K(+) ions and K(+) channels in the proliferation and apoptosis of PASMC, with an emphasis on abnormal cell growth in human and animal models of PAH, and (2) to elaborate upon the targeting of K(+) flux pathways for pharmacological treatment of pulmonary vascular disease.

[National external quality assessment in auto-immunity: auto-antibodies against thyroid constituents]. / Contrôle national de qualité en auto-immunité: anticorps anti-thyroïdiens.

The French Health Products Safety Agency organized in 2005, for the scheme of the national external quality assessment, a survey on antibodies against thyroid constituents which included for the first time the quantitative assay. The purpose of this survey was to assess the quality of the different methods of these assays. The overall qualitative results are satisfactory. However, this survey pointed out a lower performance for immunodot which appeared to have been misused. Concerning the titer of antibodies, results show a broad dispersion between reagents. This confirms the lack of a real standardisation despite of the existence of the international MRC standards.

[Red blood cell antibody screening: error analysis in a french interlaboratory comparison program survey]. / Recherche des anticorps antiérythrocytaires: analyse d'erreurs au Contrôle national de qualité

The French quality control is organized by the French Health Products Safety Agency. In 2005, the immuno-haematology testing control included the screening of an anti KEL 1 antibody. 17 out of 2639 laboratories (0,64%) answered 'negative screening'. All laboratories received a questionnaire in order to understand the failure. In this paper the authors present the detailed laboratories' responses and failure explanations.

K+ channels in apoptosis.

A proper rate of programmed cell death or apoptosis is required to maintain normal tissue homeostasis. In disease states such as cancer and some forms of hypertension, apoptosis is blocked, resulting in hyperplasia. In neurodegenerative diseases, uncontrolled apoptosis leads to loss of brain tissue. The flow of ions in and out of the cell and its intracellular organelles is becoming increasingly linked to the generation of many of these diseased states. This review focuses on the transport of K(+) across the cell membrane and that of the mitochondria via integral K(+)-permeable channels. We describe the different types of K(+) channels that have been identified, and investigate the roles they play in controlling the different phases of apoptosis: early cell shrinkage, cytochrome c release, caspase activation, and DNA fragmentation. Attention is also given to K(+) channels on the inner mitochondrial membrane, whose activity may underlie anti- or pro-apoptotic mechanisms in neurons and cardiomyocytes.

Activation of a sensorimotor pathway in response to a water temperature drop in a teleost fish.

When common carp, Cyprinus carpio L., experience a rapid temperature drop, the cerebral blood volume is strongly reduced to dampen the temperature drop in the brain. Simultaneously, the preoptic area and pituitary gland are activated to launch whole-body adaptive responses. However, the preferred reaction of fish to a temperature change is an escape reaction, which implies activation of a sensorimotor pathway. Here, we used blood oxygenation level-dependent (BOLD)- and cerebral blood volume (CBV)-weighted functional magnetic resonance imaging (fMRI) to identify a sensorimotor pathway, during a 10 degrees C temperature drop in common carp. Transient activation was observed in the region where the sensory root of the trigeminal nerve enters the brain, and in the valvula cerebelli. In both regions, metabolic activity increased (increased deoxyhemoglobin content demonstrated by a decreased BOLD signal) within 30 s after the onset of the temperature drop, peaked after 2-3 min, and then decreased, even though the temperature continued to drop for another 2 min. These brain structures appear to respond to temperature change, rather than to the absolute temperature. Thus, during a temperature drop, the sensorimotor pathway consisting of the trigeminal nerve, the primary sensory trigeminal nucleus, the valvula cerebelli and some motornuclei, is active, in line with perception of temperature change in the buccal cavity, leading to motor activity for escape. This pathway operates in parallel to an acclimation pathway, which involves the preoptic area to pituitary gland pathway.

[National external quality assessment in auto-immunity: autoantibodies detected on liver, kidney and stomach tissues]. / Contrôle national de qualité en auto-immunité: anticorps anti-tissus non spécifiques d'organe.

In 2003, for the scheme of the French national external quality assessment, Afssaps organized for the first time a survey on auto-antibodies detected on liver, kidney and stomach tissues. This survey had two purposes: first to make an inventory of the methodology applied by the medical laboratories and secondly to assess the quality of the results. The survey sample contained M2 anti-mitochondrial antibodies. Overall results are satisfactory. Concerning the titer of antibodies, a broad dispersion of results was observed (12% of titers were upper than the expected titer). Delivered information to the participants, at the end of the survey, should improve analytical procedures applied by the biologists. An effort of standardization by using titrated internal controls would be suitable.

Impact of external quality assessment on antinuclear antibody detection performance.

Our objective was to evaluate performance of the clinical laboratories for the detection of antinuclear antibodies (ANA) by using indirect immunofluorescence method (IIF), in France. A national external quality assessment (EQA) on ANA detection was organized by the French health products safety agency once a year since 1998. Between 606 to 687 laboratories together with six university reference laboratories experienced in performing tests in autoimmunity participated in the six-year consecutive survey. Each laboratory had to answer to methodological procedures and give coded responses. Variability in IIF methodological procedure was observed. Use of inappropriate microscope magnifications for reading slides or nonconventional cutoff dilution of serum were pointed out to concerned laboratories. Concerning ANA measurement, the rate of good responses ranged from 92.7% to 99.5% of the laboratories when the samples contained ANA. A wide dispersion of ANA titers obtained on a same sample was repeatly observed every year. Misinterpretation of particular fluorescence pattern was noticed. On ANA negative sample, the rate of good responses was 94.3%. In conclusion, ANA detection in routine practice is far from being standardized. However, EQA may have an impact on ANA detection performance when it is conducted on several consecutive year surveys, by providing advice for participating laboratories to limit inter laboratory variations related to methodological procedures.

[Monoclonal immunoglobulin: national external quality assessment and diagnostic reasoning]. / Immunoglobuline monoclonale: contrôle national de qualité et démarche diagnostique.

In 2003, for the scheme of the French national external quality assessment, Afssaps sent to medical laboratories a sample for which two analyses could be carried out: the electrophoresis of proteins and the characterization for monoclonal immunoglobulin. The purpose of this new approach was to make it possible to the laboratories to transpose their usual diagnostic reasoning. This survey recalled to the biologists that it is necessary to check the sensitivity of the test of electrophoresis of proteins used in first intention and to use advisedly the anti-serums anti-free chains. Moreover this operation reinforced the educational aspect of the external quality assessment, pointing out the importance to ensure the coherence of the results of these two analyses carried out with a same diagnostic aim.

Plasma alpha-MSH and acetylated beta-endorphin levels following stress vary according to CRH sensitivity of the pituitary melanotropes in common carp, Cyprinus carpio.

Pituitary melanotropes release alpha-melanocyte-stimulating hormone (alpha-MSH) and acetylated beta-endorphin (NAc beta-end) during stress responses. However, effects of stressors on plasma concentrations of these hormones are highly inconsistent among fish species. Here, we show that also within a species, the common carp (Cyprinus carpio), fish sometimes respond with elevated alpha-MSH and NAc beta-end plasma levels, and at other times not. The origin of this variable response was investigated by (1) studying the effects of corticotropin-releasing hormone (CRH) on alpha-MSH and NAc beta-end release in vitro, (2) establishing where in the second messenger pathway coupled to CRH receptors melanotrope responsiveness is determined, and (3) testing modulatory actions of other hypothalamic factors (here opioid beta-endorphin). Melanotropes were in a high or low responsive state to CRH in vitro, which was especially evident when tissue was tested from fish kept at higher ambient water temperatures, and this correlates with the variability in alpha-MSH and NAc beta-end responses in vivo. Relative rates of alpha-MSH and NAc beta-end release following stimulation with CRH in vitro match plasma level changes in vivo, and this indicates that the CRH pathway does act in vivo. cAMP did not stimulate melanotropes in the low responsive state to release hormones in vitro. Thus, the mechanism that determines the cell status, occurs downstream of cAMP accumulation. Opioid beta-endorphin differentially modulated the actions of CRH, as NAc beta-end, but not alpha-MSH, release was inhibited. This response was not observed in the stress paradigms studied. We conclude that the variation in alpha-MSH and NAc beta-end stress responses in vivo correlates with many CRH responses in vitro; whether a cell is in a high or low responsive state to CRH is determined downstream of accumulation of the second messenger. We propose that melanotropes have to be in the high responsive state to be activated by CRH during stress in carp and other teleosts.

Identification of beta-endorphins in the pituitary gland and blood plasma of the common carp (Cyprinus carpio).

Carp beta-endorphin is posttranslationally modified by N-terminal acetylation and C-terminal cleavage. These processes determine the biological activity of the beta-endorphins. Forms of beta-endorphin were identified in the pars intermedia and the pars distalis of the pituitary gland of the common carp (Cyprinus carpio), as well as the forms released in vitro and into the blood. After separation and quantitation by high performance liquid chromatography (HPLC) coupled with radioimmunoassay, the beta-endorphin immunoreactive products were identified by electrospray ionisation mass spectrometry and peptide sequencing. The release of beta-endorphins by the pituitary gland was studied after stimulation with corticotrophin-releasing factor (CRF) in vitro. In the pars intermedia, eight N-acetylated truncated forms were identified. Full length N-acetyl beta-endorphin(1-33) coeluted with N-acetyl beta-endorphin(1-29) and these forms together amounted to over 50% of total immunoreactivity. These products were partially processed to N-acetyl betaendorphin(1-15) (30.8% of total immunoreactivity) and N-acetyl beta-endorphin(1-10) (3.1%) via two different cleavage pathways. The acetylated carp homologues of mammalian alpha- and gamma-endorphin were also found. N-acetyl beta-endorphin(1-15) and (1-29) and/or (1-33) were the major products to be released in vitro, and were the only acetylated beta-endorphins found in blood plasma, although never together. CRF stimulated the release of opioid beta-endorphin from the pars distalis. This non-acetylated beta-endorphin represents the full length peptide and is the most abundant form in plasma.

Evaluation of a truncated recombinant flagellin subunit vaccine against Campylobacter jejuni.

A recombinant protein comprising the maltose-binding protein (MBP) of Escherichia coli fused to amino acids 5 to 337 of the FlaA flagellin of Campylobacter coli VC167 was evaluated for immunogenicity and protective efficacy against challenge by a heterologous strain of campylobacter, Campylobacter jejuni 81-176, in two murine models. The sequence of the flaA gene of strain 81-176 revealed a predicted protein which was 98.1% similar to that of VC167 FlaA over the region expressed in the fusion protein. Mice were immunized intranasally with two doses of 3 to 50 microgram of MBP-FlaA, given 8 days apart, with or without 5 microgram of the mutant E. coli heat-labile enterotoxin (LT(R192G)) as a mucosal adjuvant. The full range of MBP-FlaA doses were effective in eliciting antigen-specific serum immunoglobulin G (IgG) responses, and these responses were enhanced by adjuvant use, except in the highest dosing group. Stimulation of FlaA-specific intestinal secretory IgA (sIgA) responses required immunization with higher doses of MBP-FlaA (>/=25 microgram) or coadministration of lower doses with the adjuvant. When vaccinated mice were challenged intranasally 26 days after immunization, the best protection was seen in animals given 50 microgram of MBP-FlaA plus LT(R192G). The protective efficacies of this dose against disease symptoms and intestinal colonization were 81.1 and 84%, respectively. When mice which had been immunized with 50 microgram of MBP-FlaA plus LT(R192G) intranasally were challenged orally with 8 x 10(10), 8 x 10(9), or 8 x 10(8) cells of strain 81-176, the protective efficacies against intestinal colonization at 7 days postinfection were 71.4, 71.4, and 100%, respectively.