Electrophysiological recording from Drosophila taste sensilla.

INTRODUCTION The chemical senses smell and taste, detect and discriminate an enormous diversity of environmental stimuli and provide fascinating but challenging models to investigate how sensory cues are represented in the brain. Important stimulus-coding events occur in peripheral sensory neurons, which express specific combinations of chemosensory receptors with defined ligand-response profiles. These receptors convert ligand recognition into spatial and temporal patterns of neural activity that are transmitted to and interpreted in central brain regions. Drosophila provides an attractive model to study chemosensory coding, because it possesses relatively simple peripheral olfactory and gustatory systems that display many organizational parallels to those of vertebrates. Moreover, virtually all of the peripheral chemosensory neurons are easily accessible for physiological analysis, as they are exposed on the surface of sensory organs in specialized sensory hairs called sensilla. In recent years, improvements in microscopy and instrumentation for electrode manipulation have opened up the much smaller Drosophila system to electrophysiological techniques, powerfully complementing many years of molecular genetic studies. As with most electrophysiological methods, there is probably no substitute for learning this technique directly from a laboratory in which it is already established. This protocol describes the basics of setting up the electrophysiology rig and stimulus delivery device, sample preparation, and performing and analyzing recordings of stimulus-evoked activity from Drosophila taste sensilla.

Characterization of the PHO1 gene family and the responses to phosphate deficiency of Physcomitrella patens.

PHO1 was previously identified in Arabidopsis (Arabidopsis thaliana) as a protein involved in loading inorganic phosphate (Pi) into the xylem of roots and its expression was associated with the vascular cylinder. Seven genes homologous to AtPHO1 (PpPHO1;1-PpPHO1;7) have been identified in the moss Physcomitrella patens. The corresponding proteins harbor an SPX tripartite domain in the N-terminal hydrophilic portion and an EXS domain in the conserved C-terminal hydrophobic portion, both common features of the plant PHO1 family. Northern-blot analysis showed distinct expression patterns for the PpPHO1 genes, both at the tissue level and in response to phosphate deficiency. Transgenic P. patens expressing the beta-glucuronidase reporter gene under three different PpPHO1 promoters revealed distinct expression profiles in various tissues. Expression of PpPHO1;1 and PpPHO1;7 was specifically induced by Pi starvation. P. patens homologs to the Arabidopsis PHT1, DGD2, SQD1, and APS1 genes also responded to Pi deficiency by increased mRNA levels. Morphological changes associated with Pi deficiency included elongation of caulonemata with inhibition of the formation of side branches, resulting in colonies with greater diameter, but reduced mass compared to Pi-sufficient plants. Under Pi-deficient conditions, P. patens also increased the synthesis of ribonucleases and of an acid phosphatase, and increased the ratio of sulfolipids over phospholipids. These results indicate that P. patens and higher plants share some common strategies to adapt to Pi deficiency, although morphological changes are distinct, and that the PHO1 proteins are well conserved in bryophyte despite the lack of a developed vascular system.

The Mont-Mort metapelites: Variscan metamorphism and geodynamic context (Brianconnais basement, Western Alps, Switzerland)

The Mont-Mort metapelites are one of the best preserved relies of the Variscan unit in the Brianconnais basement. These micaschists crystallized during a poly-phase metamorphic cycle, under amphibolite facies conditions. Mineral parageneses and geothermobarometric calculations indicate a two-stage evolution. Stage (1) (550-600 degrees C and 5-8 kbar) is documented by assemblages of zoned garnet, staurolite, kyanite(?), biotite, muscovite, quartz and pla gioclase. Stage (2) (550-600 degrees C and 2 kbar) is illustrated by assemblages of andalusite, sillimanite, muscovite, biotite. This metamorphic evolution is characterized by a nearly isothermal decompression path, terminating with the formation of andalusite-bearing veins. U-Pb monazite dates at 330 Ma and Ar-40/Ar-39 muscovite dates at 290-310 Ma (without substantial evidence of argon resetting) point to Variscan metamorphism and yield an estimate of the time interval between the thermal peak and the retrogression stage within this part of the Brianconnais basement. Restoring the Brianconnais and other Alpine basement units within an existing geodynamic model of Cordillera construction and destruction, it is possible to understand better the transition from a medium pressure/high temperature regime (collision with a peak metamorphism around 330 Ma) to low-P/high-T conditions (decompression in an extensional regime) with high geothermal gradient, as recorded by the successive Variscan parageneses within the Mont-Mort metapelites.

Simulations of action of DNA topoisomerases to investigate boundaries and shapes of spaces of knots.

The configuration space available to randomly cyclized polymers is divided into subspaces accessible to individual knot types. A phantom chain utilized in numerical simulations of polymers can explore all subspaces, whereas a real closed chain forming a figure-of-eight knot, for example, is confined to a subspace corresponding to this knot type only. One can conceptually compare the assembly of configuration spaces of various knot types to a complex foam where individual cells delimit the configuration space available to a given knot type. Neighboring cells in the foam harbor knots that can be converted into each other by just one intersegmental passage. Such a segment-segment passage occurring at the level of knotted configurations corresponds to a passage through the interface between neighboring cells in the foamy knot space. Using a DNA topoisomerase-inspired simulation approach we characterize here the effective interface area between neighboring knot spaces as well as the surface-to-volume ratio of individual knot spaces. These results provide a reference system required for better understanding mechanisms of action of various DNA topoisomerases.

Molecular analysis of sleep.

Rest or sleep in all animal species constitutes a period of quiescence necessary for recovery from activity. Whether rest and activity observed in all organisms share a similar fundamental molecular basis with sleep and wakefulness in mammals has not yet been established. In addition and in contrast to the circadian system, strong evidence that sleep is regulated at the transcriptional level is lacking. Nevertheless, several studies indicate that single genesmay regulate some specific aspects of sleep. Efforts to better understand or confirm the role of known neurotransmission pathways in sleep-wake regulation using transgenic approaches resulted so far in only limited new insights. Recent gene expression profiling efforts in rats, mice, and fruit flies are promising and suggest that only a few gene categories are differentially regulated by behavioral state. How molecular analysis can help us to understand sleep is the focus of this chapter.

Common Variable Immunodeficiency: molecular pathways and clinical manifestations

Common variable immunodeficiency (CVID), is a disease that is characterized by hypogammaglobulinemia as well as a defect in T, B and dendritic cells. This leads to recurrent bacterial infection mainly caused by Streptococcus pneumoniae, Klebsiella pneumoniae and Haemophilus influenzae, as well as inflammatory manifestations, i.e. granulomateous disease, gastro-intestinal disorders and chronic lung disease. Intravenous Immunoglobulin (IVIg) therapy reduces CVID susceptibility to bacterial infections to some extend. We analyzed clinical aspects of patients from our database. We recently showed that bacteria-specific CD4 T cells of CVID patients were impaired. We therefor postulated that CVID patients may harbor an acquired T-cell deficiency also called exhaustion. To test this hypothesis, we performed a comprehensive investigation of the functional profiles of bacteria-specific CD4 T cells isolated from 31 healthy individuals and 30 CVID patients. In the present study, we demonstrated that bacteria-specific but not virus-specific CD4 T cells in CVID patients harbored reduced proliferation capacity and expressed high level of PD-1. Interestingly, the blockade of PD-1/PD-1 ligands interactions restored partially bacteria but not virus-specific CD4 T-cell proliferation. Finally, we showed that 1) the level of endotoxins inversely correlates with IgG concentration, 2) IVIG treated CVID patients harbored reduced endotoxemia and 3) IgG concentration exceeding 7 mg/mL strongly reduces both the proportion of CVID patients with detectable endotoxemia and the concentration of endotoxins in plasma. Taken together our observations, suggest that primary B-cell defect(s) in CVID patients leads to recurrent bacterial infections that are associated to an acquired (secondary) impairment of CD4 T cells which may in turn exacerbate the lack of protection against extracellular bacteria.

Leukocyte-derived microparticles in vascular homeostasis.

Leukocyte-derived microparticles (LMPs) may originate from neutrophils, monocytes/macrophages, and lymphocytes. They express markers from their parental cells and harbor membrane and cytoplasmic proteins as well as bioactive lipids implicated in a variety of mechanisms, maintaining or disrupting vascular homeostasis. When they carry tissue factor or coagulation inhibitors, they participate in hemostasis and pathological thrombosis. Both proinflammatory and anti-inflammatory processes can be affected by LMPs, thus ensuring an appropriate inflammatory response. LMPs also play a dual role in the endothelium by either improving the endothelial function or inducing an endothelial dysfunction. LMPs are implicated in all stages of atherosclerosis. They circulate at a high level in the bloodstream of patients with high atherothrombotic risk, such as smokers, diabetics, and subjects with obstructive sleep apnea, where their prolonged contact with the vessel wall may contribute to its overall deterioration. Numbering microparticles, including LMPs, might be useful in predicting cardiovascular events. LMPs modify the endothelial function and promote the recruitment of inflammatory cells in the vascular wall, necessary processes for the progression of the atherosclerotic lesion. In addition, LMPs favor the neovascularization within the vulnerable plaque and, in the ruptured plaque, they take part in coagulation and platelet activation. Finally, LMPs participate in angiogenesis. They might represent a novel therapeutic tool to reset the angiogenic switch in pathologies with altered angiogenesis. Additional studies are needed to further investigate the role of LMPs in cardiovascular diseases. However, large-scale studies are currently difficult to set up because microparticle measurement still requires elaborate techniques which lack standardization.