A microprobe for parallel optical and electrical recordings from single neurons in vivo

Recording electrical activity from identified neurons in intact tissue is key to understanding their role in information processing. Recent fluorescence labeling techniques have opened new possibilities to combine electrophysiological recording with optical detection of individual neurons deep in brain tissue. For this purpose we developed dual-core fiberoptics-based microprobes, with an optical core to locally excite and collect fluorescence, and an electrolyte-filled hollow core for extracellular single unit electrophysiology. This design provides microprobes with tips < 10 μm, enabling analyses with single-cell optical resolution. We demonstrate combined electrical and optical detection of single fluorescent neurons in rats and mice. We combined electrical recordings and optical Ca2(+) measurements from single thalamic relay neurons in rats, and achieved detection and activation of single channelrhodopsin-expressing neurons in Thy1::ChR2-YFP transgenic mice. The microprobe expands possibilities for in vivo electrophysiological recording, providing parallel access to single-cell optical monitoring and control.     

Online nanoflow multidimensional fractionation for high efficiency phosphopeptide analysis

Despite intense, continued interest in global analyses of signaling cascades through mass spectrometry-based studies, the large-scale, systematic production of phosphoproteomics data has been hampered in-part by inefficient fractionation strategies subsequent to phosphopeptide enrichment. Here we explore two novel multidimensional fractionation strategies for analysis of phosphopeptides. In the first technique we utilize aliphatic ion pairing agents to improve retention of phosphopeptides at high pH in the first dimension of a two-dimensional RP-RP. The second approach is based on the addition of strong anion exchange as the second dimension in a three-dimensional reversed phase (RP)-strong anion exchange (SAX)-RP configuration. Both techniques provide for automated, online data acquisition, with the 3-D platform providing the highest performance both in terms of separation peak capacity and the number of unique phosphopeptide sequences identified per μg of cell lysate consumed. Our integrated RP-SAX-RP platform provides several analytical figures of merit, including: (1) orthogonal separation mechanisms in each dimension; (2) high separation peak capacity (3) efficient retention of singly- and multiply-phosphorylated peptides; (4) compatibility with automated, online LC-MS analysis. We demonstrate the reproducibility of RP-SAX-RP and apply it to the analysis of phosphopeptides derived from multiple biological contexts, including an in vitro model of acute myeloid leukemia in addition to primary polyclonal CD8(+) T-cells activated in vivo through bacterial infection and then purified from a single mouse.     

New perspectives in hemiascomycetous yeast taxonomy

DNA sequencing has revolutionized yeast taxonomy. Although initially rDNA sequences proved to be universal and convenient for assigning phylogenetic relationships, it was eventually supplanted by multigene analysis, which provided more discriminating and robust results. This led to a new classification of the major yeast clades, which is still used as a reference today. More recently, the availability of a large number of complete genome sequences has given a new perspective on the molecular taxonomy of yeasts by providing a high number of genes to compare. It also highlighted an unexpected aspect of yeast genome evolution: the existence of interspecific hybrids outside of the industrial Saccharomyces clade. Together with the loss of heterozygosity in interspecific hybrids and a reduced sexuality leading to clonal propagation, this observation obliges us to reexamine the present concept of species. In parallel, the ongoing challenge is to find a universal molecular marker, to improve fast authentication and, if possible, phylogeny of yeasts. The future of yeast taxonomy will involve the sequencing of more genomes, thorough analysis of populations to obtain a good representation of the biodiversity and integration of these data into dedicated databases.     

Diet of the nominate Southern Grey Shrike Lanius meridionalis meridionalis in the north of its range (Mediterranean France)

Capsule In this region the diet is mainly cold-blooded prey, mostly insects such as beetles.

Aims To describe the diet of this newly separated, poorly documented and endangered species.

Method Diet was inferred from pellet analysis, collected during a single breeding and winter period in the steppe of the Crau.

Results A total of 5409 prey were identified from 257 pellets. Vertebrates were seldom taken, except by adults (small passerines) during the fledgling period. High seasonal differences were found. Hymenoptera were largely consumed in autumn, Arachnida in autumn and winter, Orthoptera in summer and autumn and Lepidoptera larvae in winter and spring and by fledglings. Nevertheless, Coleoptera were ingested in large proportions all year round. Carabidae were the main prey in winter and Melolonthidae were especially important for adults during the nestling period, as were Cetoniidae for the fledglings.

Conclusion Small mammals and small birds were less exploited in France and Spain (L. m. meridionalis) than in Israel (L. m. elegans or L. m. aucheri), whereas the opposite might be expected, following a north–south climatic gradient. Thus, the nominate subspecies L. m. meridionalis differed in diet from L. m. elegans or L. m. aucheri.     

Effects of Cysteine Proteases on the Structural and Mechanical Properties of Collagen Fibers

Excessive cathepsin K (catK)-mediated turnover of fibrillar type I and II collagens in bone and cartilage leads to osteoporosis and osteoarthritis. However, little is known about how catK degrades compact collagen macromolecules. The present study is aimed to explore the structural and mechanical consequences of collagen fiber degradation by catK. Mouse tail type I collagen fibers were incubated with either catK or non-collagenase cathepsins. Methods used include scanning electron microscopy, protein electrophoresis, atomic force microscopy, and tensile strength testing. Our study revealed evidence of proteoglycan network degradation, followed by the progressive disassembly of macroscopic collagen fibers into primary structural elements by catK. Proteolytically released GAGs are involved in the generation of collagenolytically active catK-GAG complexes as shown by AFM. In addition to their structural disintegration, a decrease in the tensile properties of fibers was observed due to the action of catK. The Young''s moduli of untreated collagen fibers versus catK-treated fibers in dehydrated conditions were 3.2 ± 0.68 GPa and 1.9 ± 0.65 GPa, respectively. In contrast, cathepsin L, V, B, and S revealed no collagenase activity, except the disruption of proteoglycan-GAG interfibrillar bridges, which slightly decreased the tensile strength of fibers.     

Mechanotransduction down to individual actin filaments

The actin cytoskeleton plays an essential role in a cell's ability to generate and sense forces, both internally and in interaction with the outside world. The transduction of mechanical cues into biochemical reactions in cells, in particular, is a multi-scale process which requires a variety of approaches to be understood. This review focuses on understanding how mechanical stress applied to an actin filament can affect its assembly dynamics. Today, experiments addressing this issue at the scale of individual actin filaments are emerging and bring novel insight into mechanotransduction. For instance, recent data show that actin filaments can act as mechanosensors, as an applied tension or curvature alters their conformation and their affinity for regulatory proteins. Filaments can also transmit mechanical tension to other proteins, which consequently change the way they interact with the filaments to regulate their assembly. These results provide evidence for mechanotransduction at the scale of individual filaments, showing that forces participate in the regulation of filament assembly and organization. They bring insight into the elementary events coupling mechanics and biochemistry in cells. The experiments presented here are linked to recent technical developments, and certainly announce the advent of more exciting results in the future.     

Dissociation of the vacuolar and macroautophagic cytopathology from the cytotoxicity induced by the lipophilic local anesthetic bupivacaine

Local anesthetics, like many other cationic drugs, induce a vacuolar and macroautophagic cytopathology that has been observed in vivo and in various cell types; some also induce cytotoxicity of mitochondrial origin (apoptosis and necrosis) and it is not known whether the 2 types of toxicity overlap or interact. We compared bupivacaine with a more hydrophilic agent, lidocaine, for morphological, functional, and toxicological responses in a previously exploited nonneuronal system, primary smooth muscle cells. Bupivacaine induced little vacuolization (≥2.5?mmol/L, 4?h), but elicited autophagic accumulation (≥0.5?mmol/L, 4?h) and was massively cytotoxic at 2.5-5?mmol/L (4-24?h), the latter effect being unabated by the V-ATPase inhibitor bafilomycin A1. Lidocaine exerted little cytotoxicity at and below 5?mmol/L for 24?h, but intensely induced the V-ATPase-dependent vacuolar and autophagic cytopathology. Bupivacaine was more potent than lidocaine in disrupting mitochondrial potential, as judged by Mitotracker staining (significant proportions of cells affected in the 1-5 and 5-10?mmol/L concentration ranges, respectively). The addition of mitochondrial-inactivating toxins antimycin A and oligomycin to lidocaine (2.5?mmol/L) reproduced the profile of bupivacaine action (low intensity of vacuolization and retained autophagic accumulation). The high potency of bupivacaine as a mitochondrial toxicant eclipses the benign vacuolar and autophagic response seen with more hydrophilic local anesthetics.