French Biotech Start-Ups and Biotech Clusters in France. The Importance of Geographic Proximity

Based on a survey of the French Biotech SMEs (see annex 1), this article examines localisation effects in the biotechnology sector. It consists of two strands of analysis. The first presents a detailed statistical survey of the French biotechnology sector. Among other things, the survey shows that a) localisation effects within France are strong, and b) French firms can be grouped into four general types of firm, ranging from ‘type 1' growth-oriented product firms, to ‘type 2' niche market players, ‘type 3' subsidiaries of larger firms, and ‘type 4' firms that have been acquired. Localisation effects differ across these firms, especially across type 1 (international) and type 2 (very localised) firms. The second strand of analysis consists of a review of the localisation and related cluster literature, with implications identified for localisation and knowledge spillovers within biotech clusters. The relative effects of the proximity of scientific centres compared to public policy on start-ups is examined.

Calcium dynamics and compartmentalization in leech neurons

The aim of this project was to study how and where action potentials arise and propagate in the arborizations of identified neurons in the central nervous system of the leech. A major aim was to assess whether the entry of calcium is localized to distinct regions of the cells and to determine whether there are significant differences in calcium channel distribution between different types of neurons. A combination of electrophysiological techniques, optical recording and image analysis was used to approach these problems. I developed an experimental set-up for optical recordings of calcium transients by a fast CCD-camera. By use of calcium sensitive dyes I analysed in detail optical responses to electrical stimulation of neurons and the density of calcium channels, spatially and temporarily, in different neural cell types, including mechanosensory neurons and motoneurons. Fluorescence changes ( 06F/F) of the membrane impermeable calcium indicator Oregon Green were measured. The dye was pressure injected into the soma of neurons under investigation. 06F/F caused by a single action potential (AP) in mechanosensory neurons had approximately the same amplitude and time course in the soma and in distal processes. By contrast, in other neurons such as the Anterior Pagoda neuron, the Annulus Erector motoneuron, the L motoneuron and other motoneurons, APs evoked by passing depolarizing current in the soma produced much larger fluorescence changes in distal processes than in the soma. When APs were evoked by stimulating one distal axon through the root, 06F/F was large in all distal processes, but very small in the soma. These results confirm and extend previous electrophysiological data which demonstrate that the soma of a motoneuron in the leech, as in many other invertebrates, does not generate action potentials (Stuart, 1970; Muller and Nicholls, 2 1974; Goodman and Heitler, 1979). Impulses recorded in the soma are normally only a few millivolts in amplitude. The AP of a motoneuron propagates to muscles of the body wall along segmental nerves that emerge from ganglia. The site of impulse initiation has been found to be at a distance from the soma but within the ganglion (Melinek and Muller, 1996; Gu et al. 1991). Our experiments with fluorescent transients are in accord with the concept that they result from calcium entry through voltage sensitive channels. Thus at sites where APs are found to be large, the calcium signals are large (as in peripheral axons), while at sites where spikes are small, (as in motoneuronal cell bodies) signals were weak, or non existent

The Dynamics of Group Formation Among Leeches

Leeches exploring a new environment continuously meet each other and merge in temporary groups. After 2–3 h, leeches become attracted to each other eventually forming a large and stable group. When their number is reduced, leeches remain solitary, behaving independently. Group formation is facilitated by body injection of serotonin (5-HT) and the level of endogenous 5-HT is elevated in leeches forming a large group. In contrast, intravenous injection of 5-HT antagonists prevented injected leeches from joining a large group of conspecifics. When sensilla near the head were ablated or the supraesophageal ganglion disconnected, leeches remained solitary, but explored the environment swimming and crawling. These results suggest that group formation is initiated by a release of 5-HT triggered by sensilla stimulation and its dynamics can be explained by the establishment of a reinforcement dynamics, as observed during human group formation. As 5-HT affects social interactions also in humans, group formation in leeches and humans share a similar dynamics and hormonal control

The Role of Rac1 in the Growth Cone Dynamics and Force Generation of DRG Neurons

We used optical tweezers, video imaging, immunocytochemistry and a variety of inhibitors to analyze the role of Rac1 in the motility and force generation of lamellipodia and filopodia from developing growth cones of isolated Dorsal Root Ganglia neurons. When the activity of Rac1 was inhibited by the drug EHop-016, the period of lamellipodia protrusion/retraction cycles increased and the lamellipodia retrograde flow rate decreased; moreover, the axial force exerted by lamellipodia was reduced dramatically. Inhibition of Arp2/3 by a moderate amount of the drug CK-548 caused a transient retraction of lamellipodia followed by a complete recovery of their usual motility. This recovery was abolished by the concomitant inhibition of Rac1. The filopodia length increased upon inhibition of both Rac1 and Arp2/3, but the speed of filopodia protrusion increased when Rac1 was inhibited and decreased instead when Arp2/3 was inhibited. These results suggest that Rac1 acts as a switch that activates upon inhibition of Arp2/3. Rac1 also controls the filopodia dynamics necessary to explore the environment

Spontaneous Electrical Activity and Behavior in the Leech Hirudo Medicinalis

In the absence of external stimuli, animals explore the environment by performing irregular movements, but the neuronal mechanisms underlying this arrhythmic motion are largely unknown. In this paper, we studied the relationship between the spontaneous neuronal activity in the leech (Hirudo medicinalis) and its behavior. We analyzed the electrical activity of isolated ganglia, chains of two connected ganglia, and semi-intact preparations. The spontaneous electrical activity in ganglia was characterized by the occurrence of irregular bursts of spikes with variable duration and size. Properties of these bursts were modified by synaptic inputs arriving from the neighboring ganglia and from the two primitive brains located in the head and tail. In fact, in semi-intact preparations, unusually large bursts of spikes occurring spontaneously were recorded and caused the leech to move even in the absence of any external sensory stimulation. These large bursts appear to act as internal triggers controlling the spontaneous leech behavior and determining the duration of stereotypical motor patterns

Characterization of Embryonic Stem (ES) Neuronal Differentiation Combining Atomic Force, Confocal and DIC Microscopy Imaging

The ultimate clinical implementation of embryonic stem cells will require methods and protocols to turn these unspecialized cells into the fully functioning cell types found in a wide variety of tissues and organs. In order to achieve this, it is necessary to clearly understand the signals and cues that direct embryonic stem cell differentiation. This book provides a snapshot of current research on the differentiation of embryonic stem cells to a wide variety of cell types, including neural, cardiac, endothelial, osteogenic, and hepatic cells. In addition, induced pluripotent stem cells and other pluripotent stem cell sources are described. The book will serve as a valuable resource for engineers, scientists, and clinicians as well as students in a wide range of disciplines

Inaugural Editorial

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Reflections on STEMedicine and path forward to 2021

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Structural Heterogeneity of CNGA1 Channels Revealed by Electrophysiology and Single-Molecule Force Spectroscopy

The determination at atomic resolution of the three-dimensional molecular structure of membrane proteins such as receptors and several ion channels has been a major breakthrough in structural biology. The molecular structure of several members of the superfamily of voltage-gated ionic channels such as K+ and Na+ is now available. However, despite several attempts, the molecular structure at atomic resolution of the full cyclic nucleotide-gated (CNG) ion channel, although a member of the same superfamily of voltage-gated ion channels, has not been obtained yet, neither by X-ray crystallography nor by electron cryomicroscopy (cryo-EM). It is possible that CNG channels have a high structural heterogeneity, making difficult crystallization and single-particle analysis. To address this issue, we have combined single-molecule force spectroscopy (SMFS) and electrophysiological experiments to characterize the structural heterogeneity of CNGA1 channels expressed in Xenopus laevis oocytes. The unfolding of the cytoplasmic domain had force peaks, occurring with a probability from 0.2 to 0.96. Force peaks during the unfolding of the transmembrane domain had a probability close to 1, but the distribution of the increase in contour length between two successive force peaks had multiple maxima differing by tens of nanometers. Concomitant electrophysiological experiments showed that the rundown in mutant channels S399C is highly variable and that the effect of thiol reagents when specific residues were mutated was consistent with a dynamic structural heterogeneity. These results show that CNGA1 channels have a wide spectrum of native conformations that are difficult to detect with X-ray crystallography and cryo-EM

New views on phototransduction from atomic force microscopy and single molecule force spectroscopy on native rods

By combining atomic force microscopy (AFM) imaging and single-molecule force spectroscopy (SMFS), we analyzed membrane proteins of the rod outer segments (OS). With this combined approach we were able to study the membrane proteins in their natural environment. In the plasma membrane we identified native cyclic nucleotide-gated (CNG) channels which are organized in single file strings. We also identified rhodopsin located both in the discs and in the plasma membrane. SMFS reveals strikingly different mechanical properties of rhodopsin unfolding in the two environments. Molecular dynamic simulations suggest that this difference is likely to be related to the higher hydrophobicity of the plasma membrane, due to the higher cholesterol concentration. This increases rhodopsin mechanical stability lowering the rate of transition towards its active form, hindering, in this manner, phototransduction