Spatio-Temporal Characterization Of Causal Electrophysiological Activity Stimulated By Single Pulse Focused Ultrasound: An Ex Vivo Study On Hippocampal Brain Slices

International audienceObjective: The brain operates via generation, transmission and integration of neuronal signals and most neurological disorders are related to perturbation of these processes. Neurostimulation by Focused Ultrasound (FUS) is a promising technology with potential to rival other clinically-used techniques for the investigation of brain function and treatment of numerous neurological diseases. The purpose of this study was to characterize spatial and temporal aspects of causal electrophysiological signals directly stimulated by short, single pulses of focused ultrasound (FUS) on ex vivo mouse hippocampal brain slices. Approach: MicroElectrode Arrays (MEA) are used to study the spatio-temporal dynamics of extracellular neuronal activities both at the single neuron and neural networks scales. Hence, MEAs provide an excellent platform for characterization of electrical activity generated, modulated and transmitted in response to FUS exposure. In this study, a novel mixed FUS/MEA platform was designed for the spatio-temporal description of the causal responses generated by single 1.78 MHz FUS pulses in ex vivo mouse hippocampal brain slices. Main results: Our results show that FUS pulses can generate local field potentials (LFPs), sustained by synchronized neuronal post-synaptic potentials, and reproducing network activities. LFPs induced by FUS stimulation were found to be repeatable to consecutive FUS pulses though exhibiting a wide range of amplitudes (50-600 µV), durations (20-200 ms), and response delays (10-60 ms). Moreover, LFPs were spread across the hippocampal slice following single FUS pulses thus demonstrating that FUS may be capable of stimulating different neural structures within the hippocampus. Significance: Current knowledge on neurostimulation by ultrasound describes neuronal activity generated by trains of repetitive ultrasound pulses. This novel study details the causal neural responses produced by single-pulse FUS neurostimulation while illustrating the distribution and propagation properties of this neural activity along major neural pathways of the hippocampus

Using a vector pool containing variable-strength promoters to optimize protein production in Yarrowia lipolytica

Background The yeast Yarrowia lipolytica is an increasingly common biofactory. To enhance protein expression, several promoters have been developed, including the constitutive TEF promoter, the inducible POX2 promotor, and the hybrid hp4d promoter. Recently, new hp4d-inspired promoters have been created that couple various numbers of UAS1 tandem elements with the minimal LEU2 promoter or the TEF promoter. Three different protein-secretion signaling sequences can be used: preLip2, preXpr2, and preSuc2. Results To our knowledge, our study is the first to use a set of vectors with promoters of variable strength to produce proteins of industrial interest. We used the more conventional TEF and hp4d promoters along with five new hybrid promoters: 2UAS1-pTEF, 3UAS1-pTEF, 4UAS1-pTEF, 8UAS1-pTEF, and hp8d. We compared the production of RedStar2, glucoamylase, and xylanase C when strains were grown on three media. As expected, levels of RedStar2 and glucoamylase were greatest in the strain with the 8UAS1-pTEF promoter, which was stronger. However, surprisingly, the 2UAS1-pTEF promoter was associated with the greatest xylanase C production and activity. This finding underscored that stronger promoters are not always better when it comes to protein production. We therefore developed a method for easily identifying the best promoter for a given protein of interest. In this gateway method, genes for YFP and α-amylase were transferred into a pool of vectors containing different promoters and gene expression was then analyzed. We observed that, in most cases, protein production and activity were correlated with promoter strength, although this pattern was protein dependent. Conclusions Protein expression depends on more than just promoter strength. Indeed, promoter suitability appears to be protein dependent; in some cases, optimal expression and activity was obtained using a weaker promoter. We showed that using a vector pool containing promoters of variable strength can be a powerful tool for rapidly identifying the best producer for a given protein of interest

Secretome analysis of Trypanosoma cruzi by proteomics studies

Background: Chagas disease is a debilitating often fatal disease resulting from infection by the protozoan parasite Trypanosoma cruzi. Chagas disease is endemic in 21 countries of the Americas, and it is an emerging disease in other countries as a result of migration. Given the chronic nature of the infection where intracellular parasites persist for years, the diagnosis of T. cruzi by direct detection is difficult, whereas serologic tests though sensitive may yield false-positive results. The development of new rapid test based on the identification of soluble parasitic antigens in serum would be a real innovation in the diagnosis of Chagas disease. Methods: To identify new soluble biomarkers that may improve diagnostic tests, we investigated the proteins secreted by T. cruzi using mass spectrometric analyses of conditioned culture media devoid of serum collected during the emergence of trypomastigotes from infected Vero cells. In addition, we compared the secretomes of two T. cruzi strains from DTU Tc VI (VD and CL Brener). Results: Analysis of the secretome collected during the emergence of trypomastigotes from Vero cells led to the identification of 591 T. cruzi proteins. Three hundred sixty three proteins are common to both strains and most belong to different multigenic super families (i.e. TcS, GP63, MASP, and DGF1). Ultimately we have established a list of 94 secreted proteins, common to both DTU Tc VI strains that do not belong to members of multigene families. Conclusions: This study provides the first comparative analysis of the secretomes from two distinct T. cruzi strains of DTU TcVI. This led us to identify a subset of common secreted proteins that could potentially serve as serum markers for T. cruzi infection. Their potential could now be evaluated, with specific antibodies using sera collected from patients and residents from endemic regions.Fil: Brossas, Jean Yves. Inserm; Francia. Universite de Paris VI; Francia. Hôpital Pitié-Salpêtrière; FranciaFil: Gulin, Julián Ernesto Nicolás. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez"; Argentina. Gobierno de la Ciudad de Buenos Aires. Instituto Multidisciplinario de Investigaciones en Patologías Pediátricas. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto Multidisciplinario de Investigaciones en Patologías Pediátricas.; ArgentinaFil: Bisio, Margarita María Catalina. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez"; Argentina. Gobierno de la Ciudad de Buenos Aires. Instituto Multidisciplinario de Investigaciones en Patologías Pediátricas. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto Multidisciplinario de Investigaciones en Patologías Pediátricas.; ArgentinaFil: Chapelle, Manuel. Bruker Daltonics; FranciaFil: Marinach Patrice, Carine. Inserm; Francia. Universite de Paris VI; FranciaFil: Bordessoulles, Mallaury. Universite de Paris VI; Francia. Inserm; FranciaFil: Palazon Ruiz, George. Inserm; FranciaFil: Vion, Jeremy. Inserm; FranciaFil: Paris, Luc. Hôpital Pitié-Salpêtrière; Francia. Universite de Paris VI; FranciaFil: Altcheh, Jaime Marcelo. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez"; Argentina. Gobierno de la Ciudad de Buenos Aires. Instituto Multidisciplinario de Investigaciones en Patologías Pediátricas. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto Multidisciplinario de Investigaciones en Patologías Pediátricas.; ArgentinaFil: Mazier, Dominique. Inserm; Francia. Universite de Paris VI; Francia. Hôpital Pitié-Salpêtrière; Franci

Analyse of trans-sialidase (TcS) proteins found in secretome of 2 strains.

<p>Classification of TcS proteins for each strain into 8 group previously described [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0185504#pone.0185504.ref022" target="_blank">22</a>]. Groups IV, V and VI are less than I, II, III groups VII and VIII for two strains. On the x-axis, the number of group is indicated. The Y-axis shows the percentage of each TcS identified in our analyses.</p

List of secreted proteins that are common in both <i>T</i>.<i>cruzi</i> strains without of multigene proteins.

<p>List of secreted proteins that are common in both <i>T</i>.<i>cruzi</i> strains without of multigene proteins.</p

Functional categories classification of <i>T</i>. <i>cruzi</i> proteins from CL Brener and VD strains.

<p>Proteins were classified into 15 functional categories using literature and UniprotKB annotation. Y-axis, categories are indicated. X-axis shows the percentage of each category for each strain.</p

Overlap between secretomes of two different <i>T</i>. <i>cruzi</i> strains DTU Tc VI.

<p>A total of 591 proteins of <i>T</i>. <i>cruzi</i> were identified. We note that 78 proteins are specific to the CL Brener strain whereas 151 proteins are specific to VD strain. However, 363 proteins are common to both strains.</p