Life & Health Sciences

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Behavior, sensory reception and genomics in a vector insect


ABSTRACT

Insects vectoring human disease, like mosquitoes and kissing-bugs, endure a high risk of predation related to their life histories. Therefore, insect vectors are expected to have a finely adapted behavioral repertoire to survive in the context of their close association to vertebrate hosts. The study of molecular bases of their perception of the environment and their behavior, is relevant to understand the evolution of hematophagy as well as to promote the discovery of new targets of opportunity for developing rational control methods. Our long-lasting scientific collaboration has been dedicated to these tasks and the support of the Le Studium Foundation has been instrumental to further promote its development. We report here a series of studies that have been completed during the stay in the region Centre. Kissing-bugs are nocturnal insects that spend daylight hours hidden inside narrow shelters. Therefore, comprehending shelter choice, as well as the cues that trigger foraging decisions seems essential in order to predict bug distribution and activity precisely. We have focussed on Rhodnius prolixus, one of the two main vectors of Chagas disease in the Americas, and the way they use shelters. As an outcome of these investigations, three scientific papers have been published to report factors affecting shelter choice by bugs, the lack of a chemical marking system in bugs of this genus and the key role that host odours play in promoting bug foraging outside shelters. In parallel, we have described what seem to be diverging locomotory profiles in these bugs suggesting that foraging strategies can vary from “sitter” to “rover” individuals. As a third topic, we have developed a synthetic blend of compounds imitating the effects of the sexual pheromone attracting males to R. prolixus females. In a fourth topic we have uncovered what appears to be a local modulatory system present in the antennae of insects including the synthesis of transcripts for neuropeptides, GPCRs and nuclear receptors. Finally, we have revised the molecular bases of sensory processes in triatomine bugs vectors of Chagas disease in a review publication.


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Recombinant Intrabodies as Molecular Tools and Potential Therapeutics for Amyotrophic Lateral Sclerosis


ABSTRACT

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that has no diagnostic marker, prognosis, nor an effective treatment. Numerous physiopathological mechanisms have been described for this disease, such as glutamatergic excitotoxicity, oxidative stress, and the accumulation of protein aggregates in cells of the central nervous system, in particular the aggregation of cytoplasmic TDP-43.Our aim was targeting the protein aggregates containing TDP-43 through fragments of antibodies synthesized by the cell, termed intrabodies. In order to determine the most relevant criteria to test the protective effects of the intrabodies, we searched for different toxicity markers associated with TDP-43aggregates. During the fellowship, the fellow participated of 2 publications of the host laboratory in this field. Besides, at the end of the fellowship, the host Scientist and the Le Studium fellow organized a conference about iPS cells, a powerful tool to model in vitro neurodegenerative diseases such as ALS. In addition, the fellow generated preliminary results showing that TDP-43 overexpression in HEK 293 cells does not affect mitochondrial respiration, but causes an increase in cytoplasmic calcium levels, while impairs the mitochondrial capacity to buffer the excessive cytoplasmic calcium. Moreover, preliminary patch clamp data showed alterations in spontaneous currents in primary hippocampal and motor neurons overexpressing TDP-43. If these results are further confirmed, calcium signaling and spontaneous currents could be used as parameters to measure the efficacy of anti-TDP-43 intrabodies.


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Targeting acetylcholine receptors to enhance immunity to infection


ABSTRACT

Group 2 innate lymphoid cell (ILC2s) responses drive type 2 immunity against helminths and are initiated by host alarmin release. Here we show that in addition to signature type 2 cytokines ILC2 also synthesise and release acetylcholine (ACh).  ILC2 ACh synthesis (defined by choline acetyltransferase (ChAT) expression) following Nb or Alternaria challenge revealed pronounced ACh synthesis in ILC2 when compared to other immune cell populations. In vivo alarmin cytokine challenges selectively induced this ILC2 ACh responses. Nippostrongylus brasiliensis infection of RORCreChATLoxP mice (which have a targeted disruption of the ILC2 ACh response) resulted in higher intestinal helminth burdens than in control mice.  This impaired control of infection associated with reduced ILC2 and CD4 IL-13 production. Adoptive transfer of  RORCreChATLoxP ILC2s into RAG2-/-IL-2rg-/- resulted in subsequent infection having a higher intestinal burden than in ChATloxp recipeints. These data identify ILC2-derived ACh as a novel axis required for optimal type 2 immunity.

 

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Impact of Antibiotics on the Proliferation and Differentiation of Human Adipose-Derived Mesenchymal Stem Cells


ABSTRACT

Adipose tissue is a promising source of mesenchymal stem cells. Their potential to differentiate and regenerate other types of tissues may be affected by several factors. This may be due to in vitro cell-culture conditions, especially the supplementation with antibiotics. The aim of our study was to evaluate the effects of a penicillin-streptomycin mixture (PS), amphotericin B (AmB), a complex of AmB with copper (II) ions (AmB-Cu2+) and various combinations of these antibiotics on the proliferation and differentiation of adipose-derived stem cells in vitro. Normal human adipose-derived stem cells (ADSC, Lonza) were routinely maintained in a Dulbecco’s Modified Eagle Medium (DMEM) that was either supplemented with selected antibiotics or without antibiotics. The ADSC that were used for the experiment were at the second passage. The effect of antibiotics on proliferation was analyzed using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) and sulforhodamine-B (SRB) tests. Differentiation was evaluated based on Alizarin Red staining, Oil Red O staining and determination of the expression of ADSC, osteoblast and adipocyte markers by real-time RT-qPCR. The obtained results indicate that the influence of antibiotics on adipose-derived stem cells depends on the duration of exposure and on the combination of applied compounds. We show that antibiotics alter the proliferation of cells and also promote natural osteogenesis, and adipogenesis, and that this effect is also noticeable in stimulated osteogenesis.


Dr Kathia Zaleta

Dr María-Cristina Del Rincon-Castro

Dr Barbara Reaves

Prof. Adrian Wolstenholme

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Size determination of microbubbles in optical microscopy: a best-case scenario


ABSTRACT

Microbubble-based ultrasound contrast agents are used in clinical settings to enhance backscattered ultrasound signals from blood during perfusion and blood flow measurements. The dynamics of microbubbles contained in ultrasound contrast agents are typically studied with a high-speed camera attached to a microscope. Such microbubbles, with resting diameters between 1 µm and 7 µm, are considered in optical focus if the bubble centers are in the focal plane of the objective lens. Nonetheless, when a three-dimensional object, a stack of infinitely thin two-dimensional layers, is imaged through a microscope, the image formed onto the charge coupled device element consists of contributions from all layers. If a bubble is larger than the depth of focus, the part of the bubble above the focal plane influences the image formation and therefore the bubble size measured. If a bubble is of a size in the order of the wavelengths of the light used, the system resolution and the segmentation method influence the bubble size measured. In this study, the projections of three dimensional microbubbles (hollow spheres) were computed with an ideal, weighted three-dimensional point spread function to find out under which circumstances the optical image formation leads to a significant deviation in measurement of the actual size. The artificial images were subjected to segmentation techniques for objectively comparing original microbubble sizes with measured microbubble sizes. Results showed that a systematic error was observed for objects in focus with radius ≤ 1.65µm. Also it was concluded that even though a three-dimensional object is in focus, there is discrepancy of up to 0.66% in size measurement. In addition, size measurement of an object for the same shift above and below focus could differ by up to 3.6%. Moreover, defocusing above 25% severely deviates size measurements while defocusing up to 90% could result in mean percentage error of up to 67.96.


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Harmonic response from microscopic antibubbles


ABSTRACT

An antibubble is a gas bubble containing a liquid droplet core. Both the droplet and the gas bubble are typically surrounded by stabilising shells. Owing to electrostatic forces exerted by these shells, core droplets of micrometer diameter do not readily coalesce with the surrounding liquid medium. Owing to the incompressibility of the liquid droplet core, antibubbles will oscillate asymmetrically, i.e., the radial excursion amplitude of the surface is greater during expansion than during contraction, when subjected to diagnostic ultrasound. Consequently, the harmonic content of the ultrasound signal radiated from antibubbles must be higher than that from identical bubbles without a liquid core. Whether the harmonic signal component generated by physical antibubbles is higher than the harmonic component of identical bubbles without a core has been studied here. We subjected prefabricated antibubbles and identical bubbles without core droplets to 1-MHz ultrasound and to a commercial ultrasound system, and recorded the spectra with a broadband transducer oriented perpendicularly to the transmitter. Normalised by the acoustic response from the medium, the antibubble signal shows stronger higher harmonics than the reference signal, and negligible fundamental response. In conclusion, antibubbles are suitable candidates for harmonic imaging. The generation of higher harmonics without fundamental has been attributed to asymmetric antibubble expansion.