A New method for the capture of surface proteins in <i>Plasmodium falciparum</i> parasitized erythrocyte

Introduction: We propose a new method for the selective labeling, isolation and electrophoretic analysis of the Plasmodium falciparum protein exposed on the erythrocyte cell surface. Historically, membrane surface proteins have been isolated using a surface biotinylation followed by capture of biotin-conjugated protein via an avidin/streptavidin-coated solid support. The major drawback of the standard methods has been the labeling of internal proteins due to fast internalization of biotin. Methodology: To solve this problem, we used a biotin label that does not permeate through the membrane. As a further precaution to avoid the purification of non surface exposed proteins, we directly challenged whole labeled cells with avidin coated beads and then solubilized them using non ionic detergents. Results: A marked enrichment of most of the RBC membrane proteins known to face the external surface of the membrane validated the specificity of the method; furthermore, only small amounts of haemoglobin and cytoskeletal proteins were detected. A wide range of P. falciparum proteins were additionally described to be exposed on the erythrocyte surface. Some of them have been previously observed and used as vaccine candidates while a number of newly described antigens have been presently identified. Those antigens require further characterization and validation with additional methods. Conclusion: Surface proteins preparations were very reproducible and identification of proteins by mass spectrometry has been demonstrated to be feasible and effective.</br

Bessel beam illumination reduces random and systematic errors in quantitative functional studies using light-sheet microscopy

Light-sheet microscopy (LSM), in combination with intrinsically transparent zebrafish larvae, is a choice method to observe brain function with high frame rates at cellular resolution. Inherently to LSM, however, residual opaque objects cause stripe artifacts, which obscure features of interest and, during functional imaging, modulate fluorescence variations related to neuronal activity. Here, we report how Bessel beams reduce streaking artifacts and produce high-fidelity quantitative data demonstrating a fivefold increase in sensitivity to calcium transients and a 20 fold increase in accuracy in the detection of activity correlations in functional imaging. Furthermore, using principal component analysis, we show that measurements obtained with Bessel beams are clean enough to reveal in one-shot experiments correlations that can not be averaged over trials after stimuli as is the case when studying spontaneous activity. Our results not only demonstrate the contamination of data by systematic and random errors through conventional Gaussian illumination and but,furthermore, quantify the increase in fidelity of such data when using Bessel beams

Planetary formation in the Gamma-Cephei system

We numerically investigate under which conditions the planet detected at 2.1 AU of Gamma-Cephei could form through the core-accretion scenario despite the perturbing presence of the highly eccentric companion star. We first show that the initial stage of runaway accretion of kilometer-sized planetesimals is possible within 2.5 AU from the central star only if large amounts of gas are present. In this case, gaseous friction induces periastron alignment of the orbits which reduces the otherwise high mutual impact velocities due to the companion's secular perturbations. The following stage of mutual accretion of large embryos is also modeled. According to our simulations, the giant impacts among the embryos always lead to a core of 10 Mearth within 10 Myr, the average lifetime of gaseous discs. However, the core always ends up within 1.5 AU from the central star. Either the core grows more quickly in the inner region of the disc, or it migrates inside by scattering the residual embryosComment: 8 pages, 12 figures to appear in Astronomy and Astrophysics (accepted 08/06/2004

The ménage à trois of healthcare: the actors in after-AI era under patient consent

Introduction: Artificial intelligence has become an increasingly powerful technological instrument in recent years, revolutionizing many sectors, including public health. Its use in this field will inevitably change clinical practice, the patient-caregiver relationship and the concept of the diagnosis and treatment pathway, affecting the balance between the patient’s right to self-determination and health, and thus leading to an evolution of the concept of informed consent. The aim was to characterize the guidelines for the use of artificial intelligence, its areas of application and the relevant legislation, to propose guiding principles for the design of optimal informed consent for its use. Materials and methods: A classic review by keywords on the main search engines was conducted. An analysis of the guidelines and regulations issued by scientific authorities and legal bodies on the use of artificial intelligence in public health was carried out. Results: The current areas of application of this technology were highlighted, divided into sectors, its impact on them, as well as a summary of current guidelines and legislation. Discussion: The ethical implications of artificial intelligence in the health care system were assessed, particularly regarding the therapeutic alliance between doctor and patient, and the balance between the right to self-determination and health. Finally, given the evolution of informed consent in relation to the use of this new technology, seven guiding principles were proposed to guarantee the right to the most informed consent or dissent

Whole-brain functional imaging to highlight differences between the diurnal and nocturnal neuronal activity in zebrafish larvae

Most living organisms show highly conserved physiological changes following a 24-hour cycle which goes by the name of circadian rhythm. Among experimental models, the effects of light-dark cycle have been recently investigated in the larval zebrafish. Owing to its small size and transparency, this vertebrate enables optical access to the entire brain. Indeed, the combination of this organism with light-sheet imaging grants high spatio-temporal resolution volumetric recording of neuronal activity. This imaging technique, in its multiphoton variant, allows functional investigations without unwanted visual stimulation. Here, we employed a custom two-photon light-sheet microscope to study whole-brain differences in neuronal activity between diurnal and nocturnal periods in larval zebrafish. We describe for the first time an activity increase in the low frequency domain of the pretectum and a frequency-localised activity decrease of the anterior rhombencephalic turning region during the nocturnal period. Moreover, our data confirm a nocturnal reduction in habenular activity. Furthermore, whole-brain detrended fluctuation analysis revealed a nocturnal decrease in the self-affinity of the neuronal signals in parts of the dorsal thalamus and the medulla oblongata. Our data show that whole-brain nonlinear light-sheet imaging represents a useful tool to investigate circadian rhythm effects on neuronal activity.Comment: 18 pages, 6 figure

Inhibition of an erythrocyte tyrosine kinase with imatinib prevents <i>Plasmodium falciparum</i> egress and terminates parasitemia

With half of the world’s population at risk for malaria infection and with drug resistance on the rise, the search for mutation-resistant therapies has intensified. We report here a therapy for Plasmodium falciparum malaria that acts by inhibiting the phosphorylation of erythrocyte membrane band 3 by an erythrocyte tyrosine kinase. Because tyrosine phosphorylation of band 3 causes a destabilization of the erythrocyte membrane required for parasite egress, inhibition of the erythrocyte tyrosine kinase leads to parasite entrapment and termination of the infection. Moreover, because one of the kinase inhibitors to demonstrate antimalarial activity is imatinib, i.e. an FDA-approved drug authorized for use in children, translation of the therapy into the clinic will be facilitated. At a time when drug resistant strains of P. falciparum are emerging, a strategy that targets a host enzyme that cannot be mutated by the parasite should constitute a therapeutic mechanism that will retard evolution of resistance

Inhibition of an erythrocyte tyrosine kinase with imatinib prevents Plasmodium falciparum egress and terminates parasitemia

With half of the world's population at risk for malaria infection and with drug resistance on the rise, the search for mutation-resistant therapies has intensified. We report here a therapy for Plasmodium falciparum malaria that acts by inhibiting the phosphorylation of erythrocyte membrane band 3 by an erythrocyte tyrosine kinase. Because tyrosine phosphorylation of band 3 causes a destabilization of the erythrocyte membrane required for parasite egress, inhibition of the erythrocyte tyrosine kinase leads to parasite entrapment and termination of the infection. Moreover, because one of the kinase inhibitors to demonstrate antimalarial activity is imatinib, i.e. an FDA-approved drug authorized for use in children, translation of the therapy into the clinic will be facilitated. At a time when drug resistant strains of P. falciparum are emerging, a strategy that targets a host enzyme that cannot be mutated by the parasite should constitute a therapeutic mechanism that will retard evolution of resistance

Effects of excitation light polarization on fluorescence emission in two-photon light-sheet microscopy

Light-sheet microscopy (LSM) is a powerful imaging technique that uses a planar illumination oriented orthogonally to the detection axis. Two-photon (2P) LSM is a variant of LSM that exploits the 2P absorption effect for sample excitation. The light polarization state plays a significant, and often overlooked, role in 2P absorption processes. The scope of this work is to test whether using different polarization states for excitation light can affect the detected signal levels in 2P LSM imaging of typical biological samples with a spatially unordered dye population. Supported by a theoretical model, we compared the fluorescence signals obtained using different polarization states with various fluorophores (fluorescein, EGFP and GCaMP6s) and different samples (liquid solution and fixed or living zebrafish larvae). In all conditions, in agreement with our theoretical expectations, linear polarization oriented parallel to the detection plane provided the largest signal levels, while perpendicularly-oriented polarization gave low fluorescence signal with the biological samples, but a large signal for the fluorescein solution. Finally, circular polarization generally provided lower signal levels. These results highlight the importance of controlling the light polarization state in 2P LSM of biological samples. Furthermore, this characterization represents a useful guide to choose the best light polarization state when maximization of signal levels is needed, e.g. in high-speed 2P LSM.Comment: 16 pages, 4 figures. Version of the manuscript accepted for publication on Biomedical Optics Expres