Bone phenotype of P2X4 receptor knockout mice: implication of a P2X7 receptor mutation?

Transgenic and knockout animal models are widely used to investigate the role of receptors, signaling pathways, and other peptides and proteins. Varying results are often published on the same model from different groups, and much effort has been put into understanding the underlying causes of these sometimes conflicting results. Recently, it has been shown that a P2X4R knockout model carries a so-called passenger mutation in the P2X7R gene, potentially affecting the interpretation of results from studies using this animal model. We therefore report this case to raise awareness about the potential pitfalls using genetically modified animal models, especially within P2 receptor research. Although purinergic signaling has been recognized as an important contributor to the regulation of bone remodeling, the process that maintains the bone quality during life, little is known about the role of the P2X4 receptor (P2X4R) in regulation of bone remodeling in health and disease. To address this, we analyzed the bone phenotype of P2rx4tm1Rass (C57BL/6J) knockout mice and corresponding wildtype using microCT and biomechanical testing. Overall, we found that the P2X4R knockout mice displayed improved bone microstructure and stronger bones in an age- and gender-dependent manner. While cortical BMD, trabecular BMD, and bone volume were higher in the 6-month-old females and 3-month-old males, this was not the case for the 3-month-old females and the 6-month-old males. Bone strength was only affected in the females. Moreover, we found that P2X4R KO mice carried the P2X7 receptor 451P wildtype allele, whereas the wildtype mice carried the 451L mutant allele. In conclusion, this study suggests that P2X4R could play a role in bone remodeling, but more importantly, it underlines the potential pitfalls when using knockout models and highlights the importance of interpreting results with great caution. Further studies are needed to verify any specific effects of P2X4R on bone metabolism.

     

Toxicological Effects of the Different Substances in Tobacco Smoke on Human Embryonic Development by a Systems Chemo-Biology Approach

The physiological and molecular effects of tobacco smoke in adult humans and the development of cancer have been well described. In contrast, how tobacco smoke affects embryonic development remains poorly understood. Morphological studies of the fetuses of smoking pregnant women have shown various physical deformities induced by constant fetal exposure to tobacco components, especially nicotine. In addition, nicotine exposure decreases fetal body weight and bone/cartilage growth in addition to decreasing cranial diameter and tibia length. Unfortunately, the molecular pathways leading to these morphological anomalies are not completely understood. In this study, we applied interactome data mining tools and small compound interaction networks to elucidate possible molecular pathways associated with the effects of tobacco smoke components during embryonic development in pregnant female smokers. Our analysis showed a relationship between nicotine and 50 additional harmful substances involved in a variety of biological process that can cause abnormal proliferation, impaired cell differentiation, and increased oxidative stress. We also describe how nicotine can negatively affect retinoic acid signaling and cell differentiation through inhibition of retinoic acid receptors. In addition, nicotine causes a stress reaction and/or a pro-inflammatory response that inhibits the agonistic action of retinoic acid. Moreover, we show that the effect of cigarette smoke on the developing fetus could represent systemic and aggressive impacts in the short term, causing malformations during certain stages of development. Our work provides the first approach describing how different tobacco constituents affect a broad range of biological process in human embryonic development.     

Haemosporidian Parasites of Antelopes and Other Vertebrates from Gabon,Central Africa

Re-examination, using molecular tools, of the diversity of haemosporidian parasites (among which the agents of human malaria are the best known) has generally led to rearrangements of traditional classifications. In this study, we explored the diversity of haemosporidian parasites infecting vertebrate species (particularly mammals, birds and reptiles) living in the forests of Gabon (Central Africa), by analyzing a collection of 492 bushmeat samples. We found that samples from five mammalian species (four duiker and one pangolin species), one bird and one turtle species were infected by haemosporidian parasites. In duikers (from which most of the infected specimens were obtained), we demonstrated the existence of at least two distinct parasite lineages related to Polychromophilus species (i.e., bat haemosporidian parasites) and to sauropsid Plasmodium (from birds and lizards). Molecular screening of sylvatic mosquitoes captured during a longitudinal survey revealed the presence of these haemosporidian parasite lineages also in several Anopheles species, suggesting a potential role in their transmission. Our results show that, differently from what was previously thought, several independent clades of haemosporidian parasites (family Plasmodiidae) infect mammals and are transmitted by anopheline mosquitoes.     

Differential expression of VGLUT3 in laboratory mouse strains: Impact on drug‐induced hyperlocomotion and anxiety‐related behaviors

The atypical vesicular glutamate transporter VGLUT3 is present in subpopulations of GABAergic interneurons in the cortex and the hippocampus, in subgroups of serotoninergic neurons in raphe nuclei, and in cholinergic interneurons in the striatum. C56BL/6N mice that no longer express VGLUT3 (VGLUT3^?/?) display anxiety‐associated phenotype, increased spontaneous and cocaine‐induced locomotor activity and decreased haloperidol‐induced catalepsy. Inbred mouse strains differ markedly in their sensitivity to anxiety and behavioral responses elicited by drugs. The purpose of this study was to investigate strain differences in VGLUT3 expression levels and its potential correlates with anxiety and reward‐guided behaviors. Five inbred mouse lines were chosen according to their contrasted anxiety and drugs sensitivity: C57BL/6N, C3H/HeN, DBA/2J, 129/Sv, and BALB/c. VGLUT3 protein expression was measured in different brain areas involved in reward or mood regulation (such as the striatum, the hippocampus, and raphe nuclei) and genetic variations in Slc17a8, the gene encoding for VGLUT3, have been explored. These five inbred mouse strains express very different levels of VGLUT3, which cannot be attributed to the genetic variation of the Slc17a8 locus. Furthermore, mice behavior in the open field, elevated plus maze, spontaneous‐ and cocaine‐induced locomotor was highly heterogeneous and only partially correlated to VGLUT3 levels. These data highlight the fact that one single gene polymorphism could not account for VGLUT3 expression variations, and that region specific VGLUT3 expression level variations might play a key role in the modulation of discrete behaviors.     

Paediatric brain tissue properties measured with magnetic resonance elastography

Biomechanics and Modeling in Mechanobiology - The aim of this study is to characterise the stiffness of white and grey matter in paediatric subjects using magnetic resonance elastography (MRE) and...     

15N-metabolic labeling for comparative plasma membrane proteomics in Arabidopsis cells

An important goal for proteomic studies is the global comparison of proteomes from different genotypes, tissues, or physiological conditions. This has so far been mostly achieved by densitometric comparison of spot intensities after protein separation by 2-DE. However, the physicochemical properties of membrane proteins preclude the use of 2-DE. Here, we describe the use of in vivo labeling by the stable isotope 15N as an alternative approach for comparative membrane proteomic studies in plant cells. We confirm that 15N-metabolic labeling of proteins is possible and efficient in Arabidopsis suspension cells. Quantification of 14N versus 15N MS signals reflects the relative abundance of 14N and 15N proteins in the sample analyzed. We describe the use of 15N-metabolic labeling to perform a partial comparative analysis of Arabidopsis cells following cadmium exposure. By focusing our attention on plasma membrane proteins, we were able to confidently identify proteins showing up to 5-fold regulation compared to unexposed cells. This study provides a proof of principle that 15N-metabolic labeling is a useful technique for comparative membrane proteome studies.     

A high content in lipid-modified peripheral proteins and integral receptor kinases features in the arabidopsis plasma membrane proteome

The proteomics of plasma membrane has brought to date only scarce and partial information on the actual protein repertoire. In this work, the plant plasma membrane proteome of Arabidopsis thaliana was investigated. A highly purified plasma membrane fraction was washed by NaCl and Na2CO3 salts, and the insoluble fractions were further analyzed by nano-LC-MS/MS. With 446 proteins identified, we hereby describe the largest plasma membrane proteome diversity reported so far. Half of the proteins were predicted to display transmembrane domains and/or to be anchored to the membrane, validating a posteriori the pertinence of the approach. A fine analysis highlighted two main specific and novel features. First, the main functional category is represented by a majority of as yet unreported signaling proteins, including 11% receptor-like kinases. Second, 16% of the identified proteins are predicted to be lipid-modified, specifically involving double lipid linkage through N-terminal myristoylation, S-palmitoylation, C-terminal prenylation, or glycosylphosphatidylinositol anchors. Thus, our approach led for the first time to the identification of a large number of peripheral proteins as part of the plasma membrane and allowed the functionality of the plasma membrane in the cell context to be reconsidered.     

First efficient CRISPR‐Cas9‐mediated genome editing in Leishmania parasites

Protozoan pathogens that cause leishmaniasis in humans are relatively refractory to genetic manipulation. In this work, we implemented the CRISPR‐Cas9 system in Leishmania parasites and demonstrated its efficient use for genome editing. The Cas9 endonuclease was expressed under the control of the Dihydrofolate Reductase‐Thymidylate Synthase (DHFR‐TS) promoter and the single guide RNA was produced under the control of the U6snRNA promoter and terminator. As a proof of concept, we chose to knockout a tandemly repeated gene family, the paraflagellar rod‐2 locus. We were able to obtain null mutants in a single round of transfection. In addition, we confirmed the absence of off‐target editions by whole genome sequencing of two independent clones. Our work demonstrates that CRISPR‐Cas9‐mediated gene knockout represents a major improvement in comparison with existing methods. Beyond gene knockout, this genome editing tool opens avenues for a multitude of functional studies to speed up research on leishmaniasis.     

AIF-mediated caspase-independent necroptosis: a new chance for targeted therapeutics

Cell death has been initially divided into apoptosis, in which the cell plays an active role, and necrosis, which is considered a passive cell death program. Intense research performed in the last decades has concluded that "programmed" cell death (PCD) is a more complex physiological process than initially thought. Indeed, although in most cases the PCD process is achieved via a family of Cys proteases known as caspases, an important number of regulated PCD pathways do not involve this family of proteases. As a consequence, active forms of PCD are initially referred to as caspase-dependent and caspase-independent. More recent data has revealed that there are also active caspase-independent necrotic pathways defined as necroptosis (programmed necrosis). The existence of necroptotic forms of death was corroborated by the discovery of key executioners such as the kinase RIP1 or the mitochondrial protein apoptosis-inducing factor (AIF). AIF is a Janus protein with a redox activity in the mitochondria and a pro-apoptotic function in the nucleus. We have recently described a particular form of AIF-mediated caspase-independent necroptosis that also implicates other molecules such as PARP-1, calpains, Bax, Bcl-2, histone H2AX, and cyclophilin A. From a therapeutic point of view, the unraveling of this new form of PCD poses a question: is it possible to modulate this necroptotic pathway independently of the classical apoptotic paths? Because the answer is yes, a wider understanding of AIF-mediated necroptosis could theoretically pave the way for the development of new drugs that modulate PCD. To this end, we present here an overview of the current knowledge of AIF and AIF-mediated necroptosis. We also summarize the state of the art in some of the most interesting therapeutic strategies that could target AIF or the AIF-mediated necroptotic pathway.     

Melatonin as a central molecule connecting neural development and calcium signaling

Melatonin (MEL) is a neuroendocrine hormone secreted by the pineal gland in association with the suprachiasmatic nucleus and peripheral tissues. MEL has been observed to play a critical role in the reproductive process and in the fetomaternal interface. Extrapineal synthesis has been reported in mammalian models during pregnancy, especially by the placenta tissue. MEL can regulate intracellular processes (e.g., G-proteins) and the activity of second messengers (e.g., cAMP, IP_3, Ca²⁺). During neurodevelopment, these activities regulated by melatonin have an important role as an intracellular signaling for gene expression regulation. To review the role of MEL in neurodevelopment, we built interactome networks of different proteins that act in these processes using systems biology tools. The analyses of interactome networks revealed that MEL could modulate neurodevelopment through the regulation of Ca²⁺ intracellular levels and influencing BMP/SMAD signaling, thus affecting neural gene responses and neuronal differentiation.