Localization and Relative Quantification of Carbon Nanotubes in Cells with Multispectral Imaging Flow Cytometry

Carbon-based nanomaterials, like carbon nanotubes (CNTs), belong to this type of nanoparticles which are very difficult to discriminate from carbon-rich cell structures and de facto there is still no quantitative method to assess their distribution at cell and tissue levels. What we propose here is an innovative method allowing the detection and quantification of CNTs in cells using a multispectral imaging flow cytometer (ImageStream, Amnis). This newly developed device integrates both a high-throughput of cells and high resolution imaging, providing thus images for each cell directly in flow and therefore statistically relevant image analysis. Each cell image is acquired on bright-field (BF), dark-field (DF), and fluorescent channels, giving access respectively to the level and the distribution of light absorption, light scattered and fluorescence for each cell. The analysis consists then in a pixel-by-pixel comparison of each image, of the 7,000-10,000 cells acquired for each condition of the experiment. Localization and quantification of CNTs is made possible thanks to some particular intrinsic properties of CNTs: strong light absorbance and scattering; indeed CNTs appear as strongly absorbed dark spots on BF and bright spots on DF with a precise colocalization.This methodology could have a considerable impact on studies about interactions between nanomaterials and cells given that this protocol is applicable for a large range of nanomaterials, insofar as they are capable of absorbing (and/or scattering) strongly enough the light.     

A second-generation diagnostic single nucleotide polymorphism (SNP)-based assay, optimized to distinguish among eight poplar (Populus L.) species and their early hybrids

Rapid identification of Populus L. species and hybrids can be achieved with relatively little effort through the use of primer extension-based single nucleotide polymorphism (SNP) genotyping assays. We present an optimized set of 36 SNP markers from 28 gene regions that diagnose eight poplar species (Populus angustifolia James, Populus balsamifera L., Populus deltoides Bartram, Populus fremontii Watson, Populus laurifolia Ledeb., Populus maximowiczii Henry, Populus nigra L., and Populus trichocarpa Torr. & Gray). A total of 700 DNA sequences from six Populus species (1–15 individuals per species) were used to construct the array. A set of flanking and probe oligonucleotides was developed and tested. The accuracy of the SNP assay was validated by genotyping 448 putatively “pure” individuals from 14 species of Populus. Overall, the SNP assay had a high success rate (97.6 %) and will prove useful for the identification of all Aigeiros Duby and Tacamahaca Spach. species and their early-generation hybrids within natural populations and breeding programs. Null alleles and intraspecific polymorphisms were detected for a few locus/species combinations in the Aigeiros and Tacamahaca sections. When we attempted to genotype aspens of the section Populus (Populus alba L., Populus grandidentata Michx., Populus tremula L., and Populus tremuloides Michx.), the success rate of the SNP array decreased by 13 %, demonstrating moderate cross-sectional transferability.     

Crop seed oil bodies: From challenges in protein identification to an emerging picture of the oil body proteome

Oleaginous seeds store lipids in specialized structures called oil bodies (OBs). These organelles consist of a core of neutral lipids bound by proteins embedded in a phospholipid monolayer. OB proteins are well conserved in plants and have long been grouped into only two categories: structural proteins or enzymes. Recent work, however, which identified other classes of proteins associated with OBs, clearly shows that this classification is obsolete. Proteomics‐mediated OB protein identification is facilitated in plants for which the genome is sequenced and annotated. However, it is not clear whether this knowledge can be dependably transposed to less well‐characterized plants, including the well‐established commercial sources of seed oil as well as the many others being proposed as novel sources for biodiesel, especially in Africa and Asia. Toward an update of the current data available on OB proteins this review discusses (i) the specific difficulties for proteomic studies of organelles; (ii) a 2012 census of the proteins found in seed OBs from various crops; (iii) the oleosin composition of OBs and their role in organelle stability; (iv) PTM of OB proteins as an emerging field of investigation; and finally we describe the emerging model of the OB proteome from oilseed crops.     

A Type IV Pilus Mediates DNA Binding during Natural Transformation in Streptococcus pneumoniae

Natural genetic transformation is widely distributed in bacteria and generally occurs during a genetically programmed differentiated state called competence. This process promotes genome plasticity and adaptability in Gram-negative and Gram-positive bacteria. Transformation requires the binding and internalization of exogenous DNA, the mechanisms of which are unclear. Here, we report the discovery of a transformation pilus at the surface of competent Streptococcus pneumoniae cells. This Type IV-like pilus, which is primarily composed of the ComGC pilin, is required for transformation. We provide evidence that it directly binds DNA and propose that the transformation pilus is the primary DNA receptor on the bacterial cell during transformation in S. pneumoniae. Being a central component of the transformation apparatus, the transformation pilus enables S. pneumoniae, a major Gram-positive human pathogen, to acquire resistance to antibiotics and to escape vaccines through the binding and incorporation of new genetic material.     

The Interactomes of Influenza Virus NS1 and NS2 Proteins Identify New Host Factors and Provide Insights for ADAR1 Playing a Supportive Role in Virus Replication

Influenza A NS1 and NS2 proteins are encoded by the RNA segment 8 of the viral genome. NS1 is a multifunctional protein and a virulence factor while NS2 is involved in nuclear export of viral ribonucleoprotein complexes. A yeast two-hybrid screening strategy was used to identify host factors supporting NS1 and NS2 functions. More than 560 interactions between 79 cellular proteins and NS1 and NS2 proteins from 9 different influenza virus strains have been identified. These interacting proteins are potentially involved in each step of the infectious process and their contribution to viral replication was tested by RNA interference. Validation of the relevance of these host cell proteins for the viral replication cycle revealed that 7 of the 79 NS1 and/or NS2-interacting proteins positively or negatively controlled virus replication. One of the main factors targeted by NS1 of all virus strains was double-stranded RNA binding domain protein family. In particular, adenosine deaminase acting on RNA 1 (ADAR1) appeared as a pro-viral host factor whose expression is necessary for optimal viral protein synthesis and replication. Surprisingly, ADAR1 also appeared as a pro-viral host factor for dengue virus replication and directly interacted with the viral NS3 protein. ADAR1 editing activity was enhanced by both viruses through dengue virus NS3 and influenza virus NS1 proteins, suggesting a similar virus-host co-evolution.     

Zinc Piracy as a Mechanism of Neisseria meningitidis for Evasion of Nutritional Immunity

The outer membrane of Gram-negative bacteria functions as a permeability barrier that protects these bacteria against harmful compounds in the environment. Most nutrients pass the outer membrane by passive diffusion via pore-forming proteins known as porins. However, diffusion can only satisfy the growth requirements if the extracellular concentration of the nutrients is high. In the vertebrate host, the sequestration of essential nutrient metals is an important defense mechanism that limits the growth of invading pathogens, a process known as “nutritional immunity.” The acquisition of scarce nutrients from the environment is mediated by receptors in the outer membrane in an energy-requiring process. Most characterized receptors are involved in the acquisition of iron. In this study, we characterized a hitherto unknown receptor from Neisseria meningitidis, a causative agent of sepsis and meningitis. Expression of this receptor, designated CbpA, is induced when the bacteria are grown under zinc limitation. We demonstrate that CbpA functions as a receptor for calprotectin, a protein that is massively produced by neutrophils and other cells and that has been shown to limit bacterial growth by chelating Zn²⁺ and Mn²⁺ ions. Expression of CbpA enables N. meningitidis to survive and propagate in the presence of calprotectin and to use calprotectin as a zinc source. Besides CbpA, also the TonB protein, which couples energy of the proton gradient across the inner membrane to receptor-mediated transport across the outer membrane, is required for the process. CbpA was found to be expressed in all N. meningitidis strains examined, consistent with a vital role for the protein when the bacteria reside in the host. Together, our results demonstrate that N. meningitidis is able to subvert an important defense mechanism of the human host and to utilize calprotectin to promote its growth.     

Masculinization of the X Chromosome in the Pea Aphid

Evolutionary theory predicts that sexually antagonistic mutations accumulate differentially on the X chromosome and autosomes in species with an XY sex-determination system, with effects (masculinization or feminization of the X) depending on the dominance of mutations. Organisms with alternative modes of inheritance of sex chromosomes offer interesting opportunities for studying sexual conflicts and their resolution, because expectations for the preferred genomic location of sexually antagonistic alleles may differ from standard systems. Aphids display an XX/X0 system and combine an unusual inheritance of the X chromosome with the alternation of sexual and asexual reproduction. In this study, we first investigated theoretically the accumulation of sexually antagonistic mutations on the aphid X chromosome. Our results show that i) the X is always more favourable to the spread of male-beneficial alleles than autosomes, and should thus be enriched in sexually antagonistic alleles beneficial for males, ii) sexually antagonistic mutations beneficial for asexual females accumulate preferentially on autosomes, iii) in contrast to predictions for standard systems, these qualitative results are not affected by the dominance of mutations. Under the assumption that sex-biased gene expression evolves to solve conflicts raised by the spread of sexually antagonistic alleles, one expects that male-biased genes should be enriched on the X while asexual female-biased genes should be enriched on autosomes. Using gene expression data (RNA-Seq) in males, sexual females and asexual females of the pea aphid, we confirm these theoretical predictions. Although other mechanisms than the resolution of sexual antagonism may lead to sex-biased gene expression, we argue that they could hardly explain the observed difference between X and autosomes. On top of reporting a strong masculinization of the aphid X chromosome, our study highlights the relevance of organisms displaying an alternative mode of sex chromosome inheritance to understanding the forces shaping chromosome evolution.     

Semen CD4+ T Cells and Macrophages Are Productively Infected at All Stages of SIV infection in Macaques

The mucosal events of HIV transmission have been extensively studied, but the role of infected cells present in the genital and rectal secretions, and in the semen, in particular, remains a matter of debate. As a prerequisite to a thorough in vivo investigation of the early transmission events through infected cells, we characterized in detail by multi-parameter flow cytometry the changes in macaque seminal leukocytes during SIVmac251 infection, focusing on T cells, macrophages and dendritic cells. Using immunocytofluorescence targeting SIV proteins and real-time quantitative PCR targeting SIV DNA, we investigated the nature of the infected cells on sorted semen leukocytes from macaques at different stages of infection. Finally, we cocultured semen CD4⁺ T cells and macrophages with a cell line permissive to SIV infection to assess their infectivity in vitro. We found that primary infection induced strong local inflammation, which was associated with an increase in the number of leukocytes in semen, both factors having the potential to favor cell-associated virus transmission. Semen CD4⁺ T cells and macrophages were productively infected at all stages of infection and were infectious in vitro. Lymphocytes had a mucosal phenotype and expressed activation (CD69 & HLA-DR) and migration (CCR5, CXCR4, LFA-1) markers. CD69 expression was increased in semen T cells by SIV infection, at all stages of infection. Macrophages predominated at all stages and expressed CD4, CCR5, MAC-1 and LFA-1. Altogether, we demonstrated that semen contains the two major SIV-target cells (CD4+ T cells and macrophages). Both cell types can be productively infected at all stages of SIV infection and are endowed with markers that may facilitate transmission of infection during sexual exposure.     

Interactions between peptidyl tRNA hydrolase homologs and the ribosomal release factor Mrf1 in S. pombe mitochondria

Mitochondrial translation synthesizes key subunits of the respiratory complexes. In Schizosaccharomyces pombe, strains lacking Mrf1, the mitochondrial stop codon recognition factor, are viable, suggesting that other factors can play a role in translation termination. S. pombe contains four predicted peptidyl tRNA hydrolases, two of which (Pth3 and Pth4), have a GGQ motif that is conserved in class I release factors. We show that high dosage of Pth4 can compensate for the absence of Mrf1 and loss of Pth4 exacerbates the lack of Mrf1. Also Pth4 is a component of the mitochondrial ribosome, suggesting that it could help recycling stalled ribosomes.