16 research outputs found
Comparative genomics of primate CCL3L and CCL4L loci.
No full text<p>(A) Comparison of <i>CCL3L</i> and <i>CCL4L</i> in human and nonhuman primates. The top panel shows a schema of the chemokine locus at human chromosome 17q12 based on the NT_010799.14 contig. <i>CCL3</i> and <i>CCL4</i> exist as single-copy genes per haploid genome. The genes encoding the non-allelic isoforms of <i>CCL3</i> (National Center for Biotechnology Information gene ID given in parentheses) are denoted as <i>CCL3L1</i> (6349), <i>CCL3L2</i> (390788), and <i>CCL3L3</i> (414062) and those of <i>CCL4</i> are denoted as <i>CCL4L1</i> (9560) and <i>CCL4L2</i> (388372). The middle panel shows a schema of the <i>CCL3L</i> and <i>CCL4L</i> locus in chimpanzee based on the chromosome 17NW_001226927.1 contig. <i>CCL3L</i> orthologs (denoted as “1” and “2”) map ∼ 1.6 Mb apart in this contig. In contrast to the human locus, chimpanzee contigs lack <i>CCL3L2</i>. The bottom panel shows a schema of the <i>CCL3L</i> and <i>CCL4L</i> locus in rhesus monkey based on chromosome 16 NW_001103987 contig. Of note, other orthologs of <i>CCL3L</i> and <i>CCL4L</i> were found in two other rhesus contigs (NW_001103644.1 and NW_001102959). CpG islands found in primate <i>CCL3L</i> and <i>CCL4L</i> loci are also depicted. Distances between genes are approximate, and the map is not to scale. The arrows denote the orientation of the genes. k, kb; M, Mb. (B and C) Schematic representation of genomic and mRNA structure of human <i>CCL3L</i> and <i>CCL4L</i> genes that have mRNA splicing patterns that are similar (B) or dissimilar (C) to <i>CCL3</i> and <i>CCL4.</i> Exons are represented as boxes and introns as connecting lines labeled with Roman numbers; the splicing pattern is denoted by the dashed lines. <i>CCL3L1</i>, <i>CCL3L3</i>, and <i>CCL4L2</i> are each composed of three exons, and the start codon (denoted with an arrow) is located in the first exon. <i>CCL4L1</i> has a transition in the splicing acceptor site located in intron II (AG→GG, indicated in red), which results in the generation of aberrantly spliced transcripts that use alternative acceptor sites located either in the intron II or in the third exon <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1000359#pgen.1000359-Colobran1" target="_blank">[26]</a>. <i>CCL3L2</i> was previously considered as a pseudogene <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1000359#pgen.1000359-Menten1" target="_blank">[5]</a>. However, recent studies in our lab suggest that it has a four exon structure and is predicted to transcribe alternatively spliced mRNA species with open reading frames (ORFs) that contain chemokine-like domains <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1000359#pgen.1000359-ShostakovichKoretskaya1" target="_blank">[16]</a>; CCL3L2 mRNA transcripts originate from two novel upstream exons (designated as 1A and 1B) and are linked to the second and third exons, which are homologous to exons 2 and 3 found in <i>CCL3L1</i> or <i>CCL3L3</i>. (D) Nucleotide sequence of human <i>CCL3L1</i> (or <i>CCL3L3</i>) and its alignment with four distinct chimpanzee <i>CCL3L</i> (<i>chCCL3L</i>) orthologous genes from the translation initiation site until the start of intron 1. The translational start codon in <i>hCCL3L1</i> is underlined. Horizontal arrows delimit the exon–intron boundaries. Dashes indicate deletions. Polymorphic sites relative to the <i>hCCL3L1</i> are shown in red. The vertical arrow represents the site for signal peptidase cleavage. <i>chCCL3L ortholog 1</i> is predicted to encode a chemokine with amino acids that are shared with both hCCL3L1 and hCCL3. <i>chCCL3L ortholog 2</i> has a deletion of 17 nucleotides (relative to <i>hCCL3L1</i>) that may lead to loss of the signal peptide cleavage motif. Notably, two additional and different <i>CCL3L</i> orthologs were found in two independent chimpanzee contigs, denoted as NW_001227489.1 (ortholog 3) and NW_001227474.1 (ortholog 4), which have a mutation at the translation initiation site (shown in purple) and differ from each other in the splicing donor site of intron 1 (shown in blue) and other genomic regions (unpublished data). Of note, all four <i>chCCL3L1</i> orthologs had sequences that were completely homologous to the primer–probe sets used to detect <i>CCL3L</i> CNV in humans and chimpanzee previously <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1000359#pgen.1000359-Gonzalez1" target="_blank">[12]</a> and by Degenhardt et al. <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1000359#pgen.1000359-Degenhardt1" target="_blank">[9]</a>. All the chimpanzee orthologs are also predicted to encode transcripts with potential ORFs with chemokine-like domains. The accession numbers for the predicted ORFs encoded by chimpanzee <i>CCL3L</i> orthologs 1, 2, 3, and 4 are NP_001029254, XP_001152451, XP_001172388, and XP_001172226, respectively.</p
Evolution and challenges of varietal improvement strategies
No full textAgricultural production and supply chains are facing major challenges in the form of increasing demand for food products, a diversified use of agricultural products including for non-food purposes, ecologically intensive agriculture, and the necessity of taking climate change into account. The creation and adoption of new varieties that are productive, diversified, better adapted, and more environmentally friendly can help cropping systems that seek to address these issues become more efficient. Plant breeding efforts must anticipate the needs of the end users and adapt to very different agri-chains, illustrated in this chapter by two examples: first, an agri-chain that is highly structured around targeted export products, for example, the dessert banana, which requires ideotypes that meet the requirements of production and marketing systems; and second, an emerging agri-chain for multi-use sorghum, characterized by new production objectives of increased energy potential and production of biomaterials, sometimes without compromising with the requirement of high grain yields. To meet the objectives and the sustainability of agri-chains, research into genetic improvement must propose new approaches, new tools, and innovative breeding methods. The objectives, and sometimes the entire breeding process, are shared with all the actors, especially the end users, as part of an enhanced partnership within agri-chains. The breeding strategy also depends on ease of access, better use of available and useful genetic resources, in-depth knowledge of the structure and diversity of these resources, genetic determinism of desirable traits, and pre-breeding approaches. All these scientific and partnership innovations ensure the necessary responsiveness to support the execution of current breeding processes and the identification of new varieties that meet current and future uses and services
Ectopic expression of BepE<i><sub>Bhe</sub></i> in HUVECs prevents cell fragmentation.
No full text<p>(<b>A, B</b>) HUVECs of an early passage were transduced with lentiviruses for the expression of the depicted GFP-fusion proteins. The mixed culture of transduced and non-transduced cells were infected with the indicated <i>Bhe</i> strains (MOI = 200). Infected cells were either fixed and stained for microscopy or analyzed for the survival by FACS at 48 hpi. (<b>A</b>) Representative microscopy images (scale bar = 100 µm). F-actin is represented in red (Phalloidin), DNA in blue (DAPI), GFP in green. (<b>B</b>) Protection by GFP-fused BepE and its derivatives against fragmentation induced by <i>Bhe</i> Δ<i>bepDEF</i> mutant strains. GFP-positive cell were quantified by FACS and normalized to the uninfected cell population. One representative experiment (n = 3) with the mean of triplicate samples +/− SD are presented. Statistical significance was determined using Student's <i>t</i>-test. <i>P</i><0.05 was considered statistically significant.</p
BepE protects host cells from fragmentation upon translocation via T4SS.
No full text<p>(<b>A</b>) Subconfluent monolayers of HUVECs were infected with MOI = 100 of the indicated bacterial strains for 24 h. After fixation and subsequent immunocytochemical staining the specimen was analyzed by confocal laser scanning microscopy. F-actin is represented in red (Phalloidin) and DNA in Blue (DAPI). Translocation of the effector protein into the infected cells was detected by anti-Myc-staining depicted in green (scale bar = 20 µm). (<b>B</b>) Subconfluent monolayers of HUVECs were infected with MOI = 200 or MOI = 200+200 in case of mixed infection depicted in the figure. Quantification of cell fragmentation at 48 h post infection was performed as described for <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004187#ppat-1004187-g001" target="_blank">Fig. 1C and D</a> and presented as mean of triplicate samples +/− SD. Statistical significance was determined using Student's <i>t</i>-test. <i>P</i><0.05 was considered statistically significant. Data from one representative experiment (n = 2) are presented. (<b>C</b>) Protein levels of the BepE<i><sub>Bhe</sub></i> by overexpression in <i>Bartonella</i> strains. The anti-Myc western blot was obtained from total lysate of corresponding <i>Bartonella</i> strains depicted in figure.</p
BepE is essential for <i>Bartonella tribocorum</i> (<i>Btr</i>) to establish bacteremia after <i>intradermal</i> (<i>i.d.</i>) infection of the rat reservoir host.
No full text<p>(<b>A</b>) Domain organization of BepE orthologues in <i>Btr</i> and <i>Bhe</i>. The BepE homologues from <i>Bartonella</i> species depicted in the figure (BepE<i><sub>Bhe</sub></i>, BepD<i><sub>Btr</sub></i>, BepE<i><sub>Btr</sub></i>) were aligned using Geneious Pro 5.3.4. The amino acid sequence alignment with pairwise % identity is indicated. The tyrosine-containing N-termini and BID domains were aligned independently. (<b>B</b>) <i>Btr</i> Δ<i>bepDE</i> is not able to reach the blood of rats infected by the <i>i.d.</i> route. Rats (n = 5) were inoculated in the ear dermis with either <i>Btr</i> wild-type or <i>Btr</i> Δ<i>bepDE</i>. Blood was drawn at the indicated days post infection (dpi), diluted and plated on sheep blood supplemented Columbia agar plates (CBA) for counting of colony forming units (CFUs). (<b>C</b>) Complementation of the <i>Btr</i> Δ<i>bepDE</i> mutant with BepE is sufficient to restore bacteremia in rats infected by the <i>i.d.</i> route. Groups of rats (n≥3) were infected with the indicated strains by the <i>i.v.</i> or <i>i.d.</i> route. Blood was drawn at 16 dpi and CFUs were recovered as described for B. The graph represents CFUs/ml of blood for individual animals (circles) and their cohort mean (line). Statistical significance was determined using Student's <i>t</i>-test. <i>P</i><0.05 was considered statistically significant. (<b>D</b>) Heterologous complementation of <i>Btr</i> Δ<i>bepDE</i> with p<i>BIDs.E<sub>Bhe</sub></i> is sufficient to rescue the abacteremia phenotype following infection by the <i>i.d.</i> route. The infections were performed as described for (C). Data represented for BIDs.E<i><sub>Bhe</sub></i> complementation were acquired in separate experiment from the other data shown. <i>P</i><0.05 was considered statistically significant.</p
Dendritic cells are infected by <i>Bartonella</i>.
No full text<p>(<b>A</b>) Effector translocation by <i>Bhe</i> into mouse bone marrow-derived dendritic cells (BMDCs). Balb/c mouse BMDCs were infected with corresponding MOIs and strains. “Effector”, Bla-BID, translocation efficiency was assessed as the % of infected cells that converted CCF2-AM blue emission into green detected by Leica DM-IRBE inverted fluorescence microscope. The bars represent the mean of triplicate samples +/− SD. Data from one representative experiment (n = 2) are presented. (<b>B</b>) Migration of BMDCs is inhibited in a trans-well assay by <i>Bhe</i> Δ<i>bepDEF</i> infection. BMDCs were pre-infected with MOI = 50 of the indicated bacterial strains. Infected cells were embedded in collagen and mounted in a trans-well migration system that was prior seeded with a confluent monolayer of iLECs (immortalized lymphatic endothelial cells). BMDCs that migrated though the iLECs were quantified after 24 h. The data normalized to uninfected condition. The bars represent the mean of triplicate samples +/− SD. Statistical significance was determined using Student's <i>t</i>-test. <i>P</i><0.05 was considered statistically significant. Data from one representative experiment (n = 3) are presented.</p
Deletion of BepE is sufficient for <i>Bhe</i> to induce cell fragmentation.
No full text<p>(<b>A–C</b>) Subconfluent monolayers of HUVECs were infected with MOI = 200 of the indicated bacterial strains. (<b>B</b>) Infected HUVECs were fixed at 48 h post infection followed by immunocytochemical staining and confocal laser scanning microscopy. F-actin is represented in red and DNA in blue (scale bar = 50 µm). (<b>A</b>) and (<b>C</b>) quantification of cell fragmentation at 48 h post infection was performed as described for <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004187#ppat-1004187-g001" target="_blank">Fig. 1C and D</a>. The mean and SD of triplicate samples is presented. Statistical significance was determined using Student's <i>t</i>-test. <i>P</i><0.05 was considered statistically significant. Data from one representative experiment (n = 3) are presented. (<b>D</b>) Schematic view of BepE<i><sub>Bhe</sub></i> and N-terminal deletion mutants expressed in <i>Bartonella</i> from a plasmid. (<b>E</b>) Protein levels of the BepE<i><sub>Bhe</sub></i> mutants shown in figure by overexpression in <i>Bhe</i> Δ<i>bepE</i>. The anti-Myc western blot was obtained from total lysate of corresponding <i>Bartonella</i> strains.</p
