Fucosylated pentaerythrityl phosphodiester oligomers (PePOs): automated synthesis of DNA-based glycoclusters and binding to Pseudomonas aeruginosa lectin (PA-IIL)

The synthesis of propargylated pentaerythrityl phosphodiester oligomers (PePOs) was achieved using a DNA synthesizer with a bis-propargylated pentaerythritol-based phosphoramidite. An azido fucose derivative was reacted under "click" chemistry conditions activated by microwaves to construct a series of glycosylated PePOs bearing 4, 6, 8, and 10 L-fucose residues. Binding to the fucose-specific bacterial lectin (PA-IIL) was determined for the fucosylated PePOs through an enzyme-linked lectin amplification competition assay. The IC50 values measured are 10-20 times better than for monovalent l-fucose and denotate for a "macromolecular" effect rather than a "cluster" effect.     

Induced parthenogenesis in mandarin for haploid production: induction procedures and genetic analysis of plantlets

This study focused on haploid induction in mandarin through in situ gynogenesis by pollination with irradiated pollen of ‘Meyer’ lemon. Pollination was carried out for three genotypes of mandarin with four levels of gamma-ray-irradiated pollen (150, 300, 600, and 900 Gy). The resulting seeds were characterised by a small size. Embryos were rescued in vitro and the ploidy level of the plantlets was determined by flow cytometry analysis. Haploid, diploid, triploid plantlets were obtained. The haploid parthenogenetic origin was confirmed using microsatellite marker analysis and chromosome count. Diploid and triploid plants were the result of crosses between mandarin and lemon. The induction of gynogenetic haploids of ‘Fortune’ (Citrus clementina Hort ex Tan. × Citrus tangerina Hort ex Tan.) and ‘Ellendale’ (Citrus reticulata Blanco × Citrus sinensis L. Osb) is reported here for the first time.     

Long-term survival of Legionella pneumophila associated with Acanthamoeba castellanii vesicles

Legionella pneumophila, the causative agent of Legionnaires' disease, is ubiquitously found in aquatic environments, associated with free living amoebae. Trophozoite forms of the genus Acanthamoeba have been shown to support the intracellular growth of Legionella while it has been proposed that cyst forms are related to survival in harsh environments. This underlines that amoebae are of primary importance in Legionella spreading. In this study, we followed the survival of L. pneumophila Lens over 6 months in a poor medium, with or without Acanthamoeba castellanii. The results demonstrated that L. pneumophila Lens could survive for at least 6 months in association with A. castellanii and that cultivable bacteria are to be found within expelled vesicles rather than within cysts. Our findings suggest that vesicles might be further studied in order to elucidate their production and their role in the environmental spreading of Legionella.     

Gene transfer to pre-hematopoietic and committed hematopoietic precursors in the early mouse Yolk Sac: a comparative study between <Emphasis Type="Italic">in situ</Emphasis> electroporation and retroviral transduction

Background  

Hematopoietic development in vertebrate embryos results from the sequential contribution of two pools of precursors independently generated. While intra-embryonic precursors harbour the features of hematopoietic stem cells (HSC), precursors formed earlier in the yolk sac (YS) display limited differentiation and self-renewal potentials. The mechanisms leading to the generation of the precursors in both sites are still largely unknown, as are the molecular basis underlying their different potential. A possible approach to assess the role of candidate genes is to transfer or modulate their expression/activity in both sites. We thus designed and compared transduction protocols to target either native extra-embryonic precursors, or hematopoietic precursors.     

A new generation of pPRIG-based retroviral vectors

Background  

Retroviral vectors are valuable tools for gene transfer. Particularly convenient are IRES-containing retroviral vectors expressing both the protein of interest and a marker protein from a single bicistronic mRNA. This coupled expression increases the relevance of tracking and/or selection of transduced cells based on the detection of a marker protein. pAP2 is a retroviral vector containing eGFP downstream of a modified IRES element of EMCV origin, and a CMV enhancer-promoter instead of the U3 region of the 5'LTR, which increases its efficiency in transient transfection. However, pAP2 contains a limited multicloning site (MCS) and shows weak eGFP expression, which previously led us to engineer an improved version, termed pPRIG, harboring: i) the wild-type ECMV IRES sequence, thereby restoring its full activity; ii) an optimized MCS flanked by T7 and SP6 sequences; and iii) a HA tag encoding sequence 5' of the MCS (pPRIG HAa/b/c).     

The COP9 signalosome is vital for timely repair of DNA double-strand breaks

The DNA damage response is vigorously activated by DNA double-strand breaks (DSBs). The chief mobilizer of the DSB response is the ATM protein kinase. We discovered that the COP9 signalosome (CSN) is a crucial player in the DSB response and an ATM target. CSN is a protein complex that regulates the activity of cullin ring ubiquitin ligase (CRL) complexes by removing the ubiquitin-like protein, NEDD8, from their cullin scaffold. We find that the CSN is physically recruited to DSB sites in a neddylation-dependent manner, and is required for timely repair of DSBs, affecting the balance between the two major DSB repair pathways—nonhomologous end-joining and homologous recombination repair (HRR). The CSN is essential for the processivity of deep end-resection—the initial step in HRR. Cullin 4a (CUL4A) is recruited to DSB sites in a CSN- and neddylation-dependent manner, suggesting that CSN partners with CRL4 in this pathway. Furthermore, we found that ATM-mediated phosphorylation of CSN subunit 3 on S410 is critical for proper DSB repair, and that loss of this phosphorylation site alone is sufficient to cause a DDR deficiency phenotype in the mouse. This novel branch of the DSB response thus significantly affects genome stability.     

Altered retinoic acid signalling underpins dentition evolution

Small variations in signalling pathways have been linked to phenotypic diversity and speciation. In vertebrates, teeth represent a reservoir of adaptive morphological structures that are prone to evolutionary change. Cyprinid fish display an impressive diversity in tooth number, but the signals that generate such diversity are unknown. Here, we show that retinoic acid (RA) availability influences tooth number size in Cyprinids. Heterozygous adult zebrafish heterozygous for the cyp26b1 mutant that encodes an enzyme able to degrade RA possess an extra tooth in the ventral row. Expression analysis of pharyngeal mesenchyme markers such as dlx2a and lhx6 shows lateral, anterior and dorsal expansion of these markers in RA-treated embryos, whereas the expression of the dental epithelium markers dlx2b and dlx3b is unchanged. Our analysis suggests that changes in RA signalling play an important role in the diversification of teeth in Cyprinids. Our work illustrates that through subtle changes in the expression of rate-limiting enzymes, the RA pathway is an active player of tooth evolution in fish.     

Revisiting the link between breeding effort and oxidative balance through field evaluation of two sympatric sibling insect species

The idea that oxidative stress could be a major force governing evolutionary trade‐offs has recently been challenged by experimental approaches in laboratory conditions, triggering extensive debates centered on theoretical and methodological issues. Here, we revisited the link between oxidative stress and reproduction by measuring multiple antioxidant and oxidative damages in wild‐caught females of two sibling weevil species (Curculio elephas, C. glandium). The strength of our study arised from (1) studied species that were sympatric and exploited similar resource, but displayed contrasting reproductive strategies and (2) individuals were sampled throughout adult life so as to relate oxidative status to breeding effort. We found that the short‐lived C. elephas sacrifices red‐ox homeostasis for immediate reproduction upon emergence as characterized by low antioxidant defenses and elevated oxidative damage. Comparatively, C. glandium massively invests in antioxidant and maintains low oxidative damage, which may contribute to their extended prereproductive period. Intriguingly, we also reveal, for the first time in a field study, an unexpected reactivation of antioxidant defenses with the onset of reproduction. Our results thus support the existence of a strong, but complex relationship between oxidative stress and life‐history evolution and highlight the need for a finer‐scale picture of antioxidant strategies.