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The life cycle of Plasmodium falciparum, the agent responsible for malaria, depends on both cytosolic and apicoplast translation fidelity. Apicoplast aminoacyl-tRNA synthetases (aaRS) are bacterial-like enzymes devoted to organellar tRNA aminoacylation. They are all encoded by the nuclear genome and are translocated into the apicoplast only after cytosolic biosynthesis. Apicoplast aaRSs contain numerous idiosyncratic sequence insertions: An understanding of the roles of these insertions has remained elusive and they hinder efforts to heterologously overexpress these proteins. Moreover, the A/T rich content of the Plasmodium genome leads to A/U rich apicoplast tRNA substrates that display structural plasticity. Here, we focus on the P. falciparum apicoplast tyrosyl-tRNA synthetase (Pf-apiTyrRS) and its cognate tRNATyr substrate (Pf-apitRNATyr). Cloning and expression strategies used to obtain an active and functional recombinant Pf-apiTyrRS are reported. Functional analyses established that only three weak identity elements in the apitRNATyr promote specific recognition by the cognate Pf-apiTyrRS and that positive identity elements usually found in the tRNATyr acceptor stem are excluded from this set. This finding brings to light an unusual behavior for a tRNATyr aminoacylation system and suggests that Pf-apiTyrRS uses primarily negative recognition elements to direct tyrosylation specificity. published

International audience; In a pilot ProtoArray analysis, we identified 6 proteins out of 9483 recognized by autoantibodies (AAb) from patients with systemic sclerosis (SSc). We further investigated the 6 candidates by ELISA on hundreds of controls and patients, including patients with Systemic Lupus Erythematosus (SLE), known for high sera reactivity and overlapping AAb with SSc. Only 2 of the 6 candidates, Ephrin type-B receptor 2 (EphB2) and Three prime Histone mRNA EXonuclease 1 (THEX1), remained significantly recognized by sera samples from SSc compared to controls (healthy or with rheumatic diseases) with, respectively, 34% versus 14% (P = 2.10(-4)) and 60% versus 28% (P = 3.10(-8)). Above all, EphB2 and THEX1 revealed to be mainly recognized by SLE sera samples with respectively 56%, (P = 2.10(-10)) and 82% (P = 5.10(-13)). As anti-EphB2 and anti-THEX1 AAb were found in both diseases, an epitope mapping was realized on each protein to refine SSc and SLE diagnosis. A 15-mer peptide from EphB2 allowed to identify 35% of SLE sera samples (N = 48) versus only 5% of any other sera samples (N = 157), including SSc sera samples. AAb titers were significantly higher in SLE sera (P< 0.0001) and correlated with disease activity (p< 0.02). We could not find an epitope on EphB2 protein for SSc neither on THEX1 for SSc or SLE. We showed that patients with SSc or SLE have AAb against EphB2, a protein involved in angiogenesis, and THEX1, a 3'-5' exoribonuclease involved in histone mRNA degradation. We have further identified a peptide from EphB2 as a specific and sensitive tool for SLE diagnosis.

The potential use of functionalized carbon nanotubes (f-CNTs) for drug and gene delivery to the central nervous system (CNS) and as neural substrates makes the understanding of their in vivo interactions with the neural tissue essential. The aim of this study was to investigate the interactions between chemically functionalized multi-walled carbon nanotubes (f-MWNTs) and the neural tissue following cortical stereotactic administration. Two different f-MWNT constructs were used in these studies: shortened (by oxidation) amino-functionalized MWNT (oxMWNT-NH3(+)) and amino-functionalized MWNT (MWNT-NH3(+)). Parenchymal distribution of the stereotactically injected f-MWNTs was assessed by histological examination. Both f-MWNT were uptaken by different types of neural tissue cells (microglia, astrocytes and neurons), however different patterns of cellular internalization were observed between the nanotubes. Furthermore, immunohistochemical staining for specific markers of glial cell activation (GFAP and CD11b) was performed and secretion of inflammatory cytokines was investigated using real-time PCR (qRT-PCR). Injections of both f-MWNT constructs led to a local and transient induction of inflammatory cytokines at early time points. Oxidation of nanotubes seemed to induce significant levels of GFAP and CD11b over-expression in areas peripheral to the f-MWNT injection site. These results highlight the importance of nanotube functionalization on their interaction with brain tissue that is deemed critical for the development nanotube-based vector systems for CNS applications. The potential use of functionalized carbon nanotubes (f-CNTs) for drug and gene delivery to the central nervous system (CNS) and as neural substrates makes the understanding of their in vivo interactions with the neural tissue essential. The aim of this study was to investigate the interactions between chemically functionalized multi-walled carbon nanotubes (f-MWNTs) and the neural tissue following cortical stereotactic administration. Two different f-MWNT constructs were used in these studies: shortened (by oxidation) amino-functionalized MWNT (oxMWNT-NH3+) and amino-functionalized MWNT (MWNT-NH3+). Parenchymal distribution of the stereotactically injected f-MWNTs was assessed by histological examination. Both f-MWNT were uptaken by different types of neural tissue cells (microglia, astrocytes and neurons), however different patterns of cellular internalization were observed between the nanotubes. Furthermore, immunohistochemical staining for specific markers of glial cell activation (GFAP and CD11b) was performed and secretion of inflammatory cytokines was investigated using real-time PCR (qRT-PCR). Injections of both f-MWNT constructs led to a local and transient induction of inflammatory cytokines at early time points. Oxidation of nanotubes seemed to induce significant levels of GFAP and CD11b over-expression in areas peripheral to the f-MWNT injection site. These results highlight the importance of nanotube functionalization on their interaction with brain tissue that is deemed critical for the development nanotube-based vector systems for CNS applications. © 2013 Bardi et al.

Identity determinants are essential for the accurate recognition of transfer RNAs by aminoacyl-tRNA synthetases. To date, arginine determinants in the yeast Saccharomyces cerevisiae have been identified exclusively in vitro and only on a limited number of tRNA Arginine isoacceptors. In the current study, we favor a full cellular approach and expand the investigation of arginine determinants to all four tRNA Arg isoacceptors. More precisely, this work scrutinizes the relevance of the tRNA nucleotides at position 20, 35 and 36 in the yeast arginylation reaction. We built 21 mutants by site-directed mutagenesis and tested their functionality in YAL5, a previously engineered yeast knockout deficient for the expression of tRNA Arg CCG. Arginylation levels were also monitored using Northern blot. Our data collected in vivo correlate with previous observations. C35 is the prominent arginine determinant followed by G36 or U36 (G/U36). In addition, although there is no major arginine determinant in the D loop, the recognition of tRNA Arg ICG relies to some extent on the nucleotide at position 20. This work refines the existing model for tRNA Arg recognition. Our observations indicate that yeast Arginyl-tRNA synthetase (yArgRS) relies on distinct mechanisms to aminoacylate the four isoacceptors. Finally, according to our refined model, yArgRS is able to accommodate tRNA Arg scaffolds presenting N34, C/G35 and G/A/U36 anticodons while maintaining specificity. We discuss the mechanistic and potential physiological implications of these findings.