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Herein, we describe the myxobacterial natural product Corramycin isolated from Corallococcus coralloides. The linear peptide structure contains an unprecedented (2R,3S)-γ-N-methyl-β-hydroxy-histidine moiety. Corramycin exhibits anti-Gram-negative activity against Escherichia coli (E. coli) and is taken up via two transporter systems, SbmA and YejABEF. Furthermore, the Corramycin biosynthetic gene cluster (BGC) was identified and a biosynthesis model was proposed involving a 12-modular non-ribosomal peptide synthetase/polyketide synthase. Bioinformatic analysis of the BGC combined with the development of a total synthesis route allowed for the elucidation of the molecule's absolute configuration. Importantly, intravenous administration of 20 mg kg

Herein, we describe the myxobacterial natural product Corramycin (1) isolated from Corallococcus coralloides (C. coralloides). Corramycin is a linear peptide containing an unprecedented (2R,3S)  N-methyl-β-hydroxy histidine moiety and exhibiting anti-Gram-negative activity against Escherichia coli. Moreover, we describe the Corramycin biosynthetic gene cluster (BGC) and propose a biosynthesis model involving a 12-modular non-ribosomal peptide synthetase (NRPS)/polyketide synthase (PKS). Bioinformatic analysis of the BGC combined with the development of a total synthesis route allowed for the elucidation of the molecule’s absolute configuration. Furthermore, we show that the uptake of Corramycin in E. coli depends on two transporter systems, SbmA and YejABEF. Importantly, intravenous administration of 30 mg kg 1 of Corramycin in an E. coli mouse infection model resulted in significantly reduced colony forming units (CFU) and in 60 % survival of animals, with no toxic effects observed in vitro or in vivo. Corramycin is thus an intriguing innovative starting point to develop a potent antibacterial drug against hospital acquired infections.

The emergence and spread of Plasmodium falciparum resistance to first-line antimalarials creates an imperative to identify and develop potent preclinical candidates with distinct modes of action. Here, we report the identification of MMV688533, an acylguanidine that was developed following a whole-cell screen with compounds known to hit high-value targets in human cells. MMV688533 displays fast parasite clearance in vitro and is not cross-resistant with known antimalarials. In a P. falciparum NSG mouse model, MMV688533 displays a long-lasting pharmacokinetic profile and excellent safety. Selection studies reveal a low propensity for resistance, with modest loss of potency mediated by point mutations in PfACG1 and PfEHD. These proteins are implicated in intracellular trafficking, lipid utilization, and endocytosis, suggesting interference with these pathways as a potential mode of action. This preclinical candidate may offer the potential for a single low-dose cure for malaria.

The emergence and spread of Plasmodium falciparum resistance to first-line antimalarials creates an imperative to identify and develop potent preclinical candidates with distinct modes of action. Here, we report the identification of MMV688533, an acylguanidine that was developed following a whole-cell screen with compounds known to hit high-value targets in human cells. MMV688533 displays fast parasite clearance in vitro and is not cross-resistant with known antimalarials. In a P. falciparum NSG mouse model, MMV688533 displays a long-lasting pharmacokinetic profile and excellent safety. Selection studies reveal a low propensity for resistance, with modest loss of potency mediated by point mutations in PfACG1 and PfEHD. These proteins are implicated in intracellular trafficking, lipid utilization, and endocytosis, suggesting interference with these pathways as a potential mode of action. This preclinical candidate may offer the potential for a single low-dose cure for malaria.

A two-membrane system, or envelope, surrounds plastids. Because of the integration of chloroplast metabolism within the plant cell, the envelope is the site of many specific transport activities. However, only a few proteins involved in the processes of transport across the chloroplast envelope have been identified already at the molecular level. To discover new envelope transporters, we developed a subcellular proteomic approach, which is aimed to identify the most hydrophobic envelope proteins. This strategy combined the use of highly purified and characterized membrane fractions, extraction of the hydrophobic proteins with organic solvents, SDS/PAGE separation, and tandem mass spectrometry analysis. To process the large amount of MS/MS data, a blast -based program was developed for searching in protein, expressed sequence tag, and genomic plant databases. Among the 54 identified proteins, 27 were new envelope proteins, with most of them bearing multiple α-helical transmembrane regions and being very likely envelope transporters. The present proteomic study also allowed us to identify common features among the known and newly identified putative envelope inner membrane transporters. These features were used to mine the complete Arabidopsis genome and allowed us to establish a virtual plastid envelope integral protein database. Altogether, both proteomic and in silico approaches identified more than 50 candidates for the as yet previously uncharacterized plastid envelope transporters. The predictable function of some of these proteins opens up areas of investigation that may lead to a better understanding of the chloroplast metabolism. The present subcellular proteomic approach is amenable to the analysis of the hydrophobic core of other intracellular membrane systems.

Three novel human sequences showing striking homology to the recently described bovine Ca2+/calmodulin kinase II-dependent epithelial chloride channel bCaCC have been identified in an expressed sequence tags database. Full-length clones were isolated using a 5′ RACE approach. The encoded predicted proteins display 65% overall homology to bCaCC. Tissue expression patterns of the corresponding genes, designated as hCaCC-1, -2 and -3, appear to be highly restricted, with the first two genes primarily expressed in the digestive tract. Another original feature as compared to the CaCC family members is the fact that hCaCC-2 also shows expression in the brain. Taken together these findings demonstrate the existence of several CaCC-like genes in humans, some of which display distinct tissue specificity patterns within the CaCC subfamily of chloride channels.

X chromosome inactivation in both mouse and human requires the presence of a cis acting locus, the X inactivation centre. This locus is thought to be involved in the initiation and spreading of the inactivation signal in early development. In order to increase our understanding of the mouse X inactivation centre, a 94 kb region immediately distal to the Xist gene has been sequenced and analysed for the presence of transcription units and/or potential cis acting regulatory elements. We have identified a novel gene, Tsx, lying 40 kb 3' from Xist. Tsx is expressed specifically in the testis and shows no convincing homology to proteins currently in the databases. A rat homologue, also X linked, has been isolated. The mouse and rat Tsx sequences are highly divergent, suggesting that part of the X inactivation centre, including both Xist and Tsx are subject to relatively weak evolutionary constraints.

PepLine is a fully automated software which maps MS/MS fragmentation spectra of trypsic peptides to genomic DNA sequences. The approach is based on Peptide Sequence Tags (PSTs) obtained from partial interpretation of QTOF MS/MS spectra (first module). PSTs are then mapped on the six-frame translations of genomic sequences (second module) giving hits. Hits are then clustered to detect potential coding regions (third module). Our work aimed at optimizing the algorithms of each component to allow the whole pipeline to proceed in a fully automated manner using raw nucleic acid sequences (i.e., genomes that have not been "reduced" to a database of ORFs or putative exons sequences). The whole pipeline was tested on controlled MS/MS spectra sets from standard proteins and from Arabidopsis thaliana envelope chloroplast samples. Our results demonstrate that PepLine competed with protein database searching softwares and was fast enough to potentially tackle large data sets and/or high size genomes. We also illustrate the potential of this approach for the detection of the intron/exon structure of genes.

Times Cited: 4; International audience; DNA barcoding should provide rapid, accurate and automatable species identifications by using a standardized DNA region as a tag. Based on sequences available in GenBank and sequences produced for this study, we evaluated the resolution power of the whole chloroplast trnL (UAA) intron (254-767 bp) and of a shorter fragment of this intron (the P6 loop, 10-143 bp) amplified with highly conserved primers. The main limitation of the whole trnL intron for DNA barcoding remains its relatively low resolution (67.3% of the species from GenBank unambiguously identified). The resolution of the P6 loop is lower (19.5% identified) but remains higher than those of existing alternative systems. The resolution is much higher in specific contexts such as species originating from a single ecosystem, or commonly eaten plants. Despite the relatively low resolution, the whole trnL intron and its P6 loop have many advantages: the primers are highly conserved, and the amplification system is very robust. The P6 loop can even be amplified when using highly degraded DNA from processed food or from permafrost samples, and has the potential to be extensively used in food industry, in forensic science, in diet analyses based on feces and in ancient DNA studies.