Caffeine concentrations in mice plasma and testicular tissue and the effect of caffeine on the dominant lethal test.

Large groups of male Swiss mice received per os on average 100 mg caffeine per kg body weight per day for 1 or 8 weeks. The dominant lethal test was designed to achieve maximum sensitivity in order to detect any possible mutagenic effect. No mutagenic induction of dominant lethals, pre-implantation egg loss or depression of the fertility of females, caused by caffeine at the dose levels administered were observed. The half life of caffeine, which was between 2.5 and 3 h, was similar in plasma and testicular tissue. It was concluded that caffeine did not accumulate in the testicular tissue of mice. The maximum concentration of caffeine found was below 10 microgram/g testicular tissue, which is about a 100 times lower than concentrations that cause chromosome aberrations in cultured mammalian cells.     

NMR localization of the O-mycoloylation on PorH,a channel forming peptide from Corynebacterium glutamicum

PorH and PorA are two small peptides that, in complex, form a voltage-dependent ion channel in the outer membrane of Corynebacterium glutamicum. Specific post-translational modifications on PorA and PorH are required for the formation of a functional ion channel. The assignment of PorH proton NMR chemical shifts in DMSO, allowed identifying unambiguously the exact position of the PorH O-mycoloylation on Ser 56 side chain. This was further confirmed by site directed mutagenesis and mass spectrometry. Together with the previously published localization of PorA mycoloylation, this provides the complete primary structure characterization of this outer membrane porin.     

Surface Area Loss and Increased Sphericity Account for the Splenic Entrapment of Subpopulations of Plasmodium falciparum Ring-Infected Erythrocytes

Ex vivo perfusion of human spleens revealed innate retention of numerous cultured Plasmodium falciparum ring-infected red blood cells (ring-iRBCs). Ring-iRBC retention was confirmed by a microsphiltration device, a microbead-based technology that mimics the mechanical filtering function of the human spleen. However, the cellular alterations underpinning this retention remain unclear. Here, we use ImageStream technology to analyze infected RBCs’ morphology and cell dimensions before and after fractionation with microsphiltration. Compared to fresh normal RBCs, the mean cell membrane surface area loss of trophozoite-iRBCs, ring-iRBCs and uninfected co-cultured RBCs (uRBCs) was 14.2% (range: 8.3–21.9%), 9.6% (7.3–12.2%) and 3.7% (0–8.4), respectively. Microsphilters retained 100%, ∼50% and 4% of trophozoite-iRBCs, ring-iRBCs and uRBCs, respectively. Retained ring-iRBCs display reduced surface area values (estimated mean, range: 17%, 15–18%), similar to the previously shown threshold of surface-deficient RBCs retention in the human spleen (surface area loss: >18%). By contrast, ring-iRBCs that successfully traversed microsphilters had minimal surface area loss and normal sphericity, suggesting that these parameters are determinants of their retention. To confirm this hypothesis, fresh normal RBCs were exposed to lysophosphatidylcholine to induce a controlled loss of surface area. This resulted in a dose-dependent retention in microsphilters, with complete retention occurring for RBCs displaying >14% surface area loss. Taken together, these data demonstrate that surface area loss and resultant increased sphericity drive ring-iRBC retention in microsphilters, and contribute to splenic entrapment of a subpopulation of ring-iRBCs. These findings trigger more interest in malaria research fields, including modeling of infection kinetics, estimation of parasite load, and analysis of risk factors for severe clinical forms. The determination of the threshold of splenic retention of ring-iRBCs has significant implications for diagnosis (spleen functionality) and drug treatment (screening of adjuvant therapy targeting ring-iRBCs).     

High Content Analysis of Primary Macrophages Hosting Proliferating Leishmania Amastigotes: Application to Anti-leishmanial Drug Discovery

Background/Objectives

Human leishmaniases are parasitic diseases causing severe morbidity and mortality. No vaccine is available and numerous factors limit the use of current therapies. There is thus an urgent need for innovative initiatives to identify new chemotypes displaying selective activity against intracellular Leishmania amastigotes that develop and proliferate inside macrophages, thereby causing the pathology of leishmaniasis.

Methodology/Principal Findings

We have developed a biologically sound High Content Analysis assay, based on the use of homogeneous populations of primary mouse macrophages hosting Leishmania amazonensis amastigotes. In contrast to classical promastigote-based screens, our assay more closely mimics the environment where intracellular amastigotes are growing within acidic parasitophorous vacuoles of their host cells. This multi-parametric assay provides quantitative data that accurately monitors the parasitic load of amastigotes-hosting macrophage cultures for the discovery of leishmanicidal compounds, but also their potential toxic effect on host macrophages. We validated our approach by using a small set of compounds of leishmanicidal drugs and recently published chemical entities. Based on their intramacrophagic leishmanicidal activity and their toxicity against host cells, compounds were classified as irrelevant or relevant for entering the next step in the drug discovery pipeline.

Conclusions/Significance

Our assay represents a new screening platform that overcomes several limitations in anti-leishmanial drug discovery. First, the ability to detect toxicity on primary macrophages allows for discovery of compounds able to cross the membranes of macrophage, vacuole and amastigote, thereby accelerating the hit to lead development process for compounds selectively targeting intracellular parasites. Second, our assay allows discovery of anti-leishmanials that interfere with biological functions of the macrophage required for parasite development and growth, such as organelle trafficking/acidification or production of microbicidal effectors. These data thus validate a novel phenotypic screening assay using virulent Leishmania amastigotes growing inside primary macrophage to identify new chemical entities with bona fide drug potential.     

NMR secondary structure and interactions of recombinant human MOZART1 protein,a component of the gamma‐tubulin complex

Mitotic‐spindle organizing protein associated with a ring of γ‐tubulin 1 (MOZART1) is an 8.5 kDa protein linked to regulation of γ‐tubulin ring complexes (γTuRCs), which are involved in nucleation of microtubules. Despite its small size, MOZART1 represents a challenging target for detailed characterization in vitro. We described herein a protocol for efficient production of recombinant human MOZART1 in Escherichia coli and assessed the properties of the purified protein using a combination of size exclusion chromatography coupled with multiangle light scattering (SEC‐MALS), dynamic light scattering (DLS), and nuclear magnetic resonance (NMR) experiments. MOZART1 forms heterogeneous oligomers in solution. We identified optimal detergent and buffer conditions for recording well resolved NMR experiments allowing nearly full protein assignment and identification of three distinct alpha‐helical structured regions. Finally, using NMR, we showed that MOZART1 interacts with the N‐terminus (residues 1–250) of GCP3 (γ‐tubulin complex protein 3). Our data illustrate the capacity of MOZART1 to form oligomers, promoting multiple contacts with a subset of protein partners in the context of microtubule nucleation.     

Map of open and closed chromatin domains in Drosophila genome

Background

Chromatin compactness has been considered a major determinant of gene activity and has been associated with specific chromatin modifications in studies on a few individual genetic loci. At the same time, genome-wide patterns of open and closed chromatin have been understudied, and are at present largely predicted from chromatin modification and gene expression data. However the universal applicability of such predictions is not self-evident, and requires experimental verification.

Results

We developed and implemented a high-throughput analysis for general chromatin sensitivity to DNase I which provides a comprehensive epigenomic assessment in a single assay. Contiguous domains of open and closed chromatin were identified by computational analysis of the data, and correlated to other genome annotations including predicted chromatin “states”, individual chromatin modifications, nuclear lamina interactions, and gene expression. While showing that the widely trusted predictions of chromatin structure are correct in the majority of cases, we detected diverse “exceptions” from the conventional rules. We found a profound paucity of chromatin modifications in a major fraction of closed chromatin, and identified a number of loci where chromatin configuration is opposite to that expected from modification and gene expression patterns. Further, we observed that chromatin of large introns tends to be closed even when the genes are expressed, and that a significant proportion of active genes including their promoters are located in closed chromatin.

Conclusions

These findings reveal limitations of the existing predictive models, indicate novel mechanisms of epigenetic regulation, and provide important insights into genome organization and function.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-988) contains supplementary material, which is available to authorized users.     

Plant sterols: a neutron diffraction study of sitosterol and stigmasterol in soybean phosphatidylcholine membranes

Neutron scattering experiments have been performed on oriented Soybean phosphatidylcholine (SPC) bilayers, containing sitosterol or stigmasterol, two major sterols of plant plasma membranes. Sitosterol and stigmasterol were either protonated or deuterated on position C25 of the lateral chain. Incorporation of sitosterol leads to an increase of the hydrophobic thickness of SPC bilayers of 1.2 and 2 A when present, at 16 and 30 mol%, respectively. On the other hand, no change was observed when stigmasterol is present in the bilayer at its maximal solubility of 16 mol%. These results are in agreement with the fact that sitosterol is more efficient than stigmasterol to order acyl chains of SPC, as already shown with other biophysical techniques. In order to get more insight into the behavior of the lateral chains of the two sterols, the proton-deuterium contrast method was used in order to locate the (2)H25 atoms of the two sterols. For sitosterol, this atom was found close to the center of the bilayer at +/-(1.6+/-0.2 A), with a width, nu=2.5+/-0.5 A. For stigmasterol, the difference profile could be fitted in two different ways: either two possible locations are found at +/-(2.3+/-0.2 A) and +/-(10+/-0.2 A) with the same width, nu=2.5+/-0.5 A or only one broad distribution at +/-(6.1+/-0.3 A), nu=8.5+/-0.7 A. The results are discussed in terms of difference of dynamics for the lateral chain of the two sterols.     

The Cryo-EM structure of a complete 30S translation initiation complex from Escherichia coli

Formation of the 30S initiation complex (30S IC) is an important checkpoint in regulation of gene expression. The selection of mRNA, correct start codon, and the initiator fMet-tRNA(fMet) requires the presence of three initiation factors (IF1, IF2, IF3) of which IF3 and IF1 control the fidelity of the process, while IF2 recruits fMet-tRNA(fMet). Here we present a cryo-EM reconstruction of the complete 30S IC, containing mRNA, fMet-tRNA(fMet), IF1, IF2, and IF3. In the 30S IC, IF2 contacts IF1, the 30S subunit shoulder, and the CCA end of fMet-tRNA(fMet), which occupies a novel P/I position (P/I1). The N-terminal domain of IF3 contacts the tRNA, whereas the C-terminal domain is bound to the platform of the 30S subunit. Binding of initiation factors and fMet-tRNA(fMet) induces a rotation of the head relative to the body of the 30S subunit, which is likely to prevail through 50S subunit joining until GTP hydrolysis and dissociation of IF2 take place. The structure provides insights into the mechanism of mRNA selection during translation initiation.     

Leishmania parasites: could we consider them as living organisms per se?

Over the last 10 years - in Microbes and Infection - the publications dealing with protozoan parasites were mainly providing insights on the pathogenic processes leading to the local or systemic damages in the mammals, these parasitic organisms exploit/subvert as hosts. As a result, many investigators introduced the objectives of their analysis by referring to "host-pathogen" interactions. Though we, as investigators, are all determined to decipher the pathogenic processes which can indeed be coupled to the parasite uncontrolled development, I think that the parasites - alike the living organisms they subvert as hosts - need to be considered as living organisms per se, instead of being considered as "pathogens". Such a conceptual frame will promote research on the processes on which relies their perpetuation whatever the level under investigations - individual and/or population level. Only the unicellular protozoan parasites of the genus Leishmania known to be hosted by blood-feeding insects and mammals will be further considered in this brief contribution.