Visual genome-wide RNAi screening to identify human host factors required for Trypanosoma cruzi infection

The protozoan parasite Trypanosoma cruzi is the etiologic agent of Chagas disease, a neglected tropical infection that affects millions of people in the Americas. Current chemotherapy relies on only two drugs that have limited efficacy and considerable side effects. Therefore, the development of new and more effective drugs is of paramount importance. Although some host cellular factors that play a role in T. cruzi infection have been uncovered, the molecular requirements for intracellular parasite growth and persistence are still not well understood. To further study these host-parasite interactions and identify human host factors required for T. cruzi infection, we performed a genome-wide RNAi screen using cellular microarrays of a printed siRNA library that spanned the whole human genome. The screening was reproduced 6 times and a customized algorithm was used to select as hits those genes whose silencing visually impaired parasite infection. The 162 strongest hits were subjected to a secondary screening and subsequently validated in two different cell lines. Among the fourteen hits confirmed, we recognized some cellular membrane proteins that might function as cell receptors for parasite entry and others that may be related to calcium release triggered by parasites during cell invasion. In addition, two of the hits are related to the TGF-beta signaling pathway, whose inhibition is already known to diminish levels of T. cruzi infection. This study represents a significant step toward unveiling the key molecular requirements for host cell invasion and revealing new potential targets for antiparasitic therapy.     

Gonad Differentiation in the Rabbit: Evidence of Species-Specific Features

The rabbit is an attractive species for the study of gonad differentiation because of its 31-day long gestation, the timing of female meiosis around birth and the 15-day delay between gonadal switch and the onset of meiosis in the female. The expression of a series of genes was thus determined by qPCR during foetal life until adulthood, completed by a histological analysis and whenever possible by an immunohistological one. Interesting gene expression profiles were recorded. Firstly, the peak of SRY gene expression that is observed in early differentiated XY gonads in numerous mammals was also seen in the rabbit, but this expression was maintained at a high level until the end of puberty. Secondly, a peak of aromatase gene expression was observed at two-thirds of the gestation in XX gonads as in many other species except in the mouse. Thirdly, the expression of STRA8 and DMC1 genes (which are known to be specifically expressed in germ cells during meiosis) was enhanced in XX gonads around birth but also slightly and significantly in XY gonads at the same time, even though no meiosis occurs in XY gonad at this stage. This was probably a consequence of the synchronous strong NANOS2 gene expression in XY gonad. In conclusion, our data highlighted some rabbit-specific findings with respect to the gonad differentiation process.     

One-step affinity purification of fusion proteins with optimal monodispersity and biological activity: application to aggregation-prone HPV E6 proteins

Background

Bacterial expression and purification of recombinant proteins under homogeneous active form is often challenging. Fusion to highly soluble carrier proteins such as Maltose Binding Protein (MBP) often improves their folding and solubility, but self-association may still occur. For instance, HPV E6 oncoproteins, when produced as MBP-E6 fusions, are expressed as mixtures of biologically inactive oligomers and active monomers. While a protocol was previously developed to isolate MBP-E6 monomers for structural studies, it allows the purification of only one MBP-E6 construct at the time. Here, we explored a parallelizable strategy more adapted for biophysical assays aiming at comparing different E6 proteins.

Results

In this study, we took advantage of the distinct size and diffusion properties of MBP-E6 monomers and oligomers to separate these two species using a rapid batch preparation protocol on affinity resins. We optimized resin reticulation, contact time and elution method in order to maximize the proportion of monomeric MBP-E6 in the final sample. Analytical size-exclusion chromatography was used to quantify the different protein species after purification. Thus, we developed a rapid, single-step protocol for the parallel purification of highly monomeric MBP-E6 samples. MBP-fused HPV16 E6 samples obtained by this approach were validated by testing the binding to their prototypical peptide targets (the LXXLL motif from ubiquitine ligase E6AP) by BIAcore-SPR assay.

Conclusions

We have designed a rapid single-step batch affinity purification approach to isolate biologically active monomers of MBP-fused E6 proteins. This protocol should be generalizable to isolate the monomer (or the minimal biologically active oligomer) of other proteins prone to self-association.

     

m5C Methylation Guides Systemic Transport of Messenger RNA over Graft Junctions in Plants

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Locomotor activity rhythms in two sympatric parasitoid insects: Eupelmus orientalis and Eupelmus vuilleti (Hyménoptera, Eupelmidae)

With an automatic image analysis device, we studied the temporal distribution of the locomotor activity of E. orientalis and E. vuilleti during 24 h, and over several days to know whether the activity rhythms of these two Eupelmidae play a role in their competitive interactions. The analysis of locomotor activity rhythms of E. orientalis and E. vuilleti shows that the locomotor activity of both species presents daily cyclic variations. These two Eupelmidae have similar activity rhythms. Displacements of these parasitoids essentially take place during the photophase. But the activity of E. vuilleti is earlier, because the individuals of this species start their activity on average 4 to 5 h earlier than those of E. orientalis. E. vuilleti begins its displacements several hours before the onset of lighting, whereas E. orientalis is active only in the presence of the light. This shift of starting activity is thus a factor allowing these concurrent species to minimize their interactions during the cohabitation period in traditional granaries after the harvests of cowpea.     

Hydrocarbons,sterols and tocopherols in the seeds of six Adansonia species

The composition of the unsaponifiable matter of the lipids of six Adansonia species (A. grandidieri, A. za, A. fony, A. madagascariensis, A. digitata and A. suarezensis) was investigated. The total unsaponifiable content, its general composition and the identity of the components of the hydrocarbon, sterol and tocopherol fractions are presented. The unsaponifiable content in oil ranges from 0.4 to 1.1% (hexane method) and from 0.6 to 2.2% (diethyl ether method). In two species (A. grandidieri and A. suarezensis) the major components are 4-demethylsterols (23–42%) tocopherols (37-10%) and hydrocarbons (15–17%). In both species examined, eight 4-demethylsterols occur in the sterol fraction with sitosterol (81–88%) being predominant. Among the four tocopherols present, γ-tocopherol (68–98%) is the major compound. Each Adansonia species shows a characteristic gas liquid chromatography pattern for the hydrocarbon fraction. Squalene is the major component for five species (40–75%). Iso-, anteiso- and other branched hydrocarbons were not identified but were present in small amounts in comparison with n-alkanes. The dominance of odd- over even-carbon number chain length of n-alkanes was not observed in any species. The results show that C₂₂, C₂₅, C₂₆, C₂₇, C₂₈ and C₂₉ are the most frequent major constituents.     

Edaphic factors and flower colour distribution in theAnemoneae (Ranunculaceae)

Variation patterns of flower colour in some species of theAnemoneae can be correlated with edaphic variables. These examples are used in the argument that discriminative pollination through colour responsive pollen vectors is not the sole cause for non-randomness in intraspecific flower colour distributions. Some possible reasons for genetic linkage between genes controlling edaphic responses and genes controlling flower pigmentation are discussed.     

Influence de la présence de la fourmi Wasmannia auropunctata (Roger 1863) (Hymenoptera : Formicidae) sur les autres espèces de fourmis dans la réserve de la Lopé (centre du Gabon)

Résumé

Wasmannia auropunctata (Roger 1863) est une fourmi originaire d’Amérique du Sud. Elle a été introduite au Gabon vers 1920 par des agronomes qui l’ont utilisée comme agent de lutte biologique contre certains insectes parasites du cacaoyer. Aujourd’hui, cette fourmi se retrouve en zone forestière même hors des anciennes plantations de cacaoyers. Depuis 1984, sa présence a été signalée dans le parc national de la Lopé qui fait partie des réserves protégées du Gabon. Des études antérieures réalisées en Nouvelle-Calédonie ont montré que la présence de W. auropunctata a d’énormes conséquences sur la biodiversité car elle a fait disparaître l’ensemble des fourmis de cette région. Quel peut donc être l’impact de sa présence au Gabon dans la zone de la Lopé? Ces études préliminaires de l’impact de W. auropunctata sur la biodiversité de la Lopé ont consisté à évaluer la dispersion de cette espèce et la densité relative des autres espèces de fourmis en présence sur le site. Les résultats montrent que W. auropunctata se répartit selon un gradient décroissant du point d’introduction vers l’intérieur de la forêt jusqu’à une distance de 120 m. Il n’y a pas de W. auropunctata au-delà de cette distance. Du point d’introduction jusqu’à 80 m de distance, la densité relative des autres espèces de fourmis varie de 0 à 10 %. Lorsqu’il y a forte densité de W. auropunctata, les autres espèces de fourmis sont absentes.     

Suicides among cancer patients in Lithuania: A population-based census-linked study

Background: This study aims to estimate suicide risk and its socio-demographic determinants among cancer patients in the country showing the highest suicide rates among developed countries. Methods: The study is based on a unique census-linked dataset based on the linkages between the records from death and cancer registers and the 2001 population census records. Standardized mortality ratios for suicide (SMRs) were calculated for patients diagnosed with cancer in Lithuania between April 6, 2001 and December 31, 2009, relative to suicide rates in the general population. Results: We found that the relative suicide risk was elevated for both males and females, with SMRs of 1.43 (95% CI 1.23–1.66) and 1.32 (95% CI 0.95–1.80), respectively. This relationship for females became statistically significant and stronger after excluding skin cancers. The highest suicide risks were observed at older ages and during the period shortly after the diagnosis. The groups showing an increased suicide risk include lower educated, non-married, and rural male patients. Conclusion: The results of our study point to inadequacies of the health care system in dealing with mental health problems of cancer patients. Interventions allowing early detection of depression or suicidal ideation may help to prevent suicide among cancer patients in Lithuania.