Functional characterisation of cohesin subunit SMC3 and separase and their roles in the segregation of large and minichromosomes in Trypanosoma brucei

The genome of the African trypanosome, Trypanosoma brucei, presents an unusual karyotype in which two main classes of chromosomes, large and small minichromosomes, need to be faithfully replicated and segregated during the cell cycle. Although the large and minichromosomes are colocalised and segregated by association with the mitotic spindle, minichromosomes exhibit segregation patterns that differ from those observed for large chromosomes. To address whether this difference is reflected at a molecular level, two different proteins that have highly conserved functions in eukaryotic chromosomes segregation were characterised in this study. The first protein, SMC3, is a component of the chromosome cohesion apparatus that holds sister chromatids together after their replication until segregation at anaphase. The second protein, separase, is a cysteine protease that resolves sister chromatid cohesion at the onset of anaphase and has, in other organisms, additional functions during mitosis. The T. brucei cohesin subunit, TbSMC3, localised to the nucleus as a chromatin-bound protein from G1 phase until metaphase and dissociated from chromatin during anaphase until the completion of cell division. On the other hand, cytoplasmic localisation of separase with nuclear exclusion was prevalent until the onset of metaphase when the protein re-localised to the nucleus, thus providing a potential control mechanism to prevent premature cohesin cleavage. Interference with the normal expression of SMC3 and separase by RNA interference resulted in defects in growth rate, cell cycle progression and chromosomes segregation. TbSMC3 depletion produced a lethal phenotype and inhibition of cell cycle progression. Similarly, lethality with severe inhibition of cell cycle progression was the main feature of separase depletion. Using fluorescence in situ hybridisation (FISH), it was shown that SMC3 depletion had no visible effect on the symmetric segregation of the minichromosome population, but interferes with the faithful mitotic segregation of large chromosomes. In contrast, separase depletion blocks the segregation of both large and minichromosomes. In separase-depleted mitotic cells, cohesins remained bound to chromatin, which is in contrast to rapid dissociation of cohesins from chromatin in wild-type mitotic cells. The severity of segregation phenotypes after separase depletion was additionally explained by defects in the mitotic spindle assembly. In both SMC3 and separase depleted cells, cytokinesis in the absence of mitosis/karyokinesis was not inhibited in procyclic cells, resulting in the generation of anucleate 'zoid' cells. The lethality imposed on trypanosome cells after depletion of both SMC3 and separase proteins indicate that they can serve as potential drug targets for anti-parasite chemotherapy

Spatial scale influences the distribution of viral diversity in the eukaryotic virome of the mosquito Culex pipiens

Our knowledge of the diversity of eukaryotic viruses has recently undergone a massive expansion. This diversity could infuence host physiology through yet unknown phenomena of potential interest to the felds of health and food production. However, the assembly processes of this diversity remain elusive in the eukaryotic viromes of terrestrial animals. This situation hinders hypothesis-driven tests of virome infuence on host physiology. Here, we compare taxonomic diversity between different spatial scales in the eukaryotic virome of the mosquito Culex pipiens. This mosquito is a vector of human pathogens worldwide. The experimental design involved sampling in fve countries in Africa and Europe around the Mediterranean Sea and large mosquito numbers to ensure a thorough exploration of virus diversity. A group of viruses was found in all countries. This core group represented a relatively large and diverse fraction of the virome. However, certain core viruses were not shared by all host individuals in a given country, and their infection rates fuctuated between countries and years. Moreover, the distribution of coinfections in individual mosquitoes suggested random co-occurrence of those core viruses. Our results also suggested differences in viromes depending on geography, with viromes tending to cluster depending on the continent. Thus, our results unveil that the overlap in taxonomic diversity can decrease with spatial scale in the eukaryotic virome of C. pipiens. Furthermore, our results show that integrating contrasted spatial scales allows us to identify assembly patterns in the mosquito virome. Such patterns can guide future studies of virome infuence on mosquito physiology.This work was supported by the 7th Framework Programme of the European Commission (grant FP7-613996 Vmerge), the Montpellier University of Excellence Program (MUSE, ArboSud project), and the Direction Générale de l’Alimentation from the French Ministry of Agriculture and Food (DGAl grant agreement: SPA17 n0079-E). The contents of this publication are the sole responsibility of the authors and do not necessarily refect the views of the European Commission.info:eu-repo/semantics/publishedVersio

Molecular and microscopic detection of natural and experimental infections of Toxocara vitulorum in bovine milk.

Toxocara vitulorum is an Ascarid nematode infecting the small intestine of buffalo and cattle particularly neonate calves, with the postnatal route through milk is the main infection source. However, little is known about shedding rates and the optimum detection methods of T. vitulorum larvae in the milk of the infected bovine hosts. In this study, we aimed to evaluate the use of two methods, microscopy and PCR, and their detection limits both under the experimental and natural infection situations. In doing this, T. vitulorum eggs extracted from naturally occurring adult female worms were successfully subjected to experimental embryonation, and larvae were implemented in experimental infection of milk in ascending infection doses of 0, 1, 5, 10, 20, 50 larvae/2-ml milk samples. With the except of negative control, microscopy-based examination detected larvae in all samples, albeit with means, ranges, and the total number of larvae were detected in exponential rates relative to larvae densities in milk samples. PCR technique corresponded well to microscopy in detecting genomic DNA of T. vitulorum larvae in all milk samples down to a single larva/sample. On the other hand, and by applying the same methodology approach on 50 naturally-occurring bovine colostrum/milk samples, 13 (26%) and 20 (40%) samples were tested positive for T. vitulorum infection by microscopy and the PCR-based detection, respectively. Of these, 11 out of 26 buffalo samples (42.30%) and 2 out of 24 cow samples (8.33%) were tested positive by microscopy, while 16 (61.54%) and 3 (12.50%) of buffalo and cow samples were tested positive by PCR, respectively. By applying the Agreement Coefficient, substantial agreement (0.77) between molecular and microscopy detection was detected from all tested samples. In conclusion, larvae of T. vitulorum were unequivocally detected by microscopy and molecular methods in milk samples both under the experimental and natural field situations. Nevertheless, slightly higher rates by PCR than microscopy were obtained when detecting naturally-infected milk samples. To the best of our knowledge, this is the first in situ detection of larvae of T. vitulorum in the milk of the naturally infected animals

Assessment of the inhibitory effects of disinfectants on the embryonation of Ascaridia columbae eggs.

This study was set out with the aim of assessing the effects of the most commonly available commercial disinfectants on embryogenesis of A. columbae eggs. In addition to the distilled water treatment as a control group, four disinfectants were tested that included formalin, povidone iodide, TH4, and Virkon-S, in three independent experiments. In the first experiment, an overnight incubation with the working concentration of disinfectants resulted in significant inhibition of 80%, 85%, and 98% of embryonic development at day 9 post-treatment with formalin, povidone iodide, and TH4, respectively. This inhibition was continued through days 12 and 15 with the three tested disinfectants. Virkon-S did not affect embryogenesis with larval development comparable to that of the control group. In the second more dissected experiment, contact times of 10, 20, 30, and 60 minutes were set out for each disinfectant with embryogenesis inhibition results echoed that of the first experiment, with all disinfectants but Virkon-S inhibited larval development in significant proportions of eggs. Again, Virkon-S was very neutral in its effect on embryogenesis. When pigeon fecal matters were mixed with eggs and were subjected to disinfectants, discrepancies to results of the first two experiments were observed with only formalin inhibited embryogenesis in considerable proportion of eggs. Thus, and with the exception of Virkon-S, disinfectants tested at levels similar to those applicable in poultry in-houses and farms exhibited potent ovicidal activities on free eggs. To our knowledge, this is the first study involving the application of the commonly used in-poultry houses disinfectants to inactivate or delay the embryogenesis of bird Ascarids. The future perspective will potentially involve the in-field applications of the efficient disinfectants to eliminate or reduce the dissemination of infections with bird Ascarids in the chicken, turkey, pigeons, and other poultry houses

Functional characterisation of cohesin subunit SMC3 and separase and their roles in the segregation of large and minichromosomes in Trypanosoma brucei

The genome of the African trypanosome, Trypanosoma brucei, presents an unusual karyotype in which two main classes of chromosomes, large and small minichromosomes, need to be faithfully replicated and segregated during the cell cycle. Although the large and minichromosomes are colocalised and segregated by association with the mitotic spindle, minichromosomes exhibit segregation patterns that differ from those observed for large chromosomes. To address whether this difference is reflected at a molecular level, two different proteins that have highly conserved functions in eukaryotic chromosomes segregation were characterised in this study. The first protein, SMC3, is a component of the chromosome cohesion apparatus that holds sister chromatids together after their replication until segregation at anaphase. The second protein, separase, is a cysteine protease that resolves sister chromatid cohesion at the onset of anaphase and has, in other organisms, additional functions during mitosis. The T. brucei cohesin subunit, TbSMC3, localised to the nucleus as a chromatin-bound protein from G1 phase until metaphase and dissociated from chromatin during anaphase until the completion of cell division. On the other hand, cytoplasmic localisation of separase with nuclear exclusion was prevalent until the onset of metaphase when the protein re-localised to the nucleus, thus providing a potential control mechanism to prevent premature cohesin cleavage. Interference with the normal expression of SMC3 and separase by RNA interference resulted in defects in growth rate, cell cycle progression and chromosomes segregation. TbSMC3 depletion produced a lethal phenotype and inhibition of cell cycle progression. Similarly, lethality with severe inhibition of cell cycle progression was the main feature of separase depletion. Using fluorescence in situ hybridisation (FISH), it was shown that SMC3 depletion had no visible effect on the symmetric segregation of the minichromosome population, but interferes with the faithful mitotic segregation of large chromosomes. In contrast, separase depletion blocks the segregation of both large and minichromosomes. In separase-depleted mitotic cells, cohesins remained bound to chromatin, which is in contrast to rapid dissociation of cohesins from chromatin in wild-type mitotic cells. The severity of segregation phenotypes after separase depletion was additionally explained by defects in the mitotic spindle assembly. In both SMC3 and separase depleted cells, cytokinesis in the absence of mitosis/karyokinesis was not inhibited in procyclic cells, resulting in the generation of anucleate 'zoid' cells. The lethality imposed on trypanosome cells after depletion of both SMC3 and separase proteins indicate that they can serve as potential drug targets for anti-parasite chemotherapy.EThOS - Electronic Theses Online ServiceGovernment of Egypt and Ministry of Higher Education (sponsor)GBUnited Kingdo