The recent introduction by Carl Zeiss Ltd. of the Airyscan detector module for their LSM880 confocal laser-scanning microscope has enabled routine superresolution microscopy to be combined with the advantages of confocal-based fluorescence imaging. Resulting enhanced spatial resolution in X, Y, and Z provides tractable opportunity to derive new insight into protein localization(s), organelle dynamics, and thence protein function within trypanosomatids or other organisms. Here, we describe methods for preparing slides, cells, and basic microscope setup for fluorescence imaging of trypanosomatids using the LSM-880 with Airyscan platform.

Human African trypanosomiasis became a neglected disease after the 1960s, when case numbers dropped dramatically. It again became a public health problem in sub-Saharan Africa at the end of the 1990s, when new cases were reported, notably in Central Africa, and specifically in Gabon, where historic foci existed and new cases have been reported. Therefore, the present study reports on an entomological survey conducted in May 2012 to determine the pathogenic trypanosome infection rate in tsetse flies and characterize the diversity of Trypanosoma species in the Ivindo National Park (INP) in northeastern Gabon. Nine Vavoua traps were used to catch tsetse over a 7-days period. All tsetse flies captured were identified to species, dissected, and trypanosome species identified using polymerase chain reaction (PCR). In total, 160 tsetse flies were analyzed, including Glossina palpalis palpalis, Glossina fusca congolense, and Glossina tachinoïdes The trypanosome infection rate of the flies was 6.3 and 31.9% using microscopy and PCR, respectively. The species identified were Trypanosoma congolense savannah type, Trypanosoma brucei brucei, Trypanosoma brucei gambiense, Trypanosoma vivax, and Trypanosoma congolense forest type. Trypanosoma risk index was 0.75 and 7.05 for humans and for animals, respectively. This study illustrates the diversity of Trypanosoma species infecting the tsetse flies in the INP. The simultaneous occurrence of Trypanosoma and tsetse from the palpalis group may suggest that the reservoirs of African animal trypanosomiasis should be carefully monitored in this area.

BACKGROUND: Here we present a holistic screening of collapsing colonies from three professional apiaries in Spain. Colonies with typical honey bee depopulation symptoms were selected for multiple possible factors to reveal the causes of collapse.
RESULTS: Omnipresent were Nosema ceranae and Lake Sinai Virus. Moderate prevalences were found for Black Queen Cell Virus and trypanosomatids, whereas Deformed Wing Virus, Aphid Lethal Paralysis Virus strain Brookings and neogregarines were rarely detected. Other viruses, Nosema apis, Acarapis woodi and Varroa destructor were not detected. Palinologic study of pollen demonstrated that all colonies were foraging on wild vegetation. Consequently, the pesticide residue analysis was negative for neonicotinoids. The genetic analysis of trypanosomatids GAPDH gene, showed that there is a large genetic distance between Crithidia mellificae ATCC30254, an authenticated cell strain since 1974, and the rest of the presumed C. mellificae sequences obtained in our study or published. This means that the latter group corresponds to a highly differentiated taxon that should be renamed accordingly.
CONCLUSIONS: The results of this study demonstrate that the drivers of colony collapse may differ between geographic regions with different environmental conditions, or with different beekeeping and agricultural practices. The role of other pathogens in colony collapse has to bee studied in future, especially trypanosomatids and neogregarines. Beside their pathological effect on honey bees, classification and taxonomy of these protozoan parasites should also be clarified.

The literature shows that the effects of direct electric currents on biological material are numerous, including bactericidal, fungicidal, parasiticidal, and anti-tumoral, among others. Non-pathogenic trypanosomatids, such as Herpetomonas samuelpessoai, have emerged as important models for the study of basic biological processes performed by a eukaryotic cell. The present study reports a dose-dependent anti-protozoan effect of direct electric treatment with both cathodic and anodic current flows on H. samuelpessoai cells. The damaging effects can be attributable to the electrolysis products generated during electric stimulation. The pH of the cell suspension was progressively augmented from 7.4 to 10.5 after the cathodic treatment. In contrast, the anodic treatment caused a pH decrease varying from 7.4 to 6.5. Transmission electron microscopy analyses revealed profound alterations in vital cellular structures (e.g., mitochondrion, kinetoplast, flagellum, flagellar pocket, nucleus, and plasma membrane) after exposure to both cathodic and anodic current flows. Specifically, cathodic current flow treatment induced the appearance of autophagic-like structures on parasite cells, while those submitted to an anodic current flow presented marked disorganization of plasma membrane and necrotic appearance. However, parasites treated in the intermediary chamber (without contact with the electrodes) did not present significant changes in viability or morphology, and no pH variation was detected in this system. The use of H. samuelpessoai as a biological model and the direct electric current experimental approach used in our study provide important information for understanding the mechanisms involved in the cytotoxic effects of this physical agent.

Here, we describe the methodology currently used to analyze the structural organization of protozoa of the Trypanosomatidae family by atomic force microscopy. The results are compared with those obtained using light, scanning, and transmission electron microscopy.

The recent discovery of thousands of small noncoding RNAs (ncRNAs), in many different organisms, has led to the need for methods to study their function. One way to help understand their function is to determine what other RNAs interact with the ncRNAs. We have developed a novel method to investigate the RNA-RNA interactions between a small RNA and its target that we termed \"RNA walk.\" The method is based on UV-induced AMT cross-linking in vivo followed by affinity selection of the hybrid molecules and mapping the intermolecular adducts by RT-PCR. Domains carrying the cross-linked adducts are less efficiently amplified than domains that are not cross-linked. Real-time PCR is used to quantify the results. Further mapping of the interactions is performed by primer extension to determine the exact cross-linked adduct.

In this study we describe a novel method to investigate the RNA-RNA interactions between a small RNA and its target that we termed \'RNA walk\'. The method is based on UV-induced AMT cross-linking in vivo followed by affinity selection of the hybrid molecules and mapping the intermolecular adducts by RT-PCR or real-time PCR. Domains carrying the cross-linked adducts fail to efficiently amplify by PCR compared with non-cross-linked domains. This method was calibrated and used to study the interaction between a special tRNA-like molecule (sRNA-85) that is part of the trypanosome signal recognition particle (SRP) complex and the ribosome. Four contact sites between sRNA-85 and rRNA were identified by \'RNA walk\' and were further fine-mapped by primer extension. Two of the contact sites are expected; one contact site mimics the interaction of the mammalian Alu domain of SRP with the ribosome and the other contact sites include a canonical tRNA interaction. The two other cross-linked sites could not be predicted. We propose that \'RNA walk, is a generic method to map target RNA small RNAs interactions in vivo.

The glyoxalase system, comprising the metalloenzymes glyoxalase I (GLO1) and glyoxalase II (GLO2), is an almost universal metabolic pathway involved in the detoxification of the glycolytic byproduct methylglyoxal to d-lactate. In contrast to the situation with the trypanosomatid parasites Leishmania major and Trypanosoma cruzi, this trypanothione-dependent pathway is less well understood in the African trypanosome, Trypanosoma brucei. Although this organism possesses a functional GLO2, no apparent GLO1 gene could be identified in the T. brucei genome. The absence of GLO1 in T. brucei was confirmed by the lack of GLO1 activity in whole cell extracts, failure to detect a GLO1-like protein on immunoblots of cell lysates, and lack of d-lactate formation from methylglyoxal as compared to L. major and T. cruzi. T. brucei procyclics were found to be 2.4-fold and 5.7-fold more sensitive to methylglyoxal toxicity than T. cruzi and L. major, respectively. T. brucei also proved to be the least adept of the \'Tritryp\' parasites in metabolizing methylglyoxal, producing l-lactate rather than d-lactate. Restoration of a functional glyoxalase system by expression of T. cruzi GLO1 in T. brucei resulted in increased resistance to methylglyoxal and increased conversion of methylglyoxal to d-lactate, demonstrating that GLO2 is functional in vivo. Procyclic forms of T. brucei possess NADPH-dependent methylglyoxal reductase and NAD(+)-dependent l-lactaldehyde dehydrogenase activities sufficient to account for all of the methylglyoxal metabolized by these cells. We propose that the predominant mechanism for methylglyoxal detoxification in the African trypanosome is via the methylglyoxal reductase pathway to l-lactate.

Three different DNA fingerprinting techniques, the mobile genetic element (MGE)-PCR, simple sequence repeat (SSR)-PCR and random amplified polymorphic DNA (RAPD)-PCR, were used to define a large set of genetic markers to study genetic similarity within and among Trypanosoma brucei, Trypanosoma equiperdum and Trypanosoma evansi strains (n=18) from China, Africa and South America and to investigate their genetic relationships. Using the three fingerprinting techniques, >890 bands (ranging in size from 0.2 to 2kb) were defined for all 18 strains of Trypanosoma. Within each of the strains, 39-59 bands were defined. The similarity coefficients between strains ranged from approximately 41 to 94%, with a mean of 65%. There was more genetic similarity among strains within T. evansi (mean of approximately 79%) compared with T. equiperdum ( approximately 65%) and T. brucei ( approximately 59%). The similarity coefficient data were used to construct the dendrogram, which revealed that (irrespective of species) the majority of strains from China and South America grouped together to the exclusion of those from Africa. The exceptions were a T. brucei strain from Africa and a T. equiperdum strain of unknown origin. Hence, employing data sets generated using the three different fingerprinting methods, it was not possible to unequivocally distinguish among T. brucei, T. evansi and T. equiperdum, although there was a tendency for T. evansi strains to group together to the exclusion of T. brucei. The findings provide support for the hypothesis that T. evansi originated from a mutated form of T. equiperdum and stimulate further investigations of the genetic make-up and evolution of members of the subgenus Trypanozoon.

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