The toxic dinoflagellate Alexandrium catenella isolated from fjords in Southern Chile produces several analogues of saxitoxin and has been associated with outbreaks of paralytic shellfish poisoning. Three bacterial strains, which remained in close association with this dinoflagellate in culture, were isolated by inoculating the dinoflagellate onto marine agar. The phenotypically different cultivable bacterial colonies were purified. Their genetic identification was done by polymerase chain reaction amplification of the 16S rRNA genes. Partial sequence analysis suggested that the most probable affiliations were to two bacterial phyla: Proteobacteria and the Cytophaga group. The molecular identification was complemented by morphological data and biochemical profiling. The three bacterial species, when grown separately from phytoplankton cells in high-nutrient media, released algal-lytic compounds together with aminopeptidase, lipase, glucosaminidase, and alkaline phosphatase. When the same bacteria, free of organic nutrients, were added back to the algal culture they displayed no detrimental effects on the dinoflagellate cells and recovered their symbiotic characteristics. This observation is consistent with phylogenetic analysis that reveals that these bacteria correspond to species distinct from other bacterial strains previously classified as algicidal bacteria. Thus, bacterial-derived lytic activities are expressed only in the presence of high-nutrient culture media and it is likely that in situ environmental conditions may modulate their expression.

Glioblastoma (GBM) is the most prevalent type of primary brain tumor. Treatment options include maximal surgical resection and drug-radiotherapy combination. However, patient prognosis remains very poor, prompting the search for new models for drug discovery and testing, especially those that allow assessment of in vivo responses to treatment. Zebrafish xenograft models have an enormous potential to study tumor behavior, proliferation and cellular interactions. Here, an in vivo imaging and proliferation assessment method of human GBM xenograft in zebrafish larvae is introduced. Zebrafish larvae microinjected with fluorescently labeled human GBM cells were screened daily using a stereomicroscope and imaged by light sheet fluorescence microscopy (LSFM); volumetric modeling and composite reconstructions were done in single individuals. Larvae containing tumors were enzymatically dissociated, and proliferation of cancer cells was measured using dye dilution by flow cytometry. GBM micro-tumors formed mainly in the zebrafish yolk sac and perivitelline space following injection in the yolk sac, with an engraftment rate of 73%. Daily image analysis suggested cellular division, as micro-tumors progressively grew with differentiated fluorescence intensity signals. Using dye dilution assay by flow cytometry, at least three GBM cells' division cycles were identified. The combination of LSFM and flow cytometry allows assessment of proliferation and tumor growth of human GBM inside zebrafish, making it a useful model to identify effective anti-proliferative agents in a preclinical setting.

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For a deeper understanding of the phylogenetic relationships of Echinococcus genotypes and species in different intermediate hosts, we analyzed samples from human and bovine hydatid cysts. For this, segments of the cytochrome oxidase (COX1) and NADH dehydrogenase (ND1) mitochondrial genes were used. To obtain sufficient amounts of the ND1 marker to be sequenced properly, a new variant of the PCR assay was implemented. Phylogenetic analysis with both markers showed that most of the analyzed samples correspond to genotype G1. However, a sample from cysts of a bovine lung (Q21), with the COX1 marker, was grouped in a node together with a sample belonging to genotype G3. In the phylogenetic tree obtained with the ND1 marker, this sample was grouped with sequences of genotypes G3, G2, and G4. Analyzing the single nucleotide polymorphic (SNP) sites of both markers, it was observed that the Q21 sequence is almost identical to the G3 sequence and differ in only one SNP from the G2 sequence, and is completely different from G4. These results are noteworthy, since neither G2 nor G3 genotypes have been described previously in Chile, raising the possibility that the G3 genotype is present in these latitudes. This information is highly relevant; it can be employed to uncover additional unknown details of transmission cycles of this important parasite.

To better understand the evolution of the etiologic agent of Chagas disease, we cloned and sequenced 25 alleles from five Tripanosoma cruzi microsatellite markers. The study of the sequences showed highly conserved alleles present in T. cruzi clones belonging to TCI, TCIIc, and TCIIe. This result was also confirmed by the phylogenetic analysis of MCLE01 allele sequences. The examination by capillary electrophoresis of six microsatellite markers from 19 T. cruzi clones showed a high proportion of the alleles found both in the TCI and TCII sublineages. The phylogenetic reconstruction of these 19 clones produced a tree with two major clusters with bootstrap support of 100% and 95%. The first cluster includes T. cruzi clones belonging to the TCI and TCIIa lineages. The second cluster is composed of TCI, TCIIc, TCIId, and TCIIe T. cruzi clones. The analysis of five microsatellite markers in the CLBrener genome showed that almost all the microsatellite markers are synteny; non-Esmeraldo and Esmeraldo haplotypes probably come from the TCIIc and TCIIb lineages. Taken together, our results are in agreement with the two hybridization events hypothesis as the origin of current T. cruzi lineages.

To investigate whether Trypanosoma cruzi populations found in chagasic cardiopathic and non-cardiopathic patients are genetically differentiated, three molecular microsatellite markers were analysed. This analysis was also applied to compare T. cruzi samples from peripheral blood or dejections of Triatoma infestans fed on the blood of the same patients. In order to obtain the first objective, analyses of predominant T. cruzi genotypes were conducted using three approaches: a locus-by-locus analysis; a Fisher method across three loci; and analysis of molecular variance by Genepop and Arlequin programs. Only with one locus and on the blood samples was a significant differentiation detected among non-cardiopathic and cardiopathic groups, which was not confirmed by the other two methods. On the contrary, with the three approaches, it was found that T. cruzi clones present in the blood of patients are genetically differentiated from those detected in dejections of T. infestans fed on the same patients. Our results showed that the most frequent lineage both in blood as well as in triatomine dejection samples was TcI. No significant difference in T. cruzi lineage distribution was observed among chagasic cardiopathic and non-cardiopathic patients. The majority of the samples (50-60%) had only one T. cruzi clone (uniclonal) either in blood or dejection samples.