Toward resolving the eukaryotic tree: the phylogenetic positions of jakobids and cercozoans

Resolving the global phylogeny of eukaryotes has proven to be challenging. Among the eukaryotic groups of uncertain phylogenetic position are jakobids, a group of bacterivorous flagellates that possess the most bacteria-like mitochondrial genomes known. Jakobids share several ultrastructural features with malawimonads and an assemblage of anaerobic protists (e.g., diplomonads and oxymonads). These lineages together with Euglenozoa and Heterolobosea have collectively been designated "excavates". However, published molecular phylogenies based on the sequences of nuclear rRNAs and up to six nucleus-encoded proteins do not provide convincing support for the monophyly of excavates, nor do they uncover their relationship to other major eukaryotic groups. Here, we report the first large-scale eukaryotic phylogeny, inferred from 143 nucleus-encoded proteins comprising 31,604 amino acid positions, that includes jakobids, malawimonads and cercozoans. We obtain compelling support for the monophyly of jakobids, Euglenozoa plus Heterolobosea (JEH group), and for the association of cercozoans with stramenopiles plus alveolates. Furthermore, we observe a sister-group relationship between the JEH group and malawimonads after removing fast-evolving species from the dataset. We discuss the implications of these results for the concept of "excavates" and for the elucidation of eukaryotic phylogeny in general.     

SCaFoS: a tool for Selection,Concatenation and Fusion of Sequences for phylogenomics

Background

Phylogenetic analyses based on datasets rich in both genes and species (phylogenomics) are becoming a standard approach to resolve evolutionary questions. However, several difficulties are associated with the assembly of large datasets, such as multiple copies of a gene per species (paralogous or xenologous genes), lack of some genes for a given species, or partial sequences. The use of undetected paralogous or xenologous genes in phylogenetic inference can lead to inaccurate results, and the use of partial sequences to a lack of resolution. A tool that selects sequences, species, and genes, while dealing with these issues, is needed in a phylogenomics context.

Results

Here, we present SCaFoS, a tool that quickly assembles phylogenomic datasets containing maximal phylogenetic information while adjusting the amount of missing data in the selection of species, sequences and genes. Starting from individual sequence alignments, and using monophyletic groups defined by the user, SCaFoS creates chimeras with partial sequences, or selects, among multiple sequences, the orthologous and/or slowest evolving sequences. Once sequences representing each predefined monophyletic group have been selected, SCaFos retains genes according to the user's allowed level of missing data and generates files for super-matrix and super-tree analyses in several formats compatible with standard phylogenetic inference software. Because no clear-cut criteria exist for the sequence selection, a semi-automatic mode is available to accommodate user's expertise.

Conclusion

SCaFos is able to deal with datasets of hundreds of species and genes, both at the amino acid or nucleotide level. It has a graphical interface and can be integrated in an automatic workflow. Moreover, SCaFoS is the first tool that integrates user's knowledge to select orthologous sequences, creates chimerical sequences to reduce missing data and selects genes according to their level of missing data. Finally, applying SCaFoS to different datasets, we show that the judicious selection of genes, species and sequences reduces tree reconstruction artefacts, especially if the dataset includes fast evolving species.

     

ERK2 protein regulates the proliferation of human mesenchymal stem cells without affecting their mobilization and differentiation potential

Human Mesenchymal Stem Cells (hMSC), derived mainly from adult bone marrow, are valuable models for the study of processes involved in stem cell self-renewal and differentiation. As the Extracellular signal-Regulated Kinase (ERK) signalling pathway is a major contributor to cellular growth, differentiation and survival, we have studied the functions of this kinase in hMSC activity. Ablation of ERK2 gene expression (but not ERK1) by RNA interference significantly reduced proliferation of hMSC. This reduction was due to a defect in Cyclin D1 expression and subsequent arrest in the G0/G1 phase of the cell cycle. hMSC growth is enhanced through culture medium supplementation with growth factors (GFs) such as Platelet-Derived Growth Factor (PDGF), basic Fibroblast Growth Factor (bFGF) or Epidermal Growth Factor (EGF). However, these supplements could not rescue the defect observed after ERK2 knockdown, suggesting a common signalling pathway used by these GFs for proliferation. In contrast, ERK1/2 may be dissociated from chemotactic signalling induced by the same GFs. Additionally, hMSCs were capable of differentiating into adipocytes even in the absence of either ERK1 or ERK2 proteins. Our data show that hMSCs do not require cell division to enter the adipogenic differentiation process, indicating that clonal amplification of these cells is not a critical step. However, cell-cell contact seems to be an essential requirement to be able to differentiate into mature adipocytes.     

GSTT1 and GSTM1 gene copy number analysis in paraffin-embedded tissue using quantitative real-time PCR

GSTT1 and GSTM1 genes possess an inherited deletion associated with a lack of enzyme activity. The heterozygous condition of this deletion is difficult to determine in low-quality DNA with existing PCR protocols. We designed and validated a multiplex real-time PCR assay by adapting the ΔΔCt relative quantification method for the analysis of GSTT1 and GSTM1 markers to accurately differentiate the three genotypes (∗1/1, ∗1/0, and ∗0/0) in degraded DNA from formalin-fixed paraffin-embedded tissue. Gene copy number values obtained provide for unambiguous homozygous and heterozygous differentiation. The efficacy shown by the PCR assay endorses its usefulness for complete genotyping of glutathione S-transferases in archival tissues.     

Effects of radiation (Cobalt-60) on the elimination of Brevipalpus phoenicis (Acari: Tenuipalpidae) Cardinium endosymbiont

Brevipalpus phoenicis (Geijskes) (Acari: Tenuipalpidae) is a polyphagous mite with worldwide distribution and it is also a vector of several plant viruses. In citrus, B. phoenicis transmits Citrus leprosis virus (CiLV), the causal agent of leprosis, a disease that costs millions of dollars per year for its prevention and control. Brevipalpus phoenicis mites reproduce through thelytokous parthenogenesis, producing haploid females. This characteristic is attributable to the presence of an endosymbiont bacterium of the genus Cardinium; however, very little is known about the biological and ecological implications of the presence of this endosymbiont in Brevipalpus mites. In order to investigate the role of Cardinium in the transmission of CiLV to citrus plants, our goal was to eliminate the bacterium from the mite. We assessed the effectiveness of different doses of radiation from a Cobalt-60 source to cure B. phoenicis populations from Cardinium sp. The efficiency of irradiation on the elimination of the endosymbiont was determined by counting the number of females and males obtained in the F(1) generation after irradiation and confirming the presence of the endosymbiont by PCR. Both radiation treatments influenced the oviposition period and the number of eggs laid by irradiated females. Also, irradiation eliminated the Cardinium endosymbiont and increased the number of males in progeny of the exposed populations. Although macroscopic morphological abnormalities were not observed among the treated mites, the mortality was higher compared to the non-irradiated control group.     

Moderate Red Wine and Grape Juice Consumption Modulates the Hydrolysis of the Adenine Nucleotides and Decreases Platelet Aggregation in Streptozotocin-Induced Diabetic Rats

This study investigated the ex vivo effects of the moderate red wine (RW) and grape juice (GJ) consumption, and the in vitro effects of the resveratrol, caffeic acid, gallic acid, quercetin, and rutin on NTPDase (nucleoside triphosphate diphosphohydrolase), ecto-nucleotide pyrophosphatase/phosphodiesterase (E-NPP), 5′-nucleotidase, and adenosine deaminase (ADA) activities in platelets and platelet aggregation from streptozotocin-induced diabetic rats. The animals were divided into six groups (n = 10): control/saline, control/GJ, control/RW, diabetic/saline, diabetic/GJ, and diabetic/RW. RW and GJ were administered for 45 days; after this period, the blood was collected for experimental determinations. Results showed that NTPDase, E-NPP, 5′-nucleotidase, and ADA activities as well as platelet aggregation were increased in the diabetic/saline group compared to the control/saline group. Treatment with RW and GJ increased ectonucleotidases activities and prevented the increase in the ADA activity in the diabetic/GJ and diabetic/RW groups. Platelet aggregation was also decreased by the treatment with RW and GJ in the diabetic/GJ and diabetic/RW groups. In the in vitro tests, resveratrol, caffeic acid, and gallic acid increased ATP, ADP, and AMP hydrolysis, while quercetin and rutin decreased the hydrolysis of these nucleotides in platelets of diabetic rats. The ADA activity and platelet aggregation were reduced in platelets of diabetic rats in the presence of all polyphenols tested in vitro. These findings suggest that RW, GJ, and all polyphenols tested were able to modulate the ectoenzymes activities. Moreover, a decrease in the platelet aggregation was observed and it could contribute to the prevention of platelet abnormality, and consequently vascular complications in diabetic state.     

Effects of resveratrol on biomarkers of oxidative stress and on the activity of delta aminolevulinic acid dehydratase in liver and kidney of streptozotocin-induced diabetic rats

The present study investigated the effects of resveratrol (RV), a polyphenol with potent antioxidant properties, on oxidative stress parameters in liver and kidney, as well as on serum biochemical parameters of streptozotocin (STZ)-induced diabetic rats. Animals were divided into six groups (n = 8): control/saline; control/RV 10 mg/kg; control/RV 20 mg/kg; diabetic/saline; diabetic/RV10 mg/kg; diabetic/RV 20 mg/kg. After 30 days of treatment with resveratrol the animals were sacrificed and the liver, kidney and serum were used for experimental determinations. Results showed that TBARS levels were significantly increased in the diabetic/saline group and the administration of resveratrol prevented this increase in the diabetic/RV10 and diabetic/RV20 groups (P < 0.05). The activities of catalase (CAT), superoxide dismutase (SOD) and aminolevulinic acid dehydratase (δ-ALA-D) and the levels of non protein thiols (NPSH) and vitamin C presented a significant decrease in the diabetic/saline group when compared with the control/saline group (P < 0.05). The treatment with resveratrol was able to prevent these decrease improving the antioxidant defense of the diabetic/RV10 and diabetic/RV20 groups (P < 0.05). In addition, the elevation in serum alanine aminotransferase (ALT), aspartate aminotransferase (AST) and γ-glutamiltransferase (γ-GT) activities as well as in levels of urea, creatinine, cholesterol and triglycerides observed in the diabetic/saline group were reverted to levels close to normal by the administration of resveratrol in the diabetic/RV10 and diabetic/RV20 groups (P < 0.05). These findings suggest that resveratrol could have a protector effect against hepatic and renal damage induced by oxidative stress in the diabetic state, which was evidenced by the capacity of this polyphenol to modulate the antioxidant defense and to decrease the lipid peroxidation in these tissues.     

The SAR11 group of alpha-proteobacteria is not related to the origin of mitochondria

Although free living, members of the successful SAR11 group of marine alpha-proteobacteria contain a very small and A+T rich genome, two features that are typical of mitochondria and related obligate intracellular parasites such as the Rickettsiales. Previous phylogenetic analyses have suggested that Candidatus Pelagibacter ubique, the first cultured member of this group, is related to the Rickettsiales+mitochondria clade whereas others disagree with this conclusion. In order to determine the evolutionary position of the SAR11 group and its relationship to the origin of mitochondria, we have performed phylogenetic analyses on the concatenation of 24 proteins from 5 mitochondria and 71 proteobacteria. Our results support that SAR11 group is not the sistergroup of the Rickettsiales+mitochondria clade and confirm that the position of this group in the alpha-proteobacterial tree is strongly affected by tree reconstruction artefacts due to compositional bias. As a consequence, genome reduction and bias toward a high A+T content may have evolved independently in the SAR11 species, which points to a different direction in the quest for the closest relatives to mitochondria and Rickettsiales. In addition, our analyses raise doubts about the monophyly of the newly proposed Pelagibacteraceae family.     

Are the spectral Doppler indices of ovarian arteries indicative of antral follicular development and predictive of ovulatory responses and embryo yields in superovulated ewes?

Nineteen ewes received 200 mg of pFSH administered in eight decreasing doses from Days 1 to 4, starting three days before CIDR® device removal. Ten ewes received an injection of 350 μg of estradiol benzoate at CIDR® device insertion (Group E) and nine animals served as controls (Group C). B-mode and spectral Doppler ultrasonographic examinations were performed daily throughout superovulatory treatment to enumerate ovarian antral follicles and to determine ovarian blood flow indices, respectively. There were no differences (P > 0.05) in superovulatory responses between left and right ovaries/uterine horns or the two groups of animals. End-diastolic velocity (EDV) and mean velocity (Vm) values were greater (P < 0.05) on Days 1 and 2, and peak systolic velocity (SVp) was greater (P < 0.05) on Day 3 in Group C than in Group E. In Group E 15 correlations was recorded among indices (SVp, Vm, EDV, flow velocity integral-FVI, and pulsatility index-PI) and follicles numbers in different size classes on Days 1, 2 and 4, and seven correlations among indices (SVp, EDV, Vm, and vascular resistance index-RI) and superovulatory/embryo results (numbers of regressing corpora lutea, numbers/percentages of degenerated embryos and viability rates) on Days 1, 2 and 3. In Group C, there were three correlations among EDV and RI and medium-sized/large follicle numbers on Days 1 and 3, and five correlations among indices (EDV, RI and PI) and superovulatory/embryo results (numbers of luteinized unovulated follicles, degenerated embryos and unfertilized eggs) on Days 2 or 4. There was a lack of consistency in the velocimetric correlates of antral follicle numbers and superovulatory responses between the left and right side. Therefore, the usefulness of ovarian arterial indices to predict ovine superovulatory outcomes remains equivocal and requires further confirmatory studies.