Molecular phylogeny of the subgenus Drosophila (Diptera, Drosophilidae) with an emphasis on Neotropical species and groups: a nuclear versus mitochondrial gene approach

The genus Drosophila has played an essential role in many biological studies during the last 100 years but much controversy and many incompletely addressed issues still remain to be elucidated regarding the phylogeny of this genus. Because information on the Neotropical species contained in the subgenus Drosophila is particularly incomplete, with this taxonomic group being underrepresented in many studies, we designed a study to answer some evolutionary questions related to these species. We subjected at least 41 Drosophilidae taxa to a phylogenetic analysis using a 516-base pair (bp) fragment of the alpha-methyldopa (Amd) nuclear gene and a 672 bp fragment of the mitochondrial cytochrome oxidase subunit II (COII) gene both individually and in combination. We found that the subgenus Drosophila is paraphyletic and subdivided into two main clusters: the first containing species traditionally placed in the virilis-repleta radiation and the second assembling species of the immigrans-Hirtodrosophila radiation. Inside the first of these clusters we could detect the monophyly of both the flavopilosa (the sister-clade of the annulimana group) and the mesophragmatica (closely related to the repleta group) species groups. Concerning the immigrans-Hirtodrosophila lineage, Zaprionus, Liodrosophila, Samoaia, and Hirtodrosophila were the early offshoots, followed by the immigrans, quinaria, testacea, and funebris species groups. The tripunctata radiation appears to be a derived clade, composed of a paraphyletic tripunctata group, intimately interposed with members of the cardini, guarani, and guaramunu species groups. Overall, the COII gene yielded a poor phylogenetic performance when compared to the Amd gene, the evolutionary hypothesis of which agreed with the total evidence tree. This phenomenon can be explained by the fast saturation of transitional substitutions in COII, due to strong biases in both base composition and substitution patterns, as also by its great among-site rate variation heterogeneity.     

CXCR3/CXCL10 expression in the synovium of children with juvenile idiopathic arthritis

The accumulation of T cells in the synovial membrane is the crucial step in the pathophysiology of the inflammatory processes characterizing juvenile idiopathic arthritis (JIA). In this study, we evaluated the expression and the pathogenetic role in oligoarticular JIA of a CXC chemokine involved in the directional migration of activated T cells, i.e. IFNγ-inducible protein 10 (CXCL10) and its receptor, CXCR3. Immunochemistry with an antihuman CXCL10 showed that synovial macrophages, epithelial cells, and endothelial cells bear the chemokine. By flow cytometry and immunochemistry, it has been shown that CXCR3 is expressed at high density by virtually all T lymphocytes isolated from synovial fluid (SF) and infiltrating the synovial membrane. Particularly strongly stained CXCR3⁺ T cells can be observed close to the luminal space and in the perivascular area. Furthermore, densitometric analysis has revealed that the mRNA levels for CXCR3 are significantly higher in JIA patients than in controls. T cells purified from SF exhibit a definite migratory capability in response to CXCL10. Furthermore, SF exerts significant chemotactic activity on the CXCR3⁺ T-cell line, and this activity is inhibited by the addition of an anti-CXCL10 neutralizing antibody. Taken together, these data suggest that CXCR3/CXCL10 interactions are involved in the pathophysiology of JIA-associated inflammatory processes, regulating both the activation of T cells and their recruitment into the inflamed synovium.     

Regioselective glycosylation: synthesis of alpha-indoline nucleosides

Novel indoline ribonucleosides with the alpha-N-glycoside configuration are synthesized with very high regioselectivity in 90-96%yield, using TMS protected indolines and 2,3-O-(1-methylethylidene)-5-O-(triphenylmethyl)-alpha/beta-D-ribofuranose. The structures of these ribonucleosides were elucidated with X-ray crystallography as well as 2D (NOESY, COSY, and HMQC) NMR spectroscopy.     

New synthesis of PNA-3'DNA linker monomers, useful building blocks to obtain PNA/DNA chimeras

A new synthetic strategy to get the PNA-3'DNA linker with the monomethoxytrityl (Mmt) group as temporary protection of the backbone to be used for the synthesis of PNA/DNA chimeras was employed and a convenient strategy to obtain Mmt PNA monomers was developed. The synthetic strategies take advantage of the introduction of the acid-labile Mmt-protecting group in the first step.     

Chronic pain as expression of neural substrates. Issues from the neuronal dynamics and mutual relations

The Thalamo-Cortical somatosensory loop shows important synaptic re-organization in cases of chronic pain. Animal models exhibit severe functional distortions, potentially related to the anatomic rearrangements. Connectivity and information theoretic measurement represent important tools to quantify the functional disarrays. We performed electrophysiological experiments with multisite, multielectrode simultaneous recordings in the Thalamus and in the Somatosensory Cortex. The recurrent anomalies in the analytic estimates induce to hypothesize a potential neurodynamical explanation of the sensory context.     

New ruthenium(II) mixed metallocene derived complexes: Synthesis, characterization by X-ray diffraction and evaluation on DNA interaction by atomic force microscopy

A new family of Ru(II) mixed metallocene complexes of the type [Ru(η⁵-C₅H₅)(η⁶-arene)][PF₆] has been synthesized and fully characterized by NMR and UV-Vis spectroscopy. X-ray analysis of single crystal was achieved for all the complexes and revealed the presence of two enantiomers expected for planar chirality originated by the η⁶ coordination of the arene prochiral ligands. Studies of interaction of the new complexes with pBR322 DNA by atomic force microscopy showed very strong and different types of interaction. Antiproliferative tests were examined on human leukemia cancer cells (HL-60) using the MTT assay, and the IC₅₀ values revealed low antiproliferative activity compared to cisplatin.     

Heteroduplex mobility assay of the 26S rDNA D1/D2 region for differentiation of clinically relevant Candida species

The Heteroduplex Mobility Assay (HMA) method using the PCR amplified D1/D2 region of the 26S rDNA was tested for the differentiation of clinically relevant Candida species. Strains belonging to the same species are not expected to form heteroduplexes in this assay when their PCR products are mixed. D1/D2 HMA experiments between all Candida type strains tested showed heteroduplex formation, including Candida albicans and Candida dubliniensis. There was no heteroduplex formation when most clinical and non-type strains were tested against the type strain of their presumptive species, except when C. albicans WVE and C.␣dubliniensis TAI were analysed. Additional HMA experiments, phenotypic characterisation, and D1/D2 sequencing identified these isolates as Candida tropicalis and Candida parapsilosis, respectively. HMA provides a rapid and relatively simple molecular tool for the differentiation of potentially pathogenic Candida species.     

Refining the genetic portrait of Portuguese Roma through X-chromosomal markers

Due to differences in transmission between X-chromosomal and autosomal DNA, the comparison of data derived from both markers allows deeper insight into the forces that shape the patterns of genetic diversity in populations. In this study, we applied this comparative approach to a sample of Portuguese Roma (Gypsies) by analyzing 43 X-chromosomal markers and 53 autosomal markers. Portuguese individuals of non-Gypsy ancestry were also studied. Compared with the host population, reduced levels of diversity on the X chromosome and autosomes were detected in Gypsies; this result was in line with known patterns of genetic diversity typical of Roma groups. As a consequence of the complex demographic past of the Roma, during which admixture and genetic drift played major roles, the amount of linkage disequilibrium (LD) on the X chromosome in Gypsies was considerably higher than that observed in non-Gypsies. When the pattern of differentiation on the X chromosome was compared with that of autosomes, there was evidence for asymmetries in female and male effective population sizes during the admixture between Roma and non-Roma. This result supplements previous data provided by mtDNA and the Y chromosome, underlining the importance of using combined information from the X chromosome and autosomes to dissect patterns of genetic diversity. Following the out-of-India dispersion, the Roma acquired a complex genetic pattern that was influenced by drift and introgression with surrounding populations, with important contributions from both males and females. We provide evidence that a sex-biased admixture with Europeans is probably associated with the founding of the Portuguese Gypsies.