Usefulness of microsatellite typing in population genetic studies of Trypanosoma cruzi

Through microsatellite analysis of 53 monoclonal populations of Trypanosoma cruzi, we found a remarkable degree of genetic polymorphism with no single multilocus genotype being observed more than once. The microsatellite profile proved to be stable during 70 generations of the CL Brener clone in culture. The microsatellite profiling presented also high diagnostic sensitivity since DNA amplifications could be achieved with less than 100 fg DNA, corresponding to half parasite total DNA content. Based on these technical attributes the microsatellite assay turns out to be an important tool for direct typing T. cruzi in biological samples. By using this approach we were able to type T. cruzi in feces of artificially infected bugs and in single cells sorted by FACS. The microsatellites have shown to be excellent markers for T. cruzi phylogenetic reconstruction. We used maximum parsimony based on the minimum number of mutational steps to build an unrooted Wagner network, which confirms previous conclusions based on the analysis of the D7 domain of the LSU rDNA gene that T. cruzi is composed by two major groups. We also obtained evidence that strains belonging to rRNA group 2 are subdivided into two genetically distant clusters, and that one of these clusters is more related to rRNA group (1/2). These results suggest different origins for these strains.     

Late Triassic, Early and Middle Jurassic Radiolaria from ferromanganese-chert ‘nodules’ (Angelokastron, Argolis, Greece): evidence for prolonged radiolarite sedimentation in the Maliac-Vardar Ocean

In the Argolis, the Basal Sequence, constituting the eastern Pelagonian margin which bordered the Maliac-Vardar oceanic domain, includes shallow-water carbonates of Late Triassic-Early Jurassic, condensed pelagic limestones of Early-Middle Jurassic, radiolarian cherts of late Middle-Late Jurassic age and siliceous mudstones and sandstones rich in ophiolite fragments. Up-section, coarse breccias, including clasts of boninites derived from the ophiolite obducted onto the Pelagonian margin in Late Jurassic times crop out. Near Angelokastron a small quarry exposes pervasively sheared dark reddish-brown, radiolarian-bearing cherty shales with disrupted fragments of chert and chert nodules impregnated by ferro-manganese oxides. These shales occur in the footwall of a thrust bringing them into contact with the Pantokrator Limestone of the Basal Sequence. We collected more than 30 samples of the chert fragments and the shaly matrix. Thirteen nodules and one matrix sample yielded determinable radiolarians. Low to non-detectable concentrations of trace metals such as Co, Cr, Cu, Ni, Zn, and Pb indicate a hydrothermal origin of the ferro-manganese mineralization. The radiolarian taxa found indicate four age groups for the nodules that are embedded in the siliceous shale matrix that yielded a Middle Jurassic age (middle Bathonian). The first group includes a nodule of Late Triassic age (late Norian to Rhaetian); the second group nodules of Early Jurassic age (late early to late Pliensbachian and probably middle-late Toarcian); the third group nodules of early Middle Jurassic age (Aalenian–Bajocian); the last group finally includes nodules of late Middle Jurassic age (Bajocian–Bathonian). The presence of Upper Triassic to Middle Jurassic Mn-impregnated chert nodules in a Middle Jurassic matrix indicates a deep oceanic environment of deposition outside the Pelagonian realm (easternmost Adria Plate), which at that time was a shallow-water carbonate platform with a thin pelagic limestone cover. The chert nodules are with all certainty derived from the oceanic Maliac-Vardar domain and were, together with their host formation, tectonically emplaced onto the Pelagonian margin. We speculate that these nodules, more lithified than their matrix, were exhumed on the slope of an intra-oceanic accretionary wedge and were redeposited in the Middle Jurassic siliceous mudstones on the floor of the subducting Maliac-Vardar Ocean.     

The determinants of the molecular substitution process in turtles

Neutral rates of molecular evolution vary across species, and this variation has been shown to be related to biological traits. One of the first patterns to be observed in vertebrates has been an inverse relationship between body mass (BM) and substitution rates. The effects of three major life‐history traits (LHT) that covary with BM – metabolic rate, generation time and longevity (LON) – have been invoked to explain this relationship. However, most of the theoretical and empirical evidence supporting this relationship comes from endothermic vertebrates, that is, mammals and birds, in which the environmental conditions, especially temperature, do not have a direct impact on cellular and molecular biology. We analysed the variations in mitochondrial and nuclear rates of synonymous substitution across 224 turtle species and examined their correlation with two LHT (LON and BM) and two environmental variables [latitude (LAT) and habitat]. Our analyses indicate that in turtles, neutral rates of molecular evolution are hardly correlated with LON or BM. Rather, both the mitochondrial and nuclear substitution rates are significantly correlated with LAT – faster evolution in the tropics – and especially so for aquatic species. These results question the generality of the relationships reported in mammals and birds and suggest that environmental factors might be the strongest determinants of the mutation rate in ectotherms.     

Using odour traps for population monitoring and dispersal analysis of the threatened saproxylic beetles Osmoderma eremita and Elater ferrugineus in central Italy

Pheromone-based monitoring could be a very efficient method to assess the conservation status of rare and elusive insect species, but there are still few studies for which pheromone traps have been used to obtain information on presence, abundance, phenology and movements of such insects. We performed a mark-recapture study of two threatened saproxylic beetles, Osmoderma eremita (Scarabaeidae) and its predator Elater ferrugineus (Elateridae), in two beech forests of central Italy using pheromone baited window traps and unbaited pitfall traps. Two lures were used: (1) the male-produced sex pheromone of O. eremita (racemic γ-decalactone) to attract females of both species, and (2) the female-produced sex pheromone of E. ferrugineus (7-methyloctyl (Z)-4-decenoate), to attract conspecific males. In total, 13 O. eremita and 1,247 E. ferrugineus individuals were trapped. For E. ferrugineus, males were detected earlier than females, and 7-methyloctyl (Z)-4-decenoate was much more efficient lure compared to racemic γ-decalactone in detecting its presence. The population size at the two sites were estimated to 520 and 1,369 individuals, respectively. Our model suggests a sampling effort of ten traps checked for 3 days being sufficient to detect the presence of E. ferrugineus at a given site. The distribution of dispersal distances for the predator was best described by the negative exponential function with 1 % of the individuals dispersing farther than 1,600 m from their natal site. In contrast to studies on these beetles in Northern Europe, the activity pattern of the two beetle species was not influenced by variation in temperature during the season.     

Altered Protease–Activated Receptor-1 Expression and Signaling in a Malignant Pleural Mesothelioma Cell Line,NCI-H28, with Homozygous Deletion of the β-Catenin Gene

Protease activated receptors (PARs) are G-protein coupled receptors that are activated by an unique proteolytic mechanism. These receptors play crucial roles in hemostasis and thrombosis but also in inflammation and vascular development. PARs have also been implicated in tumor progression, invasion and metastasis. In this study, we investigated expression and signaling of PAR₁ in nonmalignant pleural mesothelial (Met-5A) and malignant pleural mesothelioma (NCI-H28) cells. We found that the expression level of PAR₁ was markedly higher in NCI-H28 cells compared to Met-5A and human primary mesothelial cells. Other three malignant pleural mesothelioma cell lines, i.e. REN, Ist-Mes2, and Mero-14, did not show any significant PAR₁ over-expression compared to Met-5A cell line. Thrombin and PAR₁ activating peptides enhanced Met-5A and NCI-H28 cell proliferation but in NCI-H28 cells higher thrombin concentrations were required to obtain the same proliferation increase. Similarly, thrombin caused extracellular signal-regulated kinase 1/2 activation in both cell lines but NCI-H28 cells responded at higher agonist concentrations. We also determined that PAR₁ signaling through G_q and G_12/13 proteins is severely altered in NCI-H28 cells compared to Met-5A cells. On the contrary, PAR₁ signaling through G_i proteins was persistently maintained in NCI-H28 cells. Furthermore, we demonstrated a reduction of cell surface PAR₁ expression in NCI-H28 and malignant pleural mesothelioma REN cells. Thus, our results provide evidences for dysfunctional PAR₁ signaling in NCI-H28 cells together with reduced plasma membrane localization. The role of PAR₁ in mesothelioma progression is just emerging and our observations can promote further investigations focused on this G-protein coupled receptor.     

Effects of trap baits and height on stag beetle and flower chafer monitoring: ecological and conservation implications

The implementation of conservation actions requires a reliable assessment of presence and/or abundance of targeted species. This is particularly difficult for rare and elusive species. In this study the use of bottle traps and the effects of two potential baits in relation to height in the trees were tested to detect presence and assess abundance of stag beetles (Lucanidae) and flower chafers (Scarabaeidae, Cetoniinae), an important component of forest biodiversity. The study was carried out in a flood-plain forest of northern Italy. Forty-eight handcrafted traps were assigned to two height categories (1.5–2 m and 10–20 m) and three kinds of bait: (i) red wine, white wine and sugar, (ii) red wine, beer and mashed banana, (iii) no bait, as control. Fieldwork lasted 8 weeks, with 32 surveys, from May to July. Overall, we recorded 399 captures of the following species: Dorcus parallelipipedus, Lucanus cervus, Cetonia aurata, Protaetia speciosissima, P.affinis, P. morio and P. cuprea. Traps baited with red wine, white wine and sugar showed the highest detection probabilities for all the species. A clear preference for the canopy layer (traps between 10 and 20 m high) was shown by all species except for D. parallelipipedus which was mostly captured between 1.5 and 2 m of height. The study period was long enough to improve ecological knowledge on species phenology, but not enough to include the whole phenology for all of them. The method allowed the assessment of population size only for flower chafers as the number of stag beetles captures was too low.     

A computational modelling approach to investigate different targets in deep brain stimulation for Parkinson’s disease

We investigated by a computational model of the basal ganglia the different network effects of deep brain stimulation (DBS) for Parkinson’s disease (PD) in different target sites in the subthalamic nucleus (STN), the globus pallidus pars interna (GPi), and the globus pallidus pars externa (GPe). A cellular-based model of the basal ganglia system (BGS), based on the model proposed by Rubin and Terman (J Comput Neurosci 16:211–235, 2004), was developed. The original Rubin and Terman model was able to reproduce both the physiological and pathological activities of STN, GPi, GPe and thalamo-cortical (TC) relay cells. In the present study, we introduced a representation of the direct pathway of the BGS, allowing a more complete framework to simulate DBS and to interpret its network effects in the BGS. Our results suggest that DBS in the STN could functionally restore the TC relay activity, while DBS in the GPe and in the GPi could functionally over-activate and inhibit it, respectively. Our results are consistent with the experimental and the clinical evidences on the network effects of DBS.     

Biological characterization of Trypanosoma cruzi strains from different zymodemes and schizodemes.

The development in C3H mice of thirteen strains of Trypanosoma cruzi belonging to different zymodemes and schizodemes was studied. Host mortality, virulence, histiotropism, parasitemia and polymorphism of the parasites were recorded. The strains were grouped into: a) high virulence--causing 100% mortality and characterized by predominance of very broad trypomastigotes in the bloodstream at the end of infection; b) medium virulence--causing no mortality and with a predominance of broad trypomastigotes; c) low virulence--causing no mortality with blood forms not described due to the very low parasitemia. During 18 months maintenance the parasitemia curves were kept constant for all strains except one. A direct correlation between either zymodeme or schizodeme and experimental biological properties of T. cruzi strains was not found. However, the parasitemia was subpatent and patent for strains from zymodeme C and the others respectively. Furthermore the high virulence seems to be related to one of two schizodemes found within zymodeme B strains. All strains presenting patent parasitemia independent of shizodeme and zymodeme showed a myotropism towards heart and skeletal muscle with variable inflammatory intensity. The present study confirmed the heterogeneity found by isoenzyme and k-DNA patterns among the strains of T. cruzi isolated from chagasic patients in Bambuí, Minas Gerais State, Brasil.     

Biological characterization of clones of the Y, CL and MR strains of Trypanosoma cruzi in inbred C3H mice

Ten clones of Trypanosoma cruzi isolated from Y, CL and MR strains were studied. The infectivity of culture forms, parasitemia pattern, polymorphism and mortality were studied in C3H inbred mice. Significant intra-group differences among Y and CL clones were found. MR clones showed higher homogeneity. These data indicate that T. cruzi strains can show different degrees of heterogeneity. It is suggested that conditions used to maintain T. cruzi strains may result in a selective advantage for some subpopulations (clones) after many years of laboratory maintenance.     

Isoenzyme patterns of cultured Trypanosoma cruzi: changes after prolonged subculture.

1. The isoenzyme patterns of four soluble enzymes in seven stocks of T. cruzi were determined by electrophoresis. According to their patterns they could be classified into four sets. 2. The isoenzyme patterns of two stocks were influenced by the number of subcultures. 3. Five stocks from man are distinct from those derived from a silvatic reservoir. 4. Since the isoenzyme patterns of a stock isolated from a patient with acute disease were similar to those of a silvatic reservoir, its recent introduction into the domiciliary cycle is postulated.