Characterizing the normal proteome of human ciliary body

Background

The ciliary body is the circumferential muscular tissue located just behind the iris in the anterior chamber of the eye. It plays a pivotal role in the production of aqueous humor, maintenance of the lens zonules and accommodation by changing the shape of the crystalline lens. The ciliary body is the major target of drugs against glaucoma as its inhibition leads to a drop in intraocular pressure. A molecular study of the ciliary body could provide a better understanding about the pathophysiological processes that occur in glaucoma. Thus far, no large-scale proteomic investigation has been reported for the human ciliary body.

Results

In this study, we have carried out an in-depth LC-MS/MS-based proteomic analysis of normal human ciliary body and have identified 2,815 proteins. We identified a number of proteins that were previously not described in the ciliary body including importin 5 (IPO5), atlastin-2 (ATL2), B-cell receptor associated protein 29 (BCAP29), basigin (BSG), calpain-1 (CAPN1), copine 6 (CPNE6), fibulin 1 (FBLN1) and galectin 1 (LGALS1). We compared the plasma proteome with the ciliary body proteome and found that the large majority of proteins in the ciliary body were also detectable in the plasma while 896 proteins were unique to the ciliary body. We also classified proteins using pathway enrichment analysis and found most of proteins associated with ubiquitin pathway, EIF2 signaling, glycolysis and gluconeogenesis.

Conclusions

More than 95% of the identified proteins have not been previously described in the ciliary body proteome. This is the largest catalogue of proteins reported thus far in the ciliary body that should provide new insights into our understanding of the factors involved in maintaining the secretion of aqueous humor. The identification of these proteins will aid in understanding various eye diseases of the anterior segment such as glaucoma and presbyopia.     

Ixodes ricinus, transmitted diseases and reservoirs

The tick Ixodes ricinus has been recorded in most Italian regions especially in thermo-mesophilous woods and shrubby habitats where the relative humidity allow the tick to complete its 3 year developmental cycle, as predicted for the European climatic ranges. This tick acts both as vector and reservoir for a series of wildlife zoonotic pathogens, especially the agents of Lyme diseases, Tick borne encephalitis and Human Granulocytic Ehrlichiosis, which are emerging in most of Europe. To assess the spatial distribution of these pathogens and the infection risk for humans and animals within the territory of the Province of Trento, we carried out a long term study using a combination of eco-epidemiological surveys and mathematical modelling. An extensive tick collection with a GIS based habitat suitability analysis allowed us to identify the areas where tick occurs at various density. To identify the areas with higher infection risk, we estimated the values of R0 for Borrelia burgdorferi s.l., TBE virus and Anaplasma phagocytophila under different ecological conditions. We assessed the infection prevalence in the vector and in the wildlife reservoir species that play a central role in the persistence of these infections, ie the small mammals A. flavicollis and C. glareolus. We also considered the double effect of roe deer (Capreolus capreolus) which act as reservoir for A. phagocytophila but is an incompetent host for B. burgdorferi and TBE virus, thus reducing the infection prevalence in ticks of these last two pathogens. Infection prevalence with B. burgdorferi and A. phagocytophila in the vector was assessed by PCR screening 1212 I. ricinus nymphs collected by dragging in six main study areas during 2002. The mean infection prevalence recorded was 1.32% for B. burgdorferi s.l. and 9.84% for A. phagocytophila. Infection prevalence in nymphs with TBE virus, as assessed in a previous study was 0.03%. Infection prevalence in rodents was assessed by screening (with ELISA and PCR) tissues and blood samples collected from 367 rodent individuals trapped extensively during 2002 within 6 main study areas. A. flavicollis (N=238) was found to be infected with all three pathogens investigated, with infection prevalence ranging from 3.3% for TBE virus to 11.7% for A. phagocytophila, and 16.6% with B. burgdorferi s.l. C. glareolus (N=108) showed an infection prevalence of 6.5% with A. phagocytophila and 12.7% with B. burgdorferi s.l., while no individuals were infected with TBE virus. We also screened 98 spleen samples collected from roe deer with PCR, resulting in a mean prevalence of infection with A. phagocytophila of 19.8%. Using a deterministic model we explored the condition for diseases persistence under different rodent and roe deer densities. R0 values resulted largely above 1 for B. burgdorferi s.l. in the vast majority of the areas classified as suitable for I. ricinus occurrence in Trentino, while the condition for TBE persistence appeared to be more restricted by a combination of climatic condition and host densities.     

The genetic impact of demographic decline and reintroduction in the wild boar (Sus scrofa): a microsatellite analysis

The reintroduction of wild boar from central Europe after World War II has contributed substantially to the range expansion of this species in Italy, where indiscriminate hunting in earlier times resulted in extreme demographic reduction. However, the genetic impact of such processes is not well-understood. In this study, 105 individuals from Italian and Hungarian wild boar populations were characterized for nine autosomal microsatellite loci. The Hungarian samples, and two central Italian samples from protected areas (parks) where reintroduction is not documented, were assumed to be representative of the genetic composition of the source and the target populations in the reintroduction process, respectively. Animals hunted in the wild in the Florence area of Tuscany (Italy) were then studied to identify the effects of reintroduction. The results we obtained can be summarized as follows: (i) none of the populations analysed shows genetic evidence of demographic decline; (ii) the three parental populations from Italy and Hungary are genetically distinct; however, the low level of divergence appears in conflict with the naming of the Italian and the European subspecies (Sus scrofa majori and Sus scrofa scrofa, respectively); in addition, the Italian groups appear to be as divergent from each other as they are from the Hungarian population; (iii) most of the individuals hunted near Florence are genetically intermediate between the parental groups, suggesting that hybridization has occurred in this area, the average introgression of Hungarian genotypes is 13%, but approximately 45% of the genetic pool of these individuals can not be directly attributed to any of the parental populations we analysed; (iv) analysis of microsatellite loci, though in a limited number, is an important tool for estimating the genetic effect of reintroduction in the wild boar, and therefore for the development of conservation and management strategies for this species.     

Interactions between multiple helminths and the gut microbiota in wild rodents

The gut microbiota is vital to host health and, as such, it is important to elucidate the mechanisms altering its composition and diversity. Intestinal helminths are host immunomodulators and have evolved both temporally and spatially in close association with the gut microbiota, resulting in potential mechanistic interplay. Host–helminth and host–microbiota interactions are comparatively well-examined, unlike microbiota–helminth relationships, which typically focus on experimental infection with a single helminth species in laboratory animals. Here, in addition to a review of the literature on helminth–microbiota interactions, we examined empirically the association between microbiota diversity and composition and natural infection of multiple helminth species in wild mice (Apodemus flavicollis), using 16S rRNA gene catalogues (metataxonomics). In general, helminth presence is linked with high microbiota diversity, which may confer health benefits to the host. Within our wild rodent system variation in the composition and abundance of gut microbial taxa associated with helminths was specific to each helminth species and occurred both up- and downstream of a given helminth''s niche (gut position). The most pronounced helminth–microbiota association was between the presence of tapeworms in the small intestine and increased S24–7 (Bacteroidetes) family in the stomach. Helminths clearly have the potential to alter gut homeostasis. Free-living rodents with a diverse helminth community offer a useful model system that enables both correlative (this study) and manipulative inference to elucidate helminth–microbiota interactions.     

Characterization of dental pulp stem/stromal cells of Huntington monkey tooth germs

Background

Activation by extracellular ligands of G protein-coupled (GPCRs) and tyrosine kinase receptors (RTKs), results in the generation of second messengers that in turn control specific cell functions. Further, modulation/amplification or inhibition of the initial signalling events, depend on the recruitment onto the plasma membrane of soluble protein effectors. High throughput methodologies to monitor quantitatively second messenger production, have been developed over the last years and are largely used to screen chemical libraries for drug development. On the contrary, no such high throughput methods are yet available for the other aspect of GPCRs regulation, i.e. protein translocation to the plasma membrane, despite the enormous interest of this phenomenon for the modulation of receptor downstream functions. Indeed, to date, the experimental procedures available are either inadequate or complex and expensive.

Results

Here we describe the development of a novel conceptual approach to the study of cytosolic proteins translocation to the inner surface of the plasma membrane. The basis of the technique consists in: i) generating chimeras between the protein of interests and the calcium (Ca²⁺)-sensitive, luminescent photo-protein, aequorin and ii) taking advantage of the large Ca²⁺ concentration [Ca²⁺] difference between bulk cytosolic and the sub-plasma membrane rim.

Conclusion

This approach, that keeps unaffected the translocation properties of the signalling protein, can in principle be applied to any protein that, upon activation, moves from the cytosol to the plasma membrane. Thus, not only the modulation of GPCRs and RTKs can be investigated in this way, but that of all other proteins that can be recruited to the plasma membrane also independently of receptor activation. Moreover, its automated version, which can provide information about the kinetics and concentration-dependence of the process, is also applicable to high throughput screening of drugs affecting the translocation process.     

Geographical distance and physical barriers shape the genetic structure of Eurasian red squirrels (Sciurus vulgaris) in the Italian Alps

Red squirrels (Sciurus vulgaris) are widely distributed throughout Eurasia, occurring in many types of coniferous and mixed-deciduous forests. In fragmented landscapes, small and partly isolated populations with low immigration rates show reduced genetic diversity, but reforestation can increase gene flow and restore levels of genetic variation in a few decades. No studies have so far investigated the genetic structure of red squirrel in large, continuous forests. The Italian Alps are presently characterized by almost continuous, recently reconnected forest habitats, that were affected by deep landscape changes during last glaciations but remained mostly unchanged between 10 000 and 200 years bp, when forest cover was heavily reduced. In this study we analyse patterns of genetic variability of red squirrels in and between seven sites distributed over 250 km of Alpine habitat, using mitochondrial DNA (mtDNA) and microsatellites. We use isolation-by-distance (IBD) models to investigate the relative importance that past (Pleistocene glaciations) and recent (fragmentation, bottlenecks) events had on the present genetic situation. Both nuclear and mtDNA data indicate a significant differentiation among study sites and a significant correlation between genetic and geographical distance only over a large scale. No recent bottlenecks are recorded through microsatellites and demographic models strongly support equilibrium between gene flow and drift; however, mtDNA suggests that there may have been local demographic crashes, probably in correspondence with the 19th-century forest fragmentation. These findings indicate that local landscape factors other than geographical distance per se, such as barriers of unsuitable habitat, affect gene flow and determine differentiation.     

Global species richness of hydrobiid snails determined by climate and evolutionary history

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