The conservation status of West African vultures: an updated review and a strategy for conservation

The vulture populations in West Africa are undergoing dramatic decline over the last 30 years. Their particular ecology and sociality makes them vulnerable to various risks, including environmental changes, poisoning and bioaccumulation of toxic substances from agricultural products, pesticides, and veterinary drugs used in cattle livestock. In addition, these birds are subject to direct persecution for the trade of products used in traditional medicine. This manuscript analyzes the conservation status of eight vulture species in West Africa and the threats affecting their survival. In order to assess the conservation status of vultures in West Africa, this paper analyzes all the available literature that has been published in scientific peerreviewed journals, including also technical reports and unpublished reports related to the whole West African region. Overall, and despite the high risk of extinction facing several vulture species all throughout the world, our literature surveys revealed that the scientific papers on the conservation of West African vultures are relatively few. Therefore, due to limited available literature, the main causes of vulture declines in West Africa remain relatively unclear. Apparently, all African vultures suffer from similar threats, especially poisoning, habitat alteration and conversion to agro-pastoral systems, loss of wild ungulates leading to a reduced availability of carrion, hunting for trade, for use in traditional medicine and bushmeat, persecution and human disturbance. Our review also addresses future steps that are needed for reversing the negative population trend of their free-ranging populations, including some specific conservation measures that are proposed to mitigate their decline

A method for the analysis of the oligomerization profile of the Huntington's disease-associated, aggregation-prone mutant huntingtin protein by isopycnic ultracentrifugation

: Conformational diseases, such as Alzheimer's, Parkinson's and Huntington's diseases as well as ataxias and fronto-temporal disorders, are part of common class of neurological disorders characterised by the aggregation and progressive accumulation of mutant proteins which display aberrant conformation. In particular, Huntington's disease (HD) is caused by mutations leading to an abnormal expansion in the polyglutamine (poly-Q) tract of the huntingtin protein (HTT), leading to the formation of inclusion bodies in neurons of affected patients. Furthermore, recent experimental evidence is challenging the conventional view of the disease by revealing the ability of mutant HTT to be transferred between cells by means of extracellular vesicles (EVs), allowing the mutant protein to seed oligomers involving both the mutant and wild type forms of the protein. There is still no successful strategy to treat HD. In addition, the current understanding of the biological processes leading to the oligomerization and aggregation of proteins bearing the poly-Q tract has been derived from studies conducted on isolated poly-Q monomers and oligomers, whose structural properties are still unclear and often inconsistent. Here we describe a standardised biochemical approach to analyse by isopycnic ultracentrifugation the oligomerization of the N-terminal fragment of mutant HTT. The dynamic range of our method allows one to detect large and heterogeneous HTT complexes. Hence, it could be harnessed for the identification of novel molecular determinants responsible for the aggregation and the prion-like spreading properties of HTT in the context of HD. Equally, it provides a tool to test novel small molecules or bioactive compounds designed to inhibit the aggregation of mutant HTT

Evidence of an unprecedented cytoplasmic function of DDX11, the Warsaw breakage syndrome DNA helicase, in regulating autophagy

DDX11 is a DNA helicase involved in critical cellular functions, including DNA replication/repair/ recombination as well as sister chromatid cohesion establishment. Bi-allelic mutations of DDX11 lead to Warsaw breakage syndrome (WABS), a rare genome instability disorder marked by significant prenatal and postnatal growth restriction, microcephaly, intellectual disability, and sensorineural hearing loss. The molecular mechanisms underlying WABS remain largely unclear. In this study, we uncover a novel role of DDX11 in regulating the macroautophagic/autophagic pathway. Specifically, we demonstrate that knockout of DDX11 in RPE-1 cells hinders the progression of autophagy. DDX11 depletion significantly reduces the conversion of MAP1LC3/LC3 (microtubule associated protein 1 light chain 3), suggesting a defect in autophagosome biogenesis. This is supported by imaging analysis with a LC3 reporter fused in tandem with the red and green fluorescent proteins (mRFP-GFP- LC3), which reveals fewer autophagosomes and autolysosomes in DDX11-knockout cells. Moreover, the defect in autophagosome biogenesis, observed in DDX11-depleted cells, is linked to an upstream impairment of the ATG16L1-precursor trafficking and maturation, a step critical to achieve the LC3 lipidation. Consistent with this, DDX11-lacking cells exhibit a diminished capacity to clear aggregates of a mutant HTT (huntingtin) N-terminal fragment fused to the green fluorescent protein (HTTQ74- GFP), an autophagy substrate. Finally, we demonstrate the occurrence of a functional interplay between DDX11 and SQSTM1, an autophagy cargo receptor protein, in supporting LC3 modification during autophagosome biogenesis. Our findings highlight a novel unprecedented function of DDX11 in the autophagy process with important implications for our understanding of WABS etiology

Positive demographic effects of nest surveillance campaigns to counter illegal harvest of the Bonelli's eagle in Sicily (Italy)

Illegal trade in wildlife has been identified as one of the main challenges to wildlife conservation. In 2010, an illegal trade-ring trafficking in birds of prey was uncovered in Sicily (southern Italy). This illegal trade targeted the three most endangered species in Italy: Bonelli's eagle Aquila fasciata, Lanner falcon Falco biarmicus and Egyptian vulture Neophron percnopterus, all of them long-lived territorial raptors threatened with extinction across their European distribution. Illegal harvest primarily involved young birds and eggs taken from nests. After the discovery of these activities, surveillance camps and camera traps connected to the mobile Global System for Mobile communications network were established in nine Bonelli's eagle breeding sites in which illegal harvest was reported. Surveillance activities resulted in a sharp reduction in illegal harvest that has contributed to the recent increase in population size and number of breeding pairs of Bonelli's eagle in the island. This population represents 95% of the entire Italian population and is catalogued as Critically Endangered in this country. Importantly, our results highlight the impact of illegal harvest on the population dynamics of endangered species as demonstrated by a population viability analysis. This is particularly important in the case of insular species for which demographic recovery due to immigration from other geographic areas is unlikely. Systematic patrols by forestry police authorities, a resolute application of Convention on International Trade in Endangered Species legislation via legal punishment, and the requirement of including all live captive specimens used for falconry in an obligatory DNA data bank would contribute to reducing the risk of extinction for small populations of endangered species of birds of prey

Living on the Edge: Assessing the Extinction Risk of Critically Endangered Bonelli’s Eagle in Italy

Background: The population of Bonelli’s eagle (Aquila fasciata) has declined drastically throughout its European range due to habitat degradation and unnatural elevated mortality. There are less than 1500 breeding pairs accounted for in Europe, and the species is currently catalogued as Critically Endangered in Italy, where the 22 territories of Sicily, represent nearly 95% of the entire Italian population. However, despite national and European conservation concerns, the species currently lacks a specific conservation plan, and no previous attempts to estimate the risk of extinction have been made. Methodology/Principal Findings: We incorporated the most updated demographic information available to assess the extinction risk of endangered Bonelli’s eagle in Italy through a Population Viability Analysis. Using perturbation analyses (sensitivity and elasticity), and a combination of demographic data obtained from an assortment of independent methods, we evaluated which demographic parameters have more influence on the population’s fate. We also simulated different scenarios to explore the effects of possible management actions. Our results showed that under the current conditions, Bonelli’s eagle is expected to become extinct in Italy in less than 50 years. Stand-alone juvenile mortality was the most critical demographic parameter with the strongest influence on population persistence with respect to other demographic parameters. Measures aimed at either decreasing juvenile mortality, adult mortality or decreasing both juvenile and adult mortality resulted in equivalent net positive effects on population persistence (population growth rate l.1). In contrast, changes aimed at increasing breeding success had limited positive effects on demographic trends. Conclusions/Significance: Our PVA provides essential information to direct the decision-making process and exposes gaps in our previous knowledge. To ensure the long-term persistence of the species in Italy, measures are urgently needed to decrease both adult mortality due to poaching and juvenile mortality due to nest plundering, the top ranking mortality causes.PLL is supported by a “Juan de la Cierva” postdoctoral grant of the Spanish Ministry of Economy and Competitiveness (reference JCI-2011–09588)

A method for the analysis of the oligomerization profile of the Huntington’s disease-associated, aggregation-prone mutant huntingtin protein by isopycnic ultracentrifugation

Conformational diseases, such as Alzheimer’s, Parkinson’s and Huntington’s diseases as well as ataxias and fronto-temporal disorders, are part of common class of neurological disorders characterised by the aggregation and progressive accumulation of mutant proteins which display aberrant conformation. In particular, Huntington’s disease (HD) is caused by mutations leading to an abnormal expansion in the polyglutamine (poly-Q) tract of the huntingtin protein (HTT), leading to the formation of inclusion bodies in neurons of affected patients. Furthermore, recent experimental evidence is challenging the conventional view of the disease by revealing the ability of mutant HTT to be transferred between cells by means of extracellular vesicles (EVs), allowing the mutant protein to seed oligomers involving both the mutant and wild type forms of the protein. There is still no successful strategy to treat HD. In addition, the current understanding of the biological processes leading to the oligomerization and aggregation of proteins bearing the poly-Q tract has been derived from studies conducted on isolated poly-Q monomers and oligomers, whose structural properties are still unclear and often inconsistent. Here we describe a standardised biochemical approach to analyse by isopycnic ultracentrifugation the oligomerization of the N-terminal fragment of mutant HTT. The dynamic range of our method allows one to detect large and heterogeneous HTT complexes. Hence, it could be harnessed for the identification of novel molecular determinants responsible for the aggregation and the prion-like spreading properties of HTT in the context of HD. Equally, it provides a tool to test novel small molecules or bioactive compounds designed to inhibit the aggregation of mutant HTT

Structural and functional analysis of RTEL1, an FeS helicase

The topic of my PhD thesis was the structural and functional characterisation of the human RTEL1, a DNA helicase with a pivotal role in telomere homeostasis and DNA replication. Based on a bioinformatic analysis of RTEL1, I cloned, expressed, and purified different fragments encompassing the main RTEL1 domains. To increase the chance of obtaining large amounts of pure, stable proteins, using the Ligand Independent Cloning (LIC) technique, I cloned the gene in a variety of expression vectors harbouring different fusion tags and screened a large number of expression conditions (cell strains, expression temperatures and times). Once the best expression conditions were found, I scaled up and optimised the purification of recombinant proteins by means of chromatographic methods. I started focussing on the full-length and the catalytic domain of human RTEL1, but despite many efforts I did not manage to obtain large amount of pure and soluble protein for structural studies. This was not totally unexpected, as FeS helicase are not easy to produce, and the difficulties in the assembly of the cluster can affect protein stability. I was more successful in obtaining soluble and stable protein samples from the C-terminal region, that contains a number of predicted domains interspersed with flexible and probably disordered linkers. I produced the whole C-terminal half of human RTEL1, and a number of deletion fragments, encompassing the various domains, including two repeats (RP1 and RP2, variously predicted to fold as TRP, HEAT or Harmonin Homology Domains) and a RING finger. After obtaining a good amount of well folded protein, I carried out a large number of crystallization screenings in order to obtain crystals suitable to be tested with synchrotron light. I have indeed obtained well ordered crystals from RP1; X-ray diffraction data were collected on the XRD2 macromolecular crystallography beamline at Elettra, and the structure determined. The protein folds as an Harmonin Homology Domain; the structure has been analyzed and compared with other similar folds, which suggest a possible modality of protein-protein interaction. In the meanwhile, a Small-angle X-ray Scattering (SAXS) analysis was used to reconstruct the low resolution three-dimensional shape of the C-terminal domains of RTEL1. SAXS data confirmed that all constructs are monomeric in solution; this information is discussed based on the current literature. A SAXS curve predicted from the crystal structure fits well the experimental scattering from both RP1 and RP2, confirming that both domains have the same fold. We are in the process of deriving 3D models from the longer constructs. To dissect the nucleic acid binding properties of the RTEL1 C-terminal domain, I performed Electrophoretic Mobility Shift Assays (EMSA) with the purified RTEL1 fragments, using a variety of nucleic acid substrates, in order to identify and characterise the DNA/RNA binding sites on the C-terminal half of RTEL1. As side project, in collaboration with clinical geneticists, I have characterised a DDX11 novel mutant found in patients with Warsaw Syndrome. I have thus carried out the biochemical characterisation of DDX11 variant c.2507T>C, leading to the substitution of Leucine 836 with a Proline. Using the Bac-to-Bac Baculovirus Expression System, I expressed in H5 insect cells DDX11 wild type and DDX11 L836P mutant. Once purified with a high grade of purity, I performed helicase assays and nucleic binding assays to understand whether and how much the substitution of Leucine 836 with a Proline affected the biochemical capability of DDX11 to bind and unwind DNA forks, and thus providing a molecular understanding of the disease

Basilica di San Francesco d'Assisi

Connue en italien sous le nom de Basilica di San Francesco d'Assisi; Dates de construction: 1228-1253; Dessin d'architecture par ordinateur; Photographie: Icarus AT-10

Basilica di San Francesco d'Assisi

Connue en italien sous le nom de Basilica di San Francesco d'Assisi; Dates de construction: 1228-1253; Dessin d'architecture par ordinateur; Photographie: Icarus AT-11