Pilot survey of expressed sequence tags (ESTs) from the asexual blood stages of Plasmodium vivax in human patients

Abstract Background Plasmodium vivax is the most widely distributed human malaria, responsible for 70–80 million clinical cases each year and large socio-economical burdens for countries such as Brazil where it is the most prevalent species. Unfortunately, due to the impossibility of growing this parasite in continuous in vitro culture, research on P. vivax remains largely neglected. Methods A pilot survey of expressed sequence tags (ESTs) from the asexual blood stages of P. vivax was performed. To do so, 1,184 clones from a cDNA library constructed with parasites obtained from 10 different human patients in the Brazilian Amazon were sequenced. Sequences were automatedly processed to remove contaminants and low quality reads. A total of 806 sequences with an average length of 586 bp met such criteria and their clustering revealed 666 distinct events. The consensus sequence of each cluster and the unique sequences of the singlets were used in similarity searches against different databases that included P. vivax, Plasmodium falciparum, Plasmodium yoelii, Plasmodium knowlesi, Apicomplexa and the GenBank non-redundant database. An E-value of <10-30 was used to define a significant database match. ESTs were manually assigned a gene ontology (GO) terminology Results A total of 769 ESTs could be assigned a putative identity based upon sequence similarity to known proteins in GenBank. Moreover, 292 ESTs were annotated and a GO terminology was assigned to 164 of them. Conclusion These are the first ESTs reported for P. vivax and, as such, they represent a valuable resource to assist in the annotation of the P. vivax genome currently being sequenced. Moreover, since the GC-content of the P. vivax genome is strikingly different from that of P. falciparum, these ESTs will help in the validation of gene predictions for P. vivax and to create a gene index of this malaria parasite.

The special rights other than shares in cross-border mergers of limited liability companies within the European Union. A perspective from the Spanish Law

The present research aims to study the special rights other than shares in Spanish Law and the protection of their holders in cross-border mergers of limited liability companies within the European Union frame. Special rights other than shares are recognised as an independent legal category within legal systems of some EU Member States, such as Germany or Spain, through the implementation of the Third Directive 78/855/CEE concerning mergers of public limited liability companies. The above-cited Directive contains a special regime of protection for the holders of securities, other than shares, to which special rights are attached, consisting of being given rights in the acquiring company, at least equivalent to those they possessed in the company being acquired. This safeguard is to highlight the intimate connection between this type of rights and the company whose extinction determines the existence of those. Pursuant to the Directive 2005/56/CE on cross-border mergers of limited liability companies, each company taking part in these operations shall comply with the safeguards of members and third parties provided in their respective national law to which is subject. In this regard, the protection for holders of special rights other than shares shall be ruled by the domestic M&A regime. As far as Spanish Law are concerned, holders of these special rights are recognized a right of merger information, in the same terms as shareholders, as well as equal rights in the company resulting from the cross-border merger. However, these measures are not enough guarantee for a suitable protection, thus considering those holders of special rights as special creditors, sometimes it will be necessary to go to the general protection regime for creditors. In Spanish Law, it would involve the recognition of right to the merger opposition, whose exercise would prevent the operation was completed until ensuring equal rights.

The prehistoric inhabitants of the Wadi Howar : an anthropological study of human skeletal remains from the Sudanese part of the Eastern Sahara

All currently available human skeletal remains from the Wadi Howar (Eastern Sahara, Sudan) were employed in an anthropological study. The study’s first aim was to describe this unique 5th to 2nd millennium BCE material, which comprised representatives of all three prehistoric occupation phases of the region. Detecting diachronic differences in robusticity, occupational stress levels and health within the spatially, temporally and culturally heterogeneous sample was its second objective. The study’s third goal was to reveal metric and non-metric affinities between the different parts of the series and between the Wadi Howar material and other relevant prehistoric as well as modern African populations. rnThe reconstruction and comprehensive osteological analysis of 23 as yet unpublished individuals, the bulk of the Wadi Howar series, constituted the first stage of the study. The analyses focused on each individual’s in situ position, state of preservation, sex, age at death, living height, living weight, physique, biological ancestry, epigenetic traits, robusticity, occupational stress markers, health and metric as well as morphological characteristics. Building on the results of these efforts and the re-examination of the rest of the material, the Wadi Howar series as a whole, altogether 32 individuals, could be described. rnA wide variety of robusticity, occupational stress and health variables was evaluated. The pre-Leiterband (hunter-gatherer-fisher/hunter-gatherer-fisher-herder) and the Leiterband (herder-gatherer) data of over a third of these variables differed statistically significantly or in tendency from each other. The Leiterband sub-sample was characterised by higher enamel hypoplasia frequencies, lower mean ages at death and less pronounced expressions of occupational stress traits. This pattern was interpreted as evidence that the adoption and intensification of animal husbandry did probably not constitute reactions to worsening conditions. Apart from that, the relevant observations, noteworthy tendencies and significant differences were explained as results of a broader spectrum of pre-Leiterband subsistence activities and the negative side effects of the increasingly specialised herder-gatherer economy of the Leiterband phase. rnUsing only the data which could actually be collected from it, multiple, separate, individualised discriminant function analyses were carried out for each Wadi Howar skeleton to determine which prehistoric and which modern comparative sample it was most similar to. The results of all individual analyses were then summarised and examined as a whole. Thus it became possible to draw conclusions about the affinities the Wadi Howar material shared with prehistoric as well as modern populations and to answer questions concerning the diachronic links between the Wadi Howar’s prehistoric populations. When the Wadi Howar remains were positioned in the context of the selected prehistoric (Jebel Sahaba/Tushka, A-Group, Malian Sahara) and modern comparative samples (Southern Sudan, Chad, Mandinka, Somalis, Haya) in this fashion three main findings emerged. Firstly, the series as a whole displayed very strong affinities with the prehistoric sample from the Malian Sahara (Hassi el Abiod, Kobadi, Erg Ine Sakane, etc.) and the modern material from Southern Sudan and, to a lesser extent, Chad. Secondly, the pre-Leiterband and the Leiterband sub-sample were closer to the prehistoric Malian as well as the modern Southern Sudanese material than they were to each other. Thirdly, the group of pre-Leiterband individuals approached the Late Pleistocene sample from Jebel Sahaba/Tushka under certain circumstances. A theory offering explanations for these findings was developed. According to this theory, the entire prehistoric population of the Wadi Howar belonged to a Saharo-Nilotic population complex. The Jebel Sahaba/Tushka population constituted an old Nilotic and the early population of the Malian Sahara a younger Saharan part of this complex. The pre-Leiterband groups probably colonised the Wadi Howar from the east, either during or soon after the original Saharo-Nilotic expansion. Unlike the pre-Leiterband groups, the Leiterband people originated somewhere west of the Wadi Howar. They entered the region in the context of a later, secondary Saharo-Nilotic expansion. In the process, the incoming Leiterband groups absorbed many members of the Wadi Howar’s older pre-Leiterband population. The increasing aridification of the Wadi Howar region ultimately forced its prehistoric inhabitants to abandon the wadi. Most of them migrated south and west. They, or groups closely related to them, probably were the ancestors of the majority of the Nilo-Saharan-speaking pastoralists of modern-day Southern Sudan and Eastern Chad.

In vitro characterization of human AML-reactive CTL clones generated from the naive subset of healthy donors and adoptive transfer into leukemia-engrafted NSG mice

Acute myeloid leukemia (AML) is a very aggressive cancer of the hematopoietic system. Chemotherapy and immunotherapeutical approaches including hematopoietic stem cell transplantation (HSCT) and donor lymphocyte infusion (DLI) are the only curative options available. The beneficial graft-versus-leukemia (GVL) effect of cellular immunotherapy is mostly mediated by donor-derived CD8+ T lymphocytes that recognize minor histocompatibility antigens (mHags) and leukemia-associated antigens (LAAs) presented on the surface of AML blasts (Falkenburg et al. 2008; Kolb 2008). A main complication is graft-versus-host disease (GVHD) that can be induced when cytotoxic T lymphocytes (CTLs) recognize broadly expressed antigens. To reduce the risk of GVHD, specific allogeneic T-cell therapy inducing selective GVL responses could be an option (Barrett &amp; Le Blanc 2010; Parmar et al. 2011; Smits et al. 2011). This requires efficient in vitro strategies to generate AML-reactive T cells with an early differentiation phenotype as well as vigorous effector functions and humanized mouse models to analyze the anti-leukemic potential of adoptively transferred T cells in vivo. In this study, AML-reactive CTL clones and oligoclonal T-cell lines could be reliably generated from the naive subset of healthy HLA-class I-identical donors by stimulation with primary AML blasts in mini-mixed-lymphocyte / leukemia cultures (MLLCs) in eight different patient / donor pairs. These CTLs were promising candidates for cellular immunotherapy because of their relatively early differentiation phenotype and strong proliferative and lytic capabilities. The addition of the common γ-chain cytokine IL-21 to the stimulation protocol enabled more precursors to develop into potent leukemia-reactive CTLs, presumably by its beneficial effects on cell survival and antigen-specific proliferation during the first weeks of cultures. It also strengthened the early-stage phenotype. Three long-term cultured CTLs exemplarily transferred into leukemia-engrafted immunodeficient NSG mice mediated a significant reduction of the leukemic burden after a single transfusion. These results demonstrate that CTL clones with reactivity to patient-derived AML blasts can be isolated from the naive compartment of healthy donors and show potent anti-leukemic effects in vivo. The herein described allo-MLLC approach with in vitro “programmed” naive CTL precursors independent of a HSCT setting is a valuable alternative to the conventional method of isolating in vivo primed donor CTLs out of patients after transplantation (Kloosterboer et al. 2004; Warren et al. 2010). This would make leukemia-reactive CTLs already available at the time point of HSCT, when residual leukemia disease is minimal and the chances for complete leukemia eradication are high. Furthermore, leukemia-reactive CTLs effectively expanded by this in vitro protocol can be used as screening populations to identify novel candidate LAAs and mHags for antigen-specific immunotherapy.

Genetic patterns in recovered specimens of Caretta caretta from the Adriatic Sea

The Adriatic Sea is considered a feeding and developmental area for Mediterranean loggerhead turtles, but this area is severely threatened by human impacts. In the Adriatic Sea loggerhead turtles are often found stranded or floating, but they are also recovered as by-catch from fishing activities. Nevertheless, information about population structuring and origin of individuals found in the Adriatic Sea are still limited. Cooperation with fishermen and a good network of voluntary collaborators are essential for understanding their distribution, ecology and for developing conservation strategies in the Adriatic Sea. In this study, a comparative analysis of biometric data and DNA sequence polymorphism of the long fragment of the mitochondrial control region was carried out on ninety-three loggerheads recovered from three feeding areas in the Adriatic Sea: North-western, North-eastern and South Adriatic. Differences in turtles body sizes (e.g. Straight Carapace Length) among the three recovery areas and relationship between SCL and the type of recovery were investigated. The origin of turtles from Mediterranean rookeries and the use of the Adriatic feeding habitats by loggerheads in different life-stages were assessed to understand the migratory pathway of the species. The analysis of biometric data revealed a significant difference in turtle sizes between the Southern and the Northern Adriatic. Moreover, size of captured turtles resulted significantly different from the size of stranded and floating individuals. Actually, neritic sub-adults and adults are more affected by incidental captures than juveniles because of their feeding behavior. The Bayesian mixed-stock analysis showed a strong genetic relationship between the Adriatic aggregates and Mediterranean rookeries, while a low pro¬portion of individuals of Atlantic origin were detected in the Adriatic feeding grounds. The presence of migratory pathways towards the Adriatic Sea due to the surface current system was reinforced by the finding of individuals bearing haplotypes endemic to the nesting populations of Libya, Greece and Israel. A relatively high contribution from Turkey and Cyprus to the Northwest and South Adriatic populations was identified when the three sampled areas were analyzed independently. These results have to be taken in account in a conservative perspective, since coastal hazards, affecting the population of turtles feeding in the Adriatic Sea may also affect the nesting populations of the Eastern Mediterranean with a unique genetic pattern.