Pharmacological targeting of the ephrin receptor kinase signalling by GLPG1790 in vitro and in vivo reverts oncophenotype,induces myogenic differentiation and radiosensitizes embryonal rhabdomyosarcoma cells

Background

EPH (erythropoietin-producing hepatocellular) receptors are clinically relevant targets in several malignancies. This report describes the effects of GLPG1790, a new potent pan-EPH inhibitor, in human embryonal rhabdomyosarcoma (ERMS) cell lines.

Methods

EPH-A2 and Ephrin-A1 mRNA expression was quantified by real-time PCR in 14 ERMS tumour samples and in normal skeletal muscle (NSM). GLPG1790 effects were tested in RD and TE671 cell lines, two in vitro models of ERMS, by performing flow cytometry analysis, Western blotting and immunofluorescence experiments. RNA interfering experiments were performed to assess the role of specific EPH receptors. Radiations were delivered using an x-6 MV photon linear accelerator. GLPG1790 (30 mg/kg) in vivo activity alone or in combination with irradiation (2 Gy) was determined in murine xenografts.

Results

Our study showed, for the first time, a significant upregulation of EPH-A2 receptor and Ephrin-A1 ligand in ERMS primary biopsies in comparison to NSM. GLPG1790 in vitro induced G1-growth arrest as demonstrated by Rb, Cyclin A and Cyclin B1 decrease, as well as by p21 and p27 increment. GLPG1790 reduced migratory capacity and clonogenic potential of ERMS cells, prevented rhabdosphere formation and downregulated CD133, CXCR4 and Nanog stem cell markers. Drug treatment committed ERMS cells towards skeletal muscle differentiation by inducing a myogenic-like phenotype and increasing MYOD1, Myogenin and MyHC levels. Furthermore, GLPG1790 significantly radiosensitized ERMS cells by impairing the DNA double-strand break repair pathway. Silencing of both EPH-A2 and EPH-B2, two receptors preferentially targeted by GLPG1790, closely matched the effects of the EPH pharmacological inhibition. GLPG1790 and radiation combined treatments reduced tumour mass by 83% in mouse TE671 xenografts.

Conclusions

Taken together, our data suggest that altered EPH signalling plays a key role in ERMS development and that its pharmacological inhibition might represent a potential therapeutic strategy to impair stemness and to rescue myogenic program in ERMS cells.

     

Sampling intraspecific variability in leaf functional traits: Practical suggestions to maximize collected information

The choice of the best sampling strategy to capture mean values of functional traits for a species/population, while maintaining information about traits’ variability and minimizing the sampling size and effort, is an open issue in functional trait ecology. Intraspecific variability (ITV) of functional traits strongly influences sampling size and effort. However, while adequate information is available about intraspecific variability between individuals (ITV_BI) and among populations (ITV_POP), relatively few studies have analyzed intraspecific variability within individuals (ITV_WI). Here, we provide an analysis of ITV_WI of two foliar traits, namely specific leaf area (SLA) and osmotic potential (π), in a population of Quercus ilex L. We assessed the baseline ITV_WI level of variation between the two traits and provided the minimum and optimal sampling size in order to take into account ITV_WI, comparing sampling optimization outputs with those previously proposed in the literature. Different factors accounted for different amount of variance of the two traits. SLA variance was mostly spread within individuals (43.4% of the total variance), while π variance was mainly spread between individuals (43.2%). Strategies that did not account for all the canopy strata produced mean values not representative of the sampled population. The minimum size to adequately capture the studied functional traits corresponded to 5 leaves taken randomly from 5 individuals, while the most accurate and feasible sampling size was 4 leaves taken randomly from 10 individuals. We demonstrate that the spatial structure of the canopy could significantly affect traits variability. Moreover, different strategies for different traits could be implemented during sampling surveys. We partially confirm sampling sizes previously proposed in the recent literature and encourage future analysis involving different traits.     

Forbidden versus permitted interactions: Disentangling processes from patterns in ecological network analysis

Several studies have identified the tendency for species to share interacting partners as a key property to the functioning and stability of ecological networks. However, assessing this pattern has proved challenging in several regards, such as finding proper metrics to assess node overlap (sharing), and using robust null modeling to disentangle significance from randomness. Here, we bring attention to an additional, largely neglected challenge in assessing species’ tendency to share interacting partners. In particular, we discuss and illustrate with two different case studies how identifying the set of “permitted” interactions for a given species (i.e. interactions that are not impeded, e.g. by lack of functional trait compatibility) is paramount to understand the ecological and co‐evolutionary processes at the basis of node overlap and segregation patterns.     

Genetic and Phylogeographic Structures of the Symbiotic Fungus Tuber magnatum

The quality and market price of truffles vary with the species and, traditionally, the place of origin. The premium species Tuber magnatum produces white truffles and has a patchy distribution restricted to Italy and some Balkan areas. We used polymorphic microsatellites to evaluate 316 specimens grouped into 26 populations sampled across the species' geographic range to determine if natural populations of T. magnatum are genetically differentiated. We found that the southernmost and the northwesternmost populations were significantly differentiated from the rest of the populations. The simple sequence repeat data also could be used to make inferences about the postglacial T. magnatum expansion pattern. This study is the first to identify a genetic and phylogeographic structure in T. magnatum. The presence of a genetic structure can be of practical interest in tracing truffle populations according to their geographic origin for marketing strategies. Evidence for extensive outcrossing in field populations of T. magnatum also is provided for the first time.     

Quantitative-PCR Assessment of Cryptosporidium parvum Cell Culture Infection

A quantitative TaqMan PCR method was developed for assessing the Cryptosporidium parvum infection of in vitro cultivated human ileocecal adenocarcinoma (HCT-8) cell cultures. This method, termed cell culture quantitative sequence detection (CC-QSD), has numerous applications, several of which are presented. CC-QSD was used to investigate parasite infection in cell culture over time, the effects of oocyst treatment on infectivity and infectivity assessment of different C. parvum isolates. CC-QSD revealed that cell culture infection at 24 and 48 h postinoculation was approximately 20 and 60%, respectively, of the endpoint 72-h postinoculation infection. Evaluation of three different lots of C. parvum Iowa isolate oocysts revealed that the mean infection of 0.1 N HCl-treated oocysts was only 36% of the infection obtained with oocysts treated with acidified Hanks' balanced salt solution containing 1% trypsin. CC-QSD comparison of the C. parvum Iowa and TAMU isolates revealed significantly higher levels of infection for the TAMU isolate, which agrees with and supports previous human, animal, and cell culture studies. CC-QSD has the potential to aid in the optimization of Cryptosporidium cell culture methods and facilitate quantitative evaluation of cell culture infectivity experiments.     

Parathyroid hormone-related peptide (hPTHrP) and parathyroid hormone-related peptide receptor type 1 (PTHR1) expression in human thymus.

Parathyroid hormone-related peptide (hPTHrP) is expressed in human tissues and regulates cellular proliferation, differentiation, and apoptosis by an autocrine/paracrine loop. In rodent thymus, both parathormone and parathyroid hormone-related peptide (PTHrP) are expressed by thymic epithelial cells (TECs). The present study demonstrated by RT-PCR and immunohistochemistry that hPTHrP and parathyroid hormone-related peptide receptor type 1 (PTHR1) were expressed in human thymus at both RNA and protein levels. hPTHrP was expressed mainly in the thymic medulla by epithelial (cytokeratin-positive), mature dendritic (CD40+/86+) and plasmacytoid interleukin (IL)-3Ralpha1 cells. This protein was also present in some cells forming Hassall's bodies and a few subcapsular and cortical TECs. PTHR1 was expressed by scattered subcapsular and cortical TECs and by rare TECs in the medulla. Thymocytes did not express either hPTHrP or PTHR1. Primary cultures of human TECs revealed the presence of both hPTHrP and PTHR1 mRNAs, confirming the capacity of TECs to synthesize both peptides. Moreover, synthetic (1-39) hPTHrP peptide administered on cultured TECs induced the expression of IL-6 mRNA, suggesting that hPTHrP can regulate thymic functions by inducing in TECs the expression of IL-6, which is involved in the development and maturation of thymocytes.     

The biological foundations of culture and morality

Two aspects are discussed: first, the mechanism of learning, and second, the generation of culture and morality. Both aspects are analyzed in relation to the evolution of the mind-brain system. As to the first aspect, I suggest that the use of the concept of innatism requires a re-examination of the genome-dependent effects on the mind-brain system particularly for what concerns the time scale of these effects. The reason is that the information in the genome of the parents is transmitted to the embryo during fertilization of the egg and then into the structures of neuronal cells at the very early stage of ontogenesis, whereas organization of the mind-brain system occurs at a much later time and after an extensive reorganization of the brain structure. The problem is, then, that while during the early stages of the ontogenetic development the neuronal cells maintain their genome determined properties, the full expression of these genome properties within the mind-brain organization undergo fundamental changes which depend not on the properties of the genomic information but rather on the evolution of the operational conditions which are generated in the brain organization and which determine the expression of the genomic potentialities. These operational conditions may become markedly different because of a number of on-going processes due to the formation of new synapses, axons, dendrites and neuronal networks; these phenomena are, to a large extent due to interaction with the environment. Using the Fodor’s language, the effect of the innate properties over the horizontal neuronal networks (the innatism that operates during the whole ontogenesis) assumes a much more important role in the mind-brain than in all other physiological systems. As to the second aspect, I suggest that the development of cultural andmoral worlds are products of the histories of human minds and societies, and depend on the evolutionary nature of these histories.To explain the generation of these worlds I shall discuss a basic problem. How can the neuronal networks of the mind-brain system — usually dealing with phenomena of the natural world and being themselves natural structures obeying the principles and rules of the natural phenomena, where no transition from is to ought or from facts to values is allowed — be able to generate cultural and moral concepts and generate moral behaviours? The solution here proposed is that some sort of modularitydominates in the whole structure of the mind-brain system and that the effect of this modularity is that the neurophysiological processes dealing with the processes of human cultural, moral and social life are structurally associated with emotions, intentions and values. In brief, it is suggested that the mind-brain systems contain, in addition to the division introduced by Fodor, other innate types of neurophysiological processes: some types of processes and of neuronal networks deal with the problems of the natural world and provide answers which are right or wrong, wheras other types of processes and neuronal networks deal with problems of human cultural and social life and provide answers which are adapt on non-adapt.The latter types of neuronal networks are suited to generate the cultural and moral worlds in the course of development and evolution of the Homo sapiens sapiens species and of the single individual. Finally, in view of recent findings on the patterns of mental diseases and the relations of these patterns with alterations of the frontal lobes of the mind brain system, I suggest that the particular types of processes responsible for the moral behaviour of human beings are localized in the frontal lobes. The consequence is that alterations of this type of neurophysiological processes lead to the development of various types of mental diseases.      

Regeneration in invertebrates

The mechanisms of regeneration are reviewed from a genetic, cytological and molecular biological points of view. Planarians and Hydra have been chosen and illustrated as biological examples.      

Non-organ-specific autoantibodies in renal transplant recipients: relation to BK virus infection

Polyomavirus BK reactivation is common in renal transplant recipients and may cause nephropathy with significant graft dysfunction. The induction of anti-double stranded DNA (anti-dsDNA) antibodies by BKV has been described in experimental animals and during primary infection, and has been implicated in the pathogenesis of systemic lupus erythematosus. This study evaluated the occurrence of anti-dsDNA antibodies and non-organ-specific autoantibodies (NOSA) by indirect immunofluorescence before transplantation and at 3 and 6 months post-transplantation in 90 renal transplant recipients and the association with BKV reactivation, demographic and clinical features. Moreover, the relation to HCMV infection, as detected by pp65-antigenemia, was also evaluated. Post-transplantation NOSAs were present in 23/90 (25.6%) and anti-dsDNA antibodies in 17/90 (18.9%). BK viremia was detected in at least one serum sample in 22 patients: 9 anti-dsDNA antibody-positive vs 13 negative (p<0.01). No significant correlation between the occurrence of NOSAs and anti-dsDNA antibodies and demographic and clinical features was found. No significant association with pp65-antigenemia-positivity was found, although antigenemia was positive in 6/23 NOSA-positive patients (26.1%). Although a relation seems to exist between BKV and the occurrence of anti-dsDNA antibodies in renal transplant patients, the lack of correlation with other epidemiological and clinical features does not allow any conclusion. The role of autoimmune response in this context and the relation with other patient-related factors and infectious agents should be further investigated.