Sonic hedgehog inhibition reduces in vitro tumorigenesis and alters expression of GLI1-target genes in a desmoplastic medulloblastoma cell line

Medulloblastoma is one of the most frequent and aggressive tumors of childhood. The Sonic hedgehog (Shh) pathway, related to human development, is altered in most medulloblastomas: genes like Ptch, Smo, or Sufu suffer mutations in 15% to 25% of these tumors. We tested Shh inhibition in the Daoy medulloblastoma cell line by two methods: a molecular one, direct Gli1 siRNA inhibition; and a pharmacological inhibition of Smo, upstream of Gli1, by cyclopamine. Afterwards, a comparison of cellular and molecular responses was done. In general, we proved that cell viability, cell migration and cell colony formation decreased after Shh inhibition, which might confer a less tumorigenic status to Daoy cells. Moreover, we assessed the expression of different Gli1 target genes and other genes and found that Shh shows a crosstalk with oncogenes and tumor suppressor genes that have been described in numerous tumors. All these experiments give an overview of the Shh pathway in medulloblastoma, together with the demonstration of the efficacy of cyclopamine and Gli1 siRNA Shh inhibition in vitro

Novel insights into the genomic basis of citrus canker based on the genome sequences of two strains of Xanthomonas fuscans subsp. aurantifolii

Background: Citrus canker is a disease that has severe economic impact on the citrus industry worldwide. There are three types of canker, called A, B, and C. The three types have different phenotypes and affect different citrus species. The causative agent for type A is Xanthomonas citri subsp. citri, whose genome sequence was made available in 2002. Xanthomonas fuscans subsp. aurantifolii strain B causes canker B and Xanthomonas fuscans subsp. aurantifolii strain C causes canker C. Results: We have sequenced the genomes of strains B and C to draft status. We have compared their genomic content to X. citri subsp. citri and to other Xanthomonas genomes, with special emphasis on type III secreted effector repertoires. In addition to pthA, already known to be present in all three citrus canker strains, two additional effector genes, xopE3 and xopAI, are also present in all three strains and are both located on the same putative genomic island. These two effector genes, along with one other effector-like gene in the same region, are thus good candidates for being pathogenicity factors on citrus. Numerous gene content differences also exist between the three cankers strains, which can be correlated with their different virulence and host range. Particular attention was placed on the analysis of genes involved in biofilm formation and quorum sensing, type IV secretion, flagellum synthesis and motility, lipopolysacharide synthesis, and on the gene xacPNP, which codes for a natriuretic protein. Conclusion: We have uncovered numerous commonalities and differences in gene content between the genomes of the pathogenic agents causing citrus canker A, B, and C and other Xanthomonas genomes. Molecular genetics can now be employed to determine the role of these genes in plant-microbe interactions. The gained knowledge will be instrumental for improving citrus canker control.Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)Conselho Nacional de Desenvolvimento CientIfico e Tecnologico (CNPq)Coordenacao para Aperfeicoamento de Pessoal de Ensino Superior (CAPES)Fundo de Defesa da Citricultura (FUNDECITRUS

Multiple Nuclear Gene Phylogenetic Analysis of the Evolution of Dioecy and Sex Chromosomes in the Genus Silene

In the plant genus Silene, separate sexes and sex chromosomes are believed to have evolved twice. Silene species that are wholly or largely hermaphroditic are assumed to represent the ancestral state from which dioecy evolved. This assumption is important for choice of outgroup species for inferring the genetic and chromosomal changes involved in the evolution of dioecy, but is mainly based on data from a single locus (ITS). To establish the order of events more clearly, and inform outgroup choice, we therefore carried out (i) multi-nuclear-gene phylogenetic analyses of 14 Silene species (including 7 hermaphrodite or gynodioecious species), representing species from both Silene clades with dioecious members, plus a more distantly related outgroup, and (ii) a BayesTraits character analysis of the evolution of dioecy. We confirm two origins of dioecy within this genus in agreement with recent work on comparing sex chromosomes from both clades with dioecious species. We conclude that sex chromosomes evolved after the origin of Silene and within a clade that includes only S. latifolia and its closest relatives. We estimate that sex chromosomes emerged soon after the split with the ancestor of S. viscosa, the probable closest non-dioecious S. latifolia relative among the species included in our study

Characterization of cellular and molecular responses of human glioblastoma to Transforming Growth Factor-β signalling pathway inhibition

Glioblastoma Multiforme (GBM) is the most prevalent malignant brain tumour accounting for 60-70% of all gliomas. Improvements in survival over the past 100 years can be measured only in weeks, and current achieved median survival ranges only 12-15 months. A hallmark of this malignancy is the intrinsic resistance to current therapies. Numerous efforts using molecularly targeted therapeutics have not significantly changed the near uniform lethality of this disease. The TGF-β signalling pathway plays a key role in GBM. It is implicated in progression, infiltration, and chemo/radioresistance as well as in the maintenance of stem-like phenotype of GBM CSC. Several inhibitors of different elements and regulators of the TGF-β pathway have entered to clinical trials. Among them, P17 and P144 inhibitory peptides of the TGF-β pathway have been tested for the treatment of different diseases including tumours. We decided to analyse the therapeutic potential of P144 for the treatment of GBM. We found that P144 impaired in vitro cellular processes as proliferation, migration, invasiveness and tumorigenicity. Apoptosis and anoikis were significantly increased by P144. Additionally, P144 blocked the TGF-β protective effect against apoptosis. The inhibition of TGF-β signalling by P144 affected the self-renewal capacity of a putative CSC subpopulation in vitro. These results were confirmed by the analysis on Brain Tumour Initiating Cells (BTIC) isolated from human GBM biopsies. P144 decreased in vitro proliferation, migration, and self-renewal capacity of this subpopulation. The effect of P144 was impaired by hypoxia. However, the precise underlying mechanism of hypoxia on P144 must be elucidated. We confirm the inhibition of TGF-β signalling by P144 through SMAD2 phosphorylation blockade, the pivotal initiation event of the pathway, which was translated to a reduction of P-SMAD2 nuclear translocation. Both results suggested an in vitro regulation on the transcriptional target genes of the TGF-β pathway in GBM cell lines. Furthermore, we confirmed in vitro and in vivo, the upregulation of SMAD7 and the downregulation of SKI by P144 at transcriptional and translational levels. This observation strongly suggests the implication of these factors in the molecular mechanism triggered by P144. The therapeutic potential of P144 was analysed in a mouse subcutaneous tumour model. Despite that P144 impaired tumour growth and leaded to an increase in survival, negative contradictory results were obtained in the in vivo intracranial model. We can conclude that the therapeutic potential of P144 as a treatment of GBM is clear. However, previous to potential clinical development, further studies are required in order to confirm P144 effect over GBM in the brain environment, as well as to explore P144 therapeutic potential in combination with current (TMZ and/or radiation) and emerging molecular based therapies

Characterization of cellular and molecular responses of human glioblastoma to Transforming Growth Factor-β signalling pathway inhibition

Glioblastoma Multiforme (GBM) is the most prevalent malignant brain tumour accounting for 60-70% of all gliomas. Improvements in survival over the past 100 years can be measured only in weeks, and current achieved median survival ranges only 12-15 months. A hallmark of this malignancy is the intrinsic resistance to current therapies. Numerous efforts using molecularly targeted therapeutics have not significantly changed the near uniform lethality of this disease. The TGF-β signalling pathway plays a key role in GBM. It is implicated in progression, infiltration, and chemo/radioresistance as well as in the maintenance of stem-like phenotype of GBM CSC. Several inhibitors of different elements and regulators of the TGF-β pathway have entered to clinical trials. Among them, P17 and P144 inhibitory peptides of the TGF-β pathway have been tested for the treatment of different diseases including tumours. We decided to analyse the therapeutic potential of P144 for the treatment of GBM. We found that P144 impaired in vitro cellular processes as proliferation, migration, invasiveness and tumorigenicity. Apoptosis and anoikis were significantly increased by P144. Additionally, P144 blocked the TGF-β protective effect against apoptosis. The inhibition of TGF-β signalling by P144 affected the self-renewal capacity of a putative CSC subpopulation in vitro. These results were confirmed by the analysis on Brain Tumour Initiating Cells (BTIC) isolated from human GBM biopsies. P144 decreased in vitro proliferation, migration, and self-renewal capacity of this subpopulation. The effect of P144 was impaired by hypoxia. However, the precise underlying mechanism of hypoxia on P144 must be elucidated. We confirm the inhibition of TGF-β signalling by P144 through SMAD2 phosphorylation blockade, the pivotal initiation event of the pathway, which was translated to a reduction of P-SMAD2 nuclear translocation. Both results suggested an in vitro regulation on the transcriptional target genes of the TGF-β pathway in GBM cell lines. Furthermore, we confirmed in vitro and in vivo, the upregulation of SMAD7 and the downregulation of SKI by P144 at transcriptional and translational levels. This observation strongly suggests the implication of these factors in the molecular mechanism triggered by P144. The therapeutic potential of P144 was analysed in a mouse subcutaneous tumour model. Despite that P144 impaired tumour growth and leaded to an increase in survival, negative contradictory results were obtained in the in vivo intracranial model. We can conclude that the therapeutic potential of P144 as a treatment of GBM is clear. However, previous to potential clinical development, further studies are required in order to confirm P144 effect over GBM in the brain environment, as well as to explore P144 therapeutic potential in combination with current (TMZ and/or radiation) and emerging molecular based therapies

To migrate or not: drivers of over-summering in a long-distance migratory shorebird

The phenomenon of over-summering in southern non-breeding areas by boreal-breeding birds is particularly prevalent among shorebirds. Despite its frequency, it is understudied compared with most other aspects of shorebird ecology. Our aim was to expand knowledge of this subject through a study of red knots Calidris canutus rufa over-summering at a site in Argentina during the austral winter. We measured the proportion of one-year-old and adult over-summerers and evaluated the roles of flight-feather condition and physiological status (through leukocite profile, physiological stress index and presence of blood parasites) as triggers for over-summering. We also explore sex-ratio, the evolution of body mass and extent of breeding plumage within each age-class. Over-summerers were 57% yearlings and 43% adults, 46% females and 54% males. Almost all yearlings exhibited incomplete molt of primary feathers and some were in active primary molt. This suggests that the condition of flight-feathers and the timing of molt are likely to be important factors selecting for deferred migration during the first year of life. Other factors, not associated with flight-feather molt, seem to trigger over-summering in adults, which had completed flight feather molt but had low fat loads and/or incomplete alternate plumage. We found no evidence of a weakened immune system, high loads of blood parasites or high stress levels that can explain this poor migratory conditioning and therefore over-summering. Our data indicates that our adult age-class comprises both young individuals postponing first breeding until they are at least two years old and sexually mature individuals with prior reproductive experience skipping a breeding opportunity. Breeding propensity and age at first breeding are both poorly known, but key demographic parameters that determine population growth. This study suggests that potentially they can be estimated from mark–recapture at non-breeding areas and this warrants further study.Fil: Martínez Curci, Natalia Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Marinas y Costeras. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Marinas y Costeras; ArgentinaFil: Isacch, Juan Pablo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Marinas y Costeras. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Marinas y Costeras; ArgentinaFil: D'amico, Veronica Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Centro Nacional Patagónico; ArgentinaFil: Rojas, Pablo. Gobierno de la Provincia de Buenos Aires. Organismo Provincial Para El Desarrollo Sostenible; ArgentinaFil: Castresana, Gabriel J.. Gobierno de la Provincia de Buenos Aires. Organismo Provincial Para El Desarrollo Sostenible; Argentin

Annual occurrence of Red Knot Calidris canutus rufa at Punta Rasa, Samborombón Bay, Argentina, over a 30-year period (1985–2014)

From 1985 to 2014, we monitored numbers of Red Knots Calidris canutus of the subspecies rufa occurring at Punta Rasa, Samborombón Bay, Buenos Aires province, Argentina during northward migration (March to April), and in the austral winter (mid-May to mid-August), when some knots, mainly immatures, remain in South America during the breeding season. Numbers occurring during northward migration declined over the monitoring period from a peak of 3,640 in 1987 to 168 in 2014. This is consistent with the concurrent decline of the main Tierra del Fuego wintering population (though the percentage of the Tierra del Fuego population stopping at Punta Rasa also declined from 5.4% to 1.3%). Numbers occurring at Punta Rasa during the austral winter have also declined, but not to the same extent and for the past decade more have been counted during the austral winter than during northward migration. Among those seen at Punta Rasa during the Arctic breeding season have been some with large amounts of rufous breeding plumage. The percentage of such birds has increased from 10–15% in 1987 to 19–54% during 2011–2014. Moreover, observations of individually marked birds show that some of those showing breeding plumage are mature adults, at least 3–7 years old. Conservation actions are required to manage Punta Rasa for the small but important numbers of Red Knots that occur there. Further research is needed to determine why some mature adults do not go to their Arctic breeding grounds and thereby fail to contribute to reproduction.Fil: Martínez Curci, Natalia Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Marinas y Costeras. Universidad Nacional de Mar del Plata. Facultad de Ciencia Exactas y Naturales. Instituto de Investigaciones Marinas y Costeras; ArgentinaFil: Bremer, Esteban. Fundación Vida Silvestre Argentina; ArgentinaFil: Azpiroz, Adrián B.. Instituto de Investigaciones Biológicas "Clemente Estable"; UruguayFil: Battaglia, Gabriel E.. Municipalidad del partido de La Costa; ArgentinaFil: Salerno, Javier C.. Municipalidad del partido de La Costa; ArgentinaFil: Isacch, Juan Pablo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Marinas y Costeras. Universidad Nacional de Mar del Plata. Facultad de Ciencia Exactas y Naturales. Instituto de Investigaciones Marinas y Costeras; ArgentinaFil: González, Patricia M.. Fundación Inalafquen; ArgentinaFil: Castresana, Gabriel J.. Reserva Natural Bahía Samborombón; ArgentinaFil: Rojas, Pablo. Reserva Natural Bahía Samborombón; Argentin