Orthogonal protection of peptides and peptoids for cyclization by the thiol-ene reaction and conjugation

Cyclic peptides and peptoids were prepared using the thiolene Michael-type reaction. The linear precursors were provided with additional functional groups allowing for subsequent conjugation: an orthogonally protected thiol, a protected maleimide, or an alkyne. The functional group for conjugation was placed either within the cycle or in an external position. The click reactions employed for conjugation with suitably derivatized nucleoside or oligonucleotides were either cycloadditions (Diels-Alder, Cu(I)-catalyzed azide-alkyne) or the same Michael-type reaction as for cyclization.

Pint lincRNA connects the p53 pathway with epigenetic silencing by the Polycomb repressive complex 2

BACKGROUND: The p53 transcription factor is located at the core of a complex wiring of signaling pathways that are critical for the preservation of cellular homeostasis. Only recently it has become clear that p53 regulates the expression of several long intergenic noncoding RNAs (lincRNAs). However, relatively little is known about the role that lincRNAs play in this pathway. RESULTS: Here we characterize a lincRNA named Pint (p53 induced noncoding transcript). We show that Pint is a ubiquitously expressed lincRNA that is finely regulated by p53. In mouse cells, Pint promotes cell proliferation and survival by regulating the expression of genes of the TGF-β, MAPK and p53 pathways. Pint is a nuclear lincRNA that directly interacts with the Polycomb repressive complex 2 (PRC2), and is required for PRC2 targeting of specific genes for H3K27 tri-methylation and repression. Furthermore, Pint functional activity is highly dependent on PRC2 expression. We have also identified Pint human ortholog (PINT), which presents suggestive analogies with the murine lincRNA. PINT is similarly regulated by p53, and its expression significantly correlates with the same cellular pathways as the mouse ortholog, including the p53 pathway. Interestingly, PINT is downregulated in colon primary tumors, while its overexpression inhibits the proliferation of tumor cells, suggesting a possible role as tumor suppressor. CONCLUSIONS: Our results reveal a p53 autoregulatory negative mechanism where a lincRNA connects p53 activation with epigenetic silencing by PRC2. Additionally, we show analogies and differences between the murine and human orthologs, identifying a novel tumor suppressor candidate lincRNA.

Evaluation of the Development of Metacognitive Knowledge Supported by the KnowCat System

The aim of this study was to examine the development of the metacognitive knowledge of a group of higher education students who participated actively in an experiment based on a Computer Supported Collaborative Learning environment called KnowCat. Eighteen university students participated in a 12-month learning project during which the KnowCat learning environment was used to support scaffolding process among equals during problem-solving tasks. After using KnowCat, the students were interviewed over their work in this shared workspace. Qualitative analysis revealed that the educational application of KnowCat can favour and improve the development of metacognitive knowledge.

Pint lincRNA connects the p53 pathway with epigenetic silencing by the Polycomb repressive complex 2

BACKGROUND: The p53 transcription factor is located at the core of a complex wiring of signaling pathways that are critical for the preservation of cellular homeostasis. Only recently it has become clear that p53 regulates the expression of several long intergenic noncoding RNAs (lincRNAs). However, relatively little is known about the role that lincRNAs play in this pathway. RESULTS: Here we characterize a lincRNA named Pint (p53 induced noncoding transcript). We show that Pint is a ubiquitously expressed lincRNA that is finely regulated by p53. In mouse cells, Pint promotes cell proliferation and survival by regulating the expression of genes of the TGF-β, MAPK and p53 pathways. Pint is a nuclear lincRNA that directly interacts with the Polycomb repressive complex 2 (PRC2), and is required for PRC2 targeting of specific genes for H3K27 tri-methylation and repression. Furthermore, Pint functional activity is highly dependent on PRC2 expression. We have also identified Pint human ortholog (PINT), which presents suggestive analogies with the murine lincRNA. PINT is similarly regulated by p53, and its expression significantly correlates with the same cellular pathways as the mouse ortholog, including the p53 pathway. Interestingly, PINT is downregulated in colon primary tumors, while its overexpression inhibits the proliferation of tumor cells, suggesting a possible role as tumor suppressor. CONCLUSIONS: Our results reveal a p53 autoregulatory negative mechanism where a lincRNA connects p53 activation with epigenetic silencing by PRC2. Additionally, we show analogies and differences between the murine and human orthologs, identifying a novel tumor suppressor candidate lincRNA.

Hollywood Films in the Non-Western World: What Are the Criteria Followed by the Chinese Government When Choosing Hollywood Film Imports?

This dissertation argues that the Government of the People’s Republic of China, when it made the decision to import a quota of Hollywood films in 1994 to revive the failing domestic film industry, had different possible criteria in mind. This project has studied four of them: first, importing films that gave a negative image of the United States; second, importing films that featured Chinese talent or themes; third, importing films that were box office hits in the United States; and fourth, importing films with a strong technological innovation ingredient. In order to find out the most important criteria for the Chinese Government, this dissertation offers a dataset that analyzes a population of 262 Hollywood films released in the PRC between 1994 and 2010. For each unit, a method has been developed to compile data that will determine whether the film reflects any of the four criteria, and findings in the form of yearly percentages have been drawn. Results show that, out of the four studied criteria, the two predominant reasons for China to import films were technological innovation and box office hits. This tells us that, at this point, the Chinese Government shows more interest in obtaining big revenues and learning from technically innovative American films than in delivering underlying political messages. This dissertation contributes to the existing literature by analyzing the content of all the films imported by China between 1994 and 2010, while integrating in the analysis variables based on the existing knowledge.

Predation on common tern eggs by the yellow-legged gull at the Ebro Delta

The Ebro Delta holds a large seabird community, including a common tern (Sterna hirundo) local population of 3,085 pairs in 2000 which breeds scattered in several colonies. At El Canalot colony, 1,178 (1999) and 1,156 pairs (2000) of this species bred distributed in 32 and 38 sub-colonies respectively. These sub-colonies varied in size from 1 to 223 pairs and were placed near the main breeding colonies of yellow-legged gulls (Larus cachinnans) and Audouin´s gulls (L. audouinii), which are potential egg-predators of terns. We studied egg predation during 1999 (6 sub-colonies) and 2000 (27 sub-colonies). Overall, we found that 10.6% of the nests in 1999 and 16.7% in 2000 suffered partial or total egg predation, being total in 81.1% of the predatory events. Predation was significantly higher in small sub-colonies (< 11 pairs): 49.4% in 1999 and 75.5% in 2000. Only attacks from yellow-legged gulls were observed, and defence behaviour of terns was significantly more frequent against this gull species (40.5 hours of observation), suggesting that in most cases the egg predation recorded was due to this species. Probability of egg predation was significantly and negatively correlated with distance to the nearest yellow-legged gull sub-colony, although this relationship was no more significant after adjustment for sub-colony size. On the other hand, distance to the nearest Audouin´s gull sub-colony did not show any effect. Our results suggest that the impact of large gulls (at least yellow-legged gulls) upon smaller seabirds breeding in the area might be important, especially when they are breeding in small sub-colonies. Further studies are needed to analyse the general impact of large gulls upon the breeding populations of other colonial bird species in the area.

Differential regulation of horizontally acquired and core genome genes by the bacterial modulator H-NS

Horizontal acquisition of DNA by bacteria dramatically increases genetic diversity and hence successful bacterial colonization of several niches, including the human host. A relevant issue is how this newly acquired DNA interacts and integrates in the regulatory networks of the bacterial cell. The global modulator H-NS targets both core genome and HGT genes and silences gene expression in response to external stimuli such as osmolarity and temperature. Here we provide evidence that H-NS discriminates and differentially modulates core and HGT DNA. As an example of this, plasmid R27-encoded H-NS protein has evolved to selectively silence HGT genes and does not interfere with core genome regulation. In turn, differential regulation of both gene lineages by resident chromosomal H-NS requires a helper protein: the Hha protein. Tight silencing of HGT DNA is accomplished by H-NS-Hha complexes. In contrast, core genes are modulated by H-NS homoligomers. Remarkably, the presence of Hha-like proteins is restricted to the Enterobacteriaceae. In addition, conjugative plasmids encoding H-NS variants have hitherto been isolated only from members of the family. Thus, the H-NS system in enteric bacteria presents unique evolutionary features. The capacity to selectively discriminate between core and HGT DNA may help to maintain horizontally transmitted DNA in silent form and may give these bacteria a competitive advantage in adapting to new environments, including host colonization.

Differential regulation of horizontally acquired and core genome genes by the bacterial modulator H-NS

Horizontal acquisition of DNA by bacteria dramatically increases genetic diversity and hence successful bacterial colonization of several niches, including the human host. A relevant issue is how this newly acquired DNA interacts and integrates in the regulatory networks of the bacterial cell. The global modulator H-NS targets both core genome and HGT genes and silences gene expression in response to external stimuli such as osmolarity and temperature. Here we provide evidence that H-NS discriminates and differentially modulates core and HGT DNA. As an example of this, plasmid R27-encoded H-NS protein has evolved to selectively silence HGT genes and does not interfere with core genome regulation. In turn, differential regulation of both gene lineages by resident chromosomal H-NS requires a helper protein: the Hha protein. Tight silencing of HGT DNA is accomplished by H-NS-Hha complexes. In contrast, core genes are modulated by H-NS homoligomers. Remarkably, the presence of Hha-like proteins is restricted to the Enterobacteriaceae. In addition, conjugative plasmids encoding H-NS variants have hitherto been isolated only from members of the family. Thus, the H-NS system in enteric bacteria presents unique evolutionary features. The capacity to selectively discriminate between core and HGT DNA may help to maintain horizontally transmitted DNA in silent form and may give these bacteria a competitive advantage in adapting to new environments, including host colonization.

Treatment of cancer with oral drugs: a position statement by the Spanish Society of Medical Oncology

Cancer treatment involves the participation of multiple medical specialties and, as our knowledge of the disease increases, this fact becomes even more apparent. The degree of multidisciplinarity is determined by several factors, which include the severity and type of disease, the increasing diversity in the available pharmacological and non-pharmacological therapies, and the range of specialists involved in cancer therapy, such as medical oncologists, radiotherapists, gynecologists, gastroenterologists, urologists, surgeons, and pneumologists, among others. Across Europe, the situation of cancer care can be variable due to the diversity of health systems, differences in drug reimbursement, and the degree of establishment of Medical Oncology as a medical specialty in the European Union states.

Direct Interrogation of Viral Peptides Presented by the Class I HLA of HIV-Infected T Cells

Identification of CD8+ cytotoxic T lymphocyte (CTL) epitopes has traditionally relied upon testing of overlapping peptide libraries for their reactivity with T cells in vitro. Here, we pursued deep ligand sequencing (DLS) as an alternative method of directly identifying those ligands that are epitopes presented to CTLs by the class I human leukocyte antigens (HLA) of infected cells. Soluble class I HLA-A*11:01 (sHLA) was gathered from HIV-1 NL4-3-infected human CD4+ SUP-T1 cells. HLA-A*11:01 harvested from infected cells was immunoaffinity purified and acid boiled to release heavy and light chains from peptide ligands that were then recovered by size-exclusion filtration. The ligands were first fractionated by high-pH high-pressure liquid chromatography and then subjected to separation by nano-liquid chromatography (nano-LC)–mass spectrometry (MS) at low pH. Approximately 10 million ions were selected for sequencing by tandem mass spectrometry (MS/MS). HLA-A*11:01 ligand sequences were determined with PEAKS software and confirmed by comparison to spectra generated from synthetic peptides. DLS identified 42 viral ligands presented by HLA-A*11:01, and 37 of these were previously undetected. These data demonstrate that (i) HIV-1 Gag and Nef are extensively sampled, (ii) ligand length variants are prevalent, particularly within Gag and Nef hot spots where ligand sequences overlap, (iii) noncanonical ligands are T cell reactive, and (iv) HIV-1 ligands are derived from de novo synthesis rather than endocytic sampling. Next-generation immunotherapies must factor these nascent HIV-1 ligand length variants and the finding that CTL-reactive epitopes may be absent during infection of CD4+ T cells into strategies designed to enhance T cell immunity.

Negative feedback regulation of calcineurin-dependent Prz1 transcription factor by the CaMKK-CaMK1 axis in fission yeast

Calcium signals trigger the translocation of the Prz1 transcription factor from the cytoplasm to the nucleus. The process is regulated by the calciumactivated phosphatase calcineurin, which activates Prz1 thereby maintaining active transcription during calcium signalling. When calcium signalling ceases, Prz1 is inactivated by phosphorylation and exported to the cytoplasm. In budding yeast and mammalian cells, different kinases have been reported to counter calcineurin activity and regulate nuclear export. Here, we show that the Ca2+/calmodulin-dependent kinase Cmk1 is first phosphorylated and activated by the newly identified kinase CaMKK2 homologue, Ckk2, in response to Ca2+. Then, active Cmk1 binds, phosphorylates and inactivates Prz1 transcription activity whilst at the same time cmk1 expression is enhanced by Prz1 in response to Ca2+. Furthermore, Cdc25 phosphatase is also phosphorylated by Cmk1, inducing cell cycle arrest in response to an increase in Ca2+. Moreover, cmk1 deletion shows a high tolerance to chronic exposure to Ca2+, due to the lack of cell cycle inhibition and elevated Prz1 activity. This work reveals that Cmk1 kinase activated by the newly identified Ckk2 counteracts calcineurin function by negatively regulating Prz1 activity which in turn is involved in activating cmk1 gene transcription. These results are the first insights into Cmk1 and Ckk2 function in Schizosaccharomyces pombe.

Fostering Collaborative Knowledge Building by the Effective Provision of Knowledge about the Discussion Process

Peer-reviewed