Use of the Neurospora tyrosinase gene as a reporter gene in transformation experiments

Reporter gene systems have been developed to allow investigators to visually identify transformed cells and to follow the transcriptional activity from promoter regions preceding the reporter genes. These reporter genes encode protein products which can be easily assayed and which can be strained for in the transformed cell

Early Primary Percutaneous Coronary Intervention in Patients with ST-segment Elevation Acute Myocardial Infarction from the Cluj Area

Background: The seriousness of acute myocardial infarction (AMI) and the importance of its early detection and intervention are well known. Rapid reperfusion of the infarct area positively influences the immediate and long-term prognosis of patients with ST-segment elevation AMI. Material and method: Patients with acute myocardial infarction who underwent primary percutaneous transluminal coronary angioplasty (during the first 12 hours after the onset of chest pain) in the cardiac catheterization laboratory of the Cluj-Napoca “Nicolae Stancioiu” Heart Institute between November 2008 and February 2010 were followed prospectively in order to measure time-to-treatment intervals. Results: Our sample of 321 AMI patients included mostly males (73.8% of cases, 95% CI: 68.6-78.5; p<0.001) and patients from the urban area (67.6% of cases, 95% CI: 62.1-72.6; p<0.001) aged between 50 and 79 years. Total ischemia time (from onset of precordial pain to primary angioplasty) was 338.9 minutes on average (between 100 and 720 minutes); ambulance waiting time was 22.1 minutes (3-150 minutes); transport to first hospital took 49.9 minutes (5-276 minutes) while transport to a cardiology hospital averaged 247 minutes from the onset of pain (maximum 660 minutes). The door-to-balloon time was 91.9 minutes while early intervention was possible in 27.4% (95% CI: 22.7-32.7%) of AMI cases. Conclusions: Time-to-treatment intervals allowed early reperfusion in only one third of AMI patients due to lack of access to specialised cardiology hospitals in rural areas and inconsistencies regarding the attitude towards AMI cases across counties

PIBID e a prática de inglês na escola perpassada pela literatura nas Histórias dos Irmãos Grimm

Anais do II Seminário Seminário Estadual PIBID do Paraná: tecendo saberes / organizado por Dulcyene Maria Ribeiro e Catarina Costa Fernandes — Foz do Iguaçu: Unioeste; Unila, 2014Este trabalho visa compartilhar uma experiência desenvolvida no Projeto Pibid/Letras/Inglês de incentivo a literatura como prática de ensino de inglês no Ensino Fundamental. A experiência resultou no desenvolvimento e aplicação de um Caderno Literário com base nas obras literárias dos Irmãos Grimm. O conteúdo do Caderno envolve uma breve introdução biográfica sobre os autores, seguido de várias atividades relacionadas às três obras selecionadas: Chapeuzinho Vermelho, Cinderela, e Branca de Neve e os Sete anões. As atividades foram permeadas pela abordagem denominada de Literamento (PALLU, 2012) a qual integra os estudos artísticos, literários e linguísticos em língua inglesa. A aplicação do material e as atividades foram desenvolvidas durante um período de dois meses, contabilizando o total de nove aulas. Os resultados desta experiência evidenciam pontos de reflexão sobre a contribuição positiva dos trabalhos desenvolvidos no PIBI

Motif affinity and mass spectrometry proteomic approach for the discovery of cellular AMPK targets: identification of mitochondrial fission factor as a new AMPK substrate

AMP-activated protein kinase (AMPK) is a key cellular energy sensor and regulator of metabolic homeostasis. Although it is best known for its effects on carbohydrate and lipid metabolism, AMPK is implicated in diverse cellular processes, including mitochondrial biogenesis, autophagy, and cell growth and proliferation. To further our understanding of energy homeostasis through AMPK-dependent processes, the design and application of approaches to identify and characterise novel AMPK substrates are invaluable. Here, we report an affinity proteomicstrategy for the discovery and validation of AMPK targets using an antibody to isolate proteins containing the phospho-AMPK substrate recognition motif from hepatocytes that had been treated with pharmacological AMPK activators. We identified 57 proteins that were uniquely enriched in the activator-treated hepatocytes, but were absent in hepatocytes lacking AMPK. We focused on two candidates, cingulin and mitochondrial fission factor (MFF), and further characterised/validated them as AMPK-dependent targets by immunoblotting with phosphorylation site-specific antibodies. A small-molecule AMPK activator caused transient phosphorylation of endogenous cingulin at S137 in intestinal Caco2 cells. Multiple splice-variants of MFF appear to express in hepatocytes and we identified a common AMPK-dependent phospho-site (S129) in all the 3 predominant variants spanning the mass range and a short variant-specific site (S146). Collectively, our proteomic-based approach using a phospho-AMPK substrate antibody in combination with genetic models and selective AMPK activators will provide a powerful and reliable platform for identifying novel AMPK-dependent cellular targets

Analysis of the LKB1-STRAD-MO25 complex

Mutations in the LKB1 tumour suppressor threonine kinase cause the inherited Peutz-Jeghers cancer syndrome and are also observed in some sporadic cancers. Recent work indicates that LKB1 exerts effects on metabolism, polarity and proliferation by phosphorylating and activating protein kinases belonging to the AMPK subfamily. In vivo, LKB1 forms a complex with STRAD, an inactive pseudokinase, and MO25, an armadillo repeat scaffolding-like protein. Binding of LKB1 to STRAD-MO25 activates LKB1 and re-localises it from the nucleus to the cytoplasm. To learn more about the inherent properties of the LKB1-STRAD-MO25 complex, we first investigated the activity of 34 point mutants of LKB1 found in human cancers and their ability to interact with STRAD and MO25. Interestingly, 12 of these mutants failed to interact with STRAD-MO25. Performing mutagenesis analysis, we defined two binding sites located on opposite surfaces of MO25α, which are required for the assembly of MO25α into a complex with STRADα and LKB1. In addition, we demonstrate that LKB1 does not require phosphorylation of its own T-loop to be activated by STRADα-MO25α, and discuss the possibility that this unusual mechanism of regulation arises from LKB1 functioning as an upstream kinase. Finally, we establish that STRADα, despite being catalytically inactive, is still capable of binding ATP with high affinity, but that this is not required for activation of LKB1. Taken together, our findings reinforce the functional importance of the binding of LKB1 to STRAD, and provide a greater understanding of the mechanism by which LKB1 is regulated and activated through its interaction with STRAD and MO25

Chemical genetic screen identifies Gapex-5/GAPVD1 and STBD1 as novel AMPK substrates

AMP-activated protein kinase (AMPK) is a key regulator of cellular energy homeostasis, acting as a sensor of energy and nutrient status. As such, AMPK is considered a promising drug target for treatment of medical conditions particularly associated with metabolic dysfunctions. To better understand the downstream effectors and physiological consequences of AMPK activation, we have employed a chemical genetic screen in mouse primary hepatocytes in an attempt to identify novel AMPK targets. Treatment of hepatocytes with a potent and specific AMPK activator 991 resulted in identification of 65 proteins phosphorylated upon AMPK activation, which are involved in a variety of cellular processes such as lipid/glycogen metabolism, vesicle trafficking, and cytoskeleton organisation. Further characterisation and validation using mass spectrometry followed by immunoblotting analysis with phosphorylation site-specific antibodies identified AMPK-dependent phosphorylation of Gapex-5 (also known as GTPase-activating protein and VPS9 domain-containing protein 1 (GAPVD1)) on Ser902 in hepatocytes and starch-binding domain 1 (STBD1) on Ser175 in multiple cells/tissues. As new promising roles of AMPK as a key metabolic regulator continue to emerge, the substrates we identified could provide new mechanistic and therapeutic insights into AMPK-activating drugs in the liver

Regulation of activity and localization of the WNK1 protein kinase by hyperosmotic stress

Mutations within the WNK1 (with-no-K[Lys] kinase-1) gene cause Gordon's hypertension syndrome. Little is known about how WNK1 is regulated. We demonstrate that WNK1 is rapidly activated and phosphorylated at multiple residues after exposure of cells to hyperosmotic conditions and that activation is mediated by the phosphorylation of its T-loop Ser382 residue, possibly triggered by a transautophosphorylation reaction. Activation of WNK1 coincides with the phosphorylation and activation of two WNK1 substrates, namely, the protein kinases STE20/SPS1-related proline alanine–rich kinase (SPAK) and oxidative stress response kinase-1 (OSR1). Small interfering RNA depletion of WNK1 impairs SPAK/OSR1 activity and phosphorylation of residues targeted by WNK1. Hyperosmotic stress induces rapid redistribution of WNK1 from the cytosol to vesicular structures that may comprise trans-Golgi network (TGN)/recycling endosomes, as they display rapid movement, colocalize with clathrin, adaptor protein complex 1 (AP-1), and TGN46, but not the AP-2 plasma membrane–coated pit marker nor the endosomal markers EEA1, Hrs, and LAMP1. Mutational analysis suggests that the WNK1 C-terminal noncatalytic domain mediates vesicle localization. Our observations shed light on the mechanism by which WNK1 is regulated by hyperosmotic stress

Lice (Haematopinus tuberculatus) in water buffalo farms from central Italy

The aim of the present study was to obtain information about the presence and distribution of the suckling louse Haematopinus tuberculatus in water buffalo farms in central Italy. The survey was carried out on 127 farms (epidemiological units), selected using a grid approach within a Geographical Information System, followed by proportional allocation. In each farm 6 buffaloes were examined in order to detect the louse presence. Parasitological examinations were performed on each buffalo at predilection sites. A total of 762 water buffaloes were examined. H. tuberculatus was found in the 11.0% (14/127) of the farms and in the 4.5% (34/762) of the animals. The presence H. tuberculatus should be routinely considered because it is a cause of serious health, production and economic damages in intensive breeding buffaloes

Expectations of i-Tree Eco as a tool for urban tree management in Nordic cities

While urban forests are recognized as imperative toward climate adaptation in cities and provide health and recreational benefits to citizens, municipal tree officers often struggle to find successful governance arrangements and budget support toward long-lasting investment and implementation in new planting schemes and protection of existing trees. Since its release in 2006, i-Tree Eco has helped urban tree officers worldwide to find tangible leverage in the means of quantitative mapping, numeric measures, and economic values of ecosystem services. This may in turn help ease gridlocks and potentially support constructive dialogues across sectors, with decision-makers and public engagement. With the release of i-Tree Eco v. 6 in Europe 2018, 13 Nordic cities were engaged in a larger research project with ambitions to use i-Tree Eco for the purpose of retrieving numeric and monetary data of the biophysical structures and ecosystem services of the urban forest. Based on questionnaires and semi-structured interviews, we present the results from the Nordic i-Tree project with a focus on expectations, opportunities, and potential barriers experienced in using i-Tree Eco in urban forest management. The most prominent expectation and foreseeing opportunities were recognized toward using numeric information on trees to change policies and support cross-sectoral collaboration while reaching politicians and the public. Identified barriers involved how limited resources are spent on public outreach and how information about the project to relevant stakeholders were not distributed from the beginning which may have implications on the dissemination of results. As some important ecosystem services, e.g., cultural services, are not captured by i-Tree Eco, presenting the partial value of urban trees may pose also potential risks to cross-sectoral collaboration. Other findings conclude that although numeric information on ecosystem services is seen as beneficial in terms of communicating with different stakeholders, a deeper understanding toward the criteria used in the valuation process and the potential risks of numeric approaches may provide more context-specific applications