Antimalarial drugs disrupt ion homeostasis in malarial parasites

Plasmodium chabaudi malaria parasite organelles are major elements for ion homeostasis and cellular signaling and also target for antimalarial drugs. By using confocal imaging of intraerythrocytic parasites we demonstrated that the dye acridine orange (AO) is accumulated into P. chabaudi subcellular compartments. The AO could be released from the parasite organelles by collapsing the pH gradient with the K+/H+ ionophore nigericin (20 µM), or by inhibiting the H+-pump with bafilomycin (4 µM). Similarly, in isolated parasites loaded with calcium indicator Fluo 3-AM, bafilomycin caused calcium mobilization of the acidic calcium pool that could also be release with nigericin. Interestingly after complete release of the acidic compartments, addition of thapsigargin at 10 µM was still effective in releasing parasite intracellular calcium stores in parasites at trophozoite stage. The addition of antimalarial drugs chloroquine and artemisinin resulted in AO release from acidic compartments and also affected maintenance of calcium in ER store by using different drug concentrations.

Regulatory assessment of in vitro skin corrosion and irritation data within the European framework: Workshop recommendations

Validated in vitro methods for skin corrosion and irritation were adopted by the OECD and by the European Union during the last decade. In the EU, Switzerland and countries adopting the EU legislation, these assays may allow the full replacement of animal testing for identifying and classifying compounds as skin corrosives, skin irritants, and non irritants. In order to develop harmonised recommendations on the use of in vitro data for regulatory assessment purposes within the European framework, a workshop was organized by the Swiss Federal Office of Public Health together with ECVAM and the BfR. It comprised stakeholders from various European countries involved in the process from in vitro testing to the regulatory assessment of in vitro data. Discussions addressed the following questions: (1) the information requirements considered useful for regulatory assessment; (2) the applicability of in vitro skin corrosion data to assign the corrosive subcategories as implemented by the EU Classification, Labelling and Packaging Regulation; (3) the applicability of testing strategies for determining skin corrosion and irritation hazards; and (4) the applicability of the adopted in vitro assays to test mixtures, preparations and dilutions. Overall, a number of agreements and recommendations were achieved in order to clarify and facilitate the assessment and use of in vitro data from regulatory accepted methods, and ultimately help regulators and scientists facing with the new in vitro approaches to evaluate skin irritation and corrosion hazards and risks without animal data.

What new cell biology findings could bring to therapeutics: is it time for a phenome-project in Toxoplasma gondii?

"If you know the enemy and know yourself, you need not fear the result of a hundred battles. If you know yourself but not the enemy, for every victory gained you will also suffer a defeat" (SunTzu the Art of War, 544-496 BC). Although written for the managing of conflicts and winning clear victories, this basic guideline can be directly transferred to our battle against apicomplexan parasites and how to focus future basic research in order to transfer the gained knowledge to a therapeutic intervention stratey. Over the last two decades the establishment of several key-technologies, by different groups working on Toxoplasma gondii, made this important human pathogen accessible to modern approaches in molecular cell biology. In fact more and more researchers get attracted to this easy accessible model organism to study specific biological questions, unique to apicomplexans. This fascinating, unique biology might provide us with new therapeutic options in our battle against apicomplexan parasites by finding its Achilles' heel. In this article we argue that in the absence of a powerful high throughput technology for the characterisation of essential gene of interests a coordinated effort should be undertaken to convert our knowledge of the genome into one of the phenome.

The Emergence and Governance of Euroregions: The Case of the Euregio on the Dutch-German Border

Amb una història que data de la dècada de 1950, la EUREGIO és un de les més antigues euroregions a Europa. Es pot considerar com un cas exitós d'una regió transfronterera (CBR) en el sentit que s'ha establert fermament com una agència de fronteres dins del seu tram de la frontera holandesa-alemanya. L'EUREGIO també ha estat una de les protagonistes principals darrere de l'Associació de Regions Frontereres Europees (ARFE), que en les últimes dècades va actuar per difondre el model d'euroregió a tot el territori europeu. Aquest capítol té diversos objectius. En primer lloc, es presenta el cas de la EUREGIO i presenta evidència sobre la seva història, estructura orgànica i polítiques. En segon lloc, s'analitzen les condicions del context en què la EUREGIO ha sorgit i les estructures de govern que es van crear com a resultat. Es fa especial èmfasi en la posició i el paper de l'Euroregió en el context més ampli del marc de governança europea multinivell generat per la política de cohesió de la UE. El capítol conclou amb un intent d'avaluar l'èxit i l'impacte de la EUREGIO i una discussió dels reptes relacionats amb la doble funció de l'EUREGIO com a representant dels interessos de les autoritats locals i les agències de cohesió de la UE posada en pràctica de les polítiques.

Antifungal activity of extracts from Atacama Desert fungi againstParacoccidioides brasiliensis and identification ofAspergillus felis as a promising source of natural bioactive compounds

Fungi of the genus Paracoccidioides are responsible for paracoccidioidomycosis. The occurrence of drug toxicity and relapse in this disease justify the development of new antifungal agents. Compounds extracted from fungal extract have showing antifungal activity. Extracts of 78 fungi isolated from rocks of the Atacama Desert were tested in a microdilution assay against Paracoccidioides brasiliensis Pb18. Approximately 18% (5) of the extracts showed minimum inhibitory concentration (MIC) values≤ 125.0 µg/mL. Among these, extract from the fungus UFMGCB 8030 demonstrated the best results, with an MIC of 15.6 µg/mL. This isolate was identified as Aspergillus felis (by macro and micromorphologies, and internal transcribed spacer, β-tubulin, and ribosomal polymerase II gene analyses) and was grown in five different culture media and extracted with various solvents to optimise its antifungal activity. Potato dextrose agar culture and dichloromethane extraction resulted in an MIC of 1.9 µg/mL against P. brasiliensis and did not show cytotoxicity at the concentrations tested in normal mammalian cell (Vero). This extract was subjected to bioassay-guided fractionation using analytical C18RP-high-performance liquid chromatography (HPLC) and an antifungal assay using P. brasiliensis. Analysis of the active fractions by HPLC-high resolution mass spectrometry allowed us to identify the antifungal agents present in the A. felis extracts cytochalasins. These results reveal the potential of A. felis as a producer of bioactive compounds with antifungal activity.

The quest for a message: budding yeast, a model organism to study the control of pre-mRNA splicing

Removal of introns during pre-mRNA splicing is a critical process in gene expression, and understanding its control at both single-gene and genomic levels is one of the great challenges in Biology. Splicing takes place in a dynamic, large ribonucleoprotein complex known as the spliceosome. Combining Genetics and Biochemistry, Saccharomyces cerevisiae provides insights into its mechanisms, including its regulation by RNA-protein interactions. Recent genome-wide analyses indicate that regulated splicing is broad and biologically relevant even in organisms with a relatively simple intronic structure, such as yeast. Furthermore, the possibility of coordination in splicing regulation at genomic level is becoming clear in this model organism. This should provide a valuable system to approach the complex problem of the role of regulated splicing in genomic expression.

Deciphering 3'ss selection in the yeast genome reveals an RNA thermosensor that mediates alternative splicing

Poor understanding of the spliceosomal mechanisms to select intronic 3' ends (3'ss) is a major obstacle to deciphering eukaryotic genomes. Here, we discern the rules for global 3'ss selection in yeast. We show that, in contrast to the uniformity of yeast splicing, the spliceosome uses all available 3'ss within a distance window from the intronic branch site (BS), and that in 70% of all possible 3'ss this is likely to be mediated by pre-mRNA structures. Our results reveal that one of these RNA folds acts as an RNA thermosensor, modulating alternative splicing in response to heat shock by controlling alternate 3'ss availability. Thus, our data point to a deeper role for the pre-mRNA in the control of its own fate, and to a simple mechanism for some alternative splicing.

MGMT promoter methylation in malignant gliomas.

The O(6)-methylguanine-DNA methyltransferase (MGMT) gene is located at chromosome 10q26 and codes for a DNA repair enzyme that--if active--can counteract the effects of alkylating chemotherapy. Malignant gliomas often have the MGMT gene inactivated due to aberrant methylation of its promoter region. The assessment of the MGMT promoter methylation status has become of clinical relevance as a molecular marker associated with response to alkylating chemotherapy and prolonged survival of glioblastoma patients. MGMT promoter methylation testing is also on the merge of being used as a marker for patient selection within clinical trials, e.g., the current CENTRIC trial that is specifically focusing on patients with MGMT promoter-methylated glioblastomas. In anaplastic gliomas, MGMT promoter methylation is a favorable prognostic marker independent of the type of therapy, i.e., radio- or chemotherapy. This occurrence might be associated with the high incidence of other prognostically favorable molecular markers in these tumors, such as IDH1 mutation, 1p/19q deletion or yet to be identified novel aberrations. A variety of different methods are being used to assess MGMT promoter methylation in clinical samples, which may give rise to inter-laboratory variations in test results. Immunohistochemical determination of MGMT protein expression has not proven reliable for diagnostic purposes. This brief review article aims to summarize the main aspects of MGMT promoter methylation testing in contemporary neuro-oncology, in particular its value as a clinically useful molecular marker, putting it into the context of other molecular markers of clinical use in gliomas of adult patients.

Tuotannon seuranta- ja suunnittelutyökalun kehittäminen postimyyntiyrityksessä

Tässä työssä suunniteltiin postimyyntiyrityksen palautusosastolle uusi tuotannon seuranta- ja suunnittelutyökalu. Työtä varten tutkittiin laaja otos jo kerättyä tietoa yrityksen päivittäisestä toiminnasta ja sen tehokkuudesta. Samalla tutkittiin erilaisia mahdollisuuksia vaikuttaa yrityksen toiminnan kannattavuuteen yhteiskunnallisestikin tärkeiden asioiden näkökulmasta. Tällaisia aiheita ovat muun muassa työssä jaksaminen ja työntekijöiden kannustinjärjestelmät, tuotteiden elinkaarisuunnittelu sekä yrityksen yhteiskunta- ja ympäristövastuu. Yrityksellä itsellään on se käsitys, että tuotannon seuranta on ollut epätarkkaa ja hyvä tietojen hallinta melko työlästä ja aikaa vievää, joten yritysjohto totesi tarpeelliseksi kehittää uudet työkalut tehostaakseen päivittäisen toiminnan seurantaa ja ennaltasuunnittelua. Työ tehtiin yrityksen toimeksiannosta. Osaston toimintaa seurattiin sekä yksilökohtaisesti että koko osaston tasolla, ja tulokset vastasivat hyvin odotettua. Saatujen tietojen avulla kuitenkin havaittiin, mitä asioita on tehty väärin tai puuttellisesti edellistä työkalua käyttäen ja kuinka tällaiset virheet ja puutteet olisi nyt mahdollista välttää. Suunnitteluvaiheessa tutkittiin, miten työkalun antamia tuloksia voitaisiin helposti tarkistaa ja kuinka olisi mahdollista tulostaa saatuja tietoja yksinkertaisessa muodossa paperille, jotta niitä voisi esittää tarvittaessa myös osaston ulkopuolisille tahoille. Seikkaperäisen tutkimuksen ja suunnittelun jälkeen kehitettiin uusi järjestelmä sekä tuotannon seurantaan että suunnitteluun.

Keskeytyneen uusmediatuotannon jatkaminen : kohteena 3D-Kalevala

Opinnäytetyöni aiheena on keskeytyneen uusmediatuotannon jatkaminen. Monimuototyön työosana toteutettiin 7-minuuttinen 3D animaatio, joka kertoo Suomen kansalliseepoksen Kalevalan taruhahmosta Väinämöisestä, muistelemassa menneitä. Projekti käynnistettiin alun perin vuonna 2003, mutta resurssien vähetessä se keskeytyi vuoden 2005 loppupuolella. Keväällä 2006 projekti käynnistettiin uuden projektiryhmän voimin, jossa olin itse mukana vastaten muun muassa tuotannonohjauksesta ja hahmoanimoinneista. Uusi projektiryhmä oli henkilöstöresursseiltaan pieni, joten vastuualueet olivat monipuolisia. Keskeytyneen projektin jatkamisen ja haltuunoton haasteellisuus sai minut kiinnostumaan tutkia aihetta tarkemmin. Tuotannonohjaajana vastasin hyvin pitkälle tuotannon uudesta käynnistämisestä ja projektin saattamisesta vihdoin loppuun. Keskeytyneen projektin haltuunotto oli tilanteena kaikille uusi, mikä heijastui vaikeuksina uudelleen käynnistettyyn tuotantoon. Raportin tarkoituksena ei ole olla projektinhallinnallinen käsikirja, sillä käsittelen vain tämän projektin jatkolle oleellisina pidettyjä asioita. Projekti toivottavasti kuitenkin antaa kuvan huolellisen projektinhallinnan ja onnistuneen tuotannonohjauksen tärkeydestä. Jokainen keskeytynyt projekti ei ole aina välttämättä elvytettävissä - ainakaan alkuperäisessä muodossaan. Projektin jatkamista tulisi katsoa aina tapauskohtaisesti. Keskeytymiseen on useimmiten syynsä, joten ongelmien selvittäminen ja niihin puuttuminen on tärkeää ennen jatkopäätöksen tekemistä. Myös projektityöskentelytavat kehittyvät ja pohdin työssäni uusien projektinhallintatapojen, kuten wikien käyttöä projektinhallinnan työkaluna ja projektiyhteisön välistä viestintää edistävänä työkaluna.