Guidelines for the use and interpretation of assays for monitoring autophagy.

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field.

Hypertension and microvascular remodelling

In the present review, microvascular remodelling refers to alterations in the structure of resistance vessels contributing to elevated systemic vascular resistance in hypertension. We start with some historical aspects, underscoring the importance of Folkow's contribution made half a century ago. We then move to some basic concepts on the biomechanics of blood vessels, and explicit the definitions proposed by Mulvany for specific forms of remodelling, especially inward eutrophic and inward hypertrophic. The available evidence for the existence of remodelled resistance vessels in hypertension comes next, with relatively more weight given to human, in comparison with animal data. Mechanisms are discussed. The impact of antihypertensive drug treatment on remodelling is described, again with emphasis on human data. Some details are given on the three studies to date which point to remodelling of subcutaneous resistance arteries as an independent predictor of cardiovascular risk in hypertensive patients. We terminate by considering the potential role of remodelling in the pathogenesis of end-organ damage and in the perpetuation of hypertension.

DNA/NYVAC Vaccine Regimen Induces HIV-Specific CD4 and CD8 T-Cell Responses in Intestinal Mucosa

Background: HIV vaccine-candidates based on rare adenovirus serotypes such as Ad26 and Ad35 vectors, and poxvirus vectors are important components of future promising vaccine regimens that in the near future hopefully will move into a number of efficacy clinical trials in combination with protein vaccines. For these reasons, it is important to comprehensively characterize the vaccine-induced immune responses in different anatomical compartments and particularly at mucosal sites which represent the primary port of entry for HIV.Methods: In the present study, we have investigated the anatomic distribution in blood and gut mucosal tissues (rectum and ileum) of memory poxvirus-specific CD4 and CD8 T cells in subjects vaccinated with smallpox and compared with vector (NYVAC)-specific and HIV insert-specific T-cell responses induced by an experimental DNA-C/NYVAC-C vaccine regimen.Results: Smallpox-specific CD4 T-cell responses were present in the blood of 52% of subject studied, while Smallpox-specific CD8 T cells were rarely detected (12%). With one exception, Smallpoxspecific T cells were not measurable in gut tissues. Interestingly, NYVAC vector-specific and HIV-specific CD4 and CD8 T-cell responses were detected in almost 100% of the subjects immunized with DNA-C/NYVAC-C in blood and gut tissues. The large majority (83%) of NYVAC-specific CD4 T cells expressed a4b7 integrins and the HIV co-receptor CCR5.Conclusion: These results demonstrate that the experimental DNA-C/NYVAC-C HIV vaccine regimen induces the homing of potentially protective HIV-specific CD4 and CD8 T cells in the gut, the port of entry of HIV and one of the major sites for HIV spreading and depletion of CD4 T cells.

Effects of long-term averaging of quantitative blood pressure traits on the detection of genetic associations.

Blood pressure (BP) is a heritable, quantitative trait with intraindividual variability and susceptibility to measurement error. Genetic studies of BP generally use single-visit measurements and thus cannot remove variability occurring over months or years. We leveraged the idea that averaging BP measured across time would improve phenotypic accuracy and thereby increase statistical power to detect genetic associations. We studied systolic BP (SBP), diastolic BP (DBP), mean arterial pressure (MAP), and pulse pressure (PP) averaged over multiple years in 46,629 individuals of European ancestry. We identified 39 trait-variant associations across 19 independent loci (p < 5 × 10(-8)); five associations (in four loci) uniquely identified by our LTA analyses included those of SBP and MAP at 2p23 (rs1275988, near KCNK3), DBP at 2q11.2 (rs7599598, in FER1L5), and PP at 6p21 (rs10948071, near CRIP3) and 7p13 (rs2949837, near IGFBP3). Replication analyses conducted in cohorts with single-visit BP data showed positive replication of associations and a nominal association (p < 0.05). We estimated a 20% gain in statistical power with long-term average (LTA) as compared to single-visit BP association studies. Using LTA analysis, we identified genetic loci influencing BP. LTA might be one way of increasing the power of genetic associations for continuous traits in extant samples for other phenotypes that are measured serially over time.

Musiikin sovittaminen J.V.Snellman 200-vuotta juhlakonserttiin

Opinnäytetyössäni olen tehnyt Toppilan Laulustudion tilauksesta sovituksia Kaj Chydeniuksen säveltämistä J. V. Snellmanin rakkausrunoista. Opinnäytetyöhöni kuuluu J. V. Snellman 200-vuotisjuhlakonsertin "Minkä rakkaudelle mahdoin?" dvd-tallenne, soitinsovitukset partituurina sekä raportti työstä. Opinnäytetyöni raportissa käsittelen konsertin syntyvaiheita ja sovittamisprosessia. Raporttiani varten haastattelin Kaj Chydeniusta, joka kertoi sävellysten syntymisestä, sävellysten tekotavasta sekä kokemuksiaan sovittamisesta. Sovitukset on tehty heinä-elokuussa 2006 ja ne esitettiin J.V.Snellman 200-vuotta juhlakonsertissa "Minkä rakkaudelle mahdoin?" Oulun lyseon juhlasalissa 2.9.2006. Sovituksia on 12 kappaletta ja niissä olen käyttänyt instrumentteina pianoa, kitaraa, huilua, selloa ja alttoviulua.

Ei ne suuret tulot, vaan... : riippumattoman musiikintekijän tulomuodot digitalisoituvassa maailmassa

Opinnäytetyö selvittää musiikkiteollisuuden ansaintalogiikkaa ja riippumattoman musiikintekijän tulomuotoja, sekä teknologisen kehityksen ja Internetin aiheuttaman rakennemuutoksen vaikutusta niihin. Alan nykyisenä ongelmana nähdään musiikintekijälle päätyvän rahan pieni osuus teollisuudessa liikkuvasta tulovirrasta. Työn tavoitteena on löytää kustannustehokkaita väyliä musiikin saattamiseen kuulijoille, pohtien samalla erilaisten promootiokanavien vaikutusarvoa. Lopullisena tarkoituksena on löytää tapa elättää itseään musiikin tekemisellä. Lähdeaineistona on käytetty alan kirjallisuutta, tilastoja, Internet-artikkeleita, sekä kirjoittajan omaa kokemusta alalta. Työ osoittaa, kuinka hankalaa musiikin tekemisellä tienaaminen on Suomessa. Kustannusten pitäminen mahdollisimman alhaisina ja asioiden tekeminen omatoimisesti mahdollisimman pitkälle ovat perusedellytys voitolliselle liiketoiminnalle. Digitaaliteknologia yhdessä Internetin kanssa tarjoaa mahdollisuuden tähän. Musiikintekijän merkittävimmiksi tulomuodoiksi osoittautuvat esiintymis- ja tekijänoikeuskorvaukset. Äänitteillä ansaitseminen on todennäköisintä pienlevy-yhtiöiden kanssa toimiessa, isojen levy-yhtiöiden raskas kulurakenne takaa sen että itse musiikintekijälle tuloutuu vain murto-osa tuotoista. Äänitteiden digijakelu ei vielä tarjoa varteenotettavaa tulonlähdettä musiikintekijälle, mutta sen merkitys kasvaa lähitulevaisuudessa, samoin kuin erilaisista edelleenlisensioinneista ja oheistuotteista kertyvät tulot.