Analysis of reaktivated proviruses of the human endogenous retrovirus familie HERV-K/HML2 and creation of a model for transcriptional control in melanoma and teratocarcinoma cell lines

Humane endogene Retroviren (HERVs) sind Relikte von Infektionen der Keimzellen von Primatenvorläufern mit exogenen Retroviren vor 40 Millionen Jahren. Die meisten HERV Elemente sind defekt durch die Akkumulation von Mutationen, Deletionen und Rearrangements. Lediglich einige Proviren der HERV-K/HML-2 Familie enthalte noch offenen Leserahmen für alle retroviralen Proteine. Die Expression repetitiver endogener Elemente wird in somatischen Zellen unterdrückt. Transkription und Translation von HERV-K Genprodukten tritt in Keimzelltumoren, Melanomen und Eierstockkrebs auf. In der vorliegenden Arbeit wurde die HERV-K exprimierende Melanomlinie UKRV Mel 2-C9 etabliert und charakterisiert. Es konnte gezeigt werden, dass die HERV-K Expressionen durch die dominante Aktivität des Provirus 108 bedingt ist. Die Expression und korrekte Prozessierung der viralen Proteine konnte nachgewiesen werden. Durch Elektronenmikroskopie wurde bestätigt, dass HERV-K 108 virale Partikel mit charakteristischen Strukturen der Hüllproteine produziert. Diese Partikel enthalten virale Volllängen RNA-Genome. Da HERV-K 108 in der Linie UKRV Mel 2-C9 eine Mutation im aktiven Zentrum der Reversen Transkriptase zeigt, sind die Partikel nicht infektiös. Um Einblicke in die der HERV-K Expression in Keimzelltumoren und Melanomen zu Grunde liegenden transkriptionellen Regulationsmechanismen zu erhalten, wurden mittels 5’ und 3’ RACE (rapid amplification of cDNA ends) der Transkriptionsstart und die Terminationsstelle bestimmt. Es konnte gezeigt werden, dass der HERV-K Promotor in den untersuchten Zelllinien TATA-unabhängig agiert obwohl die HERV-K LTR (long terminal repeat) ein TATA-Motiv aufweist. Die transkriptionelle Aktivität des HERV-K Promotors ist abhängig von einer GC-Box in unmittelbarer Nähe zu einem Initiatorelement am Startpunkt der Transkription. Das GC-Sequenzmotiv ist die Bindestelle für die Transkriptionsfaktoren SP1 (specific protein) und SP3. Mutationen des Bindemotivs führten zu einem drastischen Abfall der Promotoraktivität im Luziferaseassay. Durch Proteinknockdown mittels spezifischer siRNA gegen SP1 und SP3 konnte die Promotoraktivität ebenso deutlich reduziert werden. Elektrophoretic mobility shift assays und Chromatinimmunopräzipitationen bestätigten eine Bindung von SP1 und SP3 an die HERV-K LTR. Die Transkriptionsfaktoren SP1 und SP3 sind an der Initiation der basalen Transkription von HERV-K maßgeblich beteiligt.Human endogenous retroviruses (HERVs) resulted from germ line infections of primate ancestors. At present most HERV families are defective but some proviruses of the HERV-K/HML-2 family have maintained open reading frames for all viral proteins. Although usually strictly repressed, HERV-K transcription and translation is re-activated in germ cell tumours and melanoma. For these types of cancer HERV-K is a diagnostic and prognostic marker. We aim to understand the transcriptional regulation of HERV-K. The transcriptional start and termination sites were identified by 5' and 3’-rapid amplification of cDNA ends. The impact of specific transcription factors on the HERV-K LTR was analyzed using luciferase reporter assays, mutational and siRNA approaches, chromatin immuno-precipitation assays (ChIP) and electrophoretic mobility shift assays (EMSA). We demonstrate that the HERV-K LTR is a TATA independent promoter despite the presence of a TATA motif. Mutation of this motif did not abrogate promoter activity. Transcription initiation is mediated by a GC-box, the binding motif of the transcription factors SP1 (specificity protein) and SP3, located near an initiator element. Mutation of the GC-box significantly reduced HERV-K promoter activity as did a knock down of the Sp1 and Sp3 proteins. EMSA and ChIP confirmed that the GC-box represents a binding site for the transcription factors Sp1 and Sp3

The Croatian verb pričati and its »new« role among other »verbs of speaking« in Croatian

Glagoli govorenja tvore zanimljivu značenjsku skupinu leksema kako u hrvatskome tako i u mnogim drugim jezicima. U ovome radu analiziraju se značenjske bliskosti glagola govoriti, razgovarati i pričati poglavito radi mogućeg objašnjenja što omogućava glagolu pričati pojavljivanje u supstandardnim sintagmama poput pričati engleski, pričati telefonom itd.»Verbs of speaking« are an interesting semantically interrelated group of lexemes in Croatian as well as many other languages. The main aim of the present analysis is to show how the interrelationship between the Croatian verbs govoriti, razgovarati i pričati, can explain various substandard uses of the verb pričati, uses which are gaining ground not only in spoken Croatian but in the press as well

KOMUNIKACIJSKE TEHNOLOGIJE I NJIHOV UTJECAJ NA JEZIK: “PRESLAGIVANJE” GLAGOLSKIH VREMENA U SMS PORUKAMA NA HRVATSKOME JEZIKU

SMS text messaging has become one of the most dominant means of communication in Croatia. With respect to their linguistic features SMS text messages have become a language variety in their own right, exhibiting specific regularities in different aspects of language use. We investigate the use of tenses in Croatian SMS text messages in comparison to standard written Croatian. The results show a different distribution or reshuffling of past tenses, with a reappearance of tenses considered to be obsolete. We also look into the reasons that triggered off these changes as well as their impact on the Croatian tense system.SMS poruke postale su jednim od najdominantnijih sredstava komuniciranja u Hrvatskoj. U smislu postojanja vlastitih jezičnih karakteristika SMS poruke postale su zaseban jezični ‘varijetet’ sa specifičnim pravilnostima u različitim vidovima jezične uporabe. U radu se istražuje uporaba glagolskih vremena u hrvatskim SMS-tekstualnim porukama te se uspoređuje s uporabom glagolskih vremena u standardnom pisanom hrvatskom jeziku. Rezultati upućuju na različitu distribuciju, odnosno “preslagivanje” prošlih vremena, uz ponovno pojavljivanje glagolskih vremena koja se u mnogim gramatikama označavaju kao arhaična. Rad se također osvrće na uzroke takvih promjena, kao i na njihov utjecaj na sustav glagolskih vremena u hrvatskome

Are there memory effects on greenhouse gas emissions (CO2_{2}, N2_{2}O and CH4_{4}) following grassland restoration?

A 5-year greenhouse gas (GHG) exchange study of the three major gas species (CO$_{2}$, CH$_{4}$ and N$_{2}$O) from an intensively managed permanent grassland in Switzerland is presented. Measurements comprise 2 years (2010 and 2011) of manual static chamber measurements of CH$_{4}$ and N$_{2}$O, 5 years of continuous eddy covariance (EC) measurements (CO$_{2}$–H$_{2}$O – 2010–2014), and 3 years (2012–2014) of EC measurement of CH$_{4}$ and N$_{2}$O. Intensive grassland management included both regular and sporadic management activities. Regular management practices encompassed mowing (three to five cuts per year) with subsequent organic fertilizer amendments and occasional grazing, whereas sporadic management activities comprised grazing or similar activities. The primary objective of our measurements was to compare pre-plowing to post-plowing GHG exchange and to identify potential memory effects of such a substantial disturbance on GHG exchange and carbon (C) and nitrogen (N) gains and losses. In order to include measurements carried out with different observation techniques, we tested two different measurement techniques jointly in 2013, namely the manual static chamber approach and the eddy covariance technique for N$_{2}$O, to quantify the GHG exchange from the observed grassland site. Our results showed that there were no memory effects on N$_{2}$O and CH$_{4}$ emissions after plowing, whereas the CO$_{2}$ uptake of the site considerably increased when compared to pre-restoration years. In detail, we observed large losses of CO$_{2}$ and N$_{2}$O during the year of restoration. In contrast, the grassland acted as a carbon sink under usual management, i.e., the time periods 2010–2011 and 2013–2014. Enhanced emissions and emission peaks of N$_{2}$O (defined as exceeding background emissions 0.21 ± 0.55 nmol m$^{-2}$ s$^{-1}$ (SE = 0.02) for at least 2 sequential days and the 7 d moving average exceeding background emissions) were observed for almost 7 continuous months after restoration as well as following organic fertilizer applications during all years. Net ecosystem exchange of CO$_{2}$ (NEECO$_{2}$) showed a common pattern of increased uptake of CO$_{2}$ in spring and reduced uptake in late fall. NEECO$_{2}$ dropped to zero and became positive after each harvest event. Methane (CH$_{4}$) exchange fluctuated around zero during all years. Overall, CH$_{4}$ exchange was of negligible importance for both the GHG budget and the carbon budget of the site. Our results stress the inclusion of grassland restoration events when providing cumulative sums of C sequestration potential and/or global warming potential (GWP). Consequently, this study further highlights the need for continuous long-term GHG exchange observations as well as for the implementation of our findings into biogeochemical process models to track potential GHG mitigation objectives as well as to predict future GHG emission scenarios reliably

Large inter-annual variation in carbon sink strength of a permanent grassland over 16 years: Impacts of management practices and climate

Permanent grasslands cover one third of the European agricultural area and are known to store large amounts of carbon (C) in their soils. However, long-term assessments of their C sink strength are still scarce. Thus, we investigated the C budget of an intensively managed, permanent grassland in Switzerland over 16 years, compared the results to changes in soil C stocks, and determined the most important drivers of the net ecosystem CO2 exchange (NEE). Combining NEE fluxes with C imports and C exports, we quantified the grassland C budget, i.e., net biome production (NBP). We observed a large inter-annual variation in NBP, with 9 of the 16 years indicating a C sink, and 7 years indicating a C source. On average, the grassland was a small C sink to C neutral, with a NBP of -70±106 g C m$^{−2}$ yr$^{−1}$ (mean±95% confidence interval). Mean NEE fluxes were -284±115 g C m$^{−2}$ yr$^{−1}$, C exports via harvest 335±73 g C m$^{−2}$ yr$^{−1}$, and organic C imports via slurry -121±43 g C m$^{−2}$ yr$^{−1}$. Soil C stocks from 0 to 0.7 m did not change significantly (decrease of 27.5 g C m$^{−2}$ yr$^{−1}$ over 13 years). Inter-annual variation in NBP was affected by management practices and environmental conditions. In the last five years, NBP was positive (C source), most likely due to decreasing C imports in combination with extreme weather conditions. Our study demonstrated the importance of covering multiple years with different management events when assessing the C sink strength of a site. Maintaining even a small grassland C sink in the future will be challenging and will require continuous organic C imports

Cooperation and Deception Recruit Different Subsets of the Theory-of-Mind Network

The term “theory of mind” (ToM) describes an evolved psychological mechanism that is necessary to represent intentions and expectations in social interaction. It is thus involved in determining the proclivity of others to cooperate or defect. While in cooperative settings between two parties the intentions and expectations of the protagonists match, they diverge in deceptive scenarios, in which one protagonist is intentionally manipulated to hold a false belief about the intention of the other. In a functional magnetic resonance imaging paradigm using cartoons showing social interactions (including the outcome of the interaction) between two or three story characters, respectively, we sought to determine those brain areas of the ToM network involved in reasoning about cooperative versus deceptive interactions. Healthy volunteers were asked to reflect upon the protagonists' intentions and expectations in cartoons depicting cooperation, deception or a combination of both, where two characters cooperated to deceive a third. Reasoning about the mental states of the story characters yielded substantial differences in activation patterns: both deception and cooperation activated bilateral temporoparietal junction, parietal and cingulate regions, while deception alone additionally recruited orbitofrontal and medial prefrontal regions. These results indicate an important role for prefrontal cortex in processing a mismatch between a character's intention and another's expectations as required in complex social interactions

Komunikacijske tehnologije i njihov utjecaj na jezik: Pojam konvencije s gledišta jezičnih tehnologija

On the basis of a summation of topics that have emerged from research of text messaging in Croatian during the last ten years, other major questions and issues have also surfaced. In this paper we focus on the notion of convention, both from the linguistic as well as pragmatic point of view. Convention is viewed as a reflection of the complexity of language, seen not only as a cognitive phenomenon, or a cognitive ability, but also as being central to human interaction. Using text messaging as the basis for the research brought to light the importance of the technological medium, which plays a crucial role in the formation of convention. Due to the technological possibilities and limitations of the medium, new conventions appear in text messages, conventions that at the same time reflect linguistic, sociocultural and discourse/pragmatic factors. This implies that they are a reflection not only of linguistic convention, but also a reflection of the human social cognitive ability, essential in language use. On the basis of the examples analysed we also stress that the social cognitive ability is tailored to the medium of communi-cation itself.Iz desetogodišnjeg istraživanja SMS poruka u hrvatskom jeziku proizašle su teme i pitanja od šireg značaja, poput pojma konvencije. Pojam konvencije se u ovome radu analizira kako s lingvističkog, tako i pragmatičkog gledišta. Na konvenciju se gleda ne samo kao na odraz jezika viđenog kao kognitivne pojavnosti ili sposobnosti, već i kao na pojavnost od središnje važnosti za ljudsku komunikaciju. Istraživanja na temelju SMS poruka ukazuju na važnost tehnološkog medija koji igra važnu ulogu u stvaranju same konvencije. Zbog tehnoloških mogućnosti, ali i ograničenja medija, nove konvencije koje nalazimo u SMS porukama istovremeno ukazuju na bitnost jezičnih, društvenih, kulturnih, diskursnih i pragmatičkih čimbenika. To znači da konvencija nije samo ona lingvistička u užem smislu, već je jednako tako sastavni dio ljudske društvene kognitivne sposobnosti, nezaobilazne u jezičnoj uporabi. Na temelju analiziranih primjera pokazano je da se društvena kognitivna sposobnost prilagođava i samom komunikacijskom mediju

TKA following high tibial osteotomy versus primary TKA - a matched pair analysis

<p>Abstract</p> <p>Background</p> <p>High tibial osteotomy (HTO) is a well established technique for the treatment of medial osteoarthritis of the knee with varus malalignment. Results of total knee arthroplasty (TKA) after previous HTO are still discussed controversially. The aim of this study was to elucidate the clinical and radiological results as well as perioperative data of prior HTO on TKA.</p> <p>Methods</p> <p>Forty-one TKA after HTO were compared to 41 primary TKA at minimum of six years follow-up. Patients were matched according to age, gender, follow-up, etiology, and prosthetic design. Surgical data and complications were evaluated. Clinical outcome was assessed using a number of clinical scores and the visual analogue scale (VAS) for pain. X-rays were evaluated by the method of the American Knee Society. The patellar position was measured by the Insall-Salvati ratio.</p> <p>Results</p> <p>There was no significant difference in mean operation time (p = 0.47) and complication rate (p = 0.08). The Knee Score of the KSS (p = 0.0007) and the ROM (p = 0.006 for extension and p = 0.004 for flexion, respectively) were significantly better in the control group. Mid-term results of the VAS, WOMAC, Lequesne, UCLA, Feller's Patellar Score and SF-36 showed no significant difference. Femoral and tibial component alignment were similar in both groups. One tibial component showed suspect radiolucencies in the HTO group. The Insall-Salvati ratio showed three patients with patella alta and one patient with patella baja in the HTO group. At latest follow-up all implants were still in place.</p> <p>Conclusions</p> <p>Evaluating the clinical and radiological outcome, significant differences were only detected for range of motion and the Knee Score of the KSS. The present study suggests that the results of TKA with and without prior HTO are mainly identical. Although patients with a previous HTO had more complications, no statistically significant differences were noted with this group size.</p

Correction: The Endocytic Adaptor Eps15 Controls Marginal Zone B Cell Numbers.

Eps15 is an endocytic adaptor protein involved in clathrin and non-clathrin mediated endocytosis. In Caenorhabditis elegans and Drosophila melanogaster lack of Eps15 leads to defects in synaptic vesicle recycling and synapse formation. We generated Eps15-KO mice to investigate its function in mammals. Eps15-KO mice are born at the expected Mendelian ratio and are fertile. Using a large-scale phenotype screen covering more than 300 parameters correlated to human disease, we found that Eps15-KO mice did not show any sign of disease or neural deficits. Instead, altered blood parameters pointed to an immunological defect. By competitive bone marrow transplantation we demonstrated that Eps15-KO hematopoietic precursor cells were more efficient than the WT counterparts in repopulating B220⁺ bone marrow cells, CD19⁻ thymocytes and splenic marginal zone (MZ) B cells. Eps15-KO mice showed a 2-fold increase in MZ B cell numbers when compared with controls. Using reverse bone marrow transplantation, we found that Eps15 regulates MZ B cell numbers in a cell autonomous manner. FACS analysis showed that although MZ B cells were increased in Eps15-KO mice, transitional and pre-MZ B cell numbers were unaffected. The increase in MZ B cell numbers in Eps15 KO mice was not dependent on altered BCR signaling or Notch activity. In conclusion, in mammals, the endocytic adaptor protein Eps15 is a regulator of B-cell lymphopoiesis