Antisense RNA based down-regulation of RNaseE in E.coli

BACKGROUND: Messenger RNA decay is an important mechanism for controlling gene expression in all organisms. The rate of the mRNA degradation directly affects the steady state concentration of mRNAs and therefore influences the protein synthesis. RNaseE has a key importance for the general mRNA decay in E.coli. While RNaseE initiates the degradation of most mRNAs in E.coli, it is likely that the enzyme is also responsible for the degradation of recombinant RNAs. As RNaseE is essential for cell viability and knockout mutants cannot be cultured, we investigated the possibility for a down-regulation of the intracellular level of RNaseE by antisense RNAs. During this study, an antisense RNA based approach could be established which revealed a strong reduction of the intracellular level of RNaseE in E.coli. RESULTS: Despite the autoregulation of rne mRNA by its gene product, significant antisense downregulation of RNaseE is possible. The expression of antisense RNAs did not effect the cell growth negatively. The amount of antisense RNA was monitored quantitatively by a fluorescence based sandwich hybridisation assay. Induction by anhydrotetracycline was followed by a 25-fold increase of the detectable antisense RNA molecules per cell. The antisense RNA level was maintained above 400 molecules per cell until the stationary phase, which caused the level of expressed antisense RNAs to decrease markedly. Western blot experiments revealed the strongest reduction in the RNaseE protein level 90 min after antisense RNA induction. The cellular level of RNaseE could be decreased to 35% of the wild type level. When the growth entered the stationary phase, the RNaseE level was maintained still at 50 to 60% of the wild type level. CONCLUSION: In difference to eukaryotic cells, where the RNAi technology is widely used, this technology is rather unexplored in bacteria, although different natural systems use antisense RNA-based silencing, and a few studies have earlier indicated the potential of this technology also in prokaryotes. Our results show that even complicated self-regulatory systems such as RNaseE may be controlled by antisense RNA technology, indicating that systems based on antisense RNA expression may have a potential for controlling detrimental factors with plasmid-based constructs in arbitrary strains while maintaining their beneficial characteristics. The study also proved that the RNA sandwich hybridisation technique is directly applicable to quantify small RNA molecules in crude cell extracts, which may have a broader application potential as a monitoring tool in RNA inhibition applications

Ultra-high vacuum compatible induction-heated rod casting furnace

We report the design of a radio-frequency induction-heated rod casting furnace that permits the preparation of polycrystalline ingots of intermetallic compounds under ultra-high vacuum compatible conditions. The central part of the system is a bespoke water-cooled Hukin crucible supporting a casting mold. Depending on the choice of mold, typical rods have a diameter between 6 mm and 10 mm and a length up to 90 mm, suitable for single-crystal growth by means of float-zoning. The setup is all-metal sealed and may be baked out. We find that the resulting ultra-high vacuum represents an important precondition for processing compounds with high vapor pressures under a high-purity argon atmosphere up to 3 bar. Using the rod casting furnace, we succeeded to prepare large high-quality single crystals of two half-Heusler compounds, namely the itinerant antiferromagnet CuMnSb and the half-metallic ferromagnet NiMnSb.Comment: 5 pages, 4 figure

Sozialstruktur und Wahlverhalten in West- und Mitteleuropa

Die Cleavage-Theorie liefert einen Erklärungsanspruch für das kontinuierliche Wahlverhalten in der westlichen Welt. Dementsprechend oft wurde sie seit gut 40 Jahren oftmals empirisch überprüft, abgelehnt und immer weiter ergänzt. Zweifellos besitzen ein Religions-, Klassen- und Stadt/Land-Konflikt weiterhin einen stetigen Einfluss auf das Wahlverhalten, speziell für die traditionellen Volksparteien, die sich seit dem zweiten Weltkrieg oder bereits früher in ihren Prinzipien daran orientierten. Dealignment-Prozesse, in ihrer Relevanz auf das Wahlverhalten, wurden daher noch einmal untersucht mit Hilfe von Korrelationsmaßen, wie Cramer’s V, und einer multinomial Logit Regression mit fünf Indikatoren: Konfession, Kirchgang, formale Schulbildung, Class und Wohngebiet, sowie zwei Kontrollvariablen, Geschlecht und Alter. Dabei wurde mit den Sekundärdaten, EVS 1999, ESS 2004 und EVS 2008 gearbeitet, um die traditionellen Parteien der Länder, Deutschland, Niederlande und Österreich, von 1999 ausgehend, wegen ihrer unterschiedlichen religiösen Zusammensetzung und ihrer unterschiedlichen historischen Entwicklung konkordanzdemokratischer Entschlussfindungen, zu untersuchen. Dabei zeigt sich, dass der Religions-Cleavage die stärkste Konservierung der Relevanz aufweist, aber alle fünf traditionellen Indikatoren einen Überhang an Dealignment-Prozesse aufweisen, speziell, wenn sie 1999 einen hohen Einfluss aufwiesen. Transnationale Trends eines Indikators lassen sich nicht feststellen, stattdessen muss jede Ausprägung für eine Volkspartei analysiert werden. Das Class Voting ist ein äußerst komplexer und fragiler Einfluss, und zeigt in Form von Korrelationsmaßen einen stärkeren Zusammenhang in Ländern, die stark korporatistisch organisiert sind. Allerdings verliert der Klassenkonflikt an Relevanz zu Ungunsten des Alters.The well known Cleavage-Theory gives an approach for explaining continuous voting behaviour in the western world. Due this fact, the theory was proved, rejected and evolved empirically since about 40 years. Without a doubt the religious-, the class- and the rural/urban-Cleavage still matters for voting behaviour, especially for the traditional parties, which oriented themselves on those Cleavages since World War II or earlier. By means of correlations, like Cramer’s V and a multinomial logit regression, voting behaviour was analysed once again on Dealignment-tendencies by five indicators: belief, church attendance, education, Class and residential area plus two control variables, sex and age. Based on the secondary data of the EVS 1999, ESS 2004 and EVS 2008, traditional parties of the countries, Germany, Netherlands and Austria, were analysed from 1999 onwards, because of their different religious formation and the function of their welfare state evolved distinctively. In this case, the religious-cleavage preserved most, but mostly all five indicators proved more Dealignment-tendencies than a stronger Alignment, especially if they started on a higher level in 1999. Transnational trends of one indicator are not observed, so every parameter-value has to be discussed differently. Class Voting is a very complicated and fragile effect, which shows through correlation a strong coherence in countries, which are organised strongly corporatistic. But Class Voting is declining in opposite to the effect of age

Messung soziodemographischer Einflussgrößen auf das Wahlverhalten

Zentraler Aufbau dieser Arbeit war das Cleavage-Modell von Lipset & Rokkan empirisch auf das österreichische Wahlverhalten hin in den Jahren 1999, 2002 und 2006 zu überprüfen. Dieses makrosoziologische Modell, das das Wahlverhalten des Einzelnen gegenüber seiner Einstellung von (1) Kirche gegen Staat, (2) Arbeitgeber gegen Arbeitnehmer, (3) Ruralität und Urbanität und (4) Zentrum gegen Peripherie bestimmt, soll laut Dalton immer geringer das Wahlverhalten in der westlichen Welt erklären. Postmaterielle Werte, die Bildungsexpansion und weit verbreiteter Wohlstand lassen das Wahlverhalten deswegen immer schwieriger prognostizieren. Die Frage war zu klären, wie verändern sich die wichtigsten soziodemographischen Variablen, die aus der Cleavage-Theorie entnommen werden auf das Wahlverhalten heute in Österreich? Und ist ein Dealignment-Prozess der traditionellen Cleavages im Wahlverhalten in Österreich tatsächlich beobachtbar? Von 1999 als Ausgangspunkt weisen zehn Ausprägungen bei vier Parteien von 7 Variablen ein Dealignment auf, wovon lediglich bei 3 tatsächlich von Mal zu Mal ein Dealignment vorkommt. Dagegen kommt es zu 11 verstärkten Alignments der unterschiedlichen Ausprägungen und Parteien.The central aspect of this work was to proof the voting behaviour empirically in Austrian elections of 1999, 2002 and 2006 by basics of the cleavage model of Lipset & Rokkan. This macro-sociological model explains the voting behaviour by the property towards (1) Church versus State, (2) employer versus employee, (3) rurality versus urbanity and (4) center versus periphery. Based on post-materialistic values, an expanding education system and increasing wealth since the second half of the 20th century, the socio-demographic attributes loose their power to explain voting behaviour increasingly. The question is how are the most important socio-demographic attributes of the cleavage-model changing the voting behaviour today in Austrian elections? Can we observe a Dealignment in voting behaviour of the traditional cleavages today? From 1999 onwards, there were ten characteristics of a four-party-system with seven variables which caused a Dealignment. Only three of them exhibit a Dealignment gradually from 1999 to 2002 to 2006. Eleven attributes of seven variables have even an increasing alignment at different parties

Die betriebliche Integration leistungsgewandelter Mitarbeiter

Die Integration leistungsgewandelter Mitarbeiter wird vor allem auf Grund des demografischen Wandels in der Arbeitswelt ein Problem für Unternehmen. Nach Begriffsbestimmungen und der Kennzeichnung der Gruppe Leistungsgewandelter erfolgt die Einordnung des Problems in verschiedene Managementkonzepte, d. h. in das Personal-, Gesundheits-, Diversity Management und Disability Management. Sodann werden das Integrations-, Separations-, Flexibilisierungs- und Insourcing-Outsourcing-Modell zur betrieblichen Eingliederung Leistungsgewandelter diskutiert. Es erfolgt überwiegend am Beispiel des Projekts "Work2Work". Schließlich werden aus diesem Projekt Folgerungen für weitere Unternehmen abgeleitet.The integration of employees with changing capabilities and productivity levels is becoming an issue for companies against the background of demographic changes in the workplace. After defining the term and identifying the group of employees with changing capabilities and productivity levels, the issue is integrated in management concepts i.e. in human resources, health, diversity and disability management. There follows a discussion on the integration of employees with changing capabilities and productivity levels using the models for integration, separation, flexibilisation and in-sourcing/out-sourcing. The discussion is based largely on the "Work2Work" project. Conclusions are drawn from this project for other companies

Complete high-quality genome sequence of Clostridium limosum (Hathewaya limosa) isolate 14S0207, recovered from a cow with suspected blackleg in Germany

Clostridium limosum can be found in soil and the intestinal tract of animals. In 2014, C. limosum was isolated from a suspected blackleg outbreak in cattle in Schleswig-Holstein, Germany. We present a complete genome sequence of a C. limosum strain represented by a circular chromosome and three plasmids

Quantitative detection of Verticillium dahliae in soil as a basis for selection of planting sites in horticulture

Verticillium dahliae verursacht im Gartenbau, insbesondere bei Erdbeeren und Alleebäumen, große wirtschaft­liche Schäden, die vielfach auf verseuchte Standorte zurückzuführen sind. Es wird ein Nachweisverfahren beschrieben, mit dem die Mikrosklerotien des Erregers im Boden quantitativ geschätzt werden können. Auf diese Weise kann der Verseuchungsgrad einer Fläche ermittelt und daraus das Befallsrisiko für eine anfällige Kultur abgeleitet werden. Die Genauigkeit der Nachweismethode und die Grenzen ihrer Anwendung werden anhand ausgewählter Versuchsergebnisse dargestellt. Sie zeigen, dass schon geringfügige methodische Abweichungen einen signifikanten Einfluss auf die Nachweisrate ausüben können. Darüber hinaus hat die ungleichmäßige Verteilung der Mikrosklerotien im Boden einen unvermeid­baren Untersuchungsfehler zur Folge, der mit abnehmender Verseuchung größer wird. Schließlich lassen sich mit der Methode die beiden unterschiedlich virulenten Genotypen V. dahliae und V. longisporum nicht differenzieren. Aufgrund der Ungenauigkeit des Verfahrens und des Einflusses zahlreicher, noch unbekannter standortspezifischer Bodenfaktoren auf das Infektionsgeschehen, ist eine exakte Befallsprognose kaum möglich. Vor diesem Hintergrund wird die Ermittlung und Anwendung von Schadschwellen in der Praxis kritisch beurteilt. Dennoch bieten die Bodenuntersuchungen den Anbauern die Möglichkeit, eine gezielte Flächenauswahl vornehmen und Schäden vorbeugend verhindern zu können.    Verticillium dahliae causes in horticultural production, especially in strawberries and in tree nurseries, high economic damages, which arise from infested planting sites. A detection method is described, which allows the quantification of microsclerotia of the fungus in soil. So it is possible to determine the verticillium infestation level of a planting site und to estimate the disease risk for susceptible crops. Some original results are presented to illustrate the quality of performance of the method. They give evidence that small variations of the quantification procedure have an influence on the detection percentage. In addition the nonrandomly distribution of microsclerotia in soil causes a sample and analysis error, which increases with declining infestation levels. Moreover it is not possible to distinguish the two genotypes V. dahliae and V. longisporum, which show differences in their pathogenicity. Therefore an exact pre-planting disease prediction is difficult, because of the inaccuracy of the detection method and the influence of many unknown soil factors on the infection process. This is the reason why the determination and use of thresholds in practice is critical judged. Nevertheless the quantification of the soil infestation provides growers an opportunity to select verticillium-free sites and to avoid damages.   &nbsp

Reactivity of Metakaolin in Alkaline Environment: Correlation of Results from Dissolution Experiments with XRD Quantifications

Systematic investigation of filtrates and filter residues resulting from a 24 h treatment of metakaolin in different alkaline solutions were performed. On filtered metakaolin particles, inductively coupled plasma-optical emission spectrometry (ICP-OES) measurements reveal an enrichment of iron and titanium, which suggests an inhomogeneous distribution of these cations. Since the SiO2/Al2O3 ratio remains constant in all filter residues examined, the dissolution of the Si and Al monomers is congruent. Structural differences, identified by attenuated total reflection–Fourier transform infrared spectroscopy (ATR-FTIR) as a consequence of alkali uptake, influence the X-ray scattering contribution of metakaolin, and thus quantifications with the partial or no known crystal structure (PONKCS) method. This leads to deviations between the degree of reaction calculated from Si and Al solubility from filtrate and that quantified by quantitative powder X-ray diffraction (QPXRD) using the filter residue. Nevertheless, the described changes do not cause a shift in the X-ray amorphous hump in case of congruent dissolution, and thus allow the quantification of the metakaolin before and after dissolution with the same hkl-phase model

A new wireless system for decentralised measurement of physiological parameters from shake flasks

BACKGROUND: Shake flasks are widely used because of their low price and simple handling. Many researcher are, however, not aware of the physiological consequences of oxygen limitation and substrate overflow metabolism that occur in shake flasks. Availability of a wireless measuring system brings the possibilities for quality control and design of cultivation conditions. RESULTS: Here we present a new wireless solution for the measurement of pH and oxygen from shake flasks with standard sensors, which allows data transmission over a distance of more than 100 metres in laboratory environments. This new system was applied to monitoring of cultivation conditions in shake flasks. The at-time monitoring of the growth conditions became possible by simple means. Here we demonstrate that with typical protocols E. coli shake flask cultures run into severe oxygen limitation and the medium is strongly acidified. Additionally the strength of the new system is demonstrated by continuous monitoring of the oxygen level in methanol-fed Pichia pastoris shake flask cultures, which allows the optimisation of substrate feeding for preventing starvation or methanol overfeed. 40 % higher cell density was obtained by preventing starvation phases which occur in standard shake flask protocols by adding methanol when the respiration activity decreased in the cultures. CONCLUSION: The here introduced wireless system can read parallel sensor data over long distances from shake flasks that are under vigorous shaking in cultivation rooms or closed incubators. The presented technology allows centralised monitoring of decentralised targets. It is useful for the monitoring of pH and dissolved oxygen in shake flask cultures. It is not limited to standard sensors, but can be easily adopted to new types of sensors and measurement places (e.g., new sensor points in large-scale bioreactors)