Exploitation of the coil-globule plasmid DNA transition induced by small changes in temperature, pH salt, and poly(ethylene glycol) compositions for directed partitioning in aqueous two-phase systems

In this study, the interplay of two linked equilibria is examined, one concerning an aqueous two-phase system (ATPS) composed of poly(ethylene glycol) (PEG) and salt employed to partition plasmid DNA (pDNA), and the other a potential structural transition of pDNA depending on PEG and salt concentration and other system parameters. The boundary conditions for pDNA partitioning are set by PEG and salt concentrations, PEG molecular weight, pH, and temperature. While investigating these parameters, it was found that a small increase/decrease of the respective values led to a drastic and significant change in pDNA behavior. This behavior could be attributed to a coil-globule transition of the pDNA triggered by the respective phase conditions. The combination of this structural change, aggregation effects linked to the transition process, and the electrostatic potential difference found in PEG-salt systems thus offers a sensitive way to separate nucleic acid forms on the basis of their unique property to undergo coil-globule transitions under distinct system properties

The influence of cell adsorbent interactions on protein adsorption in expanded beds

Expanded bed adsorption (EBA) is a primary recovery operation allowing the adsorption of proteins directly from unclarified feedstock, e.g. culture suspensions, homogenates or crude extracts. Thus solid-liquid separation is combined with adsorptive purification in a single step. The concept of integration requires that the solid components of the feed solution are regarded as a part of the process, which influences stability, reproducibility, and overall performance. This aspect is investigated here at the example of the influence of presence and concentration of intact yeast cells (S. cerevisiae) on the adsorption of model proteins (hen egg white lysozyme and bovine serum albumin) to various stationary phases (cation and anion-exchange, hydrophobic interaction, immobilised metal affinity). The interaction of the cells with the adsorbents is determined qualitatively and quantitatively by a pulse response method as well as by a finite bath technique under different operating conditions. The consequence of these interactions for the stability of expanded beds in suspensions of varying cell concentration is measured by residence time distributions (RTDs) after tracer pulse injection (NaBr, LiCl). Analysis of the measured RTD by the PDE model allows the calculation of the fraction of perfectly fluidised bed (φ), a parameter which may be regarded as a critical quantity for the estimation of the quality of fluidisation of adsorbents in cell containing suspensions. The correlation between bed stability and performance is made by analysing the breakthrough of model proteins during adsorption from unclarified yeast culture broth. A clear relationship is found between the degree of cell/adsorbent interaction, bed stability in terms of the φ parameter, and the sorption efficiency. Only beds characterised by a φ; value larger than 0.8 in the presence of cells will show a conserved performance compared to adsorption from cell free solutions. A drop in φ, which is due to interactions of the fluidised adsorbent particles with cells from the feed, will directly result in a reduced breakthrough efficiency. The data presented highlight the importance of including the potential interaction of solid feedstock components and the expanded adsorbents into the design of EBA processes, as the interrelation found here is a key factor for the overall performance of EBA as a truly integrated operation. © 2000 Elsevier Science B.V.Fil: Fernandez Lahore, Hector. Universitat Dusseldorf; Alemania. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Geilenkirchen, S.. Universitat Dusseldorf; AlemaniaFil: Boldt, K.. Universitat Dusseldorf; AlemaniaFil: Nagel, A.. Universitat Dusseldorf; AlemaniaFil: Kula, M. R.. Universitat Dusseldorf; AlemaniaFil: Thömmes, J.. Universitat Dusseldorf; Alemani

Emissions of transboundary air pollutants in the Netherlands 1990-2012 : Informative Inventory Report 2014

Emissies Nederland blijven in 2012 onder nationale plafonds De uitstoot van stikstofoxiden (NOx), ammoniak, zwaveldioxide en niet-methaan vluchtige organische stoffen (NMVOS) is in 2012 in Nederland licht gedaald. Daarmee bleef de uitstoot onder de maxima die de Europese Unie daaraan sinds 2010 stelt. Nederland voldoet daardoor, net als in 2011, aan de vier 'nationale emissieplafonds' (NEC) voor deze stoffen. Dit blijkt uit de Nederlandse emissiecijfers van grootschalige luchtverontreinigende stoffen. Het RIVM verzamelt en analyseert deze cijfers. Behalve bovengenoemde stoffen gaat het om de uitstoot van koolmonoxide, fijn stof (PM10), zware metalen en persistente organische stoffen (POP's). De uitstoot van al deze stoffen is tussen 1990 en 2012 gedaald. Dit komt vooral door schonere auto's en brandstoffen en door emissiebeperkende maatregelen van industriële sectoren. Meer kilometers door bromfietsen Door de jaren heen zijn de methoden om de emissies te berekenen verbeterd, wat nu resulteert in nauwkeurigere cijfers. De emissies van bromfietsen en motorfietsen zijn afhankelijk van het aantal gereden kilometers per jaar en daar is nu beter inzicht in. Het totale aantal gereden kilometers door bromfietsen blijkt in de afgelopen jaren bijna twee keer zo hoog is als werd gedacht. Daarmee is de uitstoot van schadelijke stoffen navenant hoger. Ten opzichte van andere typ voertuigen blijven bromfietsen echter een relatief kleine emissiebron en dragen ze beperkt bij aan de totale nationale emissies. In steden zijn ze wel een relevante bron. Het aantal gereden kilometers door motorfietsen, en daarmee de uitstoot, blijft in lijn met eerdere inzichten. Vrachtauto's zwaarder beladen De uitstoot van schadelijke stoffen door vrachtauto's is voor het eerst berekend op basis van recente inzichten in het gewicht van vrachtauto's. Trekker-opleggers blijken zwaarder beladen dan tot nu toe werd verondersteld. Ook rijden vrachtauto's vaker met een aanhanger dan tot nu toe werd aangenomen, waardoor ze zwaarder zijn. Een hoger gewicht betekent een hoger brandstofverbruik, en veelal ook een hogere uitstoot per gereden kilometer. De uitstoot van PM10 door vrachtauto's is hierdoor circa 5 procent hoger dan in de vorige IIR-rapportage. Hogere emissies ammoniak De uitstoot van ammoniak blijkt hoger dan eerder werd verondersteld vanwege enkele nieuwe inzichten; de cijfers zijn hierdoor vanaf 1997 bijgesteld. Zo worden luchtwassers, die voornamelijk op varkensstallen zitten, niet altijd gebruikt. Ook is vanaf 2002 in melkveestallen het leefoppervlak per dier toegenomen. Door het grotere contactoppervlak van mest met lucht wordt meer ammoniak uitgestoten. Door de aangepaste aannames is het nationale totaal met 6,6 kiloton verhoogd ten opzichte van 2011.Emissions the Netherlands in 2012 remain under national ceilings Emissions of nitrogen oxides (NOx), ammonia, sulphur dioxide and non-methane volatile organic compounds (NMVOC) in the Netherlands have slightly decreased in 2012. Consequently, the emissions stayed below the caps the European Union has set from 2010. Herewith, the Netherlands comply with all four so-called emission ceilings (NEC). This has become apparent from the emission data on air pollutants from the Netherlands. RIVM collects and reports these data. Besides above-mentioned substances, emissions of carbon monoxide, particulate matter (PM10), heavy metals and persistent organic pollutants (POPs) have been reported. The emissions of all substances have decreased in the 1990 - 2012 period. The downward trend may in particular be attributed to cleaner fuels, cleaner car engines and to emission reductions in the industrial sectors. More kilometres by mopeds Over the years emission calculation methods have been improved, resulting in higher data accuracy. In 2012, the emissions from mopeds and motorcycles have been calculated, based on improved knowledge of the mileages. The total number of kilometres driven by mopeds appears to have been nearly twice as high in recent years. As a result, the emissions of pollutants are proportionally higher. In relation to the total number of vehicles, the number of mopeds however remains relatively low and their contribution to the total national emissions is limited. In cities, they are a relevant source. The mileages by motorcycles, and consequently their emissions remain in line with previous insights. Heavy-duty vehicles carry heavier loads Emissions of pollutants by heavy-duty trucks have for the first time been calculated on the basis of recent insights in truck loads. Tractor-trailer combinations appear to carry heavier loads and the fraction of trailers behind rigid trucks is larger than previously assumed. A heavier load means a higher fuel use and for most substances a higher emission per kilometre driven. PM10 emissions by heavy-duty trucks are about 5 percent higher than in the previous IIR report. Higher agricultural ammonia emissions Agricultural ammonia emissions appear to be higher than previously assumed because of new insights. Air scrubbers on animal housing (predominantly pigs) were not always in use or even employed. Since 2002, the living space per animal has increased for dairy cattle housing. This resulted in a higher contact surface manure-air and thus more ammonia emitted. The new insights have raised the national total of ammonia emissions by about 6 percent compared to 2011

Greenhouse gas emissions in The Netherlands 1990-2012 : National Inventory Report 2014

In 2012 is de totale uitstoot van broeikasgassen van Nederland, zoals CO2, methaan en lachgas, met ongeveer 1,7 procent gedaald ten opzichte van 2011. Deze daling komt vooral door een lager brandstofgebruik in de energie- en transportsector. Dit lijkt een gevolg van de economische recessie, waardoor emissies door elektriciteitsproductie en het wegtransport in Nederland zijn afgenomen. Cijfers De totale broeikasgasemissie wordt uitgedrukt in CO2-equivalenten en bedraagt in 2012 191,7 teragram (megaton of miljard kilogram) . Ten opzichte van de uitstoot in het Kyoto-basisjaar (213,2 Tg CO2-equivalenten) is dit een afname van ongeveer 10 procent. Het basisjaar, dat afhankelijk van het broeikasgas 1990 of 1995 is, dient voor het Kyoto-protocol als referentie voor de uitstoot van broeikasgassen. De uitstoot van de overige broeikasgassen zoals lachgas en methaan is sinds het basisjaar met 51 procent afgenomen. De CO2-uitstoot daarentegen is in deze periode met 4 procent gestegen. Landen zijn voor het Kyoto-protocol verplicht om de totale uitstoot van broeikasgassen op twee manieren te rapporteren: met en zonder het soort landgebruik en de verandering daarin. Dit is namelijk van invloed op de uitstoot van broeikasgassen. Voorbeelden zijn natuurontwikkeling (dat CO2 bindt) of ontbossing (waardoor CO2 wordt uitgestoten). In bovengenoemde getallen zijn deze zogeheten LULUCF-emissies (Land Use, Land Use Change and Forestry) niet meegenomen. Overige onderdelen inventarisatie Het RIVM stelt jaarlijks op verzoek van het Ministerie van Infrastructuur en Milieu (IenM) de inventarisatie van broeikasgasemissies op. De inventarisatie bevat trendanalyses om ontwikkelingen in de uitstoot van broeikasgassen tussen 1990 en 2012 te verklaren, en een analyse van de onzekerheid in deze getallen. Ook is aangegeven welke bronnen het meest aan deze onzekerheid bijdragen. Daarnaast biedt de inventarisatie documentatie van de gebruikte berekeningsmethoden, databronnen en toegepaste emissiefactoren. Met deze inventarisatie voldoet Nederland aan de nationale rapportageverplichtingen voor 2012 van het Klimaatverdrag van de Verenigde Naties (UNFCCC), van het Kyoto-Protocol en van het Bewakingsmechanisme Broeikasgassen van de Europese Unie.Total greenhouse gas emissions from the Netherlands in 2012 decreased by approximately 1.7 per cent, compared with 2011 emissions. This decrease is mainly the result of decreased fuel combustion in the Energy sector (increased electricity import) and in road transport. In 2012, total direct greenhouse gas emissions (excluding emissions from LULUCF - land use, land use change and forestry) in the Netherlands amounted to 191.7 Tg CO2 eq. This is approximately 10 per cent below the emissions in the base year (213.2 Tg CO2 eq.). The 51% reduction in the non-CO2 emissions in this period is counterbalanced by 4 per cent increase in CO2 emissions since 1990. This report documents the Netherlands' 2014 annual submission of its greenhouse gas emissions inventory in accordance with the guidelines provided by the United Nations Framework Convention on Climate Change (UNFCCC), the Kyoto Protocol and the European Union's Greenhouse Gas Monitoring Mechanism. The report comprises explanations of observed trends in emissions; a description of an assessment of key sources and their uncertainty; documentation of methods, data sources and emission factors applied; and a description of the quality assurance system and the verification activities performed on the data.Ministerie van I&

Whole Gene Capture Analysis of 15 CRC Susceptibility Genes in Suspected Lynch Syndrome Patients

Background and Aims Lynch Syndrome (LS) is caused by pathogenic germline variants in one of the mismatch repair (MMR) genes. However, up to 60% of MMR-deficient colorectal cancer cases are categorized as suspected Lynch Syndrome (sLS) because no pathogenic MMR germline variant can be identified, which leads to difficulties in clinical management. We therefore analyzed the genomic regions of 15 CRC susceptibility genes in leukocyte DNA of 34 unrelated sLS patients and 11 patients with MLH1 hypermethylated tumors with a clear family history. Methods Using targeted next-generation sequencing, we analyzed the entire non-repetitive genomic sequence, including intronic and regulatory sequences, of 15 CRC susceptibility genes. In addition, tumor DNA from 28 sLS patients was analyzed for somatic MMR variants. Results Of 1979 germline variants found in the leukocyte DNA of 34 sLS patients, one was a pathogenic variant (MLH1 c.1667+1delG). Leukocyte DNA of 11 patients with MLH1 hypermethylated tumors was negative for pathogenic germline variants in the tested CRC susceptibility genes and for germline MLH1 hypermethylation. Somatic DNA analysis of 28 sLS tumors identified eight (29%) cases with two pathogenic somatic variants, one with a VUS predicted to pathogenic and LOH, and nine cases (32%) with one pathogenic somatic variant (n = 8) or one VUS predicted to be pathogenic (n = 1). Conclusions This is the first study in sLS patients to include the entire genomic sequence of CRC susceptibility genes. An underlying somatic or germline MMR gene defect was identified in ten of 34 sLS patients (29%). In the remaining sLS patients, the underlying genetic defect explaining the MMRdeficiency in their tumors might be found outside the genomic regions harboring the MMR and other known CRC susceptibility genes

Whole Gene Capture Analysis of 15 CRC Susceptibility Genes in Suspected Lynch Syndrome Patients

This is the first study in sLS patients to include the entire genomic sequence of CRC susceptibility genes. An underlying somatic or germline MMR gene defect was identified in ten of 34 sLS patients (29%). In the remaining sLS patients, the underlying genetic defect explaining the MMR deficiency in their tumors might be found outside the genomic regions harboring the MMR and other known CRC susceptibility genes.Genome Instability and CancerMolecular Technology and Informatics for Personalised Medicine and Healt

Cell division and morphogenesis of Limnaea eggs after treatment with heat pulses at successive stages in early division cycles

Cellular reproduction is related to a number of apparently independent processes of which the integrated results are bound to produce cell division. In eggs with determinate cleavage the results of division are daughter cells of a different prospective significance. It has been observed furthermore in Limnaea eggs that morphogenesis is related to periodically recurring cell activities in the first, second and third cleavage cycle (Geilenkirchen, 1964a, b). These activities of unknown nature are dissociable from the factors involved in cell division. Obviously in the course of one division cycle the egg discriminates between processes for the preparation of the next division and processes involved in morphogenesis and differentiation later on. The data published in this paper carry the notion that successive divisions represent well-defined steps of different significance for later development and differentiation. Three questions are asked: (a) are successive cleavages of equal importance to later development; (b) are factors of morphogenetic importance present as a result of oogenesis, or do they arise during early cleavages; (c) are all steps in one cell cycle equally important to cell division and to later development? These problems are studied by the simple technique of giving eggs at successive stages of a cleavage cycle a heat pulse. The criteria for disturbance used are (a) retardation of the cleavages which follow treatment, and (b) the ultimate stage of development reached by the embryo after a pulse has been given. MATERIAL AND METHODS Eggs of Limnaea stagnalis were obtained by exposing the snails to stimulating plant leaves and a mild temperature rise In each experiment one egg mass only was used. The cleavage divisions of the eggs of one egg mass are not synchronous. In order to obtain treatment groups of synchronously dividing eggs, groups of eggs (8-10), which started first 1 Author's address: Zoologisch Laboratorium der Rijksuniversiteit, Utrecht, Holland