Tracing the Evolution of Temperature in Near Fermi Energy Heavy Ion Collisions

The kinetic energy variation of emitted light clusters has been employed as a clock to explore the time evolution of the temperature for thermalizing composite systems produced in the reactions of 26A, 35A and 47A MeV $^{64}$Zn with $^{58}$Ni, $^{92}$Mo and $^{197}$Au. For each system investigated, the double isotope ratio temperature curve exhibits a high maximum apparent temperature, in the range of 10-25 MeV, at high ejectile velocity. These maximum values increase with increasing projectile energy and decrease with increasing target mass. The time at which the maximum in the temperature curve is reached ranges from 80 to 130 fm/c after contact. For each different target, the subsequent cooling curves for all three projectile energies are quite similar. Temperatures comparable to those of limiting temperature systematics are reached 30 to 40 fm/c after the times corresponding to the maxima, at a time when AMD-V transport model calculations predict entry into the final evaporative or fragmentation stage of de-excitation of the hot composite systems. Evidence for the establishment of thermal and chemical equilibrium is discussed.Comment: 9 pages, 5 figure

Development of analytical-molecularbiological methods for the construction of a plasmid gene bank in Escheriachia coli using soil-DNA and its screening for new enzymes for technical application

Das große Artenreichtum mikrobieller Organismen in der Natur hat im Verlauf der Evolution zu einer Vielzahl unterschiedlicher Enzyme geführt. Da weniger als ein Prozent der Bodenmikroorganismen mit gängigen Methoden kultivierbar ist, wird in neueren Verfahren versucht, die genomische DNA aus Bodenproben zu isolieren, in einen Expressionswirt zu klonieren und die Genbank nach neuen Enzymen für die technische Anwendung zu durchmustern. Diese Vorgehensweise ist aber mit einer Reihe von technologischen Problemen verbunden. Ziel dieser Arbeit war daher die Entwicklung von Methoden zur Charakterisierung, Isolierung und Reinigung von Boden-DNA sowie die Erstellung einer Genbank, um diese nach Hydrolasen und Oxidoreduktasen zu untersuchen. Zunächst galt das Interesse der Entwicklung einer Methode zur DNA-Quantifizierung in Boden-Extrakten mittels SYBR-Green-I-(SG)-Fluoreszenz, die durch co-extrahierte Huminsäuren gestört wird. Die Aufklärung der Struktur von SG bildete die Basis für Interaktionsstudien zwischen dem Farbstoff, DNA und Huminsäuren. Es zeigte sich, dass SG in Abhängigkeit vom Farbstoff/Basenpaar-(Fbp)-Verhältnis einen biphasischen Bindungsmodus an DNA aufweist. Einerseits konnte mit hydrodynamischen Messungen (Semi-)Interkalation nachgewiesen werden. Auf der anderen Seite deuteten Untersuchungen mit den Homopolymeren Poly(dA)Poly(dT) und Poly(dG)Poly(dC) darauf hin, dass SG sich bei hohen Fbp-Verhältnissen in die kleine Furche der DNA einlagert. Ausgehend von diesen Ergebnissen konnte die durch Huminsäuren verursachte Fluoreszenz-Löschung auf Störmechanismen wie den inneren Filtereffekt, dynamische Fluoreszenz-Löschung und kompetitive Bindung von SG zurückgeführt werden. Darauf aufbauend wurde ein neuer Test entwickelt und umfassend validiert. Ein weiterer Teil der Arbeit bestand darin, Techniken zur Gewinnung, Reinigung und enzymatischen Spaltung von Boden-DNA zu untersuchen. Die DNA-Extraktion aus Bodenproben erfolgte in Anlehnung an ein in der Literatur beschriebenes Verfahren. Die durch Huminstoffe kontaminierten DNA-Extrakte konnten mit Hilfe von Ausschlusschromatographie-Säulen gereinigt werden. Die nachfolgende DNA-Spaltung war mit dem Enzym EcoRI möglich, wobei der Einfluss nicht-abgetrennter Huminstoffe durch Zugabe von Rinderserumalbumin minimiert wurde. Anschließend folgte die Ligation der DNA in den Vektor pJOE930. Nach Transformation in E. coli wurden etwa 43000 Transformanden auf Tributyrin-Platten ausplattiert, wobei 19 Klone eine lipolytische Aktivität zeigten. Die Untersuchung des Substratspektrums ergab einen Klon (Klon HZ04) mit interessanten Eigenschaften bei der Hydrolyse verschiedener para-Nitrophenyl-(pNP)-Ester, wie z.B. pNP-Butyrat, -Caproat, -Caprylat, -Laurat, -Palmitat, -Acrylat, und -Methacrylat sowie in geringem Umfang pNP-Trimethylacetat. Das abgeleitete Protein zeichnet sich durch ein GDSAG- und ein GGGx-Motiv aus. Letzteres ist charakteristisch für die sog. Oxyanionbindungstasche von Hydrolasen. Zirka 13000 der Transformanden wurden mit weiteren Testsystemen untersucht. Auf stärkehaltigen Agarmedien konnten 16 Amylase-aktive Klone identifiziert werden. Keiner der Klone zeigte eine proteolytische Aktivität auf kombinierten 5-Brom-4-chlor-3-indoylphosphat-Magermilch-Agarmedien, während ein Klon in der Lage war, Phytat zu hydrolysieren. Für die Identifizierung von Oxidoreduktasen wurden Testverfahren eingesetzt, die auf dem Nachweis von Carbonylgruppen infolge der Oxidation des eingesetzten Substrates 1,2-Propandiol beruhen. Von den etwa 20000 getesteten Klonen zeigten 19 Transformanden eine Carbonyl-bildende Aktivität. Im Verlauf der Arbeiten zeichneten sich zwei Klone durch eine Braunfärbung aus, die vermutlich auf eine Katalase/Peroxidase- bzw. Aminolävulinsäure-Synthase-Aktivität zurückzuführen ist. Zusammenfassend ist festzustellen, dass die in dieser Arbeit entwickelten Verfahren den Zugang zu einer erheblichen Anzahl an neuen Genen aus Mikroorganismen ermöglicht haben, die bisher noch nicht kultiviert worden sind.The immense reservoir of untapped microbial diversity is regarded as a source for new enzymes. Traditional approaches to isolate novel enzymes by cultivation of microorganisms from soil samples revealed to be incomplete as only a minor proportion of microorganisms are culturable under standard laboratory conditions. Thus, the access to genomes of microbial communities via gene banks is an important key for the identification of new enzymes. In the present work, the principle approach for the generation of genomic preparations from microbial communities involved the recovery of DNA from soil samples, the quantification of DNA, the purification of the soil-DNA, and its subsequent cloning into E. coli in order to screen the gene bank for hydrolases and oxidoreductases useful for technical application. DNA extracts from sediment samples are often contaminated by humic acids which interfere with many molecular biological applications, e.g. DNA quantification, enzymatic DNA modification. In order to develop a novel method for DNA quantification in crude extracts, the interaction between DNA and the fluorescent dye SYBR Green I (SG) was studied first. The analysis of the structure of SG (2-[N-(3-dimethylaminopropyl)-N-propylamino]-4-(2,3-dihydro-3-methyl-(benzo-1,3-thiazol-2-yl)-methylidene)-1-phenylquinolinium) and of the molar absorption coefficient (about 73000 (M·cm)-1 at 494 nm) enabled interaction studies with DNA which revealed the occurrence of (semi-)intercalation, followed by surface binding at dye/base pair ratios above 0,15. Only the latter binding led to a significant increase in fluorescence. Studies with poly(dA)poly(dT) and poly(dG)poly(dC) homopolymers indicated minor groove binding. On the basis of these results mechanisms underlying the impact of humic acids on DNA quantification by SG and consequences for the analysis of soils and aquatic sediments were investigated. According to these studies the effect of humic acids was based on an inner filter effect, collisional quenching, and competitive binding of SG by humic acids and DNA. Consequently, the standard assay was modified and its performance was verified by determination of DNA recoveries and concentrations of standards as well as environmental samples in comparison to the PicoGreen and Hoechst 33258 assays. The consistency of the determined DNA concentrations with respect to the data found in literature and the high recoveries indicated the reliability of the novel assay. For the generation of the gene bank soil-DNA was extracted according to a modified method described in literature, purified with gel filtration columns for removal of humic substances and digested by EcoRI. In order to prevent negative effects of residual impurities, bovine serum albumin was added to digestion reactions. After digestion the DNA molecules were size fractionated, ligated into the plasmid pJOE930 and the recombinant plasmids were transferred to E. coli. Around 43000 recombinant clones were plated directly on tributyrin agar plates. 19 lipolytic clones were discovered. The substrate specificity was investigated for four clones where clone HZ04 showed interesting activity towards hydrolysis of different para-nitrophenyl-(pNP)-esters like pNP-butyrate, -caproate, -caprylate, -laurate and -palmitate, -acrylate, -methacrylate and to a minor extent pNP-trimethylacetate. Around 13000 clones were screened for protease, amylase and phytase activities. Clones expressing protease activity were not found, whereas the initial screening for amylases revealed 16 positive clones. One clone showed phytase activity. Roughly 20000 colonies were screened for carbonyl-forming activity using 1,2-propanediol as substrate. Here, 19 clones showed a positive reaction. During the activity-based screening of the gene bank, two clones were noticed because of their brown-coloured phenotype. Analysis of the plasmids revealed gene products with amino acids similarities to a catalase/peroxidase and an aminolevulinic acid synthase. Thus, the recombinant approach showed the feasibility of direct cloning of soil-DNA to exploit microbial biodiversity for the discovery of new genes conferring hydrolytic and oxidoreductase activities using the plasmid pJOE930 and E. coli as host. This approach enabled the access to an enormous pool of genes from microorganisms that have not been cultivated yet. It should be mentioned that studies dealing with quantification and purification of DNA were of considerable importance

Investigations on DNA intercalation and surface binding by SYBR Green I, its structure determination and methodological implications

The detection of double-stranded (ds) DNA by SYBR Green I (SG) is important in many molecular biology methods including gel electrophoresis, dsDNA quantification in solution and real-time PCR. Biophysical studies at defined dye/base pair ratios (dbprs) were used to determine the structure–property relationships that affect methods applying SG. These studies revealed the occurrence of intercalation, followed by surface binding at dbprs above ∼0.15. Only the latter led to a significant increase in fluorescence. Studies with poly(dA) · poly(dT) and poly(dG) · poly(dC) homopolymers showed sequence-specific binding of SG. Also, salts had a marked impact on SG fluorescence. We also noted binding of SG to single-stranded (ss) DNA, although SG/ssDNA fluorescence was at least ∼11-fold lower than with dsDNA. To perform these studies, we determined the structure of SG by mass spectrometry and NMR analysis to be [2-[N-(3-dimethylaminopropyl)-N-propylamino]-4-[2,3-dihydro-3-methyl-(benzo-1,3-thiazol-2-yl)-methylidene]-1-phenyl-quinolinium]. For comparison, the structure of PicoGreen (PG) was also determined and is [2-[N-bis-(3-dimethylaminopropyl)-amino]-4-[2,3-dihydro-3-methyl-(benzo-1,3-thiazol-2-yl)-methylidene]-1-phenyl-quinolinium](+). These structure–property relationships help in the design of methods that use SG, in particular dsDNA quantification in solution and real-time PCR

Mechanisms underlying the impact of humic acids on DNA quantification by SYBR Green I and consequences for the analysis of soils and aquatic sediments

DNA quantification of soils and sediments is useful for the investigation of microbial communities and for the acquisition of their genomes that are exploited for the production of natural products. However, in such samples DNA quantification is impaired by humic acids (HA). Due to its lack of specificity and sensitivity, UV spectrophotometry cannot be applied. Consequently, fluorimetric assays applying Hoechst (H) 33258 or PicoGreen (PG) are used. Here, we investigated the SYBR Green I (SG) assay, which was also affected by HA, but was found to be 25- and 1.7-fold more sensitive compared to the H 33258 and PG assays, respectively. Spectrophotometric, fluorimetric and quenching studies as well as gel mobility shift assays suggested that the effect of HA on the SG assay was based on an inner filter effect, collisional quenching and binding of SG to HA. As to the latter finding, the standard 6250-fold dilution of the SG reagent was optimised to a 2000-fold dilution. Although the sensitivity of the optimised SG assay was reduced by a factor of 1.3, the interfering effect of HA could be reduced up to 22-fold. A significant reduction of HA interferences by lowering the pH of the assay was not observed. Finally, the performance of the modified SG assay and the corresponding evaluation methods were verified by the determination of DNA recoveries and concentrations of standards and environmental samples in comparison to the PG assay

Nonresonant central exclusive production of charged-hadron pairs in proton-proton collisions at s\sqrt{s} = 13 TeV

International audienceThe central exclusive production of charged-hadron pairs in pp collisions at a centre-of-mass energy of 13\TeV is examined, based on data collected in a special high-$\beta^*$ run of the LHC. The nonresonant continuum processes are studied with the invariant mass of the centrally produced two-pion system in the resonance-free region, $m_{\pi^+\pi^-}$$\lt$ 0.7 GeV or $m_{\pi^+\pi^-}$$\gt$ 1.8 GeV. Differential cross sections as functions of the azimuthal angle between the surviving protons, squared exchanged four-momenta, and $m_{\pi^+\pi^-}$ are measured in a wide region of scattered proton transverse momenta, between 0.2 and 0.8 GeV, and for pion rapidities $\lvert y\rvert$$\lt$ 2. A rich structure of interactions related to double-pomeron exchange is observed. A parabolic minimum in the distribution of the two-proton azimuthal angle is observed for the first time. It can be interpreted as an effect of additional pomeron exchanges between the protons from the interference between the bare and the rescattered amplitudes. After model tuning, various physical quantities are determined that are related to the pomeron cross section, proton-pomeron and meson-pomeron form factors, pomeron trajectory and intercept, and coefficients of diffractive eigenstates of the proton