Characterization of the CRISPR-Cas subtype I-B proteins Cas6b and Cas8b of Methanococcus maripaludis C5

The CRISPR-Cas system is an adaptive immune system found in archaea and bacteria to defend themselves against mobile genetic elements (e.g. phages). The system employs base complementarity of small RNA species (crRNAs) to target the foreign nucleic acids for degradation. The hallmark of the system is the CRISPR array or locus, which is composed of repetitive DNA sequences (repeats) that are interspersed by unique sequences (spacers). Spacer sequences can be derived from earlier encounters with viruses and, as part of the crRNAs, confer the base complementarity during a reoccurring attack. During the ongoing battle between prokaryotes and viruses diverse CRISPR-Cas systems evolved into three main types that are further subdivided. This thesis shows the first characterization of a subtype I-B CRISPR-Cas system. RNA-Seq data proved the in vivo activity of this CRISPR-Cas system in Methanococcus maripaludis C5. The data further revealed that the crRNAs are always composed of a complete spacer sequence flanked by an 8 nt 5' repeat tag and a 2 nt 3' repeat tag. Eigth cas genes were identified for M. maripaludis. Two Cas proteins, Cas8b and an annotated hypothetical protein were characterized in more detail. The hypothetical protein was shown to be the endoribonuclease responsible for the single-turnover catalysis of precursor crRNA into mature crRNA and was termed Cas6b. The reaction performed by Cas6b yields the 8 nt 5' terminal tag of the mature crRNAs. Despite sharing only low sequence identity of 11 %, the two Cas6 proteins of M. maripaludis and Pyrococcus furiosus could be well aligned using a structural model of Cas6b and the crystal structure of P. furiosus Cas6. Cas6b mutant analysis was used to determine four amino acid residues (lysine 30, histidine 38, histidine 40 and tyrosine 47) that comprise the catalytic site of Cas6b. The RNA binding properties of Cas6b were determined and showed a dimerization upon binding to a non-cleavable substrate. Further analyses including RNA crosslinking experiments followed by mass spectrometry identified a methionine residue (M185) that tightly coordinated to a uridine (U15) of the repeat sequence. Cas6b activity assays employing differently structured repeat variants of M. maripaludis and a 37 nt repeat sequence of Clostridium thermocellum could show, that the processing reaction performed by Cas6b does not recognize a secondary structure of the substrate. In addition to the verification to the in vivo activity of the CRISPR-Cas system, the RNA-Seq data also revealed a varying abundance pattern of crRNAs. To assess the crRNA abundance a experimental procedure was designed, which was aimed to analyse the influence of spacer sequences on a) the processing by Cas6 and b) the stability of crRNAs. With the help of this global approach influences of the spacer length and spacer sequence on the crRNA maturation and in vitro stability were recognized. In this context, future experiments will also determine further possible influences on crRNA abundance including i) crRNA loading into the Cas protein interference complex (Cascade) and ii) possible regulatory effects in terms of crRNA utilization dependent regulation. The characterization of the subtype-specific protein Cas8b revealed a splitting of the recombinant protein into two defined fragments. The exact point of cleavage was determined by Edman sequencing and provides evidence for a proteolytic cleavage of the full-length protein (either autocatalytically or by a protease). Other CRISPR-Cas subtypes were reported to contain two proteins serving as small and big subunit of the interference complex Cascade. For subtype I-B on the other hand Cas8b was found to be the only equivalent to these two proteins and it was proposed that the identified cleavage generates the large and small Cascade subunit. A biochemical analysis of Cas8b with respect to its putative roles during CRISPR-Cas immunity showed an unspecific binding to nucleic acids while no nucleolytic cleavage was observed. Possible functions of Cas8b are discussed and future studies will focus on the analysis of the protein functions in the context of a complete Cascade

Diagonalizations over polynomial time computable sets

AbstractA formal notion of diagonalization is developed which allows to enforce properties that are related to the class of polynomial time computable sets (the class of polynomial time computable functions respectively), like, e.g., p-immunity. It is shown that there are sets—called p-generic— which have all properties enforceable by such diagonalizations. We study the behaviour and the complexity of p-generic sets. In particular, we show that the existence of p-generic sets in NP is oracle dependent, even if we assume P ≠ NP

Differences in the performance tests of the fast and slow stretch and shortening cycle among professional, amateur and elite youth soccer players

The purpose of this study was to establish whether physical attributes can differentiate between professional, amateur and elite youth soccer players; such a distinction could aid in the selection process for youth soccer. Therefore, this investigation evaluated a suspected difference in the performance tests of the slow and fast stretch and the shortening cycle (squat jump [SJ], countermovement jump [CMJ], and drop jump from varying heights [DJ]) among professional, amateur and elite youth soccer players. Cross-sectional data were collected. The results indicate that higher performance in the SJ and CMJ seem to depend on the level of player because the mean performance of the PRO was 38.7 ± 4.0 cm in the SJ and 41.2 ± 3.8 in the CMJ, which were significantly (p<0.05) different compared with all other groups. In the DJ, there were significant (p<0.05) differences between the professional players (PRO) and lower-level players as well as between the PRO and youth soccer players. The results suggest that jump performance can differentiate between elite, sub-elite, and youth soccer players and highlights the importance of appropriate conditioning for developing strength and power in youth soccer players

Periodate-oxidized ATP stimulates the permeability transition of rat liver mitochondria

AbstractPeriodate-oxidized ADP (oADP) and periodate-oxidized ATP (oATP) stimulate the permeability transition in energized rat liver mitochondria measured as the Ca2+-efflux induced by Ca2+ and Pi. In the presence of Mg2+ and Pi, mitochondria lose intramitochondrial adenine nucleotides at a slow rate. oATP induces a strong decrease of the matrix adenine nucleotides which is inhibited by carboxyatractyloside. Under these conditions, Mg2+ prevents the opening of the permeability transition pore. EGTA prevents the Pi-induced slow efflux of adenine nucleotides, but is without effect on the oATP-induced strong decrease of adenine nucleotides. This oATP-induced strong adenine nucleotide efflux is inhibited by ADP. oATP reduces the increase of matrix adenine nucleotides occurring when the mitochondria are incubated with Mg2+ and ATP. This effect of oATP is also prevented by carboxyatractyloside. oATP is not taken up by the mitochondria. It is suggested that oATP induces a strong efflux of matrix adenine nucleotides by the interaction with the ADP/ATP carrier from the cytosolic side. The induction of the mitochondrial permeability transition by oADP and oATP is attributed to two mechanisms—a strong decrease in the intramitochondrial adenine nucleotide content, especially that of ADP, and a stabilization of the c-conformation of the ADP/ATP carrier

Green preparation and spectroscopic characterization of plasmonic silver nanoparticles using fruits as reducing agents

Chemicals typically available in plants have the capability to reduce silver and gold salts and to create silver and gold nanoparticles. We report the preparation of silver nanoparticles with sizes between 10 and 300 nm from silver nitrate using fruit extract collected from pineapples and oranges as reducing agents. The evolvement of a characteristic surface plasmon extinction spectrum in the range of 420 nm to 480 nm indicates the formation of silver nanoparticles after mixing silver nitrate solution and fruit extract. Shifts in plasmon peaks over time indicate the growth of nanoparticles. Electron microscopy shows that the shapes of the nanoparticles are different depending on the fruit used for preparation. The green preparation process can result in individual nanoparticles with a very poor tendency to form aggregates with narrow gaps even when aggregation is forced by the addition of NaCl. This explains only modest enhancement factors for near-infrared-excited surface enhanced Raman scattering. In addition to the surface plasmon band, UV–visible absorption spectra show features in the UV range which indicates also the presence of small silver clusters, such as Ag42+. The increase of the plasmon absorption correlates with the decrease of absorption band in the UV. This confirms the evolution of silver nanoparticles from silver clusters. The presence of various silver clusters on the surface of the “green” plasmonic silver nanoparticles is also supported by a strong multicolor luminesce signal emitted by the plasmonic particles during 473 nm excitation

Relation between pulse pressure and the pulsation strength in camera-based photoplethysmograms

Abstract Camera-based photoplethysmography (cbPPG) is an innovative measuring technique that enables the remote extraction of vital signs using video cameras. Most studies in the field focus on heart rate detection while other physiological quantities are often ignored. In this work, we analyzed the relation between the pulse pressure and the pulsation strengths of cbPPG signals for 70 patients after surgery. Our results show a high correlation between the two measures (r = 0.54). Furthermore, the influence of technical and medical factors was tested. The controlled impact of these factors proved to enhance the correlation by between 9 and 27 %.</jats:p

Application of Photogrammetry in Geology: 3D Investigation of Rock Fracture Distributions

Geology as a science has an important visual component and the knowledge of any geologist is deeply linked to visual experience of rock outcrops, thin sections and analytical images. One of the shortcomings of most geological images such as maps, cross sections and outcrop photographs is that they are 2D, while processes geologists are interested in are typically occurring in 3D space. The 3D geometry of faults, fractures and joints is crucial to quantify geological processes related to fracture mechanics, such as hydrothermal mineralization and ground water flow, but also geotechnical problems such as rock mass stability. A number of studies have shown that some geological structures can be described with a scale invariant, fractal distribution. So far these observations on which these findings are based were restricted to one and two dimensions and has been difficult to obtain a full spatial geometric picture of fracture sets from rock outcrops, because much of the rock is not directly accessible. However, without taking into account the spatial distribution of geological structures the true geometry of joint patterns cannot be fully described and scaling laws, fractal or not, cannot be derived. We present images of joint patterns based on datasets acquired by digital photographs which are processed to three dimensional images using the photogrammetry software Siro3D. This technique allows to obtain a highly accurate 3D picture of the visible outcrop. The spatial pattern of joints in nature is investigated using the software SiroJoint. For the analysis of joint systems a large data set was collected from the Heavitree Quarzite at Ormiston Gorge, near Alice Springs. The Heavitree Quartzite is fragmented by a spectacularly regular three-dimensional joint pattern, which is repeated at different scales and therefore represents a perfect laboratory for our investigations (Hobbs 1993). Siro3D generates a spatially fully referenced 3D image from overlapping digital images, such that each pixel of the image is assigned spatial coordinates. The software SiroJoint routinely constructs planes from the intersection of the rock-face with the linear trace of planar features (Poropat 2001). It provides stereographic plots of structural elements and additionally measures joint persistence, area, and joint spacing. Our measurements allow to analyse geometrical scaling relationships of joint sets with high accuracy and will help explore the character of their 3D complexity. Several hundred joint planes were defined with SiroJoint in an Ormiston Gorge outcrop. Three different joint sets can be distinguished. Joint set one and two are characterized by steeply inclined planes with joint spacings ranging between 2 cm to 40 cm and 2 cm to 10m respectively. Both joints sets depict a power law distribution in joint spacing/frequency plots. The third set is defined by a subhorizontal orientation. It shows a very regular spacing in the meter scale and lacks an exponential distribution. We intend to use the results as a basis to compare observed fracture pattern with those generated by computational methods like Iterated Function Systems. This might help to understand how physical rock properties influence the spatial complexity of fracture systems and develop constitutive scaling relationships for certain rock types.conferenc

A location aware mobile tourist guide selecting and interpreting sights and services by context matching

Most tourists exploring a destination either join a guided tour or walk on their own using maps. Neither are their individual preferences nor the actual situation considered. These tours are strongly inflexible. The ideal is an intelligent guide taking care of the whole tour organisation and execution in time. This is the main objective of the Dynamic Tour Guide (DTG). The DTG is a mobile agent that selects attractions, plans an individual tour, provides navigational guidance and offers location based interpretation. This kind of ambient intelligence is based on the analysis of all available context information to support the tourist in any possible way with the help of a mobile device