Hierarchical self-assembly of the DNA-grafted supramolecular polymers

Conjugation of functional entities with a specific set of optical, mechanical or biological properties to DNA strands allows engineering of sophisticated DNA-containing architectures. Among various hybrid systems, DNA-grafted polymers occupy an important place in modern materials science. In this contribution we present the non-covalent synthesis and properties of DNA-grafted linear supramolecular polymers (SPs), which are assembled in a controllable manner from short chimeric DNA-pyrene oligomers. The synthetic oligomers consist of two parts: a 10 nucleotides long DNA chain and a covalently attached segment of variable number of phosphodiester-linked pyrenes. The temperature-dependent formation of DNA-grafted SPs is described by a nucleation-elongation mechanism. The high tendency of pyrenes to aggregate in water, leads to the rapid formation of SPs. The core of the assemblies consists of stacked pyrenes. They form a 1D platform, to which the DNA chains are attached. Combined spectroscopic and microscopic studies reveal that the major driving forces of the polymerization are π-stacking of pyrenes and hydrophobic interactions, and DNA pairing contributes to a lesser extent. AFM and TEM experiments demonstrate that the 1D SPs appear as elongated ribbons with a length of several hundred nanometers. They exhibit an apparent helical structure with a pitch-to-pitch distance of 50±15 nm. Since DNA pairing is a highly selective process, the ongoing studies are aimed to utilize DNA-grafted SPs for the programmable arrangement of functional entities. For example, the addition of non-modified complementary DNA strands to the DNA-grafted SPs leads to the cooperative formation of higher-order assemblies. Also, our experiments suggest that the fluorescent pyrene core of 1D ribbons serves as an efficient donor platform for energy transfer applications.

Granular flow in polymineralic rocks bearing sheet silicates: New evidence from natural examples

Natural deformation in carbonate mylonites bearing sheet silicates occurs via a complex interaction of granular flow and solution transfer processes and involves continuous cycles of dissolution, grain boundary diffusion, nucleation and growth. In this way, new sheet silicates (a) nucleate within voids formed by grain boundary sliding of calcite grains. (b) grow, and (c) rotate towards the shear plane. As a consequence, small mica grains show a wide range of orientations with respect to the shear plane, but moderate to large grains are subparallel both to each other and to the shear plane. Increases of average grain sizes with increasing temperature of sheet silicates in mica-rich layers is more pronounced than in mica-poor layers. In the calcitic matrix however, sheet silicates can only grow via solution-precipitation and mass transfer processes. Therefore, the observed grain size variability indicates drastic differences in mass transfer behavior between the individual layers, which might be related to differences in the fluid flux. Based on these observations, a conceptual model for the microfabric evolution in sheet silicate bearing mylonites is presented. © 2001 Elsevier Science B.V. All rights reserved.

Dissolution-Precipitation in Porous Media: Experiments and Modelling

The evolution of porosity due to dissolution/precipitation processes of minerals and the associated change of transport parameters are of major interest for natural geological environments and engineered underground structures. We designed a reproducible and fast to conduct 2D experiment, which is flexible enough to investigate several process couplings implemented in the numerical code OpenGeosys-GEM (OGS-GEM). We investigated advective-diffusive transport of solutes, effect of liquid phase density on advective transport, and kinetically controlled dissolution/precipitation reactions causing porosity changes. In addition, the system allowed to investigate the influence of microscopic (pore scale) processes on macroscopic (continuum scale) transport. A Plexiglas tank of dimension 10 × 10 cm was filled with a 1 cm thick reactive layer consisting of a bimodal grain size distribution of celestite (SrSO4) crystals, sandwiched between two layers of sand. A barium chloride solution was injected into the tank causing an asymmetric flow field to develop. As the barium chloride reached the celestite region, dissolution of celestite was initiated and barite precipitated. Due to the higher molar volume of barite, its precipitation caused a porosity decrease and thus also a decrease in the permeability of the porous medium. The change of flow in space and time was observed via injection of conservative tracers and analysis of effluents. In addition, an extensive post-mortem analysis of the reacted medium was conducted. We could successfully model the flow (with and without fluid density effects) and the transport of conservative tracers with a (continuum scale) reactive transport model. The prediction of the reactive experiments initially failed. Only the inclusion of information from post-mortem analysis gave a satisfactory match for the case where the flow field changed due to dissolution/precipitation reactions. We concentrated on the refinement of post-mortem analysis and the investigation of the dissolution/precipitation mechanisms at the pore scale. Our analytical techniques combined scanning electron microscopy (SEM) and synchrotron X-ray micro-diffraction/micro-fluorescence performed at the XAS beamline (Swiss Light Source). The newly formed phases include an epitaxial growth of barite micro-crystals on large celestite crystals (epitaxial growth) and a nano-crystalline barite phase (resulting from the dissolution of small celestite crystals) with residues of celestite crystals in the pore interstices. Classical nucleation theory, using well-established and estimated parameters describing barite precipitation, was applied to explain the mineralogical changes occurring in our system. Our pore scale investigation showed limits of the continuum scale reactive transport model. Although kinetic effects were implemented by fixing two distinct rates for the dissolution of large and small celestite crystals, instantaneous precipitation of barite was assumed as soon as oversaturation occurred. Precipitation kinetics, passivation of large celestite crystals and metastability of supersaturated solutions, i.e. the conditions under which nucleation cannot occur despite high supersaturation, were neglected. These results will be used to develop a pore scale model that describes precipitation and dissolution of crystals at the pore scale for various transport and chemical conditions. Pore scale modelling can be used to parameterize constitutive equations to introduce pore-scale corrections into macroscopic (continuum) reactive transport models. Microscopic understanding of the system is fundamental for modelling from the pore to the continuum scale.

Temporal and spatial regulation of cell division by the nucleoid in Escherichia coli

Selection of division sites and coordination of cytokinesis with other cell cycle events are critical for every organism to proliferate. In E. coli, the nucleoid is proposed to exclude division from the site of the chromosome (nucleoid occlusion model). We studied the effect of the nucleoid on timing and placement of cell division. An early cell division protein, FtsZ, was used to follow development of the division septum. FtsZ forms a ring structure (Z ring) at potential division sites. The dynamics of Z ring was visualized in live cells by fusing FtsZ with a green fluorescent protein (GFP). Emanating FtsZ-GFP polymers from the constricted septum or aggregates in daughter cells were also observed, probably representing the FtsZ depolymerization and immature FtsZ nucleation processes. We next examined the nucleoid occlusion model. Mutants carrying abnormally positioned chromosomes were employed. In chromosomal partition mutants, replicated chromosomes cannot segregate. The Z ring was excluded from midcell to the edge of the nucleoid. This negative effect of nucleoids was further confirmed in replication deficient dnaA mutants, in which only a single chromosome is present in the cell center. These results suggest that the nucleoid, replicating or not, inhibits division in the area where the chromosome occupies. In addition, increasing the level of FtsZ does not overcome nucleoid inhibition. Interestingly in anucleate cells produced by both mutants, the Z ring was localized in the central part of the cell, which indicates that the nucleoid is not required for FtsZ assembly. Relaxation of chromosomes by reducing the gyrase activity or disruption of protein translation/translocation did not abolish the division inhibition capacity of the nucleoid. However, preventing transcription did compromise the nucleoid occlusion effect, leading to formation of multiple FtsZ rings above the nucleoid. In summary, we demonstrate that nucleoids negatively regulate the timing and position of division by inhibiting FtsZ assembly at unselected sites. Relief of this inhibition at midcell is coincident with the completion of DNA replication. On the other hand, FtsZ assembly does not require the nucleoid. ^

THE MOLECULAR INTERACTION BETWEEN TYPE II DIABETES AND ALZHEIMER’S DISEASE THROUGH CROSS-SEEDING OF PROTEIN MISFOLDING

With the population of the world aging, the prominence of diseases such as Type II Diabetes (T2D) and Alzheimer’s disease (AD) are on the rise. In addition, patients with T2D have an increased risk of developing AD compared to age-matched individuals, and the number of AD patients with T2D is higher than among aged-matched non-AD patients. AD is a chronic and progressive dementia characterized by amyloid-beta (Aβ) plaques, neurofibrillary tangles (NFTs), neuronal loss, brain inflammation, and cognitive impairment. T2D involves the dysfunctional use of pancreatic insulin by the body resulting in insulin resistance, hyperglycemia, hyperinsulinemia, pancreatic beta cell (β-cell) death, and other complications. T2D and AD are considered protein misfolding disorders (PMDs). PMDs are characterized by the presence of misfolded protein aggregates, such as in T2D pancreas (islet amyloid polypeptide - IAPP) and in AD brain (amyloid– Aβ) of affected individuals. The misfolding and accumulation of these proteins follows a seeding-nucleation model where misfolded soluble oligomers act as nuclei to propagate misfolding by recruiting other native proteins. Cross-seeding occurs when oligomers composed by one protein seed the aggregation of a different protein. Our hypothesis is that the pathological interactions between T2D and AD may in part occur through cross-seeding of protein misfolding. To test this hypothesis, we examined how each respective aggregate (Aβ or IAPP) affects the disparate disease pathology through in vitro and in vivo studies. Assaying Aβ aggregates influence on T2D pathology, IAPP+/+/APPSwe+/- double transgenic (DTg) mice exhibited exacerbated T2D-like pathology as seen in elevated hyperglycemia compared to controls; in addition, IAPP levels in the pancreas are highest compared to controls. Moreover, IAPP+/+/APPSwe+/- animals demonstrate abundant plaque formation and greater plaque density in cortical and hippocampal areas in comparison to controls. Indeed, IAPP+/+/APPSwe+/- exhibit a colocalization of both misfolded proteins in cerebral plaques suggesting IAPP may directly interact with Aβ and aggravate AD pathology. In conclusion, these studies suggest that cross-seeding between IAPP and Aβ may occur, and that these protein aggregates exacerbate and accelerate disease pathology, respectively. Further mechanistic studies are necessary to determine how these two proteins interact and aggravate both pancreatic and brain pathologies.

(Table 1) Summary of general lithology in IODP Exp302 cores

The Cenozoic ice-rafted debris (IRD) history of the central Arctic is reconstructed utilizing the terrigenous coarse sand fraction in IODP 302 cores from 0 to 273 meters composite depth. This Holocene - middle Eocene quantitative record of terrigenous sand accumulation on the Lomonosov Ridge, along with qualitative information on grain texture and composition, confirms the interpretation that ice initiation (sea ice and glacial ice) occurred ~46 Ma in the Arctic, and provides a long-term pattern of Arctic ice expansion and decay since the middle Eocene. IRD mass accumulation rates range from 0 to 0.13 g/cm2/ka in the middle Eocene and from 0 to 0.36 g/cm2/ka in the Neogene. IRD mass accumulation rate (MAR) maxima in the Miocene and Pliocene cooccur with either glacial initiation or intensification in the sub-Arctic. The 46.25 Ma IRD onset in the central Arctic slightly precedes the earliest evidence of ice in the Antarctic, and compares in timing with a >1000 ppm decrease in atmospheric concentrations of CO2. The decline of pCO2 in the middle Eocene may have driven both poles across the temperature threshold that enabled the nucleation of glaciers on land and partial freezing of the surface Arctic Ocean, especially during times of low insolation.

Continuous condensation particle (CP) observations from 1984 through 2009 at Neumayer Station, Antacrtica

Continuous condensation particle (CP) observations were conducted from 1984 through 2009 at Neumayer Station under stringent contamination control. During this period, the CP concentration (median 258 1/cm**3) showed no significant long term trend but exhibited a pronounced seasonality characterized by a stepwise increase starting in September and reaching its annual maximum of around 10**3/cm**3 in March. Minimum values below 10**2/cm**3 were observed during June/July. Dedicated time series analyses in the time and frequency domain revealed no significant correlations between inter-annual CP concentration variations and atmospheric circulation indices like Southern Annular Mode (SAM) or Southern Ocean Index (SOI). The impact of the Pinatubo volcanic eruption and strong El Niño events did not affect CP concentrations. From thermodenuder experiments we deduced that the portion of volatile (at 125 °C) and semi-volatile (at 250 °C) particles which could be both associated with biogenic sulfur aerosol, was maximum during austral summer, while during winter non-volatile sea salt particles dominated. During September through April we could frequently observe enhanced concentrations of ultrafine particles within the nucleation mode (between 3 nm and 7 nm particle diameter), preferentially in the afternoon.

Measurements of velocity-dependent and slip-dependent frictional strength in laboratory friction experiments on natural samples from IODP Expedition 316 and ODP Hole 190-1174B

Slowslip forms part of the spectrum of fault behaviour between stable creep and destructive earthquakes. Slow slip occurs near the boundaries of large earthquake rupture zones and may sometimes trigger fast earthquakes. It is thought to occur in faults comprised of rocks that strengthen under fast slip rates, preventing rupture as a normal earthquake, or on faults that have elevated pore-fluid pressures. However, the processes that control slow rupture and the relationship between slow and normal earthquakes are enigmatic. Here we use laboratory experiments to simulate faulting in natural rock samples taken from shallow parts of the Nankai subduction zone, Japan, where very low-frequency earthquakes - a form of slow slip - have been observed.We find that the fault rocks exhibit decreasing strength over millimetre-scale slip distances rather than weakening due to increasing velocity. However, the sizes of the slip nucleation patches in our laboratory simulations are similar to those expected for the very lowfrequency earthquakes observed in Nankai. We therefore suggest that this type of fault-weakening behaviour may generate slow earthquakes. Owing to the similarity between the expected behaviour of slow earthquakes based on our data, and that of normal earthquakes during nucleation, we suggest that some types of slow slip may represent prematurely arrested earthquakes.

Coarse-sand ice-rafted debris components and mass accumulation rates of ODP Site 152-918

To understand the late Cenozoic glacial history of the Northern Hemisphere, continuous long-term proxy records from climatically sensitive regions must be examined. Ice-rafted debris (IRD) from Ocean Drilling Program (ODP) Site 918, located in the Irminger Basin, is one such record. IRD in marine sediments is a direct indicator of the presence of glacial ice extending to sea level on adjacent landmasses, and, therefore, is an important paleoclimatic signal from the mid- to high latitudes. The IRD record at Site 918 is the first long-term ice-rafting record available for southeast Greenland, a region that may have been a key nucleation area for widespread glaciation during the late Cenozoic (Larsen et al, 1994, doi:10.2973/odp.proc.ir.152.1994). This data report presents the results of coarse sand-size IRD mass accumulation rate (MAR) analyses for Site 918 from the late Miocene through the Pleistocene. In addition, a preliminary analysis of IRD compositions is included. Detailed discussions of the local, regional, and global paleoclimatic implications of this data, and of the companion Site 919 Pleistocene IRD MAR data (Krissek, 1999, doi:10.2973/odp.proc.sr.163.118.1999), are in preparation. Such future work will include comparisons of these IRD MAR data sets to the Site 919 oxygen isotope stratigraphy developed by Flower (1998, doi:10.2973/odp.proc.sr.152.219.1998).

Geochemistry of minerals in medium-grained diabase from ODP Holes 137-504B and 140-504B

Samples recovered from Hole 504B during Leg 140 include a number of medium-grained, holocrystalline diabases that appear to represent the cores of thick dikes. The plagioclase and pyroxene in these samples occur in a variety of crystal morphologies. Plagioclase occurs as phenocrysts, microphenocrysts, elongate crystals, skeletal crystals, and branching radial clusters. Pyroxene occurs as phenocrysts, microphenocrysts, ophitic crystals, and poikilitic crystals. Plagioclase compositions became progressively poorer in anorthite and MgO and progressively richer in FeO as crystallization proceeded, while the average grain volume decreased and the aspect ratio of individual grains increased. Pyroxene compositions are largely independent of crystal morphology. The diabase dikes recovered from Hole 504B during Leg 140 appear to have crystallized in situ. Crystal compositions and morphologies are consistent with a rapid cooling rate and solidification times for individual dikes on the order of hours or days. The crystallization rate and nucleation rate of plagioclase lagged behind the cooling rate so that the degree of undercooling progressively increased as crystallization proceeded. Plagioclase crystal morphologies indicate much greater degrees of supersaturation than do pyroxene or olivine crystal morphologies. The 504B diabase magmas appear to have been emplaced with abundant preexisting pyroxene and olivine nuclei, but with few preexisting plagioclase nuclei. The suppression of plagioclase nucleation and crystallization relative to that of pyroxene and olivine could provide a mechanism by which the actual fractionation assemblage is more pyroxene-rich and plagioclase-poor than that predicted from thermodynamic models, or that observed in isothermal crystallization experiments.

Geochemistry of clinopyroxene megacrysts, xenocrysts, and phenocrysts in diabase dikes from ODP Hole 140-504B

We report the major, rare earth, and other trace element compositions of clinopyroxenes from two Leg 140, Hole 504B diabase dikes. These pyroxenes reflect a complex history of crystal growth and magma evolution. The large ranges of composition found reflect incorporation of exotic phenocrysts into the melt, the early formation of crystal clots before dike intrusion during an undercooling event, and in-situ fractionation of melt during and following dike emplacement. Some of the pyroxenes occur in coarse two- and three-phase glomerocrysts, which may be ôprotogabbrosö representing early stages of melt crystallization in the lower crust. Large variations in trace element composition are found. These likely reflect heterogeneous nucleation and growth of plagioclase and pyroxene in the melt, as well as complex interface kinetics that may affect partition coefficients during rapid crystal growth expected during undercooling. This can explain the formation of irregular chemical sector zoning in some equant anhedral phenocrysts. Undercooling of magmas in the lower crust most likely reflects input of fresh hot melt into a stagnating melt-storage zone. Dikes intruded upward from an inflated melt-storage zone during such a cycle are likely to be larger than those intruded from the storage zone between such cycles, when it would be deflated, consistent with the greater overall thickness of the phyric dikes in the Leg 140 section of Hole 504B.

Shear experiments from IODP Expedition 322 and 333 samples

The mechanical behavior of the plate boundary fault zone is of paramount importance in subduction zones, because it controls megathrust earthquake nucleation and propagation as well as the structural style of the forearc. In the Nankai area along the NanTroSEIZE (Kumano) drilling transect offshore SW Japan, a heterogeneous sedimentary sequence overlying the oceanic crust enters the subduction zone. In order to predict how variations in lithology, and thus mechanical properties, affect the formation and evolution of the plate boundary fault, we conducted laboratory tests measuring the shear strengths of sediments approaching the trench covering each major lithological sedimentary unit. We observe that shear strength increases nonlinearly with depth, such that the (apparent) coefficient of friction decreases. In combination with a critical taper analysis, the results imply that the plate boundary position is located on the main frontal thrust. Further landward, the plate boundary is expected to step down into progressively lower stratigraphic units, assisted by moderately elevated pore pressures. As seismogenic depths are approached, the décollement may further step down to lower volcaniclastic or pelagic strata but this requires specific overpressure conditions. High-taper angle and elevated strengths in the toe region may be local features restricted to the Kumano transect.

Silicate mineralogy of basalts at DSDP Leg 54 Holes

Basalts drilled from the East Pacific Rise, OCP Ridge, and Siqueiros fracture zone during Leg 54 are texturally diverse. Dolerites are equigranular at Sites 422 and 428 and porphyritic, with phenocrysts of plagioclase (An69.73) and Ca-rich clinopyroxene (Ca42Mg48Fe10) at Site 427. The East Pacific Rise lavas and some of those from the OCP Ridge are fine-grained and porphyritic. The majority of the large crystals are clustered skeletal glomerocrysts of plagioclase An64-77), together with olivine (Fo80-87), Ca-rich clinopyroxene, or both. Euhedral phenocrysts of plagioclase, together with olivine, Carich clinopyroxene, and Cr-Al spinel in some cases, occur in most of the fine-grained lavas. These phenocrysts are small (maximum dimension <1 mm in all but one sample), sparse (combined modal amount <1% in all samples), and distinctive from the megacrysts which characterize many ocean-floor lavas. In two East Pacific Rise lavas, zoned plagioclase (An83 cores) is the sole phenocryst phase. In other porphyritic lavas from all the main East Pacific Rise and OCP Ridge units drilled during Leg 54, the plagioclase phenocrysts contain cores of bytownite (An79-87) surrounded by more-sodic feldspar (An67-77). Core/rim relationships vary from continuous normal zoning, through discontinuous zoning, to extensive resorption of the calcic cores in some samples. The compositions of the plagioclase calcic cores are systematically related to those of the glomerophyric plagioclase and olivine in the lavas containing them. Furthermore, only one compositional population of calcic cores occurs in each rock. The possible causes of these relationships are far from clear. Magma mixing, although superficially applicable, is inconsistent with important aspects of the phenocryst mineralogy of these particular lavas. A more satisfactory model to explain both phenocryst zoning and rapid glomerocryst growth immediately before extrusion may be constructed by postulating influx of water into the upwelling magmas within Layer 3 of the oceanic crust beneath the East Pacific Rise, and subsequent loss of part of this water during effervescence within feeder dykes between Layer 3 and the ocean floor. It is shown that this model is fully consistent with published data on water and carbon dioxide contents and ratios in the pillow-margin glasses, vesicles, and phenocryst inclusions of ocean-floor basalts. The evidence for the precipitation of plagioclase- dominated crystalline assemblages from these magmas in the upper part of Layer 3 is concordant with recent geophysically based modeling of the structure of the East Pacific Rise. Calcium-rich clinopyroxenes in dolerites from the OCP Ridge and Siqueiros fracture zone show radial, oscillatory, and sector-zoning. In Sample 428A-5-2 (Piece 5a), the compositional trends resulting from this zoning closely resemble those of the pyroxenes in some lunar lavas. The controls on crystallization of interstitial pigeonite - epitaxial upon augite - in this rock are discussed. Both sector-zoning of the augite and nucleation of pigeonite within microvolumes of magma with a low Ca(Mg + Fe) ratio appear to be important factors.

Distribution of foraminifera and other components in ODP Leg 188 sites

During Leg 188 of the Ocean Drilling Program (ODP), employing JOIDES Resolution, we drilled holes at three sites in the southern Indian Ocean in and near Prydz Bay, East Antarctica, between 28 January and 29 February 2000. The objectives of the voyage were to: - Core through sediments deposited when Antarctica underwent the transition from "greenhouse" to the modern "icehouse" state late in the Eocene or early in the Oligocene, at sites obtaining their sediment from the currently subglacial Gamburtsev Mountains that probably were the site of nucleation of the ice sheet (principally Site 1166); - Obtain a sediment record from times at which major changes in the ice sheet volume and characteristics took place as judged from oxygen isotope records, especially at ~23.7 Ma (Oligocene/Miocene boundary), 12-16 Ma (middle Miocene), and 2.7 Ma (late Pliocene) (mainly Site 1165); and - Sample through the upper Pliocene and Quaternary in an attempt to document fluctuations in the extent of the ice sheet over the continental shelf during the Quaternary (especially Site 1167). Paleogene foraminifer-bearing marine sections were not intersected, and thus discussion of marine sections is restricted to the Neogene. Foraminifers are not major contributors to Leg 188 chronostratigraphy but contribute to paleoenvironmental interpretation, to issues such as carbonate compensation depth (CCD) effects and source and history of sediment, and provide a basis for Sr and d18O studies. Chronostratigraphy for the various sections was compiled from diatoms, radiolarians, and paleomagnetism (Shipboard Scientific Party, 2001, doi:10.2973/odp.proc.ir.188.101.2001). Foraminifers were sporadic rather than continuous except in short intervals; however, the Neogene foraminifers from the region are very poorly known and the new records proved to be of significant value in paleoenvironmental interpretation. Only at Site 1167 did drilling intersect a section that yielded foraminifers virtually throughout. Other than for the very young section at each site, there is virtually no continuity of assemblages between sites and thus each section is treated here as separate and unrelated.

Crystal size variations in Eemian-age ice from the GRIP ice core, Central Greenland

Continuous measurements of ice crystal size have been carried out on an 80 m sequence between 2790 and 2870 m depth in the GRIP ice core from Central Greenland. The ice in this interval is at present considered to orginate from the Eemian interglacial period. The record reveals that the crystal size in ice older than 100,000 yr is highly dependent on climatic conditions at the time of snowfall. This dependence shows up as a strong correlation between ?18O values and crystal size throughout the Eemian, as well as a negative correlation between crystal size and several soluble and insoluble impurities. Although high-resolution impurity records are available from selected parts of the Eemian ice, the study is not conclusive on which impurities are most effective in slowing grain growth. It is shown that the normal grain-growth process, commonly observed in the upper few hundred metres of polar ice sheets, does not yield grain sizes compatible with observed ones at this depth in the ice sheet, even in those parts of the Eemian ice where impurity drag effects are not present. Polygonization of crystals within the ice sheet and the nucleation and rapid growth of new grains at relatively high temperatures in the lowest part probably play an important role in producing the observed grain-size variations. The relevance of possible flow disturbances of the GRIP Eemian climatic record for the results presented is discussed briefly.