New palynological and tephrostratigraphical investigations of two salt lagoons on the island of Mljet, south Dalmatia, Croatia

In the sediments of both of the investigated lakes, the tephra from the Mercato-Ottaviano eruption (Vesuvius, southern Italy) (ca. 7900 B.P.) could be identified. The palynological investigations show that from ca. 9000-7200 B.P. (8000-6000 cal B.C.) deciduous oak forests predominated, with only a few representatives of Mediterranean vegetation. At the transition to the central European Atlantic Period those forests changed to an open vegetation type, dominated byJuniperus andPhillyrea. At about 5500 B.P. (4400 cal B.C.), theJuniperus-Phillyrea vegetation was replaced byQuercus ilex woodland that still occurs on the island of Mljet today and is considered to be the natural vegetation of the Dalmatian coastland. The associated vegetation of theQ. ilex forests changed several times. At the beginning of theQ. ilex period,Juniperus values were still high, but soon they decreased andErica spread. In more recent times theQ. ilex forests were partially replaced by plantations ofPinus halcpensis. Indicators of human impact are sparse throughout the pollen record. Clear evidence for human influence exists only from ca. 3100 B.P. (1300 cal B.C.) whenJuglans andPinus halepensis were introduced to the area. Later,Olea andSecale cultivation can be suggested and further spreading ofJuniperus indicates use of the land as pasture.     

A new method for flat-embedding large native cryostat sections for targeting small preneoplastic lesions in comparative ultrastructural and ultracytochemical investigations

 Ultrastructural studies of rare and small cellular lesions in pathologically altered tissue are difficult to perform by applying conventional electron microscopic preparation. The search for lesions, often consisting of only a few cells in randomly obtained small specimen blocks, is time consuming and often without success. The methodological requirements for comparative enzyme cytochemical and morphological studies, i.e., preservation of both enzyme activity and ultrastructure, are divergent. By processing large native cryostat sections for electron microscopy, small preneoplastic focal lesions were successfully targeted in liver and kidney. Glucose-6-phosphatase, alkaline phosphatase, acid phosphatase, catalase, and cytochrome c oxidase activities were distinctly localized to endoplasmic reticulum, canalicular membrane, lysosomes, peroxisomes, and mitochondria, respectively, in the morphologically altered cells. Fixation of serial cryostat sections and enzyme reactions were both carried out through a semipermeable membrane except those for cytochrome c oxidase, which was demonstrated after fixation through the membrane by floating the section in incubation medium containing cytochrome c. Thereafter, the sections were flat embedded and polymerized between epoxy resin disks and aluminum dishes fitting exactly together. The objects of interest were identified in the light microscope, cut out, and reembedded in reversed gelatine capsules. By using this technique an ultrastructural preservation was achieved similar to that seen after immersion fixation. The enzyme activities were clearly localized without diffusion of the reaction product or unspecific deposits. The procedure permits precise targeting and complex studies of rare and small lesions, and opens new perspectives for the use of cryo-preserved tissue. Accepted: 10 March 1998     

Rapid, automated nucleic acid probe assays using silicon microstructures for nucleic acid concentration

A system for rapid point-of-use nucleic acid (NA) analysis based on PCR techniques is described. The extraction and concentration of DNA from test samples has been accomplished utilizing silicon fluidic microchips with high surface-area-to-volume ratios. Short (500 bp) and medium size (48,000 bp) DNA have been captured, washed, and eluted using the silicon dioxide surfaces of these chips. Chaotropic (GuHCl) salt solutions were used as binding agents. Wash and elution agents consisted of ethanol-based solutions and water, respectively. DNA quantities approaching 40 ng/cm2 of binding area were captured from input solutions in the 100-1000 ng/mL concentration range. For dilute samples of interest for pathogen detection, PCR and gel electrophoresis were used to demonstrate extraction efficiencies of about 50 percent, and concentration factors of about 10x using bacteriophage lambda DNA as the target. Rapid, multichannel PCR thermal cycling modules with integrated solid-state detection components have also been demonstrated. These results confirm the viability of utilizing these components as elements of a compact, disposable cartridge system for the detection of NA in applications such as clinical diagnostics, biowarfare agent detection, food quality control, and environmental monitoring.     

Fine sequence analysis of 60 kb around the Arabidopsis thaliana AtEm1 locus on chromosome III

The Arabidopsis thaliana Em1 gene has been mapped to the lower arm of chromosome III. Fine analysis of 60 kb around this gene, based largely on identification and sequencing of cognate cDNAs, has allowed us to identify 15 genes or putative genes. Cognate cDNAs exist for ten of these genes, indicating that they are effectively expressed. Analysis by sequence alignment and intracellular localization prediction programs allows attribution of a potential protein product to these genes which show no obvious functional relationship. Comparison of the true exon/intron structure based on cDNA sequences with that proposed by three commonly used prediction programs shows that, in the absence of further information, the results of these predictions on anonymous genomic sequences should be interpreted with caution. Examination of the non-coding sequence showed the presence of a novel repeated, palindromic element. The results of this detailed analysis show that in-depth studies will be necessary to exploit correctly the complete A. thaliana genome sequence.     

Large-scale deployment of grass in crop rotations as a multifunctional climate mitigation strategy

The agriculture sector can contribute to climate change mitigation by reducing its own greenhouse gas (GHG) emissions, sequestering carbon in vegetation and soils, and providing biomass to substitute for fossil fuels and other GHG-intensive products. The sector also needs to address water, soil, and biodiversity impacts caused by historic and current practices. Emerging EU policies create incentives for cultivation of perennial plants that provide biomass along with environmental benefits. One such option, common in northern Europe, is to include grass in rotations with annual crops to provide biomass while remediating soil organic carbon (SOC) losses and other environmental impacts. Here, we apply a spatially explicit model on >81,000 sub-watersheds in EU27 + UK (Europe) to explore the effects of widespread deployment of such systems. Based on current accumulated SOC losses in individual sub-watersheds, the model identifies and quantifies suitable areas for increased grass cultivation and corresponding biomass- and protein supply, SOC sequestration, and reductions in nitrogen emissions to water as well as wind and water erosion. The model also provides information about possible flood mitigation. The results indicate a substantial climate mitigation potential, with combined annual GHG savings from soil-carbon sequestration and displacement of natural gas with biogas from grass-based biorefineries, equivalent to 13%–48% of current GHG emissions from agriculture in Europe. The environmental co-benefits are also notable, in some cases exceeding the estimated mitigation needs. Yield increases for annual crops in modified rotations mitigate the displacement effect of increasing grass cultivation. If the grass is used as feedstock in lieu of annual crops, the displacement effect can even be negative, that is, a reduced need for annual crop production elsewhere. Incentivizing widespread deployment will require supportive policy measures as well as new uses of grass biomass, for example, as feedstock for green biorefineries producing protein concentrate, biofuels, and other bio-based products.     

Simultaneous assessment of migration and proliferation of murine fibrosarcoma cells, as affected by hydroxyurea, vinblastine, cytochalasin B, Razoxane and interferon

Abstract. Using porous cell culture chambers, we have simultaneously assessed growth and locomotion of cancer cells to investigate whether certain agents affect cell motility in addition to cell division. First, cells from a murine fibrosarcoma cell line, 1.0/L1, were grown in ordinary flask cultures to determine appropriate cell inocula. Doses of agents were selected to reduce the final 4 day culture cellularity to about 50%, when present during the last two days of culturing. Secondly, the effects of these agents on cell numbers in the porous chambers and on cell migration out of the chambers ('emigration fraction') were recorded. We also examined, using a similar type of porous chamber, whether the agents could affect leucocyte chemotaxis. Hydroxyurea (an inhibitor of DNA synthesis) reduced cancer cell emigration as well as cell growth, without interfering with leucocyte chemotaxis. Cytochalasin B (a microfilament disrupting agent) inhibited cancer cell motility and growth, as well as leucocyte chemotaxis. Vinblastine (a microtubule disrupting agent), at the very low dose chosen, reduced cancer cell growth, but did not consistently affect the migration of either cell type. The experimental anti-metastasis agent Razoxane reduced growth, but had no detectable effects on motility. High doses of natural murine interferon–α/β weakly inhibited both cancer cell growth and locomotion. This motivates for further studies of these and other cytokines, as treatment with agents inhibiting cancer cell locomotion might possibly prevent peri-operative spread of cancer in patients.     

An approximate Bayesian approach for estimation of the instantaneous reproduction number under misreported epidemic data

In epidemic models, the effective reproduction number is of central importance to assess the transmission dynamics of an infectious disease and to orient health intervention strategies. Publicly shared data during an outbreak often suffers from two sources of misreporting (underreporting and delay in reporting) that should not be overlooked when estimating epidemiological parameters. The main statistical challenge in models that intrinsically account for a misreporting process lies in the joint estimation of the time-varying reproduction number and the delay/underreporting parameters. Existing Bayesian approaches typically rely on Markov chain Monte Carlo algorithms that are extremely costly from a computational perspective. We propose a much faster alternative based on Laplacian-P-splines (LPS) that combines Bayesian penalized B-splines for flexible and smooth estimation of the instantaneous reproduction number and Laplace approximations to selected posterior distributions for fast computation. Assuming a known generation interval distribution, the incidence at a given calendar time is governed by the epidemic renewal equation and the delay structure is specified through a composite link framework. Laplace approximations to the conditional posterior of the spline vector are obtained from analytical versions of the gradient and Hessian of the log-likelihood, implying a drastic speed-up in the computation of posterior estimates. Furthermore, the proposed LPS approach can be used to obtain point estimates and approximate credible intervals for the delay and reporting probabilities. Simulation of epidemics with different combinations for the underreporting rate and delay structure (one-day, two-day, and weekend delays) show that the proposed LPS methodology delivers fast and accurate estimates outperforming existing methods that do not take into account underreporting and delay patterns. Finally, LPS is illustrated in two real case studies of epidemic outbreaks.     

Echolocation Performance of the Vampire Bat (Desmodus rotundus)

The neotropical vampire bats (Desmodus rotundus) echolocate using ultrasonic pulses like those of the Latin American phyllostomatid bats. In this paper the orally produced echolocation sounds of Desmodus are analysed and the performance of the echolocation system is studied in two-choice training experiments on two vampire bats. Ability to detect objects is relatively limited; both animals were capable of discerning the presence of a 1 cm wide metal strip at a distance of 50 cm, but they failed with 0.5 cm wide strips. The ultrasonic pulses produced at a distance of 50 cm appear to sample an area with a diameter of 2.5 to 3.0 cm (i. e., the solid angle tested with each pulse is 3° to 3° 40′ in extent).