Aerobic glucose metabolism of Saccharomyces kluyveri: growth, metabolite production, and quantification of metabolic fluxes.

The growth and product formation of Saccharomyces kluyveri was characterized in aerobic batch cultivation on glucose. At these conditions it was found that ethyl acetate was a major overflow metabolite in S. kluyveri. During the exponential-growth phase on glucose ethyl acetate was produced at a constant specific rate of 0.12 g ethyl acetate per g dry weight per hour. The aerobic glucose metabolism in S. kluyveri was found to be less fermentative than in S. cerevisiae, as illustrated by the comparably low yield of ethanol on glucose (0.08 +/- 0.02 g/g), and high yield of biomass on glucose (0.29 +/- 0.01 g/g). The glucose metabolism of S. kluyveri was further characterized by the new and powerful techniques of metabolic network analysis. Flux distributions in the central carbon metabolism were estimated for respiro-fermentative growth in aerobic batch cultivation on glucose and respiratory growth in aerobic glucose-limited continuous cultivation. It was found that in S. kluyveri the flux into the pentose phosphate pathway was 18.8 mmole per 100 mmole glucose consumed during respiratory growth in aerobic glucose-limited continuous cultivation. Such a low flux into the pentose phosphate pathway cannot provide the cell with enough NADPH for biomass formation which is why the remaining NADPH will have to be provided by another pathway. During batch cultivation of S. kluyveri the tricarboxylic acid cycle was working as a cycle with a considerable flux, that is in sharp contrast to what has previously been observed in S. cerevisiae at the same growth conditions, where the tricarboxylic acid cycle operates as two branches. This indicates that the respiratory system was not significantly repressed in S. kluyveri during batch cultivation on glucose.     

Two new species of seahorses (Syngnathidae, Hippocampus) from the Middle Miocene (Sarmatian) Coprolitic Horizon in Tunjice Hills, Slovenia: The oldest fossil record of seahorses

Two new and the only known extinct seahorse species Hippocampus sarmaticus and Hippocampus slovenicus are described from the Middle Miocene beds (Lower Sarmatian) in Slovenia, representing the oldest known fossil record of seahorses. Hippocampus sarmaticus was most similar to the extant seahorse species Hippocampus trimaculatus, while H. slovenicus can be most easily compared to the extant pygmy seahorses H. bargibanti, H. denise, and H. colemani. These Sarmatian seahorses lived among seagrasses and macroalgae in the temperate shallow costal waters of the western part of the Central Paratethys Sea.     

Superior Performance of Microporous Aluminophosphate with LTA Topology in Solar‐Energy Storage and Heat Reallocation

Hydrophilic porous materials are recognized as very promising materials for water‐sorption‐based energy storage and transformation. In this study, a porous, zeolite‐like aluminophosphate with LTA (Linde Type A) topology is inspected as an energy‐storage material. The study is motivated by the material's high predicted pore volume. According to sorption and calorimetric tests, the aluminophosphate outperforms all other zeolite‐like and metal‐organic porous materials tested so far. It adsorbs water in an extremely narrow relative‐pressure interval (0.10 < p /p ₀ < 0.15) and exhibits superior water uptake (0.42 g g^?1) and energy‐storage capacity (527 kW h m^?3). It also shows remarkable cycling stability; after 40 cycles of adsorption/desorption its capacity drops by less than 2%. Desorption temperature for this material, which is one of crucial parameters in applications, is lower from desorption temperatures of other tested materials by 10–15 °C. Furthermore, its heat‐pump performance is very high, allowing efficient cooling in demanding conditions (with cooling power up to 350 kW h m^?3 even at 30 °C temperature difference between evaporator and environment). On the microscopic scale, sorption mechanism in AlPO₄‐LTA is elucidated by X‐ray diffraction, nuclear magnetic resonance measurements, and first‐principles calculations. In this aluminophosphate, energy is stored predominately in hydrogen‐bonded network of water molecules within the pores.     

Quantitative agar-invasion assay

A new method for quantification of yeast invasion into the agar medium was developed. Classical agar-invasion assays have been the methods of choice for determination of yeast invasion, but their main disadvantage is the lack of quantification. Our new Quantitative yeast agar-invasion test allows for quantitative measurements and enables sorting strains by their degree of invasiveness. The invasion abilities were measured for 10 clinical and non-clinical Saccharomyces cerevisiae strains and a strain of Candida albicans. Finally, the correlation between the degrees of strains invasiveness and their reported virulence was observed, proposing our assay as a method for quick determination of yeast virulence potential.     

Acetylcholinesterase alterations reveal the fitness cost of mutations conferring insecticide resistance

Background

Insecticide resistance is now common in insects due to the frequent use of chemicals to control them, which provides a useful tool to study the adaptation of eukaryotic genome to new environments. Although numerous potential mutations may provide high level of resistance, only few alleles are found in insect natural populations. Then, we hypothesized that only alleles linked to the highest fitness in the absence of insecticide are selected.

Results

To obtain information on the origin of the fitness of resistant alleles, we studied Drosophila melanogaster acetylcholinesterase, the target of organophosphate and carbamate insecticides. We produced in vitro 15 possible proteins resulting from the combination of the four most frequent mutations and we tested their catalytic activity and enzymatic stability. Mutations affected deacetylation of the enzyme, decreasing or increasing its catalytic efficiency and all mutations diminished the stability of the enzyme. Combination of mutations result to an additive alteration.

Conclusion

Our findings suggest that the alteration of activity and stability of acetylcholinesterase are at the origin of the fitness cost associated with mutations providing resistance. Magnitude of the alterations was related to the allelic frequency in Drosophila populations suggesting that the fitness cost is the main driving force for the maintenance of resistant alleles in insecticide free conditions.

     

Yeast beta-alanine synthase shares a structural scaffold and origin with dizinc-dependent exopeptidases

beta-Alanine synthase (beta AS) is the final enzyme of the reductive pyrimidine catabolic pathway, which is responsible for the breakdown of pyrimidine bases, including several anticancer drugs. In eukaryotes, beta ASs belong to two subfamilies, which exhibit a low degree of sequence similarity. We determined the structure of beta AS from Saccharomyces kluyveri to a resolution of 2.7 A. The subunit of the homodimeric enzyme consists of two domains: a larger catalytic domain with a dizinc metal center, which represents the active site of beta AS, and a smaller domain mediating the majority of the intersubunit contacts. Both domains exhibit a mixed alpha/beta-topology. Surprisingly, the observed high structural homology to a family of dizinc-dependent exopeptidases suggests that these two enzyme groups have a common origin. Alterations in the ligand composition of the metal-binding site can be explained as adjustments to the catalysis of a different reaction, the hydrolysis of an N-carbamyl bond by beta AS compared with the hydrolysis of a peptide bond by exopeptidases. In contrast, there is no resemblance to the three-dimensional structure of the functionally closely related N-carbamyl-d-amino acid amidohydrolases. Based on comparative structural analysis and observed deviations in the backbone conformations of the eight copies of the subunit in the asymmetric unit, we suggest that conformational changes occur during each catalytic cycle.     

Further development of the cassette-based pYC plasmid system by incorporation of the dominant hph, nat and AUR1-C gene markers and the lacZ reporter system

Dominant selection markers encoding hygromycin B phosphotransferase (hph), nourseothricin N-acetyltransferase (nat) and a mutant inositol phosphoceramide synthase (AUR1-C) were all incorporated into the pYC yeast plasmid vector system, thus expanding this system with possible alternatives to the use of G418 resistance. We found the markers to be of use not only in standard laboratory strains of Saccharomyces cerevisiae but also in an industrial strain of S. carlsbergensis (syn. of S. pastorianus) brewing yeast as well as in Saccharomyces kluyveri. As the pYC system contains means of counter-selection for plasmid loss and loop-out of integrated plasmids, it now provides ample opportunities for genetic manipulation of industrial and non-conventional yeasts when the URA3 marker and FOA counter-selection is not an option. Furthermore, the lacZ system for analyzing gene expression was included in the system.     

Selective radiography of 10B distribution in organs using cold and thermal neutron beams

The investigation of boron biodistribution by neutron induced autoradiography was performed using a CR-39 etched track detector. Calibration samples, made of boronated chicken liver, and freeze-dried mouse tissue samples were irradiated both with thermal and cold neutrons. Digital images were made on the basis of track densities, with the selection of tracks due to 10B(n, alpha)7Li reaction. A comparison of results obtained both with thermal and cold neutrons demonstrated that it is possible to obtain equivalent results with both neutron sources, although the background noise due to recoil protons from the fast neutrons of the thermal neutron source is 15 % higher than that of the cold neutron source. 10B concentrations in the range of 0.5 ppm to 150 ppm were determined, with a standard deviation of 13 % and 8 % for the mouse tissue samples and calibration samples, respectively.     

Examination of fragment dose contribution in heavy ion radiotherapy

Heavy-ion radiotherapy is an efficient method for the treatment of deep-seated tumors, because the stopping of ions in a tissue delivers the maximal absorbed dose to the tumor-affected areas with minimal damage to the healthy tissues. However, heavy ions can undergo nuclear reactions, giving products with lower Z-values and hence a longer range in the tissue. This causes a dose increase beyond the mean range of the primary beam. The contribution of such reaction products was examined in an experiment where a stack of tissue-like targets interleaved with CR-39 etched track detectors (ETD) was irradiated with heavy ions. The analysis was performed using a recently developed technique of trajectory tracing, which enables the spectroscopy of fragments with different Z-values.