Examination of the Virulence of Actinobacillus pleuropneumoniae Serovar 16 in Pigs.

Different virulence variants of A. pleuropneumoniae are involved in the etiology of porcine pleuropneumonia. The purpose of the present trial was examination of the virulence of the Actinobacillus pleuropneumoniae A-85/14 strain, the type strain of serovar 16, in an animal challenge experiment. Thirty 12-week-old piglets seronegative for A. pleuropneumoniae were allocated into three trial groups each of 10 animals, and they were infected intranasally with 106, 107, or 108 colony forming units (cfu) of the strain, respectively. Clinical signs were recorded twice a day, and the animals were euthanized 6 days after the infection. Typical clinical signs and postmortem lesions of porcine pleuropneumonia were seen in the animals of each trial group; however, they were generally mild, and no significant differences could be seen between the three groups. Even 106 colony forming units of A. pleuropneumoniae A-85/14 strain could induce clinical signs and lesions. Based on these results, the type strain of serovar 16 of A. pleuropneumoniae must be regarded as a typical pathogenic strain of the species.

Subcycle surface electron emission driven by strong-field terahertz waveforms.

The advent of intense terahertz (THz) sources opened a new era when the demonstration of the acceleration and manipulation of free electrons by THz pulses became within reach. THz-field-driven electron emission was predicted to be confined to a single burst due to the single-cycle waveform. Here we demonstrate the confinement of single-cycle THz-waveform-driven electron emission to one of the two half cycles from a solid surface emitter. Either the leading or the trailing half cycle was active, controlled by reversing the field polarity. THz-driven single-burst surface electron emission sources, which do not rely on field-enhancement structures, will impact the development of THz-powered electron acceleration and manipulation devices, all-THz compact electron sources, THz waveguides and telecommunication, THz-field-based measurement techniques and solid-state devices.

Contrasting response of microeukaryotic and bacterial communities to the interplay of seasonality and local stressors in shallow soda lakes.

Seasonal environmental variation is a leading driver of microbial planktonic community assembly and interactions. However, departures from usual seasonal trends are often reported. To understand the role of local stressors in modifying seasonal succession, we sampled fortnightly, throughout three seasons, five nearby shallow soda lakes exposed to identical seasonal and meteorological changes. We characterised their microeukaryotic and bacterial communities by amplicon sequencing of the 16S and 18S rRNA gene, respectively. Biological interactions were inferred by analyses of synchronous and time-shifted interaction networks, and the keystone taxa of the communities were topologically identified. The lakes showed similar succession patterns during the study period with spring being characterised by the relevance of trophic interactions and a certain level of community stability followed by a more dynamic and variable summer-autumn period. Adaptation to general seasonal changes happened through shared core microbiome of the lakes. Stochastic events such as desiccation disrupted common network attributes and introduced shifts from the prevalent seasonal trajectory. Our results demonstrated that, despite being extreme and highly variable habitats, shallow soda lakes exhibit certain similarities in the seasonality of their planktonic communities, yet local stressors such as droughts instigate deviations from prevalent trends to a greater extent for microeukaryotic than for bacterial communities.

Habitat distribution of the genus Belliella in continental waters and the description of Belliella alkalica sp. nov., Belliella calami sp. nov. and Belliella filtrata sp. nov.

The genus Belliella belongs to the family Cyclobacteriaceae (order Cytophagales, phylum Bacteroidota) and harbours aerobic chemoheterotrophic bacteria. Members of this genus were isolated from various aquatic habitats, and our analysis based on global amplicon sequencing data revealed that their relative abundance can reach up to 5-10 % of the bacterioplankton in soda lakes and pans. Although a remarkable fraction of the most frequent genotypes that we identified from continental aquatic habitats is still uncultured, five new alkaliphilic Belliella strains were characterized in detail in this study, which were isolated from three different soda lakes and pans of the Carpathian Basin (Hungary). Cells of all strains were Gram-stain-negative, obligate aerobic, rod-shaped, non-motile and non-spore-forming. The isolates were oxidase- and catalase-positive, red-coloured, but did not contain flexirubin-type pigments; they formed bright red colonies that were circular, smooth and convex. Their major isoprenoid quinone was MK-7 and the predominant fatty acids were iso-C15 : 0, iso-C17 : 0 3-OH and summed feature 3 containing C16 : 1 ω6c and/or C16 : 1 ω7c. The polar lipid profiles contained phosphatidylethanolamine, an unidentified aminophospholipid, an unidentified glycolipid, and several unidentified lipids and aminolipids. Based on whole-genome sequences, the DNA G+C content was 37.0, 37.1 and 37.8 mol % for strains R4-6T, DMA-N-10aT and U6F3T, respectively. The distinction of three new species was confirmed by in silico genomic comparison. Orthologous average nucleotide identity (<85.4 %) and digital DNA-DNA hybridization values (<38.9 %) supported phenotypic, chemotaxonomic and 16S rRNA gene sequence data and, therefore, the following three novel species are proposed: Belliella alkalica sp. nov. (represented by strains R4-6T=DSM 111903T=JCM 34281T=UCCCB122T and S4-10), Belliella calami sp. nov. (DMA-N-10aT=DSM 107340T=JCM 34280T=UCCCB121T) and Belliella filtrata sp. nov. (U6F3T=DSM 111904T=JCM 34282T=UCCCB123T and U6F1). Emended descriptions of species Belliella aquatica, Belliella baltica, Belliella buryatensis, Belliella kenyensis and Belliella pelovolcani are also presented.

Spectral and Redox Properties of a Recombinant Mouse Cytochrome b561 Protein Suggest Transmembrane Electron Transfer Function.

Cytochrome b561 proteins (CYB561s) are integral membrane proteins with six trans-membrane domains, two heme-b redox centers, one on each side of the host membrane. The major characteristics of these proteins are their ascorbate reducibility and trans-membrane electron transferring capability. More than one CYB561 can be found in a wide range of animal and plant phyla and they are localized in membranes different from the membranes participating in bioenergization. Two homologous proteins, both in humans and rodents, are thought to participate-via yet unidentified way-in cancer pathology. The recombinant forms of the human tumor suppressor 101F6 protein (Hs_CYB561D2) and its mouse ortholog (Mm_CYB561D2) have already been studied in some detail. However, nothing has yet been published about the physical-chemical properties of their homologues (Hs_CYB561D1 in humans and Mm_CYB561D1 in mice). In this paper we present optical, redox and structural properties of the recombinant Mm_CYB561D1, obtained based on various spectroscopic methods and homology modeling. The results are discussed in comparison to similar properties of the other members of the CYB561 protein family.

Environmental changes associated with drying climate are expected to affect functional groups of pro- and microeukaryotes differently in temporary saline waters.

Temporary ponds are among the most sensitive aquatic habitats to climate change. Their microbial communities have crucial roles in food webs and biogeochemical cycling, yet how their communities are assembled along environmental gradients is still understudied. This study aimed to reveal the environmental drivers of diversity (OTU-based richness, evenness, and phylogenetic diversity) and community composition from a network of saline temporary ponds, soda pans, in two consecutive spring seasons characterized by contrasting weather conditions. We used DNA-based molecular methods to investigate microbial community composition. We tested the effect of environmental variables on the diversity of prokaryotic (Bacteria, Cyanobacteria) and microeukaryotic functional groups (ciliates, heterotrophic flagellates and nanoflagellates, fungi, phytoplankton) within and across the years. Conductivity and the concentration of total suspended solids and phosphorus were the most important environmental variables affecting diversity patterns in all functional groups. Environmental conditions were harsher and they also had a stronger impact on community composition in the dry spring. Our results imply that these conditions, which are becoming more frequent with climate change, have a negative effect on microbial diversity in temporary saline ponds. This eventually might translate into community-level shifts across trophic groups with changing local conditions with implications for ecosystem functioning.

Effectiveness of the Entomopathogenic Fungal Species Metarhizium anisopliae Strain NCAIM 362 Treatments against Soil Inhabiting Melolontha melolontha Larvae in Sweet Potato (Ipomoea batatas L.).

The effect of fungal entomopathogen M. anisopliae strain NCAIM 362 against M. melolontha larvae in sweet potato was tested under open field conditions when crop management included compost supply and soil cover (agro-foil or agro-textile). Additionally, the effect of M. anisopliae same strain against M. melolontha was compared with the effect of α-cypermethrin under greenhouse conditions. Soil microbial community using Illumina sequencing and soil biological activity were tested as possible parameter influencing M. anisopliae effect. According to the results, compost supply and textile cover may enhance the effectiveness of M. anisopliae under open field conditions, while no effect of fungal treatment was detected under greenhouse conditions. Even if soil parameters (chemical composition, bacterial, and biological activity) were identical, the effect of α-cypermethrin against M. melolontha larvae was significant: lower ratio of larval survival and less damaged tubers were detected after the chemical treatment. Our results suggest that M. anisopliae strain NCAIM 362 is not effective to control M. melolontha larvae, further pieces of research are needed to test other species of the Metarhizium genus to find an effective agent for sustainable pest control in sweet potato.

Single-cycle scalable terahertz pulse source in reflection geometry.

A tilted-pulse-front pumped terahertz pulse source is proposed for the generation of extremely high field single-cycle terahertz pulses. The very simple and compact source consists of a single crystal slab having a blazed reflection grating grooved in its back surface. Its further important advantages are the energy scalability and the symmetric THz beam profile. Generation of ∼50 MV/cm focused field with 10.8 mJ terahertz pulse energy is predicted for a 7 cm diameter LiNbO 3 crystal, if the pump pulse is of 870 mJ energy, 1030 nm central wavelength and 1 ps pulse duration. Such sources can decisively promote the realization of THz driven electron and proton accelerators and open the way for a new generation concept of terahertz pulses having extreme high field.

Siculibacillus lacustris gen. nov., sp. nov., a new rosette-forming bacterium isolated from a freshwater crater lake (Lake St. Ana, Romania).

A new aerobic alphaproteobacterium, strain SA-279T, was isolated from a water sample of a crater lake. The 16S rRNA gene sequence analysis revealed that strain SA-279T formed a distinct lineage within the family Ancalomicrobiaceae and shared the highest pairwise similarity values with Pinisolibacterravus E9T (96.4 %) and Ancalomicrobiumadetum NBRC 102456T (94.2 %). Cells of strain SA-279T were rod-shaped, motile, oxidase and catalase positive, and capable of forming rosettes. Its predominant fatty acids were C18 : 1ω7c (69.0 %) and C16 : 1ω7c (22.7 %), the major respiratory quinone was Q-10, and the main polar lipids were phosphatidylethanolamine, phosphatidylmonomethylethanolamine, phosphatidylcholine, phosphatidylglycerol, an unidentified aminophospholipid and an unidentified lipid. The G+C content of the genomic DNA of strain SA-279T was 69.2 mol%. On the basis of the phenotypic, chemotaxonomic and molecular data, strain SA-279T is considered to represent a new genus and species within the family Ancalomicrobiaceae, for which the name Siculibacillus lacustris gen. nov., sp. nov. is proposed. The type strain is SA-279T (=DSM 29840T=JCM 31761T).

Plasmon-plasmon coupling probed by ultrafast, strong-field photoemission with <7 Å sensitivity.

The coupling of propagating surface plasmon waves and localized plasmon oscillations in nanostructures is an essential phenomenon determining electromagnetic field enhancement on the nanoscale. Here, we use our recently developed ultrafast photoemission near-field probing technique to investigate the fundamental question of plasmon-plasmon coupling and its effect on large field enhancement factors. By measuring and analyzing plasmon field enhancement values at different nanostructured surfaces, we can separate the contributions from propagating and localized plasmons. When resonance conditions are met, a significant field enhancement factor can be attributed to the generation of localized plasmons on surface nanostructures, acting as dipole sources resonantly driven by the propagating plasmon field. Our plasmon-plasmon coupling results can contribute directly to applications in surface-enhanced Raman scattering (SERS) and the development of plasmonic sensors and nanostructured photocathodes.

Numerical investigation of a scalable setup for efficient terahertz generation using a segmented tilted-pulse-front excitation.

A hybrid-type terahertz pulse source is proposed for high energy terahertz pulse generation. It is the combination of the conventional tilted-pulse-front setup and a transmission stair-step echelon-faced nonlinear crystal with a period falling in the hundred-micrometer range. The most important advantage of the setup is the possibility of using plane parallel nonlinear optical crystal for producing good-quality, symmetric terahertz beam. Another advantage of the proposed setup is the significant reduction of imaging errors, which is important in the case of wide pump beams that are used in high energy experiments. A one dimensional model was developed for determining the terahertz generation efficiency, and it was used for quantitative comparison between the proposed new hybrid setup and previously introduced terahertz sources. With lithium niobate nonlinear material, calculations predict an approximately ten-fold increase in the efficiency of the presently described hybrid terahertz pulse source with respect to that of the earlier proposed setup, which utilizes a reflective stair-step echelon and a prism shaped nonlinear optical crystal. By using pump pulses of 50 mJ pulse energy, 500 fs pulse length and 8 mm beam spot radius, approximately 1% conversion efficiency and 0.5 mJ terahertz pulse energy can be reached with the newly proposed setup.

Identification of Noncompetitive Inhibitors of Cytosolic 5'-Nucleotidase II Using a Fragment-Based Approach.

We used a combined approach based on fragment-based drug design (FBDD) and in silico methods to design potential inhibitors of the cytosolic 5'-nucleotidase II (cN-II), which has been recognized as an important therapeutic target in hematological cancers. Two subgroups of small compounds (including adenine and biaryl moieties) were identified as cN-II binders and a fragment growing strategy guided by molecular docking was considered. Five compounds induced a strong inhibition of the 5'-nucleotidase activity in vitro, and the most potent ones were characterized as noncompetitive inhibitors. Biological evaluation in cancer cell lines showed synergic effect with selected anticancer drugs. Structural studies using X-ray crystallography lead to the identification of new binding sites for two derivatives and of a new crystal form showing important domain swapping. Altogether, the strategy developed herein allowed identifying new original noncompetitive inhibitors against cN-II that act in a synergistic manner with well-known antitumoral agents.

Structure-activity relationships of ß-hydroxyphosphonate nucleoside analogues as cytosolic 5'-nucleotidase II potential inhibitors: synthesis, in vitro evaluation and molecular modeling studies.

The cytosolic 5'-nucleotidase II (cN-II) has been proposed as an attractive molecular target for the development of novel drugs circumventing resistance to cytotoxic nucleoside analogues currently used for treating leukemia and other malignant hemopathies. In the present work, synthesis of ß-hydroxyphosphonate nucleoside analogues incorporating modifications either on the sugar residue or the nucleobase, and their in vitro evaluation towards the purified enzyme were carried out in order to determine their potency towards the inhibition of cN-II. In addition to the biochemical investigations, molecular modeling studies revealed important structural features for binding affinities towards the target enzyme.

Identification and characterization of inhibitors of cytoplasmic 5'-nucleotidase cN-II issued from virtual screening.

Clinical and preclinical observations have lead to the hypothesis that 5'-nucleotidase cN-II could constitute a therapeutic target in oncology, either per se or to increase the activity of cytotoxic nucleoside analogs. To identify potential cN-II inhibitors, we performed in silico screening of freely available chemical databases, in vitro enzymatic assays with recombinant cN-II, soaking experiments with crystals of truncated cN-II as well as biological evaluation of selected compounds, alone or in combination with cytotoxic nucleoside analogs, on cancer cells. The top ranked compounds from virtual screening included an anthraquinone derivative (AdiS) that were shown to block the enzyme activity with a K(i) of 2.0mM. Soaking experiments performed with crystals of truncated cN-II allowed to obtain crystallographic data at a resolution of 2.9 Å and indicating interaction between AdiS and F354/I152 situated in the effector site 1 of cN-II. In addition, this compound exhibited different levels of cytotoxicity in vitro on several cancer cell lines and increased the induction of apoptosis in RL cells incubated with 0.5 or 1.5 µM cladribine, 0.05 µM clofarabine or 30 µM fludarabine. Finally, AdiS showed synergy with cladribine and additivity with clofarabine. This study showed that virtual screening is a useful tool for the identification of potent cN-II inhibitors, and our biological results indicated interesting activity for one lead compound that can be further developed as therapeutics.

Effects of PACAP in UV-A radiation-induced retinal degeneration models in rats.

The retina is constantly exposed to ultraviolet (UV) light with different wavelengths, which may lead to chronic UV-induced retinal injury. In our previous studies, we have shown the protective effects of pituitary adenylate cyclase activating polypeptide (PACAP) in toxic and ischemic retinal injuries. The aim of the present study was to investigate the effects of PACAP in UV-A-induced retinal lesion. We used diffuse UV-A radiation (315-400 nm) to induce acute retinal damage over a short period of exposure. Using standard histological (morphological and morphometrical) analysis, we assessed the actions of intravitreal PACAP (100 pmol/5 µl) treatment on acute UV-A-induced retinal damage. We measured the thickness of nuclear and plexiform layers as well as the number of cells in the outer nuclear and inner nuclear layers and in the ganglion cell layer. Outer limiting membrane-inner limiting membrane distances in the cross-section of the retina were also examined. Our results show that UV-A light-induced retinal damage led to severe degeneration in the photoreceptor layer, and in the outer and inner nuclear layers. Alteration in the plexiform layers was also observed. We found that post-irradiation PACAP treatment significantly attenuated the UV-A-induced retinal damage. Our results provide the basis for future clinical application of PACAP treatment in retinal degeneration and may have clinical implications in several ophthalmic diseases.

Lipase hydration state in the gas phase: sorption isotherm measurements and inverse gas chromatography.

The adsorption of water and substrate on immobilized Candida antarctica lipase B was studied by performing adsorption isotherm measurements and using inverse gas chromatography (IGC). Water adsorption isotherm of the immobilized enzyme showed singular profile absorption incompatible with the Brunauer-Emmet-Teller model, probably due to the hydrophobic nature of the support, leading to very low interactions with water. IGC allowed determining the evolution with water thermodynamic activity (a(W)) of both dispersive surface energies and acidity and basicity constants of immobilized enzyme. These results showed that water molecules progressively covered immobilized enzyme, when increasing a(W), leading to a saturation of polar groups above a(W) 0.1 and full coverage of the surface above a(W) 0.25. IGC also enabled relevant experiments to investigate the behavior of substrates under a(W) that they will experience, in a competitive situation with water. Results indicated that substrates had to displace water molecules in order to adsorb on the enzyme from a(W) values ranging from 0.1 to 0.2, depending on the substrate. As the conditions used for these adsorption studies resemble the ones of the continuous enzymatic solid/gas reactor, in which activity and selectivity of the lipase were extensively studied, it was possible to link adsorption results with particular effects of water on enzyme properties.

Lipase-catalyzed regioselective monoacetylation of unsymmetrical 1,5-primary diols.

Lipase B from Candida antarctica (CALB) has been selected as the most suitable enzyme to catalyze the regioselective monoacetylation of 1,5-diol isoprostane intermediate, using vinyl acetate as an acyl transfer reagent in THF. We next applied this reaction on linear 2-substituted, 2,2'-disubstituted-1,5-pentanediols, and cyclic 2,3-disubstituted-1,5-pentanediols. To rationalize the regioselectivity observed, molecular docking simulations were performed.

Aggregation of the 636 nm emitting monomeric protochlorophyllide form into flash-photoactive, oligomeric 644 and 655 nm emitting forms in vitro.

Artificial formation of flash-photoactive oligomeric protochlorophyllide complexes was found in etiolated pea (Pisum sativum L. cv. Zsuzsi) epicotyl homogenates containing glycerol (40% v/v) and sucrose (40% m/v). The 77 K fluorescence emission spectra indicated that the ratio of the 644 and 655 nm emitting forms to the 636 nm form increased during 3 to 5-day incubation in the dark at -14 degrees C. Electron micrographs showed the presence of well-organized prolamellar bodies in the homogenates. The same phenomena were found when the homogenates were frozen into liquid nitrogen and thawed to room temperature in several cycles. Similar treatments of intact epicotyl pieces caused significant membrane destructions. In homogenates, the in vitro produced 644 and 655 nm emitting protochlorophyllide forms were flash-photoactive; the extent of phototransformation increased compared to that in native epicotyls. The newly appeared 692 nm chlorophyllide band showed a blue shift (similar to the Shibata shift in leaves), however this process took place only partially due to the effect of the isolation medium. These results prove that the in vitro accumulated 644 and 655 nm protochlorophyllide forms were produced from the flash-photoactive 636 nm emitting monomeric NADPH:protochlorophyllide oxidoreductase units via aggregation, in connection with structure stabilization properties of glycerol and sucrose.

Theory of spectroscopic devices.

A fundamental formalism featuring the common working principle of different spectroscopic devices is introduced. General formulas for angular dispersion, free spectral range, and spectral resolution are deduced from both the impulse response function and the spatial transmission function of the device, based on the assumption that these functions can be written up as the product of a finite width, real-aperture function, and a complex periodic function. The method will also be shown to work in specific cases.