Effects of γ-polyglutamic acid on grassland sandy soil properties and plant functional traits exposed to drought stress.

The current study provides field experimental data that support the use of γ-polyglutamic acid (γ-PGA) in drought stress and proposes its application in grassland management. We hypothesized that water treatment combined with PGA application to sandy soil would reduce drought stress in grasslands more effectively than watering alone. A randomized block design was used, with three replicate watering blocks (no watering, weekly watering, and monthly watering) and PGA treatments at four different concentrations (0%, 0.3%, 1%, and 2% PGA). The results showed that PGA acts as a biostimulant, alleviating the effects of stress in plants by: (1) increasing the availability of ions, especially K+, Zn2+, Mn2+, Fe2+/3+, Ca2+, and Mg2+, as well as N-NH4+, and N-NO3-, (2) elongating plant roots, (3) increasing the aboveground biomass, (4) improving the resprouting capacity of the dominant grass Nardus stricta, and (5) improving the regeneration of dicotyledons. In the case of meadows on sandy soils, the use of low PGA concentrations (0.3% or 1%) was the most beneficial for the availability of macro- and microelements and improving the functional traits of plants. Irrigation had a greater effect than using PGA only for the dicotyledon to monocotyledon ratio.

Characterization of a Tigecycline-, Linezolid- and Vancomycin-Resistant Clinical Enteroccoccus faecium Isolate, Carrying vanA and vanB Genes.

INTRODUCTION: Increasing incidence of Enterococcus faecium resistant to key antimicrobials used in therapy of hospitalized patients is a worrisome phenomenon observed worldwide. Our aim was to characterize a tigecycline-, linezolid- and vancomycin-resistant E. faecium isolate with the vanA and vanB genes, originating from a hematoma of a patient hospitalized in an intensive care unit in Poland. METHODS: Antimicrobial susceptibility (a broad panel) was tested using gradient tests with predefined antibiotic concentrations. The complete genome sequence was obtained from a mixed assembly of Illumina MiSeq and Oxford Nanopore's MinION reads. The genome was analyzed with appropriate tools available at the Center for Genomic Epidemiology, PubMLST and GenBank. Transferability of oxazolidinone, tigecycline and vancomycin resistance genes was investigated by conjugation, followed by PCR screen of transconjugants for antimicrobial resistance genes and plasmid rep genes characteristic for the donor and genomic sequencing of selected transconjugants. RESULTS: The isolate was resistant to most antimicrobials tested; susceptibility to daptomycin, erythromycin and chloramphenicol was significantly reduced, and only oritavancin retained the full activity. The isolate represented sequence type 18 (ST18) and carried vanA, vanB, poxtA, fexB, tet(L), tet(M), aac(6')-aph(2''), ant(6)-Ia and ant(6')-Ii. The vanA, poxtA and tet(M) genes located on ~ 40-kb plasmids were transferable by conjugation yielding transconjugants resistant to vancomycin, linezolid and tigecycline. The substitutions in LiaS, putative histidine kinase, SulP, putative sulfate transporter, RpoB and RpoC were potential determinants of an elevated daptomycin MIC. Comparative analyses of the studied isolate with E. faecium isolates from other countries revealed its similarity to ST18 isolates from Ireland and Uganda from human infections. CONCLUSIONS: We provide the detailed characteristics of the genomic determinants of antimicrobial resistance of a clinical E. faecium demonstrating the concomitant presence of both vanA and vanB and resistance to vancomycin, linezolid, tigecycline and several other compounds and decreased daptomycin susceptibility. This isolate is a striking example of an accumulation of resistance determinants involving various mechanisms by a single hospital strain.

Hydrocarbon Degradation and Microbial Survival Improvement in Response to γ-Polyglutamic Acid Application.

To improve the environmental sustainability of cleanup activities of contaminated sites there is a need to develop technologies that minimize soil and habitat disturbances. Cleanup technologies, such as bioremediation, are based on biological products and processes, and they are important for the future of our planet. We studied the potential of γ-poly glutamic acid (PGA) as a natural component of biofilm produced by Bacillus sp. to be used for the decomposition of petroleum products, such as heavy naphtha (N), lubricating oil (O), and grease (G). The study aimed to assess the impact of the use of different concentrations of PGA on the degradation process of various fractions of petroleum hydrocarbons (PH) and its effect on bacterial population growth in harsh conditions of PH contamination. In laboratory conditions, four treatments of PGA with each of the petroleum products (N, O, and G) were tested: PGA0 (reference), PGA1 (1% PGA), PGA1B (1% PGA with Bacillus licheniformis), and PGA10 (10% PGA). After 7, 28, 56, and 112 days of the experiment, the percentage yield extraction, hydrocarbon mass loss, geochemical ratios, pH, electrical conductivity, and microorganisms survival were determined. We observed an increase in PH removal, reflected as a higher amount of extraction yield (growing with time and reaching about 11% in G) and loss of hydrocarbon mass (about 4% in O and G) in all treatments of the PGA compared to the reference. The positive degradation impact was intensive until around day 60. The PH removal stimulation by PGA was also reflected by changes in the values of geochemical ratios, which indicated that the highest rate of degradation was at the initial stage of the process. In general, for the stimulation of PH removal, using a lower (1%) concentration of PGA resulted in better performance than a higher concentration (10%). The PH removal facilitated by PGA is related to the anionic homopoliamid structure of the molecule and its action as a surfactant, which leads to the formation of micelles and the gradual release of PH absorbed in the zeolite carrier. Moreover, the protective properties of PGA against the extinction of bacteria under high concentrations of PH were identified. Generally, the γ-PGA biopolymer helps to degrade the hydrocarbon pollutants and stabilize the environment suitable for microbial degraders development.

Evolution of tunnels in α/ß-hydrolase fold proteins-What can we learn from studying epoxide hydrolases?

The evolutionary variability of a protein's residues is highly dependent on protein region and function. Solvent-exposed residues, excluding those at interaction interfaces, are more variable than buried residues whereas active site residues are considered to be conserved. The abovementioned rules apply also to α/ß-hydrolase fold proteins-one of the oldest and the biggest superfamily of enzymes with buried active sites equipped with tunnels linking the reaction site with the exterior. We selected soluble epoxide hydrolases as representative of this family to conduct the first systematic study on the evolution of tunnels. We hypothesised that tunnels are lined by mostly conserved residues, and are equipped with a number of specific variable residues that are able to respond to evolutionary pressure. The hypothesis was confirmed, and we suggested a general and detailed way of the tunnels' evolution analysis based on entropy values calculated for tunnels' residues. We also found three different cases of entropy distribution among tunnel-lining residues. These observations can be applied for protein reengineering mimicking the natural evolution process. We propose a 'perforation' mechanism for new tunnels design via the merging of internal cavities or protein surface perforation. Based on the literature data, such a strategy of new tunnel design could significantly improve the enzyme's performance and can be applied widely for enzymes with buried active sites.

Evaluation of the Effectiveness of the Biopreparation in Combination with the Polymer γ-PGA for the Biodegradation of Petroleum Contaminants in Soil.

Biodegradation is a method of effectively removing petroleum hydrocarbons from the natural environment. This research focuses on the biodegradation of aliphatic hydrocarbons, monoaromatic hydrocarbons such as benzene, toluene, ethylbenzene, and all three xylene isomers (BTEX) and polycyclic aromatic hydrocarbons (PAHs) as a result of soil inoculation with a biopreparation A1 based on autochthonous microorganisms and a biopreparation A1 with the addition of γ-PGA. The research used biopreparation A1 made of the following strains: Dietzia sp. IN133, Gordonia sp. IN138 Mycolicibacterium frederiksbergense IN53, Rhodococcus erythropolis IN119, Rhodococcus sp. IN136 and Pseudomonas sp. IN132. The experiments were carried out in laboratory conditions (microbiological tests, respirometric tests, and in semi-technical conditions (ex-situ prism method). The biodegradation efficiency was assessed on the basis of respirometric tests, chromatographic analyses and toxicological tests. As a result of inoculation of AB soil with the biopreparation A1 within 6 months, a reduction of total petroleum hydrocarbons (TPH) (66.03%), BTEX (80.08%) and PAHs (38.86%) was achieved and its toxicity was reduced. Inoculation of AB soil with the biopreparation A1 with the addition of γ-PGA reduced the concentration of TPH, BTEX and PAHs by 79.21%, 90.19%, and 51.18%, respectively, and reduced its toxicity. The conducted research has shown that the addition of γ-PGA affects the efficiency of the biodegradation process of petroleum pollutants, increasing the degree of TPH biodegradation by 13.18%, BTEX by 10.11% and PAHs by 12.32% compared to pure biopreparation A1.

Simple Selection Procedure to Distinguish between Static and Flexible Loops.

Loops are the most variable and unorganized elements of the secondary structure of proteins. Their ability to shift their shape can play a role in the binding of small ligands, enzymatic catalysis, or protein-protein interactions. Due to the loop flexibility, the positions of their residues in solved structures show the largest B-factors, or in a worst-case scenario can be unknown. Based on the loops' movements' timeline, they can be divided into slow (static) and fast (flexible). Although most of the loops that are missing in experimental structures belong to the flexible loops group, the computational tools for loop reconstruction use a set of static loop conformations to predict the missing part of the structure and evaluate the model. We believe that these two loop types can adopt different conformations and that using scoring functions appropriate for static loops is not sufficient for flexible loops. We showed that common model evaluation methods, are insufficient in the case of flexible solvent-exposed loops. Instead, we recommend using the potential energy to evaluate such loop models. We provide a novel model selection method based on a set of geometrical parameters to distinguish between flexible and static loops without the use of molecular dynamics simulations. We have also pointed out the importance of water network and interactions with the solvent for the flexible loop modeling.

Hydromorphologically-driven variability of thermal and oxygen conditions at the block ramp hydraulic structure: The Porebianka River, Polish Carpathians.

Growing anthropopressure in mountain streams aimed at limiting erosion and flood protection often caused adverse effects on the natural environment. In recent years, great attention has been paid to the restoration and conservation of natural habitats in mountain streams using environmentally friendly solutions such as the Block Ramp (BR) Hydraulic Structures. In this study we investigated the factors responsible for spatial variability in thermal and oxygen conditions at the single BR structure in the growing season, and the relation between water temperature and dissolved oxygen (DO) concentration. This has been done by measurements of hydraulic characteristics along with physicochemical properties of water, such as water temperature and DO concentration, at two different discharges. The redundancy analysis has been applied in order to describe the relationships among hydraulic parameters and physicochemical variables, and extract potential sources of water temperature and DO variability within the BR hydraulic structure. Results have shown that DO and water temperature distributions within the BR hydraulic structure depend on discharge conditions and are associated with the submergence of the block ramp. The highest heterogeneity in hydraulic, DO and water temperature conditions occurs at low flow and is associated with the presence of crevices between protruding cobbles at the block ramp. The lowest variability, in turn, occurs at high discharge, when the block ramp is completely submerged. The results indicated that thermal and oxygen conditions within the BR hydraulic structure are independent of hydraulic parameters at low flow. Moreover, the relation between DO concentration and water temperature is positive at low flow indicating potential impact of biological processes. On the contrary, at high discharge both, the DO concentrations and water temperature within the BR structure, depend on bed shear velocity and maximum Reynolds number.

Riparian ground beetles in gravel bed rivers: validation of Invertebrate Bankfull Assessment method.

Bankfull Assessment Method (IBA method) applies riverine invertebrate community of ground beetles as a biotic indicator of bankfull variation in a dynamic river. IBA index is calculated as a proportion of small sized individuals (1-6 mm) to large size individuals (18-24 mm) of ground beetles in given sample site. The aim of the study was to validate the IBA bankfull discharge method with 135 carabid assemblages collected in gravel-bed rivers of various modifications of channel morphology in the Polish Carpathians. Constrained canonical correspondence analysis revealed that biotic factors such as IBA, plant cover and plant height described most of the variation of small and large sized species abundance. The IBA index variation confirmed earlier expectation of the mean IBA values. Mean value of the index for the lowest bench is about 5, for the middle bench about 2 and it never exceeds 1 for the highest bench. The bankfull calculated using morphometric methods (Wolmann) was located between the first and second bench on each river. Values of the IBA for the lowest bench depend significantly on channel incision whereas modifications of vegetation affect the values for all three benches. The study presents high value of IBA method in estimating bankfull discharge which is considered to be important supporting flow predicting morphological changes responsible for river channel shape with direct connection to living organisms occupying river banks as well as to river environmental quality. Proposed simple method could be important for local watershed managers for prediction of bank embankments level (e.g. maximum level of rip-rap or gabions) or for maximum level of low head hydraulic structures when they are necessary in terms of river engineering works but when they are still built as environmental friendly hydraulic structures.

Eleutherococcus Species Cultivated in Europe: A New Source of Compounds with Antiacetylcholinesterase, Antihyaluronidase, Anti-DPPH, and Cytotoxic Activities.

Secondary metabolites of the roots of Eleutherococcus spp. cultivated in Poland, or the bioactivity, are not fully known. The 75% methanol extracts of five Eleutherococcus spp. (E. senticosus, E. divaricatus, E. sessiliflorus, E. gracilistylus, and E. henryi) were examined for the content of polyphenols and phenolic acids as well as for antiacetylcholinesterase, antihyaluronidase, anti-DPPH∗, and cytotoxic activities. The richest in polyphenols were the roots of E. henryi (10.4 mg/g DW), while in flavonoids the roots of E. divaricatus (6.5 mg/g DW). The richest in phenolic acids occurred the roots of E. henryi [protocatechuic acid (1865 µg/g DE), caffeic acid (244 µg/g DE), and p-coumaric and ferulic acids (55 µg/g DE)]. The highest inhibition of AChE was observed for E. gracilistylus and E. sessiliflorus (32%), at the concentration of 100 µg/0.19 mL of the reaction mixture, while that of Hyal for the roots of E. henryi (40.7%), at the concentration of 100 µg/0.16 mL of the reaction mixture. Among five species tested, the E. henryi extract exhibited the strongest HL-60 cell line growth's inhibition (IC50 270 µg/mL). The extracts reduced DPPH∗ in a time-dependent mode, at the concentration of 0.8 mg/mL. After 90 min from 14.7 to 26.2%, DPPH∗was reduced. A phytochemical composition and activity of the Eleutherococcus species, cultivated in Poland, are still under research; however, on the basis of the results obtained, it may be concluded that they may become a source of phytochemicals and be useful for Europe's citizens.

Effects of Chronic Metal Exposure on the Morphology of Beetles Species Representing Different Ecological Niches.

To test effects of metal contamination on beetles morphology, specimens from species representing herbivores (Strophosoma capitatum), carnivores (Carabus arcensis and C. violaceus) and detritivores (Anoplotrupes stercorosus) were collected from an area polluted with zinc, lead and cadmium and from a control site. Both the length and width of elytra and pronotum were compared. Females of all species from the polluted area were smaller than those from the control site with the average difference of 2.7% (range 0.7%-6%). In contrast, males responded less consistently among species: A. stercorosus showed lower size of elytra and pronotum at the polluted area, while in C. arcensis only the elytra length and pronotum width were smaller. C. violaceus males exhibited smaller elytra length and pronotum length and width at the polluted area. In contrast, no differences between the two sites were found for S. capitatum males. Sex differences may originate from different energy investment strategies in females and males related to the reproduction needs. Even if the observed differences in body size were small, the smaller body size in females of all studied species, irrespectively of the guild, is striking and may indicate on lower fitness of a range of species inhabiting metal polluted areas.

Species Relationships in the Genus Bryodaemon (Coleoptera: Curculionidae).

Establishing reliable taxonomy and phylogeny of similar, evolutionarily young species is among the greatest challenges in biology. Clearly the best approach is to use a combination of informative traits, including molecular markers and morphometric measurements. The objective of this study was to verify the taxonomy and phylogeny of four morphologically similar Carpathian species of Bryodaemon Podlussany, 1998 (Coleoptera: Curculionidae). Species relationships were studied using three molecular markers: two nuclear (ITS-2 and EF1-α) and one mitochondrial (COI, barcoding marker). We also took morphometric measurements of 35 taxonomically derived characteristics of body parts and genital apparatus. The potential presence of apomorphic features also was determined. We then compared our results with data concerning the ecology and geography of previously studied species. Our analyses confirmed the monophyly ofthis group and established a phylogeny for the genus. We propose that B. hanakii is the earliest derived species, based on morphometric measurements, apomorphies and the EF-lα phylogeny. The pattern ofnucleotide variation in this marker also indicates that B. rozneri and B. boroveci are the youngest species. This hypothesis is consistent with geographical ranges and ecological preferences of Carpathian Bryodaemon species. We also considered an alternative hypothesis based on the COI gene tree which indicated that B. rozneri was the oldest species. However, this arrangement is inconsistent with our morphological data.

Orthosomella lipae sp. n. (Microsporidia) a parasite of the weevil, Liophloeus lentus Germar, 1824 (Coleoptera: Curculionidae).

A new microsporidium, Orthosomella lipae sp. n., was isolated from the outer ovariole sheath, trophic chambers, oocytes, somatic tissues and eggs of adults of the weevil, Liophloeus lentus Germar, 1824 (Coleoptera: Curculionidae) from southern Poland. Morphological and life cycle characteristics revealed using light and electron microscopy, place this new species within the Unikaryonidae. However, the 16S rDNA phylogeny indicates that it is associated with the genus Orthosomella.

[Occurrence and susceptibility to antibiotics of carbapenem-resistant Pseudomonas aeruginosa strains between 1998 and 2009]. / Wystepowanie oraz lekowrazliwosc opornych na karbapenemy szczepów Pseudomonas aeruginosa izolowanych z materialu klinicznego w latach 1998-2009.

P. aeruginosa rods are dangerous pathogens mainly responsible for nosocomial infections of different localization. Resistance to carbapenems, observed among them, is a serious threat due to ability to be transmitted between bacterial species. The aim of our study was to retrospectively evaluate the frequency of isolation and susceptibility to antibiotics of imipenem- and meropenem-resistant P. aeruginosa strains isolated between 1998 and 2009 from patients of University Hospital No 1 of dr A. Jurasz in Bydgoszcz. Study shows increasing number of isolation that type of strains from 19 in 1998 to 144 in 2009. Among all isolated P. aeruginosa strains majority was obtained from patients of the Intensive Care Units, Rehabilitation and Surgery Clinics. Examined strains of P. aeruginosa rods were mainly isolated from urine (20.5%), bronchoalveolar lavage (17.7%) and wound swabs (14.5%) samples. The isolates demonstrated frequently resistance to carbenicillin (> or 66.7%), ticarcillin (> or = 72.7%) and cefotaxime (> or = 75.6%). The lowest rate of resistant strains was observed in case of ceftazidime (< or = 68.8%), aztreonam (< or = 47.4%) and colistin (< or = 1.7%) suggesting the highest activity of that antimicrobials against infections caused by examined strains.