Poly(2-hydroxyethyl methacrylate) for enzyme immobilization: impact on activity and stability of horseradish peroxidase

On the basis of their versatile structure and chemistry as well as tunable mechanical properties, polymer brushes are well-suited as supports for enzyme immobilization. However, a robust surface design is hindered by an inadequate understanding of the impact on activity from the coupling motif and enzyme distribution within the brush. Herein, horseradish peroxidase C (HRP C, 44 kDa), chosen as a model enzyme, was immobilized covalently through its lysine residues on a N-hydroxysuccinimidyl carbonate-activated poly(2-hydroxyethyl methacrylate) (PHEMA) brush grafted chemically onto a flat impenetrable surface. Up to a monolayer coverage of HRP C is achieved, where most of the HRP C resides at or near the brush-air interface. Molecular modeling shows that lysines 232 and 241 are the most probable binding sites, leading to an orientation of the immobilized HRP C that does not block the active pocket of the enzyme. Michaelis-Menten kinetics of the immobilized HRP C indicated little change in the K(m) (Michaelis constant) but a large decrease in the V(max) (maximum substrate conversion rate) and a correspondingly large decrease in the k(cat) (overall catalytic rate). This indicates a loss in the percentage of active enzymes. Given the relatively ideal geometry of the HRPC-PHEMA brush, the loss of activity is most likely due to structural changes in the enzyme arising from either secondary constraints imposed by the connectivity of the N-hydroxysuccinimidyl carbonate linking moiety or nonspecific interactions between HRP C and DSC-PHEMA. Therefore, a general enzyme-brush coupling motif must optimize reactive group density to balance binding with neutrality of surroundings.     

Geographic distribution modelling for ruminant liver flukes (Fasciola hepatica) in south-eastern Europe

Maximum entropy ecological niche modelling was utilised to predict the geographic range for fluke genotypes and haplotypes in south-eastern Europe, using the Maxent program. The lowest (0.832) and the highest (0.947) area under the curve values were observed in the models for the haplotypes CtCmt1 and CtCmt2.2, respectively. Precipitation and temperature contribute equally to model building of the genotypes based on the 28S rDNA gene. In regard to the mtDNA gene region, precipitation is the most important factor in modelling the CtCmt1 haplotype range, while temperature appears to be the most important factor in modelling the CtCmt2.1 and CtCmt2.2 haplotype ranges. The highest level of probability for the geographic distribution of Fasciola hepatica genotypes and haplotypes covered the regions of southern Bulgaria and central and northern Greece which contain a high concentration of potential ruminant hosts.     

Using regression trees to predict alpha diversity based upon geographical and habitat characteristics

Different environmental factors act as driving forces of diversity at different scales of analysis; and also the effect of one environmental factor changes as the scale of analysis changes. Most studies rely on multiple regression models, and such models tend to mix-up the effect of all factors and assume that factors effects are additive. We believe that the effect of environment on diversity should be characterized by a hierarchical structure with coarse scale factors, like geographical tropics to poles gradients, defining the envelope of possible diversity conditions, and other more local factors, like habitat structure, being responsible for the fine tuning of diversity. This structure is most efficiently modeled with regression trees. We show that for six habitat types in Greek protected areas regression tree models were able to describe plant species richness based upon environmental factors considerably more efficiently than multiple regression models. More importantly when the models were extrapolated to other sites in Greece, outside their domain, the differences between the predictive ability of the two approaches was magnified. The tree models picked up important ecological characteristics, and a hierarchical structure that used coarse scale factors, like latitude and longitude, for the coarse scale estimate of alpha diversity, and finer scale factors like fragmentation, for the fine-tuning of the estimation. Therefore, we advocate that the regression tree methodology is most appropriate for modeling the relationship between diversity and environmental factors, and the use of the classical regression approaches might be misleading.     

Retrospective Characterization of a Vaccine-Derived Poliovirus Type 1 Isolate from Sewage in Greece

Retrospective molecular and phenotypic characterization of a vaccine-derived poliovirus (VDPV) type 1 isolate (7/b/97) isolated from sewage in Athens, Greece, in 1997 is reported. VP1 sequencing of this isolate revealed 1.87% divergence from the VP1 region of reference strain Sabin 1, while further genomic characterization of isolate 7/b/97 revealed a recombination event in the nonstructural part of the genome between a vaccine strain and a nonvaccine strain probably belonging to Enterovirus species C. Amino acid substitutions commonly found in previous studies were identified in the capsid coding region of the isolate, while most of the attenuation and temperature sensitivity determinants were reverted. The ultimate source of isolate 7/b/97 is unknown. The recovery of such a highly divergent derivative of a vaccine strain emphasizes the need for urgent implementation of environmental surveillance as a supportive procedure in the polio surveillance system even in countries with high rates of OPV coverage in order to prevent cases or even outbreaks of poliomyelitis that otherwise would be inevitable.     

Effects of chilling stress on leaf morphology,anatomy, ultrastructure,gas exchange,and essential oils in the seasonally dimorphic plant Teucrium polium (Lamiaceae)

The Mediterranean region (and globally also other regions) is characterized by the presence of phryganic plants, i.e. subshrubs that grow under hot and arid environmental conditions. These plants are reported to be affected by summer drought stress. However, in the present study the phryganic plant Teucrium polium (mountain germander) appears to be affected by winter chilling stress rather than by summer drought stress in a specific area. Winter leaves of the plant are smaller and thicker compared to summer leaves, have more stomata and glandular hairs, and their chloroplasts are larger, more numerous, with voluminous starch grains. Moreover, epidermal and mesophyll cells of winter leaves contain in their vacuoles dark phenolics and calcium oxalate crystals. Summer leaves are devoid of vacuolar phenolics and their chloroplasts possess many large plastoglobuli. Leaf gas exchange parameters (photosynthesis, transpiration, stomatal conductance) are significantly higher in winter leaves. Concentrations of osmoprotectors (stress indicators) like proline and soluble sugars are similarly higher in winter leaves. Essential oil assessments showed a significantly higher oil yield of winter leaves compared to summer leaves. Percentages of the major oil components (linalool, terpinen-4-ol, germacrene D, and spathulenol) are remarkably higher in winter oils than in summer oils. In conclusion, low environmental temperatures (1–10 °C) appear to decisively influence the structure and function of winter leaves compared to summer leaves. Winter plants undergo chilling stress to which they respond by developing various mechanical and chemical defensive strategies.     

Survey of gastrointestinal parasitic infections of beef cattle in regions under Mediterranean weather in Greece

Gastrointestinal (GI) parasitic infections of beef cattle were investigated in 15 farms from 3 different regions of Greece, characterized by temperate Mediterranean climate, during two years. A total of 262 faecal samples were collected. Helminth eggs were detected in 42 (16%) samples. Strongyle-type eggs were found in 28 (10.7%) samples, Strongyloides spp. and Toxocara spp. eggs in 8 (3.1%) samples each, and Capillaria spp. and Moniezia spp. eggs in 1 (0.4%) sample each. Coccidian oocysts were found in 123 (46.9%) samples. A logistic regression model showed that the age of animals, the number of employees, and the region of animals were associated with coccidian infections. There was almost a four-fold increase in the risk of coccidian infections in calves less than 12 months old compared with animals that were more than 36 months old (p < 0.05). The risk of coccidian infections was 0.07, 0.18 and 0.27-fold lower for farms with 1, 2 and 3 employees respectively compared to farms with 4 employees; farms with less than 4 employees had a protective effect (p < 0.05 in all cases). Almost a four-fold increased risk of coccidian infections was shown for animals from Epirus compared with those from Thessaly (p < 0.05). These findings are useful to formulate appropriate control strategies for coccidian parasites of beef cattle in Greece and other areas with similar climatic conditions.     

The influence of Zn on signaling pathways and attachment of Mytilus galloprovincialis haemocytes to extracellular matrix proteins

The present study investigates the cytotoxic mechanisms induced by zinc (Zn) in haemocytes of mussel Mytilus galloprovincialis. Haemocytes play a key role in the immune defence of mussels. Micromolar concentration of Zn (50 microM) play an important role in the elevation of pHi and increase in Na+ influx in haemocytes. The observed effects were inhibited by the Na+/H+ exchanger (NHE) inhibitor, ethyl-N-isopropyl-amiloride (EIPA). Furthermore, our results showed that Zn caused an increase in O(-)(2) production that was reversed after NHE inhibition. Phorbol ester (PMA) caused a significant rise both in pHi and Na+ influx as well as in O(-)(2) production. These effects were reversed by calphostin C. Our results indicated that Zn also enhanced haemocyte attachment to both BSA and laminin which was reversed by EIPA and calphostin C. The enhancement of haemocytes attachment to both BSA and laminin after Zn suggests that it is likely to play a signal role in cytoskeleton-dependent process of cell growth and migration in mussel M. galloprovincialis haemocytes. We conclude that Zn induces a signaling pathway with the involvement of NHE, PKC, O(-)(2) and alpha1- and beta-adrenergic receptors.     

Towards a functional-structural plant model of cut-rose: simulation of light environment, light absorption, photosynthesis and interference with the plant structure

Background and Aims

The production system of cut-rose (Rosa × hybrida) involves a complex combination of plant material, management practice and environment. Plant structure is determined by bud break and shoot development while having an effect on local light climate. The aim of the present study is to cover selected aspects of the cut-rose system using functional–structural plant modelling (FSPM), in order to better understand processes contributing to produce quality and quantity.

Methods

The model describes the production system in three dimensions, including a virtual greenhouse environment with the crop, light sources (diffuse and direct sun light and lamps) and photosynthetically active radiation (PAR) sensors. The crop model is designed as a multiscaled FSPM with plant organs (axillary buds, leaves, internodes, flowers) as basic units, and local light interception and photosynthesis within each leaf. A Monte-Carlo light model was used to compute the local light climate for leaf photosynthesis, the latter described using a biochemical rate model.

Key Results

The model was able to reproduce PAR measurements taken at different canopy positions, different times of the day and different light regimes. Simulated incident and absorbed PAR as well as net assimilation rate in upright and bent shoots showed characteristic spatial and diurnal dynamics for different common cultivation scenarios.

Conclusions

The model of cut-rose presented allowed the creation of a range of initial structures thanks to interactive rules for pruning, cutting and bending. These static structures can be regarded as departure points for the dynamic simulation of production of flower canes. Furthermore, the model was able to predict local (per leaf) light absorption and photosynthesis. It can be used to investigate the physiology of ornamental plants, and provide support for the decisions of growers and consultants.     

Seroprevalence and risk factors associated with zoonotic parasitic infections in small ruminants in the Greek temperate environment

A cross-sectional serological study was carried out to screen the sheep and goat population of Thessaly, Greece for evidence of infection with Toxoplasma, Toxocara, Leishmania, and Echinococcus and to determine the risk factors related to herd characteristics, herd management practices, farmer status, and the bioclimatic variables associated with these zoonotic parasitic infections. A total of 540 sheep and goat serum samples were examined. The seroprevalence of infection in all examined animals was 24.5% for Toxoplasma, 32% for Toxocara, 0% for Leishmania and 85.9% for Echinococcus. The final logistic regression model showed that the species of small ruminant, herd size, anthelmintic treatment, class of anthelmintic treatment, grazing with other herds, educational level of farmer, elevation of farm location, and generalized land cover were associated with Toxoplasma gondii infections, while the species of small ruminant, farm type, anthelmintic treatment, class of anthelmintic treatment, rotation of grazing, age of farmer, elevation of farm location, and generalized land cover were associated with Toxocara canis infections. Antibodies to T. gondii were detected in 102 (28.3%) of 360 sheep and in 30 (16.8%) of 179 goats. Animals in small flocks (150–300 animals) had an approximately 0.42-fold lower risk of having positive cases of T. gondii among animals compared with large flocks (> 300 animals). Antibodies to T. canis were found in 155 (42.9%) of 361 sheep and 18 (10.1%) of 179 goats. The later finding constitutes the first report of seropositive goats to Toxocara. The risk of positivity for T. canis was 7.71-fold higher in sheep than in goats. Geographically, animals from plain areas had 2.9 and 2.01-fold higher risk of having positive cases of T. gondii and T. canis respectively. The significant bioclimatic variables (p < 0.05) associated with the occurrence locations of T. gondii infection were related to higher temperature, lower precipitation, and lower elevation compared to the absence locations of T. gondii. The significant bioclimatic variables (p < 0.05) associated with occurrence locations of T. canis infection were related to lower temperature and higher precipitation compared to absence locations of T. canis. These findings are useful to formulate appropriate control strategies for zoonotic parasites of sheep and goats in Greece and other areas with similar climatic conditions.