Hierarchical modeling of protein interactions

A novel approach to hierarchical peptide-protein and protein-protein docking is described and evaluated. Modeling procedure starts from a reduced space representation of proteins and peptides. Polypeptide chains are represented by strings of alpha-carbon beads restricted to a fine-mesh cubic lattice. Side chains are represented by up to two centers of interactions, corresponding to beta-carbons and the centers of mass of the remaining portions of the side groups, respectively. Additional pseudoatoms are located in the centers of the virtual bonds connecting consecutive alpha carbons. These pseudoatoms support a model of main-chain hydrogen bonds. Docking starts from a collection of random configurations of modeled molecules. Interacting molecules are flexible; however, higher accuracy models are obtained when the conformational freedom of one (the larger one) of the assembling molecules is limited by a set of weak distance restraints extracted from the experimental (or theoretically predicted) structures. Sampling is done by means of Replica Exchange Monte Carlo method. Afterwards, the set of obtained structures is subject to a hierarchical clustering. Then, the centroids of the resulting clusters are used as scaffolds for the reconstruction of the atomic details. Finally, the all-atom models are energy minimized and scored using classical tools of molecular mechanics. The method is tested on a set of macromolecular assemblies consisting of proteins and peptides. It is demonstrated that the proposed approach to the flexible docking could be successfully applied to prediction of protein-peptide and protein-protein interactions. The obtained models are almost always qualitatively correct, although usually of relatively low (or moderate) resolution. In spite of this limitation, the proposed method opens new possibilities of computational studies of macromolecular recognition and mechanisms of assembly of macromolecular complexes.     

Netted crop covers reduce honeybee foraging activity and colony strength in a mass flowering crop

The widespread use of protective covers in horticulture represents a novel landscape‐level change, presenting the challenges for crop pollination. Honeybees (Apis mellifera L) are pollinators of many crops, but their behavior can be affected by conditions under covers. To determine how netting crop covers can affect honeybee foraging dynamics, colony health, and pollination services, we assessed the performance of 52 nucleus honeybee colonies in five covered and six uncovered kiwifruit orchards. Colony strength was estimated pre‐ and postintroduction, and the foraging of individual bees (including pollen, nectar, and naïve foragers) was monitored in a subset of the hives fitted with RFID readers. Simultaneously, we evaluated pollination effectiveness by measuring flower visitation rates and the number of seeds produced after single honeybee visits. Honeybee colonies under cover exhibited both an acute loss of foragers and changes in the behavior of successful foragers. Under cover, bees were roughly three times less likely to return after their first trip outside the hive. Consequently, the number of adult bees in hives declined at a faster rate in these orchards, with colonies losing on average 1,057 ± 274 of their bees in under two weeks. Bees that did forage under cover completed fewer trips provisioning their colony, failing to reenter after a few short‐duration trips. These effects are likely to have implications for colony health and productivity. We also found that bee density (bees/thousand flowers) and visitation rates to flowers were lower under cover; however, we did not detect a resultant change in pollination. Our findings highlight the need for environment‐specific management techniques for pollinators. Improving honeybee orientation under covers and increasing our understanding of the effects of covers on bee nutrition and brood rearing should be primary objectives for maintaining colonies and potentially improving pollination in these systems.     

Association study of folate-related enzymes (MTHFR,MTR, MTRR) genetic variants with non-obstructive male infertility in a Polish population

Spermatogenesis is a process where an important contribution of genes involved in folate-mediated one-carbon metabolism is observed. The aim of the present study was to investigate the association between male infertility and the MTHFR (677C > T; 1298A > C), MTR (2756A > G) and MTRR (66A > G) polymorphisms in a Polish population. No significant differences in genotype or allele frequencies were detected between the groups of 284 infertile men and of 352 fertile controls. These results demonstrate that common polymorphisms in folate pathway genes are not major risk factors for non-obstructive male infertility in the Polish population.     

The influence of terpinen-4-ol and eucalyptol – The essential oil components - on fungi and plant sterol monolayers

Antifungal and herbicidal activity of terpenes, being the components of the essential oils, is directly related to the incorporation of these compounds into cellular membranes. Thus, the differences in the lipid composition of various pathogenic membranes may be the factor determining the activity of these molecules. One of the class of lipids, which form the membrane environment are sterols. The aim of this work was to compare the effect of two terpenes: terpinen-4-ol and eucalyptol on the monolayers formed by ergosterol and β – sitosterol, which are the components of fungi and plant membranes, respectively. The modifications in the sterol monolayer properties were investigated in the surface pressure-area measurements and penetration studies as well as in a micrometer scale (Brewster angle microscopy experiments) and in nanoscale (GIXD technique). It was evidenced that although at higher surface pressure the terpene molecules are in part removed from the interface, they are able to substantially modify the condensation, morphology and molecular organization of the sterol film. It was also found that the incorporation of terpenes into sterol films is comparable for both sterols, however, β – sitosterol monolayers properties are affected more strongly than ergosterol films. Finally, the analysis of the results of the studies performed on model membrane systems and the results of antimicrobial studies reported in literature, enabled us to suggest that the activity of terpenes depends on the membrane composition and that the sterol concentration may be important from the point of view of antifungal effect of terpinen-4-ol and eucalyptol.     

Local and systemic regulation of PSII efficiency in triticale infected by the hemibiotrophic pathogen Microdochium nivale

Microdochium nivale is a fungal pathogen that causes yield losses of cereals during winter. Cold hardening under light conditions induces genotype‐dependent resistance of a plant to infection. We aim to show how photosystem II (PSII) regulation contributes to plant resistance. Using mapping population of triticale doubled haploid lines, three M. nivale strains and different infection assays, we demonstrate that plants that maintain a higher maximum quantum efficiency of PSII show less leaf damage upon infection. The fungus can establish necrotrophic or biotrophic interactions with susceptible or resistant genotypes, respectively. It is suggested that local inhibition of photosynthesis during the infection of sensitive genotypes is not balanced by a supply of energy from the tissue surrounding the infected cells as efficiently as in resistant genotypes. Thus, defence is limited, which in turn results in extensive necrotic damage. Quantitative trait loci regions, involved in the control of both PSII functioning and resistance, were located on chromosomes 4 and 6, similar to a wide range of PSII‐ and resistance‐related genes. A meta‐analysis of microarray experiments showed that the expression of genes involved in the repair and de novo assembly of PSII was maintained at a stable level. However, to establish a favourable energy balance for defence, genes encoding PSII proteins resistant to oxidative degradation were downregulated to compensate for the upregulation of defence‐related pathways. Finally, we demonstrate that the structural and functional integrity of the plant is a factor required to meet the energy demand of infected cells, photosynthesis‐dependent systemic signalling and defence responses.     

The influence of fatty acids on model cholesterol/phospholipid membranes

The aim of this work was to verify the influence of the saturated (SFA) (stearic acid) and the unsaturated (UFA) (oleic and alpha-linolenic) fatty acids on model cholesterol/phospholipid membranes. The experiments were based on the Langmuir monolayer technique. Cholesterol and phospholipid were mixed in the molar ratio that corresponds to the proportion of these lipids in the majority of natural human membranes. Into the binary cholesterol/phospholipid monolayers, various amounts of fatty acids were incorporated. Our investigations were based on the analysis of the interactions between molecules in ternary (cholesterol/phospholipids/fatty acid) mixtures, however, also binary (cholesterol/fatty acid and phospholipids/fatty acid) mixed system were examined. It was concluded that the influence of the fatty acids on model cholesterol/phospholipid membrane is closely connected with the shape of the fatty acid molecule, resulting from the saturation degree of the hydrocarbon chain. It was found that the saturated fatty acid makes the model membrane more rigid, while the presence of unsaturated fatty acid increases its fluidity. The increasing amount of stearic acid gradually destabilizes model membrane, however, this effect is the weakest at low content of SFA in the mixed monolayer. Unsaturated fatty acids in a small proportion make the membrane thermodynamically more stable, while higher content of UFA decreases membrane stability. This explains low proportion of the free fatty acids to other lipids in natural membrane.     

The Knowledge of the Role of Papillomavirus—Related Head and Neck Pathologies among General Practitioners,Otolaryngologists and Trainees. A Survey-Based Study

Objectives

The aim of the survey was to introduce knowledge of HPV''s role in head and neck pathologies to general physicians (GPs), otorhinolaryngologists (ENTs) and newly graduated doctors, as well as to promote HPV-related diseases prevention.

Study Design

Cross-sectional study.

Methods

Self-designed questionnaire was sent to 2100 doctors. A total of 404 doctors, including 144 ENTs, 192 GPs and 68 trainees, responded.

Results

The majority of ENTs (86.8%) had contact with recurrent respiratory papillomatosis (RRP) and oropharyngeal cancers (OPCs) patients; in contrast, the majority of GPs (55.7%) did not (p = 0.00). The knowledge of HPV aetiology of cervical cancer versus OPCs and RRP was statistically higher. 7% of ENTs, 20% of GPs and 10% of trainees had not heard about HPV in oropharyngeal diseases. Women had greater knowledge than men. Both in the group of GPs and ENTs, 100% of respondents had heard about the impact of vaccination on the reduction of cervical cancer incidence. Only 39.11% of respondents had heard about the possibility of using vaccination against HPV in RRP—ENT doctors significantly more often than GPs and trainees (p = 0.00). Only 28.96% of physicians had heard about the potential value of HPV vaccination in preventing OPCs, including 44.44% of ENT doctors, 23.44% of GPs and 11.76% of trainees (p = 0.00). The doctors from district hospitals showed lower level of knowledge compared with clinicians (p = 0.04).

Conclusions

The different levels of knowledge and awareness of HPV issues highlight the need for targeted awareness strategies in Poland with implementation of HPV testing and vaccination. The information should be accessible especially to those with lower education levels: ENTs from small, provincial wards, GPs from cities of < 200 000 inhabitants and older physicians. The incorporation of HPV issues into the studies curriculum would be fruitful in terms of improving the knowledge of trainees.     

Dog Tear Film Proteome In-Depth Analysis

In this study, mass spectrometry was used to explore the canine tear proteome. Tear samples were obtained from six healthy dogs, and one-dimensional sodium dodecyl sulphate polyacrylamide gel electrophoresis (1D SDS-PAGE) was used as a first step to separate intact proteins into 17 bands. Each fraction was then trypsin digested and analysed by matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry (MALDI-TOF-MS/MS) to characterize the protein components in each fraction. In total, 125 tear proteins were identified, with MCA (Major Canine Allergen), Serum albumin, UPF0557 protein C10orf119 homolog, Collagen alpha-2(I) chain, Tyrosine -protein kinase Fer, Keratine type II cytoskeletal, Beta-crystallin B2, Interleukin-6 and Desmin occuring as the most confident ones with the highest scores. The results showed that the proteomic strategy used in this study was successful in the analysis of the dog tear proteome. To the best of our knowledge, this study is the first to report the comprehensive proteome profile of tears from healthy dogs by 1D SDS PAGE and MALDI-TOF. Data are available via ProteomeXchange with identifier PXD003124.     

Contribution to the Prediction of the Fold Code: Application to Immunoglobulin and Flavodoxin Cases

Background

Folding nucleus of globular proteins formation starts by the mutual interaction of a group of hydrophobic amino acids whose close contacts allow subsequent formation and stability of the 3D structure. These early steps can be predicted by simulation of the folding process through a Monte Carlo (MC) coarse grain model in a discrete space. We previously defined MIRs (Most Interacting Residues), as the set of residues presenting a large number of non-covalent neighbour interactions during such simulation. MIRs are good candidates to define the minimal number of residues giving rise to a given fold instead of another one, although their proportion is rather high, typically [15-20]% of the sequences. Having in mind experiments with two sequences of very high levels of sequence identity (up to 90%) but different folds, we combined the MIR method, which takes sequence as single input, with the “fuzzy oil drop” (FOD) model that requires a 3D structure, in order to estimate the residues coding for the fold. FOD assumes that a globular protein follows an idealised 3D Gaussian distribution of hydrophobicity density, with the maximum in the centre and minima at the surface of the “drop”. If the actual local density of hydrophobicity around a given amino acid is as high as the ideal one, then this amino acid is assigned to the core of the globular protein, and it is assumed to follow the FOD model. Therefore one obtains a distribution of the amino acids of a protein according to their agreement or rejection with the FOD model.

Results

We compared and combined MIR and FOD methods to define the minimal nucleus, or keystone, of two populated folds: immunoglobulin-like (Ig) and flavodoxins (Flav). The combination of these two approaches defines some positions both predicted as a MIR and assigned as accordant with the FOD model. It is shown here that for these two folds, the intersection of the predicted sets of residues significantly differs from random selection. It reduces the number of selected residues by each individual method and allows a reasonable agreement with experimentally determined key residues coding for the particular fold. In addition, the intersection of the two methods significantly increases the specificity of the prediction, providing a robust set of residues that constitute the folding nucleus.     

Steps towards flexible docking: modeling of three-dimensional structures of the nuclear receptors bound with peptide ligands mimicking co-activators' sequences

We developed a fully flexible docking method that uses a reduced lattice representation of protein molecules, adapted for modeling peptide–protein complexes. The CABS model (Carbon Alpha, Carbon Beta, Side Group) employed here, incorporates three pseudo-atoms per residue—C, Cβ and the center of the side group instead of full-atomic protein representation. Force field used by CABS was derived from statistical analysis of non-redundant database of protein structures. Application of our method included modeling of the complexes between various nuclear receptors (NRs) and peptide co-activators, for which three-dimensional structures are known. We tried to rebuild the native state of the complexes, starting from separated components. Accuracy of the best obtained models, calculated as coordinate root-mean-square deviation (cRMSD) between the target and the modeled structures, was under 1 Å, which is competitive with experimental methods, such as crystallography or NMR. Forthcoming modeling study should lead to better understanding of mechanisms of macromolecular assembly and will explain co-activators’ effects on receptors activity, especially on vitamin D receptor and other nuclear receptors.