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.     

7-Methylguanosine monophosphate analogues with 5′-(1,2,3-triazoyl) moiety: Synthesis and evaluation as the inhibitors of cNIIIB nucleotidase

The hydrolysis of nucleoside 5′-monophosphates to the corresponding nucleosides and inorganic phosphate is catalysed by 5′-nucleotidases, thereby contributing to the control of endogenous nucleotide turnover and affecting the fate of exogenously delivered nucleotide- and nucleoside-derived therapeutics in cells. A recently identified nucleotidase cNIIIB shows preference towards 7-methylguanosine monophosphate (m⁷GMP) as a substrate, which suggests its potential involvement in mRNA degradation. However, the extent of biological functions and the significance of cNIIIB remains to be elucidated. Here, we synthesised a series of m⁷GMP analogues carrying a 1,2,3-triazole moiety at the 5′ position as the potential inhibitors of human cNIIIB. The compounds were synthesised by using the copper-catalysed azide-alkyne cycloaddition (CuAAC) between 5′-azido-5′-deoxy-7-methylguanosine and different phosphate or phosphonate derivatives carrying terminal alkyne. The analogues were evaluated as cNIIIB inhibitors using HPLC and malachite green assays, demonstrating that compound 1a, carrying a 1,2,3-triazoylphosphonate moiety, inhibits cNIIIB activity at micromolar concentrations (IC₅₀ 87.8?±?7.5?µM), while other analogues showed no activity. In addition, compound 1d was identified as an artifical substrate for ^HscNIIIB. Further characterization of inhibitor 1a revealed that it is poorly recognised by other m⁷G-binding proteins, eIF4E and DcpS, indicating its selectivity towards cNIIIB. The first inhibitor (1a) and unnatural substrate (1d) of cNIIIB, identified here, can be used as molecular probes for the elucidation of biological roles of cNIIIB, including the verification of its proposed function in mRNA metabolism.     

True deception during extra‐pair courtship feeding: cheating whiskered tern Chlidonias hybrida females perform better

Males of many bird species feed their mates during the pre‐incubation period. The food provisioned by males during these courtship feedings (CFs) represents the key source of energy for the female during egg formation. Non‐pair males may trade food for extra pair copulations (EPC) with females during extra pair courtship feeding (EPCF), while females may trade copulations for food with non‐pair males to obtain additional resources. Because EPCs can be costly to the females, they are expected to behave in ways that will deceive non‐pair males to obtain additional resources at no cost to themselves. We investigated EPCFs in whiskered terns Chlidonias hybrida breeding in food‐rich conditions, on carp ponds in southern Poland. Almost all CFs (n = 2751) took place during the female's fertile period and peaked just before clutch initiation. 10% of all CFs were performed by non‐pair males. Females tried to obtain food from the non‐pair male during 39% of EPCFs, by swindling (the female solicits a non‐social male for copulation and tries to swindle food with no cloacal contact) or by snatching (the female tries to take the gift without engaging in copulation). In the remaining 61% of EPCFs, females did not react or chased the visiting male away. The probability of a female's obtaining food during EPCF was much higher (0.69, 95% CI: 0.47–0.85) if she swindled rather than snatched (0.08, 95% CI: 0.02–0.22). Only 0.7% of EPCFs were followed by EPCs. The high availability of food in the study area allows males to perform frequent EPCFs, despite the very low probability of obtaining EPCs. This is the first time that ‘true deception’ during EPCFs has been reported in birds: swindling females obtain food from non‐pair males at no immediate detectable cost to themselves.     

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.     

Fluorescence-based sensors to monitor localization and functions of linear and k63-linked ubiquitin chains in cells

Ubiquitin chains modify a major subset of the proteome, but detection of ubiquitin signaling dynamics and localization is limited due to a lack of appropriate tools. Here, we employ ubiquitin-binding domain (UBD)-based fluorescent sensors to monitor linear and K63-linked chains in?vitro and in?vivo. We utilize the UBD in NEMO and ABIN (UBAN) for detection of linear chains, and RAP80 ubiquitin-interacting motif (UIM) and TAB2 Npl4 zinc finger (NZF) domains to detect K63 chains. Linear and K63 sensors decorated the ubiquitin coat surrounding cytosolic Salmonella during bacterial autophagy, whereas K63 sensors selectively monitored Parkin-induced mitophagy and DNA damage responses in fixed and living cells. In addition, linear and K63 sensors could be used to monitor endogenous signaling pathways, as demonstrated by their ability to differentially interfere with TNF- and IL-1-induced NF-κB pathway. We propose that UBD-based biosensors could serve as prototypes to track and trace other chain types and ubiquitin-like signals in?vivo.     

Light- and dark-induced action potentials in Physcomitrella patens

Glass microelectrodes were inserted into Physcomitrella patens gametophyte leaves and action potentials (APs) were recorded in response to sudden illumination as well as after darkening, i.e., when the dark-induced membrane depolarization crossed a threshold. Application of 5 mM La³⁺ (a calcium channel inhibitor), 10 mM TEA⁺ (a potassium channel inhibitor) and increased free Ca²⁺ resulted in a loss of excitability. Lack of Ca²⁺ in the external medium did not prevent APs from occurring. It was concluded that during light- dark-induced excitation of Physcomitrella patens, APs might rely upon calcium influxes from the intracellular compartments. APs were not blocked by the proton pump inhibitors (DES, DCCD), although the resting potential (RP) diminished significantly.Key words: action potential, calcium, moss, Physcomitrella patens, plant     

Osmosensing by WNK Kinases

With No Lysine (K) WNK kinases regulate electro-neutral cotransporters that are controlled by osmotic stress and chloride. We showed previously that autophosphorylation of WNK1 is inhibited by chloride, raising the possibility that WNKs are activated by osmotic stress. Here we demonstrate that unphosphorylated WNK isoforms 3 and 1 autophosphorylate in response to osmotic pressure in vitro, applied with the crowding agent polyethylene glycol (PEG)400 or osmolyte ethylene glycol (EG), and that this activation is opposed by chloride. Small angle x-ray scattering of WNK3 in the presence and absence of PEG400, static light scattering in EG, and crystallography of WNK1 were used to understand the mechanism. Osmosensing in WNK3 and WNK1 appears to occur through a conformational equilibrium between an inactive, unphosphorylated, chloride-binding dimer and an autophosphorylation-competent monomer. An improved structure of the inactive kinase domain of WNK1, and a comparison with the structure of a monophosphorylated form of WNK1, suggests that large cavities, greater hydration, and specific bound water may participate in the osmosensing mechanism. Our prior work showed that osmolytes have effects on the structure of phosphorylated WNK1, suggestive of multiple stages of osmotic regulation in WNKs.     

Animals in Iodine Deficiency or Sulfadimethoxine Models of Thyroid Damage Are Differently Affected by the Consumption of Brassica Sprouts

Biological Trace Element Research - The study was primarily aimed at investigating the effect of brassica sprout consumption, namely rutabaga (Brassica napus L. var. napobrassica) sprouts (R)...     

Seasonal dynamics of shoot biomass of dominant clonal herb species in an oak–hornbeam forest herb layer

Seasonal fluctuations of light availability, nutrient concentrations, and moisture affect plant population traits like density, standing biomass, and flowering. We analyzed seasonal changes of density and shoot biomass of the four most frequent herb species growing in an oak–hornbeam forest community, i.e., Anemone nemorosa, Ficaria verna, Galeobdolon luteum, and Galium odoratum. In 2010 and 2011 plant biomass was harvested from 7 to 10 randomly situated square sample plots (0.36 m²) in the homogenous oak–hornbeam forest community every week in the spring and every two weeks in the summer and autumn. The highest abundance of Anemone nemorosa reached over 1000 shoots per m², of Ficaria verna 459.5 shoots per m², of Galium odoratum 83.6 shoots per m², and of Galeobdolon luteum 98.4 shoots per m² (means for 2010 and 2011, based on all sample plots). We did not observe negative correlation between density and shoot biomass. Growth rates of vegetative shoot biomass amounted to 0.857 mg day^?1 for Anemone nemorosa, 0.467 mg day^?1 for Ficaria verna, 0.722 mg day^?1 for Galium odoratum, and 0.448 mg day^?1 for Galeobdolon luteum (means for 2010 and 2011). Spring ephemerals had much higher densities of shoots than summer-greens. Summer-greens reached higher biomass of individual shoots than spring ephemerals. Flowering shoots constituted only 4, 2, and 11% of all shoots for A. nemorosa, F. verna, and G. odoratum, respectively. More resource availability resulting in high shoot biomass did not translate to higher share of flowering shoots.