Honeybee Colony Vibrational Measurements to Highlight the Brood Cycle

Insect pollination is of great importance to crop production worldwide and honey bees are amongst its chief facilitators. Because of the decline of managed colonies, the use of sensor technology is growing in popularity and it is of interest to develop new methods which can more accurately and less invasively assess honey bee colony status. Our approach is to use accelerometers to measure vibrations in order to provide information on colony activity and development. The accelerometers provide amplitude and frequency information which is recorded every three minutes and analysed for night time only. Vibrational data were validated by comparison to visual inspection data, particularly the brood development. We show a strong correlation between vibrational amplitude data and the brood cycle in the vicinity of the sensor. We have further explored the minimum data that is required, when frequency information is also included, to accurately predict the current point in the brood cycle. Such a technique should enable beekeepers to reduce the frequency with which visual inspections are required, reducing the stress this places on the colony and saving the beekeeper time.     

Mechanisms of ligand transfer by the hepatic tocopherol transfer protein

alpha-Tocopherol is a member of the vitamin E family that functions as the principal fat-soluble antioxidant in vertebrates. Body-wide distribution of tocopherol is regulated by the hepatic alpha-tocopherol transfer protein (alphaTTP), which stimulates secretion of the vitamin from hepatocytes to circulating lipoproteins. This biological activity of alphaTTP is thought to stem from its ability to facilitate the transfer of vitamin E between membranes, but the mechanism by which the protein exerts this activity remains poorly understood. Using a fluorescence energy transfer methodology, we found that the rate of tocopherol transfer from lipid vesicles to alphaTTP increases with increasing alphaTTP concentration. This concentration dependence indicates that ligand transfer by alphaTTP involves direct protein-membrane interaction. In support of this notion, equilibrium analyses employing filtration, dual polarization interferometry, and tryptophan fluorescence demonstrated the presence of a stable alphaTTP-bilayer complex. The physical association of alphaTTP with membranes is markedly sensitive to the presence of vitamin E in the bilayer. Some naturally occurring mutations in alphaTTP that cause the hereditary disorder ataxia with vitamin E deficiency diminish the effect of tocopherol on the protein-membrane association, suggesting a possible mechanism for the accompanying pathology.     

High-performance thin-layer chromatography-bioautography for multiple antibiotic residues in cow's milk

An analytical method to identify and quantify multiple antibiotic residues (chloramphenicol, ampicillin, benzylpenicillin, dicloxacillin and erythromycin) in cow's milk by high-performance thin-layer chromatography (HPTLC) combined with bioautography was developed. The test microorganism used for bioautography was Bacillus subtilis ATCC 6633. Antibiotic residues were extracted with acetonitrile, fat eliminated with petroleum ether and residues isolated with dichloromethane The sensitivity of the method guarantees the detection of the above-mentioned antibiotics at levels below maximum residue limits (MRL) allowed for milk. Percentage recoveries ranged between 90 and 100%, with coefficients of variation between 7.2 and 21.3%. Some advantages of this methodology over thin-layer chromatography (TLC)/bioautography are also discussed.     

A conserved aspartate residue located at the extracellular end of the binding pocket controls cation interactions in brain glutamate transporters

In the brain, transporters of the major excitatory neurotransmitter glutamate remove their substrate from the synaptic cleft to allow optimal glutamatergic neurotransmission. Their transport cycle consists of two sequential translocation steps, namely cotransport of glutamic acid with three Na(+) ions, followed by countertransport of K(+). Recent studies, based on several crystal structures of the archeal homologue Glt(Ph), indicate that glutamate translocation occurs by an elevator-like mechanism. The resolution of these structures was not sufficiently high to unambiguously identify the sites of Na(+) binding, but functional and computational studies suggest some candidate sites. In the Glt(Ph) structure, a conserved aspartate residue (Asp-390) is located adjacent to a conserved tyrosine residue, previously shown to be a molecular determinant of ion selectivity in the brain glutamate transporter GLT-1. In this study, we characterize mutants of Asp-440 of the neuronal transporter EAAC1, which is the counterpart of Asp-390 of Glt(Ph). Except for substitution by glutamate, this residue is functionally irreplaceable. Using biochemical and electrophysiological approaches, we conclude that although D440E is intrinsically capable of net flux, this mutant behaves as an exchanger under physiological conditions, due to increased and decreased apparent affinities for Na(+) and K(+), respectively. Our present and previous data are compatible with the idea that the conserved tyrosine and aspartate residues, located at the external end of the binding pocket, may serve as a transient or stable cation binding site in the glutamate transporters.     

Using multilayer network analysis to explore the temporal dynamics of collective behavior

Social organisms often show collective behaviors such as group foraging or movement.Collective behaviors can emerge from interactions between group members and may depend on the behavior of key individuals.When social interactions change over time,collective behaviors may change because these behaviors emerge from interactions among individuals.Despite the importance of,and growing interest in,the temporal dynamics of social interactions,it is not clear how to quantify changes in interactions over time or measure their stability.Furthermore,the temporal scale at which we should observe changes in social networks to detect biologically meaningful changes is not always apparent.Here we use multilayer network analysis to quantify temporal dynamics of social networks of the social spider Stegodyphus dumicola and determine how these dynamics relate to individual and group behaviors.We found that social interactions changed over time at a constant rate.Variation in both network structure and the identity of a keystone individual was not related to the mean or variance of the collective prey attack speed.Individuals that maintained a large and stable number of connections,despite changes in network structure,were the boldest individuals in the group.Therefore,social interactions and boldness are linked across time,but group collective behavior is not influenced by the stability of the social network.Our work demonstrates that dynamic social networks can be modeled in a multilayer framework.This approach may reveal biologically important temporal changes to social structure in other systems.     

The gibberellin-induced, cysteine-rich protein GIP2 from Petunia hybrida exhibits in planta antioxidant activity

Numerous GAST-like genes have been identified in various plant species. All code for small proteins with a conserved C-terminal region in which 12 cysteines are located in exactly the same positions. We have previously identified five gibberellin (GA)-induced GAST1-like genes in petunia, GIP1-5. GIP2 is expressed in elongating zones, and its suppression in transgenic petunia plants inhibits stem elongation, suggesting a role for the protein in GA-induced cell growth. However, nothing is known about the biochemical activity of GIP2 or any other GAST-like protein. As all contain putative catalytic disulfide bonds (putative redox-active cysteines), we speculated that they might be involved in redox regulation. Expression analysis of GIP2, GIP4 and GIP5 revealed that they are induced by H(2)O(2). To study whether GIP2 modulates H(2)O(2) levels, we generated transgenic petunia plants expressing GIP2 under the regulation of the ubiquitous CaMV 35S promoter. The transgene reduced H(2)O(2) levels in leaves following wounding. It also reduced the levels of H(2)O(2) in guard cells following osmotic stress and ABA treatments, leading to the suppression of stomatal closure. In addition, the transgene promoted stem and corolla elongation. As reactive oxygen species (ROS) are involved in cell elongation, we suggest that GIP2 affects growth by regulating the levels of ROS. As all known GAST-like proteins contain putative redox-active cysteines, they may all act as antioxidants.     

Engineered vascular beds provide key signals to pancreatic hormone-producing cells

The mechanisms underlying early islet graft failure are not entirely clear, but are thought to involve ischemic injury due to delayed vascularization. We hypothesize that blood vessels play an active role in cell-cell communications supporting islet survival and engraftment. To test this hypothesis and to uncouple endothelial cell (EC)-generated signaling stimuli from their nutritional and gas exchange functions, we developed three dimensional (3D) endothelial vessel networks in engineered pancreatic tissues prepared from islets, fibroblasts and ECs. The tri-culture setup, seeded on highly porous biocompatible polymeric scaffolds closely mimics the natural anatomical context of pancreatic vasculature. Enhanced islet survival correlating with formation of functional tube-like endothelial vessels was demonstrated. Addition of foreskin fibroblasts to islet-endothelial cultures promoted tube-like structure formation, which further supported islet survival as well as insulin secretion. Gene expression profiles of EC growth factors, extracellular matrix (ECM), morphogenes and differentiation markers were significantly different in 2D versus 3D culture systems and were further modified upon addition of fibroblasts. Implantation of prevascularized islets into diabetic mice promoted survival, integration and function of the engrafted engineered tissue, supporting the suggested role of ECs in islet survival. These findings present potential strategies for preparation of transplantable islets with increased survival prospects.     

Base-CP proteasome can serve as a platform for stepwise lid formation

26S proteasome, a major regulatory protease in eukaryotes, consists of a 20S proteolytic core particle (CP) capped by a 19S regulatory particle (RP). The 19S RP is divisible into base and lid sub-complexes. Even within the lid, subunits have been demarcated into two modules: module 1 (Rpn5, Rpn6, Rpn8, Rpn9 and Rpn11), which interacts with both CP and base sub-complexes and module 2 (Rpn3, Rpn7, Rpn12 and Rpn15) that is attached mainly to module 1. We now show that suppression of RPN11 expression halted lid assembly yet enabled the base and 20S CP to pre-assemble and form a base-CP. A key role for Regulatory particle non-ATPase 11 (Rpn11) in bridging lid module 1 and module 2 subunits together is inferred from observing defective proteasomes in rpn11–m1, a mutant expressing a truncated form of Rpn11 and displaying mitochondrial phenotypes. An incomplete lid made up of five module 1 subunits attached to base-CP was identified in proteasomes isolated from this mutant. Re-introducing the C-terminal portion of Rpn11 enabled recruitment of missing module 2 subunits. In vitro, module 1 was reconstituted stepwise, initiated by Rpn11–Rpn8 heterodimerization. Upon recruitment of Rpn6, the module 1 intermediate was competent to lock into base-CP and reconstitute an incomplete 26S proteasome. Thus, base-CP can serve as a platform for gradual incorporation of lid, along a proteasome assembly pathway. Identification of proteasome intermediates and reconstitution of minimal functional units should clarify aspects of the inner workings of this machine and how multiple catalytic processes are synchronized within the 26S proteasome holoenzymes.     

Actigraphic estimates of sleep and the sleep-wake rhythm,and 6-sulfatoxymelatonin levels in healthy Dutch children

ABSTRACT

Sleep and the sleep-wake rhythm are essential for children’s health and well-being, yet reference values are lacking. This study therefore aimed to assess actigraphic estimates of sleep and the 24-h sleep-wake rhythm, as well as 6-sulfatoxymelatonin (aMT6s) levels in healthy children of different age groups. Additionally, relationships between the outcomes and sex, highest parental educational level (as an indication of socioeconomic status (SES)), and body-mass-index (BMI) were explored. In this cross-sectional study, healthy Dutch children (2–18 years) wore an actigraph (GT3x) for 7 consecutive days, collected first-morning void urine and completed a sleep log and sociodemographic questionnaire. Actigraphically estimated sleep variables were sleep onset latency (SOL), sleep efficiency (SE), total sleep time (TST), and wake after sleep onset (WASO). Non-parametric sleep-wake rhythm variables were intradaily variability (IV); interdaily stability (IS); the activity counts and timing of the least active 5-h period (L5counts and midpoint) and of the most active 10-h period (M10 counts and midpoint); and the relative amplitude (RA), i.e. the ratio of the difference and the sum of M10 and L5 counts. Finally, creatinine-corrected aMT6s levels were obtained by isotope dilution mass spectrometry. Effects of age group (preschool 2–5 years/school-aged 6–12 years/teenager 13–18 years), sex, highest parental educational level and BMI (Z-scores) were explored. Ninety-four children participated, equally divided across age groups (53% boys). Teenagers slept less, but more efficiently, than younger children, while their 24 h sleep-wake rhythm was the least stable and most fragmented (likely due to fragmentation of daytime activity). Additionally, aMT6s levels significantly declined over the age groups. Children from highly educated parents had lower sleep efficiency, but a more stable sleep-wake rhythm. Finally, sex or increase in BMI was not associated with any of the outcomes in this study. In conclusion, this study provides reference values of healthy children across different age groups and different sociodemographic factors. In the future, this information may help to better interpret outcomes in clinical populations.