Cells on the run: shear-regulated integrin activation in leukocyte rolling and arrest on endothelial cells

The arrest of rolling leukocytes on various target vascular beds is mediated by specialized leukocyte integrins and their endothelial immunoglobulin superfamily (IgSF) ligands. These integrins are kept in largely inactive states and undergo in situ activation upon leukocyte-endothelial contact by both biochemical and mechanical signals from flow-derived shear forces. In vivo and in vitro studies suggest that leukocyte integrin activation involves conformational alterations through inside-out signaling followed by ligand-induced rearrangements accelerated by external forces. This activation process takes place within fractions of seconds by in situ signals transduced to the rolling leukocyte as it encounters specialized endothelial-displayed chemoattractants, collectively termed arrest chemokines. In neutrophils, selectin rolling engagements trigger intermediate affinity integrins to support reversible adhesions before chemokine-triggered arrest. Different leukocyte subsets appear to use different modalities of integrin activation during rolling and arrest at distinct endothelial sites.     

Reciprocal changes in calcification of the gastrolith and cuticle during the molt cycle of the red claw crayfish Cherax quadricarinatus

Mobilization of calcium during the molt cycle from the cuticle to transient calcium deposits is widely spread in crustaceans. The dynamics of calcium transport to transient calcium deposits called gastroliths and to the cuticle over the course of the molt cycle were studied in the crayfish Cherax quadricarinatus. In this species, calcium was deposited in the gastroliths during premolt and transported back to the cuticle during postmolt, shown by digital X-ray radiograph analysis. The predominant mineral in the crayfish is amorphous calcium carbonate embedded in an organic matrix composed mainly of chitin. Scanning electron micrographs of the cuticle during premolt showed that the endocuticle and parts of the exocuticle were the source of most of the labile calcium, while the epicuticle did not undergo degradation and remained mineralized throughout the molt cycle. The gastroliths are made of concentric layers of amorphous calcium carbonate intercalated between chitinous lamella. Measurements of pH and calcium levels during gastrolith deposition showed that calcium concentrations in the gastroliths, stomach, and muscle were about the same (10 to 11 mmol l(-1)). On the other hand, pH varied greatly, from 8.7+/-0.15 in the gastrolith cavity through 7.6+/-0.2 in muscle to 6.9+/-0.5 in the stomach.     

Characterization of the Novel Fusobacterium nucleatum Plasmid pKH9 and Evidence of an Addiction System

Fusobacterium nucleatum is an important oral anaerobic pathogen involved in periodontal and systemic infections. Studies of the molecular mechanisms involved in fusobacterial virulence and adhesion have been limited by lack of systems for efficient genetic manipulation. Plasmids were isolated from eight strains of F. nucleatum. The smallest plasmid, pKH9 (4,975 bp), was characterized and used to create new vectors for fusobacterial genetic manipulation. DNA sequence analysis of pKH9 revealed an open reading frame (ORF) encoding a putative autonomous rolling circle replication protein (Rep), an ORF predicted to encode a protein homologous to members of the FtsK/SpoIIIE cell division-DNA segregation protein family, and an operon encoding a putative toxin-antitoxin plasmid addiction system (txf-axf). Deletion analysis localized the pKH9 replication region in a 0.96-kbp fragment. The pKH9 rep gene is not present in this fragment, suggesting that pKH9 can replicate in fusobacteria independently of the Rep protein. A pKH9-based, compact Escherichia coli-F. nucleatum shuttle plasmid was constructed and found to be compatible with a previously described pFN1-based fusobacterial shuttle plasmid. Deletion of the pKH9 putative addiction system (txf-axf) reduced plasmid stability in fusobacteria, indicating its addiction properties and suggesting it to be the first plasmid addiction system described for fusobacteria. pKH9, its genetic elements, and its shuttle plasmid derivatives can serve as useful tools for investigating fusobacterial properties important in biofilm ecology and pathogenesis.     

Human quiescin-sulfhydryl oxidase, QSOX1: probing internal redox steps by mutagenesis

The flavoprotein quiescin-sulfhydryl oxidase (QSOX) rapidly inserts disulfide bonds into unfolded, reduced proteins with the concomitant reduction of oxygen to hydrogen peroxide. This study reports the first heterologous expression and enzymological characterization of a human QSOX1 isoform. Like QSOX isolated from avian egg white, recombinant HsQSOX1 is highly active toward reduced ribonuclease A (RNase) and dithiothreitol but shows a >100-fold lower k cat/ K m for reduced glutathione. Previous studies on avian QSOX led to a model in which reducing equivalents were proposed to relay through the enzyme from the first thioredoxin domain (C70-C73) to a distal disulfide (C509-C512), then across the dimer interface to the FAD-proximal disulfide (C449-C452), and finally to the FAD. The present work shows that, unlike the native avian enzyme, HsQSOX1 is monomeric. The recombinant expression system enabled construction of the first cysteine mutants for mechanistic dissection of this enzyme family. Activity assays with mutant HsQSOX1 indicated that the conserved distal C509-C512 disulfide is dispensable for the oxidation of reduced RNase or dithiothreitol. The four other cysteine residues chosen for mutagenesis, C70, C73, C449, and C452, are all crucial for efficient oxidation of reduced RNase. C452, of the proximal disulfide, is shown to be the charge-transfer donor to the flavin ring of QSOX, and its partner, C449, is expected to be the interchange thiol, forming a mixed disulfide with C70 in the thioredoxin domain. These data demonstrate that all the internal redox steps occur within the same polypeptide chain of mammalian QSOX and commence with a direct interaction between the reduced thioredoxin domain and the proximal disulfide of the Erv/ALR domain.     

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.     

Fatty Acid-binding Protein 5 (FABP5) Regulates Cognitive Function Both by Decreasing Anandamide Levels and by Activating the Nuclear Receptor Peroxisome Proliferator-activated Receptor β/δ (PPARβ/δ) in the Brain

Endocannabinoids modulate multiple behaviors, including learning and memory. We show that the endocannabinoid anandamide (AEA) can alter neuronal cell function both through its established role in activation of the G-protein-coupled receptor CB1, and by serving as a precursor for a potent agonist of the nuclear receptor PPARβ/δ, in turn up-regulating multiple cognition-associated genes. We show further that the fatty acid-binding protein FABP5 controls both of these functions in vivo. FABP5 both promotes the hydrolysis of AEA into arachidonic acid and thus reduces brain endocannabinoid levels, and directly shuttles arachidonic acid to the nucleus where it delivers it to PPARβ/δ, enabling its activation. In accordance, ablation of FABP5 in mice results in excess accumulation of AEA, abolishes PPARβ/δ activation in the brain, and markedly impairs hippocampus-based learning and memory. The data indicate that, by controlling anandamide disposition and activities, FABP5 plays a key role in regulating hippocampal cognitive function.     

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.