The Effect of Old Age on the Free-Running Period of Circadian Rhythms in Rat

The free-running period is regarded to be an exclusive feature of the endogenous circadian clock. Changes during aging in the free-running period may therefore reflect age-related changes in the internal organization of this clock. However, the literature on alterations in the free-running period in aging is not unequivocal. In the present study, with various confounding factors kept to a minimum, it was found that the free-running periods for active wakefulness, body temperature, and drinking behavior were significantly shorter (by 12-17 min) in old than in young rats. In addition, it was found that the day-to-day stability of the different sleep states was reduced in old rats, whereas that of the drinking rhythm was enhanced. Transient cycles were not observed, nor were there any age-related differences in daily totals of the various sleep-wake states. The amplitudes of the circadian rhythms of active wakefulness, quiet sleep, and temperature were reduced, whereas those of paradoxical sleep and quiet wakefulness remained unchanged.     

Neogene origins and implied warmth tolerance of Amazon tree species

Tropical rain forest has been a persistent feature in South America for at least 55 million years. The future of the contemporary Amazon forest is uncertain, however, as the region is entering conditions with no past analogue, combining rapidly increasing air temperatures, high atmospheric carbon dioxide concentrations, possible extreme droughts, and extensive removal and modification by humans. Given the long‐term Cenozoic cooling trend, it is unknown whether Amazon forests can tolerate air temperature increases, with suggestions that lowland forests lack warm‐adapted taxa, leading to inevitable species losses. In response to this uncertainty, we posit a simple hypothesis: the older the age of a species prior to the Pleistocene, the warmer the climate it has previously survived, with Pliocene (2.6–5 Ma) and late‐Miocene (8–10 Ma) air temperature across Amazonia being similar to 2100 temperature projections under low and high carbon emission scenarios, respectively. Using comparative phylogeographic analyses, we show that 9 of 12 widespread Amazon tree species have Pliocene or earlier lineages (>2.6 Ma), with seven dating from the Miocene (>5.6 Ma) and three >8 Ma. The remarkably old age of these species suggest that Amazon forests passed through warmth similar to 2100 levels and that, in the absence of other major environmental changes, near‐term high temperature‐induced mass species extinction is unlikely.     

Forms and Functions of Meso and Micro-niches of Carbon within Soil Aggregates

Soil aggregates include sand/silt/clay, water, ion and organic matter contents combined with natural dry/wet (D/W) cycling alters both the formation and function of intra-aggregate pore continuity, connectivity, dead-end storage volumes, and tortuosity. Surface aggregates in the 0-5 cm depths of most soils experience from 34 to 57 D/W cycles that exceed differences in water contents >10%. Both the rates of drying or wetting, (intensity) and the D/W range of soil water contents (severity) alter the transport of water, C and N through micro and mesofaunal habitats among multiple size domains. This report identifies micro-niche locations of accumulating soil C within soil aggregate regions that may affect nematode residence sites and migration pathways. Recent advances in X-ray microtomography enable the examination of intact pore networks within soil aggregates at resolutions as small as 4 microns. Geostatistical and multi-fractal methods provide concise characteristics of pore spatial distributions within the aggregates and are useful for comparing these alterations among soils. Aggregates subjected to multiple D/W cycles developed greater spatial correlations that parallel increases in the (13)C sorption within aggregate interiors were compared with locations of soil microbial communities. Past research indicates microbial activities within the soil aggregate matrix are spatially heterogeneous due to complex pore geometries within aggregates. Illumination of the "blackbox" interiors of soil aggregates includes a discussion of natural and anthropogenic alterations of solution flow and carbon sequestration by soil aggregates containing biophysical gradients.     

Neurophysiological Effects of Sleep Deprivation in Healthy Adults,a Pilot Study

Total sleep deprivation (TSD) may induce fatigue, neurocognitive slowing and mood changes, which are partly compensated by stress regulating brain systems, resulting in altered dopamine and cortisol levels in order to stay awake if needed. These systems, however, have never been studied in concert. At baseline, after a regular night of sleep, and the next morning after TSD, 12 healthy subjects performed a semantic affective classification functional magnetic resonance imaging (fMRI) task, followed by a [¹¹C]raclopride positron emission tomography (PET) scan. Saliva cortisol levels were acquired at 7 time points during both days. Affective symptoms were measured using Beck Depression Inventory (BDI), Spielberger State Trait Anxiety Index (STAI) and visual analogue scales. After TSD, perceived energy levels, concentration, and speed of thought decreased significantly, whereas mood did not. During fMRI, response speed decreased for neutral words and positive targets, and accuracy decreased trendwise for neutral words and for positive targets with a negative distracter. Following TSD, processing of positive words was associated with increased left dorsolateral prefrontal activation. Processing of emotional words in general was associated with increased insular activity, whereas contrasting positive vs. negative words showed subthreshold increased activation in the (para)hippocampal area. Cortisol secretion was significantly lower after TSD. Decreased voxel-by-voxel [¹¹C]raclopride binding potential (BP_ND) was observed in left caudate. TSD induces widespread cognitive, neurophysiologic and endocrine changes in healthy adults, characterized by reduced cognitive functioning, despite increased regional brain activity. The blunted HPA-axis response together with altered [¹¹C]raclopride binding in the basal ganglia indicate that sustained wakefulness requires involvement of additional adaptive biological systems.     

Improved enantioselective conversion of styrene epoxides and meso-epoxides through epoxide hydrolases with a mutated nucleophile-flanking residue

In epoxide hydrolase from Agrobacterium radiobacter (EchA), phenylalanine 108 flanks the nucleophilic aspartate and forms part of the substrate-binding pocket. The influence of mutations at this position on the activity and enantioselectivity of the enzyme was investigated. Screening for improved enantioselectivity towards para-nitrophenyl glycidyl ether (pNPGE) using spectrophotometric progress curve analysis yielded five different mutants with 3- to 7-fold improved enantioselectivity. The increase in enantioselectivity was in most cases the result of an enhanced catalytic efficiency toward the preferred enantiomer. Several mutations at position F108 resulted in a higher activity toward cis-disubstituted meso-epoxides, which were converted to a single product enantiomer. Mutant F108C converted cis-2,3-epoxybutane to (2R,3R)-2,3-butanediol of >99% ee with a 7-fold improved activity, and mutant F108A hydrolyzed cyclohexene oxide to (1R,2R)-1,2-cyclohexanediol of >99% ee with a more than 150-fold higher activity than wild-type enzyme. It is concluded that single amino acid substitutions in the active site of epoxide hydrolase can result in enzyme variants with catalytic properties that are suitable for preparative scale production of (S)-epoxides and chiral vicinal diols in high yield and with excellent ee.     

Predicting the response of the deep-ocean microbiome to geochemical perturbations by hydrothermal vents

Submarine hydrothermal vents perturb the deep-ocean microbiome by injecting reduced chemical species into the water column that act as an energy source for chemosynthetic organisms. These systems thus provide excellent natural laboratories for studying the response of microbial communities to shifts in marine geochemistry. The present study explores the processes that regulate coupled microbial-geochemical dynamics in hydrothermal plumes by means of a novel mathematical model, which combines thermodynamics, growth and reaction kinetics, and transport processes derived from a fluid dynamics model. Simulations of a plume located in the ABE vent field of the Lau basin were able to reproduce metagenomic observations well and demonstrated that the magnitude of primary production and rate of autotrophic growth are largely regulated by the energetics of metabolisms and the availability of electron donors, as opposed to kinetic parameters. Ambient seawater was the dominant source of microbes to the plume and sulphur oxidisers constituted almost 90% of the modelled community in the neutrally-buoyant plume. Data from drifters deployed in the region allowed the different time scales of metabolisms to be cast in a spatial context, which demonstrated spatial succession in the microbial community. While growth was shown to occur over distances of tens of kilometers, microbes persisted over hundreds of kilometers. Given that high-temperature hydrothermal systems are found less than 100 km apart on average, plumes may act as important vectors between different vent fields and other environments that are hospitable to similar organisms, such as oil spills and oxygen minimum zones.     

Cartilage proteoglycan aggregates. Electronmicroscopic studies of native and fragmented molecules

1. Proteoglycan aggregates from bovine nasal cartilage were studied by using electron microscopy of proteoglycan/cytochrome c monolayers. 2. The aggregates contained a variably long central filament of hyaluronic acid with an average length of 1037nm. The proteoglycan monomers attached to the hyaluronic acid appeared as side chain filaments varying in length (averaging 249nm). They were distributed along the central filament at an average distance of about 36nm. 3. Chondroitin sulphate side chains were removed from the proteoglycan monomers of the aggregates by partial chondroitinase digestion. The molecules obtained had the same general appearance as intact aggregates. 4. Proteoglycan aggregates were treated with trypsin and the largest fragment, which contains the hyaluronic acid, link protein and hyaluronic acid-binding region, was recovered and studied with electron microscopy. Filaments that lacked the side chain extensions and had the same length as the central filament in the intact aggregate were observed. 5. Hyaluronic acid isolated after papain digestion of cartilage extracts gave filaments with similar length and size distribution as observed for the central filament both in the intact aggregate and in the trypsin digests. 6. Umbilical-cord hyaluronic acid was also studied and gave electron micrographs similar to those described for hyaluronic acid from cartilage. However, the length of the filament was somewhat shorter. 7. The electron micrographs of both intact and selectively degraded proteoglycans corroborate the current model of cartilage proteoglycan structure.     

Methicillin Resistant Staphylococcus aureus ST398 in Veal Calf Farming: Human MRSA Carriage Related with Animal Antimicrobial Usage and Farm Hygiene

Introduction

Recently a specific MRSA sequence type, ST398, emerged in food production animals and farmers. Risk factors for carrying MRSA ST398 in both animals and humans have not been fully evaluated. In this cross-sectional study, we investigated factors associated with MRSA colonization in veal calves and humans working and living on these farms.

Methods

A sample of 102 veal calf farms were randomly selected and visited from March 2007–February 2008. Participating farmers were asked to fill in a questionnaire (n = 390) to identify potential risk factors. A nasal swab was taken from each participant. Furthermore, nasal swabs were taken from calves (n = 2151). Swabs were analysed for MRSA by selective enrichment and suspected colonies were confirmed as MRSA by using slide coagulase test and PCR for presence of the mecA-gene. Spa types were identified and a random selection of each spa type was tested with ST398 specific PCR. The Sequence Type of non ST398 strains was determined. Data were analyzed using logistic regression analysis.

Results

Human MRSA carriage was strongly associated with intensity of animal contact and with the number of MRSA positive animals on the farm. Calves were more often carrier when treated with antibiotics, while farm hygiene was associated with a lower prevalence of MRSA.

Conclusion

This is the first study showing direct associations between animal and human carriage of ST398. The direct associations between animal and human MRSA carriage and the association between MRSA and antimicrobial use in calves implicate prudent use of antibiotics in farm animals.     

Development of a charcoal paper adenosine triphosphate:pyrophosphate exchange assay: Kinetic characterization of NEDD8 activating enzyme

Ubiquitin activating enzyme (UAE, UBE1, or E1) and seven known homologous “E1s” initiate the conjugation pathways for ubiquitin and 16 other ubiquitin-like modifiers (ULMs) found in humans. The initial step catalyzed by E1s uses adenosine triphosphate (ATP) to adenylate the C terminus of the appropriate ULM and results in the production of inorganic pyrophosphate (PPi). The mechanism of these enzymes can be studied with assays that measure the rate of ULM-dependent ATP:PPi exchange. The traditional method follows the initial velocity of [³²P]PPi incorporation into ATP by capturing the nucleotide on activated charcoal powder to separate it from excess [³²P]PPi and then measuring [³²P]ATP in a scintillation counter. We have modified the method by using charcoal paper to capture the nucleotide and a phosphorimager to quantify the [³²P]ATP. The significant increase in throughput that these modifications provide is accomplished without any sacrifice in sensitivity or accuracy compared with the traditional method. To demonstrate this, we reproduce and extend the characterization of the NEDD8 activating enzyme.