Real‐time monitoring of greenhouse gas emissions with tall chambers reveals diurnal N2O variation and increased emissions of CO2 and N2O from Miscanthus following compost addition

Miscanthus x giganteus's efficacy as an energy crop relies on maintaining low greenhouse gas (GHG) emissions. As demand for Miscanthus is expected to rise to meet bioenergy targets, fertilizers and composts may be employed to increase yields, but will also increase GHG emissions. Manipulation experiments are vital to investigate the consequences of any fertilizer additions, but there is currently no way to measure whole‐plant GHG fluxes from crops taller than 2.5 m, such as Miscanthus, at the experimental plot scale. We employed a unique combination of eddy covariance (EC), soil chambers and an entirely new automated chamber system, SkyBeam, to measure high frequency (ca. hourly) fluxes of carbon dioxide (CO₂), methane (CH₄) and nitrous oxide (N₂O) from a Miscanthus crop amended with green compost. Untreated controls were also monitored in a fully replicated experimental design. Net ecosystem exchange (NEE) of CO₂ was partitioned into soil respiration (R_s), gross primary productivity (GPP) and ecosystem respiration, and the crop was harvested to determine the effect of compost on crop productivity. Compost increased NEE emissions by 100% (p < .05), which was the result of a 20% increase of R_s (p < .06) and a 32% reduction in GPP (p < .05) and biomass of 37% (p < .06). Methane fluxes were small and unaffected by compost addition. N₂O emissions increased 34% under compost during an emission event; otherwise, fluxes were low and often negative, even under dry conditions. Diurnal variation in N₂O fluxes, with uptake during the day and emission at night was observed. These fluxes displayed a negative relationship with soil temperature and a hitherto undescribed diurnal temperature hysteresis. We conclude that compost addition negatively affected the productivity and environmental effects of Miscanthus cultivation during the first year following application.     

Sustained effects of atmospheric [CO2] and nitrogen availability on forest soil CO2 efflux

Soil CO₂ efflux (F_soil) is the largest source of carbon from forests and reflects primary productivity as well as how carbon is allocated within forest ecosystems. Through early stages of stand development, both elevated [CO₂] and availability of soil nitrogen (N; sum of mineralization, deposition, and fixation) have been shown to increase gross primary productivity, but the long‐term effects of these factors on F_soil are less clear. Expanding on previous studies at the Duke Free‐Air CO₂ Enrichment (FACE) site, we quantified the effects of elevated [CO₂] and N fertilization on F_soil using daily measurements from automated chambers over 10 years. Consistent with previous results, compared to ambient unfertilized plots, annual F_soil increased under elevated [CO₂] (ca. 17%) and decreased with N (ca. 21%). N fertilization under elevated [CO₂] reduced F_soil to values similar to untreated plots. Over the study period, base respiration rates increased with leaf productivity, but declined after productivity saturated. Despite treatment‐induced differences in aboveground biomass, soil temperature and water content were similar among treatments. Interannually, low soil water content decreased annual F_soil from potential values – estimated based on temperature alone assuming nonlimiting soil water content – by ca. 0.7% per 1.0% reduction in relative extractable water. This effect was only slightly ameliorated by elevated [CO₂]. Variability in soil N availability among plots accounted for the spatial variability in F_soil, showing a decrease of ca. 114 g C m^?2 yr^?1 per 1 g m^?2 increase in soil N availability, with consistently higher F_soil in elevated [CO₂] plots ca. 127 g C per 100 ppm [CO₂] over the +200 ppm enrichment. Altogether, reflecting increased belowground carbon partitioning in response to greater plant nutritional needs, the effects of elevated [CO₂] and N fertilization on F_soil in this stand are sustained beyond the early stages of stand development and through stabilization of annual foliage production.     

A high quality nuclear magnetic resonance solution structure of peptide deformylase from Escherichia coli: Application of an automated assignment strategy using GARANT

The NMR structure of the peptide deformylase (PDF) (1–150) from Escherichia coli, which is an essential enzyme that removes the formyl group from nascent polypeptides and represents a potential target for drug discovery, was determined using 15N/13C doubly labeled protein. Nearly completely automated assignment routines were employed to assign three-dimensional triple resonance, 15N-resolved and 13C-resolved NOESY spectra using the program GARANT. This assignment strategy, demonstrated on a 17 kDa protein, is a significant advance in the automation of NMR data assignment and structure determination that will accelerate future work. A total of 2302 conformational constraints were collected as input for the distance geometry program DYANA. After restrained energy minimization with the program X-PLOR the 20 best conformers characterize a high quality structure with an average of 0.43 Å for the root-mean-square deviation calculated from the backbone atoms N, C and C, and 0.81 Å for all heavy atoms of the individual conformers relative to the mean coordinates for residues 1 to 150. The globular fold of PDF contains two -helices comprising residues 25–40, 125–138, six -strands 57–60, 70–77, 85–88, 98–101, 105–111, 117–123 and one 310 helix comprising residues 49–51. The C-terminal helix contains the HEXXH motif positioning a zinc ligand in a similar fashion to other metalloproteases, with the third ligand being cysteine and the fourth presumably a water. The three-dimensional structure of PDF affords insight into the substrate recognition and specificity for N-formylated over N-acetylated substrates and is compared to other PDF structures.     

A direct approach to the measurement of genetic variation in fish populations: applications of the polymerase chain reaction to studies of Atlantic cod, Gadus morhua L.

We used the polymerase chain reaction (PCR) and direct DNA sequencing to study genetic variation within and among populations of Atlantic cod, Gadus morhua , in the western North Atlantic. In a 307 bp region of the mitochondrial cytochrome b gene, 24 variable nucleotide positions define 24 genotypes, which differ by from one to six nucleotide substitutions. Greenland cod ( G. ogac ) differs from the most similar G. morhua genotype by an additional 12 nucleotide substitutions. Silent transitions dominate both intra- and interspecific comparisons, however four nucleotide substitutions within morhua result in amino acid replacements. Direct sequencing of DNA reveals substantially more of the genetic variation that exists within and between species than do previous indirect methods based on restriction fragment length polymorphisms, and thus has far greater potential to quantify such differences as may exist among fish stocks. Preliminary experiments also indicate that automation of DNA sequencing provides an efficient, rapid, and accurate means for detection of genetic variation in natural populations offish.     

On-line monitoring of enzymes in downstream processing by flow injection analysis (FIA)

Flow injection analysis (FIA) has been employed to automate enzyme assays for formate dehydrogenase (FDH) and l-leucine dehydrogenase (l-LeuDH). Coupled to a special sampling device the FIA assays were used to monitor on-line downstream processes, e.g. disintegration of microbial cells and cross-flow filtration of cell homogenates.     

Permeable collagen-glycosaminoglycan cross-linked copolymers for the study of biological responses of coculturede Sertoli and spermatogenic cells

A novel collagen-glycosaminoglycan (C-GAG) substrate was developed to overcome the optical opacity of a HATF nitrocellulose substrate and to provide a more physiological permeable substrate for cocultured Sertoli and spermatogenic cells. Cocultures were prepared on optically transparent C-GAG discs attached to a polyester mesh to facilitate handling. Sertoli cells displayed a cuboidal-to-columnar shape; a large number of spermatogonia and primary spermatocytes connected by intercellular bridges were associated with basolateral and apical surfaces of Sertoli cells up to 12 days after plating. Rat Sertoli-spermatogenic cell cocultures have been used for testing the effect of toxicants on rat spermatogenesis in vitro. In our initial studies, we tested the effects of the toxicant gossypol on spermatogenic cells cocultured with Sertoli cells on nonpermeable (plastic) and permeable substrates (HATF nitrocellulose) under both standard culture conditions and during perifusion after achieving a continuous electrical-resistant cell monolayer. A selective mitochondrial structural damage was observed in spermatogenic cells (spermatogonia and spermatocytes) but not in the coexisting Sertoli cells. This damage was time- (15–60 min) and dose-dependent (0.1–10µM) and developed more rapidly under perifusion conditions. Similar mitochondrial damage was reported in the intact animal but required higher concentrations (mg) and longer administration time (months) for detection. Studies are in progress to evaluate the effect of additional toxic chemical agents on functional properties of Sertoli and spermatogenic cells in cocultures prepared on various classes of C-GAG substrates.Abbreviations C-GAG, collagen type I-glycosaminoglycans - C-C6S, collagen type I-chondroitin-6-sulfate - C-H, collagen type I-heparin     

Solution structure and dynamics of melanoma inhibitory activity protein

Melanoma inhibitory activity (MIA) is a small secreted protein that is implicated in cartilage cell maintenance and melanoma metastasis. It is representative of a recently discovered family of proteins that contain a Src Homologous 3 (SH3) subdomain. While SH3 domains are normally found in intracellular proteins and mediate protein-protein interactions via recognition of polyproline helices, MIA is single-domain extracellular protein, and it probably binds to a different class of ligands.Here we report the assignments, solution structure, and dynamics of human MIA determined by heteronuclear NMR methods. The structures were calculated in a semi-automated manner without manual assignment of NOE crosspeaks, and have a backbone rmsd of 0.38 Å over the ordered regions of the protein. The structure consists of an SH3-like subdomain with N- and C-terminal extensions of approximately 20 amino acids each that together form a novel fold. The rmsd between the solution structure and our recently reported crystal structure is 0.86 Å over the ordered regions of the backbone, and the main differences are localized to the most dynamic regions of the protein. The similarity between the NMR and crystal structures supports the use of automated NOE assignments and ambiguous restraints to accelerate the calculation of NMR structures.     

Rhesus monkey (Macaca mulatta) complex learning skills reassessed

Results from three experiments on basic learning and transfer in rhesus monkeys (Macaca mulatta) are reported in which fully automated testing paradigms, afforded by the Language Research Center's Computerized Test System (LRC-CTS), were employed. Performance levels for discrimination learning set, transfer index, and mediational-learning testing were uniformly higher than was predicted from the literature, in contrast to previous reports of compromised learning under similar conditions (automated apparatus, planimetric stimuli, spatial discontiguity between stimuli and response loci). Analyses reveal relatively advanced learning set performance, transfer-index ratios, and positive transfer of learning even at stringent criterion levels. Moreover, the data suggest that rhesus monkeys tested in these experiments exhibit mediational instead of associative learning strategies, as do great apes and in contrast to previous reports of rhesus learning. We argue that the LRC-CTS enhances learning by nonhuman primate subjects, obviating those factors, reported in the literature from experiments in which manual or other automated systems were employed, that compromise learning.     

Challenges and prospects in the telemetry of insects

Radio telemetry has been widely used to study the space use and movement behaviour of vertebrates, but transmitter sizes have only recently become small enough to allow tracking of insects under natural field conditions. Here, we review the available literature on insect telemetry using active (battery‐powered) radio transmitters and compare this technology to harmonic radar and radio frequency identification (RFID) which use passive tags (i.e. without a battery). The first radio telemetry studies with insects were published in the late 1980s, and subsequent studies have addressed aspects of insect ecology, behaviour and evolution. Most insect telemetry studies have focused on habitat use and movement, including quantification of movement paths, home range sizes, habitat selection, and movement distances. Fewer studies have addressed foraging behaviour, activity patterns, migratory strategies, or evolutionary aspects. The majority of radio telemetry studies have been conducted outside the tropics, usually with beetles (Coleoptera) and crickets (Orthoptera), but bees (Hymenoptera), dobsonflies (Megaloptera), and dragonflies (Odonata) have also been radio‐tracked. In contrast to the active transmitters used in radio telemetry, the much lower weight of harmonic radar and RFID tags allows them to be used with a broader range of insect taxa. However, the fixed detection zone of a stationary radar unit (< 1 km diameter) and the restricted detection distance of RFID tags (usually < 1–5 m) constitute major constraints of these technologies compared to radio telemetry. Most of the active transmitters in radio telemetry have been applied to insects with a body mass exceeding 1 g, but smaller species in the range 0.2–0.5 g (e.g. bumblebees and orchid bees) have now also been tracked. Current challenges of radio‐tracking insects in the field are related to the constraints of a small transmitter, including short battery life (7–21 days), limited tracking range on the ground (100–500 m), and a transmitter weight that sometimes approaches the weight of a given insect (the ratio of tag mass to body mass varies from 2 to 100%). The attachment of radio transmitters may constrain insect behaviour and incur significant energetic costs, but few studies have addressed this in detail. Future radio telemetry studies should address (i) a larger number of species from different insect families and functional groups, (ii) a better coverage of tropical regions, (iii) intraspecific variability between sexes, ages, castes, and individuals, and (iv) a larger tracking range via aerial surveys with helicopters and aeroplanes equipped with external antennae. Furthermore, field and laboratory studies, including observational and experimental approaches as well as theoretical modelling, could help to clarify the behavioural and energetic consequences of transmitter attachment. Finally, the development of commercially available systems for automated tracking and potential future options of insect telemetry from space will provide exciting new avenues for quantifying movement and space use of insects from local to global spatial scales.