Palaeoclimate evidence of vulnerable permafrost during times of low sea ice.

Climate change in the Arctic is occurring rapidly, and projections suggest the complete loss of summer sea ice by the middle of this century1. The sensitivity of permanently frozen ground (permafrost) in the Northern Hemisphere to warming is less clear, and its long-term trends are harder to monitor than those of sea ice. Here we use palaeoclimate data to show that Siberian permafrost is robust to warming when Arctic sea ice is present, but vulnerable when it is absent. Uranium-lead chronology of carbonate deposits (speleothems) in a Siberian cave located at the southern edge of continuous permafrost reveals periods in which the overlying ground was not permanently frozen. The speleothem record starts 1.5 million years ago (Ma), a time when greater equator-to-pole heat transport led to a warmer Northern Hemisphere2. The growth of the speleothems indicates that permafrost at the cave site was absent at that time, becoming more frequent from about 1.35 Ma, as the Northern Hemisphere cooled, and permanent after about 0.4 Ma. This history mirrors that of year-round sea ice in the Arctic Ocean, which was largely absent before about 0.4 Ma (ref. 3), but continuously present since that date. The robustness of permafrost when sea ice is present, as well as the increased permafrost vulnerability when sea ice is absent, can be explained by changes in both heat and moisture transport. Reduced sea ice may contribute to warming of Arctic air4-6, which can lead to warming far inland7. Open Arctic waters also increase the source of moisture and increase autumn snowfall over Siberia, insulating the ground from low winter temperatures8-10. These processes explain the relationship between an ice-free Arctic and permafrost thawing before 0.4 Ma. If these processes continue during modern climate change, future loss of summer Arctic sea ice will accelerate the thawing of Siberian permafrost.

Functional divergence in gastrointestinal microbiota in physically-separated genetically identical mice.

Despite the fundamental contribution of the gut microbiota to host physiology, the extent of its variation in genetically-identical animals used in research is not known. We report significant divergence in both the composition and metabolism of gut microbiota in genetically-identical adult C57BL/6 mice housed in separate controlled units within a single commercial production facility. The reported divergence in gut microbiota has the potential to confound experimental studies using mammalian models.

A protein-based electrochemical biosensor array platform for integrated microsystems.

This paper elucidates challenges in integrating different classes of proteins into a microsystem and presents an electrochemical array strategy for heterogeneous protein-based biosensors. The overlapping requirements and limitations imposed by biointerface formation, electrochemical characterization, and microsystem fabrication are identified. A planar electrode array is presented that synergistically resolves these requirements using thin film Au and Ag/AgCl electrodes on a dielectric substrate. Using molecular self-assembly, electrodes were modified by nano-structures of two diverse proteins, alkali ion-channel protein and alcohol dehydrogenase enzyme. Electrochemical impedance spectroscopy and cyclic voltammetry measurements were performed to characterize sensor response to alkali ion and alcohol, respectively. This work demonstrates the viability of the electrochemical microsystem platform for heterogeneous protein-based biosensor interfaces.

Speleothems reveal 500,000-year history of Siberian permafrost.

Soils in permafrost regions contain twice as much carbon as the atmosphere, and permafrost has an important influence on the natural and built environment at high northern latitudes. The response of permafrost to warming climate is uncertain and occurs on time scales longer than those assessed by direct observation. We dated periods of speleothem growth in a north-south transect of caves in Siberia to reconstruct the history of permafrost in past climate states. Speleothem growth is restricted to full interglacial conditions in all studied caves. In the northernmost cave (at 60°N), no growth has occurred since Marine Isotopic Stage (MIS) 11. Growth at that time indicates that global climates only slightly warmer than today are sufficient to thaw extensive regions of permafrost.

CMOS Amperometric Instrumentation and Packaging for Biosensor Array Applications.

An integrated CMOS amperometric instrument with on-chip electrodes and packaging for biosensor arrays is presented. The mixed-signal integrated circuit supports a variety of electrochemical measurement techniques including linear sweep, constant potential, cyclic and pulse voltammetry. Implemented in 0.5 µm CMOS, the 3 × mm(2) chip dissipates 22.5 mW for a 200 kHz clock. The highly programmable chip provides a wide range of user-controlled stimulus rate and amplitude settings with a maximum scan range of 2 V and scan rates between 1 mV/sec and 400 V/sec. The amperometric readout circuit provides ±500 fA linear resolution and supports inputs up to ±47 µA. A 2 × 2 gold electrode array was fabricated on the surface of the CMOS instrumentation chip. An all-parylene packaging scheme was developed for compatibility with liquid test environments as well as a harsh piranha electrode cleaning process. The chip was tested using cyclic voltammetry of different concentrations of potassium ferricyanide at 100 mV/s and 200 mV/s, and results were identical to measurements using commercial instruments.

Amperometric electrochemical microsystem for a miniaturized protein biosensor array.

Protein-based bioelectrochemical interfaces offer great potential for rapid detection, continuous use, and miniaturized sensor arrays. This paper introduces a microsystem platform that enables multiple bioelectrochemical interfaces to be interrogated simultaneously by an onchip amperometric readout system. A post-complementary metal-oxide semiconductor (CMOS) fabrication procedure is described that permits the formation of planar electrode arrays and self assembly of biosensor interfaces on the electrodes. The onchip, 0.5-mum CMOS readout electronics include a compact potentiostat that supports a very broad range of input currents-6 pA to 10 muA-to accommodate diverse biosensor interfaces. The 2.3 times 2.2-mm chip operates from a 5-V supply at 0.6 mA. A prototype electrochemical sensor platform, including an onchip potentiostat and miniaturized biosensor array, was characterized by using cyclic voltammetry. The linear relationship between the oxidization peak values and the concentrations of target analytes in the solution verifies functionality of the system and demonstrates the potential for future implementations of this platform in high sensitivity, low cost, and onchip protein-based sensor arrays.

CMOS Baseline Tracking and Cancellation Instrumentation for Nanoparticle-Coated Chemiresistors.

Chemiresistor (CR) sensors and sensor arrays coated with thiolate-monolayer-protected gold nanoparticle (MPN) interfaces show great promise as detectors in gas-chromatographic microsystems with applications in biomedical and environmental analysis including breath biomarkers of disease. This paper describes a new readout circuit that overcomes the wide range of baseline resistances and drift in baseline values inherent to MPN-coated CRs to achieve a 57 ppm readout resolution. The 0.5-mum CMOS circuit operates at 5 V and provides a response resolution of 74 muV. It can cancel baseline voltages from 0.3 to 4.3 V with an accuracy of 4.2 mV and can track and compensate for drifts up to 30 mV/min. Performance was verified with MPN-coated CRs, where drift was measured and effectively cancelled. The circuit topology and size support an on-chip MPN-coated CR sensor array.

Rational design of vector and antibiotic peptides using solid-state NMR.

The application of 2H solid-state NMR in determining structure activity relationships and mechanism of action of membrane active peptides is discussed. The enhancement of the disruption of anionic lipids in the membrane by new lead compounds is shown to be a key determinant of both DNA vector and antimicrobial activity.

Separated local field NMR experiments on oriented samples rotating at the magic angle.

Biophysical studies on membrane proteins by solid state NMR (SSNMR) can be carried out directly in a membrane environment. Samples are usually prepared in form of multi-lamellar dispersions for magic angle sample spinning or as aligned multi-layers for orientation dependent NMR experiments without sample rotation. A new development is the application of MAS NMR to aligned samples (MAOSS; Magic Angle Oriented Sample Spinning). In combination with separated local field (SLF) experiments, size and orientation of heteronuclear dipolar couplings may be extracted from two-dimensional experiments which correlate dipolar couplings with isotropic chemical shifts. The orientation of these (1)H-X dipolar couplings can be directly related to the orientation of molecular groups in the sample. Here, we demonstrate the feasibility of these experiments on highly ordered polyethylene fibers which serve as model compound. Based on these data, the experiment is also applied to ordered multi-layers of bacteriorhodopsin (purple membrane) which is used as a model for aligned membrane proteins. We present a detailed analysis of different experimental designs with respect to angular sensitivity and the influence of residual sample disorder ("mosaic spread"). The results of the MAOSS-SLF experiment are discussed within the context of established solid state NMR experiments which are usually performed without sample rotation and we compare the data to orientation information obtained from X-ray diffraction.

Motion coherence thresholds in infants--different tasks identify at least two distinct motion systems.

Optokinetic nystagmus (OKN) can be demonstrated from birth, but behavioural discrimination tasks such as habituation and preferential looking do not reveal any sensitivity to motion direction until a few weeks of age. This study compared coherence threshold for motion direction for OKN and preferential looking responses using closely comparable stimuli, in infants between 6 and 27 weeks of age. Infants were tested with two random dot motion displays, a uniform area of moving dots for OKN responses and a display in which a region was segmented on one side by differential motion direction for preferential looking responses. Coherence thresholds for each response were determined by a staircase method. For OKN responses, mean coherence thresholds were between 20% and 25%, with no significant improvement in OKN performance throughout the age range. Preferential looking thresholds were significantly higher than OKN thresholds. Preferential looking thresholds improved significantly with age, but remained higher than OKN thresholds throughout the age range tested. Experiments varying direction reversal frequency and stimulus area indicated that these differences were not simply a consequence of the spatial and temporal non-uniformity of the preferential looking stimulus. The differences in sensitivity levels and age trends for OKN and preferential looking responses we have found suggest that different directional mechanisms are involved in the two responses. We discuss the possibility that, in early infancy, OKN and preferential looking reflect the performance of subcortical and cortical directional mechanisms respectively.

Directional motion asymmetry in infant VEPs--which direction?

Monocular viewing during early infancy reveals asymmetries in optokinetic nystagmus (OKN) and visual evoked potentials (VEPs). This study investigates the VEP asymmetry to see if it is consistent in direction with the OKN asymmetry. Steady-state VEPs were recorded from infants (5-21 weeks) viewing gratings that underwent successive displacements in the same direction, leftward or rightward. In addition, transient VEPs were recorded to the two directions of an oscillating stimulus. Both tests produced larger VEP amplitudes for nasal-to-temporal compared to temporal-to-nasal movement. Horizontal eye movements were monitored by EOG while viewing these stimuli to test whether the asymmetry was a consequence of eye movements. No difference in eye movements as a function of the stimulus was found, excluding differences in retinal slip as an explanation of the asymmetry. The stronger neural response for nasal-to-temporal displacements is opposite to the asymmetry of OKN. Oculomotor and VEP asymmetries may be related; however this relationship is not simply that the stronger neural response, indicated by the VEP, leads to a stronger optokinetic response.

Observations of light-induced structural changes of retinal within rhodopsin.

Photo-isomerization of the 11-cis retinal chromophore activates the mammalian light-receptor rhodopsin, a representative member of a major superfamily of transmembrane G-protein-coupled receptor proteins (GPCRs) responsible for many cell signal communication pathways. Although low-resolution (5 A) electron microscopy studies confirm a seven transmembrane helix bundle as a principal structural component of rhodopsin, the structure of the retinal within this helical bundle is not known in detail. Such information is essential for any theoretical or functional understanding of one of the fastest occurring photoactivation processes in nature, as well as the general mechanism behind GPCR activation. Here we determine the three-dimensional structure of 11-cis retinal bound to bovine rhodopsin in the ground state at atomic level using a new high-resolution solid-state NMR method. Significant structural changes are observed in the retinal following activation by light to the photo-activated M(I) state of rhodopsin giving the all-trans isomer of the chromophore. These changes are linked directly to the activation of the receptor, providing an insight into the activation mechanism of this class of receptors at a molecular level.

Are there separate first-order and second-order mechanisms for orientation discrimination?

In a series of experiments we compared orientation discrimination performance for Gabor stimuli in which the stimulus profile was either matched to the receptive field profile of single V1 simple cells ('simple'), or in which the carrier and envelope orientations were different ('tigertails'). In the first Experiment, using small, high spatial frequency, peripheral stimuli to minimise the number of detectors involved, we found that simple stimuli were more detectable than tigertails of the same contrast energy, and that orientation discrimination thresholds for simple stimuli were lower than for tigertails of equal detectability. In later experiments with larger stimuli we measured thresholds for detecting tilts of the envelope with the carrier fixed in orientation. Envelope thresholds were similar for different carrier orientations, but carrier orientation had a strong biasing effect upon perceived envelope orientation. When the orientation difference between envelope and carrier was small, the carrier orientation was attracted to that of the envelope; when the difference was large (>10 degrees ) repulsion was found. The biases were reduced by half-wave rectifying the stimuli, putatively making the envelope visible to a first-order filter (Experiment 2). Discrimination thresholds for envelope orientation were higher than those for carrier orientation, and this difference was greater for briefly-presented parafoveal stimuli than for long duration foveal stimuli (Experiments 3 and 4). We conclude from these results that there are separate mechanisms for envelope and carrier orientation discriminations for large stimuli, but that first- and second-order mechanisms are not independent in the discrimination of orientation.

Localization of activin beta(A)-, beta(B)-, and beta(C)-subunits in humanprostate and evidence for formation of new activin heterodimers of beta(C)-subunit.

Activin ligands are formed by dimerization of activin ss(A)- and/or ss(B)-subunits to produce activins A, AB, or B. These ligands are members of the transforming growth factor-ss superfamily and act as growth and differentiation factors in many cells and tissues. New additions to this family include activin ss(C)-, ss(D)-, and ss(E)-subunits. The aim of this investigation was to examine the localization of and dimerization among activin subunits; the results demonstrate that activin ss(C) can form dimers with activin ss(A) and ss(B) in vitro, but not with the inhibin alpha-subunit. Using a specific antibody, activin ss(C) protein was localized to human liver and prostate and colocalized with ss(A)- and ss(B)-subunits to specific cell types in benign and malignant prostate tissues. Activin C did not alter DNA synthesis of the prostate tumor cell line, LNCaP, or the liver tumor cell line, HepG2, in vitro when added alone or with activin A. Therefore, the capacity to form novel activin heterodimers (but not inhibin C) resides in the human liver and prostate. Activin A, AB, and B have diverse actions in many tissues, including liver and prostate, but there is no known biological activity for activin C. Thus, the evidence of formation of activin AC or BC heterodimers may have significant implications in the regulation of levels and/or biological activity of other activins in these tissues.

A sensitive and specific in vitro bioassay for activin using a mouse plasmacytoma cell line, MPC-11.

A new in vitro bioassay for activin was developed using the mouse plasmacytoma cell line, MPC-11. Human recombinant (hr) activin A dose-dependently inhibited the proliferation of these cells, whereas a range of other factors, including inhibin, follistatin and transforming growth factor-beta1, -beta2 and -beta3 had no effect. Conditioned medium containing activin B induced an inhibition similar to hr-activin A. The inhibitory influence of activin A could be blocked by follistatin, but not by hr-inhibin A. This bioassay had a sensitivity for activin A of around 0.4 ng/ml, an ED50 response of 3.5 ng/ml, and an intra-assay coefficient of variation of <11%. It offers substantial advantages over existing in vitro activin bioassays in terms of ease of use, specificity and throughput. The utility of the MPC-11 bioassay was demonstrated in the purification of activin from amniotic fluid, where an almost identical profile of bioactive activin A was detected compared with the pituitary cell bioassay of activin. Bioactive activin could also be detected in unpurified ovine allantoic and amniotic fluids and bovine follicular fluid. Measuring activin in untreated and heat-treated human sera or seminal plasma was hampered by a non-specific inhibitory effect, so that several serum samples did not run parallel with the hr-activin A standard. This inhibitory effect by serum could not be overcome by addition of follistatin, suggesting it is not activin-like bioactivity. This new bioassay for activin demonstrates widespread applicability for monitoring of purified or partially purified samples during purification procedures, bioactivity measurements, receptor-binding studies and assays of cell culture medium.

Differential expression of NADPH-diaphorase between electrophysiologically-defined classes of pyramidal neurons in rat ventral subiculum, in vitro.

The subiculum is the major output region of the hippocampal formation. We have studied pyramidal neurons in slices of rat ventral subiculum to determine if there is a correlation between nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) activity and electrophysiological phenotype. The majority of NADPH-d-positive pyramidal neurons were found in the superficial cell layer (i.e. nearest to the hippocampal fissure) of the subiculum and appreciable NADPH-d activity was absent from pyramidal neurons in area CA1. This distribution of NADPH-d activity was mimicked by that of immunoreactivity for the neuronal isoform of nitric oxide synthase. Subicular pyramidal neurons were classified, electrophysiologically, as intrinsically burst-firing or regular spiking. After electrophysiological characterization, neurons were filled with Neurobiotin and revealed using fluorescence immunocytochemistry. The slices containing these neurons were also processed for NADPH-d. NADPH-d activity was found in six out of eight regular spiking neurons but was not found in any of 13 intrinsically burst-firing neurons (P=0.0008, Fisher's Exact Test). We conclude that in rat ventral subiculum, NADPH-d activity is present in a proportion of pyramidal neurons and indicates the presence of the neuronal isoform of nitric oxide synthase. Furthermore, amongst pyramidal neurons, NADPH-d activity is distributed preferentially to those with the regular spiking phenotype. The distribution of regular spiking neurons suggests that they may not be present to the same extent in all subicular output pathways. Thus, the actions of nitric oxide may be relatively specific to particular hippocampal connections.

Characterization and determination of the biological activities of noncleavable high molecular weight forms of inhibin A and activin A.

Recombinant expression of human alpha- and beta A-inhibin subunit cDNAs in mammalian 293 cells results in the secretion of 20-53K free alpha-subunit-derived products, 30-105K alpha beta A-inhibin dimers, and 24-110K beta A-activin dimers. The present study verifies that the wide variation in the size of these products is due to incomplete cleavage of the proteolytic processing sites and the differential glycosylation of the N-linked glycosylation site at amino acid number 302 in the alpha C-subunit. The identity of each of these products was established by mutagenesis of proteolytic processing sites and N-linked glycosylation sites, combined with the analysis of transfection products by immunoprecipitation and one- and two-dimensional SDS-PAGE (SDS/SDS-beta-ME). Transient expression of processing site mutants of the alpha- and beta A-subunits in 293 cells was used to generate microgram quantities of noncleavable 55K and 65K inhibin dimers, and noncleavable 110K activin A dimers. The 55K and 65K inhibin A forms were purified and found to be fully biologically active in a rat pituitary cell bioassay. The 110K high molecular weight (HMW) form of human activin A failed to show any FSH-releasing activity in the pituitary assay. Since radioactively labeled 55K and 65K inhibin A and 110K activin A remained intact after incubation with rat pituitary cells for 72 h, there appears to be no conversion of these dimers to lower molecular weight forms by proteolytic cleavage at additional sites. These results show for the first time that 55K and 65K inhibit A are intrinsically biologically active and do not require cleavage to the 32K form for activation. In contrast, cleavage of the 110K activin A precursor to the 24K form would appear to be necessary for activity.

Enzyme linked immunosorbent assay for detecting benzodiazepines in urine.

A relatively simple ELISA technique was developed for the detection of a range of benzodiazepines (BZs) in urine. The assay employs a mouse anti-oxazepam antibody that is highly specific for the BZs. The limit of detection using 10 microliters samples of urine was 0.3 microgram ml-1 oxazepam. N-Desmethyldiazepam showed equal cross-reactivity to oxazepam, 11 BZs cross-reacted weakly and flurazepam and chlordiazepoxide did not cross-react at levels reported to be found in urine. No cross-reactivity was observed with drugs of abuse and a range of therapeutic drugs commonly found in urine. The assay was used as a screen to detect the presence of BZs in urine from 88 addicts that had been screened by the EMIT technique and a radioreceptor assay (RRA) for BZs. The ELISA produced two false negatives that were EMIT and RRA positive whereas the EMIT produced four different false negatives that were positive by both ELISA and RRA. Thirty-three positives were common to all three assays. The ELISA was also used to monitor nitrazepam-like activity in the urine of a greyhound receiving 5 mg oral medication and the results were compared with those obtained by RRA. Both assays were able to detect nitrazepam-like activity for up to 10 h post-administration.

Functional analysis of the cysteine residues of activin A.

Site-directed mutagenesis and mammalian cell expression was used to analyze the function of each of the 13 cysteine residues in the human activin A beta-subunit precursor. Substitution of the four cysteine residues in the proregion with alanine residues did not affect the function of the proregion in facilitating the dimerization and secretion of activin A homodimers. A series of activin mutants were constructed in which the nine cysteine residues (amino acids 4, 11, 12, 40, 44, 80, 81, 113, and 115) in the mature 116-amino acid beta-subunit were individually altered to alanine residues. Alanine substitution at either cysteine residues 4 or 12 did not interfere with homodimer formation, but the mutant activin A molecules had reduced biological and receptor binding activity (2- to 3-fold). Activin A monomers were produced when cysteine mutants 44, 80, and 113 were expressed in tissue culture cells. Monomers of cys mutants 44 and 80 had approximately 2% of the biological and receptor binding activity of wild type activin A. Cys 113 monomers had undetectable levels of biological activity. No detectable activin monomers or dimers were secreted from cells transfected with plasmids containing cys mutants 11, 40, 81, and 115. The data presented here suggest that a low level of noncovalent dimer formation of cysteine mutant monomers 44 and 80 may explain their low level of biological activity. Therefore, dimer formation is suggested to be an essential prerequisite for high affinity receptor binding and biological potency.(ABSTRACT TRUNCATED AT 250 WORDS)