The power to detect regional declines in common bird populations using continental monitoring data.

Anthropogenic environmental change is driving the rapid loss of biodiversity. Large declines in the abundance of historically common species are now emerging as a major concern. Identifying declining populations through long-term biodiversity monitoring is vital for implementing timely conservation measures. It is, therefore, critical to evaluate the likelihood that persistent long-term population trends of a given size could be detected using existing monitoring data and methods. Here, we test the power to detect declines in Australia's common landbirds using long-term citizen science monitoring. We use spatially explicit simulations of occupancy dynamics and virtual sampling, designed to mimic bird monitoring in better-sampled regions of Australia, to assess likely power in these data to detect trends relevant for conservation. We predict the statistical power for 326 common species that meet minimum requirements for monitoring data across 10 regions of Australia, estimating the number of species for which we would have a high (≥80%) chance of detecting declines of different sizes. The power to detect declines of ≥30% per decade was predicted to be high for at least one-third of the common species in 7 of 10 regions, with a total of 103 (32% of 326) unique species sufficiently monitored in at least one region. These species spanned 12 taxonomic orders, four orders of magnitude in body mass, and a broad diversity of dietary guilds, suggesting the current species pool will likely serve as robust indicators for a broad range of environmental states and pressures. Power was strongly affected by species' detectability, and power to detect even large declines was negligible when species are detected on ≤50% of visits to an occupied site. Predicted power for many species fell just short of the 80% threshold in one or more regions, which suggests an increase in effort targeting these species could greatly enhance the species and regional representation of these data. Against the backdrop of unprecedented biodiversity losses, this study shows how critical evaluation of existing monitoring schemes is valuable both for assessing the contribution of citizen science schemes to biodiversity monitoring and for designing strategic monitoring to significantly improve the knowledge these schemes provide.

Age-associated changes in the blood-brain barrier: comparative studies in human and mouse.

AIMS: While vascular pathology is a common feature of a range of neurodegenerative diseases, we hypothesized that vascular changes occur in association with normal ageing. Therefore, we aimed to characterize age-associated changes in the blood-brain barrier (BBB) in human and mouse cohorts. METHODS: Immunohistochemistry and Evans blue assays were used to characterize BBB dysfunction (tight junction protein expression and serum plasma protein accumulation), vascular pathology (pericyte loss and vascular density) and glial pathology (astrocyte and microglial density) in ageing neurological control human prefrontal cortex (a total of 23 cases from 5 age groups representing the spectrum of young adult to old age: 20-30 years, 31-45 years, 46-60 years, 61-75 years and 75+) and C57BL/6 mice (3 months, 12 months, 18 months and 24 months, n = 5/6 per group). RESULTS: Quantification of the tight junction protein ZO-1 within the cortex and cerebellum of the mouse cohort showed a significant trend to both increased number (cortex P < 0.001, cerebellum P < 0.001) and length (cortex P < 0.001, cerebellum P < 0.001) of junctional breaks associated with increasing age. GFAP expression significantly correlated with ageing in the mice (P = 0.037). In the human cohort, assessment of human protein accumulation (albumin, fibrinogen and human IgG) demonstrated cells morphologically resembling clasmatodendritic astrocytes, indicative of BBB dysfunction. Semiquantitative assessment of astrogliosis in the cortex expression revealed an association with age (P = 0.003), while no age-associated changes in microglial pathology, microvascular density or pericyte coverage were detected. CONCLUSIONS: This study demonstrates BBB dysfunction in normal brain ageing, both in human and mouse cohorts.

Density gradients in a rotating stratified fluid: experimental evidence for a new instability.

Velocity gradients induced in a rapidly rotaiting, density-stratified saltwater solution by a slowly rotating disk produce sharp vertical gradients of density, which appear as regularly spaced, curved horizontal sheets when the ratio of angular velocities exceeds a critical value. The existence of the sheets is apparently a finite amplitude manifestationl of a recently proposed viscous instability.

The production and characterisation of an immobilised chaperonin system.

Here we report a method of immobilising the chaperonins GroEL and GroES to a glass matrix. The immobilised chaperone system has been used to successfully refold target proteins denatured by guanidine hydrochloride and produce substantially higher levels of active protein than occur on dilution into aqueous solution alone. The chaperone system has been shown to refold proteins from each of the three categories of GroEL substrate. The refolding of the enzyme glycerol dehydrogenase from Bacillus stearothermophilus shows a two-fold increase in activity in the presence of immobilised GroEL compared to that in free solution. The lactate dehydrogenase from B. stearothermophilus also shows a two-fold higher yield of activity in the presence of the immobilised GroEL and ATP. The presence of immobilised GroEL in the absence of ATP arrests the refolding of LDH. The enzyme citrate synthetase from porcine heart demonstrates a three-fold increase in activity when refolded in the presence of immobilised GroEL, ATP and free GroES. Similar results are obtained in the presence of free GroEL, immobilised GroES and ATP. The matrix-bound chaperone can be removed from the refolding mixture by centrifugation, producing a reusable system that can be easily isolated and purified from the refolded substrate.

Stalling of human DNA (cytosine-5) methyltransferase at single-strand conformers from a site of dynamic mutation.

Single-strand conformers (SSCs) from the C-rich strand of the triplet repeat at the FMR-1 locus are rapidly and selectively methylated by the human DNA (cytosine-5) methyltransferase. The apparent affinity of the enzyme for the FMR-1 SSC is about tenfold higher than it is for a control Watson-Crick paired duplex. The de novo methylation rate for the SSC is over 150-fold higher than the de novo rate for the control duplex. Methylation of what is generally called a hemi-methylated duplex occurs with a rate enhancement of over 100-fold, while methylation of what can be viewed as a hemi-methylated FMR-1 SSC is actually slower than the de novo rate. The pronounced inhibition of the methyltransferase by the methylated SSC suggests that the enzyme has a higher affinity for the methylated product of its reaction with the SSC than it has for the unmethylated SSC substrate. Gel retardation studies show that the methyltransferase binds selectively to SSCs from the C-rich strand of the FMR-1 triplet repeat. This suggests a two-step stalling process in which the human methyltransferase first selectively methlyates and subsequently stalls at the C-rich strand SSC. Stalling may reflect the inability of the enzyme to release a DNA product that is fixed in a conformation resembling its transition state by the unusual structure of the substrate. In particular, the data suggest that DNA methyltransferase may physically participate in biological processes that lead to dynamic mutation at FMR-1. In general, the data raise the possibility that a two-step stalling process occurs at secondary structures associated with chromosome instability, chromosome remodelling, viral replication or viral integration and may account for the local hypermethylation and global hypomethylation associated with viral and non-viral tumorigenesis.

Recognition of unusual DNA structures by human DNA (cytosine-5)methyltransferase.

The symmetry of the responses of the human DNA (cytosine-5)methyltransferase to alternative placements of 5-methylcytosine in model oligodeoxynucleotide duplexes containing unusual structures has been examined. The results of these experiments more clearly define the DNA recognition specificity of the enzyme. A simple three-nucleotide recognition motif within the CG dinucleotide pair can be identified in each enzymatically methylated duplex. The data can be summarized by numbering the four nucleotides in the dinucleotide pair thus: 1 4/2 3. With reference to this numbering scheme, position 1 can be occupied by cytosine or 5-methylcytosine; position 2 can be occupied by guanosine or inosine; position 3, the site of enzymatic methylation, can be occupied only by cytosine; and position 4 can be occupied by guanosine, inosine, O6-methylguanosine, cytosine, adenosine, an abasic site, or the 3' hydroxyl group at the end of a gapped molecule. Replacing the guanosine normally found at position 4 with any of the moieties introduces unusual (non-Watson-Crick) pairing at position 3 and generally enhances methylation of the cytosine at that site. The exceptional facility of the enzyme in actively methylating unusual DNA structures suggests that the evolution of the DNA methyltransferase, and perhaps DNA methylation itself, may be linked to the biological occurrence of unusual DNA structures.

Hypermethylation of telomere-like foldbacks at codon 12 of the human c-Ha-ras gene and the trinucleotide repeat of the FMR-1 gene of fragile X.

Runs of G residues on the G-rich strands of 30mers from the region spanning codon 12 of c-Ha-ras appear to be protected against chemical modification by dimethylsulfate. This suggests that the G-rich strand might spontaneously form a Hoogsteen-paired quadruplex, which is characteristic of telomere-like DNA sequences. In this report we show that the predominant species in 1:1 mixtures of complementary 30mers from this region are duplex DNA and a smaller amount of unimolecular foldback formed by the C-rich strand. Foldbacks of this type resemble structures first observed in the C-rich strand of telomeric DNA and also occur at the CCG triplet repeat present in the FMR-1 gene of human fragile X syndrome. Foldbacks from the C-rich strand of c-Ha-ras and the FMR-1 triplet repeat are exceptional substrates for the human methyltransferase in isolation. Substituting inosine for guanosine alters the secondary structure of the folded oligomers and dramatically reduces their ability to serve as substrates for the human methyltransferase, suggesting that secondary structure is required for recognition by the enzyme. These findings suggest that one mechanism by which methyl groups accumulate in the c-Ha-ras region of chromosome 11 during carcinogenesis and at the FMR-1 locus during repeat expansion at fragile X may be structurally induced de novo methylation at sites undergoing local conformational change. Such methylation might serve to mark unusual structures for repair. In the absence of repair, asymmetrically methylated duplexes produced by resolution of the unusual structures would be rapidly converted to symmetrically methylated duplexes through the methyl-directed activity also carried by the human methyltransferase.

The response of M.HpaII to heteroduplexes.

Synthetic oligodeoxyribonucleotide duplexes have been used to study the methylation specificity of M.HpaII, a bacterial DNA methyltransferase. Substrates of four types were compared. A 30-mer containing a Watson-Crick paired CCGG recognition sequence was rapidly methylated at the central cytosine on each strand in the recognition sequence. A 30-mer containing an asymmetrically methylated recognition sequence, of the type transiently produced by DNA replication, was rapidly methylated at the central cytosine on the unmethylated strand. A heteroduplex containing an A.C mispair in the recognition sequence (CCGG/CCAG) was rapidly methylated at the cytosine in the mispair. A heteroduplex containing an A.C and an adjacent C.C mispair in the recognition sequence (CCGG/CCCA) was not methylated at a significant rate. The results show that M.HpaII can tolerate a single mispair at its recognition site in a heteroduplex without loss of activity or specificity.

Receptor-based design of dihydrofolate reductase inhibitors: comparison of crystallographically determined enzyme binding with enzyme affinity in a series of carboxy-substituted trimethoprim analogues.

By the use of molecular models of Escherichia coli dihydrofolate reductase (DHFR), analogues of trimethoprim (TMP) were designed which incorporated various 3'-carboxyalkoxy moieties in order to acquire ionic interactions with positively charged active-site residues. Certain of these compounds have shown exceptionally high affinity for this enzyme. For example, the 3'-(carboxypentyl)oxy analogue was found to be 55-fold more inhibitory than TMP toward E. coli DHFR (Ki = 0.024 nM vs. 1.32 nM for TMP). X-ray crystallographic studies of E. coli DHFR in binary complexes with TMP and two members of this acid-containing series of compounds defined the binding of these inhibitors and showed the carboxyl group of the latter two inhibitors to be ionically bound to Arg-57. These observations were in agreement with postulated binding modes that were based on receptor modeling.

Presynaptic alpha-adrenoceptors: do exogenous and neuronally released noradrenaline act at different sites?

The effects of dopamine receptor and alpha-adrenoceptor agonists and antagonists on the stimulation-evoked overflow of radioactivity from strips of dog saphenous vein previously loaded with [3H]-noradrenaline have been examined alone and in combination. In the presence of neuronal and extraneuronal catecholamine uptake inhibitors, noradrenaline (0.1-1 X 10(-6)M) and dopamine (0.01-1 X 10(-6)M) both inhibited the stimulation-evoked overflow of radioactivity. Sulpiride (1 X 10(-6)M) was without effect and prazosin (1 X 10(-7)M) had little effect on stimulation-evoked overflow but yohimbine enhanced it approximately 2 fold; the effect of yohimbine was similar at concentrations of 1 X 10(-7) and 1 X 10(-6)M. Sulpiride abolished the inhibitory effect of dopamine on stimulation-evoked overflow, but was without effect against noradrenaline. When allowance was made for the effects of yohimbine, alone, on overflow, yohimbine (1 X 10(-7)M) had no effect against dopamine and minimal effects against noradrenaline. A similar result was obtained when the concentration of yohimbine was increased to 1 X 10(-6)M. Prazosin did not antagonize the effect of noradrenaline. In the absence of the uptake inhibitors, clonidine (0.01-1 X 10(-5)M) inhibited stimulation-evoked overflow of radioactivity. Yohimbine (1 X 10(-6)M) was without effect on its own and antagonized the effects of clonidine at a concentration of 0.1 X 10(-5)M, but not at 0.01 or 1.0 X 10(-5)M. These findings suggest that dopamine inhibits overflow by stimulating presynaptic dopamine receptors on the terminals of the noradrenergic nerves supplying the dog saphenous vein. The interaction between yohimbine and noradrenaline is discussed in terms of the current concepts of control of transmitter release mediated via presynaptic alpha 2-adrenoceptors.

Marketed sperm: use and regulation in the United States.

OBJECTIVE: To collate information relating to the current use of marketed semen for therapeutic donor insemination (DI) in the United States. DATA IDENTIFICATION: Literature was identified by Medline search and government document review. LITERATURE SELECTION: Materials selected for review included empirical studies, policy reviews, legal documents, and government surveys relating to use of donor sperm. RESULTS: Use of marketed (donor) sperm is associated with significant medical, genetic, and psychological risk. These risks directly affect the individuals involved in therapeutic DI. Also, the public's health is involved because these risks include transmission of infectious disease and genetic anomalies. Legal and social concerns associated with therapeutic DI include offspring's knowledge of genetic endowment, parental responsibility, and donor confidentiality. This analysis shows that policies currently in place regarding the use of marketed semen in therapeutic DI do not ensure consumer safety and do not protect society's interest. CONCLUSIONS: Current policies need to be improved to protect those directly involved in the therapeutic DI process and to address public health and societal concerns. Recommendations include: [1] programs to assure quality and safety of marketed sperm, [2] implementation of a central registry to collect information about the use and outcomes of therapeutic DI, and [3H] expansion of available therapeutic DI guidelines to address psychological and social support for persons involved in therapeutic DI.

Effect of opioid peptides and potassium on the release of vasoactive intestinal polypeptide and thyrotropin releasing hormone from perifused rat hypothalami.

Opioid peptides have been demonstrated to stimulate prolactin secretion, and it has been postulated that this is mediated, at least in part, by an effect on hypothalamic prolactin releasing and release-inhibiting factors and neurotransmitters. The aim of this study was to investigate the effect of opioid peptides and depolarizing concentrations of K+ on the release of both vasoactive intestinal polypeptide (VIP) and thyrotropin releasing hormone (TRH) from perifused rat hypothalami. Both met-enkephalin and beta-endorphin stimulated the release of VIP significantly whilst not affecting the release of TRH. In addition, leu-enkephalin was found to have no effect on the release of either VIP or TRH. In contrast, depolarizing concentrations of K+ (50 mM) were found to cause the immediate release of TRH, but not VIP, from the same perifusion. The results suggest a role for VIP, but not TRH, in opioid peptide stimulated release of prolactin. In addition, the data indicates that a substance may be released in response to K+ depolarization which is inhibitory to the release of VIP.

Evidence for hyaluronan production in the air pouch model in rats.

OBJECTIVE: The aim of our work was to investigate the presence of hyaluronan (HA) in the rat air pouch and its behaviour in response to inflammatory stimuli. METHODS: HA levels (by a microplate assay) and the leucocyte count were determined in the fluid obtained from air pouches in which acute or subacute inflammation had been induced by the injection of monosodium urate crystals (MSU) or high density polyethylene (HDPE) debris respectively and in relative controls. RESULTS: In control pouches of both groups, remarkable levels of HA were found; these levels were higher in the very first hours (2475 and 1850 micrograms/l at 6 hrs) and then gradually decreased. In pouches injected with MSU, HA moderately increased (p < 0.001) after 6 hrs, reached a peak after 12 hrs (p < 0.001) and began to taper at 24 hrs (p < 0.001). The leucocyte count was also increased at 6 hrs (p < 0.001), became higher at 12 hrs (p < 0.001) and tapered at 24 hrs (p < 0.001). In the HDPE pouches, HA levels were significantly reduced with respect to controls after 6 hours (p < 0.001), increasing later (p < 0.001) to reach a peak at 24 hrs (p < 0.001), and returning to the original levels, or even below, in the following 72 hours. CONCLUSIONS: These data confirm that the pouch lining produces fair amounts of HA and provide evidence that, in this system, HA levels seem to be influenced by the degree of inflammation even if with variable behaviour in relation to the different characteristics and phases of phlogosis. The present data suggest that the air pouch is a useful experimental model for studies on HA metabolism in either acute or chronic inflammation.

Management of respiratory failure in toxic disasters.

Accidental or deliberate release of toxic substances may be a cause of mass casualties, some of which may involve partial or complete respiratory failure. Life support measures may be delayed by the need for containment of the toxic release and decontamination. Advanced life support (ALS) may be provided in such circumstances using the TOXALS protocol. With proper training and equipment, emergency medical personnel can provide effective prehospital care during decontamination of chemical casualties.

The pre-hospital management of injury following mass toxic release; a comparison of military and civil approaches.

Mass release of toxic substances may occur either accidentally or deliberately in both peace and war. Different approaches to the management of casualties from such an event have been developed by civil and military emergency medical teams, and reflect the different circumstances in which they operate. The nature and classification of toxic hazards is considered and the civil and military operational and medical responses compared. Both systems have different aspects that can contribute to early casualty management in a contaminated environment.

Clinical analogies for the management of toxic trauma.

The management of injuries from toxic release (HAZMAT) incidents is unfamiliar to many emergency medical responders owing to the relative rarity of such incidents. However, the risks from toxic release in both industrial and other metropolitan areas are increasing and emergency medical services (EMS) personnel should be trained to be aware of the dangers to both victims and responders alike. Many examples exist of analogies for the management of HAZMAT situations from conventional prehospital and hospital emergency medicine. Application of the lessons learned in more familiar situations will be of benefit for the preparation of an effective EMS response for HAZMAT.

A computer-based interview system for patients with back pain. A validation study.

A microcomputer-based system has been designed to interview patients with a view to investigating and establishing common syndromes of back and leg pain. In a randomized crossover validation study, 50 consecutive outpatients were interviewed by the computer and had a conventional clerking by a doctor. The conventional clerking made minor errors in 3.75% of questions answered and major errors in 0.90%. The computer made minor errors in 6.75% of questions and major errors in 5.45%. The majority of the computer errors were due to inadequate question design. These have been corrected, and it is anticipated that the computer will now have an overall rate of 94% correct answers and be sufficiently accurate to pursue the aim of clinical syndrome identification.

Risk factors for equine influenza serum antibody titres in young thoroughbred racehorses given an inactivated vaccine.

Young Thoroughbred racehorses (222 yearlings entering training and 246 2-year-old horses already in training) from eight flat-training yards in Newmarket, UK were used to monitor serological responses to vaccination with an inactivated influenza virus vaccine. Blood samples taken prior to and after vaccination were tested by single radial haemolysis (SRH) to determine antibody titres (expressed as area of haemolysis in mm(2)). Prior to vaccination, yearlings had mean antibody titres (64+/-4 mm(2)) that were approximately half of those of 2-year-olds (115+/-3 mm(2)) and 89% of yearlings and 73% of 2-year-olds had SRH titres <140 mm(2). Extrapolation from experimental and field studies suggests that these levels would not protect against homologous influenza virus infection. Both age-groups showed anamnestic responses to vaccination resulting in similar peak mean titres ( approximately 160+/-2mm(2)) with 67% of yearlings and 73% of 2-year-olds achieving levels > or =140 mm(2). A second dose of vaccine administered a month after the first in yearlings did not increase the mean titre but 75% of horses had levels of antibody > or =140 mm(2). The vaccination history in the official passport of yearlings showed that 23% had no record of previous vaccination and were probably fully susceptible to infection. For yearlings entering training, the important predictors from multiple-regression analyses of SRH titres prior to vaccination were "Time since last vaccination," "Total number of previous vaccines" and "Age at first vaccination." In 2-year-olds and following two doses of vaccine in yearlings, there was no significant relationship between these factors and SRH titre.

Advanced life support for acute toxic injury (TOXALS).

The need to consider the problem of acute toxic injury in the prehospital context emphasized by the recent use of highly toxic agents of warfare in terrorist attacks. Toxic agents differ widely in their nature but may be considered to have four distinct properties: toxicity, latency, persistency and transmissibility. Toxicity and latency determine the onset and pathophysiology of the poisoning and therefore the clinical management. Persistency and transmissibility determine the level of hazard to rescue personnel and the evacuation system and therefore the rationale of logistic management. Previously, special emphasis has been given to the importance of isolation and decontamination of the patient before any medical intervention can occur. This approach, however, although essential for the safety of medical responders may not be in the best interests of the patient who may be in a life-threatening situation within a contaminated zone (CONZONE). Toxic injury may require more rapid help than traumatic injury; moreover, traumatic and toxic injury may co-exist, as in the case of explosion with toxic emission. The special skills required are defined in the TOXALS programme and must now become a standard part of the training and practice of prehospital care medical care.