Further investigation of lead exposure as a potential threatening process for a scavenging marsupial species.

There is a growing recognition of the harmful effects of lead exposure on avian and mammalian scavengers. This can lead to both lethal and non-lethal effects which may negatively impact wildlife populations. Our objective was to assess medium-term lead exposure in wild Tasmanian devils (Sarcophilus harrisii). Frozen liver samples (n = 41), opportunistically collected in 2017-2022, were analysed using inductively coupled plasma mass spectrometry (ICP-MS) to determine liver lead concentrations. These results were then used to calculate the proportion of animals with elevated lead levels (>5 mg/kg dry weight) and examine the role of explanatory variables that may have influenced the results. The majority of samples analysed were from the south-east corner of Tasmania, within 50 km of Hobart. No Tasmanian devil samples were found to have elevated lead levels. The median liver lead concentration was 0.17 mg/kg (range 0.05-1.32 mg/kg). Female devils were found to have significantly higher liver lead concentrations than males (P = 0.013), which was likely related to lactation, but other variables (age, location, body mass) were not significant. These results suggest that wild Tasmanian devil populations currently show minimal medium-term evidence of exposure to lead pollution, although samples were concentrated in peri-urban areas. The results provide a baseline level which can be used to assess the impact of any future changes in lead use in Tasmania. Furthermore, these data can be used as a comparison for lead exposure studies in other mammalian scavengers, including other carnivorous marsupial species.

Hydroureteronephrosis and fetal mummification secondary to uterine torsion in a Merino ewe.

A 6 year old pluriparous Merino ewe was presented for investigation of a large intra-abdominal mass. Post-mortem examination revealed a 360° clockwise uterine torsion was present with a mummifying fetus. The torsion involved the left ureter resulting in a severe hydroureteronephrosis. Uterine torsion is uncommon in the ewe, occurring in less than 0.1% of pregnancies in one report (Mahmoud et al. Livest Res Rural Dev 2018;30), but cases are likely to be undiagnosed, particularly under the extensive management conditions typical of Australia. The chronicity of the condition in this ewe would support this statement. To the authors' knowledge this is the first reported case of hydroureteronephrosis secondary to uterine torsion in any species.

Invasive melanoma in vivo can be distinguished from basal cell carcinoma, benign naevi and healthy skin by canine olfaction: a proof-of-principle study of differential volatile organic compound emission.

BACKGROUND: Volatile organic compounds (VOCs) are continuously released by the body during normal metabolic processes, but their profiles change in the presence of cancer. Robust evidence that invasive melanoma in vivo emits a characteristic VOC signature is lacking. OBJECTIVES: To conduct a canine olfactory, proof-of-principle study to investigate whether VOCs from invasive melanoma are distinguishable from those of basal cell carcinoma (BCC), benign naevi and healthy skin in vivo. METHODS: After a 13-month training period, the dog's ability to discriminate melanoma was evaluated in 20 double-blind tests, each requiring selection of one melanoma sample from nine controls (three each of BCC, naevi and healthy skin; all samples new to the dog). RESULTS: The dog correctly selected the melanoma sample on nine (45%) occasions (95% confidence interval 0·23-0·68) vs. 10% expected by chance alone. A one-sided exact binomial test gave a P-value of < 0·01, supporting the hypothesis that samples were not chosen at random but that some degree of VOC signal from the melanoma samples significantly increased the probability of their detection. Use of a discrete-choice model confirmed melanoma as the most influential of the recorded medical/personal covariates in determining the dog's choice of sample. Accuracy rates based on familiar samples during training were not a reliable indicator of the dog's ability to distinguish melanoma, when confronted with new, unknown samples. CONCLUSIONS: Invasive melanoma in vivo releases odorous VOCs distinct from those of BCC, benign naevi and healthy skin, adding to the evidence that the volatile metabolome of melanoma contains diagnostically useful biomarkers.

Bayesian analysis of culture and PCR methods for detection of Campylobacter spp. in broiler caecal samples.

The objective of this study was to estimate the sensitivity and specificity of a culture method and a polymerase chain reaction (PCR) method for detection of two Campylobacter species: C. jejuni and C. coli. Data were collected during a 3-year survey of UK broiler flocks, and consisted of parallel sampling of caeca from 436 batches of birds by both PCR and culture. Batches were stratified by season (summer/non-summer) and whether they were the first depopulation of the flock, resulting in four sub-populations. A Bayesian approach in the absence of a gold standard was adopted, and the sensitivity and specificity of the PCR and culture for each Campylobacter subtype was estimated, along with the true C. jejuni and C. coli prevalence in each sub-population. Results indicated that the sensitivity of the culture method was higher than that of PCR in detecting both species when the samples were derived from populations infected with at most one species of Campylobacter. However, from a mixed population, the sensitivity of culture for detecting both C. jejuni or C. coli is reduced while PCR is potentially able to detect both species, although the total probability of correctly identifying at least one species by PCR is similar to that of the culture method.

Risk factors for antimicrobial resistance in Escherichia coli found in GB turkey flocks.

Four models are presented investigating risks present on Great Britain (GB) turkey farms in breeding and fattening flocks for ciprofloxacin and cephalosporin resistance. Risk factors for ciprofloxacin resistance in fattening flocks were sourcing of feed from national compounders, antimicrobial use in the flock and evidence of mice. Disinfection of floors and walls at depopulation, older flocks and division of the flock with partitions reduced the risk. In breeding farms holding over 10,000 birds, administration of fluoroquinolones within the last year and horses on the neighbouring farm all increased the risk, whereas replenishing foot dips more than once a week reduced the risk. For cephalosporin-resistant Escherichia coli on fattening farms, being an independent farm, having a watercourse near the poultry houses, dividing the flock with partitions and providing staff with gloves reduced the risk. Factors that increased the risk included if staff worked with other livestock and if there were pigs on neighbouring farms. This work suggests that good hygiene and biosecurity, rodent control and responsible use of antimicrobials on turkey farms might help minimise the prevalence of fluoroquinolone and cephalosporin resistance in E coli, and restrict the spread of resistance genes to other organisms.

Comparison of micro-CT and cone beam CT-based assessments for relative difference of grey level distribution in a human mandible.

OBJECTIVES: The purpose of this study was to examine the ability of CT to assess the relative difference of degree of bone mineralization (grey level) parameters in a human mandible. METHODS: Ten mandibular sections from cadavers (81.5 ± 12.1 years) were scanned using micro-CT with 27.2 µm voxel size and cone beam CT (CBCT) with 200 µm, 300 µm, and 400 µm voxel sizes. In addition, 15 clinical CBCT images from young patients (mean age 18.9 ± 3.3 years) were identified. After segmentation of bone voxels, alveolar bone and basal cortical bone regions were digitally isolated. A histogram of grey level, which is equivalent to degree of bone mineralization, was obtained from each region of the CT images. Mean, standard deviation (SD), coefficient of variation (COV), fifth percentile low (Low(5)) and high (High(5)) of alveolar bone and basal cortical bone regions were obtained. Percentage differences of grey level parameters between alveolar and basal cortical bones were computed. RESULTS: The alveolar bone region had significantly lower Mean, Low(5) and High(5) values but significantly higher SD and COV than the basal cortical bone region for all CT images (p < 0.05). All parameters were significantly lower for the old cadaver group than for the young patient group (p < 0.05). CONCLUSIONS: CBCT and micro-CT provide comparable results in the assessment of relative difference in grey level distribution between alveolar and basal cortical bone regions in the human mandible. The percentage difference relative to an internal reference (basal cortical bone) can be a reliable method when assessing the degree of bone mineralization using CBCT images for both cross-sectional and longitudinal comparisons.

On the choice of parameterisation and priors for the Bayesian analyses of Mendelian randomisation studies.

Mendelian randomisation is a form of instrumental variable analysis that estimates the causal effect of an intermediate phenotype or exposure on an outcome or disease in the presence of unobserved confounding, using a genetic variant as the instrument. A Bayesian approach allows current knowledge to be incorporated into the analysis in the form of informative prior distributions, and the unobserved confounder can be modelled explicitly. We consider Bayesian methods for Mendelian randomisation in the case where all relationships are linear and there are no interactions. A 'full' model in which the unobserved confounder is included explicitly is not completely identifiable, although the causal parameter can be estimated. We compare inferences from this general but non-identified model with a reduced parameter model that is identifiable. We show that, theoretically, additional information about the causal parameter can be obtained by using the non-identifiable full model, rather than the identifiable reduced model, but that this is advantageous only when realistically informative priors are used and when the instrument is weak or the sample size is small. Furthermore, we consider the impact of using 'vague' versus 'informative' priors.

hERG subunit composition determines differential drug sensitivity.

BACKGROUND AND PURPOSE: The majority of human ether-a-go-go-related gene (hERG) screens aiming to minimize the risk of drug-induced long QT syndrome have been conducted using heterologous systems expressing the hERG 1a subunit, although both hERG 1a and 1b subunits contribute to the K+ channels producing the repolarizing current I(Kr) . We tested a range of compounds selected for their diversity to determine whether hERG 1a and 1a/1b channels exhibit different sensitivities that may influence safety margins or contribute to a stratified risk analysis. EXPERIMENTAL APPROACH: We used the IonWorks™ plate-based electrophysiology device to compare sensitivity of hERG 1a and 1a/1b channels stably expressed in HEK293 cells to 50 compounds previously shown to target hERG channels. Potency was determined as IC50 values (µM) obtained from non-cumulative, eight-point concentration-effect curves of normalized data, fitted to the Hill equation. To minimize possible sources of variability, compound potency was assessed using test plates arranged in alternating columns of cells expressing hERG 1a and 1a/1b. KEY RESULTS: Although the potency of most compounds was similar for the two targets, some surprising differences were observed. Fluoxetine (Prozac) was more potent at blocking hERG 1a/1b than 1a channels, yielding a corresponding reduction in the safety margin. In contrast, E-4031 was a more potent blocker of hERG 1a compared with 1a/1b channels, as previously reported, as was dofetilide, another high-affinity blocker. CONCLUSIONS AND IMPLICATIONS: The current assays may underestimate the risk of some drugs to cause torsades de pointes arrhythmia, and overestimate the risk of others.

Experimental bovine spongiform encephalopathy: detection of PrP(Sc) in the small intestine relative to exposure dose and age.

European regulations for the control of bovine spongiform encephalopathy (BSE) decree destruction of the intestines from slaughtered cattle, therefore producers have been obliged to import beef casings from countries with a negligible BSE risk. This study applies immunohistochemical and biochemical approaches to investigate the occurrence and distribution of disease-associated prion protein (PrP(Sc)) in the duodenum, jejunum and ileum of cattle orally exposed to a 1 g or 100 g dose of a titrated BSE brainstem homogenate. Samples were derived from animals at various times post exposure. Lymphoid follicles were counted and the frequency of affected follicles recorded. No PrP(Sc) was detected in the duodenum or jejunum of animals exposed to a 1 g dose or in the duodenum of animals receiving a 100 g dose. PrP(Sc) was detected in the lymphoid tissue of the ileum of 1/98 (1.0%) animals receiving the 1 g dose and in the jejunum and ileum of 8/58 (13.8%) and 45/99 (45.5%), respectively, of animals receiving the 100 g dose. The frequency of PrP(Sc)- positive follicles was less than 1.5% per case and biochemical tests appeared less sensitive than immunohistochemistry. The probability of detecting lymphoid follicles in the ileum declined with age and for the 100 g exposure the proportion of positive follicles increased, while the proportion of positive animals decreased with age. Detection of PrP(Sc) in intestinal neural tissue was rare. The results suggest that the jejunum and duodenum of BSE-infected cattle contain considerably less BSE infectivity than the ileum, irrespective of exposure dose. In animals receiving the low exposure dose, as in most natural cases of BSE, the rarity of PrP(Sc) detection compared with high-dose exposure, suggests a very low BSE risk from food products containing the jejunum and duodenum of cattle slaughtered for human consumption.

Cadmium, copper, iron, and zinc concentrations in kidneys of grey wolves, Canis lupus, from Alaska, Idaho, Montana (USA) and the Northwest Territories (Canada).

Cadmium, copper, iron, and zinc levels were measured in the kidneys of 115 grey wolves (Canis lupus) from Idaho, Montana and Alaska (United States), and from the Northwest Territories (Canada). No significant differences in the levels of iron or copper were observed between locations, but wolf kidneys from more northern locations had significantly higher cadmium levels (Alaska > Northwest Territories > Montana ≈ Idaho), and wolves from Alaska showed significantly higher zinc than other locations. Additionally, female wolves in Alaska had higher iron levels than males, and adult wolves in Montana had higher copper levels than subadults.

GABA receptor rho1 subunit interacts with a novel splice variant of the glycine transporter, GLYT-1.

Ionotropic gamma-aminobutyric acid (GABA(A) and GABA(C)) receptors mediate fast synaptic inhibition in the central nervous system. GABA(C) receptors are expressed predominantly in the retina on bipolar cell axon terminals, and are thought to mediate feedback inhibition from GABAergic amacrine cells. Utilizing the yeast two-hybrid system, we previously identified MAP1B as a binding partner of the GABA(C) receptor rho1 subunit. Here we describe the isolation of an additional rho1 interacting protein: a novel C-terminal variant of the glycine transporter GLYT-1. We show that GLYT-1 exists as four alternatively spliced mRNAs which encode proteins expressing one of two possible intracellullar N- and C-terminal domains. Variants containing the novel C terminus efficiently transport glycine when expressed in COS cells, but with unusual kinetics. We have confirmed the interaction between the novel C terminus and rho1 subunit and demonstrated binding in heterologous cells. This interaction may be crucial for the integration of GABAergic and glycinergic neurotransmission in the retina.

Molecular cloning and pharmacological characterization of a somatostatin receptor subtype in the gymnotiform fish Apteronotus albifrons.

The actions of the various forms of somatostatin (SRIF), including those of the tetradecapeptide SRIF(14), are mediated by specific receptors. In mammals, five subtypes of SRIF receptors, termed sst(1-5), have been cloned. Using a combination of reverse transcriptase-polymerase chain reaction and genomic library screening in the gymnotiform fish Apteronotus albifrons, a gene encoding the first-known nonmammalian SRIF receptor has been isolated. The deduced amino acid sequence displays 59% identity with the human sst(3) receptor protein; hence, the gene is termed "Apteronotus sst(3)." The predicted protein consists of 494 amino acid residues exhibiting a putative seven-transmembrane domain topology typical of G protein-coupled receptors. A signal corresponding to the Apteronotus sst(3) receptor was detected in brain after amplification of poly(A)(+)-RNA by reverse transcriptase-polymerase chain reaction, but not by Northern blot analysis or in situ hybridization, suggesting a low level of expression. Membranes prepared from CCL39 cells stably expressing the Apteronotus sst(3) receptor gene bound [(125)I][Leu(8),d-Trp(22), (125) I-Tyr(25)]SRIF(28) with high affinity and in a saturable manner (B(max) = 4470 fmol/mg protein; pK(D) = 10.5). SRIF(14) and various synthetic SRIF receptor agonists produced a dose-dependent inhibition of radioligand binding, with the following rank order of potency: SRIF(14) approximately SRIF(28) > BIM 23052 > octreotide > BIM 23056. Under low stringency conditions, an Apteronotus sst(3) probe hybridized to multiple DNA fragments in HindIII or EcoRI digests of A. albifrons DNA, indicating that the Apteronotus sst(3) receptor is a member of a larger family of Apteronotus SRIF receptors.

The role of Ca2+-activated K+ channel spliced variants in the tonotopic organization of the turtle cochlea.

1. Turtle auditory hair cells contain multiple isoforms of the pore-forming alpha-subunit of the large-conductance Ca2+-activated K+ (KCa) channel due to alternative splicing at two sites. Six splice variants were studied by expression in Xenopus oocytes. 2. The isoforms possessed differences in apparent Ca2+ sensitivity and kinetics. The lowest Ca2+ sensitivity was observed in a novel variant resulting from a 26 amino acid deletion around one of the splice sites. 3. Co-expression of a bovine beta-subunit slowed the current relaxation 10-fold compared with channels formed from alpha-subunits alone but preserved the original order of kinetic differences. The beta-subunit also increased the Ca2+ sensitivity of isoforms to bring them nearer the range of sensitivity of the native KCa channels of the hair cell. 4. With channels formed from alpha-subunits or alpha + beta-subunits, the half-activation voltage in a fixed Ca2+ concentration, and the time constant of the current relaxation, varied linearly with the combined size of the insertions/deletions at the splice sites. 5. Experiments in which the beta/alpha concentration ratio was varied indicated that the beta-subunit exerts an all-or-none effect on the Ca2+ sensitivity and kinetics of the channel. 6. Co-expression of an avian beta2-subunit had effects on kinetics and Ca2+ sensitivity of several alpha-isoforms which were qualitatively similar to those produced by the bovine beta-subunit. 7. We conclude that differential expression of alternatively spliced alpha-subunit variants and a non-uniform distribution of a beta-subunit can produce a range of KCa channel properties needed to explain the tonotopic organization of the turtle cochlea.

The functional role of alternative splicing of Ca(2+)-activated K+ channels in auditory hair cells.

Turtle auditory hair cells are frequency tuned by the activity of large-conductance calcium-activated potassium (KCa) channels, the frequency range being dictated primarily by the channel kinetics. Seven alternatively spliced isoforms of the KCa channel alpha-subunit, resulting from exon insertion at two splice sites, were isolated from turtle hair cells. These, when expressed in Xenopus oocytes, produced KCa channels with a range of apparent calcium sensitivities and channel kinetics. However, most expressed channels were less calcium sensitive than the hair cells' native KCa channels. Coexpression of alpha-subunit with a bovine beta-subunit substantially increased the channel's calcium sensitivity while markedly slowing its kinetics, but kinetic differences between isoforms were preserved. These data suggest a molecular mechanism for hair cell frequency tuning involving differential expression of different KCa channel alpha-subunits in conjunction with an expression gradient of a regulatory beta-subunit.

Transgenic expression of epidermal growth factor and keratinocyte growth factor in beta-cells results in substantial morphological changes.

The upregulation of a limited number of growth factors in our interferon-gamma transgenic model for regeneration within the pancreas lead us to propose that these factors are important during pancreatic regeneration. In this study, we have assessed the influence of two growth factors within the pancreas, epidermal growth factor (EGF) and keratinocyte growth factor (KGF), by ectopically expressing these proteins under the control of the human insulin promoter in transgenic mice. This beta-cell-targeted expression of either EGF or KGF resulted in significant morphological changes, including cellular proliferation and disorganized islet growth. Intercrossing the individual Ins-EGF and Ins-KGF transgenic mice resulted in more profound changes in pancreatic morphology including proliferation of pancreatic cells and extensive intra-islet fibrosis. Insulin-producing beta-cells were found in some of the ducts of older Ins-EGF and Ins-EGFxKGF transgenic mice, and amylase-producing cells were observed within the islet structures of the double transgenic mice. These data suggest that both EGF and KGF are capable of affecting pancreatic differentiation and growth, and that co-expression of these molecules in islets has a more substantial impact on the pancreas than does expression of either growth factor alone.

Pancreatic expression of keratinocyte growth factor leads to differentiation of islet hepatocytes and proliferation of duct cells.

Keratinocyte growth factor, (KGF), a member of the fibroblast growth factor (FGF) family, is involved in wound healing. It also promotes the differentiation of many epithelial tissues and proliferation of epithelial cells as well as pancreatic duct cells. Additionally, many members of the highly homologous FGF family (including KGF), influence both growth and cellular morphology in the developing embryo. We have previously observed elevated levels of KGF in our interferon-gamma transgenic mouse model of pancreatic regeneration. To understand the role of KGF in pancreatic differentiation, we generated insulin promoter-regulated KGF transgenic mice. Remarkably, we have found that ectopic KGF expression resulted in the emergence of hepatocytes within the islets of Langerhans in the pancreas. Additionally, significant intra-islet duct cell proliferation in the pancreata of transgenic KGF mice was observed. The unexpected appearance of hepatocytes and proliferation of intra-islet duct cells in the pancreata of these mice evidently stemmed directly from local exposure to KGF.

Identification of Ca(2+)-activated K+ channel splice variants and their distribution in the turtle cochlea.

Turtle auditory-hair cells are frequency-tuned by the activity of calcium-activated potassium (KCa) channels, a cell's characteristic frequency being determined by the KCa channel density and kinetics which both vary systematically along the cochlea. As a first step towards identifying the source of KCa channel variation, we have isolated, by reverse-transcription polymerase chain reaction on dissociated hair cells, the main cDNAs homologous to the slo gene which encodes the channel's alpha-subunit. A total of six alternatively spliced variants were identified, the smallest of which is 94% identical to a mouse Slo sequence. Variation occurs by insertion of exons at only two splice sites, two of these exons encoding novel 31- and 61-amino acid sequences. As we were unable to detect splicing at other potential sites, we infer that the six variants correspond to naturally occurring combinations. The spatial distribution of the variants, defined by isolating hair cells from different regions of the cochlea, indicated that some isoforms were non-uniformly distributed. Those containing large inserts in the first splice site were notably absent from the highest-frequency region. We suggest that alternative splicing of the slo gene may contribute to variation in KCa channel properties.