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.

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.

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.

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.

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.

Reduced brain glucose with normal plasma glucose in salicylate poisoning.

After the intraperitoneal injection into young mice of 700-800 mg/kg of salicylate, brain glucose fell to one-third or less of control values despite normal plasma glucose levels; brain lactate was nearly doubled and there were small decreases in phosphocreatine (18%) and in glycogen (17%). ATP, pyruvate, alpha-ketoglutarate, and glutamate were unchanged. In liver, glycogen was reduced 79% and lactate was five times higher than in control animals; glucose, glucose-6-phosphate, and ATP were unchanged. Since salicylate uncouples oxidative phosphorylation, it is postulated that high energy phosphate in the brain is maintained near normal levels by a compensatory increase in cerebral glycolysis. Apparently the brain glucose level falls because the rate of utilization exceeds the rate at which glucose can be supplied from the blood. Concurrent administration of glucose with salicylate elevated brain glucose concentration and was associated with striking improvement in the condition and the increased survival of the animals. These findings stress the fact that in salicylate poisoning the supply of glucose to the brain may be inadequate even when blood glucose levels are normal.

Sources of carbon for hepatic glycogen synthesis in the conscious dog.

To identify the source(s) of carbon for the indirect pathway of hepatic glycogen synthesis, we studied nine 42-h fasted conscious dogs given a continuous intraduodenal infusion of glucose, labeled with [1-13C]glucose and [3-3H]glucose, at 8 mg.kg-1.min-1 for 240 min. Glycogen formation by the direct pathway was measured by 13C-NMR. Net hepatic balances of glucose, gluconeogenic amino acids, lactate, and glycerol were determined using the arteriovenous difference technique. During the steady-state period (the final hour of the infusion), 81% of the glucose infused was absorbed as glucose. Net gut output of lactate and alanine accounted for 5% and 3% of the glucose infused, respectively. The cumulative net hepatic uptakes were: glucose, 15.5 +/- 3.8 g; gluconeogenic amino acids, 32.2 +/- 2.2 mmol (2.9 +/- 0.2 g of glucose equivalents); and glycerol, 6.1 +/- 0.9 mmol (0.6 +/- 0.1 g of glucose equivalents). The liver produced a net of 29.2 +/- 9.6 mmol of lactate (2.6 +/- 0.8 g of glucose equivalents). Net hepatic glycogen synthesis totaled 9.3 +/- 2.5 g (1.8 +/- 0.4 g/100 g liver), with the direct pathway being responsible for 57 +/- 10%. Thus, net hepatic glucose uptake was sufficient to account for all glycogen formed by both the direct and indirect pathways. Total net hepatic uptake of gluconeogenic precursors (gluconeogenic amino acids, glycerol, and lactate) was able to account for only 20% of net glycogen synthesis by the indirect pathway. In a net sense, our data are consistent with an intrahepatic origin for most of the three-carbon precursors used for indirect glycogen synthesis.

Counterregulation during hypoglycemia is directed by widespread brain regions.

Previous studies have demonstrated the importance of the brain in directing counterregulation during insulin-induced hypoglycemia in dogs. The capability of selective carotid or vertebrobasilar hypoglycemia in triggering counterregulation was assessed in this study using overnight-fasted dogs. Insulin (21 pM.kg-1.min-1) was infused for 3 h to create peripheral hypoglycemia in the presence of 1) selective carotid hypoglycemia (vertebral glucose infusion, n = 5), 2) selective vertebrobasilar hypoglycemia (carotid glucose infusion, n = 5), 3) the absence of brain hypoglycemia (carotid and vertebral glucose infusion, n = 4), or 4) total brain hypoglycemia (no head glucose infusion, n = 5). Glucose was infused via a leg vein as needed in each group to minimize the differences in peripheral glucose levels (2.6 +/- 0.1, 3.0 +/- 0.2, 2.7 +/- 0.1, and 2.5 +/- 0.1 mM, respectively). The humoral responses (cortisol, glucagon, catecholamines, and pancreatic polypeptide) to hypoglycemia were minimally attenuated (< 40%) by selective carotid or vertebrobasilar euglycemia. In addition, the increase in hepatic glucose production, as assessed using [3-3H]glucose, was attenuated by only 41 and 34%, respectively, during selective carotid or vertebrobasilar hypoglycemia. These observations offer support for the hypothesis that more than one center is important in hypoglycemic counterregulation in the dog and that they are located in brain regions supplied by the carotid and vertebrobasilar arteries, because significant counterregulation occurred when hypoglycemia developed in either of these circulations. Counterregulation during hypoglycemia, therefore, is probably directed by widespread brain regions that contain glucose-sensitive neurons such that the sensing sites are redundant.

Adenosine diphosphate ribosylation of fibroblast growth factor-2.

Basic fibroblast growth factor (FGF-2) is posttranslationally modified by the enzymatic transfer of ADP-ribose from nicotinamide adenine dinucleotide (NAD). When sonicated nuclei of adrenal capillary endothelial or SK-Hep1 cells are incubated with [32P]NAD, FGF-2 is rapidly ADP-ribosylated in a dose- and time-dependent fashion. Proteins structurally related to FGF-2 (FGF-6 and -7) are readily modified, suggesting that they share a common substrate motif. Yet, FGF-1, the most structurally homologous member of the FGF family, is a poor substrate. The reaction is also specific; interleukin-1 alpha, transforming growth factor-alpha, nerve growth factor, insulin-like growth factor-I, and granulocyte macrophage-colony stimulating factor are not substrates for ribosylation. Because the ADP ribosylation of FGF-2 is acid resistant but base and hydroxylamine sensitive, the linkage appears to be mediated through arginine. Most importantly, however, we also establish that endogenous FGF-2 is a substrate for ribosylation. As such, an immunoreactive ADP-ribosylated FGF-2 is detected in extracts of SK-Hep1 nuclei when they are incubated with [32P]NAD. Taken together, these findings suggest that the role played by ADP ribosylation in signal transduction, DNA repair, the control of the cell cycle, and cell differentiation may involve its ability to target molecules such as FGF-2.

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.

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.

Ocular penetration of topical chloramphenicol in humans.

Commercial ophthalmic chloramphenicol solutions and ointments were applied to human eyes, and the concentrations of chloramphenicol in aqueous humor and tear samples were analyzed using gas-liquid chromatography. Within several minutes after a single application of 0.5% chloramphenicol solution, the tear concentrations of the drug had fallen below 1 mg/liter, the minimal bacteriostatic concentration for many ocular pathogens. Repeated drops of 0.5% chloramphenicol solutions during several hours were required to produce an aqueous humor concentration of 1 mg/liter. A single application of 1% chloramphenicol ointment gave prolonged drug concentrations in the tears and aqueous humor, falling to 1 mg/liter in two to four hours. The repeated use of chloramphenicol solution or ointment was well tolerated by the patients. We conclude that the penetration of chloramphenicol in the anterior segment of the eye is best carried out by the repeated application of chloramphenicol ointment.

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.

Expression of major histocompatibility complex antigens and adhesion molecules in hearts of patients with chronic Chagas' disease.

We have previously reported that heart lesions in patients with chronic cardiac Chagas' disease are composed predominantly of granzyme A+, cytolytic CD8+ T lymphocytes. We now pursue this study in the immunopathology of chronic chagasic cardiomyopathy by investigation of the expression of HLA antigens, and adhesion molecules in the hearts of seven chagasic patients with cardiac disease, two asymptomatic chagasic patients, and seven normal controls. Comparative immunohistochemical analyses show that HLA-ABC antigen expression is enhanced on the myocardial cells of chagasic patients with chronic cardiomyopathy, suggesting a possible role for these cells as targets for the CD8+ cytolytic lymphocytes dominant in these lesions. The HLA-DR antigens are not observed on myocardial cells, but are consistently upregulated on the endothelial cells in the hearts of patients with chronic chagasic cardiomyopathy. Intercellular adhesion molecule is expressed by endothelial cells of both chagasic and nonchagasic individuals, but E-selectin was detected only on vessels of hearts from chagasic patients who had chronic cardiomyopathy. Most of the lymphocytes in these lesions express lymphocyte function antigen-1 (LFA-1), CD44, and very late antigen-4, and a few display weak expression of LFA-3. We propose that the expression of these adhesion molecules and major histocompatibility complex antigens by endothelial cells, myocardial cells, and lymphoid cells in these lesions contribute to the pathogenesis of chronic chagasic cardiomyopathy.

Amplification of a Trypanosoma cruzi DNA sequence from inflammatory lesions in human chagasic cardiomyopathy.

The major cause of morbidity and mortality in Chagas' disease is a chronic inflammatory cardiomyopathy, which presents ten or more years following initial infection. Demonstration of Trypanosoma cruzi in cardiac tissue by routine microscopy or culture is difficult in these patients, which has suggested that persistent organisms are not required for chronic disease. Consequently, studies have focused on elucidating an autoimmune pathogenesis of chronic injury. To further assess the persistence of T. cruzi in host tissue, DNA extracted from formalin-fixed, paraffin-embedded autopsy specimens from seronegative or seropositive patients was amplified by the polymerase chain reaction using T. cruzi-specific primers. Trypanosoma cruzi DNA sequences were not consistently amplified from four seropositive patients who lacked evidence of fatal chronic chagasic cardiomyopathy (CCC) (0 positive of 12 heart samples, 0 positive of four gonadal samples, and 0 positive of four adrenal samples) or nine seronegative patients (0 positive of 27 heart samples, 0 positive of nine gonadal samples and 0 positive of nine adrenal samples). In seven seropositive patients with severe CCC, cardiac tissue adjacent to inflammatory infiltrates yielded amplified T. cruzi DNA sequences in 18 of 21 heart samples. Parallel testing of gonadal and adrenal tissues from these same patients produced detectable T. cruzi DNA in none of the gonadal tissue samples and one of the seven adrenals. Our studies demonstrate that T. cruzi, or a portion of its genome, is present in the inflammatory lesion of chronic cardiac Chagas' disease.

Characterization of inflammatory infiltrates in chronic chagasic myocardial lesions: presence of tumor necrosis factor-alpha+ cells and dominance of granzyme A+, CD8+ lymphocytes.

The inflammatory infiltrates in the heart lesions of chronic chagasic cardiomyopathy are composed predominantly of small lymphocytes with admixed macrophages, plasma cells, and segmented leukocytes. The phenotypes of the lymphoid cells in these infiltrates of human Chagas' disease have not been previously detailed. We used a panel of monoclonal and polyclonal antibodies to immunohistochemically characterize the inflammatory cells in frozen and fixed cardiac tissues from autopsied patients with severe chronic chagasic cardiomyopathy. In all cases, the inflammatory lesions were dominated by CD8+ lymphocytes, many of which expressed granzyme A. A few macrophage-like cells that expressed tumor necrosis factor-alpha were observed in each case. Relatively few natural killer cells or B lymphocytes were found in the lesions. These findings in human chagasic lesions are compatible with concepts that involve cytolysis and fibrosis, and new experimental findings that emphasize potential roles for CD8+ T cells in Chagas' disease.

Salt tolerance of EMRSA-16 and its effect on the sensitivity of screening cultures.

The salt (NaCl) tolerance of methicillin-resistant Staphylococcus aureus (EMRSA)-16 was compared with 18 other MRSA isolates by an agar incorporation technique. The NaCl minimum inhibitory concentration (MIC) of EMRSA-16 was 7% which compared with an MIC50 of 7%, MIC90 of 10%, range (5.5-10.5%) for the other isolates. Study of the growth kinetics in broth containing NaCl at concentrations up to 10% indicated complete inhibition of growth by 7 and 10% NaCl and partial inhibition by 5%. Addition of EMRSA-16 at inocula of < or = 1 cfu/mL into salt broths revealed lower than expected EMRSA recovery from broths containing 5, 7.5 and 10% NaCl. Two and a half per cent NaCl broths were not inhibitory. Selective broth containing 2.5% NaCl should be considered for use when screening for EMRSA-16.

Control of varicella-zoster infection on renal and other specialist units.

The introduction of chickenpox onto our renal unit recently raised several issues surrounding the management of patient and staff contracts. This paper describes the action taken and makes various recommendations for future management of similar cases. Guidelines are proposed for the management of patients and staff as well as the role of the infection control team in handling a chickenpox problem. Future developments, including the use of VZ vaccine for patient and staff, are also discussed.

Age differences in conversational source monitoring.

The present investigation simulated a group conversation in which participants asked (inquirer) and answered (responder) questions, as well as listened to others exchange information. Source (inquirer; responder) identification accuracy was evaluated immediately or after 1 week. Older adults were less adept at source identification, although this difference was reduced with personal (Experiment 2) rather than categorical (Experiment 1) topics. The age difference was independent of explicit memory (cued recall and recognition), suggesting that memory for source and information are separable. Older adults were comparable to younger adults in responder identification but worse at inquirer identification. Responder identification was better than inquirer identification, with the latter dropping to chance at 1 week. Source identification was most accurate when participants were in the responder role; there was little difference between the inquirer and listener roles.