Mental health in the veterinary profession: an individual or organisational focus?

The veterinary profession is experiencing a shortage of veterinarians, with attrition recognised as a substantial contributor. Research has also indicated increased levels of mental ill health and alarming suicide rates in practitioners. The primary aim of this study was to investigate the impact of eleven modifiable workplace factors on mental health outcomes, job appreciation and intention to leave the veterinary profession. The second aim was to ascertain whether workplace factors influence mental health outcomes after controlling for individual resilience. An online survey was completed by 73 practising Australian veterinarians. Unfavourable workplace factors correlated with adverse outcomes including depression, stress, reduced job appreciation and increased likelihood of leaving both the role and the profession. Workplace factors remained linked with the outcomes of job appreciation, depression and stress whilst controlling for practitioner resilience. Job appreciation was a significant predictor of intention to leave both the current role and the profession. Via multiple linear regression, two categories were identified as associated with improved psychological outcomes and job appreciation. These were workplace factors that represent breaks from workload and control or decision latitude in the workplace. Whilst resilience represents a key area for intervention, workplace factors potentially represent an easier-to-modify area for intervention.

Review: Physiological growth trend of current meat broilers and dietary protein and energy management approaches for sustainable broiler production.

The food production system needs to be sustainable including poultry sector to feed the increasing global population. An accepted economical and environmental approach of broiler production is to produce larger broilers faster while using less feed. Broiler production is aimed at producing consumable meat and meat products. The global broiler meat market has evolved over the years with increasing selection pressure shifted toward attaining yield characteristics for increased cut-up parts such as breast and thighs. There is a shift toward a big bird market in the U.S. with approximately 70% of the broiler meat produced from large birds (>2.72 kg). Genetic selection of broilers for quantitative traits such as growth rate and lean muscle mass without increasing the fat mass has altered broiler physiological homeostasis to adapt toward the larger rates of muscle protein turnover. Physiological stresses created due to selection pressures in broilers have produced several muscle myopathies including an emerging one called woody breast myopathy. The sustainable broiler production practice may require humane consideration of raising broilers in less stressful grow-out regimes that will have minimal impact on broiler metabolic health. Another sustainability approach of broiler production toward feed efficiency lies on understanding dietary formulation approach of amino acids and energy that promote optimal nutrient utilization and minimal nutrient output to environment while also fulfilling the growth demands and body composition changes associated with increased protein gain in current meat broilers brought by the genetic progress.

Non-cellulosic polysaccharide content in feed ingredients and ileal and total tract non-cellulosic polysaccharide digestibility in 21- and 42-day-old broilers fed diets with and without added composite enzymes.

An initial study profiled non-cellulosic polysaccharide (NCP) levels in feed ingredient samples-corn, soybean meal (SBM), whole soybean, and distillers dried grains with solubles (DDGS). A separate NCP digestibility assay was performed in broilers at day 21 (grower phase) and day 42 (finisher phase) fed corn-soy based diets with and without composite enzymes (phytases, multi-carbohydrases, and proteases). Negative control (NC), NC + composite enzymes (NC+E), and positive control (PC) diets were tested. Negative control and NC + E diets were isocaloric, 3,020 kcal/kg ME at grower phase and 3,026 kcal/kg ME at finisher phase. Positive control diet was formulated to meet the Cobb standard nutrient specifications. Diets, pooled digesta, and excreta samples from all treatment diets were collected from 21- and 42-day-old birds and NCP content analyzed as soluble NCP (S-NCP) and insoluble NCP (I-NCP) fractions. Digestibility coefficient (DC) values were determined for all dietary treatments for both the feeding periods. Results from the ingredient analysis showed NCP levels of ∼7 to 10% in corn samples, ∼8 to 11% in SBM samples, ∼11 to 14% (including pectin level) in whole soybean, and ∼12 to 17% in DDGS samples, suggesting variation (P < 0.05) in NCP levels existed within ingredient samples. Digestibility assays showed that enzyme treated (NC + E) diet improved DC values at day 21 from 6 to 10 units and 6 to 9 units for ileal and total tract collection, respectively, for I-NCP fraction (P < 0.05) compared to DC values for NC or PC diets. The ileal DC values at day 42 were not different between treatment diets (∼0.6) but total tract DC values improved 9 to 11 units for broilers fed NC + E diet compared to NC or PC diets. Higher DC values for S-NCP were observed for all treatments for both feeding periods (∼0.7-ileal and ∼0.8-total tract) compared to DC values for I-NCP. Overall, the use of supplemental enzymes in corn-soy-based diets showed improvement in total NCP digestion.

A patient tumour-on-a-chip system for personalised investigation of radiotherapy based treatment regimens.

Development of personalised cancer models to predict response to radiation would benefit patient care; particularly in malignancies where treatment resistance is prevalent. Herein, a robust, easy to use, tumour-on-a-chip platform which maintains precision cut head and neck cancer for the purpose of ex vivo irradiation is described. The device utilises sintered discs to separate the biopsy and medium, mimicking in vivo microvascular flow and diffusion, maintaining tissue viability for 68 h. Integrity of tissues is demonstrated by the low levels of lactate dehydrogenase release and retained histology, accompanied by assessment of cell viability by trypan blue exclusion and flow cytometry; fluid dynamic modelling validates culture conditions. An irradiation jig is described for reproducible delivery of clinically-relevant doses (5 × 2 Gy) to newly-presenting primary tumours (n = 12); the addition of concurrent cisplatin is also investigated (n = 8) with response analysed by immunohistochemistry. Fractionated irradiation reduced proliferation (BrdU, p = 0.0064), increased DNA damage (Æ´H2AX, p = 0.0043) and caspase-dependent apoptosis (caspase-cleaved cytokeratin-18) compared to control; caspase-dependent apoptosis was further increased by concurrent cisplatin compared to control (p = 0.0063). This is a proof of principle study showing the response of cancer tissue to irradiation ex vivo in a bespoke system. The novel platform described has the potential to personalise treatment for patients in a cost-effective manner with applicability to any solid tumour.

Identification of Cytauxzoon felis antigens via protein microarray and assessment of expression library immunization against cytauxzoonosis.

BACKGROUND: Cytauxzoonosis is a disease of felids in North America caused by the tick-transmitted apicomplexan parasite Cytauxzoon felis. Cytauxzoonosis is particularly virulent for domestic cats, but no vaccine currently exists. The parasite cannot be cultivated in vitro, presenting a significant limitation for vaccine development. METHODS: Recent sequencing of the C. felis genome has identified over 4300 putative protein-encoding genes. From this pool we constructed a protein microarray containing 673 putative C. felis proteins. This microarray was probed with sera from C. felis-infected and naïve cats to identify differentially reactive antigens which were incorporated into two expression library vaccines, one polyvalent and one monovalent. We assessed the efficacy of these vaccines to prevent of infection and/or disease in a tick-challenge model. RESULTS: Probing of the protein microarray resulted in identification of 30 differentially reactive C. felis antigens that were incorporated into the two expression library vaccines. However, expression library immunization failed to prevent infection or disease in cats challenged with C. felis. CONCLUSIONS: Protein microarray facilitated high-throughput identification of novel antigens, substantially increasing the pool of characterized C. felis antigens. These antigens should be considered for development of C. felis vaccines, diagnostics, and therapeutics.

NNT reverse mode of operation mediates glucose control of mitochondrial NADPH and glutathione redox state in mouse pancreatic ß-cells.

OBJECTIVE: The glucose stimulation of insulin secretion (GSIS) by pancreatic ß-cells critically depends on increased production of metabolic coupling factors, including NADPH. Nicotinamide nucleotide transhydrogenase (NNT) typically produces NADPH at the expense of NADH and ΔpH in energized mitochondria. Its spontaneous inactivation in C57BL/6J mice was previously shown to alter ATP production, Ca2+ influx, and GSIS, thereby leading to glucose intolerance. Here, we tested the role of NNT in the glucose regulation of mitochondrial NADPH and glutathione redox state and reinvestigated its role in GSIS coupling events in mouse pancreatic islets. METHODS: Islets were isolated from female C57BL/6J mice (J-islets), which lack functional NNT, and genetically close C57BL/6N mice (N-islets). Wild-type mouse NNT was expressed in J-islets by adenoviral infection. Mitochondrial and cytosolic glutathione oxidation was measured with glutaredoxin 1-fused roGFP2 probes targeted or not to the mitochondrial matrix. NADPH and NADH redox state was measured biochemically. Insulin secretion and upstream coupling events were measured under dynamic or static conditions by standard procedures. RESULTS: NNT is largely responsible for the acute glucose-induced rise in islet NADPH/NADP+ ratio and decrease in mitochondrial glutathione oxidation, with a small impact on cytosolic glutathione. However, contrary to current views on NNT in ß-cells, these effects resulted from a glucose-dependent reduction in NADPH consumption by NNT reverse mode of operation, rather than from a stimulation of its forward mode of operation. Accordingly, the lack of NNT in J-islets decreased their sensitivity to exogenous H2O2 at non-stimulating glucose. Surprisingly, the lack of NNT did not alter the glucose-stimulation of Ca2+ influx and upstream mitochondrial events, but it markedly reduced both phases of GSIS by altering Ca2+-induced exocytosis and its metabolic amplification. CONCLUSION: These results drastically modify current views on NNT operation and mitochondrial function in pancreatic ß-cells.

Demographic and mortality analysis of hospitalized children at a referral hospital in Addis Ababa, Ethiopia.

BACKGROUND: Global childhood mortality rates remain high. Millennium Development Goal 4 focused efforts on reducing rates by two-thirds between 1990 and 2015. In Ethiopia, child mortality rates dropped 71 % from 1990 to 2015, however it is estimated that 184,000 Ethiopian children die each year. There is limited information about pediatric hospital admissions in Ethiopia. Our aims were to examine the temporal relationship of mortality to admission, describe the demographics, and identify cause mortality of children admitted to the Zewditu Memorial Hospital (ZMH). METHODS: A four-year retrospective review of pediatric admissions was conducted at the pediatric emergency room and pediatric hospital ward at ZMH in Addis Ababa, Ethiopia. Admission entries from 2011-2014 of children age 29 days-14 years were reviewed. Age, gender, admission date, disease classification, discharge status and date were obtained. Patient gender was compared using Chi-square analysis. A descriptive analysis was used for age and cause mortality. RESULTS: A total of 6866 patient entries were reviewed. The proportion of admissions younger than age 5 was 0.747 (95 % CI 0.736-0.757). Overall mortality was 0.042 (95 % CI, 0.037-0.047). The proportion of recorded deaths occurring within 2 days of admission was 0.437 (95 % CI 0.380-0.494). The proportion of male admissions was significantly higher than female admissions in all age groups (male 0.575, p < 0.0001, 95 % CI 0.562-0.586). The main causes of mortality were pneumonia (0.253, 95 % CI, 0.203-0.303), severe acute malnutrition (0.222, 95 % CI 0.174-0.27), HIV/AIDS-related complications (0.056, 95 % CI 0.029-0.083), spina bifida (0.049, 95 % CI 0.024-0.074), and hydrocephalus (0.045, 95 % CI 0.021-0.069). CONCLUSIONS: Our study revealed a lower mortality rate than previously reported in Ethiopia. Despite this, 44 % of pediatric hospital mortality occurred early during hospitalization, higher than reported at other Ethiopian hospitals. This adds further evidence that systematic efforts should be dedicated to improve pediatric emergency care. Admissions included 58 % male patients, similar to other reports in Ethiopia implying that this may be a nation-wide phenomenon. The observed disparity may be due to societal factors regarding care-seeking behaviors or male predilection for respiratory illness warranting further investigation. Cause mortality patterns were similar to reports in analogous settings.

Acute nutrient regulation of the mitochondrial glutathione redox state in pancreatic ß-cells.

The glucose stimulation of insulin secretion by pancreatic ß-cells depends on increased production of metabolic coupling factors, among which changes in NADPH and ROS (reactive oxygen species) may alter the glutathione redox state (EGSH) and signal through changes in thiol oxidation. However, whether nutrients affect EGSH in ß-cell subcellular compartments is unknown. Using redox-sensitive GFP2 fused to glutaredoxin 1 and its mitochondria-targeted form, we studied the acute nutrient regulation of EGSH in the cytosol/nucleus or the mitochondrial matrix of rat islet cells. These probes were mainly expressed in ß-cells and reacted to low concentrations of exogenous H2O2 and menadione. Under control conditions, cytosolic/nuclear EGSH was close to -300 mV and unaffected by glucose (from 0 to 30 mM). In comparison, mitochondrial EGSH was less negative and rapidly regulated by glucose and other nutrients, ranging from -280 mV in the absence of glucose to -299 mV in 30 mM glucose. These changes were largely independent from changes in intracellular Ca(2+) concentration and in mitochondrial pH. They were unaffected by overexpression of SOD2 (superoxide dismutase 2) and mitochondria-targeted catalase, but were inversely correlated with changes in NAD(P)H autofluorescence, suggesting that they indirectly resulted from increased NADPH availability rather than from changes in ROS concentration. Interestingly, the opposite regulation of mitochondrial EGSH and NAD(P)H autofluorescence by glucose was also observed in human islets isolated from two donors. In conclusion, the present study demonstrates that glucose and other nutrients acutely reduce mitochondrial, but not cytosolic/nuclear, EGSH in pancreatic ß-cells under control conditions.

Dynamic measurements of mitochondrial hydrogen peroxide concentration and glutathione redox state in rat pancreatic ß-cells using ratiometric fluorescent proteins: confounding effects of pH with HyPer but not roGFP1.

Using the ROS (reactive oxygen species)-sensitive fluorescent dyes dichlorodihydrofluorescein and dihydroethidine, previous studies yielded opposite results about the glucose regulation of oxidative stress in insulin-secreting pancreatic ß-cells. In the present paper, we used the ratiometric fluorescent proteins HyPer and roGFP1 (redox-sensitive green fluorescent protein 1) targeted to mitochondria [mt-HyPer (mitochondrial HyPer)/mt-roGFP1 (mitochondrial roGFP1)] to monitor glucose-induced changes in mitochondrial hydrogen peroxide concentration and glutathione redox state in adenovirus-infected rat islet cell clusters. Because of the reported pH sensitivity of HyPer, the results were compared with those obtained with the mitochondrial pH sensors mt-AlpHi and mt-SypHer. The fluorescence ratio of the mitochondrial probes slowly decreased (mt-HyPer) or increased (mt-roGFP1) in the presence of 10 mmol/l glucose. Besides its expected sensitivity to H2O2, mt-HyPer was also highly pH sensitive. In agreement, changes in mitochondrial metabolism similarly affected mt-HyPer, mt-AlpHi and mt-SypHer fluorescence signals. In contrast, the mt-roGFP1 fluorescence ratio was only slightly affected by pH and reversibly increased when glucose was lowered from 10 to 2 mmol/l. This increase was abrogated by the catalytic antioxidant Mn(III) tetrakis (4-benzoic acid) porphyrin but not by N-acetyl-L-cysteine. In conclusion, due to its pH sensitivity, mt-HyPer is not a reliable indicator of mitochondrial H2O2 in ß-cells. In contrast, the mt-roGFP1 fluorescence ratio monitors changes in ß-cell mitochondrial glutathione redox state with little interference from pH changes. Our results also show that glucose acutely decreases rather than increases mitochondrial thiol oxidation in rat ß-cells.

Is childhood OCD a risk factor for eating disorders later in life? A longitudinal study.

BACKGROUND: It has been suggested that childhood obsessive-compulsive disorder (OCD) may be a risk factor for the development of an eating disorder (ED) later in life, but prospective studies are lacking. We aimed to determine the prevalence of ED at follow-up and clinical predictors in a longitudinal clinical sample of adolescents/young adults diagnosed with OCD in childhood. METHOD: All contactable (n=231) young people with OCD assessed over 9 years at a national and specialist paediatric OCD clinic were included in this study. At follow-up, 126 (57%) young people and parents completed the ED section of the Developmental and Well-being Assessment. Predictors for ED were investigated using logistic regression. RESULTS: In total, 16 participants (12.7%) had a diagnosis of ED at follow-up. Having an ED was associated with female gender and persistent OCD at follow-up. There was a trend for family history of ED being predictive of ED diagnosis. Five (30%) of those who developed an ED at follow-up had ED symptoms or food-related obsessions/compulsions at baseline. A difference in predictors for an ED versus other anxiety disorders at follow-up was identified. CONCLUSIONS: This study provides initial evidence that baseline clinical predictors such as female gender and family history of ED might be specific to the later development of ED in the context of childhood OCD. Clinicians should be alert to ED subthreshold symptoms in young girls presenting with OCD. Future longitudinal studies are needed to clarify the relationship between childhood OCD and later ED.

Arachidonic acid actions on functional integrity and attenuation of the negative effects of palmitic acid in a clonal pancreatic ß-cell line.

Chronic exposure of pancreatic ß-cells to saturated non-esterified fatty acids can lead to inhibition of insulin secretion and apoptosis. Several previous studies have demonstrated that saturated fatty acids such as PA (palmitic acid) are detrimental to ß-cell function compared with unsaturated fatty acids. In the present study, we describe the effect of the polyunsaturated AA (arachidonic acid) on the function of the clonal pancreatic ß-cell line BRIN-BD11 and demonstrate AA-dependent attenuation of PA effects. When added to ß-cell incubations at 100 µM, AA can stimulate cell proliferation and chronic (24 h) basal insulin secretion. Microarray analysis and/or real-time PCR indicated significant AA-dependent up-regulation of genes involved in proliferation and fatty acid metabolism [e.g. Angptl (angiopoietin-like protein 4), Ech1 (peroxisomal Δ3,5,Δ2,4-dienoyl-CoA isomerase), Cox-1 (cyclo-oxygenase-1) and Cox-2, P<0.05]. Experiments using specific COX and LOX (lipoxygenase) inhibitors demonstrated the importance of COX-1 activity for acute (20 min) stimulation of insulin secretion, suggesting that AA metabolites may be responsible for the insulinotropic effects. Moreover, concomitant incubation of AA with PA dose-dependently attenuated the detrimental effects of the saturated fatty acid, so reducing apoptosis and decreasing parameters of oxidative stress [ROS (reactive oxygen species) and NO levels] while improving the GSH/GSSG ratio. AA decreased the protein expression of iNOS (inducible NO synthase), the p65 subunit of NF-κB (nuclear factor κB) and the p47 subunit of NADPH oxidase in PA-treated cells. These findings indicate that AA has an important regulatory and protective ß-cell action, which may be beneficial to function and survival in the 'lipotoxic' environment commonly associated with Type 2 diabetes mellitus.

Long-term outcomes of obsessive-compulsive disorder: follow-up of 142 children and adolescents.

BACKGROUND: Obsessive-compulsive disorder (OCD) often starts in childhood and adolescence and can be a chronic disorder with high persistence rates. There are few prospective long-term follow-up studies. AIMS: To follow up young people with OCD to clarify persistence rates and relevant predictors, presence of other psychiatric disorders, functional impairment, service utilisation and perceived treatment needs. METHOD: All young people with OCD assessed over 9 years at the National and Specialist Paediatric OCD clinic, Maudsley Hospital, London, were included. Sixty-one per cent (142 of 222) of all contactable young people and parents completed computerised diagnostic interviews and questionnaires. RESULTS: We found a persistence rate of OCD of 41%; 40% of participants had a psychiatric diagnosis other than OCD at follow-up. The main predictor for persistent OCD was duration of illness at assessment. High levels of baseline psychopathology predicted other psychiatric disorders at follow-up. Functional impairment and quality of life were mildly to moderately affected. Approximately 50% of participants were still receiving treatment and about 50% felt a need for further treatment. CONCLUSIONS: This study confirms that paediatric OCD can be a chronic condition that persists into adulthood. Early recognition and treatment might prevent chronicity. Important challenges for services are ensuring adequate treatment and a smooth transition from child to adult services.

Alpha-synuclein deficiency in the C57BL/6JOlaHsd strain does not modify disease progression in the ME7-model of prion disease.

We previously detailed how intrahippocampal inoculation of C57BL/6J mice with murine modified scrapie (ME7) leads to chronic neurodegeneration (Cunningham C, Deacon R, Wells H, Boche D, Waters S, Diniz CP, Scott H, Rawlins JN, Perry VH (2003) Eur J Neurosci 17:2147-2155.). Our characterization of the ME7-model is based on inoculation of this murine modified scrapie agent into C57BL/6J mice from Harlan laboratories. This agent in the C57BL/6J host generates a disease that spans a 24-week time course. The hippocampal pathology shows progressive misfolded prion (PrP(Sc)) deposition, astrogliosis and leads to behavioural dysfunction underpinned by the early synaptic loss that precedes neuronal death. The Harlan C57BL/6J, although widely used as a wild type mouse, are a sub-strain harbouring a spontaneous deletion of alpha-synuclein with the full description C57BL/6JOlaHsd. Recently alpha-synuclein has been shown to ameliorate the synaptic loss in a mouse model lacking the synaptic chaperone CSP-alpha. This opens a potential confound of the ME7-model, particularly with respect to the signature synaptic loss that underpin the physiological and behavioural dysfunction. To investigate if this strain-selective loss of a candidate disease modifier impacts on signature ME7 pathology, we compared cohorts of C57BL/6JOlaHsd (alpha-synuclein negative) with the founder strain from Charles Rivers (C57BL/6JCrl, alpha-synuclein positive). There were subtle changes in behaviour when comparing control animals from the two sub-strains indicating potentially significant consequences for studies assuming neurobiogical identity of both strains. However, there was no evidence that the absence of alpha-synuclein modifies disease. Indeed, accumulation of PrP(Sc), synaptic loss and the behavioural dysfunction associated with the ME7-agent was the same in both genetic backgrounds. Our data suggest that alpha-synuclein deficiency does not contribute to the compartment specific processes that give rise to prion disease mediated synaptotoxicity and neurodegeneration.

Saturated fatty acids produce an inflammatory response predominantly through the activation of TLR4 signaling in hypothalamus: implications for the pathogenesis of obesity.

In animal models of diet-induced obesity, the activation of an inflammatory response in the hypothalamus produces molecular and functional resistance to the anorexigenic hormones insulin and leptin. The primary events triggered by dietary fats that ultimately lead to hypothalamic cytokine expression and inflammatory signaling are unknown. Here, we test the hypothesis that dietary fats act through the activation of toll-like receptors 2/4 and endoplasmic reticulum stress to induce cytokine expression in the hypothalamus of rodents. According to our results, long-chain saturated fatty acids activate predominantly toll-like receptor 4 signaling, which determines not only the induction of local cytokine expression but also promotes endoplasmic reticulum stress. Rats fed on a monounsaturated fat-rich diet do not develop hypothalamic leptin resistance, whereas toll-like receptor 4 loss-of-function mutation and immunopharmacological inhibition of toll-like receptor 4 protects mice from diet-induced obesity. Thus, toll-like receptor 4 acts as a predominant molecular target for saturated fatty acids in the hypothalamus, triggering the intracellular signaling network that induces an inflammatory response, and determines the resistance to anorexigenic signals.

Effect of lipid infusion on metabolism and force of rat skeletal muscles during intense contractions.

The hypothesis that during intense muscle contraction induced by electrical stimulation, long chain fatty acids (LCFA) might reduce mitochondrial ATP/ADP ratio, raising the contribution of glycolysis for ATP production was examined. The effect of a lipid infusion (Lipovenus emulsion) on UCP-3 mRNA level, lactate, glucose-6-phosphate (G-6P) and glycogen content was investigated in rat. Blood samples for determination of free fatty acids and lactate were collected at 0, 30 and 60 min during rest and at 0, 10 and 20 min during muscle contraction. The content of lactate, glycogen and G-6P was also determined in soleus (SO), red gastrocnemius (RG) and white gastrocnemius (WG) muscles collected immediately after muscle contraction period. In addition, the force level was determined during muscle contractions. The effect of Lipovenus emulsion on respiration of mitochondria isolated from rat skeletal muscle, and content of UCP-3 and lactate in cultured skeletal muscle cells was also determined. The in vivo experiments showed that Lipovenus induced a significant increase of UCP-3 mRNA levels. After Lipovenus infusion, lactate level was increased in RG muscle only, whereas the contents of glycogen and G-6P were decreased in both RG and WG muscles (P < 0.05). Lipovenus infusion failed to exert any effect on muscle force performance (P > 0.05). The in vitro experiments showed that Lipovenus infusion induced a significant increase in mitochondrial respiration, but had no effect on UCP-3 content. Lactate concentration was significantly increased in the culture medium of stimulated cells in the control and Lipovenus groups compared with the respective not-stimulated cells (P< 0.05). We concluded that as mitochondrial function becomes limited by the FFA-uncoupling effect, the ATP demand is mainly supplied by anaerobic glucose metabolism preventing an expected decrease in muscle contraction performance.

Imprinted expression of neuronatin from modified BAC transgenes reveals regulation by distinct and distant enhancers.

Neuronatin (Nnat) is an imprinted gene that is expressed exclusively from the paternal allele while the maternal allele is silent and methylated. The Nnat locus exhibits some unique features compared with other imprinted domains. Unlike the majority of imprinted genes, which are organised in clusters and coordinately regulated, Nnat does not appear to be closely linked to other imprinted genes. Also unusually, Nnat is located within an 8-kb intron of the Bc10 gene, which generates a biallelically expressed, antisense transcript. A similar organisation is conserved at the human NNAT locus on chromosome 20. Nnat expression is first detected at E8.5 in rhombomeres 3 and 5, and subsequently, expression is widespread within postmitotic neuronal tissues. Using modified BAC transgenes, we show that imprinted expression of Nnat at ectopic sites requires, at most, an 80-kb region around the gene. Furthermore, reporter transgenes reveal distinct and dispersed cis-regulatory elements that direct tissue-specific expression and these are predominantly upstream of the region that confers allele-specific expression.

Normal values for distortion product otoacoustic emissions in children: a study using primary levels previously demonstrated to be optimum for identification of hearing loss.

No published data exist for normal values of distortion product otoacoustic emissions (DPOAE) in children at primary levels f1 = 65 dB and f2 = 55 dB SPL. These primary levels have been previously demonstrated to be optimal for identification of hearing impaired ears in adults. A total of 102 normal children underwent audiological assessment, including exclusion of middle ear disease, pure tone audiometry and DPOAE DP-grams (primaries L1/L2 = 65/55 dB SPL, f1:f2 = 1.22). There was a statistically significant decrease in DPOAE amplitude with increasing age. DPOAE amplitude was also dependent on the frequency of f2. However, there was wide inter- and intra-individual variation in DPOAE amplitude at different frequencies of f2. There was also a large overlap between the range of values of DPOAE amplitude between the adjacent age groups. Detailed assessment of DPOAE in children is feasible in the clinical setting. These normal values should prove invaluable in future studies; however, the large range of normal values means that cross-sectional studies may not be able to detect small variations in cochlear function.

Deletion of a silencer element disrupts H19 imprinting independently of a DNA methylation epigenetic switch.

The H19 imprinted gene is silenced when paternally inherited and active only when inherited maternally. This is thought to involve a cis-acting control region upstream of H19 that is responsible for regulating a number of functions including DNA methylation, asynchronous replication of parental chromosomes and an insulator. Here we report on the function of a 1.2 kb upstream element in the mouse, which was previously shown to function as a bi-directional silencer in Drosophila. The cre-loxP-mediated targeted deletion of the 1.2 kb region had no effect on the maternal allele. However, there was loss of silencing of the paternal allele in many endodermal and other tissues. The pattern of expression was very similar to the expression pattern conferred by the enhancer elements downstream of H19. We could not detect an effect on the expression of the neighbouring imprinted Igf2 gene, suggesting that the proposed boundary element insulating this gene from the downstream enhancers was unaffected. Despite derepression of the paternal H19 allele, the deletion surprisingly did not affect the differential DNA methylation of the locus, which displayed an appropriate epigenetic switch in the parental germlines. Furthermore, the characteristic asynchronous pattern of DNA replication at H19 was also not disrupted by the deletion, suggesting that the sequences that mediate this were also intact. The silencer is therefore part of a complex cis-regulatory region upstream of the H19 gene and acts specifically to ensure the repression of the paternal allele, without a predominant effect on the epigenetic switch in the germline.

A silencer element identified in Drosophila is required for imprinting of H19 reporter transgenes in mice.

The H19 gene is subject to genomic imprinting because it is methylated and repressed after paternal inheritance and is unmethylated and expressed after maternal inheritance. We recently identified a 1.1-kb control element in the upstream region of the H19 gene that functions as a cis-acting silencer element in Drosophila. Here we investigate the function of this element in mice. We demonstrate that both H19-lacZ and H19-PLAP reporter transgenes can undergo imprinting with repression and hypermethylation after paternal transmission at many integration sites. However, transgenes that were deleted for the 1.1-kb silencer element showed loss of paternal repression, but they did not show marked changes in the paternal methylation of the remaining upstream region. This study demonstrates that the 1.1-kb control element identified in Drosophila is required to silence paternally transmitted H19 minitransgenes in mice.

Developmental potential of mouse primordial germ cells.

There are distinctive and characteristic genomic modifications in primordial germ cells that distinguish the germ cell lineage from somatic cells. These modifications include, genome-wide demethylation, erasure of allele-specific methylation associated with imprinted genes, and the re-activation of the X chromosome. The allele-specific differential methylation is involved in regulating the monoallelic expression, and thus the gene dosage, of imprinted genes, which underlies functional differences between parental genomes. However, when the imprints are erased in the germ line, the parental genomes acquire an equivalent epigenetic and functional state. Therefore, one of the reasons why primordial germ cells are unique is because this is the only time in mammals when the distinction between parental genomes ceases to exist. To test how the potentially imprint-free primordial germ cell nuclei affect embryonic development, we transplanted them into enucleated oocytes. Here we show that the reconstituted oocyte developed to day 9.5 of gestation, consistently as a small embryo and a characteristic abnormal placenta. The embryo proper also did not progress much further even when the inner cell mass was 'rescued' from the abnormal placenta by transfer into a tetraploid host blastocyst. We found that development of the experimental conceptus was affected, at least in part, by a lack of gametic imprints, as judged by DNA methylation and expression analysis of several imprinted genes. The evidence suggests that gametic imprints are essential for normal development, and that they can neither be initiated nor erased in mature oocytes; these properties are unique to the developing germ line.