Assessing the quality and value of metabolic chart data for capturing core outcomes for pediatric medium-chain acyl-CoA dehydrogenase (MCAD) deficiency.

BACKGROUND: Generating rigorous evidence to inform care for rare diseases requires reliable, sustainable, and longitudinal measurement of priority outcomes. Having developed a core outcome set for pediatric medium-chain acyl-CoA dehydrogenase (MCAD) deficiency, we aimed to assess the feasibility of prospective measurement of these core outcomes during routine metabolic clinic visits. METHODS: We used existing cohort data abstracted from charts of 124 children diagnosed with MCAD deficiency who participated in a Canadian study which collected data from birth to a maximum of 11 years of age to investigate the frequency of clinic visits and quality of metabolic chart data for selected outcomes. We recorded all opportunities to collect outcomes from the medical chart as a function of visit rate to the metabolic clinic, by treatment centre and by child age. We applied a data quality framework to evaluate data based on completeness, conformance, and plausibility for four core MCAD outcomes: emergency department use, fasting time, metabolic decompensation, and death. RESULTS: The frequency of metabolic clinic visits decreased with increasing age, from a rate of 2.8 visits per child per year (95% confidence interval, 2.3-3.3) among infants 2 to 6 months, to 1.0 visit per child per year (95% confidence interval, 0.9-1.2) among those ≥ 5 years of age. Rates of emergency department visits followed anticipated trends by child age. Supplemental findings suggested that some emergency visits occur outside of the metabolic care treatment centre but are not captured. Recommended fasting times were updated relatively infrequently in patients' metabolic charts. Episodes of metabolic decompensation were identifiable but required an operational definition based on acute manifestations most commonly recorded in the metabolic chart. Deaths occurred rarely in these patients and quality of mortality data was not evaluated. CONCLUSIONS: Opportunities to record core outcomes at the metabolic clinic occur at least annually for children with MCAD deficiency. Methods to comprehensively capture emergency care received at outside institutions are needed. To reduce substantial heterogeneous recording of core outcome across treatment centres, improved documentation standards are required for recording of recommended fasting times and a consensus definition for metabolic decompensations needs to be developed and implemented.

Implementation of Epilepsy Multigene Panel Testing in Ontario, Canada.

BACKGROUND: Epilepsy is a common neurological condition that shows a marked genetic predisposition. The advent of next-generation sequencing (NGS) has transformed clinical genetic testing by allowing the rapid screen for causative variants in multiple genes. There are currently no NGS-based multigene panel diagnostic tests available for epilepsy as a licensed clinical diagnostic test in Ontario, Canada. Eligible patient samples are sent out of country for testing by commercial laboratories, which incurs significant cost to the public healthcare system. OBJECTIVE: An expert Working Group of medical geneticists, pediatric neurologists/epileptologists, biochemical geneticists, and clinical molecular geneticists from Ontario was formed by the Laboratories and Genetics Branch of the Ontario Ministry of Health and Long-Term Care to develop a programmatic approach to implementing epilepsy panel testing as a provincial service. RESULTS: The Working Group made several recommendations for testing to support the clinical delivery of care in Ontario. First, an extension of community healthcare outcomes-based program should be incorporated to inform and educate ordering providers when requesting and interpreting a genetic panel test. Second, any gene panel testing must be "evidence-based" and takes into account varied clinical indications to reduce the chance of uncertain and secondary results. Finally, an ongoing evaluative process was recommended to ensure continued test improvement for the future. CONCLUSION: This epilepsy panel testing implementation plan will be a model for genetic care directed toward a specific set of conditions in the province and serve as a prototype for genetic testing for other genetically heterogeneous diseases.

Mise en œuvre d'un test diagnostique permettant en Ontario l'analyse d'un panel de plusieurs gènes liés à l'épilepsie.Contexte:L'épilepsie demeure un trouble neurologique fréquent dont la prédisposition génétique apparaît notable. L'émergence du séquençage de nouvelle génération (SNG) a aussi transformé les tests génétiques en permettant un dépistage rapide des variantes causales que l'on retrouve dans de nombreux gènes. À l'heure actuelle, il n'existe pas, pour l'épilepsie, de tests diagnostiques homologués qui permettent en Ontario l'analyse d'un panel de gènes en vertu du SNG. Les échantillons de patients admissibles sont alors envoyés à l'extérieur du Canada afin d'être analysés par des laboratoires commerciaux, ce qui pèse lourd dans les budgets des systèmes publics de santé. Objectif : Un groupe de travail formé d'experts (généticiens médicaux, neurologues pédiatriques et spécialistes en épileptologie, généticiens biochimiques et généticiens moléculaires cliniques) a été formé par le service des laboratoires et de la génétique des ministères de la Santé et des Soins de Longue durée de l'Ontario afin d'élaborer une démarche programmatique visant à mettre en œuvre des tests diagnostiques basés sur un panel de plusieurs gènes. Ces tests seraient ensuite reconnus à titre de service public. Résultats:En matière de dépistage, ce groupe de travail a ainsi émis plusieurs recommandations visant à accompagner la prestation clinique en Ontario. Tout d'abord, un programme s'inspirant du projet « ECHO ¼ (Extension of Community Healthcare Outcomes) devrait être ajouté dans le but de renseigner et de sensibiliser les prestataires de soins de santé qui demandent et qui interprètent ces tests basés sur un panel de plusieurs gènes. Ensuite, tout test de ce type doit reposer sur des preuves et tenir compte d'une panoplie d'indications cliniques afin de réduire les possibilités d'incertitude et de résultats secondaires. Enfin, il a été recommandé de procéder à un processus continu d'évaluation pour s'assurer que ces tests puissent être améliorés dans le futur. Conclusion:Ce plan de mise en œuvre de tests basés sur un panel de plusieurs gènes deviendra un modèle pour les soins destinés à un ensemble spécifique de problèmes de santé en Ontario. Outre l'épilepsie, il pourra servir comme prototype pour le dépistage d'autres maladies hétérogènes sur le plan génétique.

Herbivory and Attenuated UV Radiation Affect Volatile Emissions of the Invasive Weed Calluna vulgaris.

Calluna vulgaris (heather) is an aggressive invasive weed on the Central Plateau, North Is., New Zealand (NZ), where it encounters different environmental factors compared to its native range in Europe, such as high ultraviolet radiation (UV) and a lack of specialist herbivores. The specialist herbivore Lochmaea suturalis (heather beetle) was introduced from the United Kingdom (UK) in 1996 as a biocontrol agent to manage this invasive weed. Like other plant invaders, a novel environment may be challenging for heather as it adjusts to its new conditions. This process of "adjustment" involves morphological and physiological changes often linked to phenotypic plasticity. The biochemical responses of exotic plants to environmental variables in their invaded range is poorly understood. The production and release of volatile organic compounds (VOCs) is essential to plant communication and highly susceptible to environmental change. This study therefore aimed to explore the VOC emissions of heather in response to different levels of UV exposure, and to feeding damage by L. suturalis. Using tunnel houses clad with UV-selective filters, we measured VOCs produced by heather under NZ ambient, 20% attenuated, and 95% attenuated solar UV treatments. We also compared VOC emissions in the field at adjacent sites where L. suturalis was present or absent. Volatiles produced by the same target heather plants were measured at four different times in the spring and summer of 2018-2019, reflecting variations in beetle's abundance, feeding stage and plant phenology. Heather plants under 95% attenuated UV produced significantly higher amounts of (E)-ß-farnesene, decanal, benzaldehyde, and benzeneacetaldehyde compared to 25% attenuated and ambient UV radiation. We also found significant differences in volatiles produced by heather plants in beetle-present versus beetle-absent sites on most sampling occasions. We also recorded a lower number of generalist herbivores on heather at sites where L. suturalis was present. Interactions between invasive plants, a novel environment, and the native communities they invade, are discussed.

Metabolomics for improved treatment monitoring of phenylketonuria: urinary biomarkers for non-invasive assessment of dietary adherence and nutritional deficiencies.

Management of phenylketonuria (PKU) requires lifelong restriction of phenylalanine (Phe) intake using specialized medical foods to prevent neurocognitive impairment in affected patients. However, dietary adherence is challenging to maintain while ensuring adequate nutrition, which can lead to sub-optimal clinical outcomes. Metabolomics offers a systematic approach to identify new biomarkers of disease progression in PKU when using urine as a surrogate for blood specimens that is more accurate than self-reported diet records. Herein, the plasma and urine metabolome of a cohort of classic PKU patients (median age = 11 years; n = 22) mainly prescribed (78%) a Phe-restricted diet were characterized using multisegment injection-capillary electrophoresis-mass spectrometry (MSI-CE-MS). Overall, there was good mutual agreement between plasma Phe and tyrosine (Tyr) concentrations measured from PKU patients when using an amino acid analyzer based on UPLC-UV as compared to MSI-CE-MS with a mean bias of 12% (n = 82). Longitudinal measurements of recently diagnosed PKU infants (n = 3) revealed good long-term regulation of blood Phe with dietary management, and only occasional episodes exceeding the recommended therapeutic range (>360 µM) unlike older PKU patients. Plasma metabolomic studies demonstrated that non-adherent PKU patients had lower circulating concentrations of Tyr, arginine, 2-aminobutyric acid, and propionylcarnitine (q < 0.05, FDR) that were inversely correlated to Phe (r ≈ -0.600 to -0.830). Nontargeted metabolite profiling also revealed urinary biomarkers associated with poor dietary adherence among PKU patients, including elevated concentrations of catabolites indicative of Phe intoxication (e.g., phenylpyruvic acid, phenylacetylglutamine, hydroxyphenylacetic acid). Additionally, PKU patients with poor blood Phe control had lower excretion of urinary compounds derived from co-metabolism of Tyr due to microbiota activity (e.g., cresol sulfate, phenylsulfate), as well as several metabolites associated with inadequate nutrient intake, including low carnitine and B vitamin status (e.g., folic acid, vitamin B12). Interestingly, an unknown urinary metabolite was strongly correlated with Phe excretion in PKU patients (r = 0.861), which was subsequently identified as imidazole lactic acid when using high resolution MS/MS. Overall, urine profiling offers a non-invasive approach for better treatment monitoring of individual PKU patients, which can also guide the design of novel therapies that improve adherence to Phe-restricted diets without acquired nutritional deficiencies.

Health Care for Mitochondrial Disorders in Canada: A Survey of Physicians.

BACKGROUND: An improved understanding of diagnostic and treatment practices for patients with rare primary mitochondrial disorders can support benchmarking against guidelines and establish priorities for evaluative research. We aimed to describe physician care for patients with mitochondrial diseases in Canada, including variation in care. METHODS: We conducted a cross-sectional survey of Canadian physicians involved in the diagnosis and/or ongoing care of patients with mitochondrial diseases. We used snowball sampling to identify potentially eligible participants, who were contacted by mail up to five times and invited to complete a questionnaire by mail or internet. The questionnaire addressed: personal experience in providing care for mitochondrial disorders; diagnostic and treatment practices; challenges in accessing tests or treatments; and views regarding research priorities. RESULTS: We received 58 survey responses (52% response rate). Most respondents (83%) reported spending 20% or less of their clinical practice time caring for patients with mitochondrial disorders. We identified important variation in diagnostic care, although assessments frequently reported as diagnostically helpful (e.g., brain magnetic resonance imaging, MRI/MR spectroscopy) were also recommended in published guidelines. Approximately half (49%) of participants would recommend "mitochondrial cocktails" for all or most patients, but we identified variation in responses regarding specific vitamins and cofactors. A majority of physicians recommended studies on the development of effective therapies as the top research priority. CONCLUSIONS: While Canadian physicians' views about diagnostic care and disease management are aligned with published recommendations, important variations in care reflect persistent areas of uncertainty and a need for empirical evidence to support and update standard protocols.

Les soins de santé prodigués au Canada à des individus atteints de troubles mitochondriaux : une enquête menée auprès de médecins. Contexte: Dans le cas de patients atteints de troubles mitochondriaux rares, il est permis de croire qu'une meilleure compréhension des pratiques en matière de diagnostic et de traitement peut contribuer, au moyen des lignes directrices, à l'étalonnage et à l'établissement de priorités en ce qui regarde la recherche évaluative. Notre intention a été de décrire les soins prodigués au Canada par des médecins, notamment leur variabilité, dans le cas de ces patients. Méthodes: Pour ce faire, nous avons effectué une enquête transversale auprès de médecins canadiens qui posent des diagnostics de troubles mitochondriaux et qui prodiguent des soins continus aux patients qui en sont atteints. À cet effet, nous avons fait appel à la méthode d'enquête dite « en boule de neige ¼ (snowball sampling) afin d'identifier des participants possiblement admissibles. Ces derniers ont été ensuite contactés par la poste, et ce, à cinq reprises au maximum. Ils ont été invités à remplir un questionnaire et à le retourner par la poste ou en ligne. Ce questionnaire abordait les aspects suivants : leur expérience personnelle à titre de prestataire de soins ; leurs pratiques en matière de diagnostic et de traitement ; les défis se présentant à eux au moment d'avoir accès à des tests ou à des traitements ; et finalement leurs points de vue en ce qui regarde les priorités de la recherche. Résultats: Dans le cadre de cette enquête, nous avons reçu 58 réponses, ce qui représente un taux de 52 %. Une majorité de répondants (83 %) ont indiqué allouer 20 % ou moins de leur temps de pratique clinique aux soins de patients atteints de ces troubles. Nous avons également noté d'importantes variations concernant les soins et les diagnostics, et ce, même si les outils d'évaluation fréquemment considérés utiles sur le plan diagnostic (p. ex. : des IRM du cerveau/la spectroscopie par RM) étaient également recommandés dans des lignes directrices déjà publiées. Environ la moitié de nos répondants (49 %) recommanderaient volontiers un « cocktail ¼ de vitamines pour tous leurs patients ou la plupart d'entre eux. Quand il est question de vitamines spécifiques et de cofacteurs, nous avons cependant identifié une variation dans leurs réponses. Interrogés quant à la priorité numéro un en matière de recherche, une majorité de répondants a dit recommander la poursuite d'études portant sur la mise sur pied de traitements thérapeutiques efficaces. Conclusions: Bien que les points de vue de ces médecins canadiens en ce qui regarde les diagnostics et la prise en charge des troubles mitochondriaux soient en phase avec des recommandations publiées, d'importantes variations reflètent la persistance d'aspects incertains ainsi qu'un besoin de données empiriques afin de renforcer et de mettre à jour les protocoles de rééférence.

Approach to the interpretation of unexpected laboratory results arising in the care of patients with inborn errors of metabolism (IEM).

Endocrinologists may encounter abnormal results in routine laboratory tests while caring for patients with inborn errors of metabolism. This article provides a framework for understanding these abnormalities as: a) part of the pathophysiology of the exceptional disease, b) exceptional laboratory errors related to the exceptional disease, or c) routine laboratory errors to which any patient sample is susceptible.

Individual variation and repeatability of corticosterone responses of little penguins (Eudyptula minor) sampled in two successive years at Oamaru, New Zealand.

Plasma corticosterone concentrations increase when birds experience a stressor, and plasma corticosterone responses to a capture and handling stressor have been measured in many species of birds. Whilst it is assumed that the reported corticosterone responses reflect the inherent sensitivity of each bird to the stressor, responses of the same birds have rarely been measured at intervals of one or more years. The current study was conducted to measure the repeatability in two successive years of corticosterone responses of little penguins (Eudyptula minor) at Oamaru, New Zealand. There was a wide range of individual corticosterone responses to capture and restraint in 96 little penguins in 2012 and 50 penguins sampled at the same time of year in 2013. There were significant repeatabilities for the ranks of corticosterone at 15, 30 and 60min (r=0.416±0.160, r=0.636±0.115 and r=0.380±0.166 respectively) and for the ranks of integrated corticosterone responses (r=0.594±0.126) for 23 birds sampled in both years. There were no significant relationships between the size of corticosterone responses and age, body weight or condition index. Mean corticosterone concentrations at 60min were 114.22±6.65ng/ml in 2012 and 116.94±6.42ng/ml in 2013. Mean corticosterone responses did not differ between two successive years and were greater than responses of other penguin species. Penguins are well suited to long term studies in which corticosterone responses are measured annually as potential measures of changing marine environmental conditions.

Biotinidase deficiency: Spectrum of molecular, enzymatic and clinical information from newborn screening Ontario, Canada (2007-2014).

Untreated profound biotinidase deficiency results in a wide range of clinical features, including optic atrophy, cutaneous abnormalities, hearing loss and developmental delay. Ontario, Canada incorporated this treatable deficiency in newborn screening over the past 8years. This study elucidates the molecular, biochemical, and clinical findings from the pilot project. Information from initial screens, serum biotinidase activity level assays, molecular testing, and family history for 246 positive newborns screens were analyzed. A mutation spectrum was created for the province of Ontario, including common mutations such as D444H, D444H/A171T, Q456H, C33fs, and R157H. Individuals with partial deficiency were separated into 3 groups: D444H homozygotes (Group 1); compound heterozygotes for D444H with another profound allele (Group 2); compound heterozygotes with two non-D444H alleles (Group 3). Biochemical phenotype-genotype associations in partial deficiency showed a significant difference in serum biotinidase activity in between any given two groups. Three children with partial deficiency discontinued biotin for varied lengths of time. Two of whom became symptomatic with abnormal gait, alopecia, skin rashes and developmental delay. A need for more congruency in diagnostic, treatment and educational practices was highlighted across the province. Heterogeneity and variation in clinical presentations and management was observed in patients with the partial deficiency.

ß-Alanine Supplementation Does Not Augment the Skeletal Muscle Adaptive Response to 6 Weeks of Sprint Interval Training.

Sprint interval training (SIT), repeated bouts of high-intensity exercise, improves skeletal muscle oxidative capacity and exercise performance. ß-alanine (ß-ALA) supplementation has been shown to enhance exercise performance, which led us to hypothesize that chronic ß-ALA supplementation would augment work capacity during SIT and augment training-induced adaptations in skeletal muscle and performance. Twenty-four active but untrained men (23 ± 2 yr; VO2peak = 50 ± 6 mL · kg(-1) · min(-1)) ingested 3.2 g/day of ß-ALA or a placebo (PLA) for a total of 10 weeks (n = 12 per group). Following 4 weeks of baseline supplementation, participants completed a 6-week SIT intervention. Each of 3 weekly sessions consisted of 4-6 Wingate tests, i.e., 30-s bouts of maximal cycling, interspersed with 4 min of recovery. Before and after the 6-week SIT program, participants completed a 250-kJ time trial and a repeated sprint test. Biopsies (v. lateralis) revealed that skeletal muscle carnosine content increased by 33% and 52%, respectively, after 4 and 10 weeks of ß-ALA supplementation, but was unchanged in PLA. Total work performed during each training session was similar across treatments. SIT increased markers of mitochondrial content, including cytochome c oxidase (40%) and ß-hydroxyacyl-CoA dehydrogenase maximal activities (19%), as well as VO2peak (9%), repeated-sprint capacity (5%), and 250-kJ time trial performance (13%), but there were no differences between treatments for any measure (p < .01, main effects for time; p > .05, interaction effects). The training stimulus may have overwhelmed any potential influence of ß-ALA, or the supplementation protocol was insufficient to alter the variables to a detectable extent.

Toxicological responses of the earthworm Eisenia fetida to 18-crown-6 under laboratory conditions.

The earthworm Eisenia fetida was exposed to artificial soil supplemented with 18-crown-6 (1,4,7,10,13,16-hexaoxacyclooctadecane) to investigate its effects on earthworm mortality, growth, avoidance, burrowing behavior and respiration. The results revealed that 18-crown-6 had the potential to negatively affect the behavior of earthworms. The 7-d LC50 was 585 mg kg(-1) soil. Avoidance behavior was the most sensitive endpoint, with a 48-h EC50 of 120 mg kg(-1) soil. Growth, burrow length and respiration showed general decreases with increasing 18-crown-6 concentrations. Behavioral endpoints and respiration may be regarded as sensitive parameters in evaluating the toxicity of this chemical to earthworms.

Development of a second-tier method for C4, C5 and C2 acylcarnitine analysis in plasma.

INTRODUCTION: Acylcarnitines are typically analyzed using either a flow injection analysis (FIA) method or liquid chromatography-mass spectrometry (LC-MS/MS) methods. The FIA method is a fast, efficient method, however it does not have the capability to separate compounds with the same molecular weight. These isobaric interferences can be removed by chromatographic separation with LC-MS/MS. In this study, we aimed to develop and optimize a qualitative LC-MS/MS method to separate the isobaric interferences for two-, four- and five-carbon acylcarnitines. METHODS: The samples were first prepared by acylcarnitine derivatization with butanolic HCl. The developed LC-MS/MS method is a combination of isocratic and gradient elution used to separate acylcarnitines. Multiple reaction monitoring was used for determination of precursor and product ions for each acylcarnitine species as well as known interferences used in our study. We used this method to analyze quality assurance and patient samples with elevated two-, four- and five-carbon acylcarnitines. RESULTS: Butyryl- and isobutyrylcarnitines as well as valeryl- and isovalerylcarnitines were successfully separated using the developed method. This method was able also to separate and distinguish acetylcarnitine from glutamate interference that has been causing overestimation of acetylcarnitine. In patients, the dominant five-carbon acylcarnitine was found to be isovalerylcarnitine. We confirmed that the majority of analyzed patient samples had additional carnitine adducts present but not valerylcarnitine. Butyryl- and isobutyrylcarnitines, in variable ratios, were present in every patient sample. CONCLUSION: We developed a qualitative LC-MS/MS method for butyl-ester derivatized acylcarnitines, which can be used as a second-tier method for diagnosis and monitoring of various inborn errors of metabolism in our hospital network.

Characterization of hatch-size and growth rates of captive and wild-reared brown kiwi (Apteryx mantelli) chicks.

Avian growth rate patterns represent a trade off between a tissue's functional maturity and its capacity for growth. At the time of hatch, the brown kiwi (Apteryx mantelli) limb has a high level of maturity in order for the chick to be able to kick its way out of the shell and walk and forage independently from an early age. Growth curves of limb segments, bill length and bodyweight are presented for captive-reared, BNZ Operation Nest Egg™ chicks over a period of 3 months from the point of hatch. Some parameters were slightly larger in the females than in males at time of hatch, including the bill length. Growth in bodyweight began to slow earlier in males than in females. Regressions of limb and bill measurements over time showed linear patterns of growth instead of a sigmoidal curve as seen in other birds, probably due to the short period of observation. Bodyweight and bill length were then compared to these morphometrics in a wild population of kiwi. Captive-reared chicks were found to hatch with shorter bills and to increase in bodyweight at a faster rate than the wild birds. Rapid weight gain has been implicated in developmental limb deformities in other precocial and long-legged birds and should be avoided in captive kiwi.

Prolonged glucocorticoid administration affects oocyte morphology in cats (Felis catus) undergoing an ovarian stimulation protocol.

While captivity-related stress and the associated rise in baseline glucocorticoid (GC) concentrations have been linked to ovarian quiescence in some felid species, no study has examined the effects of elevated GC on oocyte quality. This study examined the effects of exogenous GC administration on the ovarian response and oocyte quality of domestic cats after an ovarian stimulation protocol. Entire mature female cats were divided into treatment (n = 6) and control (n = 6) groups. Cats in the GC treatment (GCT) group were given 1 mg kg-1 oral prednisolone daily from Day 0-45. All cats (n = 12) were given 0.088 mg kg-1 day-1 progesterone orally from Day 0-37, before treatment with 75 IU eCG im to induce follicular growth on Day 40, followed by 50 IU hCG im 80 h later to induce ovulation. Cats were ovariohysterectomised 30 h after the hCG treatment. Blood samples were collected on Days 0, 10, 30 and 40 (prior to eCG treatment), 80 h after eCG treatment, and on Day 45 for cortisol, glucose, prednisolone, oestradiol, and progesterone analysis. Cortisol concentrations did not differ between treatment groups throughout the study. Mean glucose concentrations were higher in the GCT cats (P = 0.004). Prednisolone was undetectable in all samples. Oestradiol and progesterone concentrations confirmed that the eCG treatment stimulated follicular activity and ovulation in all cats. Following ovariohysterectomy, the ovarian responses were graded (1 = excellent, 4 = poor) and oocytes retrieved from the oviducts. Each oocyte was given a total oocyte score (TOS: using an 9-point scale, 8 = best) based on four parameters: oocyte morphology, size, ooplasm uniformity and granularity, and zona pellucida (ZP) thickness and variation. Ovulation was confirmed in all cats, with a mean of 10.5 ± 1.1 ovulations per cat. Ovarian mass, ovarian response, number of ovulations, and oocyte recovery did not differ between groups. Oocyte diameter did not differ between the groups, but the ZP was thinner in the GCT group (3.1 ± 0.3 µm vs. 4.1 ± 0.3 µm, P = 0.03). The TOS was similar between treatment and control cats, but the ooplasm grade was lower (1.5 ± 0.1 vs. 1.9 ± 0.1, P = 0.01) and there was a tendency for ZP grade to be poorer (0.8 ± 0.1 vs. 1.2 ± 0.2; P = 0.08) in the treatment group. In conclusion, the GC treatment resulted in morphological changes to oocytes collected following ovarian stimulation. Whether these changes would affect fertility warrants further investigation.

Accelerometry and infrared thermography show potential for assessing ovarian activity in domestic cats (Felis catus).

Accurate and reliable monitoring of ovarian activity is challenging in many felids as current methods are either invasive or not amenable to real-time assessments. This 45-day study assessed whether accelerometry and infrared (IR) thermography can be used to address these limitations. Intact female domestic cats (n = 12) were given 0.088 mg kg-1 day-1 altrenogest (progestin) orally for 37 days to suppress follicular growth. On Day 40, cats were given 75 IU eCG im to induce follicular growth and 50 IU hCG im 80 h later to induce ovulation. Cats were ovariohysterectomised 30-31 h after the hCG treatment. Actical® accelerometers were fitted to the cats' collars and activity monitored continuously from the start of the altrenogest treatment until ovariohysterectomy. Infrared images of the perivulvar, perianal, and gluteal area were taken of each cat on Day 30 and daily from Days 36-45 of the study. Perivulvar temperature (PVT), PVT relative to gluteal temperatures (PVT-GT), and PVT relative to perianal temperature (PVT-PAT) were recorded for each image. Blood samples were collected on Days 0, 10, 30, and 40, immediately prior to the hCG treatment, and at the time of ovariohysterectomy. Serum oestradiol and progesterone concentrations indicated complete ovarian suppression by Day 30 and, together with morphological assessment of the ovaries, confirmed the induction of follicular growth and ovulation in all cats. Daily activity counts differed among cats (P < 0.001), so the daily activity counts of each cat were converted to a proportional change from the average daily activity count from Days 30-39 (defined as the 'proportional daily activity'). Proportional daily activity counts increased after the stimulation of follicular growth with eCG, with peak levels (2.03 ± 0.29-fold higher than pre-treatment levels; P = 0.006) occurring three days after the eCG treatment. The PVT-GT showed the greatest sensitivity to detect subtle changes in body temperature, increasing from early to late follicular growth (1.96 ± 0.33 °C increase from Day 41-43; P < 0.001) and decreasing after hCG-induced ovulation (1.24 ± 0.41 °C decrease from Day 43-45; P = 0.01). In conclusion, both accelerometry and IR thermography show potential as non-invasive, real-time methods for assessing ovarian activity in cats, but further research is required to determine if these methods could be used to monitor natural/non-stimulated oestrous cycles.

Exogenous glucocorticoid treatment affects Sertoli cell load and epididymal sperm quality in domestic cats (Felis catus).

Elevated glucocorticoid (GC) concentrations associated with captivity-related stress have been linked to impaired testicular function and low sperm quality in felids, but direct physiological evidence is lacking. This study assessed the effects of exogenous GC treatment on felid testicular function using the domestic cat (Felis catus) as a model species. Sixteen intact male cats aged 2.4 ± 0.8 years (mean ± SEM) were divided randomly into treatment (n = 8) and control (n = 8) groups. Treatment cats were given 1 mg kg-1 oral prednisolone daily for 50 days. Blood samples were taken on Days 0 (first prednisolone treatment), 2, 4, 7, 10, 20, 30, 40, 50 (prior to neutering) and 60 of the trial. All cats were orchiectomised on day 50, epididymal sperm assessed, and the testes fixed for histological assessment. Testosterone concentrations did not differ between the two groups. While sperm motility was similar between the treatment and control groups, cats given prednisolone had a higher proportion of morphologically abnormal sperm in both the caput (72.5% vs. 59.6%, P < 0.001) and cauda (56.7% vs. 35.8%, P < 0.001) epididymis. Testicular histomorphometric data and total number of germ cells per seminiferous tubule cross section did not differ between groups, nor did the relative abundance of spermatogonia, spermatocytes, and spermatids. Cats given prednisolone had fewer Sertoli cells per tubule cross-section than those in the control group (17.1 ± 0.9 vs. 19.7 ± 0.8, P = 0.04), which was likely related to higher rates of Sertoli cell apoptosis in treatment versus control cats (0.25 ± 0.02 vs. 0.10 ± 0.02 apoptotic Sertoli cells per tubule, respectively; P < 0.001). Sertoli cell load (number of germ cells per Sertoli cell) was also higher in the treatment group than in the control group (11.5 ± 0.8 vs. 9.4 ± 1.2 germ cells per Sertoli cell, respectively; P < 0.001), and was positively correlated with the percentage of morphologically abnormal sperm in the epididymis (r2 = 0.78, P < 0.001). Prednisolone treatment resulted in an increase in the proportion of abnormal sperm in the epididymis, which may be explained by an increased nurturing demand on a reduced Sertoli cell population. These findings provide novel evidence to support the hypothesis that elevated GC concentrations, such as those resulting from captivity-related stress, have the potential to impair testicular function and sperm quality in felids.

Families' healthcare experiences for children with inherited metabolic diseases: protocol for a mixed methods cohort study.

INTRODUCTION: Children with inherited metabolic diseases (IMDs) often have complex and intensive healthcare needs and their families face challenges in receiving high-quality, family centred health services. Improvement in care requires complex interventions involving multiple components and stakeholders, customised to specific care contexts. This study aims to comprehensively understand the healthcare experiences of children with IMDs and their families across Canada. METHODS AND ANALYSIS: A two-stage explanatory sequential mixed methods design will be used. Stage 1: quantitative data on healthcare networks and encounter experiences will be collected from 100 parent/guardians through a care map, 2 baseline questionnaires and 17 weekly diaries over 5-7 months. Care networks will be analysed using social network analysis. Relationships between demographic or clinical variables and ratings of healthcare experiences across a range of family centred care dimensions will be analysed using generalised linear regression. Other quantitative data related to family experiences and healthcare experiences will be summarised descriptively. Ongoing analysis of quantitative data and purposive, maximum variation sampling will inform sample selection for stage 2: a subset of stage 1 participants will participate in one-on-one videoconference interviews to elaborate on the quantitative data regarding care networks and healthcare experiences. Interview data will be analysed thematically. Qualitative and quantitative data will be merged during analysis to arrive at an enhanced understanding of care experiences. Quantitative and qualitative data will be combined and presented narratively using a weaving approach (jointly on a theme-by-theme basis) and visually in a side-by-side joint display. ETHICS AND DISSEMINATION: The study protocol and procedures were approved by the Children's Hospital of Eastern Ontario's Research Ethics Board, the University of Ottawa Research Ethics Board and the research ethics boards of each participating study centre. Findings will be published in peer-reviewed journals and presented at scientific conferences.

Chronic gastrointestinal inflammation induces anxiety-like behavior and alters central nervous system biochemistry in mice.

BACKGROUND & AIMS: Clinical and preclinical studies have associated gastrointestinal inflammation and infection with altered behavior. We investigated whether chronic gut inflammation alters behavior and brain biochemistry and examined underlying mechanisms. METHODS: AKR mice were infected with the noninvasive parasite Trichuris muris and given etanercept, budesonide, or specific probiotics. Subdiaphragmatic vagotomy was performed in a subgroup of mice before infection. Gastrointestinal inflammation was assessed by histology and quantification of myeloperoxidase activity. Serum proteins were measured by proteomic analysis, circulating cytokines were measured by fluorescence activated cell sorting array, and serum tryptophan and kynurenine were measured by liquid chromatography. Behavior was assessed using light/dark preference and step-down tests. In situ hybridization was used to assess brain-derived neurotrophic factor (BDNF) expression in the brain. RESULTS: T muris caused mild to moderate colonic inflammation and anxiety-like behavior that was associated with decreased hippocampal BDNF messenger RNA (mRNA). Circulating tumor necrosis factor-α and interferon-γ, as well as the kynurenine and kynurenine/tryptophan ratio, were increased. Proteomic analysis showed altered levels of several proteins related to inflammation and neural function. Administration of etanercept, and to a lesser degree of budesonide, normalized behavior, reduced cytokine and kynurenine levels, but did not influence BDNF expression. The probiotic Bifidobacterium longum normalized behavior and BDNF mRNA but did not affect cytokine or kynurenine levels. Anxiety-like behavior was present in infected mice after vagotomy. CONCLUSIONS: Chronic gastrointestinal inflammation induces anxiety-like behavior and alters central nervous system biochemistry, which can be normalized by inflammation-dependent and -independent mechanisms, neither of which requires the integrity of the vagus nerve.

Murine ß-galactosidase stability is not dependent on temperature or protective protein/cathepsin A.

GM1 gangliosidosis, a neurodegenerative disorder, and Morquio B disease, a skeletal disorder, are lysosomal storage disorders caused by inherited defects in the enzyme ß-galactosidase (GLB1; EC 3.1.2.23; MIM #611458). Enzyme replacement therapy (ERT), a standard of care for a number of non-neuronopathic lysosomal storage disorders, is not yet available for GLB1 deficiency. Although functionally active recombinant human and feline GLB1 precursors have been purified, ERT has not yet been demonstrated in GM1 gangliosidosis or Morquio B disease models. A major obstacle to developing effective therapy may be the stability of human GLB1. We show here that mouse GLB1 has greater stability when compared to human GLB1, and that human GLB1 activity is temperature and protective-dependent on protein cathepsin A, while that of mouse GLB1 is not. These findings may impact on the eventual development of ERT for GLB1 deficiency. Despite our attempts to improve the extracellular stability of human GLB1 through sequence modification and the use of chemical chaperone N-butyldeoxygalactonojirimycin, the specific enzyme activity remained well below that of mGLB1.

Analysis of serum tranexamic acid in patients undergoing open heart surgery.

BACKGROUND: Tranexamic acid is a drug used during open cardiac surgery to prevent blood loss. The blood levels of 10-100 µg/mL are reported to be in the therapeutic range and higher levels are linked to increased incidence of adverse effects. The aim of this study was to optimize and validate an LC-MS/MS method for serum tranexamic acid and measure its levels in patients from the DEPOSITION Pilot trial in order to prove the concept that topical administration will yield lower serum concentration. METHODS: The method development was carried out in several steps including sample preparation, and optimization of chromatography and tandem mass spectrometry parameters. Method validation including day-to-day precision with 4 QC levels, limit of detection, sample stability, carryover, and concentration-signal linearity was carried out. Ninety patient samples were analyzed using the validated method. RESULTS: Fast and efficient LC-MS/MS method for analysis of tranexamic acid in serum was developed. The run time was 7 min with the total time of one hour including the sample preparation. The method precision was acceptable (%CV = 10.5-12.6%) with no sample carryover observed. The matrix effect on the analytical sensitivity was negligible and the lower limit of detection was 0.5 µg/mL. The difference in the mean adjusted concentrations between topical (45 patients) and intravenous (45 patients) groups was statistically significant (0.1154 µg/mL/kg vs. 0.2542 µg/mL/kg, p < 0.0001) CONCLUSIONS: Rapid and simple LC-MS/MS method for analysis of tranexamic acid was optimized and validated. The laboratory has played a crucial role in proving the concept that topical administration yields significantly lower systemic levels of tranexamic acid, and thus decreases the risk of adverse outcomes in patients undergoing open cardiac surgery.