Child Fatality Review.

Understanding why children die is necessary to implement strategies to prevent future deaths and improve the health of any community. Child fatality review teams (CFRTs) have existed since the 1970s and provide a necessary framework to ensure that proper questions are asked about a child's death. CFRTs provide a vital function in a community to ensure that preventable causes of deaths are identified. Pediatricians are necessary members of CFRTs because they provide medical expertise and context around a child's death. All CFRTs should have pediatric physician representation, and results from team meetings should inform public policy at all levels of government. Pediatricians should be supported in their efforts to be present on CFRTs, and they should use data from team meetings to help advocate for implementing prevention strategies.

Unintentional Firearm Injury Deaths Among Children and Adolescents Aged 0-17 Years - National Violent Death Reporting System, United States, 2003-2021.

In the United States, unintentional injury is the fourth leading cause of death among infants (i.e., children aged <1 year) and is the top cause of death among children and adolescents aged 1-17 years; firearms are a leading injury method. Unsecured firearms (e.g., unlocked and loaded) are associated with risk for unintentional childhood firearm injury death. Data recorded during 2003-2021 by the National Violent Death Reporting System (NVDRS) from 49 states, the District of Columbia, and Puerto Rico were used to characterize unintentional firearm injury deaths of U.S. infants, children, and adolescents aged 0-17 years (referred to as children in this report). NVDRS identified 1,262 unintentional firearm injury deaths among children aged 0-17 years: the largest percentage (33%) of these deaths were among children aged 11-15 years, followed by 29% among those aged 0-5 years, 24% among those aged 16-17 years, and 14% among persons aged 6-10 years. Overall, 83% of unintentional firearm injury deaths occurred among boys. The majority (85%) of victims were fatally injured at a house or apartment, including 56% in their own home. Approximately one half (53%) of fatal unintentional firearm injuries to children were inflicted by others; 38% were self-inflicted. In 9% of incidents, it was unknown whether the injury was self- or other-inflicted. Approximately two thirds (67%) of shooters were playing with or showing the firearm to others when it discharged. Overall, firearms used in unintentional injury deaths were often stored loaded (74%) and unlocked (76%) and were most commonly accessed from nightstands and other sleeping areas (30%). Unintentional firearm injury deaths of children are preventable. Secured firearm storage practices (e.g., storing firearms locked, unloaded, and separate from ammunition) have been identified as protective factors against child firearm injuries and deaths, underscoring the importance of policymakers, health care professionals (e.g., pediatricians), and others partnering with parents, caregivers, and firearm owners to promote secure firearm storage.

Comparisons between human and rodent hepatic glutathione S-Transferase activities reveal sex and species differences.

Glutathione S-transferases (GSTs) are conjugating enzymes involved in drug metabolism, antioxidant defence, and cell signalling. Herein, we investigated hepatic GST conjugation in several mouse and rat strains, including both sexes, with a direct comparison to humans.Using general and isoform-selective substrates, all mouse strains had significantly greater activities than humans for total cytosolic GST, GST-M, GST-T, and microsomal GST activities. Some strains had significantly greater GST-P activities compared to humans. Sex differences between males and females were evident in all strains for total cytosolic GST, GST-M, and GST-P, and sex differences in GST-T and microsomal GST activities within strains were noted.All rats had significantly greater activities than humans for GST-M and GST-T; only some strains were significantly greater than humans for GST-P, total cytosolic GST, and microsomal GST. Sex differences within strains showed significantly greater GST-M and GST-T activities in males compared to females. Select strains showed sex differences for total cytosolic and microsomal GST activities; there were no sex differences in GST-P activities.Significant differences in glutathione conjugation between humans and rodents exist, including sex differences. This highlights the need for careful animal selection in pre-clinical studies where GSTs are the primary metabolic pathway.

The Need for Improved Collaboration between Schools and Child Death Review Teams.

BACKGROUND: Schools are essential public health partners for safeguarding students' health. Child Death Review (CDR) is one public health activity where collaboration with schools is integral for developing strategies to prevent child death but the degree of collaboration is unknown. This study assessed school participation in CDR and the prevalence of school problems in reviewed child death cases, comparing non-suicide and suicide-related deaths. METHODS: Using the National Fatality Review-Case Reporting System, we created a dataset of school-aged children (5 to 20 years) whose death was reviewed from 2005 to 2017 and used frequencies, proportions, and chi-squared statistics on selected measures. RESULTS: Educational representatives infrequently participated in CDRs (24.9%). School records were rarely accessed for reviewed deaths (5.2%). Less than half (41.2%) of reviewed deaths had any school information and of these, 35.5% of children were indicated as having problems in school. Compared with non-suicide deaths, a larger proportion of suicide deaths had school representatives attend CDRs (28.4% vs 24.1%, P < .0001), and access to educational records (4.2% vs 9.2%, P < .0001). IMPLICATIONS: Efforts are needed to address potential barriers to systems integration, including state policies and federal educational privacy laws. CONCLUSIONS: School participation in CDRs is lacking for both suicide and non-suicide deaths.

Protective placental inflammatory and oxidative stress responses are attenuated in the context of twin pregnancy and chorioamnionitis in assisted reproduction.

PURPOSE: Assisted reproduction technologies (ART) are associated with increased risks of pregnancy complications and obstetric interventions. Here, we aimed to determine if ART affects placental inflammation and oxidative stress as a mechanism for unfavorable pregnancy outcomes. METHODS: The levels of six cytokines (IFN-γ, IL-1ß, IL-6, IL-8, IL-10, TNFα) were measured using multiplex ELISA. The activity of four antioxidant enzymes (glutathione S-transferase (GST), glutathione peroxidase (GPx), glutathione reductase, superoxide dismutase) and levels of two antioxidants (GSH, vitamin E) were measured using commercial/in-house assays. Markers were compared between ART and unassisted pregnancies, and then groups were stratified using ICD9/10 codes to determine differences in specific clinical contexts. RESULTS: In unassisted twin pregnancies, there was a trend of decreased cytokine levels (IL-1ß, IL-6, IL-8, TNFα, p < 0.05), but cytokines in ART twins were the same or higher. Additionally, GST and GPx activities were lower in unassisted twins, and vitamin E levels were higher in ART twins (p < 0.05). In pregnancies complicated by chorioamnionitis, there was a trend of increased cytokine levels in unassisted pregnancies (IL-1ß, IL-6, and IL-8, p < 0.05). No increase was observed in ART, and IFN-γ and TNFα were decreased (p < 0.05). Placental GST and GPx activities were higher in unassisted pregnancies with chorioamnionitis compared to ART (p < 0.05). CONCLUSION: Attenuation of protective placental inflammatory and oxidative stress responses may play a role in the underlying pathogenesis of negative birth outcomes in ART, expanding our understanding of adverse pregnancy outcomes when ART is used to conceive.

Maternal Serotonin Reuptake Inhibitor Antidepressants Have Acute Effects on Fetal Heart Rate Variability in Late Gestation.

Background: Prenatal exposure to serotonin reuptake inhibitor (SRI) antidepressants increases risk for adverse neurodevelopmental outcomes, yet little is known about whether effects are present before birth. In relation to maternal SRI pharmacokinetics, this study investigated chronic and acute effects of prenatal SRI exposure on third-trimester fetal heart rate variability (HRV), while evaluating confounding effects of maternal depressed mood. Methods: At 36-weeks' gestation, cardiotocograph measures of fetal HR and HRV were obtained from 148 pregnant women [four groups: SRI-Depressed (n = 31), SRI-Non-Depressed (n = 18), Depressed (unmedicated; n = 42), and Control (n = 57)] before, and ~5-h after, typical SRI dose. Maternal plasma drug concentrations were quantified at baseline (pre-dose) and four time-points post-dose. Mixed effects modeling investigated group differences between baseline/pre-dose and post-dose fetal HR outcomes. Post hoc analyses investigated sex differences and dose-dependent SRI effects. Results: Maternal SRI plasma concentrations were lowest during the baseline/pre-dose fetal assessment (trough) and increased to a peak at the post-dose assessment; concentration-time curves varied widely between individuals. No group differences in fetal HR or HRV were observed at baseline/pre-dose; however, following maternal SRI dose, short-term HRV decreased in both SRI-exposed fetal groups. In the SRI-Depressed group, these post-dose decreases were displayed by male fetuses, but not females. Further, episodes of high HRV decreased post-dose relative to baseline, but only among SRI-Non-Depressed group fetuses. Higher maternal SRI doses also predicted a greater number of fetal HR decelerations. Fetuses exposed to unmedicated maternal depressed mood did not differ from Controls. Conclusions: Prenatal SRI exposure had acute post-dose effects on fetal HRV in late gestation, which differed depending on maternal mood response to SRI pharmacotherapy. Importantly, fetal SRI effects were sex-specific among mothers with persistent depressive symptoms, as only male fetuses displayed acute HRV decreases. At trough (pre-dose), chronic fetal SRI effects were not identified; however, concurrent changes in maternal SRI plasma levels suggest that fetal drug exposure is inconsistent. Acute SRI-related changes in fetal HRV may reflect a pharmacologic mechanism, a transient impairment in autonomic functioning, or an early adaption to altered serotonergic signaling, which may differ between males and females. Replication is needed to determine significance with postnatal development.

Characterization of Hepatic UDP-Glucuronosyltransferase Enzyme Abundance-Activity Correlations and Population Variability Using a Proteomics Approach and Comparison with Cytochrome P450 Enzymes.

The expression of ten major drug-metabolizing UDP-glucuronosyltransferase (UGT) enzymes in a panel of 130 human hepatic microsomal samples was measured using a liquid chromatography-tandem mass spectrometry-based approach. Simultaneously, ten cytochromes P450 and P450 reductase were also measured, and activity-expression relationships were assessed for comparison. The resulting data sets demonstrated that, with the exception of UGT2B17, 10th to 90th percentiles of UGT expression spanned 3- to 8-fold ranges. These ranges were small relative to ranges of reported mean UGT enzyme expression across different laboratories. We tested correlation of UGT expression with enzymatic activities using selective probe substrates. A high degree of abundance-activity correlation (Spearman's rank correlation coefficient > 0.6) was observed for UGT1As (1A1, 3, 4, 6) and cytochromes P450. In contrast, protein abundance and activity did not correlate strongly for UGT1A9 and UGT2B enzymes (2B4, 7, 10, 15, and 17). Protein abundance was strongly correlated for UGTs 2B7, 2B10, and 2B15. We suggest a number of factors may contribute to these differences including incomplete selectivity of probe substrates, correlated expression of these UGT2B isoforms, and the impact of splice and polymorphic variants on the peptides used in proteomics analysis, and exemplify this in the case of UGT2B10. Extensive correlation analyses identified important criteria for validating the fidelity of proteomics and enzymatic activity approaches for assessing UGT variability, population differences, and ontogenetic changes. SIGNIFICANCE STATEMENT: Protein expression data allow detailed assessment of interindividual variability and enzyme ontogeny. This study has observed that expression and enzyme activity are well correlated for hepatic UGT1A enzymes and cytochromes P450. However, for the UGT2B family, caution is advised when assuming correlation of expression and activity as is often done in physiologically based pharmacokinetic modeling. This can be due to incomplete probe substrate specificities, but may also be related to presence of inactive UGT protein materials and the effect of splicing variations.

Chronic Exposure to 4-Nonylphenol Alters UDP-Glycosyltransferase and Sulfotransferase Clearance of Steroids in the Hard Coral, Pocillopora damicornis.

The effects of the xenoestrogen 4-nonylphenol (4NP) on endocrine and metabolic homeostasis in the reef building coral, Pocillopora damicornis were investigated. The aim was to understand if ubiquitous nonylphenol ethoxylate contaminants in the marine environment result in altered homeostatic function. Coral colonies were chronically exposed (6 weeks) to a sublethal concentration (1 ppb) of 4NP and sampled over the coral's lunar reproductive cycle. Although activity of steroidogenic enzymes [cytochrome P450 (CYP) 17, CYP 19, and 3-ß-Hydroxysteroid dehydrogenase] and the conjugation enzyme glutathione-S-transferase was not altered, significant increases in the activity of the steroid clearing enzyme UDP-glycosyltransferase (UGT) were observed. The natural fluctuation of UGT activity with the lunar cycle was replaced with consistently high UGT activity throughout the reproductive cycle during 4NP exposure. No effect of 4NP on the reverse reaction, mediated by ß-glucuronidase, was observed. Thus, 4NP shifts the UGT:ß-glucuronidase ratio toward greater clearance at points in the lunar cycle where retention of compounds is typically favored. Additionally, 4NP reduced activity of the steroid regeneration enzyme steroid sulfatase, further shifting the system toward clearance rather than regeneration. These data imply that environmentally relevant levels of 4NP may be impacting the reproductive health of corals and threatening the persistence of coral reefs.

Parameterization of Microsomal and Cytosolic Scaling Factors: Methodological and Biological Considerations for Scalar Derivation and Validation.

Mathematical models that can predict the kinetics of compounds have been increasingly adopted for drug development and risk assessment. Data for these models may be generated from in vitro experimental systems containing enzymes contributing to metabolic clearance, such as subcellular tissue fractions including microsomes and cytosol. Extrapolation from these systems is facilitated by common scaling factors, known as microsomal protein per gram (MPPG) and cytosolic protein per gram (CPPG). Historically, parameterization of MPPG and CPPG has employed the use of recovery factors, commonly benchmarked to cytochromes P450 which work well in some contexts, but could be problematic for other enzymes. Here, we propose absolute quantification of protein content and supplementary assays to evaluate microsomal/cytosolic purity that should be employed. Examples include calculation of microsomal latency by mannose-6-phosphatase activity and immunoblotting of subcellular fractions with fraction-specific markers. Further considerations include tissue source, as disease states can affect enzyme expression and activity, and the methodology used for scalar parameterization. Regional- and organ-specific expression of enzymes, in addition to differences in organ physiology, is another important consideration. Because most efforts have focused on the liver that is, for the most part, homogeneous, derived scalars may not capture the heterogeneity of other major tissues contributing to xenobiotic metabolism including the kidneys and small intestine. Better understanding of these scalars, and how to appropriately derive them from extrahepatic tissues can provide support to the inferences made with physiologically based pharmacokinetic modeling, increase its accuracy in characterizing in vivo drug pharmacokinetics, and improve confidence in go-no-go decisions for clinical trials.

Detection and quantitation of non-steroidal anti-inflammatory drug use close to the time of birth using umbilical cord tissue.

BACKGROUND: Nonsteroidal anti-inflammatory drugs are contraindicated in the third trimester of pregnancy due to negative effects including alteration of uteroplacental blood flow, premature ductus arteriosus closure, and adverse effects on the fetal kidney. However, many women are unaware of these risks, and commonly report their use in pregnancy. We aimed to determine if umbilical cord was a reliable matrix for detecting NSAID use, determine incidence of use close to labour, and uncover associations with obstetric/neonatal outcomes. METHODS: We developed a UHPLC-MS/MS method to simultaneously detect diclofenac, ibuprofen, indomethacin, naproxen, and salicylic acid in plasma and umbilical cord lysate. Using this method, we screened 380 lysates to determine the prevalence of NSAID use. Results were compared to the clinical outcomes in pregnancy using ICD9/10 chart codes (n = 21). RESULTS: The UHPLC-MS/MS method has excellent linearity, accuracy, and precision in solvent and plasma, but lower sensitivity in umbilical cord lysate. We report a 3 % rate of NSAID ingestion within days of labour - the pharmacokinetically-determined window for active ingestion. There were no significant differences observed for maternal, obstetric, or neonatal outcomes between the NSAID positive group (n = 11) and NSAID negative group (n = 369). CONCLUSIONS: Because NSAID use in third trimester is contraindicated, even a 3% usage rate is alarmingly high. Based on UHPLC-MS/MS performance of umbilical cord lysate, 3% is likely a conservative estimate. Recent adoption of NSAIDs under clinical supervision to support in vitro fertilisation and prevent pre-eclampsia indicates future work should focus on determining safe dosages of NSAIDs and the correct therapeutic window in pregnancy.

Homology Modeling of Human Uridine-5'-diphosphate-glucuronosyltransferase 1A6 Reveals Insights into Factors Influencing Substrate and Cosubstrate Binding.

The elimination of numerous endogenous compounds and xenobiotics via glucuronidation by uridine-5'-diphosphate glycosyltransferase enzymes (UGTs) is an essential process of the body's chemical defense system. UGTs have distinct but overlapping substrate preferences, but the molecular basis for their substrate specificity remains poorly understood. Three-dimensional protein structures can greatly enhance our understanding of the interactions between enzymes and their substrates, but because of the inherent difficulties in purifying and crystallizing integral endoplasmic reticulum membrane proteins, no complete mammalian UGT structure has yet been produced. To address this problem, we have created a homology model of UGT1A6 using I-TASSER to explore, in detail, the interactions of human UGT1A6 with its substrates. Ligands were docked into our model in the presence of the cosubstrate uridine-5'-diphosphate-glucuronic acid, interacting residues were examined, and poses were compared to those cocrystallized with various plant and bacterial glycosyltransferases (GTs). Our model structurally resembles other GTs, and docking experiments replicated many of the expected UGT-substrate interactions. Some bias toward the template structures' protein-substrate interactions and binding preferences was evident.

Umbilical cord as an analytical matrix - A technical note.

The umbilical cord (UC) connects the fetal blood supply to the placenta, so is exposed to all systemic endo- and xenobiotics. We have extensive experience using UC as an analytical matrix for detecting and/or quantitating drugs, chemicals and endogenous compounds. This technical note describes advantages (large amount available, ease of collection, small sample needed for use, rapid availability) and challenges (clinical relationships, processing difficulties, matrix effects on analytes and detection technologies) of UC as an analytical matrix in ELISA and LC/MS platforms, and provides guidance for successfully working with this tissue.

SCN1A variants from bench to bedside-improved clinical prediction from functional characterization.

Variants in the SCN1A gene are associated with a wide range of disorders including genetic epilepsy with febrile seizures plus (GEFS+), familial hemiplegic migraine (FHM), and the severe childhood epilepsy Dravet syndrome (DS). Predicting disease outcomes based on variant type remains challenging. Despite thousands of SCN1A variants being reported, only a minority has been functionally assessed. We review the functional SCN1A work performed to date, critically appraise electrophysiological measurements, compare this to in silico predictions, and relate our findings to the clinical phenotype. Our results show, regardless of the underlying phenotype, that conventional in silico software correctly predicted benign from pathogenic variants in nearly 90%, however was unable to differentiate within the disease spectrum (DS vs. GEFS+ vs. FHM). In contrast, patch-clamp data from mammalian expression systems revealed functional differences among missense variants allowing discrimination between disease severities. Those presenting with milder phenotypes retained a degree of channel function measured as residual whole-cell current, whereas those without any whole-cell current were often associated with DS (p = .024). These findings demonstrate that electrophysiological data from mammalian expression systems can serve as useful disease biomarker when evaluating SCN1A variants, particularly in view of new and emerging treatment options in DS.

Coexpression of Human Hepatic Uridine Diphosphate Glucuronosyltransferase Proteins: Implications for Ontogenetic Mechanisms and Isoform Coregulation.

Uridine diphosphate glucuronosyltransferases (UGTs) catalyze glucuronidation to facilitate systemic and local clearance of numerous chemicals and drugs. To investigate whether UGT expression is coregulated in human liver, we analyzed the protein expression of UGTs 1A1, 1A3, 1A4, 1A6, 1A9, 2B7, 3A1, and 3A2 using western blots from 164 healthy human liver samples, comparing expression with age and sex. UGT1A6 levels were significantly higher in children than adults, and UGT3A1 and 3A2 expression significantly increased with age from childhood to age >65 yearas. In children aged <18 years, UGT1A4/1A9 protein expression was significantly correlated, but not for adults aged >18 years. UGT1A3 expression was always significantly correlated with other UGT1A isoforms in all adults aged >18 years. In individuals aged ≥12 years, expression of UGT1A1/1A4, UGT1A1/1A6, UGT1A1/1A9, and UGT1A4/1A6 significantly correlated, which was not observed in children aged <12 years. In contrast, UGT1A4/2B7 showed significant correlation in children aged <12 years, but not in individuals aged ≥12 years, and this was observed in female but not male individuals. Expression of UGT1A6/1A9 and UGT3A1/3A2 correlated in the entire sample population, but UGT3As did not correlate with other UGTs. These correlations were sex dependent, as UGT1A3/1A1, UGT1A4/2B7 and UGT3A1/3A2 correlated more highly in male than female individuals, while UGT1A4/1A6 protein correlated more significantly in female than male individuals. This is the first report on the ontogeny of UGT3A isoforms, showing maximal expression in the elderly, and is the first demonstration that UGT isoforms commonly coexpress in vivo, in both age-dependent and sex-dependent manners.

Dysregulation of inflammatory cytokines and inhibition of VEGFA in the human umbilical cord are associated with negative pregnancy outcomes.

INTRODUCTION: Cytokines and vascular endothelial growth factors (VEGF) are involved in all aspects of pregnancy: from placentation, through fetal development, parturition and neonatal well-being. Umbilical cord inflammatory cytokines and/or VEGF have not been well studied with respect to dysregulation associated with disorders of pregnancy or maternal/neonatal outcomes. METHODS: Here we have used multiplex ELISA to screen umbilical cord lysates (comprising cord blood, endothelia and Wharton's jelly, n = 380), for levels of IFN-γ, IL1-ß, IL-6, IL-8, IL-10, TNF-α and VEGFs A, C and D and associations with 46 ICD9/10 codes encompassing obstetric, maternal and neonatal variables. RESULTS: No significant differences were observed for IFNγ, VEGFC or VEGFD with any clinical outcomes. The cytokines IL1-ß, IL-6, IL-8, IL-10, and TNF-α showed varying levels of induction and suppression with primarily fetal-placental and neonatal complications. The largest number of significant differences between umbilical cytokines and clinical outcomes were observed for chorioamnionitis (IL1-ß, IL-6, IL-8, TNF-α), and meconium passage during birth (IL1-ß, IL-6, IL-8) where significant pro-inflammatory responses occurred and sex differences in IL-8 expression were noted. In contrast, gonococcal infection showed suppressed immune response significantly lowering IL1-ß, IL-6, IL-8, IL-10 and TNF-α. For 12/46 negative pregnancy outcomes, strong suppression of VEGFA occurred. DISCUSSION: Angiogenic and inflammatory changes in the umbilical cord could be detrimental by increasing vascular permeability in the umbilical artery or vein and/or altering vascular tone, either of which would alter blood flow affecting delivery and removal of compounds. Further elucidation of inflammatory responses in the umbilical cord may provide mechanistic understanding of adverse pregnancy outcomes.

Characterization of the Ontogeny of Hepatic UDP-Glucuronosyltransferase Enzymes Based on Glucuronidation Activity Measured in Human Liver Microsomes.

An understanding of the postnatal development of hepatic UDP-glucuronosyltransferase (UGT) enzymes is required for accurate prediction of the age-dependent changes in pharmacokinetics of many drugs used in children. However, the maturation rate of hepatic UGT isoforms remains a major knowledge gap. This study aimed to establish the age-associated changes in glucuronidation activity of 10 major hepatic UGT isoforms in humans, namely, UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A9, UGT2B4, UGT2B7, UGT2B10, UGT2B15, and UGT2B17. Human liver microsomes from pediatric and adult donors were incubated under optimized incubation conditions to assess the activity rates of hepatic UGT isoforms using a panel of 19 in vitro UGT probe substrates and clinically used drugs. Statistically strong correlations of glucuronidation activities allowed the ontogeny of UGT1A1, UGT1A4, UGT2B7, UGT2B10, and UGT2B15 to be established using multiple selective UGT substrates and matched human liver microsome samples. The postnatal development of hepatic UGTs is isoform-dependent using either individual or cross-correlated selective isoform substrates. Maximal adult activity was reached at different times ranging from within a month (UGT1A1, UGT2B4, UGT2B7, UGT2B10, and UGT2B15), during infancy (UGT1A3, UGT1A4, and UGT1A9), to adolescence (UGT1A6 and UGT2B17). This study provides an extensive characterization of the postnatal ontogeny profiles of hepatic UGT enzymes that are instrumental for predicting drug disposition via in vitro-in vivo extrapolation algorithms and verifying pharmacokinetic predictions against in vivo observations via pediatric physiologically based pharmacokinetic modeling in pediatric patients.

Antioxidant enzyme cycling over reproductive lunar cycles in Pocillopora damicornis.

The impacts of continued degradation of watersheds on coastal coral reefs world-wide is alarming, and action addressing anthropogenic stressors and subsequent rehabilitation of watersheds and adjacent reefs is an urgent priority. The aim of this study is to develop and improve the use of antioxidant enzymes as bioindicators of stress in coral species. In order to fully develop such tools, it is necessary to first understand baseline cycling of these enzymes within coral tissues. Due to inherent links between reproduction and oxidative stress, these aims may be facilitated by sampling coral tissues over reproductively-linked lunar cycles to determine variations from baseline. By developing a greater understanding of biochemical markers of stress in corals, specifically antioxidant defense enzymes catalase (CAT), glutathione reductase (GR), glutathione peroxidase (GPx), and superoxide dismutase (SOD) in Hawaiian Pocillopora damicornis, we have provided molecular tools that identify thresholds of stress on coral reefs. Our results suggest that the coral reproductive state is a significant factor affecting the activity of antioxidant enzymes. Specifically, CAT and GR display maximum activity during peak reproductive state. Whereas significant maximal Se-independent GPx and SOD activity was measured during off-peak reproductive cycles. Such insight into the cyclical variation of the activity of these enzymes should be applied towards differentiating the influence of natural biological activity cycling in diagnostic tests identifying the effects of different physical environmental factors and chemical pollutants on coral health. Through the development and application of these molecular biomarkers of stress, we look to improve our ability to identify problems at the sub-lethal level, when action can be taken to mitigate a/biotic impacts.

The Ontogeny of UDP-glucuronosyltransferase Enzymes, Recommendations for Future Profiling Studies and Application Through Physiologically Based Pharmacokinetic Modelling.

Limited understanding of drug pharmacokinetics in children is one of the major challenges in paediatric drug development. This is most critical in neonates and infants owing to rapid changes in physiological functions, especially in the activity of drug-metabolising enzymes. Paediatric physiologically based pharmacokinetic models that integrate ontogeny functions for cytochrome P450 enzymes have aided our understanding of drug exposure in children, including those under the age of 2 years. Paediatric physiologically based pharmacokinetic models have consequently been recognised by the European Medicines Agency and the US Food and Drug Administration as innovative tools in paediatric drug development and regulatory decision making. However, little is currently known about age-related changes in UDP-glucuronosyltransferase-mediated metabolism, which represents the most important conjugation reaction for xenobiotics. Therefore, the objective of the review was to conduct a thorough literature survey to summarise our current understanding of age-related changes in UDP-glucuronosyltransferases as well as associated clinical and experimental sources of variance. Our findings indicate that there are distinct differences in UDP-glucuronosyltransferase expression and activity between isoforms for different age groups. In addition, there is substantial variability between individuals and laboratories reported for human liver microsomes, which results in part from a lack of standardised experimental conditions. Therefore, we provide a number of best practice recommendations for experimental conditions, which ultimately may help improve the quality of data used for quantitative clinical pharmacology approaches, and thus for safe and effective pharmacotherapy in children.

Optimization of Experimental Conditions of Automated Glucuronidation Assays in Human Liver Microsomes Using a Cocktail Approach and Ultra-High Performance Liquid Chromatography-Tandem Mass Spectrometry.

UDP-glucuronosyltransferase (UGT)-mediated metabolism is possibly the most important conjugation reaction for marketed drugs. However, there are currently no generally accepted standard incubation conditions for UGT microsomal assays, and substantial differences in experimental design and methodology between laboratories hinder cross-study comparison of in vitro activities. This study aimed to define optimal experimental conditions to determine glucuronidation activity of multiple UGT isoforms simultaneously using human liver microsomes. Hepatic glucuronidation activities of UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A9, UGT2B4, UGT2B7, UGT2B10, UGT2B15, and UGT2B17 were determined using cocktail incubations of 10 UGT probe substrates. Buffer components and cosubstrates were assessed over a range of concentrations including magnesium chloride (MgCl2; 0-10 mM) and uridine 5'-diphosphoglucuronic acid (UDPGA; 1-25 mM) with either Tris-HCl or potassium phosphate buffer (100 mM, pH 7.4). Greater microsomal glucuronidation activity by different hepatic UGT isoforms was obtained using 10 mM MgCl2 and 5 mM UDPGA with 100 mM Tris-HCl buffer. The influence of bovine serum albumin (BSA; 0.1%-2% w/v) on glucuronidation activity was also assessed. Enzyme- and substrate-dependent effects of BSA were observed, resulting in decreased total activity of UGT1A1, UGT1A3, and UGT2B17 and increased total UGT1A9 and UGT2B7 activity. The inclusion of BSA did not significantly reduce the between-subject variability of UGT activity. Future in vitro UGT profiling studies under the proposed optimized experimental conditions would allow high-quality positive control data to be generated across laboratories, with effective control of a high degree of between-donor variability for UGT activity and for chemical optimization toward lower-clearance drug molecules in a pharmaceutical drug discovery setting.