Dysregulation of Glucocorticoid Receptor Homeostasis and Glucocorticoid-Associated Genes in Umbilical Cord Endothelial Cells of Diet-Induced Obese Pregnant Sheep.

Maternal obesity (MO) is associated with offspring cardiometabolic diseases that are hypothesized to be partly mediated by glucocorticoids. Therefore, we aimed to study fetal endothelial glucocorticoid sensitivity in an ovine model of MO. Rambouillet/Columbia ewes were fed either 100% (control) or 150% (MO) National Research Council recommendations from 60 d before mating until near-term (135 days gestation). Sheep umbilical vein and artery endothelial cells (ShUVECs and ShUAECs) were used to study glucocorticoid receptor (GR) expression and function in vitro. Dexamethasone dose-response studies of gene expression, activation of a glucocorticoid response element (GRE)-dependent luciferase reporter vector, and cytosolic/nuclear GR translocation were used to assess GR homeostasis. MO significantly increased basal GR protein levels in both ShUVECs and ShUAECs. Increased GR protein levels did not result in increased dexamethasone sensitivity in the regulation of key endothelial gene expression such as endothelial nitric oxide synthase, plasminogen activator inhibitor 1, vascular endothelial growth factor, or intercellular adhesion molecule 1. In ShUVECs, MO increased GRE-dependent transactivation and FKBP prolyl isomerase 5 (FKBP5) expression. ShUAECs showed generalized glucocorticoid resistance in both dietary groups. Finally, we found that ShUVECs were less sensitive to dexamethasone-induced activation of GR than human umbilical vein endothelial cells (HUVECs). These findings suggest that MO-mediated effects in the offspring endothelium could be further mediated by dysregulation of GR homeostasis in humans as compared with sheep.

Maternal hepatic adaptations during obese pregnancy encompass lobe-specific mitochondrial alterations and oxidative stress.

Maternal obesity (MO) is rising worldwide, affecting half of all gestations, constituting a possible risk-factor for some pregnancy-associated liver diseases (PALD) and hepatic diseases. PALD occur in approximately 3% of pregnancies and are characterized by maternal hepatic oxidative stress (OS) and mitochondrial dysfunction. Maternal hepatic disease increases maternal and fetal morbidity and mortality. Understanding the role of MO on liver function and pathophysiology could be crucial for better understanding the altered pathways leading to PALD and liver disease, possibly paving the way to prevention and adequate management of disease. We investigated specific hepatic metabolic alterations in mitochondria and oxidative stress during MO at late-gestation. Maternal hepatic tissue was collected at 90% gestation in Control and MO ewes (fed 150% of recommended nutrition starting 60 days before conception). Maternal hepatic redox state, mitochondrial respiratory chain (MRC), and OS markers were investigated. MO decreased MRC complex-II activity and its subunits SDHA and SDHB protein expression, increased complex-I and complex-IV activities despite reduced complex-IV subunit mtCO1 protein expression, and increased ATP synthase ATP5A subunit. Hepatic MO-metabolic remodeling was characterized by decreased adenine nucleotide translocator 1 and 2 (ANT-1/2) and voltage-dependent anion channel (VDAC) protein expression and protein kinase A (PKA) activity (P<0.01), and augmented NAD+/NADH ratio due to reduced NADH levels (P<0.01). MO showed an altered redox state with increased OS, increased lipid peroxidation (P<0.01), decreased GSH/GSSG ratio (P=0.005), increased superoxide dismutase (P=0.03) and decreased catalase (P=0.03) antioxidant enzymatic activities, lower catalase, glutathione peroxidase (GPX)-4 and glutathione reductase protein expression (P<0.05), and increased GPX-1 abundance (P=0.03). MO-related hepatic changes were more evident in the right lobe, corroborated by the integrative data analysis. Hepatic tissue from obese pregnant ewes showed alterations in the redox state, consistent with OS and MRC and metabolism remodeling. These are hallmarks of PALD and hepatic disease, supporting MO as a risk-factor and highlighting OS and mitochondrial dysfunction as mechanisms responsible for liver disease predisposition.

Maternal obesity in sheep impairs foetal hepatic mitochondrial respiratory chain capacity.

BACKGROUND: Changes in the nutritional environment in utero induced by maternal obesity (MO) lead to foetal metabolic dysfunction predisposing offspring to later-life metabolic diseases. Since mitochondria play a crucial role in hepatic metabolism and function, we hypothesized that MO prior to conception and throughout pregnancy programmes foetal sheep liver mitochondrial phenotype. MATERIAL AND METHODS: Ewes ate an obesogenic diet (150% requirements; MO), or 100% requirements (CTR), from 60 days prior to conception. Foetal livers were removed at 0.9 gestation. We measured foetal liver mitochondrial DNA copy number, activity of superoxide dismutase, cathepsins B and D and selected protein content, total phospholipids and cardiolipin and activity of mitochondrial respiratory chain complexes. RESULTS: A significant decrease in activities of mitochondrial complexes I, II-III and IV, but not aconitase, was observed in MO. In the antioxidant machinery, there was a significant increase in activity of total superoxide dismutase (SOD) and SOD2 in MO. However, no differences were found regarding autophagy-related protein content (p62, beclin-I, LC3-I, LC3-II and Lamp2A) and cathepsin B and D activities. A 21.5% decrease in total mitochondrial phospholipid was observed in MO. CONCLUSIONS: The data indicate that MO impairs foetal hepatic mitochondrial oxidative capacity and affects total mitochondrial phospholipid content. In addition, MO affects the regulation of foetal liver redox pathways, indicating metabolic adaptations to the higher foetal lipid environment. Consequences of in utero programming of foetal hepatic metabolism may persist and compromise mitochondrial bioenergetics in later life, and increase susceptibility to metabolic diseases.

Simultaneous Quantification of Protein Expression and Modifications by Top-down Targeted Proteomics: A Case of the Sarcomeric Subproteome.

Determining changes in protein expression and post-translational modifications (PTMs) is crucial for elucidating cellular signal transduction and disease mechanisms. Conventional antibody-based approaches have inherent problems such as the limited availability of high-quality antibodies and batch-to-batch variation. Top-down mass spectrometry (MS)-based proteomics has emerged as the most powerful method for characterization and quantification of protein modifications. Nevertheless, robust methods to simultaneously determine changes in protein expression and PTMs remain lacking. Herein, we have developed a straightforward and robust top-down liquid chromatography (LC)/MS-based targeted proteomics platform for simultaneous quantification of protein expression and PTMs with high throughput and high reproducibility. We employed this method to analyze the sarcomeric subproteome from various muscle types of different species, which successfully revealed skeletal muscle heterogeneity and cardiac developmental changes in sarcomeric protein isoform expression and PTMs. As demonstrated, this targeted top-down proteomics platform offers an excellent 'antibody-independent' alternative for the accurate quantification of sarcomeric protein expression and PTMs concurrently in complex mixtures, which is generally applicable to different species and various tissue types.

Maternal obesity impairs fetal cardiomyocyte contractile function in sheep.

Obesity is a major public health problem worldwide. In the United States, one-third of women of reproductive age are obese. Human studies show that maternal obesity (MO) predisposes offspring to cardiovascular disease. However, the underlying mechanisms remain unclear. Given the similarities between pregnancy in sheep and humans, we studied sheep to examine the impact of MO on fetal cardiomyocyte contractility at term. We observed that MO impaired cardiomyocyte contractility by reducing peak shortening and shortening/relengthening velocity, prolonging time to relengthening. MO disrupted Ca2+ homeostasis in fetal cardiomyocytes, increasing intracellular Ca2+ and inducing cellular Ca2+ insensitivity. The Ca2+-release channel was impaired, but Ca2+ uptake was unaffected by MO. The upstream kinases that phosphorylate the Ca2+-release channel-ryanodine receptor-2, PKA, and calmodulin-dependent protein kinase II-were activated in MO fetuses. Contractile dysfunction was associated with an increased ratio of myosin heavy chain (MHC)-ß to MHC-α and upregulated cardiac troponin (cTn)-T and tropomyosin, as well as cTn-I phosphorylation. In summary, this is the first characterization of the effects of MO on fetal cardiomyocyte contractility. Our findings indicate that MO impairs fetal cardiomyocyte contractility through altered intracellular Ca2+ handling, overloading fetal cardiomyocyte intracellular Ca2+ and aberrant myofilament protein composition. These mechanisms may contribute to developmental programming by MO of offspring cardiac function and predisposition to later life cardiovascular disease in the offspring.-Wang, Q., Zhu, C., Sun, M., Maimaiti, R., Ford, S. P., Nathanielsz, P. W., Ren, J., Guo, W. Maternal obesity impairs fetal cardiomyocyte contractile function in sheep.

Lack of involvement of chitinase in direct toxicity of Beauveria bassiana cultures to the aphid Myzus persicae.

The fungal insect pathogen Beauveria bassiana produces a range of insecticidal metabolites and enzymes, including chitinases and proteases, which may assist the disease progression. The enzymes often play a predominant role in the pathogenicity pathway and both chitinases and proteases have previously been shown to be important in host infection. Spray application of supernatants of B. bassiana broth cultures of an isolate from New Zealand caused significant mortality in the green peach aphid, Myzus persicae, within 24 h, demonstrating an apparent contact toxicity. Three-day-old broth cultures were the most effective, with less insect mortality seen using six-day-old broth. However, aphicidal activity increased again when treating aphids with seven-day-old broth. Cultures grew substantially better and produced more potent aphicidal cultures when cultured in media with an initial pH above 5.5. Chitinase was produced a day earlier than the serine protease Pr1, but the peak production periods of these enzymes did not correlate with the aphicidal activities of three- or six-day-old cultures. Cultures treated with EDTA or heated to inactivate the enzymes still showed strong insecticidal activity. Neither beauvericin nor bassianolide, two known insecticidal metabolites, were detected in the supernatants. Therefore the key aphicidal components of B. bassiana cultures were not associated with chitinase nor Pr1 and are yet to be identified.

Z-band and M-band titin splicing and regulation by RNA binding motif 20 in striated muscles.

Titin (TTN) has multifunctional roles in sarcomere assembly, mechanosignaling transduction, and muscle stiffness. TTN splicing generates variable protein sizes with different functions. Therefore, understanding TTN splicing is important to develop a novel treatment for TTN-based diseases. The I-band TTN splicing regulated by RNA binding motif 20 (RBM20) has been extensively studied. However, the Z- and M-band splicing and regulation remain poorly understood. Herein, we aimed to define the Z- and M-band splicing in striated muscles and determined whether RBM20 regulates the Z- and M-band splicing. We discovered four new Z-band TTN splicing variants, and one of them dominates in mouse, rat, sheep, and human hearts. But only one form can be detected in frog and chicken hearts. In skeletal muscles, three new Z repeats (Zr) were detected, and Zr4 to 6 exclusion dominates in the fast muscles, whereas Zr4 skipping dominates in the slow muscle. No developmental changes were detected in the Z-band. In the M-band, two new variants were discovered with alternative 3' splice site in exon363 (Mex5) and alternative 5' splice site in intron 362. However, only the sheep heart expresses two new variants rather than other species. Skeletal muscles express three M-band variants with altered ratios of Mex5 inclusion to Mex5 exclusion. Finally, we revealed that RBM20 does not regulate the Z- and M-band splicing in the heart, but does in skeletal muscles. Taken together, we characterized the Z- and M-band splicing and provided the first evidence of the role of RBM20 in the Z- and M-band TTN splicing.

Differential contribution of chronic binge alcohol and antiretroviral therapy to metabolic dysregulation in SIV-infected male macaques.

The incidence of alcohol use disorder (AUD) is higher among people living with HIV (PLWH). The advent and continued development of antiretroviral therapy (ART) has significantly reduced mortality, shifting the course of HIV infection to a chronic illness. However, this is associated with an increased incidence of comorbid conditions, including type 2 diabetes mellitus, insulin resistance, and cardiovascular complications. Using a nonhuman primate model of simian immunodeficiency virus (SIV) infection, previous studies have demonstrated that chronic binge alcohol (CBA) administration decreases whole body insulin responsiveness, irrespective of ART administration. The objective of the current study was to determine the effects of CBA and ART on insulin-sensitive peripheral tissues before the development of overt clinical symptoms of SIV disease. Our results show that CBA reduced omental adipocyte cell size, increased collagen expression, and decreased the in vitro differentiation potential of adipose-derived stem cells. In contrast, it did not alter skeletal muscle or omental or hepatic expression of insulin signaling proteins. However, ART significantly decreased skeletal muscle expression of phosphatase and tensin homolog, total mechanistic target of rapamycin, and ribosomal protein S6. In addition, ART increased hepatic phosphorylation of AMP-activated protein kinase α and increased gene expression of key enzymes required for gluconeogenesis and fatty acid synthesis. These findings suggest that CBA and ART differentially promote adverse metabolic effects in an organ-specific manner that may underlie insulin resistance associated with alcohol, SIV, and ART. Whether this is translated in PLWH with AUD remains to be determined.

Chronic binge alcohol administration impairs glucose-insulin dynamics and decreases adiponectin in asymptomatic simian immunodeficiency virus-infected macaques.

Alcohol use disorders (AUDs) frequently exist among persons living with HIV/AIDS. Chronic alcohol consumption, HIV infection, and antiretroviral therapy (ART) are independently associated with impairments in glucose-insulin dynamics. Previous studies from our laboratory have shown that chronic binge alcohol (CBA) administration decreases body mass index, attenuates weight gain, and accentuates skeletal muscle wasting at end-stage disease in non-ART-treated simian immunodeficiency virus (SIV)-infected male rhesus macaques. The aim of this study was to investigate whether CBA and ART alone or in combination alter body composition or glucose-insulin dynamics in SIV-infected male rhesus macaques during the asymptomatic phase of SIV infection. Daily CBA or sucrose (SUC) administration was initiated 3 mo before intrarectal SIV inoculation and continued until the study end point at 11 mo post-SIV infection. ART or placebo was initiated 2.5 mo after SIV infection and continued until study end point. Four treatment groups (SUC/SIV ± ART and CBA/SIV ± ART) were studied. CBA/SIV macaques had significantly decreased circulating adiponectin and resistin levels relative to SUC/SIV macaques and reduced disposition index and acute insulin response to glucose, insulin, and C-peptide release during frequently sampled intravenous glucose tolerance test, irrespective of ART status. No statistically significant differences were observed in homeostatic model assessment-insulin resistance values, body weight, total body fat, abdominal fat, or total lean mass or bone health among the four groups. These findings demonstrate CBA-mediated impairments in glucose-insulin dynamics and adipokine profile in asymptomatic SIV-infected macaques, irrespective of ART.

Chronic binge alcohol administration accentuates expression of pro-fibrotic and inflammatory genes in the skeletal muscle of simian immunodeficiency virus-infected macaques.

BACKGROUND: Chronic binge alcohol (CBA) administration exacerbates skeletal muscle (SKM) wasting at the terminal stage of simian immunodeficiency virus (SIV) infection in rhesus macaques. This is associated with a pro-inflammatory and oxidative milieu which we have previously shown to be associated with a disrupted balance between anabolic and catabolic mechanisms. In this study, we attempted to characterize the SKM gene expression signature in CBA-administered SIV-infected macaques, using the same animals from the previous study. METHODS: Administration of intragastric alcohol or sucrose to male rhesus macaques began 3 months prior to SIV infection and continued throughout the duration of study. Gene transcriptomes of SKM excised at necropsy (~10 months post-SIV) from healthy na\xEFve control (Control), sucrose-administered, SIV-infected (SUC-SIV), and CBA-administered, SIV-infected (CBA-SIV) macaques were evaluated in microarray data sets. The Protein Analysis Through Evolutionary Relationships classification tool was used to filter differentially regulated genes based on their predicted function into select biological processes relevant to SKM wasting which were inflammation, extracellular matrix (ECM) remodeling, and metabolism. RESULTS: In total, 1,124 genes were differentially regulated between SUC-SIV and Controls, 2,022 genes were differentially expressed between the CBA-SIV and Controls, and 836 genes were differentially expressed between CBA-SIV and SUC-SIV animals. The relevance of altered gene expression was reflected in the up-regulation of pro-inflammatory CCL-2, CCL-8, CX3CL1, SELE, HP, and TNFRS10A mRNA expression. In addition, ECM remodeling was reflected in the up-regulation of TIMP-1, MMP 2, and MMP 9 mRNA expression and transforming growth factor-beta 1 protein expression. In addition, hydroxyproline content and picrosirius staining reflected increased collagen deposition in the CBA-SIV muscle tissue. CONCLUSIONS: The results of the study demonstrate SKM inflammation as an important underlying mechanism for muscle wasting. In addition, the study provides evidence of SKM fibrotic transformation as a factor in CBA-induced accentuation of SIV-associated muscle wasting.

Maternal obesity, lipotoxicity and cardiovascular diseases in offspring.

Maternal obesity has risen dramatically over the past 20 years, by nearly 42% in African-Americans and 29% in Caucasians. Maternal obesity is afflicted with many maternal obstetric complications in the offspring including high blood pressure, obesity, gestational diabetes and increased perinatal morbidity. Maternal nutritional environment plays a rather important role in the programming of the health set-points in the offspring such as glucose and insulin metabolism, energy balance and predisposition to metabolic disorders. In particular, maternal obesity is associated with elevated prevalence of cardiovascular diseases in the offspring. Evidence from human and experimental studies including rodents and nonhuman primates has indicated that maternal obesity or overnutrition programs offspring for an increased risk of adult obesity. Maternal obesity or fat diet exposure predisposes the onset and development of obesity, insulin resistance, cardiac hypertrophy and myocardial contractile anomalies in the offspring. A number of mechanisms including elevated hormones (leptin, insulin), nutrients (fatty acids, triglycerides and glucose) and inflammatory cytokines have been postulated to play a key role in maternal obesity-induced postnatal cardiovascular sequelae. In addition, lipotoxicity (accumulation of lipid metabolites) resulting from maternal obesity is capable of activating a number of stress signaling cascades including pro-inflammatory cytokines and oxidative stress to exacerbate maternal obesity-induced cardiovascular complications later on in adult life. This mini-review summarizes the recent knowledge with regard to the role of lipotoxicity in maternal obesity-induced change in cardiovascular function in the offspring. This article is part of a Special Issue entitled "Focus on Cardiac Metabolism".

Diet reduction to requirements in obese/overfed ewes from early gestation prevents glucose/insulin dysregulation and returns fetal adiposity and organ development to control levels.

Obesity at conception and excess gestational weight gain pose significant risks for adverse health consequences in human offspring. This study evaluated the effects of reducing dietary intake of obese/overfed ewes beginning in early gestation on fetal development. Sixty days prior to conception, ewes were assigned to a control diet [CON: 100% of National Research Council (NRC) recommendations], a diet inducing maternal obesity (MO: 150% of NRC recommendations), or a maternal obesity intervention diet (MOI: 150% of NRC recommendations to day 28 of gestation, then 100% NRC) until necropsy at midgestation (day 75) or late (day 135) gestation. Fetal size and weight, as well as fetal organ weights, were greater (P < 0.05) at midgestation in MO ewes than those of CON and MOI ewes. By late gestation, whereas fetal size and weight did not differ among dietary groups, cardiac ventricular weights and wall thicknesses as well as liver and perirenal fat weights remained elevated in fetuses from MO ewes compared with those from CON and MOI ewes. MO ewes and fetuses exhibited elevated (P < 0.05) plasma concentrations of triglycerides, cholesterol, insulin, glucose, and cortisol at midgestation compared with CON and MOI ewes and fetuses. In late gestation, whereas plasma triglycerides and cholesterol, insulin, and cortisol remained elevated in MO vs. CON and MOI ewes and fetuses, glucose concentrations were elevated in both MO and MOI fetuses compared with CON fetuses, which was associated with elevated placental GLUT3 expression in both groups. These data are consistent with the concept that reducing maternal diet of obese/overfed ewes to requirements from early gestation can prevent subsequent alterations in fetal growth, adiposity, and glucose/insulin dynamics.

Maternal nutrient restriction in the ewe from early to midgestation programs reduced steroidogenic enzyme expression and tended to reduce progesterone content of corpora lutea, as well as circulating progesterone in nonpregnant aged female offspring.

BACKGROUND: Previously we reported decreased circulating progesterone and fertility in one and two year old ewes born to undernourished mothers. This study was designed to investigate if this reduction in progesterone persisted into old age, and if it did, what mechanisms are involved. METHODS: Ewes were fed a nutrient restricted (NR, 50% of NRC recommendations) or control (C, 100% of NRC) diets from day 28 to 78 of gestation, then all were fed to requirements through parturition and weaning. Female offspring (4 per treatment group) were maintained as a group and fed to requirements from weaning until assigned to this study at 6 years of age. Ewes were synchronized for estrus (day 0) and blood samples were collected daily from day 0 to day 11 before necropsy on day 12. Blood serum and luteal tissue were assayed for progesterone concentrations by validated radioimmunoassay. RESULTS: Circulation progesterone concentrations tended to be lower (P = 0.06) in NR than C offspring from day 0 to 11 of the estrous cycle. While total luteal weight was similar across groups, total progesterone content also tended to be reduced (P = 0.07) in luteal tissue of NR than C offspring. Activity of hepatic progesterone catabolizing enzymes and selected angiogenic factors in luteal tissue were similar between groups. Messenger RNA expression of steroidogenic enzymes StAR and P450scc were reduced (P < 0.05), while protein expression of StAR tended to be reduced (P < 0.07) and P450scc was reduced (P < 0.05) in luteal tissue of NR versus C offspring. CONCLUSIONS: There appears to be no difference in hepatic steroid catabolism that could have led to the decreased serum progesterone. However, these data are consistent with the programming of decreased steroidogenic enzyme expression in CL of NR offspring, leading to reduced synthesis and secretion of progesterone.

Multigenerational effects of fetal dexamethasone exposure on the hypothalamic-pituitary-adrenal axis of first- and second-generation female offspring.

OBJECTIVE: Synthetic glucocorticoid (sGC) administration to women threatening preterm delivery increases neonatal survival. Evidence shows that fetal exposure to glucocorticoid levels higher than appropriate for current maturation programs offspring development. We examined fetal sGC multigenerational effects on F1 and F2 female offspring hypothalamo-pituitary-adrenal axis (HPAA) function. STUDY DESIGN: At 0.7 gestation, pregnant F0 ewes received 4 dexamethasone injections (2 mg, approximately 60 µg/kg(-1) per day(-1), 12 hours apart) or saline (control). F1 female offspring were bred to produce F2 female offspring. Postpubertal HPAA function was tested in F1 and F2 ewes. RESULTS: F1 and F2 ewe lambs showed reduced birthweight and morphometrics. Dexamethasone increased baseline but reduced stimulated HPAA activity in F1 and F2 female offspring. CONCLUSION: This is the first demonstration that sGC doses in the clinical range have multigenerational effects on hypothalamo-pituitary-adrenal activity in a precocial species, indicating the need for the study of long-term effects of fetal sGC exposure.

Elevated glucocorticoids during ovine pregnancy increase appetite and produce glucose dysregulation and adiposity in their granddaughters in response to ad libitum feeding at 1 year of age.

OBJECTIVE: Synthetic glucocorticoids (sGCs) are administered to women threatening preterm labor. We have shown multigenerational endocrine and metabolic effects of fetal sGC exposure. We hypothesized that sGC exposure would alter the second filial generation (F2) offspring neonatal leptin peak that controls development of appetitive behavior with metabolic consequences. STUDY DESIGN: F0 nulliparous ewes were bred to a single ram. Beginning at day 103 of gestation (term 150 days), dexamethasone (DEX) ewes received 4 injections of 2 mg DEX intramuscularly, 12 hours apart. Control ewes received saline. Ewes lambed naturally. At 22 months of age, F1 offspring were mated to produce F2 offspring. At 10 months of age, F2 female offspring were placed on an ad libitum feeding challenge for 12 weeks. RESULTS: DEX F2 female offspring did not show a postnatal leptin peak and their plasma cortisol concentration was elevated in the first days of life. During the feeding challenge, DEX F2 offspring consumed 10% more feed and gained 20% more weight compared with control F2 offspring. At the end of the feeding challenge, DEX F2 offspring had greater adiposity compared with control F2 offspring. F2 sGC offspring showed impaired insulin secretion in response to an intravenous glucose tolerance test. CONCLUSION: sGC administration to F0 mothers eliminates the neonatal leptin peak in F2 female offspring potentially by inhibition caused by elevated cortisol in the DEX F2 offspring. F2 offspring showed increased appetite, weight gain, and adiposity during an ad libitum feeding challenge accompanied by decreased insulin response to an intravenous glucose tolerance test.

The dental technician as a member of the hypodontia multidisciplinary team, with practical considerations for anterior restoration design.

Most patients seeking treatment for hypodontia will require prosthetic replacement of their missing teeth. This will be in the form of dentures, bridges and implant restorations. As these are created by one or more dental technicians who supports the clinical team, a close working relationship between these colleagues is likely to improve the quality of treatment outcome. This interaction will usually occur towards the end of the patient's treatment process, when definitive restorations are prescribed. However, appropriately trained and experienced dental technicians should be involved throughout the patient's treatment process as an integral part of the multidisciplinary team approach to effectively manage these patients.This paper describes the contribution of dental technicians to patient care with particular focus on communication between the restorative dentistry clinical team and the dental technician to improve the quality of anterior restorations. As missing maxillary lateral incisor teeth are a common presentation for this patient group, further technical detail relating to planning resin-bonded bridges for replacement of these teeth is included.

A genome resource to address mechanisms of developmental programming: determination of the fetal sheep heart transcriptome.

The pregnant sheep has provided seminal insights into reproduction related to animal and human development (ovarian function, fertility, implantation, fetal growth, parturition and lactation). Fetal sheep physiology has been extensively studied since 1950, contributing significantly to the basis for our understanding of many aspects of fetal development and behaviour that remain in use in clinical practice today. Understanding mechanisms requires the combination of systems approaches uniquely available in fetal sheep with the power of genomic studies. Absence of the full range of sheep genomic resources has limited the full realization of the power of this model, impeding progress in emerging areas of pregnancy biology such as developmental programming. We have examined the expressed fetal sheep heart transcriptome using high-throughput sequencing technologies. In so doing we identified 36,737 novel transcripts and describe genes, gene variants and pathways relevant to fundamental developmental mechanisms. Genes with the highest expression levels and with novel exons in the fetal heart transcriptome are known to play central roles in muscle development. We show that high-throughput sequencing methods can generate extensive transcriptome information in the absence of an assembled and annotated genome for that species. The gene sequence data obtained provide a unique genomic resource for sheep specific genetic technology development and, combined with the polymorphism data, augment annotation and assembly of the sheep genome. In addition, identification and pathway analysis of novel fetal sheep heart transcriptome splice variants is a first step towards revealing mechanisms of genetic variation and gene environment interactions during fetal heart development.

Early maternal undernutrition programs increased feed intake, altered glucose metabolism and insulin secretion, and liver function in aged female offspring.

Insulin resistance and obesity are components of the metabolic syndrome that includes development of cardiovascular disease and diabetes with advancing age. The thrifty phenotype hypothesis suggests that offspring of poorly nourished mothers are predisposed to the various components of the metabolic syndrome due to adaptations made during fetal development. We assessed the effects of maternal nutrient restriction in early gestation on feeding behavior, insulin and glucose dynamics, body composition, and liver function in aged female offspring of ewes fed either a nutrient-restricted [NR 50% National Research Council (NRC) recommendations] or control (C: 100% NRC) diet from 28 to 78 days of gestation, after which both groups were fed at 100% of NRC from day 79 to lambing and through lactation. Female lambs born to NR and C dams were reared as a single group from weaning, and thereafter, they were fed 100% NRC recommendations until assigned to this study at 6 yr of age. These female offspring were evaluated by a frequently sampled intravenous glucose tolerance test, followed by dual-energy X-ray absorptiometry for body composition analysis prior to and after ad libitum feeding of a highly palatable pelleted diet for 11 wk with automated monitoring of feed intake (GrowSafe Systems). Aged female offspring born to NR ewes demonstrated greater and more rapid feed intake, greater body weight gain, and efficiency of gain, lower insulin sensitivity, higher insulin secretion, and greater hepatic lipid and glycogen content than offspring from C ewes. These data confirm an increased metabolic "thriftiness" of offspring born to NR mothers, which continues into advanced age, possibly predisposing these offspring to metabolic disease.

Growth and insulin dynamics in two generations of female offspring of mothers receiving a single course of synthetic glucocorticoids.

OBJECTIVE: Synthetic glucocorticoid administration to women threatening preterm delivery increases neonatal survival. However, mounting evidence shows that fetal exposure to glucocorticoid levels higher than appropriate for current maturation adversely programs offspring development. We examined fetal synthetic glucocorticoid multigenerational metabolic effects on F1 and F2 female offspring. STUDY DESIGN: At 0.7 gestation, pregnant F0 ewes received 4 injections of dexamethasone (2 mg, approximately 60 ug.kg(-1) day(-1) 12 hours apart) or saline (control). F1 female offspring were bred to produce F2 female offspring. Postpubertal pancreatic ß-cell function was tested in F1 and F2 by intravenous glucose tolerance test. RESULTS: F1 and F2 ewe lambs showed reduced birthweight and morphometrics, and similar increased fasting glucose and decreased intravenous glucose tolerance test ß-cell response. CONCLUSION: This is the first demonstration of multigenerational programming of later life ß-cell response by clinically relevant doses of synthetic glucocorticoid indicating the need for study of long-term effects of fetal exposure to synthetic glucocorticoid.

Do integrated care pathways have a place in critical care?

PURPOSE OF REVIEW: Recent high-profile campaigns have endeavoured to improve the quality of critical care provision. Within other specialities, integrated care pathways (ICPs) have been advocated as an effective means of improving practice. We review the published research regarding their efficacy and potential role in critical care. RECENT FINDINGS: ICPs have been used in many specialities to good effect, reducing morbidity, increasing adherence to practice guidelines and improving multidisciplinary communication and efficiency. Many aspects of ICPs are already used in critical and perioperative care. SUMMARY: ICPs show promise as a mechanism for improving efficiency and care quality in specific circumstances. However, little research specific to critical care has been undertaken and it may be challenging to protocolize the multisystem care required in many critical illnesses. Although further evaluation is required to find the most appropriate way of incorporating ICPs into critical care, we commend their thoughtful adoption.