Maturation of lipid metabolism in the fetal and newborn sheep heart.

At birth, the fetus experiences a dramatic change in environment that is accompanied by a shift in myocardial fuel preference from lactate and glucose in fetal life to fatty acid oxidation after birth. We hypothesized that fatty acid metabolic machinery would mature during fetal life in preparation for this extreme metabolic transformation at birth. We quantified the pre- (94-day and 135-day gestation, term ∼147 days) and postnatal (5 ± 4 days postnatal) gene expression and protein levels for fatty acid transporters and enzymes in hearts from a precocial species, the sheep. Gene expression of fatty acid translocase (CD36), acyl-CoA synthetase long-chain 1 (ACSL1), carnitine palmitoyltransferase 1 (CPT1), hydroxy-acyl dehydrogenase (HADH), acetyl-CoA acetyltransferase (ACAT1), isocitrate dehydrogenase (IDH), and glycerol phosphate acyltransferase (GPAT) progressively increased through the perinatal period, whereas several genes [fatty acid transport protein 6 (FATP6), acyl-CoA synthetase long chain 3 (ACSL3), long-chain acyl-CoA dehydrogenase (LCAD), very long-chain acyl-CoA dehydrogenase (VLCAD), pyruvate dehydrogenase kinase (PDK4), phosphatidic acid phosphatase (PAP), and diacylglycerol acyltransferase (DGAT)] were stable in fetal hearts and had high expression after birth. Protein expression of CD36 and ACSL1 progressively increased throughout the perinatal period, whereas protein expression of carnitine palmitoyltransferase 1a (fetal isoform) (CPT1a) decreased and carnitine palmitoyltransferase 1b (adult isoform) (CPT1b) remained constitutively expressed. Using fluorescent-tagged long-chain fatty acids (BODIPY-C12), we demonstrated that fetal (125 ± 1 days gestation) cardiomyocytes produce 59% larger lipid droplets (P < 0.05) compared with newborn (8 ± 1 day) cardiomyocytes. These results provide novel insights into the perinatal maturation of cardiac fatty acid metabolism in a precocial species.NEW & NOTEWORTHY This study characterized the previously unknown expression patterns of genes that regulate the metabolism of free fatty acids in the perinatal sheep myocardium. This study shows that the prenatal myocardium prepares for the dramatic switch from carbohydrate metabolism to near complete reliance on free fatty acids postnatally. Fetal and neonatal cardiomyocytes also demonstrate differing lipid storage mechanisms where fetal cardiomyocytes form larger lipid droplets compared with newborn cardiomyocytes.

Intrauterine growth restriction elevates circulating acylcarnitines and suppresses fatty acid metabolism genes in the fetal sheep heart.

At birth, the mammalian myocardium switches from using carbohydrates as the primary energy substrate to free fatty acids as the primary fuel. Thus, a compromised switch could jeopardize normal heart function in the neonate. Placental embolization in sheep is a reliable model of intrauterine growth restriction (IUGR). It leads to suppression of both proliferation and terminal differentiation of cardiomyocytes. We hypothesized that the expression of genes regulating cardiac fatty acid metabolism would be similarly suppressed in IUGR, leading to compromised processing of lipids. Following 10 days of umbilicoplacental embolization in fetal sheep, IUGR fetuses had elevated circulating long-chain fatty acylcarnitines compared with controls (C14: CTRL 0.012 ± 0.005 nmol/ml vs. IUGR 0.018 ± 0.005 nmol/ml, P < 0.05; C18: CTRL 0.027 ± 0.009 nmol/mol vs. IUGR 0.043 ± 0.024 nmol/mol, P < 0.05, n = 12 control, n = 12 IUGR) indicative of impaired fatty acid metabolism. Uptake studies using fluorescently tagged BODIPY-C12-saturated free fatty acid in live, isolated cardiomyocytes showed lipid droplet area and number were not different between control and IUGR cells. mRNA levels of sarcolemmal fatty acid transporters (CD36, FATP6), acylation enzymes (ACSL1, ACSL3), mitochondrial transporter (CPT1), ß-oxidation enzymes (LCAD, HADH, ACAT1), tricarboxylic acid cycle enzyme (IDH), esterification enzymes (PAP, DGAT) and regulator of the lipid droplet formation (BSCL2) gene were all suppressed in IUGR myocardium (P < 0.05). However, protein levels for these regulatory genes were not different between groups. This discordance between mRNA and protein levels in the stressed myocardium suggests an adaptive protection of key myocardial enzymes under conditions of placental insufficiency. KEY POINTS: The fetal heart relies on carbohydrates in utero and must be prepared to metabolize fatty acids after birth but the effects of compromised fetal growth on the maturation of this metabolic system are unknown. Plasma fatty acylcarnitines are elevated in intrauterine growth-restricted (IUGR) fetuses compared with control fetuses, indicative of impaired fatty acid metabolism in fetal organs. Fatty acid uptake and storage are not different in IUGR cardiomyocytes compared with controls. mRNA levels of genes regulating fatty acid transporter and metabolic enzymes are suppressed in the IUGR myocardium compared with controls, while protein levels remain unchanged. Mismatches in gene and protein expression, and increased circulating fatty acylcarnitines may have long-term implications for offspring heart metabolism and adult health in IUGR individuals. This requires further investigation.

Down-regulation of MEIS1 promotes the maturation of oxidative phosphorylation in perinatal cardiomyocytes.

Fetal cardiomyocytes shift from glycolysis to oxidative phosphorylation around the time of birth. Myeloid ecotropic viral integration site 1 (MEIS1) is a transcription factor that promotes glycolysis in hematopoietic stem cells. We reasoned that MEIS1 could have a similar role in the developing heart. We hypothesized that suppression of MEIS1 expression in fetal sheep cardiomyocytes leads to a metabolic switch as found at birth. Expression of MEIS1 was assayed in left ventricular cardiac tissue and primary cultures of cardiomyocytes from fetal (100- and 135-d gestation, term = 145 d), neonatal, and adult sheep. Cultured cells were treated with short interfering RNA (siRNA) to suppress MEIS1. Oxygen consumption rate was assessed with the Seahorse metabolic flux analyzer, and mitochondrial activity was assessed by staining cells with MitoTracker Orange. Cardiomyocyte respiratory capacity increased with advancing age concurrently with decreased expression of MEIS1. MEIS1 suppression with siRNA increased maximal oxygen consumption in fetal cells but not in postnatal cells. Mitochondrial activity was increased and expression of glycolytic genes decreased when MEIS1 expression was suppressed. Thus, we conclude that MEIS1 is a key regulator of cardiomyocyte metabolism and that the normal down-regulation of MEIS1 with age underlies a gradual switch to oxidative metabolism.-Lindgren, I. M., Drake, R. R., Chattergoon, N. N., Thornburg, K. L. Down-regulation of MEIS1 promotes the maturation of oxidative phosphorylation in perinatal cardiomyocytes.

Respiratory chain protein turnover rates in mice are highly heterogeneous but strikingly conserved across tissues, ages, and treatments.

The mitochondrial respiratory chain (RC) produces most of the cellular ATP and requires strict quality-control mechanisms. To examine RC subunit proteostasis in vivo, we measured RC protein half-lives (HLs) in mice by liquid chromatography-tandem mass spectrometry with metabolic [(2)H3]-leucine heavy isotope labeling under divergent conditions. We studied 7 tissues/fractions of young and old mice on control diet or one of 2 diet regimens (caloric restriction or rapamycin) that altered protein turnover (42 conditions in total). We observed a 6.5-fold difference in mean HL across tissues and an 11.5-fold difference across all conditions. Normalization to the mean HL of each condition showed that relative HLs were conserved across conditions (Spearman's ρ = 0.57; P < 10(-4)), but were highly heterogeneous between subunits, with a 7.3-fold mean range overall, and a 2.2- to 4.6-fold range within each complex. To identify factors regulating this conserved distribution, we performed statistical analyses to study the correlation of HLs to the properties of the subunits. HLs significantly correlated with localization within the mitochondria, evolutionary origin, location of protein-encoding, and ubiquitination levels. These findings challenge the notion that all subunits in a complex turnover at comparable rates and suggest that there are common rules governing the differential proteolysis of RC protein subunits under divergent cellular conditions.

Pre-Hospital Spinal Immobilization: Neurological Outcomes for Spinal Motion Restriction Versus Spinal Immobilization.

Introduction: New recommendations for emergency medical services spinal precautions limit long spinal board use to extrication purposes only and are to be removed immediately. Outcomes for spinal motion restriction against spinal immobilization were studied. Methods: A retrospective chart review of trauma patients was conducted over a six-month period at a level I trauma center. Injury severity details and neurologic assessments were collected on 277 patients. Results: Upon arrival, 25 (9.0%) patients had a spine board in place. Patients placed on spine boards were more likely to be moderately or severely injured [injury severity score (ISS) > 15: 36.0% vs. 9.9%, p = 0.001] and more likely to have neurological deficits documented by emergency medical services (EMS; 30.4% vs. 8.8%, p = 0.01) and the trauma team (29.2% vs. 10.9%, p = 0.02). Conclusions: This study suggested that the long spine board was being used properly for more critically injured patients. Further research is needed to compare neurological outcomes using a larger sample size and more consistent documentation.

Improving the nutritional value of Golden Rice through increased pro-vitamin A content.

"Golden Rice" is a variety of rice engineered to produce beta-carotene (pro-vitamin A) to help combat vitamin A deficiency, and it has been predicted that its contribution to alleviating vitamin A deficiency would be substantially improved through even higher beta-carotene content. We hypothesized that the daffodil gene encoding phytoene synthase (psy), one of the two genes used to develop Golden Rice, was the limiting step in beta-carotene accumulation. Through systematic testing of other plant psys, we identified a psy from maize that substantially increased carotenoid accumulation in a model plant system. We went on to develop "Golden Rice 2" introducing this psy in combination with the Erwinia uredovora carotene desaturase (crtI) used to generate the original Golden Rice. We observed an increase in total carotenoids of up to 23-fold (maximum 37 microg/g) compared to the original Golden Rice and a preferential accumulation of beta-carotene.

Hospital Inpatient Admissions With Dehydration and/or Malnutrition in Medicare Beneficiaries Receiving Enteral Nutrition: A Cohort Study.

BACKGROUND: Enteral nutrition (EN) supports many older and disabled Americans. This study describes the frequency and cost of acute care hospitalization with dehydration and/or malnutrition of Medicare beneficiaries receiving EN, focusing on those receiving home EN. METHODS: Medicare 5% Standard Analytic Files were used to determine Medicare spending for EN supplies and the proportion and cost of beneficiaries receiving EN, specifically home EN, admitted to the hospital with dehydration and/or malnutrition. RESULTS: In 2013, Medicare paid $370,549,760 to provide EN supplies for 125,440 beneficiaries, 55% of whom were also eligible for Medicaid. Acute care hospitalization with dehydration and/or malnutrition occurred in 43,180 beneficiaries receiving EN. The most common principal diagnoses were septicemia (21%), aspiration pneumonitis (9%), and pneumonia (5%). In beneficiaries receiving EN at home, >one-third (37%) were admitted with dehydration and/or malnutrition during a mean observation interval of 231 ± 187 days. Admitted patients were usually hospitalized more than once with dehydration and/or malnutrition (1.73 ± 1.30 admissions) costing $23,579 ± 24,966 per admitted patient, totaling >$129,685,622 during a mean observation interval of 276 ± 187 days. Mortality in the year following enterostomy tube placement was significantly higher for admitted compared with nonadmitted patients (40% vs 33%; P = .05). CONCLUSION: Acute care hospitalizations with dehydration and/or malnutrition in Medicare beneficiaries receiving EN were common and expensive. Additional strategies to reduce these, with particular focus on vulnerable populations such as Medicaid-eligible patients, are needed.

Analysis of patients ≥65 with predominant cervical spine fractures: Issues of disposition and dysphagia.

BACKGROUND: Cervical spine fractures occur in 2.6% to 4.7% of trauma patients aged 65 years or older. Mortality rates in this population ranges from 19% to 24%. A few studies have specifically looked at dysphagia in elderly patients with cervical spine injury. AIMS: The aim of this study is to evaluate dysphagia, disposition, and mortality in elderly patients with cervical spine injury. SETTINGS AND DESIGN: Retrospective review at an the American College of Surgeons-verified level 1 trauma center. METHODS: Patients 65 years or older with cervical spine fracture, either isolated or in association with other minor injuries were included in the study. Data included demographics, injury details, neurologic deficits, dysphagia evaluation and treatment, hospitalization details, and outcomes. STATISTICAL ANALYSIS: Categorical and continuous data were analyzed using Chi-square analysis and one-way analysis of variance, respectively. RESULTS: Of 136 patients in this study, 2 (1.5%) had a sensory deficit alone, 4 (2.9%) had a motor deficit alone, and 4 (2.9%) had a combined sensory and motor deficit. Nearly one-third of patients (n = 43, 31.6%) underwent formal swallow evaluation, and 4 (2.9%) had a nasogastric tube or Dobhoff tube placed for enteral nutrition, whereas eight others (5.9%) had a gastrostomy tube or percutaneous endoscopic gastrostomy tube placed. Most patients were discharged to a skilled nursing unit (n = 50, 36.8%), or to home or home with home health (n = 48, 35.3%). Seven patients (5.1%) died in the hospital, and eight more (5.9%) were transferred to hospice. CONCLUSION: Cervical spine injury in the elderly patient can lead to significant consequences, including dysphagia and need for skilled nursing care at discharge.

Pediatric Farm Injuries: Morbidity and Mortality.

BACKGROUND: Agriculture is an industry where family members often live and work on the same premises. This study evaluated injury patterns and outcomes in children from farm-related accidents. METHODS: A 10-year retrospective review of farm-accident related injuries was conducted of patients 17 years and younger. Data collected included demographics, injury mechanism, accident details, injury severity and patterns, treatments required, hospitalization details, and discharge disposition. RESULTS: Sixty-five patients were included; 58.5% were male and the mean age was 9.7 years. Median Injury Severity Score and Glasgow Coma Scale were 5 and 15, respectively. Accident mechanisms included animal-related (43.1%), fall (21.5%), and motor vehicle (21.5%). Soft tissue injuries, concussions and upper extremity fractures were the most common injuries observed (58.5%, 29.2%, and 26.2%, respectively). Twenty-six patients (40%) required surgical intervention. Mean hospital length of stay was 3.4 ± 4.7 days. The majority of patients were discharged to home (n = 62, 95.4%) and two patients suffered permanent disability. CONCLUSIONS: Overall, outcomes for this population were favorable, but additional measures to increase safety, such as fall prevention, animal handling, and driver safety training should be advocated.

Biological features of placental programming.

The placenta is a key organ in programming the fetus for later disease. This review outlines nine of many structural and physiological features of the placenta which are associated with adult onset chronic disease. 1) Placental efficiency relates the placental mass to the fetal mass. Ratios at the extremes are related to cardiovascular disease risk later in life. 2) Placental shape predicts a large number of disease outcomes in adults but the regulators of placental shape are not known. 3) Non-human primate studies suggest that at about mid-gestation, the placenta becomes less plastic and less able to compensate for pathological stresses. 4) Recent studies suggest that lipids have an important role in regulating placental metabolism and thus the future health of offspring. 5) Placental inflammation affects nutrient transport to the fetus and programs for later disease. 6) Placental insufficiency leads to inadequate fetal growth and elevated risks for later life disease. 7) Maternal height, fat and muscle mass are important in combination with placental size and shape in predicting adult disease. 8) The placenta makes a host of hormones that influence fetal growth and are related to offspring disease. Unfortunately, our knowledge of placental growth and function lags far behind that of other organs. An investment in understanding placental growth and function will yield enormous benefits to human health because it is a key player in the origins of the most expensive and deadly chronic diseases that humans face.

A detailed assessment of the human coronary venous system using contrast computed tomography of perfusion-fixed specimens.

BACKGROUND: Access to the coronary venous system is required for the delivery of several cardiac therapies including cardiac resynchronization therapy, coronary sinus ablation, and coronary drug delivery. Therefore, characterization of the coronary venous anatomy will provide insights to gain improved access to these vessels and subsequently improved therapies. For example, cardiac resynchronization therapy has a 30% nonresponder rate, partially due to suboptimal lead placement within the coronary veins. OBJECTIVE: To understand the implications of coronary venous anatomy for the development of devices deployed within these vessels. METHODS: We cannulated the coronary sinus of 121 perfusion-fixed human hearts with a venogram balloon catheter and injected contrast into the venous system while obtaining computed tomographic images. For each major coronary vein, distance to the coronary sinus, branching angle, arc length, tortuosity, number of branches, and ostial diameter were assessed from the reconstructed anatomy. RESULTS: Twenty-nine percent (35/121) specimens did not have a venous branch overlying the inferolateral side of the heart large enough to fit a 5F pacing lead. No significant differences in anatomy were found between subgroups with varying cardiac medical histories. CONCLUSION: The anatomical approach employed in this study has allowed for the development of a unique database of human coronary venous anatomy that can be used for the optimization of design and delivery of cardiac devices.

Estimation of burn depth at burn centers in the United States: a survey.

Accurate burn depth estimation remains one of the foundations of optimal burn care. The method by which burn depth is determined has traditionally been clinical examination alone. This continues to hold true in the United States, despite a plethora of literature supporting the use of more accurate modalities such as laser Doppler imaging (LDI). LDI has widespread use in burn centers in the United Kingdom and around the world. Thus, the reason for a lack of use in U.S. burn centers remains elusive. A survey of U.S. burn center directors was conducted to assess their current practices and attitudes with regard to burn depth estimation at U.S. burn centers in an effort to answer this question. Surveys were returned from 68 burn center directors (49% response rate). All respondents reported using clinical examination in their current practice for the daily evaluation of acute burns, with a biopsy being the next most commonly used modality. The most preferred modality was also clinical examination (60%), followed by LDI (6%) and biopsy (4%). The top three modalities ranked as "most promising" for daily use were clinical examination, LDI, and noncontact/high-frequency ultrasound. Directors identified the top three limitations to the use of new technology as cost (72%), availability (63%), and lack of support by evidence to date (35%). Future studies may need to focus on overcoming these perceived limitations before the widespread use of LDI or other new modalities will be realized at burn centers in the United States.

Evaluation of chest tube administration of tissue plasminogen activator to treat retained hemothorax.

BACKGROUND: When retained hemothorax occurs, video-assisted thoracoscopy or thoracotomy is performed, but recently, tissue plasminogen activator (tPA) has been used. This study evaluated intrapleural tPA use for retained traumatic hemothoraces. METHODS: A retrospective review was conducted of trauma patients treated with intrapleural tPA for retained hemothorax. Data included demographics, past medical and surgical histories, injury details, treatment details, and outcomes. RESULTS: Seven patients (median age = 47 years, male = 6, blunt trauma = 6) met study criteria. All patients received a chest tube. Six patients later received computed tomography-guided drains for tPA infusion. Number of tPA treatments per patient varied from 1 to 5. Median total tPA dosage was 24 mg. Median time from injury to chest tube placement was 11 days and from chest tube placement to first tPA treatment was 4 days. No patients required a video-assisted thoracoscopy; however, 1 patient required thoracotomy. There were no deaths or bleeding complications attributed to intrapleural tPA. CONCLUSION: Although future studies are needed to identify optimum treatment guidelines, intrapleural tPA appears to be a safe and efficacious treatment option.

Do medical student's surgical examination scores correlate with performance markers?

BACKGROUND: Some medical school training consists of oral examinations. METHODS: We conducted a 9-year review of third-year medical student examinations including oral examinations, National Board of Medical Examiners Surgery Subject Examination (SSE, ie, shelf), and United States Medical Licensing Examinations Step 1 and Step 2. RESULTS: Step 1 showed a moderate to strong association with Period 1 orals (Somers' D = .297, P < .001), but not Period 2 orals (Somers' D = .048, P = .053). Period 1 orals (percentage) had a strong association with SSE (Somers' D = .356, P < .001) and Step 2 (Somers' D = .368, P < .001). Period 2 orals (pass/fail) suggested a positive, but not statistically significant, association with SSE (Somers' D = .334, P = .085) and Step 2 (Somers' D = .370, P = .055). Step 1 shows a strong association with SSE (Somers' D = .490, P < .001). SSE showed a strong association with Step 2 (Somers' D = .506, P < .001). CONCLUSIONS: Orals can be used to identify students who may have difficulty passing the SSE. Step 1 can be used to identify students at risk of poor performance on the SSE, and SSE can be used to identify students at risk for poor performance on Step 2.

Achieving endoscopic competency in a general surgery residency.

BACKGROUND: In 2006, the Residency Review Committee for Surgery increased the total number of required endoscopy cases for graduating residents. Our goal was to evaluate general surgery resident competency in endoscopy, focusing on quality measures. METHODS: A 9-year retrospective review was conducted of 29 residents. Total number of endoscopies performed throughout residency was recorded. Procedures performed as fifth-year residents with indirect supervision were evaluated for quality measures. RESULTS: An average of 76 esophagogastroduodenoscopies and 147 colonoscopies were performed through their first 4 years of residency. Chief residents performed an average of 16 esophagogastroduodenoscopies and 22 colonoscopies. Of colonoscopies performed during their fifth year, 191 were performed while the resident had only indirect supervision. During these cases, cecal intubation was achieved in 90.6% of cases, an average of .48 polyps were identified, and average scope withdrawal time was 13.4 ± 7.1 minutes. CONCLUSIONS: Our data indicate that surgery residents achieve competency in colonoscopy before performing 140 colonoscopies, supporting the concept that surgery residencies can function as an excellent training ground for endoscopy.

Why is golden rice golden (yellow) instead of red?

The endosperm of Golden Rice (Oryza sativa) is yellow due to the accumulation of beta-carotene (provitamin A) and xanthophylls. The product of the two carotenoid biosynthesis transgenes used in Golden Rice, phytoene synthase (PSY) and the bacterial carotene desaturase (CRTI), is lycopene, which has a red color. The absence of lycopene in Golden Rice shows that the pathway proceeds beyond the transgenic end point and thus that the endogenous pathway must also be acting. By using TaqMan real-time PCR, we show in wild-type rice endosperm the mRNA expression of the relevant carotenoid biosynthetic enzymes encoding phytoene desaturase, zeta-carotene desaturase, carotene cis-trans-isomerase, beta-lycopene cyclase, and beta-carotene hydroxylase; only PSY mRNA was virtually absent. We show that the transgenic phenotype is not due to up-regulation of expression of the endogenous rice pathway in response to the transgenes, as was suggested to be the case in tomato (Lycopersicon esculentum) fruit, where CRTI expression resulted in a similar carotenoid phenomenon. This means that beta-carotene and xanthophyll formation in Golden Rice relies on the activity of constitutively expressed intrinsic rice genes (carotene cis-trans-isomerase, alpha/beta-lycopene cyclase, beta-carotene hydroxylase). PSY needs to be supplemented and the need for the CrtI transgene in Golden Rice is presumably due to insufficient activity of the phytoene desaturase and/or zeta-carotene desaturase enzyme in endosperm. The effect of CRTI expression was also investigated in leaves of transgenic rice and Arabidopsis (Arabidopsis thaliana). Here, again, the mRNA levels of intrinsic carotenogenic enzymes remained unaffected; nevertheless, the carotenoid pattern changed, showing a decrease in lutein, while the beta-carotene-derived xanthophylls increased. This shift correlated with CRTI-expression and is most likely governed at the enzyme level by lycopene-cis-trans-isomerism. Possible implications are discussed.

A MADS-box gene necessary for fruit ripening at the tomato ripening-inhibitor (rin) locus.

Tomato plants harboring the ripening-inhibitor (rin) mutation yield fruits that fail to ripen. Additionally, rin plants display enlarged sepals and loss of inflorescence determinacy. Positional cloning of the rin locus revealed two tandem MADS-box genes (LeMADS-RIN and LeMADS-MC), whose expression patterns suggested roles in fruit ripening and sepal development, respectively. The rin mutation alters expression of both genes. Gene repression and mutant complementation demonstrate that LeMADS-RIN regulates ripening, whereas LeMADS-MC affects sepal development and inflorescence determinacy. LeMADS-RIN demonstrates an agriculturally important function of plant MADS-box genes and provides molecular insight into nonhormonal (developmental) regulation of ripening.