The pluripotent regulatory circuitry connecting promoters to their long-range interacting elements.

The mammalian genome harbors up to one million regulatory elements often located at great distances from their target genes. Long-range elements control genes through physical contact with promoters and can be recognized by the presence of specific histone modifications and transcription factor binding. Linking regulatory elements to specific promoters genome-wide is currently impeded by the limited resolution of high-throughput chromatin interaction assays. Here we apply a sequence capture approach to enrich Hi-C libraries for >22,000 annotated mouse promoters to identify statistically significant, long-range interactions at restriction fragment resolution, assigning long-range interacting elements to their target genes genome-wide in embryonic stem cells and fetal liver cells. The distal sites contacting active genes are enriched in active histone modifications and transcription factor occupancy, whereas inactive genes contact distal sites with repressive histone marks, demonstrating the regulatory potential of the distal elements identified. Furthermore, we find that coregulated genes cluster nonrandomly in spatial interaction networks correlated with their biological function and expression level. Interestingly, we find the strongest gene clustering in ES cells between transcription factor genes that control key developmental processes in embryogenesis. The results provide the first genome-wide catalog linking gene promoters to their long-range interacting elements and highlight the complex spatial regulatory circuitry controlling mammalian gene expression.

Transcription factories: genetic programming in three dimensions.

Among the most intensively studied systems in molecular biology is the eukaryotic transcriptional apparatus, which expresses genes in a regulated manner across hundreds of different cell types. Several studies over the past few years have added weight to the concept that transcription takes place within discrete 'transcription factories' assembled inside the cell nucleus. These studies apply innovative technical approaches to gain insights into the molecular constituents, dynamical behaviour and organizational regulators of transcription factories, providing exciting insights into the spatial dimension of transcriptional control.

Pioglitazone attenuates acute cocaine toxicity in rat isolated heart: potential protection by metabolic modulation.

BACKGROUND: The authors tested whether cocaine depresses mitochondrial acylcarnitine exchange and if a drug that enhances glucose metabolism could protect against cocaine-induced cardiac dysfunction. METHODS: Oxygen consumption with and without cocaine was compared in rat cardiac mitochondria using octanoylcarnitine (lipid) or pyruvate (nonlipid) substrates. Isolated hearts from rats with or without a pioglitazone-supplemented diet were exposed to cocaine. RESULTS: The 0.5 mM cocaine inhibited respiration supported by octanoylcarnitine (82 ± 10.4 and 45.7 ± 4.24 ngatomO min⁻¹ · mg⁻¹ · protein ± SEM, for control and cocaine treatment, respectively; P < 0.02) but not pyruvate-supported respiration (281 ± 12.5 and 267 ± 12.7 ngatomO min⁻¹ · mg⁻¹ · protein ± SEM; P = 0.45). Cocaine altered contractility, lusitropy, coronary resistance, and lactate production in isolated heart. These effects were each blunted in pioglitazone-treated hearts. The pioglitazone diet attenuated the drop in the rate-pressure product (P = 0.002), cocaine-induced diastolic dysfunction (P = 0.04), and myocardial vascular resistance (P = 0.05) compared with that of controls. Lactate production was higher in pretreated hearts (P = 0.008) and in ventricular myocytes cultured with pioglitazone (P = 0.0001). CONCLUSIONS: Cocaine inhibited octanoylcarnitine-supported mitochondrial respiration. A pioglitazone diet significantly attenuated the effects of cocaine on isolated heart. The authors postulate that inhibition of acylcarnitine exchange could contribute to cocaine-induced cardiac dysfunction and that metabolic modulation warrants additional study.

In silico models of cancer.

Cancer is a complex disease that involves multiple types of biological interactions across diverse physical, temporal, and biological scales. This complexity presents substantial challenges for the characterization of cancer biology, and motivates the study of cancer in the context of molecular, cellular, and physiological systems. Computational models of cancer are being developed to aid both biological discovery and clinical medicine. The development of these in silico models is facilitated by rapidly advancing experimental and analytical tools that generate information-rich, high-throughput biological data. Statistical models of cancer at the genomic, transcriptomic, and pathway levels have proven effective in developing diagnostic and prognostic molecular signatures, as well as in identifying perturbed pathways. Statistically inferred network models can prove useful in settings where data overfitting can be avoided, and provide an important means for biological discovery. Mechanistically based signaling and metabolic models that apply a priori knowledge of biochemical processes derived from experiments can also be reconstructed where data are available, and can provide insight and predictive ability regarding the behavior of these systems. At longer length scales, continuum and agent-based models of the tumor microenvironment and other tissue-level interactions enable modeling of cancer cell populations and tumor progression. Even though cancer has been among the most-studied human diseases using systems approaches, significant challenges remain before the enormous potential of in silico cancer biology can be fully realized.

Safety of high volume lipid emulsion infusion: a first approximation of LD50 in rats.

BACKGROUND: Lipid infusion reverses systemic local anesthetic toxicity. The acceptable upper limit for lipid administration is unknown and has direct bearing on clinical management. We hypothesize that high volumes of lipid could have undesirable effects and sought to identify the dose required to kill 50% of the animals (LD(50)) of large volume lipid administration. METHODS: Intravenous lines and electrocardiogram electrodes were placed in anesthetized, male Sprague-Dawley rats. Twenty percent lipid emulsion (20, 40, 60, or 80 mL/kg) or saline (60 or 80 mL/kg), were administered over 30 mins; lipid dosing was assigned by the Dixon "up-and-down" method. Rats were recovered and observed for 48 hrs then euthanized for histologic analysis of major organs. Three additional rats were administered 60 mL/kg lipid emulsion and euthanized at 1, 4, and 24 hrs to identify progression of organ damage. RESULTS: The maximum likelihood estimate for LD(50) was 67.72 (SE, 10.69) mL/kg. Triglycerides were elevated immediately after infusion but returned to baseline by 48 hrs when laboratory abnormalities included elevated amylase, aspartate aminotransferase, and serum urea nitrogen for all lipid doses. Histologic diagnosis of myocardium, brain, pancreas, and kidneys was normal at all doses. Microscopic abnormalities in lung and liver were observed at 60 and 80 mL/kg; histopathology in the lung and liver was worse at 1 hr than at 4 and 24 hrs. CONCLUSIONS: The LD(50) of rapid, high volume lipid infusion is an order of magnitude greater than doses typically used for lipid rescue in humans and supports the safety of lipid infusion at currently recommended doses for toxin-induced cardiac arrest. Lung and liver histopathology was observed at the highest infused volumes.

Systems biology of embryogenesis.

The development of a complete organism from a single cell involves extraordinarily complex orchestration of biological processes that vary intricately across space and time. Systems biology seeks to describe how all elements of a biological system interact in order to understand, model and ultimately predict aspects of emergent biological processes. Embryogenesis represents an extraordinary opportunity (and challenge) for the application of systems biology. Systems approaches have already been used successfully to study various aspects of development, from complex intracellular networks to four-dimensional models of organogenesis. Going forward, great advancements and discoveries can be expected from systems approaches applied to embryogenesis and developmental biology.

Two-transcript gene expression classifiers in the diagnosis and prognosis of human diseases.

BACKGROUND: Identification of molecular classifiers from genome-wide gene expression analysis is an important practice for the investigation of biological systems in the post-genomic era--and one with great potential for near-term clinical impact. The 'Top-Scoring Pair' (TSP) classification method identifies pairs of genes whose relative expression correlates strongly with phenotype. In this study, we sought to assess the effectiveness of the TSP approach in the identification of diagnostic classifiers for a number of human diseases including bacterial and viral infection, cardiomyopathy, diabetes, Crohn's disease, and transformed ulcerative colitis. We examined transcriptional profiles from both solid tissues and blood-borne leukocytes. RESULTS: The algorithm identified multiple predictive gene pairs for each phenotype, with cross-validation accuracy ranging from 70 to nearly 100 percent, and high sensitivity and specificity observed in most classification tasks. Performance compared favourably with that of pre-existing transcription-based classifiers, and in some cases was comparable to the accuracy of current clinical diagnostic procedures. Several diseases of solid tissues could be reliably diagnosed through classifiers based on the blood-borne leukocyte transcriptome. The TSP classifier thus represents a simple yet robust method to differentiate between diverse phenotypic states based on gene expression profiles. CONCLUSION: Two-transcript classifiers have the potential to reliably classify diverse human diseases, through analysis of both local diseased tissue and the immunological response assayed through blood-borne leukocytes. The experimental simplicity of this method results in measurements that can be easily translated to clinical practice.

Epinephrine impairs lipid resuscitation from bupivacaine overdose: a threshold effect.

BACKGROUND: Lipid emulsion infusion reverses local anesthetic-induced cardiac toxicity, but the effect of adding epinephrine has not been studied. We compared escalating doses of epinephrine on recovery with lipid infusion in a rat model of bupivacaine overdose. METHODS: Rats anesthetized with isoflurane received an IV bolus of 20 mg/kg bupivacaine, producing asystole (zero time) in all animals. Ventilation (100% oxygen) and chest compressions were started immediately, and at 3 min the rats received one of six IV treatments (n = 5 for all groups): 5 ml/kg saline followed by infusion for 2 min at 1.0 ml x kg x min, and a second 5 ml/kg bolus at 5 min; or the same bolus and infusion treatment using 30% lipid emulsion plus a single injection of epinephrine at one of five doses: 0 (lipid control), 1, 2.5, 10, or 25 mcg/kg. An electrocardiogram and arterial pressure were monitored continuously, and arterial blood gas was measured at 7.5 and 15 min. RESULTS: Epinephrine improved initial return of spontaneous circulation (rate-pressure product > 30% baseline) but only 3 of 5 rats at 10 mcg/kg and 1 of 5 rats at 25 mcg/kg sustained return of spontaneous circulation by 15 min. Lipid alone resulted in slower but more sustained recovery. Epinephrine doses above a threshold near 10 mcg/kg increased lactate, worsened acidosis, and resulted in poor recovery at 15 min, as compared with lipid controls. There was tight correlation of epinephrine dose to serum lactate at 15 min. CONCLUSIONS: Epinephrine over a threshold dose near 10 mcg/kg impairs lipid resuscitation from bupivacaine overdose, possibly by inducing hyperlactatemia.

Resuscitation with lipid versus epinephrine in a rat model of bupivacaine overdose.

BACKGROUND: Lipid emulsion infusion reverses cardiovascular compromise due to local anesthetic overdose in laboratory and clinical settings. The authors compared resuscitation with lipid, epinephrine, and saline control in a rat model of bupivacaine-induced cardiac toxicity to determine whether lipid provides a benefit over epinephrine. METHODS: Bupivacaine, 20 mg/kg, was infused in rats anesthetized with isoflurane, producing asystole in all subjects. Ventilation with 100% oxygen and chest compressions were begun immediately, along with intravenous treatment with 30% lipid emulsion or saline (5-ml/kg bolus plus continuous infusion at 0.5 ml . kg . min) or epinephrine (30 microg/kg). Chest compressions were continued and boluses were repeated at 2.5 and 5 min until the native rate-pressure product was greater than 20% baseline. Electrocardiogram and arterial pressure were monitored continuously and at 10 min, arterial blood gas, central venous oxygen saturation, and blood lactate were measured. Effect size (Cohen d) was determined for comparisons at 10 min. RESULTS: Lipid infusion resulted in higher rate-pressure product (P < 0.001, d = 3.84), pH (P < 0.01, d = 3.78), arterial oxygen tension (P < 0.05, d = 2.8), and central venous oxygen saturation (P < 0.001, d = 4.9) at 10 min than did epinephrine. Epinephrine treatment caused higher lactate (P < 0.01, d = 1.48), persistent ventricular ectopy in all subjects, pulmonary edema in four of five rats, hypoxemia, and a mixed metabolic and respiratory acidosis by 10 min. CONCLUSIONS: Hemodynamic and metabolic metrics during resuscitation with lipid surpassed those with epinephrine, which were no better than those seen in the saline control group. Further studies are required to optimize the clinical management of systemic local anesthetic toxicity.

Metabolic context affects hemodynamic response to bupivacaine in the isolated rat heart.

Previous studies have demonstrated that the local anesthetic bupivacaine selectively inhibits oxidative metabolism of fatty acids in isolated cardiac mitochondria. In the present investigation, we compare the development of bupivacaine cardiotoxicity during fatty acid and carbohydrate metabolism. Hearts from adult male Sprague-Dawley rats were excised and retrograde perfused with a solution containing fatty acid (oleate or octanoate) or carbohydrate substrates for cardiac metabolism. An infusion of bupivacaine was initiated and sustained until asystole, after which full cardiac recovery was allowed. During fatty acid metabolism, substantially lower bupivacaine doses induced both arrhythmia (60.4+/-11.5 microg oleate and 106.8+/-14.8 octanoate versus 153.4+/-21.4 carbohydrate; P<0.05) and asystole (121.0+/-30.1 microg and 171.5+/-20.2 versus 344.7+/-34.6; P<0.001). Dose-response analysis revealed significantly increased sensitivity to bupivacaine toxicity during fatty acid metabolism, indicated by lower V50 doses for both heart rate (70.6+/-5.6 microg oleate and 122.3+/-6.2 octanoate versus 152.6+/-8.6) and rate-pressure product (63.4+/-5.1 microg and 133.7+/-7.9 versus 165.1+/-12.2). Time to recovery following bupivacaine exposure was elevated in the fatty acid group (24.3+/-2.0 s versus 15.8+/-3.1; P<0.04). Fatty acid metabolism was shown to predispose the isolated heart to bupivacaine toxicity, confirming that the local anesthetic exerts specific effects on lipid processes in cardiomyocytes.

Adding bupivacaine to high-potassium cardioplegia improves function and reduces cellular damage of rat isolated hearts after prolonged, cold storage.

BACKGROUND: Bupivacaine retards myocardial acidosis during ischemia. The authors measured function of rat isolated hearts after prolonged storage to determine whether bupivacaine improves cardiac protection compared with standard cardioplegia alone. METHODS: After measuring cardiac function on a Langendorff apparatus, hearts were perfused with cardioplegia alone (controls), cardioplegia containing 500 microm bupivacaine, or cardioplegia containing 2 mm lidocaine; were stored at 4 degrees C for 12 h; and were then reperfused. Heart rate and left ventricular developed pressures were measured for 60 min. Maximum positive rate of change in ventricular pressure, oxygen consumption, and lactate dehydrogenase release were also measured. RESULTS: All bupivacaine-treated, four of five lidocaine-treated, and no control hearts beat throughout the 60-min recovery period. Mean values of heart rate, left ventricular developed pressure, maximum positive rate of change in ventricular pressure, rate-pressure product, and efficiency in bupivacaine-treated hearts exceeded those of the control group (P < 0.001 at 60 min for all). Mean values of the lidocaine group were intermediate. Oxygen consumption of the control group exceeded the other groups early in recovery, but not at later times. Lactate dehydrogenase release from the bupivacaine group was less than that from the control group (P < 0.001) but did not differ from baseline. CONCLUSIONS: Adding bupivacaine to a depolarizing cardioplegia solution reduces cell damage and improves cardiac function after prolonged storage. Metabolic inhibition may contribute to this phenomenon, which is not entirely explained by sodium channel blockade.

Lipid infusion accelerates removal of bupivacaine and recovery from bupivacaine toxicity in the isolated rat heart.

BACKGROUND AND OBJECTIVES: Infusion of a lipid emulsion has been advocated for treatment of severe bupivacaine cardiac toxicity. The mechanism of lipid rescue is unknown. These studies address the possibility that lipid infusion reduces cardiac bupivacaine content in the context of cardiac toxicity. METHODS: We compared the effects of a 1% lipid emulsion with standard Krebs buffer after inducing asystole in isolated rat heart with 500 micromol/L bupivacaine. We compared times to first heart beat and recovery of 90% of baseline rate pressure product (RPP = heart rate x [left ventricular systolic pressure - left ventricular diastolic pressure]) between controls and hearts receiving 1% lipid immediately after bupivacaine. We also used minibiopsies to compare control bupivacaine tissue content with hearts getting lipid immediately after an infusion of radiolabeled bupivacaine. We then compared bupivacaine efflux from hearts with and without lipid infusion started 75 seconds after radiolabeled bupivacaine was administered. RESULTS: Infusion of lipid resulted in more rapid return of spontaneous contractions and full recovery of cardiac function. Average (+/- SEM) times to first beat and to 90% recovery of rate pressure product were 44.6 +/- 3.5 versus 63.8 +/- 4.3 seconds (P < .01) and 124.7 +/- 12.4 versus 219.8 +/- 25.6 seconds (P < .01) for lipid and controls, respectively. Lipid treatment resulted in more rapid loss of bupivacaine from heart tissue (P < .0016). Late lipid infusion, 75 seconds after bupivacaine infusion ended, increased the release of bupivacaine measured in effluent for the first 15-second interval compared with controls (183 vs. 121 nmol, n = 5 for both groups, P < .008). CONCLUSIONS: Lipid emulsion speeds loss of bupivacaine from cardiac tissue while accelerating recovery from bupivacaine-induced asystole. These findings are consistent with the hypothesis that bupivacaine partitions into the emulsion and supports the concept of a "lipid sink." However, the data do not exclude other possible mechanisms of action.