Visualizing electromagnetic fields at the nanoscale by single molecule localization.

Coupling of light to the free electrons at metallic surfaces allows the confinement of electric fields to subwavelength dimensions, far below the optical diffraction limit. While this is routinely used to manipulate light at the nanoscale, in electro-optic devices and enhanced spectroscopic techniques, no characterization technique for imaging the underlying nanoscopic electromagnetic fields exists, which does not perturb the field or employ complex electron beam imaging. Here, we demonstrate the direct visualization of electromagnetic fields on patterned metallic substrates at nanometer resolution, exploiting a strong "autonomous" fluorescence-blinking behavior of single molecules within the confined fields allowing their localization. Use of DNA-constructs for precise positioning of fluorescence dyes on the surface induces this distance-dependent autonomous blinking thus completely obviating the need for exogenous agents or switching methods. Mapping such electromagnetic field distributions at nanometer resolution aids the rational design of nanometals for diverse photonic applications.

Portopulmonary hypertension: imatinib as a novel treatment and the Emory experience with this condition.

Portopulmonary hypertension (PoPH) is a common and feared complication of end-stage liver disease, and imposes increased risk of perioperative morbidity in the liver transplant patient. Herein, we present the first successful use of Imatinib in the perioperative management of a patient who was not responding to conventional treatments as well as our institution's experience with this devastating complication. Of patients evaluated for transplant, 4.1% were identified with PoPH, half of which were listed, and one quarter of which were transplanted. Patients with PoPH were twice as likely to be transplanted than all other candidates (48% vs 25%), though less likely to survive their first year (69% vs 86.4%).

Antifibrinolytic agents reduce tissue plasminogen activator-mediated neuronal toxicity in vitro.

INTRODUCTION: Serine proteases and their inhibitors play an important role in physiological homeostasis including neuronal activity, hemostasis, and wound healing. Tissue plasminogen activator (tPA) is involved in normal neuronal plasticity and memory formation but can also be neurotoxic. We hypothesized that the serine protease inhibitor aprotinin confers neuronal protection by inhibiting tPA activity. METHODS: Using cultured rat dopaminergic neuroblasts (N27 line), tPA-induced cytotoxicity was quantitated by an MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay and flow cytometry using propidium iodide DNA staining. The anti-apoptotic effects of aprotinin and other protease inhibitors were also evaluated using these systems. RESULTS: Treatment of cultured neuroblasts with tPA (10-20 microg/ml) caused a dose-dependent decrease in cell viability (71.3+/-2.4 at 10 microg/ml down to 52.7+/-2.5% at 20 microg/m tPA, 24-h treatment), which was potentiated in the absence of serum in the culture medium (59.5+/-6.3% at 10 microg/ml down to 47.9+/-4.7% at 20 microg/ml). Aprotinin was effective in ameliorating cell death when administered 30 min before tPA exposure as shown by increased cell viability (91.8+/-0.6% at tPA at 20 microg/ml), but this protection was significantly reduced when aprotinin was administered after tPA. The efficacy of aprotinin as a neuroprotectant was equivalent or superior to other direct tPA antagonist peptides Glu-Gly-Arg-chlormethylketone (EGRck) and Phe-Pro-Arg-chlormethylketone (FPRck) in this setting. CONCLUSION: These data suggest that one of the mechanisms of neuroprotection afforded by aprotinin may be inhibition of tPA-mediated neurotoxicity.

Genetic modification of the manganese superoxide dismutase/glutathione peroxidase 1 pathway influences intracellular ROS generation in quiescent, but not contracting, skeletal muscle cells.

Increased amounts of reactive oxygen species (ROS) are generated by skeletal muscle during contractile activity, but their intracellular source is unclear. The oxidation of 2',7'-dichlorodihydrofluorescein (DCFH) was examined as an intracellular probe for reactive oxygen species in skeletal muscle myotubes derived from muscles of wild-type mice and mice that were heterozygous knockout for manganese superoxide dismutase (Sod2(+/-)), homozygous knockout for glutathione peroxidase 1 (GPx1(-/-)), or MnSOD transgenic overexpressors (Sod2-Tg). Myoblasts were stimulated to fuse and loaded with DCFH 5-7 days later. Intracellular DCF epifluorescence was measured and myotubes were electrically stimulated to contract for 15 min. Quiescent myotubes with decreased MnSOD or GPx1 showed a significant increase in the rate of DCFH oxidation whereas those with increased MnSOD did not differ from wild type. Following contractions, myotubes from all groups showed an equivalent increase in DCF fluorescence. Thus the oxidation of DCFH in quiescent skeletal muscle myotubes is influenced by the content of enzymes that regulate mitochondrial superoxide and hydrogen peroxide content. In contrast, the increase in DCFH oxidation following contractions was unaffected by reduced or enhanced MnSOD or absent GPx1, indicating that reactive oxygen species produced by contractions were predominantly generated by nonmitochondrial sources.

Highly optimised global organisation of metabolic networks.

High-level, mathematically precise descriptions of the global organisation of complex metabolic networks are necessary for understanding the global structure of metabolic networks, the interpretation and integration of large amounts of biologic data (sequences, various -omics) and ultimately for rational design of therapies for disease processes. Metabolic networks are highly organised to execute their function efficiently while tolerating wide variation in their environment. These networks are constrained by physical requirements (e.g. conservation of energy, redox and small moieties) but are also remarkably robust and evolvable. The authors use well-known features of the stoichiometry of bacterial metabolic networks to demonstrate how network architecture facilitates such capabilities, and to develop a minimal abstract metabolism which incorporates the known features of the stoichiometry and respects the constraints on enzymes and reactions. This model shows that the essential functionality and constraints drive the tradeoffs between robustness and fragility, as well as the large-scale structure and organisation of the whole network, particularly high variability. The authors emphasise how domain-specific constraints and tradeoffs imposed by the environment are important factors in shaping stoichiometry. Importantly, the consequence of these highly organised tradeoffs and tolerances is an architecture that has a highly structured modularity that is self-dissimilar and scale-rich.

Oxygen-mediated regulation of skeletal muscle satellite cell proliferation and adipogenesis in culture.

Major problems in stem cell biology revolve around defining the developmental potential of cell populations and understanding how their potential is maintained or progressively restricted. Oxygen (O(2)) is an obvious environmental factor which has received little attention in culturing skeletal muscle progenitor cells. In this work, we examine the effects of O(2) levels on the developmental potential, proliferative capacity, and phenotype of the adult skeletal muscle fiber progenitor population (satellite cells), and cell lines that model multipotential embryonic paraxial mesoderm from which skeletal muscle develops. Both satellite cell proliferation and survival of mature fibers increased in physiologic (6%) O(2) vs. non-physiologic 20% O(2) used in virtually all traditional cell culture. Six percent O(2) conditions also accelerated the up-regulation of multiple MyoD family myogenic regulatory factors (MRFs). An unexpected finding was that fiber-adherent satellite cells could assume a non-myogenic phenotype. By the criteria of molecular markers and gross lipid accumulation, satellite cells were found to assume an adipocyte phenotype, and did so more prominently in 20% O(2) than in physiologic O(2). Selection of the adipogenic fate and execution of adipogenesis by multipotential mesenchymal cell lines was also dramatically higher in traditional 20 vs. 6% O(2), and decreased adipogenesis in physiologic O(2) was associated with significantly less expression of the adipogenic regulator, PPAR gamma. These results suggest that regulatory pathways affected by O(2) are important for satellite cell proliferation, execution of cell fate, and parent muscle survival in culture, and so may play a role in vivo under normal or pathologic conditions.

Long-term proliferation and dopaminergic differentiation of human mesencephalic neural precursor cells.

We report on generation of dopamine neurons from long-term cultures of human fetal mesencephalic precursor cells. These CNS precursor cells were successfully expanded in vitro using the mitogens epidermal growth factor (EGF) and fibroblast growth factor-2 (FGF-2). Incubation of these cultures in 3% atmospheric oxygen resulted in higher cellular yields than room air. Following incubation in differentiation media containing interleukin (IL)-1b (IL-1b), IL-11, leukemia inhibitory factor (LIF), and glial cell line-derived neurotrophic factor (GDNF), up to 1% of the precursor cells converted into cells immunoreactive for tyrosine hydroxylase (TH), a marker for dopamine neurons. The TH immunoreactive cells exhibited morphological and functional properties characteristic of dopamine neurons in culture. These precursor cells might serve as a useful source of human dopamine neurons for studying the development and degeneration of human dopamine neurons and may further serve as a continuous, on-demand source of cells for therapeutic transplantation in patients with Parkinson's disease.

Culture in reduced levels of oxygen promotes clonogenic sympathoadrenal differentiation by isolated neural crest stem cells.

Isolated neural crest stem cells (NCSCs) differentiate to autonomic neurons in response to bone morphogenetic protein 2 (BMP2) in clonal cultures, but these neurons do not express sympathoadrenal (SA) lineage markers. Whether this reflects a developmental restriction in NCSCs or simply inappropriate culture conditions was not clear. We tested the growth and differentiation potential of NCSCs at approximately 5% O(2), which more closely approximates physiological oxygen levels. Eighty-three percent of p75(+)P(0-) cells isolated from embryonic day 14.5 sciatic nerve behaved as stem cells under these conditions, suggesting that this is a nearly pure population. Furthermore, addition of BMP2 plus forskolin in decreased oxygen cultures elicited differentiation of thousands of cells expressing tyrosine hydroxylase, dopamine-beta-hydroxylase, and the SA lineage marker SA-1 in nearly all colonies. Such cells also synthesized and released dopamine and norepinephrine. These data demonstrate that isolated mammalian NCSCs uniformly possess SA lineage capacity and further suggest that oxygen levels can influence cell fate. Parallel results indicating that reduced oxygen levels can also promote the survival, proliferation, and catecholaminergic differentiation of CNS stem cells (Studer et al., 2000) suggests that neural stem cells may exhibit a conserved response to reduced oxygen levels.

Enhanced proliferation, survival, and dopaminergic differentiation of CNS precursors in lowered oxygen.

Standard cell culture systems impose environmental oxygen (O(2)) levels of 20%, whereas actual tissue O(2) levels in both developing and adult brain are an order of magnitude lower. To address whether proliferation and differentiation of CNS precursors in vitro are influenced by the O(2) environment, we analyzed embryonic day 12 rat mesencephalic precursor cells in traditional cultures with 20% O(2) and in lowered O(2) (3 +/- 2%). Proliferation was promoted and apoptosis was reduced when cells were grown in lowered O(2), yielding greater numbers of precursors. The differentiation of precursor cells into neurons with specific neurotransmitter phenotypes was also significantly altered. The percentage of neurons of dopaminergic phenotype increased to 56% in lowered O(2) compared with 18% in 20% O(2). Together, the increases in total cell number and percentage of dopaminergic neurons resulted in a ninefold net increase in dopamine neuron yield. Differential gene expression analysis revealed more abundant messages for FGF8, engrailed-1, and erythropoietin in lowered O(2). Erythropoietin supplementation of 20% O(2) cultures partially mimicked increased dopaminergic differentiation characteristic of CNS precursors cultured in lowered O(2). These data demonstrate increased proliferation, reduced cell death, and enhanced dopamine neuron generation in lowered O(2), making this method an important advance in the ex vivo generation of specific neurons for brain repair.

Efficacy and safety of heat exchanger added to venovenous bypass circuit during orthotopic liver transplantation.

Hypothermia during orthotopic liver transplantation (OLT) is common despite measures to prevent this complication. We retrospectively analyzed two groups of patients; those managed with (n = 113) or without (n = 109) a heat exchanger (HE) incorporated in the venovenous bypass (VVB) circuit to test the hypothesis that normothermia before liver reperfusion minimizes hypotension during reperfusion and decreases neohepatic transfusion requirements. Use of the HE resulted in significantly warmer patients during reperfusion and at the end of surgery (P < .001). An increase in neohepatic transfusion requirement was observed in patients with HE use: packed red blood cells, 4 +/- 4 versus 3 +/- 3 units; fresh-frozen plasma, 5 +/- 5 versus 4 +/- 4 units; platelets, 8 +/- 8 versus 6 +/- 7 units; and cryoprecipitate, 5 +/- 7 versus 3 +/- 5 units. There was no difference between the two groups in the untoward hemodynamic events during reperfusion of the liver (P = .31). We conclude that during OLT, the use of an HE in a nonheparinized VVB circuit helps maintain normothermia. Our limited experience suggests that its use is safe but does not improve hemodynamic stability during reperfusion or decrease blood loss during the neohepatic period.

p27Kip1 is expressed transiently in developing myotomes and enhances myogenesis.

Vertebrate skeletal muscle development is characterized by tight coupling of muscle differentiation with cell cycle arrest in G1/G0. Key regulators of G1 progression are the G1 cyclin-dependent kinases, their positive regulators, the G1 cyclins, and their negative regulators, the cyclin-dependent kinase inhibitors (CDIs). Here we show that p27Kip1 protein, a G1 CDI, is expressed in a prominent but transient wave in the developing myotomes of the mouse embryo. We relate its expression to expression of MyoD and myogenin proteins, which are determination and differentiation class myogenic regulatory factors, respectively. Functional assays showed that ectopic p27 expression can powerfully enhance the efficiency of MyoD-initiated muscle differentiation in cell culture. When considered together with the myotomal expression patterns of p18, p21, and p57, these results suggest a model in which p27 acts as a "trigger" CDI while myoblasts are exiting the cell cycle and initiating differentiation. At later times, when p27 protein has been down-regulated, it is proposed that accumulation of p18, p21, and p57 maintain the differentiated myocytes in a postmitotic state.

Arteriovenous obstruction of arm due to venovenous bypass during liver transplantation.

Two cases of arteriovenous obstruction of the right arm due to venovenous bypass during orthotopic liver transplantation are reported. Possible explanation and risk factors for the development of this complication are discussed.

Early tracheal extubation after liver transplantation.

OBJECTIVE: To assess the value and safety of tracheal extubation in the operating room at the end of liver transplantation. DESIGN: Retrospective chart review. SETTING: University Medical Center. PARTICIPANTS: Eighteen adult patients extubated in the operating room at the end of liver transplantation (study patients) compared with 17 patients who were not extubated and had < or = 3 U of blood transfused during liver transplantation (control patients). INTERVENTIONS: Data collected include severity of preoperative liver disease, anesthetic technique, use of venovenous bypass, surgical time, intraoperative blood replacement, core temperature and arterial blood gases on admission to the intensive care unit (ICU), times to discharge from ICU and the hospital. MEASUREMENTS AND MAIN RESULTS: Except for age (43.9 +/- 2.7 in study patients v 52.4 +/- 2.5 years; p = 0.03), patients were similar with regard to preoperative Child's-Pugh class and liver function tests. Study patients received more crystalloid in the OR (5,306 +/- 561 v 3,771 +/- 454 mL; p = 0.04), were warmer (36.6 degrees C +/- 0.2 degree C v 35.6 degrees C +/- 0.3 degree C; p = 0.01), had a lower arterial pH (7.29 +/- 0.01 v 7.36 +/- 0.02; p = 0.003) and higher arterial carbon dioxide tension (45 +/- 1 v 35 +/- 2 mmHg; p < 0.001) on admission to ICU than controls. There were no significant differences between groups with regard to discharge times from the ICU (50.6 +/- 2.7 hours in the study group v 61.2 +/- 4.7 in control group; p = 0.06), or discharge from the hospital (14.8 +/- 1.6 in the study group v 21.3 +/- 3 days in control group; p = 0.06). CONCLUSIONS: Tracheal extubation of selected patients at the end of liver transplant surgery in the operating room is safe but did not result in decreased ICU or hospital stay.

Plano-concave microcuvette for measuring the absorption coefficient of highly absorbing liquids.

A powerful and simple method based on the use of a plano-concave microcuvette was investigated for measuring the absorption coefficient of highly absorbing liquids. A plano-convex lens put on a plane-parallel plate formed a microcuvette with small, continuously varying thicknesses. This microcuvette was filled with liquid and illuminated by a homogeneous beam. The parabolic variation of the liquid thickness generates a Gaussian spatial intensity distribution behind the cuvette. This Gaussian profile, detected by a CCD camera, was used to determine the absorption coefficient of the liquid. An absorption coefficient as high as 1.54 x 10(4) cm(-1) was measured by use of high-concentration malachite green dye solutions. A comparison of the results with data extrapolated from those of conventional methods showed good agreement.

Randomized controlled trial to evaluate flush and reperfusion techniques in liver transplantation.

To determine the impact of different flush and reperfusion techniques on postreperfusion syndrome (PRS) and postoperative graft function, 100 transplants were randomly assigned into four groups as follows: group 1 (n=31), portal vein flush, no vena caval venting; group 2 (n=21), hepatic arterial flush, no vena caval venting; group 3 (n=29), portal vein flush with vena caval venting; and group 4 (n=19), hepatic artery flush with vena caval venting. Donor and recipient characteristics were similar. Extensive intraoperative and postoperative monitoring was performed and measurements were documented immediately before reperfusion and at 1, 5, 15, and 30 min after reperfusion. PRS was defined by three criteria: mean arterial pressure (MAP) <60 mmHg at 1 min after reperfusion, MAP <60 mmHg at 5 min after reperfusion, and a decrease of 30% or more for the MAP percent area under the curve during the initial 5 min after reperfusion (%AUC). Using these definitions, the overall incidence of PRS was 21%, 8%, and 43%, respectively. Group 1 was the most hemodynamically stable; the incidence of PRS in group 1 was 2/31 (7%) at 1 min and 8/31 (25%) using %AUC criteria compared with 7/21 (33%) at 1 min and 12/21 (57%) using %AUC criteria for group 2 (P<0.05). The patients in groups 3 and 4 (vena caval venting) demonstrated smaller percentage increases in serum potassium levels (as determined by %AUC; 4.3+/-6.8 and 0.3+/-5.4, vs. 15.1+/-8.1 for group 1 and 22.9+/-8.2 for group 2). The difference between group 4 and group 2 was statistically significant (P<0.05). The increases in serum potassium did not translate into increased cardiac or hemodynamic instability. Combining all data obtained over the first 30 min after reperfusion, there was no statistically significant difference in hemodynamic or biochemical changes noted among the four groups. Postoperative liver function was similar among the four groups. We conclude that portal vein flush without vena caval venting provided a lower incidence of PRS than any other technique. Vena caval venting decreased the release of potassium into the circulation. Postoperative graft function was not significantly affected by flush and reperfusion techniques.

Decreased alloreactivity to human islets secreting recombinant viral interleukin 10.

The objective of this study was to analyze allogeneic lymphocyte proliferative responses to cultured human pancreatic islets after gene transfer of viral interleukin (IL)-10 to the islets using replication-defective adenoviral vector. Human islets, either whole or dispersed into single cells, were cocultured with adenovector containing an expression cassette encoding the viral IL-10 gene under control of an SV40 promoter, this sequence replacing viral E1A and part of E1B early viral protein sequences. Subsequent production of recombinant protein by islets was determined by ELISA, and was found dependent on the multiplicity of infection (or ratio of vector to target cells). Protein was secreted by transfected islets at high levels 3-7 days after gene transfer. At high multiplicity of infection (100:1), islet viability was normal, but insulin secretion in response to glucose stimulation was blunted by 50%. Low-level recombinant viral IL-10 secretion by the islets was associated with increased allogeneic lymphocyte proliferation in mixed islet lymphocyte reactions. At protein levels in islet supernatant above 5 ng/ml, lymphocyte proliferation was significantly reduced. This pattern of viral IL-10 effect on lymphocyte proliferation correlated well with mixed lymphocyte reaction assays using purified protein. We conclude that transferred cytokine sequences are secreted by transfected islets as a function of the initial vector inoculum. The functional effect of the secreted cytokine viral IL-10 on allogeneic lymphocyte proliferation is dose dependent. Low-level recombinant protein secretion tended to augment lymphocyte proliferation, whereas high-level secretion significantly down-regulates this response.