Reporting Outcomes of Pediatric Intensive Care Unit Patients to Referring Physicians via an Electronic Health Record-Based Feedback System.

BACKGROUND: Many critically ill children are initially evaluated in front-line settings by clinicians with variable pediatric training before they are transferred to a pediatric intensive care unit (PICU). Because clinicians learn from past performance, communicating outcomes of patients back to front-line clinicians who provide pediatric emergency care could be valuable; however, referring clinicians do not consistently receive this important feedback. OBJECTIVES: Our aim was to determine the feasibility, usability, and clinical relevance of a semiautomated electronic health record (EHR)-supported system developed at a single institution to deliver timely and relevant PICU patient outcome feedback to referring emergency department (ED) physicians. METHODS: Guided by the Health Information Technology Safety Framework, we iteratively designed, implemented, and evaluated a semiautomated electronic feedback system leveraging the EHR in one institution. After conducting interviews and focus groups with stakeholders to understand the PICU-ED health care work system, we designed the EHR-supported feedback system by translating stakeholder, organizational, and usability objectives into feedback process and report requirements. Over 6 months, we completed three cycles of implementation and evaluation, wherein we analyzed EHR access logs, reviewed feedback reports sent, performed usability testing, and conducted physician interviews to determine the system's feasibility, usability, and clinical relevance. RESULTS: The EHR-supported feedback process is feasible with timely delivery and receipt of feedback reports. Usability testing revealed excellent Systems Usability Scale scores. According to physicians, the process was well-integrated into their clinical workflows and conferred minimal additional workload. Physicians also indicated that delivering and receiving consistent feedback was relevant to their clinical practice. CONCLUSION: An EHR-supported system to deliver timely and relevant PICU patient outcome feedback to referring ED physicians was feasible, usable, and important to physicians. Future work is needed to evaluate impact on clinical practice and patient outcomes and to investigate applicability to other clinical settings involved in similar care transitions.

In vitro exposure of vitellogenic rainbow trout ovarian follicles to endocrine disrupting chemicals can alter basal estradiol-17ß production and responsiveness to a gonadotropin challenge.

Endogenous estrogens play major roles in many aspects of female reproductive development in fish. In order to develop a relatively high-throughput assay to determine the potential impact on reproductive development, vitellogenic rainbow trout ovarian follicles were exposed to a suite of contaminants in vitro and then assessed for the ability to produce estradiol-17ß (E2) after a 500 ng/ml salmon gonadotropin (sGTH) challenge. There was a positive correlation between ovarian follicle size and E2 production, but an inverse correlation between size and responsiveness to sGTH. Significant impacts on E2 levels were observed following treatment with different endocrine disrupting chemicals, such as 17α-ethinylestradiol (EE2), prochloraz, or trenbolone. EE2 was remarkably potent and significantly reduced ovarian follicle responsiveness to sGTH at concentrations as low as 0.1 nM. Of the other contaminants tested, only tamoxifen impacted E2 levels, and only at concentrations near the limits of solubility. Flutamide, fluoxetine, 4-hydroxy tamoxifen, hydroxyflutamide, and norfluoxetine had little or no impact. Quantitative PCR analyses of steroidogenesis-related genes were carried out on EE2 treated ovarian follicles, but significant transcriptional responses to EE2 were not observed. Overall, this study suggests that xenoestrogens and anti-estrogens are more likely to interfere with ovarian E2 synthesis than other classes of EDCs. This also provides a template for further testing of the effects of EDCs on ovarian function.

A teleost androgen promotes development of primary ovarian follicles in coho salmon and rapidly alters the ovarian transcriptome.

Recent studies using several teleost models have revealed that androgens increase the size of previtellogenic (primary and/or early secondary) ovarian follicles. To explore our hypothesis that androgens drive the development of primary follicles into early secondary follicles, and to determine the mechanisms underlying these androgenic effects, we exposed juvenile coho salmon to near-physiological and relatively sustained levels of the nonaromatizable androgen 11-ketotestosterone (11-KT). This resulted in significant growth of primary ovarian follicles after 10 and 20 days, with follicles after 20 days displaying a morphological phenotype characteristic of early secondary follicles (presence of cortical alveoli). Utilizing the same experimental approach, we then analyzed how 11-KT rapidly altered the ovarian transcriptome after 1 and 3 days of treatment. RNA-Seq analysis revealed that 69 (day 1) and 1,022 (day 3) contiguous sequences (contigs) were differentially expressed relative to controls. The differentially expressed contigs mapped to genes including those encoding proteins involved in gonadotropin, steroid hormone, and growth factor signaling, and in cell and ovarian development, including genes with putative androgen-response elements. Biological functions and canonical pathways identified as potentially altered by 11-KT include those involved in ovarian development, tissue differentiation and remodeling, and lipid metabolism. We conclude that androgens play a major role in stimulating primary ovarian follicle development and the transition into secondary growth.

Preserved transmembrane protein mobility in polymer-supported lipid bilayers derived from cell membranes.

Supported lipid bilayers (SLBs) have contributed invaluable information about the physiochemical properties of cell membranes, but their compositional simplicity often limits the level of knowledge that can be gained about the structure and function of transmembrane proteins in their native environment. Herein, we demonstrate a generic protocol for producing polymer-supported lipid bilayers on glass surfaces that contain essentially all naturally occurring cell-membrane components of a cell line while still retaining transmembrane protein mobility and activity. This was achieved by merging vesicles made from synthetic lipids (PEGylated lipids and POPC lipids) with native cell-membrane vesicles to generate hybrid vesicles which readily rupture into a continuous polymer-supported lipid bilayer. To investigate the properties of these complex hybrid SLBs and particularly the behavior of their integral membrane-proteins, we used total internal reflection fluorescence imaging to study a transmembrane protease, ß-secretase 1 (BACE1), whose ectoplasmic and cytoplasmic domains could both be specifically targeted with fluorescent reporters. By selectively probing the two different orientations of BACE1 in the resulting hybrid SLBs, the role of the PEG-cushion on transmembrane protein lateral mobility was investigated. The results reveal the necessity of having the PEGylated lipids present during vesicle adsorption to prevent immobilization of transmembrane proteins with protruding domains. The proteolytic activity of BACE1 was unadulterated by the sonication process used to merge the synthetic and native membrane vesicles; importantly it was also conserved in the SLB. The presented strategy could thus serve both fundamental studies of membrane biophysics and the production of surface-based bioanalytical sensor platforms.

Coupling supported lipid bilayer electrophoresis with matrix-assisted laser desorption/ionization-mass spectrometry imaging.

Herein, we describe a new analytical platform utilizing advances in heterogeneous supported lipid bilayer (SLB) electrophoresis and matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) imaging. This platform allowed for the separation and visualization of both charged and neutral lipid membrane components without the need for extrinsic labels. A heterogeneous SLB was created using vesicles containing monosialoganglioside GM1, disialoganglioside GD1b, POPC, as well as the ortho and para isomers of Texas Red-DHPE. These components were then separated electrophoretically into five resolved bands. This represents the most complex separation by SLB electrophoresis performed to date. The SLB samples were flash frozen in liquid ethane and dried under vacuum before imaging with MALDI-MS. Fluorescence microscopy was employed to confirm the position of the Texas Red labeled lipids, which agreed well with the MALDI-MS imaging results. These results clearly demonstrate this platform's ability to isolate and identify nonlabeled membrane components within an SLB.

A zebrafish model of congenital disorders of glycosylation with phosphomannose isomerase deficiency reveals an early opportunity for corrective mannose supplementation.

Individuals with congenital disorders of glycosylation (CDG) have recessive mutations in genes required for protein N-glycosylation, resulting in multi-systemic disease. Despite the well-characterized biochemical consequences in these individuals, the underlying cellular defects that contribute to CDG are not well understood. Synthesis of the lipid-linked oligosaccharide (LLO), which serves as the sugar donor for the N-glycosylation of secretory proteins, requires conversion of fructose-6-phosphate to mannose-6-phosphate via the phosphomannose isomerase (MPI) enzyme. Individuals who are deficient in MPI present with bleeding, diarrhea, edema, gastrointestinal bleeding and liver fibrosis. MPI-CDG patients can be treated with oral mannose supplements, which is converted to mannose-6-phosphate through a minor complementary metabolic pathway, restoring protein glycosylation and ameliorating most symptoms, although liver disease continues to progress. Because Mpi deletion in mice causes early embryonic lethality and thus is difficult to study, we used zebrafish to establish a model of MPI-CDG. We used a morpholino to block mpi mRNA translation and established a concentration that consistently yielded 13% residual Mpi enzyme activity at 4 days post-fertilization (dpf), which is within the range of MPI activity detected in fibroblasts from MPI-CDG patients. Fluorophore-assisted carbohydrate electrophoresis detected decreased LLO and N-glycans in mpi morphants. These deficiencies resulted in 50% embryonic lethality by 4 dpf. Multi-systemic abnormalities, including small eyes, dysmorphic jaws, pericardial edema, a small liver and curled tails, occurred in 82% of the surviving larvae. Importantly, these phenotypes could be rescued with mannose supplementation. Thus, parallel processes in fish and humans contribute to the phenotypes caused by Mpi depletion. Interestingly, mannose was only effective if provided prior to 24 hpf. These data provide insight into treatment efficacy and the broader molecular and developmental abnormalities that contribute to disorders associated with defective protein glycosylation.

Phosphatidylserine reversibly binds Cu2+ with extremely high affinity.

Phosphatidylserine (PS) embedded within supported lipid bilayers was found to bind Cu(2+) from solution with extraordinarily high affinity. In fact, the equilibrium dissociation constant was in the femtomolar range. The resulting complex formed in a 1:2 Cu(2+)-to-PS ratio and quenches a broad spectrum of lipid-bound fluorophores in a reversible and pH-dependent fashion. At acidic pH values, the fluorophores were almost completely unquenched, while at basic pH values significant quenching (85-90%) was observed. The pH at which the transition occurred was dependent on the PS concentration and ranged from approximately pH 5 to 8. The quenching kinetics was slow at low Cu(2+) concentrations and basic pH values (up to several hours), while the unquenching reaction was orders of magnitude more rapid upon lowering the pH. This was consistent with diffusion-limited complex formation at basic pH but rapid dissociation under acidic conditions. The tight binding of Cu(2+) to PS may have physiological consequences under certain circumstances.

UHRF1 phosphorylation by cyclin A2/cyclin-dependent kinase 2 is required for zebrafish embryogenesis.

Ubiquitin-like, containing PHD and RING finger domains 1 (uhrf1) is regulated at the transcriptional level during the cell cycle and in developing zebrafish embryos. We identify phosphorylation as a novel means of regulating UHRF1 and demonstrate that Uhrf1 phosphorylation is required for gastrulation in zebrafish. Human UHRF1 contains a conserved cyclin-dependent kinase 2 (CDK2) phosphorylation site at Ser-661 that is phosphorylated in vitro by CDK2 partnered with cyclin A2 (CCNA2), but not cyclin E. An antibody specific for phospho-Ser-661 recognizes UHRF1 in both mammalian cancer cells and in nontransformed zebrafish cells, but not in zebrafish bearing a mutation in ccna2. Depleting Uhrf1 from zebrafish embryos by morpholino injection causes arrest before gastrulation and early embryonic death. This phenotype is rescued by wild-type UHRF1, but not by UHRF1 in which the phospho-acceptor site is mutated, demonstrating that UHRF1 phosphorylation is essential for embryogenesis. UHRF1 was detected in the nucleus and cytoplasm, whereas nonphosphorylatable UHRF1 is unable to localize to the cytoplasm, suggesting the importance of localization in UHRF1 function. Together, these data point to an essential role for UHRF1 phosphorylation by CDK/CCNA2 during early vertebrate development.

Protein separation by electrophoretic-electroosmotic focusing on supported lipid bilayers.

An electrophoretic-electroosmotic focusing (EEF) method was developed and used to separate membrane-bound proteins and charged lipids based on their charge-to-size ratio from an initially homogeneous mixture. EEF uses opposing electrophoretic and electroosmotic forces to focus and separate proteins and lipids into narrow bands on supported lipid bilayers (SLBs). Membrane-associated species were focused into specific positions within the SLB in a highly repeatable fashion. The steady-state focusing positions of the proteins could be predicted and controlled by tuning experimental conditions, such as buffer pH, ionic strength, electric field, and temperature. Careful tuning of the variables should enable one to separate mixtures of membrane proteins with only subtle differences. The EEF technique was found to be an effective way to separate protein mixtures with low initial concentrations, and it overcame diffusive peak broadening to allow four bands to be separated simultaneously within a 380 µm wide isolated supported membrane patch.

Supported bilayer electrophoresis under controlled buffer conditions.

A pH controlled flow cell device was constructed to allow electrophoretic movement of charged lipids and membrane associated proteins in supported phospholipid bilayers. The device isolated electrolysis products near the electrodes from the electrophoresis process within the bilayer. This allowed the pH over the bilayer region to remain within ±0.2 pH units or better over many hours at salt concentrations up to 10 mM. Using this setup, it was found that the electrophoretic mobility of a dye conjugated lipid (Texas Red 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (TR-DHPE)) was essentially constant between pH 3.3 and 9.3. In contrast, streptavidin, which was bound to biotinylated lipids, shifted from migrating cathodically at acidic pH values to migrating anodically under basic conditions. This shift was due to the modulation of the net charge on the protein, which changed the electrophoretic forces experienced by the macromolecule. The addition of a polyethylene glycol (PEG) cushion beneath the bilayer or the increase in the ionic strength of the buffer solution resulted in a decrease of the electroosmotic force experienced by the streptavidin with little effect on the Texas Red-DHPE. As such, it was possible in part to control the electrophoretic and electroosmotic contributions to streptavidin independently of one another.

Klf6/copeb is required for hepatic outgrowth in zebrafish and for hepatocyte specification in mouse ES cells.

Krüppel-like factor 6 (Klf6; copeb in zebrafish) is a zinc-finger transcription factor and tumor suppressor gene. Klf6(-)(/)(-) mice have defects in hematopoiesis and angiogenesis and do not form a liver. However, the vascular abnormalities in Klf6(-/-) mice obfuscate its role in liver development since these two processes are linked in mammals. We utilized zebrafish and mouse ES cells to investigate the role of copeb in endoderm specification and hepatogenesis separate from its function in angiogenesis. During zebrafish development, copeb expression is enriched in digestive organs. Morpholino knockdown of copeb blocks expansion of the liver, pancreas and intestine, but does not affect their specification, differentiation or the vascularization of the liver. Decreased hepatocyte proliferation in copeb morphants is accompanied by upregulation of the cell cycle inhibitor, cdkn1a, a Copeb transcriptional target. A cell autonomous role for Klf6 in endoderm and hepatic development was investigated by manipulating Klf6 expression in mouse ES cells driven to differentiate along the hepatic lineage. Expression of the endoderm markers Hnf3beta, Gata4, Sox17, and CxCr4 is not induced in Klf6(-/-) cells but is upregulated in ES cells over-expressing Klf6. Collectively, these findings indicate that copeb/Klf6 is essential for the development of endoderm-derived organs.

Inbreeding depression and outbreeding depression are evident in wild-type zebrafish lines.

Maintaining wild-type (WT) zebrafish stocks for research while preserving viability within the lines used presents significant challenges to zebrafish husbandry practices. Genetic homogeneity is established through inbreeding to provide continuity across experiments. This, however, leads to decreased fitness through inbreeding depression. In the laboratory setting, it is imperative that researchers consistently obtain a large number of viable embryos; thus, inbreeding depression must be suppressed. Genetic variation can be established by creating hybrid lines; however, crosses between genetically distinct lines can cause an outbreeding depression as well. There is little data describing the effects of inbreeding depression or outbreeding depression from such crosses in zebrafish. Additionally, there is a need to establish breeding standards within the zebrafish field. This study examines the susceptibility to inbreeding and outbreeding depression in crosses between four WT zebrafish lines: the inbred lines AB and Tab 14, and the F1 generation of hybrid lines TuAB and TLAB. We report that mating frequency and clutch size were significantly greater in hybrid female crosses than in inbred female crosses. Inbreeding depression in common zebrafish lines such as AB and Tab 14 used here results in fewer successful matings and smaller clutch sizes. Further, outbreeding depression caused by crossing distantly related lines, such as the inbred Tab 14 and the hybrid TLAB lines, can also influence successful zebrafish mating. These data provide evidence needed to further characterize commonly used WT zebrafish lines. We suggest that to maintain lines that mate frequently and yield large clutches, hybrid females of known backgrounds should be used.

Antibody-antigen exchange equilibria in a field of an external force: design of reagentless biosensors.

This correspondence presents a new strategy for detecting biological molecules that relies on competitive exchange interactions of an analyte with two-component molecular tethers attaching superparamagnetic microspheres (4 microm in diameter) to a sensor surface. The individual tethers consist of an antibody-antigen complex and are designed to selectively detect antigenic proteins in a sensitive reagentless fashion. In order to impart a driving force to the otherwise free energy neutral antibody-antigen exchange equilibrium, a small mechanical force of approximately 10 pN was applied to stretch the antibody-antigen tethers using a massively parallel magnetic tweezers device. The experimental work was carried out with human cardiac troponin I. This serum heart attack marker was used as an example of analytes of credible relevance to biomedical diagnostics. The initial results illustrate the functioning of a cardiotroponin sensor and offer a preliminary estimate of its sensitivity of 16 pM.

A graphical model for evolutionary optimization.

We present a statistical model of empirical optimization that admits the creation of algorithms with explicit and intuitively defined desiderata. Because No Free Lunch theorems dictate that no optimization algorithm can be considered more efficient than any other when considering all possible functions, the desired function class plays a prominent role in the model. In particular, this provides a direct way to answer the traditionally difficult question of what algorithm is best matched to a particular class of functions. Among the benefits of the model are the ability to specify the function class in a straightforward manner, a natural way to specify noisy or dynamic functions, and a new source of insight into No Free Lunch theorems for optimization.

The role of insulin receptor signaling in zebrafish embryogenesis.

Insulin receptor (IR) signaling is considered to be important in growth and development in addition to its major role in metabolic homeostasis. The metabolic role of insulin in carbohydrate and lipid metabolism is extensively studied. In contrast, the role of IR activation during embryogenesis is less understood. To address this, we examined the function of the IR during zebrafish development. Zebrafish express two isoforms of IR (insra and insrb). Both isoforms were cloned and show high homology to the human insulin receptor and can functionally substitute for the human IR in fibroblasts derived from insr gene-deleted mice. Gene expression studies reveal that these receptors are expressed at moderate levels in the central nervous system during development. Morpholino-mediated selective knockdown of each of the IR isoforms causes growth retardation and profound morphogenetic defects in the brain and eye. These results clearly demonstrate that IR signaling plays essential roles in vertebrate embryogenesis and growth.

Ion diffusion in channels containing random arrays of microspheres: an electrochemical time-of-flight method.

Rates of chloride ion diffusion in narrow (ca. 3 microm thick), rectangular (ca. 0.1 x 1.0 mm(2)) channels partially filled with polystyrene microspheres are investigated by a potentiometric electrochemical time-of-flight (P-ETOF) method. Lithographically fabricated on glass slides, P-ETOF devices consist of a centrally positioned 10 microm wide, ca. 1 mm long generator microelectrode and two sensor microelectrodes of the same dimensions symmetrically positioned on both side of the generator at a distance of 50 microm. The electrodes are silver-plated and partially oxidized in a chloride electrolyte to form Ag/AgCl deposits. Constant current reduction of AgCl on the generator electrode is used to produce chloride ions at a constant rate. Ag/AgCl deposited on the sensor microelectrodes allows time-dependent potentiometric monitoring of the increasing concentration of chloride ions diffusing across the interelectrode gap. The device is enclosed with a parallel glass plate to form a narrow channel with the polystyrene microbeads serving as spacers. The packing density of the microspheres expressed in terms of the fractional void volume (rho) varied from ca. 0.6 to 1.0. Using rho, we modified a diffusion equation describing the change of chloride ion concentration at the sensor microelectrode to include the effect of the microspheres restricting the void volume. We rely on digital simulations as well as on direct P-ETOF experiments to show that the proposed equation does accurately account for the effect of rho on the diffusion processes. We thus demonstrate that P-ETOF can be used to measure the number of identical microspheres in the active region of a narrow channel device. In the latter context, a future application of P-ETOF as a signal transduction mechanism in biosensors is outlined.

Electrochemistry of TEMPO in the aqueous liquid/vapor interfacial region: measurements of the lateral mobility and kinetics of surface partitioning.

A new method is described to simultaneously determine the kinetics of surface partitioning and the lateral diffusion constant of redox active amphiphiles. It concerns water-soluble amphiphiles for which the surface adsorption equilibrium constant and the solution diffusion constant are measured independently. The method involves cyclic voltammetric experiments carried out at the air/water interface with microband electrodes aligned with the plane of the water surface. Typically, 100 nm wide, 1.0 cm long microband electrodes are fabricated by the vacuum vapor deposition of gold films on glass. The front face of the electrode substrates are coated with impermeable, dimensionally stable, polymer barrier films with thickness L in the range of approximately 0.1-1.0 microm. Fracturing such gold-coated glass substrates exposes gold microbands. The recorded voltammetric current sensitively depends on the barrier film thickness, the surfactant surface diffusion constant, Dsurf, and its rate constant of desorption, kdes. For a given surfactant, such as the nitroxyl piperidine free radical TEMPO featured in this report, large currents are observed with microband electrodes that do not carry a barrier film (L = 0). This is because the surfactant surface population diffusing along the air/water interface can be directly electro-oxidized at the edge of the microband. Smaller currents are measured in the presence of a barrier film, since, in those instances, the surface population may contribute to the voltammetric current only via a mechanism involving surfactant desorption from the water surface into bulk, where it contributes to the three-dimensional solution diffusion processes. The quantitative interpretation of the voltammetric experiments was made possible with finite element simulations with FEMLAB. These produce a set of calibration curves, Dsurf versus log kdes, for each value of the barrier film thickness. The intersection of the calibration curves determines the unique values of Dsurf and kdes. For TEMPO, Dsurf = 4.4 +/- 1.2 x 10(-5) cm2/s and kdes >/= 2 x 10(4) s(-1). Surfactant desorption rate constants of this magnitude have not been previously experimentally accessible. Since, in our earlier report (Wu, D. G.; Malec, A. D.; Head-Gordon, M.; Majda, M. J. Am. Chem. Soc. 2005, 27, 4490-4496), we showed that TEMPO is not immersed in water and that it diffuses along the interface hydrogen-bonded to just one or two water molecules, its Dsurf value approximates the water diffusion constant in the aqueous liquid-vapor interfacial region.