Evaluation of the Maquet Neonatal and Pediatric Quadrox I with an integrated arterial line filter during cardiopulmonary bypass.

BACKGROUND: Perfusion equipment has evolved since its introduction into clinical practice more than fifty years ago to include smaller cardiopulmonary bypass (CPB) circuits and components. Perfusionists are now exploring the function of new oxygenators with an integrated arterial line filter (IALF). The purpose of this trial was to examine the Maquet Quadrox-I Neonatal and Pediatric oxygenators with IALF in a clinical setting, with respect to gas transfer, heat exchange co-efficiency (HEC), trans-membrane pressure (TMP) gradient and clinical experience. METHODS: The Maquet Quadrox-I Neonatal oxygenator was used on 30 patients ranging from 2.2-13.1 kg. The Maquet Quadrox-I Pediatric oxygenator was used on 15 patients ranging from 12.7-24.5 kg. Arterial and venous blood gases were taken once the patient was stable on CPB and, subsequently, every 30 minutes afterwards, as per institution protocol. The values for gas transfer rates, HEC and TMP gradient were stratified into three main categories with each oxygenator: normothermia, cooling and re-warming. RESULTS: During all conditions, the gas transfer rate with both oxygenators was efficient. The HEC values showed efficient heat exchanger performance during all conditions with both oxygenators. While maintaining CPB flow within the manufacturer's recommended flow rate for each oxygenator, the TMP gradient range for the Neonatal Quadrox-I was 10-40 mmHg and the Pediatric Quadrox-I was 10-60 mmHg. During the clinical trial, foam was shown to break through the cardiotomy on several occasions when high sucker return was required. CONCLUSION: This new line of oxygenators performed well with regards to gas transfer, HEC and TMP gradient, but there were clinical experiences that did not meet expectations. There were repeated incidences with the venous reservoir which ultimately cast a negative light on the design of this new product from Maquet. In the future, the authors would like to evaluate updated versions of this product from Maquet and any other pediatric perfusion devices that could help the patient in the clinical arena.

Dipole coupling of a double quantum dot to a microwave resonator.

We demonstrate the realization of a hybrid solid-state quantum device, in which a semiconductor double quantum dot is dipole coupled to the microwave field of a superconducting coplanar waveguide resonator. The double dot charge stability diagram extracted from measurements of the amplitude and phase of a microwave tone transmitted through the resonator is in good agreement with that obtained from transport measurements. Both the observed frequency shift and linewidth broadening of the resonator are explained considering the double dot as a charge qubit coupled with a strength of several tens of MHz to the resonator.

A systematic evaluation of the core communication skills expected of a perfusionist.

BACKGROUND: The objective of this systematic evaluation was to identify the sentinel standards necessary to obtain a core level of communication required of a clinical perfusionist during cardiopulmonary bypass (CPB). Once these sentinel standards were identified and a core level of communication was established (via four simulated case scenarios), a team of cardiac healthcare professionals was assembled to interpret both the accuracy of response and the speed of response encountered in each case scenario. METHODS: Four simulated case scenarios were utilized in order to replicate the typical patterns of verbal exchange that occur during surgeries using extracorporeal technology. The simulated case scenarios included CPB interactions associated with preparation, initiation, maintenance, termination and post CPB. For all CPB interactions, two variables were measured: accuracy of the perfusionist's response and speed of the perfusionist's response. The cases took place in a controlled setting within an empty operating room at The Children's Hospital of Philadelphia. Four clinical perfusionists each represented the role of the "perfusionist" in all simulated case scenarios. RESULTS: When analyzing the accuracy and speed of the responses, each clinical perfusionist recorded an average score of 96.3% or higher with all case scenarios. Since the clinical perfusionists who participated in the scenarios were primarily pediatric perfusionists, the scores were best during the pediatric case scenario, 99.3% (Case Scenario #4). The lowest scores were captured during Case Scenario #3 (96.3%) which involved a more intense adult patient scenario. CONCLUSION: The systematic evaluation of both response accuracy and response time (presented in various adult and pediatric patient case scenarios) can be beneficial within the realm of perfusion education. Students will be introduced to core communication concepts within the clinical realm. This study supports the idea that simulation and evaluation may ease the transition for students from the didactic to clinical realm in terms of communication. Further studies need to be developed in order to define "standard" CPB communication guidelines for perfusion students.

Tensile forces and shape entropy explain observed crista structure in mitochondria.

We present a model from which the observed morphology of the inner mitochondrial membrane can be inferred as minimizing the system's free energy. In addition to the usual energetic terms for bending, surface area, and pressure difference, our free energy includes terms for tension that we hypothesize to be exerted by proteins and for an entropic contribution due to many dimensions worth of shapes available at a given energy. We also present measurements of the structural features of mitochondria in HeLa cells and mouse embryonic fibroblasts using three-dimensional electron tomography. Such tomograms reveal that the inner membrane self-assembles into a complex structure that contains both tubular and flat lamellar crista components. This structure, which contains one matrix compartment, is believed to be essential to the proper functioning of mitochondria as the powerhouse of the cell. Interpreting the measurements in terms of the model, we find that tensile forces of ∼20 pN would stabilize a stress-induced coexistence of tubular and flat lamellar cristae phases. The model also predicts a pressure difference of -0.036 ± 0.004 atm (pressure higher in the matrix) and a surface tension equal to 0.09 ± 0.04 pN/nm.

Spin states in graphene quantum dots.

We investigate ground and excited state transport through small (d≈70 nm) graphene quantum dots. The successive spin filling of orbital states is detected by measuring the difference between ground-state energies as a function of a magnetic field. For a magnetic field in-plane of the quantum dot the Zeeman splitting of spin states is measured. The results are compatible with a g factor of 2, and we detect a spin-filling sequence for a series of states which is reasonable given the strength of exchange interaction effects expected by comparing Coulomb interaction energy and kinetic energy of charge carriers in graphene.

Electron-hole crossover in graphene quantum dots.

We investigate the addition spectrum of a graphene quantum-dot in the vicinity of the electron-hole crossover as a function of perpendicular magnetic field. Coulomb-blockade resonances of the 50 nm wide dot are visible at all gate voltages across the transport gap ranging from hole to electron transport. The magnetic field dependence of more than 50 states displays the unique complex evolution of the diamagnetic spectrum of a graphene dot from the low-field regime to the Landau regime with the n=0 Landau level situated in the center of the transport gap marking the electron-hole crossover. The average peak spacing in the energy region around the crossover decreases with increasing magnetic field. In the vicinity of the charge neutrality point we observe a well resolved and rich excited state spectrum.

Radiofrequency ablation with a high-power generator: device efficacy in an in vivo porcine liver model.

PURPOSE: The purpose of this study was to test the feasibility and efficacy of using a high-power generator with nondeployable electrodes to create large zones of coagulation in an in vivo porcine liver model. METHODS: With approval from our institution's research animal care and use committee, 12 female swine (mean weight = 55 kg) were anesthetized and received RF ablation at laparotomy. Twenty-nine ablations were performed in four groups using: (i) a conventional 200-W generator and cluster electrode (n = 4), or an experimental prototype 250-W generator and (ii) a single, 17-gauge electrode (n = 9), (iii) a cluster electrode (n = 8) or (iv) three electrodes spaced 2.0 cm apart in a triangular configuration (n = 8). In the three-electrode group, power was applied by switching between electrodes using a prototype switching device. All electrodes were internally cooled. Ablation zone size, shape and generator data from each group were compared using a mixed-linear model with animals modeled as random effects. RESULTS: The high-power generator was able to increase significantly the zone of coagulation. Mean (+/-SD) ablation diameter was largest in the switched group (4.31 +/- 0.7 cm) followed by the cluster (3.98 +/- 0.5 cm) and single-electrode (3.26 +/- 0.5 cm) groups. Mean diameter in the high-power single-electrode group was no different than the low-power cluster group (3.25 +/- 0.4 cm, p = 0.98). Circularity measures were high (>0.75) in all groups. CONCLUSIONS: Coupling a high-power generator and switching device is feasible. At higher powers, the switching device creates larger zones of ablation than cluster or single electrodes. Single-electrode ablations created with the prototype high-power generator were equivalent to those produced with the cluster electrode at conventional lower powers.

Evaluation of cis-acting elements in the rubella virus subgenomic RNA that play a role in its translation.

The subgenomic (SG) mRNA of rubella virus (RUB) contains the structural protein open reading frame (SP-ORF) that is translated to produce the three virion structural proteins: capsid (C) and glycoproteins E2 and E1. RUB expression vectors have been developed that express heterologous genes from the SG RNA, including replicons which replace the SP-ORF with a heterologous gene, and these expression vectors are candidate vaccine vectors. In the related alphaviruses, translational enhancing elements have been identified in both the 5' untranslated region (UTR) of the SG RNA and the N-terminal region of the C gene. To optimize expression from RUB vectors, both the 5'UTR of the SG RNA and the C gene were surveyed for translational enhancing elements using both plasmids and replicons expressing reporter genes from the SG RNA. In replicons, the entire 5'UTR was necessary for translation; interestingly, when plasmids were used the 5'UTR was dispensable for optimal translation. The RUB C gene contains a predicted long stem-loop starting 62 nts downstream from the initiation codon (SLL) that has a structure and stability similar to SL's found in the C genes of two alphaviruses, Sindbis virus (SIN) and Semliki Forest virus, that have been shown to enhance translation of the SG RNA in infected cells. However, a series of fusions of various lengths of the N-terminus of the RUB C protein with reporter genes showed that the SLL had an attenuating effect on translation that was overcome by mutagenesis that destabilized the SLL or by adding downstream sequences of the C gene to the fusion. Thus, for optimal expression efficiency from RUB expression vectors, only the 5'UTR of the SG RNA is required. Further investigation of the differing effects of the SLL on RUB and alphavirus SG RNA translation revealed that the SIN and RUB SLLs could enhance translation when expressed from a SIN cytopathic replicon, but not when expressed from a plasmid, a RUB replicon, or a SIN noncytopathic replicon. Thus, the SLL only functions in a "cytopathic environment" in which cell translation has been altered.

Modeling tubular shapes in the inner mitochondrial membrane.

The inner mitochondrial membrane has been shown to have a novel structure that contains tubular components whose radii are of the order of 10 nm as well as comparatively flat regions. The structural organization of mitochondria is important for understanding their functionality. We present a model that can account, thermodynamically, for the observed size of the tubules. The model contains two lipid constituents with different shapes. They are allowed to distribute in such a way that the composition differs on the two sides of the tubular membrane. Our calculations make two predictions: (1) there is a pressure difference of 0.2 atmospheres across the inner membrane as a necessary consequence of the experimentally observed tubule radius of 10 nm, and (2) migration of differently shaped lipids causes concentration variations of the order of 7% between the two sides of the tubular membrane.

Characterization of genotype II Rubella virus strains.

Two genotypes of Rubella virus have been described that differ by 8-9% at the nucleotide level in the E1 glycoprotein gene. Of these, genotype II (RGII) was only recently reported and in this study two RGII viruses, the BRDII vaccine strain and BR1 wild type strain, were characterized. Monoclonal antibodies against each of the virion proteins (capsid [C], glycoproteins E1 and E2) and polyclonal anti-rubella virus sera reacted similarly with purified virions from the RGII and reference RGI strains on Western gels, with the exception of one anti-E2 Mab, and thus the two genotypes are closely related antigenically. The genomic sequences of two genotype II (RGII) rubella virus strains were determined and compared with the six previously reported RGI sequences. The genomes of these viruses all contained 9762 nts and the lengths of the three untranslated regions (UTRs) and two open reading frames (ORF's) were identical. The overall difference between the RGI and RGII sequences at the nt level was approximately 8% and this difference was maintained across most of the genome. At the amino acid level, the RGI and RGII sequences differed overall by approximately 4%, however this difference was not uniform across the ORF's as the N-terminal third of P150 and the entirety of P90, both replicase proteins, were more conserved (<1% difference) while the C-terminal two thirds of P150 exhibited greater variation ( approximately 8% difference), including a hypervariable region between residues 771-801 within which divergence as great as 20-30% was detected. The parent wt virus of the BRDII vaccine was not available and its sequence was compared with the BR1 sequence to identify potential attenuating mutations. The BRDII and BR1 sequences varied at 252 residues (2.59%), including twelve in the UTRs and thirty coding differences in the ORF's. None of these differences in the BRDII sequence was vaccine-specific when compared with RGI wt and vaccine sequences and, therefore, there appeared to be no common pathway in the generation of live, attenuated rubella vaccines.

Insight into mitochondrial structure and function from electron tomography.

In recent years, electron tomography has provided detailed three-dimensional models of mitochondria that have redefined our concept of mitochondrial structure. The models reveal an inner membrane consisting of two components, the inner boundary membrane (IBM) closely apposed to the outer membrane and the cristae membrane that projects into the matrix compartment. These two components are connected by tubular structures of relatively uniform size called crista junctions. The distribution of crista junction sizes and shapes is predicted by a thermodynamic model based upon the energy of membrane bending, but proteins likely also play a role in determining the conformation of the inner membrane. Results of structural studies of mitochondria during apoptosis demonstrate that cytochrome c is released without detectable disruption of the outer membrane or extensive swelling of the mitochondrial matrix, suggesting the formation of an outer membrane pore large enough to allow passage of holo-cytochrome c. The possible compartmentation of inner membrane function between the IBM and the cristae membrane is also discussed.

Membrane architecture of mitochondria in neurons of the central nervous system.

Electron tomography was used to help redefine the membrane architecture of mitochondria in neurons of the brain. Investigations were conducted on unexplored questions of structural homogeneity between mitochondria in the four intensely studied regions of the brain and in the functionally distinct neuronal sub-compartments. These mitochondria have the majority of cristae composed of both tubular and lamellar segments with the tubes arranged more peripherally and the lamellae more centrally located. Cristae that are entirely tubular were not commonly seen and those that are entirely lamellar were rare. It was determined that cristae connect through narrow, sometimes very long tubular regions to the peripheral surface of the inner membrane. A structurally distinct type of contact site was revealed in brain mitochondria, which we named the bridge contact site. These bridges may play a role in the structural integrity of the outer and inner membrane systems. It was found that the membrane architecture in the various brain regions and neuronal compartments was strikingly uniform, including consistently tubular crista junctions. The functional consequences of this junctional architecture are discussed in relation to the segregation of proteins between the inner boundary membrane and the cristae membranes, and in relation to the model of microcompartmentation of macromolecules inside cristae.

Rubella virus DI RNAs and replicons: requirement for nonstructural proteins acting in cis for amplification by helper virus.

A rubella virus (RUB) replicon was constructed by replacing the 3' proximal structural protein ORF (SP-ORF) in Robo402, a RUB infectious cDNA clone, with a reporter gene, green fluorescent protein (GFP). This replicon, RUBrep/GFP, mimics naturally occurring RUB defective-interfering (DI) RNAs generated during serial undiluted passage that maintain the 5' proximal nonstructural protein ORF (NS-ORF) but contain deletions in the SP-ORF. Following transfection of Vero cells with in vitro RNA transcripts from RUBrep/GFP, replicon replication occurred and the replicon was amplified and spread to other cells in the presence of standard helper virus. GFP expression was a much more sensitive indicator of replicon replication than was Northern analysis to detect replicon-specific RNAs. Most of a series of RUBrep/GFP constructs with deletions in the NS-ORF not only were incapable of self-replication, but were not amplified by standard helper virus. The only exception was a construct with an in-frame deletion between two NotI sites that removed nucleotides 1685-2192 of the genome; this construct did not express GFP by itself, but did express GFP in the presence of standard helper RUB and was spread to other cells. Thus, with the exception of this region, the NS-ORF is required in cis for amplification of RUB replicons by standard helper virus, explaining the selection of DI RNAs that maintain the NS-ORF. Surprisingly, when the NotI deletion was introduced into Robo402, a viable virus resulted that replicated only threefold less efficiently than did Robo402 virus. Thus, the NotI region of the NS-ORF is not necessary for virus replication. This deletion covers a region of the NS-ORF without predicted function, which therefore may function as a spacer or hinge between functional domains. Nevertheless, it was an unexpected finding that a small virus such as RUB could dispense with approximately 10% of its genome.

Analysis of TAF90 mutants displaying allele-specific and broad defects in transcription.

Yeast TAF90p is a component of at least two transcription regulatory complexes, the general transcription factor TFIID and the Spt-Ada-Gcn5 histone acetyltransferase complex (SAGA). Broad transcription defects have been observed in mutants of other TAF(II)s shared by TFIID and SAGA but not in the only two TAF90 mutants isolated to date. Given that the numbers of mutants analyzed thus far are small, we isolated and characterized 11 temperature-sensitive mutants of TAF90 and analyzed their effects on transcription and integrity of the TFIID and SAGA complexes. We found that the mutants displayed a variety of allele-specific defects in their ability to support transcription and maintain the structure of the TFIID and SAGA complexes. Sequencing of the alleles revealed that all have mutations corresponding to the C terminus of the protein, with most clustering within the conserved WD40 repeats; thus, the C terminus of TAF90p is required for its incorporation into TFIID and function in SAGA. Significantly, inactivation of one allele, taf90-20, caused the dramatic reduction in the levels of total mRNA and most specific transcripts analyzed. Analysis of the structure and/or activity of both TAF90p-containing complexes revealed that this allele is the most disruptive of all. Our analysis defines the requirement for the WD40 repeats in preserving TFIID and SAGA function, demonstrates that the defects associated with distinct mutations in TAF90 vary considerably, and indicates that TAF90 can be classified as a gene required for the transcription of a large number of genes.

Electron tomography of mitochondria after the arrest of protein import associated with Tom19 depletion.

In a mutant form of Neurospora crassa, in which sheltered RIP (repeat induced point mutation) was used to deplete Tom19, protein transport through the TOM/TIM pathway is arrested by the addition of p-fluorophenylalanine (FPA). Using intermediate-voltage electron tomography, we have generated three-dimensional reconstructions of 28 FPA-treated mitochondria at four time points (0-32 h) after the addition of FPA. We determined that the cristae surface area and volume were lost in a roughly linear manner. A decrease in mitochondrial volume was not observed until after 16 h of FPA treatment. The inner boundary membrane did not appear to shrink or contract away from the outer membrane. Interestingly, the close apposition of these membranes remained over the entire periphery, even after all of the cristae had disappeared. The different dynamics of the shrinkage of cristae membrane and inner boundary membrane has implications for compartmentalization of electron transport proteins. Two structurally distinct types of contact sites were observed, consistent with recently published work. We determined that the cristae in the untreated (control) mitochondria are all lamellar. The cristae of FPA-treated mitochondria retain the lamellar morphology as they reduce in size and do not adopt tubular shapes. Importantly, the crista junctions exhibit tubular as well as slot-like connections to the inner boundary membrane, persisting until the cristae disappear, indicating that their stability is not dependent on continuous protein import through the complex containing Tom19.

Evidence for phase-separated quantum dots in cubic InGaN layers from resonant raman scattering

The emission of light in the blue-green region from cubic InxGa1-xN alloys grown by molecular beam epitaxy is observed at room temperature and 30 K. By using selective resonant Raman spectroscopy (RRS) we demonstrate that the emission is due to quantum confinement effects taking place in phase-separated In-rich quantum dots formed in the layers. RRS data show that the In content of the dots fluctuates across the volume of the layers. We find that dot size and alloy fluctuation determine the emission wavelengths.

Development of a rubella virus vaccine expression vector: use of a picornavirus internal ribosome entry site increases stability of expression.

Rubella virus (RUB) is a small plus-strand RNA virus classified in the Rubivirus genus of the family Togaviridae. Live, attenuated RUB vaccines have been successfully used in vaccination programs for over 25 years, making RUB an attractive vaccine vector. In this study, such a vector was constructed using a recently developed RUB infectious cDNA clone (Robo). Using a standard strategy employed to produce expression and vaccine vectors with other togaviruses, the subgenomic promoter was duplicated to produce a recombinant construct (termed dsRobo) that expressed reporter genes such as chloramphenicol acetyltransferase and green fluorescent protein (GFP) under control of the second subgenomic promoter. However, expression of the reporter genes, as exemplified by GFP expression by dsRobo/GFP virus, was unstable during passaging, apparently due to homologous recombination between the subgenomic promoters leading to deletion of the GFP gene. To improve the stability of the vector, the internal ribosome entry site (IRES) of a picornavirus, encephalomyocarditis virus, was used instead of the second subgenomic promoter to eliminate homology. Construction was initiated by first replacing the subgenomic promoter in the parent Robo infectious clone with the IRES. Surprisingly, viable virus resulted; this virus did not synthesize a subgenomic RNA. The subgenomic promoter was then reintroduced in an orientation such that a single subgenomic RNA was produced, GFP was the initial gene on this RNA, while the RUB structural protein open reading frame was downstream and under control of the IRES element. GFP expression by this vector was significantly improved in comparison to dsRobo/GFP. This strategy should be applicable to increase the stability of other togavirus vectors.

Expression of the hepatitis E virus ORF1.

Hepatitis E virus (HEV) is an unclassified, plus-strand RNA virus whose genome contains three open reading frames (ORFs). ORF1, the 5' proximal ORF of HEV, encodes nonstructural proteins involved in RNA replication which share homology with the products of the corresponding ORF of members of the alphavirus-like superfamily of plus-strand RNA viruses. Among animal virus members of this superfamily (the alphavirus and rubivirus genera of the family Togaviridae), the product of this ORF is a nonstructural polyprotein (NSP) that is cleaved by a papain-like cysteine protease (PCP) within the NSP. To determine if the NSP of HEV is similarly processed, ORF1 was introduced into a plasmid vector which allowed for expression both in vitro using a coupled transcription/translation system and in vivo using a vaccinia virus-driven transient expression system. A recombinant vaccinia virus expressing ORF1 was also constructed. Both in vitro and in vivo expression under standard conditions yielded only the full-length 185 kDa polyprotein. Addition of co-factors in vitro, such as divalent cations and microsomes which have been shown to activate other viral proteases, failed to change this expression pattern. However, in vivo following extended incubations (24-36 hours), two potential processing products of 107 kDa and 78 kDa were observed. N- and C-terminus-specific immunoprecipitation and deletion mutagenesis were used to determine that the order of these products within the NSP is NH2-78 kDa-107 kDa-COOH. However, site-specific mutagenesis of Cys483, predicted by computer alignment to be one member of the catalytic dyad of a PCP within the NSP, failed to abolish this cleavage. Additionally, sequence alignment across HEV strains revealed that the other member of the proposed catalytic dyad of this PCP, His590, was not conserved. Thus, the cleavage of the NSP observed following prolonged in vivo expression was not mediated by this protease and it is doubtful that a functional PCP exists within the NSP. Attempts to detect NSP expression and processing in HEV-infected primary monkey hepatocytes were not successful and therefore this proteolytic cleavage could not be authenticated. Overall, the results of this study indicate that either the HEV NSP is not processed or that it is cleaved at one site by a virally-encoded protease novel among alpha-like superfamily viruses or a cellular protease.