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Oxygen (O2) delivery, which is fundamental to supporting patients with critical illness, is a function of blood O2 content and flow. This article reviews red blood cell (RBC) physiology and dysfunction relevant to disordered O2 delivery in the critically ill. Flow is the focus of O2 delivery regulation: O2 content is relatively fixed, whereas flow fluctuates greatly. Thus, blood flow volume and distribution vary to maintain coupling between O2 delivery and demand. This article reviews conventional RBC physiology influencing O2 delivery and introduces a paradigm for O2 delivery homeostasis based on coordinated gas transport and vascular signaling by RBCs.

Individuals with sickle cell disease (SCD) face ongoing risk of multi-organ ischemia resulting in chronic disability, frequent hospitalizations, and early mortality. The relationship between hemoglobin (Hb) S polymerization, erythrocyte sickling, and tissue ischemia has been of great interest. Oxygen off-loading and increasing deoxy-Hb concentration promote HbS polymerization, the latter, which has been linked to early erythrocyte deformation or “reversible” sickling. Eventually, severe polymerization weakens the cell membrane, leading to “irreversibly” sickled cells. Whether the degree of polymerization and the cell's morphologic state, in turn, influence oxygen binding and thus, tissue oxygen availability has been of interest, but technically challenging to study in patients. Over two decades, Wang and colleagues developed an imaging platform, photoacoustic microscopy (PAM), which offers two unique aspects compared to other intravital microscopy systems: (1) high resolution in vivo human imaging using the cuticle as the window to a highly organized vascular bed capable of imaging single capillary loops, and (2) measurements of oxy- and deoxy-Hb levels within single capillaries and single erythrocytes. In this study, we aimed to: (1) characterize capillary morphology and hemodynamic/oxygen metabolic properties in the cuticle nailbed of individuals with SCD compared to healthy controls and (2) track single erythrocytes along the capillary loop to obtain measurements of erythrocyte elongation (ellipticity index, EI) and oxygen saturation before and after tissue oxygen exchange. We hypothesized that erythrocyte EI, as an index of HbS polymerization, would be associated with decreased arteriolar oxygen saturation and/or increased oxygen extraction fraction (OEF) across capillaries—representing compromised “oxygen reserve.”

Supplemental Digital Content is available in the text., Background: Current paradigms suggest that nitric oxide (NO) produced by endothelial cells (ECs) through endothelial nitric oxide synthase (eNOS) in the vessel wall is the primary regulator of blood flow and blood pressure. However, red blood cells (RBCs) also carry a catalytically active eNOS, but its role is controversial and remains undefined. This study aimed to elucidate the functional significance of RBC eNOS compared with EC eNOS for vascular hemodynamics and nitric oxide metabolism. Methods: We generated tissue-specific loss- and gain-of-function models for eNOS by using cell-specific Cre-induced gene inactivation or reactivation. We created 2 founder lines carrying a floxed eNOS (eNOSflox/flox) for Cre-inducible knockout (KO), and gene construct with an inactivated floxed/inverted exon (eNOSinv/inv) for a Cre-inducible knock-in (KI), which respectively allow targeted deletion or reactivation of eNOS in erythroid cells (RBC eNOS KO or RBC eNOS KI mice) or in ECs (EC eNOS KO or EC eNOS KI mice). Vascular function, hemodynamics, and nitric oxide metabolism were compared ex vivo and in vivo. Results: The EC eNOS KOs exhibited significantly impaired aortic dilatory responses to acetylcholine, loss of flow-mediated dilation, and increased systolic and diastolic blood pressure. RBC eNOS KO mice showed no alterations in acetylcholine-mediated dilation or flow-mediated dilation but were hypertensive. Treatment with the nitric oxide synthase inhibitor Nγ-nitro-l-arginine methyl ester further increased blood pressure in RBC eNOS KOs, demonstrating that eNOS in both ECs and RBCs contributes to blood pressure regulation. Although both EC eNOS KOs and RBC eNOS KOs had lower plasma nitrite and nitrate concentrations, the levels of bound NO in RBCs were lower in RBC eNOS KOs than in EC eNOS KOs. Reactivation of eNOS in ECs or RBCs rescues the hypertensive phenotype of the eNOSinv/inv mice, whereas the levels of bound NO were restored only in RBC eNOS KI mice. Conclusions: These data reveal that eNOS in ECs and RBCs contribute independently to blood pressure homeostasis.

BACKGROUND: During storage, red blood cells (RBCs) undergo significant biochemical and morphologic changes, referred to collectively as the “storage lesion”. It was hypothesized that these defects may arise from disrupted oxygen-based regulation of RBC energy metabolism, with resultant depowering of intrinsic antioxidant systems. STUDY DESIGN AND METHODS: As a function of storage duration, the dynamic range in RBC metabolic response to three models of biochemical oxidant stress (methylene blue, hypoxanthine/xanthine oxidase, and diamide) was assessed, comparing glycolytic flux by NMR and UHPLC–MS methodologies. Blood was processed/stored under standard conditions (AS-1 additive solution) with leukoreduction. Over a 6-week period, RBC metabolic and antioxidant status were assessed at baseline and following exposure to the three biochemical oxidant models. Comparison was made of glycolytic flux ((1)H-NMR tracking of [2-(13)C]-glucose and metabolomic phenotyping with [1,2,3-(13)C(3)] glucose), reducing equivalent (NADPH/NADP(+)) recycling, and thiol-based (GSH/GSSG) antioxidant status. RESULTS: As a function of storage duration, we observed the following: (1) a reduction in baseline hexose monophosphate pathway (HMP) flux, the sole pathway responsible for the regeneration of the essential reducing equivalent NADPH; with (2) diminished stress-based dynamic range in both overall glycolytic as well as proportional HMP flux. In addition, progressive with storage duration, RBCs showed (3) constraint in reducing equivalent (NADPH) recycling capacity, (4) loss of thiol based (GSH) recycling capacity, and (5) dysregulation of metabolon assembly at the cytoplasmic domain of Band 3 membrane protein (cdB3). CONCLUSION: Blood storage disturbs normal RBC metabolic control, depowering antioxidant capacity and enhancing vulnerability to oxidative injury.

BACKGROUND: Characteristics of red blood cells (RBCs) are influenced by donor variability. This study assessed quality and metabolomic variables of RBC subpopulations of varied biologic age in red blood cell concentrates (RCCs) from male and female donors to evaluate their contribution to the storage lesion. STUDY DESIGN AND METHODS: Red blood cell concentrates from healthy male (n = 6) and female (n = 4) donors were Percoll separated into less dense (“young”, Y-RCCs) and dense (“old”, O-RCCs) subpopulations, which were assessed weekly for 28 days for changes in hemolysis, mean cell volume (MCV), hemoglobin concentration (MCHC), hemoglobin autofluorescence (HGB), morphology index (MI), oxygen affinity (p50), rigidity, intracellular reactive oxygen species (ROS), calcium ([Ca(2+)]), and mass spectrometry–based metabolomics. RESULTS: Young RCCs having disc-to-discoid morphology showed higher MCV and MI, but lower MCHC, HGB, and rigidity than O-RCCs, having discoid-to-spheroid shape. By Day 14, Y-RCCs retained lower hemolysis and rigidity and higher p50 compared to O-RCCs. Donor sex analyses indicated that females had higher MCV, HGB, ROS, and [Ca(2+)] and lower hemolysis than male RBCs, in addition to having a decreased rate of change in hemolysis by Day 28. Metabolic profiling indicated a significant sex-related signature across all groups with increased markers of high membrane lipid remodeling and antioxidant capacity in Y-RCCs, whereas O-RCCs had increased markers of oxidative stress and decreased coping capability. CONCLUSION: The structural, functional, and metabolic dissimilarities of Y-RCCs and O-RCCs from female and male donors demonstrate RCC heterogeneity, where RBCs from females contribute less to the storage lesion and age slower than males.

One of the challenges of quantitative Photoacoustic (PA) imaging is unmixing the optical absorption (μa) of the tissue from system response (C) and Gruneisen parameter (Γ). In this study, we have calculated the absorption coefficient and functional parameters, i.e. total hemoglobin (tHb) and oxygen saturation (sO2) of 5 blood tubes with sO2 values ranging from 24.9% to 97.6% at different depths in intralipid solution. Beer’s law is used to calculate the optical fluence in the target area. Initial values for μa and C×Γ are found by fitting a line to the log of PA beam data. These initial values are iteratively updated using a conjugate gradient method. This process is repeated for all 11 wavelengths. The absorption coefficient spectrum follows the molar extinction coefficient spectrum of deoxy hemoglobin for lower sO2 percentages, and it becomes closer to the spectrum of oxy hemoglobin when the sO2 percentage increases. The calculated absorption coefficients at 11 wavelengths are used to estimate the absolute value of the tHb and sO2 of each blood sample at different depths. The mean error of the estimated tHb values for blood tubes at all depths with respect to the real values are less than 13%. Moreover, the largest sO2 estimation error is 7.5% for the blood sample with sO2 of 24.9%. Our quantitative PA method performed well for the data collected from blood samples. We are investigating this method on our clinical data.

Background Antimicrobial Photodynamic therapy (aPDT) has demonstrated efficacy in situations where conventional antibiotic therapies can be challenged such as biofilms, gram-negative bacteria, and antimicrobial resistant organisms. Surface characteristics can affect biofilm adherence and integrity and so may modify the effectiveness of aPDT. This study investigates the killing efficacy of aPDT on S. mutans biofilms grown on relevant dental substrata, examining the killing efficacy and specifically the effects of aPDT on the biofilm matrix architecture. Materials and Methods S. mutans (NCTC 10449) was grown in 48 hours biofilms on different substrata, specifically glass, titanium, and denture acrylic. During aPDT assays, the biofilms were treated with a purpurin based sensitizer ([25 ug/ml] in DMSO) for 30 minutes, then exposed to a 664 nm diode laser at light doses of 15, 30, and 45 J/cm2. Colony forming unit assays were performed to determine survival following treatment. Controls for comparison in survival assays consisted of (No light/No PS; No light/PS; and No light/DMSO). MAIR-IR spectroscopy analysis was performed to investigate aPDT effects on biofilm composition before and after jet impingement. Results Survival was greatly reduced in the biofilm cultures following the aPDT assays. All light doses achieved a greater then 3-log inactivation on 48 hours biofilms grown on polished denture acrylic. The higher light doses (45 and 30 J) achieved greater than 3-log inactivation in 48 hours biofilms grown on glass. The higher light doses (30 and 45 J/cm2) produced a 2-log inactivation in 48 hours biofilms grown on titanium. Multiple attenuated internal reflection infrared (MAIR-IR) spectroscopy data demonstrates enhanced loss of exopolysaccharide (EPS) and Amide in the aPDT treated biofilms following jet impingement. Conclusion Antimicrobial PDT experiments using a purpurin based sensitizer and laser light doses of 15, 30, and 45 J/cm2, against S. mutans biofilm grown on different surfaces, show the effectiveness of this therapy. In CFU survival assays, a dose response to the laser is evident. While considerable disinfection was achieved on all surfaces compared to the controls, not all surfaces could be disinfected equally. MAIR-IR spectroscopy showed that aPDT groups lost more EPS and Amide versus controls, suggesting aPDT induced biofilm embrittlement, which was revealed by jet impingement. With demonstrated efficacy against various microbes and on different substrata, antimicrobial aPDT shows potential for clinical application in biofilm-mediated diseases such as peri-implantitis and periodontitis. Lasers Surg. Med. © 2016 Wiley Periodicals, Inc.

Background Dental implants are commonly used today for the treatment of partially and fully edentulous patients. Despite the high success rate they are not resistant to complications and failure due to a variety of problems including peri-implantitis or peri-mucositis due to bacterial biofilm formation on the implant surface. The use of non-surgical and surgical treatment procedure to promote healing in cases with peri-implantitis have limited efficacy. Here we studied the ability of photodynamic therapy to destroy a known bacterial pathogen and the extracellular matrix architecture of biofilm attached to titanium plates and germanium prisms. Methods Titanium plates or germanium prisms were incubated for 24 h with Fusobacterium nucleatum a fusiform, gram-negative bacterium was used to enable biofilm formation. Photodynamic therapy was carried out by incubating the biofilm samples on each substrata with porfimer sodium. Treatment was carried out using a diode laser at 630 nm, 150 mW/cm 2 for light doses ranging from 25–100 J/cm 2 . Evaluation of killing efficacy was done by counting colony forming units compared to controls. Multiple attenuated internal reflection-infrared spectroscopy (MAIR-IR) and SEM were used to analyze the samples pre and post PDT for validation. Results F. nucleatum was significantly reduced in a dose dependent manner by treatment with PDT. Changes in biofilm components and strength of bioadhesion were examined with MAIR-IR following jet impingement using calibrated water jets. SEM demonstrates significant morphological alterations in the bacteria, consistent with damage associated with exposure to reactive oxygen species. Conclusion The results are indicative that aPDT is a method that can be used to eradicate micro-organisms associated with biofilm in peri-implantitis on relevant substrata. Data shows that the slime layer of the biofilm is removed and that further methods need to be employed to completely remove weakened or destroyed biofilm matrix components. Reactive oxygen species (ROS) mediated oxidative damage results in morphologic changes as a consequence of changes in cell membrane integrity.

BACKGROUND: We set out to define the impact of collection, processing, and storage on plasma product microparticle (MP) abundance, potential for nitric oxide (NO) scavenging, and vasoactivity. STUDY DESIGN AND METHODS: Three currently US licensed products were tested: liquid plasma (LP), fresh frozen plasma (FFP), and solvent detergent plasma (SDP), along with a product under development, spray-dried solvent detergent plasma (SD-SDP) with/without beads. Vasoactivity was assessed in vitro using rabbit aortic vascular rings; MP abundance was determined by flow cytometry; and NO scavenging capacity/rate was determined using a biochemical NO consumption assay. All samples were analyzed unprocessed and following centrifugation at two speeds (2,500× g to remove platelets, and 25,000× g to remove microparticles). RESULTS: Significant differences in vasoactivity were observed, with SD-SDP minus beads demonstrating the greatest constriction and FFP the lowest constriction response. All products exhibited the same total NO scavenging capacity; however, significant differences were observed in the maximal rate of scavenging, with SD-SDP minus beads and FFP reacting fastest and SDP the slowest. Across all products, platelet and microparticle depletion had no effect on vasoactivity or NO scavenging (total or rate). Microparticles (RBC derived) were found only in FFP and LP, with relative abundance (LP > FFP). Additionally, storage had no effect on total or RBC-derived MP abundance, NO scavenging, or vasoactivity. CONCLUSION: Although vasoactivity differed between plasma products, we did not find similar differences in either total or RBC-derived MP abundance or NO scavenging capacity/rate.

Oxygen (O2) delivery, the maintenance of which is fundamental to supporting those with critical illness, is a function of blood O2 content and flow. Here, we review red blood cell (RBC) physiology relevant to disordered O2 delivery in the critically ill.Flow (rather than content) is the focus of O2 delivery regulation. O2 content is relatively fixed, whereas flow fluctuates by several orders of magnitude. Thus, blood flow volume and distribution vary to maintain coupling between O2 delivery and demand. The trapping, processing and delivery of nitric oxide (NO) by RBCs has emerged as a conserved mechanism through which regional blood flow is linked to biochemical cues of perfusion sufficiency. We will review conventional RBC physiology that influences O2 delivery (O2 affinityrheology) and introduce a new paradigm for O2 delivery homeostasis based on coordinated gas transport and vascular signaling by RBCs.By coordinating vascular signaling in a fashion that links O2 and NO flux, RBCs couple vessel caliber (and thus blood flow) to O2 need in tissue. Malfunction of this signaling system is implicated in a wide array of pathophysiologies and may be explanatory for the dysoxia frequently encountered in the critical care setting.

This chapter summarizes the principles of RSNO measurement in the gas phase, utilizing ozone-based chemiluminescence and the copper cysteine (2C) ± carbon monoxide (3C) reagent. Although an indirect method for quantifying RSNOs, this assay represents one of the most robust methodologies available. It exploits the NO• detection sensitivity of ozone based chemiluminscence, which is within the range required to detect physiological concentrations of RSNO metabolites. Additionally, the specificity of the copper cysteine (2C and 3C) reagent for RSNOs negates the need for sample pretreatment, thereby minimizing the likelihood of sample contamination (false positive results), NO species inter-conversion, or the loss of certain highly labile RSNO species. Herein, we outline the principles of this methodology, summarizing key issues, potential pitfalls and corresponding solutions.

Energy metabolism in RBCs is characterized by O2-responsive variations in flux through the Embden Meyerhof pathway (EMP) or the hexose monophosphate pathway (HMP). Therefore, the generation of ATP, NADH, and 2,3-DPG (EMP) or NADPH (HMP) shift with RBC O2 content because of competition between deoxyhemoglobin and key EMP enzymes for binding to the cytoplasmic domain of the Band 3 membrane protein (cdB3). Enzyme inactivation by cdB3 sequestration in oxygenated RBCs favors HMP flux and NADPH generation (maximizing glutathione-based antioxidant systems). We tested the hypothesis that sickle hemoglobin disrupts cdB3-based regulatory protein complex assembly, creating vulnerability to oxidative stress. In RBCs from patients with sickle cell anemia, we demonstrate in the present study constrained HMP flux, NADPH, and glutathione recycling and reduced resilience to oxidative stress manifested by membrane protein oxidation and membrane fragility. Using a novel, inverted membrane-on-bead model, we illustrate abnormal (O2-dependent) association of sickle hemoglobin to RBC membrane that interferes with sequestration/inactivation of the EMP enzyme GAPDH. This finding was confirmed by immunofluorescent imaging during RBC O2 loading/unloading. Moreover, selective inhibition of inappropriately dispersed GAPDH rescues antioxidant capacity. Such disturbance of cdB3-based linkage between O2 gradients and RBC metabolism suggests a novel mechanism by which hypoxia may influence the sickle cell anemia phenotype.

The ability of hemoglobin to scavenge the potent vasodilator nitric oxide (NO) in the blood has been well established as a mechanism of vascular tone homeostasis. In endothelial cells, the alpha chain of hemoglobin (hereafter, alpha globin) and endothelial NO synthase form a macromolecular complex, providing a sink for NO directly adjacent to the production source. We have developed an alpha globin mimetic peptide (named HbαX) that displaces endogenous alpha globin and increases bioavailable NO for vasodilation. Here we show that, in vivo, HbαX administration increases capillary oxygenation and blood flow in arterioles acutely and produces a sustained decrease in systolic blood pressure in normal and angiotensin II–induced hypertensive states. HbαX acts with high specificity and affinity to endothelial NO synthase, without toxicity to liver and kidney and no effect on p50 of O 2 binding in red blood cells. In human vasculature, HbαX blunts vasoconstrictive response to cumulative doses of phenylephrine, a potent constricting agent. By binding to endothelial NO synthase and displacing endogenous alpha globin, HbαX modulates important metrics of vascular function, increasing vasodilation and flow in the resistance vasculature.

The major problem in building QSAR/QSPR models for mixtures lies in their characterization. It has been shown that it is possible to construct QSPR models for the density of binary liquid mixtures using simple mole fraction weighted physicochemical descriptors. Such parameters are unsatisfactory; however, from the point of view of interpretation of the resultant models. In this paper, an alternative mechanism-based approach to the characterization of mixtures has been investigated. It has been shown that while it is not possible to build significant linear models using these descriptors, it has been possible to construct satisfactory artificial neural network models. The performance of these models and the importance of the individual descriptors are discussed.

Objectives The theory of a red blood cell derived nitric oxide (NO) reserve conserving NO bioactivity and delivering NO as a function of oxygen demand has been the subject of much interest. We identified the human coronary circulation as an ideal model system in which to analyse NO metabolites because of its large physiological oxygen gradient. Our objective was to identify whether oxygen drove apportion between various NO metabolite species across a single vascular bed. Methods Plasma and red blood cell NO metabolites were assessed from the left main coronary artery, coronary sinus and pulmonary artery (providing cross heart and cross pulmonary analysis) of healthy subjects under resting conditions and following administration of an inhibitor of NO biosynthesis. Physiological parameters and angiographic data were monitored throughout the study. Results Under baseline conditions we observed significant metabolite flux upon the transit of blood across the coronary and pulmonary vascular beds. Whilst there was no net loss of NO through the coronary circulation ( p =0.0759), plasma nitrite/protein NO (excluding nitrate) ( p =0.0279) and red blood cell sulphanilamide labile signal ( p =0.0143) decreased whereas haemoglobin-bound NO increased three-fold ( p =0.005). These changes across the coronary circulation were reversed through the pulmonary circuit with red blood cell sulphanilamide labile signal ( p =0.0143) and plasma nitrite/protein NO ( p =0.0279) increasing and haemoglobin-bound NO decreasing. Blockade of NO synthesis increased mean arterial blood pressure ( p

Major disparities in reported levels of basal human nitric oxide metabolites have resulted in a recent literature focusing almost exclusively on methods. We chose to analyze triiodide chemiluminescence, drawn by the prospect of identifying why the most commonly employed assay in nitric oxide biology typically yielded lower metabolite values, compared with several other techniques. We found that the sensitivity of triiodide was greatly affected by the auto-capture of nitric oxide by deoxygenated cell-free heme in the reaction chamber. Potential contaminants and signal losses were also associated with standard sample purification procedures and the chemistry involved in nitrite removal. To inhibit heme nitric oxide auto-capture, we added potassium ferricyanide to the triiodide reagent, reasoning this would provide a more complete detection of any liberated nitric oxide. From human venous blood samples, we established nitric oxide levels ranging from 0.000178 to 0.00024 mol nitric oxide/mol hemoglobin. We went on to find significantly elevated nitric oxide levels in venous blood taken from diabetic patients in comparison to healthy controls (p < 0.0001). We concluded that the lack of signals reported of late by several groups using triiodide chemiluminescence for the detection of hemoglobin-bound nitric oxide may not represent levels on the border of assay sensitivity but rather underestimated values because of methodological limitations. We therefore stress the need for assay systems to be developed that differentiate between individual nitric oxide metabolite species and overcome the limitations we outline, allowing accurate conclusions to be drawn regarding physiological nitric oxide metabolite levels.

We describe the work of the European project QUASI (Quantum Simulation in Industry, project EP25047) which has sought to develop a flexible QM/MM scheme and to apply it to a range of industrial problems. A number of QM/MM approaches were implemented within the computational chemistry scripting system, ChemShell, which provides the framework for deploying a variety of independent program packages. This software was applied in several large-scale QM/MM studies which addressed the catalytic decomposition of N 2 O by Cu-containing zeolites, the methanol synthesis reaction catalysed by Cu clusters supported on ZnO surfaces, and the modelling of enzyme structure and reactivity.

Hydrogenated zinc oxide gives rise to complex vibrational spectra with many prominent features that remain unexplained. Our calculations have unambiguously shown that the presence of vacant oxygen and zinc interstitial surface sites is the only way to rationalize the observed spectra, notably the 1710 cm−1 zinc hydride stretching mode. The large number of such sites, which expose low-coordinated surface ions, are inherent at ionically reconstructed polar surfaces. The thermal stability of the sorbed hydrogen and the infrared activity of the resulting species are correlated with site coordination and coverage.

BACKGROUND: There is need for an artificial oxygen (O2) carrier for use when: stored blood is unavailable or undesirable. To date, efforts to develop hemoglobin (Hb) based oxygen carriers (HBOCs) have failed, because of design flaws which do not preserve physiologic interactions of Hb with: O2 (they capture O2 in lungs, but do not release O2 effectively to tissue) and nitric oxide (NO) (they trap NO, causing vasoconstriction). EM design surmounts these weaknesses by: encapsulating Hb, controlling O2 capture/release with a novel 2,3-DPG shuttle and attenuating NO uptake through shell properties. METHODS: The EM prototype and its lyophilized form were analyzed: (1) structurally (dynamic light scattering (DLS), transmission electron microscopy (TEM) and atomic force microscopy (AFM)), as well as for: (2) payload retention (Drabkin), (3) biocompatibility (ex vivo complement activation), (4) O2 affinity (p50, Hill n, Adair), (5) rheology (cone and plate viscometer in rabbit plasma), (6) NO consumption (chemiluminescence), (7) pharmacokinetic (PK) profile (tracking 99mTc-labeled EM in rats), and (8) in vivo O2 delivery (two rodent models: hemorrhagic shock [rats, instrumented for tissue pO2] and hemodilution [bioluminescent HIF-1α reporter mice]). RESULTS: EM was structurally stable (size: 175±10 nm; polydispersity: 0.26±0.0 by DLS, confirmed by TEM and AFM; zeta potential: 12±2 mV). After 3 months storage, we observed nominal change (0.96); which is likely to translate to a t1/2 in humans of ~ 3h. EM NO sequestration varied as a function of shell crosslinking and was below the rate observed for RBCs. In our hemorrhagic shock model in fully instrumented SD Rats (400g), 40% blood volume was removed; animals were then resuscitated with an equal volume of EM (N=6) or normal saline (N=6). EM was suspended at 40 wt/vol%, [Hb]=4mM. EM infusion rapidly stabilized hemodynamics. During the 1st hour, we observed resolution of both lactic acidosis (3.2±1.5 v 8.2±2.1 mM) [for EM and NS, respectively, throughout] and elevated AV O2 difference (24±11 v 67±23%) as well as improved brain pO2 (30.5±1.4 v 17.2±1.3 Torr); p CONCLUSIONS: The ErythroMer prototype has passed rigorous initial ex vivo and in vivo "proof of concept" testing and bench testing, which suggests this design surmounts prior challenges (by HBOCs) in emulating normal RBC physiologic interactions with O2 and NO. In models of major bleeding/anemia, EM reconstitutes normal hemodynamics and O2 delivery, observed at the system, tissue, and cellular level. EM potential for extended ambient dry storage has significant implications for portability and use. Next steps include formulation scaling, detailed study of pharmacokinetics, biodistribution and safety, as well as evaluation in large animal models of hemorrhagic shock. Disclosures Pan: KaloCyte, Inc.: Equity Ownership; Children's Discovery Institute: Research Funding; National Institutes of Health: Research Funding. Spinella:KaloCyte, Inc.: Equity Ownership; Children's Discovery Institute: Research Funding; National Institutes of Health: Research Funding. Hare:Children's Discovery Institute: Research Funding. Lanza:KaloCyte, Inc.: Membership on an entity's Board of Directors or advisory committees; National Institutes of Health: Research Funding. Doctor:KaloCyte, Inc.: Equity Ownership; Children's Discovery Institute: Research Funding; National Institutes of Health: Research Funding.

Ab initio molecular orbital calculations have been performed on the transition state of the addition of methyl radical to twelve vinyl monomers using the SV 3-21G basis set. A linear relationship has been found between the calculated energies of activation and previously calculated energies of reaction. This supports the assumptions of an Evans-Polanyi type rule in previous work which attempted to correlate reactivity with calculated energies of reaction. The activation energies obtained for methyl addition to butadiene and styrene were calculated to be negative. This is caused by errors introduced by a number of sources, viz. basis set superposition error, spin contamination and zero point energy. These errors are discussed. Previous authors have reported reasonable agreement between calculated activation energies at SV3-21G and experimental values for methyl addition to ethylene, this work suggests that this agreement was coincidental and results from the fortuitos cancellation of errors. The nature of the transition state for these radical addition reactions is discussed and the limitations of the SV3-21G basis set are highlighted. The theoretical prediction of activation energies for radical addition reactions would require much larger calculations, beyond the computational means of most research laboratories.

BACKGROUND: : In response to high rates of teen motor vehicle crashes (MVCs) many states have enacted graduated driver licensing (GDL) systems. GDL delays full licensure and allows beginners to obtain experience under lower risk conditions. The purpose of this study is to evaluate the impact over the past 10 years to determine its effect on teen MVCs. METHODS: : Connecticut MVC data from 1999 to 2008 were analyzed. Percent change (1999 vs. 2008) in MVC rates per 10,000 registered drivers was calculated by age, gender, during the night restriction (11:00 pm and 5:00 am), and MVCs with passengers. Linear regression analysis estimated the decrease of MVC rates each year. RESULTS: : The MVC rate decreased by 40% for 16-year-old and 30% for 17-year-old drivers. In comparison, rates among 18-year-old, 19-year-old, 25- to 29-year-old, and 30- to 59-year-old drivers were reduced by 16%, 7%, 8%, and 11%, respectively. The MVC rate for 20- to 24-year-old drivers increased by 1%. During nighttime restricted driving times, MVC rates decreased by 54% among 16-year-old and 49% among 17-year-old drivers. The MVC rate with passengers decreased by 65% for 16-year-old and 53% for 17-year-old drivers. In comparison, rates of nighttime and with passenger MVCs among older drivers were significantly less. CONCLUSIONS: : Implementation of Connecticut's GDL system has resulted in significant reductions in MVC rates among novice drivers. This analysis provides a method for other states to examine the impact of their GDL system. Language: en

Thienopyridines (ticlopidine, clopidogrel and prasugrel) are pro-drugs that require metabolism to exhibit a critical thiol group in the active form that binds to the P2Y₁₂ receptor to inhibit platelet activation and prevent thrombus formation in vivo. We investigated whether these thienopyridines participate in S-nitrosation (SNO) reactions that might exhibit direct anti-platelet behaviour. Optimum conditions for in vitro formation of thienopyridine-SNO formation were studied by crushing ticlopidine, clopidogrel or prasugrel into aqueous solution and adding sodium nitrite, or albumin-SNO. Ozone-based chemiluminescence techniques were utilised to specifically detect NO release from the SNO produced. Effect on agonist-induced platelet aggregation was monitored using light transmittance in a 96 well microplate assay. Pharmaceutical grade preparations of ticlopidine, clopidogrel and prasugrel were found to exhibit significant free thiol and formed SNO derivatives directly from anionic nitrite in water under laboratory conditions without the need for prior metabolism. Thienopyridine-SNO formation was dependent on pH, duration of mixing and nitrite concentration, with prasugrel-SNO being more favourably formed. The SNO moiety readily participated in trans-nitrosation reactions with albumin and plasma. Prasugrel-SNO showed significantly better inhibition of platelet aggregation compared with clopidogrel-SNO, however when compared on the basis of SNO concentration these were equally effective (IC₅₀=7.91 ± 1.03 v/s 10.56 ± 1.43 μM, ns). Thienopyridine-derived SNO is formed directly from the respective base drug without the need for prior in vivo metabolism and therefore may be an important additional contributor to the pharmacological effectiveness of thienopyridines not previously considered.

Molecular dynamics calculations are reported on the sorption and diffusion of small gas molecules (He, H2, and O2) in amorphous polyisobutylene. An all‐atom force field (with explicit hydrogen atoms) was used. In addition to the standard method of obtaining the diffusion coefficient from an equilibrium calculation via the mean‐square displacement we used a nonequilibrium technique that applies a fictitious external field selectively to the gas molecules. To our knowledge, nonequilibrium MD is, for the first time, applied to the problem of gas diffusion in polymers. Results of both techniques are compared. Energy profiles of the jump events underlying gas diffusion through polymers are studied. We also discuss the possible presence of anomalous (non‐Einstein) behavior of gas molecules diffusing through an amorphous polymer. Gas solubilities in polyisobutylene are calculated by particle‐insertion techniques.

In our earlier work, we have demonstrated that it is possible to characterize binary mixtures using single component descriptors by applying various mixing rules. We also showed that these methods were successful in building predictive QSPR models to study various mixture properties of interest. Here in, we developed a QSPR model of an excess thermodynamic property of binary mixtures i.e. excess molar volume (V(E) ). In the present study, we use a set of mixture descriptors which we earlier designed to specifically account for intermolecular interactions between the components of a mixture and applied successfully to the prediction of infinite-dilution activity coefficients using neural networks (part 1 of this series). We obtain a significant QSPR model for the prediction of excess molar volume (V(E) ) using consensus neural networks and five mixture descriptors. We find that hydrogen bond and thermodynamic descriptors are the most important in determining excess molar volume (V(E) ), which is in line with the theory of intermolecular forces governing excess mixture properties. The results also suggest that the mixture descriptors utilized herein may be sufficient to model a wide variety of properties of binary and possibly even more complex mixtures.

Results of molecular dynamics simulations of oxygen molecules and helium atoms in amorphous polyisobutylene are presented. The systems are allowed to evolve for several nanoseconds. In these rather long simulations it is found that the diffusion of He and O2 through the polymer matrix exhibits anomalous (non-Einstein) behaviour for time scales of at least 0.1 and 6 nanoseconds for He and O2, respectively. This contrasts with previous much shorter molecular dynamics studies of gas molecules in polymers which found that Einstein diffusion sets in after a few picoseconds.

This study uses an organ chamber bioactivity assay to characterise the direct effect of sodium nitrite upon rabbit blood vessels (aorta (Ao), inferior vena cava (IVC) and pulmonary artery (PA)) in a haemoglobin independent/variable oxygen environment. In 95% oxygen constriction to 8g (Ao), 6g (PA) and 4g (IVC) was achieved using 1 microM phenylephrine. The same constriction in 1% oxygen required 3 microM. During 95% oxygen constriction was consistent and sustained for all vessels. However under 1% oxygen PA was quick to constrict but rapidly gave up this tension whereas Ao was slower to constrict but exhibited a more sustained response. Relaxation of each vessel was assessed post constriction using 10 microM sodium nitrite. Results were expressed as a percentage loss in tension compared to the maximum achieved and corrected by controls which received no nitrite. At 95% oxygen PA relaxed greater than Ao (10.04% +/- 2.28% vs. 5.25% +/- 1.51%). IVC response was varied (2.26% +/- 9.43%). At 1% oxygen all vessels relaxed more. However the pattern was reversed with both IVC (14.20% +/- 3.63%) and PA (16.55% +/- 0.93%) relaxing less than Ao (42.20% +/- 5.21%). These results suggest that relatively low concentrations of sodium nitrite can vasodilate blood vessels. This effect is independent of haemoglobin and tissue specific.

The vasorelaxant properties of red blood cells (RBCs) have been implicated in both the control of normal vascular tone and the protection of tissues from ischemic events. The identity of the vasorelaxant released from RBCs has yet to be elucidated, however growing evidence suggests that nitric oxide bound to the beta 93 cysteine residue of haemoglobin (SNO-Hb) may be responsible. The vasorelaxant moiety is released during the transition of haemoglobin from its R (oxygenated) to T (deoxygenated) state. We subsequently chose to assess the significance of haemoglobin saturation on the capacity of RBCs to mediate hypoxic vasorelaxation. Human RBC samples suspended in saline were manipulated in a thin film rotating tonometer, designed to rapidly change haemoglobin saturation within the time frame of circulatory transit. Various cycles of oxygenation and deoxygenation were performed. The vasorelaxant properties of the RBCs were analysed using an aortic ring bioactivity assay, wherein changes in isometric tension were recorded to study vessel relaxation. The rabbit aortic rings were preconstricted with phenylephrine under hypoxic conditions (approximately 1% O2) prior to RBC addition. Highly saturated RBCs (98.22% +/- 0.45 HbO2) elicited significantly (P0.001) more relaxation of hypoxic blood vessels compared to those partially saturated (20.40% +/- 5.28 HbO2). Upon re-oxygenation, previously de-oxygenated RBCs were also capable of eliciting vessel relaxation, which was not significantly different from that observed with the original oxygenated RBC relaxation response. Interestingly, the relaxant capability was not simply returned from extracellular milieu upon re-oxygenation. This data provides further evidence that the conformational switch of haemoglobin from the R-state (oxygenated) to the T-state (deoxygenated) is essential for the release of the vasoactive moiety contained within red blood cells.

A plethora of publications on techniques and methodologies for measuring nitric oxide (NO) or reaction products of NO (NO metabolites) has served in recent years to complicate and confuse the majority of researchers interested in this field. Here, we provide a practical approach and summarize the key issues and corresponding solutions regarding quantification with the use of ozone-based chemiluminescence, which is the most accurate, sensitive, and widely used NO detection method. We have drawn on the vast experience of leaders in the field to produce this consensus, but the views and implications presented herein represent our own, and we limit our advice to those techniques with which we have direct experience. Hopefully, this guide will allow authors to make more informed decisions regarding NO metabolite measurement methodology, without the need for each subsequent group to rediscover previously observed advantages and pitfalls.

This study uses an organ chamber bioactivity assay to characterise the direct effect of sodium nitrite upon rabbit blood vessels (aorta (Ao), inferior vena cava (IVC) and pulmonary artery (PA)) in a haemoglobin independent/variable oxygen environment. In 95% oxygen constriction to 8g (Ao), 6g (PA) and 4g (IVC) was achieved using 1μM phenylephrine. The same constriction in 1% oxygen required 3μM. During 95% oxygen constriction was consistent and sustained for all vessels. However under 1% oxygen PA was quick to constrict but rapidly gave up this tension whereas Ao was slower to constrict but exhibited a more sustained response.

The vasorelaxant properties of red blood cells (RBCs) have been implicated in both the control of normal vascular tone and the protection of tissues from ischemic events. The identity of the vasorelaxant released from RBCs has yet to be elucidated, however growing evidence suggests that nitric oxide bound to the s93 cysteine residue of haemoglobin (SNO-Hb) may be responsible. The vasorelaxant moiety is released during the transition of haemoglobin from its R (oxygenated) to T (deoxygenated) state. We subsequently chose to assess the significance of haemoglobin saturation on the capacity of RBCs to mediate hypoxic vasorelaxation.

A plethora of publications on techniques and methodologies for measuring nitric oxide (NO) or reaction products of NO (NO metabolites) has served in recent years to complicate and confuse the majority of researchers interested in this field. Here, we provide a practical approach and summarize the key issues and corresponding solutions regarding quantification with the use of ozone-based chemiluminescence, which is the most accurate, sensitive, and widely used NO detection method. We have drawn on the vast experience of leaders in the field to produce this consensus, but the views and implications presented herein represent our own, and we limit our advice to those techniques with which we have direct experience. Hopefully, this guide will allow authors to make more informed decisions regarding NO metabolite measurement methodology, without the need for each subsequent group to rediscover previously observed advantages and pitfalls.

The concept and practice of using information as a tool in military operations to support political objectives has gained increased notoriety and emphasis within U.S. political and military arenas over the course of the last decade. Two particular publications of 2001, the Quadrennial Defense Review Report (QDR) and the United States Army Field Manual 3-0, Operations, highlight this fundamental shift in the growing importance of using information in warfare. Of particular importance, the 2001 QDR mandated that information operations be treated not merely as an enabling function, but as a core capability of future forces. Recent military operations conducted during Operation Iraqi Freedom (OIF), however, indicate that the application of this newly christened element of combat power has fallen well short of desired effectiveness. Without comprehensive changes in training, equipment distribution and personnel management, the Army will continue to struggle to employ information operations and fail to achieve the directives of the 2001 QDR. Fortunately, the Army has begun several studies in an effort to make the necessary changes to improve information operations. Yet one model that the Army has not yet studied is that of international marketing and advertising agencies. These firms have developed effective organizational structures, techniques, and procedures to share their ideas with people across a vast array of cultures, all with the intent of inducing a predetermined action. Gleaning the pertinent lessons from the international marketing model can help the Army empower tactical and operational commanders with the tools necessary to better understand the culture of a country, region, or area of operations. With this knowledge, these commanders could greatly improve the effectives of their information operations.

EPR spectroscopic techniques have been developed for the measurement of oxygen and nitric oxide in vivo. Specifically, the methods for in vivo measurement of these molecules has been applied to the study of septic shock, utilising an experimental murine model developed in our laboratory. Oxygen was measured as pO2 by the particlulate probes Gloxy and LiPc, which were surgically implanted at specific sites in tissues, and the soluble probe Trityl, which was administered intravenously. Nitric oxide was measured as the NO-Fe-(DETC)2 complex after administration of Fe2+ and DETC. LPS was seen to significantly decrease liver oxygen measured across the lobule and at the sinusoids by the Gloxy probe; there was a corresponding increase in nitric oxide both in the liver and systemically. The nitric oxide most likely originated from increased iNOS enzyme in the liver as demonstrated by Western blotting and the localisation of nitric oxide to the liver was confirmed with EPR imaging. LPS also caused a decrease in cerebral blood and tissue oxygenation, the rate of which was found to be dependent on the blood oxygenation. The development and applications of these in vivo EPR techniques for biomedical research and diagnostics is discussed.

The recent interest in the catalytic properties of lanthanum perovskites for methane combustion and three way catalysis has led to considerable debate as to their structure and defect chemistry. We have investigated the doping of LaCoO3 with the tetravalent cerium cation using atomistic simulation techniques. We have compared three routes for cerium insertion and identified the favoured doping mechanism, which explain experimental observations relating to the effect of cerium on catalytic activity.

A new solid-state embedding approach has been developed which focuses on modelling the surfaces of polar materials. The method is applied to investigate the chemisorption of pre- methanol species on the polar (000-1) surface of zincite (a major phase of zinc oxide having the wurtzite structure). Initial results include the geometries of active sites and adsorbates in different charge states.