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Human protein C (PC) is a natural anticoagulant, antithrombotic, anti-inflammatory, and anti-apoptotic in the bloodstream. PC deficiency can lead to abnormal blood clot formation inside blood vessels, possibly causing heart attack, stroke, skin necrosis, or even death. PC can be, therefore, a valuable therapeutic with little side effect, unlike the currently used anti-coagulants. To reduce the cost involved in immuno purification of PC from blood plasma, single chain variable fragments (mini-Mab) are being produced by recombinantE. coli using phagemid technique. As an economic means of purifying the PC specific mini-Mab, metal affinity chromatography (IMAC) purification process was also investigated. Then using the purified mini-Mab, the feasibility of PC purification from the Cohn Fraction IV-1 was examined. Cohn Fraction IV-1 is usually a discarded side-stream from the blood plasma fractionation of human serum albumin. It holds 90% of PC in plasma, but is very cheap. Preliminary study of PC purification from the Cohn Fraction IV-1 showed 16% purification yield using mini-Mab immobilized NHS-activated Sepharose. The economic analysis for PC purification using mini-Mab showed that the overall process was found to be tens of times cheaper than that using Mab. [ABSTRACT FROM AUTHOR]

Cohn Fraction IV-1 (CFIV-1) is a by-product (often discarded) of a plasma fractionation process. It retains 90% of Protein C (PC) of plasma but contains several coagulants structurally homologous to PC. Of these coagulants, Factor II (FII) has the longest half-life (12 times of PC) and largest quantity (9 times of PC) in CFIV-1. Current purification process for PC is by immunoaffinity chromatography using monoclonal antibodies, which is very expensive. Immobilized metal affinity chromatography (IMAC) is an inexpensive process that uses metal ions to adsorb proteins via their surface histidines. Affinity of PC to the metal ions in IMAC is higher than that of FII because PC has 15 surface histidines and FII has 5. Two important factors in an IMAC process are pH and imidazole concentration. PH controls protonation of histidine, and imidazole, a histidine analog, competitively reacts with metal ions. The effects of pH and imidazole on adsorption and elution of PC and FII during IMAC process were studied. The effect of pH on PC and FII adsorption was similar within the range of 6.0 and 8.0. At concentrations below 15 mM imidazole, little PC or FII eluted. At 15 and 20 mM imidazole 2.5% of PC was eluted, while 20-30% of FII was eluted. [ABSTRACT FROM AUTHOR]

Low heat (42∼ 45oC) hyperthermia is an effective cancer therapy with few side effects. Well engineered nanoparticles can effectively guide heat to the tumor without damaging the normal tissue. When nano-sized particles are injected to an organ with a tumor, they tend to accumulate in the tumor due to the unorganized nature of its vasculature. Magnetic nanoparticles, such as Fe3O4, are heated by a well selected alternating electromagnetic frequency. At a frequency of 450 KHz or lower, Fe3O4 nanoparticles at a size of 10∼ 30 nm were heated effectively, without heating tissue main components. Gold nanoparticles are known as strong near infrared (NIR) absorbers. Nanogold particles at a diameter of 150 nm were added at 0.01wt% to the tumor model, placed at depths of 1∼ 2.5 cm, in an optically equivalent experimental breast model. Then the surface of the breast model was scanned with NIR light at 788 nm. The particles in the tumor model increased the optical contrast of the tumor by 1∼ 3.5dB. Considering that some of the FDA approved MRI contrast agents are made of Fe3O4, gold-coated Fe3O4 particles have a potential to be used as safe optical and thermal markers, allowing seamless breast cancer detection and cancer-specific hyperthermic treatment. [ABSTRACT FROM AUTHOR]

Cardiovascular disease (CVD) is the leading cause of death in US. Early and accurate diagnosis of CVD is crucial to save many lives, especially for the patients suffering the heart attack. Accurate and fast quantification of cardiac muscle specific biomarkers in the blood enables accurate diagnosis and prognosis and timely treatment of the patients. A prototype of fiber-optic, multi-analyte, immuno-biosensing system integrated with an automatic flow control unit has been in development to quantify four important cardiac markers in blood plasma accurately, rapidly and simultaneously. The validity of the sensor was, however, challenged because the concentrations of two markers are only at tens of picomolar level. Here, plasmon rich nano-metallic particles and selected biocompatible solvents were developed for fluorescence enhancement to improve the sensitivity of our fluorophore mediated biosensing. By applying the nano-metal particle and the solvent, the sensitivity of single cardiac marker sensors were increased by 1.5 ∼ 3 times. By using the fluorescence enhancing nano-metallic particle reagents, simultaneous quantification of four cardiac markers in plasma is currently possible, using 3-cm-sensor within 10 minutes at an average signal-to-noise ratio of 20. [ABSTRACT FROM AUTHOR]

A single compartment model of the arterial circulation was used to generate an arterial blood pressure waveform from pre-determined stroke volume (SV) and arterial resistance (R). With fixed stroke volume and varying resistances blood pressure waveforms showed mean values proportional to resistance but amplitude lessening with higher pressure; the amplitude of the hypothetical volume waveform of the arterial system was the same for all resistance values. Where SV varied and R changed reciprocally, the waveform when analysed with the PulseCO™ algorithm gave estimates slightly higher than the input stroke volumes (r 0.9998; y=0.99x + 5.28 ml). Where SV varied with fixed R mean blood pressure varied with stroke volume; SV estimates were, again, slightly higher than the input stroke volumes (r 0.9994; y=0.986x + ml). Estimates of SV andRfromValsalva manoeuvre BP were used in the model to generate arterial blood pressure. SV estimates closely resembled the original model values (r 0.988; y = 1.0802x − 3.9251). The model appears capable of generating BP waveforms compatible with real BP waveforms since stroke volume estimates closely resemble the original stroke volumes used in the model. [ABSTRACT FROM AUTHOR]

A new Oxyphor (Oxyphor G3) has been used to selectively determine the oxygen pressure in interstitial (pericellular) spaces. Oxyphor G3 is a Pd-tetrabenzoporphyrin, encapsulated inside generation 2 poly-arylglycine (AG) dendrimer, and therefore is a true near infrared oxygen sensor, having a strong absorption band at 636nm and emission near 800nm. The periphery of the dendrimer is modified with oligoethylene glycol residues (Av. MW 350) to make the probe water soluble and biologically inert. Oxyphor G3 was injected along 'tracks' in the tissue using a small needle (30gage or less) and remained in the pericellular space, allowing oxygen measurements for several hours with a single injection. The oxygen pressure distributions (histograms) were compared with those for Oxyphor G2 in the intravascular (blood plasma) space. In normal muscle, in the lower oxygen pressure region of the histograms (capillary bed) the oxygen pressure difference was small. At higher oxygen pressures in the histograms there were differences consistent with the presence of high flow vessels with oxygen pressures substantially above those of the surrounding interstitial space. In tumors, the oxygen pressures in the two spaces were similar but with large differences among tumors. In mice, anesthesia with ketamine plus xylazine markedly decreased oxygen pressures in the interstitial and intravascular spaces compared to awake or isoflurane anesthetized mice. [ABSTRACT FROM AUTHOR]

Near-infrared spectroscopy (NIRS) has been used to measure changes in cerebral oxy- and deoxy- haemoglobin (δ[HbO2], δ[HHb]) in response to functional activation. It has been previously reported that during functional activation of the motor cortex heart rate increases. The aim of this study was to investigate systemic changes during functional activation of the frontal cortex. The responses to anagram presentations with varying difficulty (4-Letters and 7-Letters) over a 6 minute period were recorded. A Hamamatsu NIRO 200 NIRS system recorded δ[HbO2] and δ[HHb] using the modified Beer Lambert law (MBL) and tissue oxygenation index (TOI) employing spatial resolved spectroscopy (SRS) over the left and right frontal hemisphere. Mean blood pressure (MBP) and heart rate (HR) were measured continuously. Nine young healthy volunteers (mean age 23) were included in the analysis. Significant task related changes were observed in both the NIRS and systemic signals during the anagram solving with increases in [HbO2] and [HHb] accompanied by changes in MBP and HR. The [HbO2] and [HHb] signals measured over the frontal region were found to have a varying association with the MBP signal across different volunteers. The effect of these systemic changes on measured NIRS signals must be considered [ABSTRACT FROM AUTHOR]

Recent advances in near-infrared spectroscopy (NIRS) allow measurements of absolute tissue oxygen saturation (TOI) using spatially resolved spectroscopy (SRS), while enabling better depth sensitivity. However concerns remain regarding the relative contribution of the extracranial circulation to the cerebral NIRS TOI signal. In this study we investigated this during a period of selective rise in cerebral blood flow (CBF) produced by the administration of acetazolamide (ACZ) in 10 healthy volunteers. A two channel spectrometer (NIRO 300, Hamamatsu Photonics KK) was used to measure absolute cerebral TOI over the frontal cortex using the SRS technique using an optode spacing of 5 cm and 1.5 cm for channel 1 and 2 respectively. After ACZ administration we were able to observe a significant increase in the velocity of middle cerebral artery (Vmca, measured with the transcranial Doppler (TCD)) which was accompanied by an increase in TOI as monitored by the NIRO 300 with an optode spacing of 5 cm but not with an optode spacing of 1.5 cm. Furthermore a direct relationship was seen between the Vmca and the TOI measured at 5 cm optode spacing. This work suggests that using this commercial NIRS instrument with an optode spacing of 5 cm one is able to detect the intracranial changes. [ABSTRACT FROM AUTHOR]

Exposure of rats to mild hypoxia initially increases cerebral blood flow (CBF) as much as two-fold which maintains the arterial oxygen delivery rate. Several days after continued hypoxia, CBF decreases toward its baseline level as the blood oxygen carrying capacity is increased through increased hemoglobin content [1]. Evidently, CBF regulation and the oxygen carrying capacity of blood are correlated. To quantitatively analyze the CBF control mechanisms associated with chronic hypoxia, a mathematical model was developed that describes the concentration dynamics of O2 and CO2 transport and metabolic processes in blood and brain tissue. In capillary blood, species transport processes were represented by a one-dimensional convection-dispersion model with diffusion between blood and tissue cells in the cortex and brain stem. Three possible control mechanisms for CBF in response to chronic hypoxia were analyzed: 1) local PO2 change in cerebral tissue; 2) reduced blood flow associated with elevated blood viscosity from increased Hct; 3) neurogenic input from O2 receptors in the brain stem. Our hypothesis is that increased PO2 in the brain stem is the signal for the return of CBF to its baseline condition. This PO2 increase results from an increase in arterial oxygen carrying capacity and a decrease in local energy metabolism. Model simulations quantify the relative contributions of each of these control mechanisms during 4 days of hypoxic exposure. These simulations are consistent with experimental data that show CBF returns to its baseline even though the cerebral cortical tissue remains hypoxic as indicated by increased levels of the transcription factor Hypoxia Inducible Factor-1 (HIF-1). [ABSTRACT FROM AUTHOR]

Aged rat brain is more sensitive to reperfusion injury induced by cardiac arrest and resuscitation. The mitochondrial respiratory chain, the major source of free radicals during reperfusion, is likely to be the target of lipid peroxidation. Previous work has shown a higher mortality and lower hippocampal neuronal survival in older rats. 4-hydroxy-2-nonenal (HNE), a major product of lipid peroxidation, was found to be elevated in cortex and brainstem after resuscitation. In this study we investigated the acute changes of mitochondrial function in aging rat brain following cardiac arrest and resuscitation; the effect of an antioxidant, alpha-phenyl-tert-butyl-nitrone (PBN) was also tested. Fischer 344 rats, 6 and 24-month old, were subjected to cardiac arrest (7-10 minutes) and allowed to recover 1 hour after resuscitation. Mitochondria of cortex and brainstem were isolated and assayed for respiratory function. Compared to their respective non-arrested control group, 1h untreated groups (both 6 month and 24 month) had similar state 3 (ADP-stimulated) but higher state 4 (resting state) respiratory rates. The respiratory control ratio (state 3/state 4) of cortex in the 1h untreated group was 26% lower than the non-arrested control group; similar results were found in brainstem. The decreased mitochondrial respiratory function was improved by PBN treatment. HNE-modified mitochondrial proteins were elevated 1h after resuscitation, with an evident change in the aged. Treatment with PBN reduced the elevated HNE production in mitochondria of cortex. The data suggest (i) there is increased sensitivity to lipid peroxidation with aging, (ii) mitochondrial respiratory function related to coupled oxidation decreases following cardiac arrest and resuscitation, and (iii) treatment with antioxidant, such as PBN, reduces the oxidative damage following cardiac arrest and resuscitation. [ABSTRACT FROM AUTHOR]

Regulation of brain metabolism and cerebral blood flow involves complex control systems with several interacting variables at both cellular and organ levels. Quantitative understanding of the spatially and temporally heterogeneous brain control mechanisms during internal and external stimuli requires the development and validation of a computational (mathematical) model of metabolic processes in brain. This paper describes a computational model of cellular metabolismin blood-perfused brain tissue,which considers the astrocyteneuron lactate-shuttle (ANLS) hypothesis. The model structure consists of neurons, astrocytes, extra-cellular space, and a surrounding capillary network. Each cell is further compartmentalized into cytosol and mitochondria. Inter-compartment interaction is accounted in the form of passive and carrier-mediated transport. Our model was validated against experimental data reported by Crumrine and LaManna, who studied the effect of ischemia and its recovery on various intra-cellular tissue substrates under standard diet conditions. The effect of ketone bodies on brain metabolism was also examined under ischemic conditions following cardiac resuscitation through our model simulations. The influence of ketone bodies on lactate dynamics on mammalian brain following ischemia is studied incorporating experimental data. [ABSTRACT FROM AUTHOR]

To define some of the specific cellular effects of chronic hypoxia on the small intestine, we measured the concentration of glucose transporter 2 (GLUT2) at two sites, the jejunum and ileum. Wister rats were subjected to 21-day normoxia (n=6) or to continuous 21-day hypobaric hypoxia approximately 0.5 ATM (n=5). Western blot analysis was performed and the abundance of GLUT2 protein was quantified as relative densitometric units and normalized to actin. GLUT2 content was similar in the jejunum and ileum under normoxic (jejunum = 0.65±0.13 mean±SD; ileum = 0.56±0.22 OD; mean difference 0.09, p=0.09) and hypoxic conditions (jejunum=0.56±0.14 OD mean±SD; ileum = 0.58±0.16; mean difference −0.01, p =0.42). GLUT2 decreased by 14% of the mean normoxic jejunal levels whereas ileal GLUT2 was slightly increased. A maximum decline in weight of 15% occurred at day 4 followed by a blunted rate of weight gain for rats in the hypoxic group. Thus, sustained exposure to hypobaric hypoxia reduced the availability of GLUT2 for glucose transport in the jejunum. Regulating small intestinal content of glucose transporters may be an important mechanism for tissue adaptation to chronic hypoxia. This finding provides initial insight into hypoxic tolerance of the gut to chronic hypobaric hypoxic exposure. [ABSTRACT FROM AUTHOR]

The binding and buffering of O2 and CO2 in the blood influence their exchange in lung and tissues and their transport through the circulation. To investigate the binding and buffering effects, a model of blood-tissue gas exchange is used. The model accounts for hemoglobin saturation, the simultaneous binding of O2, CO2 , H+, 2,3-DPG to hemoglobin, and temperature effects [1,2]. Invertible Hill-type saturation equations facilitate rapid calculation of respiratory gas redistribution among the plasma, red blood cell and tissue that occur along the concentration gradients in the lung and in the capillary-tissue exchange regions. These equations are well-suited to analysis of transients in tissue metabolism and partial pressures of inhaled gas. The modeling illustrates that because red blood cell velocities in the flowing blood are higher than plasma velocities after a transient there can be prolonged differences between RBC and plasma oxygen partial pressures. The bloodtissue gas exchange model has been incorporated into a higher level model of the circulatory system plus pulmonary mechanics and gas exchange using the RBC and plasma equations to account for pH and CO2 buffering in the blood. [ABSTRACT FROM AUTHOR]

An anatomically realistic mathematical model of oxygen transport in cardiac tissue was developed to help in deciding what angiogenic strategies should be used to rebuild the vasculature post myocardial infarction (MI). Model predictions closely match experimental measurements from a previous study, and can be used to predict distributions of oxygen concentration in normal and infarcted rat hearts. Furthermore, the model can accurately predict tissue oxygen levels in infarcted tissue treated with pro-angiogenic compounds. Immunoliposome (IL) targeting to areas of inflammation after MI could provide the means by which pro-angiogenic compounds can be selectively targeted to the infarcted region. The adhesion of model drug carriers and immunoliposomes coatedwith antibody to P-selectin was quantified in a MI rat model. Anti-P-selectin coated model drug carriers showed a 140% and 180% increase in adhesion in the boarder zone of the MI 1 and 4 hours post-MI, respectively. Circulating for 24 hrs, radiolabeled anti-P-selectin immunoliposomes showed an 83% and 92% increase in targeting to infarcted myocardium when injected 0 and 4 hrs post-MI, respectively. Targeting to upregulated adhesion molecules on the endothelium provides a promising strategy for selectively delivering compounds to the infarct region of the myocardium using our liposomal based drug delivery vehicle. [ABSTRACT FROM AUTHOR]

Relating external to internal respiration during exercise requires quantitative modeling analysis for reliable inferences with respect to metabolic rate. Often, oxygen transport and metabolism based on steady-state mass balances (Fick principle) and passive diffusion between blood and tissue are applied to link pulmonary to cellular respiration. Indeed, when the work rate does not change rapidly, a quasi-steady-state analysis based on the Fick principle is sufficient to estimate the rate of O2 consumption in working muscle. During exercise when the work rate changes quickly, however, non-invasive in vivo measurements to estimate muscle O2 consumption are not sufficient to characterize cellular respiration of working muscle. To interpret transient changes of venous O2 concentration, blood flow, and O2 consumption in working muscle, a mathematical model of O2 transport and consumption based on dynamic mass balances is required. In this study, a comparison is made of the differences between simulations of O2 uptake and O2 consumption within working skeletal muscle based on a dynamic model and quasi-steady-state approximations. The conditions are specified under which the quasi-steadystate approximation becomes invalid. [ABSTRACT FROM AUTHOR]

A novel evanescent-based biosensor (Endotect™, ThreeFold Sensors, Inc.) was developed with laser-based fiber optics using fluorescent dyelabeled recombinant human estrogen receptor-α (rhERα) and hERβ as probes. A three-tiered approach evaluating various steps in the formation of the estrogen- receptor complex and its subsequent activity was developed for instrument calibration to detect estrogen mimics in biological samples, water and soil. Using this approach, binding affinities and activities of certain known estrogen mimics were determined for their use as calibrator molecules. Results indicated rhERα and rhERβ may be employed as probes to distinguish estrogen mimics with a broad range of affinities. In addition, application of the biosensor for detecting DNA-binding proteins in human tissue extracts was demonstrated. The later studies suggest the biosensor may be used as a clinical laboratory tool for assessing tumor marker proteins. [ABSTRACT FROM AUTHOR]

The aim of this study was to establish if patients with hemodynamically relevant or irrelevant stenoses of the extracranial internal carotid artery have different intracranial arterial oxygen saturation as measured by transcranial pulse oximetry using near infrared spectroscopy. Patients with unilateral stenosis ≥70% according to North American Symptomatic Carotid Endarterectomy Trial (NASCET) were included. Hemodynamic relevance was assessed using ultrasound criteria. Transcranial spectroscopy recordings were taken before and after surgical or interventional treatment of the stenosis. Optodes were placed bilaterally on the intact frontoparietal aspect of the skull. Oxygen saturation and diversion angle alpha from the hemoglobin plane were measured. There were no significant differences regarding arterial oxygen saturation between the two groups. Oxygen saturation ranged from 0.910 ± 0.08 to 0.957 ± 0.028 in the subgroups (all values asmean± S.E.). These values are consistent with previous studies and theoretical values. In smokers we found a significantly shifted diversion angle from the hemoglobin plane to the negative side. This indicates the presence of an absorber other than oxy- and desoxyhemoglobin in the optical field. Weconclude that transcranial pulse oximetry cannot distinguish between patients with hemodynamically relevant and irrelevant stenosis of the internal carotid artery. However it seems to be capable of distinguishing smokers from non-smokers. [ABSTRACT FROM AUTHOR]

A three-tiered approach was developed to determine the influence of a chemically-diverse group of compounds exhibiting estrogen mimicry using recombinant human estrogen receptor (rhER) activity to calibrate a receptor protein-based biosensor. In the initial tier, a ligand competition array was developed to evaluate compounds inhibiting [3H]estradiol-17β binding to rhER. Each of six different concentrations of [3H]estradiol-17β was mixed with increasing concentrations of an unlabeled putative mimic. Each of these mixtures was incubated with a constant amount of rhERα and then receptorbound [[3H]estradiol-17β was measured. This array protocol analyzes ligand binding affinities of hERα with a potential inhibitor over the entire range of receptor protein saturation. When either hERα or hERβ binds to an estrogenic ligand, the receptor monomer forms both homo- and hetero-dimers. Then the ligand-receptor dimer complex activates transcription by associating with an estrogen response element (ERE), which is a specific DNA sequence located upstream of estrogenresponsive genes. The second tier for ligand evaluation utilized an electrophoretic mobility shift assay (EMSA), which was performed with an ERE sequence labeled with [α[32]P]dATP and incubated with rhER in the presence or absence of unlabeled ligand. ERE-hER complexes were separated by electrophoresis and analyzed using phosphor imaging technology. To assess biological effects of an estrogen mimic on expression of an ER-target gene, a yeast cell-based bioassay was constructed with recombinant DNA technology using Saccharomyces cerevisiae. Each of these engineered yeast cells contained a rhERα expression plasmid (YEpE12) and a separate reporter plasmid (YRG2) containing an ERE sequence upstream of a β-galactosidase reporter gene. Incubation of these yeast cells with an estrogenic compound allows formation of ligand-hERα complexes, which recognize the ERE sequence regulating β-galactosidase expression. Estrogenic compounds, which were evaluated as calibrators for ligand-based and EREbased biosensors, elicit varying responses in each of the three tiers of the protocol. [ABSTRACT FROM AUTHOR]

Breast cancer remains the most common cancer among women, with an estimated 212,920 new cases and 40,970 deaths in the United States in 2006. The present work extends the studies of nanoparticles targeted to the luteinizing hormone-releasing hormone (LHRH) receptor which is overexpressed in breast, ovarian, endometrial and prostate cancer cells. In contrast, LHRH receptors are not expressed, or expressed at a low level in most visceral organs. In our studies, we conjugated Fe3O4 nanoparticles (20-30 nm) with [D-Trp6]LHRH (Triptorelin), a decapeptide analog of LHRH currently used for treatment of sex-hormone-dependent tumors. Conjugation of [D-Trp6]LHRH to Fe3O4 particles retained its binding affinity and biological activity for the LHRH receptor. Treatment of two separate breast tumor cell lines (MCF-7 and MDA-MB231) with these conjugated nanoparticles resulted in 95-98% cell death and loss of viability within 24 h whereas no change in cell proliferation or cell apoptosis was observed in cells treated with equal amounts of either [D-Trp6]LHRH or unconjugated Fe3O4 nanoparticles. These studies provide critical and important information regarding use of LHRH receptor targeted therapy for breast cancer. [ABSTRACT FROM AUTHOR]

Cardiovascular diseases (CVDs) have been the leading threat to human life. An effective way for sensitive and accurate CVD diagnosis is to measure the biochemical markers released from the damaged myocardial cells in the bloodstream. Here, a multi-analyte, fluorophore mediated, fiber-optic immuno-biosensing system is being developed to simultaneously and rapidly quantify four clinically important cardiac markers, myoglobin, C-reactive protein, cardiac troponin I, and B-type natriuretic peptide. To quantify these markers at a pico-molar level, novel nanoparticle reagents enhancing fluorescence were used and signal enhancement was obtained as high as ~230%. Micro-electro-mechanical system (MEMS) was integrated to this system to ensure a reliable and fully-automated sensing performance. A point-of-care, automatic microfluidic sensing system for four cardiac marker quantification was developed with the properties of 3 cm sensor size, 300 μL sample volume, 9-minute assay time, and an average signal-to-noise ratio of 35. [ABSTRACT FROM AUTHOR]

The ultimate goal of this study is to develop a tumor-specific multi-functional, nano-entity that can be used for both cancer detection and treatment. Low heat (42~45°C) hyperthermia is an effective cancer treatment method with little side effect. Magnetic nanoparticles, such as Fe3O4, can be heated by alternating electromagnetic (AEM) fields at well selected frequencies, without heating normal tissue. Nanogold particles (NGPs) are effective optical absorbers and also excellent fluorescent enhancers. Therefore, coating gold on Fe3O4 particles can enhance the optical contrast as well as keeping the particle property for hyperthermia. Indocyanine green (ICG), a FDA approved fluorophore, has a very low quantum yield, and its fluorescence can be enhanced by linking ICGto gold-coated Fe3O4 nanoparticles. Luteinizing hormone releasing hormone (LHRH), which has high affinity to breast cancer, can be used for tumor-specific targeting. Our study results showed: Fe3O4 particles at a size range of 10~30 nm can be heated well by an AEM field at a rate of 18°C/wt%-minute; the fluorescence of ICG was extensively enhanced by NGPs; LHRH-coated gold nanoparticles provided as much cancer specificity as LHRHalone. Combining these properties in one entity, i.e.,LHRH/ICGlinked, gold-coated Fe3O4 nanoparticles, can be a tumor-specific nano-agent for optical detection and electro-magnetically induced hyperthermia for breast cancer. [ABSTRACT FROM AUTHOR]

The main objective was to evaluate a Scanning Laser Ophthalmoscope (SLO) based particle tracking method as a means of quantitative assessment of retinal blood velocity and vessel diameter changes in response to hypoxia and hyperoxia. Retinal blood velocities were measured by tracking fluorescent microspheres (1.0 μm diameter) in anesthetized adult pigmented rats. Velocities were calculated based on microsphere position changes and the recording frame rate. Hypoxia was induced by inspiring a mixture of nitrogen and air and hyperoxia was induced by inspiring 100% oxygen. Average blood velocities during hypoxia obtained for arteries, veins, and small vessels (diameter < 40 μm) were 39.9 ± 9.9, 34.9 ± 2.7, and 8.8 ± 1.8 mm/sec, respectively, whereas during hyperoxia, the average blood velocities obtained were 23.7 ± 6.2, 28.2 ± 2.7, and 7.6 ± 0.7 mm/sec. Hypoxia was found to increase the diameters of arteries by 25%but did not change the diameters of veins; whereas, hyperoxia was found to decrease their diameters by 25% and 18%. Changes detected in vessel diameter and blood velocity suggest that the level of oxygen tension alters retinal hemodynamics. Dynamics of retinal hemodynamics in response to hypoxia and hyperoxia can be assessed using the SLO imaging method. [ABSTRACT FROM AUTHOR]

Factor V Leiden (FVL) is an abnormality with a single amino acid mutation of Factor V (FV) and is the most common, hereditary blood coagulation disorder. FVL is currently diagnosed by DNA analysis, which takes a long assay time, high cost, and a specially trained person. We are developing a rapid, accurate, and cost-effective biosensing system to quantify both FV and FVL in blood plasma, to diagnose FVL and also to evaluate the seriousness of the disease status. This system is based on a sandwich immuno-reaction on an optical fiber. To produce the monoclonal antibody against only FV or only FVL without cross-reacting with the other molecule and with a higher probability, a 20 amino acid sequence (20-mer) of FV or FVL around the mutation region was injected into mice and then hybridoma cell lines specific to each 20-mer were selected. When these antibodies were tested with native FV or FVL molecules, they were found to be cross-reacting with the other molecules, but some with higher affinity to FV (FV preferred) and some to FVL (FVL preferred). Using these antibodies, two different sensors were developed: FV preferred and FVL preferred sensors. These two sensors allowed us to quantify FV and FVL in plasma with a maximum error of 4%. The plasma levels of both molecules provide us not only FVL diagnosis but also the level of the seriousness. The same principles may be used for developing diagnostic tools for other diseases with a single point mutation. [ABSTRACT FROM AUTHOR]

Oxy- (HbO2) and deoxy- (HHb) haemoglobin signalsmeasured by near infrared (NIR) spectroscopy over the human frontal lobes frequently contain respiratory and low frequency oscillations (LFOs). It has been suggested previously that venous oxygen saturation (SvO2) can be calculated from these respiratory oscillations. In this paper, we investigated the use of a Fourier transform based algorithm to calculate an oxygen saturation measure known as SoscO2 which may be a close estimate of the underlying SvO2. SoscO2 was calculated using three different frequency ranges, (1) respiratory oscillations only, (2) LFOs only, and (3) both respiratory oscillations and LFOs. At each frequency range SoscO2 was calculated using either (1) the modified Beer-Lambert law (MBL) or (2) spatially resolved spectroscopy (SRS). In total six different measurements of SoscO2 were investigated here. Experiments were performed in six adult ventilated patients with traumatic brain injury. The patients' inspired oxygen fraction (FiO2) was raised in two hyperoxic phases. The calculated SoscO2 values were compared with other cerebral oxygenation measures including an intraparenchymal catheter based brain tissue oxygen tension (PbrO2) and the NIR based tissue oxygenation index (TOI). It was found that the SoscO2 calculated using the combined respiratory and LFO frequency range and the SRS method resulted in the highest detection rates of hyperoxic changes.This measure of SoscO2 may provide a viable, continuous, non invasive, bedside measure of cerebral venous oxygen saturation. [ABSTRACT FROM AUTHOR]

Use of Visible Light Spectrophotometry (VLS) and Laser Doppler Flowmetry (LDF) is currently being studied by the authors to assess the viability of tissue margins in colon resection and to assess mucosal oxygenation in the colon. Thus, as a preliminary study it was necessary to evaluate whether there is any systematic inter-probe variability of the measurements by VLS and LDF. The oral mucosa was used as a model. Methods SO2 with VLS (Whitland Research RM200) and blood flow with LDF (Moor Instruments DRT4) were measured at 10 sites each on the tongue and oral mucosa of 10 healthy volunteers at 0, 6 and 24 hours using 3 different probes for VLS and 2 probes for LDF. Results The results showed that the SO2 measurements by VLS using the different probes on the tongue and mucosa were significantly correlated (P<0.05). SO2 values at 6 hours were significantly higher than at 0 and 24 hours (P<0.05) in all but one case. SO2 measurements were not correlated with LDF. LDF measurements by the 2 probes were correlated significantly (P<0.05) but the standard deviations were very large. Conclusions SO2 measurements on the oral mucosa are reproducible. Due to the large variations in LDF, VLS is likely to be the more clinically useful tool for identifying mucosal ischaemia [ABSTRACT FROM AUTHOR]

It has been suggested that changes in oxidised cytochrome c oxidase concentration ([oxCCO]) measured using cerebral near infrared spectroscopy (NIRS) may be algorithm artefacts. We examine the change in near infrared (NIR) attenuation by the healthy adult brain (n=10) during hypoxaemia. Broadband spectroscopic data were collected during normoxia, and hypoxaemia. The UCLn algorithm was used to fit (a) oxy- (HbO2) and deoxy-haemoglobin (HHb) spectra (2 component fit), and (b) HbO2, HHb and oxidised-reduced cytochrome c oxidase difference spectra (3 component fit) to themean change inNIR attenuation between baseline and hypoxaemia. The sum of squares of the residuals was 100×10−7 OD2 for the 2 component fit and 8×10−7 OD2 for the 3 component fit, and the two sets of residuals differed from each other (p=0.0003). We compare experimental and simulated data and suggest that the 2 component residuals indicate a change in [oxCCO]. Changes in near infrared attenuation by the healthy adult brain during hypoxaemia cannot be accounted for solely by changes in oxyand deoxy-haemoglobin concentrations. Including [oxCCO] in the algorithm improves its fit quality. These data suggest that changes in cerebral cytochrome c oxidase redox occur during hypoxaemia and that they can be detected using NIRS. [ABSTRACT FROM AUTHOR]

The survival of three-dimensional tissue requires a vascular network to provide transport of oxygen and metabolic byproduct. Here, we report a new approach to create capillary blood vessels in vitro on biomaterials suitable for use as scaffolds in engineering tissues. Endothelial cells were cultured on chemical and topographical patterns of micro-sized grooves on gelatin. Selective attachment and spreading of cells within the grooves was ensured by microcontact printing the plateau regions with cell resistant PEG/PLA (polyethyleneglycol-L-polylacticacid). Human microvascular endothelial cells plated on these patterned biomaterials attached and spread exclusively within the grooves. These topographical features promote endothelial cells to form capillary tube-like structures. The results demonstrated that capillary structures formed on biomaterials are useful for engineering vascularized tissues. [ABSTRACT FROM AUTHOR]

Multi-spectral systems consisting of a small number of wavelengths are increasingly using light emitting diodes (LEDs) to reduce the overall costs of the system. However, LEDs typically have broad spectral bandwidths and cannot be modeled as having a single discrete wavelength. This paper puts forth a simple model to analyze the effects of using LEDs to illuminate a single layer of homogenous tissue. Monte Carlo simulations are used to approximate photon propagation through a semi-infinite turbid medium oximetry system using two light emitting diodes with broad spectra for varying oxygen saturations. The results are then compared against diffusion solutions for narrowband illumination at the same two center wavelengths. [ABSTRACT FROM AUTHOR]

A new computational model, using computational fluid dynamics (CFD), is presented that describes fluid behavior in cylindrical cell culture dishes resulting from motion imparted by an orbital shaker apparatus. This model allows for the determination of wall shear stresses over the entire area of the bottom surface of a dish (representing the growth surface for cells in culture) which was previously too complex for accurate quantitative analysis. Two preliminary cases are presented that show the complete spatial resolution of the shear on the bottom of the dishes. The maximum shear stress determined from the model is compared to an existing simplified point function that provides only the maximum value. Furthermore, this new model incorporates seven parameters versus the four in the previous technique, providing improved accuracy. Optimization of computational parameters is also discussed. [ABSTRACT FROM AUTHOR]

Antioxidants have been studied for their capacity to reduce the cytotoxic effects of radiation in normal tissues for at least 50 years. Early research identified sulfur-containing antioxidants as those with the most beneficial therapeutic ratio, even though these compounds have substantial toxicity when given in-vivo. Other antioxidant molecules (small molecules and enzymatic) have been studied for their capacity to prevent radiation toxicity both with regard to reduction of radiation-related cytotoxicity and for reduction of indirect radiation effects including long-term oxidative damage. Finally, categories of radiation protectors that are not primarily antioxidants, including those that act through acceleration of cell proliferation (e.g. growth factors), prevention of apoptosis, other cellular signaling effects (e.g. cytokine signal modifiers), or augmentation of DNA repair, all have direct or indirect effects on cellular redox state and levels of endogenous antioxidants. In this review we discuss what is known about the radioprotective properties of antioxidants, and what those properties tell us about the DNA and other cellular targets of radiation. [ABSTRACT FROM AUTHOR]

Resveratrol, a phytoalexin found in the skin of grapes, is believed to have multiple bioactivities including anti-cancer, anti-carcinogenesis and antiinflammatory. The mechanisms by which resveratrol might produce these effects are not well understood. In this study, malignant human pancreatic cancer cells were treated without or with resveratrol in combination with ionizing radiation (IR), and then the mitochondrial function of treated cells was evaluated using several standardized assays. They include the CalceinAMmethod for mitochondria transition pore; the JC-1 staining method for mitochondria membrane potential; the CM-H2DCFDA method for reactive oxygen species; and the Annexin V/propidium iodide (PI) method for apoptosis/cell death. Our results indicated that (1) pore function was partially intact after resveratrol, but resveratrol probably interfered with the accumulation of intracellular Calcein AM; (2) depolarization of the mitochondria membrane was increased in the resveratrol treated cells, consistent with mitochondrial dysfunction; (3) ROS was slightly increased with resveratrol, a phenomenon that was greatly increased when this agent was combined with IR; and (4) in parallel with the above changes in mitochondrial and drug transport, cells treated with resveratrol showed increased apoptosis as measured by Annexin V/PI staining. In summary, the anti-cancer effect of resveratrol is associated with the damage of mitochondrial function that leads to increased ROS, apoptosis, and possibly intracellular drug accumulation via inhibition of proteins involved in multi-drug resistance (MDR). [ABSTRACT FROM AUTHOR]

Tumor cells exhibit mechanisms by which chemotherapeutic drugs can be actively pumped out of the cell (e.g., p-glycoprotein pGP, MRP1), resulting in a multidrug resistant phenotype. Many human tumors show pronounced hypoxia which can result in a local ATP depletion which in turn may compromise the efficacy of these transporters. The aim of this study was therefore to assess the transport activity and expression of drug transporters under hypoxic conditions. Prostate carcinoma cells (R3327-AT1) were exposed to hypoxia (pO2≶0.5 mmHg) for up to 24h and pump activity was determined by an efflux assay. The results showed that exposing cells to hypoxia for 3-6 h led to a moderate increase in pGP activity. After 24 h pGP activity was reduced by 44% compared to control levels. Hypoxia reduced the MRP1 activity to a lesser extent (by 25%). However, the expression of pGP and MRP1 was almost independent of the medium pO2. In conclusion, pronounced hypoxia had only minor effects on the activity of drug transporters with the activity decreasing only after 12-24 h under hypoxia, possibly as a result of ATP depletion. Instead, indirect effects of hypoxia leading to extracellular acidosis seem to have a much more pronounced effect on pGP activity. [ABSTRACT FROM AUTHOR]

The usual mechanisms for biochemical events are steady-state systems without dynamic simulation. Our study is to simulate lipogenesis from the breakdown of glucose coupled with oxidative phosphorylation in mitochondria by using JSim (for Java Simulator) as software development environment, which enables non-linear differential equations to be used in a simulation giving a time course through a variety of non-steady-state conditions. Glycolysis and lipogenesis coupled with oxidative phosphorylation in mitochondria non-linear differential model is built in this paper. Simulation and discussion on lipogenesis by carbohydrate responsive element-binding protein (ChREBP) are given. Our model provides a potential way to analyze the experimental databank. [ABSTRACT FROM AUTHOR]

It is a widely held paradigm in molecular biology that a change in the third base of a codon is silent in terms of expression. In this investigation, results are presented that challenge that paradigm, at least in terms of one polymorphism in KCNJ11, which is one of five genes that have been implicated in the disorder Hyperinsulinism of Infancy. In two cohorts of Australian patients, an uneven distribution of KCNJ11 SNP's was observed. A silent polymorphism at codon 190 was over-represented in the patients who responded well to medical treatment and under-represented in those that required radical surgical intervention. In an attempt to investigate this polymorphism, it was expressed in vitro and western blot analysis showed that there were virtually no bands from the homozygous variant samples, while strong bands were seen in normal controls. The human genome is highly redundant in terms of tRNA species for each amino acids but enigmatically under-represents a number of specific codons. The polymorphism in question occurs within one such codon. We propose that the presence of a base change at the third position of codon that is not represented by a corresponding anti-codon within the human nuclear tRNA leads to a decreased rate of expression of the protein. [ABSTRACT FROM AUTHOR]

Endogenously generated reactive oxygen species and genotoxic carcinogens can covalently modify bases in cellular DNA. If not recognized and removed prior to S-phase of the cell cycle, such modifications can block DNA replication fork progression. If blocked forks are not are not resolved, they result in double strand breaks and cell death. Recent data indicate that the process of translesion DNA synthesis (TLS) is a highly conserved mechanism for bypassing lesions in template DNA. Although not fully understood, in yeast a ubiquitin ligase (RAD18) signals error-prone Y family polymerases to the blocked fork to bypass the damage with potentially mutagenic consequences. Homologs of the yeast proteins are found in higher eukaryotic cells, including human. We are examining the hypothesis that RAD18 acts as a proximal signal to Y-family polymerases to bypass damage, in a manner analogous to yeast but with additional layers of complexity. Here we report that RAD18 accumulates in nuclear foci after UV irradiation only in cells entering S-phase with DNA damage. These foci co-localize with proliferating cell nuclear antigen (PCNA). In addition, a newly described DNA polymerase, pol iota, also forms nuclear foci in a damage- and S-phase dependent manner. These data support our overall hypothesis that RAD18 accumulates at blocked forks and initiates the signal to recruit TLS polymerases. [ABSTRACT FROM AUTHOR]

The pivotal role of hypoxia within the pathophysiological framework of solid malignant tumors is now considered to be indisputable. The fact that hypoxia can cause resistance to various cancer therapies and promote malignant progression is reflected in its adverse impact on prognosis which is repeatedly shown for various tumor entities. Knowledge in this area is based on direct assessment of the oxygenation status using O2-sensitive microsensors. However, weaknesses of this standard method are its invasiveness and limitation to accessible tumor entities. Hypoxia-inducible factor (HIF)-1α, the master transcriptional regulator of the hypoxic response, as well as certain downstream genes, e.g., glucose transporter (GLUT)-1 and carbonic anhydrase (CA) IX, have been considered to be suitable as surrogate biomarkers of hypoxia due to their tight regulation by O2 levels under certain, well-defined in vitro conditions. The fact that statistical correlations between the expression of these proteins and direct pO2 measurements in the clinic have been sporadically reported seemed to support their role as "endogenous hypoxia markers". Remaining disparities were mainly attributed to the influence of tumor heterogeneity. In a series of studies, we have addressed this question by examining the expression of HIF-1α, GLUT-1 and CA IX in tissue microareas where direct O2 measurements had previously been carried out, so that the influence of tumor heterogeneity could be reduced to a minimum. Using this methodology, no correlation between the expression of "endogenous hypoxia markers" and direct pO2 measurements could be found. In conclusion, while there may be a stringent association between these markers and the oxygenation status under standardized in vitro conditions, this is not transferable to the clinical assessment of oxygenation status in patients. The term "endogenous hypoxia markers" should therefore be avoided, at least in the clinical oncology setting. [ABSTRACT FROM AUTHOR]

Conspicuously high respiratory quotients (RQs) are found in solid tumors in vivo. RQs in the range between 1.29 and 1.95 neither reflect the degree of substrate oxidation by tumor cells nor indicate the types of fuels involved in metabolic processes. Instead, such tumor RQs most probably are caused by (a) channeling of glycolytic end-products into lipogenesis, and by (b) CO2 release from the tumor following extracellular buffering of H+ -inos by bicarbonate. H+ -inos exported from the intracellular space into the interstitial compartment titrate extracellular bicarbonate to CO2 and H2O with the aid of the ectoenzyme carbonic anhydrase IX, which is activated at low pH. Strikingly high (RQs) may thus be a further characteristic of the tumor microenvironment and of the tumor (patho-)physiome, the latter quantitatively describing the pathophysiologic characteristics of tumor cells and solid tumors. [ABSTRACT FROM AUTHOR]

Factor V (FV) is a coagulant in plasma. The FV molecule consists of a heavy chain and a light chain, and Factor V Leiden (FVL) is mutated FV at a single amino acid in the heavy chain. FVL patients are in a dangerous hyper-coagulation state in their body. Current FVL diagnosis is done by DNA analysis, which is expensive and time consuming. Our group has been developing a real-time, cost effective immuno-optical biosensor for FVL diagnosis. For the sensor development, pure FVL, which is not currently available, is needed. Here, we have attempted FVL purification from FVL patient's plasma. Since plasma contains many proteins and some proteins are structurally homologous to FV, the purification must be done by a very specific method, such as immuno-affinity chromatography. However, an antibody that does not react with FV is not currently available. Because the mutation is in the heavy chain and the amino acid sequence of the light chain of FVL is identical to that of FV, antibodies generated against the light chain of FV were tested for purifying FVL. Plasma was obtained from a homozygous FVL patient. First, the plasma was pretreated by barium citrate and polyethylene glycol 6000, to remove the vitamin K-dependent proteins, alpha globulins, and other smaller than 6 kDa molecular weight proteins. The yield in the process was 54%. Immuno-affinity purification of FVL from patient plasma was then performed using an anti-FV light chain antibody immobilized CNBr-Sepharose, and the purification yield was 25%. In summary, the antibody against the light chain of FV was able to purify the single point mutated form of FV (FVL) from plasma with an overall yield of 14%. The same principle can probably be used for purification of the other single point mutated proteins. [ABSTRACT FROM AUTHOR]

The protein C (PC) pathway plays an important role in vascular function, and acquired deficiency during sepsis is associated with increased mortality. We have explored the role of PC suppression in modulating early inflammatory events in a model of polymicrobial sepsis. We show that increased levels of organ damage and dysfunction are associated with decreased levels of endogenous PC. Notably, animals with low PC had correspondingly high levels of pulmonary iNOS expression, which correlated with chemokines KC/Gro and MIP2, previously shown to predict outcome in thismodel. Treatment with activated protein C (aPC) not only reduced the pathology score, leukocyte infiltration and markers of organ dysfunction, but also suppressed the induction of iNOS, and the chemokine response (including KC/Gro, MIP2, IP-10, RANTES, GCP-2 and lymphotactin), and increased apoA1. aPC treatment also suppressed the induction of VEGF, a marker recently suggested to play a pathophysiological role in sepsis. These data demonstrate a clear link between low protein C and degree of organ damage and dysfunction in sepsis, as well as the early reversal with aPC treatment. Moreover, our data show a direct role of aPC in broadly modulating monocyte and T-cell chemokines following systemic inflammatory response. [ABSTRACT FROM AUTHOR]

Protein C (PC) is an important anticoagulant in blood plasma. Cohn Fraction IV-1 (CFIV-1) is an inexpensive PC source but contains a large amount of factor II (FII). Immobilized metal affinity chromatography (IMAC) utilizes metal ions to adsorb proteins primarily via their surface histidine. Two major operation parameters for IMAC are imidazole concentration and pH: imidazole is a histidine analog and pH controls the protein surface protonation level. The effects of these two parameters on the adsorption and elution of PC and FII were studied for each protein individually and also together as a mixture. For the individual proteins, low FII (16%) and high PC (98%) adsorption were achieved at 8 mM imidazole, pH 8.0. At 11 mM imidazole, 92% of the adsorbed FII was eluted, with only a 3% PC loss. At 40 mM, 97% of the adsorbed PC was recovered. For the protein mixture, very similar adsorption and elution results were obtained, but slightly greater PC loss (16%) during elution at 11 mM imidazole. This result shows that there is a high potential for the PC purification from CFIV-1 by appropriately adjusting the imidazole concentration and pH in the IMAC process. [ABSTRACT FROM AUTHOR]

Lactate has been reconsidered! As we now know, most is produced aerobically We report that lactate accumulation commonly occurs in the presence of oxygen and is sufficient to instigate signals for angiogenesis and connective tissue deposition. These include vascular endothelial growth factor (VEGF), transforming growth factor beta (TGF beta), interleukin-1 (IL-1), and hypoxia-inducible factor (hif-1alpha). This paper, a mini-review, is occasioned by new data showing increased presence of VEGF and angiogenesis in an oxygenated site by adding a slow-release source of lactate into Matrigel® and implanting the Matrigel subcutaneously in mice. [ABSTRACT FROM AUTHOR]

Authors contributed equally to this manuscript Natural adjuvants, such as bacterial lipopolysaccharide (LPS), activate antigen presenting cells via Toll-like receptors and, indirectly, increase the survival of antigenactivated T cells. The molecular mechanisms leading to increased survival remain poorly defined. Because T cell clonal expansion leads to high energy demands, we hypothesized that increased glucose uptake and/or utilization in adjuvant-activated T cells could be important molecular event(s) that would lead to adjuvant-associated T cell survival advantage. Using a fluorescent analog of 2-deoxyglucose, 2-NBDG, we measured glucose accumulation and rate of uptake in T cells from mice treated with antigen in the absence or presence of LPS. Although adjuvant activated T cells increased the accumulation of 2-NBDG, the rate of uptake was unchanged compared to cells activated with only antigen. Furthermore, glucose transport inhibitors, cytochalasin B or phloretin, decreased the accumulation of glucose in adjuvant- treated T cells, but this decrease did not impair adjuvant-associated survival advantages. Together, these data indicate that increased glucose uptake through glucose transporters is not required for increased survival of activated T cells. [ABSTRACT FROM AUTHOR]

We have developed a noninvasive EPR (electron paramagnetic resonance) oximetry, based on a new class of oxygen-sensing nano-particulate probe (LiNc-BuO), for simultaneous monitoring of stem-cell therapy and in situ oxygenation (partial pressure of oxygen, pO2) in a mouse model of acute myocardial infarction (AMI). AMI was induced by a permanent occlusion of left-anterior-descending (LAD) coronary artery. Skeletal myoblast (SM) cells were used for therapy. The oximetry probe was implanted in the midventricular region using a needle. Tissue histological studies after 3 weeks of implantation of the probe revealed significant fibrosis, which was solely due to the needle track and not due to the probe particles. The feasibility of long-term monitoring of pO2 was established in control (non-infarct) group of hearts (> 3 months; pO2=15.0±1.2 mmHg,). A mixture of the probe with/without SM cells (1±105) was implanted as a single injection in the infarcted region and the myocardial tissue pO2 at the site of cell therapy was measured for 4 weeks. The pO2 was significantly higher in infarcted hearts treated with SM cells (pO2=3.5±0.9 mmHg) compared to untreated hearts (pO2=1.6±0.7 mmHg). We have demonstrated, for the first time, the feasibility of monitoring pO2 in mouse hearts after stem cell therapy. [ABSTRACT FROM AUTHOR]