Immunoliposomes are potent carriers for targeting of therapeutic drugs to specific cells. Membrane type-1 matrix metalloproteinase (MT1-MMP), which plays an important role in angiogenesis, is expressed on angiogenic endothelium cells as well as tumor cells. Then, the MT1-MMP might be useful as a target molecule for tumor and neovascularity. In the present study, we addressed a utility of antibodies against the MT1-MMP as a targeting ligand of liposomal anticancer drug. Fab' fragments of antibody against the MT1-MMP were modified at distal end of polyethylene glycol (PEG) of doxorubicin (DXR)-encapsulating liposomes, DXR-sterically stabilized immunoliposomes (DXR-SIL[anti-MT1-MMP(Fab')]). Modification with the antibody significantly enhanced cellular uptake of DXR-SIL[anti-MT1-MMP(Fab')] into the HT1080 cells, which highly express MT1-MMP, compared with the non-targeted liposomes (DXR-stealthliposomes (DXR-SL)), suggesting that MT1-MMP antibody (Fab') is a potent targeting ligand for the MT1-MMP expressed cells. In vivo systemic administration of DXR-SIL[anti-MT1-MMP(Fab')] into the tumor-bearing mice showed significant suppression of tumor growth compared to DXR-SL. This is presumably due to the active targeting of immunoliposomes for tumor and neovascularity. However, tumor accumulation of DXR-SIL[anti-MT1-MMP(Fab')] and DXR-SL were comparable, suggesting that both liposomal formulations accumulated in tumor via enhanced permeation and retention (EPR) effect, but not via targeting to the MT1-MMP expressed on both the endothelial and tumor cells. It appears that the enhanced antitumor activity of DXR-SIL[anti-MT1-MMP(Fab')] resulted from acceleration of cellular uptake of lioposomes owing to the incorporated antibody after extravasation from capillaries in tumor.

Most plant-derived polyphenols exhibit strong antioxidant potentials, established by various assay procedures. With pulse radiolysis experiments, absolute scavenging rate constants can be obtained with a variety of oxidizing radicals which allow further structure-activity correlations and, combined with EPR spectroscopy, detailed insight into the mechanisms governing these antioxidant reactions. The most striking difference occurs between regular flavonoids and both condensed and hydrolyzable tannins. The tannins are considered superior antioxidants as their eventual oxidation may lead to oligomerization via phenolic coupling and enlargement of the number of reactive sites, a reaction which has never been observed with the flavonoids themselves.

The formation and reactivity of ferryl haemoglobin (and myoglobin), which occurs on addition of H2O2, has been proposed as a mechanism contributing to oxidative stress associated with human diseases. However, relatively little is known of the reaction between hydrogen peroxide and human haemoglobin. We have studied the reaction between hydrogen peroxide and purified (catalase free) human metHbA. Addition of H2O2 resulted in production of both ferryl haem iron (detected by optical spectroscopy) and an associated protein radical (detected by EPR spectroscopy). Titrating metHbA with H2O2 showed that maximum ferryl levels could be obtained at a 1:1 stoichiometric ratio of haem to H2O2. No oxygen was evolved during the reaction, indicating that human metHbA does itself not possess catalytic activity. The protein radicals obtained in this reaction reached a steady state concentration, during hydrogen peroxide decomposition, but started to decay once the hydrogen peroxide had been completely exhausted. The presence of catalase, at concentrations around 10(4) fold lower than metHb, increased the apparent stoichiometry of the reaction to 1 mol metHb: approximately 20 mol H2O2 and abolished the protein radical steady state. The biological implications for these results are discussed.

The mechanisms by which cytosolic proteins reversibly bind the membrane and induce the curvature for membrane trafficking and remodeling remain elusive. The epsin N-terminal homology (ENTH) domain has potent vesicle tubulation activity despite a lack of intrinsic molecular curvature. EPR revealed that the N-terminal alpha-helix penetrates the phosphatidylinositol 4,5-bisphosphate-containing membrane at a unique oblique angle and concomitantly interacts closely with helices from neighboring molecules in an antiparallel orientation. The quantitative fluorescence microscopy showed that the formation of highly ordered ENTH domain complexes beyond a critical size is essential for its vesicle tubulation activity. The mutations that interfere with the formation of large ENTH domain complexes abrogated the vesicle tubulation activity. Furthermore, the same mutations in the intact epsin 1 abolished its endocytic activity in mammalian cells. Collectively, these results show that the ENTH domain facilitates the cellular membrane budding and fission by a novel mechanism that is distinct from that proposed for BAR domains.

In hypertension, the blood pressure response to exercise is exaggerated. We demonstrated previously that this heightened pressor response to physical activity is mediated by an overactive skeletal muscle exercise pressor reflex (EPR), with important contributions from its metaboreflex and mechanoreflex components. However, the mechanisms driving the abnormal blood pressure response to EPR activation are largely unknown. Recent evidence in humans suggests that the muscle metaboreflex partially mediates the enhanced EPR-induced pressor response via abnormally large changes in sympathetic nerve activity (SNA). Whether the muscle mechanoreflex induces similarly exaggerated alterations in SNA in hypertension remains unknown, as does the role of the mechanoreceptors mediating muscle reflex activity. To address these issues, the EPR was selectively activated by electrically inducing hindlimb muscle contraction in decerebrate normotensive Wistar-Kyoto (WKY) and spontaneously hypertensive (SHR) rats. Stimulation of the EPR evoked significantly larger increases in mean arterial pressure (MAP) and renal SNA (RSNA) in SHR compared with WKY (ΔRSNA from baseline: 140 ± 11 vs. 48 ± 8%). The mechanoreflex was stimulated by stretching hindlimb muscle which likewise elicited significantly greater elevations in MAP and RSNA in SHR than WKY (ΔRSNA from baseline: 105 ± 11 vs. 35 ± 7%). Blockade of mechanoreceptors in muscle with gadolinium significantly attenuated the MAP and RSNA responses to contraction and stretch in SHR. These data suggest that 1) the exaggerated pressor response to activation of the EPR and muscle mechanoreflex in hypertension is mediated by abnormally large reflex-induced augmentations in SNA and 2) this accentuated sympathetic responsiveness is evoked, in part, by stimulation of muscle mechanoreceptors.

Near infrared fluorescence-guidance can be used for the detection of small cancer metastases and can aid in the endoscopic management of cancer. Indocyanine green (ICG) is a Food and Drug Administration (FDA)-approved fluorescence agent. Through non-specific interactions with serum proteins, ICG achieves enhanced permeability and retention (EPR) effects. Yet, ICG demonstrates rapid clearance from the circulation. Therefore, ICG may be an ideal contrast agent for real-time fluorescence imaging of tumors. To evaluate the usefulness of real-time dual fluorescence and white light endoscopic optical imaging to detect tumor implants using the contrast agent ICG, fluorescence-guided laparoscopic procedures were performed in mouse models of peritoneally disseminated ovarian cancers. Animals were administered intravenous ICG or a control contrast agent, IR800-conjugated to albumin. The ability to detect small ovarian cancer implants was then compared. Using the dual view microendoscope, ICG clearly enabled visualization of peritoneal ovarian cancer metastatic nodules derived from SHIN3 and OVCAR5 cells at 6 and 24 hr after injection with significantly higher tumor-to-background ratio than the control agent, IR800-albumin (p < 0.001). In conclusion, ICG has the desirable properties of having both EPR effects and rapid clearance for the real-time endoscopic detection of tiny ovarian cancer peritoneal implants compared to a control macromolecular agent with theoretically better EPR effects but longer circulatory retention. Given that ICG is already FDA-approved and has a long track record of human use, this method could be easily translated to the clinic as a robust tool for fluorescence-guided endoscopic procedures for the management and treatment of cancer.

By low temperature electron paramagnetic resonance we have detected the formation of a free radical signal during incubation of partially oxygenated hemoglobin at 235 K. The observed signal has g parallel = 2.0565 and g perpendicular = 2.0043, consistent with the previously reported values for superoxide. The presence of additional EPR signals for oxygen-17 bound hemoglobin, with (017-017)A perpendicular = 63 G and (017-016)A perpendicular = 94 G under identical conditions, confirms the presence of a radical containing two nonequivalent oxygens as required for a superoxide in magnetically inequivalent environments. The superoxide radical has not previously been directly detected during hemoglobin autoxidation because of its rapid dismutation. Our ability to follow the formation of superoxide for more than 15 min is attributed to its production in the hydrophobic heme pocket where dismutation is slow. The enhanced production of this free radical at intermediate oxygen pressures is shown to coincide with enhanced rates of hemoglobin autoxidation for partially oxygenated intermediates. The formation of superoxide in the heme pocket under these conditions is attributed to enhanced heme pocket flexibility. Greater flexibility facilitates distal histidine interactions which destabilize the iron-oxygen bond resulting in the release of superoxide radical into the heme pocket.

We report the inhibition of the Aquifex aeolicus IspH enzyme (LytB, (E)-4-hydroxy-3-methyl-but-2-enyl diphosphate reductase, EC 1.17.1.2) by a series of diphosphates and bisphosphonates. The most active species was an alkynyl diphosphate having IC(50) = 0.45 microM (K(i) approximately 60 nM), which generated a very large change in the 9 GHz EPR spectrum of the reduced protein. On the basis of previous work on organometallic complexes, together with computational docking and quantum chemical calculations, we propose a model for alkyne inhibition involving pi (or pi/sigma) "metallacycle" complex formation with the unique fourth Fe in the Fe(4)S(4) cluster. Aromatic species had less activity, and for these we propose an inhibition model based on an electrostatic interaction with the active site E126. Overall, the results are of broad general interest since they not only represent the first potent IspH inhibitors but also suggest a conceptually new approach to inhibiting other Fe(4)S(4)-cluster-containing proteins that are of interest as drug and herbicide targets.

Multifunctional nanoparticles combining therapy and imaging have the potential to improve cancer treatment by allowing personalized therapy. Herein, we aimed to compare in vivo different strategies in terms of targeting capabilities: (1) passive targeting via the EPR effect, (2) active targeting of αvβ3 integrin via RGD grafting, (3) magnetic targeting via a magnet placed on the tumor and (4) the combination of magnetic targeting and active targeting of αvβ3 integrin. For a translational approach, PLGA-based nanoparticles loaded with paclitaxel and superparamagnetic iron oxides were used. Electron Spin Resonance spectroscopy and Magnetic Resonance Imaging (MRI) were used to both quantify and visualize the accumulation of multifunctional nanoparticles into the tumors. We demonstrate that compared to untargeted or single targeted nanoparticles, the combination of both active strategy and magnetic targeting drastically enhanced (i) nanoparticle accumulation into the tumor tissue with an 8-fold increase compared to passive targeting (1.12% and 0.135% of the injected dose, respectively), (ii) contrast in MRI (imaging purpose) and (iii) anti-cancer efficacy with a median survival time of 22 days compared to 13 for the passive targeting (therapeutic purpose). Double targeting of nanoparticles to tumors by different mechanisms could be a promising translational approach for the management of therapeutic treatment and personalized therapy.

Despite several advancements in chemotherapy, the real therapy of cancer still remains a challenge. The development of new anti-cancer drugs for the treatment of cancer has not kept pace with the progress in cancer therapy, because of the nonspecific drug distribution resulting in low tumour concentrations and systemic toxicity. The main hindrance for the distribution of anti-cancer agents to the tumour site is the highly disorganized tumour vasculature, high blood viscosity in the tumour, and high interstitial pressure within the tumour tissue. Recently, several approaches such as drug modifications and development of new carrier systems for anti-cancer agents have been attempted to enhance their tumour reach. Approaches such as drug delivery through enhanced permeability and retention (EPR) effect have resulted in a significant improvement in concentration in tumours, while approaches such as drug-carrier implants and microparticles have resulted in improvement in local chemotherapy of cancer. This review discusses different strategies employed for the delivery of anti-cancer agents to tumours, such as through EPR effect, local chemotherapeutic approaches using drug delivery systems, and special strategies such as receptor-mediated delivery, pH-based carriers, application of ultrasound and delivery to resistant tumour cells and brain using nanoparticles.

A robust core-shell-corona micelle bearing redox-responsive shell-specific cross-links was evaluated as a carrier of docetaxel (DTX) for cancer therapy. The polymer micelles of poly(ethylene glycol)-b-poly(L-lysine)-b-poly(L-phenylalanine) (PEG-PLys-PPhe) in the aqueous phase provided the three distinct functional domains: the PEG outer corona for prolonged circulation, the PLys middle shell for disulfide cross-linking, and the PPhe inner core for DTX loading. The shell cross-linking was performed by the reaction of disulfide-containing cross-linkers with Lys moieties in the middle shells. The shell cross-linking did not change the micelle size or the spherical morphology. The shell cross-linked micelles exhibited enhanced serum stability. The DTX release from the DTX-loaded disulfide cross-linked micelles (DTX-SSCLM) was facilitated by increasing the concentration of glutathione (GSH). At an intracellular GSH level, DTX release was facilitated due to the reductive cleavage of the disulfide cross-links in the shell domains. The in vivo tissue distribution and tumor accumulation of the DTX-SSCLM that were labeled with a near-infrared fluorescence (NIRF) dye, Cy5.5, were monitored in MDA-MB231 tumor-bearing mice. Non-invasive real-time optical imaging results indicated that the DTX-SSCLM exhibited enhanced tumor specificity due to the prolonged stable circulation in blood and the enhanced permeation and retention (EPR) effect compared with the DTX-loaded non-cross-linked micelles (DTX-NCLM). The DTX-SSCLM exhibited enhanced therapeutic efficacy in tumor-bearing mice compared with free DTX and DTX-NCLM. The domain-specific shell cross-linking that is described in this work may serve as a useful guidance for enhancing the antitumor therapeutic efficacy of various polymer micelles and nano-aggregates.

Nanoparticle based delivery formulations have become a leading delivery strategy for cancer imaging and therapy. The success of nanoparticle-based therapy relies heavily on their ability to utilize the enhanced permeability and retention (EPR) effect and active targeting moieties to their advantage. However, these methods often fail to enable a uniform NP distribution across the tumor, and lead to insufficient local concentrations of drug. Oftentimes, this heterogeneous drug distribution is one of the primary reasons for suboptimal treatment efficacy in NP delivery platforms. Herein, we seek to examine the biophysical causes of heterogeneous NP distribution in stroma-rich desmoplastic tumors; namely the abnormal tumor vasculature, deregulated extracellular matrix and high interstitial hypertension associated with these tumors. It is suggested that these factors help explain the discrepancy between promising outlooks for many NP formulations in preclinical studies, but suboptimal clinical outcomes for most FDA approved nanoformulations. Furthermore, examination into the role of the physicochemical properties of NPs on successful drug delivery was conducted in this review. In light of the many formidable barriers against successful NP drug delivery, we provided possible approaches to mitigate delivery issues from the perspective of stromal remodeling and NP design. In all, this review seeks to provide guidelines for optimizing nanoparticle-based cancer drug delivery through both modified nanoparticle design and alleviation of biological barriers to successful therapy.

The distribution of the lipid-attached doxyl electron paramagnetic resonance (EPR) spin label in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine membranes has been studied by (1)H and (13)C magic angle spinning nuclear magnetic resonance relaxation measurements. The doxyl spin label was covalently attached to the 5th, 10th, and 16th carbons of the sn-2 stearic acid chain of a 1-palmitoyl-2-stearoyl-(5/10/16-doxyl)-sn-glycero-3-phosphocholine analog. Due to the unpaired electron of the spin label, (1)H and (13)C lipid relaxation rates are enhanced by paramagnetic relaxation. For all lipid segments the influence of paramagnetic relaxation is observed even at low probe concentrations. Paramagnetic relaxation rates provide a measure for the interaction strength between lipid segments and the doxyl group. Plotted along the membrane director a transverse distribution profile of the EPR probe is obtained. The chain-attached spin labels are broadly distributed in the membrane with a maximum at the approximate chain position of the probe. Both (1)H and (13)C relaxation measurements show these broad distributions of the doxyl group in the membrane indicating that (1)H spin diffusion does not influence the relaxation measurements. The broad distributions of the EPR label result from the high degree of mobility and structural heterogeneity in liquid-crystalline membranes. Knowing the distribution profiles of the EPR probes, their influence on relaxation behavior of membrane inserted peptide and protein segments can be studied by (13)C magic angle spinning nuclear magnetic resonance. As an example, the location of Ala residues positioned at three sites of the transmembrane WALP-16 peptide was investigated. All three doxyl-labeled phospholipid analogs induce paramagnetic relaxation of the respective Ala site. However, for well ordered secondary structures the strongest relaxation enhancement is observed for that doxyl group in the closest proximity to the respective Ala. Thus, this approach allows study of membrane insertion of protein segments with respect to the high molecular mobility in liquid-crystalline membranes.

One of the most important biological reactions of nitric oxide (nitrogen monoxide, *NO) is its reaction with transition metals, of which iron is the major target. This is confirmed by the ubiquitous formation of EPR-detectable g=2.04 signals in cells, tissues, and animals upon exposure to both exogenous and endogenous *NO. The source of the iron for these dinitrosyliron complexes (DNIC), and its relationship to cellular iron homeostasis, is not clear. Evidence has shown that the chelatable iron pool (CIP) may be at least partially responsible for this iron, but quantitation and kinetic characterization have not been reported. In the murine cell line RAW 264.7, *NO reacts with the CIP similarly to the strong chelator salicylaldehyde isonicotinoyl hydrazone (SIH) in rapidly releasing iron from the iron-calcein complex. SIH pretreatment prevents DNIC formation from *NO, and SIH added during the *NO treatment "freezes" DNIC levels, showing that the complexes are formed from the CIP, and they are stable (resistant to SIH). DNIC formation requires free *NO, because addition of oxyhemoglobin prevents formation from either *NO donor or S-nitrosocysteine, the latter treatment resulting in 100-fold higher intracellular nitrosothiol levels. EPR measurement of the CIP using desferroxamine shows quantitative conversion of CIP into DNIC by *NO. In conclusion, the CIP is rapidly and quantitatively converted to paramagnetic large molecular mass DNIC from exposure to free *NO but not from cellular nitrosothiol. These results have important implications for the antioxidative actions of *NO and its effects on cellular iron homeostasis.

We present a rather general and efficient method of simulating electron-spin echo spectra for spin systems where the microwave frequency does not simultaneously excite EPR transitions that share a common level. The approach can handle arbitrary pulse sequences with microwave pulses of arbitrary length and strength. The signal is computed as a sum over signals from the electron coherence transfer pathways contributing to the detected echo. For each pathway, amplitudes and frequencies of the signal components are computed and used to construct a spectral histogram from which the time-domain signal is obtained. For multinuclear spin systems, the nuclear subspace is factorized to accelerate the computation. The method is also applicable to high electron spin systems with significant zero-field splitting and to pulse electron-nuclear double resonance experiments. The method is implemented in the software package EasySpin, and several illustrative calculations are shown.

Inefficiencies in systemic drug delivery and tumor residence as well as micro-environmental selection pressures contribute to the development of multidrug resistance (MDR) in cancer. Characteristics of MDR include abnormal vasculature, regions of hypoxia, up-regulation of ABC-transporters, aerobic glycolysis, and an elevated apoptotic threshold. Nano-sized delivery vehicles are ideal for treating MDR cancer as they can improve the therapeutic index of drugs and they can be engineered to achieve multifunctional parameters. The multifunctional ability of nanocarriers makes them more adept at treating heterogeneous tumor mass than traditional chemotherapy. Nanocarriers also have preferential tumor accumulation via the EPR effect; this accumulation can be further enhanced by actively targeting the biological profile of MDR cells. Perhaps the most significant benefit of using nanocarrier drug delivery to treat MDR cancer is that nanocarrier delivery diverts the effects of ABC-transporter mediated drug efflux; which is the primary mechanism of MDR. This review discusses the capabilities, applications, and examples of multifunctional nanocarriers for the treatment of MDR. This review emphasizes multifunctional nanocarriers that enhance drug delivery efficiency, the application of RNAi, modulation of the tumor apoptotic threshold, and physical approaches to overcome MDR.

Liposomal formulations have been used to encapsulate and deliver a wide variety of therapeutic and diagnostic agents. Their circulation can be prolonged by the addition of neutral, hydrophilic polymers such as poly(ethylene glycol) (PEG) to the outer surface. An extended circulation lifetime allows them to take advantage of the enhanced permeability and retention effect (EPR), resulting in increased delivery to target sites. Incorporation of PEG also prevents aggregation and aids in the formation of uniform, small mono-disperse particles. This is often accomplished with the use of PEG-lipid conjugates, PEG molecules with a hydrophobic domain to anchor them into the liposomal bilayer upon formulation. Here we present data showing that some commonly used PEG-lipids are chemically unstable due to the presence of carboxylic ester bonds. This instability limits their utility in aqueous environments common to many liposomal preparations. To address this problem, we designed and synthesized three alternative PEG-lipids. Using SPLP (PEG-stabilized liposomal vesicles encapsulating plasmid DNA) as a model system, we investigated the properties of the novel PEG-lipids. An accelerated stability study was conducted at 37 degrees C for 42 days to confirm chemical stability and an in vivo model was used to assess the pharmacokinetics, toxicity and activity of the SPLP. We show that the novel PEG-lipids are more stable in liposomal formulation, less toxic upon systemic administration, and accordingly, are suitable replacements for the PEG-lipids described previously.

BACKGROUND

We aimed to evaluate the clinical usefulness of qSOFA as a risk stratification tool for patients admitted with infection compared to traditional SIRS criteria or our triage system; the Rapid Emergency Triage and Treatment System (RETTS).

METHODS

The study was an observational cohort study performed at one Emergency Department (ED) in an urban university teaching hospital in Norway, with approximately 20,000 visits per year. All patients >16 years presenting with symptoms or clinical signs suggesting an infection (n = 1535) were prospectively included in the study from January 1 to December 31, 2012. At arrival in the ED, vital signs were recorded and all patients were triaged according to RETTS vital signs, presenting infection, and sepsis symptoms. These admission data were also used to calculate qSOFA and SIRS. Treatment outcome was later retrieved from the patients' electronic records (EPR) and mortality data from the Norwegian population registry.

RESULTS

Of the 1535 admitted patients, 108 (7.0%) fulfilled the Sepsis2 criteria for severe sepsis. The qSOFA score ≥2 identified only 33 (sensitivity 0.32, specificity 0.98) of the patients with severe sepsis, whilst the RETTS-alert ≥ orange identified 92 patients (sensitivity 0.85, specificity 0.55). Twenty-six patients died within 7 days of admission; four (15.4%) of them had a qSOFA ≥2, and 16 (61.5%) had RETTS ≥ orange alert. Of the 68 patients that died within 30 days, only eight (11.9%) scored ≥2 on the qSOFA, and 45 (66.1%) had a RETTS ≥ orange alert.

CONCLUSIONS

In order to achieve timely treatment for sepsis, a sensitive screening tool is more important than a specific one. Our study is the fourth study were qSOFA finds few of the sepsis cases in prehospital or at arrival to the ED. We add information on the RETTS triage system, the two highest acuity levels together had a high sensitivity (85%) for identifying sepsis at arrival to the ED - and thus, RETTS should not be replaced by qSOFA as a screening and trigger tool for sepsis at arrival.

CONCLUSIONS

In this observational cohort study, qSOFA failed to identify two thirds of the patients admitted to an ED with severe sepsis. Further, qSOFA failed to be a risk stratification tool as the sensitivity to predict 7-day and 30-day mortality was low. The sensitivity was poorer than the other warning scores already in use at the study site, RETTS-triage and the SIRS criteria.

The dismal prognosis of patients with malignant brain tumors such as glioblastoma multiforme (GBM) is attributed mostly to their diffuse growth pattern and early microscopic tumor spread to distant regions of the brain. Because the microscopic tumor foci cannot be visualized with current imaging modalities, it remains impossible to direct treatments optimally. Here we explored the ability of integrin-targeted surface-enhanced resonance Raman spectroscopy (SERRS) nanoparticles to depict the true tumor extent in a GBM mouse model that closely mimics the pathology in humans. The recently developed SERRS-nanoparticles have a sensitivity of detection in the femtomolar range. An RGD-peptide-conjugated version for integrin-targeting (RGD-SERRS) was compared directly to its non-targeted RAD-SERRS control in the same mice via Raman multiplexing. Pre-blocking with RGD peptide before injection of RGD-SERRS nanoparticles was used to verify the specificity of integrin-targeting. In contrast to the current belief that the enhanced permeability and retention (EPR) effect results in a baseline uptake of nanoparticles regardless of their surface chemistry, integrin-targeting was shown to be highly specific, with markedly lower accumulation after pre-blocking. While the non-targeted SERRS particles enabled delineation of the main tumor, the RGD-SERRS nanoparticles afforded a major improvement in visualization of the true extent and the diffuse margins of the main tumor. This included the detection of unexpected tumor areas distant to the main tumor, tracks of migrating cells of 2-3 cells in diameter, and even isolated distant tumor cell clusters of less than 5 cells. This Raman spectroscopy-based nanoparticle-imaging technology holds promise to allow high precision visualization of the true extent of malignant brain tumors.

OBJECTIVE

This study evaluated a computerized method for extracting numeric clinical measurements related to diabetes care from free text in electronic patient records (EPR) of general practitioners.

METHODS

Accuracy of this number-oriented approach was compared to manual chart abstraction. Audits measured performance in clinical practice for two commonly used electronic record systems.

RESULTS

Numeric measurements embedded within free text of the EPRs constituted 80% of relevant measurements. For 11 of 13 clinical measurements, the study extraction method was 94%-100% sensitive with a positive predictive value (PPV) of 85%-100%. Post-processing increased sensitivity several points and improved PPV to 100%. Application in clinical practice involved processing times averaging 7.8 minutes per 100 patients to extract all relevant data.

CONCLUSIONS

The study method converted numeric clinical information to structured data with high accuracy, and enabled research and quality of care assessments for practices lacking structured data entry.

Desulfoferrodoxin, a non-heme iron protein, was purified previously from extracts of Desulfovibrio desulfuricans (ATCC 27774) (Moura, I., Tavares, P., Moura, J. J. G., Ravi, N., Huynh, B. H., Liu, M.-Y., and LeGall, J. (1990) J. Biol. Chem. 265, 21596-21602). The as-isolated protein displays a pink color (pink form) and contains two mononuclear iron sites in different oxidation states: a ferric site (center I) with a distorted tetrahedral sulfur coordination similar to that found in desulforedoxin from Desulfovibrio gigas and a ferrous site (center II) octahedrally coordinated with predominantly nitrogen/oxygen-containing ligands. A new form of desulfoferrodoxin which displays a gray color (gray form) has now been purified. Optical, electron paramagnetic resonance (EPR), and Mössbauer data of the gray desulfoferrodoxin indicate that both iron centers are in the high-spin ferric states. In addition to the EPR signals originating from center I at g = 7.7, 5.7, 4.1, and 1.8, the gray form of desulfoferrodoxin exhibits a signal at g = 4.3 and a shoulder at g = 9.6, indicating a high-spin ferric state with E/D approximately 1/3 for the oxidized center II. Redox titrations of the gray form of the protein monitored by optical spectroscopy indicate midpoint potentials of +4 +/- 10 and +240 +/- 10 mV for centers I and II, respectively. Mössbauer spectra of the gray form of the protein are consistent with the EPR finding that both centers are high-spin ferric and can be analyzed in terms of the EPR-determined spin Hamiltonian parameters. The Mössbauer parameters for both the ferric and ferrous forms of center II are indicative of a mononuclear high spin iron site with octahedral coordination and predominantly nitrogen/oxygen-containing ligands. Resonance Raman studies confirm the structural similarity of center I and the distorted tetrahedral FeS4 center in desulforedoxin and provide evidence for one or two cysteinyl-S ligands for center II. On the basis of the resonance Raman results, the 635 nm absorption band that is responsible for the gray color of the oxidized protein is assigned to a cysteinyl-S->>Fe(III) charge transfer transition localized on center II. The novel properties and possible function of center II are discussed in relation to those of mononuclear iron centers in other enzymes.

BACKGROUND

In heart failure, there is a sympathetically mediated hyperkinetic cardiovascular response to exercise that limits tolerance to physical activity. Alterations in skeletal muscle morphology and metabolism have led to the hypothesis that the exercise pressor reflex (EPR) becomes hyperactive after the development of cardiomyopathy and contributes to the exaggerated circulatory response elicited.

RESULTS

To test this hypothesis, Sprague-Dawley rats were divided into the following groups: control, sham, and dilated cardiomyopathy (DCM, induced by ischemic injury). Using transthoracic echocardiography, left ventricular fractional shortening was 47+/-2%, 44+/-1%, and 24+/-2% in control, sham, and DCM rats, respectively. Activation of the EPR by electrically induced static muscle contraction resulted in significantly larger increases in mean arterial pressure and heart rate in DCM animals (32+/-2 mm Hg, 13+/-1 bpm) compared with control (20+/-1 mm Hg, 8+/-1 bpm) and sham (20+/-2 mm Hg, 8+/-1 bpm) rats. Comparable results were obtained with selective stimulation of the mechanically sensitive component of the EPR by passive muscle stretch. The augmentations in EPR and mechanoreflex activity in DCM occurred progressively over a 10-week period, becoming greater as the severity of left ventricular dysfunction increased.

CONCLUSIONS

In DCM, the potentiated cardiovascular response to static muscle contraction is mediated, in part, by an exaggerated EPR. The muscle mechanoreflex contributes significantly to the EPR dysfunction that develops.

A mononuclear (1:1) copper complex of curcumin, a phytochemical from turmeric, was synthesized and examined for its superoxide dismutase (SOD) activity. The complex was characterized by elemental analysis, IR, NMR, UV-VIS, EPR, mass spectroscopic methods and TG-DTA, from which it was found that a copper atom is coordinated through the keto-enol group of curcumin along with one acetate group and one water molecule. Cyclic voltammetric studies of the complex showed a reversible Cu(2+)/Cu(+) couple with a potential of 0.402 V vs NHE. The Cu(II)-curcumin complex is soluble in lipids and DMSO, and insoluble in water. It scavenges superoxide radicals with a rate constant of 1.97 x 10(5) M(-1) s(-1) in DMSO determined by stopped-flow spectrometer. Subsequent to the reaction with superoxide radicals, the complex was found to be regenerated completely, indicating catalytic activity in neutralizing superoxide radicals. Complete regeneration of the complex was observed, even when the stoichiometry of superoxide radicals was 10 times more than that of the complex. This was further confirmed by EPR monitoring of superoxide radicals. The SOD mimicking activity of the complex was determined by xanthine/xanthine oxidase assay, from which it has been found that 5 microg of the complex is equivalent to 1 unit of SOD. The complex inhibits radiation-induced lipid peroxidation and shows radical-scavenging ability. It reacts with DPPH radicals with rate constant 10 times less than that of curcumin. Pulse radiolysis-induced one-electron oxidation of the complex by azide radicals in TX-100 micellar solutions produced strongly absorbing ( approximately 500 nm) phenoxyl radicals, indicating that the phenolic moiety of curcumin remained intact on complexation with copper. The results confirm that the new Cu(II)-curcumin complex possesses SOD activity, free radical neutralizing ability, and antioxidant potential. Quantum chemical calculations with density functional theory have been performed to support the experimental observations.

Oxomanganese(V) species have been implicated in a variety of biological and synthetic processes, including their role as a key reactive center within the oxygen-evolving complex in photosynthesis. Nearly all mononuclear Mn(V)-oxo complexes have tetragonal symmetry, producing low-spin species. A new high-spin Mn(V)-oxo complex that was prepared from a well-characterized oxomanganese(III) complex having trigonal symmetry is now reported. Oxidation experiments with [FeCp(2)](+) were monitored with optical and electron paramagnetic resonance (EPR) spectroscopies and support a high-spin oxomanganese(V) complex formulation. The parallel-mode EPR spectrum has a distinctive S = 1 signal at g = 4.01 with a six-line hyperfine pattern having A(z) = 113 MHz. The presence of an oxo ligand was supported by resonance Raman spectroscopy, which revealed O-isotope-sensitive peaks at 737 and 754 cm(-1) assigned as a Fermi doublet centered at 746 cm(-1)(Δ(18)O = 31 cm(-1)). Mn Kβ X-ray emission spectra showed Kβ' and Kβ(1,3) bands at 6475.92 and 6490.50 eV, respectively, which are characteristic of a high-spin Mn(V) center.