MaxCal can infer models from coupled stochastic trajectories of gene expression and cell division.

Gene expression is inherently noisy due to small numbers of proteins and nucleic acids inside a cell. Likewise, cell division is stochastic, particularly when tracking at the level of a single cell. The two can be coupled when gene expression affects the rate of cell division. Single-cell time-lapse experiments can measure both fluctuations by simultaneously recording protein levels inside a cell and its stochastic division. These information-rich noisy trajectory data sets can be harnessed to learn about the underlying molecular and cellular details that are often not known a priori. A critical question is: How can we infer a model given data where fluctuations at two levels-gene expression and cell division-are intricately convoluted? We show the principle of maximum caliber (MaxCal)-integrated within a Bayesian framework-can be used to infer several cellular and molecular details (division rates, protein production, and degradation rates) from these coupled stochastic trajectories (CSTs). We demonstrate this proof of concept using synthetic data generated from a known model. An additional challenge in data analysis is that trajectories are often not in protein numbers, but in noisy fluorescence that depends on protein number in a probabilistic manner. We again show that MaxCal can infer important molecular and cellular rates even when data are in fluorescence, another example of CST with three confounding factors-gene expression noise, cell division noise, and fluorescence distortion-all coupled. Our approach will provide guidance to build models in synthetic biology experiments as well as general biological systems where examples of CSTs are abundant.

How to Better Value EMS Clinicians as Key Care Team Members.

Emergency medical services (EMS) clinicians, including emergency medical technicians and paramedics, are skilled professionals whose expertise is leveraged routinely to meet a wide range of patient needs. Collaborative interdisciplinary care requires mutual understanding, trust, and respect. Yet, among EMS clinicians and in- and out-of-hospital clinicians, these values are too often not expressed in working relationships. This article offers guidance on how to nourish successful partnerships with EMS clinicians and motivate good care.

Thromboelastography During Rewarming for Management of Pediatric Cardiac Surgery Patients.

BACKGROUND: Thromboelastography (TEG) predicts bleeding in pediatric patients undergoing cardiac surgical procedure. We hypothesize that TEG indicators at rewarming correlate with postprotamine values and that rewarming TEG is associated with surrogate end points for postoperative bleeding in pediatric patients undergoing complex cardiac surgical procedure. METHODS: In a retrospective study of 703 pediatric (≤18 years) patients undergoing complex cardiac surgical procedures, TEG results obtained during rewarming and after protamine administration were compared using linear regression. A composite end point of extended blood product transfusion or surgical reexploration for bleeding was used as a surrogate for postoperative bleeding. RESULTS: By multivariable analysis, longer cardiopulmonary bypass time and lower TEG maximal amplitude (MA) during rewarming were independently associated with the risk of the composite end point in the operating room or in the intensive care unit (P < .05). Among patients with an MA of less than 45 mm during rewarming, those who received a platelet transfusion in the operating room compared with those who did not were less likely to reach the composite end point within the subsequent 24 hours (8% vs 32%, respectively; P < .01). Good correlation was observed between TEG variables at rewarming vs after protamine administration (Pearson r ≥ 0.7). The relationship between platelet transfusion volume (mL/kg) and the percentage change in the MA was determined using linear regression, and a platelet transfusion calculator was generated. CONCLUSIONS: A lower MA during rewarming is associated with an increased risk of perioperative bleeding. In patients with a rewarming MA of less than 45 mm, an intraoperative platelet transfusion may reduce the risk of subsequent bleeding. Individualized platelet transfusion therapy based on rewarming TEG may reduce the risk of bleeding while minimizing unnecessary platelet transfusion.

High fat diet causes inferior vertebral structure and function without disc degeneration in RAGE-KO mice.

Back pain and spinal pathologies are associated with obesity in juveniles and adults, yet studies identifying causal relationships are lacking and none investigate sex differences. This study determined if high fat (HF) diet causes structural and functional changes to vertebrae and intervertebral discs (IVDs); if these changes are modulated in mice with systematic ablation for the receptor for advanced glycation endproducts (RAGE-KO); and if these changes are sex-dependent. Wild-type (WT) and RAGE-KO mice were fed a low fat (LF) or HF diet for 12 weeks starting at 6 weeks, representing the juvenile population. HF diet led to weight/fat gain, glucose intolerance, and increased cytokine levels (IL-5, MIG, and RANTES); with less fat gain in RAGE-KO females. Most importantly, HF diet reduced vertebral trabecular bone volume fraction and compressive and shear moduli, without a modifying effect of RAGE-KO, but with a more pronounced effect in females. HF diet caused reduced cortical area fraction only in WT males. Neither HF diet nor RAGE-KO affected IVD degeneration grade. Biomechanical properties of coccygeal motion segments were affected by RAGE-KO but not diet, with some interactions identified. In conclusion, HF diet resulted in inferior vertebral structure and function with some sex differences, no IVD degeneration, and few modifying effects of RAGE-KO. These structural and functional deficiencies with HF diet provide further evidence that diet can affect spinal structures and may increase the risk for spinal injury and degeneration with aging and additional stressors. Back pain and spinal pathologies are associated with obesity in juveniles and adults, yet studies identifying causal relationships are lacking and none investigate sex differences.

Electrical stimulation of whole blood for growth factor release and potential clinical implications.

Clinicians have increasingly applied platelet-rich plasma (PRP) for wound healing treatments. Topical treatments commonly require biochemical agents such as bovine thrombin to activate PRP ex vivo for clotting and growth factor release to facilitate healing upon application to the wound of interest. Recent studies have explored electrical stimulation as an alternative to bovine thrombin for PRP activation due to the former's cost, workflow complexity and potentially significant side effects; however, both approaches require separating the PRP from whole blood (WB) prior to activation. Eliminating the separation (typically centrifugation) step would reduce the cost and duration of the clinical procedure, which may be critical in trauma and surgical applications. We hypothesize that electric pulses (EPs) can release growth factors from WB, as they do from PRP, without requiring centrifugation of WB into PRP. A pilot study for two donors demonstrates the potential for EP stimulated growth factor release from WB. This motivates future experiments assessing EP parameter optimization for WB activation and in vivo studies to determine the clinical benefits for topical treatments and, especially, for injections in orthopedic applications that already utilize non-treated/non-activated WB.

Tunable activation of therapeutic platelet-rich plasma by pulse electric field: Differential effects on clot formation, growth factor release, and platelet morphology.

BACKGROUND: Activation of platelet-rich plasma (PRP) by pulse electric field (PEF) releases growth factors which promote wound healing (e.g., PDGF, VEGF for granulation, EGF for epithelialization). AIMS: To determine after PEF activation of therapeutic PRP: 1) platelet gel strength; 2) profile of released growth factors; 3) alpha- and T-granule release; 4) platelet morphology. METHODS: Concentrated normal donor PRP was activated by 5 µsec (long) monopolar pulse, ~4000 V/cm (PEF A) or 150 nsec (short) bipolar pulse, ~3000 V/cm (PEF B) in the presence of 2.5 mM (low) or 20 mM (high) added CaCl2. Clot formation was evaluated by thromboelastography (TEG). Surface exposure of alpha granule (P-selectin) and T-granule (TLR9 and protein disulfide isomerase [PDI]) markers were assessed by flow cytometry. Factors in supernatants of activated PRP were measured by ELISA. Platelet morphology was evaluated by transmission electron microscopy (TEM). RESULTS: Time to initial clot formation was shorter with thrombin (<1 min) than with PEF A and B (4.4-8.7 min) but clot strength (elastic modulus, derived from TEG maximum amplitude) was greater with PEF B than with either thrombin or PEF A (p<0.05). Supernatants of PRP activated with PEF A had higher EGF levels than supernatants from all other conditions. In contrast, levels of PF4, PDGF, and VEGF in supernatants were not significantly different after PEF A, PEF B, and thrombin activation. T-granule markers (TLR9 and PDI) were higher after thrombin than after PEF A or B with low or high CaCl2. By TEM, platelets in PEF-treated samples retained a subset of granules suggesting regulated granule release. CONCLUSION: Pulse length and polarity can be modulated to produce therapeutic platelet gels as strong or stronger than those produced by thrombin, and this is tunable to produce growth factor profiles enhanced in specific factors important for different stages of wound healing.

Design, characterization and experimental validation of a compact, flexible pulsed power architecture for ex vivo platelet activation.

Electric pulses can induce various changes in cell dynamics and properties depending upon pulse parameters; however, pulsed power generators for in vitro and ex vivo applications may have little to no flexibility in changing the pulse duration, rise- and fall-times, or pulse shape. We outline a compact pulsed power architecture that operates from hundreds of nanoseconds (with the potential for modification to tens of nanoseconds) to tens of microseconds by modifying a Marx topology via controlling switch sequences and voltages into each capacitor stage. We demonstrate that this device can deliver pulses to both low conductivity buffers, like standard pulsed power supplies used for electroporation, and higher conductivity solutions, such as blood and platelet rich plasma. We further test the effectiveness of this pulse generator for biomedical applications by successfully activating platelets ex vivo with 400 ns and 600 ns electric pulses. This novel bioelectrics platform may provide researchers with unprecedented flexibility to explore a wide range of pulse parameters that may induce phenomena ranging from intracellular to plasma membrane manipulation.

Modification of Pulsed Electric Field Conditions Results in Distinct Activation Profiles of Platelet-Rich Plasma.

BACKGROUND: Activated autologous platelet-rich plasma (PRP) used in therapeutic wound healing applications is poorly characterized and standardized. Using pulsed electric fields (PEF) to activate platelets may reduce variability and eliminate complications associated with the use of bovine thrombin. We previously reported that exposing PRP to sub-microsecond duration, high electric field (SMHEF) pulses generates a greater number of platelet-derived microparticles, increased expression of prothrombotic platelet surfaces, and differential release of growth factors compared to thrombin. Moreover, the platelet releasate produced by SMHEF pulses induced greater cell proliferation than plasma. AIMS: To determine whether sub-microsecond duration, low electric field (SMLEF) bipolar pulses results in differential activation of PRP compared to SMHEF, with respect to profiles of activation markers, growth factor release, and cell proliferation capacity. METHODS: PRP activation by SMLEF bipolar pulses was compared to SMHEF pulses and bovine thrombin. PRP was prepared using the Harvest SmartPreP2 System from acid citrate dextrose anticoagulated healthy donor blood. PEF activation by either SMHEF or SMLEF pulses was performed using a standard electroporation cuvette preloaded with CaCl2 and a prototype instrument designed to take into account the electrical properties of PRP. Flow cytometry was used to assess platelet surface P-selectin expression, and annexin V binding. Platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), endothelial growth factor (EGF) and platelet factor 4 (PF4), and were measured by ELISA. The ability of supernatants to stimulate proliferation of human epithelial cells in culture was also evaluated. Controls included vehicle-treated, unactivated PRP and PRP with 10 mM CaCl2 activated with 1 U/mL bovine thrombin. RESULTS: PRP activated with SMLEF bipolar pulses or thrombin had similar light scatter profiles, consistent with the presence of platelet-derived microparticles, platelets, and platelet aggregates whereas SMHEF pulses primarily resulted in platelet-derived microparticles. Microparticles and platelets in PRP activated with SMLEF bipolar pulses had significantly lower annexin V-positivity than those following SMHEF activation. In contrast, the % P-selectin positivity and surface P-selectin expression (MFI) for platelets and microparticles in SMLEF bipolar pulse activated PRP was significantly higher than that in SMHEF-activated PRP, but not significantly different from that produced by thrombin activation. Higher levels of EGF were observed following either SMLEF bipolar pulses or SMHEF pulses of PRP than after bovine thrombin activation while VEGF, PDGF, and PF4 levels were similar with all three activating conditions. Cell proliferation was significantly increased by releasates of both SMLEF bipolar pulse and SMHEF pulse activated PRP compared to plasma alone. CONCLUSIONS: PEF activation of PRP at bipolar low vs. monopolar high field strength results in differential platelet-derived microparticle production and activation of platelet surface procoagulant markers while inducing similar release of growth factors and similar capacity to induce cell proliferation. Stimulation of PRP with SMLEF bipolar pulses is gentler than SMHEF pulses, resulting in less platelet microparticle generation but with overall activation levels similar to that obtained with thrombin. These results suggest that PEF provides the means to alter, in a controlled fashion, PRP properties thereby enabling evaluation of their effects on wound healing and clinical outcomes.

A Proposed Computed Tomography Contrast Agent Using Carboxybetaine Zwitterionic Tantalum Oxide Nanoparticles: Imaging, Biological, and Physicochemical Performance.

OBJECTIVES: The aim of this study was to produce and evaluate a proposed computed tomography (CT) contrast agent based on carboxybetaine zwitterionic (CZ)-coated soluble tantalum oxide (TaO) nanoparticles (NPs). We chose tantalum to provide superior imaging performance compared with current iodine-based clinical CT contrast agents. We developed the CZ coating to provide biological and physical performance similar to that of current iodinated contrast agents. In addition, the aim of this study was to evaluate the imaging, biological, and physicochemical performance of this proposed contrast agent compared with clinically used iodinated agents. MATERIALS AND METHODS: We evaluated CT imaging performance of our CZ-TaO NPs compared with that of an iodinated agent in live rats, imaged centrally located within a tissue-equivalent plastic phantom that simulated a large patient. To evaluate vascular contrast enhancement, we scanned the rats' great vessels at high temporal resolution during and after contrast agent injection. We performed several in vivo CZ-TaO NP studies in healthy rats to evaluate tolerability. These studies included injecting the agent at the anticipated clinical dose (ACD) and at 3 times and 6 times the ACD, followed by longitudinal hematology to assess impact to blood cells and organ function (from 4 hours to 1 week). Kidney histological analysis was performed 48 hours after injection at 3 times the ACD. We measured the elimination half-life of CZ-TaO NPs from blood, and we monitored acute kidney injury biomarkers with a kidney injury assay using urine collected from 4 hours to 1 week. We measured tantalum retention in individual organs and in the whole carcass 48 hours after injection at ACD. Carboxybetaine zwitterionic TaO NPs were synthesized and analyzed in detail. We used multidimensional nuclear magnetic resonance to determine surface functionality of the NPs. We measured NP size and solution properties (osmolality and viscosity) of the agent over a range of tantalum concentrations, including the high concentrations required for standard clinical CT imaging. RESULTS: Computed tomography imaging studies demonstrated image contrast improvement of approximately 40% to 50% using CZ-TaO NPs compared with an iodinated agent injected at the same mass concentration. Blood and organ analyses showed no adverse effects after injection in healthy naive rats at 3 times the ACD. Retention of tantalum at 48 hours after injection was less than 2% of the injected dose in the whole carcass, which very closely matched the reported retention of existing commercial iodine-based contrast agents. Urine analysis of sensitive markers for acute kidney injury showed no responses at 1 week after injection at 3 times the ACD; however, a moderate response in the neutrophil gelatinase-associated lipocalin biomarker was measured at 24 and 48 hours. Compared with other TaO NPs reported in the literature, CZ-TaO NPs had relatively low osmolality and viscosity at concentrations greater than 200 mg Ta/mL and were similar in these physical properties to dimeric iodine-based contrast agents. CONCLUSIONS: We found that a CZ-TaO NP-based contrast agent is potentially viable for general-purpose clinical CT imaging. Our results suggest that such an agent can be formulated with clinically viable physicochemical properties, can be biologically safe and cleared rapidly in urine, and can provide substantially improved image contrast at CT compared with current iodinated agents.

In vivo comparison of tantalum, tungsten, and bismuth enteric contrast agents to complement intravenous iodine for double-contrast dual-energy CT of the bowel.

To assess the ability of dual-energy CT (DECT) to separate intravenous contrast of bowel wall from intraluminal contrast, we scanned 16 rabbits on a clinical DECT scanner: n = 3 using only iodinated intravenous contrast, and n = 13 double-contrast enhanced scans using iodinated intravenous contrast and experimental enteric non-iodinated contrast agents in the bowel lumen (five bismuth, four tungsten, and four tantalum based). Representative image pairs from conventional CT images and DECT iodine density maps of small bowel (116 pairs from 232 images) were viewed by four abdominal imaging attending radiologists to independently score each comparison pair on a visual analog scale (-100 to +100%) for (1) preference in small bowel wall visualization and (2) preference in completeness of intraluminal enteric contrast subtraction. Median small bowel wall visualization was scored 39 and 42 percentage points (95% CI 30-44% and 36-45%, both p < 0.001) higher for double-contrast DECT than for conventional CT with enteric tungsten and tantalum contrast, respectively. Median small bowel wall visualization for double-contrast DECT was scored 29 and 35 percentage points (95% CI 20-35% and 33-39%, both p < 0.001) higher with enteric tungsten and tantalum, respectively, than with bismuth contrast. Median completeness of intraluminal enteric contrast subtraction in double-contrast DECT iodine density maps was scored 28 and 29 percentage points (95% CI 15-31% and 28-33%, both p < 0.001) higher with enteric tungsten and tantalum, respectively, than with bismuth contrast. Results suggest that in vivo double-contrast DECT with iodinated intravenous and either tantalum- or tungsten-based enteric contrast provides better visualization of small bowel than conventional CT. Copyright © 2016 John Wiley & Sons, Ltd.

CT Image Contrast of High-Z Elements: Phantom Imaging Studies and Clinical Implications.

PURPOSE: To quantify the computed tomographic (CT) image contrast produced by potentially useful contrast material elements in clinically relevant imaging conditions. MATERIALS AND METHODS: Equal mass concentrations (grams of active element per milliliter of solution) of seven radiodense elements, including iodine, barium, gadolinium, tantalum, ytterbium, gold, and bismuth, were formulated as compounds in aqueous solutions. The compounds were chosen such that the active element dominated the x-ray attenuation of the solution. The solutions were imaged within a modified 32-cm CT dose index phantom at 80, 100, 120, and 140 kVp at CT. To simulate larger body sizes, 0.2-, 0.5-, and 1.0-mm-thick copper filters were applied. CT image contrast was measured and corrected for measured concentrations and presence of chlorine in some compounds. RESULTS: Each element tested provided higher image contrast than iodine at some tube potential levels. Over the range of tube potentials that are clinically practical for average-sized and larger adults-that is, 100 kVp and higher-barium, gadolinium, ytterbium, and tantalum provided consistently increased image contrast compared with iodine, respectively demonstrating 39%, 56%, 34%, and 24% increases at 100 kVp; 39%, 66%, 53%, and 46% increases at 120 kVp; and 40%, 72%, 65%, and 60% increases at 140 kVp, with no added x-ray filter. CONCLUSION: The consistently high image contrast produced with 100-140 kVp by tantalum compared with bismuth and iodine at equal mass concentration suggests that tantalum could potentially be favorable for use as a clinical CT contrast agent.

Blood protein predictors of brain amyloid for enrichment in clinical trials?

BACKGROUND: Measures of neocortical amyloid burden (NAB) identify individuals who are at substantially greater risk of developing Alzheimer's disease (AD). Blood-based biomarkers predicting NAB would have great utility for the enrichment of AD clinical trials, including large-scale prevention trials. METHODS: Nontargeted proteomic discovery was applied to 78 subjects from the Australian Imaging, Biomarkers and Lifestyle Flagship Study of Ageing with a range of NAB values. Technical and independent replications were performed by immunoassay. RESULTS: Seventeen discovery candidates were selected for technical replication. α2-Macroglobulin, fibrinogen γ-chain (FGG), and complement factor H-related protein 1 were confirmed to be associated with NAB. In an independent cohort, FGG plasma levels combined with age predicted NAB had a sensitivity of 59% and specificity of 78%. CONCLUSION: A single blood protein, FGG, combined with age, was shown to relate to NAB and therefore could have potential for enrichment of clinical trial populations.

Complementary contrast media for metal artifact reduction in dual-energy computed tomography.

Metal artifacts have been a problem associated with computed tomography (CT) since its introduction. Recent techniques to mitigate this problem have included utilization of high-energy (keV) virtual monochromatic spectral (VMS) images, produced via dual-energy CT (DECT). A problem with these high-keV images is that contrast enhancement provided by all commercially available contrast media is severely reduced. Contrast agents based on higher atomic number elements can maintain contrast at the higher energy levels where artifacts are reduced. This study evaluated three such candidate elements: bismuth, tantalum, and tungsten, as well as two conventional contrast elements: iodine and barium. A water-based phantom with vials containing these five elements in solution, as well as different artifact-producing metal structures, was scanned with a DECT scanner capable of rapid operating voltage switching. In the VMS datasets, substantial reductions in the contrast were observed for iodine and barium, which suffered from contrast reductions of 97% and 91%, respectively, at 140 versus 40 keV. In comparison under the same conditions, the candidate agents demonstrated contrast enhancement reductions of only 20%, 29%, and 32% for tungsten, tantalum, and bismuth, respectively. At 140 versus 40 keV, metal artifact severity was reduced by 57% to 85% depending on the phantom configuration.

Platelet activation using electric pulse stimulation: growth factor profile and clinical implications.

BACKGROUND: Autologous platelet gel therapy using platelet-rich plasma has emerged as a promising alternative for chronic wound healing, hemostasis, and wound infection control. A critical step for this therapeutic approach is platelet activation, typically performed using bovine thrombin (BT) and calcium chloride. However, exposure of humans to BT can stimulate antibody formation, potentially resulting in severe hemorrhagic or thrombotic complications. Electric pulse stimulation using nanosecond PEFs (pulse electric fields) is an alternative, nonbiochemical platelet activation method, thereby avoiding exposure to xenogeneic thrombin and associated risks. METHODS: In this study, we identified specific requirements for a clinically relevant activator instrument by dynamically measuring current, voltage, and electric impedance for platelet-rich plasma samples. From these samples, we investigated the profile of growth factors released from human platelets with electric pulse stimulation versus BT, specifically platelet-derived growth factor, transforming growth factor ß, and epidermal growth factor, using commercial enzyme-linked immunosorbent assay kits. RESULTS: Electric pulse stimulation triggers growth factor release from platelet α-granules at the same or higher level compared with BT. CONCLUSION: Electric pulse stimulation is a fast, inexpensive, easy-to-use platelet activation method for autologous platelet gel therapy.

Biological performance of a size-fractionated core-shell tantalum oxide nanoparticle x-ray contrast agent.

OBJECTIVES: Metal-containing nanoparticles show great promise as x-ray contrast media and could enable reduced radiation dose, increased contrast, and the visualization of smaller anatomic features. In this study, we report progress toward these goals using a size-fractionated core-shell tantalum oxide nanoparticle contrast agent. MATERIALS AND METHODS: A core-shell tantalum oxide nanoparticle contrast agent was synthesized and size fractionated for preclinical investigation of biodistribution, blood half-life, organ retention, and histopathology. Fractionated agent was injected at anticipated clinical dose and at 3 times the anticipated clinical dose to evaluate biological performance. Computed tomography (CT) imaging studies were also performed to evaluate short-term clearance kinetics and new imaging applications. RESULTS: Improved control of 2-diethylphosphatoethylsilane-TaO nanoparticle size resulted in significantly reduced retention of injected tantalum. In vivo and in vitro CT imaging studies demonstrated short-term biodistribution differences in the kidney between small-molecule iodinated contrast media and fractionated 2-diethylphosphatoethylsilane-TaO, as well as preliminary data about new "Ta-only" imaging applications using multienergy CT image acquisition. CONCLUSIONS: Size-fractionated core-shell tantalum oxide nanoparticles with a well-defined particle size distribution have several key features required of clinically viable vascular imaging compounds and may be used in developing multienergy CT imaging applications.

Preclinical assessment of a zwitterionic tantalum oxide nanoparticle X-ray contrast agent.

Tantalum oxide nanoparticles show great potential as the next generation of X-ray contrast media. Recently, we reported advances in tantalum oxide nanoparticles and identified improvements that were required for such particles to progress further. Namely, the viscosity of concentrated particles, the amount of retention in reticuloendothelial (RES) tissues, and the effect of large quantities of particles on the kidneys after administration were all identified as critical factors which needed further study, understanding, and development. Here, we report on a zwitterionic siloxane polymer nanoparticle coating that reduced the viscosity of concentrated solutions of particles by a factor of 5, decreased tissue retention of injected particles by a factor of 10, and, importantly, did not induce pathological responses in the kidneys.

Synthesis, characterization, and computed tomography imaging of a tantalum oxide nanoparticle imaging agent.

Water-soluble ≤6 nm tantalum oxide nanoparticles have been synthesized and characterized in solution using HPLC-ICP, DLS, and multinuclear NMR. Nanoparticle formulation permitted intravenous injection, in vivo imaging, and subsequent renal clearance. A clinical CT scanner provided excellent resolution following agent injection, and distribution to the arterial system was visualized. In vitro CT imaging studies indicated that at equal molar concentration of tantalum and iodine, tantalum produced greater image contrast than iodine across the diagnostic X-ray spectrum with contrast benefit increasing with peak X-ray energy.

MicroRNA target detection and analysis for genes related to breast cancer using MDLcompress.

We describe initial results of miRNA sequence analysis with the optimal symbol compression ratio (OSCR) algorithm and recast this grammar inference algorithm as an improved minimum description length (MDL) learning tool: MDLcompress. We apply this tool to explore the relationship between miRNAs, single nucleotide polymorphisms (SNPs), and breast cancer. Our new algorithm outperforms other grammar-based coding methods, such as DNA Sequitur, while retaining a two-part code that highlights biologically significant phrases. The deep recursion of MDLcompress, together with its explicit two-part coding, enables it to identify biologically meaningful sequence without needlessly restrictive priors. The ability to quantify cost in bits for phrases in the MDL model allows prediction of regions where SNPs may have the most impact on biological activity. MDLcompress improves on our previous algorithm in execution time through an innovative data structure, and in specificity of motif detection (compression) through improved heuristics. An MDLcompress analysis of 144 over expressed genes from the breast cancer cell line BT474 has identified novel motifs, including potential microRNA (miRNA) binding sites that are candidates for experimental validation.

Oxygen-induced maturation of SOD1: a key role for disulfide formation by the copper chaperone CCS.

The antioxidant enzyme Cu,Zn-superoxide dismutase (SOD1) has the distinction of being one of the most abundant disulfide-containing protein known in the eukaryotic cytosol; however, neither catalytic nor physiological roles for the conserved disulfide are known. Here we show that the disulfide status of Saccharomyces cerevisiae SOD1 significantly affects the monomer-dimer equilibrium, the interaction with the copper chaperone CCS, and the activity of the enzyme itself. Disulfide formation in SOD1 by O2 is slow but is greatly accelerated by the Cu-bound form of CCS (Cu-CCS) in vivo and in vitro even in the presence of excess reductants; once formed, this disulfide is kinetically stable. Biochemical assays reveal that Cu-CCS facilitates Cys oxidation and disulfide isomerization in the stepwise conversion of the immature form of the enzyme to the active state. The immature form of SOD1 is most susceptible to oxidative insult and to aggregation reminiscent of that observed in amyotrophic lateral sclerosis. Thus Cu-CCS mediation of correct disulfide formation in SOD1 is important for regulation of enzyme activity and for prevention of misfolding or aggregation.

Oxygen and the copper chaperone CCS regulate posttranslational activation of Cu,Zn superoxide dismutase.

Oxidative stress leads to the up-regulation of many antioxidant enzymes including Cu,Zn superoxide dismutase (SOD1) via transcriptional mechanisms; however, few examples of posttranslational regulation are known. The copper chaperone for SOD1 (CCS) is involved in physiological SOD1 activation, and its primary function is thought to be delivery of copper to the enzyme. Data presented here are consistent with a previously uncharacterized function for CCS in the SOD1 pathway, namely mediating enzyme activation in response to increases in oxygen tension. Activity assays with pure proteins and cell extracts reveal that O(2) (or superoxide) is required for activation of SOD1 by CCS. Dose-response studies with a translational blocking agent demonstrate that the cellular oxidative response to O(2) is multitiered: existing apo-pools of SOD1 are activated by CCS in the early response, followed by increasing expression of SOD1 protein with persistent oxidative stress. This CCS function provides oxidant-responsive posttranslational regulation of SOD1 activity and may be relevant to a wide array of physiological stresses that involve a sudden elevation of oxygen availability.