Using a Graph database to identify NFV domain monitors during run-time

This disclosure relates to the field of Infrastructure in virtualized performance monitoring of Virtual Network Functions’ standard resources built on ETSI Guidelines. In real-time, the monitoring requirements are identified using a graph database with a faster, more efficient and monitoring identification made easier using graph data-modelling techniques

Voltage-driven Biocatalysis by Membrane-bound Liver Enzymes

Human liver microsomes (HLMs) contain the major drug metabolizing cytochrome P450 (CYP) enzyme, and their redox partner protein, CYP-NADPH reductase (CPR). Due to the broad range of biocatalytic reactions catalyzed by CYP enzymes, chemists have focused on the structure-function, biosensing, and catalytic applications of this unique class of enzymes. For drug development, HLMs are being used as the in vitro system to study drug metabolism, inhibition, isoform and metabolite profiling, biosensing applications, and drug-drug interactions. My research focus is to understand the electrocatalytic pathways of human and bacterial (bactosomes) membrane-bound liver CYP enzymes as a new bioelectronics platform for drug development and biosensing. We designed a biologically active bactosomal film on a self-assembled monolayer surface, and compared the electrocatalytic properties of CYP, CPR, and the combination of these two expressed in bactosomes. In addition, we developed a human liver microsomal bioreactor constructed on carbon nanostructure electrodes for applications in stereoselective drug metabolite synthesis. We extended this carbon nanostructure approach with magnetic nanomaterials to develop sensitive biosensors and volume-efficient electrocatalytic systems. My other short-term project was to utilize electrochemistry to understand meat color attributes based on redox properties of myoglobin from different species studied under various meat relevant pH conditions.Chemistr

Magnetite-Quantum Dot Immunoarray for Plasmon-Coupled-Fluorescence Imaging of Blood Insulin and Glycated Hemoglobin

New microarray chip strategies that are sensitive and selective and that can measure low levels of important biomarkers directly in a blood sample are significant for improving human health by allowing timely diagnosis of an abnormal condition. Herein, we designed an antibody–aptamer immunoarray chip to demonstrate simultaneous measurement of blood insulin and glycated hemoglobin (HbA1c) levels relevant to diabetic and prediabetic disorders using a surface plasmon microarray with validation by fluorescence imaging. To accomplish both surface plasmon and fluorescence imaging on the same sample, we decorated magnetite nanoparticles with quantum dots for covalent immobilization of aptamers for subsequent capture and isolation of the aptamers specific for insulin and HbA1c markers from 20-times diluted whole blood samples. Direct clinically relevant analysis, along with fluorescent imaging of the two markers, was achieved by this new immunoarray platform. The limit of detection was 4 pM for insulin and 1% for HbA1c. Examination of cross-talk using thrombin and platelet-derived growth factor confirmed that the designed immunoarray was highly selective for insulin and HbA1c. Surface plasmon kinetic analysis provided apparent binding constants of 0.24 (±0.08) nM and 37 (±3) μM, respectively, for the binding of insulin and HbA1c onto their surface immobilized monoclonal antibodies. Thus, quantitative imaging of ultralow levels of blood biomarker levels with binding kinetics is uniquely obtained in the designed immunoarray chip. In conclusion, this report demonstrates considerable significance of the developed magnetite-quantum dot-bioconjugate strategy for clinical diagnostics of whole blood biomarkers with characterization of molecular binding interactions

Species-Specificity in Myoglobin Oxygenation and Reduction Potential Properties

The objective was to compare oxygenation and reduction potential properties of bovine and porcine myoglobins in-vitro. Cyclic voltammetry and homology-based myoglobin modeling were used to determine the species-specific effects on myoglobin reduction potential and oxygenation properties at pH 5.6, 6.4, and 7.4. At all pHs, porcine myoglobin had greater (P = 0.04) oxygen affinity than bovine myoglobin. For both species, oxygen affinity was higher at pH 6.4 > pH 7.4 > 5.6 (P = 0.0002). Myoglobin reduction potential for both species was affected by pH (P < 0.0001). The redox potentials became more negative as pH increased, indicating a proton-coupled electron transfer. There were no differences (P = 0.51) between species in reduction potential properties of heme. Homology-based myoglobin modeling indicated that the porcine myoglobin has a shorter distance between the distal histidine and heme than does bovine myoglobin. The variation in amino acid composition between bovine and porcine myoglobin could be partially responsible for differences in oxygen affinity

Mechanistic Insights into Voltage-Driven Biocatalysis of a Cytochrome P450 Bactosomal Film on a Self-Assembled Monolayer

Simple construction of biocatalytically active films of cytochrome P450 (CYP) bactosomes is quite useful for low-cost, stereoselective, and nicotinamide adenine dinucleotide phosphate hydride-free drug metabolism assays, biosensing, and biocatalytic applications. We report here real-time monitoring of the formation of biocatalytically active films of membrane-bound human CYP 2C9 or 3A4 expressed with CYP reductase (CPR) in <i>Escherichia coli</i> (so-called bactosomes) on a cysteamine self-assembled monolayer of gold-infused quartz crystals. The CYP 2C9+CPR-containing bactosomes exhibited oxygen reduction currents and metabolite yields greater than those of the CYP 3A4+CPR film. The electrocatalytic property correlated with the greater levels of CPR activity and the amount of CYP 2C9 in the CYP 2C9+CPR bactosomes than in the CYP 3A4+CPR bactosomes. The electron mediating role of CPR in the CYP 2C9 bactosomal film (<i>E</i>°′ = −450 mV vs Ag/AgCl) toward electrocatalytic oxygen reduction and hydroxylation of diclofenac was experimentally identified by comparing the film with bactosomes expressed with either CYP 2C9 (<i>E</i>°′ = −310 mV) or CPR (<i>E</i>°′ = −450 mV). The onset of oxygen reduction potentials correlated with the formal potentials of CYP and CYP+CPR films and revealed the electrocatalysis by CYP alone or in association with CPR. Furthermore, an ∼2-fold increase in the level of 4-hydroxydiclofenac product formation supported the favorable role of added catalase (hydrogen peroxide scavenger) in preventing damage by reactive oxygen species to the membrane-bound CYP or CYP+CPR bactosomes. The insignificant role of a peroxide shunt pathway for electrocatalysis in the case of the membrane-bound CYP film alone (unlike membrane-free isolated soluble CYP enzymes) and the electron mediation by CPR from the electrode to initiate CYP catalysis in the CYP+CPR bactosomes were discovered in this study. In conclusion, this report describes voltage-driven biocatalysis by bactosomal CYP films with new mechanistic insights into the formal potentials and electrocatalytic pathways of membrane-bound CYP films either alone or in association with CPR in the membrane

A Simple Construction of Electrochemical Liver Microsomal Bioreactor for Rapid Drug Metabolism and Inhibition Assays

In order to design a green microsomal bioreactor on suitably identified carbon electrodes, it is important to understand the direct electrochemical properties at the interfaces between various carbon electrode materials and human liver microsomes (HLM). The novelty of this work is on the investigation of directly adsorbed HLM on different carbon electrodes with the goal to develop a simple, rapid, and new bioanalytical platform of HLM useful for drug metabolism and inhibition assays. These novel biointerfaces are designed in this study by a one step adsorption of HLM directly onto polished basal plane pyrolytic graphite (BPG), edge plane pyrolytic graphite (EPG), glassy carbon (GC), or high-purity graphite (HPG) electrodes. The estimated direct electron transfer (ET) rate constant of HLM on the smooth GC surface was significantly greater than that of the other electrodes. On the other hand, the electroactive surface coverage and stability of microsomal films were greater on highly surface defective, rough EPG and HPG electrodes compared to the smooth GC and less defective hydrophobic BPG surfaces. The presence of significantly higher oxygen functionalities and flatness of the GC surface is attributed to favoring faster ET rates of the coated layer of thin HLM film compared to other electrodes. The cytochrome P450 (CYP)-specific bioactivity of the liver microsomal film on the catalytically superior, stable HPG surface was confirmed by monitoring the electrocatalytic conversion of testosterone to 6β-hydroxytestosterone and its inhibition by the CYP-specific ketoconazole inhibitor. The identification of optimal HPG and EPG electrodes to design biologically active interfaces with liver microsomes is suggested to have immense significance in the design of one-step, green bioreactors for stereoselective drug metabolite synthesis and drug metabolism and inhibition assays