Revealing the mechanistic interactions of profenofos and captan pesticides with serum protein via biophysical and computational investigations.

Profenofos (PF) and captan (CT) are among the most utilized organophosphorus insecticides and phthalimide fungicides, respectively. To elucidate the physicochemical and influential toxicokinetic factors, the mechanistic interactions of serum albumin and either PF or CT were carried out in the current study using a series of spectroscopy and computational analyses. Both PF and CT could bind to bovine serum albumin (BSA), a representative serum protein, with moderate binding constants in a range of 103-104 M-1. The bindings of PF and CT did not induce noticeable BSA's structural changes. Both pesticides bound preferentially to the site I pocket of BSA, where the hydrophobic interaction was the main binding mode of PF, and the electrostatic interaction drove the binding of CT. As a result, PF and CT may not only induce direct toxicity by themselves, but also compete with therapeutic drugs and essential substances to sit in the Sudlow site I of serum albumin, which may interfere with the pharmacokinetics and equilibrium of drugs and other substances causing consequent adverse effects.

Aminochalcones Attenuate Neuronal Cell Death under Oxidative Damage via Sirtuin 1 Activity.

Encouraged by the lack of effective treatments and the dramatic growth in the global prevalence of neurodegenerative diseases along with various pharmacological properties of chalcone pharmacophores, this study focused on the development of aminochalcone-based compounds, organic molecules characterized by a chalcone backbone (consisting of two aromatic rings connected by a three-carbon α,ß-unsaturated carbonyl system) with an amino group attached to one of the aromatic rings, as potential neuroprotective agents. Thus, the aminochalcone-based compounds in this study were designed by bearing a -OCH3 moiety at different positions on the ring and synthesized by the Claisen-Schmidt condensation. The compounds exhibited strong neuroprotective effects against hydrogen peroxide-induced neuronal death in the human neuroblastoma (SH-SY5Y) cell line (i.e., by improving cell survival, reducing reactive oxygen species production, maintaining mitochondrial function, and preventing cell membrane damage). The aminochalcone-based compounds showed mild toxicity toward a normal embryonic lung cell line (MRC-5) and a human neuroblastoma cell line, and were predicted to have preferable pharmacokinetic profiles with potential for oral administration. Molecular docking simulation indicated that the studied aminochalcones may act as competitive activators of the well-known protective protein, SIRT1, and provided beneficial knowledge regarding the essential key chemical moieties and interacting amino acid residues. Collectively, this work provides a series of four promising candidate agents that could be developed for neuroprotection.

One-step impedimetric NT-proBNP aptasensor targeting cardiac insufficiency in artificial saliva.

Currently, sensitive and accurate approaches for diagnosis, rapid assessment, and cardiac biomarker monitoring in patients with heart failure are needed. In this context, the advantages of aptamers over traditional antibodies have been employed to fabricate a single-step impedimetric N-terminal pro b-type natriuretic peptide (NT-proBNP)-modified gold microelectrode array. The development of an electrochemical aptasensing platform was based on the coimmobilization of alkanethiol self-assembled monolayers and amine-terminated aptamer that specifically recognized cardiac NT-proBNP protein resulting in charge electron transfer. Electroimpedimetric signals of the sensor were observed to be linear to the NT-proBNP concentrations in the range of 5.0 × 10-3 to 1.0 pg mL-1 (R2 = 0.9624), while achieving a low detection limit of 5.0 × 10-3 pg mL-1. Clinically relevant detection levels for NT-proBNP were achieved in a simple, rapid, and label-free measurement using artificial saliva, which was highlighted to be specific, regenerative, and selective over potential interferers occurring during the processes of cardiac insufficiency, Therefore, the novel NT-proBNP aptasensor is a promising point-of-care tool exhibiting safe, non-invasive, affordable, and non-prescription home use accessible to overcome the limitations associated with conventional ELISA and previous aptasensing.

Neuroprotective Properties of Bis-Sulfonamide Derivatives Against 6-OHDA-Induced Parkinson's Model via Sirtuin 1 Activity and in silico Pharmacokinetic Properties.

Parkinson's disease (PD) is considered one of the health problems in the aging society. Due to the limitations of currently available drugs in preventing disease progression, the discovery of novel neuroprotective agents has been challenged. Sulfonamide and its derivatives were reported for several biological activities. Herein, a series of 17 bis-sulfonamide derivatives were initially tested for their neuroprotective potential and cytotoxicity against the 6-hydroxydopamine (6-OHDA)-induced neuronal death in SH-SY5Y cells. Subsequently, six compounds (i.e., 2, 4, 11, 14, 15, and 17) were selected for investigations on underlying mechanisms. The data demonstrated that the pretreatment of selected compounds (5 µM) can significantly restore the level of cell viability, protect against mitochondrial membrane dysfunction, decrease the activity of lactate dehydrogenase (LDH), decrease the intracellular oxidative stress, and enhance the activity of NAD-dependent deacetylase sirtuin-1 (SIRT1). Molecular docking was also performed to support that these compounds could act as SIRT1 activators. In addition, in silico pharmacokinetic and toxicity profile prediction was also conducted for guiding the potential development. Thus, the six neuroprotective bis-sulfonamides were highlighted as potential agents to be further developed for PD management.

In silico and multi-spectroscopic analyses on the interaction of 5-amino-8-hydroxyquinoline and bovine serum albumin as a potential anticancer agent.

5-Amino-8-hydroxyquinoline (5A8HQ), an amino derivative of 8-hydroxyquinoline, has become a potential anticancer candidate because of its promising proteasome inhibitory activity to overcome and yet synergize bortezomib for fighting cancers. Therefore, in this study, its physicochemical properties and interaction activities with serum protein have extensively been elucidated by both in vitro and in silico approaches to fulfill the pharmacokinetic and pharmacodynamic gaps. 5A8HQ exhibited the drug-likeness properties, where oral administration seems to be a route of choice owing to its high-water solubility and intestinal absorptivity. Multi-spectroscopic investigations suggested that 5A8HQ tended to associate with bovine serum albumin (BSA), a representative of serum protein, via the ground-state complexation. It apparently bound in a protein cleft between subdomains IIA and IIIA of BSA as suggested by the molecular docking and molecular dynamics simulations. The binding was mainly driven by hydrogen bonding and electrostatic interactions with a moderate binding constant at 104 M-1, conforming with the predicted free fraction in serum at 0.484. Therefore, 5A8HQ seems to display a good bioavailability in plasma to reach target sites and exerts its potent pharmacological activity. Likewise, serum albumin is a good candidate to be reservoir and transporter of 5A8HQ in the circulatory system.

Pesticide Aptasensors-State of the Art and Perspectives.

Contamination by pesticides in the food chain and the environment is a worldwide problem that needs to be actively monitored to ensure safety. Unfortunately, standard pesticide analysis based on mass spectrometry takes a lot of time, money and effort. Thus, simple, reliable, cost-effective and field applicable methods for pesticide detection have been actively developed. One of the most promising technologies is an aptamer-based biosensor or so-called aptasensor. It utilizes aptamers, short single-stranded DNAs or RNAs, as pesticide recognition elements to integrate with various innovative biosensing technologies for specific and sensitive detection of pesticide residues. Several platforms for aptasensors have been dynamically established, such as colorimetry, fluorometry, electrochemistry, electrochemiluminescence (ECL) and so forth. Each platform has both advantages and disadvantages depending on the purpose of use and readiness of technology. For example, colorimetric-based aptasensors are more affordable than others because of the simplicity of fabrication and resource requirements. Electrochemical-based aptasensors have mainly shown better sensitivity than others with exceedingly low detection limits. This paper critically reviews the progression of pesticide aptasensors throughout the development process, including the selection, characterization and modification of aptamers, the conceptual frameworks of integrating aptamers and biosensors, the ASSURED (affordable, sensitive, specific, user-friendly, rapid and robust, equipment-free and deliverable to end users) criteria of different platforms and the future outlook.

Unravelling the interaction mechanism between clioquinol and bovine serum albumin by multi-spectroscopic and molecular docking approaches.

Clioquinol has recently been proposed for the treatment of Alzheimer's disease. It is able to diminish ß-amyloid protein aggregation and to restore cognition of Alzheimer's mice. However, its therapeutic benefits for Alzheimer's disease in human remain controversy and need further confirmation. Herein, we have explored the interaction mechanism of clioquinol toward bovine serum albumin (BSA) by means of multi-spectroscopic and docking simulation approaches. Clioquinol interacts with BSA by a combined mechanism of static and dynamic processes. Application of the Hill's equation to fluorescence quenching experiment revealed that the binding constant of the BSA-clioquinol complex is extremely high at 108 M-1 level. Competitive displacement and docking analysis consistently suggested that there are the multiple binding modes of clioquinol toward BSA. Competitive binding study showed that clioquinol shares the binding sites with ibuprofen and digitoxin on albumin, referring to be site II and site III binding compounds. Besides, partial binding in site I was also observed. Docking simulation confirmed that clioquinol favors to bind in site I, site II, site III, fatty acid binding site 5, and the protein cleft between subdomain IB and IIIB of the BSA. Due to its small size and electric dipole property, clioquinol may easily fit in multiple pockets of the BSA. Our finding suggests the potential role of BSA as a clioquinol carrier in the vascular system. Nonetheless, clioquinol-induced BSA aggregation has been observed by the three-dimensional fluorescence technique. This phenomenon may not only impair the BSA, but may also affect other endogenous proteins, which eventually causes adverse effects to human. Therefore, the redesigned or modified molecular structure of clioquinol may reduce its toxicity and improve its bioavailability.

Insight into the Molecular Interaction of Cloxyquin (5-chloro-8-hydroxyquinoline) with Bovine Serum Albumin: Biophysical Analysis and Computational Simulation.

Cloxyquin is a potential therapeutic compound possessing various bioactivities, especially antibacterial, antifungal, cardioprotective, and pain relief activities. Herein, the interaction mechanism between cloxyquin and bovine serum albumin (BSA) has been elucidated in order to fulfill its pharmacokinetic and pharmacodynamic gaps essential for further development as a therapeutic drug. Multi-spectroscopic and biophysical model analysis suggested that cloxyquin interacts with BSA via a static process by ground-state complex formation. Its binding behavior emerged as a biphasic fashion with a moderate binding constant at the level of 104 M-1. Thermodynamic analysis and molecular docking simulation concurrently revealed that hydrophobic interaction is a major driving force for BSA-cloxyquin complexation. Binding of cloxyquin tends to slightly enlarge the monomeric size of BSA without a significant increase of aggregate fraction. Cloxyquin preferentially binds into the fatty acid binding site 5 (FA5) of the BSA via hydrophobic interaction amongst its quinoline scaffold and Phe550, Leu531, and Leu574 residues of BSA. The quinoline ring and hydroxyl moiety of cloxyquin also form the π-π interaction and the hydrogen bond with Phe506. Our data indicate a potential function of serum albumin as a carrier of cloxyquin in blood circulation.

Lifestyle behaviors and serum vitamin C in the Thai population in Bangkok Metropolitan.

This study aimed to investigate the influence of lifestyle behaviors on the vitamin C levels in the circulating blood of the Thai population in Bangkok Metropolitan. The participants (n=250) included community workers (i.e., construction and business office workers) from the Bangkok Metropolitan, and the participants were placed in various behavior and lifestyle groups (Group I: reference; Group II: alcohol drinkers; Group III: outdoor workers; Group IV: smokers; and Group V: combined). The results showed that the lowest and highest vitamin C intakes were 7 and 27 mg/day in Groups IV and III, respectively. Group I (indoor workers free of smoking and drinking), had the highest total serum vitamin C level (39.7 µmol/L), while Group V (outdoor workers with smoking and drinking), had the lowest value (12.5 µmol/L). Furthermore, Group V had the highest prevalence (44 %) of total serum vitamin C deficiency (<11 µmol/L), while Group I had the lowest deficient indication (8 %). The vitamin C dietary intake and total serum levels were positively correlated in the reference group (Spearman's correlation=0.402, p < 0.05) but not in the other four groups. The significant adjusted odds ratio of inadequate total serum vitamin C (< 23 µmol/L) was 2.90 (CI: 1.15, 7.31) in Group IV and 3.73 (CI: 1.42, 9.81) in Group V. Moreover, the tendency to have an inadequate total serum vitamin C level was demonstrated in the following order: Group I < II < III < IV < V. Our results indicated that outdoor workers (Group III) and smokers (Group IV) had a greater likelihood of having a vitamin C deficiency than the reference group. A high percentage of deficiency was clearly observed among the outdoor workers with smoking and drinking behaviors (Group V).

Probing the origin of estrogen receptor alpha inhibition via large-scale QSAR study.

Estrogen is an important component for the sustenance of normal physiological functions of the mammary glands, particularly for growth and differentiation. Approximately, two-thirds of breast cancers are positive for estrogen receptor (ERs), which is a predisposing factor for the growth of breast cancer cells. As such, ERα represents a lucrative therapeutic target for breast cancer that has attracted wide interest in the search for inhibitory agents. However, the conventional laboratory processes are cost- and time-consuming. Thus, it is highly desirable to develop alternative methods such as quantitative structure-activity relationship (QSAR) models for predicting ER-mediated endocrine agitation as to simplify their prioritization for future screening. In this study, we compiled and curated a large, non-redundant data set of 1231 compounds with ERα inhibitory activity (pIC50). Using comprehensive validation tests, it was clearly observed that the model utilizing the substructure count as descriptors, performed well considering two objectives: using less descriptors for model development and achieving high predictive performance (R Tr 2 = 0.94, Q CV 2 = 0.73, and Q Ext 2 = 0.73). It is anticipated that our proposed QSAR model may become a useful high-throughput tool for identifying novel inhibitors against ERα.

Exploring the chemical space of influenza neuraminidase inhibitors.

The fight against the emergence of mutant influenza strains has led to the screening of an increasing number of compounds for inhibitory activity against influenza neuraminidase. This study explores the chemical space of neuraminidase inhibitors (NAIs), which provides an opportunity to obtain further molecular insights regarding the underlying basis of their bioactivity. In particular, a large set of 347 and 175 NAIs against influenza A and B, respectively, was compiled from the literature. Molecular and quantum chemical descriptors were obtained from low-energy conformational structures geometrically optimized at the PM6 level. The bioactivities of NAIs were classified as active or inactive according to their half maximum inhibitory concentration (IC50) value in which IC50 < 1µM and ≥ 10µM were defined as active and inactive compounds, respectively. Interpretable decision rules were derived from a quantitative structure-activity relationship (QSAR) model established using a set of substructure descriptors via decision tree analysis. Univariate analysis, feature importance analysis from decision tree modeling and molecular scaffold analysis were performed on both data sets for discriminating important structural features amongst active and inactive NAIs. Good predictive performance was achieved as deduced from accuracy and Matthews correlation coefficient values in excess of 81% and 0.58, respectively, for both influenza A and B NAIs. Furthermore, molecular docking was employed to investigate the binding modes and their moiety preferences of active NAIs against both influenza A and B neuraminidases. Moreover, novel NAIs with robust binding fitness towards influenza A and B neuraminidase were generated via combinatorial library enumeration and their binding fitness was on par or better than FDA-approved drugs. The results from this study are anticipated to be beneficial for guiding the rational drug design of novel NAIs for treating influenza infections.

Paper-based acetylcholinesterase inhibition assay combining a wet system for organophosphate and carbamate pesticides detection.

A dramatic increase in pesticide usage in agriculture highlights the need for on-site monitoring for public health and safety. Here, a paper-based sensor combined with a wet system was developed for the simple and rapid screening of organophosphate (OP) and carbamate (CM) pesticides based on the inhibition of acetylcholinesterase (AChE). The paper-based sensor was designed as a foldable device consisting of a cover and detection sheets pre-prepared with indoxyl acetate and AChE, respectively. The paper-based sensor requires only the incubation of a sample on the test zone for 10 minutes, followed by closing of the foldable sheet to initiate the enzymatic reaction. Importantly, the buffer loading hole was additionally designed on the cover sheet to facilitate the interaction of the coated substrate and the immobilized enzyme. This subsequently facilitates the mixing of indoxyl acetate with AChE, resulting in the improved analytical performance of the sensor. The absence or decrease in blue color produced by the AChE hydrolysis of indoxyl acetate can be observed in the presence of OPs and CMs. Under optimized conditions and using image analysis, the limit of detection (LOD) of carbofuran, dichlorvos, carbaryl, paraoxon, and pirimicarb are 0.003, 0.3, 0.5, 0.6, and 0.6 ppm, respectively. The assay could be applied to determine OP and CM residues in spiked food samples. Visual interpretation of the color signal was clearly observed at the concentration of 5 mg/kg. Furthermore, a self-contained sample pre-concentration approach greatly enhanced the detection sensitivity. The paper-based device developed here is low-cost, requires minimal reagents and is easy to handle. As such, it would be practically useful for pesticide screening by non-professional end-users.

Polyacrylamide hydrogel encapsulated E. coli expressing metal-sensing green fluorescent protein as a potential tool for copper ion determination.

A simple, inexpensive and field applicable metal determination system would be a powerful tool for the efficient control of metal ion contamination in various sources e.g. drinking-water, water reservoir and waste discharges. In this study, we developed a cell-based metal sensor for specific and real-time detection of copper ions. E. coli expressing metal-sensing green fluorescent protein (designated as TG1/(CG)6GFP and TG1/H6CdBP4GFP) were constructed and served as a metal analytical system. Copper ions were found to exert a fluorescence quenching effect, while zinc and cadmium ions caused minor fluorescence enhancement in the engineered bacterial suspension. To construct a user-friendly and reagentless metal detection system, TG1/H6CdBP4GFP and TG1/(CG)6GFP were encapsulated in polyacrylamide hydrogels that were subsequently immobilized on an optical fiber equipped with a fluorescence detection module. The sensor could be applied to measure metal ions by simply dipping the encapsulated bacteria into a metal solution and monitoring fluorescence changes in real time as a function of the metal concentration in solution. The sensor system demonstrated high specificity toward copper ions. The fluorescence intensities of the encapsulated TG1/(CG)6GFP and TG1/H6CdBP4GFP were quenched by approximately 70 % and 80 % by a high-dose of copper ions (50 mM), respectively. The level of fluorescence quenching exhibited a direct correlation with the copper concentration, with a linear correlation coefficient (r) of 0.99. The cell-based metal sensor was able to efficiently monitor copper concentrations ranging between 5 M and 50 mM, encompassing the maximum allowed copper contamination in drinking water (31.15 M) established by the WHO. Furthermore, the cell-based metal sensor could undergo prolonged storage for at least 2 weeks without significantly influencing the copper sensitivity.

Engineering of a novel chimera of superoxide dismutase and Vitreoscilla hemoglobin for rapid detoxification of reactive oxygen species.

The genes encoding human manganese superoxide dismutase (MnSOD) and Vitreoscilla hemoglobin (VHb) were fused in-frame to generate a bifunctional enzyme that possessed MnSOD and peroxidase-like activities. At neutral pH, the coupling of the SOD and peroxidase reactions revealed that the bifunctional enzyme exhibited a 2.5 times shorter transient period and a 1.67 times higher reaction rate at steady-state conditions. Furthermore, the catalytic rate of the bifunctional enzyme was not affected as much by the external H2O2 scavenger catalase. This indicates that the bifunctional protein possesses a greater antioxidant capability, which is possibly due to the close proximity between the active site of MnSOD and the heme moiety of VHb. Our findings not only provide insight into the synergistic functions of SOD and peroxidase but also could potentially be used to develop novel therapeutic agents with more efficient O2 carrying capability.

Fluorescent protein-based optical biosensor for copper ion quantitation.

In the present study, spectroscopic determinations of copper ions using chimeric metal-binding green fluorescent protein (His6GFP) as an active indicator have been explored. Supplementation of copper ions to the GFP solution led to a remarkable decrease of fluorescent intensity corresponding to metal concentrations. For circumstances, rapid declining of fluorescence up to 60% was detected in the presence of 500 microM copper. This is in contrast to those observed in the case of zinc and calcium ions, in which approximately 10-20% of fluorescence was affected. Recovery of its original fluorescence up to 80% was mediated by the addition of ethylenediamine tetraacetic acid. More importantly, in the presence of metal ions, the emission wavelength maximum remains unchanged while reduction of the optical density of the absorption spectrum has been observed. This indicates that the chromophore's ground state was possibly affected by the static quenching process. Results from circular dichroism measurements revealed that the overall patterns of circular dichroism spectra after exposure to copper ions were not significantly different from that of the control, where the majority of sharp positive band around 195-196 nm in combination with a broad negative deflection around 215-216 nm was obtained. Taken together, it can be presumed that copper ions exerted their static quenching on the fluorescence rather than structural or conformational alteration. However, notification has to be made that some peptide rearrangements may also occur in the presence of metal ions. Further studies were conducted to investigate the feasibility of using the His6GFP as a sensing unit for copper ions. The His6GFP was encapsulated in Sol-gel and immobilized onto the optical fiber connected with a fluorescence detecting device. The Sol-gel was doped into the metal solution where the quenching of fluorescence could be monitored in real time. The sensing unit provided a high sensitivity of detection in the range of 0.5 microM to 50 mM with high selectivity for copper ions. All these findings open up a high potential to apply the fluorescent protein-based bioanalytical tool for copper determination in the future.

Data mining of magnetocardiograms for prediction of ischemic heart disease.

Ischemic Heart Disease (IHD) is a major cause of death. Early and accurate detection of IHD along with rapid diagnosis are important for reducing the mortality rate. Magnetocardiogram (MCG) is a tool for detecting electro-physiological activity of the myocardium. MCG is a fully non-contact method, which avoids the problems of skin-electrode contact in the Electrocardiogram (ECG) method. However, the interpretation of MCG recordings is time-consuming and requires analysis by an expert. Therefore, we propose the use of machine learning for identification of IHD patients. Back-propagation neural network (BPNN), the Bayesian neural network (BNN), the probabilistic neural network (PNN) and the support vector machine (SVM) were applied to develop classification models for identifying IHD patients. MCG data was acquired by sequential measurement, above the torso, of the magnetic field emitted by the myocardium using a J-T interval of 125 cases. The training and validation data of 74 cases employed 10-fold cross-validation methods to optimize support vector machine and neural network parameters. The predictive performance was assessed on the testing data of 51 cases using the following metrics: accuracy, sensitivity, and specificity and area under the receiver operating characteristic (ROC) curve. The results demonstrated that both BPNN and BNN displayed the highest and the same level of accuracy at 78.43 %. Furthermore, the decision threshold and the area under the ROC curve was -0.2774 and 0.9059, respectively, for BPNN and 0.0470 and 0.8495, respectively, for BNN. This indicated that BPNN was the best classification model, BNN was the best performing model with sensitivity of 96.65 %, and SVM employing the radial basis function kernel displayed the highest specificity of 86.36 %.

Modeling the LPS neutralization activity of anti-endotoxins.

Bacterial lipopolysaccharides (LPS), also known as endotoxins, are major structural components of the outer membrane of Gram-negative bacteria that serve as a barrier and protective shield between them and their surrounding environment. LPS is considered to be a major virulence factor as it strongly stimulates the secretion of pro-inflammatory cytokines which mediate the host immune response and culminating in septic shock. Quantitative structure-activity relationship studies of the LPS neutralization activities of anti-endotoxins were performed using charge and quantum chemical descriptors. Artificial neural network implementing the back-propagation algorithm was selected for the multivariate analysis. The predicted activities from leave-one-out cross-validation were well correlated with the experimental values as observed from the correlation coefficient and root mean square error of 0.930 and 0.162, respectively. Similarly, the external testing set also yielded good predictivity with correlation coefficient and root mean square error of 0.983 and 0.130. The model holds great potential for the rational design of novel and robust compounds with enhanced neutralization activity.

Identification of ischemic heart disease via machine learning analysis on magnetocardiograms.

Ischemic heart disease (IHD) is predominantly the leading cause of death worldwide. Early detection of IHD may effectively prevent severity and reduce mortality rate. Recently, magnetocardiography (MCG) has been developed for the detection of heart malfunction. Although MCG is capable of monitoring the abnormal patterns of magnetic field as emitted by physiologically defective heart, data interpretation is time-consuming and requires highly trained professional. Hence, we propose an automatic method for the interpretation of IHD pattern of MCG recordings using machine learning approaches. Two types of machine learning techniques, namely back-propagation neural network (BNN) and direct kernel self-organizing map (DK-SOM), were applied to explore the IHD pattern recorded by MCG. Data sets were obtained by sequential measurement of magnetic field emitted by cardiac muscle of 125 individuals. Data were divided into training set and testing set of 74 cases and 51 cases, respectively. Predictive performance was obtained by both machine learning approaches. The BNN exhibited sensitivity of 89.7%, specificity of 54.5% and accuracy of 74.5%, while the DK-SOM provided relatively higher prediction performance with a sensitivity, specificity and accuracy of 86.2%, 72.7% and 80.4%, respectively. This finding suggests a high potential of applying machine learning approaches for high-throughput detection of IHD from MCG data.

Rational design of analyte channels of the green fluorescent protein for biosensor applications.

A novel solvent-exposed analyte channel, generated by F165G substitution, on the surface of green fluorescent protein (designated His(6)GFPuv/F165G) was successfully discovered by the aid of molecular modeling software (PyMOL) in conjunction with site-directed mutagenesis. Regarding the high predictive performance of PyMOL, two pore-containing mutants namely His(6)GFPuv/H148G and His(6)GFPuv/H148G/F165G were also revealed. The pore sizes of F165G, H148G, and the double mutant H148G/F165G were in the order of 4, 4.5 and 5.5 A, respectively. These mutants were subjected to further investigation on the effect of small analytes (e.g. metal ions and hydrogen peroxide) as elucidated by fluorescence quenching experiments. Results revealed that the F165G mutant exhibited the highest metal sensitivity at physiological pH. Meanwhile, the other 2 mutants lacking histidine at position 148 had lower sensitivity against Zn(2+) and Cu(2+) than those of the template protein (His(6)GFPuv). Hence, a significant role of this histidine residue in mediating metal transfer toward the GFP chromophore was proposed and evidently demonstrated by testing in acidic condition. Results revealed that at pH 6.5 the order of metal sensitivity was found to be inverted whereby the H148G/F165G became the most sensitive mutant. The dissociation constants (K(d)) to metal ions were in the order of 4.88 x 10(-6) M, 16.67 x 10(-6) M, 25 x 10(-6) M, and 33.33 x 10(-6) M for His(6)GFPuv/F165G, His(6)GFPuv, His(6)GFPuv/H148G/F165G and His(6)GFPuv/H148G, respectively. Sensitivity against hydrogen peroxide was in the order of H148G/F165G > H148G > F165G indicating the crucial role of pore diameters. However, it should be mentioned that H148G substitution caused a markedly decrease in pH- and thermo-stability. Taken together, our findings rendered the novel pore of GFP as formed by F165G substitution to be a high impact channel without adversely affecting the intrinsic fluorescent properties. This opens up a great potential of using F165G mutant in enhancing the sensitivity of GFP in future development of biosensors.

EDTA-induced membrane fluidization and destabilization: biophysical studies on artificial lipid membranes.

The molecular mechanism of ethylenediaminetetraacetic acid (EDTA)-induced membrane destabilization has been studied using a combination of four biophysical techniques on artificial lipid membranes. Data from Langmuir film balance and epifluorescence microscopy revealed the fluidization and expansion effect of EDTA on phase behavior of monolayers of either 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) or mixtures of DPPC and metal-chelating lipids, such as N(alpha),N(alpha)-Bis[carboxymethyl]-N(epsilon)-[(dioctadecylamino)succinyl]-L-lysine or 1,2-dioleoyl-sn-glycero-3-[N-(5-amino-1-carboxypentyl iminodiacetic acid) succinyl]. A plausible explanation could be drawn from the electrostatic interaction between negatively charged groups of EDTA and the positively charged choline head group of DPPC. Intercalation of EDTA into the lipid membrane induced membrane curvature as elucidated by atomic force microscopy. Growth in size and shape of the membrane protrusion was found to be time-dependent upon exposure to EDTA. Further loss of material from the lipid membrane surface was monitored in real time using a quartz crystal microbalance. This indicates membrane restabilization by exclusion of the protrusions from the surface. Loss of lipid components facilitates membrane instability, leading to membrane permeabilization and lysis.