Antifungal Activity and Molecular Mechanisms of Copper Nanoforms against Colletotrichum gloeosporioides.

In this work, we have synthesized copper nanoforms (Cu NFs) using ascorbic acid as a reducing agent and polyvinylpyrrolidone as a stabilizer. Elemental characterization using EDS has shown the nanostructure to be of high purity and compare well with commercially sourced nanoforms. SEM images of both Cu NFs show some agglomeration. The in-house NFs had a better even distribution and size of the nanostructures. The XRD peaks represented a face-centered cubic structure of Cu2O. The commercially sourced Cu NFs were found to be a mixture of Cu and Cu2O. Both forms had a crystalline structure. Using these two types of Cu NFs, an antimicrobial study against Colletotrichum gloeosporioides, a devastating plant pathogen, showed the in-house Cu NFs to be most effective at inhibiting growth of the pathogen. Interestingly, at low concentrations, both Cu NFs increased fungal growth, although the mycelia appeared thin and less dense than in the control. SEM macrographs showed that the in-house Cu NFs inhibited the fungus by flattening the mycelia and busting some of them. In contrast, the mycelia were short and appeared clustered when exposed to commercial Cu NFs. The difference in effect was related to the size and/or oxidation state of the Cu NFs. Furthermore, the fungus produced a defense mechanism in response to the NFs. The fungus produced melanin, with the degree of melanization directly corresponding to the concentration of the Cu NFs. Localization of aggregated Cu NFs could be clearly observed outside of the model membranes. The large agglomerates may only contribute indirectly by a hit-and-bounce-off effect, while small structures may adhere to the membrane surface and/or internalize. Spatio-temporal membrane dynamics were captured in real time. The dominant dynamics culminated into large fluctuations. Some of the large fluctuations resulted in vesicular transformation. The major transformation was exo-bud/exo-cytosis, which may be a way to excrete the foreign object (Cu NFs).

Black tea polyphenol theaflavin as promising antioxidant and potential copper chelator.

BACKGROUND: In this work, we investigated the antioxidant and copper chelating abilities of theaflavin, a polyphenol responsible for astringency, color, and sensation in black tea. Using voltammetric techniques, the analyses were conducted with disposable electrochemical printed carbon chips in conjunction with a portable hand-held potentiostat. RESULTS: Voltammograms of theaflavin showed five separate oxidation peaks, corresponding to the oxidation of five individual functional groups. Electroanalytical data indicated that, after interaction with copper, theaflavin had higher antioxidant potential and was a better copper chelator than epigallocatechin gallate, a major polyphenol present in green tea and a well-known antioxidant. This could be attributed to the extra fused ring and larger number of OH groups in theaflavin. CONCLUSIONS: Our findings introduce another natural compound as a potential nutraceutical in oxidation- and copper-modulated illnesses. This simple and fast approach would also be highly pertinent to the inspection of the health benefits of natural food products. To the best of our knowledge, this is the first report of the electrochemical analysis of Cu (II) chelation with theaflavin. © 2020 Society of Chemical Industry.

Effects of Capsaicin on Biomimetic Membranes.

Capsaicin is a natural compound that produces a warm sensation and is known for its remarkable medicinal properties. Understanding the interaction between capsaicin with lipid membranes is essential to clarify the molecular mechanisms behind its pharmacological and biological effects. In this study, we investigated the effect of capsaicin on thermoresponsiveness, fluidity, and phase separation of liposomal membranes. Liposomal membranes are a bioinspired technology that can be exploited to understand biological mechanisms. We have shown that by increasing thermo-induced membrane excess area, capsaicin promoted membrane fluctuation. The effect of capsaicin on membrane fluidity was dependent on lipid composition. Capsaicin increased fluidity of (1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) membranes, while it rigidified DOPC and cholesterol-based liposomes. In addition, capsaicin tended to decrease phase separation of heterogeneous liposomes, inducing homogeneity. We imagine this lipid re-organization to be associated with the physiological warming sensation upon consumption of capsaicin. Since capsaicin has been reported to have biological properties such as antimicrobial and as antiplatelet, the results will help unravel these biological properties.

Polyphenols Modulate Alzheimer's Amyloid Beta Aggregation in a Structure-Dependent Manner.

Some polyphenols, which are common natural compounds in fruits, vegetables, seeds, and oils, have been considered as potent inhibitors of amyloid beta (Aß) aggregation, one critical pathogenic event in Alzheimer's disease (AD). However, the mechanisms by which polyphenols affect aggregation are not fully understood. In this study, we aimed to investigate the effect of two classes of polyphenols (flavonoids and stilbenes) on the self-assembly of Aß_42, in particular, how this relates to structure. We found that the flavonoids gallocatechin gallate (GCG) and theaflavin (TF) could completely inhibit Aß aggregation, while two stilbenes, resveratrol and its glucoside derivative piceid, could also suppress Aß aggregation, but to a much lesser extent. Intriguingly, resveratrol accelerated the formation of Aß fibrils before its decreasing effect on fibrillation was detected. Atomic force microscopy (AFM) images showed a huge mass of long and thin Aß fibrils formed in the presence of resveratrol. Although the morphology was the same in the presence of piceid, the fibrils were sparse in the presence of picead. In the presence of flavonoids, Aß morphology was unchanged from prior to incubation (0 h), in agreement with amyloid beta kinetics analysis using thioflavin-T fluorescence assay. The electrochemical data showed a higher ability of GCG and TF to interact with Aß than resveratrol and piceid, which could be attributed to the presence of more aromatic rings and hydroxyl groups. In addition, the two flavonoids exhibited a similar propensity for Aß aggregation, despite having some differences in their structure. However, in the case of stilbenes, the addition of a glucoside at C-7 slightly decreased anti-Aß aggregation property compared to resveratrol. These findings contribute to a better understanding of the essential structural features of polyphenols required for inhibiting Aß aggregation, and the possible mechanisms for modulating aggregation.

Strikingly different effects of cholesterol and 7-ketocholesterol on lipid bilayer-mediated aggregation of amyloid beta (1-42).

Oxidized cholesterol has been widely reported to contribute to the pathogenesis of Alzheimer's disease (AD). However, the mechanism by which they affect the disease is not fully understood. Herein, we aimed to investigate the effect of 7-ketocholesterol (7keto) on membrane-mediated aggregation of amyloid beta (Aß-42), one of the critical pathogenic events in AD. We have shown that when cholesterol is present in lipid vesicles, kinetics of Aß nuclei formation is moderately hindered while that of fibril growth was considerably accelerated. The partial substitution of cholesterol with 7keto slightly enhanced the formation of Aß-42 nuclei and remarkably decreased fibril elongation, thus maintaining the peptide in protofibrillar aggregates, which are reportedly the most toxic species. These findings add in understanding of how cholesterol and its oxidation can affect Aß-induced cytotoxicity.

Carbon-Based Nanomaterials in Biomass-Based Fuel-Fed Fuel Cells.

Environmental and sustainable economical concerns are generating a growing interest in biofuels predominantly produced from biomass. It would be ideal if an energy conversion device could directly extract energy from a sustainable energy resource such as biomass. Unfortunately, up to now, such a direct conversion device produces insufficient power to meet the demand of practical applications. To realize the future of biofuel-fed fuel cells as a green energy conversion device, efforts have been devoted to the development of carbon-based nanomaterials with tunable electronic and surface characteristics to act as efficient metal-free electrocatalysts and/or as supporting matrix for metal-based electrocatalysts. We present here a mini review on the recent advances in carbon-based catalysts for each type of biofuel-fed/biofuel cells that directly/indirectly extract energy from biomass resources, and discuss the challenges and perspectives in this developing field.

Raft-dependent endocytic movement and intracellular cluster formation during T cell activation triggered by concanavalin A.

Certain food ingredients can stimulate the human immune system. A lectin, concanavalin A (ConA), from Canavalia ensiformis (jack bean) is one of the most well-known food-derived immunostimulants and mediates activation of cell-mediated immunity through T cell proliferation. Generally, T cell activation is known to be triggered by the interaction between T cells and antigen-presenting cells (APCs) via a juxtacrine (contact-dependent) signaling pathway. The mechanism has been well characterized and is referred to as formation of the immunological synapse (IS). We were interested in the mechanism behind the T cell activation by food-derived ConA which might be different from that of T cell activation by APCs. The purpose of this study was to characterize T cell activation by ConA with regard to (i) movement of raft domain, (ii) endocytic vesicular transport, (iii) the cytoskeleton (actin and microtubules), and (iv) cholesterol composition. We found that raft-dependent endocytic movement was important for T cell activation by ConA and this movement was dependent on actin, microtubules, and cholesterol. The T cell signaling mechanism triggered by ConA can be defined as endocrine signaling which is distinct from the activation process triggered by interaction between T cells and APCs by juxtacrine signaling. Therefore, we hypothesized that T cell activation by ConA includes both two-dimensional superficial raft movement on the membrane surface along actin filaments and three-dimensional endocytic movement toward the inside of the cell along microtubules. These findings are important for developing new methods for immune stimulation and cancer therapy based on the function of ConA.

Printable Electrochemical Biosensors: A Focus on Screen-Printed Electrodes and Their Application.

In this review we present electrochemical biosensor developments, focusing on screen-printed electrodes (SPEs) and their applications. In particular, we discuss how SPEs enable simple integration, and the portability needed for on-field applications. First, we briefly discuss the general concept of biosensors and quickly move on to electrochemical biosensors. Drawing from research undertaken in this area, we cover the development of electrochemical DNA biosensors in great detail. Through specific examples, we describe the fabrication and surface modification of printed electrodes for sensitive and selective detection of targeted DNA sequences, as well as integration with reverse transcription-polymerase chain reaction (RT-PCR). For a more rounded approach, we also touch on electrochemical immunosensors and enzyme-based biosensors. Last, we present some electrochemical devices specifically developed for use with SPEs, including USB-powered compact mini potentiostat. The coupling demonstrates the practical use of printable electrode technologies for application at point-of-use. Although tremendous advances have indeed been made in this area, a few challenges remain. One of the main challenges is application of these technologies for on-field analysis, which involves complicated sample matrices.

Modified screen printed electrode for development of a highly sensitive label-free impedimetric immunosensor to detect amyloid beta peptides.

Alzheimer's disease (AD) is a fatal neurodegenerative disease affecting approximately 26 million people world-wide, and the number is increasing as life expectancy increases. Since the only reliable diagnosis for the pathology is histochemical post-mortem examination, there is a rather urgent need for reliable, sensitive and quick detection techniques. Amyloid beta, being one of the established and widely accepted biomarkers of AD is a target biomolecule. Herein, we present fabrication of a labelless impedimetric amyloid beta immunosensor on carbon DEP (disposable electrochemical printed) chip. Three types of amyloid ß impedimetric immunosensors were fabricated in a systematic step-wise manner in order to understand the effects that each surface modification chemistry had on detection sensitivity. We found that compared to a bare electrode, surface modification through formation of SAM of AuNPs increased sensitivity by approximately three orders of magnitude (LoD from 2.04 µM to 2.65 nM). A further modification using protein G, which helps orientate antibodies to an optimum position for interaction with antigen, lowered the LoD further to 0.57 nM. We have demonstrated that the presence of one of the most abundance proteins in biological fluids, bovine serum albumin (BSA), did not interfere with the sensitivity of the sensor. Since the DEP chips are disposable and the detection platform label-free, the developed sensor is relatively fast and cheap. These methods could easily be applied for detection of other antigens, with selection of the detection platform based on the desired for sensitivity.

Localization of amyloid beta (Aß1-42) protofibrils in membrane lateral compartments: effect of cholesterol and 7-Ketocholesterol.

Cholesterol plays an important role in the interaction of Alzheimer's amyloid beta (Aß) with cell membranes, an important event in Aß-induced cytotoxicity. However, it is not fully understood how cholesterol influences the association of Aß with membrane lateral compartments. We have shown that by modulating membrane fluidity, cholesterol decreased peptide localization in solid-ordered domains and increased that in liquid-ordered domains. It changed the amount of Aß associating with liquid-disordered (Ld) phase with different tendencies depending on the composition of heterogeneous membrane systems. 7-Ketocholesterol, an oxidized derivative of cholesterol, majorly enhanced the fluidity of and Aß interaction with Ld phase. These findings are useful for clarifying the impact of cholesterol and its oxidation in Aß-induced toxicity.

Structure-dependent interactions of polyphenols with a biomimetic membrane system.

Polyphenols are naturally-occurring compounds, reported to be biologically active, and through their interactions with cell membranes. Although association of the polyphenols with the bilayer has been reported, the detailed mechanism of interaction is not yet well elucidated. We report on spatio-temporal real-time membrane dynamics observed in the presence of polyphenols. Two distinct membrane dynamics, corresponding to the two classes of polyphenols used, were observed. Flavonoids (epi-gallocatechin-3-gallate, gallocatechin, theaflavin and theaflavin-3-gallate) caused lipid membrane aggregation and rigidification. As simple structural modification through opening of the aromatic C-ring into an olefin bond, present in trans-stilbenes (resveratrol and picead), completely changed the membrane properties, increasing fluidity and inducing fluctuation. There were differences in the membrane transformations within the same class of polyphenols. Structure-dependent classification of membrane dynamics may contribute to a better understanding of the physicochemical mechanism involved in the bioactivity of polyphenols. In general, an increase in the number of hydrophilic side chains (galloyl, hydroxyl, glucoside, gallate) increased the reactivity of the polyphenols. Most notable was the difference observed through a simple addition of the gallate group. Unraveling the importance of these polyphenols, at a functional group level further opens the key to tailored design of bioactive compounds as potential drug candidates.

Endo- and exocytic budding transformation of slow-diffusing membrane domains induced by Alzheimer's amyloid beta.

Cell-sized liposomes are a powerful tool for clarifying physicochemical mechanisms that govern molecular interactions. Herein, budding transformation of membrane domains was induced by amyloid beta peptides. The peptides increased the membrane viscosity as demonstrated by the Brownian motion of membrane domains. These results could aid in understanding the physicochemical mechanism of Alzheimer's disease.

The effect of oxysterols on the interaction of Alzheimer's amyloid beta with model membranes.

The interaction of amyloid beta (Aß) peptide with cell membranes has been shown to be influenced by Aß conformation, membrane physicochemical properties and lipid composition. However, the effect of cholesterol and its oxidized derivatives, oxysterols, on Aß-induced neurotoxicity to membranes is not fully understood. We employed here model membranes to investigate the localization of Aß in membranes and the peptide-induced membrane dynamics in the presence of cholesterol and 7-ketocholesterol (7keto) or 25-hydroxycholesterol (25OH). Our results have indicated that oxysterols rendered membranes more sensitive to Aß, in contrast to role of cholesterol in inhibiting Aß/membrane interaction. We have demonstrated that two oxysterols had different impacts owing to distinct positions of the additional oxygen group in their structures. 7keto-containing cell-sized liposomes exhibited a high propensity toward association with Aß, while 25OH systems were more capable of morphological changes in response to the peptide. Furthermore, we have shown that 42-amino acid Aß (Aß-42) pre-fibril species had higher association with membranes, and caused membrane fluctuation faster than 40-residue isoform (Aß-40). These findings suggest the enhancing effect of oxysterols on interaction of Aß with membranes and contribute to clarify the harmful impact of cholesterol on Aß-induced neurotoxicity by means of its oxidation.

Membrane fusion and vesicular transformation induced by Alzheimer's amyloid beta.

Amyloid beta (Aß) peptides, produced through endo-proteolytic cleavage of amyloid precursor protein, are thought to be involved in the death of neural cells in Alzheimer's disease (AD). Although the mechanisms are not fully known, it has been suggested that disruption of cellular activity due to Aß interactions with the cell membrane may be one of the underlying causes. Here in, we have investigated the interaction between Aß-42 and biomimetic lipid membranes and the resulting perturbations in the lipid vesicles. We have shown that Aß oligomeric species localized closer to the membrane surface. Localization of the fibrillar species of Aß-42, although varied, was not as closely associated with the membrane surface. We have demonstrated that the presence of Aß-42 leads to an increase in membrane surface area, inducing lipid temporal vesicular transformation. Furthermore, we have unequivocally shown that Aß-peptides mediate membrane fusion. Although membrane fusion induced by Aß has been hypothesized/proposed, this is the first time it has been visually captured. This fusion may be one of the mechanisms behind the membrane increase in surface area and the resulting vesicular transformation. We have shown that the longer 'amyloidogenic' isoform causes vesicular transformation more readily, and has a higher membrane fusogenic potential than Aß-40. Although not core to this study, it is hugely interesting to observe the high agreement between membrane dynamics and the reported amyloidogenicity of the peptides and aggregation species opening up the potential role of vesicular dynamics for profiling and biosensing of Aß-induced neuro-toxicity.

Dynamic Morphological Changes Induced By GM1 and Protein Interactions on the Surface of Cell-Sized Liposomes.

It is important to understand the physicochemical mechanisms that are responsible for the morphological changes in the cell membrane in the presence of various stimuli such as osmotic pressure. Lipid rafts are believed to play a crucial role in various cellular processes. It is well established that Ctb (Cholera toxin B subunit) recognizes and binds to GM1 (monosialotetrahexosylganglioside) on the cell surface with high specificity and affinity. Taking advantage of Ctb-GM1 interaction, we examined how Ctb and GM1 molecules affect the dynamic movement of liposomes. GM1 a natural ligand for cholera toxin, was incorporated into liposome and the interaction between fluorescent Ctb and the liposome was analyzed. The interaction plays an important role in determining the various surface interaction phenomena. Incorporation of GM1 into membrane leads to an increase of the line tension leading to either rupture of liposome membrane or change in the morphology of the membrane. This change in morphology was found to be GM1 concentration specific. The interaction between Ctb-GM1 leads to fast and easy rupture or to morphological changes of the liposome. The interactions of Ctb and the glycosyl chain are believed to affect the surface and the curvature of the membrane. Thus, the results are highly beneficial in the study of signal transduction processes.

Size-dependent partitioning of nano/microparticles mediated by membrane lateral heterogeneity.

It is important that we understand the physical, chemical, and biological mechanisms that govern the interaction between nanoparticles (NPs) and heterogeneous cellular surfaces because of the possible cytotoxicity of engineered nanomaterials. In this study, we investigated the lateral localization of nano/microparticles within a biomimetic heterogeneous membrane interface using cell-sized two-phase liposomes. We found that lateral heterogeneity in the membrane mediates the partitioning of nano/microparticles in a size-dependent manner: small particles with a diameter of ≤200 nm were localized in an ordered phase, whereas large particles preferred a fluidic disordered phase. This partitioning behavior was verified by temperature-controlled membrane miscibility transition and laser-trapping of associated particles. In terms of the membrane elastic energy, we present a physical model that explains this localization preference of nano/microparticles. The calculated threshold diameter of particles that separates the particle-partitioning phase was 260 nm, which is in close agreement with our observation (200 nm). These findings may lead to a better understanding of the basic mechanisms that underlie the association of nanomaterials within a cell surface.

Thermo-induced vesicular dynamics of membranes containing cholesterol derivatives.

Membrane structural organization is an intrinsic property of a cell membrane. Any changes in lipid composition, and/or any stimuli that affect molecular packing induce structural re-organization. It membrane dynamics provide a means by which changes in structure organization can be determined, upon a change in the membrane internal or external environment. Here, we report on the effect of thermo-stress on membranes containing cholesterol liquid crystal (LC) compounds cholesterol benzoate (BENZO) and oxidized cholesterols. We have (1) revealed that lipid vesicles containing this artificial cholesterol derivative (BENZO) is thermo-responsive, and that this thermo-sensitivity is significantly similar to naturally oxy-cholesterols (2) elucidated the mechanism behind the membrane perturbation. Using Langmuir monolayer experiments, we have demonstrated that membrane perturbation was due to an increase in the molecular surface area, (3) discussed the similarities between cholesterol benzoate in the cholesterol LC state and in lipid bilayer membranes. Last, (4) drawing from previously reported findings, our new data on membrane dynamics, and the discussion above, we propose that artificial cholesterol derivatives such as BENZO, open new possibilities for controlled and tailored design using model membrane systems. Examples could include the development of membrane technology and provide a trigger for progress in thermo-tropical liquid crystal engineering.

Damage in brain development by morpholino knockdown of zebrafish dax1.

DAX1 is an orphan nuclear receptor and involved in development of steroidogenic organs, which activates transcription of genes involved in steroidogenesis. In this study, we analyzed the function of the zebrafish dax1 during early development of central nervous systems to appear unidentified aspects of DAX1 and decrease confusions concerned with functions of DAX1 in early development of vertebrates. By whole-mount in situ hybridization of embryo at the 32 h post fertilization (hpf), expression of zebrafish dax1 was detected around the forebrain, midbrain, hindbrain, and the extending tail tip. Embryos injected with zebrafish dax1 morpholino antisense nucleotide (MO) exhibited delayed development. When the developmental stage of wild type embryos was at Prim-15 (32 hpf), zebrafish dax1MO injected embryos were at Prim-5 (24 hpf). Concurrently with developmental delay, the MO injected embryos showed high mortality. At 48 hpf, the MO injected embryos exhibited abnormal development in the central nervous systems. The enlarged tectum and the protruded rhombomeres were observed. Moreover, development of central nervous systems, especially midbrain-hindbrain boundary, became narrower. At 5 day post fertilization, the MO injected embryos formed edemas around head, pericardial sac and abdomen. Collectively, our results indicated that the zebrafish dax1 is important for brain development.

Physicochemical Profiling of Surfactant-Induced Membrane Dynamics in a Cell-Sized Liposome.

We used a cell-sized model system, giant liposomes, to investigate the interaction between lipid membranes and surfactants, and the membrane transformation during the solubilization process was captured in real time. We found that there are four distinct dynamics in surfactant-induced membrane deformation: an episodic increase in the membrane area prior to pore-forming associated shrinkage (Dynamics A), fission into many small liposomes (Dynamics B), the formation of multilamellar vesicles and peeling (Dynamics C), and bursting (Dynamics D). Classification of the diversity of membrane dynamics may contribute to a better understanding of the physicochemical mechanism of membrane solubilization induced by various surfactants.

A carbon nanotube structured biomimetic catalyst for polysaccharide degradation.

A unique artificial catalyst that mimics the structure of active sites in real enzymes using functionalized carbon nanotubes is presented. This concept will allow for the potential construction of a library of biomimetic catalysts for enzyme active centers, for which the structure-catalysis relationships are well defined.