Electrochemical Detection of Gallic Acid-Capped Gold Nanoparticles Using a Multiwalled Carbon Nanotube-Reduced Graphene Oxide Nanocomposite Electrode.

Recently, a plethora of ecofriendly methods have been developed for the synthesis of AuNPs using a multitude of biogenic agents. Polyphenols from plants are particularly attractive for producing AuNPs because in addition to helping with the synthesis of AuNPs, the polyphenol capping of the NPs can be used as a platform for versatile applications. Polyphenol-capped AuNPs could also make the detection of AuNPs possible, should they be released into the environment. Because polyphenols are redox-active, they can be used as a probe to detect AuNPs using electrochemical techniques. In this work, we have developed an MWCNT-rGO nanocomposite electrode for the sensitive detection of AuNPs capped with gallic acid (GA, a green-tea-derived polyphenol) using differential pulse voltammetry (DPV). The reduction of gallic acid-capped AuNPs was used as the quantification signal, and the calibration curve displayed a detection limit of 2.57 pM. Using cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS), we have shown that the modification of the electrode surface with an MWCNT-rGO hybrid nanocomposite resulted in a 10-fold increase in current response leading to the sensitive detection of GA-AuNPs compared to unmodified electrodes. We have also demonstrated the applicability of the electrochemical sensor in detecting GA-AuNPs in various analytical matrixes such as human serum and natural creek water (Highland Creek, ON) with good recovery.

Probing the antioxidant activity of Δ9-tetrahydrocannabinol and cannabidiol in Cannabis sativa extracts.

Herein, we report the antioxidant activity of cannabidiol (CBD) and Δ9-tetrahydrocannabinol (THC) in pure and mixed solutions at different ratios, as well as of six different Cannabis sativa extracts containing various proportions of CBD and THC by using spectrophotometric (reducing power assay, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), 2,2-diphenyl-1-picrylhydrazyl (DPPH), hypochlorous acid (HOCl) scavenging assays) and electrochemical methods (cyclic voltammetry and differential pulse voltammetry). The isolated cannabinoids, the different stoichiometric ratios of CBD and THC, and the natural extracts proved to have remarkable antioxidant properties in all the methods employed in this work. The antioxidant activity of CBD and THC was compared against that of the well-defined antioxidants such as ascorbic acid (AA), resveratrol (Resv) and (-)-epigallocatechin-3-gallate (EGCG). Clear evidence of the synergistic and antagonistic effects between CBD and THC regarding to their antioxidant activities was observed. Moreover, a good correlation was obtained between the optical and electrochemical methods, which proved that the reported experimental procedures can easily be adapted to determine the antioxidant activity of extracts from various Cannabis sativa species and related compounds.

Localization of heat shock protein HSPA6 (HSP70B') to sites of transcription in cultured differentiated human neuronal cells following thermal stress.

Heat shock proteins (Hsps) are a set of highly conserved proteins that are involved in cellular repair and protective mechanisms. In order to identify potential stress-sensitive sites in differentiated SH-SY5Y human neuronal cells, localization of two inducible members of the HSPA (HSP70) family was investigated, namely HSPA6 (HSP70B') and HSPA1A (HSP70-1). Following heat shock, yellow fluorescent protein (YFP)-tagged HSPA6 and HSPA1A proteins localized to nuclear speckles that are enriched in RNA splicing factors (identified by SC35 and SON marker proteins) and then to the granular component of the nucleolus (identified by nucleophosmin). Subsequently, YFP-HSPA6 protein, but not YFP-HSPA1A, localized to the periphery of nuclear speckles that are sites of RNA transcription. The HSPA6 gene is present in the human genome but not in genomes of rat and mouse. Hence, current animal models of neurodegenerative diseases are lacking a potentially protective member of the HSPA family. Potential stress-sensitive sites were identified in differentiated human SH-SY5Y cells by the localization of HSPA6 (HSP70B') and HSPA1A (HSP70-1) to nuclear components following heat shock. HSPA6 and HSPA1A rapidly moved to nuclear speckles, enriched in RNA splicing factors, then to the granular layer of the nucleolus. Subsequently, HSPA6 exhibited a novel localization not observed for the more widely studied HSPA1A, namely association with the periphery of nuclear speckles that are sites of transcription. HS = heat shock; HSPA6 = HSP70B' protein; HSPA1A = HSP70-1 protein.

Electrochemical immunosensors for effective evaluation of amyloid-beta modulators on oligomeric and fibrillar aggregation processes.

A novel electrochemical immunosensor fabricated from gold compact disc electrodes was designed for rapid evaluation of aggregation processes that lead to the formation of oligomeric and fibrillar states of amyloid-beta(1-42) (Aß(1-42)) during Alzheimer's disease. Conformation-specific antibodies were immobilized on the surface of the gold electrode using a 3,3'-dithiobis (sulfosuccinimidyl) propionate (DTSSP) linker. Surface binding events were analyzed by electrochemical impedance spectroscopy (EIS) in which the formation of an antigen-antibody complex was quantified as a function of charge transfer resistance using a [Fe(CN)6](3-/4-) redox probe. The effectiveness of novel sym-triazine-derived aggregation modulators (TAE-1, TAE-2) to reduce the population of toxic oligomers was evaluated. Aß fibril formation was validated by thioflavin T (ThT) fluorescence, whereas oligomer formation was investigated by MALDI. Antigen detection by EIS was further supported by immuno dot blot assays for oligomeric and fibrillar components. Docking simulations of the aggregation modulators TAE-1 and TAE-2 with Aß(1-42) fibrils performed using Autodock Vina suggest a mechanism for the improved aggregation inhibition observed for TAE-2. The results demonstrate the utility and convenience of impedance immunosensing as an analytical tool for rapid and comprehensive evaluation of effective Aß aggregation modulating agents.

Quantum dot based fluorometric detection of cancer TF-antigen.

Cancer is a major global health challenge that would benefit from advances in screening methods for early detection that are rapid and low cost. TF-antigen is a tumor-associated antigen displayed on cell surface proteins of a high percentage of human carcinomas. Here we present a fluorometric bioassay for TF-antigen (galactose-ß-(1→3)-N-acetyl-d-galactosamine) that utilizes quantum dot (QD) technology coupled with magnetic beads for rapid detection of TF-antigen at high sensitivity (10(-7) M range). In the competitive bioassay, 4-aminophenyl ß-d-galactopyranoside (4-APG) conjugated to QDs competes with TF-antigen for binding sites on peanut agglutinin (PNA) that is immobilized on magnetic beads. The bioassay is specific and ultrasensitive in the environment of complex protein mixtures, demonstrating its potential applicability for the screening of clinical samples.

Hsp60 protein pattern in coral is altered by environmental changes in light and temperature.

The heat shock protein Hsp60 exhibited marked oscillation during a 12-hour day period when the coral Turbinaria reniformis was maintained in the laboratory under constant conditions of light (200µE) and temperature (27°C). A biphasic pattern of Hsp60 was apparent, punctuated by a low protein level at the midpoint of the 12-hour day period. Oscillation of Hsp60 was also apparent when coral was kept in darkness in lieu of a scheduled light period. The pattern of Hsp60 was altered when coral was exposed to increased light intensity (400µE) or temperature elevation (32°C). These observations suggest that Hsp60 in coral exhibits oscillation that is altered by increased light and temperature elevation.

Hsp72 chaperone function is dispensable for protection against stress-induced apoptosis.

In addition to its role as a molecular chaperone, heat shock protein 72 (Hsp72) protects cells against a wide range of apoptosis inducing stresses. However, it is unclear if these two roles are functionally related or whether Hsp72 inhibits apoptosis by a mechanism independent of chaperone activity. The N-terminal adenosine triphosphatase domain, substrate-binding domain and the C-terminal EEVD regulatory motif of Hsp72 are all essential for chaperone activity. In this study, we show that Hsp72 mutants with a functional substrate-binding domain but lacking chaperone activity retain their ability to protect cells against apoptosis induced by heat and tumor necrosis factor alpha. In contrast, a deletion mutant lacking a functional substrate-binding domain has no protective capacity. The ability of the Hsp72 substrate-binding domain to inhibit apoptosis independent of the regulatory effects of the adenosine triphosphate-binding domain indicates that the inhibition of apoptosis may involve a stable binding interaction with a regulatory substrate rather than Hsp72 chaperone activity.

Diazonium-Modified Screen-Printed Electrodes for Immunosensing Growth Hormone in Blood Samples.

Altered growth hormone (GH) levels represent a major global health challenge that would benefit from advances in screening methods that are rapid and low cost. Here, we present a miniaturized immunosensor using disposable screen-printed carbon electrodes (SPCEs) for the detection of GH with high sensitivity. The diazonium-based linker layer was electrochemically deposited onto SPCE surfaces, and subsequently activated using covalent agents to immobilize monoclonal anti-GH antibodies as the sensing layer. The surface modifications were monitored using contact angle measurements and X-ray photoelectron spectroscopy (XPS). The dissociation constant, Kd, of the anti-GH antibodies was also determined as 1.44 (±0.15) using surface plasmon resonance (SPR). The immunosensor was able to detect GH in the picomolar range using a 20 µL sample volume in connection with electrochemical impedance spectroscopy (EIS). The selectivity of the SPCE-based immunosensors was also challenged with whole blood and serum samples collected at various development stages of rats, demonstrating the potential applicability for detection in biological samples. Our results demonstrated that SPCEs provided the development of low-cost and single-use electrochemical immunosensors in comparison with glassy carbon electrode (GCE)-based ones.

Induction of heat shock proteins in differentiated human and rodent neurons by celastrol.

Neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis have been termed protein misfolding disorders that are characterized by the neuronal accumulation of protein aggregates. Manipulation of the cellular stress-response involving induction of heat shock proteins (Hsps) in differentiated neurons offers a therapeutic strategy to counter conformational changes in neuronal proteins that trigger pathogenic cascades resulting in neurodegenerative diseases. Hsps are protein repair agents that provide a line of defense against misfolded, aggregation-prone proteins. These proteins are not induced in differentiated neurons by conventional heat shock. We have found that celastrol, a quinine methide triterpene, induced expression of a wider set of Hsps, including Hsp70B', in differentiated human neurons grown in tissue culture compared to cultured rodent neuronal cells. Hence the beneficial effect of celastrol against human neurodegenerative diseases may exceed its potential in rodent models of these diseases.

Stress-induced localization of HSPA6 (HSP70B') and HSPA1A (HSP70-1) proteins to centrioles in human neuronal cells.

The localization of yellow fluorescent protein (YFP)-tagged HSP70 proteins was employed to identify stress-sensitive sites in human neurons following temperature elevation. Stable lines of human SH-SY5Y neuronal cells were established that expressed YFP-tagged protein products of the human inducible HSP70 genes HSPA6 (HSP70B') and HSPA1A (HSP70-1). Following a brief period of thermal stress, YFP-tagged HSPA6 and HSPA1A rapidly appeared at centrioles in the cytoplasm of human neuronal cells, with HSPA6 demonstrating a more prolonged signal compared to HSPA1A. Each centriole is composed of a distal end and a proximal end, the latter linking the centriole doublet. The YFP-tagged HSP70 proteins targeted the proximal end of centrioles (identified by γ-tubulin marker) rather than the distal end (centrin marker). Centrioles play key roles in cellular polarity and migration during neuronal differentiation. The proximal end of the centriole, which is involved in centriole stabilization, may be stress-sensitive in post-mitotic, differentiating human neurons.

Localization of heat shock proteins in cerebral cortical cultures following induction by celastrol.

Hsp70, Hsp32, and Hsp27 were induced by celastrol in rat cerebral cortical cultures at dosages that did not affect cell viability. Pronounced differences were observed in the cellular localization of these heat shock proteins in cell types of cerebral cortical cultures. Celastrol-induced Hsp70 localized to the cell body and cellular processes of neurons that were identified by neuron-specific ßIII-tubulin. Hsp70 was not detected in adjacent GFAP-positive glial cells that demonstrated a strong signal for Hsp27 and Hsp32 in both glial cell bodies and cellular processes. Cells in the cerebral cortex region of the brain are selectively impacted during the progression of Alzheimer's disease which is a "protein misfolding disorder." Heat shock proteins provide a line of defense against misfolded, aggregation-prone proteins. Celastrol is a potential agent to counter this neurodegenerative disorder as recent evidence indicates that in vivo administration of celastrol in a transgenic model of Alzheimer's reduces an important neuropathological hallmark of this disease, namely, amyloid beta pathology that involves protein aggregation.

Induction of heat shock proteins in cerebral cortical cultures by celastrol.

Alzheimer's disease, Parkinson's disease and amyotrophic lateral sclerosis (ALS) are 'protein misfolding disorders' of the mature nervous system that are characterized by the accumulation of protein aggregates and selective cell loss. Different brain regions are impacted, with Alzheimer's affecting cells in the cerebral cortex, Parkinson's targeting dopaminergic cells in the substantia nigra and ALS causing degeneration of cells in the spinal cord. These diseases differ widely in frequency in the human population. Alzheimer's is more frequent than Parkinson's and ALS. Heat shock proteins (Hsps) are 'protein repair agents' that provide a line of defense against misfolded, aggregation-prone proteins. We have suggested that differing levels of constitutively expressed Hsps (Hsc70 and Hsp27) in neural cell populations confer a variable buffering capacity against 'protein misfolding disorders' that correlates with the relative frequencies of these neurodegenerative diseases. The high relative frequency of Alzheimer's may due to low levels of Hsc70 and Hsp27 in affected cell populations that results in a reduced defense capacity against protein misfolding. Here, we demonstrate that celastrol, but not classical heat shock treatment, is effective in inducing a set of neuroprotective Hsps in cultures derived from cerebral cortices, including Hsp70, Hsp27 and Hsp32. This set of Hsps is induced by celastrol at 'days in vitro' (DIV) 13 when cultured cortical cells reached maturity. The inducibility of a set of neuroprotective Hsps in mature cortical cultures at DIV13 suggests that celastrol is a potential agent to counter Alzheimer's disease, a neurodegenerative 'protein misfolding disorder' of the adult brain that targets cells in the cerebral cortex.

Biological activity of sym-triazines with acetylcholine-like substitutions as multitarget modulators of Alzheimer's disease.

The bioactivities of two novel compounds (TAE-1 and TAE-2) that contain a sym-triazine scaffold with acetylcholine-like substitutions are examined as promising candidate agents against Alzheimer's disease. Inhibition of amyloid-ß fibril formation in the presence of Aß1-42, evaluated by Thioflavin T fluorescence, demonstrated comparable or improved activity to a previously reported pentapeptide-based fibrillogenesis inhibitor, iAß5p. Destabilization of Aß1-42 assemblies by TAE-1 and TAE-2 was confirmed by scanning electron microscopy imaging. sym-Triazine inhibition of acetylcholinesterase (AChE) activity was observed in cytosol extracted from differentiated human SH-SY5Y neuronal cells and also using human erythrocyte AChE. The sym-triazine derivatives were well tolerated by these cells and promoted beneficial effects on human neurons, upregulating expression of synaptophysin, a synaptic marker protein, and MAP2, a neuronal differentiation marker.

sym-Triazines for directed multitarget modulation of cholinesterases and amyloid-ß in Alzheimer's disease.

Alzheimer's disease (AD) is a complex neurodegenerative disorder marked by numerous causative factors of disease progression, termed pathologies. We report here the synthesis of a small library of novel sym-triazine compounds designed for targeted modulation of multiple pathologies related to AD, specifically human acetylcholinesterase (AChE), butyrylcholinesterase (BuChE), and Aß aggregation. Rational targeting of AChE was achieved by the incorporation of acetylcholine substrate analogues into a sym-triazine core in either a mono-, di-, or trisubstituted regime. A subset of these derivatives demonstrated improved activity compared to several commercially available cholinesterase inhibitors. High AChE/BuChE selectivity was characteristic of all derivatives, and AChE steady-state kinetics indicated a mixed-type inhibition mechanism. Further integration of multiple hydrophobic phenyl units allowed for improved ß-sheet intercalation into amyloid aggregates. Several highly effective structures exhibited fibril inhibition greater than the previously reported ß-sheet-disrupting penta-peptide, iAß5p, evaluated by thioflavin T fluorescence spectroscopy and transmission electron microscopy. Highly effective sym-triazines were shown to be well tolerated by differentiated human neuronal cells, as demonstrated by the absence of adverse effects on cellular viability at a wide range of concentrations. Parallel targeting of multiple pathologies using sym-triazines is presented here as an effective strategy to address the complex, multifactorial nature of AD progression.

Oxidative stress effect of dopamine on α-synuclein: electroanalysis of solvent interactions.

The interaction of dopamine (DA) and α-synuclein (α-S) can lead to protein misfolding and neuronal death triggered by oxidative stress relevant to the progression of Parkinson's disease (PD). In this study, interfacial properties associated with DA-induced α-S aggregation under various solution conditions (i.e., pH, ionic strength) were investigated in vitro. The electrochemical oxidation of tyrosine (Tyr) residues in α-S was detected in the presence of DA. DA concentration dependence was analyzed and found to significantly affect α-S aggregation pathways. At low pH, DA was shown to be stable and produced no observable difference in interfacial properties. Between pH 7 and 11, DA promoted α-S aggregation. Significant differences in oxidation current signals in response to high pH and ionic strength suggested the importance of initial interactions in the stabilization of toxic oligomeric structures and subsequent off-pathways of α-S. Our results demonstrate the importance of solution interactions with α-S and the unique information that electrochemical techniques can provide for the investigation of α-S aggregation at early stages, an important step toward the development of future PD therapeutics.

Heteromeric complexes of heat shock protein 70 (HSP70) family members, including Hsp70B', in differentiated human neuronal cells.

Human neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis have been termed "protein misfolding disorders." Upregulation of heat shock proteins that target misfolded aggregation-prone proteins has been proposed as a potential therapeutic strategy to counter neurodegenerative disorders. The heat shock protein 70 (HSP70) family is well characterized for its cytoprotective effects against cell death and has been implicated in neuroprotection by overexpression studies. HSP70 family members exhibit sequence and structural conservation. The significance of the multiplicity of HSP70 proteins is unknown. In this study, coimmunoprecipitation was employed to determine if association of HSP70 family members occurs, including Hsp70B' which is present in the human genome but not in mouse and rat. Heteromeric complexes of Hsp70B', Hsp70, and Hsc70 were detected in differentiated human SH-SY5Y neuronal cells. Hsp70B' also formed complexes with Hsp40 suggesting a common co-chaperone for HSP70 family members.

Increased light intensity induces heat shock protein Hsp60 in coral species.

The effect of increased light intensity and heat stress on heat shock protein Hsp60 was examined in two coral species using a branched coral and a laminar coral, selected for their different resistance to environmental perturbation. Transient Hsp60 induction was observed in the laminar coral following either light or thermal stress. Sustained induction was observed when these stresses were combined. The branched coral exhibited comparatively weak transient Hsp60 induction after heat stress and no detectable induction following light stress, consistent with its susceptibility to bleaching in native environments compared to the laminar coral. Our observations also demonstrate that increased light intensity and heat stress exhibited a greater negative impact on the photosynthetic capacity of environmentally sensitive branched coral than the more resistant laminar coral. This supports a correlation between stress induction of Hsp60 and (a) ability to counter perturbation of photosynthetic capacity by light and heat stress and (b) resistance to environmentally induced coral bleaching.