Absence Of Dopamine Research Articles

A fluorescence assay mediated peroxidase-like activity of magnetic nanoparticles for dopamine detection in human blood serum with a smartphone-based portable device

Dopamine (DA) concentration in the blood is a critical diagnostic indicator for human illnesses. Thus, the platform-based fluorescence method for DA detection with a smartphone was proven. The detection assay used the complimentary properties of 3-aminopropyl triethoxysilane-functionalized magnetic nanoparticles (MNPs@APTES). In the absence of DA, MNPs@APTES is capable of catalyzing the oxidation of o-phenylenediamine (OPD) by H2O2 to produce the fluorescent species (2,3-diaminophenazine; DAP). The smartphone-based portable fluorescence device can measure the fluorescence intensity of the DAP, which can then be translated into the RGB signal corresponding to the DAP concentration via the smartphone application. However, the RGB intensity was diminished in the presence of DA as a result of the oxidation of DA in an alkaline medium to produce polydopamine (PDA) passivated onto the surface of the MNPs@APTES, which inhibited catalytic activity and produced less DAP. Thus, there is a correlation between the concentration of DA and the degree of fluorescence decrease. This platform demonstrated a high degree of selectivity for DA relative to other analogous substances and potential interferences. In addition, it successfully detected DA levels in human blood serum samples over a linear working range of 1–50 μM with a detection limit of 0.48 μM.

Effect of Dopamine on Growth, Some Biochemical Attributes, and the Yield of Crisphead Lettuce under Nitrogen Deficiency

Nitrogen (N) represents the most important nutrient for plant growth and productivity, but extreme and ineffective usage of N fertilizer results in boosted plant production expenditures and environmental contamination. For the world’s sustainable food production and environmental profits, there has been increased research interest in reducing the use of N fertilization along with improving plant N deficiency (ND) tolerance. Dopamine (DA), a potential antioxidant, mediates several physio-biochemical processes in plants under normal or stressful conditions. However, their roles in increasing ND tolerance in crisphead lettuce are not well-documented. We investigate the role of DA concentration (0.50 and 100 µM) on the growth and yield of crisphead lettuce plants under ND. Under normal conditions (100% recommended N fertilizer dose), DA (50 and 100 μM) application significantly enhanced growth, chlorophyll concentration, N%, antioxidant enzymes activity, as well as yield and its components, decreased nitrate accumulation and oxidative biomarkers compared to untreated plants (0 μM DA). ND significantly decreased plant growth and yield attributes as well as evoked oxidative impairment and nitrate accumulation as compared to 100% recommended N fertilizer dose in the absence of DA. However, within ND conditions, the application of DA concentrations significantly mitigated ND-induced oxidative burst and improved plant growth, chlorophyll concentration, N%, nitrate concentration, peroxidase, catalase, superoxide dismutase, total soluble solid, vitamin C, dry matter %, and total sugars, over 0 μM DA treated plants. Current findings highlighted that exogenous application of 100 μM DA could reinforce the crisphead lettuce plant’s resilience to ND by minimizing reactive oxygen species accumulation and promoting enzymatic antioxidants alongside growth, yield, and quality improvement. The beneficial effects of DA in lessening ND’s drastic impacts on crisphead lettuce resulted from upregulating antioxidant enzyme activity, impairment of oxidative biomarkers, and maintaining chlorophyll levels. The current findings open pioneering prospects to reduce nitrogen fertilization by DA application without any drastic effect on plant productivity. But further research is needed to fully understand DA effects and their mechanisms in inducing ND tolerance in different plant species, including crisphead lettuce.

The rostromedial tegmental nucleus RMTg is not a critical site for ethanol-induced motor activation in rats.

Opioid drugs indirectly activate dopamine (DA) neurons in the ventral tegmental area (VTA) through a disinhibition mechanism mediated by mu opioid receptors (MORs) present both on the GABA projection neurons located in the medial tegmental nucleus/tail of the VTA (RMTg/tVTA) and on the VTA GABA interneurons. It is well demonstrated that ethanol, like opioid drugs, provokes VTA DA neuron disinhibition by interacting (through its secondary metabolite, salsolinol) with MORs present in VTA GABA interneurons, but it is not known whether ethanol could disinhibit VTA DA neurons through the MORs present in the RMTg/tVTA. The objective of the present study was to determine whether ethanol, directly microinjected into the tVTA/RMTg, is also able to induce VTA DA neurons disinhibition. Disinhibition of VTA DA neurons was indirectly assessed through the analysis of the motor activity of rats. Cannulae were placed into the tVTA/RMTg to perform microinjections of DAMGO (0.13 nmol), ethanol (150 or 300 nmol) or acetaldehyde (250 nmol) in animals pre-treated with either aCSF or the irreversible antagonist of MORs, beta-funaltrexamine (beta-FNA; 2.5 nmol). After injections, spontaneous activity was monitored for 30 min. Neither ethanol nor acetaldehyde directly administered into the RMTg/tVTA were able to increase the locomotor activity of rats at doses that, in previous studies performed in the posterior VTA, were effective in increasing motor activities. However, microinjections of 0.13 nmol of DAMGO into the tVTA/RMTg significantly increased the locomotor activity of rats. These activating effects were reduced by local pre-treatment of rats with beta-FNA (2.5 nmol). The tVTA/RMTg does not appear to be a key brain region for the disinhibiting action of ethanol on VTA DA neurons. The absence of dopamine in the tVTA/RMTg extracellular medium, the lack of local ethanol metabolism or both could explain the present results.

Mutual Cooperativity of Three Allosteric Sites on the Dopamine D1 Receptor.

An amine-containing molecule called Compound A has been reported by a group from Bristol-Myers Squibb to act as a positive allosteric modulator (PAM) at the dopamine D1 receptor. We synthesized the more active enantiomer of Compound A (BMS-A1) and compared it with the D1 PAMs DETQ and MLS6585, which are known to bind to intracellular loop 2 and the extracellular portion of transmembrane helix 7, respectively. Results from D1/D5 chimeras indicated that PAM activity of BMS-A1 tracked with the presence of D1 sequence in the N-terminal/extracellular region of the D1 receptor, a unique location compared with either of the other PAMs. In pairwise combinations, BMS-A1 potentiated the small allo-agonist activity of each of the other PAMs, while the triple PAM combination (in the absence of dopamine) produced a cAMP response about 64% of the maximum produced by dopamine. Each of the pairwise PAM combinations produced a much larger leftward shift of the dopamine EC50 than either single PAM alone. All three PAMs in combination produced a 1000-fold leftward shift of the dopamine curve. These results demonstrate the presence of three non-overlapping allosteric sites that cooperatively stabilize the same activated state of the human D1 receptor. SIGNIFICANCE STATEMENT: Deficiencies in dopamine D1 receptor activation are seen in Parkinson disease and other neuropsychiatric disorders. In this study, three positive allosteric modulators of the dopamine D1 receptor were found to bind to distinct and separate sites, interacting synergistically with each other and dopamine, with the triple combination causing a 1000-fold leftward shift of the response to dopamine. These results showcase multiple opportunities to modulate D1 tone and highlight new pharmacological approaches for allosteric modulation of G-protein-coupled receptors.

2-(Fluoromethoxy)-4'-(S-methanesulfonimidoyl)-1,1'-biphenyl (UCM-1306), an Orally Bioavailable Positive Allosteric Modulator of the Human Dopamine D1 Receptor for Parkinson's Disease.

Tolerance development caused by dopamine replacement with l-DOPA and therapeutic drawbacks upon activation of dopaminergic receptors with orthosteric agonists reveal a significant unmet need for safe and effective treatment of Parkinson’s disease. In search for selective modulators of the D1 receptor, the screening of a chemical library and subsequent medicinal chemistry program around an identified hit resulted in new synthetic compound 26 [UCM-1306, 2-(fluoromethoxy)-4′-(S-methanesulfonimidoyl)-1,1′-biphenyl] that increases the dopamine maximal effect in a dose-dependent manner in human and mouse D1 receptors, is inactive in the absence of dopamine, modulates dopamine affinity for the receptor, exhibits subtype selectivity, and displays low binding competition with orthosteric ligands. The new allosteric modulator potentiates cocaine-induced locomotion and enhances l-DOPA recovery of decreased locomotor activity in reserpinized mice after oral administration. The behavior of compound 26 supports the interest of a positive allosteric modulator of the D1 receptor as a promising therapeutic approach for Parkinson’s disease.

Forecasting gait freezing event in Parkinson's patients utilizing machine learning approach from accelerometer signals

Neurological illnesses are one of the most common medical conditions affecting human races and societies worldwide. Parkinson's disease is a neurological ailment caused by absence of dopamine in the human brain and has an impact on the afflicted person's daily routine. Gait freezing event is the most concerning symptom of Parkinson's disease, and it affects around half of people with severe Parkinson's. Machine learning methods are used in this study to detect and forecast gait freezing events. Two hundred thirty seven gait freezing instances from eight patients were collected from tri-axial accelerometer data set and used to train four machine learning classification models. After comparing different performance measures of the four classification models it was found that the Random forest classification model was the most suitable one for predicting gait freezing events in Parkinson disease as it had the best accuracy, sensitivity ,selectivity and least error among the four models. Keywords: Parkinson’s disease , Gait freezing , machine learning , tri-axial accelerometer , Random forest classification model.

A facile aptamer-based sensing strategy for dopamine detection through the fluorescence energy transfer between dye and single-wall carbon nanohorns

Dopamine (DBA) as an important biomarker, plays a crucial role in disease diagnosis. In this study, we have developed a fast and simple aptamer-based fluorescence strategy which used single-wall carbon nanohorns (SWCNHs) as a quencher for dopamine detection. SWCNHs were negatively charged after pretreated, which improved its dispersion in solution. 5-carboxy-fluorescein (FAM) was used to label dopamine aptamer. In the absence of dopamine, FAM-modified aptamer could be absorbed onto the SWCNHs surface due to π-π interaction, resulting in the fluorescence intensity decreased. Dopamine could specifically bind with FAM-DNA to form G-quadruplex, which could not be absorbed onto the surface of SWCNHs. Hence, the fluorescence of FAM-DNA recovered, and the fluorescent intensity as a function of different concentrations of dopamine was measured. We obtained a detection limit of 5 μM for this detection system with a linear detection range of 0.02–2.20 mM. Furthermore, the feasibility of the innovative detection system has been verified by detecting dopamine in spiked serum samples.

Bidirectional Optogenetic Modulation of the Subthalamic Nucleus in a Rodent Model of Parkinson's Disease.

Parkinson’s disease (PD) is a neurodegenerative disorder characterized by a range of motor symptoms. Treatments are focused on dopamine replacement therapy or deep brain stimulation (DBS). The subthalamic nucleus (STN) is a common target for DBS treatment of PD. However, the function of the STN in normal conditions and pathology is poorly understood. Here, we show in rats that optogenetic modulation of STN neuronal activity exerts bidirectional control of motor function, where inhibition of the STN increases movement and STN activation decreases movement. We also examined the effect of bidirectional optogenetic manipulation STN neuronal activity under dopamine depleted condition using the bilateral rodent 6-hydroxydopamine (6-OHDA) model of Parkinson’s disease. Optogenetic inhibition of the STN in the absence of dopamine had no impact on motor control yet STN excitation led to pronounced abnormal involuntary movement. Administration of levodopa rescued the abnormal involuntary movements induced by STN excitation. Although dopamine and STN dysfunction are well established in PD pathology, here we demonstrate simultaneous STN over activity and loss of dopamine lead to motor deficits. Moreover, we show the dysfunction of the STN is dependent on dopamine. This study provides evidence that the loss of dopamine and the over activity of the STN are key features of PD motor deficits. These results provide insight into the STN pathology in PD and therapeutic mechanism of targeting the STN for the treatment for PD.

The Dopamine Assisted Synthesis of MoO3/Carbon Electrodes With Enhanced Capacitance in Aqueous Electrolyte.

A capacitance increase phenomenon is observed for MoO3 electrodes synthesized via a sol-gel process in the presence of dopamine hydrochloride (Dopa HCl) as compared to α-MoO3 electrodes in 5M ZnCl2 aqueous electrolyte. The synthesis approach is based on a hydrogen peroxide-initiated sol-gel reaction to which the Dopa HCl is added. The powder precursor (Dopa)xMoOy, is isolated from the metastable gel using freeze-drying. Hydrothermal treatment (HT) of the precursor results in the formation of MoO3 accompanied by carbonization of the organic molecules; designated as HT-MoO3/C. HT of the precipitate formed in the absence of dopamine in the reaction produced α-MoO3, which was used as a reference material in this study (α-MoO3-ref). Scanning electron microscopy (SEM) images show a nanobelt morphology for both HT-MoO3/C and α-MoO3-ref powders, but with distinct differences in the shape of the nanobelts. The presence of carbonaceous content in the structure of HT-MoO3/C is confirmed by FTIR and Raman spectroscopy measurements. X-ray diffraction (XRD) and Rietveld refinement analysis demonstrate the presence of α-MoO3 and h-MoO3 phases in the structure of HT-MoO3/C. The increased specific capacitance delivered by the HT-MoO3/C electrode as compared to the α-MoO3-ref electrode in 5M ZnCl2 electrolyte in a −0.25–0.70 V vs. Ag/AgCl potential window triggered a more detailed study in an expanded potential window. In the 5M ZnCl2 electrolyte at a scan rate of 2 mV s−1, the HT-MoO3/C electrode shows a second cycle capacitance of 347.6 F g−1. The higher electrochemical performance of the HT-MoO3/C electrode can be attributed to the presence of carbon in its structure, which can facilitate electron transport. Our study provides a new route for further development of metal oxides for energy storage applications.

A Simple Fluorescent Aptasensing Platform Based on Graphene Oxide for Dopamine Determination.

Dopamine (DA) is a catecholamine neurotransmitter playing an important role in different biological functions including central nervous, renal, cardiovascular, and hormonal systems. The sensitive and selective detection of this neurotransmitter plays a key role in the early diagnosis of various diseases related to abnormal levels of dopamine. Therefore, it is of great importance to explore rapid, simple, and accurate methods for detection of dopamine with high sensitivity and specificity. We propose in this work a fluorescent aptasensor based on graphene oxide (GO) as a quencher, for the rapid determination of dopamine. The principle of this aptasensor is based on fluorescence resonance energy transfer (FRET), where GO was used as energy donor, and a carboxy fluorescein (FAM)-labeled aptamer as acceptor. In the absence of DA, FAM-aptamer was adsorbed on the surface of GO through π-π stacking interactions between nucleotide bases and the carbon network, leading to a weak FRET and a quenching of the FAM fluorescence. However, by adding the target, the aptamer undergoes a conformational change to bind to DA with high affinity, resulting in a fluorescence recovery. Under the optimal experimental conditions, the fluorescence recovery was linearly proportional to the concentration of DA in the range of 3-1680 nM, with a limit of detection of 0.031 nM and a limit of quantification of 0.1 nM. Moreover, the developed assay exhibited minor response in the presence of various interferents and it revealed a satisfactory applicability in human serum samples.

Bile Acids Gate Dopamine Transporter Mediated Currents.

Bile acids (BAs) are molecules derived from cholesterol that are involved in dietary fat absorption. New evidence supports an additional role for BAs as regulators of brain function. Sterols such as cholesterol interact with monoamine transporters, including the dopamine (DA) transporter (DAT) which plays a key role in DA neurotransmission and reward. This study explores the interactions of the BA, obeticholic acid (OCA), with DAT and characterizes the regulation of DAT activity via both electrophysiology and molecular modeling. We expressed murine DAT (mDAT) in Xenopus laevis oocytes and confirmed its functionality. Next, we showed that OCA promotes a DAT-mediated inward current that is Na+-dependent and not regulated by intracellular calcium. The current induced by OCA was transient in nature, returning to baseline in the continued presence of the BA. OCA also transiently blocked the DAT-mediated Li+-leak current, a feature that parallels DA action and indicates direct binding to the transporter in the absence of Na+. Interestingly, OCA did not alter DA affinity nor the ability of DA to promote a DAT-mediated inward current, suggesting that the interaction of OCA with the transporter is non-competitive, regarding DA. Docking simulations performed for investigating the molecular mechanism of OCA action on DAT activity revealed two potential binding sites. First, in the absence of DA, OCA binds DAT through interactions with D421, a residue normally involved in coordinating the binding of the Na+ ion to the Na2 binding site (Borre et al., J. Biol. Chem., 2014, 289, 25764–25773; Cheng and Bahar, Structure, 2015, 23, 2171–2181). Furthermore, we uncover a separate binding site for OCA on DAT, of equal potential functional impact, that is coordinated by the DAT residues R445 and D436. Binding to that site may stabilize the inward-facing (IF) open state by preventing the re-formation of the IF-gating salt bridges, R60-D436 and R445-E428, that are required for DA transport. This study suggests that BAs may represent novel pharmacological tools to regulate DAT function, and possibly, associated behaviors.

Dopamine‐Assisted Coral Films of Cobalt as Bifunctional Electrodes for Overall Water Splitting

Coral‐type cobalt nanostructures are electrodeposited on a dopamine‐modified nickel plate under ordinary conditions. The deposited electrodes are studied for both oxygen evolution reaction (OER) and hydrogen evolution reaction (HER) as bifunctional electrodes for overall water splitting. These electrodes show overpotentials of 300 and 290 mV for OER and HER respectively, at a benchmark current density of 10 mA cm−2. In case of OER, the absence of dopamine gives higher overpotential and exfoliation of Co coating. In the presence of dopamine, the electrodes show lower overpotential and better long‐term stability for both HER and OER for overall water splitting. Hence, the present Ni−dopamine−Co electrode interface can be a better bifunctional electrode for the overall water splitting reaction.

Role of Free Catecholamine in Thiol-Ene Crosslinking for Hyaluronic Acid Hydrogels with High Loading Efficiency of Anticancer Drugs.

Hyaluronic acid (HA) is a widely used polysaccharide in biomedical field because of its excellent biocompatibility. Its chemical structure can be modified with various functional groups. Recently, dopamine has been tethered onto the polymeric backbone to ensure long-term stability and tissue adhesiveness of HA hydrogel. However, the radical scavenging effect of dopamine on typical photo-induced crosslinking for hydrogels has not been specifically studied. Photo-crosslinkable norbornene-modified HA (NorHA) was synthesized and crosslinked by dithiothreitol containing dopamine at different concentrations. During in situ ultraviolet light-triggered crosslinking, storage moduli were monitored using an oscillatory rheometer. Additionally, the amount of thiol utilized for HA crosslinking was investigated under the presence and absence of dopamine. Finally, doxorubicin was encapsulated in the hydrogels, and the drug loading efficiency and release kinetics were measured. Adding dopamine into the NorHA pre-gel solution delayed the gelation time, yet the final storage modulus of the hydrogel remained constant. That is, dopamine might partially consume the energy required for thiol-ene reaction to generate semiquinone radicals. Furthermore, the residual thiols which were not involved in the crosslinking decreased when the hydrogel was formed at a high concentration of dopamine, indicating the formation of Michael adducts of semiquinone and thiols. Interestingly, the presence of dopamine in the hydrogel increased the loading efficiency of the hydrophobic drugs due to π-π stacking and hydrogen bonding between dopamine and drugs. The presence of free catecholamines in a photo-crosslinkable polymer can delay the gelation time but improve the drug loading efficiency.

Multi-Electrode Array Analysis Identifies Complex Dopamine Responses and Glucose Sensing Properties of Substantia Nigra Neurons in Mouse Brain Slices.

Dopaminergic (DA) midbrain neurons within the substantia nigra (SN) display an autonomous pacemaker activity that is crucial for dopamine release and voluntary movement control. Their progressive degeneration is a hallmark of Parkinson's disease. Their metabolically demanding activity-mode affects Ca2+ homeostasis, elevates metabolic stress, and renders SN DA neurons particularly vulnerable to degenerative stressors. Accordingly, their activity is regulated by complex mechanisms, notably by dopamine itself, via inhibitory D2-autoreceptors and the neuroprotective neuronal Ca2+ sensor NCS-1. Analyzing regulation of SN DA neuron activity-pattern is complicated by their high vulnerability. We studied this activity and its control by dopamine, NCS-1, and glucose with extracellular multi-electrode array (MEA) recordings from midbrain slices of juvenile and adult mice. Our tailored MEA- and spike sorting-protocols allowed high throughput and long recording times. According to individual dopamine-responses, we identified two distinct SN cell-types, in similar frequency: dopamine-inhibited and dopamine-excited neurons. Dopamine-excited neurons were either silent in the absence of dopamine, or they displayed pacemaker-activities, similar to that of dopamine-inhibited neurons. Inhibition of pacemaker-activity by dopamine is typical for SN DA neurons, and it can undergo prominent desensitization. We show for adult mice, that the number of SN DA neurons with desensitized dopamine-inhibition was increased (~60–100%) by a knockout of NCS-1, or by prevention of NCS-1 binding to D2-autoreceptors, while time-course and degrees of desensitization were not altered. The number of neurons with desensitized D2-responses was also higher (~65%) at high glucose-levels (25 mM), compared to lower glucose (2.5 mM), while again desensitization-kinetics were unaltered. However, spontaneous firing-rates were significantly higher at high glucose-levels (~20%). Moreover, transient glucose-deprivation (1 mM) induced a fast and fully-reversible pacemaker frequency reduction. To directly address and quantify glucose-sensing properties of SN DA neurons, we continuously monitored their electrical activity, while altering extracellular glucose concentrations stepwise from 0.5 mM up to 25 mM. SN DA neurons were excited by glucose, with EC50 values ranging from 0.35 to 2.3 mM. In conclusion, we identified a novel, common subtype of dopamine-excited SN neurons, and a complex, joint regulation of dopamine-inhibited neurons by dopamine and glucose, within the range of physiological brain glucose-levels.

Chronic and Acute Manipulation of Cortical Glutamate Transmission Induces Structural and Synaptic Changes in Co-cultured Striatal Neurons.

In contrast to the prenatal topographic development of sensory cortices, striatal circuit organization is slow and requires the functional maturation of cortical and thalamic excitatory inputs throughout the first postnatal month. While mechanisms regulating synapse development and plasticity are quite well described at excitatory synapses of glutamatergic neurons in the neocortex, comparatively little is known of how this translates to glutamate synapses onto GABAergic neurons in the striatum. Here we investigate excitatory striatal synapse plasticity in an in vitro system, where glutamate can be studied in isolation from dopamine and other neuromodulators. We examined pre-and post-synaptic structural and functional plasticity in GABAergic striatal spiny projection neurons (SPNs), co-cultured with glutamatergic cortical neurons. After synapse formation, medium-term (24 h) TTX silencing increased the density of filopodia, and modestly decreased dendritic spine density, when assayed at 21 days in vitro (DIV). Spine reductions appeared to require residual spontaneous activation of ionotropic glutamate receptors. Conversely, chronic (14 days) TTX silencing markedly reduced spine density without any observed increase in filopodia density. Time-dependent, biphasic changes to the presynaptic marker Synapsin-1 were also observed, independent of residual spontaneous activity. Acute silencing (3 h) did not affect presynaptic markers or postsynaptic structures. To induce rapid, activity-dependent plasticity in striatal neurons, a chemical NMDA receptor-dependent “long-term potentiation (LTP)” paradigm was employed. Within 30 min, this increased spine and GluA1 cluster densities, and the percentage of spines containing GluA1 clusters, without altering the presynaptic signal. The results demonstrate that the growth and pruning of dendritic protrusions is an active process, requiring glutamate receptor activity in striatal projection neurons. Furthermore, NMDA receptor activation is sufficient to drive glutamatergic structural plasticity in SPNs, in the absence of dopamine or other neuromodulators.

Morphological Changes in Astrocytes by Self-Oxidation of Dopamine to Polydopamine and Quantification of Dopamine through Multivariate Regression Analysis of Polydopamine Images.

Astrocytes, also known as astroglia, are important cells for the structural support of neurons as well as for biochemical balance in the central nervous system (CNS). In this study, the polymerization of dopamine (DA) to polydopamine (PDA) and its effect on astrocytes was investigated. The polymerization of DA, being directly proportional to the DA concentration, raises the prospect of detecting DA concentration from PDA optically using image-processing techniques. It was found here that DA, a naturally occurring neurotransmitter, significantly altered astrocyte cell number, morphology, and metabolism, compared to astrocytes in the absence of DA. Along with these effects on astrocytes, the polymerization of DA to PDA was tracked optically in the same cell culture wells. This polymerization process led to a unique methodology based on multivariate regression analysis that quantified the concentration of DA from optical images of astrocyte cell culture media. Therefore, this developed methodology, combined with conventional imaging equipment, could be used in place of high-end and expensive analytical chemistry instruments, such as spectrophotometry, mass spectrometry, and fluorescence techniques, for quantification of the concentration of DA after polymerization to PDA under in vitro and potentially in vivo conditions.

Fluorescent silver nanoclusters stabilized in guanine-enhanced DNA hybridization for recognizing different small biological molecules

Fluorescent DNA-stabilized Ag nanoclusters (DNA/Ag NCs) plays significant roles in biochemical detections due to their high fluorescence quantum yield, high stability and good biocompatibility. In this paper, two guanine-enhanced fluorescence DNA-stabilized silver nanoclusters were designed to study the effect of guanine coating degree on silver nanoclusters fluorescence and their interactions with small biological molecules. The results indicated that the degree of guanine coating had a slight influence on the fluorescence of the two DNA/Ag NCs, however, the two DNA/Ag NCs had different fluorescence response when interacting with different small biological molecules. The fluorescence of the DNA/Ag NCs wrapped in the guanine-rich strand could be quenched by small biomolecules such as ascorbic acid, dopamine and cysteine, while the fluorescence of the DNA/Ag NCs approached to the guanine-rich strand could only be quenched by dopamine and cysteine. Based on this interesting phenomenon, an analytical method for recognition of ascorbic acid, dopamine and cysteine from other small biological molecules can be established by measuring the fluorescence change of DNA/Ag NCs and a method for selective detection of ascorbic acid in the absence of dopamine and cysteines can be achieved by combining the two DNA/Ag NCs probe.

Determination of dopamine by a label-free fluorescent aptasensor based on AuNPs and carbon quantum dots

In order to reduce the complex modification process and obtain a fast and label-free aptasensor, a label-free fluorescent aptasensor based on gold nanoparticles (AuNPs) and carbon quantum dots (CQDs) was constructed for rapid determination of dopamine (DA). In the absence of DA, CQDs were adsorbed on the surface of AuNPs through the electrostatic force between CQDs and aptamer, which led to the fluorescence quenching of CQDs. When DA were added, they combined with the corresponding aptamers with higher affinity and selectivity, resulting in CQDs far from the surface of AuNPs and thus the fluorescence intensity of CQDs was recovered. Under the optimal condition, the fluorescence intensity of the released CQDs was linearly proportional to the concentration of DA in a range of 5×10-8˜2.5×10-4mol/L with a detection limit of 1×10-8mol/L. Moreover, the aptasensor exhibited a satisfactory detection result of DA in human serum samples. This study provided a more effective method to detect a broad range of targets by using appropriate aptamers, holding great potential in the field of food safety and biomedical diagnostics.

Comparative Pharmacology and Structure–Activity Relationships of D1 Dopamine Receptor Positive Allosteric Modulators

The D1 dopamine receptor (D1R) is a key component of regulating dopaminergic signaling and, as such, is associated with many important neurological processes and diseases. It is an attractive target to treat a wide range of neuropsychiatric disorders, such as Parkinson's disease and the cognitive decline associated with schizophrenia and Alzheimer's disease. Positive allosteric modulators (PAMs) of the D1R have emerged as a drug development strategy, as D1R orthosteric agonists have clinical liabilities curtailing their use. We discovered two D1R PAMs via a high‐throughput screen, MLS1082 and MLS6585. The two PAMs act similarly, potentiating agonist‐stimulated G‐protein and β‐arrestin‐mediated signaling and increasing dopamine's affinity for the D1R. Neither compound has any inherent agonist activity in the absence of dopamine. MLS1082 and MLS6585 are structurally distinct chemical series and we have found they act at two separate binding sites. MLS1082 shares a similar binding site with two known D1 PAMs, Compound B and DETQ, which involves the R130 amino acid in the second intracellular loop of the D1R. MLS6585 displays additive activity with MLS1082, Compound B, and DETQ and its activity is not affected by a R130Q point mutation, strongly suggesting that MLS6585 acts at a separate binding site than these other D1R PAMs. Though D1R PAMs have been described by our group and others, the pharmacology and structure–activity relationships (SAR) underlying D1R allosteric modulation is not well understood. We used several analog sets of both MLS1082 and MLS6585 in order to begin addressing these questions as well as to improve on the D1R PAM activity of the parent compounds. Many of the analogs of both MLS1082 and MLS6585 retained PAM activity, validating these drug scaffolds as D1R modulators. The analogs of one series displayed additive activity with the opposite series, suggesting that the analogs are binding to similar sites as their parent compounds. The largest improvement on the parent compounds came via better selectivity for the D1R over the closely related D5 dopamine receptor. This has important implications for understanding the homology of allosteric sites between related receptor types. A small number of seemingly inactive analogs appear to act as silent allosteric modulators (SAMs), in that they blocked the activity of the parent PAM compound, presumably by competing for the same binding site. Further analog studies are underway to better understand the chemical SAR needed for D1R allosteric modulation as well as the differences between the two D1R PAM binding sites.Support or Funding InformationFunded by NINDS Intramural Research Program and Eshelman Institute for InnovationThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Intracellular Binding Site for a Positive Allosteric Modulator of the Dopamine D1 Receptor.

The binding site for DETQ [2-(2,6-dichlorophenyl)-1-((1S,3R)-3-(hydroxymethyl)-5-(2-hydroxypropan-2-yl)-1-methyl-3,4-dihydroisoquinolin-2(1H)-yl)ethan-1-one], a positive allosteric modulator (PAM) of the dopamine D1 receptor, was identified and compared with the binding site for CID 2886111 [N-(6-tert-butyl-3-carbamoyl-4,5,6,7-tetrahydro-1-benzothiophen-2-yl)pyridine-4-carboxamide], a reference D1 PAM. From D1/D5 chimeras, the site responsible for potentiation by DETQ of the increase in cAMP in response to dopamine was narrowed down to the N-terminal intracellular quadrant of the receptor; arginine-130 in intracellular loop 2 (IC2) was then identified as a critical amino acid based on a human/rat species difference. Confirming the importance of IC2, a β2-adrenergic receptor construct in which the IC2 region was replaced with its D1 counterpart gained the ability to respond to DETQ. A homology model was built from the agonist-state β2-receptor structure, and DETQ was found to dock to a cleft created by IC2 and adjacent portions of transmembrane helices 3 and 4 (TM3 and TM4). When residues modeled as pointing into the cleft were mutated to alanine, large reductions in the potency of DETQ were found for Val119 and Trp123 (flanking the conserved DRY sequence in TM3), Arg130 (located in IC2), and Leu143 (TM4). The D1/D5 difference was found to reside in Ala139; changing this residue to methionine as in the D5 receptor reduced the potency of DETQ by approximately 1000-fold. None of these mutations affected the activity of CID 2886111, indicating that it binds to a different allosteric site. When combined, DETQ and CID 2886111 elicited a supra-additive response in the absence of dopamine, implying that both PAMs can bind to the D1 receptor simultaneously.