Fine-tuning GPCR-mediated neuromodulation by biasing signaling through different G protein subunits.

G-protein-coupled receptors (GPCRs) mediate neuromodulation through the activation of heterotrimeric G proteins (Gαßγ). Classical models depict that G protein activation leads to a one-to-one formation of Gα-GTP and Gßγ species. Each of these species propagates signaling by independently acting on effectors, but the mechanisms by which response fidelity is ensured by coordinating Gα and Gßγ responses remain unknown. Here, we reveal a paradigm of G protein regulation whereby the neuronal protein GINIP (Gα inhibitory interacting protein) biases inhibitory GPCR responses to favor Gßγ over Gα signaling. Tight binding of GINIP to Gαi-GTP precludes its association with effectors (adenylyl cyclase) and, simultaneously, with regulator-of-G-protein-signaling (RGS) proteins that accelerate deactivation. As a consequence, Gαi-GTP signaling is dampened, whereas Gßγ signaling is enhanced. We show that this mechanism is essential to prevent the imbalances of neurotransmission that underlie increased seizure susceptibility in mice. Our findings reveal an additional layer of regulation within a quintessential mechanism of signal transduction that sets the tone of neurotransmission.

Coexistence of chronic hyperalgesia and multilevel neuroinflammatory responses after experimental SCI: a systematic approach to profiling neuropathic pain.

BACKGROUND: People with spinal cord injury (SCI) frequently develop neuropathic pain (NP) that worsens disability and diminishes rehabilitation efficacy. Chronic NP is presently incurable due to poor understanding of underlying mechanisms. We hypothesized that multilocus neuroinflammation (NIF) might be a driver of SCI NP, and tested it by investigating whether NP coexisted with central NIF, neurotransmission (NTM), neuromodulation (NML) and neuroplasticity (NPL) changes post-SCI. METHODS: Female Sprague-Dawley rats (230-250 g) with T10 compression or laminectomy were evaluated for physical conditions, coordinated hindlimb functions, neurological reflexes, and mechanical/thermal sensitivity thresholds at 1 day post-injury (p.i.) and weekly thereafter. Eight weeks p.i., central nervous system tissues were histochemically and immunohistochemically characterized for parameters/markers of histopathology and NIF/NTM/NML/NPL. Also analyzed was the correlative relationship between levels of selected biomarkers and thermosensitivity thresholds via statistical linear regression. RESULTS: SCI impaired sensorimotor functions, altered reflexes, and produced spontaneous pain signs and hypersensitivity to evoked nociceptive, mechanical, and thermal inputs. Only injured spinal cords exhibited neural lesion, microglia/astrocyte activation, and abnormal expression of proinflammatory cytokines, as well as NIF/NTM/NML/NPL markers. Brains of SCI animals displayed similar pathophysiological signs in the gracile and parabrachial nuclei (GrN and PBN: sensory relay), raphe magnus nucleus and periaqueduct gray (RMN and PAG: pain modulation), basolateral amygdala (BLA: emotional-affective dimension of pain), and hippocampus (HPC: memory/mood/neurogenesis). SCI augmented sensory NTM/NPL (GrN and PBN); increased GAD67 (PAG) level; reduced serotonin (RMN) and fear-off neuronal NTR2 (BLA) expressions; and perturbed neurogenesis (HPC). CONCLUSION: T10 compression caused chronic hyperalgesia that coexisted with NIF/NTM/NML/NPL responses at multilevel neuroaxis centers. The data have provided multidimensional biomarkers as new mechanistic leads to profile SCI NP for therapeutic/therapy development.

Tilapia fish microbial spoilage monitored by a single optical gas sensor.

As consumption of fish and fish-based foods increases, non-destructive monitoring of fish freshness also becomes more prominent. Fish products are very perishable and prone to microbiological growth, not always easily detected by organoleptic evaluation. The analysis of the headspace of fish specimens through gas sensing is an interesting approach to monitor fish freshness. Here we report a gas sensing method for monitoring Tilapia fish spoilage based on the application of a single gas sensitive gel material coupled to an optical electronic nose. The optical signals of the sensor and the extent of bacterial growth were followed over time, and results indicated good correlation between the two determinations, which suggests the potential application of this simple and low cost system for Tilapia fish freshness monitoring.

Tunable Gas Sensing Gels by Cooperative Assembly.

The cooperative assembly of biopolymers and small molecules can yield functional materials with precisely tunable properties. Here, the fabrication, characterization, and use of multicomponent hybrid gels as selective gas sensors are reported. The gels are composed of liquid crystal droplets self-assembled in the presence of ionic liquids, which further coassemble with biopolymers to form stable matrices. Each individual component can be varied and acts cooperatively to tune gels' structure and function. The unique molecular environment in hybrid gels is explored for supramolecular recognition of volatile compounds. Gels with distinct compositions are used as optical and electrical gas sensors, yielding a combinatorial response conceptually mimicking olfactory biological systems, and tested to distinguish volatile organic compounds and to quantify ethanol in automotive fuel. The gel response is rapid, reversible, and reproducible. These robust, versatile, modular, pliant electro-optical soft materials possess new possibilities in sensing triggered by chemical and physical stimuli.

Highly Potent and Selective Dopamine D4 Receptor Antagonists Potentially Useful for the Treatment of Glioblastoma.

To better understand the role of dopamine D4 receptor (D4R) in glioblastoma (GBM), in the present paper, new ligands endowed with high affinity and selectivity for D4R were discovered starting from the brain penetrant and D4R selective lead compound 1-(3-(4-phenylpiperazin-1-yl)propyl)-3,4-dihydroquinolin-2(1H)-one (6). In particular, the D4R antagonist 24, showing the highest affinity and selectivity over D2R and D3R within the series (D2/D4 = 8318, D3/D4 = 3715), and the biased ligand 29, partially activating D4R Gi-/Go-protein and blocking ß-arrestin recruitment, emerged as the most interesting compounds. These compounds, evaluated for their GBM antitumor activity, induced a decreased viability of GBM cell lines and primary GBM stem cells (GSC#83), with the maximal efficacy being reached at a concentration of 10 µM. Interestingly, the treatment with both compounds 24 and 29 induced an increased effect in reducing the cell viability with respect to temozolomide, which is the first-choice chemotherapeutic drug in GBM.

Effects of Magnetite Nanoparticles and Static Magnetic Field on Neural Differentiation of Pluripotent Stem Cells.

Neurodevelopmental processes of pluripotent cells, such as proliferation and differentiation, are influenced by external natural forces. Despite the presence of biogenic magnetite nanoparticles in the central nervous system and constant exposure to the Earth's magnetic fields and other sources, there is scant knowledge regarding the role of electromagnetic stimuli in neurogenesis. Moreover, emerging applications of electrical and magnetic stimulation to treat neurological disorders emphasize the relevance of understanding the impact and mechanisms behind these stimuli. Here, the effects of magnetic nanoparticles (MNPs) in polymeric coatings and the static external magnetic field (EMF) were investigated on neural induction of murine embryonic stem cells (mESCs) and human induced pluripotent stem cells (hiPSCs). The results show that the presence of 0.5% MNPs in collagen-based coatings facilitates the migration and neuronal maturation of mESCs and hiPSCs in vitro. Furthermore, the application of 0.4 Tesla EMF perpendicularly to the cell culture plane, discernibly stimulates proliferation and guide fate decisions of the pluripotent stem cells, depending on the origin of stem cells and their developmental stage. Mechanistic analysis reveals that modulation of ionic homeostasis and the expression of proteins involved in cytostructural, liposomal and cell cycle checkpoint functions provide a principal underpinning for the impact of electromagnetic stimuli on neural lineage specification and proliferation. These findings not only explore the potential of the magnetic stimuli as neural differentiation and function modulator but also highlight the risks that immoderate magnetic stimulation may affect more susceptible neurons, such as dopaminergic neurons.

Chirality of Novel Bitopic Agonists Determines Unique Pharmacology at the Dopamine D3 Receptor.

The dopamine D2/D3 receptor (D2R/D3R) agonists are used as therapeutics for Parkinson's disease (PD) and other motor disorders. Selective targeting of D3R over D2R is attractive because of D3R's restricted tissue distribution with potentially fewer side-effects and its putative neuroprotective effect. However, the high sequence homology between the D2R and D3R poses a challenge in the development of D3R selective agonists. To address the ligand selectivity, bitopic ligands were designed and synthesized previously based on a potent D3R-preferential agonist PF592,379 as the primary pharmacophore (PP). This PP was attached to various secondary pharmacophores (SPs) using chemically different linkers. Here, we characterize some of these novel bitopic ligands at both D3R and D2R using BRET-based functional assays. The bitopic ligands showed varying differences in potencies and efficacies. In addition, the chirality of the PP was key to conferring improved D3R potency, selectivity, and G protein signaling bias. In particular, compound AB04-88 exhibited significant D3R over D2R selectivity, and G protein bias at D3R. This bias was consistently observed at various time-points ranging from 8 to 46 min. Together, the structure-activity relationships derived from these functional studies reveal unique pharmacology at D3R and support further evaluation of functionally biased D3R agonists for their therapeutic potential.

Midbrain Dopaminergic Neurons Differentiated from Human-Induced Pluripotent Stem Cells.

The work with midbrain dopaminergic neurons (mDAN) differentiation might seem to be hard. There are about 40 different published protocols for mDAN differentiation, which are eventually modified according to the respective laboratory. In many cases, protocols are not fully described, failing to provide essential tips for researchers starting in the field. Considering that commercial kits produce low mDAN percentages (20-50%), we chose to follow a mix of four main protocols based on Kriks and colleagues' protocol, from which the resulting mDAN were engrafted with success in three different animal models of Parkinson's disease. We present a differential step-by-step methodology for generating mDAN directly from human-induced pluripotent stem cells cultured with E8 medium on Geltrex, without culture on primary mouse embryonic fibroblasts prior to mDAN differentiation, and subsequent exposure of neurons to rock inhibitor during passages for improving cell viability. The protocol described here allows obtaining mDAN with phenotypical and functional characteristics suitable for in vitro modeling, cell transplantation, and drug screening.

Novel Conducting and Biodegradable Copolymers with Noncytotoxic Properties toward Embryonic Stem Cells.

Electroactive biomaterials that are easily processed as scaffolds with good biocompatibility for tissue regeneration are difficult to design. Herein, the synthesis and characterization of a variety of novel electroactive, biodegradable biomaterials based on poly(3,4-ethylenedioxythiphene) copolymerized with poly(d,l lactic acid) (PEDOT-co-PDLLA) are presented. These copolymers were obtained using (2,3-dihydrothieno[3,4-b][1,4]dioxin-2-yl)methanol (EDOT-OH) as an initiator in a lactide ring-opening polymerization reaction, resulting in EDOT-PDLLA macromonomer. Conducting PEDOT-co-PDLLA copolymers (in three different proportions) were achieved by chemical copolymerization with 3,4-ethylenedioxythiophene (EDOT) monomers and persulfate oxidant. The PEDOT-co-PDLLA copolymers were structurally characterized by 1H NMR and Fourier transform infrared spectroscopy. Cyclic voltammetry confirmed the electroactive character of the materials, and conductivity measurements were performed via electrochemical impedance spectroscopy. In vitro biodegradability was evaluated using proteinase K over 35 days, showing 29-46% (w/w) biodegradation. Noncytotoxicity was assessed by adhesion, migration, and proliferation assays using embryonic stem cells (E14.tg2a); excellent neuronal differentiation was observed. These novel electroactive and biodegradable PEDOT-co-PDLLA copolymers present surface chemistry and charge density properties that make them potentially useful as scaffold materials in different fields of applications, especially for neuronal tissue engineering.

Magnetic hydrogels for levodopa release and cell stimulation triggered by external magnetic field.

Magnetic responsive hydrogels composed of alginate (Alg) and xanthan gum (XG), crosslinked with Ca2+ ions, were modified by in situ magnetic nanoparticles (MNP) formation. In comparison to magnetic Alg hydrogels, magnetic Alg-XG hydrogels presented superior mechanical and swelling properties, due to the high charge density and molecular weight of XG. The loading efficiency of levodopa (LD), an important antiparkinson drug, in the Alg-XG/MNP hydrogels was the highest (64%), followed by Alg/MNP (56%), Alg-XG (53%) and Alg (28%). A static external magnetic field (EMF) of 0.4 T stimulated the release of LD from Alg-XG/MNP hydrogels achieving 64 ±â€¯6% of the initial loading after 30 h. The viability, proliferation and expression of dopaminergic markers of human neuroblastoma SH-SY5Y cell on the LD loaded magnetic hydrogels were successful, particularly under EMF, which stimulated the release of LD. Overall, the results of this study provided the rational design of magnetic hydrogels for the delivery of drugs, which combined with external magnetic stimulus, might improve cell proliferation and specific differentiation.