Abstract
MicroRNAs (miRNAs) are small non-coding RNAs (typically consisting of 18–25 nucleotides) that negatively control expression of target genes at the post-transcriptional level. Owing to the ...biological significance of miRNAs, miRTarBase was developed to provide comprehensive information on experimentally validated miRNA–target interactions (MTIs). To date, the database has accumulated >13,404 validated MTIs from 11,021 articles from manual curations. In this update, a text-mining system was incorporated to enhance the recognition of MTI-related articles by adopting a scoring system. In addition, a variety of biological databases were integrated to provide information on the regulatory network of miRNAs and its expression in blood. Not only targets of miRNAs but also regulators of miRNAs are provided to users for investigating the up- and downstream regulations of miRNAs. Moreover, the number of MTIs with high-throughput experimental evidence increased remarkably (validated by CLIP-seq technology). In conclusion, these improvements promote the miRTarBase as one of the most comprehensively annotated and experimentally validated miRNA–target interaction databases. The updated version of miRTarBase is now available at http://miRTarBase.cuhk.edu.cn/.
Signaling across cellular membranes, the 826 human G protein-coupled receptors (GPCRs) govern a wide range of vital physiological processes, making GPCRs prominent drug targets. X-ray crystallography ...provided GPCR molecular architectures, which also revealed the need for additional structural dynamics data to support drug development. Here, nuclear magnetic resonance (NMR) spectroscopy with the wild-type-like A2A adenosine receptor (A2AAR) in solution provides a comprehensive characterization of signaling-related structural dynamics. All six tryptophan indole and eight glycine backbone 15N–1H NMR signals in A2AAR were individually assigned. These NMR probes provided insight into the role of Asp522.50 as an allosteric link between the orthosteric drug binding site and the intracellular signaling surface, revealing strong interactions with the toggle switch Trp 2466.48, and delineated the structural response to variable efficacy of bound drugs across A2AAR. The present data support GPCR signaling based on dynamic interactions between two semi-independent subdomains connected by an allosteric switch at Asp522.50.
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•Comprehensive view of GPCR signaling pathways using NMR probes•Allosteric signaling monitored by NMR probes distributed throughout A2AAR•Function-related conformational polymorphisms at intracellular A2AAR surface•Strong coupling between allosteric switch and signaling-activation motif
Monitoring dynamics of GPCR signaling using stable isotope NMR reveals the path of communication enabling an allosteric response to ligand binding.
The κ-opioid receptor (KOP) mediates the actions of opioids with hallucinogenic, dysphoric, and analgesic activities. The design of KOP analgesics devoid of hallucinatory and dysphoric effects has ...been hindered by an incomplete structural and mechanistic understanding of KOP agonist actions. Here, we provide a crystal structure of human KOP in complex with the potent epoxymorphinan opioid agonist MP1104 and an active-state-stabilizing nanobody. Comparisons between inactive- and active-state opioid receptor structures reveal substantial conformational changes in the binding pocket and intracellular and extracellular regions. Extensive structural analysis and experimental validation illuminate key residues that propagate larger-scale structural rearrangements and transducer binding that, collectively, elucidate the structural determinants of KOP pharmacology, function, and biased signaling. These molecular insights promise to accelerate the structure-guided design of safer and more effective κ-opioid receptor therapeutics.
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•κ-opioid receptor active-state crystal structure disclosed•κ-opioid receptor structure reveals features involved in biased signaling•Structure provides template for the creation of safe and effective analgesics
A crystal structure of the active κ-opioid receptor provides a guide for the development of safe and effective new analgesics.
Cardiac disease remains the leading cause of morbidity and mortality worldwide. The β1-adrenergic receptor (β1-AR) is a major regulator of cardiac functions and is downregulated in the majority of ...heart failure cases. A key physiological process is the activation of heterotrimeric G-protein Gs by β1-ARs, leading to increased heart rate and contractility. Here, we use cryo-electron microscopy and functional studies to investigate the molecular mechanism by which β1-AR activates Gs. We find that the tilting of α5-helix breaks a hydrogen bond between the sidechain of His373 in the C-terminal α5-helix and the backbone carbonyl of Arg38 in the N-terminal αN-helix of Gαs. Together with the disruption of another interacting network involving Gln59 in the α1-helix, Ala352 in the β6-α5 loop, and Thr355 in the α5-helix, these conformational changes might lead to the deformation of the GDP-binding pocket. Our data provide molecular insights into the activation of G-proteins by G-protein-coupled receptors.
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•Cryo-EM structure of β1-adrenergic receptor and Gs at 2.6-Å resolution•Network of interactions within Gαs are disrupted by β1-AR•Rotational opening of the α-helical domain of Gαs during its activation•Functional studies of critical residues on β1-AR involved in the activation of Gs
Su et al. report the cryo-EM structure of the complex of isoproterenol-bound β1-adrenergic receptor and heterotrimeric Gs-protein. The structural and functional studies reveal insights into the activation of Gs by β1-adrenergic receptor. This work advances our understanding of the control of heart rate and contractility by the nervous system and hormones.
The present study was designed to investigate the rostral-caudal effect of spinal magnetic stimulation on diaphragmatic motor-evoked potentials after cervical spinal cord injury. The diaphragm ...electromyogram was recorded in rats that received a laminectomy or a left midcervical contusion at the acute (1 day), subchronic (2 weeks), or chronic (8 weeks) injury stages. The center of a figure-eight coil was placed at 30 mm lateral to bregma on the left side, and the effect of magnetic stimulation was evaluated by stimulating the rostral, middle, and caudal cervical regions in spontaneously breathing rats. The results demonstrated that cervical magnetic stimulation induced intensity-dependent motor-evoked potentials in the bilateral diaphragm in both uninjured and contused rats; however, the left diaphragm exhibited a higher amplitude and earlier onset than the right diaphragm. Moreover, the intensity-response curve was shifted upward in the rostral-to-caudal direction of magnetic stimulation, suggesting that caudal cervical magnetic stimulation produced more robust diaphragmatic motor-evoked potentials compared with rostral cervical magnetic stimulation. Interestingly, the diaphragmatic motor-evoked potentials were similar between uninjured and contused rats during cervical magnetic stimulation despite weaker inspiratory diaphragmatic activity in contused rats. In addition, in contused animals but not uninjured animals, diaphragmatic motor-evoked potential amplitudes were greater at the chronic stage than during earlier injury stages. These results demonstrated that cervical magnetic stimulation can excite the residual phrenic motor circuit to activate the diaphragm in the presence of a significant lesion in the cervical spinal cord. These findings indicate that this non-invasive approach is effective for modulating diaphragmatic excitability after cervical spinal cord injury.
The present study was designed to examine the effect of trans‐spinal magnetic stimulation on bilateral respiratory and forelimb muscles in healthy subjects. Two wings of a figure‐of‐eight magnetic ...coil were placed on the dorsal vertebrae, from the fifth cervical to the second thoracic dorsal vertebra with a center at the seventh cervical vertebra. The surface electromyograms of bilateral diaphragm and biceps were recorded in response to trans‐spinal magnetic stimulation with 20%–100% maximum output of the stimulatory device in male (n = 12) and female participants (n = 8). Trans‐spinal magnetic stimulation can induce a co‐activation of bilateral diaphragm and biceps when the stimulation intensity is above 60%. The onset latency was comparable between the left and right sides of the muscles, suggesting bilateral muscles could be simultaneously activated by trans‐spinal magnetic stimulation. In addition, the intensity–response curve of the biceps was shifted upward compared with that of the diaphragm in males, indicating that the responsiveness of the biceps was greater than that of the diaphragm. This study demonstrated the feasibility of utilizing trans‐spinal magnetic stimulation to co‐activate the bilateral diaphragm and biceps. We proposed that this stimulatory configuration can be an efficient approach to activate both respiratory and forelimb muscles.
Trans‐spinal magnetic stimulation is a feasible method to activate the diaphragm and biceps and produce the motor evoked potential recorded by surface electromyogram. Coactivation of bilateral diaphragm and biceps could be induced by trans‐spinal magnetic stimulation simultaneously when the intensity is over 60% maximal stimulus output.
The lipidic cubic phase (LCP) technique has proved to facilitate the growth of high-quality crystals that are otherwise difficult to grow by other methods. However, the crystal size optimization ...process could be time and resource consuming, if it ever happens. Therefore, improved techniques for structure determination using these small crystals is an important strategy in diffraction technology development. Microcrystal electron diffraction (MicroED) is a technique that uses a cryo-transmission electron microscopy to collect electron diffraction data and determine high-resolution structures from very thin micro- and nanocrystals. In this work, we have used modified LCP and MicroED protocols to analyze crystals embedded in LCP converted by 2-methyl-2,4-pentanediol or lipase, including Proteinase K crystals grown in solution, cholesterol crystals, and human adenosine A2A receptor crystals grown in LCP. These results set the stage for the use of MicroED to analyze microcrystalline samples grown in LCP, especially for those highly challenging membrane protein targets.
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•LCP phase conversion enables the study of LCP-embedded crystals by MicroED•Lipase hydrolysis can liberate crystals from LCP for MicroED studies•LCP-MicroED opens a path for structure studies of membrane protein microcrystals
Zhu et al. demonstrate modified microcrystal electron diffraction (MicroED) and lipidic cubic phase (LCP) sample preparation procedures, which can be used to facilitate MicroED analysis of LCP-embedded microcrystals. These results set the stage for the use of MicroED to analyze microcrystals that have been crystallized within LCP.
Bexarotene is an FDA-approved drug for the treatment of cutaneous T-cell lymphoma (CTCL); however, its use provokes or disrupts other retinoid-X-receptor (RXR)-dependent nuclear receptor pathways and ...thereby incites side effects including hypothyroidism and raised triglycerides. Two novel bexarotene analogs, as well as three unique CD3254 analogs and thirteen novel NEt-TMN analogs, were synthesized and characterized for their ability to induce RXR agonism in comparison to bexarotene (
). Several analogs in all three groups possessed an isochroman ring substitution for the bexarotene aliphatic group. Analogs were modeled for RXR binding affinity, and EC
as well as IC
values were established for all analogs in a KMT2A-MLLT3 leukemia cell line. All analogs were assessed for liver-X-receptor (LXR) activity in an LXRE system to gauge the potential for the compounds to provoke raised triglycerides by increasing LXR activity, as well as to drive LXRE-mediated transcription of brain ApoE expression as a marker for potential therapeutic use in neurodegenerative disorders. Preliminary results suggest these compounds display a broad spectrum of off-target activities. However, many of the novel compounds were observed to be more potent than
. While some RXR agonists cross-signal the retinoic acid receptor (RAR), many of the rexinoids in this work displayed reduced RAR activity. The isochroman group did not appear to substantially reduce RXR activity on its own. The results of this study reveal that modifying potent, selective rexinoids like bexarotene, CD3254, and NEt-TMN can provide rexinoids with increased RXR selectivity, decreased potential for cross-signaling, and improved anti-proliferative characteristics in leukemia models compared to
.
The present study was designed to evaluate the rostrocaudal and lateral-midline effects of trans-spinal magnetic stimulation on diaphragmatic motor evoked potential by utilizing a figure-of-eight ...coil. The bilateral diaphragm electromyograms were recorded during trans-spinal magnetic stimulation from 60% to 100% of maximum output in 21 healthy subjects. The rostrocaudal effect of trans-spinal magnetic stimulation was evaluated by comparing diaphragmatic motor evoked potential when the coil was placed at the midline of the fifth (C5) and seventh (C7) cervical vertebrae and the second thoracic vertebra (T2). The diaphragmatic motor evoked potential was also examined during midline and lateral (± 15 mm) trans-spinal magnetic stimulation to examine the lateral-midline effect. The results demonstrated that the amplitude of diaphragmatic motor evoked potential was not significantly different in response to C5, C7, or T2 trans-spinal magnetic stimulation. In addition, the sensitivity of the left and right diaphragms to trans-spinal magnetic stimulation was different, as reflected by a greater amplitude of the right diaphragmatic motor evoked potential during midline trans-spinal magnetic stimulation. Moreover, although midline trans-spinal magnetic stimulation could induce coactivation of the bilateral diaphragm, lateral trans-spinal magnetic stimulation can induce a greater motor evoked potential in the ipsilateral than the contralateral diaphragm. Finally, there was no significant sex effect on the diaphragmatic motor evoked potential induced by trans-spinal magnetic stimulation. These results suggest that trans-spinal magnetic stimulation using a figure-of-eight coil is feasible to induce diaphragmatic motor evoked potential, and there is a lateral-midline effect of trans-spinal magnetic stimulation on the bilateral diaphragm.
The present study investigated position effect of trans-spinal magnetic stimulation using figure-of-eight coil on diaphragm in healthy humans. The result demonstrated that midline trans-spinal magnetic stimulation induces coactivation of bilateral diaphragm, whereas lateral trans-spinal magnetic stimulation induces greater motor evoked potentials in the ipsilateral than the contralateral diaphragm. These results suggest that trans-spinal magnetic stimulation is feasible to induce diaphragmatic motor evoked potential, and there is a lateral-midline effect of trans-spinal magnetic stimulation on diaphragm.
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