Graphene oxide-polyamine preprogrammable nanoreactors with sensing capability for corrosion protection of materials.

Corrosion is one of the major issues for sustainable manufacturing globally. The annual global cost of corrosion is US$2.5 trillion (approximately 3.4% of the world's GDP). The traditional ways of corrosion protection (such as barriers or inhibiting) are either not very effective (in the case of barrier protection) or excessively expensive (inhibiting). Here, we demonstrate a concept of nanoreactors, which are able to controllably release or adsorb protons or hydroxides directly on corrosion sites, hence, selectively regulating the corrosion reactions. A single nanoreactor comprises a nanocompartment wrapped around by a pH-sensing membrane represented, respectively, by a halloysite nanotube and a graphene oxide/polyamine envelope. A nanoreactor response is determined by the change of a signaling pH on a given corrosion site. The nanoreactors are self-assembled and suitable for mass-line production. The concept creates sustainable technology for developing smart anticorrosion coatings, which are nontoxic, selective, and inexpensive.

Endophilin functions as a membrane-bending molecule and is delivered to endocytic zones by exocytosis.

Two models have been proposed for endophilin function in synaptic vesicle (SV) endocytosis. The scaffolding model proposes that endophilin's SH3 domain recruits essential endocytic proteins, whereas the membrane-bending model proposes that the BAR domain induces positively curved membranes. We show that mutations disrupting the scaffolding function do not impair endocytosis, whereas those disrupting membrane bending cause significant defects. By anchoring endophilin to the plasma membrane, we show that endophilin acts prior to scission to promote endocytosis. Despite acting at the plasma membrane, the majority of endophilin is targeted to the SV pool. Photoactivation studies suggest that the soluble pool of endophilin at synapses is provided by unbinding from the adjacent SV pool and that the unbinding rate is regulated by exocytosis. Thus, endophilin participates in an association-dissociation cycle with SVs that parallels the cycle of exo- and endocytosis. This endophilin cycle may provide a mechanism for functionally coupling endocytosis and exocytosis.

CASK and FARP localize two classes of post-synaptic ACh receptors thereby promoting cholinergic transmission.

Changes in neurotransmitter receptor abundance at post-synaptic elements play a pivotal role in regulating synaptic strength. For this reason, there is significant interest in identifying and characterizing the scaffolds required for receptor localization at different synapses. Here we analyze the role of two C. elegans post-synaptic scaffolding proteins (LIN-2/CASK and FRM-3/FARP) at cholinergic neuromuscular junctions. Constitutive knockouts or muscle specific inactivation of lin-2 and frm-3 dramatically reduced spontaneous and evoked post-synaptic currents. These synaptic defects resulted from the decreased abundance of two classes of post-synaptic ionotropic acetylcholine receptors (ACR-16/CHRNA7 and levamisole-activated AChRs). LIN-2's AChR scaffolding function is mediated by its SH3 and PDZ domains, which interact with AChRs and FRM-3/FARP, respectively. Thus, our findings show that post-synaptic LIN-2/FRM-3 complexes promote cholinergic synaptic transmission by recruiting AChRs to post-synaptic elements.

A unique C2 domain at the C terminus of Munc13 promotes synaptic vesicle priming.

Neurotransmitter release during synaptic transmission comprises a tightly orchestrated sequence of molecular events, and Munc13-1 is a cornerstone of the fusion machinery. A forward genetic screen for defects in neurotransmitter release in Caenorhabditis elegans identified a mutation in the Munc13-1 ortholog UNC-13 that eliminated its unique and deeply conserved C-terminal module (referred to as HC2M) containing a Ca2+-insensitive C2 domain flanked by membrane-binding helices. The HC2M module could be functionally replaced in vivo by protein domains that localize to synaptic vesicles but not to the plasma membrane. HC2M is broadly conserved in other Unc13 family members and is required for efficient synaptic vesicle priming. We propose that the HC2M domain evolved as a vesicle/endosome adaptor and acquired synaptic vesicle specificity in the Unc13ABC protein family.

Long-Acting Insulin-Zwitterionic Polymer Conjugate Mitigates Hypoglycemia.

Insulin, a main medication to control glycemia of type 1 and advanced type 2 diabetes, faces problems of a short half-life and poor stability during its clinical use. Zwitterionic polymer shows unique properties of antifouling and low immunogenicity. Here, we have synthesized a new insulin-zwitterionic polymer conjugate (INS-PMPC) through grafting-from strategy by controlled radical polymerization. Apart from showing excellent stability upon mechanical agitation, the resulting INS-PMPC conjugate provided over 20 h of glycemic control due to improved pharmacokinetics in diabetic mice with one single subcutaneous injection. Most importantly, this insulin-zwitterionic polymer conjugate significantly decreases the incidence of hypoglycemia.

mir-234 controls neuropeptide release at the Caenorhabditis elegans neuromuscular junction.

miR-137 is a highly conserved microRNA (miRNA) that is associated with the control of brain function and the etiology of psychiatric disorders including schizophrenia and bipolar disorder. The Caenorhabditis elegans genome encodes a single miR-137 ortholog called mir-234, the function of which is unknown. Here we show that mir-234 is expressed in a subset of sensory, motor and interneurons in C. elegans. Using a mir-234 deletion strain, we systematically examined the development and function of these neurons in addition to global C. elegans behaviors. We were however unable to detect phenotypes associated with loss of mir-234, possibly due to genetic redundancy. To circumvent this issue, we overexpressed mir-234 in mir-234-expressing neurons to uncover possible phenotypes. We found that mir-234-overexpression endows resistance to the acetylcholinesterase inhibitor aldicarb, suggesting modification of neuromuscular junction (NMJ) function. Further analysis revealed that mir-234 controls neuropeptide levels, therefore positing a cause of NMJ dysfunction. Together, our data suggest that mir-234 functions to control the expression of target genes that are important for neuropeptide maturation and/or transport in C. elegans. SIGNIFICANCE STATEMENT: The miR-137 family of miRNAs is linked to the control of brain function in humans. Defective regulation of miR-137 is associated with psychiatric disorders that include schizophrenia and bipolar disorder. Previous studies have revealed that miR-137 is required for the development of dendrites and for controlling the release of fast-acting neurotransmitters. Here, we analyzed the function a miR-137 family member (called mir-234) in the nematode animal model using anatomical, behavioral, electrophysiological and neuropeptide analysis. We reveal for the first time that mir-234/miR-137 is required for the release of slow-acting neuropeptides, which may also be of relevance for controlling human brain function.

Recruitment of sphingosine kinase to presynaptic terminals by a conserved muscarinic signaling pathway promotes neurotransmitter release.

Sphingolipids are potent lipid second messengers that regulate cell differentiation, migration, survival, and secretion, and alterations in sphingolipid signaling have been implicated in a variety of diseases. However, how sphingolipid levels are regulated, particularly in the nervous system, remains poorly understood. Here, we show that the generation of sphingosine-1-phosphate by sphingosine kinase (SphK) promotes neurotransmitter release. Electrophysiological, imaging, and behavioral analyses of Caenorhabditis elegans mutants lacking sphingosine kinase sphk-1 indicate that neuronal development is normal, but there is a significant defect in neurotransmitter release from neuromuscular junctions. SPHK-1 localizes to discrete, nonvesicular regions within presynaptic terminals, and this localization is critical for synaptic function. Muscarinic agonists cause a rapid increase in presynaptic SPHK-1 abundance, whereas reduction of endogenous acetylcholine production results in a rapid decrease in presynaptic SPHK-1 abundance. Muscarinic regulation of presynaptic SPHK-1 abundance is mediated by a conserved presynaptic signaling pathway composed of the muscarinic acetylcholine receptor GAR-3, the heterotrimeric G protein Gαq, and its effector, Trio RhoGEF. SPHK-1 activity is required for the effects of muscarinic signaling on synaptic transmission. This study shows that SPHK-1 promotes neurotransmitter release in vivo and identifies a novel muscarinic pathway that regulates SphK abundance at presynaptic terminals.

The Claudin-like Protein HPO-30 Is Required to Maintain LAChRs at the C. elegans Neuromuscular Junction.

Communications across chemical synapses are primarily mediated by neurotransmitters and their postsynaptic receptors. There are diverse molecular systems to localize and regulate the receptors at the synapse. Here, we identify HPO-30, a member of the claudin superfamily of membrane proteins, as a positive regulator for synaptic localization of levamisole-dependent AChRs (LAChRs) at the Caenorhabditis elegans neuromuscular junction (NMJ). The HPO-30 protein localizes at the NMJ and shows genetic and physical association with the LAChR subunits LEV-8, UNC-29, and UNC-38. Using genetic and electrophysiological assays in the hermaphrodite C. elegans, we demonstrate that HPO-30 functions through Neuroligin at the NMJ to maintain postsynaptic LAChR levels at the synapse. Together, this work suggests a novel function for a tight junction protein in maintaining normal receptor levels at the NMJ.SIGNIFICANCE STATEMENT Claudins are a large superfamily of membrane proteins. Their role in maintaining the functional integrity of tight junctions has been widely explored. Our experiments suggest a critical role for the claudin-like protein, HPO-30, in maintaining synaptic levamisole-dependent AChR (LAChR) levels. LAChRs contribute to <20% of the acetylcholine-mediated conductance in adult Caenorhabditis elegans; however, they play a significant functional role in worm locomotion. This study provides a new perspective in the study of LAChR physiology.

SNT-1 Functions as the Ca2+ Sensor for Tonic and Evoked Neurotransmitter Release in Caenorhabditis Elegans.

Synaptotagmin-1 (Syt1) binds Ca2+ through its tandem C2 domains (C2A and C2B) and triggers Ca2+-dependent neurotransmitter release. Here, we show that snt-1, the homolog of mammalian Syt1, functions as the Ca2+ sensor for both tonic and evoked neurotransmitter release at the Caenorhabditis elegans neuromuscular junction. Mutations that disrupt Ca2+ binding in double C2 domains of SNT-1 significantly impaired tonic release, whereas disrupting Ca2+ binding in a single C2 domain had no effect, indicating that the Ca2+ binding of the two C2 domains is functionally redundant for tonic release. Stimulus-evoked release was significantly reduced in snt-1 mutants, with prolonged release latency as well as faster rise and decay kinetics. Unlike tonic release, evoked release was triggered by Ca2+ binding solely to the C2B domain. Moreover, we showed that SNT-1 plays an essential role in the priming process in different subpopulations of synaptic vesicles with tight or loose coupling to Ca2+ entry.SIGNIFICANCE STATEMENT We showed that SNT-1 in Caenorhabditis elegans regulates evoked neurotransmitter release through Ca2+ binding to its C2B domain in a similar way to Syt1 in the mouse CNS and the fly neuromuscular junction. However, the largely decreased tonic release in snt-1 mutants argues SNT-1 has a clamping function. Indeed, Ca2+-binding mutations in the C2 domains in SNT-1 significantly reduced the frequency of the miniature EPSC, indicating that SNT-1 also acts as a Ca2+ sensor for tonic release. Therefore, revealing the differential mechanisms between invertebrates and vertebrates will provide significant insights into our understanding how synaptic vesicle fusion is regulated.

Sensory Neurons Arouse C. elegans Locomotion via Both Glutamate and Neuropeptide Release.

C. elegans undergoes periods of behavioral quiescence during larval molts (termed lethargus) and as adults. Little is known about the circuit mechanisms that establish these quiescent states. Lethargus and adult locomotion quiescence is dramatically reduced in mutants lacking the neuropeptide receptor NPR-1. Here, we show that the aroused locomotion of npr-1 mutants results from the exaggerated activity in multiple classes of sensory neurons, including nociceptive (ASH), touch sensitive (ALM and PLM), and stretch sensing (DVA) neurons. These sensory neurons accelerate locomotion via both neuropeptide and glutamate release. The relative contribution of these sensory neurons to arousal differs between larval molts and adults. Our results suggest that a broad network of sensory neurons dictates transitions between aroused and quiescent behavioral states.

NLP-12 engages different UNC-13 proteins to potentiate tonic and evoked release.

A neuropeptide (NLP-12) and its receptor (CKR-2) potentiate tonic and evoked ACh release at Caenorhabditis elegans neuromuscular junctions. Increased evoked release is mediated by a presynaptic pathway (egl-30 Gαq and egl-8 PLCß) that produces DAG, and by DAG binding to short and long UNC-13 proteins. Potentiation of tonic ACh release persists in mutants deficient for egl-30 Gαq and egl-8 PLCß and requires DAG binding to UNC-13L (but not UNC-13S). Thus, NLP-12 adjusts tonic and evoked release by distinct mechanisms.

RIC-7 promotes neuropeptide secretion.

Secretion of neurotransmitters and neuropeptides is mediated by exocytosis of distinct secretory organelles, synaptic vesicles (SVs) and dense core vesicles (DCVs) respectively. Relatively little is known about factors that differentially regulate SV and DCV secretion. Here we identify a novel protein RIC-7 that is required for neuropeptide secretion in Caenorhabditis elegans. The RIC-7 protein is expressed in all neurons and is localized to presynaptic terminals. Imaging, electrophysiology, and behavioral analysis of ric-7 mutants indicates that acetylcholine release occurs normally, while neuropeptide release is significantly decreased. These results suggest that RIC-7 promotes DCV-mediated secretion.

Phytic Acid-Gallium Network on a Polyimide Fiber Woven Fabric as an Artificial Ligament for Boosting Ligament-Bone Healing and Infection Treatment.

The development of an artificial ligament with a multifunction of promoting bone formation, inhibiting bone resorption, and preventing infection to obtain ligament-bone healing for anterior cruciate ligament (ACL) reconstruction still faces enormous challenges. Herein, a novel artificial ligament based on a PI fiber woven fabric (PIF) was fabricated, which was coated with a phytic acid-gallium (PA-Ga) network via a layer-by-layer assembly method (PFPG). Compared with PIF, PFPG with PA-Ga coating significantly suppressed osteoclastic differentiation, while it boosted osteoblastic differentiation in vitro. Moreover, PFPG obviously inhibited fibrous encapsulation and bone absorption while accelerating new bone regeneration for ligament-bone healing in vivo. PFPG remarkably killed bacteria and destroyed biofilm, exhibiting excellent antibacterial properties in vitro as well as anti-infection ability in vivo, which were ascribed to the release of Ga ions from the PA-Ga coating. The cooperative effect of the surface characteristics (e.g., hydrophilicity/surface energy and protein absorption) and sustained release of Ga ions for PFPG significantly enhanced osteogenesis while inhibiting osteoclastogenesis, thereby achieving ligament-bone integration as well as resistance to infection. In summary, PFPG remarkably facilitated osteoblastic differentiation, while it suppressed osteoclastic differentiation, thereby inhibiting osteoclastogenesis for bone absorption while accelerating osteogenesis for ligament-bone healing. As a novel artificial ligament, PFPG represented an appealing option for graft selection in ACL reconstruction and displayed considerable promise for application in clinics.

Phasic/tonic glial GABA differentially transduce for olfactory adaptation and neuronal aging.

Phasic (fast) and tonic (sustained) inhibition of γ-aminobutyric acid (GABA) are fundamental for regulating day-to-day activities, neuronal excitability, and plasticity. However, the mechanisms and physiological functions of glial GABA transductions remain poorly understood. Here, we report that the AMsh glia in Caenorhabditis elegans exhibit both phasic and tonic GABAergic signaling, which distinctively regulate olfactory adaptation and neuronal aging. Through genetic screening, we find that GABA permeates through bestrophin-9/-13/-14 anion channels from AMsh glia, which primarily activate the metabolic GABAB receptor GBB-1 in the neighboring ASH sensory neurons. This tonic action of glial GABA regulates the age-associated changes of ASH neurons and olfactory responses via a conserved signaling pathway, inducing neuroprotection. In addition, the calcium-evoked, vesicular glial GABA release acts upon the ionotropic GABAA receptor LGC-38 in ASH neurons to regulate olfactory adaptation. These findings underscore the fundamental significance of glial GABA in maintaining healthy aging and neuronal stability.

Functional Roles of UNC-13/Munc13 and UNC-18/Munc18 in Neurotransmission.

Neurotransmitters are released from synaptic and secretory vesicles following calcium-triggered fusion with the plasma membrane. These exocytotic events are driven by assembly of a ternary SNARE complex between the vesicle SNARE synaptobrevin and the plasma membrane-associated SNAREs syntaxin and SNAP-25. Proteins that affect SNARE complex assembly are therefore important regulators of synaptic strength. In this chapter, we review our current understanding of the roles played by two SNARE interacting proteins: UNC-13/Munc13 and UNC-18/Munc18. We discuss results from both invertebrate and vertebrate model systems, highlighting recent advances, focusing on the current consensus on molecular mechanisms of action and nanoscale organization, and pointing out some unresolved aspects of their functions.

Chemically recyclable polyolefin-like multiblock polymers.

Polyolefins are the most important and largest volume plastics produced. Unfortunately, the enormous use of plastics and lack of effective disposal or recycling options have created a plastic waste catastrophe. In this work, we report an approach to create chemically recyclable polyolefin-like materials with diverse mechanical properties through the construction of multiblock polymers from hard and soft oligomeric building blocks synthesized with ruthenium-mediated ring-opening metathesis polymerization of cyclooctenes. The multiblock polymers exhibit broad mechanical properties, spanning elastomers to plastomers to thermoplastics, while integrating a high melting transition temperature (Tm) and low glass transition temperature (Tg), making them suitable for use across diverse applications (Tm as high as 128°C and Tg as low as -60°C). After use, the different plastics can be combined and efficiently deconstructed back to the fundamental hard and soft building blocks for separation and repolymerization to realize a closed-loop recycling process.

The C2 and PH domains of CAPS constitute an effective PI(4,5)P2-binding unit essential for Ca2+-regulated exocytosis.

Ca2+-dependent activator proteins for secretion (CAPSs) are required for Ca2+-regulated exocytosis in neurons and neuroendocrine cells. CAPSs contain a pleckstrin homology (PH) domain that binds PI(4,5)P2-membrane. There is also a C2 domain residing adjacent to the PH domain, but its function remains unclear. In this study, we solved the crystal structure of the CAPS-1 C2PH module. The structure showed that the C2 and PH tandem packs against one another mainly via hydrophobic residues. With this interaction, the C2PH module exhibited enhanced binding to PI(4,5)P2-membrane compared with the isolated PH domain. In addition, we identified a new PI(4,5)P2-binding site on the C2 domain. Disruption of either the tight interaction between the C2 and PH domains or the PI(4,5)P2-binding sites on both domains significantly impairs CAPS-1 function in Ca2+-regulated exocytosis at the Caenorhabditis elegans neuromuscular junction (NMJ). These results suggest that the C2 and PH domains constitute an effective unit to promote Ca2+-regulated exocytosis.

CaMKII mediates sexually dimorphic synaptic transmission at neuromuscular junctions in C. elegans.

Sexually dimorphic behaviors are ubiquitous throughout the animal kingdom. Although both sex-specific and sex-shared neurons have been functionally implicated in these diverse behaviors, less is known about the roles of sex-shared neurons. Here, we discovered sexually dimorphic cholinergic synaptic transmission in C. elegans occurring at neuromuscular junctions (NMJs), with males exhibiting increased release frequencies, which result in sexually dimorphic locomotion behaviors. Scanning electron microscopy revealed that males have significantly more synaptic vesicles (SVs) at their cholinergic synapses than hermaphrodites. Analysis of previously published transcriptome identified the male-enriched transcripts and focused our attention on UNC-43/CaMKII. We ultimately show that differential accumulation of UNC-43 at cholinergic neurons controls axonal SV abundance and synaptic transmission. Finally, we demonstrate that sex reversal of all neurons in hermaphrodites generates male-like cholinergic transmission and locomotion behaviors. Thus, beyond demonstrating UNC-43/CaMKII as an essential mediator of sex-specific synaptic transmission, our study provides molecular and cellular insights into how sex-shared neurons can generate sexually dimorphic locomotion behaviors.

UNC-43/CaMKII-triggered anterograde signals recruit GABAARs to mediate inhibitory synaptic transmission and plasticity at C. elegans NMJs.

Disturbed inhibitory synaptic transmission has functional impacts on neurodevelopmental and psychiatric disorders. An essential mechanism for modulating inhibitory synaptic transmission is alteration of the postsynaptic abundance of GABAARs, which are stabilized by postsynaptic scaffold proteins and recruited by presynaptic signals. However, how GABAergic neurons trigger signals to transsynaptically recruit GABAARs remains elusive. Here, we show that UNC-43/CaMKII functions at GABAergic neurons to recruit GABAARs and modulate inhibitory synaptic transmission at C. elegans neuromuscular junctions. We demonstrate that UNC-43 promotes presynaptic MADD-4B/Punctin secretion and NRX-1α/Neurexin surface delivery. Together, MADD-4B and NRX-1α recruit postsynaptic NLG-1/Neuroligin and stabilize GABAARs. Further, the excitation of GABAergic neurons potentiates the recruitment of NLG-1-stabilized-GABAARs, which depends on UNC-43, MADD-4B, and NRX-1. These data all support that UNC-43 triggers MADD-4B and NRX-1α, which act as anterograde signals to recruit postsynaptic GABAARs. Thus, our findings elucidate a mechanism for pre- and postsynaptic communication and inhibitory synaptic transmission and plasticity.

Catalytically Controlled Ring-Opening Polymerization of 2-Oxo-15-crown-5 for Degradable and Recyclable PEG-Like Polyesters.

Poly(ethylene glycol) (PEG) has been extensively used in diverse applications. However, it is not biodegradable and shows abnormal immune responses. Herein, a fast, controlled, ring-opening polymerization (ROP) of 2-oxo-15-crown-5 (O-15C5) is reported to prepare well-defined PEG-like polyesters, poly(O-15C5). This approach relies on a coordination between the macrocyclic monomer and Na+ that increases the electrophilicity of the carbonyl group of O-15C5 and leads to a fast controlled ROP (dispersity, DM < 1.2). Both computational and mechanistic studies show that the selective Na+ binding to the monomer over poly(O-15C5) allows the ring-opening initiation and propagation to be more energetically favorable than side transesterifications. This is the key to control the challenging entropy-driven ROP of O-15C5. Moreover, with the aid of Na+ and organic base, poly(O-15C5) depolymerized readily into O-15C5 in 2 h. Also, it degraded in a buffer of pH 7.4 by hydrolysis.