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Agents that elevate intracellular cyclic AMP (cAMP) levels promote neuronal survival in a manner independent of neurotrophic factors. Inhibitors of phosphatidylinositol 3 kinase and dominant-inactive mutants of the protein kinase Akt do not block the survival effects of cAMP, suggesting that another signaling pathway is involved. In this report, we demonstrate that elevation of intracellular cAMP levels in rat cerebellar granule neurons leads to phosphorylation and inhibition of glycogen synthase kinase 3␤ (GSK-3␤). The increased phosphorylation of GSK-3␤ by protein kinase A (PKA) occurs at serine 9, the same site phosphorylated by Akt. Purified PKA is able to phosphorylate recombinant GSK-3␤ in vitro. Inhibitors of GSK-3 block apoptosis in these neurons, and transfection of neurons with a GSK-3␤ mutant that cannot be phosphorylated interferes with the prosurvival effects of cAMP. These data suggest that activated PKA directly phosphorylates GSK-3␤ and inhibits its apoptotic activity in neurons.Neurons require continuous exposure to extracellular trophic factors for survival, and those that fail to receive sufficient trophic factor support undergo apoptotic cell death (34). Among the extracellular factors shown to influence neuronal survival are the neurotrophins, which include nerve growth factor, brain-derived neurotrophic factor, neurotrophin 3 and neurotrophin 4, the fibroblast growth factors, ciliary growth factor, insulin, and insulin-like growth factors (2, 31). Agents that elevate intracellular cyclic AMP (cAMP) also promote neuronal survival in a manner independent of neurotrophic factors (24, 38). Substantial progress has been made over the last several years in delineating signal transduction pathways that mediate trophic factor-induced cell survival. Less is known about the survival pathways activated by cAMP in neurons.Recent reports have established a key role for phosphatidylinositol (PI)-3 kinase in regulating trophic factor-dependent survival of neurons (18,22,23). The Akt protein kinase (also termed proteins kinase B [PKB] and Rac) has been implicated as the transducer of PI-3 kinase-dependent survival signals generated by serum and certain growth factors (6,22,28). In response to PI-3 kinase activation, Akt binds to phosphorylated membrane lipids via its pleckstrin homology domain and is phosphorylated at threonine 308 and serine 473 (17). Phosphorylation of Akt at these two sites leads to its activation and the propagation of an antiapoptotic signal. Several downstream targets of Akt implicated in cell survival include the Bcl-2 family member BAD (13), caspase 9 (8), and FKHRL1, a member of the Forkhead family of transcription factors (5). Another Akt substrate recently implicated in cell fate decisions is glycogen synthase kinase 3 (GSK-3). Mammalian GSK-3 exists as two isoforms termed ␣ (51 kDa) and ␤ (47 kDa), each encoded by a distinct gene (45-47). The GSK-3 isoforms share 85% homology at the amino acid level and are ubiquitously expressed (45-47). Although GSK-3 was originally identifie...

We report advances in nanoimprint lithography, its application in nanogap metal contacts, and related fabrication yield. We have demonstrated 5 nm linewidth and 14 nm linepitch in resist using nanoimprint lithography at room temperature with a pressure less than 15 psi. We fabricated gold contacts (for the application of single macromolecule devices) with 5 nm separation by nanoimprint in resist and lift-off of metal. Finally, the uniformity and manufacturability of nanoimprint over a 4 in. wafer were demonstrated.The field of nanotechnology is advancing rapidly. Applications of nanotechnology include subwavelength optical elements, biochemical analysis devices, photonic crystals, high-density single-domain magnetic storage, and singlemolecule devices, to name a few. Yet, key to the commercial success of these nanotechnology applications are low cost and high throughput manufacturing capabilities. State-of-theart manufacturing photolithography patterning tools are both too expensive and incapable of producing the necessary pitch and feature sizes of these applications. Thus, presently, researchers have been largely constrained to using lowthroughput lithography tools, such as electron-beam lithography (EBL), atomic force microscopy (AFM), and ion-beam lithography. For high-throughput and low-cost lithography, various "nanoprinting" technologies have been developed. 1-3 Here, we report our investigation of the resolution limit of nanoimprint lithography, where we demonstrated a nanoimprint record of 5 nm linewidth features and 14 nm pitch over a large area, its applications in nanogap metal contacts, and a study of fabrication yields.In photocurable nanoimprint lithography (P-NIL) (shown in Fig. 1), a mold is pressed into a low viscosity photocurable resist liquid to physically deform the resist shape such that it conforms to the topology of the mold. The resist is cured with exposure to UV light, crosslinking the various components in the resist liquid, producing a uniform, relatively rigid polymer network. The mold is then separated from the cured resist. Finally, an anisotropic reactive ion etch (RIE) removes the residual resist in the compressed area, exposing the substrate surface.In order to explore the performance of P-NIL, a variety of molds were fabricated to test specific attributes, including minimum pitch (maximum density), minimum feature size, and large-area uniformity patterning. Previously, 10 nm dots and 40 nm pitch have been demonstrated by NIL 1 with the resolution limited by our ability to fabricate the mold, as proximity effects inherent with EBL make sub-35 nm pitch patterning very difficult. To produce a mold with a pitch resolution surpassing EBL capabilities, we fabricated a NIL mold by selectively wet etching Al 0.7 Ga 0.3 As from a cleaved edge of a GaAs/ Al 0.7 Ga 0.3 As superlattice [grown by molecular-beam epitaxy (MBE)] with a dilute solution of hydrofluoric acid (HF). 4,5 This mold fabrication process offers many advantages, specifically very dense sub-50 nm pitch topologies can be ...

Oxygen reduction reaction/oxygen evolution reaction (ORR/OER) catalytic activities of p-orbital heteroatom-doped carbon nanomaterials are demonstrated to correlate to the combination of the electron affinity and electronegativity of doping elements, which serves as an activity descriptor for the entire family of p-block element dopants. Such a descriptor has predictive power and enables effective design of new bifunctional catalysts with enhanced ORR/OER activities.

Density functional theory (DFT) was applied to study sulfur-doped graphene clusters as oxygen reduction reaction (ORR) cathode catalysts for fuel cells. Several sulfurdoped graphene clusters with/without Stone−Wales defects were investigated and their electronic structures, reaction free energy, transition states, and energy barriers were calculated to predict their catalytic properties. The results show that sulfur atoms could be adsorbed on the graphene surface, substitute carbon atoms at the graphene edges in the form of sulfur/ sulfur oxide, or connect two graphene sheets by forming a sulfur cluster ring. These sulfur-doped graphene clusters with sulfur or sulfur oxide locating at graphene edges show electrocatalytic activity for ORR. Catalytic active sites distribute at the zigzag edge or the neighboring carbon atoms of doped sulfur oxide atoms, which possess large spin or charge density. For those being the active catalytic sites, sulfur atoms with the highest charge density take a two-electron transfer pathway while the carbon atoms with high spin or charge density follow a four-electron transfer pathway. It was predicted from the reaction energy barriers that the sulfur-doped graphene could show ORR catalytic properties comparable to platinum. The prediction is consistent with the experimental results on S-doped graphene.

Myocyte enhancer factor 2 (MEF2) proteins are important regulators of gene expression during the development of skeletal, cardiac, and smooth muscle. MEF2 proteins are also present in brain and recently have been implicated in neuronal survival and differentiation. In this study we examined the cellular mechanisms regulating the activity of MEF2s during apoptosis of cultured cerebellar granule neurons, an established in vitro model for studying depolarization-dependent neuronal survival. All four MEF2 isoforms (A, B, C, and D) were detected by immunoblot analysis in cerebellar granule neurons. Endogenous MEF2A and MEF2D, but not MEF2B or MEF2C, were phosphorylated with the induction of apoptosis. The putative sites that were phosphorylated during apoptosis are functionally distinct from those previously reported to enhance MEF2 transcription. The increased phosphorylation of MEF2A and MEF2D was followed by decreased DNA binding, reduced transcriptional activity, and caspase-dependent cleavage to fragments containing N-terminal DNA binding domains and C-terminal transactivation domains. Expression of the highly homologous N terminus of MEF2A (1-131 amino acids) antagonized the transcriptional activity and prosurvival effects of a constitutively active mutant of MEF2D (MEF2D-VP16). We conclude that MEF2A and MEF2D are prosurvival factors with high transcriptional activity in postmitotic cerebellar granule neurons. When these neurons are induced to undergo apoptosis by lowering extracellular potassium, MEF2A and MEF2D are phosphorylated, followed by decreased DNA binding and cleavage by a caspase-sensitive pathway to N-terminal fragments lacking the transactivation domains. The degradation of MEF2D and MEF2A and the generation of MEF2 fragments that have the potential to act as dominant-inactive transcription factors lead to apoptotic cell death.

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Graphical AbstractHighlights d HFS triggers synaptic plasticity of CGRP afferents and chronic pain d LTP-inducible HFS activates spinal microglia through CSF1 signaling d Microglial BDNF is essential for HFS-induced spinal LTP and chronic pain SUMMARY Spinal long-term potentiation (LTP) at C-fiber synapses is hypothesized to underlie chronic pain. However, a causal link between spinal LTP and chronic pain is still lacking. Here, we report that high-frequency stimulation (HFS; 100 Hz, 10 V) of the mouse sciatic nerve reliably induces spinal LTP without causing nerve injury. LTP-inducible stimulation triggers chronic pain lasting for more than 35 days and increases the number of calcitonin gene-related peptide (CGRP) terminals in the spinal dorsal horn. The behavioral and morphological changes can be prevented by blocking NMDA receptors, ablating spinal microglia, or conditionally deleting microglial brain-derived neurotrophic factor (BDNF). HFS-induced spinal LTP, microglial activation, and upregulation of BDNF are inhibited by antibodies against colony-stimulating factor 1 (CSF-1). Together, our results show that microglial CSF1 and BDNF signaling are indispensable for spinal LTP and chronic pain. The microglia-dependent transition of synaptic potentiation to structural alterations in pain pathways may underlie pain chronicity.