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Iron-catalyzed C−C coupling reactions of pyrrole provide a unique alternative to the traditional Pd-catalyzed counterpart. However, many details regarding the actual mechanism remain unknown. A series of macrocyclic iron(III) complexes were used to evaluate specifics related to the role of O(2), radicals, and μ-oxodiiron-complex participation in the catalytic cycle. It was determined that the mononuclear tetra-azamacrocyclic complex is a true catalyst and not a stoichiometric reagent, while more than one equivalent of a sacrificial oxidant is needed. Furthermore, the reaction does not proceed through an organic radical pathway. μ-Oxodiiron complexes are not involved in the main catalytic pathway, and the dimers are, in fact, off-cycle species that decrease catalytic efficiency.

The release of calcium ions (Ca2+) from the endoplasmic reticulum (ER) and related store-operated calcium entry (SOCE) regulate maturation of normal megakaryocytes. The N-methyl-D-aspartate (NMDA) receptor (NMDAR) provides an additional mechanism for Ca2+ influx in megakaryocytic cells, but its role remains unclear. We created a model of NMDAR hypofunction in Meg-01 cells using CRISPR-Cas9 mediated knockout of the GRIN1 gene, which encodes an obligate, GluN1 subunit of the NMDAR. We found that compared with unmodified Meg-01 cells, Meg-01-GRIN1 −/− cells underwent atypical differentiation biased toward erythropoiesis, associated with increased basal ER stress and cell death. Resting cytoplasmic Ca2+ levels were higher in Meg-01-GRIN1 −/− cells, but ER Ca2+ release and SOCE were lower after activation. Lysosome-related organelles accumulated including immature dense granules that may have contributed an alternative source of intracellular Ca2+. Microarray analysis revealed that Meg-01-GRIN1 −/− cells had deregulated expression of transcripts involved in Ca2+ metabolism, together with a shift in the pattern of hematopoietic transcription factors toward erythropoiesis. In keeping with the observed pro-cell death phenotype induced by GRIN1 deletion, memantine (NMDAR inhibitor) increased cytotoxic effects of cytarabine in unmodified Meg-01 cells. In conclusion, NMDARs comprise an integral component of the Ca2+ regulatory network in Meg-01 cells that help balance ER stress and megakaryocytic-erythroid differentiation. We also provide the first evidence that megakaryocytic NMDARs regulate biogenesis of lysosome-related organelles, including dense granules. Our results argue that intracellular Ca2+ homeostasis may be more important for normal megakaryocytic and erythroid differentiation than currently recognized; thus, modulation may offer therapeutic opportunities.

The number of substituted pyridine pyridinophanes found in the literature is limited due to challenges associated with 12-membered macrocycle and modified pyridine synthesis. Most notably, the electrophilic character at the 4-position of pyridine in pyridinophanes presents a unique challenge for introducing electrophilic chemical groups. Likewise, of the few reported, most substituted pyridine pyridinophanes in the literature are limited to electron-donating functionalities. Herein, new synthetic strategies for four new macrocycles bearing the electron-withdrawing groups CN, Cl, NO(2), and CF(3) are introduced. Potentiometric titrations were used to determine the protonation constants of the new pyridinophanes. Further, the influence of such modifications on the chemical behavior is predicted by comparing the potentiometric results to previously reported systems. X-ray diffraction analysis of the 4-Cl substituted species and its Cu(II) complex are also described to demonstrate the metal binding nature of these ligands. DFT analysis is used to support the experimental findings through energy calculations and ESP maps. These new molecules serve as a foundation to access a range of new pyridinophane small molecules and applications in future work.

Summary Ionotropic glutamate receptors (iGluRs) are ligand-gated ion channels that play crucial roles in the central nervous system. iGluR homologs, termed glutamate receptor-like channels (GLRs), have been found in plants. Investigating the structural and functional relationship between iGluRs and GLRs was limited by GLR protein expression, purification, and structural characterization. Here, we provide a detailed protocol for Arabidopsis thaliana GLR3.4 (AtGLR3.4) expression in a mammalian cell line and purification for structure determination by cryogenic electron microscopy (cryo-EM). For the complete details on the use and execution of this protocol, please refer to Green et al. (2021)., Graphical abstract We provide a detailed protocol for expression and purification of Arabidopsis thaliana GLR3.4 protein that can be used for structure determination by cryo-EM. The image has been created using BioRender (https://biorender.com/)., Highlights • Protocol for expression of plant glutamate receptor-like channel (GLR) in HEK cells • Purification protocol that yields pure monodisperse tetrameric GLR protein • Purified protein subjected to cryo-EM analysis produces high-resolution GLR structure, Ionotropic glutamate receptors (iGluRs) are ligand-gated ion channels that play crucial roles in the central nervous system. iGluR homologs, termed glutamate receptor-like channels (GLRs), have been found in plants. Investigating the structural and functional relationship between iGluRs and GLRs was limited by GLR protein expression, purification, and structural characterization. Here, we provide a detailed protocol for Arabidopsis thaliana GLR3.4 (AtGLR3.4) expression in a mammalian cell line and purification for structure determination by cryogenic electron microscopy (cryo-EM).

Owing to the increasing importance of manganese(II) complexes in the field of magnetic resonance imaging (MRI), large efforts have been devoted to find an appropriate ligand for Mn(II) ion encapsulation by providing balance between the seemingly contradictory requirements (i.e., thermodynamic stability and kinetic inertness vs low ligand denticity enabling water molecule(s) to be coordinated in its metal center). Among these ligands, a large number of pyridine or pyridol based open-chain and macrocyclic chelators have been investigated so far. As a next step in the development of these chelators, 15-pyN(3)O(2)Ph and its transition metal complexes were synthesized and characterized using established methods. The 15-pyN(3)O(2)Ph ligand incorporates both pyridine and ortho-phenylene units to decrease ligand flexibility. The thermodynamic properties, protonation and stability constants, were determined using pH-potentiometry; the solid-state structures of two protonation states of the free ligand and its manganese complex were obtained by single crystal X-ray diffractometry. The results show a seven-coordinate metal center with two water molecules in the first coordination sphere. The longitudinal relaxivity of [Mn(15-pyN(3)O(2)Ph)](2+) was found to be 5.16 mM(−1) s(−1) at 0.49 T (298 K). Furthermore, the [Formula: see text] value of 11.72 mM(−1) s(−1) (0.49 T), which is doubled at 1.41 T field, suggests that design of this Mn(II) complex does achieve some characteristics required for contrast imaging. In addition,(17)O NMR measurements were performed in order to access the microscopic parameters governing this key feature (e.g., water exchange rate). Finally, manganese complexes of ligands with analogous polyaza macrocyclic scaffold have been investigated as low molecular weight Mn(CAT) mimics. Here, we report the H(2)O(2) disproportionation study of [Mn(15-pyN(3)O(2)Ph)](2+) to demonstrate the versatility of this ligand scaffold as well.

Activity-driven changes in the neuronal surface glycoproteome are known to occur with synapse formation, plasticity, and related diseases, but their mechanistic basis and significance are unclear. Here, we observed that N-glycans on surface glycoproteins of dendrites shift from immature to mature forms containing sialic acid in response to increased neuronal activation. In exploring the basis of these N-glycosylation alterations, we discovered that they result from the growth and proliferation of Golgi satellites scattered throughout the dendrite. Golgi satellites that formed during neuronal excitation were in close association with endoplasmic reticulum (ER) exit sites and early endosomes and contained glycosylation machinery without the Golgi structural protein, GM130. They functioned as distal glycosylation stations in dendrites, terminally modifying sugars either on newly synthesized glycoproteins passing through the secretory pathway or on surface glycoproteins taken up from the endocytic pathway. These activities led to major changes in the dendritic surface of excited neurons, impacting binding and uptake of lectins, as well as causing functional changes in neurotransmitter receptors such as nicotinic acetylcholine receptors. Neural activity thus boosts the activity of the dendrite’s satellite micro-secretory system by redistributing Golgi enzymes involved in glycan modifications into peripheral Golgi satellites. This remodeling of the neuronal surface has potential significance for synaptic plasticity, addiction, and disease.

The use of tetra-aza pyridinophanes is of increasing interest in the fields of bioinorganic modeling, catalysis, and imaging. However, a full study of how modifications to the pyridyl moiety affect the characteristics of the daughter metal complexes, has not been explored. In this study, six tetra-aza macrocyclic ligands were metalated with Fe(iii) and were characterized for the first time. The pyridyl functional groups studied include: 4-hydroxyl (L1), 4-H (L2), 4-chloro (L3), 4-trifluoromethyl (L4), 4-nitrile (L5), and 4-nitro (L6) modified pyridyl on a pyclen base structure. The resulting iron complexes were characterized by X-ray diffraction analysis, cyclic voltammetry, and metal-binding affinities (log β) were determined. Analysis of these results indicate that such functionalizations introduce a handle by which electrochemical properties and thermodynamic stability of daughter complexes with transition metal ions can be tuned, which in turn, could potentially impact the reactivity of these complexes in future studies.

The catalase family of enzymes, which include a variety with a binuclear manganese active site, mitigate the risk from reactive oxygen species by facilitating the disproportionation of hydrogen peroxide into molecular oxygen and water. In this work, hydrogen peroxide disproportionation using complexes formed between manganese and cyclen or pyclen were investigated due to the spectroscopic similarities with the native MnCAT enzyme. Potentiometric titrations were used to construct speciation diagrams that identify the manganese complex compositions at different pH values. Each complex behaves as a functional mimic of catalase enzymes. UV-visible spectroscopic investigations of the H(2)O(2) decomposition reaction yielded information about the structure of the initial catalyst and intermediates that include monomeric and dimeric species. The results indicate that rigidity imparted by the pyridine ring of pyclen is a key factor in increased TON and TOF values measured compared to cyclen.

Alzheimer’s and other neurodegenerative diseases are chronic conditions affecting millions of individuals worldwide. Oxidative stress is a consistent component described in the development of many neurodegenerative diseases. Therefore, innovative strategies to develop drug candidates that overcome oxidative stress in the brain are needed. To target these challenges, a new, water-soluble 12-membered tetraaza macrocyclic pyridinophane L4 was designed and produced using a building-block approach. Potentiometric data show that the neutral species of L4 provides interesting zwitterionic behavior at physiological pH, akin to amino acids, and a nearly ideal isoelectric point of 7.3. The copper(II) complex of L4 was evaluated by X-ray diffraction and cyclic voltammetry to show the potential modes of antioxidant activity derived, which was also demonstrated by 2,2-diphenyl-1-picrylhydrazyl and coumarin carboxylic acid antioxidant assays. L4 was shown to have dramatically enhanced antioxidant activity and increased biological compatibility compared to parent molecules reported previously. L4 attenuated hydrogen peroxide (H(2)O(2))-induced cell viability loss more efficiently than precursor molecules in the mouse hippocampal HT-22 cell model. L4 also showed potent (fM) level protection against H(2)O(2) cell death in a BV2 microglial cell culture. Western blot studies indicated that L4 enhanced the cellular antioxidant defense capacity via Nrf2 signaling activation as well. Moreover, a low-cost analysis and high metabolic stability in phase I and II models were observed. These encouraging results show how the rational design of lead compounds is a suitable strategy for the development of treatments for neurodegenerative diseases where oxidative stress plays a substantial role.

A number of challenges arise when using ferrocene as a component of electrochemical biosensors, including solubility in aqueous solutions. Therefore, entrapment of the biotin-ferrocene molecules within the chitosan film provides a route for immobilization on an electroactive surface such as an electrode while making the system water compatible. The use of the chitosan-ferrocene bioconjugate thin film on the electrode surface produces a signal that can be monitored in aqueous media. Herein, we discuss a series of modified ferrocene molecules that contain various linkers that provide non-covalent entanglement points to the chitosan medium. The electrochemical analysis and electron microscopy results show marked differences in the ferrocene loaded chitosan polymers when the termini of the ferrocene-linker vary between –SH and –NH2. The –SH modified systems showed increased reversible and robust electrochemical signals relative to the –NH2 congeners. Further studies showed that non-covalent impregnation strategy used is robust to degradation and less than 1% of the ferrocene molecules were leached over time. These results indicate that there are specific considerations needed when using chitosan-ferrocene systems as components in sensor arrays in future studies.

Biological cells routinely reconfigure their shape using dynamic signalling and regulatory networks that direct self-assembly processes in time and space, through molecular components that sense, process and transmit information from the environment. A similar strategy could be used to enable life-like behaviours in synthetic materials. Nucleic acid nanotechnology offers a promising route towards this goal through a variety of sensors, logic and dynamic components and self-assembling structures. Here, by harnessing both dynamic and structural DNA nanotechnology, we demonstrate dynamic control of the self-assembly of DNA nanotubes-a well-known class of programmable DNA nanostructures. Nanotube assembly and disassembly is controlled with minimal synthetic gene systems, including an autonomous molecular oscillator. We use a coarse-grained computational model to capture nanotube length distribution dynamics in response to inputs from nucleic acid circuits. We hope that these results may find use for the development of responsive nucleic acid materials, with potential applications in biomaterials science, nanofabrication and drug delivery.

Classically, endoplasmic reticulum (ER) retention signals in secreted integral membrane proteins impose the requirement to assemble with other cognate subunits to form functional assemblies before they can exit the ER. We report that GluK5 has two ER retention signals in its cytoplasmic C-terminus: an arginine-based signal and a di-leucine motif previously thought to be an endocytic motif. GluK5 assembles with GluK2, but surprisingly GluK2 association does little to block the ER retention signals. We find instead that the ER retention signals are blocked by two proteins involved in intracellular trafficking, SAP97 and CASK. We show that SAP97, in the presence of CASK and the receptor complex, assumes an extended conformation. In the extended conformation, SAP97 makes its SH3 and GuK domains available to bind and sterically mask the ER retention signals in the GluK5 C-terminus. SAP97 and CASK are also necessary for sorting receptor cargoes into the local dendritic secretory pathway in neurons. We show that the ER retention signals of GluK5 play a vital role in sorting the receptor complex in the local dendritic secretory pathway in neurons. These data suggest a new role for ER retention signals in trafficking integral membrane proteins in neurons. SIGNIFICANCE: We present evidence that the ER retention signals in the kainate receptors containing GluK5 impose a requirement for sorting into local dendritic secretory pathways in neurons, as opposed to traversing the somatic Golgi apparatus. There are two ER retention signals in the C-terminus of GluK5. We show that both are blocked by physical association with SAP97 and CASK. The SH3 and GuK domains of SAP97, in the presence of CASK, bind directly to each ER retention signal and form a complex. These results support an entirely new function for ER retention signals in the C-termini of neuronal receptors, such as NMDA and kainate receptors, and define a mechanism for selective entry of receptors into local secretory pathways.

Activity-driven changes in the neuronal surface glycoproteome are known to occur with synapse formation, plasticity and related diseases, but their mechanistic basis and significance are unclear. Here, we observed that N-glycans on surface glycoproteins of dendrites shift from immature to mature forms containing sialic acid in response to increased neuronal excitation. In exploring the basis of these N-glycosylation alterations, we discovered they result from the growth and proliferation of Golgi satellites scattered throughout the dendrite. Golgi satellites that formed with neuronal excitation were in close association with ER exit sites and early endosomes and contained glycosylation machinery without the Golgi structural protein, GM130. They functioned as distal glycosylation stations in dendrites, terminally modifying sugars either on newly synthesized glycoproteins passing through the secretory pathway, or on surface glycoproteins taken up from the endocytic pathway. These activities led to major changes in the dendritic surface of excited neurons, impacting binding and uptake of lectins, as well as causing functional changes in neurotransmitter receptors such as nicotinic acetylcholine receptors. Neural activity thus boosts the activity of the dendrite’s satellite micro-secretory system by redistributing Golgi enzymes involved in glycan modifications into peripheral Golgi satellites. This remodeling of the neuronal surface has potential significance for synaptic plasticity, addiction and disease.

Membrane traffic can be studied by imaging a cargo protein as it transits the secretory pathway. The best tools for this purpose initially block exit of the secretory cargo from the endoplasmic reticulum (ER), and then release the block to generate a cargo wave. However, previously developed regulatable secretory cargoes are often tricky to use or specific for a single model organism. To overcome these hurdles for budding yeast, we recently optimized an artificial fluorescent secretory protein that exits the ER with the aid of the Erv29 cargo receptor, which is homologous to mammalian Surf4. The fluorescent secretory protein forms aggregates in the ER lumen and can be rapidly disaggregated by addition of a ligand to generate a nearly synchronized cargo wave. Here we term this regulatable secretory protein ESCargo (Erv29/Surf4-dependentSecretoryCargo) and demonstrate its utility not only in yeast cells, but also in cultured mammalian cells,Drosophilacells, and the ciliateTetrahymena thermophila. Kinetic studies indicate that rapid transport out of the ER requires recognition by Erv29/Surf4. By choosing an appropriate ER signal sequence and expression vector, this simple technology can likely be used with many model organisms.

This work develops a new multifunctional biocompatible anticancer nanoformulation to provide targeted image-guided cancer-selective therapeutics. It consists of three active covalently bound components: (1) biocompatible nitrogen-doped graphene quantum dots (GQDs) as a multifunctional delivery and imaging platform, (2) hyaluronic acid (HA) unit targeted to the CD44 receptors on a variety of cancer cells, and (3) oxidative stress-based cancer-selective ferrocene (Fc) therapeutic. The biocompatible GQD platform synthesized from glucosamine exhibits high-yield intrinsic fluorescence. It is utilized for tracking Fc-GQD-HA formulation in vitro indicating internalization enhancement in HeLa cells targeted by the HA over non-cancer HEK-293 cells not overexpressing CD44 receptor. Fc-GQD-HA, non-toxic at 1 mg/mL to HEK-293 cells, induces cytotoxic response in HeLa enhanced over time, while therapeutic ROS generation by Fc-GQD-HA is ~3 times greater than that of Fc alone. This outlines the targeted delivery, imaging, and cancer-specific treatment capabilities of the new Fc-GQD-HA formulation enabling desired cancer-focused nanotherapeutic approach.

The pyridinophane molecule L2 (3,6,9,15-tetraazabicyclo[9.3.1]penta-deca-1(15),11,13-trien-13-ol) has shown promise as a therapuetic for neurodegenerative diseases involving oxidative stress and metal ion misregulation. Protonation and metal binding stability constants with Mg(2+), Ca(2+), Cu(2+), and Zn(2+) ions were determined to further explore the therapeutic and pharmacological potential of this water soluble small molecule. These studies show that incorporation of an –OH group in position 4 of the pyridine ring decreases the pI values compared to cyclen and L1 (3,6,9,15-tetraazabicyclo[9.3.1]penta-deca-1(15),11,13-triene). Furthermore, this approach tunes the basicity of the tetra-aza macrocyclic ligand through the enhanced resonance stabilization of the –OH in position 4 and rigidity of the pyridine ring such that L2 has increased basicity compared to previously reported tetra-aza macrocycles. A metal binding preference for Cu(2+), a redox cycling agent known to produce oxidative stress, indicates that this would be the in vivo metal target of L2. However, the binding constant of L2 with Cu(2+) is moderated compared to cyclen due to the rigidity of the ligand and shows how ligand design can be used to tune metal selectivity. An IC(50)=298.0 µM in HT-22 neuronal cells was observed. Low metabolic liability was determined in both Phase I and II in vitro models. Throughout these studies other metal binding systems were used for comparison and as appropriate controls. The reactivity reported to date and pharmacological features described herein warrant further studies in vivo and the pursuit of L2 congeners using the knowledge that pyridine substitution in a pyridinophane can be used to tune the structure of the ligand and retain the positive therapeutic outcomes.

All-trans retinoic acid (ATRA) and arsenic trioxide (ATO) are effective induction therapy for acute promyelocytic leukaemia (APL). However, early thrombo-haemorrhagic complications and mortality remain high. We aimed to investigate how the timing of ATRA initiation and the inclusion of ATO influence patient outcomes. Clinical records were retrospectively reviewed for all patients treated for APL in a single, tertiary centre during 2000-2017. Among 70 patients with APL, 36 (51.4%) presented with thrombo-haemorrhagic complications, and four (5.8%) died within 30 days. The median time to ATRA initiation was 11.2 (range 0-104) h from the time of admission. Patients requiring more transfusions started on ATRA sooner (P = 0.04). Patients with adverse early events did not start ATRA later (P = 0.99). Nevertheless, patients that required additional tests for diagnosis (PML immunofluorescence or molecular) started on ATRA later (28.5 versus 5.3 h; P 0.0001), and had more thrombo-haemorrhagic complications (P = 0.04). Long-term survival was actually better in patients who started ATRA later (P = 0.03), which is likely explained by higher proportion of low risk patients in this group. Patients treated with ATO (n = 23) maintained higher fibrinogen levels and required less transfusions during induction (P 0.05), with no disease-related deaths in this group over a median follow-up time of 37.8 months (interquartile range 44.9 months). In summary, fast ATRA initiation reduces early but not late adverse events in APL patients, and the inclusion of ATO helps further improve both early and late outcomes in APL.

How nicotine exposure produces long-lasting changes that remodel neural circuits with addiction is unknown. Here, we report that long-term nicotine exposure alters the trafficking of α4β2-type nicotinic acetylcholine receptors (α4β2Rs) by dispersing and redistributing the Golgi apparatus. In cultured neurons, dispersed Golgi membranes were distributed throughout somata, dendrites and axons. Small, mobile vesicles in dendrites and axons lacked standard Golgi markers and were identified by other Golgi enzymes that modify glycans. Nicotine exposure increased levels of dispersed Golgi membranes, which required α4β2R expression. Similar nicotine-induced changes occurred in vivo at dopaminergic neurons at mouse nucleus accumbens terminals, consistent with these events contributing to nicotine’s addictive effects. Characterization in vitro demonstrated that dispersal was reversible, that dispersed Golgi membranes were functional, and that membranes were heterogenous in size, with smaller vesicles emerging from larger “ministacks”, similar to Golgi dispersal induced by nocadazole. Protocols that increased cultured neuronal synaptic excitability also increased Golgi dispersal, without the requirement of α4β2R expression. Our findings reveal novel activity- and nicotine-dependent changes in neuronal intracellular morphology. These changes regulate levels and location of dispersed Golgi membranes at dendrites and axons, which function in local trafficking at subdomains.

Pincer molecules are of increasing interest due to the modular nature of modification and range of reactivity observed when coordinated to metal ions. A subset within the family of pincer molecules use a pyridine group to bridge the outer two arms as well as provide a N-donor atom for metal binding. While the arm appendages have been studied extensively, little research has been conducted on the electronic effects of the central, substituted pyridine systems. Therefore, a series of NNN pincer-type ligands with substitution on the 4-position of the pyridine ring with −OH, −OBn, −H, −Cl, and −NO(2) functional groups were synthesized and characterized through NMR spectroscopy and ESI-HRMS. Each pincer was metalated with Cu(ii) salts and evaluated through X-ray diffraction analysis, cyclic voltammetry, and density functional theory analysis. The results indicate that the relatively unstudied −OBn group demonstrates both electron-withdrawing (XRD bond lengths) and electron-donating (NMR spectroscopy) properties. The −NO(2) pincer ligand shows a redox event within experimental windows evaluated, in contrast to the other congeners studied. In addition, electron-donating groups increase the electron density around the Cu(ii) center based on DFT studies and cyclic voltammetry. These findings can be applied to other pyridine-based pincer systems when considering ligand design and warrants future characterization of 4-position substituted pyridines.

Macrocyclic ligands have been explored extensively as scaffolds for transition metal catalysts for oxygen and hydrogen atom transfer reactions. C-C reactions facilitated using earth abundant metals bound to macrocyclic ligands have not been well-understood but could be a green alternative to replacing the current expensive and toxic precious metal systems most commonly used for these processes. Therefore, the yields from direct Suzuki-Miyaura C-C coupling of phenylboronic acid and pyrrole to produce 2-phenylpyrrole facilitated by eight high-spin iron complexes ([Fe(3+)L1(Cl)(2)](+), [Fe(3+)L4(Cl)(2)](+), [Fe(2+)L5(Cl)](+), [Fe(2+)L6(Cl)(2)], [Fe(3+)L7(Cl)(2)](+), [Fe(3+)L8(Cl)(2)](+), [Fe(2+)L9(Cl)](+), and [Fe(2+)L10(Cl)](+)) were compared to identify the effect of structural and electronic properties on catalytic efficiency. Specifically, catalyst complexes were compared to evaluate the effect of five properties on catalyst reaction yields: 1. the coordination requirements of the catalyst, 2. Redox half-potential of each complex, 3. topological constraint/rigidity, 4. N-atom modification(s) increasing oxidative stability of the complex, and 5. geometric parameters. The need for two labile cis-coordination sites was confirmed based on a 42% decrease in catalytic reaction yield observed when complexes containing penta-dentate ligands were used in place of complexes with tetra-dentate ligands. A strong correlation between iron(III/II) redox potential and catalytic reaction yields was also observed, with [Fe(2+)L6(Cl)(2)] providing the highest yield (81%, −405 mV). A Lorentzian fitting of redox potential versus yields predicts that these catalysts can undergo more fine tuning to further increase yields. Interestingly, the remaining properties explored did not show a direct, strong relationship to catalytic reaction yields. Altogether, these results show that modifications to the ligand scaffold using fundamental concepts of inorganic coordination chemistry can be used to control the catalytic activity of macrocyclic iron complexes by controlling redox chemistry of the iron center. Furthermore, the data provides direction for the design of improved catalysts for this reaction and strategies to understand the impact of a ligand scaffolds on catalytic activity of other reactions.

Herein we describe the synthesis, characterization, and role of three dichloric iron(III) complexes, [L1Fe(III)(Cl)2]ClO4 (L1Fe), [L2Fe(III)(Cl)2]ClO4 (L2Fe), and [L3Fe(III)(Cl)2]ClO4 (L3Fe) [L1 (Pyclen) = 1,4,7,10-tetra-aza-2,6-pyridinophane; L2 = 1,4,7,10-tetra-aza-2,6-pyridinophane-14-ol; L3 = 1,4,7,10-tetra-aza-2,6-pyridinophane-13-ol, in the coupling of pyrrole and phenylboronic acid to form 2-phenylpyrrole. The oxidation- and spin state of the iron complexes were characterized using X-ray crystallography, UV–vis absorbance spectroscopy, electron paramagnetic resonance spectroscopy, cyclic voltammetry, and mass spectrometry. Electrochemistry results rank ligand L1–L3 as moderate tetra-azamacrocycle donors to iron between cyclen and Me2EBC-12. Characterization of the iron(III) complexes and subsequent catalytic testing indicates that the complexes enter the C–C coupling catalytic cycle in the high-spin iron(III) oxidation state. Furthermore, the results indicate that the iron(III) complexes are essential for catalytic and regioselective production of the 2-phenylpyrrole product.

The use of proteins that bind and catalyze reactions with DNA alongside DNA nanostructures has broadened the functionality of DNA devices. DNA binding proteins have been used to specifically pattern and tune structural properties of DNA nanostructures and polymerases have been employed to directly and indirectly drive structural changes in DNA structures and devices. Despite these advances, undesired and poorly understood interactions between DNA nanostructures and proteins that bind DNA continue to negatively affect the performance and stability of DNA devices used in conjunction with enzymes. A better understanding of these undesired interactions will enable the construction of robust DNA nanostructure-enzyme hybrid systems. Here, we investigate the undesired disassembly of DNA nanotubes in the presence of viral RNA polymerases (RNAPs) under conditions used for in vitro transcription. We show that nanotubes and individual nanotube monomers (tiles) are non-specifically transcribed by T7 RNAP, and that RNA transcripts produced during non-specific transcription disassemble the nanotubes. Disassembly requires a single-stranded overhang on the nanotube tiles where transcripts can bind and initiate disassembly through strand displacement, suggesting that single-stranded domains on other DNA nanostructures could cause unexpected interactions in the presence of viral RNA polymerases.

The neutrophil enzyme, myeloperoxidase, by converting hydrogen peroxide (H2O2) and chloride to hypochlorous acid (HOCl), provides important defense against ingested micro-organisms. However, there is debate about how efficiently HOCl is produced within the phagosome and whether its reactions with phagosomal constituents influence the killing mechanism. The phagosome is a small space surrounding the ingested organism, into which superoxide, H2O2 and high concentrations of proteins from cytoplasmic granules are released. Previous studies imply that HOCl is produced in the phagosome, but a large proportion should react with proteins before reaching the microbe. To mimic these conditions, we subjected neutrophil granule extract to sequential doses of H2O2. Myeloperoxidase in the extract converted all the H2O2 to HOCl, which reacted with the granule proteins. 3-Chlorotyrosine, protein carbonyls and large amounts of chloramines were produced. At higher doses of H2O2, the extract developed potent bactericidal activity against Staphylococcus aureus. This activity was due to ammonia monochloramine, formed as a secondary product from protein chloramines and dichloramines. Isolated myeloperoxidase and elastase also became bactericidal when modified with HOCl and antibacterial activity was seen with a range of species. Comparison of levels of protein modification in the extract and in phagosomes implies that a relatively low proportion of phagosomal H2O2 would be converted to HOCl, but there should be sufficient for substantial protein chloramine formation and some breakdown to ammonia monochloramine. It is possible that HOCl could kill ingested bacteria by an indirect mechanism involving protein oxidation and monochloramine formation.

Essentials Intracellular calcium pathways regulate megakaryopoiesis but details are unclear.We examined effects of NMDAR‐mediated calcium influx on normal and leukemic cells in culture.NMDARs facilitated differentiation of normal but proliferation of leukemic megakaryocytes.NMDAR inhibitors induced differentiation of leukemic Meg‐01 cells. Background N‐methyl‐d‐aspartate receptors (NMDARs) contribute calcium influx in megakaryocytic cells but their roles remain unclear; both pro‐ and anti‐differentiating effects have been shown in different contexts. Objectives The aim of this study was to clarify NMDAR contribution to megakaryocytic differentiation in both normal and leukemic cells. Methods Meg‐01, Set‐2, and K‐562 leukemic cell lines were differentiated using phorbol‐12‐myristate‐13‐acetate (PMA, 10 nmol L−1) or valproic acid (VPA, 500 μmol L−1). Normal megakaryocytes were grown from mouse marrow‐derived hematopoietic progenitors (lineage‐negative and CD41a‐enriched) in the presence of thrombopoietin (30‐40 nmol L−1). Marrow explants were used to monitor proplatelet formation in the native bone marrow milieu. In all culture systems, NMDARs were inhibited using memantine and MK‐801 (100 μmol L−1); their effects compared against appropriate controls. Results The most striking observation from our studies was that NMDAR antagonists markedly inhibited proplatelet formation in all primary cultures employed. Proplatelets were either absent (in the presence of memantine) or short, broad and intertwined (with MK‐801). Earlier steps of megakaryocytic differentiation (acquisition of CD41a and nuclear ploidy) were maintained, albeit reduced. In contrast, in leukemic Meg‐01 cells, NMDAR antagonists inhibited differentiation in the presence of PMA and VPA but induced differentiation when applied by themselves. Conclusions NMDAR‐mediated calcium influx is required for normal megakaryocytic differentiation, in particular proplatelet formation. However, in leukemic cells, the main NMDAR role is to inhibit differentiation, suggesting diversion of NMDAR activity to support leukemia growth. Further elucidation of the NMDAR and calcium pathways in megakaryocytic cells may suggest novel ways to modulate abnormal megakaryopoiesis.

The Eu3+/2+ redox couple provides a convenient design platform for responsive pO2 sensors for magnetic resonance imaging (MRI). Specifically the Eu2+ ion provides T1w contrast enhancement under hypoxic conditions in tissues, whereas, under normoxia, the Eu3+ ion can produce contrast from chemical exchange saturation transfer in MRI. The oxidative stability of the Eu3+/2+ redox couple for a series of tetraaza macrocyclic complexes was investigated in this work using cyclic voltammetry. A series of Eu-containing cyclen-based macrocyclic complexes revealed positive shifts in the Eu3+/2+ redox potentials with each replacement of a carboxylate coordinating arm of the ligand scaffold with glycinamide pendant arms. The data obtained reveal that the complex containing four glycinamide coordinating pendant arms has the highest oxidative stability of the series investigated.

Berenil, a minor groove DNA binding molecule, has been extensively used in veterinary medicine. Modeling studies have suggested that berenil binds to A/T rich regions on the DNA and the product of this interaction causes the formation of crosslinks between opposite DNA strands. These crosslinks could potentially inhibit fundamental biological processes including transcription and DNA replication. We had previously used the pBR322 genome as a model system to investigate the role of A/T sequences on berenil activity. We reported that the insertion of poly(dA)poly(dT) sequences into the pBR322 genome causes replication inhibition of the recombinant plasmids when cultures were exposed to berenil. However, we noticed that even in the absence of these sequences the parental plasmid replication was also inhibited, albeit less than the recombinants. This observation led us to the present study were we attempted to identify the location of natural berenil target sites in the pBR322 genome. Through a combination of deletion analysis, recombinant DNA and a replication assay we uncovered a 378 bp DNA fragment that has all the hallmarks of a berenil target site. A recombinant plasmid lacking this region is more refractive to the drug than the parental plasmid, and another variant containing and extra copy of this region increases the susceptibility of the plasmid towards berenil. The 378 bp region is about 60% A/T rich and contains about 21 potential berenil binding sites.

Glutamate-like receptors (GLRs) in plants play an important role in a number of physiological processes, including wound response, stomatal aperture control, seed germination, root development, innate immune responses, pollen tube growth and morphogenesis. GLRs share amino acid sequence similarity with ionotropic glutamate receptors (iGluRs) that mediate neurotransmission in the nervous system of vertebrates. In contrast to iGluRs, however, for which numerous full-length structures are available, the structural information about the plant GLRs has been missing. Here we determine crystal structures of Arabidopsis thaliana GLR3.2 ligand-binding domain (LBD) in complex with glycine and methionine to 1.57 and 1.86 Å resolution, respectively. Our structures show a fold similar to iGluRs, with several secondary structure elements either missing or different. The closed clamshell conformation of GLR3.2 LBD suggests that both glycine and methionine act as agonists. The structures reveal molecular determinants of ligand binding and explain the promiscuity of GLRs’ ligand activation compared to iGluRs. Structural similarities of LBDs confirm an evolutionary relationship between GLRs and iGluRs and predict common molecular principles of their gating mechanisms that are driven by the bilobed clamshell-like LBDs.

The primary function of neutrophils is to engulf and destroy invading pathogens. If the bactericidal capacity of neutrophils is defective, an individual may suffer from enhanced susceptibility to potentially fatal microbial infection. To identify such defects, and to investigate the mechanisms used to kill bacteria, the bactericidal activity of neutrophils must be accurately quantified. This chapter provides details of a comprehensive microbiological technique that quantifies neutrophil bactericidal activity by measuring the loss of viability of ingested bacteria over time. Two variations of this technique are presented: a simple "one-step" protocol providing a composite measure of phagocytosis and killing, and a more advanced "two-step" protocol that allows calculation of separate rate constants for both of these processes.

SUMMARY Stable transmission of genetic material during cell division requires accurate chromosome segregation. PLK1 dynamics at kinetochores control establishment of correct kinetochore-microtubule attachments and subsequent silencing of the spindle checkpoint. However, the regulatory mechanism responsible for PLK1 activity in prometaphase has not yet been affirmatively identified. Here we identify Apolo1, which tunes PLK1 activity for accurate kinetochore-microtubule attachments. Apolo1 localizes to kinetochores during early mitosis, and suppression of Apolo1 results in misaligned chromosomes. Using the fluorescence resonance energy transfer (FRET)-based PLK1 activity reporter, we found that Apolo1 sustains PLK1 kinase activity at kinetochores for accurate attachment during prometaphase. Apolo1 is a cognate substrate of PLK1, and the phosphorylation enables PP1γ to inactivate PLK1 by dephosphorylation. Mechanistically, Apolo1 constitutes a bridge between kinase and phosphatase, which governs PLK1 activity in prometaphase. These findings define a previously uncharacterized feedback loop by which Apolo1 provides fine-tuning for PLK1 to guide chromosome segregation in mitosis., Graphical abstract, In brief Xu et al. identify Apolo1, which governs PLK1 activity and promotes faithful chromosome segregation in prometaphase by bridging kinase and phosphatase activities.

Glutamate receptor-like channels (GLRs) play important roles in numerous plant physiological processes. GLRs are homologous to ionotropic glutamate receptors (iGluRs) that mediate neurotransmission in vertebrates. Here we determine crystal structures of Arabidopsis thaliana GLR3.2 ligand-binding domain (LBD) in complex with glycine and methionine to 1.58- and 1.75-Å resolution, respectively. Our structures show a fold similar to that of iGluRs, but with several secondary structure elements either missing or different. The closed clamshell conformation of GLR3.2 LBD suggests that both glycine and methionine act as agonists. The mutation R133A strongly increases the constitutive activity of the channel, suggesting that the LBD mutated at the residue critical for agonist binding produces a more stable closed clamshell conformation. Furthermore, our structures explain the promiscuity of GLR activation by different amino acids, confirm evolutionary conservation of structure between GLRs and iGluRs, and predict common molecular principles of their gating mechanisms driven by bilobed clamshell-like LBDs.

Alzheimer’s disease currently affects over 5.4 million Americans with $236 billion spent annually on the direct costs of patient care. Health experts expect this price tag to rise to $1.1 trillion ...

Microwave heating has been used to substitute carbonyl ligands for 1,3,5-triaza-7-phosphaadamantane (PTA) ligands on the Os3(CO)12 molecule. Os3(CO)11(PTA) (1) has been prepared in 66% yield by the reaction of Os3(CO)12 with MeCN, followed by reaction with one mole equivalent of PTA. Os3(CO)10(PTA)2 (2) has been prepared in 65% yield via a three-step reaction. First, Os3(CO)12 was heated in MeCN. Next, one mole equivalent of PTA was added to this reaction mixture. Finally, one mole equivalent of trimethylamine-N-oxide was added followed by another mole equivalent of PTA. Additionally, Os3(CO)9(PTA)3 (3) has been prepared in 85% yield by the reaction of Os3(CO)12 with 3.5 mol equivalents of PTA in 1,2-dichlorobenzene, and Os3(CO)8(PTA)4 (4) has been prepared in 36% yield by the reaction of Os3(CO)12 with 8 mol equivalents of PTA in a mixture of acetonitrile and 1,2-dichlorobenzene. The solubilities of clusters 1–4 in water at various pH levels have been studied, and 4 was found to be soluble in water with a neutral pH. X-ray diffraction has been used to determine the crystal structures of clusters 1 and 3. The X-ray crystal structure of a protonated form of 3, [Os3(CO)9(PTAH)3]Cl3 (5), has also been determined. All PTA ligands occupy equatorial coordination sites on the triangular Os3 units.

Correction for ‘Functionalized pyridine in pyclen-based iron(iii) complexes: evaluation of fundamental properties’ by Magy A. Mekhail et al., RSC Adv., 2020, 10, 31165–31170. DOI: 10.1039/d0ra05756h.

EuDOTA-glycine derivatives have been explored as alternatives to typical gadolinium-containing complexes for MRI agents used in diagnostic imaging. Different imaging modalities can be accessed (T(1) or PARACEST) dependent on the oxidation state of the europium ion. Throughout the past 30 years, there have been significant manipulations and additions made to the DOTA scaffold; yet, characterizations related to electrochemistry and structure determined through XRD analysis have not been fully analyzed. In this work, electrochemical analysis using cyclic voltammetry was carried out on EuDOTA derivatives, including the free ligand DOTAGly(4) (4) and the complexes. Effects of glycinate substitution on the DOTA scaffold, specifically, ligand interactions with the glassy carbon electrode were observed. A range of electrochemical investigations were carried out to show that increased glycinate substitution led to increased interaction with the electrode surface, thus implicating a new factor to consider when evaluating the electrochemistry of glycinate substituted ligands. In addition, the solid-state structure of EuDOTAGly(4) (Eu4) was determined by X-ray diffraction and a brief analysis is presented compared to known Ln(3+) structures found within literature. The Eu4 complex crystalizes in a rare polymer type arrangement via bridging side-arms between adjacent complexes.

Nicotinic acetylcholine α4β2∗ receptors (nAChRs) are implicated in various neurodegenerative diseases and smoking addiction. Imaging of brain high-affinity α4β2∗ nAChRs at the cellular and subcellular levels would greatly enhance our understanding of their functional role. Since better resolution could be achieved with fluorescent probes, using our previously developed positron emission tomography (PET) imaging agent [18F]nifrolidine, we report here design, synthesis and evaluation of two fluorescent probes, nifrodansyl and nifrofam for imaging α4β2∗ nAChRs. The nifrodansyl and nifrofam exhibited nanomolar affinities for the α4β2∗ nAChRs in [3H]cytisine-radiolabeled rat brain slices. Nifrofam labeling was observed in α4β2∗ nAChR-expressing HEK cells and was upregulated by nicotine exposure. Nifrofam co-labeled cell-surface α4β2∗ nAChRs, labeled with antibodies specific for a β2 subunit extracellular epitope indicating that nifrofam labels α4β2∗ nAChR high-affinity binding sites. Mouse brain slices exhibited discrete binding of nifrofam in the auditory cortex showing promise for examining cellular distribution of α4β2∗ nAChRs in brain regions.

Nicotinamides are a class of compounds with a wide variety of applications, from use as antimicrobial agents to inhibitors of biological processes. These compounds are also cofactors, which are necessary components of metabolic processes. Structural modification gives rise to the activities observed. Similarly, 1,3,4-thiadiazoles have been shown to possess antioxidant, antimicrobial, or anti-inflammatory biological activity. To take advantage of each of the inherent characteristics of the two aforementioned functional groups, 2-nicotinamido-1,3,4-thiadiazole, C8H6N4OS, was synthesized. Since defining chemical connectivity is paramount in understanding biological activity, in this report, the structural characterization of 2-nicotinamido-1,3,4-thiadiazole has been carried out using X-ray crystallographic methods. The NMR-derived assignments were made possible by utilizing one- (1D) and two-dimensional (2D) NMR techniques. In addition, UV–Visible and IR spectroscopies, and elemental analysis were used to fully characterize the product synthesized by the one-step reaction between nicotinoyl chloride hydrochloride and 2-amino-1,3,4-thiadiazole. Computational parameters related to blood–brain barrier permeability are also presented.

The reaction of copper(II) perchlorate with the hydrochloride salt of 3,6,9,15-tetra-azabicyclo[9.3.1]penta-deca-1,11,13-triene (L1) in acetonitrile forms two macrocyclic complexes that can be characterized: [L1CuIICl][ClO4] (1) and [L1CuIICl]2[CuCl4] (2). The structural, electronic, and redox properties of these complexes were studied using spectroscopy (EPR and UV–visible) and electrochemistry. In addition, the solid-state structure of 1 was obtained using X-ray diffraction. The copper(II) is five-coordinate ligated by four N-atoms of the macrocycle and a chloride atom. EPR studies of 1 both in DMF and aqueous solution indicate the presence of a single copper(II) species. In contrast, EPR studies of 2 performed in frozen DMF and in the solid-state reveal the presence of two spectroscopically distinct copper(II) complexes assigned as [L1CuIICl]+ and [CuIICl4]2−. Lastly, electrochemical studies demonstrate that both [L1CuIICl]+ and [CuIICl4]2− are redox active. Specifically, the [L1CuIICl]+ undergoes a quasi-reversible Cu(II)/(I) redox reaction in the absence of excess chloride. In the presence of chloride, however, the chemical irreversibility of this couple becomes evident at concentrations of chloride that exceed 50 mM. As a result, the presence of chloride from the chemical equilibrium of this latter species impedes the reversibility of the reduction of [L1CuIICl]+ to [L1CuICl]0.

A library of ferrocene–biotin bioconjugates has been produced using solid phase methods and evaluated for a change in redox activity in the presence of avidin. This was carried out as a means to ex...

This work reports on a new random ternary polymerization method for the synthesis of multidentate imidazole polymers. The polymers can behave as ligands for the functionalization of cadmium sulfide nanowires. Due to the intrinsic differences in the electronegativity of the groups next to the vinyl bond, the vinyl groups displayed unique NMR signals, and allowed for the measurement of the individual monomer conversions in the random terpolymer system. The activated ester bearing terpolymer was postmodified with N-alkyl imidazole units, followed by the boc deprotection of the amine terminal groups. The resulting poly(imid-PEGMA-MAMamine) provided water solubility, dye loading capability as well as the ability to coordinate with metal chalcogenide surfaces using the imidazole units. Upon the attachment of a rhodamine dye, studies were performed to analyze the potential of such polymers to modify CdS nanowires using fluorescence microscopy. The fluorescence microscopy results provided confirmation of the polymeric ligand attachment, and sets the foundation for further optical studies using this system.

Previous studies tracking AMPA receptor (AMPAR) diffusion at synapses observed a large mobile extrasynaptic AMPAR pool. Using super-resolution microscopy, we examined how fluorophore size and photostability affected AMPAR trafficking outside of, and within, post-synaptic densities (PSDs) from rats. Organic fluorescent dyes (≈4 nm), quantum dots, either small (≈10 nm diameter; sQDs) or big (>20 nm; bQDs), were coupled to AMPARs via different-sized linkers. We find that >90% of AMPARs labeled with fluorescent dyes or sQDs were diffusing in confined nanodomains in PSDs, which were stable for 15 min or longer. Less than 10% of sQD-AMPARs were extrasynaptic and highly mobile. In contrast, 5–10% of bQD-AMPARs were in PSDs and 90–95% were extrasynaptic as previously observed. Contrary to the hypothesis that AMPAR entry is limited by the occupancy of open PSD ‘slots’, our findings suggest that AMPARs rapidly enter stable ‘nanodomains’ in PSDs with lifetime >15 min, and do not accumulate in extrasynaptic membranes., eLife digest Forgetting is a common experience in our everyday life. Yet much remains unknown about how we remember, and about why our memories sometimes fail us. The brain contains 80 to 100 billion nerve cells or neurons, which communicate with one another at junctions called synapses. At a synapse, one neuron releases a chemical message, which must diffuse across a small gap, and then activate proteins called receptors on another neuron. If the first neuron activates the second repeatedly, the second cell responds by inserting additional receptors into its membrane at the synapse. This strengthens the connection between the two neurons. Strengthening of synapses is thought to be one of the key mechanisms underlying learning. To confirm this, it would be helpful to be able to monitor the movement and position of individual receptors at synapses. However, the space between the two nerve cells at at synapse, called the synaptic cleft, is no more than 40 nanometers wide. This is about 25 times thinner than a human hair, and too small to be seen with light microscopy. Electron microscopy can visualize synapses, but does not work in living tissue. The only other option is to attach a fluorescent label – either a dye or a man-made crystal called a quantum dot – to a protein found in synapses and monitor the resulting fluorescence. Though the probe must be small enough to pass through the synaptic cleft to do this. Using fluorescence microscopy, researchers have examined the distribution in synapses of proteins called AMPA receptors, which have a key role in memory. Multiple studies have shown groups of AMPA receptors gathered outside synapses. This has led to the suggestion that during learning, AMPA receptors wait outside the synapse until a space becomes available within the synapse’s membrane. However, this has yet to be confirmed directly, in part because conventional fluorescent dyes and quantum dots are too bulky to enter synaptic clefts when bound to a receptor. Lee et al. have now developed a quantum dot that is only 10 nanometers wide and therefore small enough to enter the synaptic cleft with an AMPA receptor attached. These small quantum dots were then used to label AMPA receptors in neurons collected from rats and then grown in a petri dish, which provided a completely new view of synapses. The images show that the majority of AMPA receptors in neurons circulate within confined domains – a little like holding pens – inside the synapse, rather than waiting outside as previously assumed. Labeling the receptors with smaller 4-nanometer-wide fluorescent tags produces a similar picture. Further work is still need to determine how AMPA receptors get into the synapse and contribute to new memories.

Metal-ion misregulation and oxidative stress continue to be components of the continually evolving hypothesis describing the molecular origins of Alzheimer's disease. Therefore, these features are viable targets for synthetic chemists to explore through hybridizations of metal-binding ligands and antioxidant units. To date, the metal-binding unit in potential therapeutic small molecules has largely been inspired by clioquinol with the exception of a handful of heterocyclic small molecules and open-chain systems. Heterocyclic small molecules such as cyclen (1,4,7,10-tetraazacyclododecane) have the advantage of straightforward N-based modifications, allowing the addition of functional groups. In this work, we report the synthesis of a triazine bridged system containing two cyclen metal-binding units and an antioxidant coumarin appendage inspired by nature. This new potential therapeutic molecule shows the ability to bind copper in a unique manner compared to other chelates proposed to treat Alzheimer's disease. DPPH and TEAC assays exploring the activity of N-(2-((4,6-di(1,4,7,10-tetraazacyclododecan-1-yl)-1,3,5-triazin-2-yl)amino)ethyl)-2-oxo-2H-chromene-3-carboxamide (molecule 1) show that the molecule is antioxidant. Cellular studies of molecule 1 indicate a low toxicity (EC

To better understand smoking cessation, we examined the actions of varenicline (Chantix) during long-term nicotine exposure. Varenicline reduced nicotine upregulation of α4β2-type nicotinic receptors (α4β2Rs) in live cells and neurons, but not for membrane preparations. Effects on upregulation depended on intracellular pH homeostasis and were not observed if acidic pH in intracellular compartments was neutralized. Varenicline was trapped as a weak base in acidic compartments and slowly released, blocking 125I-epibatidine binding and desensitizing α4β2Rs. Epibatidine itself was trapped; 125I-epibatidine slow release from acidic vesicles was directly measured and required the presence of α4β2Rs. Nicotine exposure increased epibatidine trapping by increasing the numbers of acidic vesicles containing α4β2Rs. We conclude that varenicline as a smoking cessation agent differs from nicotine through trapping in α4β2R-containing acidic vesicles that is selective and nicotine-regulated. Our results provide a new paradigm for how smoking cessation occurs and suggest how more effective smoking cessation reagents can be designed. DOI: http://dx.doi.org/10.7554/eLife.25651.001, eLife digest Tobacco continues to be widely used worldwide, primarily via cigarette smoking, and is a leading cause of preventable deaths. Stopping smoking is difficult because the nicotine in tobacco is highly addictive, and so several drugs have been developed to help people break their addiction. Varenicline (also known by the trade name Chantix) is a commonly prescribed anti-smoking drug, but it is not fully understood how it works. Nicotine affects the brain by binding to proteins called nicotinic acetylcholine receptors (nAChRs) that sit on the surface of neurons. This binding releases a number of chemical signals, including some that produce feelings of pleasure. Over time, the receptors become less sensitive to nicotine and produce more “high-affinity” binding sites for nicotine to bind to. This adaptation is one reason why stopping smoking can produce strong feelings of withdrawal. Previously, varenicline was thought to partially activate nAChRs, preventing nicotine from binding to the receptors. However, this can only explain how varenicline counteracts the rapid-acting effects of nicotine, not nicotine’s longer-term effects. Furthermore, it was not known how nAChR signaling responds to long-term exposure to a combination of both drugs (as occurs when people try to quit smoking with the aid of varenicline). Now, Govind et al. reveal how varenicline reverses the effect of long-term nicotine exposure on nAChR signaling. Both varenicline and nicotine accumulate in acidic compartments – called vesicles – inside cells, where they become charged and less able to move through the cell membrane. When the vesicles also contain high-affinity nAChRs, varenicline becomes trapped inside them and is only slowly released. By contrast, nicotine is not trapped because it exits the vesicles more rapidly. Long-term exposure to nicotine greatly increased the number of vesicles that contained high-affinity nAChRs, thereby trapping more varenicline. One consequence of trapping varenicline was that the activity of the nAChRs on the surface of the neuron was diminished, apparently through the slow release of the trapped varenicline from the acidic vesicles. This slow release causes the receptors to enter a “desensitized” state in which they do not signal. Understanding how varenicline counteracts the long-term effects of nicotine on nAChR signaling will help us to design more effective anti-smoking drugs. Govind et al. also found that compounds similar to varenicline become trapped in vesicles, but it is not clear how the degree of trapping of a compound correlates with how effectively it combats nicotine addiction. The results may also help us to understand and treat addictions to other drugs of abuse, such as opioids, amphetamines and cocaine, which have chemical properties that mean they might also be selectively trapped in acidic vesicles. DOI: http://dx.doi.org/10.7554/eLife.25651.002

Neutrophils ingest and kill bacteria within phagocytic vacuoles. We investigated where they produce hypochlorous acid (HOCl) following phagocytosis by measuring conversion of protein tyrosine residues to 3-chlorotyrosine. We also examined how varying chloride availability affects the relationship between HOCl formation in the phagosome and bacterial killing. Phagosomal proteins, isolated following ingestion of opsonized magnetic beads, contained 11.4 Cl-Tyr per thousand tyrosine residues. This was 12 times higher than the level in proteins from the rest of the neutrophil and ~6 times higher than previously recorded for protein from ingested bacteria. These results indicate that HOCl production is largely localized to the phagosomes and a substantial proportion reacts with phagosomal protein before reaching the microbe. This will in part detoxify the oxidant but should also form chloramines which could contribute to the killing mechanism. Neutrophils were either suspended in chloride-free gluconate buffer or pretreated with formyl-Met-Leu-Phe, a procedure that has been reported to deplete intracellular chloride. These treatments, alone or in combination, decreased both chlorination in phagosomes and killing of Staphylococcus aureus by up to 50%. There was a strong positive correlation between the two effects. Killing was predominantly oxidant and myeloperoxidase dependent (88% inhibition by diphenylene iodonium and 78% by azide). These results imply that lowering the chloride concentration limits HOCl production and oxidative killing. They support a role for HOCl generation, rather than an alternative myeloperoxidase activity, in the killing process.

Immunofluorescence, a powerful technique to detect specific targets using fluorescently labeled antibodies, has been widely used in both scientific research and clinical diagnostics. The probes should be made with small antibodies and high brightness. We conjugated GFP binding protein (GBP) nanobodies, small single-chain antibodies from llamas, with new ∼7 nm quantum dots. These provide simple and versatile immunofluorescence nanoprobes with nanometer accuracy and resolution. Using the new probes we tracked the walking of individual kinesin motors and measured their 8 nm step sizes; we tracked Piezo1 channels, which are eukaryotic mechanosensitive channels; we also tracked AMPA receptors on living neurons. Finally, we used a new super-resolution algorithm based on blinking of (small) quantum dots that allowed ∼2 nm precision.

Oxidative stress resulting from metal-ion misregulation plays a role in the development of Alzheimer's disease (AD). This process includes the production of tissue-damaging reactive oxygen species and amyloid aggregates. Herein we describe the synthesis, characterization and protective capacity of the small molecule, lipoic cyclen, which has been designed to target molecular features of AD. This construct utilizes the biologically compatible and naturally occurring lipoic acid as a foundation for engendering low cellular toxicity in multiple cell lines, radical scavenging capacity, tuning the metal affinity of the parent cyclen, and results in an unexpected affinity for amyloid without inducing aggregation. The hybrid construct thereby shows protection against cell death induced by amyloid aggregates and copper ions. These results provide evidence for the rational design methods used to produce this fused molecule as a potential strategy for the development of lead compounds for the treatment of neurodegenerative disorders.