Structural basis for cooperative interactions of substituted 2-aminopyrimidines with the acetylcholine binding protein

The nicotinic acetylcholine receptor (nAChR) and the acetylcholine binding protein (AChBP) are pentameric oligomers in which binding sites for nicotinic agonists and competitive antagonists are found at selected subunit interfaces. The nAChR spontaneously exists in multiple conformations associated with its activation and desensitization steps, and conformations are selectively stabilized by binding of agonists and antagonists. In the nAChR, agonist binding and the associated conformational changes accompanying activation and desensitization are cooperative. AChBP, which lacks the transmembrane spanning and cytoplasmic domains, serves as a homology model of the extracellular domain of the nAChRs. We identified unique cooperative binding behavior of a number of 4,6-disubstituted 2-aminopyrimidines to Lymnaea AChBP, with different molecular variants exhibiting positive, n_H > 1.0, and negative cooperativity, n_H < 1.0. Therefore, for a distinctive set of ligands, the extracellular domain of a nAChR surrogate suffices to accommodate cooperative interactions. X-ray crystal structures of AChBP complexes with examples of each allowed the identification of structural features in the ligands that confer differences in cooperative behavior. Both sets of molecules bind at the agonist-antagonist site, as expected from their competition with epibatidine. An analysis of AChBP quaternary structure shows that cooperative ligand binding is associated with a blooming or flare conformation, a structural change not observed with the classical, noncooperative, nicotinic ligands. Positively and negatively cooperative ligands exhibited unique features in the detailed binding determinants and poses of the complexes.Nicotinic acetylcholine receptors (nAChRs) function as allosteric pentamers of identical or homologous transmembrane spanning subunits. Ligand binding at two or more of the five intersubunit sites, located radially in the extracellular domain, drives a conformational change that results in the opening of a centrosymmetric transmembrane channel, internally constructed among the five subunits (SI Appendix, Fig. S2A) (1–4). Up to five potential agonist-competitive antagonist sites on the pentamer are found at the outer perimeter of the subunit interfaces. Amino acid side-chain determinants on both subunit interfaces dictate selectivity among the many subtypes of nAChRs. The interconversion between resting, active, and desensitized states occurs in the absence of ligands, and partial occupation of the binding sites suffices for agonist activation of the receptor and its antagonism (5–7). Cooperativity of agonist association and its coupling to channel gating likely play important roles in the dynamics of nicotinic responses and in sharpening the concentration and temporal windows for activation.As revealed in functional studies, most nAChRs are hetero-oligomeric, where the sites of ligand occupation are not identical (1–4). This arrangement arises when a common α-subunit pairs with one or more nonidentical subunit partners, termed non–α-subunits (7, 8). Nonidentity of the subunit interface complementary to the α-subunit may also give rise to heterogeneity in binding constants typically seen for antagonists and mask partially the degree of agonist cooperativity. An exception to this is the α7-neuronal nAChR composed of five identical subunits and exhibiting a high degree of cooperativity for agonist activation (9). Recently, sequence alignments identified genes coding for pentameric ligand-gated ion channels in prokaryotes led to the resolution of the first structure by X-ray crystallography on 3D crystals of a pentameric receptor protein from Erminia chrysanthemi (ELIC) (10) and Gloeobacter violaceus (GLIC) (11, 12) and provided high-resolution structures of the two end point states of the cooperative gating mechanism in the same pentameric ligand-gated ion channel (GLIC) (13). Recently, the first structure of a eukaryotic member of the family, the anionic glutamate receptor from Caenorhabditis elegans (GluCl), was solved at atomic resolution (14), revealing remarkable identity of 3D structure with GLIC.The acetylcholine binding protein (AChBP) was characterized from mollusks (15–17) and consists of only a homologous extracellular domain of the nAChR. Assembled as a homomeric pentamer, AChBP exhibits a similar profile of ligand selectivity toward the classical nicotinic agonists and antagonists of quaternary amine, tertiary and secondary amine (alkaloid), imine, and peptide origin that bind nicotinic receptors (18–25). If looked at solely on the basis of ligand-binding capacities, AChBP could be considered as a distinct subtype of nAChR. Although its homomeric composition and ligand selectivity best resemble the α7-subtype of nAChR, when the concentration dependence of ligand occupation has been examined, no evidence of cooperativity emerged (21). Accordingly the cooperative behavior for both activation and desensitization of receptors, seen for the classical nicotinic agonists with nAChRs, might arise from a cooperative torsional motion driven by the transmembrane spanning domain of the receptor (26).We demonstrate here a set of ligands that bind to the AChBP in a cooperative fashion, whereby binding to a single subunit affects the binding energy at identical interfaces in the pentamer. Hence, interactions within the extracellular domain of this family of homologous pentameric proteins establish a circumferential linkage between subunit interfaces which results in cooperative behavior.     

Bilateral polymicrogyria associated with dystonia: A new neurogenetic syndrome?

The clinical presentation of bilateral perisylvian polymicrogyria (PMG) is highly variable, including oromotor dysfunction, epilepsy, intellectual disability, and pyramidal signs. Extrapyramidal features are extremely rare. We present four apparently unrelated patients with a unique association of PMG with dystonia. The clinical, genetic, and radiologic features are described and possible mechanisms of dystonia are discussed. All patients were female and two were born to consanguineous families. All presented with early childhood onset dystonia. Other neurologic symptoms and signs classically seen in bilateral perisylvian PMG were observed, including oromotor dysfunction and speech abnormalities ranging from dysarthria to anarthria (4/4), pyramidal signs (3/4), hypotonia (3/4), postnatal microcephaly (1/4), and seizures (1/4). Neuroimaging showed a unique pattern of bilateral PMG with an infolded cortex originating primarily from the perisylvian region in three out of four patients. Whole exome sequencing was performed in two out of four patients and did not reveal pathogenic variants in known genes for cortical malformations or movement disorders. The dystonia seen in our patients is not described in bilateral PMG and suggests an underlying mechanism of impaired connectivity within the motor network or compromised cortical inhibition. The association of bilateral PMG with dystonia in our patients may represent a new neurogenetic disorder.     

Characterization and antibacterial properties of N-halamine-derivatized cross-linked polymethacrylamide nanoparticles

N-halamine-derivatized cross-linked polymethacrylamide nanoparticles with sizes ranging between 18 ± 2.0 and 460 ± 60 nm were prepared via surfactant-free dispersion co-polymerization of methacrylamide (MAA) and the cross-linking monomer N,N-methylenebisacrylamide (MBAA) in an aqueous continuous phase, followed by a chlorination process using sodium hypochlorite. The effect of various polymerization parameters (monomer concentration, initiator type and concentration, polymerization duration, polymerization temperature, and the weight ratio [MBAA]/[MAA]) on the size and size distribution of the produced cross-linked P(MAA–MBAA) nanoparticles was elucidated. The effect of various chlorination parameters (hypochlorite concentration, chlorination period and temperature) on the bound oxidative chlorine atom (Cl) content of the P(MAA–MBAA) nanoparticles was also investigated. The bactericidal activity of these chloramine-derivatized nanoparticles was tested against two common bacterial pathogens (Escherichia coli and Staphylococcus aureus), and they were found to be highly potent. Furthermore, these nanoparticles also exerted their antimicrobial activity against multi-drug resistant (MDR) bacteria, further demonstrating their efficacy.     

Hippocampus-specific deficiency in RNA editing of GluA2 in Alzheimer's disease

Adenosine to inosine (A-to-I) RNA editing is a base recoding process within precursor messenger RNA, catalyzed by members of the adenosine deaminase acting on RNA (ADAR) family. A notable example occurs at the Q/R site of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid glutamate receptor subunit GluA2. Abnormally, low editing at this site leads to excessive calcium influx and cell death. We studied hippocampus and caudate samples from Alzheimer's disease (AD) patients and age-matched healthy controls, using direct sequencing and a high accuracy primer-extension technique to assess RNA editing at the Q/R GluA2 site. Both techniques revealed lower, more variable RNA editing in AD, specific to the hippocampus and the GluA2 site. Deficient editing also characterized the hippocampus of apolipoprotein ε4 allele carriers, regardless of clinical diagnosis. In AD, messenger RNA expression of neuronal markers was decreased in the hippocampus, and expression of the Q/R-site editing enzyme ADAR2 was decreased in caudate. These findings provide a link between neurodegenerative processes and deficient RNA editing of the GluA2 Q/R site, and may contribute to both diagnosis and treatment of AD.     

Prexasertib (LY2606368) reduces clonogenic survival by inducing apoptosis in primary patient-derived osteosarcoma cells and synergizes with cisplatin and talazoparib

Progress in the systemic control of osteosarcoma has been limited over the past decades thus indicating the urgent clinical need for the development of novel treatment strategies. Therefore, we have recently developed new preclinical models to study promising novel agents for the treatment of pediatric osteosarcoma. The checkpoint kinase (chk) inhibitor prexasertib (LY2606368) and its salt form (LSN2940930) have recently been shown to be active in adult and pediatric malignancies, including sarcoma. We have now tested the potency of prexasertib in clonogenic survival assays in two new lines of primary patient-derived osteosarcoma cells and in two established osteosarcoma cell lines as a single agent and in combination with cisplatin and the poly ADP-ribose polymerase (PARP) inhibitor talazoparib. Prexasertib alone results in strongly reduced clonogenic survival at low nanomolar concentrations and acts by affecting cell cycle progression, induction of apoptosis and induction of double-stranded DNA breakage at concentrations that are well below clinically tolerable and safe plasma concentrations. In combination with cisplatin and talazoparib, prexasertib acts in a synergistic fashion. Chk1 inhibition by prexasertib and its combination with the DNA damaging agent cisplatin and the PARP-inhibitor talazoparib thus emerges as a potential new treatment option for pediatric osteosarcoma which will now have to be tested in preclinical primary patient derived in vivo models and clinical studies.     

Heat Source Modeling and Residual Stress Analysis for Metal Directed Energy Deposition Additive Manufacturing

The advancement in additive manufacturing encourages the development of simplified tools for deep and swift research of the technology. Several approaches were developed to reduce the complexity of multi-track modeling for additive manufacturing. In the present work, a simple heat source model called concentrated heat source was evaluated for single- and multi-track deposition for directed energy deposition. The concentrated heat source model was compared with the widely accepted Goldak heat source model. The concentrated heat source does not require melt pool dimension measurement for thermal model simulation. Thus, it reduces the considerable time for preprocessing. The shape of the melt pool and temperature contour around the heat source was analyzed for single-track deposition. A good agreement was noticed for the concentrated heat source model melt pool, with an experimentally determined melt pool, using an optical microscope. Two heat source models were applied to multi-track 3D solid structure thermo-mechanical simulation. The results of the two models, for thermal history and residual stress, were compared with experimentally determined data. A good agreement was found for both models. The concentrated heat source model reported less than the half the computational time required for the Goldak model. The validated model, for 3D solid structure thermo-mechanical simulation, was used to analyze thermal stress evolution during the deposition process. The material deposition on the base plate at room temperature results in lower peak temperatures in the layers near the base plate. Consequently, the higher thermal stress in the layers near the base plate was found, compared to the upper layers during the deposition process.