Advancing Heteroanionicity in Zintl Phases: Crystal Structures, Thermoelectric and Magnetic Properties of Two Quaternary Semiconducting Arsenide Oxides, Eu8Zn2As6O and Eu14Zn5As12O.

Two novel quaternary oxyarsenides, Eu8Zn2As6O and Eu14Zn5As12O, were synthesized through metal flux reactions, and their crystal structures were established by single-crystal X-ray diffraction methods. Eu8Zn2As6O crystallizes in the orthorhombic space group Pbca, featuring polyanionic ribbons composed of corner-shared triangular [ZnAs3] units, running along the [100] direction. The structure of Eu14Zn5As12O crystallizes in the monoclinic space group P2/m and its anionic substructure can be described as an infinite "ribbonlike" chain comprised of [ZnAs3] trigonal-planar units, although the structural complexity here is greater and also amplified by disorder on multiple crystallographic positions. In both structures, the O2- anion occupies an octahedral void with six neighboring Eu2+ cations. Formal electron counting, electronic structure calculations, and transport properties reveal the charge-balanced semiconducting nature of these heteroanionic Zintl phases. High-temperature thermoelectric transport properties measurements on Eu14Zn5As12O reveal relatively high resistivity (ρ500K = 8 Ω·cm) and Seebeck coefficient values (S500K = 220 µV K-1), along with a low concentration and mobility of holes as the dominant charge-carriers (n500K = 8.0 × 1017 cm-3, µ500K = 6.4 cm2/V s). Magnetic studies indicate the presence of divalent Eu2+ species in Eu14Zn5As12O and complex magnetic ordering, with two transitions observed at T1 = 21.6 K and T2 = 9 K.

Novel ternary Zintl phosphide halides Ba3P5X (X = Cl, Br) with 1D helical phosphorus chains: synthesis, crystal and electronic structure.

Black single crystals of two novel ternary phosphide halides, Ba3P5Cl and Ba3P5Br, were grown using molten metal Pb-flux high-temperature reactions. These compounds were structurally characterized with the aid of the single-crystal X-ray diffraction (SCXRD) method at 100(2) K. The SCXRD shows that both compounds are isostructural and adopt a new structure type (space group R3̄c, No. 167, Z = 6) with unit cell parameters a = 14.9481(16) Å, c = 7.3954(11) Å and a = 15.045(4) Å, c = 7.537(3) Å for Ba3P5Cl and Ba3P5Br, respectively. Cl- and Br- anions are octahedrally coordinated by Ba2+ cations, thus composing a face-sharing 1D infinite chain 1∞[XBa3]5+ running along the [001] direction. Moreover, the crystal structures feature peculiar one-dimensional disordered infinite helical chains of 1∞P-, composed of partially occupied phosphorous atoms, each being a superposition of three symmetrical copies of the ordered phosphorus chain, with continuity along the c-axis. Ba3P5X (X = Cl, Br) compounds are charge-balanced heteroanionic Zintl phases according to the charge-partitioning scheme (Ba2+)3[P-]5X-. The presumed semiconducting behavior of both compounds corroborates well with the results of the electronic structure calculations performed with the aid of the TB-LMTO-ASA code.

Halogen, chalcogen, and hydrogen bonding in organoiodine cocrystals of heterocyclic thiones: imidazolidine-2-thione, 2-mercaptobenzimidazole, 2-mercapto-5-methylbenzimidazole, 2-mercaptobenzoxazole, and 2-mercaptobenzothiazole.

Through the combination of heterocyclic thiones with variation in the identity of the heterocyclic elements, namely, imidazolidine-2-thione, 2-mercaptobenzimidazole, 2-mercapto-5-methylbenzimidazole, 2-mercaptobenzoxazole, and 2-mercaptobenzothiazole with the common halogen-bond donors 1,2-, 1,3-, and 1,4-diiodotetrafluorobenzene, 1,3,5-trifluorotriiodobenzene, and tetraiodoethylene, a series of 18 new crystalline structures were characterized. In most cases, N-H...S hydrogen bonding was observed, with these interactions in imidazole-containing structures typically resulting in two-dimensional motifs (i.e. ribbons). Lacking the second N-H group, the thiazole and oxazole hydrogen bonding resulted in only dimeric pairs. C-I...S and C-I...I halogen bonding, as well as C=S...I chalcogen bonding, served to consolidate the packing by linking the hydrogen-bonding ribbons or dimeric pairs.

Clonal Decomposition and DNA Replication States Defined by Scaled Single-Cell Genome Sequencing.

Accurate measurement of clonal genotypes, mutational processes, and replication states from individual tumor-cell genomes will facilitate improved understanding of tumor evolution. We have developed DLP+, a scalable single-cell whole-genome sequencing platform implemented using commodity instruments, image-based object recognition, and open source computational methods. Using DLP+, we have generated a resource of 51,926 single-cell genomes and matched cell images from diverse cell types including cell lines, xenografts, and diagnostic samples with limited material. From this resource we have defined variation in mitotic mis-segregation rates across tissue types and genotypes. Analysis of matched genomic and image measurements revealed correlations between cellular morphology and genome ploidy states. Aggregation of cells sharing copy number profiles allowed for calculation of single-nucleotide resolution clonal genotypes and inference of clonal phylogenies and avoided the limitations of bulk deconvolution. Finally, joint analysis over the above features defined clone-specific chromosomal aneuploidy in polyclonal populations.

Cysteine transport through excitatory amino acid transporter 3 (EAAT3).

Excitatory amino acid transporters (EAATs) limit glutamatergic signaling and maintain extracellular glutamate concentrations below neurotoxic levels. Of the five known EAAT isoforms (EAATs 1-5), only the neuronal isoform, EAAT3 (EAAC1), can efficiently transport the uncharged amino acid L-cysteine. EAAT3-mediated cysteine transport has been proposed to be a primary mechanism used by neurons to obtain cysteine for the synthesis of glutathione, a key molecule in preventing oxidative stress and neuronal toxicity. The molecular mechanisms underlying the selective transport of cysteine by EAAT3 have not been elucidated. Here we propose that the transport of cysteine through EAAT3 requires formation of the thiolate form of cysteine in the binding site. Using Xenopus oocytes and HEK293 cells expressing EAAT2 and EAAT3, we assessed the transport kinetics of different substrates and measured transporter-associated currents electrophysiologically. Our results show that L-selenocysteine, a cysteine analog that forms a negatively-charged selenolate ion at physiological pH, is efficiently transported by EAATs 1-3 and has a much higher apparent affinity for transport when compared to cysteine. Using a membrane tethered GFP variant to monitor intracellular pH changes associated with transport activity, we observed that transport of either L-glutamate or L-selenocysteine by EAAT3 decreased intracellular pH, whereas transport of cysteine resulted in cytoplasmic alkalinization. No change in pH was observed when cysteine was applied to cells expressing EAAT2, which displays negligible transport of cysteine. Under conditions that favor release of intracellular substrates through EAAT3 we observed release of labeled intracellular glutamate but did not detect cysteine release. Our results support a model whereby cysteine transport through EAAT3 is facilitated through cysteine de-protonation and that once inside, the thiolate is rapidly re-protonated. Moreover, these findings suggest that cysteine transport is predominantly unidirectional and that reverse transport does not contribute to depletion of intracellular cysteine pools.

Amphetamine modulates excitatory neurotransmission through endocytosis of the glutamate transporter EAAT3 in dopamine neurons.

Amphetamines modify the brain and alter behavior through mechanisms generally attributed to their ability to regulate extracellular dopamine concentrations. However, the actions of amphetamine are also linked to adaptations in glutamatergic signaling. We report here that when amphetamine enters dopamine neurons through the dopamine transporter, it stimulates endocytosis of an excitatory amino acid transporter, EAAT3, in dopamine neurons. Consistent with this decrease in surface EAAT3, amphetamine potentiates excitatory synaptic responses in dopamine neurons. We also show that the process of internalization is dynamin- and Rho-mediated and requires a unique sequence in the cytosolic C terminus of EAAT3. Introduction of a peptide based on this motif into dopamine neurons blocks the effects of amphetamine on EAAT3 internalization and its action on excitatory responses. These data indicate that the internalization of EAAT3 triggered by amphetamine increases glutamatergic signaling and thus contributes to the effects of amphetamine on neurotransmission.

A sensitive membrane-targeted biosensor for monitoring changes in intracellular chloride in neuronal processes.

BACKGROUND: Regulation of chloride gradients is a major mechanism by which excitability is regulated in neurons. Disruption of these gradients is implicated in various diseases, including cystic fibrosis, neuropathic pain and epilepsy. Relatively few studies have addressed chloride regulation in neuronal processes because probes capable of detecting changes in small compartments over a physiological range are limited. METHODOLOGY/PRINCIPAL FINDINGS: In this study, a palmitoylation sequence was added to a variant of the yellow fluorescent protein previously described as a sensitive chloride indicator (YFPQS) to target the protein to the plasma membrane (mbYFPQS) of cultured midbrain neurons. The reporter partitions to the cytoplasmic face of the cellular membranes, including the plasma membrane throughout the neurons and fluorescence is stable over 30-40 min of repeated excitation showing less than 10% decrease in mbYFPQS fluorescence compared to baseline. The mbYFPQS has similar chloride sensitivity (k(50) =  41 mM) but has a shifted pKa compared to the unpalmitoylated YFPQS variant (cytYFPQS) that remains in the cytoplasm when expressed in midbrain neurons. Changes in mbYFPQS fluorescence were induced by the GABA(A) agonist muscimol and were similar in the soma and processes of the midbrain neurons. Amphetamine also increased mbYFPQS fluorescence in a subpopulation of cultured midbrain neurons that was reversed by the selective dopamine transporter (DAT) inhibitor, GBR12909, indicating that mbYFPQS is sensitive enough to detect endogenous DAT activity in midbrain dopamine (DA) neurons. CONCLUSIONS/SIGNIFICANCE: The mbYFPQS biosensor is a sensitive tool to study modulation of intracellular chloride levels in neuronal processes and is particularly advantageous for simultaneous whole-cell patch clamp and live-cell imaging experiments.

Large collective motions regulate the functional properties of glutamate transporter trimers.

Glutamate transporters clear synaptically released glutamate to maintain precise communication between neurons and limit glutamate neurotoxicity. Although much progress has been made on the topology, structure, and function of these carriers, few studies have addressed large-scale structural motions collectively associated with substrate transport. Here we show that a series of single cysteine substitutions in the helical hairpin HP2 of excitatory amino acid transporter 1 form intersubunit disulfide cross-links within the trimer. After cross-linking, substrate uptake, but not substrate-activated anion conductance, is completely inhibited in these mutants. These disulfide bridges link residue pairs > 40 Å apart in the outward-facing crystal structure, and can be explained by concerted subunit movements predicted by the anisotropic network model (ANM). The existence of these global motions is further supported by the observation that single cysteine substitutions at the extracellular part of the transmembrane domain 8 can also be cross-linked by copper phenanthroline as predicted by the ANM. Interestingly, the transport domain in the un-cross-linked subunit of the trimer assumes an inward-facing orientation, suggesting that individual subunits potentially undergo separate transitions between outward- and inward-facing forms, rather than an all-or-none transition of the three subunits, a mechanism also supported by ANM-predicted intrinsic dynamics. These results shed light on how large collective motions contribute to the functional dynamics of glutamate transporters.

Enhancing glutamate transport: mechanism of action of Parawixin1, a neuroprotective compound from Parawixia bistriata spider venom.

Previous studies have shown that a compound purified from the spider Parawixia bistriata venom stimulates the activity of glial glutamate transporters and can protect retinal tissue from ischemic damage. To understand the mechanism by which this compound enhances transport, we examined its effects on the functional properties of glutamate transporters after solubilization and reconstitution in liposomes and in transfected COS-7 cells. Here, we demonstrate in both systems that Parawixin1 promotes a direct and selective enhancement of glutamate influx by the EAAT2 transporter subtype through a mechanism that does not alter the apparent affinities for the cosubstrates glutamate or sodium. In liposomes, we observed maximal enhancement by Parawixin1 when extracellular sodium and intracellular potassium concentrations are within physiological ranges. Moreover, the compound does not enhance the reverse transport of glutamate under ionic conditions that favor efflux, when extracellular potassium is elevated and the sodium gradient is reduced, nor does it alter the exchange of glutamate in the absence of internal potassium. These observations suggest that Parawixin1 facilitates the reorientation of the potassium-bound transporter, the rate-limiting step in the transport cycle, a conclusion further supported by experiments showing that Parawixin1 does not stimulate uptake by an EAAT2 transport mutant (E405D) defective in the potassium-dependent reorientation step. Thus, Parawixin1 enhances transport through a novel mechanism targeting a step in the transport cycle distinct from substrate influx or efflux and provides a basis for the design of new drugs that act allosterically on transporters to increase glutamate clearance.

Structural rearrangements at the translocation pore of the human glutamate transporter, EAAT1.

Structure-function studies of mammalian and bacterial excitatory amino acid transporters (EAATs), as well as the crystal structure of a related archaeal glutamate transporter, support a model in which TM7, TM8, and the re-entrant loops HP1 and HP2 participate in forming a substrate translocation pathway within each subunit of a trimer. However, the transport mechanism, including precise binding sites for substrates and co-transported ions and changes in the tertiary structure underlying transport, is still not known. In this study, we used chemical cross-linking of introduced cysteine pairs in a cysteine-less version of EAAT1 to examine the dynamics of key domains associated with the translocation pore. Here we show that cysteine substitution at Ala-395, Ala-367, and Ala-440 results in functional single and double cysteine transporters and that in the absence of glutamate or dl-threo-beta-benzyloxyaspartate (dl-TBOA), A395C in the highly conserved TM7 can be cross-linked to A367C in HP1 and to A440C in HP2. The formation of these disulfide bonds is reversible and occurs intra-molecularly. Interestingly, cross-linking A395C to A367C appears to abolish transport, whereas cross-linking A395C to A440C lowers the affinities for glutamate and dl-TBOA but does not change the maximal transport rate. Additionally, glutamate and dl-TBOA binding prevent cross-linking in both double cysteine transporters, whereas sodium binding facilitates cross-linking in the A395C/A367C transporter. These data provide evidence that within each subunit of EAAT1, Ala-395 in TM7 resides close to a residue at the tip of each re-entrant loop (HP1 and HP2) and that these residues are repositioned relative to one another at different steps in the transport cycle. Such behavior likely reflects rearrangements in the tertiary structure of the translocation pore during transport and thus provides constraints for modeling the structural dynamics associated with transport.