Distinct Affinities Research Articles

Mild and severe cereal yellow dwarf viruses differ in silencing suppressor efficiency of the P0 protein

Viral pathogenicity has often been correlated to the expression of the viral encoded-RNA silencing suppressor protein (SSP). The silencing suppressor activity of the P0 protein encoded by cereal yellow dwarf virus-RPV (CYDV-RPV) and -RPS (CYDV-RPS), two poleroviruses differing in their symptomatology was investigated. CYDV-RPV displays milder symptoms in oat and wheat whereas CYDV-RPS is responsible for more severe disease. We showed that both P0 proteins (P0CY-RPV and P0CY-RPS) were able to suppress local RNA silencing induced by either sense or inverted repeat transgenes in an Agrobacterium tumefaciens-mediated expression assay in Nicotiana benthamiana. P0CY-RPS displayed slightly higher activity. Systemic spread of the silencing signal was not impaired. Analysis of short-interfering RNA (siRNA) abundance revealed that accumulation of primary siRNA was not affected, but secondary siRNA levels were reduced by both CYDV P0 proteins, suggesting that they act downstream of siRNA production. Correlated with this finding we showed that both P0 proteins partially destabilized ARGONAUTE1. Finally both P0CY-RPV and P0CY-RPS interacted in yeast cells with ASK2, a component of an E3-ubiquitin ligase, with distinct affinities.

Carbohydrate-Based Nanocarriers Exhibiting Specific Cell Targeting with Minimum Influence from the Protein Corona.

Whenever nanoparticles encounter biological fluids like blood, proteins adsorb on their surface and form a so-called protein corona. Although its importance is widely accepted, information on the influence of surface functionalization of nanocarriers on the protein corona is still sparse, especially concerning how the functionalization of PEGylated nanocarriers with targeting agents will affect protein corona formation and how the protein corona may in turn influence the targeting effect. Herein, hydroxyethyl starch nanocarriers (HES-NCs) were prepared, PEGylated, and modified on the outer PEG layer with mannose to target dendritic cells (DCs). Their interaction with human plasma was then studied. Low overall protein adsorption with a distinct protein pattern and high specific affinity for DC binding were observed, thus indicating an efficient combination of "stealth" and targeting behavior.

KIR and HLA genotypes predictive of low-affinity interactions are associated with lower relapse in autologous hematopoietic cell transplantation for acute myeloid leukemia.

Killer cell Ig-like receptors (KIRs) bind cognate HLA class I ligands with distinct affinities, affecting NK cell licensing and inhibition. We hypothesized that differences in KIR and HLA class I genotypes predictive of varying degrees of receptor-ligand binding affinities influence clinical outcomes in autologous hematopoietic cell transplantation (AHCT) for acute myeloid leukemia (AML). Using genomic DNA from a homogeneous cohort of 125 AML patients treated with AHCT, we performed KIR and HLA class I genotyping and found that patients with a compound KIR3DL1(+) and HLA-Bw4-80Thr(+), HLA-Bw4-80Ile(-) genotype, predictive of low-affinity interactions, had a low incidence of relapse, compared with patients with a KIR3DL1(+) and HLA-Bw4-80Ile(+) genotype, predictive of high-affinity interactions (hazard ratio [HR], 0.22; 95% confidence interval [CI], 0.06-0.78; p = 0.02). This effect was influenced by HLA-Bw4 copy number, such that relapse progressively increased with one copy of HLA-Bw4-80Ile (HR, 1.6; 95% CI, 0.84-3.1; p = 0.15) to two to three copies (HR, 3.0; 95% CI, 1.4-6.5; p = 0.005) and progressively decreased with one to two copies of HLA-Bw4-80Thr (p = 0.13). Among KIR3DL1(+) and HLA-Bw4-80Ile(+) patients, a predicted low-affinity KIR2DL2/3(+) and HLA-C1/C1 genotype was associated with lower relapse than a predicted high-affinity KIR2DL1(+) and HLA-C2/C2 genotype (HR, 0.25; 95% CI, 0.09-0.73; p = 0.01). Similarly, a KIR3DL1(+) and HLA-Bw4-80Thr(+), HLA-Bw4-80Ile(-) genotype, or lack of KIR3DL1(+) and HLA-Bw4-80Ile(+) genotype, rescued KIR2DL1(+) and HLA-C2/C2 patients from high relapse (p = 0.007). These findings support a role for NK cell graft-versus-leukemia activity modulated by NK cell receptor-ligand affinities in AHCT for AML.

Development of regulatory T cells in the thymus: two paths to the same goal? (HEM2P.237)

Abstract Mature thymic regulatory T cells (Tregs) are defined by dual expression of Foxp3 and CD25 on CD4SP thymocytes. However, the cellular pathways by which CD4 thymocytes differentiate into Tregs remain incompletely understood. Previous reports demonstrated that Treg development occurred via a two-step process. In step one, strong TCR signaling induces expression of CD25 leading to a CD25+Foxp3- Treg progenitor (TRP). In step two, exposure of this TRP to IL2 results in conversion to a CD25+Foxp3+ Treg. Recently, work by Singer and colleagues have defined a CD25-Foxp3lo TRP that can also be converted by IL2 into CD25+Foxp3+ Tregs. A key question is why two distinct cellular pathways involving distinct TRP populations are needed to reach the same endpoint. Using a combination of maturation marker analysis and RAG2-GFP reporter mice we observed that CD25+Foxp3- TRP arise more rapidly than the CD25-Foxp3lo TRP following positive selection. Analysis of Nur77-GFP reporter mice revealed that these two progenitor populations also display distinct affinity spectrums for self-peptide. The CD25+Foxp3- TRP have a high and relatively narrow range of TCR affinities for antigen, while the CD25-Foxp3lo TRP have a lower but broader range of TCR affinity for antigen. Our results suggest that these two different Treg developmental pathways generate Tregs with distinct TCR repertoires that may have distinct function in protecting against autoimmunity and reactivity to commensal organisms.

Molecular Mechanism of Flocculation Self-Recognition in Yeast and Its Role in Mating and Survival

ABSTRACTWe studied the flocculation mechanism at the molecular level by determining the atomic structures of N-Flo1p and N-Lg-Flo1p in complex with their ligands. We show that they have similar ligand binding mechanisms but distinct carbohydrate specificities and affinities, which are determined by the compactness of the binding site. We characterized the glycans of Flo1p and their role in this binding process and demonstrate that glycan-glycan interactions significantly contribute to the cell-cell adhesion mechanism. Therefore, the extended flocculation mechanism is based on the self-interaction of Flo proteins and this interaction is established in two stages, involving both glycan-glycan and protein-glycan interactions. The crucial role of calcium in both types of interaction was demonstrated: Ca2+ takes part in the binding of the carbohydrate to the protein, and the glycans aggregate only in the presence of Ca2+. These results unify the generally accepted lectin hypothesis with the historically first-proposed “Ca2+-bridge” hypothesis. Additionally, a new role of cell flocculation is demonstrated; i.e., flocculation is linked to cell conjugation and mating, and survival chances consequently increase significantly by spore formation and by introduction of genetic variability. The role of Flo1p in mating was demonstrated by showing that mating efficiency is increased when cells flocculate and by differential transcriptome analysis of flocculating versus nonflocculating cells in a low-shear environment (microgravity). The results show that a multicellular clump (floc) provides a uniquely organized multicellular ultrastructure that provides a suitable microenvironment to induce and perform cell conjugation and mating.

Parameters influencing ciprofloxacin, ofloxacin, amoxicillin and sulfamethoxazole retention by natural and converted calcium phosphates

The retention of four antibiotics, ciprofloxacin, ofloxacin, amoxicillin and sulfamethoxazole by a natural phosphate rock (francolite) was studied and compared with a converted hydroxyapatite powder. The maximum sorption capacities were found to correlate with the molecular weight of the molecules. The mechanisms of sorption depended mostly on the charge of the antibiotic whereas the kinetics of the process was sensitive to their hydrophobic/hydrophilic character. The two materials showed slightly distinct affinities for the various antibiotics but exhibited similar maximum sorption capacities despite different specific surface areas. This was mainly attributed to the more pronounced hydrophobic character of the francolite phase constituting the natural phosphate. These data enlighten that the retention properties of these mineral phases depend on a complex interplay between the inter-molecular and molecule-solid interactions. These findings are relevant to understand better the contribution of calcium phosphates in the fate and retention of antibiotics in soils.

Adsorption of Cu(II) on humic acids derived from different organic materials

The adsorption of Cu(II) from aqueous solution onto humic acid (HA) which was isolated from cattle manure (CHA), peat (PHA), and leaf litter (LHA) as a function of contact time, pH, ion strength, and initial concentration was studied using the batch method. X-ray absorption spectroscopy (XAS) was used to examine the coordination environment of the Cu(II) adsorbed by HA at a molecular level. Moreover, the chemical compositions of the isolated HA were characterized by elemental analysis and solid-state 13C nuclear magnetic resonance spectroscopy (NMR). The kinetic data showed that the adsorption equilibrium can be achieved within 8 h. The adsorption kinetics followed the pseudo-second-order equation. The adsorption isotherms could be well fitted by the Langmuir model, and the maximum adsorption capacities of Cu(II) on CHA, PHA, and LHA were 229.4, 210.4, and 197.7 mg g−1, respectively. The adsorption of Cu(II) on HA increased with the increase in pH from 2 to 7, and maintained a high level at pH>7. The adsorption of Cu(II) was also strongly influenced by the low ionic strength of 0.01 to 0.2 mol L−1 NaNO3, but was weakly influenced by high ionic strength of 0.4 to 1 mol L−1 NaNO3. The Cu(II) adsorption on HA may be mainly attributed to ion exchange and surface complexation. XAS results revealed that the binding site and oxidation state of Cu adsorbed on HA surface did not change at the initial Cu(II) concentrations of 15 to 40 mg L−1. For all the Cu(II) adsorption samples, each Cu atom was surrounded by 4 O/N atoms at a bond distance of 1.95 Å in the first coordination shell. The presence of the higher Cu coordination shells proved that Cu(II) was adsorbed via an inner-sphere covalent bond onto the HA surface. Among the three HA samples, the adsorption capacity and affinity of CHA for Cu(II) was the greatest, followed by that of PHA and LHA. All the three HA samples exhibited similar types of elemental and functional groups, but different contents of elemental and functional groups. CHA contained larger proportions of methoxyl C, phenolic C and carbonyl C, and smaller proportions of alkyl C and carbohydrate C than PHA and LHA. The structural differences of the three HA samples are responsible for their distinct adsorption capacity and affinity toward Cu(II). These results are important to achieve better understanding of the behavior of Cu(II) in soil and water bodies in the presence of organic materials.

Understanding Selectivity of the Na+/K+ -ATPase using a Computational Approach

The Na+/K+-ATPase (NKA) is a membrane protein that transports Na+ ions out of the cell and K+ ions into it against their concentration gradient. The high selectivity of NKA for Na+ or K+ can be explained by distinct affinities of the two ionic species for the binding sites, by different kinetic barriers encountered to reach them, or by both. Understanding the exact location of binding sites is the first step in determining the origin of selectivity in NKA. We used molecular dynamics simulations and free energy calculations to study ion binding. We show that the exact location of the binding sites, the thermodynamic affinity of the two ions and the cooperativity of the binding depend crucially on the protonation state of the binding residues. Importantly, this protonation state changes when the NKA shifts between the sodium and potassium occluded states.

Aberrant expression of the embryonic transcription factor brachyury in human tumors detected with a novel rabbit monoclonal antibody.

The embryonic transcription factor brachyury is overexpressed in a variety of human tumors, including lung, breast, colon and prostate carcinomas, chordomas and hemangioblastomas. In human carcinoma cells, overexpression of brachyury associates with the occurrence of the phenomenon of epithelial-mesenchymal transition (EMT), acquisition of metastatic propensity and resistance to a variety of anti-cancer therapeutics. Brachyury is preferentially expressed in human tumors vs. normal adult tissues, and high levels of this molecule associate with poor prognosis in patients with lung, colon and prostate carcinomas, and in breast cancer patients treated with adjuvant tamoxifen. Brachyury is immunogenic in humans and vaccines against this novel oncotarget are currently undergoing clinical investigation. While our group and others have employed various anti-brachyury antibodies to interrogate the above findings, we report here on the development and thorough characterization of a novel rabbit monoclonal antibody (MAb 54-1) that reacts with distinct high affinity and specificity with human brachyury. MAb 54-1 was successfully used in ELISA, western blot, immunofluorescence and immunohistochemistry assays to evaluate expression of brachyury in various human tumor cell lines and tissues. We propose the use of this antibody to assist in research studies of EMT and in prognostic studies for a range of human tumors.

C-Reactive Protein: An In-Depth Look into Structure, Function, and Regulation

Cardiovascular disease is the leading cause of morbidity and mortality in the adult population worldwide, with atherosclerosis being its key pathophysiologic component. Atherosclerosis possesses a fundamental chronic inflammatory aspect, and the involvement of numerous inflammatory molecules has been studied in this scenario, particularly C-reactive protein (CRP). CRP is a plasma protein with strong phylogenetic conservation and high resistance to proteolysis, predominantly synthesized in the liver in response to proinflammatory cytokines, especially IL-6, IL-1β, and TNF. CRP may intervene in atherosclerosis by directly activating the complement system and inducing apoptosis, vascular cell activation, monocyte recruitment, lipid accumulation, and thrombosis, among other actions. Moreover, CRP can dissociate in peripheral tissue—including atheromatous plaques—from its native pentameric form into a monomeric form, which may also be synthesized de novo in extrahepatic sites. Each form exhibits distinct affinities for ligands and receptors, and exerts different effects in the progression of atherosclerosis. In view of epidemiologic evidence associating high CRP levels with cardiovascular risk—reflecting the biologic impact it bears on atherosclerosis—measurement of serum levels of high-sensitivity CRP has been proposed as a tool for assessment of cardiovascular risk.

Upper Cretaceous (Maastrichtian) Charophyte Gyrogonites from the Lameta Formation of Jabalpur, Central India: Palaeobiogeographic and Palaeoecological Implications

Abstract A charophyte gyrogonite assemblage consisting of Platychara cf. sahnii, Nemegtichara grambastii and Microchara sp. is reported herein from two localities (Bara Simla Hill and Chui Hill sections) of the Lameta Formation at Jabalpur. he Lameta Formation locally underlying the Deccan traps has been shown to be pedogenically modified alluvial plain deposits containing one of the most extensive dinosaur nesting sites in the world. They are associated with dinosaur bones and freshwater ostracod assemblages that suggest a Late Cretaceous (Maastrichtian) age. This is the first detailed systematic account of charophyte gyrogonites from the Lameta Formation. This charophyte assemblage is compatible with the biostratigraphic attribution provided by the ostracods. From a biogeographic viewpoint, it exhibits considerable similarity to other infratrappean assemblages of the Nand, Dongargaon, and Dhamni-Pavna sections (Maharashtra), and some intertrappean assemblages of Kora in Gujarat, Rangapur in Andhra Pradesh and Gurmatkal in South India. Globally, the genus Microchara is well distributed throughout Eurasia, whereas the genus Platychara occurs richly in the Upper Cretaceous deposits of Europe, Asia, America and Africa. However, at the specific level, Platychara cf. sahnii shows close affinities with charophytes from the Maastrichtian of Iran whilst Nemegtichara grambastii shows distinct affinities with two species of Early Palaeogene deposits of China and Mongolia. The presence of charophyte gyrogonites in the Lameta sediments is attributed to local lacustrine and palustrine conditions within a flood plain environment.

Molecular Dynamics Simulation: The Behavior of Asphaltene in Crude Oil and at the Oil/Water Interface

Carboxyl asphaltene is commonly discussed in the petroleum industry. In most conditions, electroneutral carboxyl asphaltene molecules can be deprotonated to become carboxylate asphaltenes. Both in crude oil and at the oil/water interface, the characteristics of anionic carboxylate asphaltenes are different than those of the carboxyl asphaltenes. In this paper, molecular dynamics (MD) simulations are utilized to study the structural features of different asphaltene molecules, namely, C5 Pe and anionic C5 Pe, at the molecular level. In crude oil, the electroneutral C5 Pe molecules prefer to form a steady face-to-face stacking, while the anionic C5 Pe molecules are inclined to form face-to-face stacking and T-shaped II stacking because of the repulsion of the anionic headgroups. Anionic C5 Pe has a distinct affinity to the oil/water interface during the simulation, while the C5 Pe molecules persist in the crude oil domain. A three-stage model of anionic C5 Pe molecules adsorbed at the oil/water interface is finally developed.

OR25: DYSREGULATION OF INNATE IMMUNE RESPONSES DUE TO PROMISCOUS PEPTIDE REPERTOIRE OF HLA-E∗01:01

Over the last years, HLA-E has gained attention as an intermediate molecule for the innate and adaptive immune responses. Structural analysis of pHLA-E/NKG2 or pHLA-E/TCR complexes visualized fine-tuning of immune responses through single AA interaction between pHLA-E and its cognate receptor. Hence, it becomes important to understand the impact of subtle peptide differences especially in the context of viral infections. While HLA-E was thought to monitor class I expression, little is known about its peptide repertoire and mode of peptide recruitment. Innate immune response represented by NK cells is dysregulated during cellular stress or viral infections. We recently showed that certain repertoire of self peptides were selected and presented and these pHLA-E molecules presented ligands of the innate immune response. Cellular and biochemical assays showed the balance between single peptides bound to HLA-E∗01:01 and their distinct affinity to NKG2A/C receptor complexes. In this regard, we used soluble HLA technology to obtain HLA-E∗01:01 bound self-peptides, sequenced by mass-spectrometry. Peptides obtained from healthy cells or cells mimicking CMV infection showed different lengths, sequences and immune responses. Following peptide synthesis, empty HLA-E∗01:01 molecules on transduced T2 cells were loaded with the distinct peptides, IC50 values were determined and cytotoxicity assays were performed using CD56+ or CD8+ effector cells. CD94 and NKG2C or NKG2A were recombinantly expressed, heterogenic CD94/NKG2 complexes purified and binding affinities to pHLA-E complexes were determined. In a pseudo viral infection system, where Tapasin is non-functional, we detected promiscuity of peptides, that in cellular assays showed no binding to NKG2C or NKG2A, but interactions and cytotoxic effects with T cells. The balance shift from innate to adaptive immunity was very distinct and suggests a post transplant role of HLA-E as well as its specialized function in anti-viral-immunity.

Forestry impacts on the hidden fungal biodiversity associated with bryophytes.

Recent studies have revealed an unexpectedly high, cryptic diversity of fungi associated with boreal forest bryophytes. Forestry practices heavily influence the boreal forest and fundamentally transform the landscape. However, little is known about how bryophyte-associated fungal communities are affected by these large-scale habitat transformations. This study assesses to what degree bryophyte-associated fungal communities are structured across the forest successional stages created by current forestry practices. Shoots of Hylocomium splendens were collected in Picea abies dominated forests of different ages, and their associated fungal communities were surveyed by pyrosequencing of ITS2 amplicons. Although community richness, diversity and evenness were relatively stable across the forest types and all were consistently dominated by ascomycete taxa, there was a marked shift in fungal community composition between young and old forests. Numerous fungal operational taxonomic units (OTUs) showed distinct affinities for different forest ages. Spatial structure was also detected among the sites, suggesting that environmental gradients resulting from the topography of the study area and dispersal limitations may also significantly affect bryophyte-associated fungal community structure. This study confirms that Hylocomium splendens hosts an immense diversity of fungi and demonstrates that this community is structured in part by forest age, and as such is highly influenced by modern forestry practices.

InlC of L. monocytogenes Binds Human Tuba for Bacterial Cell-Cell Spreading

The human pathogen Listeria monocytogenes is able to directly spread to neighboring cells of host tissues, a process recently linked to the virulence factor InlC. InlC targets the sixth SH3 domain (SH3-6) of human Tuba, disrupting its physiological interaction with the cytoskeletal protein N-WASP. The resulting loss of cortical actin tension proposedly slackens the junctional membrane allowing protrusion formation by motile Listeria. Complexes of Tuba SH3-6 with physiological partners N-WASP and Mena reveal equivalent binding modes but distinct affinities. The interaction surface of the infection complex InlC/Tuba SH3-6 is centered on phenylalanine146 of InlC stacking upon asparagine1569 of Tuba. Replacing Phe146 by alanine largely abrogates molecular affinity and in vivo mimics deletion of inlC. Collectively, our findings indicate that InlC hijacks Tuba through its LRR domain, blocking the peptide binding groove to prevent recruitment of its physiological partners.

Marine communities on oil platforms in Gabon, West Africa: high biodiversity oases in a low biodiversity environment.

The marine biodiversity of Gabon, West Africa has not been well studied and is largely unknown. Our examination of marine communities associated with oil platforms in Gabon is the first scientific investigation of these structures and highlights the unique ecosystems associated with them. A number of species previously unknown to Gabonese waters were recorded during our surveys on these platforms. Clear distinctions in benthic communities were observed between older, larger platforms in the north and newer platforms to the south or closer to shore. The former were dominated by a solitary cup coral, Tubastraea sp., whereas the latter were dominated by the barnacle Megabalanus tintinnabulum, but with more diverse benthic assemblages compared to the northerly platforms. Previous work documented the presence of limited zooxanthellated scleractinian corals on natural rocky substrate in Gabon but none were recorded on platforms. Total estimated fish biomass on these platforms exceeded one ton at some locations and was dominated by barracuda (Sphyraena spp.), jacks (Carangids), and rainbow runner (Elagatis bipinnulata). Thirty-four percent of fish species observed on these platforms are new records for Gabon and 6% are new to tropical West Africa. Fish assemblages closely associated with platforms had distinct amphi-Atlantic affinities and platforms likely extend the distribution of these species into coastal West Africa. At least one potential invasive species, the snowflake coral (Carijoa riisei), was observed on the platforms. Oil platforms may act as stepping stones, increasing regional biodiversity and production but they may also be vectors for invasive species. Gabon is a world leader in terrestrial conservation with a network of protected areas covering >10% of the country. Oil exploration and biodiversity conservation currently co-exist in terrestrial and freshwater ecosystems in Gabon. Efforts to increase marine protection in Gabon may benefit by including oil platforms in the marine protected area design process.

Heterogeneity in the inter-tumor transcriptome of high risk prostate cancer.

BackgroundGenomic analyses of hundreds of prostate tumors have defined a diverse landscape of mutations and genome rearrangements, but the transcriptomic effect of this complexity is less well understood, particularly at the individual tumor level. We selected a cohort of 25 high-risk prostate tumors, representing the lethal phenotype, and applied deep RNA-sequencing and matched whole genome sequencing, followed by detailed molecular characterization.ResultsTen tumors were exposed to neo-adjuvant hormone therapy and expressed marked evidence of therapy response in all except one extreme case, which demonstrated early resistance via apparent neuroendocrine transdifferentiation. We observe high inter-tumor heterogeneity, including unique sets of outlier transcripts in each tumor. Interestingly, outlier expression converged on druggable cellular pathways associated with cell cycle progression, translational control or immune regulation, suggesting distinct contemporary pathway affinity and a mechanism of tumor stratification. We characterize hundreds of novel fusion transcripts, including a high frequency of ETS fusions associated with complex genome rearrangements and the disruption of tumor suppressors. Remarkably, several tumors express unique but potentially-oncogenic non-ETS fusions, which may contribute to the phenotype of individual tumors, and have significance for disease progression. Finally, one ETS-negative tumor has a striking tandem duplication genotype which appears to be highly aggressive and present at low recurrence in ETS-negative prostate cancer, suggestive of a novel molecular subtype.ConclusionsThe multitude of rare genomic and transcriptomic events detected in a high-risk tumor cohort offer novel opportunities for personalized oncology and their convergence on key pathways and functions has broad implications for precision medicine.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-014-0426-y) contains supplementary material, which is available to authorized users.

Sedimentary and faunal signatures of the post-glacial marine drowning of the Pontocaspian Gemlik “lake” (Sea of Marmara)

A Late Pleistocene (Neoeuxinian) Pontocaspian fauna is documented from sediments cored at̴ ∼75 m present water depth in the shallow-silled (∼50 m) Gulf of Gemlik, Sea of Marmara. As determined by AMS-14C dating of Pontocaspian gastropods (15,860 ± 90 BP uncal.), the age of such fauna is consistent with known episodes of Caspian overflow into the Black Sea Basin (Early Hvalynian floods). The fossil mollusc fauna is relatively diverse (12 non-marine taxa), made up of neritid and hydrobioid gastropods, and dreissenid and lymnocardiine bivalves. The presence of hitherto poorly known Pontocaspian taxa in the Sea of Marmara, further documented by the record in Gulf of Gemlik, seems to document that the Neoeuxinian Sea extended south of the Bosporus in the latest Pleistocene. Further, they provide insight into the origin of the modern Black Sea Pontocaspian taxa. The Gemlik Neoeuxinian lacustrine-brackish fauna under scrutiny is the westernmost known expansion of the Pontocaspian domain during the Late Pleistocene. This distinct biogeographic faunal affinity, with all its eastbound implications in terms of landscape and seaway connections, came to an end with the abrupt marine inundation that accompanied the postglacial sea-level rise from the Mediterranean Sea.

Functional insight into development of positive allosteric modulators of AMPA receptors

Positive allosteric modulators of α-amino-3-hydroxy-5-methyl-isoxazole-propionic acid (AMPA) ionotropic glutamate receptors facilitate synaptic plasticity and contribute essentially to learning and memory, properties which make AMPA receptors targets for drug discovery and development. One region at which several different classes of positive allosteric modulators bind lies at the dimer interface between the ligand-binding core of the second, membrane-proximal, extracellular domain of AMPA receptors. This solvent-accessible binding pocket has been the target of drug discovery efforts, leading to the recent delineation of five “subsites” which differentially allow access to modulator moieties, and for which distinct modulator affinities and apparent efficacies are attributed. Here we use the voltage-clamp technique in conjunction with rapid drug application to study the effects of mutants lining subsites “A” and “B” of the allosteric modulator pocket to assess affinity and efficacy of allosteric modulation by cyclothiazide, CX614, CMPDA and CMPDB. A novel analysis of the decay of current produced by the onset of desensitization has allowed us to estimate both affinity and efficacy from single concentrations of modulator. Such an approach may be useful for effective high throughput screening of new target compounds.

Aqueous self-assembly and cation selectivity of cobaltabisdicarbollide dianionic dumbbells.

The anion [3,3'-Co(C2B9H11)2](-) ([COSAN](-)) produces aggregates in water. These aggregates are interpreted to be the result of C-H⋅⋅⋅H-B interactions. It is possible to generate aggregates even after the incorporation of additional functional groups into the [COSAN](-) units. The approach is to join two [COSAN](-) anions by a linker that can adapt itself to act as a crown ether. The linker has been chosen to have six oxygen atoms, which is the ideal number for K(+) selectivity in crown ethers. The linker binds the alkaline metal ions with different affinities; thus showing a distinct degree of selectivity. The highest affinity is shown towards K(+) from a mixture containing Li(+), Na(+), K(+), Rb(+) and Cs(+); this can be indicative of pseudo-crown ether performance of the dumbbell. One interesting possibility is that the [COSAN](-) anions at the two ends of the linker can act as a hook-and-loop fastener to close the ring. This facet is intriguing and deserves further consideration for possible applications. The distinct affinity towards alkaline metal ions is corroborated by solubility studies and isothermal calorimetry thermograms. Furthermore, cryoTEM micrographs, along with light scattering results, reveal the existence of small self-assemblies and compact nanostructures ranging from spheres to single-/multi-layer vesicles in aqueous solutions. The studies reported herein show that these dumbbells can have different appearances, either as molecules or aggregates, in water or lipophilic phases; this offers a distinct model as drug carriers.