Distinct Affinities Research Articles

Abstract A28: Imaged-based computational predictions of imaging agent efficacy in pancreatic tumors expressing TLR2

Abstract The current therapies continuously fail to provide successful results for pancreatic adenocarcinoma, one of the most deadly cancers with only 6% overall 5-year survival rate. The improvement of techniques for early detection, predicting of treatment efficacy and monitoring of tumor response during and after therapy remain under focused research. By combining computational modeling with intravital fluorescence microscopy we developed a novel method to assess intratumoral and intracellular distribution of imaging agents targeted to pancreatic cancer cells. Our long-standing goal is to provide a tool for optimizing efficacy of targeted agents based on individual patient data. Our team reported previously that, in addition to the important role the toll-like receptor 2 (TLR2) plays in the immune system response, TLR2 is also a bona fide cell-surface marker for targeting pancreatic cancer cells. TLR2 recognizes a vast number of biomolecules, including lipopeptides, such as a novel TLR2 ligand designed in our laboratory (TLR2L). We used intravital fluorescence microscopy methods for the real time in vivo imaging of TLR2L conjugated to near-infrared fluorescent dye, Cyanine 5 (TLR2L-Cy5), and its distribution in the tissue of pancreatic adenocarcinoma tumor xenografts in mice with endogenous expression of TLR2. In order to quantify the space- and time-dependent dynamics of TLR2L we combined intravital dorsal window chamber experiments with computational modeling of TLR2L-Cy5 diffusion and internalization following its intravenous administration. Our computational model is calibrated to the tissue histology of a particular tumor and accounts for explicitly defined individual tumor cells and expression of cell membrane receptors, as well as the extracellular matrix interpenetrated by the interstitial fluid, and tumor vasculature. The fluorescent imaging agent is also modeled as individual discrete molecules that extravasate via influx from blood capillaries, move through the tumor and become internalized by the cells. Our microscopic level computational model allowed for quantitative assessment of targeted imaging agent extravasation, interstitial diffusion and intracellular accumulation on a cell-to-cell basis. When accounting for various values of diffusion and binding affinity, we identified a cross-dependence between these physical characteristics of targeted agents. Our computational results revealed that agents of distinct affinities can reach similar efficacies of binding and internalization. Moreover, we identified the optimized dose-response protocols appropriate for any targeted agent with a priori known physical and biochemical properties. Finally, when considering different extravasation schemes of the agent we also showed that the time of crossing the capillary wall by the ligand plays an important role in effective binding and saturation of cell receptors and intracellular distribution. In addition to improvement in drug delivery schemes, our model predicts which biochemical and physical properties of targeted agents could be tuned for the maximum effect in patient-specific extracellular matrix environments, tumor topologies and receptor expression levels. We present an interdisciplinary approach to quantify diffusion and cellular uptake of an imaging agent targeted to pancreatic cancer cell lines expressing the TLR2 receptor. This integrated method can be used for optimizing the administration schedules and time points for data collection from individual human tumor xenografts in order to personalize the treatment and improve its efficacy. In the future, the model can be extended other targeted imaging or therapeutic agents, to other solid tumors, and to an individual patient's tumor histology. Citation Format: Aleksandra Karolak, Veronica Estrella, Tingan Chen, Amanda Huynh, David Morse, Katarzyna Rejniak. Imaged-based computational predictions of imaging agent efficacy in pancreatic tumors expressing TLR2. [abstract]. In: Proceedings of the AACR Special Conference on Engineering and Physical Sciences in Oncology; 2016 Jun 25-28; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2017;77(2 Suppl):Abstract nr A28.

Occurrence, reproductive rate and identification of the non-native Noble false widow spider Steatoda nobilis (Thorell, 1875) in Ireland

The noble false widow Steatoda nobilis (Thorell, 1875) has established thriving populations in urban centres throughout England and Wales since it was accidentally imported from the Canary Islands and Madeira to Britain over a century ago. In recent years, Steatoda nobilis has colonised parts of Western Europe, California, Chile and the Middle East. In Ireland, Steatoda nobilis was first recorded in 1999 from a single location in Co Wicklow. The present study examines the current range and main habitats of Steatoda nobilis in Ireland and assesses its potential as an invasive species by documenting its reproductive rate. Additionally, we present photographic material illustrating the intraspecific phenotypic variations exhibited by Steatoda nobilis to assist in correct identification of this species by the public. Our data shows that Steatoda nobilis is an extremely prolific, resilient species with distinct synanthropic affinities. This species currently occurs in at least sixteen Irish counties with the largest populations observed in the greater Dublin area, where it has become widespread in buildings and on street furniture. Steatoda nobilis seems to be currently absent from natural, undisturbed habitats such as woodlands, bogs and grassland. We suggest that due to its comparatively fast reproductive rate, long life span and year-round activity, Steatoda nobilis might have a detrimental impact on native urban-dwelling spiders.

Sigma-2 receptor and progesterone receptor membrane component 1 (PGRMC1) are two different proteins: Proofs by fluorescent labeling and binding of sigma-2 receptor ligands to PGRMC1

A controversial relationship between sigma-2 and progesterone receptor membrane component 1 (PGRMC1) proteins, both representing promising targets for the therapy and diagnosis of tumors, exists since 2011, when the sigma-2 receptor was reported to be identical to PGRMC1. Because a misidentification of these proteins will lead to biased future research hampering the possible diagnostic and therapeutic exploitation of the two targets, there is the need to solve the debate on their identity. With this aim, we have herein investigated uptake and distribution of structurally different fluorescent sigma-2 receptor ligands by flow cytometry and confocal microscopy in MCF7 cells, where together with intrinsic sigma-2 receptors, PGRMC1 was constitutively present or alternatively silenced or overexpressed. HCT116 cells, with constitutive or silenced PGRMC1, were also studied. These experiments showed that the fluorescent sigma-2 ligands bind to their receptor irrespective of PGRMC1 expression. Furthermore, isothermal titration calorimetry was conducted to examine if DTG and PB28, two structurally distinct nanomolar affinity sigma-2 ligands, bind to purified PGRMC1 proteins that have recently been revealed to form both apo-monomeric and heme-mediated dimeric forms. While no binding to apo-PGRMC1 monomer was detected, a micromolar affinity to heme-mediated dimerized PGRMC1 was demonstrated in DTG but not in PB28. The current data provide evidence that sigma-2 receptor and PGRMC1 are not identical, paving the pathway for future unbiased research in which these two attractive targets are treated as different proteins while the identification of the true sigma-2 protein further needs to be pursued.

Tamoxifen Isomers and Metabolites Exhibit Distinct Affinity and Activity at Cannabinoid Receptors: Potential Scaffold for Drug Development.

Tamoxifen (Tam) is a selective estrogen receptor (ER) modulator (SERM) that is an essential drug to treat ER-positive breast cancer. Aside from known actions at ERs, recent studies have suggested that some SERMs like Tam also exhibit novel activity at cannabinoid subtype 1 and 2 receptors (CB1R and CB2Rs). Interestingly, cis- (E-Tam) and trans- (Z-Tam) isomers of Tam exhibit over a 100-fold difference in affinity for ERs. Therefore, the current study assessed individual isomers of Tam and subsequent cytochrome P450 metabolic products, 4-hydroxytamoxifen (4OHT) and 4-hydroxy-N-desmethyl tamoxifen (End) for affinity and activity at CBRs. Results showed that Z-4OHT, but not Z-Tam or Z-End, exhibits higher affinity for both CB1 and CB2Rs relative to the E-isomer. Furthermore, Z- and E-isomers of Tam and 4OHT show slightly higher affinity for CB2Rs, while both End isomers are relatively CB1R-selective. When functional activity was assessed by G-protein activation and regulation of the downstream effector adenylyl cyclase, all isomers examined act as full CB1 and CB2R inverse agonists. Interestingly, Z-Tam appears to be more efficacious than the full inverse agonist AM630 at CB2Rs, while both Z-Tam and Z-End exhibit characteristics of insurmountable antagonism at CB1 and CB2Rs, respectively. Collectively, these results suggest that the SERMs Tam, 4OHT and End elicit ER-independent actions via CBRs in an isomer-specific manner. As such, this novel structural scaffold might be used to develop therapeutically useful drugs for treatment of a variety of diseases mediated via CBRs.

Distinct Immunogenetic Signatures in IgA Versus IgG Multiple Myeloma

Immunogenetic analysis of MM has proven instrumental in elucidating disease ontogeny e.g. by revealing the clonal relationship between switch variants expressed by the bone marrow plasma cells and myeloma progenitors in the marrow and blood; demonstrating the marked under-representation of the inherently autoreactive IGHV4-34 gene; and, identifying patterns of somatic hypermutation (SHM) indicative of post-germinal center derivation. Yet, limited information exists about the composition of the immunoglobulin (IG) gene repertoire in MM cases expressing different heavy chain isotype, in particular A versus G. This is relevant in light of studies showing an overall higher SHM impact in CD27+IgA+ compared to CD27+IgG+ normal memory B cells, perhaps reflecting a distinct location of the immune response, especially considering that IgA class switching mostly occurs in mucosa-associated lymphoid tissues. From a clinical perspective, it is also relevant to note that IgA patients exhibit a higher incidence of the t(4;14) translocation, shorter progression-free survival and worse median overall survival compared to IgG patients. Here, we explored potential differences in the immunoprofiles of IgA versus IgG MM focusing on IG gene repertoire and SHM characteristics. In total, 428 patients with a diagnosis of MM following the IMWG criteria from collaborating institutions in Greece, Italy and Spain (n=355) or retrieved from the LIGM-DB (n=73) were included in the study. Of these, 135 and 293 belonged to IgA and IgG MM groups, respectively. Amongst the evaluated productive IG rearrangements, IGHV3 subgroup genes predominated in both groups (IgA: 58.5%; IgG: 52.2%). However, at the individual gene level, major asymmetries were noted, since only 7 IGHV genes accounted for 41.6% of the IgA and 46.7% of the IgG cases, respectively. Of these, 3 genes were shared between IgA and IgG MM cases: IGHV3-30 (IgA: 11.9% - IgG: 13.3%), IGHV3-23 (IgA: 5.2% - IgG: 6.8%) and IGHV3-9 (IgA: 6.7% - IgG: 4.4%), whereas the remaining 4 of the 7 most frequent genes were specific for each group with significant (p<0.05) differences regarding the IGHV3-7 (5.2% in IgA versus 1.7% in IgG) and IGHV3-21 gene (0.7% in IgA 4.1% in IgG). IGHD3 predominated in both groups (IgA: 37% - IgG: 39.6%) followed by IGHD2 in IgG MM (18.4%) and IGHD6 in IgA MM (20.7%). IGHJ4 and IGHJ6 were the most frequent IGHJ genes with no significant differences in relative frequency. Searching for restricted IGHV-IGHJ combinations, we noted that the IGHV3-9 gene preferentially paired with the IGHJ6 gene in IgA MM versus the IGHJ4 gene in IgG MM (66.7% and 46.2% of all IGHV3-9 rearrangements, respectively). The median complementarity-determining region 3 (CDR3) length was identical in both IgA and IgG MM (15 amino acids, aa), yet differences were identified for specific CDR3 lengths as in the case of 19 aa, concerning 10.4% of all IgA versus 4.8% of all IgG rearrangements (p<0.05). Turning to SHM, the vast majority of rearrangements (IgA: 90.4%, IgG: 85%) were heavily mutated (IGHV germline identity (GI) <95%) with median GI of 91.8% for IgA and 92.2% for IgG. To study the topology of SHM, we compared the ratios of replacement (R) to silent (S) mutations in the framework (FR) and complementarity determining regions (CDRs) in cases expressing common frequent IGHV genes, namely IGHV3-23, IGHV3-30 and IGHV3-9 and identified distinct SHM patterns in all 3 instances: (i) IGHV3-23: the highest R/S ratios in IgA versus IgG MM were observed in FR2 (3.88) and CDR1 (3.9), respectively; (ii) IGHV3-30: overall "normal" SHM topology with higher R/S in CDRs rather than FRs, however, compared to IgG, IgA cases also showed a very high R/S in FR2 (5.3 versus 1.4); and, (iii) IGHV3-9: significantly (p<0.05) higher R/S ratios in CDR1 and CDR2 in IgG versus IgA cases (10.2 and 8.3 versus 1 and 2.9, respectively). Overall, in-depth immunogenetic analysis in the largest to-date series of IgA MM and IgG MM patients reveals differences regarding IGH gene repertoires, CDR3 characteristics and the topology of SHM. These findings suggest distinct antigen exposure histories and/or affinity maturation processes for IgA versus IgG MM, further highlighting the importance of microenvironmental stimuli in disease pathogenesis. DisclosuresTerpos:Celgene: Honoraria; Novartis: Honoraria; Genesis: Consultancy, Honoraria, Other: Travel expenses; BMS: Consultancy, Honoraria; Amgen: Consultancy, Honoraria, Other: Travel expenses, Research Funding; Takeda: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Other: Travel expenses, Research Funding. Stamatopoulos:Gilead: Consultancy, Honoraria, Research Funding; Abbvie: Honoraria, Other: Travel expenses; Novartis: Honoraria, Research Funding; Janssen: Honoraria, Other: Travel expenses, Research Funding.

Differential Effect of Membrane Composition on the Pore-Forming Ability of Four Different Sea Anemone Actinoporins.

Sea anemone actinoporins constitute a protein family of multigene pore-forming toxins (PFT). Equinatoxin II (EqtII), fragaceatoxin C (FraC), and sticholysins I and II (StnI and StnII, respectively), produced by three different sea anemone species, are the only actinoporins whose molecular structures have been studied in depth. These four proteins show high sequence identities and practically coincident three-dimensional structures. However, their pore-forming activity can be quite different depending on the model lipid system employed, a feature that has not been systematically studied before. Therefore, the aim of this work was to evaluate and compare the influence of several distinct membrane conditions on their particular pore-forming behavior. Using a complex model membrane system, such as sheep erythrocytes, StnII showed hemolytic activity much higher than those of the other three actinoporins studied. In lipid model systems, pore-forming ability when assayed against 4:1 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)/sphingomyelin (SM) vesicles, with the membrane binding being the rate-limiting step, decreased in the following order: StnI > StnII > EqtII > FraC. When using 1:1:1 DOPC/SM/cholesterol LUVs, the presence of Chol not only enhanced membrane binding affinities by ∼2 orders of magnitude but also revealed how StnII was much faster than the other three actinoporins in producing calcein release. This ability agrees with the proposal that explains this behavior in terms of their high sequence variability along their first 30 N-terminal residues. The influence of interfacial hydrogen bonding in SM- or dihydro-SM-containing bilayers was also shown to be a generalized feature of the four actinoporins studied. It is finally hypothesized that this observed variable ability could be explained as a consequence of their distinct specificities and/or membrane binding affinities. Eventually, this behavior can be modulated by the nature of their natural target membranes or the interaction with not yet characterized isotoxin forms from the same sea anemone species.

Probing the structure of the uncoupled nicotinic acetylcholine receptor

In the absence of activating anionic lipids and cholesterol, the nicotinic acetylcholine receptor (nAChR) from Torpedo adopts an uncoupled conformation that does not usually gate open in response to agonist. The uncoupled conformation binds both agonists and non-competitive channel blockers with a lower affinity than the desensitized state, consistent with both the extracellular agonist-binding and transmembrane channel-gating domains individually adopting resting-state like conformations. To test this hypothesis, we characterized the binding of the agonist, acetylcholine, and two fluorescent channel blockers, ethidium and crystal violet, to resting, desensitized and uncoupled nAChRs in reconstituted membranes. The measured Kd for acetylcholine binding to the uncoupled nAChR is similar to that for the resting state, confirming that the agonist binding site adopts a resting-state like conformation. Although both ethidium and crystal violet bind to the resting and desensitized channel pores with distinct affinities, no binding of either probe was detected to the uncoupled nAChR. Our data suggest that the transmembrane domain of the uncoupled nAChR adopts a conformation distinct from that of the resting and desensitized states. The lack of binding is consistent with a more constricted channel pore, possibly along the lines of what is observed in crystal structures of the prokaryotic homolog, ELIC.

Novel indole-based compounds that differentiate alkylindole-sensitive receptors from cannabinoid receptors and microtubules: Characterization of their activity on glioma cell migration

Indole-based compounds, such as the alkyl-indole (AI) compound WIN55212-2, activate the cannabinoid receptors, CB1 and CB2, two well-characterized G protein-coupled receptors (GPCR). Reports indicate that several indole-based cannabinoid agonists, including WIN55212-2, lack selectivity and interact with at least two additional targets: AI-sensitive GPCRs and microtubules. Studying how indole-based compounds modulate the activity of these 4 targets has been difficult as selective chemical tools were not available. Here we report the pharmacological characterization of six newly-developed indole-based compounds (ST-11, ST-23, ST-25, ST-29, ST-47 and ST-48) that exhibit distinct binding affinities at AI-sensitive receptors, cannabinoid CB1 and CB2 receptors and the colchicine site of tubulin. Several compounds exhibit some level of selectivity for AI-sensitive receptors, including ST-11 that binds AI-sensitive receptors with a Kd of 52nM and appears to have a weaker affinity for the colchicine site of tubulin (Kd=3.2μM) and does not bind CB1/CB2 receptors. Leveraging these characteristics, we show that activation of AI-sensitive receptors with ST-11 inhibits both the basal and stimulated migration of the Delayed Brain Tumor (DBT) mouse glioma cell line. Our study describes a new series of indole-based compounds that enable the pharmacological and functional differentiation of alkylindole-sensitive receptors from cannabinoid receptors and microtubules.

Influenza A virus nucleoprotein targets subnuclear structures.

The Influenza A virus nucleoprotein (NP) is the major protein component of the genomic viral ribonucleoprotein (vRNP) complexes, which are the replication- and transcription-competent units of Influenza viruses. Early during infection, NP mediates import of vRNPs into the host cell nucleus where viral replication and transcription take place; also newly synthesized NP molecules are targeted into the nucleus, enabling coreplicational assembly of progeny vRNPs. NP reportedly acts as regulatory factor during infection, and it is known to be involved in numerous interactions with host cell proteins. Yet, the NP-host cell interplay is still poorly understood. Here, we report that NP significantly interacts with the nuclear compartment and displays distinct affinities for different subnuclear structures. NP subnuclear behavior was studied by expression of fluorescent NP fusion proteins - including obligate monomeric NP - and site-specific fluorescence photoactivation measurements. We found that NP constructs accumulate in subnuclear domains frequently found adjacent to or overlapping with promyelocytic leukemia bodies and Cajal bodies. Targeting of NP to Cajal bodies could further be demonstrated in the context of virus infection. We hypothesize that by targeting functional nuclear organization, NP might either link viral replication to specific cellular machinery or interfere with host cell processes.

A geotraverse across two paleo-subduction zones in Tien Shan, Tajikistan

We present first LA-ICP-MS U–Pb zircon ages as well as geochemical and Sr–Nd–Pb isotope data for 14 magmatic rocks collected along ca. 400km profile across the Chatkal-Kurama terrane in the Mogol-Tau and Kurama ranges and the Gissar Segment of the Tien Shan orogen in Tajikistan. These new data from supra-subduction and post-collisional magmatic rocks of two Late Paleozoic active margins constrain a tectonic model for terrane motions across two paleo-subduction zones: (1) The 425Ma old Muzbulak granite of the Mogol-Tau range formed in a supra-subduction setting at the northern margin of the Turkestan Ocean. The north-dipping plate was subducted from the Early Silurian to the earliest Middle Devonian. Thereafter the northern side of the Turkestan Ocean remained a passive margin until the Early Carboniferous. (2) In the Early Carboniferous, subduction under the northern margin of the Turkestan Ocean resumed and the 315 to 305Ma old Kara-Kiya, Muzbek, and Karamazar intrusions formed in a supra-subduction setting in the Mogol-Tau and Kurama ranges. (3) At the same time, in the Early Carboniferous, rifting of the southern passive margin of the Turkestan Ocean formed the short-lived Gissar Basin, separated from the Turkestan Ocean by the Gissar micro-continent. North-dipping subduction in the Gissar Basin is documented by the 315Ma Kharangon plagiogranite and the voluminous ca. 321–312Ma Andean-type supra-subduction Gissar batholith. The Kharangon and Khanaka gabbro-plagiogranite intrusions of the southern Gissar range have geochemical and Sr–Nd isotopic compositions (87Sr/86Sr(t) 0.7047–0.7056, εNd of +1.5 to +2.3) compatible with mantle-derived origin typical for plagiogranites associated with ophiolites. The supra-subduction rocks from the Gissar batholith and from the Mogol-Tau Kurama ranges have variably mixed Sr–Nd–Pb isotopic signatures (87Sr/86Sr(t) 0.7057–0.7064, εNd of −2.1 to −5.0) typical for continental arcs where mantle-derived magmas interact with continental crust. (4) In the latest Carboniferous, the Turkestan Ocean and the Gissar Basin were closed. The Early Permian Chinorsay (288Ma) and Dara-i-pioz (267Ma) post-collisional intrusions, emplaced in the northern part of the Gissar micro-continent after a long period of amagmatic evolution, have intraplate geochemical affinities and isotopic Sr–Nd–Pb isotopic compositions (87Sr/86Sr(t) 0.7074–0.7086, εNd of −5.5 to −7.4) indicating derivation from Precambrian continental crust which is supported by old Nd model ages (1.5 and 1.7Ga), and by the presence of inherited zircon grains with ages 850–500Ma in the Chinorsay granodiorite. The post-collisional intrusions in the southern Gissar and in the Mogol-Tau and Kurama ranges (297–286Ma), emplaced directly after supra-subduction magmatic series, have geochemical and isotopic signatures of arc-related magmas. The distinct shoshonitic affinities of post-collisional intrusions in the Mogol-Tau and Kurama ranges are explained by the interaction of hot asthenospheric material with subduction-enriched wedge of lithospheric mantle due to slab break-off at post-collisional stage. Despite origination from different tectonic environments, all magmatic rocks have relatively old Nd model ages (1.7–1.0Ga) indicating a significant proportion of Paleoproterozoic or older crustal material in their sources and their model ages are similar to those of post-collisional intrusions from the Alai and Kokshaal Segments of the South Tien Shan.

The conservation of phosphate-binding residues among PHT1 transporters suggests that distinct transport affinities are unlikely to result from differences in the phosphate-binding site

The plant PHosphate Transporter 1 (PHT1) family of membrane proteins belongs to the major facilitator super family and plays a major role in the acquisition of inorganic phosphate (Pi) from the soil and its transport within the plant. These transporters have been well characterized for expression patterns, localization, and in some cases affinity. Furthermore, the crystal structure of a high-affinity eukaryotic phosphate transporter from the fungus Piriformospora indica (PiPT) has revealed important information on the residues involved in Pi transport. Using multiple-sequence alignments and homology modelling, the phosphate-binding site residues were shown to be well conserved between all the plant PHT1 proteins, Saccharomyces cerevisiae PHO84 and PiPT. For example, Asp 324 in PiPT is conserved in the equivalent position in all plant PHT1 and yeast transporters analyzed, and this residue in ScPHO84 was shown by mutagenesis to be important for both the binding and transport of Pi. Moreover, Asp 45 and Asp 149, which are predicted to be involved in proton import, and Lys 459, which is putatively involved in Pi-binding, are all fully conserved in PHT1 and ScPHO84 transporters. The conserved nature of the residues that play a key role in Pi-binding and transport across the PHT1 family suggests that the differing Pi affinities of these transporters do not reside in differences in the Pi-binding site. Recent studies suggest that phosphate transporters could possess dual affinity and that post-translational modifications may be important in regulating affinity for phosphate.

Effects of substratum on the growth and survivorship of Montipora capitata and Porites lobata transplants

Artificial transplantation of corals is a common method used to restore damaged or unhealthy coral assemblages. Though a number of studies have successfully transplanted coral fragments, there is no general consensus on the type of substratum to be used. The present study focused on the growth and survivorship of Montipora capitata (rice coral) and Porites lobata (lobe coral) fragments, which were transplanted onto different natural and synthetic substrata. No significant differences in coenosarc tissue growth or survivorship were observed between the species. Measurements after 184days of growth, found transplant growth to be significantly higher on rhyolite breccia and amorphous coral skeletons than on black ʻAʻā lava. Nevertheless, no significant differences were observed between any of the other substrata. After 365days of growth, survivorship was also observed to not be significantly different between substrata; with the only exception of being lower on glass substratum. It is hypothesized that success in a coral reef restoration project is largely determined by the actual coral fragmentation and transplantation process; as no distinct substratum affinity was observed for M. capitata and P. lobata transplants.

Absolute binding free energies for octa-acids and guests in SAMPL5 : Evaluating binding free energies for octa-acid and guest complexes in the SAMPL5 blind challenge.

As part of the SAMPL5 blind prediction challenge, we calculate the absolute binding free energies of six guest molecules to an octa-acid (OAH) and to a methylated octa-acid (OAMe). We use the double decoupling method via thermodynamic integration (TI) or Hamiltonian replica exchange in connection with the Bennett acceptance ratio (HREM-BAR). We produce the binding poses either through manual docking or by using GalaxyDock-HG, a docking software developed specifically for this study. The root mean square deviations for our most accurate predictions are 1.4kcalmol-1 for OAH with TI and 1.9kcalmol-1 for OAMe with HREM-BAR. Our best results for OAMe were obtained for systems with ionic concentrations corresponding to the ionic strength of the experimental solution. The most problematic system contains a halogenated guest. Our attempt to model the σ-hole of the bromine using a constrained off-site point charge, does not improve results. We use results from molecular dynamics simulations to argue that the distinct binding affinities of this guest to OAH and OAMe are due to a difference in the flexibility of the host. We believe that the results of this extensive analysis of host-guest complexes will help improve the protocol used in predicting binding affinities for larger systems, such as protein-substrate compounds.

Stereoselective induction of developmental toxicity and immunotoxicity by acetochlor in the early life stage of zebrafish

Acetochlor (ACT) has been frequently detected in the aquatic environment and implicated in disruption of the immune system in fish, the mechanisms of which, especially at enantiomeric levels, remains unclear. In the present study, embryonic zebrafish were exposed to ACT and its enantiomers at concentrations of 0, 2, 8, 15, 30 and 60 μM from 2 h post-fertilization (hpf) to 72 hpf. We demonstrated that ACT and its enantiomers could cause time- and concentration-dependent mortality (72 h LC50 ranged from 48.4 to 53.1 μM) and developmental malformations (e.g., 48 h EC50 for yolk sac edema ranged from 36.7 to 54.1 μM), as well as increase transcription of the key genes involved in the innate immune system. A consistent enantioselectivity in these endpoints was observed with (−)-R-ACT showed stronger effects than (+)-S-ACT, and the transcription levels of il-1β exhibited significant enantioselectivity at concentrations as low as 8 μM. Further Western blot analysis revealed that significant elevations of Il-1β protein expression in all (−)-R-ACT treatment groups. According to the molecular docking and molecular dynamics simulations, the enantioselectivity between ACT enantiomers was attributed to the distinct binding affinity to Il-1β. Overall, our in vivo and in silico studies uniquely disclosed the enantioselective immunotoxicity of ACT and its underlying mechanisms and highlighted the need to evaluate the environmental risk of chiral chloroacetamide herbicide in aquatic organisms at enantiomeric levels.

Bashō In Brazil, or Zen and the Art of Concrete Poetry

By exploring the ubiquity of Bashō's frog haiku in Brazil as the naturalization of a poetics of nature and the ideographic sign, this essay traces distinct affinities between the arts of Zen and concrete poetry, in both theory and practice. It begins by observing the spiritual significance of Zen for the haiku, especially present in the emblematic poetry and figure of Bashō, and proceeds by considering the various interrelations between haiku poetics and concrete poetry, particularly evident in transcreations of Bashō's poem by the Noigandres group of concrete poets. It concludes by positing a direct correspondence between concrete poetry and Zen, aptly represented in the work of Paulo Leminski. Along the way, the essay explores questions regarding the inscription of reality in the natural sign, the transcription (or translation) of experience in a language of words, and the poetic communication of an incommunicable beyond within.

Antiparallel protocadherin homodimers use distinct affinity- and specificity-mediating regions in cadherin repeats 1-4.

Protocadherins (Pcdhs) are cell adhesion and signaling proteins used by neurons to develop and maintain neuronal networks, relying on trans homophilic interactions between their extracellular cadherin (EC) repeat domains. We present the structure of the antiparallel EC1-4 homodimer of human PcdhγB3, a member of the γ subfamily of clustered Pcdhs. Structure and sequence comparisons of α, β, and γ clustered Pcdh isoforms illustrate that subfamilies encode specificity in distinct ways through diversification of loop region structure and composition in EC2 and EC3, which contains isoform-specific conservation of primarily polar residues. In contrast, the EC1/EC4 interface comprises hydrophobic interactions that provide non-selective dimerization affinity. Using sequence coevolution analysis, we found evidence for a similar antiparallel EC1-4 interaction in non-clustered Pcdh families. We thus deduce that the EC1-4 antiparallel homodimer is a general interaction strategy that evolved before the divergence of these distinct protocadherin families.

Petrogenesis and tectonic implications of Triassic mafic complexes with MORB/OIB affinities from the western Garzê-Litang ophiolitic mélange, central Tibetan Plateau

Although numerous Paleo-Tethyan ophiolites with mid-oceanic ridge basalts (MORB) and/or oceanic-island basalt (OIB) affinities have been reported in the central Tibetan Plateau (CTP), the origin and tectonic nature of these ophiolites are not well understood. The petrogenesis, mantle sources and geodynamic setting of the mafic rocks from these ophiolites are unclear, which is the main reason for this uncertainty. In this paper, we present new geochronological, mineralogical and Sr-Nd isotopic data for the Chayong and Xiewu mafic complexes in the western Garzê-Litang suture zone (GLS), a typical Paleo-Tethyan suture crossing the CTP. Zircon LA-ICP-MS U–Pb ages of 234±3Ma and 236±2Ma can be interpreted as formation times of the Chayong and Xiewu mafic complexes, respectively. The basalts and gabbros of the Chayong complex exhibit enriched MORB (E-MORB) compositional affinities except for a weak depletion of Nb, Ta and Ti relative to the primitive mantle, whereas the basalts and gabbros of the Xiewu complex display distinct E-MORB and OIB affinities. The geochemical features suggest a probable fractionation of olivine ± clinopyroxene ± plagioclase as well as insignificant crustal contamination. The geochemical and Sr-Nd isotopic data reveal that the Chayong mafic rocks may have been derived from depleted MORB-type mantle metasomatized by crustal components and Xiewu mafic rocks from enriched lithospheric mantle metasomatized by OIB-like components. The ratios of Zn/Fet, La/Yb and Sm/Yb indicate that these mafic melts were produced by the partial melting of garnet + minor spinel-bearing peridotite or spinel ± minor garnet-bearing peridotite. We propose that back-arc basin spreading associated with OIB/seamount recycling had occurred in the western GLS at least since the Middle Triassic times, and the decompression melting of the depleted MORB-type asthenosphere mantle and partial melting of sub-continental lithosphere were metasomatized by plume-related melts, such as OIB s, which led to the generation of the Chayong and Xiewu mafic melts.

Changes in Healthy Human IgG Fc-Glycosylation after Birth and during Early Childhood.

Glycosylation on the fragment crystallizable (Fc) region of immunoglobulin G (IgG) has a large influence on the interaction of the antibody with Fc gamma receptors (FcγRs). IgG consists of four subclasses that all have distinct affinities for the different FcγRs. Knowledge about the Fc-glycosylation in healthy human is valuable as reference for new biomarkers and in the design of biopharmaceuticals that rely on IgG Fc-glycosylation. Previously, subclass-specific characterization of IgG Fc-glycosylation was performed for healthy adults, pregnant women, and newborns. For young healthy children, however, the subclass-specific description of IgG Fc-glycosylation is still lacking. Therefore, we performed the IgG subclass-specific analysis of the Fc-glycosylation of 130 healthy humans between birth and 40 years of age, including 22 samples derived from the umbilical cords of newborns. The analysis was performed by a previously published matrix-assisted laser desorption/ionization (MALDI)-time-of-flight (TOF)-mass spectrometry (MS) workflow, including a derivatization step for the linkage-specific stabilization of sialic acids. The characterization revealed that when children start to produce their own IgG they have a decreased galactosylation, sialylation, and bisection and an increased fucosylation compared with newborns. During childhood, the fucosylation and sialylation decrease, whereas bisection increases and galactosylation stays constant.

Development of regulatory T cells in the thymus - two paths to the same goal?

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. Further, TCR sequencing has provided evidence that these 2 progenitor populations contain largely distinct TCR repertoires, suggesting these different TRP populations may be required to provide tolerance to different antigens. Indeed, our preliminary data suggests that these TRPs home to different locations upon adoptive transfer. Our results suggests a model whereby cells are driven by high affinity or high avidity interactions with self-peptide through two discrete pathways to form a comprehensive Treg repertoire capable of protecting against autoimmunity and reactivity to commensal organisms.

A Multivalent Marine Lectin from Crenomytilus grayanus Possesses Anti-cancer Activity through Recognizing Globotriose Gb3.

In this study, we report the structure and function of a lectin from the sea mollusk Crenomytilus grayanus collected from the sublittoral zone of Peter the Great Bay of the Sea of Japan. The crystal structure of C. grayanus lectin (CGL) was solved to a resolution of 1.08 Å, revealing a β-trefoil fold that dimerizes into a dumbbell-shaped quaternary structure. Analysis of the crystal CGL structures bound to galactose, galactosamine, and globotriose Gb3 indicated that each CGL can bind three ligands through a carbohydrate-binding motif involving an extensive histidine- and water-mediated hydrogen bond network. CGL binding to Gb3 is further enhanced by additional side-chain-mediated hydrogen bonds in each of the three ligand-binding sites. NMR titrations revealed that the three binding sites have distinct microscopic affinities toward galactose and galactosamine. Cell viability assays showed that CGL recognizes Gb3 on the surface of breast cancer cells, leading to cell death. Our findings suggest the use of this lectin in cancer diagnosis and treatment.