Evolution of allostery in the cyclic nucleotide binding module

Analysis of cyclic nucleotide binding (CNB) domains shows that they have evolved to sense a wide variety of second messenger signals; a mechanism for allosteric regulation by CNB domains is proposed

Synergistic Allostery in Multiligand-Protein Interactions

Amide hydrogen-deuterium exchange mass spectrometry is powerful for describing combinatorial coupling effects of a cooperative ligand pair binding at noncontiguous sites: adenosine at the ATP-pocket and a docking peptide (PIFtide) at the PIF-pocket, on a model protein kinase PDK1. Binding of two ligands to PDK1 reveal multiple hotspots of synergistic allostery with cumulative effects greater than the sum of individual effects mediated by each ligand. We quantified this synergism and ranked these hotspots using a difference in deuteration-based approach, which showed that the strongest synergistic effects were observed at three of the critical catalytic loci of kinases: the αB-αC helices, and HRD-motif loop, and DFG-motif. Additionally, we observed weaker synergistic effects at a distal GHI-subdomain locus. Synergistic changes in deuterium exchange observed at a distal site but not at the intermediate sites of the large lobe of the kinase reveals allosteric propagation in proteins to operate through two modes. Direct electrostatic interactions between polar and charged amino acids that mediate targeted relay of allosteric signals, and diffused relay of allosteric signals through soft matter-like hydrophobic core amino acids. Furthermore, we provide evidence that the conserved β-3 strand lysine of protein kinases (Lys111 of PDK1) functions as an integrator node to coordinate allosteric coupling of the two ligand-binding sites. It maintains indirect interactions with the ATP-pocket and mediates a critical salt bridge with a glutamate (Glu130) of αC helix, which is conserved across all kinases. In summary, allosteric propagation in cooperative, dual-liganded enzyme targets is bidirectional and synergistic and offers a strategy for combinatorial drug development.Fil: Ghode, Abhijeet. National University Of Singapore; SingapurFil: Gross, Lissy Zoe Florens. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigación en Biomedicina de Buenos Aires - Instituto Partner de la Sociedad Max Planck; ArgentinaFil: Tee, Wei Ven. National University Of Singapore; SingapurFil: Guarnera, Enrico. Agency For Science, Technology And Research; SingapurFil: Berezovsky, Igor N.. Agency For Science, Technology And Research; SingapurFil: Biondi, Ricardo Miguel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigación en Biomedicina de Buenos Aires - Instituto Partner de la Sociedad Max Planck; ArgentinaFil: Anand, Ganesh S.. National University Of Singapore; Singapu

Ethyl 4-(2-fur­yl)-2-oxochroman-3-carboxyl­ate

The title compound, C16H14O5, was prepared from the reaction of 3-carbethoxy­coumarin with furan in the presence of AlCl3 as catalyst. In the crystal, inter­molecular C—H⋯O hydrogen-bonding inter­actions between four mol­ecules lead to a tetra­mer in the unit cell. The furan ring is anti­periplanar [C—C—C—O = 167.9 (13)°] and the ethoxy­carbonyl group is (−)anti­clinal [C—C—C—O = −128.6 (14)°] to the lactone ring

Ethyl gallate isolated from phenol-enriched fraction of Caesalpinia mimosoides Lam. Promotes cutaneous wound healing: a scientific validation through bioassay-guided fractionation

The tender shoots of Caesalpinia mimosoides Lam. are used ethnomedically by the traditional healers of Uttara Kannada district, Karnataka (India) for the treatment of wounds. The current study was aimed at exploring phenol-enriched fraction (PEF) of crude ethanol extract of tender shoots to isolate and characterize the most active bio-constituent through bioassay-guided fractionation procedure. The successive fractionation and sub-fractionation of PEF, followed by in vitro scratch wound, antimicrobial, and antioxidant activities, yielded a highly active natural antioxidant compound ethyl gallate (EG). In vitro wound healing potentiality of EG was evidenced by a significantly higher percentage of cell migration in L929 fibroblast cells (97.98 ± 0.46% at 3.81 μg/ml concentration) compared to a positive control group (98.44 ± 0.36%) at the 48th hour of incubation. A significantly higher rate of wound contraction (98.72 ± 0.41%), an elevated tensile strength of the incised wound (1,154.60 ± 1.42 g/mm2), and increased quantity of connective tissue elements were observed in the granulation tissues of the 1% EG ointment treated animal group on the 15th post-wounding day. The accelerated wound healing activity of 1% EG was also exhibited by histopathological examinations through Hematoxylin and Eosin, Masson’s trichome, and Toluidine blue-stained sections. Significant up-regulation of enzymatic and non-enzymatic antioxidant contents (reduced glutathione, superoxide dismutase, and catalase) and down-regulation of oxidative stress marker (lipid peroxidation) clearly indicates the effective granular antioxidant activity of 1% EG in preventing oxidative damage to the skin tissues. Further, in vitro antimicrobial and antioxidant activities of EG supports the positive correlation with its enhanced wound-healing activity. Moreover, molecular docking and dynamics for 100 ns revealed the stable binding of EG with cyclooxygenase-2 (−6.2 kcal/mol) and matrix metalloproteinase-9 (−4.6 kcal/mol) and unstable binding with tumor necrosis factor-α (−7.2 kcal/mol), suggesting the potential applicability of EG in inflammation and wound treatment

Central nervous system immune interactome is a function of cancer lineage, tumor microenvironment, and STAT3 expression.

BACKGROUNDImmune cell profiling of primary and metastatic CNS tumors has been focused on the tumor, not the tumor microenvironment (TME), or has been analyzed via biopsies.METHODSEn bloc resections of gliomas (n = 10) and lung metastases (n = 10) were analyzed via tissue segmentation and high-dimension Opal 7-color multiplex imaging. Single-cell RNA analyses were used to infer immune cell functionality.RESULTSWithin gliomas, T cells were localized in the infiltrating edge and perivascular space of tumors, while residing mostly in the stroma of metastatic tumors. CD163+ macrophages were evident throughout the TME of metastatic tumors, whereas in gliomas, CD68+, CD11c+CD68+, and CD11c+CD68+CD163+ cell subtypes were commonly observed. In lung metastases, T cells interacted with CD163+ macrophages as dyads and clusters at the brain-tumor interface and within the tumor itself and as clusters within the necrotic core. In contrast, gliomas typically lacked dyad and cluster interactions, except for T cell CD68+ cell dyads within the tumor. Analysis of transcriptomic data in glioblastomas revealed that innate immune cells expressed both proinflammatory and immunosuppressive gene signatures.CONCLUSIONOur results show that immunosuppressive macrophages are abundant within the TME and that the immune cell interactome between cancer lineages is distinct. Further, these data provide information for evaluating the role of different immune cell populations in brain tumor growth and therapeutic responses.FUNDINGThis study was supported by the NIH (NS120547), a Developmental research project award (P50CA221747), ReMission Alliance, institutional funding from Northwestern University and the Lurie Comprehensive Cancer Center, and gifts from the Mosky family and Perry McKay. Performed in the Flow Cytometry & Cellular Imaging Core Facility at MD Anderson Cancer Center, this study received support in part from the NIH (CA016672) and the National Cancer Institute (NCI) Research Specialist award 1 (R50 CA243707). Additional support was provided by CCSG Bioinformatics Shared Resource 5 (P30 CA046592), a gift from Agilent Technologies, a Research Scholar Grant from the American Cancer Society (RSG-16-005-01), a Precision Health Investigator Award from University of Michigan (U-M) Precision Health, the NCI (R37-CA214955), startup institutional research funds from U-M, and a Biomedical Informatics & Data Science Training Grant (T32GM141746)

Eef1a2 Promotes Cell Growth, Inhibits Apoptosis and Activates JAK/STAT and AKT Signaling in Mouse Plasmacytomas

The canonical function of EEF1A2, normally expressed only in muscle, brain, and heart, is in translational elongation, but recent studies suggest a non-canonical function as a proto-oncogene that is overexpressed in a variety of solid tumors including breast and ovary. Transcriptional profiling of a spectrum of primary mouse B cell lineage neoplasms showed that transcripts encoding EEF1A2 were uniquely overexpressed in plasmacytomas (PCT), tumors of mature plasma cells. Cases of human multiple myeloma expressed significantly higher levels of EEF1A2 transcripts than normal bone marrow plasma cells. High-level expression was also a feature of a subset of cell lines developed from mouse PCT and from the human MM.Heightened expression of EEF1A2 was not associated with increased copy number or coding sequence mutations. shRNA-mediated knockdown of Eef1a2 transcripts and protein was associated with growth inhibition due to delayed G1-S progression, and effects on apoptosis that were seen only under serum-starved conditions. Transcriptional profiles and western blot analyses of knockdown cells revealed impaired JAK/STAT and PI3K/AKT signaling suggesting their contributions to EEF1A2-mediated effects on PCT induction or progression.EEF1A2 may play contribute to the induction or progression of some PCT and a small percentage of MM. Eef1a2 could also prove to be a useful new marker for a subset of MM and, ultimately, a possible target for therapy

Prolonged Normothermic Ex Vivo Kidney Perfusion Is Superior to Cold Nonoxygenated and Oxygenated Machine Perfusion for the Preservation of DCD Porcine Kidney Grafts

The increased usage of marginal grafts has triggered interest in perfused kidney preservation to minimize graft injury. We used a donation after circulatory death (DCD) porcine kidney autotransplantation model to compare 3 of the most frequently used ex vivo kidney perfusion techniques: nonoxygenated hypothermic machine perfusion (non-oxHMP), oxygenated hypothermic machine perfusion (oxHMP), and normothermic ex vivo kidney perfusion (NEVKP). Methods: Following 30 min of warm ischemia, grafts were retrieved and preserved with either 16 h of non-oxHMP, oxHMP, or NEVKP (n = 5 per group). After contralateral nephrectomy, grafts were autotransplanted and animals were followed for 8 d. Kidney function and injury markers were compared between groups. Results: NEVKP demonstrated a significant reduction in preservation injury compared with either cold preservation method. Grafts preserved by NEVKP showed superior function with lower peak serum creatinine (NEVKP versus non-oxHMP versus oxHMP: 3.66 ± 1.33 mg/dL, 8.82 ± 3.17 mg/dL, and 9.02 ± 5.5 mg/dL) and more rapid recovery. The NEVKP group demonstrated significantly increased creatinine clearance on postoperative day 3 compared with the cold perfused groups. Tubular injury scores on postoperative day 8 were similar in all groups. Conclusions: Addition of oxygen during HMP did not reduce preservation injury of DCD kidney grafts. Grafts preserved with prolonged NEVKP demonstrated superior initial graft function compared with grafts preserved with non-oxHMP or oxHMP in a model of pig DCD kidney transplantation

Recommendations for performing, interpreting and reporting hydrogen deuterium exchange mass spectrometry (HDX-MS) experiments.

Hydrogen deuterium exchange mass spectrometry (HDX-MS) is a powerful biophysical technique being increasingly applied to a wide variety of problems. As the HDX-MS community continues to grow, adoption of best practices in data collection, analysis, presentation and interpretation will greatly enhance the accessibility of this technique to nonspecialists. Here we provide recommendations arising from community discussions emerging out of the first International Conference on Hydrogen-Exchange Mass Spectrometry (IC-HDX; 2017). It is meant to represent both a consensus viewpoint and an opportunity to stimulate further additions and refinements as the field advances