Toll-like Receptor 3-mediated Necrosis via TRIF,RIP3, and MLKL

Toll-like receptor (TLR) signaling is triggered by pathogen-associated molecular patterns that mediate well established cytokine-driven pathways, activating NF-κB together with IRF3/IRF7. In addition, TLR3 drives caspase 8-regulated programmed cell death pathways reminiscent of TNF family death receptor signaling. We find that inhibition or elimination of caspase 8 during stimulation of TLR2, TLR3, TLR4, TLR5, or TLR9 results in receptor interacting protein (RIP) 3 kinase-dependent programmed necrosis that occurs through either TIR domain-containing adapter-inducing interferon-β (TRIF) or MyD88 signal transduction. TLR3 or TLR4 directly activates programmed necrosis through a RIP homotypic interaction motif-dependent association of TRIF with RIP3 kinase (also called RIPK3). In fibroblasts, this pathway proceeds independent of RIP1 or its kinase activity, but it remains dependent on mixed lineage kinase domain-like protein (MLKL) downstream of RIP3 kinase. Here, we describe two small molecule RIP3 kinase inhibitors and employ them to demonstrate the common requirement for RIP3 kinase in programmed necrosis induced by RIP1-RIP3, DAI-RIP3, and TRIF-RIP3 complexes. Cell fate decisions following TLR signaling parallel death receptor signaling and rely on caspase 8 to suppress RIP3-dependent programmed necrosis whether initiated directly by a TRIF-RIP3-MLKL pathway or indirectly via TNF activation and the RIP1-RIP3-MLKL necroptosis pathway.     

Single-Molecule characterization of oligomerization kinetics and equilibria of the tumor suppressor p53

The state of oligomerization of the tumor suppressor p53 is an important factor in its various biological functions. It has a well-defined tetramerization domain, and the protein exists as monomers, dimers and tetramers in equilibrium. The dissociation constants between oligomeric forms are so low that they are at the limits of measurement by conventional methods in vitro. Here, we have used the high sensitivity of single-molecule methods to measure the equilibria and kinetics of oligomerization of full-length p53 and its isolated tetramerization domain, p53tet, at physiological temperature, pH and ionic strength using fluorescence correlation spectroscopy (FCS) in vitro. The dissociation constant at 37 °C for tetramers dissociating into dimers for full-length p53 was 50 ± 7 nM, and the corresponding value for dimers into monomers was 0.55 ± 0.08 nM. The half-lives for the two processes were 20 and 50 min, respectively. The equivalent quantities for p53tet were 150 ± 10 nM, 1.0 ± 0.14 nM, 2.5 ± 0.4 min and 13 ± 2 min. The data suggest that unligated p53 in unstressed cells should be predominantly dimeric. Single-molecule FCS is a useful procedure for measuring dissociation equilibria, kinetics and aggregation at extreme sensitivity.     

Mechanisms of atrial-selective block of Na⁺ channels by ranolazine: I. Experimental analysis of the use-dependent block

Atrial-selective inhibition of cardiac Na(+) channel current (I(Na)) and I(Na)-dependent parameters has been shown to contribute to the safe and effective management of atrial fibrillation. The present study examined the basis for the atrial-selective actions of ranolazine. Whole cell I(Na) was recorded at 15°C in canine atrial and ventricular myocytes and in human embryonic kidney (HEK)-293 cells expressing SCN5A. Tonic block was negligible at holding potentials from -140 to -100 mV, suggesting minimal drug interactions with the closed state. Trains of 40 pulses were elicited over a range of holding potentials to determine use-dependent block. Guarded receptor formalism was used to analyze the development of block during pulse trains. Use-dependent block by ranolazine increased at more depolarized holding potentials, consistent with an interaction of the drug with either preopen or inactivated states, but was unaffected by longer pulse durations between 5 and 200 ms, suggesting a weak interaction with the inactivated state. Block was significantly increased at shorter diastolic intervals between 20 and 200 ms. Responses in atrial and ventricular myocytes and in HEK-293 cells displayed a similar pattern. Ranolazine is an open state blocker that unbinds from closed Na(+) channels unusually fast but is trapped in the inactivated state. Kinetic rates of ranolazine interactions with different states of atrial and ventricular Na(+) channels were similar. Our data suggest that the atrial selectivity of ranolazine is due to a more negative steady-state inactivation curve, less negative resting membrane potential, and shorter diastolic intervals in atrial cells compared with ventricular cells at rapid rates.     

Multi-species occupancy models: review,roadmap, and recommendations

Recent technological and methodological advances have revolutionized wildlife monitoring. Although most biodiversity monitoring initiatives are geared towards focal species of conservation concern, researchers are increasingly studying entire communities, specifically the spatiotemporal drivers of community size and structure and interactions among species. This has resulted in the emergence of multi-species occupancy models (MSOMs) as a promising and efficient approach for the study of community ecology. Given the potential of MSOMs for conservation and management action, it is critical to know whether study design and model assumptions are consistent with inference objectives. This is especially true for studies that are designed for a focal species but can give insights about a community. Here, we review the recent literature on MSOMs, identify areas of improvement in the multi-species study workflow, and provide a reference model for best practices for focal species and community monitoring study design. We reviewed 92 studies published between 2009 and early 2018, spanning 27 countries and a variety of taxa. There is a consistent under-reporting of details that are central to determining the adequacy of designs for generating data that can be used to make inferences about community-level patterns of occupancy, including the spatial and temporal extent, types of detectors used, covariates considered, and choice of field methods and statistical tools. This reporting bias could consequently result in skewed estimates, affecting conservation actions and management plans. On the other hand, comprehensive reporting is likely to help researchers working on MSOMs assess the robustness of inferences, in addition to making strides in terms of reproducibility and reusability of data. We use our literature review to inform a roadmap with best practices for MSOM studies, from simulations to design considerations and reporting, for the collection of new data as well as those involving existing datasets.     

Functional relevance of Gedunin as a bona fide ligand of NADPH oxidase 5 and ROS scavenger: An in silico and in vitro assessment in a hyperglycemic RBC model

Clinical evidence suggests that type 2 diabetes therapy can greatly benefit from the suppression of reactive oxygen species generation and the activation or restoration of cellular antioxidant mechanisms. In human, NADPH oxidase (NOX) is the main producer of reactive oxygen species (ROS) that supress the activity of endogenous antioxidant enzymes. In the present study, the antioxidant potential of Gedunin was studied. In silico findings reveal its strong binding affinity with NOX5 C terminal HSP90 binding site that disrupts NOX5 stability and its ability to generate ROS, leading to restoration antioxidant enzymes activities. It was found that Gedunin suppressed hyperglycaemia induced oxidative stress in an in vitro RBC model and markedly reversed glucose induced changes including haemoglobin glycosylation and lipid peroxidation. A significant restoration of activities of cellular antioxidant enzymes; superoxide dismutase, catalase and glutathione peroxidase in the presence of Gedunin revealed its ability to reduce oxidative stress. These results substantiated Gedunin as a bona fide inhibitor of human NOX5 and a ROS scavenging antioxidant with promising therapeutic attributes including its natural origin and inhibition of multiple diabetic targets.     

B-Ring-Aryl Substituted Luotonin A Analogues with a New Binding Mode to the Topoisomerase 1-DNA Complex Show Enhanced Cytotoxic Activity

Topoisomerase 1 inhibition is an important strategy in targeted cancer chemotherapy. The drugs currently in use acting on this enzyme belong to the family of the camptothecins, and suffer severe limitations because of their low stability, which is associated with the hydrolysis of the δ-lactone moiety in their E ring. Luotonin A is a natural camptothecin analogue that lacks this functional group and therefore shows a much-improved stability, but at the cost of a lower activity. Therefore, the development of luotonin A analogues with an increased potency is important for progress in this area. In the present paper, a small library of luotonin A analogues modified at their A and B rings was generated by cerium(IV) ammonium nitrate-catalyzed Friedländer reactions. All analogues showed an activity similar or higher than the natural luotonin A in terms of topoisomerase 1 inhibition and some compounds had an activity comparable to that of camptothecin. Furthermore, most compounds showed a better activity than luotonin A in cell cytotoxicity assays. In order to rationalize these results, the first docking studies of luotonin-topoisomerase 1-DNA ternary complexes were undertaken. Most compounds bound in a manner similar to luotonin A and to standard topoisomerase poisons such as topotecan but, interestingly, the two most promising analogues, bearing a 3,5-dimethylphenyl substituent at ring B, docked in a different orientation. This binding mode allows the hydrophobic moiety to be shielded from the aqueous environment by being buried between the deoxyribose belonging to the G(+1) guanine and Arg364 in the scissile strand and the surface of the protein and a hydrogen bond between the D-ring carbonyl and the basic amino acid. The discovery of this new binding mode and its associated higher inhibitory potency is a significant advance in the design of new topoisomerase 1 inhibitors.     

Comprehensive assay of kinase catalytic activity reveals features of kinase inhibitor selectivity

Small-molecule protein kinase inhibitors are widely used to elucidate cellular signaling pathways and are promising therapeutic agents. Owing to evolutionary conservation of the ATP-binding site, most kinase inhibitors that target this site promiscuously inhibit multiple kinases. Interpretation of experiments that use these compounds is confounded by a lack of data on the comprehensive kinase selectivity of most inhibitors. Here we used functional assays to profile the activity of 178 commercially available kinase inhibitors against a panel of 300 recombinant protein kinases. Quantitative analysis revealed complex and often unexpected interactions between protein kinases and kinase inhibitors, with a wide spectrum of promiscuity. Many off-target interactions occur with seemingly unrelated kinases, revealing how large-scale profiling can identify multitargeted inhibitors of specific, diverse kinases. The results have implications for drug development and provide a resource for selecting compounds to elucidate kinase function and for interpreting the results of experiments involving kinase inhibitors.     

Identification of biochemical network modules based on shortest retroactive distances

Modularity analysis offers a route to better understand the organization of cellular biochemical networks as well as to derive practically useful, simplified models of these complex systems. While there is general agreement regarding the qualitative properties of a biochemical module, there is no clear consensus on the quantitative criteria that may be used to systematically derive these modules. In this work, we investigate cyclical interactions as the defining characteristic of a biochemical module. We utilize a round trip distance metric, termed Shortest Retroactive Distance (ShReD), to characterize the retroactive connectivity between any two reactions in a biochemical network and to group together network components that mutually influence each other. We evaluate the metric on two types of networks that feature feedback interactions: (i) epidermal growth factor receptor (EGFR) signaling and (ii) liver metabolism supporting drug transformation. For both networks, the ShReD partitions found hierarchically arranged modules that confirm biological intuition. In addition, the partitions also revealed modules that are less intuitive. In particular, ShReD-based partition of the metabolic network identified a 'redox' module that couples reactions of glucose, pyruvate, lipid and drug metabolism through shared production and consumption of NADPH. Our results suggest that retroactive interactions arising from feedback loops and metabolic cycles significantly contribute to the modularity of biochemical networks. For metabolic networks, cofactors play an important role as allosteric effectors that mediate the retroactive interactions.     

Living bacterial sacrificial porogens to engineer decellularized porous scaffolds

Decellularization and cellularization of organs have emerged as disruptive methods in tissue engineering and regenerative medicine. Porous hydrogel scaffolds have widespread applications in tissue engineering, regenerative medicine and drug discovery as viable tissue mimics. However, the existing hydrogel fabrication techniques suffer from limited control over pore interconnectivity, density and size, which leads to inefficient nutrient and oxygen transport to cells embedded in the scaffolds. Here, we demonstrated an innovative approach to develop a new platform for tissue engineered constructs using live bacteria as sacrificial porogens. E.coli were patterned and cultured in an interconnected three-dimensional (3D) hydrogel network. The growing bacteria created interconnected micropores and microchannels. Then, the scafold was decellularized, and bacteria were eliminated from the scaffold through lysing and washing steps. This 3D porous network method combined with bioprinting has the potential to be broadly applicable and compatible with tissue specific applications allowing seeding of stem cells and other cell types.