A Modeling-Derived Hypothesis on Chronicity in Respiratory Diseases: Desensitized Pathogen Recognition Secondary to Hyperactive IRAK/TRAF6 Signaling

Several chronic respiratory diseases exhibit hyperactive immune responses in the lung: abundant inflammatory mediators; infiltrating neutrophils, macrophages, lymphocytes and other immune cells; and increased level of proteases. Such diseases include cystic fibrosis (CF), chronic obstructive pulmonary disease (COPD) and severe/neutrophilic asthma. Paradoxically, patients with these diseases are also susceptible to detrimental bacterial infection and colonization. In this paper, we seek to explain how a positive feedback mechanism via IL-8 could lead to desensitization of epithelial cells to pathogen recognition thus perpetuating bacterial colonization and chronic disease states in the lung. Such insight was obtained from mathematical modeling of the IRAK/TRAF6 signaling module, and is consistent with existing clinical evidence. The potential implications for targeted treatment regimes for these persistent respiratory diseases are explored

Continuous adsorption process of CO2/N2/H2O from CH4 flow using type A zeolite adsorbents in the presence of ultrasonic waves

In this study, the adsorption capacity of different types of zeolite (3A, 4A, and 5A) for adsorbing CO2 from a mixture of CH4, N2, and water vapor was investigated with and without the use of ultrasonic waves. In this study, functional groups of O–H, Si–O–Al and Si–O–Si bands were identified in Fourier transform infrared (FTIR) spectroscopy of these adsorbents. X-ray diffraction patterns (XRD) for zeolite 3A and 5A showed the structure of chabazite and for zeolite 4A the structure of sodalite. In the Brunauer-Emmett-Teller (BET) test, the specific surface area of zeolites was measured as 16, 11.96 and 437 m2g−1, respectively. Ultrasonic waves increased the adsorption capacity of zeolites to adsorb CO2 at lower temperatures 80.64 to 175.44 mgg−1. Zeolite 5A has a higher affinity for CO2 than CH4 or N2, the selectivity of CO2/ N2 and CO2/ CH4 were 19.55 and 24.17, respectively. Data from adsorption experiments were used to learn an artificial neural network (ANN), and the ANN provided acceptable results for predicting the adsorption process

A requirement for replication in activation of the ATR-dependent DNA damage checkpoint

Using the Xenopus egg extract system, we investigated the involvement of DNA replication in activation of the DNA damage checkpoint. We show here that DNA damage slows replication in a checkpoint-independent manner and is accompanied by replication-dependent recruitment of ATR and Rad1 to chromatin. We also find that the replication proteins RPA and Polα accumulate on chromatin following DNA damage. Finally, damage-induced Chk1 phosphorylation and checkpoint arrest are abrogated when replication is inhibited. These data indicate that replication is required for activation of the DNA damage checkpoint and suggest a unifying model for ATR activation by diverse lesions during S phase

Structure of a kinesin microtubule depolymerization machine

With their ability to depolymerize microtubules (MTs), KinI kinesins are the rogue members of the kinesin family. Here we present the 1.6 Å crystal structure of a KinI motor core from Plasmodium falciparum, which is sufficient for depolymerization in vitro. Unlike all published kinesin structures to date, nucleotide is not present, and there are noticeable differences in loop regions L6 and L10 (the plus-end tip), L2 and L8 and in switch II (L11 and helix4); otherwise, the pKinI structure is very similar to previous kinesin structures. KinI-conserved amino acids were mutated to alanine, and studied for their effects on depolymerization and ATP hydrolysis. Notably, mutation of three residues in L2 appears to primarily affect depolymerization, rather than general MT binding or ATP hydrolysis. The results of this study confirm the suspected importance of loop 2 for KinI function, and provide evidence that KinI is specialized to hydrolyze ATP after initiating depolymerization

Expression, Purification and Characterization of Inactive and Active Forms of ERK2 from Insect Expression System

Extracellular signal-regulated kinase 2 (ERK2) is a serine/threonine protein kinase involved in many cellular programs, such as cell proliferation, differentiation, motility and programed cell-death. It is therefore considered an important target in the treatment of cancer. In an effort to support biochemical screening and small molecule drug discovery, we established a robust system to generate both inactive and active forms of ERK2 using insect expression system. We report here, for the first time, that inactive ERK2 can be expressed and purified with 100% homogeneity in the unphosphorylated form using insect system. This resulted in a significant 20-fold yield improvement compared to that previously reported using bacterial expression system. We also report a newly developed system to generate active ERK2 in insect cells through in vivo co-expression with a constitutively active MEK1 (S218D S222D). Isolated active ERK2 was confirmed to be doubly phosphorylated at the correct sites, T185 and Y187, in the activation loop of ERK2. Both ERK2 forms, inactive and active, were well characterized by biochemical activity assay for their kinase function. Inactive and active ERK2 were the two key reagents that enabled successful high through-put biochemical assay screen and structural drug discovery studies

Biochemical characterization of respiratory syncytial virus RNA dependent RNA polymerase complex

RNA dependent RNA polymerases (RdRP) from non-segmented negative strand (NNS) RNA viruses perform both mRNA transcription and genome replication and these activities are regulated by their interactions with RNA and other accessory proteins within the ribonucleoprotein (RNP) complex. Detailed biochemical characterization of these enzymatic activities and their regulation is essential for understanding the life cycles of many pathogenic RNA viruses and for antiviral drug discovery. We developed biochemical and biophysical kinetic methods to study the RNA synthesis and RNA binding activities of respiratory syncytial virus (RSV) L/P RdRP. We determined that the intact L protein is essential for RdRP activity and in truncated L protein constructs RdRP activity is abrogated due to their deficiency in RNA template binding. These results are in agreement with the observation of a RNA template-binding tunnel at the interface of RdRP and capping domains in RSV and vesicular stomatitis virus (VSV) L protein cryo-EM structures. We also describe a non-radiometric assay for measuring RNA polymerization activity of RSV RdRP that is amenable to compound screening and profiling

RAF Inhibitors Activate the MAPK Pathway by Relieving Inhibitory Autophosphorylation

ATP competitive inhibitors of the BRAFV600E oncogene paradoxically activate downstream signaling in cells bearing wild-type BRAF (BRAFWT). In this study, we investigate the biochemical mechanism of wild-type RAF (RAFWT) activation by multiple catalytic inhibitors using kinetic analysis of purified BRAFV600E and RAFWT enzymes. We show that activation of RAFWT is ATP dependent and directly linked to RAF kinase activity. These data support a mechanism involving inhibitory autophosphorylation of RAF's phosphate-binding loop that, when disrupted either through pharmacologic or genetic alterations, results in activation of RAF and the mitogen-activated protein kinase (MAPK) pathway. This mechanism accounts not only for compound-mediated activation of the MAPK pathway in BRAFWT cells but also offers a biochemical mechanism for BRAF oncogenesis. © 2013 Elsevier Inc

Inhibition of prenylated KRAS in a lipid environment

<div><p>RAS mutations lead to a constitutively active oncogenic protein that signals through multiple effector pathways. In this chemical biology study, we describe a novel coupled biochemical assay that measures activation of the effector BRAF by prenylated KRAS^G12V in a lipid-dependent manner. Using this assay, we discovered compounds that block biochemical and cellular functions of KRAS^G12V with low single-digit micromolar potency. We characterized the structural basis for inhibition using NMR methods and showed that the compounds stabilized the inactive conformation of KRAS^G12V. Determination of the biophysical affinity of binding using biolayer interferometry demonstrated that the potency of inhibition matches the affinity of binding only when KRAS is in its native state, namely post-translationally modified and in a lipid environment. The assays we describe here provide a first-time alignment across biochemical, biophysical, and cellular KRAS assays through incorporation of key physiological factors regulating RAS biology, namely a negatively charged lipid environment and prenylation, into the <i>in vitro</i> assays. These assays and the ligands we discovered are valuable tools for further study of KRAS inhibition and drug discovery.</p></div