I. Comparison of Translesion Bypass of Guanine–N2 Monoadducts of Mitomycin C and Guanine-N7 Monoadducts of 2,7-diaminomitosene by T7 exo-, Klenow exo-, eta and Klenow exo+ DNA Polymerases. II. Structure-based Design, Synthesis, Structure-conformation and Structure-activity Relationships Studies of D-Phe-Pro-D-Arg-P1’-CONH2 Tetrapeptides with Inhibitory Activity for Thrombin.

The guanine (G)-N2 DNA monoadduct of mitomycin C (MC), a cytotoxic anticancer drug, inhibits translesion bypass by DNA polymerases. 2,7-Diaminomitosene (2,7-DAM) is the major metabolite of MC in tumor cells, generated by the reduction of MC. 2,7-DAM alkylates DNA in the cell in situ, forming an adduct at the N7 position of 2\u27-deoxyguanosine (2,7-DAM-dG) and is noncytotoxic. In part I of this study we tested a potential correlation between the lack of cytotoxicity of 2,7-DAM and the relative ease of bypass of this adduct as compared with the MC adduct. 24-mer and 27-mer templates, adducted at a single guanine either with MC or 2,7-DAM were synthesized and submitted to extension of primers by T7 exo-, Klenow exo-, Klenow exo+, and eta DNA polymerases. The G-N7-2,7-DAM adduct was bypassed by all four polymerases, resulting in the production of a fully extended primer. In sharp contrast, the G-N2-MC monoadduct was not bypassed beyond the adduct position under the same conditions by any of the four polymerases. In parallel experiments in cell free systems, template oligonucleotides containing a single 2,7-DAM-dG-N7 adduct directed selective incorporation of cytosine in the 5\u2732P-labeled primer strands opposite the adducted guanine, catalyzed by Klenow (exo-) DNA polymerase. These results showed for the first time that the dG-N7-2, 7-DAM lesion is non-mutagenic in cell-free systems. In part II of this research structure-based design and molecular docking were employed to design in silico libraries of peptides as potential reversible inhibitors of thrombin. The candidate inhibitors were selected from two original classes of amino acids sequences (1)-D-Phe-Pro-Arg (P1)-D-Pro(P1\u27)-P2\u27-P3\u27-CONH2 and (2)-D-Phe-Pro-D-Arg(P1)-P1\u27-P2\u27-P3\u27-CONH2. For the first time in the field of peptides inhibitors for thrombin we showed that the presence of D-Pro at P1\u27 Position and the use of D-Arg instead of L-Arg at P1 Position is responsible for inhibiting hydrolysis of these of peptides by thrombin, causing these sequences to be inhibitors. In vitro kinetics of thrombin inhibition showed a specific structure-activity relationship at P1\u27 position in the peptide sequence space (2)-D-Phe-Pro-D-Arg(P1)-P1\u27-CONH2. The lead peptides (D-Phe-Pro-D-Arg-D-Ala-CONH2, D-Phe-Pro-D-Arg-D-Thr-CONH2, D-Phe-Pro-D-Arg-D-Cys-CONH2, D-Phe-Pro-D-Arg-D-Ser-CONH2) had competitive or mixed inhibition with respect to thrombin and are characterized by inhibitory constant in the 20-0.8 micromolar range

A protocol for qualitative and quantitative measurement of endosomal processing using hot spot analysis

A detailed quantification of antigen processing by endosomal compartments provides important information on the pattern of protein fragmentation. Here, we describe a protocol that combines gradient purified endosomes, incubated with antigens, followed by hot spot analysis of MS/MS-sequenced peptides. The analysis identifies differences in endosomal antigen processing by dendritic cells under diverse experimental conditions. For complete details on the use and execution of this protocol, please refer to Clement et al. (2021)

An Expanded Self-Antigen Peptidome Is Carried by the Human Lymph As Compared to the Plasma

The pre-nodal afferent lymph is the fluid which directly derives from the extracellular milieu from every parenchymal organ and, as it continues to circulate between the cells, it collects products deriving from the organ metabolism/catabolism. A comprehensive qualitative and quantitative investigation of the self-antigenic repertoire transported by the human lymph is still missing.A major difference between lymph and plasma could be visualized by FPLC and 2D gel in the amount of low molecular weight products corresponding to peptide fragments. Naturally processed peptides in normal pre-nodal human lymph were then fractionated by HPLC and characterized by multidimensional mass spectrometry. Analysis of more then 300 sequences identified self-peptides derived from both intracellular and extracellular proteins revealing the variety of catabolic products transported by human lymph. Quantitative analysis established that at least some of these peptides are present in the circulating lymph in nanomolar concentration.The peptidome, generated by physiological tissue catabolism and transported by the pre-nodal lymph, is in addition to the self-peptidome generated in endosomal compartment. Unlike self antigen processed by local or nodal APC, which mostly produce epitopes constrained by the endosomal processing activity, self antigens present in the lymph could derived from a wider variety of processing pathways; including caspases, involved in cellular apoptosis, and ADAM and other metalloproteinases involved in surface receptor editing, cytokines processing and matrix remodeling. Altogether, expanding the tissue-specific self-repertoire available for the maintenance of immunological tolerance

A Novel Prodrug of a nNOS Inhibitor with Improved Pharmacokinetic Potential

Under different pathological conditions, aberrant induction of neuronal nitric oxide synthase (nNOS) generates overproduction of NO that can cause irreversible cell damage. The aim of this study was to develop an amidoxime prodrug of a potent nNOS inhibitor, the benzhydryl acetamidine. We synthesized the benzhydryl acetamidoxime, which was evaluated in vitro to ascertain the potential NOS inhibitory activity, as well as conducting bioconversion into the parent acetamidine. The prodrug was also profiled for in vitro physicochemical properties, by determining the lipophilicity, passive permeation through the human gastrointestinal tract and across the blood-brain barrier by PAMPA, and chemical, enzymatic, and plasma stability. The obtained data demonstrate that the amidoxime prodrug shows an improved pharmacokinetic profile with respect to the acetamidine nNOS inhibitor, thus suggesting that it could be a promising lead compound to treat all those pathological conditions in which nNOS activity is dysregulated

Quantitative Profiling of the Lymph Node Clearance Capacity

Transport of tissue-derived lymphatic fluid and clearance by draining lymph nodes are pivotal for maintenance of fluid homeostasis in the body and for immune-surveillance of the self- and non-self-proteomes. Yet a quantitative analysis of nodal filtration of the tissue-derived proteome present in lymphatic fluid has not been reported. Here we quantified the efficiency of nodal clearance of the composite proteomic load using label-free and isotope-labeling proteomic analysis of pre-nodal and post-nodal samples collected by direct cannulation. These results were extended by quantitation of the filtration efficiency of fluorophore-labeled proteins, bacteria, and beads infused at physiological flow rates into pre-nodal lymphatic collectors and collected by post-nodal cannulation. We developed a linear model of nodal filtration efficiency dependent on pre-nodal protein concentrations and molecular weight, and uncovered criteria for disposing the proteome incoming from defined anatomical districts under physiological conditions. These findings are pivotal to understanding the maximal antigenic load sustainable by a draining node, and promote understanding of pathogen spreading and nodal filtration of tumor metastasis, potentially helping to improve design of vaccination protocols, immunization strategies and drug delivery

Strong suppression of nuclear-charge changing interactions for 18 TeV/ c In ions channeled through a bent Si crystal

Abstract We present experimental results giving evidence for the strong reduction—a factor of more than 20—of nuclear-charge changing interactions for 18 TeV In 49+ ions channeled through a silicon crystal bent to 7.5, 11.9 and 19.8 mrad. A very small fraction of the deflected ions suffer electromagnetic or nuclear interactions leading to proton loss while traversing the 60 mm long crystal, even though its thickness corresponds to about 0.13 nuclear interaction lengths for an amorphous material. By considering the deflected ions only, we show experimentally that the nuclear-charge pickup reaction believed to be induced by virtual photons is a short-range phenomenon

Gene expression signature-based chemical genomic prediction identifies a novel class of HSP90 pathway modulators

SummaryAlthough androgen receptor (AR)-mediated signaling is central to prostate cancer, the ability to modulate AR signaling states is limited. Here we establish a chemical genomic approach for discovery and target prediction of modulators of cancer phenotypes, as exemplified by AR signaling. We first identify AR activation inhibitors, including a group of structurally related compounds comprising celastrol, gedunin, and derivatives. To develop an in silico approach for target pathway identification, we apply a gene expression-based analysis that classifies HSP90 inhibitors as having similar activity to celastrol and gedunin. Validating this prediction, we demonstrate that celastrol and gedunin inhibit HSP90 activity and HSP90 clients, including AR. Broadly, this work identifies new modes of HSP90 modulation through a gene expression-based strategy

Implementation of AI/Deep Learning Disruption Predictor into a Plasma Control System

This paper reports on advances to the state-of-the-art deep-learning disruption prediction models based on the Fusion Recurrent Neural Network (FRNN) originally introduced a 2019 Nature publication. In particular, the predictor now features not only the disruption score, as an indicator of the probability of an imminent disruption, but also a sensitivity score in real-time to indicate the underlying reasons for the imminent disruption. This adds valuable physics-interpretability for the deep-learning model and can provide helpful guidance for control actuators now that it is fully implemented into a modern Plasma Control System (PCS). The advance is a significant step forward in moving from modern deep-learning disruption prediction to real-time control and brings novel AI-enabled capabilities relevant for application to the future burning plasma ITER system. Our analyses use large amounts of data from JET and DIII-D vetted in the earlier NATURE publication. In addition to when a shot is predicted to disrupt, this paper addresses reasons why by carrying out sensitivity studies. FRNN is accordingly extended to use many more channels of information, including measured DIII-D signals such as (i) the n1rms signal that is correlated with the n =1 modes with finite frequency, including neoclassical tearing mode and sawtooth dynamics, (ii) the bolometer data indicative of plasma impurity content, and (iii) q-min, the minimum value of the safety factor relevant to the key physics of kink modes. The additional channels and interpretability features expand the ability of the deep learning FRNN software to provide information about disruption subcategories as well as more precise and direct guidance for the actuators in a plasma control system

Interplay between edge states and simple bulk defects in graphene nanoribbons

We study the interplay between the edge states and a single impurity in a zigzag graphene nanoribbon. We use tight-binding exact diagonalization techniques, as well as density functional theory calculations to obtain the eigenvalue spectrum, the eigenfunctions, as well the dependence of the local density of states (LDOS) on energy and position. We note that roughly half of the unperturbed eigenstates in the spectrum of the finite-size ribbon hybridize with the impurity state, and the corresponding eigenvalues are shifted with respect to their unperturbed values. The maximum shift and hybridization occur for a state whose energy is inverse proportional to the impurity potential; this energy is that of the impurity peak in the DOS spectrum. We find that the interference between the impurity and the edge gives rise to peculiar modifications of the LDOS of the nanoribbon, in particular to oscillations of the edge LDOS. These effects depend on the size of the system, and decay with the distance between the edge and the impurity.Comment: 10 pages, 15 figures, revtex

In vivo data: treatment with the F11R/JAM-A peptide 4D decreases mortality and reduces the generation of atherosclerotic plaques in ApoE-deficient mice

The data in this article focus on the F11 Receptor (F11R/JAM-A; Junctional Adhesion Molecule-A; JAM-A, F11R), a cell adhesion protein constitutively expressed on the membrane surface of circulating platelets and localized within the tight junctions of healthy endothelial cells (ECs). Previous reports have shown that F11R/JAM-A plays a critical role in the adhesion of platelets to an inflamed endothelium due to its’ pathological expression on the luminal surface of the cytokine-inflamed endothelium. Since platelet adhesion to an inflamed endothelium is an early step in the development of atherosclerotic plaque formation, and with time, resulting in heart attacks and stroke, we conducted a long-term, study utilizing the atherosclerosis-prone ApoE-/- mice to attempt a blockade of the formation of atherosclerotic plaques by preventing the adhesion of platelets to the inflamed vasculature in vivo. Utilizing a nonhydrolyzable peptide derived from an amino acid sequence of F11R/JAM-A, peptide 4D, we have shown in culture that the adhesion of platelets to the inflamed endothelial cells could be blocked by peptide 4D. The present data demonstrate the positive health benefits of chronic peptide 4D administration to the atherosclerosis-prone ApoE-/- mice, and provides new information for potential use of this F11R derived peptide in the prevention of atherosclerosis. The data presented in this article provide further experimental support for the study presented in Babinska et al., Atherosclerosis 284 (2019) 92-101