Reversal of DNA damage induced Topoisomerase 2 DNA–protein crosslinks by Tdp2

Mammalian Tyrosyl-DNA phosphodiesterase 2 (Tdp2) reverses Topoisomerase 2 (Top2) DNA–protein crosslinks triggered by Top2 engagement of DNA damage or poisoning by anticancer drugs. Tdp2 deficiencies are linked to neurological disease and cellular sensitivity to Top2 poisons. Herein, we report X-ray crystal structures of ligand-free Tdp2 and Tdp2-DNA complexes with alkylated and abasic DNA that unveil a dynamic Tdp2 active site lid and deep substrate binding trench well-suited for engaging the diverse DNA damage triggers of abortive Top2 reactions. Modeling of a proposed Tdp2 reaction coordinate, combined with mutagenesis and biochemical studies support a single Mg2+-ion mechanism assisted by a phosphotyrosyl-arginine cation-π interface. We further identify a Tdp2 active site SNP that ablates Tdp2 Mg2+ binding and catalytic activity, impairs Tdp2 mediated NHEJ of tyrosine blocked termini, and renders cells sensitive to the anticancer agent etoposide. Collectively, our results provide a structural mechanism for Tdp2 engagement of heterogeneous DNA damage that causes Top2 poisoning, and indicate that evaluation of Tdp2 status may be an important personalized medicine biomarker informing on individual sensitivities to chemotherapeutic Top2 poisons

Bioinformatic tools for single-cell data analysis in clinical studies

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Chemical Engineering, February, 2020Cataloged from the official PDF of thesis. "February 2020."Includes bibliographical references.Mechanistic understanding of disease has been dramatically enhanced by an explosion of new high-throughput experimental techniques for profiling biological samples, including RNA-Seq, mass spectrometry, and single-cell sequencing However, the ability to gather exponentially more measurements comes with pitfalls of increased Type I error and reduced interpretability In theory, single-cell measurements can be helpful in combating this problem, since each sample of cells represents hundreds to thousands of observations But thinking is still emerging on how best to utilize single-cell data to boost statistics and generate meaningful findings This thesis represents several parallel efforts to develop and apply new bioinformatic techniques to generate robust findings from single-cell data The advances are especially pertinent for small clinical studies in which low sample numbers are limiting In the first part of the thesis, two classes of methods are introduced gene module discovery in single-cell RNA sequencing data using sparse PCA, and probability-based metrics for evaluating the degree of association between paired modalities of single-cell data (in this case, single-cell RNA sequencing and paired TCR sequencing data) The methods are shown on two different human datasets, as proof-of-concept and examples of the biological findings capable of being unearthed In the second part of the thesis, these methods are applied to larger clinical datasets with questions surrounding acquired tolerance and clinical reactivity in food allergy In the first study, T-helper cells from peanut-allergic patients undergoing oral immunotherapy were profiled to identify therapy-induced effects and baseline predictors of outcome Two distinct subsets of expanded TH2 clones were found to be suppressed, but not deleted, by the therapy In the second study, transcriptional correlates of clinical reactivity were evaluated in peanut-activated memory T-helper cells from peanut-allergic adults Cells from more reactive patients had higher expression of TH1 and MHC I gene programs, suggesting activation of auxiliary, non-TH2 cell types In each of these studies, new single-cell analysis techniques were integrated to generate clinical findings with improved robustness and interpretability.by Brinda Monian.Ph. D.Ph. D. Massachusetts Institute of Technology, Department of Chemical Engineerin

Identification of antigen-specific TCR sequences based on biological and statistical enrichment in unselected individuals

Recent advances in high-throughput T cell receptor (TCR) sequencing have allowed for new insights into the human TCR repertoire. However, methods for capturing antigen-specific repertoires remain an area of development. Here, we describe a potentially novel approach that utilizes both a biological and statistical enrichment to define putatively antigen-specific complementarity-determining region 3 (CDR3) repertoires in unselected individuals. The biological enrichment entailed FACS of in vitro antigen-activated memory CD4+ T cells, followed by TCRβ sequencing. The resulting TCRβ sequences were then filtered by selecting those that are statistically enriched when compared with their frequency in the autologous resting T cell compartment. Applying this method to define putatively peanut protein-specific repertoires in 27 peanut-allergic individuals resulted in a library of 7345 unique CDR3β amino acid sequences that had similar characteristics to other validated antigen-specific repertoires in terms of homology and diversity. In-depth analysis of these CDR3βs revealed 36 public sequences that demonstrated high levels of convergent recombination. In a network analysis, the public CDR3βs were shown to be core sequences with more edges than their private counterparts. This method has the potential to be applied to a wide range of T cell-mediated disorders and to yield new biomarkers and biological insights

Alginate–Polyethylene Oxide Blend Nanofibers and the Role of the Carrier Polymer in Electrospinning

We present here a systematic investigation to understand why aqueous sodium alginate can only be electrospun into fibers through a blend with another polymer; specifically, polyethylene oxide (PEO). We seek to examine and understand the role of PEO as the “carrier polymer”. The addition of PEO favorably reduces electrical conductivity and surface tension of the alginate solution, aiding in fiber formation. While PEO has the ability to coordinate through its ether group (−COC−) with metal cation like the sodium cation of sodium alginate, we demonstrate in this study using PEO as well as polyvinyl alcohol (PVA) that coordination may have little effect on electrospinnability. More importantly, we show that PEO as carrier polymer provides molecular entanglement that is required for electrospinning. Since the selected carrier polymer provides the necessary entanglement, this carrier polymer must be electrospinnable, entangled and of a high molecular weight (more than 600 kDa for PEO). On the basis of these requirements, we stipulate that the PEO–PEO interaction of the high molecular-weight entangled PEO is key to “carrying” the alginate from solution to fibers during electrospinning. Further, using the resulting understanding of the role of PEO, we were able to increase the alginate concentration by employing a higher molecular-weight PEO: up to 70 wt % alginate using 2000 kDa PEO and, with, the addition of Triton X-100 surfactant, up to 85 wt % alginate, higher than previously reported

Preservation of Cell Viability and Protein Conformation on Immobilization within Nanofibers via Electrospinning Functionalized Yeast

We investigate the immobilization of a model system of functionalized yeast that surface-display enhanced green fluorescent protein (eGFP) within chemically crosslinked polyvinyl alcohol (PVA) nanofibers. Yeast is incorporated into water insoluble nanofibrous materials by direct electrospinning with PVA followed by vapor phase chemical crosslinking of the polymer. Incorporation of yeast into the fibers is confirmed by elemental analysis and the viability is indicated by live/dead staining. Following electrospinning and crosslinking, we confirm that the yeast maintains its viability as well as the ability to express eGFP in the correct conformation. This method of processing functionalized yeast may thus be a powerful tool in the direct immobilization of properly folded, active enzymes within electrospun nanofibers with potential applications in biocatalysis

Expansion of the CD4+ effector T-cell repertoire characterizes peanut-allergic patients with heightened clinical sensitivity

© 2019 American Academy of Allergy, Asthma & Immunology Background: Individuals with peanut allergy range in clinical sensitivity: some can consume grams of peanut before experiencing any symptoms, whereas others suffer systemic reactions to 10 mg or less. Current diagnostic testing only partially predicts this clinical heterogeneity. Objective: We sought to identify characteristics of the peanut-specific CD4+ T-cell response in peanut-allergic patients that correlate with high clinical sensitivity. Methods: We studied the T-cell receptor β-chain (TCRβ) usage and phenotypes of peanut-activated, CD154+ CD4+ memory T cells using fluorescence-activated cell sorting, TCRβ sequencing, and RNA-Seq, in reactive and hyporeactive patients who were stratified by clinical sensitivity. Results: TCRβ analysis of the CD154+ and CD154− fractions revealed more than 6000 complementarity determining region 3 sequences and motifs that were significantly enriched in the activated cells and 17% of the sequences were shared between peanut-allergic individuals, suggesting strong convergent selection of peanut-specific clones. These clones were more numerous among the reactive patients, and this expansion was identified within effector, but not regulatory T-cell populations. The transcriptional profile of CD154+ T cells in the reactive group skewed toward a polarized TH2 effector phenotype, and expression of TH2 cytokines strongly correlated with peanut-specific IgE levels. There were, however, also non–TH2-related differences in phenotype. Furthermore, the ratio of peanut-specific clones in the effector versus regulatory T-cell compartment, which distinguished the clinical groups, was independent of specific IgE concentration. Conclusions: Expansion of the peanut-specific effector T-cell repertoire is correlated with clinical sensitivity, and this observation may be useful to inform our assessment of disease phenotype and to monitor disease longitudinally

Reversal of DNA damage induced Topoisomerase 2 DNA–protein crosslinks by Tdp2

Mammalian Tyrosyl-DNA phosphodiesterase 2 (Tdp2) reverses Topoisomerase 2 (Top2) DNA–protein crosslinks triggered by Top2 engagement of DNA damage or poisoning by anticancer drugs. Tdp2 deficiencies are linked to neurological disease and cellular sensitivity to Top2 poisons. Herein, we report X-ray crystal structures of ligand-free Tdp2 and Tdp2-DNA complexes with alkylated and abasic DNA that unveil a dynamic Tdp2 active site lid and deep substrate binding trench well-suited for engaging the diverse DNA damage triggers of abortive Top2 reactions. Modeling of a proposed Tdp2 reaction coordinate, combined with mutagenesis and biochemical studies support a single Mg(2+)-ion mechanism assisted by a phosphotyrosyl-arginine cation-π interface. We further identify a Tdp2 active site SNP that ablates Tdp2 Mg(2+) binding and catalytic activity, impairs Tdp2 mediated NHEJ of tyrosine blocked termini, and renders cells sensitive to the anticancer agent etoposide. Collectively, our results provide a structural mechanism for Tdp2 engagement of heterogeneous DNA damage that causes Top2 poisoning, and indicate that evaluation of Tdp2 status may be an important personalized medicine biomarker informing on individual sensitivities to chemotherapeutic Top2 poisons