scholarly journals A universal fluorescence-based toolkit for real-time quantification of DNA and RNA nuclease activity

2019 ◽  
Author(s):  
Emily C. Sheppard ◽  
Sally Rogers ◽  
Nicholas J. Harmer ◽  
Richard Chahwan
Keyword(s):  
Dna Repair ◽  
Real Time ◽  
Critical Role ◽  
Immune Surveillance ◽  
Cost Effective ◽  
Nuclease Activity ◽  
Cellular Mechanisms ◽  
Substrate Preferences ◽  
Dna And Rna ◽  
Kinetics Of

AbstractDNA and RNA nucleases play a critical role in a growing number of cellular processes ranging from DNA repair to immune surveillance. Nevertheless, many nucleases have unknown or poorly characterized activities. Elucidating nuclease substrate specificities and co-factors can support a more definitive understanding of cellular mechanisms in physiology and disease. Using fluorescence-based methods, we present a quick, safe, cost-effective, and real-time versatile nuclease assay, which uniquely studies nuclease enzyme kinetics. In conjunction with a substrate library we can now analyse nuclease catalytic rates, directionality, and substrate preferences. The assay is sensitive enough to detect kinetics of repair enzymes when confronted with DNA mismatches or DNA methylation sites. We have also extended our analysis to study the kinetics of human single-strand DNA nuclease TREX2, DNA polymerases, RNA, and RNA:DNA nucleases. These nucleases are involved in DNA repair, immune regulation, and have been associated with various diseases, including cancer and immune disorders.

scholarly journals Evolving neoantigen profiles in colorectal cancers with DNA repair defects

2019 ◽  
Author(s):  
Giuseppe Rospo ◽  
Annalisa Lorenzato ◽  
Nabil Amirouchene-Angelozzi ◽  
Alessandro Magrì ◽  
Carlotta Cancelliere ◽  
...  
Keyword(s):  
Dna Repair ◽  
Clonal Evolution ◽  
Immune Surveillance ◽  
Cellular Mechanisms ◽  
Patient Response ◽  
Genomic Signatures ◽  
Immune Therapies ◽  
Repair Pathways ◽  
Over Time

AbstractBackgroundNeoantigens that arise as a consequence of tumour-specific mutations can be recognized by T lymphocytes leading to effective immune surveillance. In colorectal cancer (CRC) and other tumour types, a high number of neoantigens is associated with patient response to immune therapies. The molecular processes governing the generation of neoantigens and their turnover in cancer cells are poorly understood. We exploited CRC as a model system to understand how alterations in DNA repair pathways modulate neoantigen profiles over time.MethodsWe performed Whole Exome Sequencing (WES) and RNA sequencing (RNAseq) in CRC cell lines, in vitro and vivo, and in CRC patient-derived xenografts (PDXs) to track longitudinally genomic profiles, clonal evolution, mutational signatures and predicted neoantigens.ResultsThe majority of CRC models showed remarkably stable mutational and neoantigen profiles, however those carrying defects in DNA repair genes continuously diversified. Rapidly evolving and evolutionary stable CRCs displayed characteristic genomic signatures, and transcriptional profiles. Downregulation of molecules implicated in antigen presentation occurred selectively in highly mutated and rapidly-evolving CRC.ConclusionsThese results indicate that CRC carrying alterations in DNA repair pathways display dynamic neoantigen patterns that fluctuate over time. We define CRC subsets characterized by slow and fast evolvability and link this phenotype to downregulation of antigen-presenting cellular mechanisms. Longitudinal monitoring of the neoantigen landscape could be relevant in the context of precision medicine.

scholarly journals Transient kinetic analysis of oxidative dealkylation by the direct reversal DNA repair enzyme AlkB

2020 ◽  
Vol 295 (21) ◽  
pp. 7317-7326 ◽  
Author(s):  
Michael R. Baldwin ◽  
Suzanne J. Admiraal ◽  
Patrick J. O'Brien
Keyword(s):  
Dna Repair ◽  
Alkylating Agents ◽  
Transient Kinetics ◽  
Repair Enzyme ◽  
Future Studies ◽  
Dna And Rna ◽  
Double Stranded Dna ◽  
Wide Range ◽  
Kinetic Mechanisms ◽  
Kinetics Of

AlkB is a bacterial Fe(II)– and 2-oxoglutarate–dependent dioxygenase that repairs a wide range of alkylated nucleobases in DNA and RNA as part of the adaptive response to exogenous nucleic acid–alkylating agents. Although there has been longstanding interest in the structure and specificity of Escherichia coli AlkB and its homologs, difficulties in assaying their repair activities have limited our understanding of their substrate specificities and kinetic mechanisms. Here, we used quantitative kinetic approaches to determine the transient kinetics of recognition and repair of alkylated DNA by AlkB. These experiments revealed that AlkB is a much faster alkylation repair enzyme than previously reported and that it is significantly faster than DNA repair glycosylases that recognize and excise some of the same base lesions. We observed that whereas 1,N6-ethenoadenine can be repaired by AlkB with similar efficiencies in both single- and double-stranded DNA, 1-methyladenine is preferentially repaired in single-stranded DNA. Our results lay the groundwork for future studies of AlkB and its human homologs ALKBH2 and ALKBH3.

scholarly journals The Multifaceted Roles of Ku70/80

2021 ◽  
Vol 22 (8) ◽  
pp. 4134
Author(s):  
Sayma Zahid ◽  
Murielle Seif El Dahan ◽  
Florence Iehl ◽  
Paloma Fernandez-Varela ◽  
Marie-Helene Le Du ◽  
...  
Keyword(s):  
Dna Repair ◽  
Mammalian Cells ◽  
Nuclease Activity ◽  
Nonhomologous End Joining ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Strand Breaks ◽  
Dna And Rna ◽  
Central Actor ◽  
Dna Ends

DNA double-strand breaks (DSBs) are accidental lesions generated by various endogenous or exogenous stresses. DSBs are also genetically programmed events during the V(D)J recombination process, meiosis, or other genome rearrangements, and they are intentionally generated to kill cancer during chemo- and radiotherapy. Most DSBs are processed in mammalian cells by the classical nonhomologous end-joining (c-NHEJ) pathway. Understanding the molecular basis of c-NHEJ has major outcomes in several fields, including radiobiology, cancer therapy, immune disease, and genome editing. The heterodimer Ku70/80 (Ku) is a central actor of the c-NHEJ as it rapidly recognizes broken DNA ends in the cell and protects them from nuclease activity. It subsequently recruits many c-NHEJ effectors, including nucleases, polymerases, and the DNA ligase 4 complex. Beyond its DNA repair function, Ku is also involved in several other DNA metabolism processes. Here, we review the structural and functional data on the DNA and RNA recognition properties of Ku implicated in DNA repair and in telomeres maintenance.

scholarly journals A critical role for B cells in cancer immune surveillance

2020 ◽  
Author(s):  
Kavita Rawat ◽  
Anita Tewari ◽  
Claudia Jakubzick
Keyword(s):  
B Cells ◽  
B Cell ◽  
Mouse Strain ◽  
Critical Role ◽  
Immune Surveillance ◽  
Fold Increase ◽  
Neoplastic Cell ◽  
Antibody Repertoire ◽  
Deficient Mouse ◽  
Cellular Mechanisms

It is commonly believed that B cells play no role in cancer immune surveillance. However, this conclusion is based on studies using only a single B cell-deficient mouse strain, muMT mice, which does not show increased tumorigenesis compared with wild-type (WT) mice. In this study, we demonstrate a critical role for B cells in anti-tumor immunity and identify the cellular mechanisms that make muMT mice a special case. First, we replicate previous findings using a melanoma model in muMT mice. Then, we show that in muMT mice, B-cell deficiency is compensated for by a significant increase in another potent anti-tumor cell type, Type-1 interferon (IFN I)-producing plasmacytoid DCs (pDCs), resulting in normative anti-tumor responses. Depleting pDCs in muMT mice resulted in a significant increase in tumor size and burden. Conversely, adoptive transfer of antibodies from naive WT serum into pDC-depleted muMT mice significantly decreased the tumor load to WT levels. Additionally, a B cell antibody repertoire-deficient mouse strain, IghelMD4 mice, showed a 3-fold increase in tumors relative to WT mice. Overall, these findings indicate the need for a diverse antibody repertoire for early neoplastic cell recognition and the critical role B cells play in anti-cancer immunity.

scholarly journals A universal fluorescence-based toolkit for real-time quantification of DNA and RNA nuclease activity

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Emily C. Sheppard ◽  
Sally Rogers ◽  
Nicholas J. Harmer ◽  
Richard Chahwan
Keyword(s):  
Real Time ◽  
Nuclease Activity ◽  
Dna And Rna ◽  
Universal Fluorescence

JOINT ESTIMATION OF THE TRANSMISSIBILITY AND SEVERITY OF EBOLA VIRUS DISEASE IN REAL TIME

2017 ◽  
Vol 25 (04) ◽  
pp. 587-603 ◽  
Author(s):  
YUSUKE ASAI ◽  
HIROSHI NISHIURA
Keyword(s):  
Real Time ◽  
Ebola Virus ◽  
Virus Disease ◽  
Critical Role ◽  
Estimation Method ◽  
Case Fatality ◽  
Ascertainment Bias ◽  
Joint Estimation ◽  
Ebola Virus Disease ◽  
Strong Impact

The effective reproduction number [Formula: see text], the average number of secondary cases that are generated by a single primary case at calendar time [Formula: see text], plays a critical role in interpreting the temporal transmission dynamics of an infectious disease epidemic, while the case fatality risk (CFR) is an indispensable measure of the severity of disease. In many instances, [Formula: see text] is estimated using the reported number of cases (i.e., the incidence data), but such report often does not arrive on time, and moreover, the rate of diagnosis could change as a function of time, especially if we handle diseases that involve substantial number of asymptomatic and mild infections and large outbreaks that go beyond the local capacity of reporting. In addition, CFR is well known to be prone to ascertainment bias, often erroneously overestimated. In this paper, we propose a joint estimation method of [Formula: see text] and CFR of Ebola virus disease (EVD), analyzing the early epidemic data of EVD from March to October 2014 and addressing the ascertainment bias in real time. To assess the reliability of the proposed method, coverage probabilities were computed. When ascertainment effort plays a role in interpreting the epidemiological dynamics, it is useful to analyze not only reported (confirmed or suspected) cases, but also the temporal distribution of deceased individuals to avoid any strong impact of time dependent changes in diagnosis and reporting.

scholarly journals Modeling Fused Filament Fabrication using Artificial Neural Networks

2021 ◽  
Author(s):  
Paul Oehlmann ◽  
Paul Osswald ◽  
Juan Camilo Blanco ◽  
Martin Friedrich ◽  
Dominik Rietzel ◽  
...  
Keyword(s):  
Real Time ◽  
Large Scale ◽  
Cost Effective ◽  
Print Quality ◽  
Ann Model ◽  
Production Environment ◽  
Fused Filament Fabrication ◽  
Artificial Neural ◽  
Using Data ◽  
Artificial Neural Network Ann

AbstractWith industries pushing towards digitalized production, adaption to expectations and increasing requirements for modern applications, has brought additive manufacturing (AM) to the forefront of Industry 4.0. In fact, AM is a main accelerator for digital production with its possibilities in structural design, such as topology optimization, production flexibility, customization, product development, to name a few. Fused Filament Fabrication (FFF) is a widespread and practical tool for rapid prototyping that also demonstrates the importance of AM technologies through its accessibility to the general public by creating cost effective desktop solutions. An increasing integration of systems in an intelligent production environment also enables the generation of large-scale data to be used for process monitoring and process control. Deep learning as a form of artificial intelligence (AI) and more specifically, a method of machine learning (ML) is ideal for handling big data. This study uses a trained artificial neural network (ANN) model as a digital shadow to predict the force within the nozzle of an FFF printer using filament speed and nozzle temperatures as input data. After the ANN model was tested using data from a theoretical model it was implemented to predict the behavior using real-time printer data. For this purpose, an FFF printer was equipped with sensors that collect real time printer data during the printing process. The ANN model reflected the kinematics of melting and flow predicted by models currently available for various speeds of printing. The model allows for a deeper understanding of the influencing process parameters which ultimately results in the determination of the optimum combination of process speed and print quality.

scholarly journals Cost-Effective Real-Time Metabolic Profiling of Cancer Cell Lines for Plate-Based Assays

Chemosensors ◽  
2021 ◽  
Vol 9 (6) ◽  
pp. 139
Author(s):  
Wiktoria Blaszczak ◽  
Zhengchu Tan ◽  
Pawel Swietach
Keyword(s):  
Real Time ◽  
Cell Lines ◽  
Acid Production ◽  
Metabolic Regulation ◽  
Cost Effective ◽  
Culture Conditions ◽  
Metabolic Inhibitors ◽  
Metabolic Phenotype ◽  
Ductal Adenocarcinoma ◽  
Oxygen Utilization

A fundamental phenotype of cancer cells is their metabolic profile, which is routinely described in terms of glycolytic and respiratory rates. Various devices and protocols have been designed to quantify glycolysis and respiration from the rates of acid production and oxygen utilization, respectively, but many of these approaches have limitations, including concerns about their cost-ineffectiveness, inadequate normalization procedures, or short probing time-frames. As a result, many methods for measuring metabolism are incompatible with cell culture conditions, particularly in the context of high-throughput applications. Here, we present a simple plate-based approach for real-time measurements of acid production and oxygen depletion under typical culture conditions that enable metabolic monitoring for extended periods of time. Using this approach, it is possible to calculate metabolic fluxes and, uniquely, describe the system at steady-state. By controlling the conditions with respect to pH buffering, O2 diffusion, medium volume, and cell numbers, our workflow can accurately describe the metabolic phenotype of cells in terms of molar fluxes. This direct measure of glycolysis and respiration is conducive for between-runs and even between-laboratory comparisons. To illustrate the utility of this approach, we characterize the phenotype of pancreatic ductal adenocarcinoma cell lines and measure their response to a switch of metabolic substrate and the presence of metabolic inhibitors. In summary, the method can deliver a robust appraisal of metabolism in cell lines, with applications in drug screening and in quantitative studies of metabolic regulation.

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