An Automated Bayesian Pipeline for Rapid Analysis of Single-Molecule Binding Data [preprint]

Single-molecule binding assays enable the study of how molecular machines assemble and function. Current algorithms can identify and locate individual molecules, but require tedious manual validation of each spot. Moreover, no solution for high-throughput analysis of single-molecule binding data exists. Here, we describe an automated pipeline to analyze single-molecule data over a wide range of experimental conditions. We benchmarked the pipeline by measuring the binding properties of the well-studied, DNA-guided DNA endonuclease, TtAgo, an Argonaute protein from the Eubacterium Thermus thermophilus. We also used the pipeline to extend our understanding of TtAgo by measuring the protein\u27s binding kinetics at physiological temperatures and for target DNAs containing multiple, adjacent binding sites

Single-Molecule Imaging Reveals that Argonaute Reshapes the Binding Properties of Its Nucleic Acid Guides

SummaryArgonaute proteins repress gene expression and defend against foreign nucleic acids using short RNAs or DNAs to specify the correct target RNA or DNA sequence. We have developed single-molecule methods to analyze target binding and cleavage mediated by the Argonaute:guide complex, RISC. We find that both eukaryotic and prokaryotic Argonaute proteins reshape the fundamental properties of RNA:RNA, RNA:DNA, and DNA:DNA hybridization—a small RNA or DNA bound to Argonaute as a guide no longer follows the well-established rules by which oligonucleotides find, bind, and dissociate from complementary nucleic acid sequences. Argonautes distinguish substrates from targets with similar complementarity. Mouse AGO2, for example, binds tighter to miRNA targets than its RNAi cleavage product, even though the cleaved product contains more base pairs. By re-writing the rules for nucleic acid hybridization, Argonautes allow oligonucleotides to serve as specificity determinants with thermodynamic and kinetic properties more typical of RNA-binding proteins than of RNA or DNA

A DNA-guided Argonaute Protein Functions in DNA Replication in Thermus thermophilus [preprint]

Argonaute proteins use nucleic acid guides to protect organisms against transposons and viruses. In the eubacterium Thermus thermophilus, the DNA-guided Argonaute TtAgo defends against transformation by DNA plasmids. Here, we report that TtAgo also participates in DNA replication. TtAgo binds small DNA guides derived from the chromosomal region where replication terminates and associates with proteins known to act in DNA replication. T. thermophilus deploys a single type II topoisomerase, gyrase. When gyrase is inhibited, T. thermophilus relies on TtAgo to complete replication of its circular genome; loss of both gyrase and TtAgo activity produces long filaments that fail to separate into individual bacteria. We propose that the primary role of TtAgo is to help T. thermophilus disentangle the catenated circular chromosomes made by DNA replication

Modeling Patient-Specific Dose-Function Response for Enhanced Characterization of Personalized Functional Damage

PURPOSE: Functional-guided radiation therapy (RT) plans have the potential to limit damage to normal tissue and reduce toxicity. Although functional imaging modalities have continued to improve, a limited understanding of the functional response to radiation and its application to personalized therapy has hindered clinical implementation. The purpose of this study was to retrospectively model the longitudinal, patient-specific dose-function response in non-small cell lung cancer patients treated with RT to better characterize the expected functional damage in future, unknown patients. METHODS AND MATERIALS: Perfusion single-photon emission computed tomography/computed tomography scans were obtained at baseline (n = 81), midtreatment (n = 74), 3 months post-treatment (n = 51), and 1 year post-treatment (n = 26) and retrospectively analyzed. Patients were treated with conventionally fractionated RT or stereotactic body RT. Normalized perfusion single-photon emission computed tomography voxel intensity was used as a surrogate for local lung function. A patient-specific logistic model was applied to each individual patient's dose-function response to characterize functional reduction at each imaging time point. Patient-specific model parameters were averaged to create a population-level logistic dose-response model. RESULTS: A significant longitudinal decrease in lung function was observed after RT by analyzing the voxelwise change in normalized perfusion intensity. Generated dose-function response models represent the expected voxelwise reduction in function, and the associated uncertainty, for an unknown patient receiving conventionally fractionated RT or stereotactic body RT. Differential treatment responses based on the functional status of the voxel at baseline suggest that initially higher functioning voxels are damaged at a higher rate than lower functioning voxels. CONCLUSIONS: This study modeled the patient-specific dose-function response in patients with non-small cell lung cancer during and after radiation treatment. The generated population-level dose-function response models were derived from individual patient assessment and have the potential to inform functional-guided treatment plans regarding the expected functional lung damage. This type of patient-specific modeling approach can be applied broadly to other functional response analyses to better capture intrapatient dependencies and characterize personalized functional damage

Bi-allelic variants in CELSR3 are implicated in central nervous system and urinary tract anomalies

CELSR3 codes for a planar cell polarity protein. We describe twelve affected individuals from eleven independent families with bi-allelic variants in CELSR3. Affected individuals presented with an overlapping phenotypic spectrum comprising central nervous system (CNS) anomalies (7/12), combined CNS anomalies and congenital anomalies of the kidneys and urinary tract (CAKUT) (3/12) and CAKUT only (2/12). Computational simulation of the 3D protein structure suggests the position of the identified variants to be implicated in penetrance and phenotype expression. CELSR3 immunolocalization in human embryonic urinary tract and transient suppression and rescue experiments of Celsr3 in fluorescent zebrafish reporter lines further support an embryonic role of CELSR3 in CNS and urinary tract formation.</p

Bi-allelic variants in CELSR3 are implicated in central nervous system and urinary tract anomalies

CELSR3 codes for a planar cell polarity protein. We describe twelve affected individuals from eleven independent families with bi-allelic variants in CELSR3. Affected individuals presented with an overlapping phenotypic spectrum comprising central nervous system (CNS) anomalies (7/12), combined CNS anomalies and congenital anomalies of the kidneys and urinary tract (CAKUT) (3/12) and CAKUT only (2/12). Computational simulation of the 3D protein structure suggests the position of the identified variants to be implicated in penetrance and phenotype expression. CELSR3 immunolocalization in human embryonic urinary tract and transient suppression and rescue experiments of Celsr3 in fluorescent zebrafish reporter lines further support an embryonic role of CELSR3 in CNS and urinary tract formation.</p

International New Ventures: Revisiting the Influences Behind the 'Born-Global' Firm

There is a small but theoretically important literature on ‘born-globals’ or international new venture firms that positions itself in contrast to the more established sequential international entry literature. In this paper we examine the pattern of entry into international markets for a set of international new ventures and show that they need not be a distinct breed of firms, as previous research has portrayed. Absent a specific technological advantage, the decision for a new venture to internationalize at inception is influenced by the size of its home market and by its production capacity, as well as by cultural and economic forces that also influence other more traditional firms that stage their entry into international markets. Most importantly, we demonstrate that the decision to internationalize or not should be considered jointly with the capacity allocation decision to specific international markets, as analysing these separately may lead to biased results. Journal of International Business Studies (2007) 38, 1113–1131. doi:10.1057/palgrave.jibs.8400308

In silico toxicology protocols

The present publication surveys several applications of in silico (i.e., computational) toxicology approaches across different industries and institutions. It highlights the need to develop standardized protocols when conducting toxicity-related predictions. This contribution articulates the information needed for protocols to support in silico predictions for major toxicological endpoints of concern (e.g., genetic toxicity, carcinogenicity, acute toxicity, reproductive toxicity, developmental toxicity) across several industries and regulatory bodies. Such novel in silico toxicology (IST) protocols, when fully developed and implemented, will ensure in silico toxicological assessments are performed and evaluated in a consistent, reproducible, and well-documented manner across industries and regulatory bodies to support wider uptake and acceptance of the approaches. The development of IST protocols is an initiative developed through a collaboration among an international consortium to reflect the state-of-the-art in in silico toxicology for hazard identification and characterization. A general outline for describing the development of such protocols is included and it is based on in silico predictions and/or available experimental data for a defined series of relevant toxicological effects or mechanisms. The publication presents a novel approach for determining the reliability of in silico predictions alongside experimental data. In addition, we discuss how to determine the level of confidence in the assessment based on the relevance and reliability of the information

Retrospective evaluation of whole exome and genome mutation calls in 746 cancer samples

Funder: NCI U24CA211006Abstract: The Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium (ICGC) curated consensus somatic mutation calls using whole exome sequencing (WES) and whole genome sequencing (WGS), respectively. Here, as part of the ICGC/TCGA Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium, which aggregated whole genome sequencing data from 2,658 cancers across 38 tumour types, we compare WES and WGS side-by-side from 746 TCGA samples, finding that ~80% of mutations overlap in covered exonic regions. We estimate that low variant allele fraction (VAF < 15%) and clonal heterogeneity contribute up to 68% of private WGS mutations and 71% of private WES mutations. We observe that ~30% of private WGS mutations trace to mutations identified by a single variant caller in WES consensus efforts. WGS captures both ~50% more variation in exonic regions and un-observed mutations in loci with variable GC-content. Together, our analysis highlights technological divergences between two reproducible somatic variant detection efforts