Chemical traits of cerebral amyloid angiopathy in familial British-, Danish-, and non-Alzheimerʼs dementias

Familial British dementia (FBD) and familial Danish dementia (FDD) are autosomal dominant forms of dementia caused by mutations in the integral membrane protein 2B (ITM2B, also known as BRI2) gene. Secretase processing of mutant BRI2 leads to secretion and deposition of BRI2-derived amyloidogenic peptides, ABri and ADan that resemble APP/β-amyloid (Aβ) pathology, which is characteristic of Alzheimer's disease (AD). Amyloid pathology in FBD/FDD manifests itself predominantly in the microvasculature by ABri/ADan containing cerebral amyloid angiopathy (CAA). While ABri and ADan peptide sequences differ only in a few C-terminal amino acids, CAA in FDD is characterized by co-aggregation of ADan with Aβ, while in contrast no Aβ deposition is observed in FBD. The fact that FDD patients display an earlier and more severe disease onset than FBD suggests a potential role of ADan and Aβ co-aggregation that promotes a more rapid disease progression in FDD compared to FBD. It is therefore critical to delineate the chemical signatures of amyloid aggregation in these two vascular dementias. This in turn will increase the knowledge on the pathophysiology of these diseases and the pathogenic role of heterogenous amyloid peptide interactions and deposition, respectively. Herein, we used matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) in combination with hyperspectral, confocal microscopy based on luminescent conjugated oligothiophene probes (LCO) to delineate the structural traits and associated amyloid peptide patterns of single CAA in postmortem brain tissue of patients with FBD, FDD as well as sporadic CAA without AD (CAA+) that show pronounced CAA without parenchymal plaques. The results show that CAA in both FBD and FDD consist of N-terminally truncated- and pyroglutamate-modified amyloid peptide species (ADan and ABri), but that ADan peptides in FDD are also extensively C-terminally truncated as compared to ABri in FBD, which contributes to hydrophobicity of ADan species. Further, CAA in FDD showed co-deposition with Aβ x-42 and Aβ x-40 species. CAA+ vessels were structurally more mature than FDD/FBD CAA and contained significant amounts of pyroglutamated Aβ. When compared with FDD, Aβ in CAA+ showed more C-terminal and less N-terminally truncations. In FDD, ADan showed spatial co-localization with Aβ3pE-40 and Aβ3-40 but not with Aβx-42 species. This suggests an increased aggregation propensity of Aβ in FDD that promotes co-aggregation of both Aβ and ADan. Further, CAA maturity appears to be mainly governed by Aβ content based on the significantly higher 500/580 patterns observed in CAA+ than in FDD and FBD, respectively. Together this is the first study of its kind on comprehensive delineation of Bri2 and APP-derived amyloid peptides in single vascular plaques in both FDD/FBD and sporadic CAA that provides new insight in non-AD-related vascular amyloid pathology. (Figure presented.

Evaluating Software Testing Techniques: A Systematic Mapping Study

Software testing techniques are crucial for detecting faults in software and reducing the risk of using it. As such, it is important that we have a good understanding of how to evaluate these techniques for their efficiency, scalability, applicability, and effectiveness at finding faults. This thesis enhances our understanding of testing technique evaluations by providing an overview of the state of the art in research. To accomplish this we utilize a systematic mapping study; structuring the field and identifying research gaps and publication trends. We then present a small case study demonstrating how our mapping study can be used to assist researchers in evaluating their own software testing techniques. We find that a majority of evaluations are empirical evaluations in the form of case studies and experiments, most of these evaluations are of low quality based on proper methodology guidelines, and that relatively few papers in the field discuss how testing techniques should be evaluated

Identification of Cytokinin-Responsive Genes Using Microarray Meta-Analysis and RNA-Seq in Arabidopsis

Cytokinins are N6-substituted adenine derivatives that play diverse roles in plant growth and development. We sought to define a robust set of genes regulated by cytokinin as well as to query the response of genes not represented on microarrays. To this end, we performed a meta-analysis of microarray data from a variety of cytokinin-treated samples and used RNA-seq to examine cytokinin-regulated gene expression in Arabidopsis (Arabidopsis thaliana). Microarray meta-analysis using 13 microarray experiments combined with empirically defined filtering criteria identified a set of 226 genes differentially regulated by cytokinin, a subset of which has previously been validated by other methods. RNA-seq validated about 73% of the up-regulated genes identified by this meta-analysis. In silico promoter analysis indicated an overrepresentation of type-B Arabidopsis response regulator binding elements, consistent with the role of type-B Arabidopsis response regulators as primary mediators of cytokinin-responsive gene expression. RNA-seq analysis identified 73 cytokinin-regulated genes that were not represented on the ATH1 microarray. Representative genes were verified using quantitative reverse transcription-polymerase chain reaction and NanoString analysis. Analysis of the genes identified reveals a substantial effect of cytokinin on genes encoding proteins involved in secondary metabolism, particularly those acting in flavonoid and phenylpropanoid biosynthesis, as well as in the regulation of redox state of the cell, particularly a set of glutaredoxin genes. Novel splicing events were found in members of some gene families that are known to play a role in cytokinin signaling or metabolism. The genes identified in this analysis represent a robust set of cytokinin-responsive genes that are useful in the analysis of cytokinin function in plants

Mutation testing in the wild: findings from GitHub

Mutation testing exploits artificial faults to measure the adequacy of test suites and guide their improvement. It has become an extremely popular testing technique as evidenced by the vast literature, numerous tools, and research events on the topic. Previous survey papers have successfully compiled the state of research, its evolution, problems, and challenges. However, the use of mutation testing in practice is still largely unexplored. In this paper, we report the results of a thorough study on the use of mutation testing in GitHub projects. Specifically, we first performed a search for mutation testing tools, 127 in total, and we automatically searched the GitHub repositories including evidence of their use. Then, we focused on the top ten most widely used tools, based on the previous results, and manually revised and classified over 3.5K GitHub active repositories importing them. Among other findings, we observed a recent upturn in interest and activity, with Infection (PHP), PIT (Java) and Humbug (PHP) being the most widely used mutation tools in recent years. The predominant use of mutation testing is development, followed by teaching and learning, and research projects, although with significant differences among mutation tools found in the literature—less adopted and largely used in teaching and research—and those found in GitHub only—more popular and more widely used in development. Our work provides a new and encouraging perspective on the state of practice of mutation testing.Junta de Andalucía US-1264651 (APOLO)Junta de Andalucía P18-FR-2895 (EKIPMENT-PLUS)Ministerio de Ciencia, Innovación y Universidades RTI2018-101204-B-C21 (HORATIO)Ministerio de Ciencia, Innovación y Universidades RTI2018-093608-BC33 (FAME

Live-cell imaging reveals subcellular localization of plant membrane compartments during oomycete infections and quantitative high-throughput imaging identifies endocytic trafficking mutants

To successfully infect plants, filamentous pathogens such as the oomycete Hyaloperonospora arabidopsidis (Hpa) are able to penetrate host tissues and form haustoria, a feeding structure, inside the host cell. Reorganization of the host cell is required to accommodate the haustoria. Formation of haustoria is accompanied by the biogenesis of the extrahaustorial membrane (EHM) which surrounds the haustorium and separates the host cell from the pathogen. In this study, available fluorescent marker protein fusions were used to monitor the re-distribution of membrane compartments at the interface between Arabidopsis and Hpa. The aquaporin PIP1;4, the ATPase ACA8, and the plasma membrane (PM) intrinsic protein NPSN12 were excluded from the EHM while the syntaxin PEN1 and the receptor-like kinase FLS2 labelled the EHM. This suggests PM-resident proteins are recruited to the EHM selectively. The nucleus is always observed adjacent to haustoria, suggesting that the presence of haustoria causes migration of the nucleus. Secretory vesicles and endosomal compartments localize around the haustoria, implicating secretory and endocytic pathways in the biogenesis of the EHM. Upon Hpa infection, haustorial encasements develop around mature haustoria. All examined plant proteins accumulate at haustorial encasements, indicating that formation of encasements is derived by default redirection of vesicle trafficking pathways. With the aim to genetically dissect endosomal trafficking regulators, I took advantage of quantitative high throughput confocal imaging system and transgenic plants containing the fluorescent biosensor GFP-2xFYVE to perform a forward genetic screen. Different numbers of GFP-2xFYVE positive endosomes were found in two reference lines, Ler/GFP-2xFYVE and Col-0/YFP-2xFYVE suggesting the endosomal levels may vary in different ecotypes of Arabidopsis. Mutants with altered numbers of FYVE Endosomal Levels (fel) have been previous identified and were re-confirmed in this study. fel1, fel2, fel3, fel6, fel9, and fel12 revealed genetically recessive mutations while fel10 could not reveal its genetic inheritance. Two mutants, fel2 and fel9 exhibited more GFP-2xFYVE compartments than wild-type reference plants. These two mutants are affected in endosome trafficking and fel2 is likely tissue specific. We identified gene loci by classical mapping and whole genome sequencing. Fel2 co-segregated with the lower arm of chromosome 4. Fel9 was mapped to two chromosome loci. Investigation of genes in the rough mapping region will unravel regulators of endocytosis or multivesicular bodies (MVBs) biogenesis. Because only few mutant phenotypes recovered in the F2 of backcrossed fel2 and fel9, identification of FEL2 and FEL9 was hampered. Additionally, basal differences of endosomal numbers in the reference lines lead to the limitation for genetic screen based on quantitative changes in endosomal numbers. Altogether, these results show that there are common elements in the subcellular changes associated with biotrophic oomycete between different pathogens. For Hpa and other fungal/oomycete pathogens, reprogramming host cell vesicle trafficking occurs to accommodate haustorial structures. A genetic screen for novel endocytosis mutants, based on quantitative measurements of endosomal numbers, was performed with advanced microscopy technology. Fel mutant plants may be further used to study molecular mechanisms for membrane trafficking, as well as subcellular rearrangement in plant-pathogen interactions

Systematic Model-based Design Assurance and Property-based Fault Injection for Safety Critical Digital Systems

With advances in sensing, wireless communications, computing, control, and automation technologies, we are witnessing the rapid uptake of Cyber-Physical Systems across many applications including connected vehicles, healthcare, energy, manufacturing, smart homes etc. Many of these applications are safety-critical in nature and they depend on the correct and safe execution of software and hardware that are intrinsically subject to faults. These faults can be design faults (Software Faults, Specification faults, etc.) or physically occurring faults (hardware failures, Single-event-upsets, etc.). Both types of faults must be addressed during the design and development of these critical systems. Several safety-critical industries have widely adopted Model-Based Engineering paradigms to manage the design assurance processes of these complex CPSs. This thesis studies the application of IEC 61508 compliant model-based design assurance methodology on a representative safety-critical digital architecture targeted for the Nuclear power generation facilities. The study presents detailed experiences and results to demonstrate the benefits of Model testing in finding design flaws and its relevance to subsequent verification steps in the workflow. Additionally, to study the impact of physical faults on the digital architecture we develop a novel property-based fault injection method that overcomes few deficiencies of traditional fault injection methods. The model-based fault injection approach presented here guarantees high efficiency and near-exhaustive input/state/fault space coverage, by utilizing formal model checking principles to identify fault activation conditions and prove the fault tolerance features. The fault injection framework facilitates automated integration of fault saboteurs throughout the model to enable exhaustive fault location coverage in the model

Emerg Infect Dis

PMC4550154611

Genomic epidemiology: from transmission to the evolution of pathogenic microorganisms

La epidemiología genómica ha revolucionado el mundo microbiano. Sus aplicaciones permiten conocer y comprender mejor el comportamiento, la transmisión y la evolución de los microorganismos. A pesar de su gran y reconocido potencial, su uso aún es limitado, especialmente en regiones en desarrollo. El principal objetivo de esta tesis fue estudiar y analizar los genomas completos de tres microorganismos patógenos y zoonóticos para comprender mejor su epidemiología, potencial patogénico y evolución. En el primer capítulo caracterizamos a nivel genómico a Salmonella enterica aislada de alimentos, fincas y muestras clínicas en Ecuador. Además, presentamos evidencia de la presencia de factores de virulencia y de un megaplásmido con genes que codifican resistencia antimicrobiana y tolerancia al estrés ambiental. Debido a la relación filogenética entre los aislamientos, se concluye la transmisión de S. enterica a través del consumo de pollo en Ecuador, y la necesidad urgente de un mejor control del uso de antimicrobianos en las granjas avícolas. En el segundo capítulo se aplicó la epidemiología genómica a aislamientos de Listeria monocytogenes, un microorganismo que ha recibido poca atención en el Ecuador a pesar de su alta tasa de hospitalización y mortalidad a nivel mundial. Comparamos los genomas de aislados clínicos y alimentarios e inferimos la relación epidemiológica a través de la cadena alimentaria con los humanos en este país. Caracterizamos aislados que, a pesar de su susceptibilidad a los antimicrobianos, tienen potencial patógeno significativo debido a la presencia de islas de patogenicidad asociadas con enfermedad severa. Además, establecimos la necesidad de implementar una vigilancia de rutina de este patógeno y buscar otros alimentos como posibles fuentes de transmisión de listeriosis en Ecuador. El tercer capítulo se centró en la evaluación de la recombinación genética entre especies patógenas de Leptospira. El análisis se realizó entre especies, intra L. interrogans y entre los genes del locus LPS, importante para el diagnóstico y desarrollo de vacunas para leptospirosis. Con los resultados de esta sección, sugerimos la existencia de una presión selectiva que promueve la variación genética de este locus y de varios genes, haciendo de Leptospira un patógeno zoonótico exitoso.Genomic epidemiology has revolutionized the microbial world. Its applications allow to know and better understand the behavior, transmission and evolution of microorganisms. Despite its great and recognized potential, its use is still limited, especially in developing regions. The main objective of this thesis was to study and analyze the complete genomes of three pathogenic and zoonotic microorganisms to better understand their epidemiology, pathogenic potential and evolution. In the first chapter, we characterized at the genomic level Salmonella enterica isolated from food, farms, and clinical samples in Ecuador. In addition, we presented evidence of the presence of virulence factors and a megaplasmid with genes encoding antimicrobial resistance and tolerance to environmental stress. Due to the phylogenetic relationship between isolates, we conclude the transmission of S. enterica through the consumption of chicken in Ecuador, and the urgent need for better control of the use of antimicrobials in poultry farms. In the second chapter, genomic epidemiology was applied to isolates of Listeria monocytogenes, a microorganism that has received little attention in Ecuador despite its high rate of hospitalization and mortality worldwide. We compared the genomes of clinical and food isolates and inferred the epidemiological relationship through the food chain to humans in this country. We characterized isolates that, despite their susceptibility to antimicrobials, have significant pathogenic potential due to the presence of pathogenicity islands associated with severe disease. In addition, we established the need to implement routine surveillance of this pathogen and to search for other foods as possible sources of transmission of listeriosis in Ecuador. The third chapter focused on the evaluation of genetic recombination among pathogenic species of Leptospira. The analysis was performed amid species, intra L. interrogans and among genes of the LPS locus, important for the diagnosis and development of vaccines for leptospirosis. With the results of this section, we suggest the existence of a selective pressure promoting genetic variation of this locus and of several genes, making of Leptospira a successful zoonotic pathogen