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

Characterization of antibiotic resistomes by reprogrammed bacteriophage-enabled functional metagenomics in clinical strains

Functional metagenomics is a powerful experimental tool to identify antibiotic resistance genes (ARGs) in the environment, but the range of suitable host bacterial species is limited. This limitation affects both the scope of the identified ARGs and the interpretation of their clinical relevance. Here we present a functional metagenomics pipeline called Reprogrammed Bacteriophage Particle Assisted Multi-species Functional Metagenomics (DEEPMINE). This approach combines and improves the use of T7 bacteriophage with exchanged tail fibres and targeted mutagenesis to expand phage host-specificity and efficiency for functional metagenomics. These modified phage particles were used to introduce large metagenomic plasmid libraries into clinically relevant bacterial pathogens. By screening for ARGs in soil and gut microbiomes and clinical genomes against 13 antibiotics, we demonstrate that this approach substantially expands the list of identified ARGs. Many ARGs have species-specific effects on resistance; they provide a high level of resistance in one bacterial species but yield very limited resistance in a related species. Finally, we identified mobile ARGs against antibiotics that are currently under clinical development or have recently been approved. Overall, DEEPMINE expands the functional metagenomics toolbox for studying microbial communities

The detection of meningococcal disease through identification of antimicrobial peptides using an in silico model creation

Philosophiae Doctor - PhDNeisseria meningitidis (the meningococcus), the causative agent of meningococcal disease (MD) was identified in 1887 and despite effective antibiotics and partially effective vaccines, Neisseria meningitidis (N. meningitidis) is the leading cause worldwide of meningitis and rapidly fatal sepsis usually in otherwise healthy individuals. Over 500 000 meningococcal cases occur every year. These numbers have made bacterial meningitis a top ten infectious cause of death worldwide. MD primarily affects children under 5 years of age, although in epidemic outbreaks there is a shift in disease to older children, adolescents and adults. MD is also associated with marked morbidity including limb loss, hearing loss, cognitive dysfunction, visual impairment, educational difficulties, developmental delays, motor nerve deficits, seizure disorders and behavioural problems. Antimicrobial peptides (AMPs) are molecules that provide protection against environmental pathogens, acting against a large number of microorganisms, including bacteria, fungi, yeast and virus. AMPs production is a major component of innate immunity against infection. The chemical properties of AMPs allow them to insert into the anionic cell wall and phospholipid membranes of microorganisms or bind to the bacteria making it easily detectable for diagnostic purposes. AMPs can be exploited for the generation of novel antibiotics, as biomarkers in the diagnosis of inflammatory conditions, for the manipulation of the inflammatory process, wound healing, autoimmunity and in the combat of tumour cells. Due to the severity of meningitis, early detection and identification of the strain of N. meningitidis is vital. Rapid and accurate diagnosis is essential for optimal management of patients and a major problem for MD is its diagnostic difficulties and experts conclude that with an early intervention the patient’ prognosis will be much improved. It is becoming increasingly difficult to confirm the diagnosis of meningococcal infection by conventional methods. Although polymerase chain reaction (PCR) has the potential advantage of providing more rapid confirmation of the presence of the bacterium than culturing, it is still time consuming as well as costly. Introduction of AMPs to bind to N. meningitidis receptors could provide a less costly and time consuming solution to the current diagnostic problems. World Health Organization (WHO) meningococcal meningitis program activities encourage laboratory strengthening to ensure prompt and accurate diagnosis to rapidly confirm the presence of MD. This study aimed to identify a list of putative AMPs showing antibacterial activity to N. meningitidis to be used as ligands against receptors uniquely expressed by the bacterium and for the identified AMPs to be used in a Lateral Flow Device (LFD) for the rapid and accurate diagnosis of MD

Lichtabhängige Regulation der Photosynthese-Gene in Dinoroseobacter shibae

The potential LOV histidine kinase Dshi_1135 was found in a high-throughput screening of the D. shibae transposon mutant library for identification of unknow regulators of photosynthesis genes in the marine model bacterium D. shibae. First experiments indicated a role in the light-dependent regulation of these genes. This study focused on the biochemical characterization of Dshi_1135 and on its role in light-dependent regulation of photosynthesis genes. Furthermore, the function of the transcription regulator PpsR in photosynthesis gene expression was investigated. Soluble Dshi_1135 was recombinantly produced using a thioredoxin tag and the E. coli Lemo21(DE3) expression strain. Purification of the Dshi_1135 protein was performed by affinity chromatography using Strep Tactin® Superflow matrix. UV/Vis spectroscopy revealed that Dshi_1135 possesses a FMN cofactor. Dshi_1135 covalently binds FMN under blue light conditions but loses it in the dark. A reversible photocycle was observed for Dshi_1135 and a photoactive cysteine residue at position 61 was identified by site-directed mutagenesis. Phosphotransfer experiments showed an autophosphorylation of Dshi_1135 upon blue light irradiation. It was therefore clearly proven that Dshi_1135 is a blue-light activated LOV protein. Analyses of a bchF-lacZ reporter gene fusion revealed, that the expression of photosynthesis gene cluster is induced under dark conditions, but not under light and blue light conditions. All collected data regarding the role of Dshi_1135 in regulation of photosynthesis genes indicate, that this protein acts as a blue light sensor but is active under dark conditions, which would be rare exception among the LOV proteins studied to date. Finally, PpsR was shown to be a strong repressor of bchF expression under blue and white light and also slightly in the dark. Additionally, the palindromic sequence TGT-N12-ACA was identified as the respective PpsR binding motif. Overall, the LOV domain protein Dshi_1135 was identified and functionally characterized as a novel blue-light sensor and regulator of photosynthesis gene expression in the marine model bacterium D. shibae.Die potentielle LOV Histidinkinase Dshi_1135 wurde in einem Hochdurchsatz-Screening der D. shibae Transposonmutantenbank gefunden für die Identifizierung von bisher unbekannten Regulatoren von Photosynthese-Genen in dem marinen Modellorganismus D. shibae. Erste Experimente deuteten auf eine lichtabhängige Regulation dieser Gene hin. Diese Arbeit fokussierte sich auf die biochemische Charakterisierung von Dshi_1135 und dessen Rolle bei der lichtabhängigen Regulation der Photosynthese-Gene. Weiterhin wurde die Funktion des Transkriptionsfaktors PpsR bei der Expression der Photosynthese-Gene untersucht. Das lösliche Dshi_1135 Protein wurde mithilfe eines Thioredoxin-Tags rekombinant in dem Expressionsstamm E. coli Lemo21(DE3) produziert. Die anschließende Reinigung des Dshi_1135 Proteins erfolgte über eine Affinitätschromatographie mittles Strep Tactin® Superflow Säulenmaterial. Über UV/Vis Spektroskopie konnte gezeigt werden, dass Dshi_1135 einen FMN-Kofaktor besitzt. Dieser bindet unter Blaulichtbedingungen kovalent an das Dshi_1135 Protein, jedoch nicht unter Dunkelbedingungen. Dshi_1135 ist in der Lage einen Photozyklus zu durchlaufen und durch eine ortsspezifische Mutagenese konnte das photoaktive Cystein an Aminosäureposition 61 bestimmt werden. Durch Phosphotranser Experimente konnte die Autophosphorylierung von Dshi_1135 gezeigt werden. Somit wurde klar bestätigt, dass es sich bei Dshi_1135 um blaulicht-aktiviertes LOV Protein handelt. Die Untersuchungen der bchF-lacZ Reportergenfusion zeigten, dass die Expression des Photosynthese-Genclusters in Dunkelheit induziert wird, jedoch nicht und Licht- und Blaulichtbedingungen. Alle generierten Daten hinsichtlich der Rolle von Dshi_1135 in der Regulation von Photosynthese-Genen deuten darauf hin, dass es sich um einen Blaulichtsensor handelt, der jedoch unter Dunkelbedingungen aktiv ist. Dies würde eine seltene Ausnahme in der Familie der bisher untersuchten LOV Photorezeptoren darstellen. Außerdem konnte gezeigt werden, dass PpsR als Repressor agiert und die bchF-Expression unter Licht- und Blaulichteinfluss stark und unter Dunkelbedingungen leicht reprimiert. Zusätzlich wurde die Palindromische Sequenz TGT-N12-ACA als das Bindemotiv für PpsR identifiziert. Zusammenfassend wurde das LOV Protein Dshi_1135 funktional charakterisiert als neuer Blaulichtsensor und Regulator der Photosynthese-Gene in dem marinen Modellorganismus D. shibae

Higher Order Mutation Testing

Mutation testing is a fault-based software testing technique that has been studied widely for over three decades. To date, work in this field has focused largely on first order mutants because it is believed that higher order mutation testing is too computationally expensive to be practical. This thesis argues that some higher order mutants are potentially better able to simulate real world faults and to reveal insights into programming bugs than the restricted class of first order mutants. This thesis proposes a higher order mutation testing paradigm which combines valuable higher order mutants and non-trivial first order mutants together for mutation testing. To overcome the exponential increase in the number of higher order mutants a search process that seeks fit mutants (both first and higher order) from the space of all possible mutants is proposed. A fault-based higher order mutant classification scheme is introduced. Based on different types of fault interactions, this approach classifies higher order mutants into four categories: expected, worsening, fault masking and fault shifting. A search-based approach is then proposed for locating subsuming and strongly subsuming higher order mutants. These mutants are a subset of fault mask and fault shift classes of higher order mutants that are more difficult to kill than their constituent first order mutants. Finally, a hybrid test data generation approach is introduced, which combines the dynamic symbolic execution and search based software testing approaches to generate strongly adequate test data to kill first and higher order mutants

Reticulate Evolution: Symbiogenesis, Lateral Gene Transfer, Hybridization and Infectious heredity

info:eu-repo/semantics/publishedVersio

Investigation of the localisation of Filamentous temperature sensitive protein Z (FtsZ) in E. coli as a biomarker for antibacterial agents

Antimicrobial resistance is a growing problem worldwide, creating a need for novel antibacterial agents and strategies. Phage therapy is re-emerging as an alternative to antibiotics, though further research is needed before phages can be used clinically. Escherichia coli is a Gram-negative pathogen responsible for infection leading to meningitis and sepsis. FtsZ is a bacterial protein which forms the Z ring and is essential for cell division. Several agents blocking cell division mislocalise FtsZ, including the phage λ Kil peptide, resulting in defective division and a filamentous phenotype. This study aimed to use an in vitro meningitis model system to investigate the effects of phages and antibiotics on FtsZ using E. coli EV36/FtsZmCherry and K12/FtsZ-mNeon strains in hCMEC human brain cells, in an effort to validate FtsZ as a biomarker for testing novel antimicrobials against. It was shown that during normal growth FtsZ localises to the midbody as a single ring in the absence and presence of human cells. When the inhibitor Kil is applied, the cells become filamentous and FtsZ is mislocalised. When the phages T7, K1F-GFP, K1E and K1-5 were applied to their host strains, it was demonstrated that these phages can inhibit FtsZ to block division in the exponential phase, leading to filamentous multi-ringed cells. ZapA is essential for filamentation by T7 phage. The T7 peptide Gp0.4 was found responsible for inhibition of FtsZ after observing a phenotype recovery with a T7Δ0.4 mutant, and this peptide is absent in K1-targeting phages. Gp0.4 delays host cell lysis and increases phage progeny titres. The Φ phages do not mislocalise FtsZ during infection of pathogenic E. coli, so not all phages have evolved this mechanism. When antibiotics were applied to the model, it was demonstrated that each class of antibiotic led to a unique phenotype: the protein synthesis inhibitors led to filamentous cells with multiple Z rings along the body, the nucleic synthesis inhibitors led to very long filaments with multiple Z rings, and the cell wall synthesis inhibitors led to filamentous cells with two distinct non-polar rings. Collectively, these results improve current knowledge of how phages interact with and inhibit host cell division, and show that different antimicrobials lead to different phenotypes of FtsZ mislocalisation, supporting its potential use as a biomarker to understand how new antimicrobials, including phages, impact cell division