Test Quality Assurance for E2E Web Test Suites: Parallelization of Dependent Test Suites and Test Flakiness Prevention

Web applications support a wide range of activities today, from e-commerce to health management, and ensuring their quality is a fundamental task. Nevertheless, testing these systems is hard because of their dynamic and asynchronous nature and their heterogeneity. Quality assurance of Web applications is usually performed through testing, performed at different levels of abstraction. At the End-to-end (E2E) level, test scripts interact with the application through the web browser, as a human user would do. This kind of testing is usually time consuming, and its execution time can be reduced by running the test suite in parallel. However, the presence of dependen- cies in the test suite can make test parallelization difficult. Best practices prescribe that test scripts in a test suite should be independent (i.e. they should not assume that the system under test is already in an expected state), but this is not always done in practice: dependent tests are a serious problem that affects end-to-end web test suites. Moreover, test dependencies are a problem because they enforce an execution order for the test suite, preventing the use of techniques like test selection, test prioritization, and test parallelization. Another issue that affects E2E Web test suites is test flakiness: a test script is called flaky when it may non-deterministically pass or fail on the same version of the Ap- plication Under Test. Test flakiness is usually caused by multiple factors, that can be very hard to determine: most common causes of flakiness are improper waiting for async operations, not respected test order dependencies and concurrency problems (e.g. race conditions, deadlocks, atomicity violations). Test flakiness is a problem that affects E2E test execution in general, but it can have a greater impact in presence of dependencies, since 1) if a test script fails due to flakiness, other test scripts that depend on it will probably fail as well, 2) most dependency-detection approaches and tools rely on multiple executions of test schedules in different orders to detect dependencies. In order to do that, execution results must be deterministic: if test scripts can pass or fail non-deterministically, those dependency detection tools can not work. This thesis proposes to improve the quality assurance for E2E Web test suites in two different directions: 1. enabling the parallel execution of dependent E2E Web test suites in a opti- mized, efficient way 2. preventing flakiness by automated refactoring of E2E Web test suites, in order to adopt the proper waiting strategies for page elements For the first research direction we propose STILE (teST suIte paralLElizer), a tool- based approach that allows parallel execution of E2E Web test suites. Our approach generates a set of test schedules that respect two important constraints: 1) every schedule respects existing test dependencies, 2) all test scripts in the test suite are executed at least once, considering all the generated schedules. For the second research direction we propose SleepReplacer, a tool-based approach to automatically refactor E2E Web test suites in order to prevent flakiness. Both of the tool-based approaches has been fully implemented in two functioning and publicly available tools, and empirically validated on different test suites

Valproic acid up-regulates p75NTR and sortilin expression to induce neuronal cell death

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Lysophosphatidic Acid Stimulates Mitogenic Activity and Signaling in Human Neuroblastoma Cells through a Crosstalk with Anaplastic Lymphoma Kinase

Lysophosphatidic acid (LPA) is a well-documented pro-oncogenic factor in different cancers, but relatively little is known on its biological activity in neuroblastoma. The LPA effects and the participation of the tyrosine kinase receptor anaplastic lymphoma kinase (ALK) in LPA mitogenic signaling were studied in human neuroblastoma cell lines. We used light microscopy and [3H]-thymidine incorporation to determine cell proliferation, Western blot to study intracellular signaling, and pharmacological and molecular tools to examine the role of ALK. We found that LPA stimulated the growth of human neuroblastoma cells, as indicated by the enhanced cell number, clonogenic activity, and DNA synthesis. These effects were curtailed by the selective ALK inhibitors NPV-TAE684 and alectinib. In a panel of human neuroblastoma cell lines harboring different ALK genomic status, the ALK inhibitors suppressed LPA-induced phosphorylation of extracellular signal-regulated kinases 1/2 (ERK1/2), which are major regulators of cell proliferation. ALK depletion by siRNA treatment attenuated LPA-induced ERK1/2 activation. LPA enhanced ALK phosphorylation and potentiated ALK activation by the ALK ligand FAM150B. LPA enhanced the inhibitory phosphorylation of the tumor suppressor FoxO3a, and this response was impaired by the ALK inhibitors. These results indicate that LPA stimulates mitogenesis of human neuroblastoma cells through a crosstalk with ALK

Differential targeting of lysophosphatidic acid LPA1, LPA2, and LPA3 receptor signalling by tricyclic and tetracyclic antidepressants

We previously reported that in different cell types antidepressant drugs activate lysophosphatidic acid (LPA) LPA1 receptor to induce proliferative and prosurvival responses. Here, we further characterize this unique action of antidepressants by examining their effects on two additional LPA receptor family members, LPA2 and LPA3. Human LPA1-3 receptors were stably expressed in HEK-293 cells (HEK-LPA1, -LPA2 and -LPA3 cells) and their functional activity was determined by Western blot and immunofluorescence. LPA effectively stimulated the phosphorylation of extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) in HEK-LPA1, -LPA2, and -LPA3 cells. The tricyclic antidepressants amitriptyline, clomipramine, imipramine and desipramine increased phospho-ERK1/2 levels in HEK-LPA1 and -LPA3 cells but were relatively poor agonists in LPA2-expressing cells. The tetracyclic antidepressants mianserin and mirtazapine were active at all three LPA receptors. When combined with LPA, both amitriptyline and mianserin potentiated Gi/o-mediated phosphorylation of ERK1/2 induced by LPA in HEK-LPA1, -LPA2 and -LPA3 cells, CHO-K1 fibroblasts and HT22 hippocampal neuroblasts. This potentiation was associated with enhanced phosphorylation of CREB and S6 ribosomal protein, two molecular targets of activated ERK1/2. The antidepressants also potentiated LPA-induced Gq/11-mediated phosphorylation of AMP-activated protein kinase in HEK-LPA1 and -LPA3 cells. Conversely, amitriptyline and mianserin were found to inhibit LPA-induced Rho activation in HEK-LPA1 and LPA2 cells. These results indicate that tricyclic and tetracyclic antidepressants can act on LPA1, LPA2 and LPA3 receptor subtypes and exert differential effects on LPA signalling through these receptors

The Neurotrophin Receptor TrkC as a Novel Molecular Target of the Antineuroblastoma Action of Valproic Acid

Neurotrophins and their receptors are relevant factors in controlling neuroblastoma growth and progression. The histone deacetylase (HDAC) inhibitor valproic acid (VPA) has been shown to downregulate TrkB and upregulate the p75NTR/sortilin receptor complex. In the present study, we investigated the VPA effect on the expression of the neurotrophin-3 (NT-3) receptor TrkC, a favorable prognostic marker of neuroblastoma. We found that VPA induced the expression of both full-length and truncated (TrkC-T1) isoforms of TrkC in human neuroblastoma cell lines without (SH-SY5Y) and with (Kelly, BE(2)-C and IMR 32) MYCN amplification. VPA enhanced cell surface expression of the receptor and increased Akt and ERK1/2 activation by NT-3. The HDAC inhibitors entinostat, romidepsin and vorinostat also increased TrkC in SH-SY5Y, Kelly and BE(2)-C but not IMR 32 cells. TrkC upregulation by VPA involved induction of RUNX3, stimulation of ERK1/2 and JNK, and ERK1/2-mediated Egr1 expression. In SH-SY5Y cell monolayers and spheroids the exposure to NT-3 enhanced the apoptotic cascade triggered by VPA. Gene silencing of both TrkC-T1 and p75NTR prevented the NT-3 proapoptotic effect. Moreover, NT-3 enhanced p75NTR/TrkC-T1 co-immunoprecipitation. The results indicate that VPA upregulates TrkC by activating epigenetic mechanisms and signaling pathways, and sensitizes neuroblastoma cells to NT-3-induced apoptosis

Identification and characterization of muscarinic receptors potentiating the stimulation of adenylyl cyclase activity by corticotropin-releasing hormone in membranes of rat frontal cortex. J Pharmacol Exp Ther.

ABSTRACT In membranes of the rat frontal cortex, acetylcholine (ACh) and other cholinergic agonists were found to potentiate the stimulation of adenylyl cyclase activity elicited by corticotropin-releasing hormone (CRH). Oxotremorine-M, carbachol and methacholine were as effective as ACh, whereas oxotremorine and arecoline were much less effective. The facilitating effect of Ach was potently blocked by the M 1 antagonists R-trihexyphenidyl, telenzepine and pirenzepine and by the M 3 antagonists hexahydro-sila-difenidol and p-fluorohexahydro-siladifenidol, whereas the M 2 and M 4 antagonists himbacine, methoctramine, AF-DX 116 and AQ-RA 741 were less potent. The mamba venom toxin MT-1, which binds with high affinity to M 1 receptors, was also a potent blocker. The pharmacological profile of the muscarinic potentiation of CRH receptor activity was markedly different from that displayed by the muscarinic inhibition of forskolin-stimulated adenylyl cyclase, which could be detected in the same membrane preparations. Moreover, the intracerebral injection of pertussis toxin impaired the muscarinic inhibition of cyclic AMP formation and reduced the Ach stimulation of [ 35 S]GTP␥S binding to membrane G proteins but failed to affect the facilitating effect on CRH receptor activity. The latter response was also insensitive to the phospholipase C inhibitor U-73122, the protein kinase inhibitor staurosporine and to the inhibitors of arachidonic acid metabolism indomethacin and nordihydroguaiaretic acid. These data demonstrate that in the rat frontal cortex, muscarinic receptors of the M 1 subtype potentiate CRH transmission by interacting with pertussis toxin-insensitive G proteins

Saliva, a bodily fluid with recognized and potential diagnostic applications

Human whole saliva is a bodily fluid that can be obtained easily by noninvasive techniques. Specimens can be collected by the patient also at home in order to monitor health status and variations of several analytes of clinical interest. The contributions to whole saliva include secretions from salivary glands and, among others, from the gingival crevicular fluid that derives from the epithelial mucosa. Therefore, saliva is currently a relevant diagnostic fluid for many substances, including steroids, nonpeptide hormones, therapeutic drugs, and drugs of abuse. This review at first briefly describes the different contributions to whole saliva. A section illustrates the procedures for the collection, handling, and storage of salivary specimens. Another section describes the present use of whole saliva for diagnostic purposes and its specific utilization for the diagnosis of several local and systemic diseases. The final sections illustrate the future opportunities offered by various not conventional techniques with a focus on the most recent –omic investigations. It describes the various issues that have to be taken into account to avoid false positives and negatives, such as the strength of the experimental plan, the adequacy of the number of samples under study, and the proper choice of controls

Salivary Cystatin D Interactome in Patients with Systemic Mastocytosis: An Exploratory Study

Mastocytosis, a rare blood disorder characterized by the proliferation of clonal abnormal mast cells, has a variegated clinical spectrum and diagnosis is often difficult and delayed. Recently we proposed the cathepsin inhibitor cystatin D-R26 as a salivary candidate biomarker of systemic mastocytosis (SM). Its C26 variant is able to form multiprotein complexes (mPCs) and since protein–protein interactions (PPIs) are crucial for studying disease pathogenesis, potential markers, and therapeutic targets, we aimed to define the protein composition of the salivary cystatin D-C26 interactome associated with SM. An exploratory affinity purification-mass spectrometry method was applied on pooled salivary samples from SM patients, SM patient subgroups with and without cutaneous symptoms (SM+C and SM−C), and healthy controls (Ctrls). Interactors specifically detected in Ctrls were found to be implicated in networks associated with cell and tissue homeostasis, innate system, endopeptidase regulation, and antimicrobial protection. Interactors distinctive of SM−C patients participate to PPI networks related to glucose metabolism, protein S-nitrosylation, antibacterial humoral response, and neutrophil degranulation, while interactors specific to SM+C were mainly associated with epithelial and keratinocyte differentiation, cytoskeleton rearrangement, and immune response pathways. Proteins sensitive to redox changes, as well as proteins with immunomodulatory properties and activating mast cells, were identified in patients; many of them were involved directly in cytoskeleton rearrangement, a process crucial for mast cell activation. Although preliminary, these results demonstrate that PPI alterations of the cystatin D-C26 interactome are associated with SM and provide a basis for future investigations based on quantitative proteomic analysis and immune validation

Top-Down Proteomics of Human Saliva Highlights Anti-inflammatory, Antioxidant, and Antimicrobial Defense Responses in Alzheimer Disease

Alzheimer disease (AD) is the most prevalent neurodegenerative disease in the elderly, characterized by accumulation in the brain of misfolded proteins, inflammation, and oxidative damage leading to neuronal cell death. By considering the viewpoint that AD onset and worsening may be influenced by environmental factors causing infection, oxidative stress, and inflammatory reaction, we investigated the changes of the salivary proteome in a population of patients with respect to that in healthy controls (HCs). Indeed, the possible use of saliva as a diagnostic tool has been explored in several oral and systemic diseases. Moreover, the oral cavity continuously established adaptative and protective processes toward exogenous stimuli. In the present study, qualitative/quantitative variations of 56 salivary proteoforms, including post-translationally modified derivatives, have been analyzed by RP-HPLC-ESI-IT-MS and MS/MS analyses, and immunological methods were applied to validate MS results. The salivary protein profile of AD patients was characterized by significantly higher levels of some multifaceted proteins and peptides that were either specific to the oral cavity or also expressed in other body districts: (i) peptides involved in the homeostasis of the oral cavity; (ii) proteins acting as ROS/RNS scavengers and with a neuroprotective role, such as S100A8, S100A9, and their glutathionylated and nitrosylated proteoforms; cystatin B and glutathionylated and dimeric derivatives; (iii) proteins with antimicrobial activity, such as α-defensins, cystatins A and B, histatin 1, statherin, and thymosin β4, this last with a neuroprotective role at the level of microglia. These results suggested that, in response to injured conditions, Alzheimer patients established defensive mechanisms detectable at the oral level. Data are available via ProteomeXchange with identifier PXD021538

A Catalog of Coding Sequence Variations in Salivary Proteins’ Genes Occurring during Recent Human Evolution

Saliva houses over 2000 proteins and peptides with poorly clarified functions, including proline-rich proteins, statherin, P-B peptides, histatins, cystatins, and amylases. Their genes are poorly conserved across related species, reflecting an evolutionary adaptation. We searched the nucleotide substitutions fixed in these salivary proteins’ gene loci in modern humans compared with ancient hominins. We mapped 3472 sequence variants/nucleotide substitutions in coding, noncoding, and 5′-3′ untranslated regions. Despite most of the detected variations being within noncoding regions, the frequency of coding variations was far higher than the general rate found throughout the genome. Among the various missense substitutions, specific substitutions detected in PRB1 and PRB2 genes were responsible for the introduction/abrogation of consensus sequences recognized by convertase enzymes that cleave the protein precursors. Overall, these changes that occurred during the recent human evolution might have generated novel functional features and/or different expression ratios among the various components of the salivary proteome. This may have influenced the homeostasis of the oral cavity environment, possibly conditioning the eating habits of modern humans. However, fixed nucleotide changes in modern humans represented only 7.3% of all the substitutions reported in this study, and no signs of evolutionary pressure or adaptative introgression from archaic hominins were found on the tested genes