Semantics of trace relations in requirements models for consistency checking and inferencing

Requirements traceability is the ability to relate requirements back to stakeholders and forward to corresponding design artifacts, code, and test cases. Although considerable research has been devoted to relating requirements in both forward and backward directions, less attention has been paid to relating requirements with other requirements. Relations between requirements influence a number of activities during software development such as consistency checking and change management. In most approaches and tools, there is a lack of precise definition of requirements relations. In this respect, deficient results may be produced. In this paper, we aim at formal definitions of the relation types in order to enable reasoning about requirements relations. We give a requirements metamodel with commonly used relation types. The semantics of the relations is provided with a formalization in first-order logic. We use the formalization for consistency checking of relations and for inferring new relations. A tool has been built to support both reasoning activities. We illustrate our approach in an example which shows that the formal semantics of relation types enables new relations to be inferred and contradicting relations in requirements documents to be determined. The application of requirements reasoning based on formal semantics resolves many of the deficiencies observed in other approaches. Our tool supports better understanding of dependencies between requirements

Scientific investigation into the water sensitivity of twentieth century oil paints

In order to develop a better understanding at the molecular level of water sensitivity in twentieth century oil paintings, water sensitive Winsor & Newton oil paint swatches and twentieth century oil paintings were characterised using gas chromatography–mass spectrometry (GC–MS) and direct injection electrospray ionisation mass spectrometry (ESI-MS), and the data were analysed using principal component analysis. Liquid chromatography coupled with tandem mass spectrometry based on quadrupole and time of flight mass detectors and electrospray interface (HPLC-ESI-Q-ToF) was also used to obtain a better insight into the molecular composition of a selection of samples. The study highlights a strong relationship between the molecular composition of the binding medium and the type of pigment present in the paint, which relates to water sensitivity. Consistently non-water sensitive lead white, titanium white, and zinc white paints [all containing zinc oxide] contained a relatively low proportion of extractable diacids, and a relatively high proportion of extractable short chain monoacids. These paints also contained a relatively low level of unsaturated and hydroxylated glycerides. Water sensitive iron oxide and ultramarine paints are associated with both a relatively high degree of oxidation and a high proportion of extractable diacids, as well as a relatively high content of unsaturated and hydroxylated glycerides. Water sensitive cadmium red, yellow and orange paints were generally not highly oxidised, but they also contained a relatively high content of unsaturated and hydroxylated glycerides. It is hypothesised that water sensitivity relates to a low degree of saponification and crosslinking and possibly, on the relative content of dicarboxylic acids

Correction to: Conservation of Modern Oil Paintings

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Low Socioeconomic Status Is Associated with Worse Outcomes After Curative Surgery for Colorectal Cancer: Results from a Large, Multicenter Study

Background: Socioeconomic status (SES) has been associated with early mortality in cancer patients. However, the association between SES and outcome in colorectal cancer patients is largely unknown. The aim of this study was to investigate whether SES is associated with short- and long-term outcome in patients undergoing curative surgery for colorectal cancer. Methods: Patients who underwent curative surgery in the region of Rotterdam for stage I–III colorectal cancer between January 2007 and July 2014 were included. Gross household income and survival status were obtained from a national registry provided by Statistics Netherlands Centraal Bureau voor de Statistiek. Patients were assigned percentiles according to the national income distribution. Logistic regression and Cox proportional hazard regression were performed to assess the association of SES with 30-day postoperative complications, overall survival and cancer-specific survival, adjusted for known prognosticators. Results: For 965 of the 975 eligible patients (99%), gross household income could be retrieved. Patients with a lower SES more often had diabetes, more often underwent an open surgical procedure, and had more comorbidities. In addition, patients with a lower SES were less likely to receive (neo) adjuvant treatment. Lower SES was independently associated with an increased risk of postoperative complications (Odds rati

Functional Studies on Primary Tubular Epithelial Cells Indicate a Tumor Suppressor Role of SETD2 in Clear Cell Renal Cell Carcinoma

SET domain-containing 2 (SETD2) is responsible for the trimethylation of histone H3 lysine36 (H3K36me3) and is one of the genes most frequently mutated in clear cell renal cell carcinoma (ccRCC). It is located at 3p21, one copy of which is lost in the majority of ccRCC tumors, suggesting that SETD2 might function as a tumor suppressor gene. However, the manner in which loss of SETD2 contributes to ccRCC development has not been studied in renal primary tubular epithelial cells (PTECs). Therefore, we studied the consequences of SETD2 knockdown through lentiviral shRNA in human PTECs. Consistent with its known function, SETD2 knockdown (SETD-KD) led to loss of H3K36me3 in PTECs. In contrast to SETD2 wild-type PTECs, which have a limited proliferation capacity; the SETD2-KD PTECs continued to proliferate. The expression profiles of SETD2-KD PTECs showed a large overlap with the expression profile of early-passage, proliferating PTECs, whereas nonproliferating PTECs showed a significantly different expression profile. Gene set enrichment analysis revealed a significant enrichment of E2F targets in SETD2-KD and proliferating PTECs as compared with nonproliferating PTECs and in proliferating PTEC compared with SETD2-KD. The SETD2-KD PTECs maintained low expression of CDKN2A and high expression of E2F1, whereas their levels changed with continuing passages in untreated PTECs. In contrast to the nonproliferating PTECs, SETD2-KD PTECs showed no β-galactosidase staining, confirming the protection against senescence. Our results indicate that SETD2 inactivation enables PTECs to bypass the senescence barrier, facilitating a malignant transformation toward ccRCC

Involvement of MicroRNAs in the Aging-Related Decline of CD28 Expression by Human T Cells

Loss of CD28 is a characteristic feature of T cell aging, but the underlying mechanisms of this loss are elusive. As differential expression of microRNAs (miRNAs) has been described between CD28+ and CD28- T cells, we hypothesized that altered miRNA expression contributes to the age-associated downregulation of CD28. To avoid the confounding effects of age-associated changes in the proportions of T cells at various differentiation stages in vivo, an experimental model system was used to study changes over time in the expression of miRNA associated with the loss of CD28 expression in monoclonal T cell populations at a lower or higher number of population doublings (PDs). This approach allows identification of age-associated miRNA expression changes in a longitudinal model. Results were validated in ex vivo samples. The cumulative number of PDs but not the age of the donor of the T cell clone was correlated with decreased expression of CD28. Principal component analysis of 252 expressed miRNAs showed clustering based on low and high PDs, irrespective of the age of the clone donor. Increased expression of miR-9-5p and miR-34a-5p was seen in clones at higher PDs, and miR-9-5p expression inversely correlated with CD28 expression in ex vivo sorted T-cells from healthy subjects. We then examined the involvement of miR-9-5p, miR-34a-5p, and the members of the miR-23a similar to 24-2 cluster, in which all are predicted to bind to the 3'UTR of CD28, in the IL-15-induced loss of CD28 in T cells. Culture of fresh naive CD28+ T cells in the presence of IL-15 resulted in a gradual loss of CD28 expression, while the expression of miR-9-5p, miR-34a-5p, and members of the miR-23a-24-2 cluster increased. Binding of miR-9-5p, miR-34a-5p, miR-24-3p, and miR-27- 3p to the 3'UTR of CD28 was studied using luciferase reporter constructs. Functional binding to the 3'UTR was shown for miR-24-3p and miR-27a-3p. Our results indicate involvement of defined miRNAs in T cells in relation to specific characteristics of T cell aging, i.e., PD and CD28 expression

MicroRNA High Throughput Loss-of-Function Screening Reveals an Oncogenic Role for miR-21-5p in Hodgkin Lymphoma

Background/Aims: Classical Hodgkin lymphoma (cHL) is among the most frequent lymphoma subtypes. The tumor cells originate from crippled germinal center (GC)-B cells that escaped from apoptosis. MicroRNAs (miRNAs) play important roles in B-cell maturation and aberrant expression of miRNAs contributes to the pathogenesis of cHL. Our aim was to identify oncogenic miRNAs relevant for growth of cHL using a high-throughput screening approach. Methods: A lentiviral pool of 63 miRNA inhibition constructs was used to identify miRNAs essential to cell growth in three cHL cell lines in duplicate. As a negative control we also infected cHL cell lines with a lentiviral barcoded empty vector pool consisting of 222 constructs. The abundance of individual constructs was followed over time by a next generation sequencing approach. The effect on growth was confirmed using individual GFP competition assays and on apoptosis using Annexin-V staining. Our previously published Argonaute 2 (Ago2) immunoprecipitation (IP) data were used to identify target genes relevant for cell growth /apoptosis. Luciferase assays and western blotting were performed to confirm targeting by miRNAs. Results: Four miRNA inhibition constructs, i.e. miR-449a-5p, miR-625-5p, let-7f-2-3p and miR-21-5p, showed a significant decrease in abundance in at least 4 of 6 infections. In contrast, none of the empty vector constructs showed a significant decrease in abundance in 3 or more of the 6 infections. The most abundantly expressed miRNA, i.e. miR-21-5p, showed significantly higher expression levels in cHL compared to GC-B cells. GFP competition assays confirmed the negative effect of miR-21-5p inhibition on HL cell growth. Annexin-V staining of cells infected with miR-21-5p inhibitor indicated a significant increase in apoptosis at day 7 and 9 after viral infection, consistent with the decrease in growth. Four miR-21-5p cell growth-and apoptosis-associated targets were AGO2-IP enriched in cHL cell lines and showed a significant decrease in expression in cHL cell lines in comparison to normal GC-B cells. For the two most abundantly expressed, i.e. BTG2 and PELI1, we confirmed targeting by miR-21-5p using luciferase assays and for PELI1 we also confirmed this at the protein level by western blotting. Conclusion: Using a miRNA loss-of-function high-throughput screen we identified four miRNAs with oncogenic effects in cHL and validated the results for the in cHL abundantly expressed miR-21-5p. MiR-21-5p is upregulated in cHL compared to GC-B cells and protects cHL cells from apoptosis possibly via targeting BTG2 and PELI1. (C) 2018 The Author(s) Published by S. Karger AG, Base

A high throughput experimental approach to identify miRNA targets in human cells

The study of human microRNAs is seriously hampered by the lack of proper tools allowing genome-wide identification of miRNA targets. We performed Ribonucleoprotein ImmunoPrecipitation—gene Chip (RIP-Chip) using antibodies against wild-type human Ago2 in untreated Hodgkin lymphoma (HL) cell lines. Ten to thirty percent of the gene transcripts from the genome were enriched in the Ago2-IP fraction of untreated cells, representing the HL miRNA-targetome. In silico analysis indicated that ∼40% of these gene transcripts represent targets of the abundantly co-expressed miRNAs. To identify targets of miR-17/20/93/106, RIP-Chip with anti-miR-17/20/93/106 treated cells was performed and 1189 gene transcripts were identified. These genes were analyzed for miR-17/20/93/106 target sites in the 5′-UTRs, coding regions and 3′-UTRs. Fifty-one percent of them had miR-17/20/93/106 target sites in the 3′-UTR while 19% of them were predicted miR-17/20/93/106 targets by TargetScan. Luciferase reporter assay confirmed targeting of miR-17/20/93/106 to the 3′-UTRs of 8 out of 10 genes. In conclusion, we report a method which can establish the miRNA-targetome in untreated human cells and identify miRNA specific targets in a high throughput manner. This approach is applicable to identify miRNA targets in any human tissue sample or purified cell population in an unbiased and physiologically relevant manner