A functional genomic approach to identify reference genes for human pancreatic beta cell real-time quantitative RT-PCR analysis

Exposure of human pancreatic beta cells to pro-inflammatory cytokines or metabolic stressors is used to model events related to type 1 and type 2 diabetes, respectively. Quantitative real-time PCR is commonly used to quantify changes in gene expression. The selection of the most adequate reference gene(s) for gene expression normalization is an important pre-requisite to obtain accurate and reliable results. There are no universally applicable reference genes, and the human beta cell expression of commonly used reference genes can be altered by different stressors. Here we aimed to identify the most stably expressed genes in human beta cells to normalize quantitative real-time PCR gene expression. We used comprehensive RNA-sequencing data from the human pancreatic beta cell line EndoC-βH1, human islets exposed to cytokines or the free fatty acid palmitate in order to identify the most stably expressed genes. Genes were filtered based on their level of significance (adjusted P-value >0.05), fold-change (|fold-change| <1.5) and a coefficient of variation <10%. Candidate reference genes were validated by quantitative real-time PCR in independent samples. We identified a total of 264 genes stably expressed in EndoC-βH1 cells and human islets following cytokines–or palmitate-induced stress, displaying a low coefficient of variation. Validation by quantitative real-time PCR of the top five genes ARF1, CWC15, RAB7A, SIAH1 and VAPA corroborated their expression stability under most of the tested conditions. Further validation in independent samples indicated that the geometric mean of ACTB and VAPA expression can be used as a reliable normalizing factor in human beta cells

An integrated multi-omics approach identifies the landscape of interferon-α-mediated responses of human pancreatic beta cells

Interferon-α (IFNα), a type I interferon, is expressed in the islets of type 1 diabetic individuals, and its expression and signaling are regulated by T1D genetic risk variants and viral infections associated with T1D. We presently characterize human beta cell responses to IFNα by combining ATAC-seq, RNA-seq and proteomics assays. The initial response to IFNα is characterized by chromatin remodeling, followed by changes in transcriptional and translational regulation. IFNα induces changes in alternative splicing (AS) and first exon usage, increasing the diversity of transcripts expressed by the beta cells. This, combined with changes observed on protein modification/degradation, ER stress and MHC class I, may expand antigens presented by beta cells to the immune system. Beta cells also up-regulate the checkpoint proteins PDL1 and HLA-E that may exert a protective role against the autoimmune assault. Data mining of the present multi-omics analysis identifies two compound classes that antagonize IFNα effects on human beta cells

Consequences Of On-Track Competition In Railways By Use Of Simulations

Institute of Transport and Logistics Studies. Faculty of Economics and Business. The University of Sydne

Deviant leisure: A criminological perspective

This article explains why an understanding of deviant leisure is significant for criminology. Through reorienting our understanding of ‘deviance’ from a contravention of norms and values to encompassing engagement in behaviour and actions that contravene a moral ‘duty to the other’, the new ‘deviant leisure’ perspective outlined here describes activities that through their adherence to cultural values inscribed by consumer capitalism, have the potential to result in harm. Using the ideological primacy of consumer capitalism as a point of departure, we explore the potential for harm that lies beneath the surface of even the most embedded and culturally accepted forms of leisure. Such an explanation requires a reading that brings into focus the subjective, socially corrosive, environmental and embedded harms that arise as a result of the commodification of leisure. In this way, this article aims to act as a conceptual foundation for diverse yet coherent research into deviant leisure

Comparing hybrid metaheuristics for the bus driver rostering problem

SearchCol is a recently proposed approach hybridizing column generation, problem specific algorithms and distinct well known metaheuristics (VNS, Tabu Search, Simulated Annealing, etc.). SearchCol allows to solve several combinatorial optimization problems by applying column generation to a given decomposition model, and using one of the available metaheuristics to search for an integer solution combining the previously generated columns, which are components of the problem. A new evolutionary algorithm (EA) was proposed as the first population based metaheuristic included in SearchCol. This EA uses a representation of individuals based on the generated columns and has been used to obtain integer solutions for a new model for the Bus Drivers Rostering problem (BDRP). Special features of this EA include local search and elitism. This paper presents a computational study evaluating the new population based heuristic (EA) versus two single solution heuristics: VNS and Simulated Annealing, exploiting different configurations of the framework on a set of benchmark instances for the BDRP.info:eu-repo/semantics/publishedVersio

A new branch-and-price approach for the kidney exchange problem

The kidney exchange problem (KEP) is an optimization problem arising in the framework of transplant programs that allow exchange of kidneys between two or more incompatible patient-donor pairs. In this paper an approach based on a new decomposition model and branch-and-price is proposed to solve large KEP instances. The optimization problem considers, hierarchically, the maximization of the number of transplants and the minimization of the size of exchange cycles. Computational comparison of different variants of branch-and-price for the standard and the proposed objective functions are presented. The results show the efficiency of the proposed approach for solving large instances.This work is financed by the ERDF — European Regional Development Fund through the COMPETE Programme (operational programm for competitiveness), by National Funds through the FCT — Fundação para a Ciência e a Tecnologia (Portuguese Foundation for Science and Technology) within project “KEP - New models for enhancing the kidney transplantation process. /FCT ref: PTDC/EGEGES/110940/2009”, by the North Portugal Regional Operational Programme (ON.2 O Novo Norte), under the National Strategic Reference Framework (NSRF), through the European Regional Development Fund (ERDF), and by national funds through FCT within project ”NORTE-07-0124-FEDER-000057”, and has also been supported by FCT through projects “SearchCol: Metaheuristic search by column generation” (PTDC/EIAEIA/100645/2008) and PEst-OE/EEI/UI0319/2014.info:eu-repo/semantics/publishedVersio

Maturity-Onset Diabetes of the Young (MODY) in Portugal: Novel GCK, HNFA1 and HNFA4 Mutations

Maturity-onset diabetes of the young (MODY) is a frequently misdiagnosed type of diabetes, which is characterized by early onset, autosomal dominant inheritance, and absence of insulin dependence. The most frequent subtypes are due to mutations of the GCK (MODY 2), HNF1A (MODY 3), and HNF4A (MODY 1) genes. We undertook the first multicenter genetic study of MODY in the Portuguese population. The GCK, HNF1A, and HNF4A genes were sequenced in 46 unrelated patients that had at least two of the three classical clinical criteria for MODY (age at diagnosis, family history, and clinical presentation). The functional consequences of the mutations were predicted by bioinformatics analysis. Mutations were identified in 23 (50%) families. Twelve families had mutations in the GCK gene, eight in the HNF1A gene, and three in the HNF4A gene. These included seven novel mutations (GCK c.494T>C, GCK c.563C>G, HNF1A c.1623G>A, HNF1A c.1729C>G, HNF4A c.68delG, HNF4A c.422G>C, HNF4A c.602A>C). Mutation-positive patients were younger at the time of diagnosis when compared to mutation-negative patients (14.3 vs. 23.0 years, p = 0.011). This study further expands the spectrum of known mutations associated with MODY, and may contribute to a better understanding of this type of diabetes and a more personalized clinical management of affected individuals

Maturity-Onset Diabetes of the Young (MODY) in Portugal: Novel GCK, HNFA1 and HNFA4 Mutations

Maturity-onset diabetes of the young (MODY) is a frequently misdiagnosed type of diabetes, which is characterized by early onset, autosomal dominant inheritance, and absence of insulin dependence. The most frequent subtypes are due to mutations of the GCK (MODY 2), HNF1A (MODY 3), and HNF4A (MODY 1) genes. We undertook the first multicenter genetic study of MODY in the Portuguese population. The GCK, HNF1A, and HNF4A genes were sequenced in 46 unrelated patients that had at least two of the three classical clinical criteria for MODY (age at diagnosis, family history, and clinical presentation). The functional consequences of the mutations were predicted by bioinformatics analysis. Mutations were identified in 23 (50%) families. Twelve families had mutations in the GCK gene, eight in the HNF1A gene, and three in the HNF4A gene. These included seven novel mutations (GCK c.494T>C, GCK c.563C>G, HNF1A c.1623G>A, HNF1A c.1729C>G, HNF4A c.68delG, HNF4A c.422G>C, HNF4A c.602A>C). Mutation-positive patients were younger at the time of diagnosis when compared to mutation-negative patients (14.3 vs. 23.0 years, p = 0.011). This study further expands the spectrum of known mutations associated with MODY, and may contribute to a better understanding of this type of diabetes and a more personalized clinical management of affected individuals