Supporting Pharmacovigilance Signal Validation and Prioritization with Analyses of Routinely Collected Health Data: Lessons Learned from an EHDEN Network Study.

INTRODUCTION: Individual case reports are the main asset in pharmacovigilance signal management. Signal validation is the first stage after signal detection and aims to determine if there is sufficient evidence to justify further assessment. Throughout signal management, a prioritization of signals is continually made. Routinely collected health data can provide relevant contextual information but are primarily used at a later stage in pharmacoepidemiological studies to assess communicated signals. OBJECTIVE: The aim of this study was to examine the feasibility and utility of analysing routine health data from a multinational distributed network to support signal validation and prioritization and to reflect on key user requirements for these analyses to become an integral part of this process. METHODS: Statistical signal detection was performed in VigiBase, the WHO global database of individual case safety reports, targeting generic manufacturer drugs and 16 prespecified adverse events. During a 5-day study-a-thon, signal validation and prioritization were performed using information from VigiBase, regulatory documents and the scientific literature alongside descriptive analyses of routine health data from 10 partners of the European Health Data and Evidence Network (EHDEN). Databases included in the study were from the UK, Spain, Norway, the Netherlands and Serbia, capturing records from primary care and/or hospitals. RESULTS: Ninety-five statistical signals were subjected to signal validation, of which eight were considered for descriptive analyses in the routine health data. Design, execution and interpretation of results from these analyses took up to a few hours for each signal (of which 15-60 minutes were for execution) and informed decisions for five out of eight signals. The impact of insights from the routine health data varied and included possible alternative explanations, potential public health and clinical impact and feasibility of follow-up pharmacoepidemiological studies. Three signals were selected for signal assessment, two of these decisions were supported by insights from the routine health data. Standardization of analytical code, availability of adverse event phenotypes including bridges between different source vocabularies, and governance around the access and use of routine health data were identified as important aspects for future development. CONCLUSIONS: Analyses of routine health data from a distributed network to support signal validation and prioritization are feasible in the given time limits and can inform decision making. The cost-benefit of integrating these analyses at this stage of signal management requires further research.

Selective microRNA-Offset RNA expression in human embryonic stem cells.

Small RNA molecules, including microRNAs (miRNAs), play critical roles in regulating pluripotency, proliferation and differentiation of embryonic stem cells. miRNA-offset RNAs (moRNAs) are similar in length to miRNAs, align to miRNA precursor (pre-miRNA) loci and are therefore believed to derive from processing of the pre-miRNA hairpin sequence. Recent next generation sequencing (NGS) studies have reported the presence of moRNAs in human neurons and cancer cells and in several tissues in mouse, including pluripotent stem cells. In order to gain additional knowledge about human moRNAs and their putative development-related expression, we applied NGS of small RNAs in human embryonic stem cells (hESCs) and fibroblasts. We found that certain moRNA isoforms are notably expressed in hESCs from loci coding for stem cell-selective or cancer-related miRNA clusters. In contrast, we observed only sparse moRNAs in fibroblasts. Consistent with earlier findings, most of the observed moRNAs derived from conserved loci and their expression did not appear to correlate with the expression of the adjacent miRNAs. We provide here the first report of moRNAs in hESCs, and their expression profile in comparison to fibroblasts. Moreover, we expand the repertoire of hESC miRNAs. These findings provide an expansion on the known repertoire of small non-coding RNA contents in hESCs.

Defined serum-free media for in vitro expansion of adipose-derived mesenchymal stem cells.

BACKGROUND: There is a growing interest in mesenchymal stem cells (MSCs) because they are regarded as good candidates for cell therapy. Adipose tissue represents an easily accessible source to derive mesenchymal stem cells (Ad-MSCs) non-invasively in large numbers. The aim of this study was to evaluate a defined serum-free medium for in vitro expansion of MSCs as a prerequisite for their clinical use. METHODS: Adipose tissue was isolated from healthy donors. Cells were isolated and expanded for five passages in serum-free medium (Mesencult-XF) and Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (DMEM-FBS). MSC morphology, marker expression, viability, population doubling time and differentiation potential toward osteogenic and adipogenic lineages were evaluated. Bone marrow MSCs were included as controls. RESULTS: Ad-MSCs cultured in Mesencult-XF had shorter population doubling time (33.3 ± 13.7 h) compared with those cultured in DMEM-FBS (54.3 ± 41.0 h, P < 0.05). Ad-MSCs cultured in Mesencult-XF displayed a stable morphology and surface marker expression and a higher differentiation potential in comparison to Ad-MSCs cultured in DMEM-FBS. CONCLUSIONS: The defined serum-free and xeno-free Mesencult-XF media appear to be a good choice for Ad-MSCs, but it is not as good in supporting culture of bone marrow MSCs when the cells are to be used for clinical purposes.

Fatty acid composition and gene expression profiles are altered in aryl hydrocarbon receptor-1 mutant Caenorhabditis elegans.

The aryl hydrocarbon receptor (AHR) is a eukaryotic transcription factor that plays an essential role in neuronal, immune, vascular, hepatic and hematopoietic development. In mammals, AHR induces metabolism-associated genes in response to xenobiotics. AHR is evolutionarily conserved, and the C. elegans AHR ortholog likely shares many physiologic functions with the mammalian version. While the role of AHR in development is known, the molecular basis of AHR action is less well understood. To understand the physiologic role of AHR in C. elegans, a combination of fatty acid profiling, transcriptomics, and phenotyping approaches was used. Fatty acid profiles from L4 larval stage whole animals indicated that C17isoA, C18:1n9t, C20:3n6 and C20:4n6 were significantly increased in an ahr-1 mutant compared to wild-type. Consistent with these changes, we observed a significant 5.8 fold increase in fat-7, and 1.7-1.9 fold increases in elo-5, nhr-49, and mdt-15 gene expression during the L4 stage. The ahr-1(ju145) mutant displayed deficits in growth and development including a reduced number of eggs laid, a higher proportion of dead embryos, delay in time to reach L4 stage, and movement deficits including a fewer number of body bends and a longer defecation cycle. To understand global effects of AHR-1 on transcription, microarray analysis was performed on L1 stage animals. Expression changes (324 under- and 238 over-expressed) were found in genes associated with metabolism, growth, and development. These results indicate a role for C. elegans AHR in regulating fatty acid composition and in contributing to some aspects of development. Since the transcriptional control of AHR targets may be evolutionarily conserved, these results provide a deeper understanding of the molecular actions of AHR in a model invertebrate system that may be informative for higher organisms.

Global microRNA expression profiling of Caenorhabditis elegans Parkinson's disease models.

MicroRNAs (miRNAs) play an important role in human brain development and maintenance. To search for miRNAs that may be involved in the pathogenesis of Parkinsons disease (PD), we utilized miRNA microarrays to identify potential gene expression changes in 115 annotated miRNAs in PD-associated Caenorhabditis elegans models that either overexpress human A53T alpha-synuclein or have mutations within the vesicular catecholamine transporter (cat-1) or parkin (pdr-1) ortholog. Here, we show that 12 specific miRNAs are differentially regulated in the animals overexpressing alpha-synuclein, five in cat-1, and three in the pdr-1 mutants. The family of miR-64 and miR-65 are co-underexpressed in the alpha-synuclein transgenic and cat-1 strains, and members of let-7 family co-underexpressed in the alpha-synuclein and pdr-1 strains; mdl-1 and ptc-1 genes are target candidates for miR-64 and miR-65 and are overexpressed in alpha-synuclein transgenic as well as miR-64/65 (tm3711) knockout animals. These results indicate that miRNAs are differentially expressed in C. elegans PD models and suggest a role for these molecules in disease pathogenesis.

Identification of phylogenetically conserved sequence motifs in microRNA 5' flanking sites from C. elegans and C. briggsae.

BACKGROUND: MicroRNAs (miRNAs) are small, noncoding RNA molecules that act as post-transcriptional regulators of gene expression. Studies concerning transcriptional regulation of miRNAs have so far concentrated on those located within the intergenic region of the genome and the search for putative promoters, thus leaving open the question of the existence of possible regulatory elements common to all miRNAs including those located in introns of protein coding genes. RESULTS: In this study, we initially searched for motifs occurring in the area 1000 bp upstream from all miRNAs independent of their genomic location. We discovered a previously unknown sequence motif GANNNNGA that displayed a conserved distribution in the nematode worms Caenorhabditis elegans and Caenorhabditis briggsae. This motif had a peak occurrence at 500 bp upstream, with a sharp drop-off toward the miRNA start site. Further analysis indicated that this motif was locally restricted and not enriched 1000-5000 bp upstream or 0-2000 bp downstream of the miRNA start site. In addition, this motif was observed to be most abundant in the upstream sequences of two important miRNAs, mir-1 and mir-124. This abundance was also conserved in phylogenetically distant species including human and mouse. CONCLUSION: The results show that the motif GANNNNGA is conserved close to miRNA precursor start sites, suggesting that it may be involved in miRNA sequence recognition or regulation. This data provides important knowledge for the identification and computational prediction of miRNA sequences.

Functional characterization of endogenous siRNA target genes in Caenorhabditis elegans.

BACKGROUND: Small interfering RNA (siRNA) molecules mediate sequence specific silencing in RNA interference (RNAi), a gene regulatory phenomenon observed in almost all organisms. Large scale sequencing of small RNA libraries obtained from C. elegans has revealed that a broad spectrum of siRNAs is endogenously transcribed from genomic sequences. The biological role and molecular diversity of C. elegans endogenous siRNA (endo-siRNA) molecules, nonetheless, remain poorly understood. In order to gain insight into their biological function, we annotated two large libraries of endo-siRNA sequences, identified their cognate targets, and performed gene ontology analysis to identify enriched functional categories. RESULTS: Systematic trends in categorization of target genes according to the specific length of siRNA sequences were observed: 18- to 22-mer siRNAs were associated with genes required for embryonic development; 23-mers were associated uniquely with post-embryonic development; 24-26-mers were associated with phosphorus metabolism or protein modification. Moreover, we observe that some argonaute related genes associate with siRNAs with multiple reads. Sequence frequency graphs suggest that different lengths of siRNAs share similarities in overall sequence structure: the 5' end begins with G, while the body predominates with U and C. CONCLUSION: These results suggest that the lengths of endogenous siRNA molecules are consequential to their biological functions since the gene ontology categories for their cognate mRNA targets vary depending upon their lengths.

Whole genome microarray analysis of C. elegans rrf-3 and eri-1 mutants.

We performed genome wide gene expression analysis on L4 stage Caenorhabditis elegans rrf-3(pk1426) and eri-1(mg366) mutant strains to study the effects caused by loss of their encoded proteins, which are required for the accumulation of endogenous secondary siRNAs. Mutant rrf-3 and eri-1 strains exhibited 72 transcripts that were co-over-expressed and 4 transcripts co-under-expressed (>2-fold, P<0.05) compared to N2 wild type strain. Ontology analysis indicated these transcripts were over represented for protein phosphorylation and sperm function genes. These results provide additional support for the hypothesis that RRF-3 and ERI-1 act together in the endo-siRNA pathway, and furthermore suggests their involvement in additional biological processes.

Selective sensitivity of Caenorhabditis elegans neurons to RNA interference.

RNA interference is a new approach to knockdown gene expression, but effectiveness varies depending on the organism, cell type or target sequence. Studies with Caenorhabditis elegans have shown that subsets of cells including neurons are often resistant to RNA interference. We measured RNA interference using green fluorescent protein reporter strains and feeding, soaking and injection delivery methods in a number of Caenorhabditis elegans neuron subtypes (dopaminergic, GABAergic, cholinergic, glutamatergic, touch). The sensitivity to RNA interference varied: GABAergic and dopaminergic neurons showed greater resistance while cholinergic, glutamatergic and touch neurons were more sensitive. Dysfunctional RRF-3, a putative RNA-directed RNA polymerase, had a significant effect on increasing neuron sensitivity in most subtypes. These results demonstrate that Caenorhabditis elegans neurons vary in their sensitivity to RNA interference.