Risk Management Plans: reassessment of safety concerns based on Good Pharmacovigilance Practices Module V (Revision 2)-a company experience.

INTRODUCTION: In the European Union (EU), a Risk Management Plan (RMP) is submitted as part of the dossier for initial marketing authorization of a medicinal product or with an application involving a significant change to an existing marketing authorization. A comprehensive revision of the EU Guideline on Good Pharmacovigilance Practices (GVP) Module V-Risk Management Systems (Revision [Rev] 2), adopted in March 2017, provides a framework for developing more focused, actionable, and risk-proportionate RMPs. This paper describes the Janssen experience with the interpretation and application of GVP Module V (Rev 2) regarding the evaluation of safety concerns in an RMP. METHODS: Janssen convened a cross-functional working group to promote consistent interpretation of the GVP Module V (Rev 2) guidance across therapeutic areas. The group created 3 algorithms to support implementation of the guidance related to removal or reclassification of safety concerns by product-specific RMP teams. RESULTS: Following implementation of the GVP Module V (Rev 2) guidance, the algorithm-driven process led to a substantial decrease in the number of safety concerns for most products. With few exceptions, EU health authorities agreed with the proposed safety concern removals or reclassifications, resulting in RMPs that were focused on only those safety concerns that required further characterization or specific risk minimization. CONCLUSIONS: The algorithm-driven process allows for consistent interpretation and application of the GVP Module V (Rev 2) guidance, which enables product teams to develop an actionable RMP using a thoughtful, evaluative, science-based approach that considers all available evidence.

Members of the ELMOD protein family specify formation of distinct aperture domains on the Arabidopsis pollen surface.

Pollen apertures, the characteristic gaps in pollen wall exine, have emerged as a model for studying the formation of distinct plasma membrane domains. In each species, aperture number, position, and morphology are typically fixed; across species they vary widely. During pollen development, certain plasma membrane domains attract specific proteins and lipids and become protected from exine deposition, developing into apertures. However, how these aperture domains are selected is unknown. Here, we demonstrate that patterns of aperture domains in Arabidopsis are controlled by the members of the ancient ELMOD protein family, which, although important in animals, has not been studied in plants. We show that two members of this family, MACARON (MCR) and ELMOD_A, act upstream of the previously discovered aperture proteins and that their expression levels influence the number of aperture domains that form on the surface of developing pollen grains. We also show that a third ELMOD family member, ELMOD_E, can interfere with MCR and ELMOD_A activities, changing aperture morphology and producing new aperture patterns. Our findings reveal key players controlling early steps in aperture domain formation, identify residues important for their function, and open new avenues for investigating how diversity of aperture patterns in nature is achieved.

Zooming in on cells reveals patterns on their outer surfaces. These patterns are actually a collection of distinct areas of the cell surface, each containing specific combinations of molecules. The outer layers of pollen grains consist of a cell wall, and a softer cell membrane that sits underneath. As a pollen grain develops, it recruits certain fats and proteins to specific areas of the cell membrane, known as 'aperture domains'. The composition of these domains blocks the cell wall from forming over them, leading to gaps in the wall called 'pollen apertures'. Pollen apertures can open and close, aiding reproduction and protecting pollen grains from dehydration. The number, location, and shape of pollen apertures vary between different plant species, but are consistent within the same species. In the plant species Arabidopsis thaliana, pollen normally develops three long and narrow, equally spaced apertures, but it remains unclear how pollen grains control the number and location of aperture domains. Zhou et al. found that mutations in two closely related A. thaliana proteins ­ ELMOD_A and MCR ­ alter the number and positions of pollen apertures. When A. thaliana plants were genetically modified so that they would produce different levels of ELMOD_A and MCR, Zhou et al. observed that when more of these proteins were present in a pollen grain, more apertures were generated on the pollen surface. This finding suggests that the levels of these proteins must be tightly regulated to control pollen aperture numbers. Further tests revealed that another related protein, called ELMOD_E, also has a role in domain formation. When artificially produced in developing pollen grains, it interfered with the activity of ELMOD_A and MCR, changing pollen aperture shape, number, and location. Zhou et al. identified a group of proteins that help control the formation of domains in the cell membranes of A. thaliana pollen grains. Further research will be required to determine what exactly these proteins do to promote formation of aperture domains and whether similar proteins control domain development in other organisms.

A species-specific functional module controls formation of pollen apertures.

Pollen apertures are an interesting model for the formation of specialized plasma-membrane domains. The plant-specific protein INP1 serves as a key aperture factor in such distantly related species as Arabidopsis, rice and maize. Although INP1 orthologues probably play similar roles throughout flowering plants, they show substantial sequence divergence and often cannot substitute for each other, suggesting that INP1 might require species-specific partners. Here, we present a new aperture factor, INP2, which satisfies the criteria for being a species-specific partner for INP1. Both INP proteins display similar structural features, including the plant-specific DOG1 domain, similar patterns of expression and mutant phenotypes, as well as signs of co-evolution. These proteins interact with each other in a species-specific manner and can restore apertures in a heterologous system when both are expressed but not when expressed individually. Our findings suggest that the INP proteins form a species-specific functional module that underlies formation of pollen apertures.

Pollen Aperture Factor INP1 Acts Late in Aperture Formation by Excluding Specific Membrane Domains from Exine Deposition.

Accurate placement of extracellular materials is a critical part of cellular development. To study how cells achieve this accuracy, we use formation of pollen apertures as a model. In Arabidopsis (Arabidopsis thaliana), three regions on the pollen surface lack deposition of pollen wall exine and develop into apertures. In developing pollen, Arabidopsis INAPERTURATE POLLEN1 (INP1) protein acts as a marker for the preaperture domains, assembling there into three punctate lines. To understand the mechanism of aperture formation, we studied the dynamics of INP1 expression and localization and its relationship with the membrane domains at which it assembles. We found that INP1 assembly occurs after meiotic cytokinesis at the interface between the plasma membrane and the overlying callose wall, and requires the normal callose wall formation. Sites of INP1 localization coincide with positions of protruding membrane ridges in proximity to the callose wall. Our data suggest that INP1 is a late-acting factor involved in keeping specific membrane domains next to the callose wall to prevent formation of exine at these sites.

INP1 involvement in pollen aperture formation is evolutionarily conserved and may require species-specific partners.

Pollen wall exine is usually deposited non-uniformly on the pollen surface, with areas of low exine deposition corresponding to pollen apertures. Little is known about how apertures form, with the novel Arabidopsis INP1 (INAPERTURATE POLLEN1) protein currently being the only identified aperture factor. In developing pollen, INP1 localizes to three plasma membrane domains and underlies formation of three apertures. Although INP1 homologs are found across angiosperms, they lack strong sequence conservation. Thus, it has been unclear whether they also act as aperture factors and whether their sequence divergence contributes to interspecies differences in aperture patterns. To explore the functional conservation of INP1 homologs, we used mutant analysis in maize and tested whether homologs from several other species could function in Arabidopsis. Our data suggest that the INP1 involvement in aperture formation is evolutionarily conserved, despite the significant divergence of INP1 sequences and aperture patterns, but that additional species-specific factors are likely to be required to guide INP1 and to provide information for aperture patterning. To determine the regions in INP1 necessary for its localization and function, we used fragment fusions, domain swaps, and interspecific protein chimeras. We demonstrate that the central portion of the protein is particularly important for mediating the species-specific functionality.

Formation of pollen apertures in Arabidopsis requires an interplay between male meiosis, development of INP1-decorated plasma membrane domains, and the callose wall.

In most plant species, surfaces of pollen grains display characteristic patterns of apertures, formed by the gaps in the pollen wall exine. The aperture patterns are species-specific and tend to be very precise, with pollen of each species usually developing a certain number of apertures placed at distinct positions and acquiring specific morphology. The precision with which pollen apertures are produced suggests that developing pollen grains possess robust mechanisms that allow them to specify particular membrane domains as the future-aperture sites and to protect these sites from exine deposition. Recently, we demonstrated that formation of apertures in Arabidopsis depends on certain membrane domains attracting a novel protein, INP1, that assembles into punctate lines and helps to anchor these membrane domains to the overlying callose wall. Here we show that in the absence of male meiosis the ability of INP1 to assemble into lines at the pollen surface is compromised. However, INP1 still arrives to the pollen surface and mediates the interactions between the plasma membrane and the callose wall, potentially contributing to the formation of grossly abnormal patterns on pollen surface.

A Ploidy-Sensitive Mechanism Regulates Aperture Formation on the Arabidopsis Pollen Surface and Guides Localization of the Aperture Factor INP1.

Pollen presents a powerful model for studying mechanisms of precise formation and deposition of extracellular structures. Deposition of the pollen wall exine leads to the generation of species-specific patterns on pollen surface. In most species, exine does not develop uniformly across the pollen surface, resulting in the formation of apertures-openings in the exine that are species-specific in number, morphology and location. A long time ago, it was proposed that number and positions of apertures might be determined by the geometry of tetrads of microspores-the precursors of pollen grains arising via meiotic cytokinesis, and by the number of last-contact points between sister microspores. We have tested this model by characterizing Arabidopsis mutants with ectopic apertures and/or abnormal geometry of meiotic products. Here we demonstrate that contact points per se do not act as aperture number determinants and that a correct geometric conformation of a tetrad is neither necessary nor sufficient to generate a correct number of apertures. A mechanism sensitive to pollen ploidy, however, is very important for aperture number and positions and for guiding the aperture factor INP1 to future aperture sites. In the mutants with ectopic apertures, the number and positions of INP1 localization sites change depending on ploidy or ploidy-related cell size and not on INP1 levels, suggesting that sites for aperture formation are specified before INP1 is brought to them.

Effect of estrogen deficiency on regional variation of a viscoelastic tissue property of bone.

Estrogen deficiency changes the regional distribution of tissue mineral density leading to alteration of the mechanical properties of bone at the tissue level. Direct measurement of the regional variation of elastic modulus and viscosity, which is the capacity to resist time-dependent viscoelastic deformation, will aid in our understanding of how estrogen deficiency alters bone quality. It was observed that, compared to bone from other anatomical sites, the jaw bone is less sensitive to estrogen deficiency. Thus, the objective of this study was to examine the effect of estrogen deficiency on (1) the regional variations of tissue modulus and viscosity of bone using nanoindentation, and (2) the modulus-viscosity relationships in jaw and vertebral bones for comparison between different anatomical sites. Mandibular and vertebral bone specimens of sham surgery and ovariectomized (OVX) rat groups were subject to nanoindentation in hydration. Indentation modulus and viscosity were measured at relatively new (less mineralized) tissue regions and at the corresponding pre-existing old (more mineralized) tissue regions of mandibular and vertebral bones. In the mandibular bones, significant regional variations of indentation modulus and viscosity were observed (p<0.039) and OVX increased the indentation viscosity. While significant positive correlations were found between indentation modulus and viscosity (p<0.001), the correlation slopes for the mandibular and vertebral bones were significant different (p<0.001). The current results indicated that changes in viscoelastic property and its regional variation should be examined to obtain a better understanding of estrogen deficiency-dependent alteration of bone quality.

Gene expression and stress response mediated by the epigenetic regulation of a transposable element small RNA.

The epigenetic activity of transposable elements (TEs) can influence the regulation of genes; though, this regulation is confined to the genes, promoters, and enhancers that neighbor the TE. This local cis regulation of genes therefore limits the influence of the TE's epigenetic regulation on the genome. TE activity is suppressed by small RNAs, which also inhibit viruses and regulate the expression of genes. The production of TE heterochromatin-associated endogenous small interfering RNAs (siRNAs) in the reference plant Arabidopsis thaliana is mechanistically distinct from gene-regulating small RNAs, such as microRNAs or trans-acting siRNAs (tasiRNAs). Previous research identified a TE small RNA that potentially regulates the UBP1b mRNA, which encodes an RNA-binding protein involved in stress granule formation. We demonstrate that this siRNA, siRNA854, is under the same trans-generational epigenetic control as the Athila family LTR retrotransposons from which it is produced. The epigenetic activation of Athila elements results in a shift in small RNA processing pathways, and new 21-22 nucleotide versions of Athila siRNAs are produced by protein components normally not responsible for processing TE siRNAs. This processing results in siRNA854's incorporation into ARGONAUTE1 protein complexes in a similar fashion to gene-regulating tasiRNAs. We have used reporter transgenes to demonstrate that the UPB1b 3' untranslated region directly responds to the epigenetic status of Athila TEs and the accumulation of siRNA854. The regulation of the UPB1b 3' untranslated region occurs both on the post-transcriptional and translational levels when Athila TEs are epigenetically activated, and this regulation results in the phenocopy of the ubp1b mutant stress-sensitive phenotype. This demonstrates that a TE's epigenetic activity can modulate the host organism's stress response. In addition, the ability of this TE siRNA to regulate a gene's expression in trans blurs the lines between TE and gene-regulating small RNAs.