Antiviral TRIMs: friend or foe in autoimmune and autoinflammatory disease?

The concept that viral sensing systems, via their ability to drive pro-inflammatory cytokine and interferon production, contribute to the development of autoimmune and autoinflammatory disease is supported by a wide range of clinical and experimental observations. Recently, the tripartite motif-containing proteins (TRIMs) have emerged as having key roles in antiviral immunity - either as viral restriction factors or as regulators of pathways downstream of viral RNA and DNA sensors, and the inflammasome. Given their involvement in these pathways, we propose that TRIM proteins contribute to the development and pathology of autoimmune and autoinflammatory conditions, thus making them potential novel targets for therapeutic manipulation.     

Contrasting responses of two Sitka spruce forest plots in Ireland to reductions in sulphur emissions: results of 20 years of monitoring

Long-term trends in ion concentrations of bulk precipitation, throughfall, forest floor leachate (humus water) and shallow and deep soil water were assessed at two Sitka spruce (Picea sitchensis) stands—one on an Atlantic peat bog in the west of Ireland (Cloosh), the other on the east coast on a peaty podzol (Roundwood). Deposition at Cloosh was dominated by marine ions (sodium, [Na⁺], chloride [Cl^?], and magnesium [Mg²⁺]), whereas bulk precipitation and throughfall at Roundwood was characterized by inputs of non-marine sulphate (nmSO₄ ^2?), acidity and inorganic nitrogen (NH₄ ⁺, NO₃ ^?). Significant declines in concentrations of nmSO₄ ^2? and acidity in bulk precipitation and throughfall were observed at both sites. The decline in throughfall nmSO₄ ^2? was significantly related to reductions in European sulphur dioxide (SO₂) emissions. At Roundwood, SO₄ ^2? declined significantly in humus, shallow and deep soil water. In deep soil water this was accompanied by a long-term increase in pH and a reduction in total aluminum (Al_tot). The recovery from acidification was delayed by high concentrations of NO₃ ^?, which strongly influenced acidity and Al_tot concentrations. At Cloosh, there was a significant decline in SO₄ ^2? in humus water but long-term trends were not evident in shallow or deep soil water; SO₄ ^2? concentrations at these depths fluctuated in response to drought-events. Marine ions strongly influenced soil water chemistry at both sites; at Cloosh soil water acidity was strongly related to Na⁺ and Cl^?, while at Roundwood, Na⁺, Cl^? and Mg²⁺ influenced Al_tot concentrations. Dissolved organic carbon increased significantly in humus and soil water at Roundwood, where it was associated with declining acidity. Soil water at both sites was influenced by a combination of anthropogenic sulphur (S) and nitrogen (N) deposition, drought and sea-salt events. The study highlights the value of long-term monitoring in assessing the response of forest soils to S and N deposition against a background of climate influences on soil water through drought and sea-salt events.     

Imaging Conditioned Fear Circuitry Using Awake Rodent fMRI

Functional magnetic resonance imaging (fMRI) is a powerful method for exploring emotional and cognitive brain responses in humans. However rodent fMRI has not previously been applied to the analysis of learned behaviour in awake animals, limiting its use as a translational tool. Here we have developed a novel paradigm for studying brain activation in awake rats responding to conditioned stimuli using fMRI. Using this method we show activation of the amygdala and related fear circuitry in response to a fear-conditioned stimulus and demonstrate that the magnitude of fear circuitry activation is increased following early life stress, a rodent model of affective disorders. This technique provides a new translatable method for testing environmental, genetic and pharmacological manipulations on emotional and cognitive processes in awake rodent models.     

MiR-7 Triggers Cell Cycle Arrest at the G1/S Transition by Targeting Multiple Genes Including Skp2 and Psme3

MiR-7 acts as a tumour suppressor in many cancers and abrogates proliferation of CHO cells in culture. In this study we demonstrate that miR-7 targets key regulators of the G1 to S phase transition, including Skp2 and Psme3, to promote increased levels of p27^KIP and temporary growth arrest of CHO cells in the G1 phase. Simultaneously, the down-regulation of DNA repair-specific proteins via miR-7 including Rad54L, and pro-apoptotic regulators such as p53, combined with the up-regulation of anti-apoptotic factors like p-Akt, promoted cell survival while arrested in G1. Thus miR-7 can co-ordinate the levels of multiple genes and proteins to influence G1 to S phase transition and the apoptotic response in order to maintain cellular homeostasis. This work provides further mechanistic insight into the role of miR-7 as a regulator of cell growth in times of cellular stress.     

A "melanopic" spectral efficiency function predicts the sensitivity of melanopsin photoreceptors to polychromatic lights

Photoreception in the mammalian retina is not restricted to rods and cones but extends to a small number of intrinsically photosensitive retinal ganglion cells expressing the photopigment melanopsin. These mRGCs are especially important contributors to circadian entrainment, the pupil light reflex, and other so-called nonimage-forming (NIF) responses. The spectral sensitivity of melanopsin phototransduction has been addressed in several species by comparing responses to a range of monochromatic stimuli. The resultant action spectra match the predicted profile of an opsin:vitamin A-based photopigment (nomogram) with a peak sensitivity (λ(max)) around 480 nm. It would be most useful to be able to use this spectral sensitivity function to predict melanopsin's sensitivity to broad-spectrum, including "white," lights. However, evidence that melanopsin is a bistable pigment with an intrinsic light-dependent bleach recovery mechanism raises the possibility of a more complex relationship between spectral quality and photoreceptor response. Here, we set out to empirically determine whether simply weighting optical power at each wavelength according to the 480-nm nomogram and integrating across the spectrum could predict melanopsin sensitivity to a variety of polychromatic stimuli. We show that pupillomotor and circadian responses of mice relying solely on melanopsin for their photosensitivity (rd/rd cl) can indeed be accurately predicted using this methodology. Our data therefore suggest that the 480-nm nomogram may be employed as the basis for a new photometric measure of light intensity (which we term "melanopic") relevant for melanopsin photoreception. They further show that measuring light in these terms predicts the melanopsin response to light of divergent spectral composition much more reliably than other methods for quantifying irradiance or illuminance currently in widespread use.     

The discovery of Antillean algal taxa described by Duchassaing in 1850

This note is to call attention to a number of marine algal taxa from the Antilles that were described by Placide Duchassaing in his 1850 work Animaux radiaires des Antilles. The mid‐19^th century was a period when the distinction between marine invertebrates and some calcified benthic marine algae was still not always clear. The names of these algal taxa were validated, although no figures or details on their specific provenance were provided, other than they were from Duchassaing's collections from the Antilles. Duchassaing assigned his new algal species to the following genera: Galaxaura, Amphiroa, Jania, Melobesia and Nullipora. (© 2013 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Mapping the Structural Topology of the Yeast 19S Proteasomal Regulatory Particle Using Chemical Cross-linking and Probabilistic Modeling

Structural characterization of proteasome complexes is an essential step toward understanding the ubiquitin-proteasome system. Currently, high resolution structures are not available for the 26S proteasome holocomplex as well as its subcomplex, the 19S regulatory particle (RP). Here we have employed a novel integrated strategy combining chemical cross-linking with multistage tandem mass spectrometry to define the proximity of subunits within the yeast 19S RP to elucidate its topology. This has resulted in the identification of 174 cross-linked peptides of the yeast 19S RP, representing 43 unique lysine-lysine linkages within 24 nonredundant pair-wise subunit interactions. To map the spatial organization of the 19S RP, we have developed and utilized a rigorous probabilistic framework to derive maximum likelihood (ML) topologies based on cross-linked peptides determined from our analysis. Probabilistic modeling of the yeast 19S AAA-ATPase ring (i.e., Rpt1–6) has produced an ML topology that is in excellent agreement with known topologies of its orthologs. In addition, similar analysis was carried out on the 19S lid subcomplex, whose predicted ML topology corroborates recently reported electron microscopy studies. Together, we have demonstrated the effectiveness and potential of probabilistic modeling for unraveling topologies of protein complexes using cross-linking data. This report describes the first study of the 19S RP topology using a new integrated strategy combining chemical cross-linking, mass spectrometry, and probabilistic modeling. Our results have provided a solid foundation to advance our understanding of the 19S RP architecture at peptide level resolution. Furthermore, our methodology developed here is a valuable proteomic tool that can be generalized for elucidating the structures of protein complexes.Basic cellular homeostasis depends on the regulated protein degradation and turnover by the ubiquitin-proteasome system (1, 2). Central to this pathway is the 26S proteasome complex, which is responsible for ubiquitin/ATP-dependent protein degradation (3–5). The 26S holocomplex is a megadalton-sized protein assembly consisting of the 20S catalytic core particle (CP)¹ and the 19S regulatory particle (RP). The eukaryotic 20S CP is composed of two copies of 14 nonidentical subunits (α_1–7 and β_1–7) arranged into four stacked heptameric rings in an order of α₇β₇β₇α₇. The crystal structure and topology of the highly ordered 20S CP has been resolved and is evolutionarily conserved (6). Although α subunits of the 20S CP are essential for the assembly of the complex and its interactions with the regulatory complex, three catalytic β subunits (β1, β2, and β5) harbor various catalytic activities responsible for regulated proteasomal degradation. The 19S RP is composed of 19 subunits, which forms two subcomplexes, the base consisting of six related AAA-ATPase (Rpt1–6) and four non-ATPase (Rpn1, Rpn2, Rpn10, and Rpn13) subunits and the lid containing nine non-ATPase subunits (Rpn3, Rpn5–9, Rpn11, Rpn12, and Rpn15/Sem1) (7, 8). In comparison with the 20S core, the function and structure of the 19S RP is much less well understood. Nevertheless, it is believed that the 19S RP is involved in multiple functions including recognition of polyubiquitinated substrates (9, 10), cleavage of the polyubiquitin chains to recycle ubiquitin (11), unfolding of substrates, assisting in opening the gate of the 20S chamber, and subsequently translocating the unfolded substrates into the catalytic chamber (4, 12–14). The six AAA-ATPase subunits (Rpt1–6), which directly interact with the 20S α-ring, function as a molecular chaperone responsible for protein unfolding and are involved in substrate translocation and modulating gating of the CP (5, 15). Although detailed functions for most of the 19S non-ATPase subunits remain elusive, Rpn11 is known to carry an Mpr1p and Pad1p N-termini (MPN) domain, which harbors an essential deubiquitination activity responsible for cleaving polyubiquitin chains from proteasomal substrates (11, 16). In addition, two proteasome subunits, Rpn10 and Rpn13, have been identified as ubiquitin receptors, which are important in docking ubiquitinated substrates to the proteasome for degradation (4). Moreover, the two largest proteasome subunits, Rpn1 and Rpn2, interact with a variety of proteins including ubiquitin receptors and deubiquitinases and thus may function as scaffolding proteins to assist proteasomal degradation. Thus far, no atomic resolution structures are available for either the 19S RP or the 26S holocomplex. New insights of the overall topology of the 19S RP will illuminate protein interactions within, thus providing evidence for its otherwise unknown functions.Although many studies have been performed to characterize the 19S structure utilizing various techniques including cryo-EM (17, 18) and native mass spectrometry (19), details on spatial interfaces and subunit interconnectivity of the 19S RP remain to be unraveled. During the course of our study, the rough topology of the 19S RP was determined by cryo-EM alone (20) or coupled with other approaches (21); nevertheless more detailed information at the peptide or atomic level is still required. In addition to technological limitations in current approaches, the highly dynamic and heterogeneous nature of the 19S RP may attribute to the difficulty in obtaining its high resolution structure. In recent years, chemical cross-linking coupled with mass spectrometry (XL-MS) has become an attractive alternative for structure analysis of proteins and protein complexes (22, 23). The ability of XL-MS to identify interaction interfaces between proteins allows us to define low resolution protein topology. In addition to protein interaction networks and the site of protein interactions at binding interfaces, cross-linking analysis can reveal information about the spatial distance between cross-linked amino acids on the surface of folded proteins. Although such knowledge only reveals the maximum distance given by the length of the cross-linker and can be influenced by protein conformational flexibility, it can be used as the distance constraint for molecular modeling of protein folds and complex topologies, i.e., the arrangement of the constituents of a complex in space. A recent study by Chen et al. (24) on yeast RNA polymerase II (RNAPII) complex has exemplified the power of XL-MS in elucidating the architecture of large multisubunit complexes. Although effective, cross-linking studies have been challenging because of the low abundance of cross-linked products and the inherent complexity of sequencing interlinked peptides by MS for unambiguous identification. To facilitate MS detection and identification of cross-linked products, we have recently developed a novel homobifunctional amine reactive, low energy MS-cleavable cross-linker, disuccinimidyl sulfoxide (DSSO), and successfully applied it to cross-link the yeast 20S proteasome for rapid, accurate, and simplified determination of protein interaction interfaces within the complex (25). The unique functionality of our cross-linking reagent and specialized bioinformatics tools significantly increase our confidence and speed in the identification of cross-linked products when compared with cross-linking studies using traditional noncleavable reagents. Current cross-linking studies have been focused on protein complexes with known crystal structures, but topological structures of protein complexes based primarily on cross-linking data have not yet been reported. This is due to the lack of computational tools that use cross-linking data to deduce the spatial organization of subunits in a given complex. To define the architecture of the yeast 19S RP, we have characterized the proximity and interconnectivity of the subunits by employing our newly developed cross-linking strategy. The resulting cross-linking information serves as a basis for a rigorous probabilistic analysis to obtain the maximum likelihood (ML) topology. This strategy is developed by first analyzing our cross-linking data for the 19S six-member AAA-ATPase base ring, as the topology ordering of yeast orthologs has been recently determined (14, 26–28). The effectiveness of this new probabilistic platform is supported by the agreement between our derived ML topology of the AAA-ATPase base ring and previous reports. When the same probabilistic approach is applied to the 19S lid subcomplex, the resulting topology is also in agreement with recently proposed models (20, 21). This work represents the first application of probabilistic modeling of protein complexes based solely on cross-link data, establishing a new workflow for future structural analysis of large protein complexes using XL-MS.     

Reply to J.N. Perry et al

The authors of “The anglerfish deception” respond to the criticism of their article.EMBO reports (2012) advanced online publication; doi: 10.1038/embor.2012.70EMBO reports (2012) 13 2, 100–105; doi: 10.1038/embor.2011.254Our respondents, eight current or former members of the EFSA GMO panel, focus on defending the EFSA''s environmental risk assessment (ERA) procedures. In our article for EMBO reports, we actually focused on the proposed EU GMO legislative reform, especially the European Commission (EC) proposal''s false political inflation of science, which denies the normative commitments inevitable in risk assessment (RA). Unfortunately the respondents do not address this problem. Indeed, by insisting that Member States enjoy freedom over risk management (RM) decisions despite the EFSA''s central control over RA, they entirely miss the relevant point. This is the unacknowledged policy—normative commitments being made before, and during, not only after, scientific ERA. They therefore only highlight, and extend, the problem we identified.The respondents complain that we misunderstood the distinction between RA and RM. We did not. We challenged it as misconceived and fundamentally misleading—as though only objective science defined RA, with normative choices cleanly confined to RM. Our point was that (i) the processes of scientific RA are inevitably shaped by normative commitments, which (ii) as a matter of institutional, policy and scientific integrity must be acknowledged and inclusively deliberated. They seem unaware that many authorities [1,2,3,4] have recognized such normative choices as prior matters, of RA policy, which should be established in a broadly deliberative manner “in advance of risk assessment to ensure that [RA] is systematic, complete, unbiased and transparent” [1]. This was neither recognized nor permitted in the proposed EC reform—a central point that our respondents fail to recognize.In dismissing our criticism that comparative safety assessment appears as a ‘first step'' in defining ERA, according to the new EFSA ERA guidelines, which we correctly referred to in our text but incorrectly referenced in the bibliography [5], our respondents again ignore this widely accepted ‘framing'' or ‘problem formulation'' point for science. The choice of comparator has normative implications as it immediately commits to a definition of what is normal and, implicitly, acceptable. Therefore the specific form and purpose of the comparison(s) is part of the validity question. Their claim that we are against comparison as a scientific step is incorrect—of course comparison is necessary. This simply acts as a shield behind which to avoid our and others'' [6] challenge to their self-appointed discretion to define—or worse, allow applicants to define—what counts in the comparative frame. Denying these realities and their difficult but inevitable implications, our respondents instead try to justify their own particular choices as ‘science''. First, they deny the first-step status of comparative safety assessment, despite its clear appearance in their own ERA Guidance Document [5]—in both the representational figure (p.11) and the text “the outcome of the comparative safety assessment allows the determination of those ‘identified'' characteristics that need to be assessed [...] and will further structure the ERA” (p.13). Second, despite their claims to the contrary, ‘comparative safety assessment'', effectively a resurrection of substantial equivalence, is a concept taken from consumer health RA, controversially applied to the more open-ended processes of ERA, and one that has in fact been long-discredited if used as a bottleneck or endpoint for rigorous RA processes [7,8,9,10]. The key point is that normative commitments are being embodied, yet not acknowledged, in RA science. This occurs through a range of similar unaccountable RA steps introduced into the ERA Guidance, such as judgement of ‘biological relevance'', ‘ecological relevance'', or ‘familiarity''. We cannot address these here, but our basic point is that such endless ‘methodological'' elaborations of the kind that our EFSA colleagues perform, only obscure the institutional changes needed to properly address the normative questions for policy-engaged science.Our respondents deny our claim concerning the singular form of science the EC is attempting to impose on GM policy and debate, by citing formal EFSA procedures for consultations with Member States and non-governmental organizations. However, they directly refute themselves by emphasizing that all Member State GM cultivation bans, permitted only on scientific grounds, have been deemed invalid by EFSA. They cannot have it both ways. We have addressed the importance of unacknowledged normativity in quality assessments of science for policy in Europe elsewhere [11]. However, it is the ‘one door, one key'' policy framework for science, deriving from the Single Market logic, which forces such singularity. While this might be legitimate policy, it is not scientific. It is political economy.Our respondents conclude by saying that the paramount concern of the EFSA GMO panel is the quality of its science. We share this concern. However, they avoid our main point that the EC-proposed legislative reform would only exacerbate their problem. Ignoring the normative dimensions of regulatory science and siphoning-off scientific debate and its normative issues to a select expert panel—which despite claiming independence faces an EU Ombudsman challenge [12] and European Parliament refusal to discharge their 2010 budget, because of continuing questions over conflicts of interests [13,14]—will not achieve quality science. What is required are effective institutional mechanisms and cultural norms that identify, and deliberatively address, otherwise unnoticed normative choices shaping risk science and its interpretive judgements. It is not the EFSA''s sole responsibility to achieve this, but it does need to recognize and press the point, against resistance, to develop better EU science and policy.