Applying refinement to the use of mice and rats in rheumatoid arthritis research

Rheumatoid arthritis (RA) is a painful, chronic disorder and there is currently an unmet need for effective therapies that will benefit a wide range of patients. The research and development process for therapies and treatments currently involves in vivo studies, which have the potential to cause discomfort, pain or distress. This Working Group report focuses on identifying causes of suffering within commonly used mouse and rat ‘models’ of RA, describing practical refinements to help reduce suffering and improve welfare without compromising the scientific objectives. The report also discusses other, relevant topics including identifying and minimising sources of variation within in vivo RA studies, the potential to provide pain relief including analgesia, welfare assessment, humane endpoints, reporting standards and the potential to replace animals in RA research

Susceptibility to tuberculosis is associated with variants in the ASAP1 gene encoding a regulator of dendritic cell migration

Human genetic factors predispose to tuberculosis (TB). We studied 7.6 million genetic variants in 5,530 people with pulmonary TB and in 5,607 healthy controls. In the combined analysis of these subjects and the follow-up cohort (15,087 TB patients and controls altogether), we found an association between TB and variants located in introns of the ASAP1 gene on chromosome 8q24 (P = 2.6 × 10−11 for rs4733781; P = 1.0 × 10−10 for rs10956514). Dendritic cells (DCs) showed high ASAP1 expression that was reduced after Mycobacterium tuberculosis infection, and rs10956514 was associated with the level of reduction of ASAP1 expression. The ASAP1 protein is involved in actin and membrane remodeling and has been associated with podosomes. The ASAP1-depleted DCs showed impaired matrix degradation and migration. Therefore, genetically determined excessive reduction of ASAP1 expression in M. tuberculosis–infected DCs may lead to their impaired migration, suggesting a potential mechanism of predisposition to TB

The microRNA-29 family in cartilage homeostasis and osteoarthritis

MicroRNAs have been shown to function in cartilage development and homeostasis, as well as in progression of osteoarthritis. The objective of the current study was to identify microRNAs involved in the onset or early progression of osteoarthritis and characterise their function in chondrocytes. MicroRNA expression in mouse knee joints post-DMM surgery was measured over 7 days. Expression of miR-29b-3p was increased at day 1 and regulated in the opposite direction to its potential targets. In a mouse model of cartilage injury and in end-stage human OA cartilage, the miR-29 family were also regulated. SOX9 repressed expression of miR-29a-3p and miR-29b-3p via the 29a/b1 promoter. TGFβ1 decreased expression of miR-29a, b and c (3p) in primary chondrocytes, whilst IL-1β increased (but LPS decreased) their expression. The miR-29 family negatively regulated Smad, NFκB and canonical WNT signalling pathways. Expression profiles revealed regulation of new WNT-related genes. Amongst these, FZD3, FZD5, DVL3, FRAT2, CK2A2 were validated as direct targets of the miR-29 family. These data identify the miR-29 family as microRNAs acting across development and progression of OA. They are regulated by factors which are important in OA and impact on relevant signalling pathways

Potential climatic transitions with profound impact on Europe

We discuss potential transitions of six climatic subsystems with large-scale impact on Europe, sometimes denoted as tipping elements. These are the ice sheets on Greenland and West Antarctica, the Atlantic thermohaline circulation, Arctic sea ice, Alpine glaciers and northern hemisphere stratospheric ozone. Each system is represented by co-authors actively publishing in the corresponding field. For each subsystem we summarize the mechanism of a potential transition in a warmer climate along with its impact on Europe and assess the likelihood for such a transition based on published scientific literature. As a summary, the ‘tipping’ potential for each system is provided as a function of global mean temperature increase which required some subjective interpretation of scientific facts by the authors and should be considered as a snapshot of our current understanding. <br/

Performance of Genotype Imputation for Rare Variants Identified in Exons and Flanking Regions of Genes

Genotype imputation has the potential to assess human genetic variation at a lower cost than assaying the variants using laboratory techniques. The performance of imputation for rare variants has not been comprehensively studied. We utilized 8865 human samples with high depth resequencing data for the exons and flanking regions of 202 genes and Genome-Wide Association Study (GWAS) data to characterize the performance of genotype imputation for rare variants. We evaluated reference sets ranging from 100 to 3713 subjects for imputing into samples typed for the Affymetrix (500K and 6.0) and Illumina 550K GWAS panels. The proportion of variants that could be well imputed (true r2>0.7) with a reference panel of 3713 individuals was: 31% (Illumina 550K) or 25% (Affymetrix 500K) with MAF (Minor Allele Frequency) less than or equal 0.001, 48% or 35% with 0.001<MAF< = 0.005, 54% or 38% with 0.005<MAF< = 0.01, 78% or 57% with 0.01<MAF< = 0.05, and 97% or 86% with MAF>0.05. The performance for common SNPs (MAF>0.05) within exons and flanking regions is comparable to imputation of more uniformly distributed SNPs. The performance for rare SNPs (0.01<MAF< = 0.05) was much more dependent on the GWAS panel and the number of reference samples. These results suggest routine use of genotype imputation for extending the assessment of common variants identified in humans via targeted exon resequencing into additional samples with GWAS data, but imputation of very rare variants (MAF< = 0.005) will require reference panels with thousands of subjects

Gain in cellular organization of inflammatory breast cancer: A 3D in vitro model that mimics the in vivo metastasis

<p>Abstract</p> <p>Background</p> <p>The initial step of metastasis in carcinomas, often referred to as the epithelial-mesenchymal transition (EMT), occurs via the loss of adherens junctions (e.g. cadherins) by the tumor embolus. This leads to a subsequent loss of cell polarity and cellular differentiation and organization, enabling cells of the embolus to become motile and invasive. However highly malignant inflammatory breast cancer (IBC) over-expresses E-cadherin. The human xenograft model of IBC (MARY-X), like IBC, displays the signature phenotype of an exaggerated degree of lymphovascular invasion (LVI) <it>in situ </it>by tumor emboli. An intact E-cadherin/α, β-catenin axis mediates the tight, compact clump of cells found both <it>in vitro </it>and <it>in vivo </it>as spheroids and tumor emboli, respectively.</p> <p>Methods</p> <p>Using electron microscopy and focused ion beam milling to acquire <it>in situ </it>sections, we performed ultrastructural analysis of both an IBC and non-IBC, E-cadherin positive cell line to determine if retention of this adhesion molecule contributed to cellular organization.</p> <p>Results</p> <p>Here we report through ultrastructural analysis that IBC exhibits a high degree of cellular organization with polar elements such as apical/lateral positioning of E-cadherin, apical surface microvilli, and tortuous lumen-like (canalis) structures. In contrast, agarose-induced spheroids of MCF-7, a weakly invasive E-cadherin positive breast carcinoma cell line, do not exhibit ultrastructural polar features.</p> <p>Conclusions</p> <p>This study has determined that the highly metastatic IBC with an exaggerated malignant phenotype challenges conventional wisdom in that instead of displaying a loss of cellular organization, IBC acquires a highly structured architecture.</p> <p>These findings suggest that the metastatic efficiency might be linked to the formation and maintenance of these architectural features. The comparative architectural features of both the spheroid and embolus of MARY-X provide an <it>in vitro </it>model with tractable <it>in vivo </it>applications.</p

Molybdenum and Phosphorus Interact to Constrain Asymbiotic Nitrogen Fixation in Tropical Forests

Biological di-nitrogen fixation (N2) is the dominant natural source of new nitrogen to land ecosystems. Phosphorus (P) is thought to limit N2 fixation in many tropical soils, yet both molybdenum (Mo) and P are crucial for the nitrogenase reaction (which catalyzes N2 conversion to ammonia) and cell growth. We have limited understanding of how and when fixation is constrained by these nutrients in nature. Here we show in tropical forests of lowland Panama that the limiting element on asymbiotic N2 fixation shifts along a broad landscape gradient in soil P, where Mo limits fixation in P-rich soils while Mo and P co-limit in P-poor soils. In no circumstance did P alone limit fixation. We provide and experimentally test a mechanism that explains how Mo and P can interact to constrain asymbiotic N2 fixation. Fixation is uniformly favored in surface organic soil horizons - a niche characterized by exceedingly low levels of available Mo relative to P. We show that soil organic matter acts to reduce molybdate over phosphate bioavailability, which, in turn, promotes Mo limitation in sites where P is sufficient. Our findings show that asymbiotic N2 fixation is constrained by the relative availability and dynamics of Mo and P in soils. This conceptual framework can explain shifts in limitation status across broad landscape gradients in soil fertility and implies that fixation depends on Mo and P in ways that are more complex than previously thought

Genes Involved in the Balance between Neuronal Survival and Death during Inflammation

Glucocorticoids are potent regulators of the innate immune response, and alteration in this inhibitory feedback has detrimental consequences for the neural tissue. This study profiled and investigated functionally candidate genes mediating this switch between cell survival and death during an acute inflammatory reaction subsequent to the absence of glucocorticoid signaling. Oligonucleotide microarray analysis revealed that following lipopolysaccharide (LPS) intracerebral administration at striatum level, more modulated genes presented transcription impairment than exacerbation upon glucocorticoid receptor blockage. Among impaired genes we identified ceruloplasmin (Cp), which plays a key role in iron metabolism and is implicated in a neurodegenative disease. Microglial and endothelial induction of Cp is a natural neuroprotective mechanism during inflammation, because Cp-deficient mice exhibited increased iron accumulation and demyelination when exposed to LPS and neurovascular reactivity to pneumococcal meningitis. This study has identified genes that can play a critical role in programming the innate immune response, helping to clarify the mechanisms leading to protection or damage during inflammatory conditions in the CNS