Using formal methods to support testing

Formal methods and testing are two important approaches that assist in the development of high quality software. While traditionally these approaches have been seen as rivals, in recent years a new consensus has developed in which they are seen as complementary. This article reviews the state of the art regarding ways in which the presence of a formal specification can be used to assist testing

MARCH6 and TRC8 facilitate the quality control of cytosolic and tail-anchored proteins.

Misfolded or damaged proteins are typically targeted for destruction by proteasome-mediated degradation, but the mammalian ubiquitin machinery involved is incompletely understood. Here, using forward genetic screens in human cells, we find that the proteasome-mediated degradation of the soluble misfolded reporter, mCherry-CL1, involves two ER-resident E3 ligases, MARCH6 and TRC8. mCherry-CL1 degradation is routed via the ER membrane and dependent on the hydrophobicity of the substrate, with complete stabilisation only observed in double knockout MARCH6/TRC8 cells. To identify a more physiological correlate, we used quantitative mass spectrometry and found that TRC8 and MARCH6 depletion altered the turnover of the tail-anchored protein heme oxygenase-1 (HO-1). These E3 ligases associate with the intramembrane cleaving signal peptide peptidase (SPP) and facilitate the degradation of HO-1 following intramembrane proteolysis. Our results highlight how ER-resident ligases may target the same substrates, but work independently of each other, to optimise the protein quality control of selected soluble and tail-anchored proteins

Plasma membrane profiling defines an expanded class of cell surface proteins selectively targeted for degradation by HCMV US2 in cooperation with UL141.

Human cytomegalovirus (HCMV) US2, US3, US6 and US11 act in concert to prevent immune recognition of virally infected cells by CD8+ T-lymphocytes through downregulation of MHC class I molecules (MHC-I). Here we show that US2 function goes far beyond MHC-I degradation. A systematic proteomic study using Plasma Membrane Profiling revealed US2 was unique in downregulating additional cellular targets, including: five distinct integrin α-chains, CD112, the interleukin-12 receptor, PTPRJ and thrombomodulin. US2 recruited the cellular E3 ligase TRC8 to direct the proteasomal degradation of all its targets, reminiscent of its degradation of MHC-I. Whereas integrin α-chains were selectively degraded, their integrin β1 binding partner accumulated in the ER. Consequently integrin signaling, cell adhesion and migration were strongly suppressed. US2 was necessary and sufficient for degradation of the majority of its substrates, but remarkably, the HCMV NK cell evasion function UL141 requisitioned US2 to enhance downregulation of the NK cell ligand CD112. UL141 retained CD112 in the ER from where US2 promoted its TRC8-dependent retrotranslocation and degradation. These findings redefine US2 as a multifunctional degradation hub which, through recruitment of the cellular E3 ligase TRC8, modulates diverse immune pathways involved in antigen presentation, NK cell activation, migration and coagulation; and highlight US2's impact on HCMV pathogenesis.This study was financially supported by grant 101-2917-I-564-035 from the Taiwan National Science Council to JLH; by a Wellcome Trust Fellowship (093966/Z/10/Z) to MPW; an MRC Project Grant and Wellcome Trust Programme Grant (G1000236, WT090323MA) to GWW and PT, European Regional Development Fund and the State Budget of Czech Republic (RECAMO, CZ.1.05/ 2.1.00/03.0101) to ER; a Wellcome Trust Principal Research Fellowship (084957/Z/08/Z) to PJL; and a Medical Research Council (MRC) grant (MC_UU_12014/3) to GSW and AJD. This study was additionally supported by the Cambridge Biomedical Research Centre, UK.This is the final published version. It first appeared at http://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1004811

Ruthenacycles and Iridacycles as Catalysts for Asymmetric Transfer Hydrogenation and Racemisation

Ruthenacycles, which are easily prepared in a single step by reaction between enantiopure aromatic amines and [Ru(arene)Cl2]2 in the presence of NaOH and KPF6, are very good asymmetric transfer hydrogenation catalysts. A range of aromatic ketones were reduced using isopropanol in good yields with ee’s up to 98%. Iridacycles, which are prepared in similar fashion from [IrCp*Cl2]2 are excellent catalysts for the racemisation of secondary alcohols and chlorohydrins at room temperature. This allowed the development of a new dynamic kinetic resolution of chlorohydrins to the enantiopure epoxides in up to 90% yield and 98% enantiomeric excess (ee) using a mutant of the enzyme Haloalcohol dehalogenase C and an iridacycle as racemisation catalyst.

Doyne lecture 2016:intraocular health and the many faces of inflammation

Dogma for reasons of immune privilege including sequestration (sic) of ocular antigen, lack of lymphatic and immune competent cells in the vital tissues of the eye has long evaporated. Maintaining tissue and cellular health to preserve vision requires active immune responses to prevent damage and respond to danger. A priori the eye must contain immune competent cells, undergo immune surveillance to ensure homoeostasis as well as an ability to promote inflammation. By interrogating immune responses in non-infectious uveitis and compare with age-related macular degeneration (AMD), new concepts of intraocular immune health emerge. The role of macrophage polarisation in the two disorders is a tractable start. TNF-alpha regulation of macrophage responses in uveitis has a pivotal role, supported via experimental evidence and validated by recent trial data. Contrast this with the slow, insidious degeneration in atrophic AMD or in neovasular AMD, with the compelling genetic association with innate immunity and complement, highlights an ability to attenuate pathogenic immune responses and despite known inflammasome activation. Yolk sac-derived microglia maintains tissue immune health. The result of immune cell activation is environmentally dependent, for example, on retinal cell bioenergetics status, autophagy and oxidative stress, and alterations that skew interaction between macrophages and retinal pigment epithelium (RPE). For example, dead RPE eliciting macrophage VEGF secretion but exogenous IL-4 liberates an anti-angiogenic macrophage sFLT-1 response. Impaired autophagy or oxidative stress drives inflammasome activation, increases cytotoxicity, and accentuation of neovascular responses, yet exogenous inflammasome-derived cytokines, such as IL-18 and IL-33, attenuate responses

Dietary Supplementation with Soluble Plantain Non-Starch Polysaccharides Inhibits Intestinal Invasion of Salmonella Typhimurium in the Chicken

Soluble fibres (non-starch polysaccharides, NSP) from edible plants but particularly plantain banana (Musa spp.), have been shown in vitro and ex vivo to prevent various enteric pathogens from adhering to, or translocating across, the human intestinal epithelium, a property that we have termed contrabiotic. Here we report that dietary plantain fibre prevents invasion of the chicken intestinal mucosa by Salmonella. In vivo experiments were performed with chicks fed from hatch on a pellet diet containing soluble plantain NSP (0 to 200 mg/d) and orally infected with S.Typhimurium 4/74 at 8 d of age. Birds were sacrificed 3, 6 and 10 d post-infection. Bacteria were enumerated from liver, spleen and caecal contents. In vitro studies were performed using chicken caecal crypts and porcine intestinal epithelial cells infected with Salmonella enterica serovars following pre-treatment separately with soluble plantain NSP and acidic or neutral polysaccharide fractions of plantain NSP, each compared with saline vehicle. Bacterial adherence and invasion were assessed by gentamicin protection assay. In vivo dietary supplementation with plantain NSP 50 mg/d reduced invasion by S.Typhimurium, as reflected by viable bacterial counts from splenic tissue, by 98.9% (95% CI, 98.1–99.7; P<0.0001). In vitro studies confirmed that plantain NSP (5–10 mg/ml) inhibited adhesion of S.Typhimurium 4/74 to a porcine epithelial cell-line (73% mean inhibition (95% CI, 64–81); P<0.001) and to primary chick caecal crypts (82% mean inhibition (95% CI, 75–90); P<0.001). Adherence inhibition was shown to be mediated via an effect on the epithelial cells and Ussing chamber experiments with ex-vivo human ileal mucosa showed that this effect was associated with increased short circuit current but no change in electrical resistance. The inhibitory activity of plantain NSP lay mainly within the acidic/pectic (homogalacturonan-rich) component. Supplementation of chick feed with plantain NSP was well tolerated and shows promise as a simple approach for reducing invasive salmonellosis

Consensus for experimental design in electromyography (CEDE) project: Application of EMG to estimate muscle force

Skeletal muscles power movement. Deriving the forces produced by individual muscles has applications across various fields including biomechanics, robotics, and rehabilitation. Since direct in vivo measurement of muscle force in humans is invasive and challenging, its estimation through non-invasive methods such as electromyography (EMG) holds considerable appeal. This matrix, developed by the Consensus for Experimental Design in Electromyography (CEDE) project, summarizes recommendations on the use of EMG to estimate muscle force. The matrix encompasses the use of bipolar surface EMG, high density surface EMG, and intra-muscular EMG (1) to identify the onset of muscle force during isometric contractions, (2) to identify the offset of muscle force during isometric contractions, (3) to identify force fluctuations during isometric contractions, (4) to estimate force during dynamic contractions, and (5) in combination with musculoskeletal models to estimate force during dynamic contractions. For each application, recommendations on the appropriateness of using EMG to estimate force and justification for each recommendation are provided. The achieved consensus makes clear that there are limited scenarios in which EMG can be used to accurately estimate muscle forces. In most cases, it remains important to consider the activation as well as the muscle state and other biomechanical and physiological factors— such as in the context of a formal mechanical model. This matrix is intended to encourage interdisciplinary discussions regarding the integration of EMG with other experimental techniques and to promote advances in the application of EMG towards developing muscle models and musculoskeletal simulations that can accurately predict muscle forces in healthy and clinical populations