Identifying Extrinsic versus Intrinsic Drivers of Variation in Cell Behavior in Human iPSC Lines from Healthy Donors

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Binding of netrin-4 to laminin short arms regulates basement membrane assembly

Netrins were first identified as neural guidance molecules, acting through receptors that are members of the DCC and UNC-5 family. All netrins share structural homology to the laminin N-terminal domains and the laminin epidermal growth factor-like domains of laminin short arms. Laminins use these domains to self-assemble into complex networks. Here we demonstrate that netrin-4 is a component of basement membranes and is integrated into the laminin polymer via interactions with the laminin gamma1 andgamma3 short arms. The binding is mediated through the laminin N-terminal domain of netrin-4. In contrast to netrin-4, other members of the netrin family do not bind to these laminin short arms. Moreover, a truncated form of netrin-4 completely inhibits laminin-111 self-assembly in vitro, and full-length netrin-4 can partially disrupt laminin self-interactions. When added to explant cultures, netrin-4 retards salivary gland branching morphogenesis.     

Mutational analysis of cytochrome b at the ubiquinol oxidation site of yeast complex III

The cytochrome bc1 complex is a dimeric enzyme of the inner mitochondrial membrane that links electron transfer from ubiquinol to cytochrome c by a protonmotive Q cycle mechanism in which ubiquinol is oxidized at one center in the enzyme, referred to as center P, and ubiquinone is rereduced at a second center, referred to as center N. To better understand the mechanism of ubiquinol oxidation, we have examined catalytic activities and pre-steady-state reduction kinetics of yeast cytochrome bc1 complexes with mutations in cytochrome b that we expected would affect oxidation of ubiquinol. We mutated two residues thought to be involved in proton conduction linked to ubiquinol oxidation, Tyr132 and Glu272, and two residues proposed to be involved in docking ubiquinol into the center P pocket, Phe129 and Tyr279. Substitution of Phe129 by lysine or arginine yielded a respiration-deficient phenotype and lipid-dependent catalytic activity. Increased bypass reactions were detectable for both variants, with F129K showing the more severe effects. Substitution with lysine leads to a disturbed coordination of a b heme as deduced from changes in the midpoint potential and the EPR signature. Removal of the aromatic side chain in position Tyr279 lowers the catalytic activity accompanied by a low level of bypass reactions. Pre-steady-state kinetics of the enzymes modified at Glu272 and Tyr132 confirmed the importance of their functional groups for electron transfer. Altered center N kinetics and activation of ubiquinol oxidation by binding of cytochrome c in the Y132F and E272D enzymes indicate long range effects of these mutations.     

A protocol for isolation and visualization of yeast nuclei by scanning electron microscopy (SEM)

This protocol details methods for the isolation of yeast nuclei from budding yeast (Saccharomyces cerevisiae) and fission yeast (Schizosaccharomyces pombe), immuno-gold labeling of proteins and visualization by field emission scanning electron microscopy (FESEM). This involves the removal of the yeast cell wall and isolation of the nucleus from within, followed by subsequent processing for high-resolution microscopy. The nuclear isolation step can be performed in two ways: enzymatic treatment of yeast cells to rupture the cell wall and generate spheroplasts (cells that have partially lost their cell wall and their characteristic shape), followed by isolation of the nuclei by centrifugation or homogenization; and whole cell freezing followed by manual cell rupture and centrifugation. This protocol has been optimized for the visualization of the yeast nuclear envelope (NE), nuclear pore complexes (NPCs) and associated cyto-skeletal structures. Samples once processed for FESEM can be stored under vacuum for weeks, allowing considerable time for image acquisition.     

Predicting spatially heterogeneous invasive spread: Pyracantha angustifolia invading a dry Andean valley in northern Argentina

Biological Invasions - Understanding the drivers of invasive species spread is key to designing optimal management programmes for controlling them. Population models, parameterized from demographic...     

Removal of adult cyathostomins alters faecal microbiota and promotes an inflammatory phenotype in horses

The interactions between parasitic helminths and gut microbiota are considered to be an important, although as yet incompletely understood, factor in the regulation of immunity, inflammation and a range of diseases. Infection with intestinal helminths is ubiquitous in grazing horses, with cyathostomins (about 50 species of which are recorded) predominating. Consequences of infection include both chronic effects, and an acute inflammatory syndrome, acute larval cyathostominosis, which sometimes follows removal of adult helminths by administration of anthelmintic drugs. The presence of cyathostomins as a resident helminth population of the equine gut (the “helminthome”) provides an opportunity to investigate the effect helminth infection, and its perturbation, has on both the immune system and bacterial microbiome of the gut, as well as to determine the specific mechanisms of pathophysiology involved in equine acute larval cyathostominosis. We studied changes in the faecal microbiota of two groups of horses following treatment with anthelmintics (fenbendazole or moxidectin). We found decreases in both alpha diversity and beta diversity of the faecal microbiota at Day 7 post-treatment, which were reversed by Day 14. These changes were accompanied by increases in inflammatory biomarkers. The general pattern of faecal microbiota detected was similar to that seen in the relatively few equine gut microbiome studies reported to date. We conclude that interplay between resident cyathostomin populations and the bacterial microbiota of the equine large intestine is important in maintaining homeostasis and that disturbance of this ecology can lead to gut dysbiosis and play a role in the aetiology of inflammatory conditions in the horse, including acute larval cyathostominosis.     

Non-native species and lake warming negatively affect Arctic char Salvelinus alpinus abundance; deep thermal refugia facilitate co-existence

This study finds that non-native species and warming temperatures have significant negative effects on Arctic char Salvelinus alpinus abundance in Irish lakes. Eutrophication was not important at the range of total phosphorus tested (0.005–0.023 mg l⁻¹). Model results predict that S. alpinus occur across the temperature range sampled (8.2–19.7°C) when non-natives are absent, but S. alpinus catch is predicted to be close to zero irrespective of temperature when non-native catch is high. This result indicates that to persist, S. alpinus may require a habitat where non-natives are at low abundance or absent. Salvelinus alpinus segregated from other species along the thermal axis, inhabiting significantly colder water and actively avoided non-native species, which appeared to limit their distribution. The thermal niche realized by S. alpinus in non-native dominated lakes was thus compressed relative to native dominated lakes and S. alpinus population density was significantly lower. These findings were consistent even when the only non-native present was Perca fluviatilis. Temperature appeared to limit the distribution of non-native species, such that the presence of deep thermal refugia is currently facilitating S. alpinus co-existence with non-natives in associated lakes. Diet analysis identified P. fluviatilis as potential predators and competitors. This study provides strong evidence that non-native species are a key driver of low S. alpinus abundance in Irish lakes. Temperature increases associated with climate change are identified as a secondary concern, as they could erode S. alpinus' thermal niche and lead to their extirpation. The lower thermal buffering capacity of shallow lakes identifies these as higher risk systems. Salvelinus alpinus conservation in Ireland should focus on preventing future illegal non-native species introductions because unlike other stressors (e.g., eutrophication etc.), species introductions are rarely reversible.     

The cis-Regulatory Atlas of the Mouse Immune System

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