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Objectives: This fourteen-centre project used professional rating scales and parent questionnaires to assess longitudinal outcomes in a large non-selected population of children receiving simultaneous and sequential bilateral cochlear implants. Methods: This was an observational non-randomised service evaluation. Data were collected at four time points: before bilateral cochlear implants or before the sequential implant, one year, two years and three years after. The measures reported are Categories of Auditory Performance II (CAPII), Speech Intelligibility Rating (SIR), Bilateral Listening Skills Profile (BLSP) and Parent Outcome Profile (POP). Results: 1001 children aged from 8 months to almost 18 years were involved, although there were many missing data. In children receiving simultaneous implants after one, two, and three years respectively, median CAP scores were 4, 5, and 6; median SIR were 1, 2, and 3. Three years after receiving simultaneous bilateral cochlear implants, 61% of children were reported to understand conversation without lip-reading and 66% had intelligible speech if the listener concentrated hard. Auditory performance and speech intelligibility were significantly better in female children than males.Parents of children using sequential implants were generally positive about their child’s well-being and behaviour since receiving the second device; those who were less positive about well-being changes also generally reported their children less willing to wear the second device. Conclusion: Data from 78% of paediatric cochlear implant centres in the United Kingdom provide a real-world picture of outcomes of children with bilateral implants in the UK. This large reference data set can be used to identify children in the lower quartile for targeted intervention.

The objective was to measure effects of 3-nitrooxypropanol (3 NP) on methane production of lactating dairy cows and any associated changes in digestion and energy and N metabolism. Six Holstein-Friesian dairy cows in mid-lactation were fed twice daily a total mixed ration with maize silage as the primary forage source. Cows received 1 of 3 treatments using an experimental design based on two 3 × 3 Latin squares with 5-wk periods. Treatments were a control placebo or 500 or 2,500 mg/d of 3 NP delivered directly into the rumen, via the rumen fistula, in equal doses before each feeding. Measurements of methane production and energy and N balance were obtained during wk 5 of each period using respiration calorimeters and digestion trials. Measurements of rumen pH (48 h) and postprandial volatile fatty acid and ammonia concentrations were made at the end of wk 4. Daily methane production was reduced by 3 NP, but the effects were not dose dependent (reductions of 6.6 and 9.8% for 500 and 2,500 mg/d, respectively). Dosing 3 NP had a transitory inhibitory effect on methane production, which may have been due to the product leaving the rumen in liquid outflow or through absorption or metabolism. Changes in rumen concentrations of volatile fatty acids indicated that the pattern of rumen fermentation was affected by both doses of the product, with a decrease in acetate:propionate ratio observed, but that acetate production was inhibited by the higher dose. Dry matter, organic matter, acid detergent fiber, N, and energy digestibility were reduced at the higher dose of the product. The decrease in digestible energy supply was not completely countered by the decrease in methane excretion such that metabolizable energy supply, metabolizable energy concentration of the diet, and net energy balance (milk plus tissue energy) were reduced by the highest dose of 3 NP. Similarly, the decrease in N digestibility at the higher dose of the product was associated with a decrease in body N balance that was not observed for the lower dose. Milk yield and milk fat concentration and fatty acid composition were not affected but milk protein concentration was greater for the higher dose of 3 NP. Twice-daily rumen dosing of 3 NP reduced methane production by lactating dairy cows, but the dose of 2,500 mg/d reduced rumen acetate concentration, diet digestibility, and energy supply. Further research is warranted to determine the optimal dose and delivery method of the product.

Objectives: To assess longitudinal outcomes in a large and varied population of children receiving bilateral cochlear implants both simultaneously and sequentially. Methods: This observational non-randomised service evaluation collected localisation and speech recognition in noise data from simultaneously and sequentially-implanted children at four time points: before bilateral cochlear implants or before the sequential implant, one year, two years and three years after bilateral implants. No inclusion criteria were applied, so children with additional difficulties, cochleovestibular anomalies, varying educational placements, 23 different home languages, a full range of outcomes and varying device use were included. Results: 1001 children were included: 465 implanted simultaneously and 536 sequentially, representing just over 50% of children receiving bilateral implants in the UK in this period. In simultaneously-implanted children the median age at implant was 2.1 years; 7% were implanted at less than one year of age. In sequentially-implanted children the interval between implants ranged from 0.1 to 14.5 years. Children with simultaneous bilateral implants localised better than those with one implant. On average children receiving a second (sequential) cochlear implant showed improvement in localisation and listening in background noise after one year of bilateral listening. The interval between sequential implants had no effect on localisation improvement although a smaller interval gave more improvement in speech recognition in noise. Children with sequential implants on average were able to use their second device to obtain spatial release from masking after two years of bilateral listening. Although ranges were large, bilateral cochlear implants on average offered an improvement in localisation and speech perception in noise over unilateral implants. Conclusion: These data represent the diverse population of children with bilateral cochlear implants in the UK from 2010-2012. Predictions of outcomes for individual patients are not possible from these data. However, there are no indications to preclude children with long inter-implant interval having the chance of a second cochlear implant.

Basolateral polymerization of cellular fibronectin (FN) into a meshwork drives endothelial cell (EC) polarity and vascular remodelling. However, mechanisms coordinating α5β1 integrin-mediated extracellular FN endocytosis and exocytosis of newly synthesized FN remain elusive. Here we show that, on Rab21-elicited internalization, FN-bound/active α5β1 is recycled to the EC surface. We identify a pathway, comprising the regulators of post-Golgi carrier formation PI4KB and AP-1A, the small GTPase Rab11B, the surface tyrosine phosphatase receptor PTPRF and its adaptor PPFIA1, which we propose acts as a funnel combining FN secretion and recycling of active α5β1 integrin from the trans-Golgi network (TGN) to the EC surface, thus allowing FN fibrillogenesis. In this framework, PPFIA1 interacts with active α5β1 integrin and localizes close to EC adhesions where post-Golgi carriers are targeted. We show that PPFIA1 is required for FN polymerization-dependent vascular morphogenesis, both in vitro and in the developing zebrafish embryo.

The adhesion nexus is the site at which integrin receptors bridge intracellular cytoskeletal and extracellular matrix networks. The connection between integrins and the cytoskeleton is mediated by a dynamic integrin adhesion complex (IAC), the components of which transduce chemical and mechanical signals to control a multitude of cellular functions. In this Cell Science at a Glance article and the accompanying poster, we integrate the consensus adhesome, a set of 60 proteins that have been most commonly identified in isolated IAC proteomes, with the literature-curated adhesome, a theoretical network that has been assembled through scholarly analysis of proteins that localise to IACs. The resulting IAC network, which comprises four broad signalling and actin-bridging axes, provides a platform for future studies of the regulation and function of the adhesion nexus in health and disease., Summary: We have generated a new depiction of the integrin adhesome network that integrates experimentally derived IAC proteomes with the literature-curated adhesome to bridge the knowledge gap between these two resources.

Dear Sir, I would like to make your readers aware of the Tri-Service Trainee Audit and Research (Tri-STAR) network. In 2013, the Research and Audit Federation of Trainees (RAFT) was formed to encourage collaboration between the burgeoning number of anaesthetic trainee research networks (TRNs) that

There is conflicting evidence in the literature on whether individuals with haemophilia in the USA have greater, reduced, or similar risks for cardiovascular disease as the general population. This study evaluated the prevalence of cardiovascular comorbidities among USA males with haemophilia A, relative to an unaffected general male population with similar characteristics. Males with haemophilia A and continuous insurance coverage were identified by ICD-9-CM code 286.0 (1 January 2007-31 December 2009) using the MarketScan Commercial and Medicare Research Databases. Individuals with haemophilia A were exact matched 1:3 with males without a diagnosis of haemophilia A. The prevalence of cardiovascular comorbidities identified by ICD-9-CM code was determined for matched cohorts. Of the study population, 2506 were grouped in the haemophilia A cohort and 7518 in the general cohort. Proportions of individuals with haemorrhagic stroke (2.0% vs. 0.5%, P < 0.001), ischemic stroke (4.7% vs. 2.7%, P < 0.001), coronary artery disease (10.7% vs. 5.8%, P < 0.001), myocardial infarction (0.8% vs. 0.3%, P = 0.003), hypertension (22.6% vs. 15.5%, P < 0.001), hyperlipidaemia (15.9% vs. 11.9%, P < 0.001), arterial thrombosis (12.1% vs. 5.9%, P < 0.001), and venous thrombosis (4.4% vs. 1.1%, P < 0.001) were significantly greater for the haemophilia A cohort. Results were consistent across most age groups, and comorbidities appeared at an earlier age in those with haemophilia A than in the general population. Among the USA haemophilia A population cardiovascular comorbidities are more prevalent and they appear earlier in life in comparison to the general male population, suggesting the need for earlier, enhanced screening for age-related comorbidities in the haemophilia community.

Matrix mechanics controls cell fate by modulating the bonds between integrins and extracellular matrix (ECM) proteins. However, it remains unclear how fibronectin (FN), type 1 collagen, and their receptor integrin subtypes distinctly control force transmission to regulate focal adhesion kinase (FAK) activity, a crucial molecular signal governing cell adhesion/migration. Here we showed, using a genetically encoded FAK biosensor based on fluorescence resonance energy transfer, that FN-mediated FAK activation is dependent on the mechanical tension, which may expose its otherwise hidden FN synergy site to integrin α5. In sharp contrast, the ligation between the constitutively exposed binding motif of type 1 collagen and its receptor integrin α2 was surprisingly tension-independent to induce sufficient FAK activation. Although integrin α subunit determines mechanosensitivity, the ligation between α subunit and the ECM proteins converges at the integrin β1 activation to induce FAK activation. We further discovered that the interaction of the N-terminal protein 4.1/ezrin/redixin/moesin basic patch with phosphatidylinositol 4,5-biphosphate is crucial during cell adhesion to maintain the FAK activation from the inhibitory effect of nearby protein 4.1/ezrin/redixin/moesin acidic sites. Therefore, different ECM proteins either can transmit or can shield from mechanical forces to regulate cellular functions, with the accessibility of ECM binding motifs by their specific integrin α subunits determining the biophysical mechanisms of FAK activation during mechanotransduction.

Coordination of the activity of multiple small GTPases is required for the regulation of many physiological processes, including cell migration. There are now several examples of functional interplay between small GTPase pairs, but the mechanisms that control GTPase activity in time and space are only partially understood. Here, we build on the hypothesis that small GTPases are part of a large, integrated network and propose that key proteins within this network integrate multiple signaling events and coordinate multiple small GTPase activities. Specifically, we identify the scaffolding protein IQGAP1 as a master regulator of multiple small GTPases, including Cdc42, Rac1, Rap1, and RhoA. In addition, we demonstrate that IQGAP1 promotes Arf6 activation downstream of β1 integrin engagement. Furthermore, following literature-curated searches and recent mass spectrometric analysis of IQGAP1-binding partners, we report that IQGAP1 recruits other small GTPases, including RhoC, Rac2, M-Ras, RhoQ, Rab10, and Rab5, small GTPase regulators, including Tiam1, RacGAP1, srGAP2 and HERC1, and small GTPase effectors, including PAK6, N-WASP, several sub-units of the Arp2/3 complex and the formin mDia1. Therefore, we propose that IQGAP1 acts as a small GTPase scaffolding platform within the small GTPase network, and recruits and/or regulates small GTPases, small GTPase regulators and effectors to orchestrate cell behavior. Finally, to identify other putative key regulators of small GTPase crosstalk, we have assembled a small GTPase network using protein-protein interaction databases.

RCP-driven α5β1 recycling suppresses Rac activity through the RacGAP1–IQGAP1 complex to permit local activation of RhoA and drive invasive migration., Inhibition of αvβ3 or expression of mutant p53 promotes invasion into fibronectin (FN)-containing extracellular matrix (ECM) by enhancing Rab-coupling protein (RCP)–dependent recycling of α5β1 integrin. RCP and α5β1 cooperatively recruit receptor tyrosine kinases, including EGFR1, to regulate their trafficking and downstream signaling via protein kinase B (PKB)/Akt, which, in turn, promotes invasive migration. In this paper, we identify a novel PKB/Akt substrate, RacGAP1, which is phosphorylated as a consequence of RCP-dependent α5β1 trafficking. Phosphorylation of RacGAP1 promotes its recruitment to IQGAP1 at the tips of invasive pseudopods, and RacGAP1 then locally suppresses the activity of the cytoskeletal regulator Rac and promotes the activity of RhoA in this subcellular region. This Rac to RhoA switch promotes the extension of pseudopodial processes and invasive migration into FN-containing matrices, in a RhoA-dependent manner. Thus, the localized endocytic trafficking of α5β1 within the tips of invasive pseudopods elicits signals that promote the reorganization of the actin cytoskeleton, protrusion, and invasion into FN-rich ECM.

Peptide quantification using MS often relies on the comparison of peptide signal intensities between different samples, which is based on the assumption that observed signal intensity has a linear relationship to peptide abundance. A typical proteomics experiment is subject to multiple sources of variance, so we focussed here on properties affecting peptide linearity under simple, well-defined conditions. Peptides from a standard protein digest were analysed by multiple reaction monitoring (MRM) MS to determine peptide linearity over a range of concentrations. We show that many peptides do not display a linear relationship between signal intensity and amount under standard conditions. Increasing the organic content of the sample solvent increased peptide linearity by increasing the accuracy and precision of quantification, which suggests that peptide non-linearity is due to concentration-dependent surface adsorption. Using multiple peptides at various dilutions, we show that peptide non-linearity is related to observed retention time and predicted hydrophobicity. Whereas the effect of adsorption on peptide storage has been investigated previously, here we demonstrate the deleterious effect of peptide adsorption on the quantification of fresh samples, highlight aspects of sample preparation that can minimise the effect, and suggest bioinformatic approaches to enhance the selection of peptides for quantification. Biological significance Accurate quantification is central to many aspects of science, especially those examining dynamic processes or comparing molecular stoichiometries. In biological research, the quantification of proteins is an important yet challenging objective. Large-scale quantification of proteins using MS often depends on the comparison of peptide intensities with only a single-level calibrant (as in stable isotope labelling and absolute quantification approaches) or no calibrants at all (as in label-free approaches). For these approaches to be reliable, it is essential that the relationship between signal intensity and concentration is linear, without a significant intercept. Here, we show that peptide adsorption can severely affect this relationship, even under controlled conditions, and we demonstrate simple methodologies that can be used to moderate and predict this effect. These findings thus enable the quantification of proteins with increased robustness and reliability., Graphical abstract, Highlights • First description of effect of peptide adsorption on linearity and quantification • Demonstration that peptide adsorption affects quantification of fresh samples • Addition of acetonitrile improves peptide solubility and reliability of quantification

Background: Interaction of stem cells with extracellular matrix (ECM) controls their fate. Results: MS reveals interacting ECM networks produced by human embryonic stem cells (hESCs) and their feeders; supportive and unsupportive hESC substrates comprise distinct ECM compositions. Conclusion: Several ECM molecules maintain hESC self-renewal. Significance: Better understanding of hESC self-renewal has applications in understanding development, generating cell therapies, and modeling diseases., Human embryonic stem cells (hESCs) are pluripotent cells that have indefinite replicative potential and the ability to differentiate into derivatives of all three germ layers. hESCs are conventionally grown on mitotically inactivated mouse embryonic fibroblasts (MEFs) or feeder cells of human origin. In addition, feeder-free culture systems can be used to support hESCs, in which the adhesive substrate plays a key role in the regulation of stem cell self-renewal or differentiation. Extracellular matrix (ECM) components define the microenvironment of the niche for many types of stem cells, but their role in the maintenance of hESCs remains poorly understood. We used a proteomic approach to characterize in detail the composition and interaction networks of ECMs that support the growth of self-renewing hESCs. Whereas many ECM components were produced by supportive and unsupportive MEF and human placental stromal fibroblast feeder cells, some proteins were only expressed in supportive ECM, suggestive of a role in the maintenance of pluripotency. We show that identified candidate molecules can support attachment and self-renewal of hESCs alone (fibrillin-1) or in combination with fibronectin (perlecan, fibulin-2), in the absence of feeder cells. Together, these data highlight the importance of specific ECM interactions in the regulation of hESC phenotype and provide a resource for future studies of hESC self-renewal.

Summary Precise spatiotemporal coordination of integrin adhesion complex dynamics is essential for efficient cell migration. For cells adherent to fibronectin, differential engagement of α5β1 and αVβ3 integrins is used to elicit changes in adhesion complex stability, mechanosensation, matrix assembly, and migration, but the mechanisms responsible for receptor regulation have remained largely obscure. We identify phosphorylation of the membrane-intercalated proteoglycan syndecan-4 as an essential switch controlling integrin recycling. Src phosphorylates syndecan-4 and, by driving syntenin binding, leads to suppression of Arf6 activity and recycling of αVβ3 to the plasma membrane at the expense of α5β1. The resultant elevation in αVβ3 engagement promotes stabilization of focal adhesions. Conversely, abrogation of syndecan-4 phosphorylation drives surface expression of α5β1, destabilizes adhesion complexes, and disrupts cell migration. These data identify the dynamic spatiotemporal regulation of Src-mediated syndecan-4 phosphorylation as an essential switch controlling integrin trafficking and adhesion dynamics to promote efficient cell migration., Graphical Abstract Highlights ► c-Src phosphorylates syndecan-4 in response to extracellular stimuli ► Syndecan-4 phosphorylation and engagement regulate Arf6 activity ► Syndecan-4-mediated Arf6 activity regulates differential integrin recycling ► Syndecan-4-mediated integrin recycling controls FA dynamics and cell migration, Efficient cell migration requires dynamic coordination of cell-matrix interactions, in part through precise spatiotemporal control of integrin adhesion dynamics. Morgan et al. show that phosphorylation of the extracellular matrix receptor syndecan-4 acts as a molecular switch, differentially directing either α5β1 or αVβ3 integrin recycling in the control of adhesion dynamics.

Cells regulate adhesion in response to internally generated and externally applied forces. Integrins connect the extracellular matrix to the cytoskeleton and provide cells with mechanical anchorages and signaling platforms. Here we show that cyclic forces applied to a fibronectin-integrin α5β1 bond switch the bond from a short-lived state with 1 s lifetime to a long-lived state with 100 s lifetime. We term this phenomenon "cyclic mechanical reinforcement," as the bond strength remembers the history of force application and accumulates over repeated cycles, but does not require force to be sustained. Cyclic mechanical reinforcement strengthens the fibronectin-integrin α5β1 bond through the RGD binding site of the ligand with the synergy binding site greatly facilitating the process. A flexible integrin hybrid domain is also important for cyclic mechanical reinforcement. Our results reveal a mechanical regulation of receptor-ligand interactions and identify a molecular mechanism for cell adhesion strengthening by cyclic forces.

When sympatric species compete, character divergence may help maintain coexistence. Snakes are often found in species-rich assemblages while exploiting similar resources; because snake body size is a relatively plastic trait that determines the range of prey sizes an individual may consume, divergence in body size between sympatric species may arise as a result of interspecific interactions. The North American racer, Coluber constrictor, and the larger coachwhip, Coluber flagellum, have a close taxonomic relationship and similar foraging strategies. Therefore, we hypothesized that C. constrictor would be smaller where they co-occur with C. flagellum, as compared to where C. flagellum is absent, throughout the southeastern extent of their range. To evaluate this hypothesis, we obtained data on body size for 2321 adult C. constrictor and 526 adult C. flagellum, along with habitat data and other potentially important factors influencing body size. Coluber constrictor was smaller than elsewhere when in peninsular Florida, in pine forests, on hydric soils and in the presence of the larger and potentially competing C. flagellum. Body size of C. flagellum did not vary by any measured habitat variables. The trends we documented are consistent with the hypothesis that C. constrictor body size is influenced by several variables, including co-occurrence with C. flagellum.

Agriculture-based reformulation initiatives, including oleic acid-rich lipid supplementation of the dairy cow diet, provide a novel means for reducing intake of saturated fatty acids (SFA) at a population level. In a blinded manner, this study evaluated the consumer acceptance of SFA-reduced, monounsaturated fatty acid-enriched (modified) milk, Cheddar cheese, and butter when compared with control and commercially available comparative samples. The effect of providing nutritional information about the modified cheese was also evaluated. Consumers (n = 115) rated samples for overall liking (appearance, flavor, and texture) using 9-point hedonic scales. Although no significant differences were found between the milk samples, the modified cheese was liked significantly less than a regular-fat commercial alternative for overall liking and liking of specific modalities and had a lower liking of texture score compared with the control cheese. The provision of health information significantly increased the overall liking of the modified cheese compared with tasting the same sample in a blinded manner. Significant differences were evident between the butter samples for overall liking and modalities of liking; all of the samples were significantly more liked than the commercial butter and sunflower oil spread. In conclusion, this study illustrated that consumer acceptance of SFA-reduced, monounsaturated fatty acid-enriched dairy products was dependent on product type. Future research should consider how optimization of the textural properties of fatty acid-modified (and fat-reduced) cheese might enhance consumer acceptance of this product.

Integrins are large, membrane-spanning, heterodimeric proteins that are essential for a metazoan existence. All members of the integrin family adopt a shape that resembles a large "head" on two "legs," with the head containing the sites for ligand binding and subunit association. Most of the receptor dimer is extracellular, but both subunits traverse the plasma membrane and terminate in short cytoplasmic domains. These domains initiate the assembly of large signaling complexes and thereby bridge the extracellular matrix to the intracellular cytoskeleton. To allowcells to sample and respond to a dynamic pericellular environment, integrins have evolved a highly responsive receptor activation mechanism that is regulated primarily by changes in tertiary and quaternary structure. This review summarizes recent progress in the structural and molecular functional studies of this important class of adhesion receptor. © 2011 Cold Spring Harbor Laboratory Press.

The crystal structure of human fibronectin (FN) type III repeats 12-14 reveals the primary heparin-binding site, a clump of positively charged residues in FN13, and a putative minor site approximately 60 A away in FN14. The IDAPS motif implicated in integrin alpha4beta1 binding is at the FN13-14 junction, rendering the critical Asp184 inaccessible to integrin. Asp184 clamps the BC loop of FN14, whose sequence (PRARI) is reminiscent of the synergy sequence (PHSRN) of FN9. Mutagenesis studies prompted by this observation reveal that both arginines of the PRARI sequence are important for alpha4beta1 binding to FN12-14. The PRARI motif may represent a new class of integrin-binding sites. The spatial organization of the binding sites suggests that heparin and integrin may bind in concert.

Adherence of microbes to host tissues is a hallmark of infectious disease and is often mediated by a class of adhesins termed MSCRAMMs (Microbial Surface Components Recognizing Adhesive Matrix Molecules). Numerous pathogens express MSCRAMMs that specifically bind the heterodimeric human glycoprotein fibronectin (Fn). In addition to roles in adhesion, Fn-binding MSCRAMMs exploit physiological Fn functions. For example, several pathogens can invade host cells by a mechanism whereby MSCRAMM-bound Fn bridges interaction with α5β1 integrin. Here, we investigate two Fn-binding MSCRAMMs, FnBPA (Staphylococcus aureus) and BBK32 (Borrelia burgdorferi) to probe structure-activity relationships of MSCRAMM-induced Fn/α5β1integrin activation. Circular dichroism, fluorescence resonance energy transfer, and dynamic light scattering techniques uncover a conformational rearrangement of Fn involving domains distant from the MSCRAMM binding site. Surface plasmon resonance experiments demonstrate a significant enhancement of Fn/α5β1 integrin affinity in the presence of FnBPA or BBK32. Detailed kinetic analysis of these interactions reveal that this change in affinity can be attributed solely to an increase in the initial Fn/α5β1 on-rate and that this rate-enhancement is dependent on high-affinity Fn-binding by MSCRAMMs. These data implicate MSCRAMM-induced perturbation of specific intramolecular contacts within the Fn heterodimer resulting in activation by exposing previously cryptic α5β1 interaction motifs. By correlating structural changes in Fn to a direct measurement of increased Fn/α5β1 affinity, this work significantly advances our understanding of the structural basis for the modulation of integrin function by Fn-binding MSCRAMMs.

In enhanced oil recovery operations, the steaming capacity of a heat recovery steam generating unit is limited by the available energy provided from the gas turbine generator, however, there are a number of other component parameters that limit the thermal and mechanical performance of any specific heat recovery steam generator. These additional parameters reside on both the flue gas side and water / steam side of the heat recovery steam generator. Repetitive failures of steam generator components are evidence of damage mechanisms that are active during operation. Some of these damage mechanisms are explored to determine the immediate impact in up-rating the steam generators to higher capacity throughput and also, on longer term reliability. A resulting finding of this study examines the efficacy of using P22 low alloy piping in lieu of carbon steel piping to address flow accelerated corrosion.

Whether β integrin ectodomains visit conformational states similarly to β and β integrins has not been characterized. Furthermore, despite a wealth of activating and inhibitory antibodies to β integrins, the conformational states that these antibodies stabilize, and the relation of these conformations to function, remain incompletely characterized. Using negative-stain electron microscopy, we show that the integrin α β ectodomain adopts extended-closed and extended-open conformations as well as a bent conformation. Antibodies SNAKA51, 8E3, N29, and 9EG7 bind to different domains in the α or β legs, activate, and stabilize extended ectodomain conformations. Antibodies 12G10 and HUTS-4 bind to the β βI domain and hybrid domains, respectively, activate, and stabilize the open headpiece conformation. Antibody TS2/16 binds a similar epitope as 12G10, activates, and appears to stabilize an open βI domain conformation without requiring extension or hybrid domain swingout. mAb13 and SG/19 bind to the βI domain and βI-hybrid domain interface, respectively, inhibit, and stabilize the closed conformation of the headpiece. The effects of the antibodies on cell adhesion to fibronectin substrates suggest that the extended-open conformation of α β is adhesive and that the extended-closed and bent-closed conformations are nonadhesive. The functional effects and binding sites of antibodies and fibronectin were consistent with their ability in binding to α β on cell surfaces to cross-enhance or inhibit one another by competitive or noncompetitive (allosteric) mechanisms., NIH Grant HL-108248

The ability to identify social partners can play a key role in the coordination of social behaviours in group-living animals. Coordinating social behaviours over long distances becomes problematic, as cues to identity are often limited to one or two sensory modalities. This limitation can often select for strong individuality in those cues used for long-distance communication. Pied babblers, Turdoides bicolor, produce a number of different types of 'loud calls' which are frequently used to signal to individuals beyond the range of visual or olfactory pathways of communication. Here, we show that three of these 'loud call' types, the v-shaped chatter, the double note ascending chatter and the atonal chatter, are each individually distinct. We hypothesise that individuality in the three loud call types tested here may represent a possible pathway to social recognition in this species that may have important consequences for social interactions. However, we also found that the atonal chatter was unstable between years suggesting that this particular call type may not be a reliable long-term indicator to identity which may affect long-term recognition in this species.

We previously demonstrated that Arg-Gly-Asp (RGD)-containing ligand-mimetic inhibitors of integrins are unable to dissociate pre-formed integrin-fibronectin complexes (IFCs). These observations suggested that amino acid residues involved in integrin-fibronectin binding become obscured in the ligand-occupied state. Because the epitopes of some function-blocking anti-integrin monoclonal antibodies (mAbs) lie near the ligand-binding pocket, it follows that the epitopes of these mAbs may become shielded in the ligand-occupied state. Here, we tested whether function-blocking mAbs directed against α5β1 can interact with the integrin after it forms a complex with an RGD-containing fragment of fibronectin. We showed that the anti-α5 subunit mAbs JBS5, SNAKA52, 16, and P1D6 failed to disrupt IFCs and hence appeared unable to bind to the ligand-occupied state. In contrast, the allosteric anti-β1 subunit mAbs 13, 4B4, and AIIB2 could dissociate IFCs and therefore were able to interact with the ligand-bound state. However, another class of function-blocking anti-β1 mAbs, exemplified by Lia1/2, could not disrupt IFCs. This second class of mAbs was also distinguished from 13, 4B4, and AIIB2 by their ability to induce homotypic cell aggregation. Although the epitope of Lia1/2 was closely overlapping with those of 13, 4B4, and AIIB2, it appeared to lie closer to the ligand-binding pocket. A new model of the α5β1-fibronectin complex supports our hypothesis that the epitopes of mAbs that fail to bind to the ligand-occupied state lie within, or very close to, the integrin-fibronectin interface. Importantly, our findings imply that the efficacy of some therapeutic anti-integrin mAbs could be limited by epitope masking.

Stille, Suzuki–Miyaura and Negishi cross‐coupling reactions of bromine‐functionalised borylated precursors enable the facile, high yielding, synthesis of borylated donor–acceptor materials that contain electron‐rich aromatic units and/or extended effective conjugation lengths. These materials have large Stokes shifts, low LUMO energies, small band‐gaps and significant fluorescence emission >700 nm in solution and when dispersed in a host polymer.

Adhesion signaling of integrin adhesion complexes (IACs) is sensitive to inhibition of key IAC kinases (FAK and/or Src), but IAC composition is not, demonstrating a separation between signaling and structural contributions of IAC components., Integrin adhesion complexes (IACs) form mechanochemical connections between the extracellular matrix and actin cytoskeleton and mediate phenotypic responses via posttranslational modifications. Here, we investigate the modularity and robustness of the IAC network to pharmacological perturbation of the key IAC signaling components focal adhesion kinase (FAK) and Src. FAK inhibition using AZ13256675 blocked FAKY397 phosphorylation but did not alter IAC composition, as reported by mass spectrometry. IAC composition was also insensitive to Src inhibition using AZD0530 alone or in combination with FAK inhibition. In contrast, kinase inhibition substantially reduced phosphorylation within IACs, cell migration and proliferation. Furthermore using fluorescence recovery after photobleaching, we found that FAK inhibition increased the exchange rate of a phosphotyrosine (pY) reporter (dSH2) at IACs. These data demonstrate that kinase-dependent signal propagation through IACs is independent of gross changes in IAC composition. Together, these findings demonstrate a general separation between the composition of IACs and their ability to relay pY-dependent signals.

Reducing the intake of dietary saturated fatty acids (SFA) to ⩽10 % of total energy intake is a key public health strategy aimed at lowering current cardiovascular disease (CVD) incidence. Consumption of dairy products (including butter) represents approximately 35 % of total dietary SFA intake(1). Supplementation of the bovine diet with oleic acid-rich plant oil has been reported to depress SFA and increase cis-monounsaturated fatty acids levels in milk, providing a strategy to lower total dietary SFA intake (2). This human intervention study aimed to investigate whether consumption of SFA-reduced, compared with conventional dairy products, would impact on the fasting lipid profile, glucose and insulin concentrations of adults at moderate CVD risk. Fifty-four adults (mean age 53 (SD 13) years, BMI 26 (SD 3) kg/m2), completed a double blind, randomised, controlled 12-week cross-over study with an 8-week washout period between treatment arms. Participants replaced habitual dairy foods/snacks with SFA-reduced or conventional UHT milk, Cheddar cheese and butter (fatty acid (FA) composition of SFA-reduced vs. conventional dairy products: total SFA: -7·0 g/d, C18:1cis: 3·0 g/d, C18:1trans: 2·4 g/d), achieving an isoenergetic daily dietary exchange (41 g/d total fat). At the beginning and end of each treatment period, fasting blood samples were collected for biochemical analysis. LDL-C was estimated using the Friedewald formula. The homeostatic model assessment of insulin resistance (HOMA-IR) was calculated using the glucose and insulin data.

Background: Recently, we demonstrated that the glycoprotein opticin is anti-angiogenic. Here, the underpining mechanism is explored. Results: By binding to collagen, opticin competitively inhibits integrin-mediated endothelial cell adhesion. Conclusion: Opticin inhibits angiogenesis by weakening endothelial cell adhesion to the surrounding extracellular matrix. Significance: Elucidating the regulatory mechanisms of angiogenesis is important for understanding pathology and drug discovery., Opticin is an extracellular matrix glycoprotein that we identified associated with the collagen network of the vitreous humor of the eye. Recently, we discovered that opticin possesses anti-angiogenic activity using a murine oxygen-induced retinopathy model: here, we investigate the underlying mechanism. Using an ex vivo chick chorioallantoic membrane assay, we show that opticin inhibits angiogenesis when stimulated by a range of growth factors. We show that it suppresses capillary morphogenesis, inhibits endothelial invasion, and promotes capillary network regression in three-dimensional matrices of collagen and MatrigelTM. We then show that opticin binds to collagen and thereby competitively inhibits endothelial cell interactions with collagen via α1β1 and α2β1 integrins, thereby preventing the strong adhesion that is required for proangiogenic signaling via these integrins.

Integrin adhesion receptors mediate cell–cell and cell–extracellular matrix interactions, which control cell morphology and migration, differentiation, and tissue integrity. Integrins recruit multimolecular adhesion complexes to their cytoplasmic domains, which provide structural and mechanosensitive signaling connections between the extracellular and intracellular milieux. The different functions of specific integrin heterodimers, such as α4β1 and α5β1, have been attributed to distinct signal transduction mechanisms that are initiated by selective recruitment of adhesion complex components to integrin cytoplasmic tails. Here, we report the isolation of ligand-induced adhesion complexes associated with wild-type α4β1 integrin, an activated α4β1 variant in the absence of the α cytoplasmic domain (X4C0), and a chimeric α4β1 variant with α5 leg and cytoplasmic domains (α4Pα5L), and the cataloguing of their proteomes by MS. Using hierarchical clustering and interaction network analyses, we detail the differential recruitment of proteins and highlight enrichment patterns of proteins to distinct adhesion complexes. We identify previously unreported components of integrin adhesion complexes and observe receptor-specific enrichment of molecules with previously reported links to cell migration and cell signaling processes. Furthermore, we demonstrate colocalization of MYO18A with active integrin in migrating cells. These datasets provide a resource for future studies of integrin receptor-specific signaling events.

The molecular basis of axonal regeneration of central nervous system (CNS) neurons remains to be fully elucidated. In part, this is due to the difficulty in maintaining CNS neurons in vitro. Here, we show that dissociated neurons from the cerebral cortex and hippocampus of adult mice may be maintained in culture for up to 9 days in defined medium without added growth factors. Outgrowth of neurites including axons was observed from both CNS sources and was significantly greater on plasma fibronectin than on other substrata such as laminin and merosin. Neurite outgrowth on fibronectin appears to be mediated by α5β1 integrin since a recombinant fibronectin fragment containing binding sites for this receptor was as effective as intact fibronectin in supporting neurite outgrowth. Conversely, function-blocking antibodies to α5 and β1 integrin sub-units inhibited neurite outgrowth on intact fibronectin. These results suggest that the axonal regeneration seen in in vivo studies using fibronectin-based matrices is due to the molecule itself and not a consequence of secondary events such as cellular infiltration. They also indicate the domains of fibronectin that may be responsible for eliciting this response., Highlights ► Fibronectin as a substratum for adult mammalian neurons. ► Increased neurite growth from hippocampal and cortical neurons grown on fibronectin. ► Increased neurite growth on fibronectin appears to involve α5β1 integrin.

A range of wastes representative of materials currently applied, or with future potential to be applied, to agricultural land in the UK as fertilisers and soil improvers or used as animal bedding in livestock production, were investigated. In addition to full physico-chemical characterization, the materials were analysed for a suite of priority organic contaminants. In general, contaminants were present at relatively low concentrations. For example, for biosolids and compost-like-output (CLO), concentrations of polychlorinated dibenzo-p-dioxins/dibenzofurans (PCDD/Fs) and polychlorinated biphenyls (PCBs) were approximately 1−10 and 5–50 times lower, respectively, than various proposed or implemented European limit values for these contaminants in biosolids or composts applied to agricultural land. However, the technical basis for these limits may require re-evaluation in some cases. Polybrominated, and mixed halogenated, dibenzo-p-dioxins/dibenzofurans are not currently considered in risk assessments of dioxins and dioxin-like chemicals, but were detected at relatively high concentrations compared with PCDD/Fs in the biosolids and CLOs and their potential contribution to the overall toxic equivalency is assessed. Other ‘emerging’ contaminants, such as organophosphate flame retardants, were detected in several of the waste materials, and their potential significance is discussed. The study is part of a wider research programme that will provide evidence that is expected to improve confidence in the use of waste-derived materials in agriculture and to establish guidelines to protect the food chain where necessary.

Strategies to mitigate greenhouse gas emissions from dairy cattle are unlikely to be adopted if production or profitability is reduced. The primary objective of this study was to examine the effects of high maize silage (MS) vs. high grass silage (GS) diets, without or with added neutral-detergent fiber (NDF) on milk production and methane emission of dairy cattle, using GreenFeed (GF) or respiration chamber (RC) techniques for methane emission measurements. Experiment 1 was 12-wks in duration with a randomized block continuous design and 40 Holstein cows (74 d in milk; DIM) in free-stall housing, assigned to 1 of 4 dietary treatments (n = 10 per treatment), according to calving date, parity and milk yield. Milk production and dry matter intake (DMI) were measured daily, and milk composition measured weekly, with methane yield (g/kg DMI) estimated using a GF unit (wks 10 to 12). Experiment 2 was a 4 × 4 Latin Square Design with 5-wk periods and 4 dairy cows (114 DIM) fed the same 4 dietary treatments as in experiment 1. Measurements of DMI, milk production and composition occurred in wk 4, and DMI, milk production and methane yield were measured for 2 d in RC during wk 5. Dietary treatments for both experiments were fed as TMRs offered ad libitum and containing 500 g silage/kg DM comprised of either 75:25 MS:GS (MS) or 25:75 MS:GS (GS), without or with added NDF from chopped straw and soy hulls (+47 g NDF/kg DM; MSNDF and GSNDF). In both experiments, compared to high GS, cows fed high MS had a higher (P = 0.01) DMI, greater (P = 0.01) milk production, and lower (P = 0.02) methane yield (24% lower in experiment 1 using GF and 8% lower in experiment 2 using RC). Added NDF increased (or tended to increase) methane yield for high MS, but not high GS diets (P = 0.02 for experiment 1 and P = 0.10 for experiment 2, forage type × NDF interaction). In the separate experiments the GF and RC methods detected similar dietary treatment effects on methane emission (expressed as g/d and g/kg DMI), although the magnitude of the difference varied between experiments for dietary treatments Overall methane emission and yield were 448 g/d and 20.9 g/kg DMI using GF for experiment 1 using GF and 458 g/d and 23.8 g/kg DMI for experiment 2 using RC, respectively.

Introduction The occurrence of a neutralizing antibody in previously untreated patients (PUPs) with haemophilia A appears to be the result of an intricate interplay of both genetic and environmental factors. Recently, the type of factor VIII (FVIII) product used in the PUPs population has been implicated as a risk factor for inhibitor development. Aim The aim of this review was to explore in a systematic manner potential hypotheses for the product-related findings in these studies (i.e. differences in the expression system of the cell lines used to produce recombinant FVIII [rFVIII], differences in the administered antigen load or changes in clinical practice over time). Results Review of the available clinical studies illustrates the high degree of variability for the risk of inhibitor development for the same products across different studies. Differences in cell lines or antigen load were not found to provide a reasonable explanation. Conclusion The possibility of changes in clinical practice over time and patient selection bias (i.e. the preferential use of one product over another in patients at higher risk for inhibitors) offers a potential explanation and should be carefully considered when evaluating the studies.

The ALPHA project is an international collaboration, based at CERN, with the experimental goal of performing precision spectroscopic measurements on antihydrogen. As part of this endeavor, the ALPHA experiment includes a silicon tracking detector. This detector consists of a three-layer array of silicon modules surrounding the antihydrogen trapping region of the ALPHA apparatus. Using this device, the antihydrogen annihilation position can be determined with a spatial resolution of better than 5 mm. Knowledge of the annihilation distribution was a critical component in the recently successful antihydrogen trapping effort. This paper will describe the methods used to reconstruct annihilation events in the ALPHA detector. Particular attention will be given to the description of the background rejection criteria.

Precision comparisons of hyperfine intervals in atomic hydrogen and antihydrogen are expected to yield experimental tests of the CPT theorem. The CERN-based ALPHA collaboration has initiated a program of study focused on microwave spectroscopy of trapped ground-state antihydrogen atoms. This paper outlines some of the proposed experiments, and summarizes measurements that characterize microwave fields that have been injected into the ALPHA apparatus.

Because of the potential benefits to hu- man health, there is interest in increasing 18:3n-3, 20:5n- 3, 22:6n-6, and cis-9,trans-11 CLA in ruminant foods. Four Aberdeen Angus steers (406 ± 8.2 kg of BW) fit- ted with ruminal and duodenal cannulas were used in a 4 × 4 Latin square experiment with 21-d periods to ex- amine the potential of fish oil (FO) and linseed oil (LO) in the diet to increase ruminal outflow of trans-11 18:1 and total n-3 PUFA in growing cattle. Treatments con- sisted of a control diet (60:40; forage:concentrate ratio, on a DM basis, respectively) based on maize silage, or the same basal ration containing 30 g/kg of DM of FO, LO, or a mixture (1:1, wt/wt) of FO and LO (LFO). Diets were offered as total mixed rations and fed at a rate of 85 g of DM/(kg of BW 0.75 /d). Oils had no ef- fect (P = 0.52) on DMI. Linseed oil had no effect (P > 0.05) on ruminal pH or VFA concentrations, whereas FO shifted rumen fermentation toward propionate at the expense of acetate. Compared with the control, LO increased (P < 0.05) 18:0, cis 18:1 (Δ9, 12-15), trans 18:1 (Δ4-9, 11-16), trans 18:2, geometric isomers of Δ9,11, Δ11,13, and Δ13,15 CLA, trans-8,cis-10 CLA, trans-10,trans-12 CLA, trans-12,trans-14 CLA, and 18:3n-3 flow at the duodenum. Inclusion of FO in the diet resulted in greater (P < 0.05) flows of cis-9 16:1, trans 16:1 (Δ6-13), cis 18:1 (Δ9, 11, and 13), trans 18:1 (Δ6-15), trans 18:2, 20:5n-3, 22:5n-3, and 22:6n-3, and decreased (P < 0.001) 18:0 at the duodenum relative to the control. For most fatty acids at the duodenum, re- sponses to LFO were intermediate of FO and LO. How- ever, LFO resulted in greater (P = 0.04) flows of total trans 18:1 than LO and increased (P < 0.01) trans-6 16:1 and trans-12 18:1 at the duodenum compared with FO or LO. Biohydrogenation of cis-9 18:1 and 18:2n-6 in the rumen was independent of treatment, but both FO and LO increased (P < 0.001) the extent of 18:3n-3 biohydrogenation compared with the control. Ruminal 18:3n-3 biohydrogenation was greater (P < 0.001) for LO and LFO than FO, whereas biohydrogenation of 20:5n-3 and 22:6n-3 in the rumen was marginally less (P = 0.05) for LFO than FO. In conclusion, LO and FO at 30 g/kg of DM altered the biohydrogenation of un- saturated fatty acids in the rumen, causing an increase in the flow of specific intermediates at the duodenum, but the potential of these oils fed alone or as a mixture to increase n-3 PUFA at the duodenum in cattle ap- pears limited.

Precision spectroscopic comparison of hydrogen and antihydrogen holds the promise of a sensitive test of the Charge-Parity-Time theorem and matter-antimatter equivalence. The clearest path towards realising this goal is to hold a sample of antihydrogen in an atomic trap for interrogation by electromagnetic radiation. Achieving this poses a huge experimental challenge, as state-of-the-art magnetic-minimum atom traps have well depths of only ∼1 T (∼0.5 K for ground state antihydrogen atoms). The atoms annihilate on contact with matter and must be ‘born’ inside the magnetic trap with low kinetic energies. At the ALPHA experiment, antihydrogen atoms are produced from antiprotons and positrons stored in the form of non-neutral plasmas, where the typical electrostatic potential energy per particle is on the order of electronvolts, more than 104 times the maximum trappable kinetic energy. In November 2010, ALPHA published the observation of 38 antiproton annihilations due to antihydrogen atoms that had been trapped for at least 172 ms and then released—the first instance of a purely antimatter atomic system confined for any length of time (Andresen et al., Nature 468:673, 2010). We present a description of the main components of the ALPHA traps and detectors that were key to realising this result. We discuss how the antihydrogen atoms were identified and how they were discriminated from the background processes. Since the results published in Andresen et al. (Nature 468:673, 2010), refinements in the antihydrogen production technique have allowed many more antihydrogen atoms to be trapped, and held for much longer times. We have identified antihydrogen atoms that have been trapped for at least 1,000 s in the apparatus (Andresen et al., Nature Physics 7:558, 2011). This is more than sufficient time to interrogate the atoms spectroscopically, as well as to ensure that they have relaxed to their ground state.

In efforts to trap antihydrogen, a key problem is the vast disparity between the neutral trap energy scale (\(\sim\!50\,\upmu\mathrm{eV}\)), and the energy scales associated with plasma confinement and space charge (~1 eV). In order to merge charged particle species for direct recombination, the larger energy scale must be overcome in a manner that minimizes the initial antihydrogen kinetic energy. This issue motivated the development of a novel injection technique utilizing the inherent nonlinear nature of particle oscillations in our traps. We demonstrated controllable excitation of the center-of-mass longitudinal motion of a thermal antiproton plasma using a swept-frequency autoresonant drive. When the plasma is cold, dense and highly collective in nature, we observe that the entire system behaves as a single-particle nonlinear oscillator, as predicted by a recent theory. In contrast, only a fraction of the antiprotons in a warm or tenuous plasma can be similarly excited. Antihydrogen was produced and trapped by using this technique to drive antiprotons into a positron plasma, thereby initiating atomic recombination. The nature of this injection overcomes some of the difficulties associated with matching the energies of the charged species used to produce antihydrogen.

Tree rearrangement operations are widely used to measure the dissimilarity between phylogenetic trees with identical leaf sets. The tree bisection and reconnection (tbr) distance for unrooted trees can be equivalently defined in terms of agreement forests. For both the tbr distance and the less general subtree prune and regraft (spr) distance, we use such forests to derive new upper and lower bounds on the maximal possible distance between two trees with n leaves.

Summary Cell migration during wound healing requires adhesion receptor turnover to enable the formation and disassembly of cell-extracellular matrix contacts. Although recent advances have improved our understanding of integrin trafficking pathways, it is not known how extracellular ligand engagement controls receptor dynamics. Using atomic force microscopy, we have measured cell avidity for fibronectin and defined a mechanism for the outside-in regulation of α5β1-integrin. Surprisingly, adhesive strength was attenuated by the syndecan-4-binding domain of fibronectin due to a rapid triggering of α5β1-integrin endocytosis. Association of syndecan-4 with PKCα was found to trigger RhoG activation and subsequent dynamin- and caveolin-dependent integrin uptake. Like disruption of syndecan-4 or caveolin, gene disruption of RhoG in mice was found to retard closure of dermal wounds due to a migration defect of the fibroblasts and keratinocytes of RhoG null mice. Thus, this syndecan-4-regulated integrin endocytic pathway appears to play a key role in tissue repair., Graphical Abstract Highlights ► Syndecan-4 engagement mobilizes cells by reducing avidity for extracellular matrix ► Syndecan-4 induces caveolin-mediated endocytosis rather than inactivation of integrin ► Matrix ligands of syndecan-4 cause RhoG activation, which triggers endocytosis ► Gene disruption of syndecan-4, RhoG, or caveolin-1 results in compromised wound healing

Regulated activation of integrins is critical for cell adhesion, motility and tissue homeostasis. Talin and kindlins activate β1-integrins, but the counteracting inhibiting mechanisms are poorly defined. We identified SHARPIN as an important inactivator of β1-integrins in an RNAi screen. SHARPIN inhibited β1-integrin functions in human cancer cells and primary leukocytes. Fibroblasts, leukocytes and keratinocytes from SHARPIN-deficient mice exhibited increased β1-integrin activity, which was fully rescued by re-expression of SHARPIN. We found that SHARPIN directly binds to a conserved cytoplasmic region of integrin α-subunits and inhibits recruitment of talin and kindlin to the integrin. Therefore, SHARPIN inhibits the critical switching of β1-integrins from inactive to active conformations.

This study investigated the effect of using the lactate-utilizing bacterium Megasphaera elsdenii NCIMB 41125 as a probiotic supplement on rumen fermentation and pH in dairy cows in the immediate postcalving period. Fourteen multiparous rumen-fistulated Holstein cows, blocked according to 305-d milk yield in the previous lactation, were used in a randomized complete block design. From d 1 to 28 postcalving, cows were fed ad libitum a total mixed ration with a forage to concentrate ratio of 392:608 and a starch concentration of 299g/kg of dry matter. Treatments consisting of a minimum of 10(10) cfu of Megasphaera elsdenii NCIMB 41125 or autoclaved M. elsdenii (placebo) were administered via the rumen cannula on d 3 and 12 of lactation (n=7 per treatment). Mid-rumen pH was measured every 15min, and eating and ruminating behaviors were recorded for 24h on d 2, 4, 6, 8, 11, 13, 15, 17, 22, and 28. Rumen fluid for volatile fatty acid and lactic acid analysis was collected at 11 time points on each of d 2, 4, 6, 13, and 15. Yields of milk and milk protein and lactose were similar, but milk fat concentration tended to be higher in cows that received the placebo. Time spent eating and ruminating and dry matter intake were similar across treatments. Ruminal lactic acid concentrations were highly variable between animals, and no cases of clinical acidosis were observed. Both treatment groups had rumen pH

Spectroscopy of antihydrogen has the potential to yield high-precision tests of the CPT theorem and shed light on the matter-antimatter imbalance in the Universe. The ALPHA antihydrogen trap at CERN's Antiproton Decelerator aims to prepare a sample of antihydrogen atoms confined in an octupole-based Ioffe trap and to measure the frequency of several atomic transitions. We describe our techniques to directly measure the antiproton temperature and a new technique to cool them to below 10 K. We also show how our unique position-sensitive annihilation detector provides us with a highly sensitive method of identifying antiproton annihilations and effectively rejecting the cosmic-ray background., 10 pages, 5 figures

Based on potential benefits to human health, there is increasing interest in altering the composition of ruminant-derived foods. Including rapeseeds in the dairy cow diet is an effective strategy for replacing medium-chain saturated fatty acids (SFA) with cis-monounsaturated fatty acids (MUFA) in bovine milk, but there is limited information on the optimum level of supplementation. Decreases in SFA due to plant oils are also accompanied by increases in milk trans fatty acid (FA) content and it is possible that high oleic acid rapeseeds may result in a higher enrichment of cis-9 18:1 and lower increases in trans FAs in milk compared with conventional varieties. Seven multiparous lactating Holstein–Friesian cows were allocated to one of seven treatments in an incomplete Latin square design with five 28-day experimental periods, to evaluate the effect of replacing calcium salts of palm oil distillate (CPO; 41 g/kg diet dry matter, DM) with 128, 168 or 207 g/kg diet DM of conventional (COR) or a high oleic acid (HOR) rapeseed fed as a supplement milled with wheat. Rapeseed variety and inclusion level had no effect (P > 0.05) on DM intake, milk yield and composition. Both rapeseed varieties decreased linearly (P < 0.001) milk fat SFA content, which was partially compensated for by a linear increase (P < 0.001) in cis-9 18:1 concentration. Reductions in milk SFA were also associated with increases (P < 0.05) in trans 18:1 and total trans FA content, with no difference (P > 0.05) between rapeseed varieties. Replacing CPO in the diet with milled rapeseeds had no effect (P > 0.05) on total milk conjugated linoleic acid (CLA) concentration. Relative to a COR, inclusion of a high oleic acid variant in the diet increased (P = 0.01) the ratio of trans-MUFA : trans-polyunsaturated fatty acids in milk that may have implications with respect to cardiovascular disease risk in humans. In conclusion, data indicated that replacing CPO with milled rapeseeds at levels up to 1150 g oil/day could be used as a nutritional strategy to lower milk SFA content without inducing adverse effects on DM intake and milk production. HOR reduced milk fat SFA content to a greater extent than a conventional variety, but did not minimise associated increases in trans FA concentrations. However, the high oleic acid variant did alter the relative abundance of specific trans 18:1, CLA and trans 18:2 isomers compared with conventional rapeseeds.