Direct visualization of electronic transport in a quantum anomalous Hall insulator.

A quantum anomalous Hall (QAH) insulator is characterized by quantized Hall and vanishing longitudinal resistances at zero magnetic field that are protected against local perturbations and independent of sample details. This insensitivity makes the microscopic details of the local current distribution inaccessible to global transport measurements. Accordingly, the current distributions that give rise to transport quantization are unknown. Here we use magnetic imaging to directly visualize the transport current in the QAH regime. As we tune through the QAH plateau by electrostatic gating, we clearly identify a regime in which the sample transports current primarily in the bulk rather than along the edges. Furthermore, we image the local response of equilibrium magnetization to electrostatic gating. Combined, these measurements suggest that the current flows through incompressible regions whose spatial structure can change throughout the QAH regime. Identification of the appropriate microscopic picture of electronic transport in QAH insulators and other topologically non-trivial states of matter is a crucial step towards realizing their potential in next-generation quantum devices.

Superfluid response of an atomically thin gate-tuned van der Waals superconductor.

A growing number of two-dimensional superconductors are being discovered in the family of exfoliated van der Waals materials. Due to small sample volume, the superfluid response of these materials has not been characterized. Here, we use a local magnetic probe to directly measure this key property of the tunable, gate-induced superconducting state in MoS2. We find that the backgate changes the transition temperature non-monotonically whereas the superfluid stiffness at low temperature and the normal state conductivity monotonically increase. In some devices, we find direct signatures in agreement with a Berezinskii-Kosterlitz-Thouless transition, whereas in others we find a broadened onset of the superfluid response. We show that the observed behavior is consistent with disorder playing an important role in determining the properties of superconducting MoS2. Our work demonstrates that magnetic property measurements are within reach for superconducting devices based on exfoliated sheets and reveals that the superfluid response significantly deviates from simple BCS-like behavior.

Proteomic analysis in primary T cells reveals IL-7 alters T cell receptor thresholding via CYTIP/cytohesin/LFA-1 localisation and activation.

The ability of the cellular immune system to discriminate self from foreign antigens depends on the appropriate calibration of the T cell receptor (TCR) signalling threshold. The lymphocyte homeostatic cytokine interleukin 7 (IL-7) is known to affect TCR thresholding, but the molecular mechanism is not fully elucidated. A better understanding of this process is highly relevant in the context of autoimmune disease therapy and cancer immunotherapy. We sought to characterise the early signalling events attributable to IL-7 priming; in particular, the altered phosphorylation of signal transduction proteins and their molecular localisation to the TCR. By integrating high-resolution proximity- phospho-proteomic and imaging approaches using primary T cells, rather than engineered cell lines or an in vitro expanded T cell population, we uncovered transduction events previously not linked to IL-7. We show that IL-7 leads to dephosphorylation of cytohesin interacting protein (CYTIP) at a hitherto undescribed phosphorylation site (pThr280) and alters the co-localisation of cytohesin-1 with the TCR and LFA-1 integrin. These results show that IL-7, acting via CYTIP and cytohesin-1, may impact TCR activation thresholds by enhancing the co-clustering of TCR and LFA-1 integrin.

Scanning SQUID microscopy in a cryogen-free dilution refrigerator.

We report a scanning superconducting quantum interference device (SQUID) microscope in a cryogen-free dilution refrigerator with a base temperature at the sample stage of at least 30 mK. The microscope is rigidly mounted to the mixing chamber plate to optimize thermal anchoring of the sample. The microscope housing fits into the bore of a superconducting vector magnet, and our design accommodates a large number of wires connecting the sample and sensor. Through a combination of vibration isolation in the cryostat and a rigid microscope housing, we achieve relative vibrations between the SQUID and the sample that allow us to image with micrometer resolution over a 150 µm range while the sample stage temperature remains at base temperature. To demonstrate the capabilities of our system, we show images acquired simultaneously of the static magnetic field, magnetic susceptibility, and magnetic fields produced by a current above a superconducting micrometer-scale device.

Adverse immunological responses against non-viral nanoparticle (NP) delivery systems in the lung.

There is a large, unmet medical need to treat chronic obstructive pulmonary disease, asthma, idiopathic pulmonary fibrosis and other respiratory diseases. New modalities are being developed, including gene therapy which treats the disease at the DNA/RNA level. Despite recent innovations in non-viral gene therapy delivery for chronic respiratory diseases, unwanted or adverse interactions with immune cells, particularly macrophages, can limit drug efficacy. This review will examine the relationship between the design and fabrication of non-viral nucleic acid nanoparticle (NP) delivery systems and their ability to trigger unwanted immunogenic responses in lung tissues. NP formulated with peptides, lipids, synthetic and natural polymers provide a robust means of delivering the genetic cargos to the desired cells. However NP, or their components, may trigger local responses such as cell damage, edema, inflammation, and complement activation. These effects may be acute short-term reactions or chronic long-term effects like fibrosis, increased susceptibility to diseases, autoimmune disorders, and even cancer. This review examines the relationship between physicochemical properties, i.e. shape, charge, hydrophobicity, composition and stiffness, and interactions of NP with pulmonary immune cells. Inhalation is the ideal route of administration for direct delivery but inhaled NP encounter innate immune cells, such as alveolar macrophages (AM) and dendritic cells (DC), that perceive them as harmful foreign material, interfere with gene delivery to target cells, and can induce undesirable side effects. Recommendations for fabrication and formulation of gene therapies to avoid adverse immunological responses are given. These include fine tuning physicochemical properties, functionalization of the surface of NP to actively target diseased pulmonary cells and employing biomimetics to increase immunotolerance.

Dichloroacetate as a possible treatment for endometriosis-associated pain: a single-arm open-label exploratory clinical trial (EPiC).

BACKGROUND: Endometriosis (where endometrial-like tissue is found outside the uterus) affects ~ 176 million women worldwide and can lead to debilitating pelvic pain. There is an unmet need for new medical treatment options for endometriosis. Pelvic peritoneal mesothelial cells of women with endometriosis exhibit detrimental metabolic reprogramming that creates an environment favouring the formation and survival of endometriosis lesions. We have generated powerful preclinical proof-of-concept data to show that it is possible to correct this metabolic phenotype using dichloroacetate (DCA), a non-hormonal compound previously used to treat rare metabolic disorders in children. We plan a single-arm, open-label, single site exploratory clinical trial to inform the design of a future randomised controlled trial (RCT) to determine the efficacy of DCA for the treatment of endometriosis-associated pain. METHODS: We will recruit 30 women with endometriosis-associated pain over a 6-month period. All participants will receive approximately 6.25 mg/kg oral DCA capsules twice daily for 6 weeks, with a dose increase to approximately 12.5 mg/kg twice daily for a further 6 weeks if their pain has not been adequately controlled on this dose regime and side-effects are acceptable. If pain is adequately controlled with minimal side-effects, the lower dose will be continued for a further 6 weeks. The primary objective is to determine whether it is possible to achieve acceptable recruitment and retention rates within the defined exclusion and inclusion criteria. Secondary objectives are to determine the acceptability of the trial to participants, including the proposed methods of recruitment, treatment, follow-up frequency and number of questionnaires. The recruitment rate will be determined by the proportion of patients recruited from the pool of eligible women. The retention rate will be determined by the proportion of participants who attended the final trial visit. DISCUSSION: This is a feasibility study to explore effectiveness and acceptability of the proposed field methodology (recruitment, retention, study processes and compliance with treatment). The results will be used to inform the design of a future RCT. TRIAL REGISTRATION: ClinicalTrials.gov, NCT04046081 Registered 6 August 2019.

Absolute and relative reliability of SCRuM test battery components assembled for schoolboy rugby players playing competitive rugby in low-resource settings: A pragmatic in-season test-retest approach.

Background: Schoolboy rugby is a popular sport which forms the bedrock of rugby development in many African countries, including Zimbabwe. With burgeoning talent identification programmes, the development of multi-dimensional, logically-validated, and reliable test batteries is essential to inform the objective selection of potentially talented young rugby athletes. Objectives: This study sought evidence on the absolute and relative test-retest reliability of the component test items in the newly-assembled SCRuM test battery. Methods: Utilising a pragmatic test-retest experimental design, a sample of 41 Under-19 schoolboy players playing competitive rugby in the elite Super Eight Schools Rugby League in Harare, Zimbabwe, participated in the study. Results: Physiological and game-specific skills tests which showed good to excellent relative reliability and acceptable absolute reliability, included: 20 m and 40 m speed, L-run, Vertical Jump (VJ), 60 s Push-Up, 2 kg Medicine Ball Chest Throw test (2 kg MBCT), Wall Sit Leg Strength test (WSLS), Repeated High Intensity Exercise test (RHIE), One Repetition Maximum Back Squat (1-RM BS) and Bench Press tests (1-RM BP), Yo-Yo Intermittent Recovery Level 1 test (Yo-Yo IRT L1), Tackling Proficiency test, Passing Ability Skill test and Running and Catching Ability skill test. Conclusion: All these tests are reliable and warrant inclusion in the SCRuM test battery for possible profiling of U19 schoolboy rugby players during the 'in-season' phase provided there is adequate participant familiarisation and test standardisation. The test-retest ICCs and measurement errors are generalisable to other young athletes in this population, making the tests useful for the evaluation of training and developmental effects of the measured constructs.

Spatial control of heavy-fermion superconductivity in CeIrIn5.

Although crystals of strongly correlated metals exhibit a diverse set of electronic ground states, few approaches exist for spatially modulating their properties. In this study, we demonstrate disorder-free control, on the micrometer scale, over the superconducting state in samples of the heavy-fermion superconductor CeIrIn5 We pattern crystals by focused ion beam milling to tailor the boundary conditions for the elastic deformation upon thermal contraction during cooling. The resulting nonuniform strain fields induce complex patterns of superconductivity, owing to the strong dependence of the transition temperature on the strength and direction of strain. These results showcase a generic approach to manipulating electronic order on micrometer length scales in strongly correlated matter without compromising the cleanliness, stoichiometry, or mean free path.

Qualities or skills discriminating under 19 rugby players by playing standards: a comparative analysis of elite, sub-elite and non-rugby players using the SCRuM test battery.

OBJECTIVE: Although schoolboy rugby is growing in popularity and played at different competitive levels in Zimbabwe, the influence of playing standard on qualities or skills of older male adolescent rugby players is unknown. Utilising a cross-sectional design, this study determined anthropometric, physiological characteristics and rugby-specific game skills defining elite under 19 (U19) schoolboy rugby players. Following development and subsequent assessment of test-retest reliability of School Clinical Rugby Measure (SCRuM) test battery, this study compared performance outcomes of elite rugby players (n = 41), sub-elite rugby players (n = 46) and non-rugby athletes (n = 26) to identify qualities or skills discriminating (i) elite from sub-elite and non-rugby players, and concomitantly (ii) sub-elite from non-rugby players. RESULTS: 40 m speed test (p < 0.001, ES = 1.78) and 2 kg Medicine Ball Chest Throw test (p < 0.001, ES = 1.69) significantly discriminated elite U19 from sub-elite and non-rugby players. These tests further differentiated sub-elite from non-rugby athletes. Additionally, 1RM back squat (p = 0.009, ES = 0.57), 1RM bench press (p = 0.005, ES = 0.61), repeated high-intensity exercise test (p < 0.001, ES = 0.88) and passing ability test (p < 0.001, ES = 0.99) discriminated elite from sub-elite counterparts. These findings highlight important attributes linked to elite U19 schoolboy rugby in Zimbabwe. However, no significant differences were observed for sum of seven skinfold (p = 0.28), tackling (p = 0.08) and catching ability (p = 0.05).

Neutrophil GM-CSF receptor dynamics in acute lung injury.

GM-CSF is important in regulating acute, persistent neutrophilic inflammation in certain settings, including lung injury. Ligand binding induces rapid internalization of the GM-CSF receptor (GM-CSFRα) complex, a process essential for signaling. Whereas GM-CSF controls many aspects of neutrophil biology, regulation of GM-CSFRα expression is poorly understood, particularly the role of GM-CSFRα in ligand clearance and whether signaling is sustained despite major down-regulation of GM-CSFRα surface expression. We established a quantitative assay of GM-CSFRα surface expression and used this, together with selective anti-GM-CSFR antibodies, to define GM-CSFRα kinetics in human neutrophils, and in murine blood and alveolar neutrophils in a lung injury model. Despite rapid sustained ligand-induced GM-CSFRα loss from the neutrophil surface, which persisted even following ligand removal, pro-survival effects of GM-CSF required ongoing ligand-receptor interaction. Neutrophils recruited to the lungs following LPS challenge showed initially high mGM-CSFRα expression, which along with mGM-CSFRß declined over 24 hr; this was associated with a transient increase in bronchoalveolar lavage fluid (BALF) mGM-CSF concentration. Treating mice in an LPS challenge model with CAM-3003, an anti-mGM-CSFRα mAb, inhibited inflammatory cell influx into the lung and maintained the level of BALF mGM-CSF. Consistent with neutrophil consumption of GM-CSF, human neutrophils depleted exogenous GM-CSF, independent of protease activity. These data show that loss of membrane GM-CSFRα following GM-CSF exposure does not preclude sustained GM-CSF/GM-CSFRα signaling and that this receptor plays a key role in ligand clearance. Hence neutrophilic activation via GM-CSFR may play an important role in neutrophilic lung inflammation even in the absence of high GM-CSF levels or GM-CSFRα expression.

Coaches' perceptions on qualities defining good adolescent rugby players and are important for player recruitment in talent identification programs: the SCRuM project.

OBJECTIVE: Competitive rugby is increasingly becoming popular among adolescent players even in countries hardly known for rugby such as Zimbabwe. Given the increased participation rates, burgeoning talent identification (TID) programs and the reportedly high injury-risk associated with competitive youth rugby, the minimal qualities or skills needed for effective performance by all young players need further clarification. Therefore, this qualitative study was conducted to explore the perceptions of high-school based rugby coaches on the key qualities or skills defining good adolescent rugby players and should be considered for player recruitment in TID programs. Currently, there is no consensus in literature from the coaches' perspective on these qualities. RESULTS: The final sample had 22 coaches (median age = 45.5 years) with years of coaching high-school rugby ranging from 6 to 17 years. Using the conventional approach to inductive content analysis four broad themes emerged suggesting the multifaceted nature of the requirements imperative for effective and optimal rugby performance among adolescent rugby players as perceived by the coaches. Themes identified included: physiological characteristics, anthropometric attributes, psychological qualities and game-specific skills. Possibly, training approaches or design of rugby-specific test-batteries should consider all these important qualities and be multi-dimensional in composition.

High-school adolescents' motivation to rugby participation and selection criteria for inclusion in school rugby teams: coaches' perspective (the SCRuM project).

OBJECTIVE: Despite increasing rugby popularity among schoolboys' worldwide, specific factors influencing their motivation to participate in rugby remain unclear. Therefore, this study was conducted in two parts with a dual purpose of exploring perceptions of rugby coaches on (i) factors motivating schoolboys to engage in competitive rugby, and (ii) criteria for selecting schoolboy rugby players for possible inclusion in school rugby teams. RESULTS: A qualitative study targeting Zimbabwean high school-based rugby coaches purposively-recruited during the 2017 Dairiboard Zimbabwe Rugby School Festival was conducted. Using the conventional approach to content analysis, the 22 recruited male coaches (median age = 45.5 years) felt that playing rugby is a choice largely influenced by either intrinsic or extrinsic motives for schoolboys. Additionally, coaches considered players' characteristics (performance during training, attitude, physical qualities and skills) and match-related factors when selecting schoolboys for possible inclusion in school rugby teams. To effectively promote competitive rugby participation among schoolboys and promote sustainable and effective talent identification programmes in Zimbabwe, more recognition should be paid to factors motivating schoolboys to participate in rugby and also on the factors coaches consider when assembling school rugby teams which indirectly informs on what coaches think should be trained among schoolboy rugby players.

Preclinical Evaluation of AZ12601011 and AZ12799734, Inhibitors of Transforming Growth Factor ß Superfamily Type 1 Receptors.

The transforming growth factor ß (TGFß) superfamily includes TGFß, activins, inhibins, and bone morphogenetic proteins (BMPs). These extracellular ligands have essential roles in normal tissue homeostasis by coordinately regulating cell proliferation, differentiation, and migration. Aberrant signaling of superfamily members, however, is associated with fibrosis as well as tumorigenesis, cancer progression, metastasis, and drug-resistance mechanisms in a variety of cancer subtypes. Given their involvement in human disease, the identification of novel selective inhibitors of TGFß superfamily receptors is an attractive therapeutic approach. Seven mammalian type 1 receptors have been identified that have context-specific roles depending on the ligand and the complex formation with the type 2 receptor. Here, we characterize the biologic effects of two transforming growth factor ß receptor 1 (TGFBR1) kinase inhibitors designed to target TGFß signaling. AZ12601011 [2-(2-pyridinyl)-4-(1H-pyrrolo[3,2-c]pyridin-1-yl)-6,7-dihydro-5H-cyclopenta[d]pyrimidine]; structure previously undisclosed] and AZ12799734 [4-({4-[(2,6-dimethyl-3-pyridinyl)oxy]-2-pyridinyl}amino)benzenesulfonamide] (IC50 = 18 and 47 nM, respectively) were more effective inhibitors of TGFß-induced reporter activity than SB-431542 [4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]benzamide] (IC50 = 84 nM) and LY2157299 [4-[2-(6-methylpyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl]quinoline-6-carboxamide monohydrate]] (galunisertib) (IC50 = 380 nM). AZ12601011 inhibited phosphorylation of SMAD2 via the type 1 receptors activin A receptor type 1B (ALK4), TGFBR1, and activin A receptor type 1C (ALK7). AZ12799734, however, is a pan TGF/BMP inhibitor, inhibiting receptor-mediated phosphorylation of SMAD1 by activin A receptor type 1L, bone morphogenetic protein receptor type 1A, and bone morphogenetic protein receptor type 1B and phosphorylation of SMAD2 by ALK4, TGFBR1, and ALK7. AZ12601011 was highly effective at inhibiting basal and TGFß-induced migration of HaCaT keratinocytes and, furthermore, inhibited tumor growth and metastasis to the lungs in a 4T1 syngeneic orthotopic mammary tumor model. These inhibitors provide new reagents for investigating in vitro and in vivo pathogenic processes and the contribution of TGFß- and BMP-regulated signaling pathways to disease states.

IL-18 associated with lung lymphoid aggregates drives IFNγ production in severe COPD.

BACKGROUND: Increased interferon gamma (IFNγ) release occurs in Chronic Obstructive Pulmonary Disease (COPD) lungs. IFNγ supports optimal viral clearance, but if dysregulated could increase lung tissue destruction. METHODS: The present study investigates which mediators most closely correlate with IFNγ in sputum in stable and exacerbating disease, and seeks to shed light on the spatial requirements for innate production of IFNγ, as reported in mouse lymph nodes, to observe whether such microenvironmental cellular organisation is relevant to IFNγ production in COPD lung. RESULTS: We show tertiary follicle formation in severe disease alters the dominant mechanistic drivers of IFNγ production, because cells producing interleukin-18, a key regulator of IFNγ, are highly associated with such structures. Interleukin-1 family cytokines correlated with IFNγ in COPD sputum. We observed that the primary source of IL-18 in COPD lungs was myeloid cells within lymphoid aggregates and IL-18 was increased in severe disease. IL-18 released from infected epithelium or from activated myeloid cells, was more dominant in driving IFNγ when IL-18-producing and responder cells were in close proximity. CONCLUSIONS: Unlike tight regulation to control infection spread in lymphoid organs, this local interface between IL-18-expressing and responder cell is increasingly supported in lung as disease progresses, increasing its potential to increase tissue damage via IFNγ.

Mutational activation of BRAF confers sensitivity to transforming growth factor beta inhibitors in human cancer cells.

Recent data implicate elevated transforming growth factor-ß (TGFß) signalling in BRAF inhibitor drug-resistance mechanisms, but the potential for targeting TGFß signalling in cases of advanced melanoma has not been investigated. We show that mutant BRAFV600E confers an intrinsic dependence on TGFß/TGFß receptor 1 (TGFBR1) signalling for clonogenicity of murine melanocytes. Pharmacological inhibition of the TGFBR1 blocked the clonogenicity of human mutant BRAF melanoma cells through SMAD4-independent inhibition of mitosis, and also inhibited metastasis in xenografted zebrafish. When investigating the therapeutic potential of combining inhibitors of mutant BRAF and TGFBR1, we noted that unexpectedly, low-dose PLX-4720 (a vemurafenib analogue) promoted proliferation of drug-naïve melanoma cells. Pharmacological or pharmacogenetic inhibition of TGFBR1 blocked growth promotion and phosphorylation of SRC, which is frequently associated with vemurafenib-resistance mechanisms. Importantly, vemurafenib-resistant patient derived cells retained sensitivity to TGFBR1 inhibition, suggesting that TGFBR1 could be targeted therapeutically to combat the development of vemurafenib drug-resistance.

Localizing the lipid products of PI3Kγ in neutrophils.

Class I phosphoinositide 3-kinases (PI3Ks) are important regulators of neutrophil migration in response to a range of chemoattractants. Their primary lipid products PtdIns(3,4,5)P3 and PtdIns(3,4)P2 preferentially accumulate near to the leading edge of migrating cells and are thought to act as an important cue organizing molecular and morphological polarization. We have investigated the distribution and accumulation of these lipids independently in mouse neutrophils using eGFP-PH reportersand electron microscopy (EM). We found that authentic mouse neutrophils rapidly polarized their Class I PI3K signalling, as read-out by eGFP-PH reporters, both at the up-gradient leading edge in response to local stimulation with fMLP as well as spontaneously and randomly in response to uniform stimulation. EM studies revealed these events occurred at the plasma membrane, were dominated by accumulation of PtdIns(3,4,5)P3, but not PtdIns(3,4)P2, and were dependent on PI3Kγ and its upstream activation by both Ras and Gßγs.

The influence of task paradigm on motor imagery ability in children with Developmental Coordination Disorder.

Children with Developmental Coordination Disorder (DCD) have difficulty imagining movements such that they conform to the customary temporal constraints of real performance. We examined whether this ability is influenced by the choice of task used to elicit motor imagery (MI). Performance of typically developing (TD) (n=30) and children with DCD (n=30) was compared on two tasks: the Visually Guided Pointing Task (VGPT) and the Computerized Virtual Radial Fitts Task (C-VRFT). Since the VGPT places higher demands on executive functions like working memory but requires less spatial planning, we reasoned that the C-VRFT would provide a purer measure of motor imagery (or simulation). Based on our earlier work, we predicted that imagery deficits in DCD would more likely manifest on the C-VRFT. Results showed high correlations between tasks in terms of executed and imagined movement time suggest that both tasks measure MI ability. However, group differences were more pronounced in the imagined condition of the radial Fitts' task. Taken together, the more spatially complex C-VRFT appears to be a more sensitive measure of motor imagery, better discriminating between DCD and TD. Implications for theory and practice are discussed.

Children with Developmental Coordination Disorder are deficient in a visuo-manual tracking task requiring predictive control.

The aim of this study was to examine how feedback, or its absence, affects children with Developmental Coordination Disorder (DCD) during a visuo-manual tracking task. This cross-sectional study included 40 children with DCD and 40 typically developing (TD) children between 6 and 10 years old. Participants were required to track a target moving along a circular path presented on a monitor by moving an electronic pen on a digitizing tablet. The task was performed under two visibility conditions (target visible throughout the trajectory and target intermittently occluded) and at two different target velocities (30° and 60° per second). Variables reflecting tracking success and tracking behavior within the target were compared between groups. Results showed that children with DCD were less proficient in tracking a moving target than TD children. Their performance deteriorated even more when the target was occluded and when the target speed increased. The mean tracking speed of the DCD group exceeded the speed at which the target rotated which was attributed to accelerations and decelerations made during tracking. This suggests that children with DCD have significant difficulties in visuo-manual tracking especially when visual feedback is reduced. It appears that their impaired ability to predict together with impairments in fine-tuning arm movements may be responsible for poor performance in the intermittently occluded visuo-manual tracking task.

Quality assurance of lower limb venous duplex scans performed by vascular surgeons.

AIM: Duplex scanning is the gold standard for investigating venous reflux; increasingly surgeons perform these scans themselves. There has been no data published analysing the accuracy of Duplex scans performed by vascular surgeons. We aimed to evaluate an objective method of comparing the results of lower limb Duplex scans performed by one consultant vascular surgeon with those performed by a vascular technologist. METHODS: We assessed 100 legs with symptomatic varicose veins. Each patient underwent two lower limb venous Duplex scans; one performed by a consultant vascular surgeon and one by a vascular technologist. Scan results were randomised and sent to two consultant vascular surgeons blinded to the identity and experience of the sonographer. They were asked to recommend treatment. A k score was calculated in each case to assess the level of agreement between the scans performed by the consultant and the technologist. RESULTS: Eighty-one patients were studied (53 females). The kappa score for assessor 1 was 0.60 (95%CI:0.44-0.75) and for assessor 2 was 0.62 (95%CI:0.48-0.75). k scores >0.60 represent a substantial strength of agreement. CONCLUSION: Duplex scans performed by this surgeon were comparable to those performed by a vascular technologist. It is possible to quality-assure duplex performed by vascular surgeons without directly observing the scanning process or reviewing digitally recorded images. We propose standardisation of training, assessment and quality assurance for vascular surgeons wishing to perform ultrasound scans.

Signaling via class IA Phosphoinositide 3-kinases (PI3K) in human, breast-derived cell lines.

We have addressed the differential roles of class I Phosphoinositide 3-kinases (PI3K) in human breast-derived MCF10a (and iso-genetic derivatives) and MDA-MB 231 and 468 cells. Class I PI3Ks are heterodimers of p110 catalytic (α, ß, δ and γ) and p50-101 regulatory subunits and make the signaling lipid, phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P3) that can activate effectors, eg protein kinase B (PKB), and responses, eg migration. The PtdIns(3,4,5)P3-3-phosphatase and tumour-suppressor, PTEN inhibits this pathway. p110α, but not other p110s, has a number of onco-mutant variants that are commonly found in cancers. mRNA-seq data shows that MCF10a cells express p110ß>>α>δ with undetectable p110γ. Despite this, EGF-stimulated phosphorylation of PKB depended upon p110α-, but not ß- or δ- activity. EGF-stimulated chemokinesis, but not chemotaxis, was also dependent upon p110α, but not ß- or δ- activity. In the presence of single, endogenous alleles of onco-mutant p110α (H1047R or E545K), basal, but not EGF-stimulated, phosphorylation of PKB was increased and the effect of EGF was fully reversed by p110α inhibitors. Cells expressing either onco-mutant displayed higher basal motility and EGF-stimulated chemokinesis.This latter effect was, however, only partially-sensitive to PI3K inhibitors. In PTEN(-/-) cells, basal and EGF-stimulated phosphorylation of PKB was substantially increased, but the p110-dependency was variable between cell types. In MDA-MB 468s phosphorylation of PKB was significantly dependent on p110ß, but not α- or δ- activity; in PTEN(-/-) MCF10a it remained, like the parental cells, p110α-dependent. Surprisingly, loss of PTEN suppressed basal motility and EGF-stimulated chemokinesis. These results indicate that; p110α is required for EGF signaling to PKB and chemokinesis, but not chemotaxis; onco-mutant alleles of p110α augment signaling in the absence of EGF and may increase motility, in part, via acutely modulating PI3K-activity-independent mechanisms. Finally, we demonstrate that there is not a universal mechanism that up-regulates p110ß function in the absence of PTEN.