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Early oesophageal adenocarcinoma (OA) and pre-neoplastic dysplasia may be treated with endoscopic resection and ablative techniques such as photodynamic therapy (PDT). Though effective, discrete areas of disease may be missed leading to recurrence. PDT further suffers from the side effects of off-target photosensitivity. A tumour specific and light targeted therapeutic agent with optimised pharmacokinetics could be used to destroy residual cancerous cells left behind after resection. A small molecule antibody-photosensitizer conjugate was developed targeting human epidermal growth factor receptor 2 (HER2). This was tested in an in vivo mouse model of human OA using a xenograft flank model with clinically relevant low level HER2 expression and heterogeneity. In vitro we demonstrate selective binding of the conjugate to tumour versus normal tissue. Light dependent cytotoxicity of the phototherapy agent in vitro was observed. In an in vivo OA mouse xenograft model the phototherapy agent had desirable pharmacokinetic properties for tumour uptake and blood clearance time. PDT treatment caused tumour growth arrest in all the tumours despite the tumours having a clinically defined low/negative HER2 expression level. This new phototherapy agent shows therapeutic potential for treatment of both HER2 positive and borderline/negative OA.

The complexity of eukaryotic cells is underscored by the compartmentalization of chemical signals by phospholipid membranes. A grand challenge of synthetic biology is building life from the ‘bottom-up’, for the purpose of generating systems simple enough to precisely interrogate biological pathways or for adapting biology to perform entirely novel functions. Achieving compartmentalization of chemistries in an addressable manner is a task exquisitely refined by nature and embodied in a unique membrane remodelling machinery that pushes membranes away from the cytosol, the ESCRT-III (endosomal sorting complex required for transport-III) complex. Here, we show efforts to engineer a single ESCRT-III protein merging functional features from its different components. The activity of such a designed ESCRT-III is shown by its ability to drive the formation of compartments encapsulating fluorescent cargo. It appears that the modular nature of ESCRT-III allows its functional repurposing into a minimal machinery that performs sophisticated membrane remodelling, therefore enabling its use to create eukaryotic-like multi-compartment architectures.

The complexity of eukaryotic cells is underscored by the compartmentalization of chemical signals by phospholipid membranes. A grand challenge of synthetic biology is building life from the ‘bottom-up’, for the purpose of generating systems simple enough to precisely interrogate biological pathways or for adapting biology to perform entirely novel functions. Achieving compartmentalization of chemistries in an addressable manner is a task exquisitely refined by nature and embodied in a unique membrane remodelling machinery that pushes membranes away from the cytosol, the ESCRT-III (endosomal sorting complex required for transport-III) complex. Here, we show efforts to engineer a single ESCRT-III protein merging functional features from its different components. The activity of such a designed ESCRT-III is shown by its ability to drive the formation of compartments encapsulating fluorescent cargo. It appears that the modular nature of ESCRT-III allows its functional repurposing into a minimal machinery that performs sophisticated membrane remodelling, therefore enabling its use to create eukaryotic-like multi-compartment architectures.

Introduction Advanced chronic kidney disease (CKD) has a major effect on the quality of life and health status of patients and requires accurate and responsive management. The use of electronic patient-reported outcome measures (ePROMs) could assist patients with advanced pre-dialysis CKD, and the clinicians responsible for their care, by identifying important changes in symptom burden in real time. We report the protocol for ‘Using Patient-Reported Outcome measures (PROMs) to promote quality of care and safety in the management of patients with Advanced Chronic Kidney Disease’ (PRO-trACK) project, which will explore the feasibility and validity of an ePROM system for use in patients with advanced CKD. Methods and analysis The project will use a mixed-methods approach in three studies: (1) usability testing of the ePROM system involving up to 30 patients and focusing on acceptability and technical performance/stability; (2) ascertaining the views of patient and clinician stakeholders on the optimal use and administration of the CKD ePROM system—this will involve qualitative face-to-face/telephone interviewing with up to 30 patients or until saturation is achieved, focus groups with up to 15 clinical staff, management and IT team members; (3) psychometric assessment of the system, within a cohort of at least 180 patients with advanced CKD, to establish the measurement properties of the ePROM. Ethics and dissemination This project was approved by the West Midlands Edgbaston Research Ethics Committee (Reference 17/WM/0010) and received Health Research Authority (HRA) approval on 24 February 2017. The findings from this project will be provided to clinicians at the Department of Renal Medicine, Queen Elizabeth Hospitals, Birmingham (QEHB), NHS England, presented at conferences and to the Kidney Patients’ Association, British Kidney Patient Association and the British Renal Society. Articles based on the findings will be written and submitted for publication in peer-reviewed journals.

Little cigars (LCs) are regulated differently than cigarettes, allowing them to be potentially targeted at youth/young adults. We exposed human bronchial epithelial cultures (HBECs) to air or whole tobacco smoke from cigarettes vs. LCs. Chronic smoke exposure increased the number of dead cells, lactate dehydrogenase release, and interleukin-8 (IL-8) secretion and decreased apical cilia, cystic fibrosis transmembrane conductance regulator (CFTR) protein levels, and transepithelial resistance. These adverse effects were significantly greater in LC-exposed HBECs than cigarette exposed cultures. LC-exposure also elicited unique gene expression changes and altered the proteomic profiles of airway apical secretions compared to cigarette-exposed HBECs. Gas chromatography-mass spectrometry (GC-MS) analysis indicated that LCs produced more chemicals than cigarettes, suggesting that the increased chemical load of LCs may be the cause of the greater toxicity. This is the first study of the biological effects of LCs on pulmonary epithelia and our observations strongly suggest that LCs pose a more severe danger to human health than cigarettes.

Magnesium chelatase (MgCH) initiates chlorophyll biosynthesis by catalysing the ATP-dependent insertion of Mg2+ into protoporphyrin. This large enzyme complex comprises ChlH, I and D subunits, with I and D involved in ATP hydrolysis, and H the protein that handles the substrate and product. The 148 kDa ChlH subunit has a globular N-terminal domain attached by a narrow linker to a hollow cage-like structure. Following deletion of this ~18 kDa domain from the Thermosynechoccus elongatus ChlH, we used single particle reconstruction to show that the apo- and porphyrin-bound forms of the mutant subunit consist of a hollow globular protein with three connected lobes; superposition of the mutant and native ChlH structures shows that, despite the clear absence of the N-terminal ‘head’ region, the rest of the protein appears to be correctly folded. Analyses of dissociation constants shows that the ΔN159ChlH mutant retains the ability to bind protoporphyrin and the Gun4 enhancer protein, although the addition of I and D subunits yields an extremely impaired active enzyme complex. Addition of the Gun4 enhancer protein, which stimulates MgCH activity significantly especially at low Mg2+ concentrations, partially reactivates the ΔN159ChlH–I–D mutant enzyme complex, suggesting that the binding site or sites for Gun4 on H do not wholly depend on the N-terminal domain., The N-terminal domain of the 148 kDa ChlH is essential for normal activity of the ChlH–I–D magnesium chelatase complex. Deleting this 18 kDa domain retains the hollow cage-like structure and porphyrin binding. Chelatase activity is partially restored by the Gun4 protein.

This article explores the work of three contemporary Northern Irish poets, Leontia Flynn, Sinead Morrissey and Alan Gillis, asking how this work relates to fragility as experienced in Northern Ireland. Is there, within this poetry, a fostering of hope with respect to Northern Ireland's fragile future? Does a new generation of poets look for continuity with the past or does it strike out on a new path? The first part of the article provides a brief introduction to the ‘first-generation peace poets’, and also gives an overview of the ways in which Northern Irish poetry has been criticized in recent decades. It then suggests that this literature might be read through the lens of the elegiac tradition, as well as by exploring the ‘post-theory’ approach of new aestheticism. The second part comprises a series of close readings, with particular emphasis on poems about Belfast, and other poems that deal with themes of loss.

Dimethylaluminum triflate-mediated activation of tetrafluorobenzoates of enantiomerically pure bromohydrins results in enantiospecific polyene cyclizations. The initiation of cyclization by enantiomerically pure bromonium ions and subsequent propagation is not subject to catastrophic erosion of enantiomeric purity by any intramolecular or intermolecular bromonium ion-to-alkene transfer. Chirality 25:692–700, 2013. © 2013 Wiley Periodicals, Inc.

Anthropometric measures relating to body size, weight and composition are increasingly being associated with cancer risk and progression. Whilst practical in epidemiologic research, where population-level associations with disease are revealed, it is important to be aware that such measures are imperfect markers of the internal physiological processes that are the actual correlates of cancer development. Body mass index (BMI), the most commonly used marker for adiposity, may mask differences between lean and adipose tissue, or fat distribution, which varies across individuals, ethnicities, and stage in the lifespan. Other measures, such as weight gain in adulthood, waist circumference and waist-to-hip ratio, contribute information on adipose tissue distribution and insulin sensitivity. Single anthropometric measures do not capture maturational events, including the presence of critical windows of susceptibility (i.e., age of menarche and menopause), which presents a challenge in epidemiologic work. Integration of experimental research on underlying dynamic genetic, hormonal, and other non-nutritional mechanisms is necessary for a confident conclusion of the overall evidence in cancer development and progression. This article discusses the challenges confronted in evaluating and interpreting the current evidence linking anthropometric factors and cancer risk as a basis for issuing recommendations for cancer prevention.

CFTR is an apical membrane anion channel that regulates fluid homeostasis in many organs including the airways, colon, pancreas and sweat glands. In cystic fibrosis, CFTR dysfunction causes significant morbidity/mortality. Whilst CFTR’s function as an ion channel has been well described, its ability to regulate other proteins is less understood. We have previously shown that plasma membrane CFTR increases the surface density of the adenosine 2B receptor (A2BR), but not of the β2 adrenergic receptor (β2AR), leading to an enhanced, adenosine-induced cAMP response in the presence of CFTR. In this study, we have found that the C-terminal PDZ-domain of both A2BR and CFTR were crucial for this interaction and that replacing the C-terminus of A2BR with that of β2AR removed this CFTR-dependency. This observation extended to intact epithelia and disruption of the actin cytoskeleton prevented A2BR-induced but not β2AR-induced airway surface liquid (ASL) secretion. We also found that CFTR expression altered the organization of the actin cytoskeleton and PDZ-binding proteins in both HEK293T cells and in well-differentiated human bronchial epithelia. Furthermore, removal of CFTR’s PDZ binding motif (ΔTRL) prevented actin rearrangement, suggesting that CFTR insertion in the plasma membrane results in local reorganization of actin, PDZ binding proteins and certain GPCRs.

The dynamins represent a superfamily of proteins that have been shown to function in a wide range of membrane fusion and fission events. An increasing number of mutations in the human classical dynamins, Dyn-1 and Dyn-2 has been reported, with diseases caused by these changes ranging from Charcot-Marie-Tooth disorder to epileptic encephalopathies. The budding yeast, Saccharomyces cerevisiae expresses a single dynamin-related protein that functions in membrane trafficking, and is considered to play a similar role to Dyn-1 and Dyn-2 during scission of endocytic vesicles at the plasma membrane. Large parts of the dynamin protein are highly conserved across species and this has enabled us in this study to select a number of disease causing mutations and to generate equivalent mutations in Vps1. We have then studied these mutants using both cellular and biochemical assays to ascertain functions of the protein that have been affected by the changes. Specifically, we demonstrate that the Vps1-G397R mutation (Dyn-2 G358R) disrupts protein oligomerization, Vps1-A447T (Dyn-1 A408T) affects the scission stage of endocytosis, while Vps1-R298L (Dyn-1 R256L) affects lipid binding specificity and possibly an early stage in endocytosis. Overall, we consider that the yeast model will potentially provide an avenue for rapid analysis of new dynamin mutations in order to understand the underlying mechanisms that they disrupt.

Anthropometric measures relating to body size, weight and composition are increasingly being associated with cancer risk and progression. Whilst practical in epidemiologic research, where population-level associations with disease are revealed, it is important to be aware that such measures are imperfect markers of the internal physiological processes that are the actual correlates of cancer development. Body mass index (BMI), the most commonly used marker for adiposity, may mask differences between lean and adipose tissue, or fat distribution, which varies across individuals, ethnicities, and stage in the lifespan. Other measures, such as weight gain in adulthood, waist circumference and waist-to-hip ratio, contribute information on adipose tissue distribution and insulin sensitivity. Single anthropometric measures do not capture maturational events, including the presence of critical windows of susceptibility (i.e., age of menarche and menopause), which presents a challenge in epidemiologic work. Integration of experimental research on underlying dynamic genetic, hormonal, and other non-nutritional mechanisms is necessary for a confident conclusion of the overall evidence in cancer development and progression. This article discusses the challenges confronted in evaluating and interpreting the current evidence linking anthropometric factors and cancer risk as a basis for issuing recommendations for cancer prevention.

The family of dynamin proteins is known to function in many eukaryotic membrane fusion and fission events. The yeast dynamin-related protein Vps1 functions at several stages of membrane trafficking, including Golgi apparatus to endosome and vacuole, peroxisomal fission, and endocytic scission. We have previously shown that in its endocytic role, Vps1 functions with the amphiphysin heterodimer Rvs161/Rvs167 to facilitate scission and release of vesicles. Phosphoproteome studies of Saccharomyces cerevisiae have identified a phosphorylation site in Vps1 at serine 599. In this study, we confirmed this phosphorylation event, and we reveal that, like Rvs167, Vps1 can be phosphorylated by the yeast cyclin-associated kinase Pho85 in vivo and in vitro. The importance of this posttranslational modification was revealed when mutagenesis of S599 to a phosphomimetic or nonphosphorylatable form caused defects in endocytosis but not in other functions associated with Vps1. Mutation to nonphosphorylatable valine inhibited the Rvs167 interaction, while both S599V and S599D caused defects in vesicle scission, as shown by both live-cell imaging and electron microscopy of endocytic invaginations. Our data support a model in which phosphorylation and dephosphorylation of Vps1 promote distinct interactions and highlight the importance of such regulatory events in facilitating sequential progression of the endocytic process.

The biosynthesis of chlorophyll, an essential cofactor for photosynthesis, requires the ATP-dependent insertion of Mg(2+) into protoporphyrin IX catalyzed by the multisubunit enzyme magnesium chelatase. This enzyme complex consists of the I subunit, an ATPase that forms a complex with the D subunit, and an H subunit that binds both the protoporphyrin substrate and the magnesium protoporphyrin product. In this study we used electron microscopy and small-angle x-ray scattering to investigate the structure of the magnesium chelatase H subunit, ChlH, from the thermophilic cyanobacterium Thermosynechococcus elongatus. Single particle reconstruction of negatively stained apo-ChlH and Chl-porphyrin proteins was used to reconstitute three-dimensional structures to a resolution of ∼30 Å. ChlH is a large, 148-kDa protein of 1326 residues, forming a cage-like assembly comprising the majority of the structure, attached to a globular N-terminal domain of ∼16 kDa by a narrow linker region. This N-terminal domain is adjacent to a 5 nm-diameter opening in the structure that allows access to a cavity. Small-angle x-ray scattering analysis of ChlH, performed on soluble, catalytically active ChlH, verifies the presence of two domains and their relative sizes. Our results provide a basis for the multiple regulatory and catalytic functions of ChlH of oxygenic photosynthetic organisms and for a chaperoning function that sequesters the enzyme-bound magnesium protoporphyrin product prior to its delivery to the next enzyme in the chlorophyll biosynthetic pathway, magnesium protoporphyrin methyltransferase.

BACKGROUND AND PURPOSE The Rho-kinase pathway regulates agonist-induced contractions in several smooth muscles, including the intestine, urinary bladder and uterus, via dynamic changes in the Ca2+ sensitivity of the contractile apparatus. However, there is evidence that Rho-kinase also modulates other cellular effectors such as ion channels. EXPERIMENTAL APPROACH We examined the regulation of colonic smooth muscle excitability by Rho-kinase using conventional microelectrode recording, isometric force measurements and patch-clamp techniques. KEY RESULTS The Rho-kinase inhibitors, Y-27632 and H-1152, decreased nerve-evoked on- and off-contractions elicited at a range of frequencies and durations. The Rho-kinase inhibitors decreased the spontaneous contractions and the responses to carbachol and substance P independently of neuronal inputs, suggesting Y-27632 acts directly on smooth muscle. The Rho-kinase inhibitors significantly reduced the depolarization in response to carbachol, an effect that cannot be due to regulation of Ca2+ sensitization. Patch-clamp experiments showed that Rho-kinase inhibitors reduce GTPγS-activated non-selective cation currents. CONCLUSIONS AND IMPLICATIONS The Rho-kinase inhibitors decreased contractions evoked by nerve stimulation, carbachol and substance P. These effects were not solely due to inhibition of the Ca2+ sensitization pathway, as the Rho-kinase inhibitors also inhibited the non-selective cation conductances activated by excitatory transmitters. Thus, Rho-kinase may regulate smooth muscle excitability mechanisms by regulating non-selective cation channels as well as changing the Ca2+ sensitivity of the contractile apparatus.

The family of dynamin proteins is known to function in many eukaryotic membrane fusion and fission events. The yeast dynamin-related protein Vps1 functions at several stages of membrane trafficking, including Golgi apparatus to endosome and vacuole, peroxisomal fission, and endocytic scission. We have previously shown that in its endocytic role, Vps1 functions with the amphiphysin heterodimer Rvs161/Rvs167 to facilitate scission and release of vesicles. Phosphoproteome studies of Saccharomyces cerevisiae have identified a phosphorylation site in Vps1 at serine 599. In this study, we confirmed this phosphorylation event, and we reveal that, like Rvs167, Vps1 can be phosphorylated by the yeast cyclin-associated kinase Pho85 in vivo and in vitro. The importance of this posttranslational modification was revealed when mutagenesis of S599 to a phosphomimetic or nonphosphorylatable form caused defects in endocytosis but not in other functions associated with Vps1. Mutation to nonphosphorylatable valine inhibited the Rvs167 interaction, while both S599V and S599D caused defects in vesicle scission, as shown by both live-cell imaging and electron microscopy of endocytic invaginations. Our data support a model in which phosphorylation and dephosphorylation of Vps1 promote distinct interactions and highlight the importance of such regulatory events in facilitating sequential progression of the endocytic process.

The family of dynamin proteins is known to function in many eukaryotic membrane fusion and fission events. The yeast dynamin-related protein Vps1 functions at several stages of membrane trafficking, including Golgi apparatus to endosome and vacuole, peroxisomal fission, and endocytic scission. We have previously shown that in its endocytic role, Vps1 functions with the amphiphysin heterodimer Rvs161/Rvs167 to facilitate scission and release of vesicles. Phosphoproteome studies of Saccharomyces cerevisiae have identified a phosphorylation site in Vps1 at serine 599. In this study, we confirmed this phosphorylation event, and we reveal that, like Rvs167, Vps1 can be phosphorylated by the yeast cyclin-associated kinase Pho85 in vivo and in vitro. The importance of this posttranslational modification was revealed when mutagenesis of S599 to a phosphomimetic or nonphosphorylatable form caused defects in endocytosis but not in other functions associated with Vps1. Mutation to nonphosphorylatable valine inhibited the Rvs167 interaction, while both S599V and S599D caused defects in vesicle scission, as shown by both live-cell imaging and electron microscopy of endocytic invaginations. Our data support a model in which phosphorylation and dephosphorylation of Vps1 promote distinct interactions and highlight the importance of such regulatory events in facilitating sequential progression of the endocytic process.

Summary Actin is critical for endocytosis in yeast cells, and also in mammalian cells under tension. However, questions remain as to how force generated through actin polymerization is transmitted to the plasma membrane to drive invagination and scission. Here, we reveal that the yeast dynamin Vps1 binds and bundles filamentous actin. Mutational analysis of Vps1 in a helix of the stalk domain identifies a mutant RR457-458EE that binds actin more weakly. In vivo analysis of Vps1 function demonstrates that the mutation disrupts endocytosis but not other functions of Vps1 such as vacuolar trafficking or peroxisome fission. The mutant Vps1 is stably expressed in cells and co-localizes with the endocytic reporters Abp1 and the amphiphysin Rvs167. Detailed analysis of individual endocytic patch behavior indicates that the mutation causes aberrant movements in later stages of endocytosis, consistent with a scission defect. Ultrastructural analysis of yeast cells using electron microscopy reveals a significant increase in invagination depth, further supporting a role for the Vps1-actin interaction during scission. In vitro analysis of the mutant protein demonstrates that—like wild-type Vps1—it is able to form oligomeric rings, but, critically, it has lost its ability to bundle actin filaments into higher-order structures. A model is proposed in which actin filaments bind Vps1 during invagination, and this interaction is important to transduce the force of actin polymerization to the membrane to drive successful scission., Graphical Abstract, Highlights • Yeast dynamin Vps1 forms oligomeric rings that bind and bundle F-actin in vitro • Mutations in the Vps1 stalk region disrupt actin binding and bundling • The Vps1 RR-EE actin binding mutant has defects in endocytic scission in vivo, Palmer et al. reveal that the yeast dynamin Vps1 is able to form oligomeric rings that can directly interact with and bundle F-actin. Defects in the Vps1-actin interaction confer endocytic scission defects in vivo.

Antibody drug conjugates now make up a significant fraction of biopharma's oncology pipeline due to great advances in the understanding of the three key components and how they should be optimised together. With this clinical success comes innovation to produce new enabling technologies that can deliver more effective antibody-drug conjugates (ADCs) with a larger therapeutic index.There are many reviews that discuss the various strategies for ADCs design but the last 5 years or so have witnessed the emergence of a number of different antibody formats compete with the standard whole immunoglobulin. Using published research, patent applications and conference disclosures, the authors review the many antibody and antibody-like formats, discussing innovations in protein engineering and how these new formats impact on the conjugation strategy and ultimately the performance. The alternative chemistries that are now available offer new linkages, stability profiles, drug:antibody ratio, pharmacokinetics and efficacy. The different sizes being considered promise to address issues, such as tumour penetration, circulatory half-life and side-effects.ADCs are at the beginning of the next stage in their evolution and as these newer formats are developed and examined in the clinic, we will discover if the predicted features have a clinical benefit. From the commercial activity, it is envisaged that smaller or fragment-based ADCs will expand oncological applications.

Evidence-based health care has become a priority in the National Health Service (NHS), with increased emphasis on clinical practices that are grounded in quality evidence rather than those that persist because of tradition. Turning and positioning of patients are well-accepted nursing activities. Appropriate positioning of the critically ill patient can dramatically improve gas exchange, resulting in a shorter stay in the critical care unit and an improved outcome. This study reviews the current published literature on the subject of positioning and gas exchange, with emphasis on oxygenation. Conclusions made from this study are that nurses need to be aware of how different positions can affect patients' oxygenation. Further research on exploring patients' experience of positioning within the critical care environment and nurses' understanding of positioning and oxygenation is needed.

Magnesium chelatase (MgCH) initiates chlorophyll biosynthesis by catalysing the ATP-dependent insertion of Mg2+ into protoporphyrin. This large enzyme complex comprises ChlH, I and D subunits, with I and D involved in ATP hydrolysis, and H the protein that handles the substrate and product. The 148 kDa ChlH subunit has a globular N-terminal domain attached by a narrow linker to a hollow cage-like structure. Following deletion of this ~18 kDa domain from the Thermosynechoccus elongatus ChlH, we used single particle reconstruction to show that the apo- and porphyrin-bound forms of the mutant subunit consist of a hollow globular protein with three connected lobes; superposition of the mutant and native ChlH structures shows that, despite the clear absence of the N-terminal ‘head’ region, the rest of the protein appears to be correctly folded. Analyses of dissociation constants shows that the ΔN159ChlH mutant retains the ability to bind protoporphyrin and the Gun4 enhancer protein, although the addition of I and D subunits yields an extremely impaired active enzyme complex. Addition of the Gun4 enhancer protein, which stimulates MgCH activity significantly especially at low Mg2+ concentrations, partially reactivates the ΔN159ChlH–I–D mutant enzyme complex, suggesting that the binding site or sites for Gun4 on H do not wholly depend on the N-terminal domain.

Photography is about moments, and the Stripper project stems from a completely random moment. Standing in my friend’s flower shop on a normal looking high street, I looked out of the window and watched with curiosity as several bronzed men, wearing very little and with near perfect physiques, were perfecting suggestive dance routines in the adjacent back garden to the shop.It was surreal, surprising, weird and unusual – the only things I’d ever seen in the backyards before were several mechanics having a crafty cigarette or pie as they worked from their commercial garages.

The first committed step in chlorophyll biosynthesis is catalysed by magnesium chelatase (E.C. 6.6.1.1), which uses the free energy of ATP hydrolysis to insert an Mg2+ ion into the ring of protoporphyrin IX. We have characterized magnesium chelatase from the thermophilic cyanobacterium Thermosynechococcus elongatus. This chelatase is thermostable, with subunit melting temperatures between 55 and 63°C and optimal activity at 50°C. The T. elongatus chelatase (kcat of 0.16 μM/min) shows a Michaelis–Menten-type response to both Mg2+ (Km of 2.3 mM) and MgATP2− (Km of 0.8 mM). The response to porphyrin is more complex; porphyrin inhibits at high concentrations of ChlH, but when the concentration of ChlH is comparable with the other two subunits the response is of a Michaelis–Menten type (at 0.4 μM ChlH, Km is 0.2 μM). Hybrid magnesium chelatases containing a mixture of subunits from the mesophilic Synechocystis and Thermosynechococcus enzymes are active. We generated all six possible hybrid magnesium chelatases; the hybrid chelatase containing Thermosynechococcus ChlD and Synechocystis ChlI and ChlH is not co-operative towards Mg2+, in contrast with the Synechocystis magnesium chelatase. This loss of co-operativity reveals the significant regulatory role of Synechocystis ChlD.

Dimethylaluminum triflate-mediated activation of tetrafluorobenzoates of enantiomerically pure bromohydrins results in enantiospecific polyene cyclizations. The initiation of cyclization by enantiomerically pure bromonium ions and subsequent propagation is not subject to catastrophic erosion of enantiomeric purity by any intramolecular or intermolecular bromonium ion-to-alkene transfer.

Enantioenriched bromohydrin derivatives (I) and (III) undergo an enantiospecific and regioselective cyclization on treatment with TfOH.

The Rho-kinase pathway regulates agonist-induced contractions in several smooth muscles, including the intestine, urinary bladder and uterus, via dynamic changes in the Ca(2+) sensitivity of the contractile apparatus. However, there is evidence that Rho-kinase also modulates other cellular effectors such as ion channels.We examined the regulation of colonic smooth muscle excitability by Rho-kinase using conventional microelectrode recording, isometric force measurements and patch-clamp techniques.The Rho-kinase inhibitors, Y-27632 and H-1152, decreased nerve-evoked on- and off-contractions elicited at a range of frequencies and durations. The Rho-kinase inhibitors decreased the spontaneous contractions and the responses to carbachol and substance P independently of neuronal inputs, suggesting Y-27632 acts directly on smooth muscle. The Rho-kinase inhibitors significantly reduced the depolarization in response to carbachol, an effect that cannot be due to regulation of Ca(2+) sensitization. Patch-clamp experiments showed that Rho-kinase inhibitors reduce GTPγS-activated non-selective cation currents.The Rho-kinase inhibitors decreased contractions evoked by nerve stimulation, carbachol and substance P. These effects were not solely due to inhibition of the Ca(2+) sensitization pathway, as the Rho-kinase inhibitors also inhibited the non-selective cation conductances activated by excitatory transmitters. Thus, Rho-kinase may regulate smooth muscle excitability mechanisms by regulating non-selective cation channels as well as changing the Ca(2+) sensitivity of the contractile apparatus.

Vps1 is the yeast dynamin-like protein that functions during several membrane trafficking events including traffic from Golgi to vacuole, endosomal recycling and endocytosis. Vps1 can also function in peroxisomal fission indicating that its ability to drive membrane fission is relatively promiscuous. It has been of interest therefore that several mutations have been identified in Vps1 that only disrupt its endocytic function. Most recently, disruption of the interaction with actin through mutation of residues in one of the central stalk α helices (RR457,458 EE) has been shown to disrupt endocytosis and cause an accumulation of highly elongated invaginations in cells. This data supports the idea that an interaction between Vps1 and actin is important to drive the scission stage in endocytosis. Another Vps1 mutant generated in the study was vps1 E461K. Here we show data demonstrating that the E461K mutation also disrupts endocytosis but at an early stage, resulting in inhibition of the invagination step itself.

Urinary catheters are associated with a number of complications, and nurses are ideally suited to minimize the associated risks by utilizing the available research in their practice. Urine tract infections caused by urine catheters are associated with increased mortality; however, urine catheter care is a nursing procedure, the importance of which is sometimes overlooked. This study reviews recommended guidelines on urine catheter care and current published literature on the subject. The aim of the study was to identify recommended practice and compare it with the current research and literature to conclude best practice. Conclusions made from this study are that existing guidelines correspond to the recommendations and findings in recent research and literature. However, more detailed guidelines and further research on how to prevent catheter-associated urine tract infections and other complications may be of benefit.

Most judicial practitioners throughout the world today would agree that criminal investigations relating to fraud and corruption are becoming more complex and multi‐national. The globalization of f...

Retinoic acid (RA) has powerful dose-dependent dysmorphogenic effects on Xenopus embryos. The defects produced are dependent upon the stage and duration of treatment. Characteristic dysmorphogenic effects occur in the visceral (branchial) arch region and the tail. These regions correspond to the domains of expression of the retinoid receptors RAR gamma and RXR beta. By expressing domain-swapped and dominant negative derivatives of RAR gamma in embryos, we have shown that the effects of RA are mediated transcriptionally. Furthermore, the expression of dominant negative receptors in the absence of exogenous ligand has no apparent harmful effect upon early development. Finally, we have identified a novel MAP kinase phosphatase whose expression pattern is localized to regions similar to RAR gamma, and which is upregulated by RA.