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Background: Understanding the cultural characteristics of healthcare organisations is widely recognised to be an important component of patient safety. A growing number of vulnerable older people are living in care homes but little attention has been paid to safety culture in this sector. In this study, we aimed to adapt the Manchester Patient Safety Framework (MaPSaF), a commonly used tool in the health sector, for use in care homes and then to test its face validity and preliminary feasibility as a tool for developing a better understanding of safety culture in the sector., Methods: As part of a wider improvement programme to reduce the prevalence of common safety incidents among residents in 90 care homes in England, we adapted MaPSaF and carried out a multimethod participatory evaluation of its face validity and feasibility for care home staff. Data were collected using participant observation, interviews, documentary analysis and a survey, and were analysed thematically., Results: MaPSaF required considerable adaptation in terms of its length, language and content in order for it to be perceived to be acceptable and useful to care home staff. The changes made reflected differences between the health and care home sectors in terms of the local context and wider policy environment, and the expectations, capacity and capabilities of the staff. Based on this preliminary study, the adapted tool, renamed 'Culture is Key', appears to have reasonable face validity and, with adequate facilitation, it is usable by front-line staff and useful in raising their awareness about safety issues., Conclusions: 'Culture is Key' is a new tool which appears to have acceptable face validity and feasibility to be used by care home staff to deepen their understanding of the safety culture of their organisations and therefore has potential to contribute to improving care for vulnerable older people., Competing Interests: Competing interests: None declared., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.)

Genetic deletion or pharmacological inhibition of cyclooxygenase (COX)-2 abrogates intestinal adenoma development at early stages of colorectal carcinogenesis. COX-2 is localised to stromal cells (predominantly macrophages) in human and mouse intestinal adenomas. Therefore, we tested the hypothesis that paracrine Cox-2-mediated signalling from macrophages drives adenoma growth and progression in vivo in the Apc Min/+ mouse model of intestinal tumorigenesis. Using a transgenic C57Bl/6 mouse model of Cox-2 over-expression driven by the chicken lysozyme locus (cLys-Cox-2), which directs integration site-independent, copy number-dependent transgene expression restricted to macrophages, we demonstrated that stromal macrophage Cox-2 in colorectal (but not small intestinal) adenomas from cLys-Cox-2 x Apc Min/+ mice was associated with significantly increased tumour size (P = 0.025) and multiplicity (P = 0.025), compared with control Apc Min/+ mice. Transgenic macrophage Cox-2 expression was associated with increased dysplasia, epithelial cell Cox-2 expression and submucosal tumour invasion, as well as increased nuclear β-catenin translocation in dysplastic epithelial cells. In vitro studies confirmed that paracrine macrophage Cox-2 signalling drives catenin-related transcription in intestinal epithelial cells. Paracrine macrophage Cox-2 activity drives growth and progression of Apc Min/+ mouse colonic adenomas, linked to increased epithelial cell β-catenin dysregulation. Stromal cell (macrophage) gene regulation and signalling represent valid targets for chemoprevention of colorectal cancer.

There has been increasing interest in complex coacervates for deriving and transporting biomaterials. Complex coacervates are a dense, polyelectrolyte-rich liquid that results from the electrostatic complexation of oppositely charged macroions. Coacervates have long been used as a strategy for encapsulation, particularly in food and personal care products. More recent efforts have focused on the utility of this class of materials for the encapsulation of small molecules, proteins, RNA, DNA, and other biomaterials for applications ranging from sensing to biomedicine. Furthermore, coacervate-related materials have found utility in other areas of biomedicine, including cartilage mimics, tissue culture scaffolds, and adhesives for wet, biological environments. Here, we discuss the self-assembly of complex coacervate-based materials, current challenges in the intelligent design of these materials, and their utility applications in the broad field of biomedicine. WIREs Nanomed Nanobiotechnol 2017, 9:e1442. doi: 10.1002/wnan.1442 For further resources related to this article, please visit the WIREs website., (© 2016 Wiley Periodicals, Inc.)

In this study, we provide the first demonstration that aqueous complex coacervates can be electrospun into chemically robust polyelectrolyte complex (PEC) fiber mats. PECs form due to electrostatic complexation between oppositely charged polymers. Here, we exploit the ability of salt to plasticize PECs, thus enabling the electrospinning of solid fibers. Electrospinning solutions were composed of a pair of strong polyelectrolytes, poly(4-styrenesulfonic acid, sodium salt) and poly(diallyldimethylammonium chloride) using potassium bromide as the plasticizing salt. We systematically investigated the effect of salt concentration and electrospinning apparatus parameters on fiber formation. Electrospun PEC fiber mats were stable over a wide range of pH values, ionic strength conditions, and many organic solvents. This study demonstrates that the electrospinning of aqueous complex coacervates can generate chemically robust, free-standing PEC fiber mats while circumventing the reliance on organic solvents, the challenge of working with entangled polyelectrolytes in solution, and the need to chemically cross-link the as-spun fibers. These PEC fiber mats hold potential in applications where environmentally benign fiber mats are imperative, such as tissue engineering scaffolds and water purification technologies.

The aim of this study was to investigate whether diurnal variation in cough reflex sensitivity exists in healthy young adults when a tidal-breathing method is used. Fifty-three participants (19-37 years) underwent cough reflex testing on two occasions: once in the morning (between 9 am - midday) and once in the afternoon (between 2-5 pm). The order of testing was counter-balanced. Within each assessment, participants inhaled successively higher citric acid concentrations via a facemask, with saline solution randomly interspersed to control for a placebo response. The lowest concentration that elicited a reflexive cough response was recorded. Morning cough thresholds (mean=0.6mol/L) were not different from afternoon cough thresholds (mean=0.6mol/L), p=0.16, T=101, r=-0.14. We found no evidence of diurnal variability in cough reflex testing. There was, however, an order effect irrespective of time of day, confirming that healthy participants are able to volitionally modulate their cough response., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Histone deacetylases (HDACs) have been extensively studied as drug targets in neurodegenerative diseases, but less is known about their role in healthy neurons. We tested zinc-dependent HDACs using RNAi in Drosophila melanogaster and found memory deficits with RPD3 and HDAC6. We demonstrate that HDAC6 is required in both the larval and adult stages for normal olfactory memory retention. Neuronal expression of HDAC6 rescued memory deficits, and we demonstrate that the N-terminal deacetylase (DAC) domain is required for this ability. This suggests that deacetylation of synaptic targets associated with the first DAC domain, such as the active-zone scaffold Bruchpilot, is required for memory retention. Finally, electrophysiological experiments at the neuromuscular junction reveal that HDAC6 mutants exhibit a partial block of homeostatic plasticity, suggesting that HDAC6 may be required for the stabilization of synaptic strength. The learning deficit we observe in HDAC6 mutants could be a behavioral consequence of these synaptic defects., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Rheology is a powerful method for material characterization that can provide detailed information about the self-assembly, structure, and intermolecular interactions present in a material. Here, we review the use of linear viscoelastic measurements for the rheological characterization of complex coacervate-based materials. Complex coacervation is an electrostatically and entropically-driven associative liquid-liquid phase separation phenomenon that can result in the formation of bulk liquid phases, or the self-assembly of hierarchical, microphase separated materials. We discuss the need to link thermodynamic studies of coacervation phase behavior with characterization of material dynamics, and provide parallel examples of how parameters such as charge stoichiometry, ionic strength, and polymer chain length impact self-assembly and material dynamics. We conclude by highlighting key areas of need in the field, and specifically call for the development of a mechanistic understanding of how molecular-level interactions in complex coacervate-based materials affect both self-assembly and material dynamics., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The mechanism of the anti-colorectal cancer (CRC) activity of the omega-3 fatty acid eicosapentaenoic acid (EPA) is not understood. We tested the hypothesis that EPA reduces expression of chemokine C-C motif ligand 2 (CCL2), a pro-inflammatory chemokine with known roles in metastasis.We measured CCL2 in clinical samples from a randomized trial of EPA in patients undergoing liver surgery for CRC liver metastasis (LM) and preclinical models. Genome-wide transcriptional profiling of tumors from EPA-treated patients was performed.EPA decreased CCL2 synthesis by CRC cells in a dose-dependent manner. CCL2 was localized to malignant epithelial cells in human CRCLM. EPA did not reduce CCL2 content in human or mouse tumors compare to control. However, EPA treatment was associated with decreased plasma CCL2 levels compared with controls (P=0.04). Reduction in plasma CCL2 following EPA treatment predicted improved disease-free survival (HR 0.32; P=0.003). Lack of 'CCL2 response' was associated with a specific CRCLM gene expression signature.In conclusion, reduction in plasma CCL2 in patients with CRCLM treated with EPA predicts better clinical outcome and a specific tumor gene expression profile. Further work is needed to validate CCL2 as a therapeutic response biomarker for omega-3 fatty acid treatment of CRC patients., Competing Interests: None of the authors have any potential conflicts of interest to disclose.

Objective: This study compares variability in measures of hyoid bone displacement and submental muscle size using 2 methods of ultrasound transducer placement: fixed and hand-held., Patients and Methods: Twenty-four healthy participants (11 males, 13 females) aged between 51 and 84 years were scanned using both methods. Repeated measures were collected within and across 3 sessions., Results: A nonlinear mixed effects model was used to estimate the effects of the method, trial, and session. The fixed transducer produced greater within- and across-session variability on several measures compared to the hand-held method. Fixed-transducer measurements of hyoid bone displacement were 9.4% smaller compared to hand-held measurements (F[1, 24.34] = 3.58, p = 0.07)., Conclusion: Results suggest that using a fixed transducer does not necessarily improve measurement accuracy of swallowing outcomes in a healthy system. The wide variability in individual measures indicates that further research is required before ultrasound is used for outcome measurement in rehabilitation., (© 2017 S. Karger AG, Basel.)

Background: Only half of patients with locally invasive rectal carcinoma respond to short-course preoperative radiotherapy. A predictive test enabling better patient selection could avoid unneccessary radiation exposure to poor responders. Macrophages within the tumour immune microenvironment with tumoricidal M1 and tumour-protective M2 phenotypes could be modulating this response. This study investigated the possible predictive value of M1 and M2 subpopulations in identifying patients' likely response to short-course preoperative radiotherapy., Methods: Biopsy samples were taken from 29 patients with locally invasive rectal carcinoma before treatment with short-course radiotherapy and surgical specimens obtained after resection following short-course preoperative radiotherapy. Dual-staining immunohistochemistry was performed with CD68 as macrophage marker, HLA-DR as M1 marker, and CD163 as M2 marker. Samples were scored for hot-and-random spots by Nuance software (version 3.0.2) and compared with patients' outcome data. Tumour response was measured by assessment of reduction of tumour-cell density., Findings: Samples revealing a low score for HLA-DR positive M1 macrophages exhibited a better response to short-course radiotherapy with up to 80% (median 80·38% [IQR 46·94-84·73]) reduction in the tumour cell density. On the other hand those with a high score exhibited a poor response with only up to 20% (20·26 [0-48·19]) reduction. The difference in response between the two groups was significant (p=0·017). No such trends were observed for CD163+ M2 macrophages. The ratio of HLA-DR+ to CD163+ macrophages for biopsy and resection samples was significantly different, showing a drop in the HLA-DR positive macrophages in the resection samples (p=0·024). The mean of the difference between the biopsy (median 2·53 [IQR 1·98-3·08]) and resection (1·38 [0·96-1·8]) was 1·15 (p=0·024)., Interpretation: Patients with a variable macrophage phenotype composition within biopsy samples from patients with locally invasive rectal carcinoma respond differently to short-course preoperative radiotherapy. Further investigation involving a panel of macrophage and other immune-cell markers could verify and validate these findings and develop them as predictive tests identifying good responders to radiotherapy in patients with locally invasive rectal carcinoma., Funding: None., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Polyelectrolyte complexes present new opportunities for self-assembled soft matter. Factors determining whether the phase of the complex is solid or liquid remain unclear. Ionic polypeptides enable examination of the effects of stereochemistry on complex formation. Here we demonstrate that chirality determines the state of polyelectrolyte complexes, formed from mixing dilute solutions of oppositely charged polypeptides, via a combination of electrostatic and hydrogen-bonding interactions. Fluid complexes occur when at least one of the polypeptides in the mixture is racemic, which disrupts backbone hydrogen-bonding networks. Pairs of purely chiral polypeptides, of any sense, form compact, fibrillar solids with a β-sheet structure. Analogous behaviour occurs in micelles formed from polypeptide block copolymers with polyethylene oxide, where assembly into aggregates with either solid or fluid cores, and eventually into ordered phases at high concentrations, is possible. Chirality is an exploitable tool for manipulating material properties in polyelectrolyte complexation.

Fluorescent imaging of colorectal tumor cells would improve tumor localization and allow intra-operative staging, facilitating stratification of surgical resections thereby improving patient outcomes. We aimed to develop and test fluorescent nanoparticles capable of allowing this in vivo. Dye-doped silica nanoparticles were synthesized. Anti-CEA (carcinoembryonic antigen) or control IgGs were conjugated to nanoparticles using various chemical strategies. Binding of CEA-targeted or control nanoparticles to colorectal cancer cells was quantified in vitro, and in vivo after systemic-delivery to murine xenografts. CEA-targeted, polyamidoamine dendrimer-conjugated, nanoparticles, but not control nanoparticles, allowed strong tumor-specific imaging. We are the first to demonstrate live, specific, in vivo imaging of colorectal cancer cells using antibody-targeted fluorescent nanoparticles. These nanoparticles have potential to allow intra-operative fluorescent visualization of tumor cells.

Hair cells are the mechanosensory cells of the inner ear. Mechanotransduction channels in hair cells are gated by tip links. The molecules that connect tip links to transduction channels are not known. Here we show that the transmembrane protein TMIE forms a ternary complex with the tip-link component PCDH15 and its binding partner TMHS/LHFPL5. Alternative splicing of the PCDH15 cytoplasmic domain regulates formation of this ternary complex. Transducer currents are abolished by a homozygous Tmie-null mutation, and subtle Tmie mutations that disrupt interactions between TMIE and tip links affect transduction, suggesting that TMIE is an essential component of the hair cell's mechanotransduction machinery that functionally couples the tip link to the transduction channel. The multisubunit composition of the transduction complex and the regulation of complex assembly by alternative splicing is likely critical for regulating channel properties in different hair cells and along the cochlea's tonotopic axis., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Proteins have gained increasing success as therapeutic agents; however, challenges exist in effective and efficient delivery. In this work, we present a simple and versatile method for encapsulating proteins via complex coacervation with oppositely charged polypeptides, poly(l-lysine) (PLys) and poly(d/l-glutamic acid) (PGlu). A model protein system, bovine serum albumin (BSA), was incorporated efficiently into coacervate droplets via electrostatic interaction up to a maximum loading of one BSA per PLys/PGlu pair and could be released under conditions of decreasing pH. Additionally, encapsulation within complex coacervates did not alter the secondary structure of the protein. Lastly the complex coacervate system was shown to be biocompatible and interact well with cells in vitro. A simple, modular system for encapsulation such as the one presented here may be useful in a range of drug delivery applications.

We consider polyelectrolyte solutions which, under suitable conditions, phase separate into a liquid-like coacervate phase and a coexisting supernatant phase that exhibit an extremely low interfacial tension. Such interfacial tension provides the basis for most coacervate-based applications, but little is known about it, including its dependence on molecular weight, charge density, and salt concentration. By combining a Debye-Hückel treatment for electrostatic interactions with the Cahn-Hilliard theory, we derive explicit expressions for this interfacial tension. In the absence of added salts, we find that the interfacial tension scales as N -3/2 (η/η c -1) 3/2 near the critical point of the demixing transition, and that it scales as η 1/2 far away from it, where N is the chain length and η measures the electrostatic interaction strength as a function of temperature, dielectric constant, and charge density of the polyelectrolytes. For the case with added salts, we find that the interfacial tension scales with the salt concentration ψ as N -1/4 (1-ψ/ψ c ) 3/2 near the critical salt concentration ψ c . Our predictions are shown to be in quantitative agreement with experiments and provide a means to design new materials based on polyelectrolyte complexation.

An analysis of 570 HIV-infected women in Swaziland using the Jadelle implant showed that age, condom use, the provider who placed the implant, and CD4 cell count had no effect on unintentional pregnancy rates. Antiretroviral regimen at the time of pregnancy, however, correlated with pregnancy outcomes (P < 0.001). None of the women on nevirapine or lopinavir/ritonavir-based regimens (n = 208 and 13, respectively) became pregnant, whereas 15 women on efavirenz (n = 121; 12.4%) became pregnant.

Using small-angle x-ray scattering (SAXS), we investigated the phase behavior of mesophases of monoolein (MO) mixed with additives commonly used for the crystallization of membrane proteins from lipidic mesophases. In particular, we examined the effect of sodium and potassium phosphate salts and the detergent β-octylglucoside (βOG) over a wide range of compositions relevant for the crystallization of membrane proteins in lipidic mesophases. We studied two types of systems: 1), ternary mixtures of MO with salt solutions above the hydration boundary; and 2), quaternary mixtures of MO with βOG and salt solutions over a wide range of hydration conditions. All quaternary mixtures showed highly regular lyotropic phase behavior with the same sequence of phases (Lα, Ia3d, and Pn3m) as MO/water mixtures at similar temperatures. The effects of additives in quaternary systems agreed qualitatively with those found in ternary mixtures in which only one additive is present. However, quantitative differences in the effects of additives on the lattice parameters of fully hydrated mesophases were found between ternary and quaternary mixtures. We discuss the implications of these findings for mechanistic investigations of membrane protein crystallization in lipidic mesophases and for studies of the suitability of precipitants for mesophase-based crystallization methods., (Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

We investigated the role of interleukin (IL)-4 receptor (IL-4R) signalling during mouse carcinogen-induced colorectal carcinogenesis and in a case-control genetic epidemiological study of IL-4Rα single nucleotide polymorphisms (SNPs). Azoxymethane-induced aberrant crypt focus (ACF; 6 weeks) and tumours (32 weeks) were analysed in wild-type (WT) BALB/c mice, as well as in IL-4Rα (-) (/-) , IL-13 (-/-) and 'double-knockout' (DKO) animals. Colorectal cancer (CRC) cases (1502) and controls (584) were genotyped for six coding IL-4Rα SNPs. The association with CRC risk and CRC-specific mortality was analysed by logistic regression. Lack of IL-4Rα expression was associated with increased ACFs [median 8.5 ACFs per mouse (IL-4Rα (-/-) ) versus 3 (WT); P = 0.007], but no difference in the number of colorectal tumours [mean 1.4 per mouse (IL-4Rα (-/-) ) versus 2 (WT)], which were smaller and demonstrated reduced nuclear/cytoplasmic β-catenin translocation compared with WT tumours. Tumour-bearing IL-4Rα (-/-) mice had fewer CD11b(+)/Gr1(+) myeloid-derived suppressor splenocytes than WT animals. IL-13 (-/-) mice developed a similar number of ACFs to IL-4Rα (-/-) and DKO mice. There was a significant increase in CRC risk associated with the functional SNP Q576R [odds ratio 1.54 (95% confidence interval 0.94-2.54), P trend 0.03 for the minor G allele]. There was no effect of IL-4Rα genotype on either CRC-specific or all-cause mortality. These combined pre-clinical and human data together demonstrate that reduced IL-4R signalling has stage-specific effects on colorectal carcinogenesis (increased CRC initiation and risk but reduced tumour progression and no effect on CRC mortality). These results should prompt evaluation of the effect of pharmacological manipulation of IL-4R signalling on future CRC risk and for CRC treatment.

Objective: We investigated the in vitro inspired dose and particle size distribution of albuterol delivered by a vibrating mesh nebulizer through the Vapotherm (Stevensville, MD) humidified high-flow nasal cannula system., Design: Albuterol (2.5 mg/3 mL) was delivered by an Aeroneb Solo (Aerogen, Galway, Ireland) nebulizer that was connected via adaptor proximal to the nasal cannula and downstream from the Vapotherm 2000i. Albuterol was collected onto an inspiratory filter mounted to a breath simulator programmed with age-appropriate breathing patterns. Particle sizing was completed by cascade impaction. Albuterol was quantified using ultraviolet spectrometry. Measurements were made using varying flow rates through infant, pediatric, and adult nasal cannulae., Setting: Aerosol research laboratory., Measurements and Main Results: The inspired dose (percent of nominal dose) for each cannula size and flow rate was 2.5%, 0.8%, 0.4%, and 0.2% for the adult cannula at 5, 10, 20, and 40 L/min, respectively; 1.2%, 0.6%, 0.1%, and 0.0% for the pediatric cannula at 3, 5, 10, and 20 L/min, respectively; and 0.6%, 0.6%, and 0.5% for the infant cannula at 3, 5, and 8 L/min, respectively. Most (62-80%) of the loaded albuterol dose accumulated within the adaptor. For each cannula size, there was a significant decrease in the inspired dose with increasing flow rates, p = 0.026 (infant), p = 0.001 (pediatric), and p < 0.001(adult). The inspired dose increased with increasing cannula size for 5, 10, and 20 L/min (p = 0.007, p < 0.001, and p = 0.005, respectively). The mass median aerodynamic diameter for all trials was less than 5 µm., Conclusion: The amount of albuterol delivered with the Vapotherm system using this model was lower than the amount expected for a clinical response for the majority of flow rates and cannula size combinations. Further studies are needed before routine use of aerosolized albuterol through a Vapotherm high-flow system can be recommended.

Mortality from locally advanced and metastatic cancer remains high despite advances in our understanding of the molecular basis of the disease and improved adjuvant therapies. Recently, there has been an increased interest in cancer metabolomics, and in particular, the potential for targeting glucose metabolism, for therapeutic gain. This interest stems from the fact that cancer cells metabolize glucose very differently from normal cells. Cancer cells preferentially switch to aerobic glycolysis rather than oxidative phosphorylation as their means of glucose metabolism. This metabolic switch is believed to enhance cancer cell survival. Several therapeutic agents that target tumor metabolism have shown significant cancer cell cytotoxicity in preclinical studies, and some have progressed to clinical trials. In this review, we discuss the alteration of carbohydrate metabolism seen in cancer cells, the underlying mechanisms, and opportunities for targeting cancer metabolism for therapeutic purposes.

As a potent inflammatory agent, endotoxin is a key analyte of interest for studies of lung ailments in domestic environments and occupational settings with organic dust. A relatively unexplored advance in endotoxin exposure assessment is the use of recombinant factor C (rFC) from the Limulus pathway in a fluorometric assay. In this study, we compared airborne endotoxin concentrations in laboratory- and field-collected parallel air samples using the kinetic Limulus amebocyte lysate (LAL) assay and the rFC assay. Air sampling was performed using paired Institute of Occupational Medicine (IOM) samplers, Button samplers, closed-face cassettes, and cyclone samplers. Field sampling was performed in 10 livestock production facilities, including those housing swine, chicken, turkey, dairy cows, cattle, and horses. Laboratory sampling was performed in exposure chambers using resuspended airborne dust collected in five livestock facilities. Paired samples were extracted in pyrogen-free water with 0.05% Tween 20 and analyzed using LAL and rFC assays. In 402 field sample pairs there was excellent agreement between endotoxin concentrations determined by LAL and rFC (r = 0.93; P < 0.0001). In 510 laboratory sample pairs there was also excellent agreement between the two assays (r = 0.86; P < 0.0001). Correlations for subgroups of facility or dust type ranged from 0.65 to 0.96. Mixed-model analysis of variance (ANOVA) for the field studies showed significant interactions of facility-sampler and facility-assay. rFC/LAL ratios of the geometric means were 0.9 to 1.14 for the samplers (not significantly different from 1.0). The data from this study demonstrate that the LAL assay and the rFC assay return similar estimates of exposure in livestock facilities. Both methods provided suitable lower limits of detection such that all but 19 of 1,824 samples were quantifiable.

Methods to efficiently determine the phase behavior of novel proteins have the potential to significantly benefit structural biology efforts. Here, we present protocols to determine both the solubility boundary and the supersolubility boundary for protein/precipitant systems using an evaporation-based crystallization platform. This strategy takes advantage of the well-defined rates of evaporation that occur in this platform to determine the state of the droplet at any point in time without relying on an equilibrium-based end point. The dynamic nature of this method efficiently traverses phase space along a known path, such that a solubility diagram can be mapped out for both soluble and membrane proteins while using a smaller amount of protein than what is typically used in optimization screens. Furthermore, a variation on this method can be used to decouple crystal nucleation and growth events, so fewer and larger crystals can be obtained within a given droplet. The latter protocol can be used to rescue a crystallization trial where showers of tiny crystals were observed. We validated both of the protocols to determine the phase behavior and the protocol to optimize crystal quality using the soluble proteins lysozyme and ribonuclease A as well as the membrane protein bacteriorhodopsin.

The release of reactive oxygen species (ROS) and cytokines by alveolar macrophages has been demonstrated in asbestos-induced pulmonary fibrosis, but the mechanism linking alveolar macrophages to the pathogenesis is not known. The GTPase Rac1 is a second messenger that plays an important role in host defense. In this study, we demonstrate that Rac1 null mice are protected from asbestos-induced pulmonary fibrosis, as determined by histological and biochemical analysis. We hypothesized that Rac1 induced pulmonary fibrosis via generation of ROS. Asbestos increased TNF-alpha and ROS in a Rac1-dependent manner. TNF-alpha was elevated only 1 day after exposure, whereas ROS generation progressively increased in bronchoalveolar lavage cells obtained from wild-type (WT) mice. To determine whether ROS generation contributed to pulmonary fibrosis, we overexpressed catalase in WT monocytes and observed a decrease in ROS generation in vitro. More importantly, administration of catalase to WT mice attenuated the development of fibrosis in vivo. For the first time, these results demonstrate that Rac1 plays a crucial role in asbestos-induced pulmonary fibrosis. Moreover, it suggests that a simple intervention may be useful to prevent progression of the disease.

Objectives: To investigate the prevalence of isolated urinary and fecal incontinence and double incontinence in community-living stroke survivors and to assess the degree of soiling., Design: Community-based postal survey., Setting: Leicestershire, United Kingdom., Participants: Sixty-four thousand seven hundred forty-nine community-dwelling residents (aged > or = 40) were randomly selected from the Leicestershire Health Authority register. Residents living in institutional settings were excluded., Measurements: Respondents were asked about previous stroke, urinary and bowel symptoms, and general health and demographic details including age, sex, and ethnicity. Urinary incontinence was defined as leakage several times a month or more often. Major fecal incontinence was defined as soiling of underwear, outer clothing, furnishings, or bedding several times a month or more often., Results: A 65% response rate to the postal survey was obtained, with the return of 39,519 eligible questionnaires; 4% (n = 1,483) reported stroke. Five percent of stroke survivors reported major fecal incontinence, with 4.3% reporting fecal and urinary incontinence and 0.8% reporting isolated fecal incontinence. Major fecal incontinence was four and a half times as prevalent in stroke survivors as in the nonstroke population, and stroke survivors were also twice as likely to report soiling of furnishings or bedding. Functional limitations influence the presence of fecal incontinence in the stroke and nonstroke population., Conclusion: Fecal incontinence is common in stroke survivors, and the degree of soiling can be considerable. Future research needs to explore the effect fecal incontinence can have on the lives of stroke survivors and on how it can best be managed in those living in the community.

Objectives: The study investigated the association between anxiety and depression and urge incontinence and the direction of causal pathways between these variables., Design: A prospective longitudinal postal survey., Method: A random sample of women aged 40 years or more, registered with a general practitioner in Leicestershire or Rutland, was mailed a postal questionnaire. The questionnaire included questions on general health, urinary symptoms and the Hospital Anxiety and Depression Scale (HADS). In total, 12,568 women responded to the baseline postal survey (65.3% response rate) and 9,596 to the first annual follow-up (79.8% response rate). The prevalence and one-year incident rates of these symptoms were compared and contrasted, whilst controlling for confounding variables., Results: A significant proportion of women with urge incontinence reported symptoms of anxiety (56.6%) and depression (37.6%). Anxiety and depression were associated with a number of urinary symptoms and were not exclusive to urge incontinence. Incident cases of anxiety and depression were predicted by the presence of urge incontinence at baseline. Incident cases of urge incontinence were predicted by anxiety at baseline, but not depression. Anxiety, urge incontinence and frequency appeared to interact and exacerbate each other., Conclusions: The findings demonstrated the relevance of emotional factors in the development and maintenance of urge incontinence. Currently, assessment and treatment protocols for urge incontinence concentrate on physical symptoms and toilet behaviours. A more integrated psychological model of urge incontinence is proposed.

Liver cancer is very common worldwide and the rates of hepatocellular carcinoma (HCC) have increased by over 70% in the last 2 decades in the US. Late diagnosis, because of the lack of clinical symptoms, and decreased hepatic function, because of underlying hepatic disease, lead to the extremely high mortality rates associated with HCC. Clearly, the identification of markers that are expressed early in the development of HCC and that are easily detected in high-risk patients would aid in early diagnosis and increased survival. We present the cloning and characterization of a novel gene, CRG-L2 (Cancer related gene-Liver 2), which displays high expression in murine and human hepatocellular carcinomas. Using in situ hybridization, we show that CRG-L2 mRNA levels are increased early during the development of liver tumors in C3H/HeJ mice, and that in normal tissues CRG-L2 mRNA is restricted to the murine testis and human placenta. Its restricted expression in normal tissues and unique early upregulation during tumor development make CRG-L2 an excellent candidate as a new clinical marker of HCC.