Mep72, a metzincin protease that is preferentially secreted by biofilms of Pseudomonas aeruginosa.

In this work, we compared the profile of proteins secreted by planktonic and biofilm cultures of Pseudomonas aeruginosa using two-dimensional difference gel electrophoresis (2D-DiGE). This revealed that a novel metzincin protease, Mep72, was secreted during biofilm growth. Subsequent Western blotting and reverse transcription-PCR (RT-PCR) analyses demonstrated that Mep72 was expressed only during biofilm growth. Mep72 has a tridomain structure comprised of a metzincin protease-like domain and two tandem carbohydrate-binding domains. Unlike the only other metzincin (alkaline protease; AprA) in P. aeruginosa, Mep72 is secreted through the type II pathway and undergoes processing during export. During this processing, the metzincin domain is liberated from the carbohydrate-binding domains. This processing may be self-catalyzed, since purified Mep72 autodegraded in vitro. This autodegradation was retarded in the presence of alginate (an extracellular matrix component of many P. aeruginosa biofilms). The expression of full-length mep72 in Escherichia coli was toxic. However, this toxicity could be alleviated by coexpression of mep72 with the adjacent gene, bamI. Mep72 and BamI were found to form a protein-protein complex in vitro. 2D-DiGE revealed that the electrophoretic mobility of several discrete protein spots was altered in the biofilm secretome of an mep72 mutant, including type III secretion proteins (PopD, PcrV, and ExoS) and a flagellum-associated protein (FliD). Mep72 was found to bind directly to ExoS and PcrV and to affect the processing of these proteins in the biofilm secretome. We conclude that Mep72 is a secreted biofilm-specific regulator that affects the processing of a very specific subset of virulence factors.This study was funded by the BBSRC, the Isaac Newton Trust (Cambridge), and a grant from the Japanese Society for Acute Infection to K.N.The paper was originally published by the American Society for Microbiology in the Journal of Bacteriology with a CC-BY licence (IJ Passmore, K Nishikawa, KS Lilley, SD Bowden, JCS Chung, M Welch, Journal of Bacteriology 2015, 197, 762–773

The soil geochemistry in the Beardmore Glacier Region, Antarctica: Implications for terrestrial ecosystem history

Although most models suggest continental Antarctica was covered by ice during the Last Glacial Maximum (LGM) it has been speculated that endemic species of soil invertebrates could have survived the Pleistocene at high elevation habitats protruding above the ice sheets. We analyzed a series of soil samples from different elevations at three locations along the Beardmore Glacier in the Transantarctic Mountains (in order of increasing elevation): Ebony Ridge (ER), Cloudmaker (CM), and Meyer Desert (MD). Geochemical analyses show the MD soils, which were exposed during the LGM, were the least weathered compared to lower elevations, and also had the highest total dissolved solids (TDS). MD soils are dominated by nitrate salts (NO₃/Cl ratios >10) that can be observed in SEM images. High δ¹⁷O and δ¹⁸O values of the nitrate indicate that its source is solely of atmospheric origin. It is suggested that nitrate concentrations in the soil may be utilized to determine a relative “wetting age” to better assess invertebrate habitat suitability. The highest elevation sites at MD have been exposed and accumulating salts for the longest times, and because of the salt accumulations, they were not suitable as invertebrate refugia during the LGM

Mass Activated Droplet Sorting (MADS) Enables Highâ Throughput Screening of Enzymatic Reactions at Nanoliter Scale

Microfluidic droplet sorting enables the highâ throughput screening and selection of waterâ inâ oil microreactors at speeds and volumes unparalleled by traditional wellâ plate approaches. Most such systems sort using fluorescent reporters on modified substrates or reactions that are rarely industrially relevant. We describe a microfluidic system for highâ throughput sorting of nanoliter droplets based on direct detection using electrospray ionization mass spectrometry (ESIâ MS). Droplets are split, one portion is analyzed by ESIâ MS, and the second portion is sorted based on the MS result. Throughput of 0.7â samplesâ sâ 1 is achieved with 98â % accuracy using a selfâ correcting and adaptive sorting algorithm. We use the system to screen â 15â 000â samples in 6â h and demonstrate its utility by sorting 25â nL droplets containing transaminase expressed in vitro. Labelâ free ESIâ MS droplet screening expands the toolbox for droplet detection and recovery, improving the applicability of droplet sorting to protein engineering, drug discovery, and diagnostic workflows.A microfluidic system for sorting nanoliter droplets based on mass spectrometry is presented. Fully automated, labelâ free sorting at 0.7â samplesâ sâ 1 is achieved with 98â % accuracy. In vitro transcription and translation (ivTT) of a transaminase enzyme in ca.â 25â nL samples is demonstrated and samples are sorted on the basis of enzyme activity.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/154315/1/anie201913203.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/154315/2/anie201913203-sup-0001-misc_information.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/154315/3/anie201913203_am.pd

Mass Activated Droplet Sorting (MADS) Enables Highâ Throughput Screening of Enzymatic Reactions at Nanoliter Scale

Microfluidic droplet sorting enables the highâ throughput screening and selection of waterâ inâ oil microreactors at speeds and volumes unparalleled by traditional wellâ plate approaches. Most such systems sort using fluorescent reporters on modified substrates or reactions that are rarely industrially relevant. We describe a microfluidic system for highâ throughput sorting of nanoliter droplets based on direct detection using electrospray ionization mass spectrometry (ESIâ MS). Droplets are split, one portion is analyzed by ESIâ MS, and the second portion is sorted based on the MS result. Throughput of 0.7â samplesâ sâ 1 is achieved with 98â % accuracy using a selfâ correcting and adaptive sorting algorithm. We use the system to screen â 15â 000â samples in 6â h and demonstrate its utility by sorting 25â nL droplets containing transaminase expressed in vitro. Labelâ free ESIâ MS droplet screening expands the toolbox for droplet detection and recovery, improving the applicability of droplet sorting to protein engineering, drug discovery, and diagnostic workflows.Ein Mikrofluidiksystem zur Sortierung von Nanolitertröpfchen basierend auf Massenspektrometrie erreicht eine vollautomatische markierungsfreie Sortierung bei 0.7 Probenâ sâ 1 mit 98â % Genauigkeit. Die Inâ vitroâ Transkription und â Translation (ivTT) eines Transaminaseâ Enzyms in Proben von etwa 25â nL wird demonstriert, und die Proben werden nach ihrer Enzymaktivität sortiert.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/154446/1/ange201913203-sup-0001-misc_information.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/154446/2/ange201913203.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/154446/3/ange201913203_am.pd

An Examination of Celtic Craft and the Creative Consciousness as a Contribution to Marketing Creativity

Examination of the Celtic craft sector identifies a creative form of marketing which has its foundations in imagination, intuition and innovation, rather than the linear prescriptions of formal marketing frameworks and language which still dominate contemporary marketing management texts. The creative marketing competencies identified in the sector are also grounded within a wider creative marketing paradigm where experimental forms of marketing are encouraged, postmodern ideals are embraced and artistic philosophy and practice encouraged. The controlled Saxon influenced Marketing Establishment is challenged by the freer, more creative fringe of Celtic marketing as the avant garde

Engaging with History after Macpherson

The Race Relations Amendment Act (2000) identifies a key role for education, and more specifically history, in promoting ‘race equality’ in Britain. In this article Ian Grosvenor and Kevin Myers consider the extent of young people’s current engagement with the history of ‘diversity, change and immigration’ which underpins the commitment to ‘race equality’. Finding that in many of Britain’s schools and universities a singular and exclusionary version of history continues to dominate the curriculum, they go on to consider the reasons for the neglect of multiculturalism. The authors identify the development of an aggressive national identity that depends on the past for its legitimacy and argue that this sense of the past is an important obstacle to future progress

Protocol for the development of guidance for stakeholder engagement in health and healthcare guideline development and implementation

Stakeholder engagement has become widely accepted as a necessary component of guideline development and implementation. While frameworks for developing guidelines express the need for those potentially affected by guideline recommendations to be involved in their development, there is a lack of consensus on how this should be done in practice. Further, there is a lack of guidance on how to equitably and meaningfully engage multiple stakeholders. We aim to develop guidance for the meaningful and equitable engagement of multiple stakeholders in guideline development and implementation. METHODS: This will be a multi-stage project. The first stage is to conduct a series of four systematic reviews. These will (1) describe existing guidance and methods for stakeholder engagement in guideline development and implementation, (2) characterize barriers and facilitators to stakeholder engagement in guideline development and implementation, (3) explore the impact of stakeholder engagement on guideline development and implementation, and (4) identify issues related to conflicts of interest when engaging multiple stakeholders in guideline development and implementation. DISCUSSION: We will collaborate with our multiple and diverse stakeholders to develop guidance for multi-stakeholder engagement in guideline development and implementation. We will use the results of the systematic reviews to develop a candidate list of draft guidance recommendations and will seek broad feedback on the draft guidance via an online survey of guideline developers and external stakeholders. An invited group of representatives from all stakeholder groups will discuss the results of the survey at a consensus meeting which will inform the development of the final guidance papers. Our overall goal is to improve the development of guidelines through meaningful and equitable multi-stakeholder engagement, and subsequently to improve health outcomes and reduce inequities in health

An RB-EZH2 Complex Mediates Silencing of Repetitive DNA Sequences

Repetitive genomic regions include tandem sequence repeats and interspersed repeats, such as endogenous retroviruses and LINE-1 elements. Repressive heterochromatin domains silence expression of these sequences through mechanisms that remain poorly understood. Here, we present evidence that the retinoblastoma protein (pRB) utilizes a cell-cycle-independent interaction with E2F1 to recruit enhancer of zeste homolog 2 (EZH2) to diverse repeat sequences. These include simple repeats, satellites, LINEs, and endogenous retroviruses as well as transposon fragments. We generated a mutant mouse strain carrying an F832A mutation in Rb1 that is defective for recruitment to repetitive sequences. Loss of pRB-EZH2 complexes from repeats disperses H3K27me3 from these genomic locations and permits repeat expression. Consistent with maintenance of H3K27me3 at the Hox clusters, these mice are developmentally normal. However, susceptibility to lymphoma suggests that pRB-EZH2 recruitment to repetitive elements may be cancer relevant

The science of clinical practice: disease diagnosis or patient prognosis? Evidence about "what is likely to happen" should shape clinical practice.

BACKGROUND: Diagnosis is the traditional basis for decision-making in clinical practice. Evidence is often lacking about future benefits and harms of these decisions for patients diagnosed with and without disease. We propose that a model of clinical practice focused on patient prognosis and predicting the likelihood of future outcomes may be more useful. DISCUSSION: Disease diagnosis can provide crucial information for clinical decisions that influence outcome in serious acute illness. However, the central role of diagnosis in clinical practice is challenged by evidence that it does not always benefit patients and that factors other than disease are important in determining patient outcome. The concept of disease as a dichotomous 'yes' or 'no' is challenged by the frequent use of diagnostic indicators with continuous distributions, such as blood sugar, which are better understood as contributing information about the probability of a patient's future outcome. Moreover, many illnesses, such as chronic fatigue, cannot usefully be labelled from a disease-diagnosis perspective. In such cases, a prognostic model provides an alternative framework for clinical practice that extends beyond disease and diagnosis and incorporates a wide range of information to predict future patient outcomes and to guide decisions to improve them. Such information embraces non-disease factors and genetic and other biomarkers which influence outcome. SUMMARY: Patient prognosis can provide the framework for modern clinical practice to integrate information from the expanding biological, social, and clinical database for more effective and efficient care

Geochemical zones and environmental gradients for soils from the central Transantarctic Mountains, Antarctica

Previous studies have established links between biodiversity and soil geochemistry in the McMurdo Dry Valleys, Antarctica, where environmental gradients are important determinants of soil biodiversity. However, these gradients are not well established in the central Transantarctic Mountains, which are thought to represent some of the least hospitable Antarctic soils. We analyzed 220 samples from 11 ice-free areas along the Shackleton Glacier (∼ 85° S), a major outlet glacier of the East Antarctic Ice Sheet. We established three zones of distinct geochemical gradients near the head of the glacier (upper), its central part (middle), and at the mouth (lower). The upper zone had the highest water-soluble salt concentrations with total salt concentrations exceeding 80 000 μ g-1, while the lower zone had the lowest water-soluble N:P ratios, suggesting that, in addition to other parameters (such as proximity to water and/or ice), the lower zone likely represents the most favorable ecological habitats. Given the strong dependence of geochemistry on geographic parameters, we developed multiple linear regression and random forest models to predict soil geochemical trends given latitude, longitude, elevation, distance from the coast, distance from the glacier, and soil moisture (variables which can be inferred from remote measurements). Confidence in our random forest model predictions was moderately high with R2 values for total water-soluble salts, water-soluble N:P, ClO-4, and ClO-3 of 0.81, 0.88, 0.78, and 0.74, respectively. These modeling results can be used to predict geochemical gradients and estimate salt concentrations for other Transantarctic Mountain soils, information that can ultimately be used to better predict distributions of soil biota in this remote region