A New Approach to Understanding Biological Control of Quinone Electrochemistry

Oxidoreductases play pivotal roles in energy capturing and converting processes of life. During these processes, quinones shuttle protons and reducing equivalents between membrane-bound oxidoreductases that generate the proton motive force during oxidative phosphorylation and photophosphorylation. A key mechanistic feature of these oxidoreductases is their ability to tune the reduction potentials of the hydroquinone, semiquinone and oxidized states of their substrate quinones. This level of control allows for maximization of conversion efficiency between the energy of the quinone reducing equivalents and the proton motive force, and prevents side reactions that may be fatal to cells. A half-century of experimental study and computational modeling of the respiratory and photosynthetic complexes has revealed little information on how this mechanistic control is accomplished. To obtain mechanistic insights into the control process, it is necessary to eliminate the biological complexity intrinsic to natural quinone oxidoreductases and create experimental systems that are simplified maquettes of quinone active sites. In this work, development of a naphthoquinone amino acid (Naq), modeled after vitamin K, allowed the creation of a range of quinone peptide maquettes designed to address uncertain mechanistic details of biological quinone control. In a simple heptamer, Naq acquires properties of quinone cofactors found in the three distinct classes of active sites of membrane oxidoreductases under different experimental conditions. Study of Naq in a lanthanide ion binding EF hand peptide allowed observation of the effect of a structural transition from coil to alpha-helix on the aqueous midpoint potential of Naq and measurement of the rate of electron transfer between reduced and oxidized Naq. Naq was also incorporated into a structured miniprotein based upon the TrpCage using a combination of the SCADS computational approach and iterative redesign by hand, creating a simple scaffold for evaluating effects of changing the local environment on Naq. Finally, using expressed protein ligation, Naq was incorporated into a single chain heme-binding maquette. Studies using this multi-cofactor protein to explore electron transfer reactions to and from Naq like those critical to respiration and photosynthesis are underway

Engineering an AB₅ protein carrier

The promise of biologic therapeutics is hindered by the challenge to deliver their activity to biochemically relevant sites within diseased cells. The favourable application of the natural protein carriers of the AB5 toxin family to this challenge has been restricted owing to still unresolved requirements for assembling non-native cargo into carrier complexes. Here, we clarify the properties of fusion peptides which allow co-assembly of a selected fluorescent protein cargo with the non-toxic B subunit of a heat-labile enterotoxin. We establish the influence of sequence length, sequence identity and secondary structure of these linking domains on the assembly and disassembly of the complexes. Through our engineering framework we identify several non-native, reduced length fusion sequences that robustly assemble with the native carriers, maintain their ability to deliver protein cargo to cells, and demonstrate substantially refined in vitro properties. Constructs based upon these sequences should prove directly applicable to a variety of protein delivery challenges, and the described design framework should find immediate application to other members of the AB5 protein carrier family

Information scraps: how and why information eludes our personal information management tools

In this paper we describe information scraps -- a class of personal information whose content is scribbled on Post-it notes, scrawled on corners of random sheets of paper, buried inside the bodies of e-mail messages sent to ourselves, or typed haphazardly into text files. Information scraps hold our great ideas, sketches, notes, reminders, driving directions, and even our poetry. We define information scraps to be the body of personal information that is held outside of its natural or We have much still to learn about these loose forms of information capture. Why are they so often held outside of our traditional PIM locations and instead on Post-its or in text files? Why must we sometimes go around our traditional PIM applications to hold on to our scraps, such as by e-mailing ourselves? What are information scraps' role in the larger space of personal information management, and what do they uniquely offer that we find so appealing? If these unorganized bits truly indicate the failure of our PIM tools, how might we begin to build better tools? We have pursued these questions by undertaking a study of 27 knowledge workers. In our findings we describe information scraps from several angles: their content, their location, and the factors that lead to their use, which we identify as ease of capture, flexibility of content and organization, and avilability at the time of need. We also consider the personal emotive responses around scrap management. We present a set of design considerations that we have derived from the analysis of our study results. We present our work on an application platform, jourknow, to test some of these design and usability findings

Reliability and Initial Validation of the Ulcerative Colitis Endoscopic Index of Severity

Background & AimsWe studied the reliability of the previously described Ulcerative Colitis Endoscopic Index of Severity (UCEIS) and validated it with an independent cohort of investigators.MethodsWe created a new library of 57 videos of flexible sigmoidoscopy and stratified them based on disease severity. Twenty-five investigators were each randomly assigned to assess 28 videos (which included 4 duplicates to assess intraobserver reliability). Investigators were blinded to clinical details except for 2 of 4 duplicated videos (to assess the impact of knowledge of symptoms on assessment). Three descriptors (“vascular pattern”, “bleeding”, and “erosions and ulcers”) comprising the UCEIS were scored with a visual analogue scale (VAS) to assess overall severity. Intrainvestigator and interinvestigator agreement was characterized by κ statistical analysis; reliability ratios were used to compare VAS and UCEIS scores.ResultsThere was a high level of correlation between UCEIS scores and overall assessment of severity (correlation coefficient, 0.93). Internal consistency (Cronbach α analysis) was 0.86. Intrainvestigator and interinvestigator reliability ratios for UCEIS scores were 0.96 and 0.88, respectively. Intrainvestigator agreement in determination of the UCEIS score was good (κ = 0.72), with individual descriptors ranging from a κ of 0.47 (for bleeding) to 0.87 (for vascular pattern). Interinvestigator agreement in determination of UCEIS scores was moderate (κ = 0.50), with descriptors ranging from a κ of 0.48 (for bleeding) to 0.54 (for vascular pattern). Intrainvestigator variability in determining UCEIS scores did not change appreciably when a video was presented with clinical details.ConclusionsThe UCEIS and its components show satisfactory intrainvestigator and interinvestigator reliability. Among investigators, the UCEIS accounted for a median of 86% of the variability in evaluation of overall severity on the VAS when assessing the endoscopic severity of UC and was unaffected by knowledge of clinical details

A Pooled Analysis of Infections, Malignancy, and Mortality in Infliximab- and Immunomodulator-Treated Adult Patients With Inflammatory Bowel Disease

The objective of this study was to analyze the safety of long-term infliximab treatment, with/without concomitant immunomodulators, across Crohn's disease (CD) and ulcerative colitis (UC) clinical trials.status: publishe

Constructing a man-made c-type cytochrome maquette in vivo:electron transfer, oxygen transport and conversion to a photoactive light harvesting maquette

The successful use of man-made proteins to advance synthetic biology requires both the fabrication of functional artificial proteins in a living environment, and the ability of these proteins to interact productively with other proteins and substrates in that environment. Proteins made by the maquette method integrate sophisticated oxidoreductase function into evolutionarily naive, non-computationally designed protein constructs with sequences that are entirely unrelated to any natural protein. Nevertheless, we show here that we can efficiently interface with the natural cellular machinery that covalently incorporates heme into natural cytochromes c to produce in vivo an artificial c-type cytochrome maquette. Furthermore, this c-type cytochrome maquette is designed with a displaceable histidine heme ligand that opens to allow functional oxygen binding, the primary event in more sophisticated functions ranging from oxygen storage and transport to catalytic hydroxylation. To exploit the range of functions that comes from the freedom to bind a variety of redox cofactors within a single maquette framework, this c-type cytochrome maquette is designed with a second, non-heme C, tetrapyrrole binding site, enabling the construction of an elementary electron transport chain, and when the heme C iron is replaced with zinc to create a Zn porphyrin, a light-activatable artificial redox protein. The work we describe here represents a major advance in de novo protein design, offering a robust platform for new c-type heme based oxidoreductase designs and an equally important proof-of-principle that cofactor-equipped man-made proteins can be expressed in living cells, paving the way for constructing functionally useful man-made proteins in vivo

Developing an instrument to assess the endoscopic severity of ulcerative colitis : The Ulcerative Colitis Endoscopic Index of Severity (UCEIS)

Full list of Investigators is given at the end of the article.Background: Variability in endoscopic assessment necessitates rigorous investigation of descriptors for scoring severity of ulcerative colitis (UC). Objective: To evaluate variation in the overall endoscopic assessment of severity, the intra- and interindividual variation of descriptive terms and to create an Ulcerative Colitis Endoscopic Index of Severity which could be validated. Design: A two-phase study used a library of 670 video sigmoidoscopies from patients with Mayo Clinic scores 0-11, supplemented by 10 videos from five people without UC and five hospitalised patients with acute severe UC. In phase 1, each of 10 investigators viewed 16/24 videos to assess agreement on the Baron score with a central reader and agreed definitions of 10 endoscopic descriptors. In phase 2, each of 30 different investigators rated 25/60 different videos for the descriptors and assessed overall severity on a 0-100 visual analogue scale. κ Statistics tested inter- and intraobserver variability for each descriptor. A general linear mixed regression model based on logit link and β distribution of variance was used to predict overall endoscopic severity from descriptors. Results: There was 76% agreement for 'severe', but 27% agreement for 'normal' appearances between phase I investigators and the central reader. In phase 2, weighted κ values ranged from 0.34 to 0.65 and 0.30 to 0.45 within and between observers for the 10 descriptors. The final model incorporated vascular pattern, (normal/patchy/ complete obliteration) bleeding (none/mucosal/luminal mild/luminal moderate or severe), erosions and ulcers (none/erosions/superficial/deep), each with precise definitions, which explained 90% of the variance (pR2, Akaike Information Criterion) in the overall assessment of endoscopic severity, predictions varying from 4 to 93 on a 100-point scale (from normal to worst endoscopic severity). Conclusion: The Ulcerative Colitis Endoscopic Index of Severity accurately predicts overall assessment of endoscopic severity of UC. Validity and responsiveness need further testing before it can be applied as an outcome measure in clinical trials or clinical practice.publishersversionPeer reviewe

Improvisation and Transformation: Yes to the Mess

The field of organizational change has chiefly been studied from a teleological perspective. Most models of change emphasize action that is rational and goal oriented. What often gets overlooked and under theorized is the continuous, iterative nature of organizational life, the unplanned and serendipitous actions by and between people that lead to new discoveries and innovation. Recent research on organizational improvisation seeks to explore this area. In this chapter we will address two questions – what is the experience of improvisation and what are the conditions that support improvisation to flourish in organizations? In the first part of this paper, we look at the phenomenology of improvisation, the actual lived experience of those who improvise in the face of the unknown or in the midst of chaotic conditions. We will explore the strategies that some professional improvisers employ to deliberately create the improvisatory moment. We will then look at the dynamics of organizational life and explore the cultural beliefs, organizational structures, and leadership practices that support improvisation. We will draw primarily upon the model from Barrett (2012) that focuses on the how the nature of jazz improvisation and the factors that support improvisation can be transferred to leadership activities. This falls in the tradition of others who draw upon arts-based metaphors, including jazz music and theatrical improvisation, to suggest insights for leadership and ways of organizing. Since this is a book devoted to individual transformation as well as organizational transformation, we will also touch on the topic of how improvisation is a developmental project and explore the potential for improvisation to lead to personal transformation. We will attempt to move back and forth between both themes – organizational and personal transformation. Ultimately the two topics are not separate. Any significant organizational transformation begins with an improvisation. And any meaningful improvisatory move by a person is potentially a moment of self-discovery and an identity-shaping event

The status of the world's land and marine mammals: diversity, threat, and knowledge

Knowledge of mammalian diversity is still surprisingly disparate, both regionally and taxonomically. Here, we present a comprehensive assessment of the conservation status and distribution of the world's mammals. Data, compiled by 1700+ experts, cover all 5487 species, including marine mammals. Global macroecological patterns are very different for land and marine species but suggest common mechanisms driving diversity and endemism across systems. Compared with land species, threat levels are higher among marine mammals, driven by different processes (accidental mortality and pollution, rather than habitat loss), and are spatially distinct (peaking in northern oceans, rather than in Southeast Asia). Marine mammals are also disproportionately poorly known. These data are made freely available to support further scientific developments and conservation action