Evolutionary History of a Specialized P450 Propane Monooxygenase

The evolutionary pressures that shaped the specificity and catalytic efficiency of enzymes can only be speculated. While directed evolution experiments show that new functions can be acquired under positive selection with few mutations, the role of negative selection in eliminating undesired activities and achieving high specificity remains unclear. Here we examine intermediates along the ‘lineage’ from a naturally occurring C12–C20 fatty acid hydroxylase (P450BM3) to a laboratory-evolved P450 propane monooxygenase (P450PMO) having 20 heme domain substitutions compared to P450BM3. Biochemical, crystallographic, and computational analyses show that a minimal perturbation of the P450BM3 fold and substrate-binding pocket accompanies a significant broadening of enzyme substrate range and the emergence of propane activity. In contrast, refinement of the enzyme catalytic efficiency for propane oxidation (not, vert, similar 9000-fold increase in kcat/Km) involves profound reshaping and partitioning of the substrate access pathway. Remodeling of the substrate-recognition mechanisms ultimately results in remarkable narrowing of the substrate profile around propane and enables the acquisition of a basal iodomethane dehalogenase activity as yet unknown in natural alkane monooxygenases. A highly destabilizing L188P substitution in a region of the enzyme that undergoes a large conformational change during catalysis plays an important role in adaptation to the gaseous alkane. This work demonstrates that positive selection alone is sufficient to completely respecialize the cytochrome P450 for function on a nonnative substrate

Engineered ascorbate peroxidase as a genetically-encoded reporter for electron microscopy

Electron microscopy (EM) is the standard method for imaging cellular structures with nanometer resolution, but existing genetic tags are inactive in most cellular compartments1 or require light and are difficult to use2. Here we report the development of a simple and robust EM genetic tag, called “APEX,” that is active in all cellular compartments and does not require light. APEX is a monomeric 28 kDa peroxidase that withstands strong EM fixation to give excellent ultrastructural preservation. We demonstrate the utility of APEX for high-resolution EM imaging of a variety of mammalian organelles and specific proteins. We also fused APEX to the N- or C-terminus of the mitochondrial calcium uniporter (MCU), a newly identified channel whose topology is disputed3,4. MCU-APEX and APEX-MCU give EM contrast exclusively in the mitochondrial matrix, suggesting that both the N-and C-termini of MCU face the matrix

Internal Executive Coaching Program: Exploring the Experiences, Benefits, and Recommendations for Participants and Organizations

Organizations are searching for ways to enhance leadership skills to meet strategic goals. Executive coaching is one method used by organizations to improve communication, job satisfaction, staff retention and overall productivity. While external coaching programs have been used for years, internal coaching programs are a more recent development and little research has been conducted to examine their effectiveness. The purpose of this study is to examine the experience of managers who participated in an internal executive coaching program in order to explore the benefits and recommendations for participants and organizations. A case study of one organization\u27s Internal Executive Coaching Program was used and data were collected from participants through interviews and surveys. Results of the study describe the program\u27s effectiveness in improving leadership skills and employee satisfaction

Interrogating marine virus-host interactions and elemental transfer with BONCAT and nanoSIMS-based methods

While the collective impact of marine viruses has become more apparent over the last decade, a deeper understanding of virus-host dynamics and the role of viruses in nutrient cycling would benefit from direct observations at the single-virus level. We describe two new complementary approaches - stable isotope probing coupled with nanoscale secondary ion mass spectrometry (nanoSIMS) and fluorescence-based biorthogonal non-canonical amino acid tagging (BONCAT) - for studying the activity and biogeochemical influence of marine viruses. These tools were developed and tested using several ecologically relevant model systems (Emiliania huxleyi/EhV207, Synechococcus sp. WH8101/Syn1, and Escherichia coli/T7). By resolving carbon and nitrogen enrichment in viral particles, we demonstrate the power of nanoSIMS tracer experiments in obtaining quantitative estimates for the total number of viruses produced directly from a particular production pathway (by isotopically labeling host substrates). Additionally, we show through laboratory experiments and a pilot field study that BONCAT can be used to directly quantify viral production (via epifluorescence microscopy) with minor sample manipulation and no dependency on conversion factors. This technique can also be used to detect newly synthesized viral proteins. Together these tools will help fill critical gaps in our understanding of the biogeochemical impact of viruses in the ocean

Finding Our Fathers

We never considered how hard it was for our fathers to go to work every day, toiling away at a job they didn’t like. And even worse, seemingly having no other choices because their families were depending on them for their very existence. Growing up, our fathers did not have time for family; spending time with us was something remote from their daily life, making them a stranger in each of their perspective homes. Worse still, We were too young to understand why our fathers were not present. This essay reveals how we have attempted to make sense of the experience this distance between us and our fathers has caused in order to detoxify our individual images of our fathers. Detoxifying the image of father means recognizing that our fathers’ have been wounded as well.</p

Revisiting the Local Scaling Hypothesis in Stably Stratified Atmospheric Boundary Layer Turbulence: an Integration of Field and Laboratory Measurements with Large-eddy Simulations

The `local scaling' hypothesis, first introduced by Nieuwstadt two decades ago, describes the turbulence structure of stable boundary layers in a very succinct way and is an integral part of numerous local closure-based numerical weather prediction models. However, the validity of this hypothesis under very stable conditions is a subject of on-going debate. In this work, we attempt to address this controversial issue by performing extensive analyses of turbulence data from several field campaigns, wind-tunnel experiments and large-eddy simulations. Wide range of stabilities, diverse field conditions and a comprehensive set of turbulence statistics make this study distinct

Arts on prescription for community‐dwelling older people with a range of health and wellness needs

Published evidence for the role of participatory art in supporting health and well‐being is growing. The Arts on Prescription model is one vehicle by which participatory art can be delivered. Much of the focus of Arts on Prescription has been on the provision of creative activities for people with mental health needs. This Arts on Prescription program, however, targeted community‐dwelling older people with a wide range of health and wellness needs. Older people were referred to the program by their healthcare practitioner. Professional artists led courses in visual arts, photography, dance and movement, drama, singing, or music. Classes were held weekly for 8–10 weeks, with six to eight participants per class, and culminated with a showing of work or a performance. Program evaluation involved pre‐ and postcourse questionnaires, and focus groups and individual interviews. Evaluation data on 127 participants aged 65 years and older were available for analysis. We found that Arts on Prescription had a positive impact on participants. Quantitative findings revealed a statistically significant improvement in the Warwick–Edinburgh Mental Well‐being Scale(WEMWBS) as well as a statistically significant increase in the level of self‐reported creativity and frequency of creative activities. Qualitative findings indicated that the program provided challenging artistic activities which created a sense of purpose and direction, enabled personal growth and achievement, and empowered participants, in a setting which fostered the development of meaningful relationships with others. This evaluation adds to the evidence base in support of Arts on Prescription by expanding the application of the model to older people with a diverse range of health and wellness needs