Promotion, Education, and Marketing of an Expanded VCU Bike Share Program

We propose to help promote, market, and provide education about an expanded bike share program at VCU. The goal of the bike share program is three-fold: 1) improve travel between campuses, 2) encourage alternate transportation to reduce traffic and parking difficulties and 3) be a green initiative on the VCU campus. The expanded bike share program will include additional bikes and bike stations, managed by an outside company. Recently, VCU’s Office of Parking and Transportation has learned that they will be receiving funds for the program, and they are looking for assistance to promote, market, and provide education about the program. We will also explore additional aspects of a bike sharing program such as encouraging the use of helmets/safety issues, using technology to track bikes, and conducting a needs assessment to determine consumer demand and preferences

Judicial Review, Irrationality, and the Limits of Intervention by the Courts

When exercising judicial review, the courts, on occasions, have intervened in circumstances where administrative decisions were not irrational. However, these low standards of judicial intervention are arguably constitutional, especially since the enactment of the Human Rights Act 1998 (HRA). To this end, this article seeks to establish a zone of executive decision-making, for reasons of democracy, where the courts are clearly excluded. But it is unable to do so. Does this mean, therefore, that judicial intervention on the grounds of irrationality exists without limit? Assuming this to be the case, it is suggested that the courts should show greater respect to the administrative branch of the state where it has genuinely sought to engage with the legal process in arriving at its decisions

Global patient outcomes after elective surgery: prospective cohort study in 27 low-, middle- and high-income countries.

BACKGROUND: As global initiatives increase patient access to surgical treatments, there remains a need to understand the adverse effects of surgery and define appropriate levels of perioperative care. METHODS: We designed a prospective international 7-day cohort study of outcomes following elective adult inpatient surgery in 27 countries. The primary outcome was in-hospital complications. Secondary outcomes were death following a complication (failure to rescue) and death in hospital. Process measures were admission to critical care immediately after surgery or to treat a complication and duration of hospital stay. A single definition of critical care was used for all countries. RESULTS: A total of 474 hospitals in 19 high-, 7 middle- and 1 low-income country were included in the primary analysis. Data included 44 814 patients with a median hospital stay of 4 (range 2-7) days. A total of 7508 patients (16.8%) developed one or more postoperative complication and 207 died (0.5%). The overall mortality among patients who developed complications was 2.8%. Mortality following complications ranged from 2.4% for pulmonary embolism to 43.9% for cardiac arrest. A total of 4360 (9.7%) patients were admitted to a critical care unit as routine immediately after surgery, of whom 2198 (50.4%) developed a complication, with 105 (2.4%) deaths. A total of 1233 patients (16.4%) were admitted to a critical care unit to treat complications, with 119 (9.7%) deaths. Despite lower baseline risk, outcomes were similar in low- and middle-income compared with high-income countries. CONCLUSIONS: Poor patient outcomes are common after inpatient surgery. Global initiatives to increase access to surgical treatments should also address the need for safe perioperative care. STUDY REGISTRATION: ISRCTN5181700

Omecamtiv mecarbil in chronic heart failure with reduced ejection fraction, GALACTIC‐HF: baseline characteristics and comparison with contemporary clinical trials

Aims: The safety and efficacy of the novel selective cardiac myosin activator, omecamtiv mecarbil, in patients with heart failure with reduced ejection fraction (HFrEF) is tested in the Global Approach to Lowering Adverse Cardiac outcomes Through Improving Contractility in Heart Failure (GALACTIC‐HF) trial. Here we describe the baseline characteristics of participants in GALACTIC‐HF and how these compare with other contemporary trials. Methods and Results: Adults with established HFrEF, New York Heart Association functional class (NYHA) ≥ II, EF ≤35%, elevated natriuretic peptides and either current hospitalization for HF or history of hospitalization/ emergency department visit for HF within a year were randomized to either placebo or omecamtiv mecarbil (pharmacokinetic‐guided dosing: 25, 37.5 or 50 mg bid). 8256 patients [male (79%), non‐white (22%), mean age 65 years] were enrolled with a mean EF 27%, ischemic etiology in 54%, NYHA II 53% and III/IV 47%, and median NT‐proBNP 1971 pg/mL. HF therapies at baseline were among the most effectively employed in contemporary HF trials. GALACTIC‐HF randomized patients representative of recent HF registries and trials with substantial numbers of patients also having characteristics understudied in previous trials including more from North America (n = 1386), enrolled as inpatients (n = 2084), systolic blood pressure < 100 mmHg (n = 1127), estimated glomerular filtration rate < 30 mL/min/1.73 m2 (n = 528), and treated with sacubitril‐valsartan at baseline (n = 1594). Conclusions: GALACTIC‐HF enrolled a well‐treated, high‐risk population from both inpatient and outpatient settings, which will provide a definitive evaluation of the efficacy and safety of this novel therapy, as well as informing its potential future implementation

Integrative "Omics"-Approach Discovers Dynamic and Regulatory Features of Bacterial Stress Responses

Bacteria constantly face stress conditions and therefore mount specific responses to ensure adaptation and survival. Stress responses were believed to be predominantly regulated at the transcriptional level. In the phototrophic bacterium Rhodobacter sphaeroides the response to singlet oxygen is initiated by alternative sigma factors. Further adaptive mechanisms include post-transcriptional and post-translational events, which have to be considered to gain a deeper understanding of how sophisticated regulation networks operate. To address this issue, we integrated three layers of regulation: (1) total mRNA levels at different time-points revealed dynamics of the transcriptome, (2) mRNAs in polysome fractions reported on translational regulation (translatome), and (3) SILAC-based mass spectrometry was used to quantify protein abundances (proteome). The singlet oxygen stress response exhibited highly dynamic features regarding short-term effects and late adaptation, which could in part be assigned to the sigma factors RpoE and RpoH2 generating distinct expression kinetics of corresponding regulons. The occurrence of polar expression patterns of genes within stress-inducible operons pointed to an alternative of dynamic fine-tuning upon stress. In addition to transcriptional activation, we observed significant induction of genes at the post-transcriptional level (translatome), which identified new putative regulators and assigned genes of quorum sensing to the singlet oxygen stress response. Intriguingly, the SILAC approach explored the stress-dependent decline of photosynthetic proteins, but also identified 19 new open reading frames, which were partly validated by RNA-seq. We propose that comparative approaches as presented here will help to create multi-layered expression maps on the system level ("expressome"). Finally, intense mass spectrometry combined with RNA-seq might be the future tool of choice to re-annotate genomes in various organisms and will help to understand how they adapt to alternating conditions

Directed evolution provides insight into conformational substrate sampling by SrtA

<div><p>The Sortase family of transpeptidases are found in numerous gram-positive bacteria and involved in divergent physiological processes including anchoring of surface proteins to the cell wall as well as pili assembly. As essential proteins, sortase enzymes have been the focus of considerable interest for the development of novel anti-microbials, however, more recently their function as unique transpeptidases has been exploited for the synthesis of novel bio-conjugates. Yet, for synthetic purposes, SrtA-mediated conjugation suffers from the enzyme’s inherently poor catalytic efficiency. Therefore, to identify SrtA variants with improved catalytic efficiency, we used directed evolution to select a catalytically enhanced SrtA enzyme. An analysis of improved SrtA variants in the context of sequence conservation, NMR and x-ray crystal structures, and kinetic data suggests a novel mechanism for catalysis involving large conformational changes that delivers substrate to the active site pocket. Indeed, using DEER-EPR spectroscopy, we reveal that upon substrate binding, SrtA undergoes a large scissors-like conformational change that simultaneously translates the sort-tag substrate to the active site in addition to repositioning key catalytic residues for esterification. A better understanding of Sortase dynamics will significantly enhance future engineering and drug discovery efforts.</p></div

Directed evolution provides insight into conformational substrate sampling by SrtA - Fig 3

<p>A) Schematic of the continuous fluorescence assay used to measure the initial acylation kinetics as previously described. B) Schematic of the fluorescence polarization assay used to follow the complete reaction coordinate. The peptide substrate is labeled at the amino terminus with the fluorescent label tetramethylrhodomain (TMR), which is efficiently excited at 557nm and emits at 576nm. In the absence of SrtA or secondary substrate (biotin labeled GGGGGDYK peptide), the fluorescence polarization is low. Alternatively, following the reaction of substrates in the presence of SrtA, avidin binding greatly increases the increases fluorescence polarization. C) Kinetic parameters for wild-type (WT) or SrtA variants.</p

Schematic representation of the protein complementation assay utilized for directed evolution of SrtA.

<p>A) Murine DHFR is cloned into pET-Duet as two independent fragments consisting of the carboxyl-terminal region fused to three amino-terminal glycines in one open reading frame and amino terminus fused to an LPETG sort-tag in a second open reading frame. Endogenous methionine aminopeptidase (MAP) cleaves the initiating methionine to expose the terminal glycines. B) SrtA enzymatically ligates the two fragments to generate an active DHFR. Endogenous bacterial DHFR is inhibited by the prokaryotic specific trimethoprim. C) Initial culture conditions testing the requirement for SrtA in the DHFR complementation assay. D) Overnight growth of bacteria containing the various assay components in the presence and absence of trimethoprim was monitored using optical density at 600nm, OD₆₀₀. Each trial was completed in duplicate. Cells carrying either the pET vector expressing only the split DHFR, or the pRSF vector driving expression of SrtA fail to grow in the presence of trimethoprim. Alternatively, bacteria expressing a positive control mDHFR (mDHFR-(PC)) with the internal LPETGG sequence, grow robustly in the absence of SrtA, but in the presence of trimethoprim. Similarly, bacteria expressing the split mDHFR gene along with SrtA also grow robustly following an overnight growth in trimethoprim. E) Growth of bacteria on LB-Agar plates containing Ampicillin, Kanamycin, IPTG, and trimethoprim. I) and III) BL21(DE3) cells containing pET-Duet C/N-mDHFR with empty pRSF vector, or II) and IV) BL21(DE3) cells containing pET-Duet C/N-mDHFR with pRSF-SrtA.</p