Catalytic Water Co-Existing with a Product Peptide in the Active Site of HIV-1 Protease Revealed by X-Ray Structure Analysis

BACKGROUND: It is known that HIV-1 protease is an important target for design of antiviral compounds in the treatment of Acquired Immuno Deficiency Syndrome (AIDS). In this context, understanding the catalytic mechanism of the enzyme is of crucial importance as transition state structure directs inhibitor design. Most mechanistic proposals invoke nucleophilic attack on the scissile peptide bond by a water molecule. But such a water molecule coexisting with any ligand in the active site has not been found so far in the crystal structures. PRINCIPAL FINDINGS: We report here the first observation of the coexistence in the active site, of a water molecule WAT1, along with the carboxyl terminal product (Q product) peptide. The product peptide has been generated in situ through cleavage of the full-length substrate. The N-terminal product (P product) has diffused out and is replaced by a set of water molecules while the Q product is still held in the active site through hydrogen bonds. The position of WAT1, which hydrogen bonds to both the catalytic aspartates, is different from when there is no substrate bound in the active site. We propose WAT1 to be the position from where catalytic water attacks the scissile peptide bond. Comparison of structures of HIV-1 protease complexed with the same oligopeptide substrate, but at pH 2.0 and at pH 7.0 shows interesting changes in the conformation and hydrogen bonding interactions from the catalytic aspartates. CONCLUSIONS/SIGNIFICANCE: The structure is suggestive of the repositioning, during substrate binding, of the catalytic water for activation and subsequent nucleophilic attack. The structure could be a snap shot of the enzyme active site primed for the next round of catalysis. This structure further suggests that to achieve the goal of designing inhibitors mimicking the transition-state, the hydrogen-bonding pattern between WAT1 and the enzyme should be replicated

A cluster randomised controlled trial of the clinical and cost-effectiveness of a 'whole systems' model of self-management support for the management of long- term conditions in primary care: trial protocol

BackgroundPatients with long-term conditions are increasingly the focus of quality improvement activities in health services to reduce the impact of these conditions on quality of life and to reduce the burden on care utilisation. There is significant interest in the potential for self-management support to improve health and reduce utilisation in these patient populations, but little consensus concerning the optimal model that would best provide such support. We describe the implementation and evaluation of self-management support through an evidence-based 'whole systems' model involving patient support, training for primary care teams, and service re-organisation, all integrated into routine delivery within primary care.MethodsThe evaluation involves a large-scale, multi-site study of the implementation, effectiveness, and cost-effectiveness of this model of self-management support using a cluster randomised controlled trial in patients with three long-term conditions of diabetes, chronic obstructive pulmonary disease (COPD), and irritable bowel syndrome (IBS). The outcome measures include healthcare utilisation and quality of life. We describe the methods of the cluster randomised trial.DiscussionIf the 'whole systems' model proves effective and cost-effective, it will provide decision-makers with a model for the delivery of self-management support for populations with long-term conditions that can be implemented widely to maximise 'reach' across the wider patient population.Trial registration numberISRCTN: ISRCTN9094004

Reframing professional development through understanding authentic professional learning

Continuing to learn is universally accepted and expected by professionals and other stakeholders across all professions. However, despite changes in response to research findings about how professionals learn, many professional development practices still focus on delivering content rather than enhancing learning. In exploring reasons for the continuation of didactic practices in professional development, this article critiques the usual conceptualization of professional development through a review of recent literature across professions. An alternative conceptualization is proposed, based on philosophical assumptions congruent with evidence about professional learning from seminal educational research of the past two decades. An argument is presented for a shift in discourse and focus from delivering and evaluating professional development programs to understanding and supporting authentic professional learning

Is exposure to formaldehyde in air causally associated with leukemia?—A hypothesis-based weight-of-evidence analysis

Recent scientific debate has focused on the potential for inhaled formaldehyde to cause lymphohematopoietic cancers, particularly leukemias, in humans. The concern stems from certain epidemiology studies reporting an association, although particulars of endpoints and dosimetry are inconsistent across studies and several other studies show no such effects. Animal studies generally report neither hematotoxicity nor leukemia associated with formaldehyde inhalation, and hematotoxicity studies in humans are inconsistent. Formaldehyde's reactivity has been thought to preclude systemic exposure following inhalation, and its apparent inability to reach and affect the target tissues attacked by known leukemogens has, heretofore, led to skepticism regarding its potential to cause human lymphohematopoietic cancers. Recently, however, potential modes of action for formaldehyde leukemogenesis have been hypothesized, and it has been suggested that formaldehyde be identified as a known human leukemogen. In this article, we apply our hypothesis-based weight-of-evidence (HBWoE) approach to evaluate the large body of evidence regarding formaldehyde and leukemogenesis, attending to how human, animal, and mode-of-action results inform one another. We trace the logic of inference within and across all studies, and articulate how one could account for the suite of available observations under the various proposed hypotheses. Upon comparison of alternative proposals regarding what causal processes may have led to the array of observations as we see them, we conclude that the case fora causal association is weak and strains biological plausibility. Instead, apparent association between formaldehyde inhalation and leukemia in some human studies is better interpreted as due to chance or confounding

Effects of Once-Weekly Exenatide on Cardiovascular Outcomes in Type 2 Diabetes.

Abstract BACKGROUND: The cardiovascular effects of adding once-weekly treatment with exenatide to usual care in patients with type 2 diabetes are unknown. METHODS: We randomly assigned patients with type 2 diabetes, with or without previous cardiovascular disease, to receive subcutaneous injections of extended-release exenatide at a dose of 2 mg or matching placebo once weekly. The primary composite outcome was the first occurrence of death from cardiovascular causes, nonfatal myocardial infarction, or nonfatal stroke. The coprimary hypotheses were that exenatide, administered once weekly, would be noninferior to placebo with respect to safety and superior to placebo with respect to efficacy. RESULTS: In all, 14,752 patients (of whom 10,782 [73.1%] had previous cardiovascular disease) were followed for a median of 3.2 years (interquartile range, 2.2 to 4.4). A primary composite outcome event occurred in 839 of 7356 patients (11.4%; 3.7 events per 100 person-years) in the exenatide group and in 905 of 7396 patients (12.2%; 4.0 events per 100 person-years) in the placebo group (hazard ratio, 0.91; 95% confidence interval [CI], 0.83 to 1.00), with the intention-to-treat analysis indicating that exenatide, administered once weekly, was noninferior to placebo with respect to safety (P<0.001 for noninferiority) but was not superior to placebo with respect to efficacy (P=0.06 for superiority). The rates of death from cardiovascular causes, fatal or nonfatal myocardial infarction, fatal or nonfatal stroke, hospitalization for heart failure, and hospitalization for acute coronary syndrome, and the incidence of acute pancreatitis, pancreatic cancer, medullary thyroid carcinoma, and serious adverse events did not differ significantly between the two groups. CONCLUSIONS: Among patients with type 2 diabetes with or without previous cardiovascular disease, the incidence of major adverse cardiovascular events did not differ significantly between patients who received exenatide and those who received placebo. (Funded by Amylin Pharmaceuticals; EXSCEL ClinicalTrials.gov number, NCT01144338 .)

0031 INHERITED VARIATION IN CIRCADIAN RHYTHM GENES AND RISKS OF PROSTATE CANCER AND THREE OTHER CANCER SITES IN COMBINED CANCER CONSORTIA

Introduction: Circadian disruption has been linked to carcinogenesis in animal models and to breast cancer in humans. However, the evidence for other types of cancer is inconclusive. Variations in genes involved in circadian rhythm provide a tool to investigate such associations. Methods: We examined associations of genetic variation in nine core circadian rhythm genes and six melatonin pathway genes with risk of colorectal, lung, ovarian and prostate cancers using genome-wide association studies (GWAS) from GAME-ON. The major results for prostate cancer were replicated in PLCO, and for colorectal cancer in GECCO. The total number of cancer cases and controls was 15,838/18,159 for colorectal, 14,818/14,227 for prostate, 12,537/17,285 for lung and 4,369/9,123 for ovary. For each cancer site, we used the SNP summary statistics for the candidate gene regions imputed to 1000 Genomes. Gene-level and pathway-level analyses were conducted using the summary-based Adaptive Rank Truncated Product method (sARTP). Results: Aggregate genetic variation in circadian rhythm and melatonin pathways were significantly associated with the risk of prostate cancer in data combining GAME-ON and PLCO, after Bonferroni correction (Ppathway<0.00625). The two most significant genes were NPAS2 (Pgene=0.0062) and AANAT (Pgene=0.00078); the latter being significant after Bonferroni correction. For colorectal cancer, we observed a suggestive association with the circadian rhythm pathway in GAME-ON (Ppathway=0.021), that was not confirmed in GECCO (Ppathway=0.76) or the combined data (Ppathway=0.17). No other association was observed for the other cancer sites. Conclusion: These findings support a potential role for circadian rhythm and melatonin pathways in prostate carcinogenesis. Further functional studies are needed to better understand the underlying biologic mechanisms