Death with dignity in Washington patients with amyotrophic lateral sclerosis.

OBJECTIVES: To describe the amyotrophic lateral sclerosis (ALS) patients who sought medication under the Washington State Death with Dignity (DWD) Act since its inception in 2009. METHODS: Chart review at 3 tertiary medical centers in the Seattle/Puget Sound region and comparison to publicly available data of ALS and all-cause DWD cohorts from Washington and Oregon. RESULTS: In Washington State, 39 patients with ALS requested DWD from the University of Washington, Virginia Mason, and Swedish Medical Centers beginning in 2009. The median age at death was 65 years (range 46-86). Seventy-seven percent of the patients used the prescriptions. All of the patients who used the medications passed away without complications. The major reasons for patients to request DWD as reported by participating physicians were loss of autonomy and dignity and decrease in enjoyable activities. Inadequate pain control, financial cost, and loss of bodily control were less commonly indicated. These findings were similar to those of the 92 patients who sought DWD in Oregon. In Washington and Oregon, the percentage of patients with ALS seeking DWD is higher compared to the cancer DWD cohort. Furthermore, compared to the all-cause DWD cohort, patients with ALS are more likely to be non-Hispanic white, married, educated, enrolled in hospice, and to have died at home. CONCLUSIONS: Although a small number, ALS represents the disease with the highest proportion of patients seeking to participate in DWD. Patients with ALS who choose DWD are well-educated and have access to palliative or life-prolonging care. The use of the medications appears to be able to achieve the patients' goals without complications.

Kinetically and Crystallographically Guided Mutations of a Benzoate CoA Ligase (BadA) Elucidate Mechanism and Expand Substrate Permissivity.

A benzoate CoA ligase (BadA), isolated from the bacterium Rhodopseudomonas palustris, catalyzes the conversion of benzoate to benzoyl CoA on the catabolic pathway of aromatic carboxylic acids. Herein, apparent Michaelis constants K(app)cat and K(app)M were determined for an expanded array of 31 substrates chosen to systematically probe the active site architecture of the enzyme and provide a baseline for expansion of wild-type substrate specificity. Acyl CoA products were observed for 25 of the 31 substrates; in general, BadA converted ortho-substituted substrates better than the corresponding meta and para regioisomers, and the turnover number was more affected by steric rather than electronic effects. The kinetic data are interpreted in relation to six crystal structures of BadA in complex with several substrates and a benzoyl-AMP reaction intermediate. In contrast to other known natural substrate-bound benzoate ligase structures, all substrate-bound BadA structures adopted the thiolation conformation instead of the adenylation conformation. We also observed all the aryl carboxylates to be uniquely oriented within the active site, relative to other structures. Together, the kinetics and structural data suggested a mechanism that involves substrate binding in the thiolation conformation, followed by substrate rotation to an active orientation upon the transition to the adenylation conformation. On the basis of this hypothesis and the structural data, sterically demanding active site residues were mutated, and the substrate specificity was expanded substantially versus that of BadA. Novel activities were seen for substrates with larger substituents, including phenyl acetate. Additionally, the mutant Lys427Ala identified this nonconserved residue as essential for the thiolation step of BadA, but not adenylation. These variously acylated CoAs can serve as novel substrates of acyl CoA-dependent acyltransferases in coupled enzyme assays to produce analogues of bioactive natural products.

Proteome and computational analyses reveal new insights into the mechanisms of hepatitis C virus-mediated liver disease posttransplantation.

UNLABELLED: Liver transplant tissues offer the unique opportunity to model the longitudinal protein abundance changes occurring during hepatitis C virus (HCV)-associated liver disease progression in vivo. In this study, our goal was to identify molecular signatures, and potential key regulatory proteins, representative of the processes influencing early progression to fibrosis. We performed global protein profiling analyses on 24 liver biopsy specimens obtained from 15 HCV(+) liver transplant recipients at 6 and/or 12 months posttransplantation. Differentially regulated proteins associated with early progression to fibrosis were identified by analysis of the area under the receiver operating characteristic curve. Analysis of serum metabolites was performed on samples obtained from an independent cohort of 60 HCV(+) liver transplant patients. Computational modeling approaches were applied to identify potential key regulatory proteins of liver fibrogenesis. Among 4,324 proteins identified, 250 exhibited significant differential regulation in patients with rapidly progressive fibrosis. Patients with rapid fibrosis progression exhibited enrichment in differentially regulated proteins associated with various immune, hepatoprotective, and fibrogenic processes. The observed increase in proinflammatory activity and impairment in antioxidant defenses suggests that patients who develop significant liver injury experience elevated oxidative stresses. This was supported by an independent study demonstrating the altered abundance of oxidative stress-associated serum metabolites in patients who develop severe liver injury. Computational modeling approaches further highlight a potentially important link between HCV-associated oxidative stress and epigenetic regulatory mechanisms impacting on liver fibrogenesis. CONCLUSION: Our proteome and metabolome analyses provide new insights into the role for increased oxidative stress in the rapid fibrosis progression observed in HCV(+) liver transplant recipients. These findings may prove useful in prognostic applications for predicting early progression to fibrosis.