Structure and backbone dynamics of a microcrystalline metalloprotein by solid-state NMR

We introduce a new approach to improve structural and dynamical determination of large metalloproteins using solid-state nuclear magnetic resonance (NMR) with (1)H detection under ultrafast magic angle spinning (MAS). The approach is based on the rapid and sensitive acquisition of an extensive set of (15)N and (13)C nuclear relaxation rates. The system on which we demonstrate these methods is the enzyme Cu, Zn superoxide dismutase (SOD), which coordinates a Cu ion available either in Cu(+) (diamagnetic) or Cu(2+) (paramagnetic) form. Paramagnetic relaxation enhancements are obtained from the difference in rates measured in the two forms and are employed as structural constraints for the determination of the protein structure. When added to (1)H-(1)H distance restraints, they are shown to yield a twofold improvement of the precision of the structure. Site-specific order parameters and timescales of motion are obtained by a gaussian axial fluctuation (GAF) analysis of the relaxation rates of the diamagnetic molecule, and interpreted in relation to backbone structure and metal binding. Timescales for motion are found to be in the range of the overall correlation time in solution, where internal motions characterized here would not be observable.     

Morphologic evidence of diffuse vascular damage in human and in the experimental model of ethylmalonic encephalopathy

Ethylmalonic encephalopathy (EE) is a rare autosomal recessive disorder characterized by early onset encephalopathy, chronic diarrhoea, petechiae, orthostatic acrocyanosis and defective cytochrome c oxidase (COX) in muscle and brain. High levels of lactic, ethylmalonic and methylsuccinic acids are detected in body fluids. EE is caused by mutations in ETHE1, a mitochondrial sulphur dioxygenase. By studying a suitable mouse model, we found that loss of ETHE1 leads to accumulation of sulphide, which is a poison for COX and other enzymatic activities thus accounting for the main features of EE. We report here the first autopsy case of a child with a genetically confirmed diagnosis of EE, and compare the histological, histochemical and immunohistochemical findings with those of the constitutive Ethe1 (-/-) mice. In addition to COX depleted cells, widespread endothelial lesions of arterioles and capillaries of the brain and gastrointestinal tract were the pathologic hallmarks in both organisms. Our findings of diffuse vascular damage of target critical organs are in keeping with the hypothesis that the pathologic effects of ETHE1 deficiency may stem from high levels of circulating hydrogen sulphide rather than the inability of specific organs to detoxify its endogenous production.     

Allogeneic stem cell transplantation for patients with chronic myeloid leukemia: Risk stratified approach with a long-term follow-up

The use of allogeneic stem cell transplantation (SCT) for chronic myeloid leukemia (CML) was almost abandoned in recent years for very effective targeted therapy with tyrosine kinase inhibitors (TKIs). However, approximately one third of patients still need another treatment including SCT. 38 consecutive CML patients were treated (most in preimatinib era) with allogeneic SCT, using partial T cell depletion (TCD) and preemptive donor lymphocyte infusion (DLI), without post‐transplant graft‐versus‐host disease (GvHD) prophylaxis. Conditioning included busulfan, cyclophosphamide, antithymocytic globulin, and fludarabine followed by donor stem cell transfusion. With a median follow up of 90.5 months (1–134), 32 patients are alive. 97% engrafted. 5‐year leukemia free survival (LFS) and overall survival (OS) were 78.95% and 84.2%, respectively. All patients are in major molecular remission and 78% in complete molecular remission. Transplant‐related mortality (TRM) was 13%. Twenty‐four patients received DLI for residual disease. Acute GvHD, mostly Grades I‐II, occurred in 18% of patients post‐transplant and in 24% of patients receiving DLI. In conclusion, the risk‐adapted approach using only partial TCD and preemptive escalated dose of DLI precluded the need for immunosuppressive medications and reduced the risk of significant GvHD without compromising engraftment and long‐term disease control. Am. J. Hematol. 2012. © 2012 Wiley Periodicals, Inc.     

Salivary secretion effects of the antipsychotic drug olanzapine in an animal model

Oral Diseases (2012) Objective: Olanzapine, introduced as an alternative to clozapine in schizophrenia therapy, is thought to display a receptor affinity similar to that of clozapine. Antipsychotics are well‐known xerogenic drugs. However, clozapine exerts both antagonistic and agonistic salivary effects (‘clozapine‐induced sialorrhea’), the latter probably via muscarinic M1 type of receptor. We hypothesise that olanzapine also has dual salivary effects. Material and methods: Effects of intravenous olanzapine were examined in rats, including those subjected to chronic preganglionic parasympathetic denervation (submandibular glands) or combined postganglionic parasympathetic and sympathetic denervation (parotid glands). Secretion was evoked reflexly, and by intravenous methacholine and the tachykinin substance P. Results: At 0.01–1 mg kg^?1, olanzapine dose dependently reduced secretion in response to methacholine or reflex stimulus but not that to substance P. At 10 mg kg^?1, olanzapine evoked a long‐lasting secretion, independent of the autonomic innervation as well as of α‐ and β‐adrenergic receptors and muscarinic receptors. The secretion was reduced, but not abolished, by a substance P receptor antagonist. Conclusions: Like clozapine, olanzapine evoked secretion. The response to olanzapine was greater and, in contrast to clozapine, involved non‐traditional gland receptors (such as substance P receptors). The findings imply that olanzapine plays an excitatory role via tachykinin receptors in humans.     

Expression of interleukin-15 and interleukin-15Rα in monocytes of HIV type 1-infected patients with different courses of disease progression

Interleukin-15 (IL-15) enhances the effector mechanisms of anti-HIV immune responses and thus is considered a potential adjuvant of HIV-1 vaccine. However, there are a lack of data concerning the relationships between IL-15 expression and regulation in HIV-1-infected patients and the course of disease progression. We found that IL-15, but not IL-15Rα, is expressed at significantly higher levels in the CD14(+) monocytes [stimulated or not with interferon (IFN)-γ] of long-term nonprogressors (LTNP) than in those of HIV-1 progressors or healthy controls. There was no between-group difference in the amounts of soluble IL-15 released from the cells. We also found that like the healthy controls, the LTNP expressed the IL-15 and IL-15Rα genes in a more coordinated manner than the progressors. Our findings show that there are significant differences in IL-15 expression between patients with different courses of HIV infection, and that the coordinated expression of the IL-15 and IL-15Rα genes is dysregulated in patients with progressive disease. They also provide important information concerning the mechanisms of infection and the potential use of IL-15 as a therapeutic agent.     

When oral anticoagulation therapy is needed in patients with cardiomyopathies: a review of literature

The question whether to anticoagulate patients with cardiomyopathy or not is over 50 years old. Multiple clinical trials have demonstrated the superior therapeutic effect of warfarin compared with placebo in the prevention of thromboembolic events amongst patients with nonvalvular atrial fibrillation. The purpose of our work is to review literature about the role of anticoagulation in the main cardiomyopathies.     

Errors and near misses in digestive endoscopy units

Background

Not much is known about errors and near misses in digestive endoscopy.

Aims

To verify whether an incident report, with certain facilitating features, gives useful information about unintended events, only excluding errors in medical diagnosis.

Method

Nine endoscopy units took part in this cross sectional, prospective, multicentre study which lasted for two weeks. Members of the staff were required to report any unintended, potentially dangerous event observed during the daily work. A form was provided with a list of “reminders” and facilitators were appointed to help.The main outcome measurements were type of event, causes, corrective interventions, stage of occurrence in the workflow and qualification of the reporters.

Results

A total of 232 errors were reported (two were not related to endoscopy). The remaining 230 amount to 10.3% of 2239 procedures; 66 (29%) were considered errors with consequences, 164 (71%) “near misses”. There were 150 pre-operative errors (65%), 22 operative (10%) and 58 post-operative (25%). Corrective interventions were provided for 60 cases of errors and 119 near misses. Most of the events were reported by the nurses (106 out of 232, 46%).

Conclusions

Short-term incident reporting focusing on near misses, using forms with lists of “reminders”, and the help of a facilitator, can give useful information on errors and near misses in digestive endoscopy.