ERK2 mitogen-activated protein kinase binding, phosphorylation, and regulation of the PDE4D cAMP-specific phosphodiesterases. The involvement of COOH-terminal docking sites and NH2-terminal UCR regions

The cAMP-specific phosphodiesterase family 4, subfamily D, isoform 3 (PDE4D3) is shown to have FQF and KIM docking sites for extracellular signal-regulated kinase 2 (ERK2) (p42(MAPK)). These straddle the target residue, Ser(579), for ERK2 phosphorylation of PDE4D3. Mutation of either or both of these docking sites prevented ERK2 from being co-immunoprecipitated with PDE4D3, ablated the ability of epidermal growth factor to inhibit PDE4D3 through ERK2 action in transfected COS cells, and attenuated the ability of ERK2 to phosphorylate PDE4D3 in vitro. The two conserved NH(2)-terminal blocks of sequence, called upstream conserved regions 1 and 2 (UCR1 and UCR2), that characterize PDE4 long isoforms, are proposed to amplify the small, inherent inhibitory effect that ERK2 phosphorylation exerts on the PDE4D catalytic unit. In contrast to this, the lone intact UCR2 region found in PDE4D1 directs COOH-terminal ERK2 phosphorylation to cause the activation of this short isoform. From the analysis of PDE4D3 truncates, it is suggested that UCR1 and UCR2 provide a regulatory signal integration module that serves to orchestrate the functional consequences of ERK2 phosphorylation. The PDE4D gene thus encodes a series of isoenzymes that are either inhibited or activated by ERK2 phosphorylation and thereby offers the potential for ERK2 activation either to increase or decrease cAMP levels in cellular compartments.     

Redox-related conformational changes in Rhodobacter capsulatus cytochrome c2

WEFT-NOESY and transfer WEFT-NOESY NMR spectra were used to determine the heme proton assignments for Rhodobacter capsulatus ferricytochrome c2. The Fermi contact and pseudo-contact contributions to the paramagnetic effect of the unpaired electron in the oxidized state were evaluated for the heme and ligand protons. The chemical shift assignments for the 1H and 15N NMR spectra were obtained by a combination of 1H-1H and 1H-15N two-dimensional NMR spectroscopy. The short-range nuclear Overhauser effect (NOE) data are consistent with the view that the secondary structure for the oxidized state of this protein closely approximates that of the reduced form, but with redox-related conformational changes between the two redox states. To understand the decrease in stability of the oxidized state of this cytochrome c2 compared to the reduced form, the structural difference between the two redox states were analyzed by the differences in the NOE intensities, pseudo-contact shifts and the hydrogen-deuterium exchange rates of the amide protons. We find that the major difference between redox states, although subtle, involve heme protein interactions, orientation of the heme ligands, differences in hydrogen bond networks and, possible alterations in the position of some internal water molecules. Thus, it appears that the general destabilization of cytochrome c2, which occurs on oxidation, is consistent with the alteration of hydrogen bonds that result in changes in the internal dynamics of the protein.     

Effect of delivery method and population size of Trichoderma harzianum on growth response of unrooted chrysanthemum cuttings

In a previous study, addition of Trichoderma harzianum Rifai isolate T-12 to a propagative medium resulted in improved performance of chrysanthemum cuttings. However, root and shoot growth of one cultivar, 'Dark Bronze Charm', were more responsive to a lower (5 g T-12/kg medium) than higher (25 g T-12/kg medium) rate of fungal propagules, suggesting potential phytotoxicity at higher concentrations. The objectives of this study were to investigate higher rates of T-12 medium amendment for phytotoxicity, and to examine an alternative method of delivering the fungus to the propagative medium in order to obtain a more uniform response from cuttings. Isolate T-12 was added to the propagative medium as either a powdered peat-bran amendment (0, 5, or 50 g T-12/kg medium) or as alginate prills (80 or 800 g T-12/kg medium). There were no differences among treatments on day seven, but by day 21, shoot fresh weight and heights were significantly greater for plants treated with prills at 800 g T-12/kg medium. Both prill treatments resulted in greater shoot height on day 14 and 21 than all other treatments, which were similar to controls. Amendment with T-12 powder at 50 g/kg increased root length, but 80 g/kg medium added as prills decreased root dry weight compared to the control. The highest rate of T-12 (800 g prills/kg medium) had no effect on root growth. This suggests that moderate, rather than high rates of T-12 are more effective in promoting rooting of unrooted chrysanthemum, and that there is a potential for phytotoxic effects on root growth with higher rates.     

Evidence of Type I and Type II Transforming Growth Factor-β Receptors in Central Nervous Tissues: Changes Induced by Focal Cerebral Ischemia

Abstract: The peptides of the transforming growth factor-β (TGF-β) family transduce their signal through ligand-induced heteromeric complexes that consist of type I and type II serine/threonine kinases. Both TGF-β receptors are abundant in many peripheral tissues, but clear evidence of their expression in cortical astrocytes and neurons has not been published so far. In this study, we investigated the expression of type I and type II TGF-β receptors and their potential ligands (TGF-β1, TGF-β2, and TGF-β3) in the CNS by using RT-PCR and immunohistochemistry. Moreover, to further the study of those cell types that exhibit TGF-β isoforms and related receptors, we examined through the use of RT-PCR whether cortical neurons and astrocytes in culture express the mRNAs for TGF-βs and their receptors. We show that the three TGF-β isoform mRNAs are present in the CNS. However, although astrocytes in culture display all three isoforms, neurons in culture express only TGF-β2. We have demonstrated that both type I and type II TGF-β receptor mRNAs and proteins are present in the CNS and in cultures of cortical neurons and astrocytes. Thus, TGF-βs may act as autocrine and paracrine signals in the CNS between both neurons and astrocytes via the same receptor systems as those found in peripheral tissues. TGF-β1 has been shown to be induced following hypoxic-ischemic brain injury and may play a critical role in the pathophysiology of degenerative processes in the CNS. In the present investigation, we confirmed that the expression of TGF-β1 was increased markedly up until 24 h and thereafter was stable over the first 3 days following permanent occlusion of the middle cerebral artery in mice. However, whereas the expression of the type I TGF-β receptor was not altered by the ischemic insult, the pattern of the type II TGF-β receptors was modified dramatically in the ischemic area 3 days after the occlusion. These data show that, even if ligands are present, they may not be able to transduce their signal. Finally, the present study clearly demonstrates that a knowledge of the expression of ligand-specific receptors following brain injury is a fundamental step in clarifying the involvement of cytokines in neurodegenerative diseases.     

The Practice of Cranial Neurosurgery and the Malpractice Liability Environment in the United States

Object

The potential imbalance between malpractice liability cost and quality of care has been an issue of debate. We investigated the association of malpractice liability with unfavorable outcomes and increased hospitalization charges in cranial neurosurgery.

Methods

We performed a retrospective cohort study involving patients who underwent cranial neurosurgical procedures from 2005-2010, and were registered in the National Inpatient Sample (NIS) database. We used data from the National Practitioner Data Bank (NPDB) from 2005 to 2010 to create measures of volume and size of malpractice claim payments. The association of the latter with the state-level mortality, length of stay (LOS), unfavorable discharge, and hospitalization charges for cranial neurosurgery was investigated.

Results

During the study period, there were 189,103 patients (mean age 46.4 years, with 48.3% females) who underwent cranial neurosurgical procedures, and were registered in NIS. In a multivariable regression, higher number of claims per physician in a state was associated with increased ln-transformed hospitalization charges (beta 0.18; 95% CI, 0.17 to 0.19). On the contrary, there was no association with mortality (OR 1.00; 95% CI, 0.94 to 1.06). We observed a small association with unfavorable discharge (OR 1.09; 95% CI, 1.06 to 1.13), and LOS (beta 0.01; 95% CI, 0.002 to 0.03). The size of the awarded claims demonstrated similar relationships. The average claims payment size (ln-transformed) (Pearson’s rho=0.435, P=0.01) demonstrated a positive correlation with the risk-adjusted hospitalization charges but did not demonstrate a correlation with mortality, unfavorable discharge, or LOS.

Conclusions

In the present national study, aggressive malpractice environment was not correlated with mortality but was associated with higher hospitalization charges after cranial neurosurgery. In view of the association of malpractice with the economics of healthcare, further research on its impact is necessary.     

Surgical Clipping versus Endovascular Intervention for the Treatment of Subarachnoid Hemorrhage Patients in New York State

Object

Randomized trials have demonstrated a survival benefit for endovascular treatment of ruptured cerebral aneurysms. We investigated the association of surgical clipping and endovascular coiling with outcomes in subarachnoid hemorrhage (SAH) patients in a real-world regional cohort.

Methods

We performed a cohort study involving patients with ruptured cerebral aneurysms, who underwent surgical clipping, or endovascular coiling from 2009–2013 and were registered in the Statewide Planning and Research Cooperative System (SPARCS) database. An instrumental variable analysis was used to investigate the association of treatment technique with outcomes.

Results

Of the 4,098 patients undergoing treatment, 2,585 (63.1%) underwent coiling, and 1,513 (36.9%) underwent clipping. Using an instrumental variable analysis, we did not identify a difference in inpatient mortality [marginal effect (ME), -0.56; 95% CI, -1.03 to 0.02], length of stay (LOS) (ME, 1.72; 95% CI, -3.39 to 6.84), or the rate of 30-day readmissions (ME, -0.30; 95% CI, -0.82 to 0.22) between the two treatment techniques for patients with SAH. Clipping was associated with a higher rate of discharge to rehabilitation (ME, 0.63; 95% CI, 0.24 to 1.01). In sensitivity analysis, mixed effect regression, and propensity score adjusted regression models demonstrated identical results.

Conclusions

Using a comprehensive all-payer cohort of patients in New York State presenting with aneurysmal SAH we did not identify an association of treatment method with mortality, LOS or 30-day readmission. Clipping was associated with a higher rate of discharge to rehabilitation.     

Multicentre prospective validation of use of the Canadian C-Spine Rule by triage nurses in the emergency department

Objectives

The Canadian C-Spine Rule for imaging of the cervical spine was developed for use by physicians. We believe that nurses in the emergency department could use this rule to clinically clear the cervical spine. We prospectively evaluated the accuracy, reliability and acceptability of the Canadian C-Spine Rule when used by nurses.

Methods

We conducted this three-year prospective cohort study in six Canadian emergency departments. The study involved adult trauma patients who were alert and whose condition was stable. We provided two hours of training to 191 triage nurses. The nurses then assessed patients using the Canadian C-Spine Rule, including determination of neck tenderness and range of motion, reapplied immobilization and completed a data form.

Results

Of the 3633 study patients, 42 (1.2%) had clinically important injuries of the cervical spine. The kappa value for interobserver assessments of 498 patients with the Canadian C-Spine Rule was 0.78. We calculated sensitivity of 100.0% (95% confidence interval [CI] 91.0%–100.0%) and specificity of 43.4% (95% CI 42.0%–45.0%) for the Canadian C-Spine Rule as interpreted by the investigators. The nurses classified patients with a sensitivity of 90.2% (95% CI 76.0%–95.0%) and a specificity of 43.9% (95% CI 42.0%–46.0%). Early in the study, nurses failed to identify four cases of injury, despite the presence of clear high-risk factors. None of these patients suffered sequelae, and after retraining there were no further missed cases. We estimated that for 40.7% of patients, the cervical spine could be cleared clinically by nurses. Nurses reported discomfort in applying the Canadian C-Spine Rule in only 4.8% of cases.

Conclusion

Use of the Canadian C-Spine Rule by nurses was accurate, reliable and clinically acceptable. Widespread implementation by nurses throughout Canada and elsewhere would diminish patient discomfort and improve patient flow in overcrowded emergency departments.Each year, Canadian emergency departments treat 1.3 million patients who have suffered blunt trauma from falls or motor vehicle collisions and who are at risk for injury of the cervical spine.¹ Most of these cases involve adults who are alert and in stable condition, and less than 1% involve fracture of the cervical spine.² Most trauma patients who have been transported in ambulances are protected by a backboard, collar and neck supports. Nurses are responsible for initial triage in the emergency department and usually send such patients to high-acuity resuscitation rooms, where they may remain fully immobilized for hours until assessment by a physician and radiography are complete. This prolonged immobilization is often unnecessary and adds considerably to patient discomfort. The delay also adds to the burden of overcrowded Canadian emergency departments in an era when they are under unprecedented pressures.^3–5 These patients occupy valuable space in resuscitation rooms, and repeated efforts to obtain satisfactory radiographs or computed tomography scans of the cervical spine use valuable time on the part of physicians, nurses and technicians.A clinical decision rule is defined as a decision-making tool incorporating three or more variables from the patient’s history, a physical examination or simple tests. Such rules are derived from original research and help clinicians with diagnostic or therapeutic decisions at the bedside. We previously developed a clinical decision rule for evaluation of the cervical spine.^6,7 The Canadian C-Spine Rule comprises simple clinical variables (Figure 1) and was designed to allow clinicians to “clear” immobilization of the cervical spine (i.e., remove neck collar and other devices) without radiography and to decrease immobilization times.⁸ We also validated the accuracy of the rule when used by physicians.⁹ We recently completed an implementation trial at 12 Canadian hospitals to evaluate the impact on patient care and outcomes of the Canadian C-Spine Rule when used by physicians.¹⁰Open in a separate windowFigure 1The Canadian C-Spine Rule to rule out cervical spine injury, adapted for use by nurses. The rule is intended for patients who have experienced trauma, who are alert (score on Glasgow Coma Scale = 15) and whose condition is stable. *The following mechanisms of injury were defined as dangerous: fall from elevation of more than 3 ft (91 cm) or five stairs, axial load to the head (e.g., diving injury), motor vehicle collision at high speed (> 100 km/h), motor vehicle collision involving a rollover or ejection, injury involving a motorized recreational vehicle, bicycle-related injury (rider struck or collision). †Simple rear-end motor vehicle collisions exclude incidents in which the patient was pushed into oncoming traffic or was hit by a bus, large truck or vehicle travelling at high speed, as well as rollovers; all such incidents would be considered high risk. ‡Neck pain with delayed onset is any pain that did not occur immediately following the precipitating incident. Adapted, with permission, from Stiell IG, Wells GA, Vandemheen K, et al. The Canadian Cervical Spine Radiography Rule for alert and stable trauma patients. JAMA 2001;286:1841–8.⁸ Copyright © 2001 American Medical Association. All rights reserved.Nurses in the emergency department usually do not evaluate the cervical spine of trauma patients, and they routinely send all immobilized patients to the emergency department’s resuscitation room. We believe that nurses could safely evaluate alert patients who have arrived by ambulance and whose condition is stable and could “clear” immobilization of the cervical spine of low-risk patients upon arrival at the triage station.¹¹ Patients could then be much more rapidly, comfortably and efficiently managed in other areas of the emergency department. An expanded decision-making role for nurses has the potential to improve the efficiency of trauma care in all Canadian hospitals. Very little research has been done to determine the ability of nurses to clear immobilization of the cervical spine.^12–15 Our objective in this study was to prospectively evaluate the accuracy, reliability and acceptability of the Canadian C-Spine Rule when used by nurses to assess patients’ need for immobilization.