Characterization of RIPK3-mediated phosphorylation of the activation loop of MLKL during necroptosis.

Mixed lineage kinase domain-like pseudokinase (MLKL) mediates necroptosis by translocating to the plasma membrane and inducing its rupture. The activation of MLKL occurs in a multimolecular complex (the 'necrosome'), which is comprised of MLKL, receptor-interacting serine/threonine kinase (RIPK)-3 (RIPK3) and, in some cases, RIPK1. Within this complex, RIPK3 phosphorylates the activation loop of MLKL, promoting conformational changes and allowing the formation of MLKL oligomers, which migrate to the plasma membrane. Previous studies suggested that RIPK3 could phosphorylate the murine MLKL activation loop at Ser345, Ser347 and Thr349. Moreover, substitution of the Ser345 for an aspartic acid creates a constitutively active MLKL, independent of RIPK3 function. Here we examine the role of each of these residues and found that the phosphorylation of Ser345 is critical for RIPK3-mediated necroptosis, Ser347 has a minor accessory role and Thr349 seems to be irrelevant. We generated a specific monoclonal antibody to detect phospho-Ser345 in murine cells. Using this antibody, a series of MLKL mutants and a novel RIPK3 inhibitor, we demonstrate that the phosphorylation of Ser345 is not required for the interaction between RIPK3 and MLKL in the necrosome, but is essential for MLKL translocation, accumulation in the plasma membrane, and consequent necroptosis.

Improving hospital doctors' working lives: online questionnaire survey of all grades.

BACKGROUND: In 2001, the Department of Health produced the Improving Working Lives (IWL) for Doctors document. This is the first national survey which asks hospital doctors what changes are needed to improve their working lives. METHODS: An online questionnaire was run over a period of six weeks and was open to all doctors of all grades. Doctors were asked to choose their top five factors from a list of 35 diverse choices or to provide alternatives in free text. Demographic data were also collected. RESULTS: 1603 hospital doctors working in the UK completed the online questionnaire. Improved secretarial or managerial support was the first IWL choice for consultants, with different aspects of clinical and non-clinical support representing their top four choices. Junior hospital doctors and staff and associate specialist grades (staff grades, associate specialists, and clinical assistants) identified improved support for education and training as their first choice, while among the female specialist registrars, it was improved support for childcare. Greater opportunities to develop new skills was an important issue for doctors in the surgical specialties and improved access to mentoring was important for all junior doctors, staff and associate specialist grades, and doctors from black and ethnic minority groups. CONCLUSIONS: Hospital doctors in the UK need more support to improve their working lives. The principle needs are better secretarial and managerial support for consultants; education, training, and mentoring for junior doctors and staff and associate specialist grades; and improved opportunities to develop new skills for those in surgical specialties. Support with childcare is an important issue for female specialist registrars. The Department of Health, NHS trusts, deaneries, and Royal Colleges need to endorse policies that promote a training and working environment that will improve working lives for all hospital doctors, ensuring that appropriate and continuing support is available from the time doctors enter the new foundation programmes and proposed run-through grades, to their time spent as consultants in today's NHS.

Expert evidence pertaining to battered women: the impact of gender of expert and timing of testimony.

The present study investigated the impact of two variables relating to general expert testimony pertaining to battered women on juror decision processes. Specifically, the gender of the expert, as well as the timing of the presentation of the testimony, were investigated in a simulated homicide trial involving a battered woman who had killed her abuser. Results indicated that when the expert was female and the testimony was presented prior to the defendant's testimony, juror's verdicts were more lenient. Moreover, across a range of case judgments, male jurors' perceptions were more favorable to the defendant when the expert was female as opposed to male. On two of the judgments this latter pattern of results was only evidenced when the testimony was presented early as opposed to late. The significance of these findings is discussed.

Post-operative central hypersensitivity and pain: the pre-emptive value of pethidine for ovariohysterectomy.

The effect of timing of analgesic drug administration on the severity of post-operative pain was investigated in dogs undergoing ovariohysterectomy using both subjective visual assessment scoring systems (VAS) and objective mechanical nociceptive threshold measurements using a novel handheld anti-nociceptiometric device. Forty dogs undergoing routine elective ovariohysterectomy were included in a randomised and double-blind study and assigned to one of three groups: (i) pre-operative analgesics; (ii) post-operative analgesics; (iii) no analgesics (saline injections). The analgesic used was pethidine (a short acting predominantly mu-opioid agonist), at a dose of 5.0 mg/kg (intramuscular). The post-operative administration of pethidine resulted in significantly higher sedation scores and significantly lower pain scores in the early post-operative period, but the dogs given pethidine pre-operatively had significantly lower pain scores than both the other groups at 8, 12 and 20 h post-extubation (P < 0.01, ANOVA). Mechanical thresholds measured at the distal tibia demonstrated the development of allodynia at 12 and 20 h post-extubation, and this was significantly prevented by the pre- (P < 0.01 at 12 h, P < 0.05 at 20 h, Kruskal-Wallis and post hoc Dunn's), but not by the post-operative administration of pethidine. Mechanical nociceptive thresholds measured at the ventral midline (site of surgery) demonstrated post-operative hyperalgesia in all groups; this hyperalgesia was least in the pre-operative pethidine group. In summary, this study clearly shows pethidine to be an effective analgesic in dogs, albeit of short duration of action, when administered post-operatively, and, importantly, that it has a positive benefit in terms of post-operative outcome measures, when administered pre-operatively, possibly as a result of blocking or preventing the development of central sensitisation following surgical stimulation.

Pharmacokinetics of carvedilol in older and younger patients.

Carvedilol, a combined beta- and alpha 1-blocking drug, was given to 8 young (age 39-47) and 21 old (age 64-79) patients with essential hypertension. Clinical and pharmacokinetic responses to 12.5, 25 and 50 mg were determined and compared. In both age groups, pharmacokinetic data were similar with all three doses of carvedilol. Peak blood levels were reached within 90 minutes and at 24 hours the trough blood level was less than 10% of the peak level. Carvedilol or its metabolite did not accumulate. Falls in systolic and diastolic BP were greater than 7 mmHg in 28 of the 29 patients. The falls in diastolic BP did not differ between groups but the older group had a greater fall in systolic BP. However, the systolic BP of the older group was higher and expressed as a percentage, the falls in BP did not differ. The time to peak fall in BP was about 4 hours and was always after the time to peak blood level. There was no correlation between blood level and BP fall. When the drug was administered 24 h after the previous dose, a further fall in BP was seen indicating a greater effect at peak drug levels. Side effects were few and, during the chronic study, there was no postural hypotension or postural hypotensive symptoms. On the study days, five patients developed postural hypotension with symptoms. These were not observed at other times and may have been due to decreased sympathetic outflow on the study days. Carvedilol lowered BP in order and younger patients. There were no significant effects of age on its pharmacokinetics. Carvedilol is an effective antihypertensive agent that can be used in people with essential hypertension in all age groups.

The use of carvedilol in elderly hypertensive patients.

Carvedilol, a beta-blocking drug with vasodilator activity, has been used in 4 studies in 107 elderly patients with essential hypertension and has reduced blood pressure effectively. In the first study the pharmacokinetics and clinical response were compared between 21 patients greater than 65 years of age and 8 patients aged 35-50 years). The peak blood levels, time to maximal concentration, area under the curve, half-life and trough level of the drug with chronic administration did not differ. The clinical responses to the drug were similar, with a greater fall in systolic blood pressure in the older group. However the initial systolic blood pressure in the older group was higher. Carvedilol was compared with metoprolol, pindolol and nitrendipine in elderly patients. The responses to carvedilol were at least equal to those obtained with the other drugs. Control was achieved in the three studies with once-daily therapy. There was no significant postural hypotensive effect. A feature of all studies was the large number of patients who responded to carvedilol. The side-effect profile of the drug was acceptable; headache and dizziness were more common than with placebo or the comparison drugs and were frequently associated. There were no adverse biochemical effects and the lipid profile was not altered. Carvedilol is an effective antihypertensive drug that lowers blood pressure equally well in the young and the old.