mTORC1 Controls Phase Separation and the Biophysical Properties of the Cytoplasm by Tuning Crowding.

Macromolecular crowding has a profound impact on reaction rates and the physical properties of the cell interior, but the mechanisms that regulate crowding are poorly understood. We developed genetically encoded multimeric nanoparticles (GEMs) to dissect these mechanisms. GEMs are homomultimeric scaffolds fused to a fluorescent protein that self-assemble into bright, stable particles of defined size and shape. By combining tracking of GEMs with genetic and pharmacological approaches, we discovered that the mTORC1 pathway can modulate the effective diffusion coefficient of particles ≥20 nm in diameter more than 2-fold by tuning ribosome concentration, without any discernable effect on the motion of molecules ≤5 nm. This change in ribosome concentration affected phase separation both in vitro and in vivo. Together, these results establish a role for mTORC1 in controlling both the mesoscale biophysical properties of the cytoplasm and biomolecular condensation.

Management of perceived devastating brain injury after hospital admission: a consensus statement from stakeholder professional organizations.

Patients with severe grades of life-threatening brain injury are commonly characterized as having devastating brain injury (DBI), which we have defined as: 'any neurological condition that is assessed at the time of hospital admission as an immediate threat to life or incompatible with good functional recovery AND where early limitation or withdrawal of therapy is being considered'. The outcome in patients with DBI is often death or severe disability, and as a consequence rapid withdrawal of life sustaining therapies is commonly contemplated or undertaken. However, accurate prognostication in life-threatening brain injury is difficult, particularly at an early stage. Evidence from controlled studies to guide decision-making is limited, and there is a risk of a 'self-fulfilling prophecy', with early prognostication leading to early withdrawal of life sustaining therapies and death. The Joint Professional Standards committee of the Faculty of Intensive Care Medicine and the Intensive Care Society convened a consensus group with representation from stakeholder professional organizations to develop clear professional guidance in this area. It recognized that the weak evidence base makes GRADE guidelines difficult to justify. We have made 12 practical, pragmatic recommendations to help clinicians deliver safe, effective, equitable, and justifiable care within resource constrained healthcare systems. In the situation where patient-centred outcomes are recognized to be unacceptable, regardless of the extent of neurological improvement, then early transition to palliative care is appropriate. These recommendations are intended to apply where the primary pathology is DBI, rather than where DBI has compounded a progressive and irreversible deterioration in other life-threatening comorbidities.

Development, validation and initial outcomes of a questionnaire to examine human factors in postgraduate surgical objective structured clinical examinations.

BACKGROUND: Human factors including stress, repetition, burnout and fatigue are associated with possible sources of error. Objective structured clinical examinations (OSCEs), where examiners concentrate for long periods, would benefit from a human factors approach to see whether these factors affect consistency of examiner behaviour, attitude and marking. Little has been published for OSCEs, in part due to the lack of a validated tool for collecting data in this setting. METHODS: A 46-item questionnaire was developed based on the Human Factors Analysis and Classification System (HFACS) domains and completed by examiners in the Intercollegiate Membership of the Royal College of Surgeons (MRCS) examination. To refine the questionnaire, an initial analysis focused on response patterns of each item. Cronbach's α was used to assess internal consistency, and a factor analysis was performed to uncover different domains emerging from the data. RESULTS: A total of 108 examiners completed the questionnaire (90·0 per cent response rate). The questionnaire, refined to 38 items based on an initial analysis of response patterns, showed good reliability for internal consistency (Cronbach's α = 0·76) and test-retest reliability (r = 0·85, n = 48, P < 0·001). Four factors had a close themed resemblance to the original HFACS domains, but were associated with different items, suggesting that the four human-factor domains might be linked to different behaviours and attitudes in an examination setting. Analyses according to sex, professional background and experience highlighted additional stress levels in examiners from one of the surgical Royal Colleges (P <0·001), matching evidence from the situation in that College at the time of this study. CONCLUSION: The recognition and further investigation of human factors in OSCEs is needed to improve examiner experience and behaviour in order to influence delivery, candidate experience and quality assurance of these examinations.

Single molecule tracking on supported membranes with arrays of optical nanoantennas.

Coupling of the localized surface plasmons between two closely apposed gold nanoparticles (nanoantenna) can cause strong enhancements of fluorescence or Raman signal intensity from molecules in the plasmonic "hot-spot". Harnessing these properties for practical applications is challenging due to the need to fabricate gold particle arrays with well-defined nanometer spacing and a means of delivering functional molecules to the hot-spot. We report fabrication of billions of plasmon-coupled nanostructures on a single substrate by a combination of colloid lithography and plasma processing. Controlled spacing of the nanoantenna gaps is achieved by taking advantage of the fact that polystyrene particles melt together at their contact point during plasma processing. The resulting polymer thread shadows a gap of well-defined spacing between each pair of gold triangles in the final array. Confocal surface-enhanced Raman spectroscopy imaging confirms the array is functionally uniform. Furthermore, a fully intact supported membrane can be formed on the intervening substrate by vesicle fusion. Trajectories of freely diffusing individual proteins are traced as they sequentially pass through, and are enhanced by, multiple gaps. The nanoantenna array thus enables enhanced observation of a fluid membrane system without static entrapment of the molecules.

Successful rehabilitation and release of a powerful owl chick with suspected rodenticide poisoning.

The successful rehabilitation and release of raptor chicks can be challenging, especially when the chicks are still in the post-fledging dependency period. Here, we report on a recently fledged powerful owl chick that was held in care for 33 days before being successfully reunited with its parents. We document the steps undertaken during the entire process from collection from the wild to post-release monitoring and recommend clinical procedures for treatment of raptors entering veterinary facilities. Success of this rehabilitation was facilitated by early care and treatment for potential rodenticide poisoning, as well as the integration of citizen scientists monitoring the family unit in the field while the chick was in care and during the post-release period. Given the emerging evidence of widespread rodenticide poisoning in raptors both in Australia and globally, it is critical to suspect all raptors may have been exposed to anticoagulant rodenticides and commence treatment with vitamin K immediately. Routine treatment for rodenticides early increases the probability of successful recovery post-trauma as well as reducing the time in treatment as much as possible.

The genome sequence of Desulfatibacillum alkenivorans AK-01: a blueprint for anaerobic alkane oxidation.

Desulfatibacillum alkenivorans AK-01 serves as a model organism for anaerobic alkane biodegradation because of its distinctive biochemistry and metabolic versatility. The D. alkenivorans genome provides a blueprint for understanding the genetic systems involved in alkane metabolism including substrate activation, CoA ligation, carbon-skeleton rearrangement and decarboxylation. Genomic analysis suggested a route to regenerate the fumarate needed for alkane activation via methylmalonyl-CoA and predicted the capability for syntrophic alkane metabolism, which was experimentally verified. Pathways involved in the oxidation of alkanes, alcohols, organic acids and n-saturated fatty acids coupled to sulfate reduction and the ability to grow chemolithoautotrophically were predicted. A complement of genes for motility and oxygen detoxification suggests that D. alkenivorans may be physiologically adapted to a wide range of environmental conditions. The D. alkenivorans genome serves as a platform for further study of anaerobic, hydrocarbon-oxidizing microorganisms and their roles in bioremediation, energy recovery and global carbon cycling.

Survival and quality of life for patients with COPD or asthma admitted to intensive care in a UK multicentre cohort: the COPD and Asthma Outcome Study (CAOS).

BACKGROUND: Non-invasive ventilation is first-line treatment for patients with acutely decompensated chronic obstructive pulmonary disease (COPD), but endotracheal intubation, involving admission to an intensive care unit, may sometimes be required. Decisions to admit to an intensive care unit are commonly based on predicted survival and quality of life, but the information base for these decisions is limited and there is some evidence that clinicians tend to be pessimistic. This study examined the outcomes in patients with COPD admitted to the intensive care unit for decompensated type II respiratory failure. METHODS: A prospective cohort study was carried out in 92 intensive care units and 3 respiratory high dependency units in the UK. Patients aged 45 years and older with breathlessness, respiratory failure or change in mental status due to an exacerbation of COPD, asthma or a combination of the two were recruited. Outcomes included survival and quality of life at 180 days. RESULTS: Of the 832 patients recruited, 517 (62%) survived to 180 days. Of the survivors, 421 (81%) responded to a questionnaire. Of the respondents, 73% considered their quality of life to be the same as or better than it had been in the stable period before they were admitted, and 96% would choose similar treatment again. Function during the stable pre-admission period was a reasonable indicator of function reported by those who survived 180 days. CONCLUSIONS: Most patients with COPD who survive to 180 days after treatment in an intensive care unit have a heavy burden of symptoms, but almost all of them-including those who have been intubated-would want similar intensive care again under similar circumstances.

Micropatterning fluid lipid bilayers on solid supports.

Lithographically patterned grids of photoresist, aluminum oxide, or gold on oxidized silicon substrates were used to partition supported lipid bilayers into micrometer-scale arrays of isolated fluid membrane corrals. Fluorescently labeled lipids were observed to diffuse freely within each membrane corral but were confined by the micropatterned barriers. The concentrations of fluorescent probe molecules in individual corrals were altered by selective photobleaching to create arrays of fluid membrane patches with differing compositions. Application of an electric field parallel to the surface induced steady-state concentration gradients of charged membrane components in the corrals. In addition to producing patches of membrane with continuously varying composition, these gradients provide an intrinsically parallel means of acquiring information about molecular properties such as the diffusion coefficient in individual corrals.

Myosin IIA and formin dependent mechanosensitivity of filopodia adhesion.

Filopodia, dynamic membrane protrusions driven by polymerization of an actin filament core, can adhere to the extracellular matrix and experience both external and cell-generated pulling forces. The role of such forces in filopodia adhesion is however insufficiently understood. Here, we study filopodia induced by overexpression of myosin X, typical for cancer cells. The lifetime of such filopodia positively correlates with the presence of myosin IIA filaments at the filopodia bases. Application of pulling forces to the filopodia tips through attached fibronectin-coated laser-trapped beads results in sustained growth of the filopodia. Pharmacological inhibition or knockdown of myosin IIA abolishes the filopodia adhesion to the beads. Formin inhibitor SMIFH2, which causes detachment of actin filaments from formin molecules, produces similar effect. Thus, centripetal force generated by myosin IIA filaments at the base of filopodium and transmitted to the tip through actin core in a formin-dependent fashion is required for filopodia adhesion.

Sex workers working within a legalised industry: their side of the story.

OBJECTIVES: To examine the characteristics and work attitudes of female sex workers working in licensed brothels in Victoria, Australia. METHODS: This was a cross-sectional study of sex workers working at 38 of the 92 licensed brothels operating in Victoria during 2006. RESULTS: Of the 108 women approached, 97 (90%) completed the questionnaire. Women working in the legal sex industry in Victoria were generally aged between 23 and 35 years (51%), had completed high school (26%) and had worked in the industry for more than 5 years (43%). Half had dependent children and one third were in a relationship. Women's primary motivation for working in the sex industry was financial, whether this was the reason for their starting (56%), or the barrier to their leaving (61%). Although women valued the higher income and flexibility of this work, many were concerned about sexually transmitted infections (STI) (55%), community attitudes towards the industry (47%), their physical safety (38%) and maintaining their anonymity (37%). Over half of the women would like to leave the industry. The majority (95%) supported the monthly STI checks that are part of the Victorian regulations, with only one fifth reporting that the cost of these tests was prohibitive. CONCLUSIONS: The findings of this study indicate that women working in licensed Victorian brothels come from a diverse range of backgrounds and circumstances and hold varying attitudes towards working in the sex industry. It is hoped that these findings go some way to redressing the assumptions commonly made about women working in the sex industry and reducing the stigma associated with this occupation.

Reducing the risk of mouth-to-mouth transmission of pathogens via re-usable, machine-read parking tickets: an observational cohort study.

The car parks at the study hospital are accessed using re-usable, machine-read tickets. In the initial phase of this study, 598 staff members were observed entering the car park, and 21.6% of them put their parking ticket in their mouth. Ultraviolet dye was used to demonstrate card-to-card cross-contamination. Swabs of the ticket machine yielded commensal bacteria: coagulase-negative staphylococci and a Bacillus sp. After placing a poster on the ticket-reading machine highlighting the potential risk of infection, a further 1366 observations demonstrated a significant and persistent decline in the proportion of staff who put their ticket in their mouth (P<0.001).

Effect of noise on tasks in operating theatres: a survey of the perceptions of healthcare staff.

Noise in the operating theatre has an adverse impact on healthcare professionals, both physically and psychologically. It can be distracting, make communication difficult, and contribute to a perceived increase in stress. Staff in theatre must deliver high quality care, and overlook noise as a potentially damaging influence. The aim of this survey was to obtain further information about the perspective of healthcare professionals on how noise can affect their practice and whether it affects their work in theatre. We distributed six closed-ended questions in the form of a Survey Monkey® questionnaire to about 50 hospitals across the UK and target groups such as medical students, the Leeds Advanced Trauma Life Support faculty group, the Court of Examiners of the Royal College of Surgeons of England, and surgical trainees sitting the Member of the Royal College of Surgeons examination. We received 519 responses of which 415 respondents (83%) thought that noise contributed to human errors. A total of 282 participants (57%) thought that the theatre was the noisiest area within the theatre suite. Both communication among staff (n=400, 80%) and concentration (n=384, 77%) were thought to be adversely affected by noise. However, 385 (78%) did not feel that music adversely affected their performance. The results provide insights into the interplay of noise and its effect on people. Although the role of music remains contentious, our results suggest that it might have a calming influence.

Sensitivity of late-latency auditory and somatosensory evoked potentials to threat of electric shock and the sedative drugs diazepam and diphenhydramine in human volunteers.

Late-latency auditory and somatosensory evoked potentials are sensitive to some centrally acting drugs and to certain psychological interventions. In this experiment we compared the effects of acute doses of a benzodiazepine, diazepam and an H(1) histamine receptor-blocking sedative, diphenhydramine, on auditory and somatosensory evoked potentials within the latency range 100-500 ms in a fear conditioning paradigm. Twelve healthy males (18-30 years) participated in three sessions at weekly intervals in which they received diazepam 10mg, diphenhydramine 75 mg and placebo in a balanced, double-blind, crossover protocol. One hundred and twenty min after diphenhydramine or 60 min after diazepam, they underwent an 8 min recording period in which auditory evoked potentials elicited by 40 ms, 95 dB[A], 1 kHz tones, and somatosensory evoked potentials elicited by a mildly painful electric shock (1.8 mA, 50 ms) were recorded at Cz (vertex). Each session consisted of four blocks of trials in which either the sound pulse or the shock was presented. Alternate blocks were designated SAFE or THREAT ('context' conditions); in THREAT blocks subjects were warned that shocks would be delivered via electrodes placed on the wrist (electrodes were removed during SAFE blocks). In one SAFE and one THREAT block, the sound stimuli and shocks (shocks were delivered only in the THREAT block) were preceded by a 2 s conditioned stimulus (CS: a red light) ('cue' condition). Diazepam, but not diphenhydramine, reduced the amplitude of the P2 auditory evoked potential. The THREAT context was associated with increased N1 and reduced N2 potential amplitudes. The CS had no effect on the amplitudes, but markedly reduced the latencies of the N1, P2 and N2 potentials under the THREAT condition. Diazepam reduced the amplitudes of the somatosensory potential evoked by the shock; the CS shortened the latencies of the later components of the response. Diazepam and diphenhydramine were approximately equi-sedative in the doses used in this experiment, as judged by visual analogue self-rating scales. The results indicate that the suppression of late-latency auditory and somatosensory evoked potentials by diazepam is not simply a reflection of sedation. Late-latency evoked potentials can be modified by an aversive CS, but the components that are sensitive to the CS are different from those that are sensitive to diazepam.

Peroxynitrite: reactive, invasive and enigmatic.

The reactive oxygen and nitrogen species superoxide ion, nitric oxide, nitrogen dioxide and peroxynitrite ion all react with biological target molecules. Some of these interactions are carefully orchestrated segments of signal transduction cascades or part of the armamentarium of the immune system, others are pathological events and may lie at the root of many diseases. As a result of these small reactive molecules, proteins, particularly metalloproteins, can be altered with loss of function, DNA can be cleaved and lipid components can be oxidized to disrupt membranes. The interactions of these species with each other and their aftermath can be sensed by the cell, resulting in a variety of responses including gene regulation and transcription. Indeed, there is recent, tantalizing evidence that the currency of reactive oxygen and nitrogen species is central to the life and death cellular decisions in homeostasis or the initiation of apoptosis. New families of metallopharmaceuticals may serve both to probe the nature and mechanisms of these events and to effect the outcome.

Nitric oxide synthase: models and mechanisms.

The overproduction or underproduction of nitric oxide has been implicated in pathological symptoms such as endotoxic shock, diabetes, allograft rejection, and myocardial ischimia/reperfusion injury. A thorough understanding of the biosynthesis of nitric oxide is necessary to probe and manipulate these signaling events. There is also considerable pharmacological interest in developing selective inhibitors of the several isoforms of nitric oxide synthase. The recently determined crystal structures of complexes between nitric oxide synthase and substrate, the mechanisms of the enzymatic reaction that generate nitric oxide and chemical precedents and models for these reactions are now coming into focus, but there are still numerous fascinating and unanswered questions regarding nitric oxide biosynthesis.

Amphiphilic peroxynitrite decomposition catalysts in liposomal assemblies.

BACKGROUND: Peroxynitrite (ONOO-), a toxic biological oxidant, has been implicated in many pathophysiological conditions. The water-soluble porphyrins 5,10,15,20-tetrakis(N-methyl-4'-pyridyl)porphinato iron(III) (FeTMPyP) and manganese(III) (MnTMPyP) have recently emerged as potential drugs for ONOO- detoxification, and FeTMPyP has demonstrated activity in models of ONOO- related disease states. We set out to develop amphiphilic analogs of FeTMPyP and MnTMPyP suitable for liposomal delivery in sterically stabilized liposomes (SLs). RESULTS: Three amphiphilic iron porphyrins (termed 1a-c.) and three manganese porphyrins (termed 2a-c.) bound to liposomes and catalyzed the decomposition of ONOO-. The polyethylene-glycol-linked metalloporphyrins 1b. and 2b. proved the most effective of these catalysts, rapidly decomposing ONOO- with second-order rate constants (kcat) of 2.9 x 10(5) M-1 s-1 and 5.0 x 10(5) M-1 s-1, respectively, in dimyristoylphosphatidylcholine liposomes. Catalysts 1b. and 2b. also bound to SLs, and these metalloporphyrin-SL constructs efficiently catalyzed ONOO- decomposition (kcat approximately 2 x 10(5) M-1 s-1). The analogous metalloporphyrins 1a. and 2a., which are not separated from the vesicle membrane surface by polyethylene glycol linkers, were significantly less effective (kcat approximately 3.5 x 10(4) M-1 s-1). CONCLUSIONS: For these amphiphilic analogs of FeTMPyP and MnTMPyP, the polarity of the environment of the metalloporphyrin headgroup is intimately related to the efficiency of the catalyst; a polar aqueous environment is essential for effective catalysis of ONOO- decomposition. Thus, catalysts 1b. and 2b. react rapidly with ONOO- and are potential therapeutic agents that, unlike their water-soluble TMPyP analogs, could be administered as liposomal formulations in SLs. These SL-bound amphiphilic metalloporphyrins may prove to be highly effective in the exploration and treatment of ONOO- related disease states.

Male genital self-mutilation.

Men who intentionally mutilate or remove their own genitals are likely to be psychotic at the time of the act, to have a number of goals and aims relating to conflicts about the male role, and to be vulnerable to sociocultural and psychological forces in a causal network as yet unknown. However, from a review of the cases of 53 male self-mutilators, it appears that a significant number involve individuals not psychotic at the time of the act but rather having character disorders and rageful feelings toward themselves or women, and transsexual males who premeditate their own gender conversion surgery. The psychiatric consultant's role in the management of such an individual in the general hospital setting includes not only care of a patient with a psychotic or impulse disorder but also involves support of the house staff, who are distressed by the fear, guilt, hopelessness, anger, and revulsion that the patient's act of genital self-mutilation causes.

The long-acting phenothiazines.

Injected intramuscularly, the enanthane and decanoate esters of the phenothiazine fluphenazine are an effective treatment of the disordered behavior and thinking of schizophrenia. The decanoate preparation is not only slightly longer-acting but also has a smaller incidence of side-effects that the enanthate. The major adverse effect of these medications is the high frequency of extrapyramidal system disturbance. Since the 50% rate of failure of schizophrenic outpatients to take prescribed oral medications decreases treatment failure to about 20% with the use of long-acting injectable phenothiazines, this route of administration offers an advantage in patient management particularly applicable to community mental health systems. Moreover, parenteral administration of long-acting fluphenazine may be useful for patients who do not attain effective serum levels with medication taken orally because of metabolic or absorption difficulties.

Training supervision as a separate faculty role.

Training supervision is a longitudinal, nonclinically focused personal relationship between a faculty member and a resident for exploring the latter's professional development. In the authors' program the training supervisor meets with the supervisee monthly over the 3-year residency. The training supervisor's role is that of a nonevaluative senior colleague who orients and advises the resident and systematically reviews training progress and problems. The authors discuss the evolution of the training supervision concept, explore potential role conflicts for the training supervisor, who may serve other functions in the residency of the supervisee, and present positive and negative resident and faculty evaluations of their program.

Emergence of hostility during alprazolam treatment.

Of 80 patients given alprazolam, eight became hostile early in treatment. The authors suggest that alprazolam-induced hostility may be more likely in patients with well-suppressed chronic anger and resentment and cite reports of hostility associated with other benzodiazepines.