Quantitative, Label-Free Proteomics in the Symptomatic Niemann-Pick, Type C1 Mouse Model Using Standard Flow Liquid Chromatography and Thermal Focusing Electrospray Ionization.

Niemann-Pick disease, type C1 (NPC1) is a fatal, autosomal recessive, neurodegenerative disorder caused by mutations in the NPC1 gene. As a result, there is accumulation of unesterified cholesterol and sphingolipids in the late endosomal/lysosomal system. This abnormal accumulation results in a cascade of pathophysiological events including progressive, cerebellar neurodegeneration, among others. While significant progress has been made to better understand NPC1, the downstream effects of cholesterol storage and the major mechanisms that drive neurodegeneration remain unclear. In the current study, a) the use of a commercial, highly efficient standard flow-ESI platform for protein biomarker identification is implemented and b) protein biomarkers are identified and evaluated at a terminal time point in the NPC1 null mouse model. In this study, alterations are observed in proteins related to fatty acid homeostasis, calcium binding and regulation, lysosomal regulation, and inositol biosynthesis and metabolism, as well as signaling by Rho family GTPases. New observations from this study include altered expression of Pcp2 and Limp2 in Npc1 mutant mice relative to control, with Pcp2 exhibiting multiple isoforms and specific to the cerebella. This study provides valuable insight into pathways altered in the late-stage pathophysiology of NPC1.

Targeted Mass Spectrometry-Based Approach for Protein-Ligand Binding Analyses in Complex Biological Mixtures Using a Phenacyl Bromide Modification Strategy.

The characterization of protein folding stability changes on the proteomic scale is useful for protein-target discovery and for the characterization of biological states. The Stability of Proteins from Rates of Oxidation (SPROX) technique is one of several mass spectrometry-based techniques recently established for the making proteome-wide measurements of protein folding and stability. A critical part of proteome-wide applications of SPROX is the identification and quantitation of methionine-containing peptides. Demonstrated here is a targeted mass spectrometry-based proteomics strategy for the detection and quantitation of methionine-containing peptides in SPROX experiments. The strategy involves the use of phenacyl bromide (PAB) for the targeted detection and quantitation of methionine-containing peptides in SPROX using selective reaction monitoring (SRM) on a triple quadrupole mass spectrometer (QQQ-MS). As proof-of-principle, the known binding interaction of Cyclosporine A with cyclophilin A protein in a yeast cell lysate is successfully detected and quantified using a targeted SRM workflow. Advantages of the described workflow over other SPROX protocols include a 20-fold reduction in the amount of total protein needed for analysis and the ability to work with the endogenous proteins in a given sample (e.g., stabile isotope labeling with amino acids in cell culture is not necessary).

Identification of Biomarkers of Exposure to FTOHs and PAPs in Humans Using a Targeted and Nontargeted Analysis Approach.

Although historic perfluorinated compounds are currently under scrutiny and growing regulatory control in the world, little is known about human exposure to other polyfluorinated compounds presently in use. Fluorotelomer alcohols (FTOHs) and polyfluoroalkyl phosphate esters (PAPs) are known to degrade to terminal perfluorinated acids and toxic reactive intermediates through metabolic pathways. Therefore, it is important to characterize their human exposure by the identification of unique biomarkers. With the use of liquid chromatography-mass spectrometry-time-of-flight analysis (LC-MS-TOF), we developed a workflow for the identification of metabolites for the 8:2 FTOH and 8:2 diPAP. Analysis of serum and urine of dosed rats indicated the 8:2 FTOH-sulfate and the 8:2 diPAP as potential biomarkers. These compounds, as well as 25 other fluorinated compounds and metabolites, were analyzed in human serum and urine samples from the general population (n = 100) and office workers (n = 30). The 8:2 FTOH-sulfate was measured for the first time in human samples in 5 to 10% of the serum samples, ranging from 50 to 80 pg/mL. The 8:2 diPAP was measured in 58% of the samples, ranging from 100 to 800 pg/mL. This study indicates the FTOH-sulfate conjugate as a biomarker of exposure to FTOHs and PAPs in humans.

Identification of Novel Perfluoroalkyl Ether Carboxylic Acids (PFECAs) and Sulfonic Acids (PFESAs) in Natural Waters Using Accurate Mass Time-of-Flight Mass Spectrometry (TOFMS).

Recent scientific scrutiny and concerns over exposure, toxicity, and risk have led to international regulatory efforts resulting in the reduction or elimination of certain perfluorinated compounds from various products and waste streams. Some manufacturers have started producing shorter chain per- and polyfluorinated compounds to try to reduce the potential for bioaccumulation in humans and wildlife. Some of these new compounds contain central ether oxygens or other minor modifications of traditional perfluorinated structures. At present, there has been very limited information published on these "replacement chemistries" in the peer-reviewed literature. In this study we used a time-of-flight mass spectrometry detector (LC-ESI-TOFMS) to identify fluorinated compounds in natural waters collected from locations with historical perfluorinated compound contamination. Our workflow for discovery of chemicals included sequential sampling of surface water for identification of potential sources, nontargeted TOFMS analysis, molecular feature extraction (MFE) of samples, and evaluation of features unique to the sample with source inputs. Specifically, compounds were tentatively identified by (1) accurate mass determination of parent and/or related adducts and fragments from in-source collision-induced dissociation (CID), (2) in-depth evaluation of in-source adducts formed during analysis, and (3) confirmation with authentic standards when available. We observed groups of compounds in homologous series that differed by multiples of CF2 (m/z 49.9968) or CF2O (m/z 65.9917). Compounds in each series were chromatographically separated and had comparable fragments and adducts produced during analysis. We detected 12 novel perfluoroalkyl ether carboxylic and sulfonic acids in surface water in North Carolina, USA using this approach. A key piece of evidence was the discovery of accurate mass in-source n-mer formation (H(+) and Na(+)) differing by m/z 21.9819, corresponding to the mass difference between the protonated and sodiated dimers.

Proteomic characterization of the cellular response to nitrosative stress mediated by s-nitrosoglutathione reductase inhibition.

The S-nitrosoglutathione-metabolizing enzyme, GSNO reductase (GSNOR), has emerged as an important regulator of protein S-nitrosylation. GSNOR ablation is protective in models of asthma and heart failure, raising the idea that GSNOR inhibitors might hold therapeutic value. Here, we investigated the effects of a small molecule inhibitor of GSNOR (GSNORi) in mouse RAW 264.7 macrophages. We found that GSNORi increased protein S-nitrosylation in cytokine-stimulated cells, and we utilized stable isotope labeling of amino acids in cell culture (SILAC) to quantify the cellular response to this "nitrosative stress". The expression of several cytokine-inducible immunomodulators, including osteopontin, cyclooxygenase-2, and nitric oxide synthase isoform 2 (NOS2), were decreased by GSNORi. In addition, selective targets of the redox-regulated transcription factor, nuclear factor (erythroid-derived 2)-like 2 (Nrf2)-including heme oxygenase 1 (HO-1) and glutamate cysteine ligase modulatory subunit-were induced by GSNORi in a NOS2- and Nrf2-dependent manner. In cytokine-stimulated cells, Nrf2 protected from GSNORi-induced glutathione depletion and cytotoxicity and HO-1 activity was required for down-regulation of NOS2. Interestingly, GSNORi also affected a marked increase in NOS2 protein stability. Collectively, these data provide the most complete description of the global effects of GSNOR inhibition and demonstrate several important mechanisms for inducible response to GSNORi-mediated nitrosative stress.

Standard-flow LC and thermal focusing ESI elucidates altered liver proteins in late stage Niemann-Pick, type C1 disease.

Aim: Mass spectrometry (MS)-based proteomics, particularly with the development of nano-ESI, have been invaluable to our understanding of altered proteins related to human disease. Niemann-Pick, type C1 (NPC1) disease is a fatal, autosomal recessive, neurodegenerative disorder. The resulting defects include unesterified cholesterol and sphingolipids accumulation in the late endosomal/lysosomal system resulting in organ dysfunction including liver disease. Materials & methods: First, we performed MS analysis of a complex mammalian proteome using both nano- and standard-flow ESI with the intent of developing a differential proteomics platform using standard-flow ESI. Next, we measured the differential liver proteome in the NPC1 mouse model via label-free quantitative MS using standard-flow ESI. Results: Using the standard-flow ESI approach, we found altered protein levels including, increased Limp2 and Rab7a in liver tissue of Npc1-/- compared to control mice. Conclusion: Standard-flow ESI can be a tool for quantitative proteomic studies when sample amount is not limited. Using this method, we have identified new protein markers of NPC1.

Predictors of adverse events in patients after discharge from the intensive care unit.

BACKGROUND: Patients discharged from the intensive care unit may be at risk of adverse events because of complex care needs. OBJECTIVE: To identify the types, frequency, and predictors of adverse events that occur in the 72 hours after discharge from an intensive care unit when no evidence of adverse events was apparent before discharge. METHODS: A predictive cohort study of 300 patients from an adult intensive care unit was undertaken. An internationally accepted protocol for chart audit was used. Frequency of adverse events was calculated, and logistic regression was used to determine independent predictors of adverse events. RESULTS: A total of 147 adverse events, 17 (11.6%) of which were defined as major, were incurred by 92 patients (30.7%). The 3 most common adverse events, hospital-incurred infection or sepsis (n = 32, 21.8%), hospital-incurred accident or injury (n = 17, 11.6%), and other complication such as deep vein thrombosis, pulmonary edema, or myocardial infarction (n = 17, 11.6%) accounted for 44.9% (n = 66) of all adverse events. Two predictors, respiratory rate less than 10/min or greater than or equal to 25/min and pulse rate exceeding 110/min, were significant independent predictors; requiring a high level of nursing care at the time of discharge was a significant predictor in univariate analysis but not in multivariate analysis. CONCLUSION: Taking, recording, and reporting vital signs are important. Nursing care requirements of patients at discharge from the intensive care unit may be worthy of further investigation in studies of patients after discharge.

Diabetic ketoacidosis and hyperglycaemic hyperosmolar syndrome - clinical guidelines.

BACKGROUND: The aim of this study was to establish a standardized approach to the initial care of patients with diabetic ketoacidosis (DKA) and hyperglycaemic hyperosmolar syndrome (HHS). DKA and HHS are metabolic emergencies. Effective and efficient management is the responsibility of the multidisciplinary team. The admission of patients to the intensive care unit (ICU) with DKA and HHS is rare, and management of patients' diverse problems is prone to error because of a lack of familiarity. AIM: The paper's aim is to set the developmental process of a clinical guideline following a review of the literature. DISCUSSION: This clinical guideline is based on a review of the evidence available within the literature in the early phase of resuscitation. Collaborative working among the multidisciplinary team through clinical practice group was the method adopted. Management of DKA and HHS is divided into three main areas: intravenous fluid replacement, insulin therapy and electrolyte management. The controversy associated with the administration of sodium bicarbonate is discussed. CONCLUSION: Effective treatment requires a rapid initial assessment of the patient based on current medical history and clinical presentation. To this end, a quick reference algorithm and guide to management were also developed. Key criteria for evaluating the effectiveness of treatment are provided and complications of treatment are addressed. The formation of the practice development group that led to this innovation is outlined, and in conclusion, the success of the group is reflected upon.

Treatment of external genital warts at Planned Parenthood Federation of America centers.

OBJECTIVE: To compare patterns and cost of treating external genital warts (EGW) at 5 major Planned Parenthood Federation of America (PPFA) affiliates. STUDY DESIGN: Charts of 422 women and 78 men treated for EGW were reviewed. Treatment must have been successful and occurred at a single clinic. Data included anatomic site, number and dates of office visits, treatment modality and cost. RESULTS: Women required average of 3.01 visits and average cost of $291.36 to reach clearance and males 2.35 visits and $301.81. Monotherapy TCA required 3.2 visits and $263.65 while cryotherapy alone required 3.3 visits and $481.97. Initial imiquimod monotherapy required 2.3 visits and $217.62. Combination therapy of imiquimod and trichloroacetic acid averaged 1.5 visits and $236.53. The largest reduction in visits and cost occurred in patients with multiple or recurrent EGW and those requiring >3 visits. From these data an EGW treatment algorithm was developed allowing more effective management and better utilization of health care resources. CONCLUSION: In the PPFA clinic setting, imiquimod alone or in combination should be initial treatment for patients with multiple or recurrent EGW or for those who do not experience complete clearance by the third clinic visit when nonimiquimod therapy is first employed.

Achieving tight glycaemic control.

The implementation of tight glycaemic control (TGC) is becoming accepted best practice within intensive care units throughout the world. It is recommended by the Surviving Sepsis Campaign and is included in the sepsis care bundle. The major impact of TGC is currently thought to be associated with reduced morbidity and mortality. The process of achieving TGC is, however, not without risk. In particular, the need for frequent, accurate blood glucose measurement and the possibility of prolonged, unrecognised hypoglycaemia are of concern. There is also the potential for patients who exhibit significant insulin resistance to require the administration of large amounts of insulin. The transfer of patients from the intensive care unit to the operating theatre or for computerised tomography during intensive insulin therapy is also hazardous. The purpose of this paper is to describe a series of nurse led pilot studies which aimed to introduce the process of TGC whilst maintaining patient safety. The results demonstrate the effectiveness of a staged approach and the achievement of TGC.

Is severity of chest pain a cue for women and men to recognize acute myocardial infarction symptoms as cardiac in origin?

Recognizing symptoms as cardiac in origin is associated with the prompt seeking of medical care in patients with acute myocardial infarction (AMI). Therefore, the authors compared the symptom attribution of men and women experiencing AMI and examined factors associated with cardiac attribution by sex. In a cross-sectional study, a total of 1059 AMI patients were consecutively recruited across 5 countries. A structured interview was performed during hospitalization. Approximately 40% of both men and women interpreted their symptoms as cardiac in origin. In men, a history of coronary heart disease (CHD) and chest pain severity were significantly associated with symptom interpretation as cardiac in origin (odds ratio [OR], 4.0; 95% confidence interval [CI], 2.9-5.6; OR, 2.0; 95% CI, 1.4-2.7, respectively). In women, a history of CHD was also significantly associated with symptom interpretation as cardiac in origin (OR, 4.95; 95% CI, 2.39-10.25), but not severity of chest pain. As opposed to men, severe chest pain may not be a cue for women to interpret their symptom as cardiac in origin. Education and counseling must take sex differences into account to be effective.

Investigating the effectiveness of critical care outreach services: a systematic review.

OBJECTIVE: We explored the impact of critical care outreach activity on patient and service outcomes and aimed to contribute to developing a typology of critical care outreach services. DESIGN: Following a sample search of Medline 15 relevant electronic databases were systematically searched from 1996 to 2004. Searches for publications from nine key authors and citations of eight key articles were performed. Hand searches of journals, bibliographies of reports and review articles, and conference abstracts were conducted. Relevant experts were contacted. A further two studies published after the review date were also included. Two reviewers assessed studies for inclusion, conducted quality assessment and extracted data. Data were synthesised using narrative techniques. MEASUREMENTS AND RESULTS: Seventeen papers and six brief reports were selected for inclusion from a list of 1,760 titles. As anticipated with a relatively new service such as critical care outreach, there were few controlled trials. There were two randomised controlled trials, 16 uncontrolled before and after studies, three quasi-experimental studies, one controlled before and after study and one post-only controlled study. The most frequent outcomes measured were mortality, cardiac arrest, unplanned critical care admissions from wards, length of stay, and critical care readmission rates. CONCLUSIONS: Although improvements in patient outcomes were found, the evidence in this review is insufficient to demonstrate this conclusively. The many differences in service delivery do not permit identification of service typology. Our findings point to a need for more comprehensive research of this expanding service in the United Kingdom.

An international perspective on gender differences in anxiety early after acute myocardial infarction.

OBJECTIVE: Higher anxiety is linked to poorer outcomes after acute myocardial infarction (AMI), including increased in-hospital reinfarction and potentially life-threatening complications. If clinicians can identify patients at greatest risk for anxiety after AMI, they can institute early treatment. Previous research on the influence of gender on the incidence of anxiety post-AMI reflects inconsistent findings, and differences across cultures have not been studied. Therefore, the purposes of this study were to determine: 1) whether there are gender differences in anxiety in a diverse international sample of AMI patients, and 2) whether there was an interaction between gender and sociodemographic and clinical variables thought to influence anxiety. METHODS: In this prospective, comparative study, 912 AMI patients were enrolled from Australia, South Korea, Japan, England, and the United States. Anxiety was assessed, using the Brief Symptom Inventory, within the first 72 hours of admission to the hospital for AMI symptoms. RESULTS: Women had higher anxiety levels than men (0.76 +/- 0.90 vs. 0.57 +/- 0.70, p =.005), and this pattern of higher anxiety in women was seen in each country studied. Neither sociodemographic nor clinical variables interacted with gender to influence anxiety. CONCLUSION: Across a variety of cultures, women have higher anxiety than men after AMI and this relationship is independent of age, education level, marital status, or presence of comorbidities or severity of AMI.

Achieving compliance with the European Working Time Directive in a large teaching hospital: a strategic approach.

This paper describes the strategy which achieved European Working Time Directive (EWTD) compliance at the Royal Free Hampstead NHS Trust in medicine and surgery. Compliance with EWTD regulations was assessed by diary card exercise, clinical care assessed through critical incident reports, electronic handover documents and nursing reports, training opportunities assessed by unit training directors, cost controls assessed by finance department analysis, and workload assessed by staff attendance on wards, in casualty and in theatres. There was a change in focus of care to a consultant-led, specialist registrar- (SpR-)driven service extending into evenings and on weekends, coupled with a move to a multi-skilled team for night cover, and to a move from traditional on-call shifts to a full shift system across both medicine and surgery. Compliance with the EWTD was achieved whilst maintaining good standards of clinical care, ensuring training opportunities for doctors in training, controlling payroll costs, removing the need for locums, and reducing workload for both junior doctors and consultants.

Update on cervical cancer screening. Current diagnostic and evidence-based management protocols.

Pap smear screening for cervical cancer has been a preventive health success. Although improved technology is increasing the accuracy of this technique, more women who have never been tested will need to undergo screening in order to further decrease the incidence of cervical cancer in the United States. The establishment of infection with high-risk genital HPV types as a causative factor in cervical cancer is a major breakthrough in understanding of this disease. Testing for the presence of high-risk HPV DNA should increase the ability to identify women who are truly at risk for cancer and true cancer precursors and to more efficiently plan further diagnostic evaluation. The 2001 revisions in TBS reflect our improved understanding of the epidemiology and natural history of cervical epithelial abnormalities and cervical cancer. These revisions are designed to facilitate communication between the clinician and the laboratory and to improve the clinician's ability to accurately interpret the cytology report and plan initial management of any abnormalities.

"Realising the potential of critical care nurses": an exploratory study of the factors that affect and comprise the nursing contribution to the recovery of critically ill patients.

BACKGROUND: This study seeks to make evident the complexity of issues associated with the delivery of care by nurses to the critically ill. Emphasis had been placed on the results and implications of these for nursing practice. For a more in-depth account, the full report can be accessed on www.lscn.co.uk. METHOD: Following multi-centre research ethics committee approval, 10 critical care units participated in the 3-month study. Data collection comprised 231 nurse interviews and 51 relative interviews during 33 observation participation periods. RESULTS: Analysis demonstrated that the context of the critical care unit, in terms of geographical layout, unit activity, case mix and skill mix of nurses, had a major effect on the ability of nurses to contribute to the recovery of the critically ill. The effectiveness of the nursing resource appeared to be a function of knowledge (theoretical and patient related), experience and exposure. Nurses who were unused to a particular environment were not seen to be as effective as those who were. A model was constructed that identified the central tenets upon which nursing care can be optimised or compromised. When nursing care was optimised the difference nurses made potentially decreased risk to patients, enabled timely patient progression and increased the potential for patient recovery. CONCLUSIONS: The results confirm that nurses have a significant contribution to make in the recovery of patients who have experienced critical illness. Recommendations are far reaching and include the need to develop a valid and reliable tool which addresses patients' need for nursing in terms of nurses' knowledge and experience, patient dependency and decreasing clinical risk across the continuum of care. Current nursing workload tools and patient:nurse ratios were seen to lack validity because they do not appraise the context in which care is delivered, define all nurses as equal and concentrate on activity rather than the effect nurses can have on the outcome of the critically ill.

Moving on from 'patient dependency' and 'nursing workload' to managing risk in critical care.

The contribution nurses make to the management of critically ill patients is usually appraised through the use of concepts such as 'patient dependency' or 'nursing workload'. These concepts fail to address the knowledge, skills and experience of nurses and consequently fail to acknowledge the risk presented by critically ill patients. This paper describes the development of a tool that attempts to measure risk and the process of risk management undertaken by nurses who coordinate the shifts and lead the nursing team. The results of this pilot study indicated that the tool was valid, but reliability has not yet been demonstrated. Thus the tool requires further refinement and testing. We chose to publish at this time because we feel the paper offers a new way of examining the contribution of nurses working in critical care.