A data-driven approach for the detection of internal standard outliers in targeted LC-MS/MS assays.

Heavy-labelled internal standard (IS) compounds are commonly used in liquid chromatography-tandem mass spectrometry (LC-MS/MS) assays to control for stochastic and systematic variation. Identifying samples that suffer from unwanted variation is critically important in order to avoid factitiously inaccurate results. Current approaches for outlier detection typically employ arbitrary thresholds and ignore systematic drift. To improve this, we applied robust linear mixed-effects models (LMMs) to capture the within- and between-run variability in IS signal and generate data-driven acceptance ranges for routine use. Data from in-house LC-MS/MS assays for 25-hydroxyvitamin D3 and D2 and prednisolone were retrospectively collected. The variation in the percentage deviation of the internal standard area from the mean of the calibrators was modelled through the use of robust LMMs. The fitted LMMs revealed significant positive drift in IS signal over the analytical runs for vitamin D, with slope coefficients of 0.118 (95% CI: 0.098, 0.138) and 0.192 (0.168, 0.215) for D3 and D2, respectively. In contrast, the models for prednisolone demonstrated a significant negative drift in IS signal, with a slope coefficient of -0.164 (-0.297, -0.036). Non-parametric, cluster bootstrap resampling enabled us to define acceptance ranges for use in future assays. Here, we have described a computational approach to extensively characterise the variation in IS signal in routinely-performed LC-MS/MS assays. This approach facilitates a robust quality assessment of IS outliers in routine practice and thus has the potential to improve patient safety. Importantly, this approach is applicable to other MS assays where linear variation in IS signal is observed.

The Development and Implementation of a Commissioned Pathway for the Identification and Stratification of Liver Disease in the Community.

OBJECTIVE: To describe the development of the Nottingham liver disease stratification pathway, present a 12-month evaluation of uptake, stratification results and compare the pathway to current British Society of Gastroenterology (BSG) guidelines. DESIGN: A referral pathway between primary and secondary care for the detection and risk stratification of liver disease. SETTING: Four Nottinghamshire Clinical Commissioning Groups (700,000 population). PATIENTS: Patients are referred to the pathway with i) raised AST/ALT ratio ii) harmful alcohol use or iii) risk or presence of non-alcoholic fatty liver disease (NAFLD). INTERVENTIONS: Clinic attendance within secondary care for transient elastography (TE) and brief lifestyle intervention. The TE result is reported back to the GP with advice on interpretation and referral guidance. MAIN OUTCOME MEASURES: Pathway uptake, patient characteristics, liver disease stratification results and stakeholder feedback. RESULTS: Over the first 12 months 968 patients attended a TE clinic appointment, with raised AST/ALT ratio being the most common single reason for referral (36.9%). Of the total, 222 (22.9%) patients had an elevated liver stiffness (≥8kPa) and in 60 (27.0%) liver stiffness was indicative of advanced chronic liver disease. If a traditional approach based on raised liver enzymes (BSG guidance) had been followed, 38.7% of those with significant liver disease (≥8kPa) would have gone undetected among those referred for either NAFLD or raised AST:ALT. CONCLUSIONS: Targeting patients with risk factors for chronic liver disease and stratifying them using TE can detect significant chronic liver disease above and beyond the approach based on liver enzyme elevation.

The application of faecal egg count results and statistical inference for clinical decision making in foals.

This study investigated the impact of variability in Parascaris spp. and strongyle faecal egg counts (FEC) from foals on treatment decision-making and detection of a patent infection. A single faecal sample was collected once daily for three days from 53 foals and a FEC was performed on three separate portions of each sample (total of nine egg counts per foal). Differences in the decision to administer an anthelmintic using the results of a single count (C1), the mean of three (X¯1-3) or nine counts (X¯1-9) and the upper 5% confidence limit of the gamma confidence interval (CI) of the estimate of the distribution mean (µ) from three (UCL1-3) and nine counts (UCL1-9) were determined for a range of egg count thresholds. The UCL1-9 was used as the best estimate of µ, hypothesis testing for treatment and the comparison of treatment decision-making using C1, X¯1-3, X¯1-9 and UCL1-3. The results of this study demonstrated that a point estimate (C1 or X¯1-3) was of limited value for estimating the distribution mean of egg counts in faeces and there was overall poor agreement in treatment decision-making for individual foals using C1 compared with UCL1-9. Of the foals with C1 of zero eggs per gram, 54% and 47% had Parascaris and strongyle eggs in subsequent counts, respectively. The egg density in faeces is inhomogeneous, resulting in considerable variability in egg count results for an individual foal: between faecal piles, different portions of a faecal pile and days. The use of the negative binomial distribution CI for µ takes this variability into account and is recommended for use when interpreting FEC data from horses.

Obesity and type 2 diabetes are important risk factors underlying previously undiagnosed cirrhosis in general practice: a cross-sectional study using transient elastography.

BACKGROUND: Rising cirrhosis incidence and mortality in the United Kingdom has been attributed predominantly to excess alcohol consumption. However, metabolic risk factors such as Type 2 diabetes and obesity may also be important. AIM: To screen at-risk individuals in general practice for undetected cirrhosis using transient elastography and study the risk factors underlying these cases. METHODS: The study was undertaken in 4 general practices (adult patient population 20 868) between February 2012 and September 2014. Patients with defined risk factors for chronic liver disease (hazardous alcohol use and/or Type 2 diabetes) were identified from the General Practice electronic records and invited for transient elastography. Elevated liver stiffness was defined as ≥8 kPa. Cirrhosis was confirmed by established histological, radiological and biochemical methods. RESULTS: Two thousand three hundred and sixty eight patients were invited for transient elastography and 899/919 who attended (97.8%) had valid measurements. Of these 230 patients had elevated liver stiffness (25.6%) and 27 had cirrhosis (2.9%). Risk factors for new cirrhosis diagnoses were obesity and/or Type 2 diabetes in 16 patients (59.3%), alcohol alone in 3 (11.1%) and both alcohol and obesity and/or diabetes in eight (29.6%). Presence of cirrhosis was significantly increased in obese patients with Type 2 diabetes or hazardous alcohol use compared to non-obese (odds ratio 9.4 [95% CI 2.2-40.9] and 5.6 [95% CI 1.6-19.7] respectively). CONCLUSIONS: The number of new cases of cirrhosis diagnosed clearly demonstrates that existing estimates of prevalence are likely to be gross underestimates. Obesity was an important risk factor for cirrhosis within both alcohol users and diabetics.

Spontaneous central venous thrombosis and shunt occlusion following peritoneovenous shunt placement for intractable ascites.

A 43-year-old man had a peritoneovenous shunt inserted for the treatment of chylous ascites secondary to myelofibrosis. Despite being on anticoagulation for superior mesenteric vein thrombosis, he developed shunt dysfunction within two weeks of insertion. Superior venacavography showed multiple filling defects in the right axillary vein, no filling of the right brachiocephalic and right subclavian vein, and thrombotic occlusion of the internal jugular veins bilaterally. The shunt was removed 11 days after insertion, and there was extensive thrombosis of the venous end of the shunt and the compressible pump chamber. Shunt thrombosis is known to occur but remains a rare complication, with 87% of such obstructions being due to a thrombus at the tip of the venous end of the shunt. Extensive thrombosis of the shunt (as in the present case) is very rare.

Oxidative stress downstream of mTORC1 but not AKT causes a proliferative defect in cancer cells resistant to PI3K inhibition.

Compounds targeting phosphatidylinositol-3-kinase/mammalian target of rapamycin (PI3K/mTOR) signaling are being investigated in multiple clinical settings, but drug resistance may reduce their benefit. Compound rechallenge after drug holidays can overcome such resistance, yet little is known about the impact of drug holidays on cell biochemistry. We found that PI3K inhibitor (PI3Ki)-resistant cells cultured in the absence of PI3Ki developed a proliferative defect, increased oxygen consumption and accumulated reactive oxygen species (ROS), leading to lactate production through hypoxia-inducible factor-1α. This metabolic imbalance was reversed by mammalian target of rapamycin complex 1 (mTORC1) inhibitors. Interestingly, neither AKT nor c-MYC was involved in mediating the metabolic phenotype, despite the latter contributing to resistant cells' proliferation. These data suggest that an AKT-independent PI3K/mTORC1 axis operates in these cells. The excessive ROS hampered cell division, and the metabolic phenotype made resistant cells more sensitive to hydrogen peroxide and nutrient starvation. Thus, the proliferative defect of PI3Ki-resistant cells during drug holidays is caused by defective metabolic adaptation to chronic PI3K/mTOR pathway inhibition. This metabolic imbalance may open the therapeutic window for challenge with metabolic drugs during drug holidays.

Inhomogeneity of the density of Parascaris spp. eggs in faeces of individual foals and the use of hypothesis testing for treatment decision making.

Faecal egg counts (FEC) are used widely for monitoring of parasite infection in animals, treatment decision-making and estimation of anthelmintic efficacy. When a single count or sample mean is used as a point estimate of the expectation of the egg distribution over some time interval, the variability in the egg density is not accounted for. Although variability, including quantifying sources, of egg count data has been described, the spatiotemporal distribution of nematode eggs in faeces is not well understood. We believe that statistical inference about the mean egg count for treatment decision-making has not been used previously. The aim of this study was to examine the density of Parascaris eggs in solution and faeces and to describe the use of hypothesis testing for decision-making. Faeces from two foals with Parascaris burdens were mixed with magnesium sulphate solution and 30 McMaster chambers were examined to determine the egg distribution in a well-mixed solution. To examine the distribution of eggs in faeces from an individual animal, three faecal piles from a foal with a known Parascaris burden were obtained, from which 81 counts were performed. A single faecal sample was also collected daily from 20 foals on three consecutive days and a FEC was performed on three separate portions of each sample. As appropriate, Poisson or negative binomial confidence intervals for the distribution mean were calculated. Parascaris eggs in a well-mixed solution conformed to a homogeneous Poisson process, while the egg density in faeces was not homogeneous, but aggregated. This study provides an extension from homogeneous to inhomogeneous Poisson processes, leading to an understanding of why Poisson and negative binomial distributions correspondingly provide a good fit for egg count data. The application of one-sided hypothesis tests for decision-making is presented.

A national survey of the provision of ultrasound surveillance for the detection of hepatocellular carcinoma.

OBJECTIVE: Hepatocellular carcinoma (HCC), the sixth most common cancer worldwide and third most common cause of cancer related death, is closely associated with the presence of cirrhosis. Survival is determined by the stage of the cancer, with asymptomatic small tumours being more amenable to treatment. Early diagnosis is dependent on regular surveillance and the primary objective of this survey was to gain a better understanding of the baseline attitudes towards and provision of ultrasound surveillance (USS) HCC surveillance in the UK. In addition, information was obtained on the stages of cancer of the patients being referred to and discussed at regional multidisciplinary team meetings. DESIGN: UK hepatologists, gastroenterologists and nurse specialists were sent a questionnaire survey regarding the provision of USS for detection of HCC in their respective hospitals. RESULTS: Provision of surveillance was poor overall, with many hospitals lacking the necessary mechanisms to make abnormal results, if detected, known to referring clinicians. There was also a lack of standard data collection and in many hospitals basic information on the number of patients with cirrhosis and how many were developing HCC was not known. For the majority of new HCC cases was currently being made only at an incurable late stage (60%). CONCLUSIONS: In the UK, the current provision of USS based HCC surveillance is poor and needs to be upgraded urgently.

Reforming maternity services in Australia: Outcomes of a private practice midwifery service.

BACKGROUND AND AIMS: recent legislative changes in Australia have enabled eligible midwives to provide private primary maternity care with fee rebates through Medicare. This paper (1) discusses these changes affecting midwifery practice; (2) describes Australia's first private midwifery service with visiting rights to hospital for labour and birth care since Medicare funding for midwives was introduced in 2010; and (3) compares outcomes with National Core Maternity Indicators. METHODS: an audit of all client records (n=323) for the survey period from September 2012 to February 2014 was undertaken. Data were extracted and compared with the 10 perinatal indicators using Chi square statistics. FINDINGS: this convenience sample of all-risk women was similar to the national birthing population for age and parity. Compared to national indicators, women were significantly more likely to have spontaneous commencement of labour (79.6% versus 54.8%) (χ(2)=79.88, p<.001), lower rates of induction (10.2% versus 26%) (χ(2)=79.88, p<.001), and not require pharmacological pain relief (54.8% versus 23.9%) (χ(2)=152.2, p<.001). The majority of women had a normal vaginal birth (70.3% versus 55.1%) (χ(2)=28.13, p<.001). The caesarean section rate (22% versus 32.3%) was significantly lower (χ(2)=15.64, p<.001) than the national rate. Average gestation of neonates was 39.3 weeks; average birth weight was 3525 gms, and fewer required transfer to the special care nursery (8.4% versus 15.3%) (χ(2)=11.89, p<.001). DISCUSSION: this is the first report of maternal and neonatal outcomes for a private midwifery service in Australia since the introduction of access to Medicare for midwives. Maternal and newborn outcomes were statistically better than national rates. Routinely reporting and publishing clinical outcomes needs to become the norm for private maternity care. CONCLUSIONS: this private midwifery caseload model has been instrumental in the ground-breaking change to primary maternity services that extends women׳s access to safe midwifery care in Australia. The potential impact of private practicing midwives to align maternity care with the best available evidence is significant.

Poor tolerability of thalidomide in end-stage oesophageal cancer.

Oesophageal cancer cachexia is a significant clinical problem, resulting in excessive morbidity and mortality. In a pilot study, 10 patients with cachexia due to advanced cancer of the oesophagus gained weight, including lean tissue, after 14-day treatment with thalidomide. Here, we present randomised placebo controlled trial data over a 6-week period to test the hypothesis that thalidomide is superior to placebo in terms of weight gain in patients with cachexia caused by oesophageal cancer. Thalidomide, 200 mg daily, or an identical placebo was given to patients with advanced oesophageal cancer. Total body weight and lean body mass were assessed in addition to drug tolerability and performance indices. Thirty-four patients were recruited. Of these, six given thalidomide and 16 given placebo completed the protocol; all withdrawals were due to adverse drug reactions or complications of disease. Thalidomide showed no benefit over placebo in participants who completed the protocol. These data suggest that thalidomide is poorly tolerated in patients with advanced cancer of the oesophagus and may not ameliorate the progression of cachexia. In the absence of hard supportive evidence, off-licence treatment with thalidomide should be used with great caution as an adjunct to nutritional support in patients with advanced cancer.

Diepoxybutane and diepoxyoctane interstrand cross-linking of the 5S DNA nucleosomal core particle.

Diepoxyalkanes form interstrand cross-links in DNA oligomers preferentially at 5'-GNC sites. We have examined cross-linking by 1,2,3,4-diepoxybutane (DEB) and 1,2,7,8-diepoxyoctane (DEO) within a fragment of the 5S RNA gene of Xenopus borealis in both the free and nucleosomal states. Sites and efficiencies of interstrand cross-linking were probed through denaturing polyacrylamide gel electrophoresis and quantitative phosphorimagery. Both agents targeted 5'-GNC sites for cross-linking in the restriction fragment in its free state, and DEO also targeted 5'-GNNC sites. Monoalkylation occurred at all deoxyguanosines. The sites for both monoalkylation and interstrand cross-linking were similar in nucleosomal and free DNA, and cross-linked DNA was cleanly incorporated into the core particle structure. These findings suggest that the 5S core particle is able to tolerate any structural abnormalities induced by diepoxide cross-linking.

cis- and trans-diamminedichloroplatinum(II) interstrand cross-linking of a defined sequence nucleosomal core particle.

Interstrand cross-linking studies with the antitumor drug cis-diamminedichloroplatinum(II) and its clinically inactive isomer, trans-diamminedichloroplatinum(II), were performed on a fragment of the 5S rRNA gene of Xenopus borealis in the free and nucleosomal state. 5S nucleosomes were formed via histone octamer exchange from chicken erythrocyte core particles. Native polyacrylamide gel electrophoresis was used to probe the ability of platinated DNA to reconstitute into core particles. Both isomers negatively impacted reconstitution when histones were present during incubation with the drug. When histones were not present during the drug treatment, platinated DNA was successfully reconstituted into core particles. These results suggest that platination of histones impedes reconstitution of free DNA. However, already-formed core particles were not disrupted upon platination. Sites of interstrand cross-linking were probed through denaturing polyacrylamide gel electrophoresis and quantitative phosphorimagery. We found both site-specific enhancement and depression of cis-diamminedichloroplatinum(II) cross-linking in the nucleosomal samples relative to free DNA at both drug concentrations that were tested (0.01 and 0.0025 mM). trans-Diamminedichloroplatinum(II) exhibited no detectable differences in the interstrand cross-linking of free and nucleosomal samples.

Enzymatic capacities for beta-oxidation of fatty fuels are low in the gill of teleost fishes despite presence of fatty acid-binding protein.

A variety of circulating fuels can support the work of the teleost gill. Previous work indicates, however, that unlike other aerobic tissues from teleosts, the gill may have a limited capacity to oxidize fatty fuels. We determined capacities for catabolism of carbohydrate, fatty acids, and amino acids in four species of temperate marine or euryhaline teleosts representing distinct lineages. In addition, we assessed the capacity for fatty acid oxidation in the gill from an Antarctic species. Activities of rate-limiting or regulatory enzymes from pathways of energy metabolism were measured at physiological temperatures (15 degrees or 1 degrees C). In the temperate species, ATP yields from glucose are 3- to 30-fold greater (varying with species) than ATP yields from a monounsaturated fatty acid, while ATP generation from glutamate is 2-50 times greater than similar capacities for the lipid fuel. Like the temperate species, capacity for beta-oxidation of fatty acids is limited in the Antarctic species. A positive linear correlation between activities of citrate synthase (central pathway of oxidative metabolism) and hexokinase (glycolysis) adds further support to the hypothesis that glucose is a preferred metabolic fuel in gill. Our results also demonstrate that fatty acid-binding protein is present in the gill of teleost fishes. It is likely that this protein plays a more important role facilitating anabolic pathways in lipid metabolism rather than fatty acid oxidation in the gill of teleost fishes.

Restaurant wheelchair accessibility.

This study was designed to determine the compliance of restaurants to the wheelchair accessibility standards set forth in the Uniform Federal Accessibility Standards. The standards that were operationalized in this study are also found in Title III of the Americans With Disabilities Act of 1990. The data were collected at 120 sites in three midwestern states. For one who uses a wheelchair, parking the car is often an obstacle to eating out. Only 53% of the restaurants surveyed provide handicapped parking. Entering the building may also be a problem. Of the restaurants that required a ramp, only 66% provided them. Inside the restaurant, the key problems were accessible rest-rooms and the height of tables. The study provided comparisons between restaurants in rural and urban settings, as well as comparisons between conventional restaurants and fast food restaurants. No notable differences emerged for these comparisons.