Comparative evaluation of different molecular methods for DNA extraction from individual Teladorsagia circumcincta nematodes.

BACKGROUND: The purpose of this study was to develop a reliable DNA extraction protocol to use on individual Teladorsagia circumcincta nematode specimens to produce high quality DNA for genome sequencing and phylogenetic analysis. Pooled samples have been critical in providing the groundwork for T. circumcincta genome construction, but there is currently no standard method for extracting high-quality DNA from individual nematodes. 11 extraction kits were compared based on DNA quality, yield, and processing time. RESULTS: 11 extraction protocols were compared, and the concentration and purity of the extracted DNA was quantified. Median DNA concentration among all methods measured on NanoDrop 2000™ ranged between 0.45-11.5 ng/µL, and on Qubit™ ranged between undetectable - 0.962 ng/µL. Median A260/280 ranged between 0.505-3.925, and median A260/230 ranged - 0.005 - 1.545. Larval exsheathment to remove the nematode cuticle negatively impacted DNA concentration and purity. CONCLUSIONS: A Schistosoma sp. DNA extraction method was determined as most suitable for individual T. circumcincta nematode specimens due to its resulting DNA concentration, purity, and relatively fast processing time.

Bioinformatic analysis of eosinophil activity and its implications for model and target species.

Eosinophils are important immune cells that have been implicated in resistance to gastrointestinal nematode (GIN) infections in both naturally and experimentally infected sheep. Proteins of particular importance appear to be IgA-Fc alpha receptor (FcαRI), C-C chemokine receptor type 3 (CCR3), proteoglycan 3 (PRG3, major basic protein 2) and EPX (eosinophil peroxidase). We used known human nucleotide sequences to search the ruminant genomes, followed by translation to protein and sequence alignments to visualize differences between sequences and species. Where a sequence was retrieved for cow, but not for sheep and goat, this was used additionally as a reference sequence. In this review, we show that eosinophil function varies among host species. Consequently, investigations into the mechanisms of ruminant immune responses to GIN should be conducted using the natural host. Specifically, we address differences in protein sequence and structure for eosinophil proteins.

Automated identification of intensive animal production locations from aerial photography.

OBJECTIVE: Successful control of an emergency animal disease outbreak requires the timely and accurate identification of properties of interest. The identification of commercial piggeries within study areas in the Goulburn-Murray Irrigation District in Victoria, Australia, is used to demonstrate the innovative application of object-based image analysis (OBIA) techniques for the identification of intensive animal production land uses, to improve the accuracy of existing datasets. METHODS: Characteristics of infrastructure and landscape features were combined to form a commercial piggery identification algorithm. These criteria were applied to recent aerial photography that had been classified using OBIA techniques. The results were then compared with three datasets containing known commercial piggery locations and visually checked by roadside surveys. RESULTS: The OBIA technique identified 21 potential piggery locations across three study areas, 14 of which were identified in existing databases. Of the 7 additional sites, 4 were dairy properties, 1 was a cropping and sheep property and 2 were previously undocumented piggery locations. CONCLUSIONS: The OBIA approach has potential of OBIA for identifying the locations of commercial piggeries. Further development and testing will determine how generic this approach is in terms of industry type and operation size. The method described is cost-effective, automated and repeatable, and could be used to regularly update existing databases by analysing newly acquired aerial imagery to identify possible land use changes. This would improve the reliability of currently available data and increase the effectiveness of a biosecurity response during an emergency animal disease outbreak.

Traumatic central cord syndrome: neurological and functional outcome at 3 years.

STUDY DESIGN: Retrospective cohort analysis with prospective follow-up. OBJECTIVES: To evaluate neurological and functional recovery following central cord syndrome. SETTING: Northern Ireland, population 1.8 million. METHODS: Twenty-seven cords were identified in 1 year. Five managed conservatively and 22 with surgery. American Spinal Injury Association (ASIA) motor scores (AMS) were calculated to assess neurological recovery. Rotterdam scores assessed functional independence at 3 years. RESULTS: Average age was 62 years. Mechanism of injury was a fall with neck hyperextension in 81% patients. Average AMS in surgical patients improved from injury, preoperatively, postoperatively, 6 months and 3 years from 51, 81, 83, 90 to 96, respectively. Conservative patients improved from time of injury to day 10 from 57 to 86 and then fell to 84 at 6 months. By 3 years, this had recovered to 91. There was no statistical significant difference in AMS (P=0.15)/change in AMS (ΔAMS) (P=0.92) or percentage of motor deficit resolution (P=0.23) between groups at 3 years. Two patients underwent surgery within 48 h and achieved full motor recovery by 3 years, but this was not significant (P=0.2). ASIA score improvement had a positive correlation with age at injury. Patients treated with surgery had better Rotterdam scores at 3 years than those managed conservatively (P=0.05). CONCLUSIONS: This study confirms the natural history of central cord syndrome. Although it demonstrates equivocal neurological recovery for both groups, patients treated with surgery regained a greater degree of functional independence.

Regulation of hindbrain Pyy expression by acute food deprivation, prolonged caloric restriction, and weight loss surgery in mice.

PYY is a gut-derived putative satiety signal released in response to nutrient ingestion and is implicated in the regulation of energy homeostasis. Pyy-expressing neurons have been identified in the hindbrain of river lamprey, rodents, and primates. Despite this high evolutionary conservation, little is known about central PYY neurons. Using in situ hybridization, PYY-Cre;ROSA-EYFP mice, and immunohistochemistry, we identified PYY cell bodies in the gigantocellular reticular nucleus region of the hindbrain. PYY projections were present in the dorsal vagal complex and hypoglossal nucleus. In the hindbrain, Pyy mRNA was present at E9.5, and expression peaked at P2 and then decreased significantly by 70% at adulthood. We found that, in contrast to the circulation, PYY-(1-36) is the predominant isoform in mouse brainstem extracts in the ad libitum-fed state. However, following a 24-h fast, the relative amounts of PYY-(1-36) and PYY-(3-36) isoforms were similar. Interestingly, central Pyy expression showed nutritional regulation and decreased significantly by acute starvation, prolonged caloric restriction, and bariatric surgery (enterogastroanastomosis). Central Pyy expression correlated with body weight loss and circulating leptin and PYY concentrations. Central regulation of energy metabolism is not limited to the hypothalamus but also includes the midbrain and the brainstem. Our findings suggest a role for hindbrain PYY in the regulation of energy homeostasis and provide a starting point for further research on gigantocellular reticular nucleus PYY neurons, which will increase our understanding of the brain stem pathways in the integrated control of appetite and energy metabolism.

Addressing the question of the effect of RBC storage on clinical outcomes: the Red Cell Storage Duration Study (RECESS) (Section 7).

The question of whether storage of red blood cells (RBCs) alters their capacity to deliver oxygen and affects patient outcomes remains in a state of clinical equipoise. Studies of the changes which occur while RBCs are stored have led to several physiologically plausible hypotheses that these changes impair RBC function when the units are transfused. Although there is some evidence of this effect in vivo from animal model experiments, the results of several largely retrospective patient studies have not been consistent. Some studies have shown an association between worse clinical outcomes and transfusion of RBC which have been stored for longer periods of time, while others have found no effect. Three multicenter, randomized, controlled trials have been developed to address this important, but currently unanswered, question. Two clinical trials, one in low birth weight neonates and the other in intensive care unit patients, are enrolling subjects in Canada (the Age of Red Blood Cells in Premature Infants; the Age of Blood Study). The third trial, which is being developed in the United States, is the Red Cell Storage Duration Study (RECESS). This is a multicenter, randomized, controlled trial in which patients undergoing complex cardiac surgical procedures who are likely to require RBC transfusion will be randomized to receive RBC units stored for either 10 or fewer days or 21 or more days. Randomization will only occur if the blood bank has enough units of RBC of both storage times to meet the crossmatch request; hence, subjects randomized to the 21 day arm will receive RBC of the same storage time as they would have following standard inventory practice of "oldest units out first". The primary outcome is the change in the Multiple Organ Dysfunction Score (MODS), a composite measure of multiorgan dysfunction, by day 7. Secondary outcomes include the change in the MODS by day 28, all-cause mortality, and several composite and single measures of specific organ system function. The estimated total sample size required will be 1434 evaluable subjects (717 per arm).

Technical issues in neonatal transfusions.

Neonatal transfusions provide challenges at several steps in the process. Neonates are often transfused with relatively small volumes at slow flow rates from syringes,whereas at other times they require relatively massive transfusions or exchange transfusions. To facilitate these specialized transfusions, blood banks often modify their procedures to provide small volumes of blood components that are sometimes dispensed in syringes or to reconstitute whole blood for exchange transfusions. Hospitals must implement policies and procedures to ensure that the blood components are transfused safely when using these specialized techniques for infants. Nevertheless, some issues remain in many hospitals, such as the difficulty in safely warming blood components for neonatal transfusions and the difficulties in using approved labels for small containers that are sometimes prepared at the bedside.

Early weaning is related to weight and rate of weight gain in infancy.

BACKGROUND: Evidence relating to the effect of early weaning on infant weight and weight gain is conflicting. The aim of this paper is to compare weight and weight gain in two groups of infants; one group weaned before 4 months (early weaned), the other weaned at 4 months or after. METHODS: Feeding practices were assessed through semistructured interviews with mothers of 1-year-old infants. Weight at birth, 8 weeks and 7 months were taken from an administrative database (Child Health System), and an additional measure of weight was taken at 14 months. RESULTS: Infants weaned early were heavier at 7 and 14 months, and gained more weight between 8 weeks and 14 months, even after breastfeeding was controlled for. CONCLUSION: Early weaning is related to rapid weight gain in infancy. This may have implications for childhood obesity.

The effects of changing temperature correction factors on measures of acidity calculated from gastric and oesophageal pH recordings.

BACKGROUND: Recently, Medtronic notified customers that new correction factors should be used for their Slimline and Zinetics24 single-use, internal-standard pH catheters. AIM AND METHODS: We selected 24-h recordings of oesophageal and gastric pH with the Zinetics24 from our archives for five healthy subjects and for five gastro-oesophageal reflux disease subjects who were studied at baseline and again after 8 days of treatment with a proton-pump inhibitor. All pH values obtained with the old correction factors were rescaled using the new correction factors. Values for median pH, integrated acidity and time pH < or = 4 were then calculated from pH values with old and new correction factors. RESULTS: The new correction factors changed values for median pH, integrated acidity and time pH < or = 4. Values for median pH and integrated acidity changed in a predictable, proportionate way, whereas values for time pH < or = 4 did not. CONCLUSIONS: The new correction factors will not change the interpretation of previously published results with median pH or integrated acidity. In contrast, values for time < or =4 cannot be converted in an obvious way with the new correction factors. Instead, the raw pH data will need to be rescaled and values for time pH < or = 4 recalculated using the rescaled pH data.

The fractal nature of human gastro-oesophageal reflux.

BACKGROUND: We are unaware of the analyses of time series data resulting from 24 h recordings of human gastric or oesophageal pH. As a result, we have no understanding of the quantitative changes in gastric or oesophageal acidity over time, the patterns that might characterize these changes, or the physiological significance of gastro-oesophageal reflux. AIM: To examine the time series for gastric and oesophageal pH. METHODS: Detrended fluctuation analysis and lag analysis were used to analyse data from 24 h recordings of oesophageal and gastric pH in five normal subjects and five subjects with gastro-oesophageal reflux disease. RESULTS: Analyses of the patterns of gastric and oesophageal pH over time in normal subjects and subjects with gastro-oesophageal reflux disease indicate that the fluctuations in pH are self-similar across different time scales and are consistent with an underlying fractal process. Furthermore, there is a significant statistical association between sequential pH values separated by as much as 2.2 h. CONCLUSIONS: We hypothesize that the self-similar, fractal pattern encodes information about gastric acidity and that the oesophagus decodes this information and, when appropriate, may signal the stomach to reduce gastric acidity. Subjects with gastro-oesophageal reflux disease might have an impaired oesophageal-gastric feedback mechanism that results in increased gastric acid, which reflux from the stomach into the oesophagus.

Oesophageal pH has a power-law distribution in control and gastro-oesophageal reflux disease subjects.

BACKGROUND: We are unaware of any solid theoretical or pathophysiological basis for selecting pH 4 or any other pH value to assess oesophageal acid exposure or to define oesophageal reflux episodes. AIM: To examine the frequency of different oesophageal pH values in control and GERD subjects. METHODS: Oesophageal pH was measured for 24 h in 57 gastro-oesophageal reflux disease subjects and 26 control subjects. Histograms were constructed using the 21,600 values from each recording and bins of 0.25 pH units. RESULTS: Compared with controls, gastro-oesophageal reflux disease subjects had significantly more low pH values and significantly fewer high pH values. In both gastro-oesophageal reflux disease and control subjects, the frequency of oesophageal pH values was characterized by a power-law distribution indicating that the same relationship that describes low pH values also describes high pH values, as well as all values in between. CONCLUSIONS: The distribution of oesophageal pH values indicates that a variety of different pH values can be used to assess oesophageal acid exposure, but raises important questions regarding how oesophageal reflux episodes are defined.

Frequency analyses of gastric pH in control and gastro-oesophageal reflux disease subjects treated with a proton-pump inhibitor.

BACKGROUND: We are unaware of any solid theoretical or pathophysiological basis for selecting pH 4 or any other pH value to assess gastric acidity. AIM: To examine the frequency of different gastric pH values in control and GERD subjects. METHODS: Gastric pH was measured for 24 h in 26 control subjects, 26 gastro-oesophageal reflux disease subjects at baseline and the same 26 gastro-oesophageal reflux disease subjects during treatment with a proton-pump inhibitor. Histograms were constructed using the 21 600 values generated from each recording and bins of 0.25 pH units. RESULTS: The distribution of gastric pH values in gastro-oesophageal reflux disease subjects was significantly different from that in controls and in some instances the distributions detected significant differences that were not detected by integrated acidity. Proton-pump inhibitor treatment significantly altered the distribution of gastric pH values and the nature of this alteration during the postprandial period was different from that during the nocturnal period. Using time pH< or =4 can significantly underestimate the magnitude of inhibition of gastric acidity caused by a proton-pump inhibitor. CONCLUSIONS: The distribution of gastric pH values provides a rationale for selecting a particular pH value to assess gastric acidity. In some instances, the distribution of gastric pH values detects significant differences between gastro-oesophageal reflux disease and normal subjects that are not detected by integrated acidity.

The basis for the decreased response to proton pump inhibitors in gastro-oesophageal reflux disease patients without erosive oesophagitis.

BACKGROUND: The reason why heartburn in gastro-oesophageal reflux disease subjects without oesophagitis is less responsive to proton pump inhibitors than heartburn in those with erosive oesophagitis is not known. METHODS: Gastric and oesophageal pH were determined in 26 subjects with gastro-oesophageal reflux disease at baseline and on days 1, 2 and 8 of treatment with 20 mg omeprazole or 20 mg rabeprazole in a randomized, two-way cross-over fashion. The presence or absence of erosive oesophagitis at baseline was documented by upper gastrointestinal endoscopy. RESULTS: At a given value of the integrated gastric acidity during treatment with a proton pump inhibitor, the probability of pathological oesophageal reflux was significantly higher in subjects with no oesophagitis than in those with erosive oesophagitis. This occurred because the post-prandial gastric acidity in subjects with no oesophagitis showed a decreased response to the antisecretory agent. CONCLUSIONS: Compared with gastro-oesophageal reflux disease subjects with erosive oesophagitis, those with no oesophagitis are relatively refractory to the pharmacodynamic effects of proton pump inhibitors on the post-prandial integrated gastric acidity.

Heartburn severity can predict pathologic oesophageal reflux in gastro-oesophageal reflux disease patients treated with a proton-pump inhibitor.

BACKGROUND: In gastro-oesophageal reflux disease (GERD) subjects treated with a gastric anti-secretory agent, it is not known whether there is a relationship between heartburn severity and oesophageal acid exposure. METHODS: Oesophageal pH and heartburn severity were determined in 27 GERD subjects at baseline and on days 1, 2 and 8 of treatment with 20 mg omeprazole or 20 mg rabeprazole in a randomized, two-way crossover fashion. RESULTS: Receiver operating characteristic (ROC) analysis was used to determine values for heartburn severity that gave optimal cut-off points for distinguishing between normal and pathologic oesophageal reflux. Using these cut-off points, we found that the probability of no pathologic oesophageal reflux (Y) could be best fitted by an exponential equation: Y = a(e-bX) + c, where a, b and c are constants and X is the value of heartburn severity. There was close agreement between predicted and observed percentages of subjects with pathologic oesophageal reflux during different days of treatment. CONCLUSIONS: In GERD subjects treated with a proton-pump inhibitor, the value of heartburn severity following a single standard meal can predict the likelihood of pathologic oesophageal reflux over the entire 24-h period.