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.

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.

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.

Neutron radiation can activate K-ras via a point mutation in codon 146 and induces a different spectrum of ras mutations than does gamma radiation.

Neutron radiation is known to produce tumors in animals and cause cell transformation. We have developed a protocol to efficiently induce thymic lymphomas in RF/J mice by a single acute dose of neutron irradiation. Activated ras genes were detected in 17% (4 of 24) of the tumors analyzed. One of the tumors contained a K-ras gene activated by a point mutation in codon 146. Activating ras mutations at position 146 have not been previously detected in any known human or animal tumors. The spectrum of ras mutations detected in neutron radiation-induced thymic lymphomas was different from that seen in thymic lymphomas induced by gamma radiation in the same strain of mice. These results may have important implications for the mechanisms by which different types of radiation damage DNA.

Phosphorylation of E47 as a potential determinant of B-cell-specific activity.

The E2A gene encodes two basic helix-loop-helix proteins designated E12 and E47. Although these proteins are widely expressed, they are required only for the B-lymphocyte lineage where DNA binding is mediated distinctively by E47 homodimers. By studying the properties of deltaE47, an N-terminal truncation of E47, we provide evidence that phosphorylation may contribute to B-cell-specific DNA binding by E47. Two serines N terminal to the deltaE47 basic helix-loop-helix domain were found to be phosphorylated in a variety of cell types but were hypophosphorylated in B cells. Phosphorylating these serines in vitro inhibited DNA binding by deltaE47 homodimers but not by deltaE47-containing heterodimers, such as deltaE47:MyoD. These results argue that hypophosphorylation may be a prerequisite for activity of E47 homodimers in B cells, suggesting the use of an inductive (nonstochastic) step in early B-cell development.

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.

Staging of T-cell receptor beta chain gene rearrangements and ras oncogene mutations in the development of murine thymic lymphomas.

While it is known that the T-cell receptor beta chain gene is rearranged in fully developed murine thymic lymphomas induced by N-nitrosomethylurea and that the ras gene is activated in approximately 50% of these tumors (L. E. Diamond et al., Mol. Cell. Biol., 8: 2233-2236, 1988), it is unknown when these events occur or where the cells committed to a malignant phenotype are first located. We have studied these questions by treating mice with N-nitrosomethylurea, extracting thymocytes and bone marrow cells from the treated mice before they would have developed tumors, transferring the cells into recipient mice, monitoring those mice until they developed lymphoid tumors, and analyzing those tumors. This analysis showed that the initial cells committed to becoming malignant can be located in either the bone marrow or thymus and that both activation of the ras oncogene and rearrangements of the T-cell receptor gene can occur earlier than 30 days after N-nitrosomethylurea treatments. Furthermore, these results suggest that the T-cell receptor beta chain gene can undergo additional rearrangements during progression of a tumor.

Flow cytometric analysis of human uterine sarcomas and cell lines.

Flow cytometric techniques were used to characterize multiple human uterine sarcomas and cell lines derived from some of these tumors. Analysis of DNA content showed that 9 of the 11 uterine sarcomas investigated were composed of at least one aneuploid population as well as a distinct diploid population. These data indicate that aneuploidy, as measured by flow cytometry, is a characteristic more common to uterine sarcomas than that previously reported for uterine adenocarcinomas. Unlike the original tumors, the cell lines established from three of the sarcomas contained predominantly diploid populations with only minor aneuploid populations. Treatment of one of the sarcoma cultures with tumor promoters did not result in an increase in the aneuploid populations. Tumors which arose in nude mice upon transplantation of two of the sarcomas did not contain the same distribution of tumor subpopulations as found in the original sarcomas. Apparently, the in vitro culture and and in vivo nude mouse conditions were not appropriate for maintaining the original equilibrium between the aneuploid and diploid subpopulations but instead provided a selective environment that resulted in the preferential growth of only certain tumor populations. Dual-parameter analysis of DNA content and alkaline phosphatase levels of one of the sarcomas were useful for distinguishing the aneuploid from the diploid population coexisting in this tumor. Our data suggest that flow cytometry is a valuable tool to analyze the characteristics of the tumor populations residing in primary uterine sarcomas as well as to determine which of these tumor subpopulations survive in culture and transplantation to nude mice.

Recognition of core binding sites by bacteriophage integrases.

Bacteriophage integrases promote recombination between DNA molecules that carry attachment sites. They are members of a large and widely distributed family of site-specific recombinases with diverse biological roles. The integrases of phages lambda and HK022 are closely related members of this family, but neither protein efficiently recombines the attachment sites of the other phage. The nucleotides responsible for this specificity difference are located close to the points of recombinational strand exchange, within an integrase binding motif called the extended core binding site. There are four imperfectly repeated copies of this motif in each set of phage attachment sites, but only two, B' and C, contain major specificity determinants. When these specificity determinants were replaced by the corresponding nucleotides from a site with the alternative specificity, the resulting mutant was recombined by both integrases. Thus, the determinants act by impeding recombination promoted by the non-cognate integrase. We found that identical nucleotide substitutions within different core site copies had different effects on recombination, suggesting that integrase does not recognize each of the extended core binding sites in the same way. Finally, substitution at several positions in lambda integrase with the corresponding HK022-specific amino acids prevents recombination of lambda attachment sites, and this defect can be suppressed in an allele-specific manner by appropriate substitutions of HK022-specific nucleotides in the extended core binding sites.

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.

The impact of nocturnal symptoms associated with gastroesophageal reflux disease on health-related quality of life.

BACKGROUND: Two types of reflux episodes have been identified: upright or daytime and supine or nocturnal. The population-based prevalence of symptoms of nocturnal gastroesophageal reflux disease (GERD) and the impact of those symptoms on health-related quality of life (HRQL) have not been established. METHODS: A national random-sample telephone survey was conducted to estimate the prevalence of frequent GERD and nocturnal GERD-like symptoms and to assess the relationship between HRQL, GERD, and nocturnal GERD symptoms. Respondents were classified as controls, subjects with symptomatic nonnocturnal GERD, and subjects with symptomatic nocturnal GERD. The HRQL was assessed using the Medical Outcomes Study Short-Form 36 Health Survey (SF-36). RESULTS: The prevalence of frequent GERD was 14%, with an overall prevalence of nocturnal GERD of 10%. Seventy-four percent of those with frequent GERD symptoms reported nocturnal GERD symptoms. Subjects with nonnocturnal GERD had significant decrements on the SF-36 physical and mental component summary scores compared with the US general population. Subjects reporting nocturnal GERD symptoms were significantly more impaired than subjects reporting nonnocturnal GERD symptoms on both the physical component summary (38.94 vs 41. 52; P<.001) and mental component summary (46.78 vs 49.51; P<.001) and all 8 subscales of the SF-36 (P<.001). Subjects with nocturnal GERD demonstrated considerable impairment compared with the US general population and chronic disease populations. Subjects with nocturnal GERD had significantly more pain than those with hypertension and diabetes (P<.001) and similar pain compared with those with angina and congestive heart failure. CONCLUSIONS: Nocturnal symptoms are commonly experienced by individuals who report frequent GERD symptoms. In addition, HRQL is significantly impaired in those persons who report frequent GERD symptoms, and HRQL impairment is exacerbated in those who report nocturnal GERD symptoms.

An alternating selection strategy for cloning phage display antibodies.

Phage display is a powerful technique that can be used to develop antibodies to target molecules. One approach for antibody phage display is to select phage from a large naive library of antibody immunoglobulin variable region fragments (Fv) expressed on the surface of phage. Phage that display antibody fragments of interest are selected by their ability to bind the target antigen immobilized on a solid support surface. A major difficulty often encountered with this approach is that phage that bind to additional antigens that are present during the phage selection steps are also selected. We have developed an alternating selection approach to minimize selection of unwanted phage. In the alternating selection approach, two selection methods are used. Each selection method contains different contaminating antigens. This approach was used to select phage that bind a phosphoryated form of the E47 transcription factor. Phage were selected based on their ability to bind a phospho-peptide in solution and alternatively a phospho-protein coated on a polyvinyl micortiter plate. This approach proved significantly better than selection with only one method. With one selection technique, 2 of 48 (4%) selected clones bound to the target antigen. With another selection technique, 15 of 48 (31%) selected clones bound to the target antigen. With alternating selection, 71 of 93 (76%) of the clones bound to the target antigen.