In vitro selection of Haemonchus contortus for benzimidazole resistance reveals a mutation at amino acid 198 of beta-tubulin

Benzimidazoles were the first broad-spectrum anthelmintics and are still in use today against gastro-intestinal nematodes of ruminants such as Haemonchus contortus. Benzimidazoles block the polymerization of nematode microtubules. However, their efficacy is jeopardized by the spread of drug-resistant parasites that carry point mutations in beta-tubulin. Here we use a novel in vitro selection-in vivo propagation protocol to breed drug-resistant H. contortus. After 8 generations of selection with thiabendazole an in vitro resistance factor of 1000 was reached that was also relevant in vivo in infected sheep. The same procedure carried out with ivermectin produced only a moderate resistance phenotype that was not apparent in sheep. Cloning and sequencing of the beta-tubulin genes from the thiabendazole-resistant H. contortus mutants revealed all of the isotype 1 alleles, and part of the isotype 2 alleles, to carry the mutation glutamate(198) to alanine (E198A). An allele-specific PCR was developed, which may be helpful in monitoring the prevalence of alanine(198) encoding alleles in the beta-tubulin isotype 1 gene pool of H. contortus in the field.

Critical Control Points for Profitability in the Cow-Calf Enterprise

Financial, economic, and biological data collected from cow-calf producers who participated in the Illinois and Iowa Standardized Performance Analysis (SPA) programs were used in this study. Data used were collected for the 1996 through 1999 calendar years, with each herd within year representing one observation. This resulted in a final database of 225 observations (117 from Iowa and 108 from Illinois) from commercial herds with a range in size from 20 to 373 cows. Two analyses were conducted, one utilizing financial cost of production data, the other economic cost of production data. Each observation was analyzed as the difference from the mean for that given year. The independent variable utilized in both the financial and economic models as an indicator of profit was return to unpaid labor and management per cow (RLM). Used as dependent variables were the five factors that make up total annual cow cost: feed cost, operating cost, depreciation cost, capital charge, and hired labor, all on an annual cost per cow basis. In the economic analysis, family labor was also included. Production factors evaluated as dependent variables in both models were calf weight, calf price, cull weight, cull price, weaning percentage, and calving distribution. Herd size and investment were also analyzed. All financial factors analyzed were significantly correlated to RLM (P < .10) except cull weight, and cull price. All economic factors analyzed were significantly correlated to RLM (P < .10) except calf weight, cull weight and cull price. Results of the financial prediction equation indicate that there are eight measurements capable of explaining over 82 percent of the farm-to-farm variation in RLM. Feed cost is the overriding factor driving RLM in both the financial and economic stepwise regression analyses. In both analyses over 50 percent of the herd-to-herd variation in RLM could be explained by feed cost. Financial feed cost is correlated (P < .001) to operating cost, depreciation cost, and investment. Economic feed cost is correlated (P < .001) with investment and operating cost, as well as capital charge. Operating cost, depreciation, and capital charge were all negatively correlated (P < .10) to herd size, and positively correlated (P < .01) to feed cost in both analyses. Operating costs were positively correlated with capital charge and investment (P < .01) in both analyses. In the financial regression model, depreciation cost was the second critical factor explaining almost 9 percent of the herd-to-herd variation in RLM followed by operating cost (5 percent). Calf weight had a greater impact than calf price on RLM in both the financial and economic regression models. Calf weight was the fourth indicator of RLM in the financial model and was similar in magnitude to operating cost. Investment was not a significant variable in either regression model; however, it was highly correlated to a number of the significant cost variables including feed cost, depreciation cost, and operating cost (P < .001, financial; P < .10, economic). Cost factors were far more influential in driving RLM than production, reproduction, or producer controlled marketing factors. Of these cost factors, feed cost had by far the largest impact. As producers focus attention on factors that affect the profitability of the operation, feed cost is the most critical control point because it was responsible for over 50 percent of the herd-to-herd variation in profit.

Development and validation of energy expenditure prediction models based on GT3X accelerometer data in 5- to 9-year-old children

Background: Accelerometry has been established as an objective method that can be used to assess physical activity behavior in large groups. The purpose of the current study was to provide a validated equation to translate accelerometer counts of the triaxial GT3X into energy expenditure in young children. Methods: Thirty-two children aged 5–9 years performed locomotor and play activities that are typical for their age group. Children wore a GT3X accelerometer and their energy expenditure was measured with indirect calorimetry. Twenty-one children were randomly selected to serve as development group. A cubic 2-regression model involving separate equations for locomotor and play activities was developed on the basis of model fit. It was then validated using data of the remaining children and compared with a linear 2-regression model and a linear 1-regression model. Results: All 3 regression models produced strong correlations between predicted and measured MET values. Agreement was acceptable for the cubic model and good for both linear regression approaches. Conclusions: The current linear 1-regression model provides valid estimates of energy expenditure for ActiGraph GT3X data for 5- to 9-year-old children and shows equal or better predictive validity than a cubic or a linear 2-regression model.

Statistical inference for the optimal approximating model

In the setting of high-dimensional linear models with Gaussian noise, we investigate the possibility of confidence statements connected to model selection. Although there exist numerous procedures for adaptive (point) estimation, the construction of adaptive confidence regions is severely limited (cf. Li in Ann Stat 17:1001–1008, 1989). The present paper sheds new light on this gap. We develop exact and adaptive confidence regions for the best approximating model in terms of risk. One of our constructions is based on a multiscale procedure and a particular coupling argument. Utilizing exponential inequalities for noncentral χ2-distributions, we show that the risk and quadratic loss of all models within our confidence region are uniformly bounded by the minimal risk times a factor close to one.

Genome-wide analysis reveals selection for important traits in domestic horse breeds.

Intense selective pressures applied over short evolutionary time have resulted in homogeneity within, but substantial variation among, horse breeds. Utilizing this population structure, 744 individuals from 33 breeds, and a 54,000 SNP genotyping array, breed-specific targets of selection were identified using an F(ST)-based statistic calculated in 500-kb windows across the genome. A 5.5-Mb region of ECA18, in which the myostatin (MSTN) gene was centered, contained the highest signature of selection in both the Paint and Quarter Horse. Gene sequencing and histological analysis of gluteal muscle biopsies showed a promoter variant and intronic SNP of MSTN were each significantly associated with higher Type 2B and lower Type 1 muscle fiber proportions in the Quarter Horse, demonstrating a functional consequence of selection at this locus. Signatures of selection on ECA23 in all gaited breeds in the sample led to the identification of a shared, 186-kb haplotype including two doublesex related mab transcription factor genes (DMRT2 and 3). The recent identification of a DMRT3 mutation within this haplotype, which appears necessary for the ability to perform alternative gaits, provides further evidence for selection at this locus. Finally, putative loci for the determination of size were identified in the draft breeds and the Miniature horse on ECA11, as well as when signatures of selection surrounding candidate genes at other loci were examined. This work provides further evidence of the importance of MSTN in racing breeds, provides strong evidence for selection upon gait and size, and illustrates the potential for population-based techniques to find genomic regions driving important phenotypes in the modern horse.

The effect of idursulfase on growth in patients with Hunter syndrome: data from the Hunter Outcome Survey (HOS)

Hunter syndrome (mucopolysaccharidosis type II) is a rare and life-limiting multisystemic disorder with an X-linked recessive pattern of inheritance. Short stature is a prominent feature of this condition. This analysis aimed to investigate the effects of enzyme replacement therapy with idursulfase on growth in patients enrolled in HOS - the Hunter Outcome Survey which is a multinational observational database. As of Jan 2012, height data before treatment were available for 567 of 740 males followed prospectively after HOS entry. Cross-sectional analysis showed that short stature became apparent after approximately 8 years of age; before this, height remained within the normal range. Age-corrected standardized height scores (z-scores) before and after treatment were assessed using piecewise regression model analysis in 133 patients (8-15 years of age at treatment start; data available on ≥ 1 occasion within +/-24 months of treatment start; growth hormone-treated patients excluded). Results showed that the slope after treatment (slope=-0.005) was significantly improved compared with before treatment (slope=-0.043) (difference=0.038, p=0.004). Analysis of covariates (age at treatment start, cognitive involvement, presence of puberty at the start of ERT, mutation type, functional classification), showed a significant influence on growth of mutation type (height deficit in terms of z-scores most pronounced in patients with deletions/large rearrangements/nonsense mutations, p<0.0001) and age (most pronounced in the 12-15-year group, p<0.0001). Cognitive involvement, pubertal status at the start of ERT and functional classification were not related to the growth deficit or response to treatment. In conclusion, the data showed an improvement in growth rate in patients with Hunter syndrome following idursulfase treatment.

Sensitive Questions in Online Surveys: An Experimental Comparison of the RRT and the Crosswise Model

Self-administered online surveys provide a higher level of privacy protection to respondents than surveys administered by an interviewer. Yet, studies show that asking sensitive questions is problematic also in self-administered mode. Because respondents might not be willing to reveal the truth and provide answers that are subject to social desirability bias, the validity of prevalence estimates of sensitive behaviors gained via online surveys can be challenged. A wellknown method to combat these problems is the Randomized Response Technique (RRT). However, convincing evidence that the RRT provides more valid estimates than direct questioning in online mode is still lacking. Moreover, an alternative approach called the Crosswise Model (CM) has recently been suggested to overcome some of the deficiencies of the RRT. We therefore conducted an experimental study in which different implementations of the RRT and the CM have been tested and compared to direct questioning. Our study is a large-scale online survey on sensitive behaviors by students such as cheating in exams and paper plagiarism. The results of the study reveal poor per-formance of the RRT, while the CM yielded significantly higher estimates of sensitive behaviors than direct questioning. We conclude that the CM is a promising approach for asking sensitive questions in self-administered surveys.

Asking Sensitive Questions in Online Surveys: An Experimental Comparison of the Randomized Response Technique and the Crosswise Model

Self-administered online surveys provide a higher level of privacy protection to respondents than surveys administered by an interviewer. Yet, studies show that asking sensitive questions is problematic also in self-administered mode. Because respondents might not be willing to reveal the truth and provide answers that are subject to social desirability bias, the validity of prevalence estimates of sensitive behaviors gained via online surveys can be challenged. A well-known method to combat these problems is the Randomized Response Technique (RRT). However, convincing evidence that the RRT provides more valid estimates than direct questioning in online mode is still lacking. Moreover, an alternative approach called the Crosswise Model (CM) has recently been suggested to overcome some of the deficiencies of the RRT. In the context of an online survey on plagiarism and cheating on exams among students of two Swiss universities (N = 6,494), we tested different implementations of the RRT and the CM and compared them to direct questioning using a randomized experimental design. Results reveal a poor performance of the RRT, which failed to elicit higher prevalence estimates than direct questioning. Using the CM however, significantly higher prevalence estimates were obtained making it a promising new alternative to the conventional RRT.

Uptake And Metabolism Of 5’-AMP In The Erythrocyte Play Key Roles In The 5’-AMP Induced Model Of Deep Hypometabolism

UPTAKE AND METABOLISM OF 5’-AMP IN THE ERYTHROCYTE PLAY KEY ROLES IN THE 5’-AMP INDUCED MODEL OF DEEP HYPOMETABOLISM Publication No. ________ Isadora Susan Daniels, B.A. Supervisory Professor: Cheng Chi Lee, Ph.D. Mechanisms that initiate and control the natural hypometabolic states of mammals are poorly understood. The laboratory developed a model of deep hypometabolism (DH) initiated by uptake of 5’-adenosine monophosphate (5’-AMP) into erythrocytes. Mice enter DH when given a high dose of 5’-AMP and the body cools readily. Influx of 5’-AMP appears to inhibit thermoregulatory control. In a 15°C environment, mice injected with 5’-AMP (0.5 mg/gw) enter a Phase I response in which oxygen consumption (VO2) drops rapidly to 1/3rd of euthermic levels. The Phase I response appears independent of body temperature (Tb). This is followed by gradual body temperature decline that correlates with VO2 decline, called Phase II response. Within 90 minutes, mouse Tb approaches 15°C, and VO2 is 1/10th of normal. Mice can remain several hours in this state, before gradually and safely recovering. The DH state translates to other mammalian species. Our studies show uptake and metabolism of 5’-AMP in erythrocytes causes biochemical changes that initiate DH. Increased AMP shifts the adenylate equilibrium toward ADP formation, consequently decreasing intracellular ATP. In turn, glycolysis slows, indicated by increased glucose and decreased lactate. 2,3-bisphosphoglycerate levels rise, allosterically reducing oxygen affinity for hemoglobin, and deoxyhemoglobin rises. Less oxygen transport to tissues likely triggers the DH model. The major intracellular pathway for AMP catabolism is catalyzed by AMP deaminase (AMPD). Multiple AMPD isozymes are expressed in various tissues, but erythrocytes only have AMPD3. Mice lacking AMPD3 were created to study control of the DH model, specifically in erythrocytes. Telemetric measurements demonstrate lower Tb and difficulty maintaining Tb under moderate metabolic stress. A more dramatic response to lower dose of 5’-AMP suggests AMPD activity in the erythrocyte plays an important role in control of the DH model. Analysis of adenylates in erythrocyte lysate shows 3-fold higher levels of ATP and ADP but similar AMP levels to wild-type. Taken together, results indicate alterations in energy status of erythrocytes can induce a hypometabolic state. AMPD3 control of AMP catabolism is important in controlling the DH model. Genetically reducing AMP catabolism in erythrocytes causes a phenotype of lower Tb and compromised ability to maintain temperature homeostasis.

A comparison of Markov and generalized linear models of hospital mortality

This paper reports a comparison of three modeling strategies for the analysis of hospital mortality in a sample of general medicine inpatients in a Department of Veterans Affairs medical center. Logistic regression, a Markov chain model, and longitudinal logistic regression were evaluated on predictive performance as measured by the c-index and on accuracy of expected numbers of deaths compared to observed. The logistic regression used patient information collected at admission; the Markov model was comprised of two absorbing states for discharge and death and three transient states reflecting increasing severity of illness as measured by laboratory data collected during the hospital stay; longitudinal regression employed Generalized Estimating Equations (GEE) to model covariance structure for the repeated binary outcome. Results showed that the logistic regression predicted hospital mortality as well as the alternative methods but was limited in scope of application. The Markov chain provides insights into how day to day changes of illness severity lead to discharge or death. The longitudinal logistic regression showed that increasing illness trajectory is associated with hospital mortality. The conclusion is reached that for standard applications in modeling hospital mortality, logistic regression is adequate, but for new challenges facing health services research today, alternative methods are equally predictive, practical, and can provide new insights. ^

Evaluation of 9 biomarkers for predicting 10-year cardiovascular risk in patients undergoing coronary angiography: findings from the LUdwigshafen RIsk and Cardiovascular Health (LURIC) study.

BACKGROUND Conventional factors do not fully explain the distribution of cardiovascular outcomes. Biomarkers are known to participate in well-established pathways associated with cardiovascular disease, and may therefore provide further information over and above conventional risk factors. This study sought to determine whether individual and/or combined assessment of 9 biomarkers improved discrimination, calibration and reclassification of cardiovascular mortality. METHODS 3267 patients (2283 men), aged 18-95 years, at intermediate-to-high-risk of cardiovascular disease were followed in this prospective cohort study. Conventional risk factors and biomarkers were included based on forward and backward Cox proportional stepwise selection models. RESULTS During 10-years of follow-up, 546 fatal cardiovascular events occurred. Four biomarkers (interleukin-6, neutrophils, von Willebrand factor, and 25-hydroxyvitamin D) were retained during stepwise selection procedures for subsequent analyses. Simultaneous inclusion of these biomarkers significantly improved discrimination as measured by the C-index (0.78, P = 0.0001), and integrated discrimination improvement (0.0219, P<0.0001). Collectively, these biomarkers improved net reclassification for cardiovascular death by 10.6% (P<0.0001) when added to the conventional risk model. CONCLUSIONS In terms of adverse cardiovascular prognosis, a biomarker panel consisting of interleukin-6, neutrophils, von Willebrand factor, and 25-hydroxyvitamin D offered significant incremental value beyond that conveyed by simple conventional risk factors.