A high-throughput pipeline for the design of real-time PCR signatures

Background  

Pathogen diagnostic assays based on polymerase chain reaction (PCR) technology provide high sensitivity and specificity. However, the design of these diagnostic assays is computationally intensive, requiring high-throughput methods to identify unique PCR signatures in the presence of an ever increasing availability of sequenced genomes.     

Effects of supplementation of maize silage diets with extruded linseed, vitamin E and plant extracts rich in polyphenols, and morning v. evening milking on milk fatty acid profiles in Holstein and Montbéliarde cows

The aim of this study was to evaluate the effects on dairy performance and milk fatty acid (FA) composition of (i) supplementation with extruded linseed (EL), (ii) supplementation with synthetic or natural antioxidants, namely vitamin E and plant extracts rich in polyphenols (PERP), (iii) cow breed (Holstein v. Montbéliarde) and (iv) time of milking (morning v. evening). After a 3-week pre-experimental period 24 lactating cows (12 Holstein and 12 Montbéliarde) were divided up into four groups of six cows: the first group received a daily control diet (diet C) based on maize silage. The second group received the same diet supplemented with EL (diet EL, fat level approximately 5% of dietary dry matter (DM)). The third group received the EL diet plus 375 IU/kg diet DM of vitamin E (diet ELE). The fourth group received the ELE diet plus 10 g/kg diet DM of a PERP mixture (diet ELEP). Compared with the diet C, feeding EL-rich diets led to lower concentrations of total saturated FA (SFA) and higher concentrations of stearic and oleic acids, each trans and cis isomer of 18:1 (except c12-18:1), non-conjugated isomers of 18:2, some isomers (c9t11-, c9c11- and t11t13-) of conjugated linoleic acid (CLA), and 18:3n-3. The vitamin E supplementation had no effect on milk yield, milk fat or protein percentage and only moderate effects on milk concentrations of FA (increase in 16:0, decreases in 18:0 and t6/7/8-18:1). The addition of PERP to vitamin E did not modify milk yield or composition and slightly altered milk FA composition (decrease in total saturated FA (SFA) and increase in monounsaturated FA (MUFA)). The minor effects of vitamin E may be partly linked to the fact that no milk fat depression occurred with the EL diet. During both periods the Holstein cows had higher milk production, milk fat and protein yields, and milk percentages of 4:0 and 18:3n-3, and lower percentages of odd-branched chain FA (OBCFA) than the Montbéliarde cows. During the experimental period the Holstein cows had lower percentages of total cis 18:1, and c9,c11-CLA, and higher percentages of 6:0, 8:0, t12-, t16/c14- and t13/14-18:1, and 18:2n-6 than Montbéliarde cows because of several significant interactions between breed and diet. Also, the total SFA percentage was higher for morning than for evening milkings, whereas those of MUFA, total cis 18:1, OBCFA and 18:2n-6 were lower. Extruded linseed supplementation had higher effect on milk FA composition than antioxidants, breed or time of milking.     

Early pregnancy detection and the hormonal characterization of embryonic-fetal mortality in fallow deer (Dama dama)

The objectives of this investigation were to 1) determine serum concentrations of progesterone (P4), estrone sulfate (E1S) and pregnancy-specific protein B (PSPB) from estrus synchronization through mid-gestation in the fallow doe (Dama dama) and 2) characterize the hormonal profiles of does whose embryos or fetuses died in utero. Ten fallow does were synchronized for 14 d with an intravaginal P4-releasing device (CIDR) and were naturally mated after CIDR removal. Blood samples were collected at CIDR insertion, CIDR removal and at intervals through Day 203 post-CIDR removal for analysis of P4, E1S and PSPB by radioimmunoassay (RIA). Ultrasonography was performed on Days 49 and 69 post-CIDR removal. Serum P4 at the time of CIDR insertion was 4.8 +/- 0.6 ng/ml, and at CIDR withdrawal it was 6.2 +/- 0.3 ng/ml. Concentrations of E1S and PSPB were nondetectable at CIDR insertion. Serum E1S was highest at Day 93, and PSPB was first detectable in pregnant does at Days 27 to 30 post-CIDR withdrawal. Ultrasonography on Day 49 revealed that 6 does were pregnant, 2 were not pregnant and 2 others were diagnosed originally as early pregnant. At Day 69, ultrasonography revealed that 6 does (60%) were pregnant and 4 (40%) were not. A comparison of the ultrasonographic and hormonal data indicated that the 2 does diagnosed as early pregnant on Day 49 had conceived but had lost the pregnancy. A third doe which was pregnant on Day 69 lost the fetus later in gestation. Hormonal profiles of does whose embryo or fetus had died were characterized by erratic P4 and E1S profiles, with PSPB becoming undetectable in the 3 does by Days 49, 65 and 80 post-CIDR removal. These data 1) demonstrate the timing for the collection of serum samples for determining early pregnancy in fallow does using 3 hormonal methods and 2) characterize the hormonal profiles of 3 fallow does with embryonic-fetal loss.     

Differential expression of iodothyronine deiodinase type 2 in growth plates of chickens divergently selected for incidence of tibial dyschondroplasia

Tibial dyschondroplasia (TD) is a genetic leg defect in broilers with a lesion of avascular, non-calcified cartilage below the growth plate of the proximal tibiatarsus. This disease is considered to result from the inability of chondrocytes to undergo terminal differentiation. Thyroid hormones are required for chondrocyte differentiation. The thyroid gland produces and secrets mostly L-thyroxine or T4 and T4 plays most of its biological activities through conversion to triiodothyronine or T3 in local tissues by iodothyronine deiodinases type 1 or type 2, which are tissue specific. In this study, no differences were found in the plasma concentrations of total T3 and T4 between two chicken lines divergently selected for the incidence of TD. Plasma T4 was higher than T3, especially in older chickens. Younger birds had much higher T3 than older birds, but there were no significant age differences in T4. The expression level of deiodinase type 2 in the growth plates of broilers with TD was one-eighth of those birds without the disease. The expression levels of deiodinase type 2 (DIO2) in commercial broilers without the disease were much higher than those with TD and lower than those without the disease in the susceptible and resistant lines, respectively. These results indicate that the inadequate expression of DIO2 in the growth plates contributes to the pathogenesis of TD in broilers and that TD is a tissue-specific hypothyroidism.     

Biochemical detection of adenosine deaminase in Trypanosoma evansi

Biochemical and molecular research on parasites has increased considerably in trypanosomes in the recent years. Many of them have the purpose of identify areas, proteins and structures of the parasite which are vulnerable and could be used in therapy against the protozoan. Based on this hypothesis this study aimed to detect biochemically the enzyme adenosine deaminase (ADA) in Trypanosoma evansi, and to adapt an assay to the measurement of its activity in trypomastigotes. Firstly, the parasites were separated from the blood of mice experimentally infected with a DEAE-cellulose column. The ADA activity in trypomastigotes was evaluated at concentrations of 0.1, 0.2, 0.5, 0.6 and 0.8 mg of protein by spectrophotometry. ADA activity was observed in the parasites at all concentrations tested and its activity was proportional to the concentration of protein, ranging between 0.64 and 2.24 U/L in the lowest and highest concentration of protein, respectively. Therefore, it is possible to detect biochemically ADA in T. evansi, an enzyme that may be associated with vital functions of the parasite, similar to what occurs in mammals. This knowledge may be useful in the association of the chemotherapic treatment with specific inhibitors of the enzyme, in future studies.     

Contribution of xanthine oxidase-derived superoxide to chronic hypoxic pulmonary hypertension in neonatal rats

Xanthine oxidase (XO)-derived reactive oxygen species (ROS) formation contributes to experimental chronic hypoxic pulmonary hypertension in adults, but its role in neonatal pulmonary hypertension has received little attention. In rats chronically exposed to hypoxia (13% O(2)) for 14 days from birth, we examined the effects of ROS scavengers (U74389G 10 mg.kg(-1).day(-1) or Tempol 100 mg.kg(-1).day(-1) ip) or a XO inhibitor, Allopurinol (50 mg.kg(-1).day(-1) ip). Both ROS scavengers limited oxidative stress in the lung and attenuated hypoxia-induced vascular remodeling, confirming a critical role for ROS in this model. However, both interventions also significantly inhibited somatic growth and normal cellular proliferation in distal air spaces. Hypoxia-exposed pups had evidence of increased serum and lung XO activity, increased vascular XO-derived superoxide production, and vascular nitrotyrosine formation. These changes were all prevented by treatment with Allopurinol, which also attenuated hypoxia-induced vascular remodeling and partially reversed inhibited endothelium-dependent arterial relaxation, without affecting normal growth and proliferation. Collectively, our findings suggest that XO-derived superoxide induces endothelial dysfunction, thus impairing pulmonary arterial relaxation, and contributes to vascular remodeling in hypoxia-exposed neonatal rats. Due to the potential for adverse effects on normal growth, targeting XO may represent a superior "antioxidant" strategy to ROS scavengers for neonates with pulmonary hypertension.