Development of a high throughput, molecular diagnostic assay for predicting telomerase activity in breast cancer cell lines and tissues

Telomerase is a cellular enzyme that helps to provide genomic stability in tumor cells by maintaining the integrity of telomeres. Telomerase is an RNA-dependent DNA polymerase that contains a protein component (hTERT) and an associated RNA (hTR), which is used as a template for telomere repeat addition. Telomerase activity, while not detectable in most normal human somatic cells, is associated with approximately 85% of malignant human cancers overall, including over 90% of breast cancers. We have optimized a novel, quantitative, high-throughput telomerase activity assay using fluorescently labelled primers and Real Time quantitation via the ABI Prism 7700 (a.k.a., the TaqMan). Using established breast cancer cell lines and a subset of breast tumors, we demonstrate that telomerase levels quantitated from the TaqMan-based assay closely correlate with values obtained using the traditional, gel-based telomerase activity assay (TRAP). In addition, we have assessed the levels of both hTERT mRNA and hTR in each of our samples via RT-PCR to determine whether relative amounts or a ratio of the two telomerase components correlate with activity in a given sample. Our ultimate goal is to develop a Real Time, fluorescent RT-PCR assay to simultaneously measure hTERT and hTR messages in breast tumor samples, in an attempt to convert the enzymatic telomerase activity assay into a quantitative nucleic acid test to predict levels of activity in routinely processed clinical specimens

Evaluation of rK39 rapid diagnostic tests for canine visceral leishmaniasis : longitudinal study and meta-analysis

Canine visceral leishmaniasis is a vector-borne disease caused by the intracellular parasite Leishmania infantum. It is an important veterinary disease, and dogs are also the main animal reservoir for human infection. The disease is widespread in the Mediterranean area, and parts of Asia and South and Central America, and is potentially fatal in both dogs and humans unless treated. Diagnosis of canine infections requires serological or molecular tests. Detection of infection in dogs is important prior to treatment, and in epidemiological studies and control programmes, and a sensitive and specific rapid diagnostic test would be very useful. Rapid diagnostic tests (RDTs) have been developed, but their diagnostic performance has been reported to be variable. We evaluated the sensitivity of a RDT based on serological detection of the rK39 antigen in a cohort of naturally infected Brazilian dogs. The sensitivity of the test to detect infection was relatively low, but increased with time since infection and the severity of infection. We then carried out a meta-analysis of published studies of rK39 RDTs, evaluating the sensitivity to detect disease and infection. The results suggest that rK39 RDTs may be useful in a veterinary clinical setting, but the sensitivity to detect infection is too low for operational control programmes

Treatment with a corticotrophin releasing factor 2 receptor agonist modulates skeletal muscle mass and force production in aged and chronically ill animals

<p>Abstract</p> <p>Background</p> <p>Muscle weakness is associated with a variety of chronic disorders such as emphysema (EMP) and congestive heart failure (CHF) as well as aging. Therapies to treat muscle weakness associated with chronic disease or aging are lacking. Corticotrophin releasing factor 2 receptor (CRF2R) agonists have been shown to maintain skeletal muscle mass and force production in a variety of acute conditions that lead to skeletal muscle wasting.</p> <p>Hypothesis</p> <p>We hypothesize that treating animals with a CRF2R agonist will maintain skeletal muscle mass and force production in animals with chronic disease and in aged animals.</p> <p>Methods</p> <p>We utilized animal models of aging, CHF and EMP to evaluate the potential of CRF2R agonist treatment to maintain skeletal muscle mass and force production in aged animals and animals with CHF and EMP.</p> <p>Results</p> <p>In aged rats, we demonstrate that treatment with a CRF2R agonist for up to 3 months results in greater extensor digitorum longus (EDL) force production, EDL mass, soleus mass and soleus force production compared to age matched untreated animals. In the hamster EMP model, we demonstrate that treatment with a CRF2R agonist for up to 5 months results in greater EDL force production in EMP hamsters when compared to vehicle treated EMP hamsters and greater EDL mass and force in normal hamsters when compared to vehicle treated normal hamsters. In the rat CHF model, we demonstrate that treatment with a CRF2R agonist for up to 3 months results in greater EDL and soleus muscle mass and force production in CHF rats and normal rats when compared to the corresponding vehicle treated animals.</p> <p>Conclusions</p> <p>These data demonstrate that the underlying physiological conditions associated with chronic diseases such as CHF and emphysema in addition to aging do not reduce the potential of CRF2R agonists to maintain skeletal muscle mass and force production.</p

A Novel Small Molecule 1,2,3,4,6-penta-O-galloyl-α-D-glucopyranose Mimics the Antiplatelet Actions of Insulin

BACKGROUND: We have shown that 1,2,3,4,6-penta-O-galloyl-α-D-glucopyranose (α-PGG), an orally effective hypoglycemic small molecule, binds to insulin receptors and activates insulin-mediated glucose transport. Insulin has been shown to bind to its receptors on platelets and inhibit platelet activation. In this study we tested our hypothesis that if insulin possesses anti-platelet properties then insulin mimetic small molecules should mimic antiplatelet actions of insulin. PRINCIPAL FINDINGS: Incubation of human platelets with insulin or α-PGG induced phosphorylation of insulin receptors and IRS-1 and blocked ADP or collagen induced aggregation. Pre-treatment of platelets with α-PGG inhibited thrombin-induced release of P-selectin, secretion of ATP and aggregation. Addition of ADP or thrombin to platelets significantly decreased the basal cyclic AMP levels. Pre-incubation of platelets with α-PGG blocked ADP or thrombin induced decrease in platelet cyclic AMP levels but did not alter the basal or PGE(1) induced increase in cAMP levels. Addition of α-PGG to platelets blocked agonist induced rise in platelet cytosolic calcium and phosphorylation of Akt. Administration of α-PGG (20 mg kg(-1)) to wild type mice blocked ex vivo platelet aggregation induced by ADP or collagen. CONCLUSIONS: These data suggest that α-PGG inhibits platelet activation, at least in part, by inducing phosphorylation of insulin receptors leading to inhibition of agonist induced: (a) decrease in cyclic AMP; (b) rise in cytosolic calcium; and (c) phosphorylation of Akt. These findings taken together with our earlier reports that α-PGG mimics insulin signaling suggest that inhibition of platelet activation by α-PGG mimics antiplatelet actions of insulin

No effect of creatine supplementation on oxidative stress and cardiovascular parameters in spontaneously hypertensive rats

Background: Exacerbated oxidative stress is thought to be a mediator of arterial hypertension. It has been postulated that creatine (Cr) could act as an antioxidant agent preventing increased oxidative stress. The aim of this study was to investigate the effects of nine weeks of Cr or placebo supplementation on oxidative stress and cardiovascular parameters in spontaneously hypertensive rats (SHR). Findings: Lipid hydroperoxidation, one important oxidative stress marker, remained unchanged in the coronary artery (Cr: 12.6 +/- 1.5 vs. Pl: 12.2 +/- 1.7 nmol.mg(-1); p = 0.87), heart (Cr: 11.5 +/- 1.8 vs. Pl: 14.6 +/- 1.1 nmol.mg(-1); p = 0.15), plasma (Cr: 67.7 +/- 9.1 vs. Pl: 56.0 +/- 3.2 nmol.mg(-1); p = 0.19), plantaris (Cr: 10.0 +/- 0.8 vs. Pl: 9.0 +/- 0.8 nmol.mg(-1); p = 0.40), and EDL muscle (Cr: 14.9 +/- 1.4 vs. Pl: 17.2 +/- 1.5 nmol.mg(-1); p = 0.30). Additionally, Cr supplementation affected neither arterial blood pressure nor heart structure in SHR (p &gt; 0.05). Conclusions: Using a well-known experimental model of systemic arterial hypertension, this study did not confirm the possible therapeutic effects of Cr supplementation on oxidative stress and cardiovascular dysfunction associated with arterial hypertension.FAPES

Parental education and perception of outdoor playing time for preschoolers

Abstract Aim: The objective of this study was to analyze whether or not socioeconomic positions influence outdoor playtime during the week (WK) and on the weekends (WEND). Methods: The sample consisted of 485 (girls; n=223) healthy preschoolers, aged from 3 to 6 years, enrolled in kindergartens from the metropolitan area of Porto, Portugal. Physical Activity (PA) was assessed for 7 consecutive days with an accelerometer. The time playing outdoors during the WK or the WEND was reported by parents. Anthropometric data (weight and height) was collected following standardized protocols. Socioeconomic position was assessed by Parental Education (PE), according to the Portuguese education system. Results: We found differences in time spent playing outdoors either for the WK or WEND, but not for Total PA (TPA), in both sexes. However, regression analysis showed that after age adjustment, BMI and TPA for both sexes, we only found significant associations between low PE (LPE) and high PE (HPE) groups on WK or WEND. However, we found no statistically significant association for boys on the WK (p=0.06). Conclusion: Our findings suggest that socioeconomic position can influence the children’s time spent in outdoor activities, especially on the weekends. This may have implications for future interventions with this age group

Associations between the time of conception and the shape of the lactation curve in early lactation in Norwegian dairy cattle

<p>Abstract</p> <p>Background</p> <p>This study was carried out to determine if an association exists between the shape of the lactation curve before it is influenced by the event of conception and the time from calving to conception in Norwegian dairy cattle. Lactation curves of Norwegian Red cows during 5 to 42 days in milk (DIM) were compared between cows conceiving between 43 and 93 DIM and cows conceiving after 93 DIM.</p> <p>Methods</p> <p>Data from 23,049 cows, represented by one lactation each, with 219,538 monthly test days were extracted from the Norwegian Dairy Herd Recording System, which represents 97% of all Norwegian dairy cows. Besides veterinary treatments, these records also included information on daily milk yield at monthly test days. The data were stratified by parity groups (1, 2, and 3 and higher) and time to conception periods (43-93 DIM and >93 DIM). The sample was selected using the following selection criteria: conception later than 42 DIM, calving season July to September, no records of veterinary treatment and the level of energy fed as concentrates between 8.69 and 12.83 MJ. The shape of the lactation curves were parameterized using a modified Wilmink-model in a mixed model analysis. Differences in the parameters of the lactation curves with different conception times were evaluated using confidence intervals.</p> <p>Results</p> <p>Lactation curves characterized by a low intercept and a steep ascending slope and a steep descending slope were associated with early conception across all parities. The peak milk yield was not associated with time of conception.</p> <p>Conclusions</p> <p>A practical application of the study results is the use of the shape of the lactation curve in future herd management. Groups of cows with impaired reproductive performance may be identified due to an unfavorable shape of the lactation curve. Monitoring lactation curves and adjusting the feeding strategy to adjust yield therefore may be useful for the improvement of reproductive performance at herd level.</p