Validation of reference genes for quantitative RT-qPCR studies of gene expression in Atlantic cod (Gadus morhua l.) during temperature stress

<p>Abstract</p> <p>Background</p> <p>One important physiological response to environmental stress in animals is change in gene expression. To obtain reliable data from gene expression studies using RT-qPCR it is important to evaluate a set of possible reference genes as normalizers for expression. The expression of these candidate genes should be analyzed in the relevant tissues during normal and stressed situations. To find suitable reference genes it was crucial that the genes were stably expressed also during a situation of physiological stress. For poikilotermic animals like cod, changes in temperature are normal, but if the changes are faster than physiological compensation, the animals respond with typical stress responses. It has previously been shown that Atlantic cod show stress responses when elevation of water temperature is faster than 1 degree/day, for this reason we chose hyperthermia as stress agent for this experiment.</p> <p>Findings</p> <p>We here describe the expression of eight candidate reference genes from Atlantic cod (<it>Gadus morhua l</it>.) and their stability during thermal stress (temperature elevation of one degree C/day for 5 days). The genes investigated were: Eukaryotic elongation factor 1 alpha, <it>ef1a</it>; 18s ribosomal RNA; <it>18s</it>, Ubiquitin conjugate protein; <it>ubiq</it>, cytoskeletal beta-actin; <it>actb</it>, major histcompatibility complex I; MHC-I light chain, beta-2 -microglobulin; <it>b2m</it>, cytoskeletal alpha-tubulin; <it>tba1c</it>, acidic ribosomal phosphoprotein; <it>rplp1</it>, glucose-6-phosphate dehydrogenase; <it>g6pd</it>. Their expression were analyzed in 6 tissues (liver, head kidney, intestine, spleen, heart and gills) from cods exposed to elevated temperature and compared to a control group. Although there were variations between tissues with respect to reference gene stability, four transcripts were more consistent than the others: <it>ubiq</it>, <it>ef1a</it>, <it>18s </it>and <it>rplp1</it>. We therefore used these to analyze the expression of stress related genes (heat shock proteins) induced during hyperthermia. We found that both transcripts were significantly upregulated in several tissues in fish exposed to increased temperature.</p> <p>Conclusion</p> <p>This is the first study comparing reference genes for RT-qPCR analyses of expression during hyperthermia in Atlantic cod. <it>ef1a, 18s, rplp1 </it>and <it>ubiq </it>transcripts were found to be well suited as reference genes during these experimental conditions.</p

Genome-Wide Analysis of Neuroblastomas using High-Density Single Nucleotide Polymorphism Arrays

BACKGROUND: Neuroblastomas are characterized by chromosomal alterations with biological and clinical significance. We analyzed paired blood and primary tumor samples from 22 children with high-risk neuroblastoma for loss of heterozygosity (LOH) and DNA copy number change using the Affymetrix 10K single nucleotide polymorphism (SNP) array. FINDINGS: Multiple areas of LOH and copy number gain were seen. The most commonly observed area of LOH was on chromosome arm 11q (15/22 samples; 68%). Chromosome 11q LOH was highly associated with occurrence of chromosome 3p LOH: 9 of the 15 samples with 11q LOH had concomitant 3p LOH (P = 0.016). Chromosome 1p LOH was seen in one-third of cases. LOH events on chromosomes 11q and 1p were generally accompanied by copy number loss, indicating hemizygous deletion within these regions. The one exception was on chromosome 11p, where LOH in all four cases was accompanied by normal copy number or diploidy, implying uniparental disomy. Gain of copy number was most frequently observed on chromosome arm 17q (21/22 samples; 95%) and was associated with allelic imbalance in six samples. Amplification of MYCN was also noted, and also amplification of a second gene, ALK, in a single case. CONCLUSIONS: This analysis demonstrates the power of SNP arrays for high-resolution determination of LOH and DNA copy number change in neuroblastoma, a tumor in which specific allelic changes drive clinical outcome and selection of therapy

Facts, values, and Attention-Deficit Hyperactivity Disorder (ADHD): an update on the controversies

The Hastings Center, a bioethics research institute, is holding a series of 5 workshops to examine the controversies surrounding the use of medication to treat emotional and behavioral disturbances in children. These workshops bring together clinicians, researchers, scholars, and advocates with diverse perspectives and from diverse fields. Our first commentary in CAPMH, which grew out of our first workshop, explained our method and explored the controversies in general. This commentary, which grows out of our second workshop, explains why informed people can disagree about ADHD diagnosis and treatment. Based on what workshop participants said and our understanding of the literature, we make 8 points. (1) The ADHD label is based on the interpretation of a heterogeneous set of symptoms that cause impairment. (2) Because symptoms and impairments are dimensional, there is an inevitable "zone of ambiguity," which reasonable people will interpret differently. (3) Many other variables, from different systems and tools of diagnosis to different parenting styles and expectations, also help explain why behaviors associated with ADHD can be interpreted differently. (4) Because people hold competing views about the proper goals of psychiatry and parenting, some people will be more, and others less, concerned about treating children in the zone of ambiguity. (5) To recognize that nature has written no bright line between impaired and unimpaired children, and that it is the responsibility of humans to choose who should receive a diagnosis, does not diminish the significance of ADHD. (6) Once ADHD is diagnosed, the facts surrounding the most effective treatment are complicated and incomplete; contrary to some popular wisdom, behavioral treatments, alone or in combination with low doses of medication, can be effective in the long-term reduction of core ADHD symptoms and at improving many aspects of overall functioning. (7) Especially when a child occupies the zone of ambiguity, different people will emphasize different values embedded in the pharmacological and behavioral approaches. (8) Truly informed decision-making requires that parents (and to the extent they are able, children) have some sense of the complicated and incomplete facts regarding the diagnosis and treatment of ADHD

Endothelial Progenitor Cell Number and Colony-forming Capacity in Overweight and Obese Adults

OBJECTIVE: To investigate whether adiposity influences endothelial progenitor cell (EPC) number and colony-forming capacity.DESIGN: Cross-sectional study of normal weight, overweight and obese adult humans.PARTICIPANTS: Sixty-seven sedentary adults (aged 45-65 years): 25 normal weight (body mass index (BMI) or=30 kg/m(2); 18 males/6 females). All participants were non-smokers and free of overt cardiometabolic disease.MEASUREMENTS: Peripheral blood samples were collected and circulating EPC number was assessed by flow cytometry. Putative EPCs were defined as CD45(-)/CD34(+)/VEGFR-2(+)/CD133(+) or CD45(-)/CD34(+) cells. EPC colony-forming capacity was measured in vitro using a colony-forming unit (CFU) assay.RESULTS: Number of circulating putative EPCs (either CD45(-)/CD34(+)/VEGFR-2(+)/CD133(+) or CD45(-)/CD34(+) cells) was lower (P\u3c0.05) in obese (0.0007±0.0001%; 0.050±0.006%) compared with overweight (0.0016±0.0004%; 0.089±0.019%) and normal weight (0.0015±0.0003%; 0.082±0.008%) adults. There were no differences in EPC number between the overweight and normal weight groups. EPC colony-formation was significantly less in the obese (6±1) and overweight (4±1) compared with normal weight (9±2) adults.CONCLUSION: These results indicate that: (1) the number of circulating EPCs is lower in obese compared with overweight and normal weight adults; and (2) EPC colony-forming capacity is blunted in overweight and obese adults compared with normal weight adults. Impairments in EPC number and function may contribute to adiposity-related cardiovascular risk

Treprostinil increases the number and angiogenic potential of endothelial progenitor cells in children with pulmonary hypertension

Background Pulmonary vasodilators in general and prostacyclin therapy in particular, have markedly improved the outcome of patients with pulmonary arterial hypertension (PAH). As endothelial dysfunction is a key feature of PAH, and as endothelial progenitor cells (EPC) may contribute to vascular repair in PAH, we suspected that prostacyclin therapy might enhance EPC numbers and functions. In the present study, objectives were to determine whether EPC may contribute to vasodilator treatment efficacy in PAH. Methods We quantified CD34+ cells, CFU-Hill and ECFC (endothelial colony forming cells) in peripheral blood from children with idiopathic PAH (n = 27) or PAH secondary to congenital heart disease (n = 52). CD34+ were enumerated by flow cytometry, CFU-Hill and ECFC by a culture assay. ECFC grown ex vivo were tested for their angiogenic capacities before and after prostacyclin analog therapy (subcutaneous treprostinil). Results ECFC counts were significantly enhanced in the 8 children treated with treprostinil, while no change was observed in children receiving oral therapy with endothelin antagonists and/or PDE5 inhibitors. CD34+ cell and CFU-Hill counts were unaffected. ECFC from patients treated with treprostinil had a hyperproliferative phenotype and showed enhanced angiogenic potential in a nude mouse preclinical model of limb ischemia. Conclusions ECFC may partly mediate the clinical benefits of prostanoids in pulmonary arterial hypertension

Disagreements with implications: diverging discourses on the ethics of non-medical use of methylphenidate for performance enhancement

<p>Abstract</p> <p>Background</p> <p>There is substantial evidence that methylphenidate (MPH; Ritalin), is being used by healthy university students for non-medical motives such as the improvement of concentration, alertness, and academic performance. The scope and potential consequences of the non-medical use of MPH upon healthcare and society bring about many points of view.</p> <p>Methods</p> <p>To gain insight into key ethical and social issues on the non-medical use of MPH, we examined discourses in the print media, bioethics literature, and public health literature.</p> <p>Results</p> <p>Our study identified three diverging paradigms with varying perspectives on the nature of performance enhancement. The beneficial effects of MPH on normal cognition were generally portrayed enthusiastically in the print media and bioethics discourses but supported by scant information on associated risks. Overall, we found a variety of perspectives regarding ethical, legal and social issues related to the non-medical use of MPH for performance enhancement and its impact upon social practices and institutions. The exception to this was public health discourse which took a strong stance against the non-medical use of MPH typically viewed as a form of prescription abuse or misuse. Wide-ranging recommendations for prevention of further non-medical use of MPH included legislation and increased public education.</p> <p>Conclusion</p> <p>Some positive portrayals of the non-medical use of MPH for performance enhancement in the print media and bioethics discourses could entice further uses. Medicine and society need to prepare for more prevalent non-medical uses of neuropharmaceuticals by fostering better informed public debates.</p

Impairment of circulating endothelial progenitors in Down syndrome

<p>Abstract</p> <p>Background</p> <p>Pathological angiogenesis represents a critical issue in the progression of many diseases. Down syndrome is postulated to be a systemic anti-angiogenesis disease model, possibly due to increased expression of anti-angiogenic regulators on chromosome 21. The aim of our study was to elucidate some features of circulating endothelial progenitor cells in the context of this syndrome.</p> <p>Methods</p> <p>Circulating endothelial progenitors of Down syndrome affected individuals were isolated, <it>in vitro </it>cultured and analyzed by confocal and transmission electron microscopy. ELISA was performed to measure SDF-1α plasma levels in Down syndrome and euploid individuals. Moreover, qRT-PCR was used to quantify expression levels of <it>CXCL12 </it>gene and of its receptor in progenitor cells. The functional impairment of Down progenitors was evaluated through their susceptibility to hydroperoxide-induced oxidative stress with BODIPY assay and the major vulnerability to the infection with human pathogens. The differential expression of crucial genes in Down progenitor cells was evaluated by microarray analysis.</p> <p>Results</p> <p>We detected a marked decrease of progenitors' number in young Down individuals compared to euploid, cell size increase and some major detrimental morphological changes. Moreover, Down syndrome patients also exhibited decreased SDF-1α plasma levels and their progenitors had a reduced expression of SDF-1α encoding gene and of its membrane receptor. We further demonstrated that their progenitor cells are more susceptible to hydroperoxide-induced oxidative stress and infection with Bartonella henselae. Further, we observed that most of the differentially expressed genes belong to angiogenesis, immune response and inflammation pathways, and that infected progenitors with trisomy 21 have a more pronounced perturbation of immune response genes than infected euploid cells.</p> <p>Conclusions</p> <p>Our data provide evidences for a reduced number and altered morphology of endothelial progenitor cells in Down syndrome, also showing the higher susceptibility to oxidative stress and to pathogen infection compared to euploid cells, thereby confirming the angiogenesis and immune response deficit observed in Down syndrome individuals.</p

Antiinflammatory Therapy with Canakinumab for Atherosclerotic Disease

Background: Experimental and clinical data suggest that reducing inflammation without affecting lipid levels may reduce the risk of cardiovascular disease. Yet, the inflammatory hypothesis of atherothrombosis has remained unproved. Methods: We conducted a randomized, double-blind trial of canakinumab, a therapeutic monoclonal antibody targeting interleukin-1β, involving 10,061 patients with previous myocardial infarction and a high-sensitivity C-reactive protein level of 2 mg or more per liter. The trial compared three doses of canakinumab (50 mg, 150 mg, and 300 mg, administered subcutaneously every 3 months) with placebo. The primary efficacy end point was nonfatal myocardial infarction, nonfatal stroke, or cardiovascular death. RESULTS: At 48 months, the median reduction from baseline in the high-sensitivity C-reactive protein level was 26 percentage points greater in the group that received the 50-mg dose of canakinumab, 37 percentage points greater in the 150-mg group, and 41 percentage points greater in the 300-mg group than in the placebo group. Canakinumab did not reduce lipid levels from baseline. At a median follow-up of 3.7 years, the incidence rate for the primary end point was 4.50 events per 100 person-years in the placebo group, 4.11 events per 100 person-years in the 50-mg group, 3.86 events per 100 person-years in the 150-mg group, and 3.90 events per 100 person-years in the 300-mg group. The hazard ratios as compared with placebo were as follows: in the 50-mg group, 0.93 (95% confidence interval [CI], 0.80 to 1.07; P = 0.30); in the 150-mg group, 0.85 (95% CI, 0.74 to 0.98; P = 0.021); and in the 300-mg group, 0.86 (95% CI, 0.75 to 0.99; P = 0.031). The 150-mg dose, but not the other doses, met the prespecified multiplicity-adjusted threshold for statistical significance for the primary end point and the secondary end point that additionally included hospitalization for unstable angina that led to urgent revascularization (hazard ratio vs. placebo, 0.83; 95% CI, 0.73 to 0.95; P = 0.005). Canakinumab was associated with a higher incidence of fatal infection than was placebo. There was no significant difference in all-cause mortality (hazard ratio for all canakinumab doses vs. placebo, 0.94; 95% CI, 0.83 to 1.06; P = 0.31). Conclusions: Antiinflammatory therapy targeting the interleukin-1β innate immunity pathway with canakinumab at a dose of 150 mg every 3 months led to a significantly lower rate of recurrent cardiovascular events than placebo, independent of lipid-level lowering. (Funded by Novartis; CANTOS ClinicalTrials.gov number, NCT01327846.

Activation of PKA via asymmetric allosteric coupling of structurally conserved cyclic nucleotide binding domains

Cyclic nucleotide-binding (CNB) domains allosterically regulate the activity of proteins with diverse functions, but the mechanisms that enable the cyclic nucleotide-binding signal to regulate distant domains are not well understood. Here we use optical tweezers and molecular dynamics to dissect changes in folding energy landscape associated with cAMP-binding signals transduced between the two CNB domains of protein kinase A (PKA). We find that the response of the energy landscape upon cAMP binding is domain specific, resulting in unique but mutually coordinated tasks: one CNB domain initiates cAMP binding and cooperativity, whereas the other triggers inter-domain interactions that promote the active conformation. Inter-domain interactions occur in a stepwise manner, beginning in intermediate-liganded states between apo and cAMP-bound domains. Moreover, we identify a cAMP-responsive switch, the N3A motif, whose conformation and stability depend on cAMP occupancy. This switch serves as a signaling hub, amplifying cAMP-binding signals during PKA activation