In vivo Identification and Specificity assessment of mRNA markers of hypoxia in human and mouse tumors

<p>Abstract</p> <p>Background</p> <p>Tumor hypoxia is linked to poor prognosis, but identification and quantification of tissue hypoxia remains a challenge. The hypoxia-specificity of HIF-1α target genes in vivo has been questioned due to the confounding influence of other microenvironmental abnormalities known to affect gene expression (e.g., low pH). Here we describe a new technique that by exploiting intratumoral oxygenation heterogeneity allows us to identify and objectively rank the most robust mRNA hypoxia biomarkers.</p> <p>Methods</p> <p>Mice carrying human (FaDu_dd) or murine (SCCVII) tumors were injected with the PET hypoxia tracer FAZA. Four hours post-injection tumors were removed, frozen, and crushed into milligram-sized fragments, which were transferred individually to pre-weighed tubes containing RNAlater and then weighed. For each fragment radioactivity per tissue mass and expression patterns of selected mRNA biomarkers were analyzed and compared.</p> <p>Results</p> <p>In both tumour models, fragmentation into pieces weighing 10 to 60 mg resulted in tissue fragments with highly variable relative content of hypoxic cells as evidenced by an up to 13-fold variation in FAZA radioactivity per mass of tissue. Linear regression analysis comparing FAZA retention with patterns of gene expression in individual tissue fragments revealed that CA9, GLUT1 and LOX mRNA levels were equally and strongly correlated to hypoxic extent in FaDu_dd. The same link between hypoxia and gene expression profile was observed for CA9 and GLUT1, but not LOX, in SCCVII tumors. Apparent in vivo hypoxia-specificity for other putative molecular markers of tissue hypoxia was considerably weaker.</p> <p>Conclusions</p> <p>The portrayed technique allows multiple pairwise measurements of mRNA transcript levels and extent of hypoxia in individual tumors at a smallest possible volumetric scale which (by limiting averaging effects inherent to whole-tumor analysis) strengthen the conclusiveness on true hypoxia-specificity of candidate genes while limiting the required number of tumors. Among tested genes, our study identified CA9, GLUT1 and possibly LOX as highly specific biomarkers of tumor hypoxia in vivo.</p

Education Modifies Genetic and Environmental Influences on BMI

Obesity is more common among the less educated, suggesting education-related environmental triggers. Such triggers may act differently dependent on genetic and environmental predisposition to obesity. In a Danish Twin Registry survey, 21,522 twins of same-sex pairs provided zygosity, height, weight, and education data. Body mass index (BMI = kg weight/ m height2) was used to measure degree of obesity. We used quantitative genetic modeling to examine how genetic and shared and nonshared environmental variance in BMI differed by level of education and to estimate how genetic and shared and nonshared environmental correlations between education and BMI differed by level of education, analyzing women and men separately. Correlations between education and BMI were −.13 in women, −.15 in men. High BMI's were less frequent among well-educated participants, generating less variance. In women, this was due to restriction of all forms of variance, overall by a factor of about 2. In men, genetic variance did not vary with education, but results for shared and nonshared environmental variance were similar to those for women. The contributions of the shared environment to the correlations between education and BMI were substantial among the well-educated, suggesting importance of familial environmental influences common to high education and lower BMI. Family influence was particularly important in linking high education and lower levels of obesity

Prenatal Stress Exposure Related to Maternal Bereavement and Risk of Childhood Overweight

BACKGROUND: It has been suggested that prenatal stress contributes to the risk of obesity later in life. In a population-based cohort study, we examined whether prenatal stress related to maternal bereavement during pregnancy was associated with the risk of overweight in offspring during school age. METHODOLOGY/PRINCIPAL FINDINGS: We followed 65,212 children born in Denmark from 1970-1989 who underwent health examinations from 7 to 13 years of age in public or private schools in Copenhagen. We identified 459 children as exposed to prenatal stress, defined by being born to mothers who were bereaved by death of a close family member from one year before pregnancy until birth of the child. We compared the prevalence of overweight between the exposed and the unexposed. Body mass index (BMI) values and prevalence of overweight were higher in the exposed children, but not significantly so until from 10 years of age and onwards, as compared with the unexposed children. For example, the adjusted odds ratio (OR) for overweight was 1.68 (95% confidence interval [CI] 1.08-2.61) at 12 years of age and 1.63 (95% CI 1.00-2.61) at 13 years of age. The highest ORs were observed when the death occurred in the period from 6 to 0 month before pregnancy (OR 3.31, 95% CI 1.71-6.42 at age 12, and OR 2.31, 95% CI 1.08-4.97 at age 13). CONCLUSIONS/SIGNIFICANCE: Our results suggest that severe pre-pregnancy stress is associated with an increased risk of overweight in the offspring in later childhood

The predictive value of microRNA-126 in relation to first line treatment with capecitabine and oxaliplatin in patients with metastatic colorectal cancer

<p>Abstract</p> <p>Background</p> <p>MicroRNA-126 is the only microRNA (miRNA) known to be endothelial cell-specific influencing angiogenesis in several ways. The aim of the present study was to analyse the possible predictive value of miRNA-126 in relation to first line capecitabine and oxaliplatin (XELOX) in patients with metastatic colorectal cancer (mCRC).</p> <p>Methods</p> <p>The study included 89 patients with mCRC. <it>In situ </it>hybridization (ISH) was performed to detect miRNA-126 in formalin-fixed paraffin embedded tissue from primary tumours. The expression of miRNA-126, area per image (μm²), was measured using image analysis. Clinical response was evaluated according to RECIST. Progression free survival (PFS) was compared using the Kaplan-Meier method and the log rank test. Tumours were classified as low or high miRNA-126 expressing tumours using the median value from the patients with response as cut-off.</p> <p>Results</p> <p>The median miRNA-126 expression level was significantly higher in patients responding to XELOX, 3629 μm²(95% CI, 2566-4846), compared to the patients not responding, 1670 μm²(95% CI, 1436-2041), <it>p </it>< 0.0001. The positive predictive value was 90%, and the negative predictive value was 71%. The median PFS of patients with high expressing tumours was 11.5 months (95% CI, 9.0-12.7 months) compared to 6.0 months (95% CI, 4.8-6.9 months) for patients with low expressing tumours, <it>p </it>< 0.0001.</p> <p>Conclusions</p> <p>Angiogenesis quantified by ISH of miRNA-126 was related to response to first line XELOX in patients with mCRC, translating to a significant difference in PFS. The predictive value of miRNA-126 remains to be further elucidated in prospective studies.</p

Intratumoral heterogeneity of microRNA expression in rectal cancer

Introduction: An increasing number of studies have investigated microRNAs (miRNAs) as potential markers of diagnosis, treatment and prognosis. So far, agreement between studies has been minimal, which may in part be explained by intratumoral heterogeneity of miRNA expression. The aim of the present study was to assess the heterogeneity of a panel of selected miRNAs in rectal cancer, using two different technical approaches. Materials and Methods: The expression of the investigated miRNAs was analysed by real-time quantitative polymerase chain reaction (RT-qPCR) and in situ hybridization (ISH) in tumour specimens from 27 patients with T3-4 rectal cancer. From each tumour, tissue from three different luminal localisations was examined. Inter- and intra-patient variability was assessed by calculating intraclass correlation coefficients (ICCs). Correlations between RT-qPCR and ISH were evaluated using Spearman's correlation. Results: ICCsingle (one sample from each patient) was higher than 50% for miRNA-21 and miRNA-31. For miRNA-125b, miRNA-145, and miRNA-630, ICCsingle was lower than 50%. The ICCmean (mean of three samples from each patient) was higher than 50% for miRNA-21(RT-qPCR and ISH), miRNA-125b (RT-qPCR and ISH), miRNA-145 (ISH), miRNA-630 (RT-qPCR), and miRNA-31 (RT-qPCR). For miRNA-145 (RT-qPCR) and miRNA-630 (ISH), ICCmean was lower than 50%. Spearman correlation coefficients, comparing results obtained by RT-qPCR and ISH, respectively, ranged from 0.084 to 0.325 for the mean value from each patient, and from -0.085 to 0.515 in the section including the deepest part of the tumour. Conclusion: Intratumoral heterogeneity may influence the measurement of miRNA expression and consequently the number of samples needed for representative estimates. Our findings with two different methods suggest that one sample is sufficient for adequate assessment of miRNA-21 and miRNA-31, whereas more samples would improve the assessment of miRNA-125b, miRNA-145, and miRNA-630. Interestingly, we found a poor correlation between the expression estimates obtained by RT-qPCR and ISH, respectively

Validity of Thermal Ramping Assays Used to Assess Thermal Tolerance in Arthropods

Proper assessment of environmental resistance of animals is critical for the ability of researchers to understand how variation in environmental conditions influence population and species abundance. This is also the case for studies of upper thermal limits in insects, where researchers studying animals under laboratory conditions must select appropriate methodology on which conclusions can be drawn. Ideally these methods should precisely estimate the trait of interest and also be biological meaningful. In an attempt to develop such tests it has been proposed that thermal ramping assays are useful assays for small insects because they incorporate an ecologically relevant gradual temperature change. However, recent model-based papers have suggested that estimates of thermal resistance may be strongly confounded by simultaneous starvation and dehydration stress. In the present study we empirically test these model predictions using two sets of independent experiments. We clearly demonstrate that results from ramping assays of small insects (Drosophila melanogaster) are not compromised by starvation- or dehydration-stress. Firstly we show that the mild disturbance of water and energy balance of D. melanogaster experienced during the ramping tests does not confound heat tolerance estimates. Secondly we show that flies pre-exposed to starvation and dehydration have “normal” heat tolerance and that resistance to heat stress is independent of the energetic and water status of the flies. On the basis of our results we discuss the assumptions used in recent model papers and present arguments as to why the ramping assay is both a valid and ecologically relevant way to measure thermal resistance in insects

Experimental probing of the interplay between ferromagnetism and localisation in (Ga,Mn)As

The question whether the Anderson-Mott localisation enhances or reduces magnetic correlations is central to the physics of magnetic alloys. Particularly intriguing is the case of (Ga,Mn)As and related magnetic semiconductors, for which diverging theoretical scenarios have been proposed. Here, by direct magnetisation measurements we demonstrate how magnetism evolves when the density of carriers mediating the spin-spin coupling is diminished by the gate electric field in metal/insulator/semiconductor structures of (Ga,Mn)As. Our findings show that the channel depletion results in a monotonic decrease of the Curie temperature, with no evidence for the maximum expected within the impurity-band models. We find that the transition from the ferromagnetic to the paramagnetic state proceeds via the emergence of a superparamagnetic-like spin arrangement. This implies that carrier localisation leads to a phase separation into ferromagnetic and nonmagnetic regions, which we attribute to critical fluctuations in the local density of states, specific to the Anderson-Mott quantum transition.Comment: 8 pages, 3 figure

Accuracy of direct genomic values in Holstein bulls and cows using subsets of SNP markers

Background: At the current price, the use of high-density single nucleotide polymorphisms (SNP) genotyping assays in genomic selection of dairy cattle is limited to applications involving elite sires and dams. The objective of this study was to evaluate the use of low-density assays to predict direct genomic value (DGV) on five milk production traits, an overall conformation trait, a survival index, and two profit index traits (APR, ASI). Methods. Dense SNP genotypes were available for 42,576 SNP for 2,114 Holstein bulls and 510 cows. A subset of 1,847 bulls born between 1955 and 2004 was used as a training set to fit models with various sets of pre-selected SNP. A group of 297 bulls born between 2001 and 2004 and all cows born between 1992 and 2004 were used to evaluate the accuracy of DGV prediction. Ridge regression (RR) and partial least squares regression (PLSR) were used to derive prediction equations and to rank SNP based on the absolute value of the regression coefficients. Four alternative strategies were applied to select subset of SNP, namely: subsets of the highest ranked SNP for each individual trait, or a single subset of evenly spaced SNP, where SNP were selected based on their rank for ASI, APR or minor allele frequency within intervals of approximately equal length. Results: RR and PLSR performed very similarly to predict DGV, with PLSR performing better for low-density assays and RR for higher-density SNP sets. When using all SNP, DGV predictions for production traits, which have a higher heritability, were more accurate (0.52-0.64) than for survival (0.19-0.20), which has a low heritability. The gain in accuracy using subsets that included the highest ranked SNP for each trait was marginal (5-6%) over a common set of evenly spaced SNP when at least 3,000 SNP were used. Subsets containing 3,000 SNP provided more than 90% of the accuracy that could be achieved with a high-density assay for cows, and 80% of the high-density assay for young bulls. Conclusions: Accurate genomic evaluation of the broader bull and cow population can be achieved with a single genotyping assays containing ∼ 3,000 to 5,000 evenly spaced SNP

Maternal Use of Antibiotics, Hospitalisation for Infection during Pregnancy, and Risk of Childhood Epilepsy: A Population-Based Cohort Study

BACKGROUND: Maternal infection during pregnancy may be a risk factor for epilepsy in offspring. Use of antibiotics is a valid marker of infection. METHODOLOGY/PRINCIPAL FINDINGS: To examine the relationship between maternal infection during pregnancy and risk of childhood epilepsy we conducted a historical cohort study of singletons born in northern Denmark from 1998 through 2008 who survived ≥29 days. We used population-based medical databases to ascertain maternal use of antibiotics or hospital contacts with infection during pregnancy, as well as first-time hospital contacts with a diagnosis of epilepsy among offspring. We compared incidence rates (IR) of epilepsy among children of mothers with and without infection during pregnancy. We examined the outcome according to trimester of exposure, type of antibiotic, and total number of prescriptions, using Poisson regression to estimate incidence rate ratios (IRRs) while adjusting for covariates. Among 191,383 children in the cohort, 948 (0.5%) were hospitalised or had an outpatient visit for epilepsy during follow-up, yielding an IR of 91 per 100 000 person-years (PY). The five-year cumulative incidence of epilepsy was 4.5 per 1000 children. Among children exposed prenatally to maternal infection, the IR was 117 per 100,000 PY, with an adjusted IRR of 1.40 (95% confidence interval (CI): 1.22-1.61), compared with unexposed children. The association was unaffected by trimester of exposure, antibiotic type, or prescription count. CONCLUSIONS/SIGNIFICANCE: Prenatal exposure to maternal infection is associated with an increased risk of epilepsy in childhood. The similarity of estimates across types of antibiotics suggests that processes common to all infections underlie this outcome, rather than specific pathogens or drugs