Ultrasound image analysis offers the opportunity to predict plasma progesterone concentrations in the estrous cycle in cows: a feasibility study

In recent years, several attempts have been made to evaluate the activity of a corpus luteum by determining its sonographic echo texture. In all of these studies the values of the echo texture parameters depended on the type and settings of the ultrasound machine. Therefore, the aim of the study was to investigate if a quantitative analysis of ultrasound (US) images of the corpus luteum (CL) after calibration of the ultrasound machine enables the assessment of the peripheral plasma progesterone (P4) level. Ten Holstein Friesian cows were examined daily at Days 4 to 8, 10 to 16, and -5 to -1 (Day 1=ovulation) of the estrous cycle. B-mode sonography of the corpora lutea was performed and blood samples were taken for plasma P4 analysis. US images were calibrated and analyzed using a software package (CAUS) developed by the authors. In addition to the area of the CL (Total Area, TotA; Tissue Area interactive, TisAi; Tissue Area Automatic, TisAa), the following US parameters were calculated from the gray level histogram and from the size of the speckles: Mean, Standard Deviation (SD) and Signal-to-Noise Ratio (SNR=Mean/SD) of echo levels, Residual Attenuation (ResAtt), Axial and Lateral speckle size (Ax and Lat, respectively). The inter-individual variability of the P4 level was expressed by the coefficient of variability (CV), averaged over all days. It appeared that the CV of the absolute P4 was high (0.65) and the P4 relative to that at Day 4 and at Day 16 was of comparable magnitude. Correlations of US parameters with P4 were highest for the P4 relative to Day 16 (P4_rel_D16). This relative P4 measure was then used for further analysis. The correlations of P4_rel_D16 with TotA, TisAa (CL area after automatic segmentation of tissue) and ResAtt were found the highest (R=0.68, 0.74, and -0.42, respectively). Multiple linear regression analysis, incorporating all US parameters revealed the formula: P4_rel_D16(pred)=-0.315+0.225TisAa-0.023ResAtt, and a goodness of fit: R(2)=0.59 (p<0.001). This formula was then used to "predict" for each image the P4_rel_D16 from the estimated US parameters. A high correlation of the predicted with the measured P4_rel_D16 was found: R=0.77. Classification of images using the predicted P4_rel_D16 to be in the range >0.80 (corresponding to 0.95 times the average_P4_rel_D16 measured during the "static" phase of the luteal cycle) by ROC analysis was correctly made in 88% of cases. In conclusion the quantitative analysis of calibrated ultrasound images may yield a good prediction of cyclic changes of P4 levels and has potential for predicting the phase in the estrous cycle of a cow.     

Limiting dilution bisulfite (pyro)sequencing reveals parent-specific methylation patterns in single early mouse embryos and bovine oocytes

To detect rare epigenetic effects associated with assisted reproduction, it is necessary to monitor methylation patterns of developmentally important genes in a few germ cells and individual embryos. Bisulfite treatment degrades DNA and reduces its complexity, rendering methylation analysis from small amounts of DNA extremely challenging. Here we describe a simple approach that allows determining the parent-specific methylation patterns of multiple genes in individual early embryos. Limiting dilution (LD) of bisulfite-treated DNA is combined with independent multiplex PCRs of single DNA target molecules to avoid amplification bias. Using this approach, we compared the methylation status of three imprinted (H19, Snrpn and Igf2r) and one pluripotency-related gene (Oct4) in three different groups of single mouse two-cell embryos. Standard in vitro fertilization of superovulated oocytes and the use of in vitro matured oocytes were not associated with significantly increased rates of stochastic single CpG methylation errors and epimutations (allele methylation errors), when compared with the in vivo produced controls. Similarly, we compared the methylation patterns of two imprinted genes (H19 and Snrpn) in individual mouse 16-cell embryos produced in vivo from superovulated and non-superovulated oocytes and did not observe major between-group differences. Using bovine oocytes and polar bodies as a model, we demonstrate that LD even allows the methylation analysis of multiple genes in single cells.     

Analysis of single nucleotide polymorphisms in three chromosomes of European sea bass Dicentrarchus labrax

Single nucleotide polymorphisms (SNPs) are believed to contain relevant information and have been therefore extensively used as genetic markers in population and conservation genetics, and molecular ecology studies. This study reports on the identification of potential SNPs in a diploid European sea bass Dicentrarchus labrax genome by using reference sequences from three assembled chromosomes and mapping all WGS datasets onto them (3× Sanger, 3× 454 and 20× SOLEXA). A total of 20,779 SNPs were identified over the 1469 gene loci and intergenic space analysed. Within chromosomes the occurrence of SNPs was the lowest in exons and higher in introns and intergenic regions, which may be explained by the fact, that coding regions are under strong selective pressure to maintain their biological function. The ratio of nonsynonymous to synonymous mutations was smaller than one for all the chromosomes, suggesting that most of deleterious nonsynonymous mutations were eliminated by negative selection. SNPs were not uniformly distributed over the chromosomes. Two chromosomes exhibited large regions with extremely low SNP density, which might represent homozygous regions in the diploid genome. The results of this study show how SNP detection can take profit from sequencing a single diploid individual, but also uncover the limits of such an approach. SNPs that have been identified will support marker development for genetic linkage mapping, population genetics and aquaculture related questions in general.     

Comparative analysis of intronless genes in teleost fish genomes: insights into their evolution and molecular function

This study assessed the relationship between the occurrence and function of intronless or single exon genes (SEG) in the genome of five teleost species and their phylogenetic distance. The results revealed that Takifugu rubripes, Tetraodon nigroviridis, Oryzias latipes, Gasterosteus aculeatus and Danio rerio genomes are respectively comprised of 2.83%, 3.42%, 4.49%, 4.35% and 4.02% SEGs. These SEGs encode for a variety of family proteins including claudins, olfactory receptors and histones that are essential for various biological functions. Subsequently, we predicted and annotated SEGs in three European sea bass, Dicentrarchus labrax chromosomes that we have sequenced, and compared results with those of stickleback (G. aculeatus) homologous chromosomes. While the annotation features of three D. labrax chromosomes revealed 78 (5.30%) intronless genes, comparisons with G. aculeatus showed that SEG composition and their order varied significantly among corresponding chromosomes, even for those with nearly complete synteny. More than half of SEGs identified in most of the species have at least one ortholog multiple exon gene in the same genome, which provides insight to their possible origin by retrotransposition. In spite of the fact that they belong to the same lineage, the fraction of predicted SEGs varied significantly between the genomes analyzed, and only a low fraction of proteins (4.1%) is conserved between all five species. Furthermore, the inter-specific distribution of SEGs as well as the functional categories shared by species did not reflect their phylogenetic relationships. These results indicate that new SEGs are continuously and independently generated after species divergence over evolutionary time as evidenced by the phylogenetic results of single exon claudins genes. Although the origin of SEGs cannot be inferred directly from the phylogeny, our results provide strong support for the idea that retrotransposition followed by tandem duplications is the most probable event that can explain the expansion of SEGs in eukaryotic organisms.     

Dietary fiber, carbohydrate quality and quantity, and mortality risk of individuals with diabetes mellitus

Background

Dietary fiber, carbohydrate quality and quantity are associated with mortality risk in the general population. Whether this is also the case among diabetes patients is unknown.

Objective

To assess the associations of dietary fiber, glycemic load, glycemic index, carbohydrate, sugar, and starch intake with mortality risk in individuals with diabetes.

Methods

This study was a prospective cohort study among 6,192 individuals with confirmed diabetes mellitus (mean age of 57.4 years, and median diabetes duration of 4.4 years at baseline) from the European Prospective Investigation into Cancer and Nutrition (EPIC). Dietary intake was assessed at baseline (1992–2000) with validated dietary questionnaires. Cox proportional hazards analysis was performed to estimate hazard ratios (HRs) for all-cause and cardiovascular mortality, while adjusting for CVD-related, diabetes-related, and nutritional factors.

Results

During a median follow-up of 9.2 y, 791 deaths were recorded, 306 due to CVD. Dietary fiber was inversely associated with all-cause mortality risk (adjusted HR per SD increase, 0.83 [95% CI, 0.75–0.91]) and CVD mortality risk (0.76[0.64–0.89]). No significant associations were observed for glycemic load, glycemic index, carbohydrate, sugar, or starch. Glycemic load (1.42[1.07–1.88]), carbohydrate (1.67[1.18–2.37]) and sugar intake (1.53[1.12–2.09]) were associated with an increased total mortality risk among normal weight individuals (BMI≤25 kg/m²; 22% of study population) but not among overweight individuals (P interaction≤0.04). These associations became stronger after exclusion of energy misreporters.

Conclusions

High fiber intake was associated with a decreased mortality risk. High glycemic load, carbohydrate and sugar intake were associated with an increased mortality risk in normal weight individuals with diabetes.     

Association of sleep duration with chronic diseases in the European Prospective Investigation into Cancer and Nutrition (EPIC)-Potsdam study

Background

In view of the reduced number of hours devoted to sleep in modern western societies the question arises what effects might result from sleep duration on occurrence of chronic diseases.

Methods

Data from 23 620 middle-aged participants of the European Prospective Investigation into Cancer and Nutrition (EPIC)-Potsdam study, that were recruited between 1994–1998, were analyzed by using Cox proportional hazard regression to examine the association between self-reported sleep duration at baseline and incidence of chronic diseases, such as diabetes, myocardial infarction, stroke, and cancer.

Results

During a mean follow-up period of 7.8 years 841 incident cases of type 2 diabetes, 197 cases of myocardial infarction, 169 incident strokes, and 846 tumor cases were observed. Compared to persons sleeping 7-<8 h/day, participants with sleep duration of <6 h had a significantly increased risk of stroke (Hazard Ratio (HR) = 2.06, 95% confidence interval (CI): 1.18–3.59), cancer (HR = 1.43, 95% CI: 1.09–1.87), and overall chronic diseases (HR = 1.31, 95% CI: 1.10–1.55) in multivariable adjusted models. Self-reported daytime sleep at baseline was not associated with incident chronic diseases in the overall study sample. However, there had been an effect modification of daytime sleep by hypertension showing that daytime sleep was inversely related to chronic disease risk among non-hypertensive participants but directly related to chronic diseases among hypertensives.

Conclusion

Sleep duration of less than 6 h is a risky behavior for the development of chronic diseases, particularly stroke and cancer, and should be therefore addressed in public health campaigns.     

Thoracic Aortic Aneurysm (TAAD)-causing Mutation in Actin Affects Formin Regulation of Polymerization

More than 30 mutations in ACTA2, which encodes α-smooth muscle actin, have been identified to cause autosomal dominant thoracic aortic aneurysm and dissection. The mutation R256H is of particular interest because it also causes patent ductus arteriosus and moyamoya disease. R256H is one of the more prevalent mutations and, based on its molecular location near the strand-strand interface in the actin filament, may affect F-actin stability. To understand the molecular ramifications of the R256H mutation, we generated Saccharomyces cerevisiae yeast cells expressing only R256H yeast actin as a model system. These cells displayed abnormal cytoskeletal morphology and increased sensitivity to latrunculin A. After cable disassembly induced by transient exposure to latrunculin A, mutant cells were delayed in reestablishing the actin cytoskeleton. In vitro, mutant actin exhibited a higher than normal critical concentration and a delayed nucleation. Consequently, we investigated regulation of mutant actin by formin, a potent facilitator of nucleation and a protein needed for normal vascular smooth muscle cell development. Mutant actin polymerization was inhibited by the FH1-FH2 fragment of the yeast formin, Bni1. This fragment strongly capped the filament rather than facilitating polymerization. Interestingly, phalloidin or the presence of wild type actin reversed the strong capping behavior of Bni1. Together, the data suggest that the R256H actin mutation alters filament conformation resulting in filament instability and misregulation by formin. These biochemical effects may contribute to abnormal histology identified in diseased arterial samples from affected patients.     

Egr2::Cre Mediated Conditional Ablation of Dicer Disrupts Histogenesis of Mammalian Central Auditory Nuclei

Histogenesis of the auditory system requires extensive molecular orchestration. Recently, Dicer1, an essential gene for generation of microRNAs, and miR-96 were shown to be important for development of the peripheral auditory system. Here, we investigated their role for the formation of the auditory brainstem. Egr2::Cre-mediated early embryonic ablation of Dicer1 caused severe disruption of auditory brainstem structures. In adult animals, the volume of the cochlear nucleus complex (CNC) was reduced by 73.5%. This decrease is in part attributed to the lack of the microneuronal shell. In contrast, fusiform cells, which similar to the granular cells of the microneural shell are derived from Egr2 positive cells, were still present. The volume reduction of the CNC was already present at birth (67.2% decrease). The superior olivary complex was also drastically affected in these mice. Nissl staining as well as Vglut1 and Calbindin 1 immunolabeling revealed that principal SOC nuclei such as the medial nucleus of the trapezoid body and the lateral superior olive were absent. Only choline acetyltransferase positive neurons of the olivocochlear bundle were observed as a densely packed cell group in the ventrolateral area of the SOC. Mid-embryonic ablation of Dicer1 in the ventral cochlear nucleus by Atoh7::Cre-mediated recombination resulted in normal formation of the cochlear nucleus complex, indicating an early embryonic requirement of Dicer1. Quantitative RT-PCR analysis of miR-96 demonstrated low expression in the embryonic brainstem and up-regulation thereafter, suggesting that other microRNAs are required for proper histogenesis of the auditory brainstem. Together our data identify a critical role of Dicer activity during embryonic development of the auditory brainstem.     

Dietary Factors Impact on the Association between CTSS Variants and Obesity Related Traits

Background/Aims

Cathepsin S, a protein coded by the CTSS gene, is implicated in adipose tissue biology–this protein enhances adipose tissue development. Our hypothesis is that common variants in CTSS play a role in body weight regulation and in the development of obesity and that these effects are influenced by dietary factors–increased by high protein, glycemic index and energy diets.

Methods

Four tag SNPs (rs7511673, rs11576175, rs10888390 and rs1136774) were selected to capture all common variation in the CTSS region. Association between these four SNPs and several adiposity measurements (BMI, waist circumference, waist for given BMI and being a weight gainer–experiencing the greatest degree of unexplained annual weight gain during follow-up or not) given, where applicable, both as baseline values and gain during the study period (6–8 years) were tested in 11,091 European individuals (linear or logistic regression models). We also examined the interaction between the CTSS variants and dietary factors–energy density, protein content (in grams or in % of total energy intake) and glycemic index–on these four adiposity phenotypes.

Results

We found several associations between CTSS polymorphisms and anthropometric traits including baseline BMI (rs11576175 (SNP N°2), p = 0.02, β = −0.2446), and waist change over time (rs7511673 (SNP N°1), p = 0.01, β = −0.0433 and rs10888390 (SNP N°3), p = 0.04, β = −0.0342). In interaction with the percentage of proteins contained in the diet, rs11576175 (SNP N°2) was also associated with the risk of being a weight gainer (p_interaction = 0.01, OR = 1.0526)–the risk of being a weight gainer increased with the percentage of proteins contained in the diet.

Conclusion

CTSS variants seem to be nominally associated to obesity related traits and this association may be modified by dietary protein intake.