Gene-specific profiling of DNA methylation and mRNA expression in bovine oocytes derived from follicles of different size categories.

Epigenetic changes, such as DNA methylation, play an essential role in the acquisition of full developmental competence by mammalian oocytes during the late follicular growth phase. Here we used the bovine model to investigate the DNA methylation profiles of seven candidate genes (imprinted: bH19, bSNRPN; non-imprinted: bZAR1, bDNMT3A, bOCT4, bDNMT3 Lo and bDNMT3 Ls) and the mRNA expression of nine candidate genes (imprinted: bSNRPN, bPEG3, bIGF2R; non-imprinted: bPRDX1, bDNMT1B, bDNMT3A, bZAR1, bHSF1 and bNLRP9) in oocytes from antral follicles of three different size classes (≤2mm, 3-5mm, ≥6mm) to unravel the epigenetic contribution to this process. We observed an increased number of aberrantly methylated alleles in bH19, bSNRPN and bDNMT3 Lo of oocytes from small antral follicles (≤2mm), correlating with lower developmental competence. Furthermore, we detected an increased frequency of CpG sites with an unclear methylation status for DNMT3 Ls, specifically in oocytes from follicles ≥6mm, predominantly at three CpG positions (CpG2, CpG7 and CpG8), of which CpG7 is a potential regulatory site. No major differences in mRNA expression were observed, indicating that the transcriptional machinery may not yet be active during the follicular growth phase. Our results support the notion that a follicle diameter of ~2mm is a critical stage for establishing DNA methylation profiles and indicate a link between DNA methylation and the acquisition of oocyte developmental competence.

Characterization of differentially methylated regions in 3 bovine imprinted genes: a model for studying human germ-cell and embryo development.

Correct imprinting is crucial for normal fetal and placental development in mammals. Experimental evidence in animal models and epidemiological studies in humans suggest that assisted reproductive technologies (ARTs) can interfere with imprinted gene regulation in gametogenesis and early embryogenesis. Bos taurus is an agriculturally important species in which ARTs are commonly employed. Because this species exhibits a similar preimplantation development and gestation length as humans, it is increasingly being used as a model for human germ-cell and embryo development. However, in contrast to humans and mice, there is relatively little information on bovine imprinted genes. Here, we characterized the bovine intergenic IGF2-H19 imprinting control region (ICR) spanning approximately 3 kb. We identified a 300-bp differentially methylated region (DMR) approximately 6 kb upstream of the H19 promoter, containing a CpG island with CTCF-binding site and high sequence similarity with the human intergenic ICR. Additional differentially methylated CpG islands lie -6 kb to -3 kb upstream of the promoter, however these are less conserved. Both classical bisulfite sequencing and bisulfite pyrosequencing demonstrated complete methylation of the IGF2-H19 ICR in sperm, complete demethylation in parthenogenetic embryos having only the female genome, and differential methylation in placental and somatic tissues. In addition, we established pyrosequencing assays for the previously reported bovine SNRPN and PEG3 DMRs. The observed methylation patterns were consistent with genomic imprinting in all analyzed tissues/cell types. The identified IGF2-H19 ICR and the developed quantitative methylation assays may prove useful for further studies on the relationship between ARTs and imprinting defects in the bovine model.

Epigenetic profile of developmentally important genes in bovine oocytes.

Assisted reproductive technologies are associated with an increased incidence of epigenetic aberrations, specifically in imprinted genes. Here, we used the bovine oocyte as a model to determine putative epigenetic mutations at three imprinted gene loci caused by the type of maturation, either in vitro maturation (IVM) in Tissue Culture Medium 199 (TCM) or modified synthetic oviduct fluid (mSOF) medium, or in vivo maturation. We applied a limiting dilution approach and direct bisulfite sequencing to analyze the methylation profiles of individual alleles (DNA molecules) for H19/IGF2, PEG3, and SNRPN, which are each associated with imprinting defects in humans and/or the mouse model, and are known to be differentially methylated in bovine embryos. Altogether, we obtained the methylation patterns of 203 alleles containing 4,512 CpG sites from immature oocytes, 213 alleles with 4,779 CpG sites from TCM-matured oocytes, 215 alleles/4,725 CpGs in mSOF-matured oocytes, and 78 alleles/1,672 CpGs from in vivo-matured oocytes. The total rate of individual CpGs and entire allele methylation errors did not differ significantly between the two IVM and the in vivo group, indicating that current IVM protocols have no or only marginal effects on these critical epigenetic marks. Furthermore, the mRNA expression profiles of the three imprinted genes and a panel of eight other genes indicative of oocyte competence were determined by quantitative real-time PCR. We found different mRNA expression profiles between in vivo-matured oocytes versus their in vitro-matured counterparts, suggesting an influence on regulatory mechanisms other than DNA methylation.

Modeling psychotic and cognitive symptoms of affective disorders: Disrupted latent inhibition and reversal learning deficits in highly stress reactive mice.

Increased stress reactivity has repeatedly been reported in patients suffering from psychiatric diseases including schizophrenia and major depression. These disorders also have other symptoms in common, such as cognitive deficits and psychotic-like behavior. We have therefore investigated if increased stress reactivity is associated with these phenotypic endpoints in an animal model of affective disorders. The stress reactivity mouse model used in this study consists of three CD-1-derived mouse lines, that have been selectively bred for high (HR), intermediate (IR) or low (LR) stress reactivity. Male mice from these three breeding lines were subjected to a reversal learning task and latent inhibition (Li) was assessed using a conditioned taste aversion paradigm. Furthermore, as the dopaminergic system is involved in both Li and reversal learning, the dopamine 1 receptor (D1R), dopamine 2 receptor (D2R) and dopamine transporter (DAT) mRNA expression levels were assessed in relevant brain areas of these animals. The results demonstrate that HR mice show perseveration in the reversal learning task and have disrupted Li. Furthermore, compared to LR mice, HR mice have decreased D2R mRNA levels in the ventral tegmental area, as well as decreased D1R mRNA levels in the cingulate cortex, and an increased expression of D2R mRNA in the nucleus accumbens. Taken together, these results demonstrate that the HR mice display cognitive deficits associated with psychotic-like behavior, similar to those observed in patients suffering from schizophrenia and major depression and could be utilized in the search for better treatment strategies for these symptoms of psychiatric disorders.

[Post-traumatic torticollis in a schoolchild: fracture, congenital anomaly or age-appropriate radiological findings of the atlas?]. / Posttraumatischer Tortikollis beim Schulkind : Fraktur, angeborene Fehlbildung oder altersentsprechender Normalbefund des Atlasbogens?

We describe the case of a 6-year-old girl with post-traumatic torticollis after falling on her head. The suspected fractures of the dens axis and/or atlas were ruled out after performing CT and MRI examinations as well as dynamic fluoroscopy. Radiological findings showed no further instability but there was a congenital non-fusion of the posterior arch and an age-appropriate non-fused anterior arch of the atlas. In addition to discoligamental injuries and fractures, congenital anomalies and normal variants of the immature anatomy of the cervical spine should also be considered in the diagnosis of the pediatric cervical spine after trauma.

[Midterm Results for Quality of Life After Surgical Treatment of Moore Type V Fractures of the Tibial Plateau]. / Mittelfristige Ergebnisse zur Lebensqualität nach operativer Versorgung von Tibiakopffrakturen vom Typ Moore V.

Background: Dislocation fractures of the tibial plateau often lead to functional restrictions and subjective complaints from the patients. Besides functional and radiological results, criteria to determine the quality of life are of increasing importance. Intermediate term restriction in quality of life was evaluated and correlated with objective radiological results in patients with Moore type V dislocation fracture of the tibial plateau. Patients and Methods: From 2003 to 2012, a multicentre retrospective cohort study in three hospitals was used to register 36 patients with 38 Moore type V dislocation fractures of the tibial plateau. The injury mechanism, the surgical treatment (one step or two step surgery, single or double plate fixation) the complication rate, the radiological result (Kellgren/Lawrence osteoarthritis score, loss of reduction, secondary deviation of the axis) after a mean follow-up of 37 months, and the quality of life (pain and function by NRS, IKDC form, EQ 5D score) after a mean follow-up of 68 months (range, 15-128 months), were analysed. Results: The mean age of the 27 men and the 9 women was 50.8 years. There were 30 cases of high impact injury. An external fixator was used for primary fracture stabilisation in 24 knees; definitive internal fixation was performed in a second step. Internal fixation using a single plate was used in 12 knees, and double plate fixation in 25 knees; one patient was treated definitively with an external fixator. Early complications (3â€Š× infection, 2â€Š× compartment syndrome, 4â€Š× implant failure) were seen in 21.1 % of patients; all could be cured surgically. The function of the affected knee joint gave a mean NRS of 4.53; the IKDC score was 50.46, and the EQ 5D 7.47. Only two patients (5 %) were free of pain, 27 (75 %) reported mild to moderate pain, and 7 patients (20 %) reported severe pain. Four patients are retired or have applied for a pension. Altogether, the quality of life was calculated as being 44 % of the initial value before the injury. Four patients required an endoprosthetic replacement at an early stage, after an average of 6 months. Signs of osteoarthritis (Kellgren/Lawrence > I) were seen in 32 of the remaining 33 fractures; 19 of these exhibited distinct signs of osteoarthritis (Kellgren/Lawrence III, IV). Loss of reduction (≥ 2 mm) was seen in 13 (34.2 %) and deviation of axis (> 10°) in 3 patients (7.8 %). There was no relation to the surgical strategy. However, there was a correlation between the subjective assessments of the quality of life and the radiological results. Conclusion: Moore type V dislocation fractures of the tibial plateau are severe knee injuries resulting in a distinct reduction in quality of life in the intermediate term. There is a correlation between the subjective assessments and the objective radiological results. Therefore, the congruency of the articular surface and the axis have to be reconstructed as precisely as possible when repairing fractures of the tibial plateau.

[Does selenium prevent peroxynitrite formation from NO in vascular surgery interventions? A clinical study]. / Verhindert Selen die Peroxynitritbildung aus NO bei gefässchirurgischen Eingriffen? Eine klinische Studie.

Nitric oxide seems to play a protective role in the pathogenesis of arteriosclerosis. It appears to protect the tissue during reperfusion in vascular surgery. High values of NO show an inverse correlation to the severity of arteriosclerosis. In our opinion selenium should be substituted in order to enhance the activity of the glutathione peroxidase. This means an effective protection against the high production of peroxinitrite in patients with arteriosclerosis. A comparison of the corresponding redox pairs SeO3(2-)/SeO4(2-) and HOONO/HONO allows the conclusion that sodium selenite can be a direct antioxidant thereby.

[The wear on enamel, amalgam and their enamel antagonists in a computer-controlled mastication simulator]. / Verschleiss von Schmelz, Amalgam und ihrer Schmelz-antagonisten im computer-gesteuerten Kausimulator.

In a long-term in-vitro test the wear of enamel fossae, amalgam restorations and their opposing enamel cusps in the occlusal contact area was evaluated, qualitatively and quantitatively. The wear of enamel cusps differed from the wear of enamel fossae. The amalgam restorations wore down more rapidly than enamel fossae, but they were less destructive against their opposing enamel cusps. The wear of all tested materials was not linear. The results will serve as reference data for future long-term in-vitro tests of posterior restorative materials.

Intrahippocampal corticosterone response in mice selectively bred for extremes in stress reactivity: a microdialysis study.

The hypothalamic-pituitary-adrenocortical (HPA) axis is one of the major stress hormone systems, and glucocorticoids (GCs) play a pivotal role in homeostatic processes throughout the body and brain. A dysregulation of the HPA axis, leading to an aberrant secretion of GCs, is associated with affective disorders such as major depression. In the present study, three mouse lines selectively bred for high (HR), intermediate (IR) or low (LR) stress reactivity were used to elucidate the temporal dynamics of intrahippocampal corticosterone (CORT) in response to a standardised stressor. In particular, we addressed the question of whether the distinct differences in HPA axis reactivity between the three mouse lines, as determined by plasma CORT measurements, are present in the central nervous system as well, and if the respective endophenotype is brought about by alterations in blood-brain barrier (BBB) functionality. We applied in vivo microdialysis in the hippocampus, demonstrating that the concentrations of CORT released from the adrenals in response to restraint stress are not only distinctly different in the plasma, but can also be found in the central nervous system, although the differences between the three mouse lines were attenuated, particularly between IR and LR animals. Additionally, a time lag of approximately 60 min was observed in all three lines regarding intrahippocampal peak concentrations of CORT after the onset of the stressor. Furthermore, we showed that the penetration and clearance of CORT in the hippocampal tissue was not affected by differences in BBB functionality because the multidrug resistance 1 P-glycoprotein (Mdr1 Pgp) was equally expressed in HR, IR and LR mice. Furthermore, we could exclude surgical damage of the BBB because peripherally-injected dexamethasone, which is a high affinity substrate for the Mdr1 Pgp and therefore restricted from entering the brain, could only be detected in the plasma and was virtually absent in the brain.

Increased stress reactivity is associated with cognitive deficits and decreased hippocampal brain-derived neurotrophic factor in a mouse model of affective disorders.

Cognitive deficits are a common feature of major depression (MD), with largely unknown biological underpinnings. In addition to the affective and cognitive symptoms of MD, a dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis is commonly observed in these patients. Increased plasma glucocorticoid levels are known to render the hippocampus susceptible to neuronal damage. This structure is important for learning and memory, creating a potential link between HPA axis dysregulation and cognitive deficits in depression. In order to further elucidate how altered stress responsiveness may contribute to the etiology of MD, three mouse lines with high (HR), intermediate (IR), or low (LR) stress reactivity were generated by selective breeding. The aim of the present study was to investigate whether increased stress reactivity is associated with deficits in hippocampus-dependent memory tests. To this end, we subjected mice from the HR, IR, and LR breeding lines to tests of recognition memory, spatial memory, and depression-like behavior. In addition, measurements of brain-derived neurotrophic factor (BDNF) in the hippocampus and plasma of these animals were conducted. Our results demonstrate that HR mice exhibit hippocampus-dependent memory deficits along with decreased hippocampal, but not plasma, BDNF levels. Thus, the stress reactivity mouse lines are a promising animal model of the cognitive deficits in MD with the unique feature of a genetic predisposition for an altered HPA axis reactivity, which provides the opportunity to explore the progression of the symptoms of MD, predisposing genetic factors as well as new treatment strategies.

Wear of ceramic inlays, their enamel antagonists, and luting cements.

The wear of ceramic inlay materials and antagonist enamel cups was measured in vitro. Per group, six MOD inlays were adhesively cemented in extracted molars. Castable glass ceramic, pressed glass ceramic, and feldspathic porcelain were used as inlay materials. A microfilled and a fine hybrid composite served as luting agents. The restorations were exposed to an in vitro wear test that corresponds to approximately 5 years of clinical service. The wear in the occlusal contact area was quantified during and at the end of the test with a three-dimensional scanner. Final wear measurements on the inlays (mean +/- SD) ranged from 21.8 +/- 8.8 microns for pressed glass ceramic to 59.0 +/- 37.9 microns for castable glass ceramic. The mean wear of the enamel antagonists ranged from 74.6 +/- 32.9 microns for pressed glass ceramic to 153.2 +/- 61.5 microns for feldspathic porcelain. The wear of the luting composites at the end of the test ranged from 4.9 +/- 5.1 microns for microfilled to 12.3 +/- 6.3 microns for fine hybrid.