Search

We present a design for silicon-compatible vertical Germanium pin photodiodes structured into all-dielectric metasurfaces. Proof-of-principle metasurfaces are fabricated on silicon-on-insulator wafers in a top-down process. Simulations and measurements of the spectroscopic properties, specifically the absorption, show high spectral selectivity, and absorption efficiencies as large as those in bulk Germanium layers with about four times the Ge layer thicknesses. Our metasurface structures can be tuned to the target wavelength through tailoring of the lateral geometry. Possible applications include spectroscopy and hyperspectral imaging, with several metasurfaces for different wavelength ranges integrated with readout circuitry into a low-cost electronic–photonic integrated circuit.

Maternal exposure to estrogens can induce long-term adverse effects in the offspring. The epigenetic programming may start as early as the period of preimplantation development. We analyzed the effects of gestational estradiol-17β (E2) exposure with two distinct low doses, corresponding to the acceptable daily intake “ADI” and close to the no-observed-effect level “NOEL”, and a high dose (0.05, 10, and 1000 µg E2/kg body weight daily, respectively). The E2 doses were orally applied to sows from insemination until sampling at day 10 of pregnancy and compared to carrier-treated controls leading to a significant increase in E2 in plasma, bile and selected somatic tissues including the endometrium in the high-dose group. Conjugated and unconjugated E2 metabolites were as well elevated in the NOEL group. Although RNA-sequencing revealed a dose-dependent effect of 14, 17, and 27 differentially expressed genes (DEG) in the endometrium, single embryos were much more affected with 982 DEG in female blastocysts of the high-dose group, while none were present in the corresponding male embryos. Moreover, the NOEL treatment caused 62 and 3 DEG in female andmale embryos, respectively. Thus, we detected a perturbed sex-specific gene expression profile leading to a leveling of the transcriptome profiles of female and male embryos. The preimplantation period therefore demonstrates a vulnerable time window for estrogen exposure, potentially constituting the cause for lasting consequences. The molecular fingerprint of low-dose estrogen exposure on developing embryos warrants a careful revisit of effect level thresholds.Summary SentenceMaternal oral low-dose estrogen exposure during the preimplantation period specifically targeted female embryos by inducing a male-like gene expression profile.

Summary Nucleosome-to-protamine exchange during mammalian spermiogenesis is essential for compaction and protection of paternal DNA. It is interesting that, depending on the species, 1% to 15% of nucleosomes are retained, but the generalizability and biological function of this retention are unknown. Here, we show concordantly in human and bovine that nucleosomes remained in sperm chromatin predominantly within distal intergenic regions and introns and associated with centromere repeats and retrotransposons (LINE1 and SINEs). In contrast, nucleosome depletion concerned particularly exons, 5′-UTR, 3′-UTR, TSS, and TTS and was associated with simple and low-complexity repeats. Overlap of human and bovine genes exhibiting nucleosome preservation in the promoter and gene body revealed a significant enrichment of signal transduction and RNA- and protein-processing factors. Our study demonstrates the genome-wide uniformity of the nucleosome preservation pattern in mammalian sperm and its connection to repetitive DNA elements and suggests a function in preimplantation processes for paternally derived nucleosomes.

Objective The prevalence of diabetes mellitus and associated complications is steadily increasing. As a resource for studying systemic consequences of chronic insulin insufficiency and hyperglycemia, we established a comprehensive biobank of long-term diabetic INSC94Y transgenic pigs, a model of mutant INS gene-induced diabetes of youth (MIDY), and of wild-type (WT) littermates. Methods Female MIDY pigs (n = 4) were maintained with suboptimal insulin treatment for 2 years, together with female WT littermates (n = 5). Plasma insulin, C-peptide and glucagon levels were regularly determined using specific immunoassays. In addition, clinical chemical, targeted metabolomics, and lipidomics analyses were performed. At age 2 years, all pigs were euthanized, necropsied, and a broad spectrum of tissues was taken by systematic uniform random sampling procedures. Total beta cell volume was determined by stereological methods. A pilot proteome analysis of pancreas, liver, and kidney cortex was performed by label free proteomics. Results MIDY pigs had elevated fasting plasma glucose and fructosamine concentrations, C-peptide levels that decreased with age and were undetectable at 2 years, and an 82% reduced total beta cell volume compared to WT. Plasma glucagon and beta hydroxybutyrate levels of MIDY pigs were chronically elevated, reflecting hallmarks of poorly controlled diabetes in humans. In total, ∼1900 samples of different body fluids (blood, serum, plasma, urine, cerebrospinal fluid, and synovial fluid) as well as ∼17,000 samples from ∼50 different tissues and organs were preserved to facilitate a plethora of morphological and molecular analyses. Principal component analyses of plasma targeted metabolomics and lipidomics data and of proteome profiles from pancreas, liver, and kidney cortex clearly separated MIDY and WT samples. Conclusions The broad spectrum of well-defined biosamples in the Munich MIDY Pig Biobank that will be available to the scientific community provides a unique resource for systematic studies of organ crosstalk in diabetes in a multi-organ, multi-omics dimension., Molecular Metabolism, 6 (8)

Summary Spermatozoa are transcriptionally inactive cells, but contain acetylated histones, normally a characteristic of transcriptionally active cells. Acetylgroups are thought to represent epigenetic marks that are transmitted to the oocyte and are involved in starting gene expression in the zygote and in regulating gene expression during early embryogenesis. We performed reverse transcription polymerase chain reaction (RT-PCR) in the common marmoset monkey (Callithrix jacchus) and in bovine spermatozoa, oocytes, zygotes, two- and four-cell embryos to evaluate the presence of specific transcripts known to play a role during fertilisation and early embryo development, namely protamine-1 (PRM1), protamine-2 (PRM2), histone H1 (H1), histone H3 (H3), histone H4 (H4), cAMP-responsive element modulator (CREM), DNA methyltransferase-1 (DNMT1), TATA box-binding protein (TBP). All transcripts tested were present in spermatozoa of the common marmoset, while bull spermatozoa lack PRM2. Marmoset oocytes exhibited transcripts for H1, H3, H4 and TBP, whereas bovine oocytes revealed H1, H3, H4, CREM, DNMT and TBP mRNAs. In zygotes, we amplified H1, H4, TBP (marmoset) and PRM1, H1, H3, H4, CREM, DNMT1 and TBP (bovine). Two-cell embryos showed PCR products for H1, H3 and TBP in the marmoset. In the bovine, all transcripts could be observed except PRM2. In four-cell embryos, PCR signals were obtained for PRM1, H1, H3, H4 and TBP in the marmoset. In the bovine, all transcripts were detected except PRM2. Our data suggest that, in both C. jacchus and Bos taurus, PRM1 transcripts are delivered by the spermatozoon to the oocyte.

In breeding programs, the application of a vitrification method suitable for direct transfer of biopsied embryos can increase the genetic improvement of cattle and help reduce the costs of embryo transfer. The aim of this study was to determine the in vitro survival of biopsied vitrified blastocysts using the new VitTrans device (Morató and Mogas 2014 Cryobiology 68, 288-293), a 1-step in-straw warming system. Immature bovine oocytes were in vitro matured, fertilized, and cultured to the blastocyst stage. A total of 110 grade 1 blastocysts (IETS codes 6 and 7) were randomly allocated to 2 groups: (1) biopsy (n=49) and (2) without biopsy, or control (n=61). Blastocysts were biopsied using a microblade mounted on a micromanipulator. A small portion of the trophoblast, approximately 15%, was cut off and a significant part of the zona pellucida was sliced away. Both groups were then vitrified using the VitTrans device. For vitrification, all blastocysts were exposed to an equilibration medium with 7.5% ethylene glycol+7.5% dimethyl sulfoxide in holding medium (HM) consisting of TCM-199 with 20% FCS, moved into a drop with 16.5% ethylene glycol+16.5% dimethyl sulfoxide+0.5M sucrose in HM, and then placed in a microdroplet on the VitTrans. The VitTrans was plunged into LN and covered with a 0.5-mL straw. For warming, the protective cover was removed from the VitTrans while still submerged in LN. Subsequently, a new 0.5-mL plastic embryo transfer straw was placed on the VitTrans while flushing the warming solution (0.3mL of 0.5M sucrose in HM at 45°C) with a syringe through the lumen of the device. By entering the warming solution into the VitTrans device, the embryo is flushed inside the plastic straw. The straw containing the embryo can then be readily used for transfer after the VitTrans is removed. To recover the embryo in the laboratory, the content of the straw was put into a Petri dish and blastocysts were placed in the culture medium and incubated at 38.5°C in 5% CO2 and 5% O2 in air. Morphology and re-expansion were evaluated 24h post-warming. The embryo survival rate was defined as the ratio of blastocysts that were able to re-expand with regards to the total number of warmed blastocysts. Due to the attachment of embryos inside the straw, a total of 18 embryos were lost during recovery (12 from the biopsied group and 6 from the nonbiopsied group). The ratio of re-expanded blastocysts from the recovered embryos was 40% in the biopsy group and 61% in the control group. In conclusion, vitrification using the VitTrans device showed good results with intact embryos compared with biopsied embryos. In addition, biopsied embryos had a tendency to adhere to the inside of the straw, which is probably due to the damage or loss of the zona pellucida. Additional research is required to minimize the loss of embryos.

Lentiviral vectors are a powerful tool for the genetic modification of livestock species. We previously generated transgenic founder cattle with lentiviral integrants carrying enhanced green fluorescent protein (EGFP) under the control of the phosphoglycerate kinase (PGK) promoter. In this study, we investigated the transmission of LV-PGK-EGFP integrants through the female and male germ line in cattle. A transgenic founder heifer (#562, Kiki) was subjected to superovulation treatment and inseminated with semen from a non-transgenic bull. Embryos were recovered and transferred to synchronized recipient heifers, resulting in the birth of a healthy male transgenic calf expressing EGFP as detected by in vivo imaging. Semen from a transgenic founder bull (#561, Jojo) was used for in vitro fertilization (IVF) of in vitro matured (IVM) oocytes from non-transgenic cows. The rates of cleavage and development to blastocyst in vitro corresponded to 52.0 +/- 4.1 and 24.5 +/- 4.4%, respectively. Expression of EGFP was observed at blastocyst stage (day 7 after IVF) and was seen in 93.0% (281/302) of the embryos. 24 EGFP-expressing embryos were transferred to 9 synchronized recipients. Analysis of 2 embryos, flushed from the uterus on day 15, two fetuses recovered on day 45, and a healthy male transgenic calf revealed consistent high-level expression of EGFP in all tissues investigated. Our study shows for the first time transmission of lentiviral integrants through the germ line of female and male transgenic founder cattle. The pattern of inheritance was consistent with Mendelian rules. Importantly, high fidelity expression of EGFP in embryos, fetuses, and offspring of founder #561 provides interesting tools for developmental studies in cattle, including interactions of gametes, embryos and fetuses with their maternal environment.

Uterine secretions have a dominant impact on the environment in which embryo development takes place. The uterine serpins (SERPINA14, previously known as UTMP) are found most abundantly during pregnancy in the uterus of ruminants. Although progesterone is currently assumed to be the major regulator of SERPINA14 expression, our recent study of transcriptome changes in bovine endometrium during the estrous cycle unexpectedly detected a marked upregulation of SERPINA14 mRNA levels at estrus. The present study describes the full-length mRNA sequence, genomic organization, and putative promoter elements of the SERPINA14 gene. The SERPINA14 mRNA abundance was quantified by real-time RT-PCR in intercaruncular endometrium at several time points during the estrous cycle and early pregnancy. Highest levels were found at estrus, followed by a dramatic decrease and a moderate expression during the luteal phase. Transcript levels were higher in pregnant endometrium compared with controls at Day 18. At estrus, immunoreactive protein was localized in deep glandular epithelium, and Western blotting concomitantly showed the 52-kDa form in uterine flushings. SERPINA14 mRNA was significantly upregulated in glandular endometrial cells in vitro after stimulation with estradiol-17beta and progesterone, but not after interferon-tau treatment. Our results clearly demonstrate that SERPINA14 appears distinctly in bovine endometrium during the estrus phase. A supporting role toward providing a well-prepared endometrial environment for passing gametes, especially sperm, is assumed.

One central effect of Active Thermal Management is the significantly faster powertrain warm up. The warm up operation strategy is divided into two parts – the first step is Zero Flow, which is the fastest passive engine warm up method, and is followed by the warm up mode with speed controlled operation of the electrical water pump (eWAPU).

Multiplication of bovine embryos by the production of aggregation chimeras is based on the concept that few blastomeres of a donor embryo form the inner cell mass (ICM) and thus the embryo proper, whereas cells of a host embryo preferentially contribute to the trophectoderm (TE), the progenitor cells of the embryonic part of the placenta. We aggregated two fluorescent blastomeres from enhanced green fluorescent protein (eGFP) transgenic Day 5 morulae with two Day 4 embryos that did not complete their first cleavage until 27 hours after IVF and tested the effect of phytohemagglutinin-L (PHA) on chimeric embryo formation. The resulting blastocysts were characterized by differential staining of cell lineages using the TE-specific factor CDX2 and confocal laser scanning microscopy to facilitate the precise localization of eGFP-positive cells. The proportions of blastocyst development of sandwich aggregates with (n = 99) and without PHA (n = 46) were 85.9% and 54.3% (P

A polymerizable-complex method (PCM) was used with automated solution deposition to generate combinatorial libraries of oxide powders that were evaluated for their ability to catalyze the combustion of soot. Chemical compositions were examined for catalytic activity in the soot combustion reaction in parallel using infrared thermography and serially using automated thermogravimetric analysis. Although infrared analysis is much faster, it is not easily suited to analyzing soot combustion in the configuration employed in the present study. Instead, the slower automated thermogravimetric analysis (TGA) was found to be much more reliable and reproducible. The results of characterizing simple oxide systems (Ce–K–O, Cu–K–O, Ce–Na–O and Cu–Na–O) are reported.

Background A mediastinitis or intraoperative bleeding are the most feared complications of the transoral laser assisted diverticulotomy of Zenker's diverticulum. To reduce these complications we developed a new endoscopic surgical technique and compared the results of this new method with the results of patients who we treated earlier. Patients and methods At the department of Otorhinolaryngology, Head and Neck Surgery, University of Luebeck 68 patients suffering from a hypopharyngeal diverticulum have been treated since 1987. In 64 patients we performed an endoscopic diverticulotomy using a CO2-Laser and the Weerda distending diverticuloscope. Four of the Zenker's diverticulua had to be removed by an external approach. From 1987 to 1994 a five millimeter bar between pouch and hypopharynx after diverticulotomy was left and sealed with fibrin glue in the first 38 patients. From 1995 to 1999 the spur of the hypopharyngeal pouch of the other 26 patients was completely separated. Afterwards we sutured the mucosa between the pouch and the hypopharynx to close the opened mediastinal space and sealed the former spur with fibrin glue. Results Comparing both endoscopic methods we had to observe concerning our first method one mediastinitis, a rise of body temperature in 29 patients (76%) and we had to perform a revision diverticulotomy in three patients (8%). Our modified technique didn't cause any mediastinitis, a rise of body temperature did only occur in five patients (18%) and no patient had to undergo revision surgery. Conclusions The transoral complete separation of the spur of the hypopharyngeal pouch with suturing the mucosa of the pouch and the hypopharynx causes good functional results and means a further reduction of complications and a further improvement of the endoscopic diverticulotomy. Our new instruments (Fa. K. Storz, Tuttlingen) will be demonstrated.

Combinatorial libraries of the mixed oxide system Cu1−xCexO3(0 ≤x≤ 1) have been generated using a modified Pechini powder synthesis process in conjunction with inkjet deposition. Mixed oxide crystalline powders were formed at temperatures below 500 °C. These powders have particle sizes in the range of 20.0 to 85.5 nm, and the powder surface area increases with cerium content. In addition, each of the mixed oxides (0

Background One of the most important criteria of malignancy of head and neck cancer are the cervical lymph metastases. Being significant for the therapeutical plan is how tumor depending parameters like T-stage, degree of differentiation and tumor localisation will influence the N-stage and therefore the extension of neck dissection. Method To evaluate the pattern of formations of metastases and the success of therapy a retrospective study was performed on 405 patients with carcinoma of the oral cavity (n = 47), the oropharynx (n = 117), the hypopharynx (n = 47) and the larynx (n = 193). Results By the time of surgery carcinoma of the hypopharynx were most frequently accompanied by cervical metastases (80%), followed by carcinoma of the oropharynx (70%), the oral cavity (52%) and the larynx (26%). Occurrence and extension of regional lymph node metastases correlated well with T-stages and degree of differentiation. After surgical therapy locoregional recurrence could be observed in 5.2% of the patients. Five-year-survival rate was reduced to 50% on patients with positive lymph nodes. The different tumour sites showed preferred patterns of metastatic spread, without complete avoidance of certain levels. Conclusion For the decision on indication and extent of neck dissection the preoperative diagnostic (ultrasound, CT-scan, MRI), localisation of tumour, T-stage, degree of differentiation and the knowledge of typical metastatic spread must be considered.

Background The intranasal allergen Provocation test (INT) is the standard procedure to verify the effect of an allergen when a nasal allergy is suspected. It may be especially useful if results from skin tests or in vitro tests conflict with those from the history. In the clinical and research setting, INT may be used to study the efficacy of different treatment modalities such as pharmacotherapy or immunotherapy. The technique of INT has not been well standardized in the past. Variations exist in planning the optimum time for testing, especially in seasonal allergies. Methods We examined influences of different test dates with regard to the pollen season on the result of INT. A total of 19 patients (eight females, 11 males, aged 26.1 +/- 4.8 years) with a history of at least two years of allergic rhinitis to birch pollen, positive skin prick test, and RAST (> or = CAP class II) to birch pollen. INT was performed during the winter-period after approximately six months without natural pollen exposition (V1), during the birch pollen season (V2) and six weeks after the birch pollen season (V3). Allergens were applied using a pump spray (0.1 ml) in concentrations of 10, 50, 100, 500, 1000, 5000, 10,000, 50,000, and 100,000 SQ units per ml (SQ/ml). Results The threshold concentrations were significantly lower at V2 (100 SQ/ml) and V3 (500 SQ/ml) if compared to V1 (1000 SQ/ ml). Conclusions Seasonal influences should be taken into account when performing INT in birch pollen allergy.

The porcine conceptus undergoes rapid differentiation and expansion of its trophoblastic membranes between Days 11 and 12 of gestation. Concomitant with trophoblast elongation, production of conceptus estrogen, the porcine embryonic pregnancy recognition signal, increases. Conceptus attachment to the uterine surface epithelium starts after Day 13, initiating epitheliochorial placentation. To analyze the transcriptome changes in the endometrium in the course of maternal recognition of pregnancy, deep sequencing of endometrial RNA samples of Day 12 pregnant animals (n = 4) and corresponding nonpregnant controls (n = 4) was performed using RNA sequencing (RNA-Seq). Between 30 000 000 and 35 000 000 sequence reads per sample were produced and mapped to the porcine genome (Sscrofa10.2). Analysis of read counts revealed 2593 differentially expressed genes (DEGs). Expression of selected genes was validated by the use of quantitative real-time RT-PCR. Bioinformatics analysis identified several functional terms specifically overrepresented for up-regulated or down-regulated genes. Comparison of the RNA-Seq data from Days 12 and 14 of pregnancy was performed at the level of all expressed genes, the level of the DEG, and the level of functional categories. This revealed specific gene expression patterns reflecting the different functions of the endometrium during these stages (i.e., recognition of pregnancy and preparation for conceptus attachment). Genes related to mitosis, immune response, epithelial cell differentiation and development, proteolysis, and prostaglandin signaling and metabolism are discussed in detail. This study identified comprehensive transcriptome changes in porcine endometrium associated with establishment of pregnancy and could be a resource for targeted studies of genes and pathways potentially involved in regulation of this process.

We have measured the spectrum of vacuum ultraviolet radiation (500 - 1700 A) arising from the impact of 625 MeV xenon ions with . The spectral features are due primarily to emission from neutral and ionized carbon atoms. These photons mask sensitivity to either the giant dipole resonance or to excited dimers. We compare with data obtained in collisions of xenon ions with methane and isobutane. Decay times of several of the lines were also measured. Some of the lines persist for times long compared to the lifetimes of the corresponding states in neutral and ionized carbon.

We generated a CRISPR/Cas9 mediated knockout (KO) of the OCT4 gene in adult fibroblasts, where biallelic deletion of a single nucleotide leads to a frameshift mutation. Through reconstruction of embryos by somatic cell NT, we were able to study the role of OCT4 during pre-implantation development until Day 7 in vitro. The presence of OCT4 protein was evaluated after immunofluorescent staining by confocal laser scanning microscopy of Day 5 morulae and Day 7 blastocysts; somatic cell NT embryos reconstructed from nontransfected cells of the same source served as control. Whereas control morulae expressed OCT4 in all cells, OCT4 KO morulae showed expression in only 67.8 ± 11.1% (mean ± SD, n = 6) of cells and overall intensity was decreased. By Day 7, no expression of OCT4 was detected in OCT4 KO blastocysts (n = 24), suggesting that maternal stores of the OCT4 protein had decayed. In contrast, control blastocysts (n = 20) showed OCT4 expression ubiquitously in both inner cell mass (ICM) and trophectoderm (TE). Simultaneously to the OCT4 staining, we differentially stained ICM and TE with the TE specific marker CDX2 and counterstained cell nuclei with 4′,6-diamidino-2-phenylindole. No significant differences between OCT4 KO Day 7 blastocysts and controls were detected in total cell numbers (89.6 ± 27.5 v. 96.3 ± 38) and percentage of CDX2 positive cells (50.7 ± 16.8% v. 59.0 ± 20.8%) (P > 0.05, mean ± SD, unpaired, two-tailed t-test). To analyse the role of OCT4 during the second lineage differentiation, we stained Day 5 morulae and Day 7 blastocysts for the epiblast and hypoblast specific markers NANOG and GATA6, respectively. In morulae, both markers were present and co-expressed in OCT4 KO and control embryos. By Day 7, control blastocysts (n = 6) already showed the typical salt and pepper distribution of NANOG and GATA6 positive cells, but expression was not mutually exclusive in all cells and also not restricted to ICM. OCT4 KO embryos lost all NANOG expression at Day 7 blastocyst stage (n = 8) and only stained positive for GATA6 in both TE and ICM. We conclude that OCT4 is not required for the quantitative allocation of cells to either the ICM or the TE during the first lineage differentiation, as total cell number and percentage of CDX2 positive cells was unchanged. Additionally, expression of NANOG seems to be OCT4 dependent and OCT4 KO embryos fail to establish the epiblast lineage—unlike mouse Oct4 KO embryos, where developmental failure was connected to loss of GATA6 expression during second lineage differentiation. This work was funded by the Bavarian Research Foundation (AZ-1031–12).

The insulin-like growth factor 2 receptor (IGF2R) is essential for prenatal growth regulation and shows gene dosage effects on fetal weight that can be affected by in-vitro embryo culture. Imprinted maternal expression of murine Igf2r is well documented for all fetal tissues excluding brain, but polymorphic imprinting and biallelic expression were reported for IGF2R in human. These differences have been attributed to evolutionary changes correlated with specific reproductive strategies. However, data from species suitable for testing this hypothesis are lacking. The domestic cow (Bos taurus) carries a single conceptus with a similar gestation length as human. We identified 12 heterozygous concepti informative for imprinting studies among 68 Bos taurus fetuses at Day 80 of gestation (28% term) and found predominantly maternal IGF2R expression in all fetal tissues but brain, which escapes imprinting. Inter-individual variation in allelic expression bias, i.e. expression of the repressed paternal allele relative to the maternal allele, ranged from 4.6−8.9% in heart, 4.3−10.2% in kidney, 6.1−11.2% in liver, 4.6−15.8% in lung and 3.2−12.2% in skeletal muscle. Allelic bias for mesodermal tissues (heart, skeletal muscle) differed significantly (P, PLoS ONE, 8 (4), ISSN:1932-6203

Interferon tau (IFNT), a type I IFN similar to alpha IFNs (IFNA), is the pregnancy recognition signal produced by the ruminant conceptus. To elucidate specific effects of bovine IFNT and of other conceptus-derived factors, endometrial gene expression changes during early pregnancy were compared to gene expression changes after intrauterine application of human IFNA2. In experiment 1, endometrial tissue samples were obtained on Day (D) 12, D15, and D18 postmating from nonpregnant or pregnant heifers. In experiment 2, heifers were treated from D14 to D16 of the estrous cycle with an intrauterine device releasing IFNA2 or, as controls, placebo lipid extrudates or PBS only. Endometrial biopsies were performed after flushing the uterus. All samples from both experiments were analyzed with an Affymetrix Bovine Genome Array. Experiment 1 revealed differential gene expression between pregnant and nonpregnant endometria on D15 and D18. In experiment 2, IFNA2 treatment resulted in differential gene expression in the bovine endometrium. Comparison of the data sets from both studies identified genes that were differentially expressed in response to IFNA2 but not in response to pregnancy on D15 or D18. In addition, genes were found that were differentially expressed during pregnancy but not after IFNA2 treatment. In experiment 3, spatiotemporal alterations in expression of selected genes were determined in uteri from nonpregnant and early pregnant heifers using in situ hybridization. The overall findings of this study suggest differential effects of bovine IFNT compared to human IFNA2 and that some pregnancy-specific changes in the endometrium are elicited by conceptus-derived factors other than IFNT.

BACKGROUND: The causes, modes, biological role and prospective significance of cell death in preimplantation development in humans and other mammals are still poorly understood. Early bovine embryos represent a very attractive experimental model for the investigation of this fundamental and important issue. METHODS AND FINDINGS: To obtain reference data on the temporal and spatial occurrence of cell death in early bovine embryogenesis, three-dimensionally preserved embryos of different ages and stages of development up to hatched blastocysts were examined in toto by confocal laser scanning microscopy. In parallel, transcript abundance profiles for selected apoptosis-related genes were analyzed by real-time reverse transcriptase-polymerase chain reaction. Our study documents that in vitro as well as in vivo, the first four cleavage cycles are prone to a high failure rate including different types of permanent cell cycle arrest and subsequent non-apoptotic blastomere death. In vitro produced and in vivo derived blastocysts showed a significant incidence of cell death in the inner cell mass (ICM), but only in part with morphological features of apoptosis. Importantly, transcripts for CASP3, CASP9, CASP8 and FAS/FASLG were not detectable or found at very low abundances. CONCLUSIONS: In vitro and in vivo, errors and failures of the first and the next three cleavage divisions frequently cause immediate embryo death or lead to aberrant subsequent development, and are the main source of developmental heterogeneity. A substantial occurrence of cell death in the ICM even in fast developing blastocysts strongly suggests a regular developmentally controlled elimination of cells, while the nature and mechanisms of ICM cell death are unclear. Morphological findings as well as transcript levels measured for important apoptosis-related genes are in conflict with the view that classical caspase-mediated apoptosis is the major cause of cell death in early bovine development.

Spermatozoa are transcriptionally inactive cells, but contain acetylated histones, normally a characteristic of transcriptionally active cells. Acetylgroups are thought to represent epigenetic marks that are transmitted to the oocyte and are involved in starting gene expression in the zygote and in regulating gene expression during early embryogenesis. We performed reverse transcription polymerase chain reaction (RT-PCR) in the common marmoset monkey (Callithrix jacchus) and in bovine spermatozoa, oocytes, zygotes, two- and four-cell embryos to evaluate the presence of specific transcripts known to play a role during fertilisation and early embryo development, namely protamine-1 (PRM1), protamine-2 (PRM2), histone H1 (H1), histone H3 (H3), histone H4 (H4), cAMP-responsive element modulator (CREM), DNA methyltransferase-1 (DNMT1), TATA box-binding protein (TBP). All transcripts tested were present in spermatozoa of the common marmoset, while bull spermatozoa lack PRM2. Marmoset oocytes exhibited transcripts for H1, H3, H4 and TBP, whereas bovine oocytes revealed H1, H3, H4, CREM, DNMT and TBP mRNAs. In zygotes, we amplified H1, H4, TBP (marmoset) and PRM1, H1, H3, H4, CREM, DNMT1 and TBP (bovine). Two-cell embryos showed PCR products for H1, H3 and TBP in the marmoset. In the bovine, all transcripts could be observed except PRM2. In four-cell embryos, PCR signals were obtained for PRM1, H1, H3, H4 and TBP in the marmoset. In the bovine, all transcripts were detected except PRM2. Our data suggest that, in both C. jacchus and Bos taurus, PRM1 transcripts are delivered by the spermatozoon to the oocyte.

For advanced out-of-hospital airway management, skilled personnel and adequate equipment are key prerequisites. There are little data on the current availability of airway management equipment and standards of medical staff on Helicopter Emergency Medical System (HEMS) helicopters in central Europe.An internet search identified all HEMS helicopters in Austria, Switzerland and Luxembourg. We identified 15 HEMS helicopter bases in Switzerland, 28 in Austria and three in Luxembourg. A questionnaire was sent to all bases, asking both for the details of the clinical background and experience of participating staff, and details of airway management equipment carried routinely on board.Replies were received from 14 helicopter bases in Switzerland (93%), 25 bases in Austria (89%) and all three bases in Luxembourg. Anaesthesiologists were by far the most frequent attending physicians (68-85%). All except one bases reported to have at least one alternative supraglottic airway device. All bases had capnometry and succinylcholine. All bases in the study except two in Austria had commercial pre-packed sets for a surgical airway. All helicopters were equipped with automatic ventilators, although not all were suitable for non-invasive ventilation (NIV; Switzerland: 43%, Austria: 12%, Luxembourg: 100%). Masks for NIV were rarely available in Switzerland (two bases; 14%) and in Austria (three bases; 12%), whereas all three bases in Luxembourg carried those masks.Most HEMS helicopters carry appropriate equipment to meet the demands of modern advanced airway management in the pre-hospital setting. Further work is needed to ensure that appropriate airway equipment is carried on all HEMS helicopters.

Bovine trophoblast cells release interferon-τ (IFNT), a type I IFN, as the pregnancy recognition signal. Since type I IFNs exert growth inhibitory and proapoptotic actions, the effect of the conceptus on components of the apoptosis pathways was determined in the bovine endometrium during the periimplantation period. Uteri of Simmental heifers were flushed post mortem at days 12, 15, and 18 of cycle or pregnancy for the recovery of conceptuses and the sampling of ipsilateral endometrial tissue at slaughter for quantitative RT-PCR, immunohistochemistry, caspase activity and TUNEL assays. Endometrium samples of pregnant animals revealed increased transcript levels for the proapoptotic genes XAF1 (day 15: 2.9-fold; day 18: 15.1-fold; p = 0.005) and CASP8 (day 18: 2.4-fold; p = 0.007). The mRNA expression increased significantly with the day of the cycle for the proapoptotic genes FASLG, TNFSF10, TNF and TNFSF1A (p = 0.004, p = 0.006, p = 0.001 and p = 0.007) and the antiapoptotic gene BIRC4 (p = 0.03). We detected high amounts of FASLG transcripts in day 18 conceptuses (16-fold higher than day 18 endometria). This finding was validated at the protein level by immunohistochemistry. To further analyse the endometrial activation of the caspase cascade, the activities of initiator caspase 8 and effector caspases 3/7 were determined luminometrically. No difference between pregnant and cyclic animals was found for either caspase activity. Additionally, a TUNEL assay showed no increase of apoptotic cells in the pregnant endometrium. In conclusion, although the bovine conceptus induces the expression of proapoptotic genes, neither an activation of a caspase cascade nor an increase of apoptotic cells was noticed. These results suggest inhibitory mechanisms preventing endometrial cells from programmed cell death.

Although somatic cell nuclear transfer (SCNT) cloning is more efficient in cattle than in any other species tested so far, there is a high rate of pregnancy failure that has been linked to structural and functional abnormalities of the placenta. We tested the hypothesis that these changes may originate from disturbed embryo–maternal interactions in the peri-implantation period. Therefore, we evaluated the response of the endometrium to SCNT embryos (produced from 7 different fetal fibroblast cell lines) as compared with embryos derived from in vitro fertilization (IVF). SCNT embryos and IVF embryos were cultured under identical conditions to the blastocyst stage (day 7) and were transferred to corresponding recipients, which were slaughtered at day 18 of pregnancy. The mRNA profiles of endometrium samples were obtained using a custom cDNA microarray enriched for transcripts differentially expressed in the endometrium and/or oviduct epithelium during the estrous cycle and/or early pregnancy. Overall, the variation in mRNA profiles was greater in the SCNT group than in the IVF group. Furthermore, 58 transcripts were differentially abundant in endometria from SCNT and IVF pregnancies. Prominent examples are orphan nuclear receptor COUP-TFII and connexin 43, both known to play important roles in uterine receptivity and conceptus placentation. These findings suggest that placental failure in bovine clone pregnancies may originate from abnormal embryo–maternal communication that develops during the peri-implantation period. Endometrium transcriptome profiles may serve as a tool to evaluate SCNT embryos for their ability to establish pregnancy and develop a functional placenta.

Mit Hilfe einer kalorimetrischen Mesanordnung wurden der Mechanismus und die Kinetik der Radikalbildung aus Dibenzoylperoxid und aromatischem tertiaren Amin untersucht. Es konnte gezeigt werden, das nicht nur die Benzoyloxi-, sondern auch Aminradikale die Polymerisation von Methacrylaten auslosen. The mechanism and the kinetics of the formation of radicals from dibenzoyl peroxide and aromatic amine were investigated by means of a calorimeter. It is shown that both the benzoyloxy radicals and the amine radicals are able to start the polymerization of methacrylates.

Cattle can serve as a model organism to resolve central questions in mammalian reproduction that cannot be clarified in the mouse model due to notable species-specific peculiarities of early mouse embryogenesis. As part of a project on structural, molecular, and functional deficiencies of bovine oocytes, we started to systematically investigate fertilization and the onset of embryo development in vitro by 3-dimensional multicolor fluorescence microscopy. We are using 3D visualisation as key approach to clarify the multiple parallel and sequential processes and events of fertilization as well as to identify and classify errors and failures. Moreover, we aim to gain insights into the mechanisms of aberrations by linking processes at the cellular and the molecular level. We studied class I and II oocytes collected from slaughterhouse ovaries and matured for 23 h in vitro. Oocytes were fixed at different times from 4 to 12 h postinsemination with formaldehyde in a microtubule-stabilising buffer containing taxol in such a way that the 3-dimensional cell architecture was maintained, and were stained for DNA, microtubules, and f-actin microfilaments. In addition, serine 10-phosphorylated histone H3 was used as a marker for chromosome condensation and the spindle midbody. For 3-dimensional imaging of the oocytes in toto, confocal serial sections were captured at 1-µm intervals using a 40× objective (NA = 1.3). For imaging details, we used a high spatial sampling density (pixel size: 50 × 50 nm, z-step size: 200 nm) close to the Nyquist criterion and image restoration by maximum likelihood estimation (MLE) deconvolution. A series of more than 500 three-dimensional snapshots of fertilized oocytes at different points in time gives a first detailed view on the spatial and temporal course of the sperm entry, the formation of the paternal pronucleus and the sperm aster, completion of oocyte meiosis and the formation of the maternal pronucleus, as well as dynamic changes of the cytoskeleton. Moreover, we can document a spectrum of abnormalities including spontaneous parthenogenetic oocyte activation, polyspermy, and aberrations of meiosis I and II. The latter include irregular spindle formation and chromosome segregation, the occurrence of chromatin bridges and abnormal spindle positioning and rotation (e.g. leading to nonextrusion of a first or a second polar body or the extrusion of two second polar bodies). Our microscopic investigation in the bovine system contributes to unraveling the origins of irregular cleavage, aneuploidy, and mosaicism in mammals. Three-dimensional high-speed microscopy of oocytes and zygotes in affordable timeframes could be of great value in improving the differential diagnosis of oocyte and sperm dysfunction, as well as in identifying and dissecting problems, limitations, and potential risks of reproductive technologies (ART).This work is supported by the Deutsche Forschungsgemeinschaft (DFG FOR 1041).

The aim of this study was to develop a reliable procedure for genomic evaluation of bovine embryos to determine gender, polled status, and hereditary defects within 24 h after collection. German Simmental animals (n = 15) were superovulated (n = 25) using a standard protocol. Embryos were recovered on Day 7 (Day 0 = oestrus). A total of 217 embryos (morula, n = 130; early blastocyst, n = 43; blastocyst, n = 44) were biopsied with a steel blade attached to a micromanipulator. Biopsied cells were immediately transferred into 1 µL TE buffer to a 500 µL reaction tube and embryos were in vitro cultured until genomic results were available. For commonly used molecular genetic methods (e.g. 5′-exonuclease genotyping, PCR or high density genotyping) DNA amounts of 2–200 ng are required. However, the DNA quantity of a single diploid cell amounts to 6 pg only. The embryo biopsies used, usually consists of 10–30 cells, necessitating an artificial amplification of the embryonic genome. Taking all vital measures to avoid external DNA contamination, isothermal whole genome amplification was performed with the REPLI-g Mini Kit (Qiagen, Valencia, CA, USA) using random hexamers and Phi29-Polymerase. Depending on the number of cells, a total DNA amount of 4–7 µg was achieved. Polled status and gender was determined using PCR with subsequent gel-electrophoresis. 5′-exonuclease assays were used to obtain genotypes for the detection of genetic defects. At present, eight, mostly Simmental-specific genetic disorders can be examined: three traits associated with severe growth retardation, dwarfism (DW), Braunvieh-haplotype 2 (BH2) and stunted growth (FH2), the lethal skin disorder zinc deficiency-like syndrome (ZDL), a fertility trait bovine male subfertility (BMS), embryonic death Fleckvieh-haplotype 4 (FH4), a bleeding disorder thrombopathia (TP) and arachnomelia (A), within 24 h. On average, 8.7 embryos were biopsied per embryo recovery, i.e. 93.9% of the total number of transferable embryos. Fourteen embryo samples (6.5%) totally failed during analysis, possibly due to the loss of samples. In successful analyses, gender was undetermined in two embryos; remaining embryos were 52.2% female and 47.8% male. Polled status could be analysed in 92.6% of the embryos. The analyses of embryos for possible inherited genetic disorders (healthy, heterozygote, or homozygote; n = 578) were successful in 90.1%. The transfer of biopsied embryos (n = 30) led to 63.3% pregnancies (Day 42). A validation of the present results has to be done as soon as the produced calves are born, demonstrating the reliability of the procedure.Research was funded by the Bayerische Forschungsstiftung (AZ-1031-12).

As a part of a study on bovine embryo genomic evaluation, effects of an intact (iZP) or opened zonae pellucidae (oZP) of in vitro-produced embryos on vitrification outcomes were assessed. In the first experiment, only iZP embryos were subjected to vitrification, using either the hollow fibre vitrification method (HFV; Matsunari et al. 2012 J. Reprod. Dev.) or the cryologic vitrification method (CVM; CryoLogic®, Blackburn, Victoria, Australia). Developmental stage (morula = M; early blastocyst = EaB; blastocyst = B) and quality (good = 1; fair = 2) before vitrification were evaluated. In a first set of experiments, quality 1 and 2 iZP embryos were vitrified either by the HFV or the CVM method. A significant difference between the 2 methods was found when comparing overall survival rates (24–48 h post-thaw; HFV 59.32% v. CVM 78.9%; P 0.1). In order to evaluate influence of embryo stage and quality on HFV or CVM outcomes, iZP embryos vitrified by the HFV method were classified regarding quality (1: n = 207; 2: n = 66; total n = 273) and embryo stage (M: n = 78; EaB: n = 74; B: n = 121). Concerning survival rates, no significant difference was found between M 1 (57.4%) and M 2 (44.6%), or EaB 1 (80%) and EaB 2 (68%). However, a significant difference was found when comparing B 1 (77%) and B 2 (29%; P 0.1) was found. Effect of ZP, either intact or opened, does not seem to affect survival rates (judged by their re-expansion 24–48 h post-thaw) of good-quality embryos.The authors thank the Bavarian Research Foundation for the financial support (AZ-1031-1; DOK-153-12).

The aim of the present study was to develop a reliable method to simultaneously split and biopsy valuable bovine embryos for a complete genomic evaluation (gender, polledness, and hereditary abnormalities) and to estimate the breeding value of progeny for traits of economic importance immediately after embryo recovery. A total of 208 good quality embryos collected from superovulated German Simmental animals were biopsied immediately after recovery using an inverse microscope (Zeiss, Germany) at 50× magnification with a single-use steel blade mounted on a holder (Bausch & Lomb, Germany) attached to a micromanipulator (Eppendorf, Germany). Biopsy was performed either by splitting the embryo and cutting of one-third of a half [G1: morulae (M), n = 50; early blastocysts (EB), n = 24; blastocysts (B), n = 16], by just splitting in equal halves (G2: M, n = 16; B, n = 2), or by cutting of just a small biopsy of the embryo (G3: M, n = 53) or of the trophoblast (G3: EB, n = 19; B, n = 28). Biopsied cells were immediately used for DNA amplification. Biopsied embryos (E) and demi-embryos (DE) were in vitro cultured in SOF, under mineral oil, at 39°C and 5% CO2, 5% O2, 90% N2 for 24 h, after which survival was recorded. Survival rate of G1 (survival of at least 1 DE: M, 98.0%; EB, 100.0%; B, 93.8%), G2 (survival of DE: M, 75.0%; B, 100.0%), and G3 (embryo survival: M, 96.3%; EB, 100.0%; B, 96.4%) were similar, but in relation to the number of original embryo the highest ratio of DE was obtained in G1 (1.67) v. G2 (0.88) and G3 (0.97; G1:G2/G3; P

The multiplication of high-value embryos by chimera formation using asynchronic aggregation is a promising alternative to embryonic cell nuclear transfer. Single blastomeres from a donor embryo are aggregated with 2 host embryos, thus several chimeras can be constructed per donor embryo. Due to the advanced developmental stage, the donor blastomeres are likely to contribute to the inner cell mass (ICM) and later give rise to the embryo proper, whereas the host embryos form extra-embryonic tissues. To test if pairs of blastomeres from Day 5 morulae are able to form the ICM when aggregated with 2 Day 4 host embryos, we produced transgenic donor embryos carrying a fluorescent reporter gene (enhanced green fluorescent protein, eGFP) by using semen from an eGFP transgenic bull (Reichenbach et al. 2010 Transgenic Res. 19, 549–556) for in vitro fertilization and in vitro host embryos produced by a standard procedure. The zona pellucida of all embryos was removed by treatment with 1 mg mL–1 pronase. Donor embryos were assessed for eGFP expression by fluorescence microscopy and disaggregated by gentle pipetting after incubation in Mg2+- and Ca2+-free medium. Pairs of blastomeres were then placed between 2 host embryos and cultured individually in a well-of-the-well culture dish. On Day 6 after aggregation, fully developed blastocysts were assessed for eGFP fluorescence. In 3 replicates, n = 30 chimeras were produced by aggregation; 13 (43%) developed to blastocysts, of which 2 (15%) showed local eGFP expression in the ICM and 7 (54%) showed a generalized expression. From the results of this study we conclude that Day 5 morulae may be multiplied in an efficient manner by using the chimera formation technique, which makes this approach applicable to ex vivo-derived embryos. In future investigations we will study the effect of using donor blastomeres from either the inside or outside of the donor morula and test the use of tetraploid host embryos to increase the rate of blastocysts with the desired genotype in the ICM. Finally, we aim to introduce this multiplication approach to the production of genotyped embryos with a genomic estimated breeding value (gEBV) and intend to produce calves with identical gEBV.Funded by the Bavarian Research Foundation (AZ-1031–1).

Despite existing security standards and security technologies, such as secure hardware, gaps between users’ demand for security and the security offered by a payment system can still remain. These security gaps imply risks for users. In this paper, we introduce a framework for the management of those risks. As a result, we present an instrument enabling users to evaluate eventual risks related with digital payment systems and to handle these risks with technical and economic instruments.

A mediastinitis or intraoperative bleeding are the most feared complications of the transoral laser assisted diverticulotomy of Zenker's diverticulum. To reduce these complications we developed a new endoscopic surgical technique and compared the results of this new method with the results of patients who we treated earlier.At the department of Otorhinolaryngology, Head and Neck Surgery, University of Luebeck 68 patients suffering from a hypopharyngeal diverticulum have been treated since 1987. In 64 patients we performed an endoscopic diverticulotomy using a CO2-Laser and the Weerda distending diverticuloscope. Four of the Zenker's diverticulua had to be removed by an external approach. From 1987 to 1994 a five millimeter bar between pouch and hypopharynx after diverticulotomy was left and sealed with fibrin glue in the first 38 patients. From 1995 to 1999 the spur of the hypopharyngeal pouch of the other 26 patients was completely separated. Afterwards we sutured the mucosa between the pouch and the hypopharynx to close the opened mediastinal space and sealed the former spur with fibrin glue.Comparing both endoscopic methods we had to observe concerning our first method one mediastinitis, a rise of body temperature in 29 patients (76%) and we had to perform a revision diverticulotomy in three patients (8%). Our modified technique didn't cause any mediastinitis, a rise of body temperature did only occur in five patients (18%) and no patient had to undergo revision surgery.The transoral complete separation of the spur of the hypopharyngeal pouch with suturing the mucosa of the pouch and the hypopharynx causes good functional results and means a further reduction of complications and a further improvement of the endoscopic diverticulotomy. Our new instruments (Fa. K. Storz, Tuttlingen) will be demonstrated.

One of the most important criteria of malignancy of head and neck cancer are the cervical lymph metastases. Being significant for the therapeutical plan is how tumor depending parameters like T-stage, degree of differentiation and tumor localisation will influence the N-stage and therefore the extension of neck dissection.To evaluate the pattern of formations of metastases and the success of therapy a retrospective study was performed on 405 patients with carcinoma of the oral cavity (n = 47), the oropharynx (n = 117), the hypopharynx (n = 47) and the larynx (n = 193).By the time of surgery carcinoma of the hypopharynx were most frequently accompanied by cervical metastases (80%), followed by carcinoma of the oropharynx (70%), the oral cavity (52%) and the larynx (26%). Occurrence and extension of regional lymph node metastases correlated well with T-stages and degree of differentiation. After surgical therapy locoregional recurrence could be observed in 5.2% of the patients. Five-year-survival rate was reduced to 50% on patients with positive lymph nodes. The different tumour sites showed preferred patterns of metastatic spread, without complete avoidance of certain levels.For the decision on indication and extent of neck dissection the preoperative diagnostic (ultrasound, CT-scan, MRI), localisation of tumour, T-stage, degree of differentiation and the knowledge of typical metastatic spread must be considered.

This study explored the personal characteristics and the health and health-related concerns reported by members of the local homeless population in order to design population-specific health programming. The study also examined whether there were significant differences between homeless who are shelter residents and those who are not. An exploratory descriptive design was used to analyze retrospective data collected by a local County Health Department in interviews of 132 homeless adults. The demographic characteristics found reflect many common patterns: marked over-representation of males, mean age in the mid-thirties, education levels comparable to similar socio-economic groups, high unemployment rates, and low health insurance rates. One third of the sample reported self-assessed health statuses of fair or poor. The most frequently identified physical health issue was joint problems, followed by cardiovascular disease. Depression was mentioned most frequently as a self-identified mental health problem. Loneliness was the number one fear identified. Chi Square analysis showed that homeless who did not stay in shelters were significantly longer term residents (p < 0.0001) of the community and reported fear of loneliness significantly more frequently (p < 0.01). This study identifies health concerns that local homeless people themselves find important and provides direction for development of sound population-specific health programming.

The intranasal allergen Provocation test (INT) is the standard procedure to verify the effect of an allergen when a nasal allergy is suspected. It may be especially useful if results from skin tests or in vitro tests conflict with those from the history. In the clinical and research setting, INT may be used to study the efficacy of different treatment modalities such as pharmacotherapy or immunotherapy. The technique of INT has not been well standardized in the past. Variations exist in planning the optimum time for testing, especially in seasonal allergies.We examined influences of different test dates with regard to the pollen season on the result of INT. A total of 19 patients (eight females, 11 males, aged 26.1 +/- 4.8 years) with a history of at least two years of allergic rhinitis to birch pollen, positive skin prick test, and RAST (or = CAP class II) to birch pollen. INT was performed during the winter-period after approximately six months without natural pollen exposition (V1), during the birch pollen season (V2) and six weeks after the birch pollen season (V3). Allergens were applied using a pump spray (0.1 ml) in concentrations of 10, 50, 100, 500, 1000, 5000, 10,000, 50,000, and 100,000 SQ units per ml (SQ/ml).The threshold concentrations were significantly lower at V2 (100 SQ/ml) and V3 (500 SQ/ml) if compared to V1 (1000 SQ/ ml).Seasonal influences should be taken into account when performing INT in birch pollen allergy.