Nuclear magnetic resonance metabolomic profiling of Day 3 and 5 embryo culture medium does not predict pregnancy outcome in good prognosis patients: a prospective cohort study on single transferred embryos.

STUDY QUESTION: Does the metabolomic profile, obtained with nuclear magnetic resonance (NMR), of spent culture media from human embryos correlate with reproductive potential in a cohort of good prognosis patients? SUMMARY ANSWER: In a large cohort of single transferred blastocysts from a homogeneous group of good prognosis patients, we find a high degree of individual variation in the metabolome that, however, has no relation to pregnancy outcome. WHAT IS KNOWN ALREADY: Differences among various specific metabolites have been linked to reproductive potential. Although results from retrospective near infrared (NIR) spectroscopy analyses of spent culture medias from transferred embryos were promising, randomized controlled trials were unable to demonstrate that NIR analysis improved pregnancy rates. Therefore, a more detailed investigation of the relation between embryo metabolism and reproductive potential is required. NMR is a powerful technique that provides detailed structural and dynamic information. STUDY DESIGN, SIZE, DURATION: A prospective cohort study was conducted at the Fertility Clinic, Aarhus University Hospital between February 2011 and July 2012. Infertile patients aged <38 years without endometriosis were offered participation and their embryos were included if greater than or equal to eight oocytes were retrieved. In total, 161 infertile patients were included in the cohort. PARTICIPANTS/MATERIALS, SETTING, METHODS: Spent culture media was collected on Days 3 and 5 after oocyte retrieval from 148 single transferred embryos. NMR spectra were obtained from 12 µl of spent media. Data were quantitatively analysed using multivariate analysis with respect to pregnancy outcome, defined as a live fetus by ultrasound in gestational Week 8, along with patient and treatment related variables such as embryo score, age, BMI, fertilization method and cause of infertility. MAIN RESULTS AND THE ROLE OF CHANCE: A total of 148 cycles were included in the analysis [embryo transfer cancelled (n = 12), no media collected (n = 1)]. Clinical pregnancy was confirmed in 47 patients (32%). We obtained high quality NMR spectra for 141 Day 3 and 137 Day 5 samples. Our spectra show a high degree of individual variation. Multivariate data analysis was performed on spectral data with several different pre-processing combinations, i.e. binning, alignment, normalization and scaling in the attempt to develop a valid prediction model. Different strategies of multivariate analysis showed, however, no correlation between the NMR profiles and pregnancy outcome, patient or treatment characteristics. No model could therefore be developed for prediction of pregnancy outcome. We conclude that within this group of good prognosis patients, large-scale metabolic variations between embryos detected with NMR have no apparent association with pregnancy outcome. LIMITATIONS, REASONS FOR CAUTION: Although this study is the largest we know of using NMR to investigate metabolomic profiles of single-transferred embryos, there may be differences that would be detected with a larger study. When analysing such a small sample volume, even small variations in the amount of media and dilution may introduce a large uncertainty in the results. WIDER IMPLICATIONS OF THE FINDINGS: Our study questions the usefulness of the entire metabolome for embryo selection, which should direct the search for viability markers in the culture media towards individual components. STUDY FUNDING/COMPETING INTERESTS: Funding was provided by Aarhus University, the Lippert Foundation, the Toyota Foundation, the Aase og Einar Danielsen foundation. Research at the Fertility Clinic, Aarhus Universtity Hospital is supported by an unrestricted grant from MSD and Ferring. The authors declare no competing interest. TRIAL REGISTRATION NUMBER: NCT01139268.

Immunological comparison of allergen immunotherapy tablet treatment and subcutaneous immunotherapy against grass allergy.

BACKGROUND: IgE-mediated allergic rhinitis to grass pollen can successfully be treated with either allergen immunotherapy tablets (SLIT tablet) or SQ-standardized subcutaneous immunotherapy (SCIT). The efficacy of these two treatment modalities for grass allergy is comparable, but the immunological mechanisms may differ. ClinicalTrials.gov ID: NCT01889875. OBJECTIVES: To compare the immunological changes induced by SQ-standardized SCIT and SLIT tablet. METHODS: We randomized 40 individuals with grass pollen rhinitis into groups receiving SCIT, SLIT tablet, or neither and followed them for 15 months with regular serum measurements of specific IgE, IgG4, IgE-blocking factor, facilitated antigen presentation (FAP), and basophil activation test (BAT). Nasal challenges were used to assess changes in nasal sensitivity. RESULTS: After 15 months of treatment IgG4, IgE-blocking factor, FAP, and BAT values differed significantly in both SCIT and SLIT-tablet treatment groups when compared to the control group. Both SCIT and SLIT-tablet groups were significantly different from the control group after 1­3 months of treatment. In general, the changes induced by SCIT reached twice that of SLIT tablet, with the exception of specific IgE where SLIT tablet induced initial threefold increase compared with SCIT. A slight but significant increase in IgE and BAT after season was seen only in the control group. Significant differences between SCIT and SLIT tablet were observed early, but the differences diminished with the length of treatment, especially for FAP inhibition. CONCLUSIONS: Both SCIT and SLIT tablet induce significant changes in specific antibodies (IgE and IgG4) and competition assays (IgE-blocking factor, FAP, and BAT). Overall, SCIT induced larger (two- to threefold) changes than SLIT tablet, with the exception of FAP, where SLIT tablet showed a gradual increase ending at the same level as SCIT. Maximal change was generally reached after 3 months' treatment.

In vitro and in vivo studies of creatine monohydrate supplementation to Duroc and Landrace pigs.

Duroc and Landrace pigs as well as primary myotubes from these breeds were used to investigate mechanisms behind differences in their response to creatine monohydrate (CMH). Pigs were supplemented with 0, 12.5, 25 or 50g CMH/d for 5 days (n=10 per treatment and breed). Plasma levels of creatine increased dose-dependently in both breeds, while muscle-creatine phosphate content increased only in the Duroc pigs. (1)H NMR metabolic profiling showed a tendency towards clustering according to CMH supplementation only among Duroc pigs, revealing a stronger response compared to Landrace pigs. The abundance of insulin-like growth factor I and myostatin mRNA was decreased by CMH supplementation while that of type 1 IGF-receptor and creatine transporter was unaffected. Protein synthesis, increased in the myotubes from both breeds, indicating protein accretion, but no effect was observed on the mRNA abundance of IGF-I, type 1 IGF-receptor, myostatin or the creatine transporter in myotubes.

Phase evolution of solitonlike optical pulses during excitonic Rabi flopping in a semiconductor.

We demonstrate that the temporal pulse phase remains essentially unaltered before separate phase characteristics are developed when propagating high-intensity pulses coherently on the exciton resonance of an optically thick semiconductor. This behavior is a clear manifestation of self-induced transmission and pulse breakup into soliton-like pulses due to Rabi flopping of the carrier density. Experiments using a novel fast-scan cross-correlation frequency-resolved optical gating (XFROG) method are in good agreement with numerical calculations based on the semiconductor Bloch equations.

Mycoplasma hyosynoviae arthritis in grower-finisher pigs.

The aetiology of acute lameness in pigs 3-5 months of age in nine Danish herds with high incidences of lameness was investigated. Eighty-seven acutely lame pigs, that exhibited lameness of varying degree in the hind legs, were selected. Non-lame pigs were matched on pen, sex and weight. The lame pigs had soft fluctuating joint swellings (odds ratio (OR), 7.21; 95% confidence interval (CI), 3.41-15.47). No indication of suppurative arthritis was observed. Joint infection with Mycoplasma hyosynoviae was found by culture in 20% (17 of 86) of the lame pigs and in 8% (seven of 83) of the non-lame pigs. Lameness and joint infection with M. hyosynoviae were significantly associated. Other ordinary bacteria were not found in any case. Macroscopic osteochondrotic lesions were observed at slaughter in 47% (37 of 78) of the previously lame pigs and in 35% (55 of 158) of an enlarged group without history of lameness. The cubital joints were most frequently affected and a history of hind leg lameness was not statistically associated with osteochondrotic lesions at slaughter (OR, 1.69; 95% CI, (1.94-3.05), or joint infection with M. hyosynoviae at slaughter (OR, 0.88; 95% CI, 0.31-2.40). Arthritis due to M. hyosynoviae infection was the primary cause of acute and severe lameness in grower-finisher pigs. Moreover, M. byosynoviae was isolated from joints of several pigs without signs of lameness. This suggests that M. hyosynoriae may be present in joints without provoking clinical illness. The mean daily incidence of treatments due to lameness in the herds was 5.4 per 1,000 pigs. Joint disease implied 30-90 min extra labour for surveillance and treatment every day per 1,000 pigs, and 5% of the affected individuals were euthanized due to lameness. The average daily weight gains in the selected pigs until slaughter seemed unaffected by the lameness.

Conformation of alamethicin in oriented phospholipid bilayers determined by (15)N solid-state nuclear magnetic resonance.

The conformation of the 20-residue antibiotic ionophore alamethicin in macroscopically oriented phospholipid bilayers has been studied using (15)N solid-state nuclear magnetic resonance (NMR) spectroscopy in combination with molecular modeling and molecular dynamics simulations. Differently (15)N-labeled variants of alamethicin and an analog with three of the alpha-amino-isobutyric acid residues replaced by alanines have been investigated to establish experimental structural constraints and determine the orientation of alamethicin in hydrated phospholipid (dimyristoylphosphatidylcholine) bilayers and to investigate the potential for a major kink in the region of the central Pro(14) residue. From the anisotropic (15)N chemical shifts and (1)H-(15)N dipolar couplings determined for alamethicin with (15)N-labeling on the Ala(6), Val(9), and Val(15) residues and incorporated into phospholipid bilayer with a peptide:lipid molar ratio of 1:8, we deduce that alamethicin has a largely linear alpha-helical structure spanning the membrane with the molecular axis tilted by 10-20 degrees relative to the bilayer normal. In particular, we find compatibility with a straight alpha-helix tilted by 17 degrees and a slightly kinked molecular dynamics structure tilted by 11 degrees relative to the bilayer normal. In contrast, the structural constraints derived by solid-state NMR appear not to be compatible with any of several model structures crossing the membrane with vanishing tilt angle or the earlier reported x-ray diffraction structure (Fox and Richards, Nature. 300:325-330, 1982). The solid-state NMR-compatible structures may support the formation of a left-handed and parallel multimeric ion channel.

Colloidal calcium phosphates in casein micelles studied by slow-speed-spinning 31P magic angle spinning solid-state nuclear magnetic resonance.

The composition of bovine casein micelles was analyzed by 31P magic angle spinning solid-state nuclear magnetic resonance spectroscopy. By looking at isotropic and anisotropic 31P chemical shift parameters, resonance line shapes, the combination of single-pulse and 1H to 31P cross-polarization spectra, and comparison with spectra for various model compounds combined with multiple-component simulation and iterative fitting procedures, we were able to identify and quantify a variety of inorganic and organic phosphates in the micelles. These include phosphates from mobile and immobile inorganic hydroxyapatite-type phosphates as well as phosphates from kappa-casein and the Ca2+-binding phosphoserines from alphas1-, alphas2-, and beta-casein. This information is discussed in relation to previous knowledge and various models for the colloid formation.

(13)C chemical shift and (13)C-(14)N dipolar coupling tensors determined by (13)C rotary resonance solid-state NMR.

This work explores the utility of simple rotary resonance experiments for the determination of the magnitude and orientation of (13)C chemical shift tensors relative to one or more (13)C--(14)N internuclear axes from (13)C magic-angle-spinning NMR experiments. The experiment relies on simultaneous recoupling of the anisotropic (13)C chemical shift and (13)C--(14)N dipole--dipole coupling interactions using 2D rotary resonance NMR with RF irradiation on the (13)C spins only. The method is demonstrated by experiments and numerical simulations for the (13)C(alpha) spins in powder samples of L-alanine and glycine with (13)C in natural abundance. To investigate the potential of the experiment for determination of relative/absolute tensor orientations and backbone dihedral angles in peptides, the influence from long-range dipolar coupling to sequential (14)N spins in a peptide chain ((14)N(i)--(13)C(alpha)(i)--(14)N(i+1) and (14)N(i+1)--(13)C'(i)--(14)N(i) three-spin systems) as well as residual quadrupolar-dipolar coupling cross-terms is analyzed numerically.

A soybean G2 glycinin allergen. 2. Epitope mapping and three-dimensional modeling.

BACKGROUND: Multiple allergens have been documented in soybean extracts. IgE from individuals allergic to soybeans, but not to peanut, has been shown by immunoblot analysis to bind to proteins with a molecular weight of approximately 22 kD. These findings suggested that this unique protein fraction from soybean might be responsible, in part, for soybean allergic reactivity. The objective of the present study was to characterize specific B cell epitopes, to determine if any amino acid was critical to IgE binding and to model the 22-kD G2 soybean allergen to the three-dimensional (3-D) phaseolin molecule. METHODS: B cell epitopes were identified using SPOTs peptide analysis. Structural orientation of the IgE-binding regions was mapped to the 3-D phaseolin molecule using molecular modeling of the protein tertiary structure. RESULTS: Eleven linear epitopes, representing 15 amino acid peptide sequences, bound to IgE in the glycinin molecule. These epitopes were predicted to be distributed asymmetrically on the surface of G2 trimers. CONCLUSIONS: Only 1 epitope could be rendered non-IgE binding by alanine substitutions in the peptide. The nonrandom distribution of the IgE binding sites provides new insight into their organization in trimers in 11S complexes of the G2 glycinin allergen.

A soybean G2 glycinin allergen. 1. Identification and characterization.

BACKGROUND: Multiple allergens have been documented in soybean extracts. IgE from individuals allergic to soybeans, but not to peanut, was shown by immunoblot analysis to bind to proteins with a molecular weight of approximately 21 kD. These findings suggested that unique proteins in soybeans might be responsible for soybean allergic reactivity. The objective of the present study was to identify unique proteins in soybean extracts that bind to specific IgE from soybean-sensitive individuals, and to characterize the allergen using physicochemical methods and IgE binding. METHODS: Two-dimensional and preparative SDS-PAGE/IgE immunoblot analysis was used to identify a 22-kD soybean-specific allergen from crude soybean extracts. N-terminal sequence analysis was used to determine the identification of the protein binding IgE from soybean-sensitive individuals. RESULTS: IgE immunoblot and amino acid sequence analysis identified the 22-kD protein as a member of the G2 glycinin soybean protein family. Further investigation revealed that the IgEs reacted with basic chains from each member of the glycinin family of soybean storage proteins. CONCLUSIONS: Each of the subunits from glycinin, the storage protein that is the most prevalent component of soybean, are major allergens.