First ovulation and ketone body status in the early postpartum period of dairy cows

The effect of ketone body status on occurrence of first ovulation during early lactation was assessed in 84 multiparous dairy cows under field conditions. Animals were equally distributed across 8 farms and were controlled by the same herd fertility monitoring program. Cows were visited twice antepartum and 6 times postpartum at weekly intervals between 5:30 and 8:30 AM. On these occasions, body condition scores and milk yields were measured, blood and milk samples were taken, cows were gynecologically examined, and parameters of reproduction were determined. The onset of first ovulation was specified by milk progesterone determination and rectal palpation. Cows starting postpartum ovarian cyclicity within or after 30 d were classified as early and late responders (ER and LR, respectively). Resumption of the estrous cycle within 30 d postpartum is considered optimal under practical conditions, and classification based on this threshold value resulted in groups of equal size and equal distribution of ER + LR cows within farms. Ketone bodies measured were beta-hydroxybutyrate in serum and acetoacetate and acetone in serum and milk. Blood serum and milk ketone body concentrations during the first 6 wk of lactation were higher in LR than in ER, whereas plasma glucose and nonesterified fatty acid and milk fat, protein and urea concentrations did not differ between groups. Maximal concentrations of ketone bodies from parturition to first ovulation were better predictors of the onset of the estrous cycle than mean or minimal concentrations over the same period. Milk acetone and serum beta-hydroxybutyrate concentrations provided the most reliable information with regard to resumption of ovarian activity of all ketone bodies.     

Hormone-stimulated calcium release is inhibited by cytoskeleton-disrupting toxins in AR4-2J cells

We have studied the role of the actin cytoskeleton in bombesin-induced inositol 1,4,5-trisphosphate (IP(3))-production and Ca(2+)release in the pancreatic acinar tumour cell line AR4-2J. Intracellular and extracellular free Ca(2+)concentrations were measured in cell suspensions, using Fura-2. Disruption of the actin cytoskeleton by pretreatment of the cells with latrunculin B (10 microM), cytochalasin D (10 microM) or toxin B from Clostridium difficile (20 ng/ml) for 5-29 h led to inhibition of both, bombesin-stimulated IP(3)-production and Ca(2+)release. The toxins had no effect on binding of bombesin to its receptor, on Ca(2+)uptake into intracellular stores and on resting Ca(2+)levels. Ca(2+)mobilization from intracellular stores, induced by thapsigargin (100 nM) or IP(3)(1 microM) was not impaired by latrunculin B. In latrunculin B-pretreated cells inhibition of both, bombesin-stimulated IP(3)- production and Ca(2+)release was partly suspended in the presence of aluminum fluoride, an activator of G-proteins. Aluminum fluoride had no effect on basal IP(3)and Ca(2+)levels of control and toxin-pretreated cells. We conclude that disruption of the actin cytoskeleton impairs coupling of the bombesin receptor to its G-protein, resulting in inhibition of phospholipase C-activity with subsequent decreases in IP(3)-production and Ca(2+)release.     

Determination of 2-hydroxypropyl-γ-cyclodextrin in plasma of cynomolgus monkeys after oral administration by gas chromatography–mass spectrometry

This report describes a specific and highly sensitive gas chromatography–mass spectrometry (GC–MS) assay for the analysis of industrially produced 2-hydroxypropyl-γ-cyclodextrin, a heterogeneous mixture of homologues and isomers, in plasma of cynomolgus monkeys. Instead of analyzing the polysaccharide mixture as a whole, in a first step the HP-γ-cyclodextrin mixture, together with the internal standard (2,6-di-O-methyl-β-cyclodextrin), was deuteromethylated, and in a second step hydrolyzed with hydrochloric acid to the respective monosaccharides. The resulting reaction mixture was trimethylsilylated to 1,4-bis(O-trimethylsilyl)-2,3-bis-O-deuteromethyl-6-O-2′-deuteromethoxypropylglucose (representative for HP-γ-CD) and 1,4-bis-(O-trimethylsilyl)-bis-2,6-O-methyl-3-O-deuteromethylglucose (representative for the internal standard), respectively, and analyzed by GC–MS. The limit of quantification of this assay was 20 nmol/l.     

Geographic structure, genetic diversity and source tracking of Spartina alterniflora

Aim To examine the distribution and structure of genetic variation among native Spartina alterniflora and to characterize the evolutionary mechanisms underlying the success of non‐native S. alterniflora. Location Intertidal marshes along the Atlantic, Gulf and Pacific coasts of North America. Methods amova , parsimony analysis, haplotype networks of chloroplast DNA (cpDNA) sequences, neighbour‐joining analysis, Bayesian analysis of population structure, and individual assignment testing were used. Results Low levels of gene flow and geographic patterns of genetic variation were found among native S. alterniflora from the Atlantic and Gulf coasts of North America. The distribution of cpDNA haplotypes indicates that Atlantic coast S. alterniflora are subdivided into ‘northern’ and ‘southern’ groups. Variation observed at microsatellite loci further suggests that mid‐Atlantic S. alterniflora are differentiated from S. alterniflora found in southern Atlantic and New England coastal marshes. Comparisons between native populations on the Atlantic and Gulf coasts and non‐native Pacific coast populations substantiate prior studies demonstrating reciprocal interspecific hybridization in San Francisco Bay. Our results corroborate historical evidence that S. alterniflora was introduced into Willapa Bay from multiple source populations. However, we found that some Willapa Bay S. alterniflora are genetically divergent from putative sources, probably as a result of admixture following secondary contact among previously allopatric native populations. We further recovered evidence in support of models suggesting that S. alterniflora has secondarily spread within Washington State, from Willapa Bay to Grays Harbor. Main conclusions Underlying genetic structure has often been cited as a factor contributing to ecological variation of native S. alterniflora. Patterns of genetic structure within native S. alterniflora may be the result of environmental differences among biogeographical provinces, of migration barriers, or of responses to historical conditions. Interactions among these factors, rather than one single factor, may best explain the distribution of genetic variation among native S. alterniflora. Comprehensive genetic comparisons of native and introduced populations can illustrate how biological invasions may result from dramatically different underlying factors – some of which might otherwise go unrecognized. Demonstrating that invasions can result from several independent or interacting mechanisms is important for improving risk assessment and future forecasting. Further research on S. alterniflora not only may clarify what forces structure native populations, but also may improve the management of non‐native populations by enabling post‐introduction genetic changes and the rapid evolution of life‐history traits to be more successfully exploited.     

Landscape Genetics of Schistocephalus solidus Parasites in Threespine Stickleback (Gasterosteus aculeatus) from Alaska

The nature of gene flow in parasites with complex life cycles is poorly understood, particularly when intermediate and definitive hosts have contrasting movement potential. We examined whether the fine-scale population genetic structure of the diphyllobothriidean cestode Schistocephalus solidus reflects the habits of intermediate threespine stickleback hosts or those of its definitive hosts, semi-aquatic piscivorous birds, to better understand complex host-parasite interactions. Seventeen lakes in the Cook Inlet region of south-central Alaska were sampled, including ten in the Matanuska-Susitna Valley, five on the Kenai Peninsula, and two in the Bristol Bay drainage. We analyzed sequence variation across a 759 bp region of the mitochondrial DNA (mtDNA) cytochrome oxidase I region for 1,026 S. solidus individuals sampled from 2009-2012. We also analyzed allelic variation at 8 microsatellite loci for 1,243 individuals. Analysis of mtDNA haplotype and microsatellite genotype variation recovered evidence of significant population genetic structure within S. solidus. Host, location, and year were factors in structuring observed genetic variation. Pairwise measures revealed significant differentiation among lakes, including a pattern of isolation-by-distance. Bayesian analysis identified three distinct genotypic clusters in the study region, little admixture within hosts and lakes, and a shift in genotype frequencies over time. Evidence of fine-scale population structure in S. solidus indicates that movement of its vagile, definitive avian hosts has less influence on gene flow than expected based solely on movement potential. Observed patterns of genetic variation may reflect genetic drift, behaviors of definitive hosts that constrain dispersal, life history of intermediate hosts, and adaptive specificity of S. solidus to intermediate host genotype.     

Quantitative variability of 342 plasma proteins in a human twin population

The degree and the origins of quantitative variability of most human plasma proteins are largely unknown. Because the twin study design provides a natural opportunity to estimate the relative contribution of heritability and environment to different traits in human population, we applied here the highly accurate and reproducible SWATH mass spectrometry technique to quantify 1,904 peptides defining 342 unique plasma proteins in 232 plasma samples collected longitudinally from pairs of monozygotic and dizygotic twins at intervals of 2–7 years, and proportioned the observed total quantitative variability to its root causes, genes, and environmental and longitudinal factors. The data indicate that different proteins show vastly different patterns of abundance variability among humans and that genetic control and longitudinal variation affect protein levels and biological processes to different degrees. The data further strongly suggest that the plasma concentrations of clinical biomarkers need to be calibrated against genetic and temporal factors. Moreover, we identified 13 cis‐SNPs significantly influencing the level of specific plasma proteins. These results therefore have immediate implications for the effective design of blood‐based biomarker studies.     

Scanning X-Ray Nanodiffraction on Dictyostelium discoideum

We have performed scanning x-ray nanobeam diffraction experiments on single cells of the amoeba Dictyostelium discoideum. Cells have been investigated in 1), freeze-dried, 2), frozen-hydrated (vitrified), and 3), initially alive states. The spatially resolved small-angle x-ray scattering signal shows characteristic streaklike patterns in reciprocal space, which we attribute to fiber bundles of the actomyosin network. From the intensity distributions, an anisotropy parameter can be derived that indicates pronounced local variations within the cell. In addition to nanobeam small-angle x-ray scattering, we have evaluated the x-ray differential phase contrast in view of the projected electron density. Different experimental aspects of the x-ray experiment, sample preparation, and data analysis are discussed. Finally, the x-ray results are correlated with optical microscopy (differential phase contrast and confocal microscopy of mutant strains with fluorescently labeled actin and myosin II), which have been carried out in live and fixed states, including optical microscopy under cryogenic conditions.     

Symmetry breakage in the vertebrate embryo: When does it happen and how does it work?

Asymmetric development of the vertebrate embryo has fascinated embryologists for over a century. Much has been learned since the asymmetric Nodal signaling cascade in the left lateral plate mesoderm was detected, and began to be unraveled over the past decade or two. When and how symmetry is initially broken, however, has remained a matter of debate. Two essentially mutually exclusive models prevail. Cilia-driven leftward flow of extracellular fluids occurs in mammalian, fish and amphibian embryos. A great deal of experimental evidence indicates that this flow is indeed required for symmetry breaking. An alternative model has argued, however, that flow simply acts as an amplification step for early asymmetric cues generated by ion flux during the first cleavage divisions. In this review we critically evaluate the experimental basis of both models. Although a number of open questions persist, the available evidence is best compatible with flow-based symmetry breakage as the archetypical mode of symmetry breakage.     

Stable adducts of nerve agents sarin,soman and cyclosarin with TRIS,TES and related buffer compounds—Characterization by LC-ESI-MS/MS and NMR and implications for analytical chemistry

Buffering compounds like TRIS are frequently used in chemical, biochemical and biomedical applications to control pH in solution. One of the prerequisites of a buffer compound, in addition to sufficient buffering capacity and pH stability over time, is its non-reactivity with other constituents of the solution. This is especially important in the field of analytical chemistry where analytes are to be determined quantitatively. Investigating the enzymatic hydrolysis of G-type nerve agents sarin, soman and cyclosarin in buffered solution we have identified stable buffer adducts of TRIS, TES and other buffer compounds with the nerve agents. We identified the molecular structure of these adducts as phosphonic diesters using 1D ¹H-³¹P HSQC NMR and LC-ESI-MS/MS techniques. Reaction rates with TRIS and TES are fast enough to compete with spontaneous hydrolysis in aqueous solution and to yield substantial amounts (up to 20–40%) of buffer adduct over the course of several hours. A reaction mechanism is proposed in which the amino function of the buffer serves as an intramolecular proton acceptor rendering the buffer hydroxyl groups nucleophilic enough for attack on the phosphorus atom of the agents. Results show that similar buffer adducts are formed with a range of hydroxyl and amino function containing buffers including TES, BES, TRIS, BIS-TRIS, BIS-TRIS propane, Tricine, Bicine, HEPES and triethanol amine. It is recommended to use alternative buffers like MOPS, MES and CHES when working with G-type nerve agents especially at higher concentrations and over prolonged times.     

Individualized medicine 2010

Molecular and cell biology have revolutionized not only diagnosis, therapy and prevention of human diseases but also greatly contributed to the understanding of their pathogenesis. Based on modern molecular and biochemical methods it is possible to identify on the one hand point mutations and single nucleotide polymorphisms. On the other hand, using high throughput array technologies, it is possible to analyse thousands of genes or gene products simultaneously, resulting in an individual gene or gene expression profile (signature). These data increasingly allow to define the individual risk for a given disease and to predict the individual prognosis of a disease as well as the efficacy of therapeutic strategies (individualized medicine). In the following sections some of the recent advances of predictive medicine and their clinical relevance will be addressed.