Automatic retrieval of single microchimeric cells and verification of identity by on-chip multiplex PCR.

The analysis of rare cells is not an easy task. This is especially true when cells representing a fetal microchimerism are to be utilized for the purpose of non-invasive prenatal diagnosis because it is both imperative and difficult to avoid contaminating the minority of fetal cells with maternal ones. Under these conditions, even highly specific biochemical markers are not perfectly reliable. We have developed a method to verify the genomic identity of rare cells that combines automatic screening for enriched target cells (based on immunofluorescence labelling) with isolation of single candidate microchimeric cells (by laser microdissection and subsequent laser catapulting) and low-volume on-chip multiplex PCR for DNA fingerprint analysis. The power of the method was tested using samples containing mixed cells of related and non-related individuals. Single-cell DNA fingerprinting was successful in 74% of the cells analysed (55/74), with a PCR efficiency of 59.2% (860/1452) for heterozygous loci. The identification of cells by means of DNA profiling was achieved in 100% (12/12) of non-related cells in artificial mixtures and in 86% (37/43) of cells sharing a haploid set of chromosomes and was performed on cells enriched from blood and cells isolated from tissue. We suggest DNA profiling as a standard for the identification of microchimerism on a single-cell basis.

External cephalic version in singleton pregnancies at term: a retrospective analysis.

BACKGROUND/AIMS: The outcomes of external cephalic versions (ECV) performed at the University Hospital Graz in Austria were analyzed to determine to what extent the cesarean section rate can be reduced by an ECV program. METHODS: All women who were admitted to the hospital with breech presentation at 37 weeks or later and underwent an attempt of ECV between 2002 and 2004 were recorded and retrospectively analyzed. RESULTS: Of 136 cases 51% were successful. Among these, the cesarean section rate was 13%. The cesarean section rate among all not successful cases was 82%. Of the successful cases which remained in cephalic presentation 92% were delivered vaginally and 5 were delivered by cesarean section. The cesarean section rate (8%) was slightly higher than in fetuses with cephalic presentation observed at the onset of contractions in 2004 (5.9%). No maternal or fetal complications or side effects occurred. CONCLUSION: Successful ECV beyond 37 weeks of gestation significantly decreases the cesarean section rate. ECV reduces the cesarean section rate among breech positions and decreases the risks related to breech delivery, when correctly performed and adequately monitored. ECV reduces higher costs connected with cesarean section.

Effect of instrument tuning on the detectabilityof biopolymers in electrospray ionization mass spectrometry.

Electrospray ionization mass spectrometry of multiply charged biopolymer ions of different molecular size revealed a strong influence of tuning parameters on their detectability in quadrupole ion trap and triple quadrupole mass spectrometers. Hence, after optimizing the ion optical parameters with the signal of the 4- charge state of (dT)(24) (low charge state tuning), a tenfold increase in the signal-to-noise ratio for a mixture of oligodeoxythymidylic acids (n = 12-18) was obtained compared with the results achieved with tune parameters optimized with a synthetic 80-mer oligodeoxynucleotide. By contrast, a detection limit in the upper femtomole region could only be reached for a 104-mer oligodeoxynucleotide utilizing the 24- charge state of the 80-mer (high charge state tuning). The same effect was observed for proteins investigated in the positive ion mode using low and high charge states of cytochrome c and carbonic anhydrase, respectively, for instrument tuning. By comparing the settings for low and high charge state tuning, it became obvious that the most significant difference was observed in the potential applied to the heated metal capillary used to transfer ions from the atmospheric pressure to the vacuum region of the ion source. Taking advantage of the optimized tuning procedure, the molecular mass of a 61 base pair product of polymerase chain reaction was accurately determined by electrospray ionization mass spectrometry on-line interfaced to ion-pair reversed-phase high-performance liquid chromatography.

Operational variables in high-performance liquid chromatography-electrospray ionization mass spectrometry of peptides and proteins using poly(styrene-divinylbenzene) monoliths.

Capillary reversed-phase high-performance liquid chromatography (RP-HPLC) utilizing monolithic poly(styrene-divinylbenzene) columns was optimized for the coupling to electrospray ionization mass spectrometry (ESI-MS) by the application of various temperatures and mobile phase additives during peptide and protein analysis. Peak widths at half height improved significantly upon increasing the temperature and ranged from 2.0 to 5.4 s for peptide and protein separations at 70 degrees. Selectivity of peptide elution was significantly modulated by temperature, whereas the effect on proteins was only minor. A comparison of 0.10% formic acid (FA), 0.050% trifluoroacetic acid (TFA), and 0.050% heptafluorobutyric acid (HFBA) as mobile phase additives revealed that highest chromatographic efficiency but poorest mass spectrometric detectabilities were achieved with HFBA. Clusters of HFBA, water, and acetonitrile were observed in the mass spectra at m/z values >500. Although the signal-to-noise ratios for the individual peptides diverged considerably both in the selected ion chromatograms and extracted mass spectra, the average mass spectrometric detectabilities varied only by a factor of less than 1.7 measured with the different additives. Limits of detection for peptides with 500 nl sample volumes injected onto a 60 mm x 0.20 mm monolithic column were in the 0.2-13 fmol range. In the analysis of hydrophobic membrane proteins, HFBA enabled highest separation selectivity at the cost of lower mass spectral quality. The use of 0.050% TFA as mobile phase additive turned out to be the best compromise between chromatographic and mass spectrometric performance in the analysis of peptides and proteins by RP-HPLC-ESI-MS using monolithic separation columns.

Monolithic capillary columns for liquid chromatography-electrospray ionization mass spectrometry in proteomic and genomic research.

Peptides, proteins, single-stranded oligonucleotides, and double-stranded DNA fragments were separated with high resolution in micropellicular, monolithic capillary columns prepared by in situ radical copolymerization of styrene and divinylbenzene. Miniaturized chromatography both in the reversed-phase and the ion-pair reversed-phase mode could be realized in the same capillary column because of the nonpolar character of the poly-(styrene/divinylbenzene) stationary phase. The high chromatographic performance of the monolithic stationary phase facilitated the generation of peak capacities for the biopolymers in the range of 50-140 within 10 min under gradient elution conditions. Employing volatile mobile phase components, separations in the two chromatographic separation modes were on-line hyphenated to electrospray ionization (tandem) mass spectrometry, which yielded intact accurate molecular masses as well as sequence information derived from collision-induced fragmentation. The inaccuracy of mass determination in a quadrupole ion trap mass spectrometer was in the range of 0.01-0.02% for proteins up to a molecular mass of 20000, and 0.02-0.12% for DNA fragments up to a molecular mass of 310000. High-performance liquid chromatography-electrospray ionization mass spectrometry utilizing monolithic capillary columns was applied to the identification of proteins by peptide mass fingerprinting, tandem mass spectrometric sequencing, or intact molecular mass determination, as well as to the accurate sizing of double-stranded DNA fragments ranging in size from 50 to 500 base pairs, and to the detection of sequence variations in DNA fragments amplified by the polymerase chain reaction.

Measurement of cell death by oxidative stress in three-dimensional spheroids from trophoblast and in fragments of decidua tissue.

We report a new morphometric method for measurement of the amount of cell death in three-dimensional multicellular spheroids of the trophoblast-like cell line AC1-M59 and of cultured pieces of decidua tissue (decidua spheroids) in response to a cytotoxic agent. The viability of the spheroids was assessed by adding propidium iodide to the culture medium at the end of the toxic treatment. On fluorescence and brightfield images of serial cryosections the areas of propidium iodide fluorescence and the entire corresponding spheroids were measured by applying digital image processing and ratiometrical quantification. As an example, we evaluated the cytotoxic effect of hydrogen peroxide on both types of spheroids. The relative potency of hydrogen peroxide to induce tissue damage was assessed quantitatively for determination of the minimal concentration that leads to an increase in cytotoxicity. The method presented suggests general applicability for in vitro determination of toxicity against tissues.

Adolescent primiparas: changes in obstetrical risk between 1983-1987 and 1999-2005.

AIMS: Teenage pregnancies have always been considered at increased risk for obstetric complications. Deliveries in adolescent primiparas in the 5-year time periods 1983-1987 and 1999-2005 were compared against each other, the general population and against primiparas aged 20-29 years in order to reveal trends and differences in obstetric outcome. METHODS: A total of 186 primiparas delivering at an age of 17 or less between October 1999 and October 2005 were compared with 353 adolescent primiparas delivered between 1983 and 1987. Type of delivery and complications such as low birthweight, pre-eclampsia, breech presentation and third stage complications were studied. RESULTS: The percentage of adolescents in the overall obstetric population decreased. The cesarean section rate remained the same in the adolescents while increasing in the general population. Rates of low birthweight and operative vaginal delivery increased in the adolescent group and overall. Third stage complications (abnormally adherent or incomplete placentas) decreased in both groups. There were no intrauterine fetal deaths in adolescent pregnancies in either time period. Other obstetric variables were unchanged in the adolescent as well as in the general population between 1999 and 2005. When comparing the adolescents' outcome with the outcome of the 20-29-year-old primiparas between 1999 and 2005, it was noted that the rates of abstracted obstetric variables were higher in the population of the 20-29-year-olds. CONCLUSIONS: The obstetric outcome of adolescent pregnancies has remained favorable over the last 18 years. We do not consider adolescence as an obstetrical risk. We suggest that adolescent pregnancy is more a public health issue than a clinical problem.

Proteomics of light-harvesting proteins in different plant species. Analysis and comparison by liquid chromatography-electrospray ionization mass spectrometry. Photosystem II.

An overview of the intact molecular masses and the hydrophobic properties of the photosystem II (PSII) light-harvesting proteins in 14 different plant species is presented. The protein separation and identification was achieved by means of reversed-phase high-performance liquid chromatography-electrospray ionization-mass spectrometry. The good correspondence of the molecular masses measured by reversed-phase high-performance liquid chromatography-electrospray ionization-mass spectrometry with those deduced from the DNA sequence (0.008%-0.016% relative deviation in Arabidopsis) enabled the identification of the different protein types. Utilizing this correlation, it was possible in several cases to spot a gene product for the previously cloned genes. In PSII, all antenna proteins show hydrophobic properties considerably different within the same as well as among various species, in contrast to observations made previously with PSI. These differences might reflect a tuning of protein-protein interactions that play a role in inducing different supramolecular organizations of PSII: within the same species as a consequence of short-term adaptations, and among species for seasonal species adaptation. The relative antenna stoichiometry was readily established on the basis of relative peak areas of the separated proteins in the ultraviolet chromatograms. The correspondence found between the high copy number of genes with the gene products reveals that the genes are not silent in their protein expression. Moreover, the high copy number of gene products as well as protein heterogeneity observed in PSII suggest a possible plant strategy to realize the high degree of organization and interconnection of the light-harvesting systems under any environmental conditions.

Multidimensional proteomic analysis of photosynthetic membrane proteins by liquid extraction-ultracentrifugation-liquid chromatography-mass spectrometry.

The membrane protein components of photosystem I (PSI) and II (PSII) from different species were prefractionated by liquid extraction and sucrose gradient ultracentrifugation and subsequently analyzed by reversed-phase high-performance liquid chromatography-electrospray ionization-mass spectrometry (RP-HPLC-ESI-MS) using poly-(styrene-divinylbenzene)-based monolithic capillary columns. The analytical method was shown to be very flexible and enabled the identification of antenna proteins as well as most of the proteins of the reaction center from PSI and PSII in various plant species with few RP-HPLC-ESI-MS analyses necessitating only minor adaptations in the gradients of acetonitrile in 0.05% aqueous trifluoroacetic acid. The membrane proteins, ranging in molecular mass (Mr) from 4196 (I protein) to more than 80,000 (PSI A/B) as well as isoforms were identified on the basis of their intact Mr and comparison with Mr deduced from known DNA or protein sequences. High quality mass spectra enabled the identification and quantitation of the nonphosphorylated and phosphorylated reaction center subunits D1, D2, and CP43 of PSII, containing five to seven membrane-spanning alpha-helices. Because of its high flexibility and suitability for proteins having a very wide range of Mr and hydrophobicities, the method is generally applicable to the analysis of complex mixtures of membrane proteins.

Liquid- and gas-phase nitration of bovine serum albumin studied by LC-MS and LC-MS/MS using monolithic columns.

Post-translational nitration of proteins was analyzed by capillary reversed-phase high-performance liquid chromatography (RP-HPLC) on-line interfaced to electrospray ionization mass spectrometry (ESI--MS) or tandem mass spectrometry (ESI--MS/MS). Both methods were compared using a tryptic digest of bovine serum albumin (BSA) and yielded sequence coverages of 95% and 33% with RP-HPLC--ESI--MS and RP-HPLC--ESI--MS/MS, respectively. At least 95% of the tyrosines were covered by the former method, whereas the latter method only detected less than 50% of the tyrosine-containing peptides. Upon liquid-phase nitration of BSA in aqueous solution using an excess of tetranitromethane, at least 16 of the 20 tyrosine residues were found to be nitrated. After exposure of solid BSA samples to gaseous nitrogen dioxide and ozone at atmospherically relevant concentration levels, only 3 nitrated peptides were detected. By use of such a model system, RP-HPLC--ESI--MS proved to be a rapid and highly efficient method for the comprehensive and quantitative detection of protein nitration.

Expression of functional Fas ligand in choriocarcinoma.

PROBLEM: In the course of pregnancy, fetal trophoblast cells and in that of choriocarcinoma-etiology, trophoblast derived tumor cells, invade the uterine mucosa without causing rejection by decidual leukocytes. Fas ligand (FasL, CD95L, APO-IL), a central regulator of the immune system, has been implicated in the maintenance of immune privileged sites, such as the eye, the testis and the pregnant uterus by inducing apoptosis in activated infiltrating leukocytes. In normal pregnancy FasL, which is expressed by trophoblast cells, appears to contribute to the immune privilege of the pregnant uterus. As choriocarcinoma derives from trophoblast we wanted to assess the expression of FasL in this tissue. METHOD OF STUDY: Immunohistochemistry, immunofluorescence, TUNEL-assay, Western blotting, coculture experiments and flourescence-associated cell sorter-analysis were the techniques used. RESULTS: Expression of FasL was found on cells of choriocarcinoma in paraffin sections in situ and on three choriocarcinoma cell lines such as JEG-3, JAR and BeWo. These results were confirmed by Western blotting. In coculture experiments choriocarcinoma cells induced apoptosis in a Jurkat cell line - sensitive to FasL mediated killing. However, when the Jurkat cells were pre-incubated with a Fas-blocking monoclonal antibody, apoptosis was abolished to a great extent. CONCLUSION: Our findings show that choriocarcinoma cells express FasL and this aforementioned molecule is biologically active. We assume that FasL expression on choriocarcinoma cells may contribute to control of anti-tumor responses by inducing apoptosis in activated Fas bearing leukocytes.

Characterization of a variant of the spinach PSII type I light-harvesting protein using kinetically controlled digestion and RP-HPLC-ESI-MS.

A previously unknown isoform of the type I major antenna protein of photosystem II of spinach was identified, and its amino-terminal sequence was characterized by a novel kinetic digestion approach, in which sequential tryptic digestion was followed by analysis of both released peptides and truncated proteins by reversed-phase high-performance liquid chromatography-electrospray ionization mass spectrometry. Using nonpolar, monolithic, 200-microm-i.d. separation columns based on poly(styrene/divinylbenzene) copolymer and applying gradients of acetonitrile in 0.05% aqueous trifluoroacetic acid, released peptides and truncated proteins could be separated and mass analyzed in a single chromatographic run. This enabled a straightforward identification of the fragments removed from the amino-terminal ends of the protein, which was essential for the characterization of the antenna isomers showing the most significant sequence variation in the amino-terminal region. The sequences of the amino termini were derived from the differences in molecular mass between intact and truncated proteins and were corroborated by sequencing using tandem mass spectrometry and database searching. The sequence of the 23 amino-terminal residues of the previously unknown isoform differed from that of the other two known isoforms only in one and three amino acids, respectively. Such subtle changes in amino acid sequence are supposed to play an important role in the supramolecular organization of photosynthetic antenna proteins.

Absence of HLA-G expression in macrophages of human decidua.

PROBLEM: Macrophages - together with natural killer (NK) cells - constitute the majority of bone marrow derived infiltrating cells in the decidua. As interferon-gamma (IFN-gamma), a cytokine produced by NK cells, has been reported to induce expression of human leukocyte antigen (HLA-G) in monocytic cells, suggesting expression of HLA-G on decidua macrophages potentially stimulated by IFN-gamma, the question arises whether decidua macrophages in normal pregnancy express HLA-G. METHOD OF STUDY: The study was based on immunohistochemistry and flow cytometry. In order to exclude that potentially elusive soluble HLA-G was not detected by immunohistochemistry, we performed in addition RT-PCR of flow-sorted decidua macrophages. RESULTS: Our findings indicate that HLA-G is not present on macrophages of first trimester or term decidua in either membrane-bound or soluble form. Transcripts for soluble HLA-G1 and -G2 were not detected. CONCLUSIONS: We exclude a role of HLA-G on the surface of decidua macrophages or of soluble HLA-G1 or -G2 as a secretory product of decidua macrophages with regard to interaction with HLA-G receptors present in or outside the decidua.