Evaluation of a Diverse, Worldwide Collection of Wild, Cultivated, and Landrace Pepper (Capsicum annuum) for Resistance to Phytophthora Fruit Rot, Genetic Diversity, and Population Structure.

Pepper is the third most important solanaceous crop in the United States and fourth most important worldwide. To identify sources of resistance for commercial breeding, 170 pepper genotypes from five continents and 45 countries were evaluated for Phytophthora fruit rot resistance using two isolates of Phytophthora capsici. Genetic diversity and population structure were assessed on a subset of 157 genotypes using 23 polymorphic simple sequence repeats. Partial resistance and isolate-specific interactions were identified in the population at both 3 and 5 days postinoculation (dpi). Plant introductions (PIs) 640833 and 566811 were the most resistant lines evaluated at 5 dpi to isolates 12889 and OP97, with mean lesion areas less than Criollo de Morelos. Genetic diversity was moderate (0.44) in the population. The program STRUCTURE inferred four genetic clusters with moderate to very great differentiation among clusters. Most lines evaluated were susceptible or moderately susceptible at 5 dpi, and no lines evaluated were completely resistant to Phytophthora fruit rot. Significant population structure was detected when pepper varieties were grouped by predefined categories of disease resistance, continent, and country of origin. Moderately resistant or resistant PIs to both isolates of P. capsici at 5 dpi were in genetic clusters one and two.

Heterogeneity in the risk of Mycobacterium bovis infection in European badger (Meles meles) cubs.

The behaviour of certain infected individuals within socially structured populations can have a disproportionately large effect on the spatio-temporal distribution of infection. Endemic infection with Mycobacterium bovis in European badgers (Meles meles) in Great Britain and Ireland is an important source of bovine tuberculosis in cattle. Here we quantify the risk of infection in badger cubs in a high-density wild badger population, in relation to the infection status of resident adults. Over a 24-year period, we observed variation in the risk of cub infection, with those born into groups with resident infectious breeding females being over four times as likely to be detected excreting M. bovis than cubs from groups where there was no evidence of infection in adults. We discuss how our findings relate to the persistence of infection at both social group and population level, and the potential implications for disease control strategies.

Long-term temporal trends and estimated transmission rates for Mycobacterium bovis infection in an undisturbed high-density badger (Meles meles) population.

We describe epidemiological trends in Mycobacterium bovis infection in an undisturbed wild badger (Meles meles) population. Data were derived from the capture, clinical sampling and serological testing of 1803 badgers over 9945 capture events spanning 24 years. Incidence and prevalence increased over time, exhibiting no simple relationship with host density. Potential explanations are presented for a marked increase in the frequency of positive serological test results. Transmission rates (R0) estimated from empirical data were consistent with modelled estimates and robust to changes in test sensitivity and the spatial extent of the population at risk. The risk of a positive culture or serological test result increased with badger age, and varied seasonally. Evidence consistent with progressive disease was found in cubs. This study demonstrates the value of long-term data and the repeated application of imperfect diagnostic tests as indices of infection to reveal epidemiological trends in M. bovis infection in badgers.

Does carbetocin for prevention of postpartum haemorrhage at caesarean section provide clinical or financial benefit compared with oxytocin?

Postpartum haemorrhage is a major cause of maternal morbidity and mortality worldwide. A recent Cochrane review of carbetocin (long-acting oxytocin analogue) concluded that its use decreased additional uterotonic requirements, however, no included studies compared its use against intravenous bolus oxytocin. The majority of studies of carbetocin have considered its use in vaginal delivery; no studies have examined the economic implications of its use. This study describes a clinical and financial evaluation undertaken at a United Kingdom District General Hospital surrounding the introduction of carbetocin for prophylaxis against postpartum haemorrhage at caesarean deliveries. A range of clinical outcomes were observed including frequency of postpartum haemorrhage, estimated blood loss, transfusion requirements, change in haemoglobin or haemodynamics, use of additional uterotonics and perioperative recovery. Finally, a composite financial analysis was performed. No clinically significant benefit was found, however associated costs increased by £18.52/patient.

Prevalence, distribution and pathological significance of the bile fluke Pseudamphistomum truncatum in Eurasian otters (Lutra lutra) in Great Britain.

Postmortem examinations on 160 otters found dead in Great Britain from 2005 to 2007 showed a high prevalence of infection with the bile fluke Pseudamphistomum truncatum in otters from Somerset and Dorset but no infection in otters from other areas, including Cornwall, the western half of Devon, north-east England and Scotland. Cases were also recorded for the first time in Gloucestershire and East Anglia. Eighteen of the 28 infected otters were male and no cubs were infected, but there was no significant correlation between infection status and sex or age. There was a strong positive correlation between bile fluke infection and gall bladder pathology. Thirty otters had thickened gall bladders resulting from hyperplasia of the epithelial and smooth muscle layers, fibrosis and infiltration by inflammatory cells and 19 of these were infected. No flukes were detected in the other 11 otters with abnormal gall bladders, possibly as a result of a successful immune response. The majority of otters with thickened gall bladders were in good physical condition but a positive association was not confirmed statistically. Fluke infection was also detected in nine of the 130 otters with apparently normal gall bladders. Liver pathology ranged from mild bile duct hyperplasia and periportal fibrosis to severe sclerosing cholangitis, hepatocyte necrosis and bile stasis. No otters were found to have died as a result of fluke infection, but there was a negative association, approaching significance, between infection and body condition.

Titrated low-dose vaginal and/or oral misoprostol to induce labour for prelabour membrane rupture: a randomised trial.

OBJECTIVE: To evaluate the clinical effectiveness and safety of titrated low-dose misoprostol for induction of labour (IOL) in the presence of prelabour rupture of membranes (PROM). DESIGN: Randomised controlled trial. SETTING: Maternity units in the UK (9) and Egypt (1). POPULATION: Women >34 weeks of gestation with PROM, singleton viable fetus and no previous caesarean section. METHODS: Subjects randomised to IOL with a titrated low-dose misoprostol regimen (oral except if unfavourable cervix, where initial dose vaginal) or a standard induction method, namely vaginal dinoprostone followed by intravenous oxytocin if the cervix was unfavourable or intravenous oxytocin alone if the cervix was favourable. MAIN OUTCOME MEASURES: Primary outcome measures were caesarean section and failure to achieve vaginal delivery within 24 hours. Analysis was by intention to treat. RESULTS: The trial did not achieve the planned sample size of 1890 due to failure in obtaining external funding. Seven hundred and fifty-eight women were randomised (375 misoprostol and 383 standard). There were less caesarean section (14 versus 18%, relative risk [RR] 0.79; 95% CI 0.57-1.09) and less women who failed to achieve vaginal delivery within 24 hours in the misoprostol group (24 versus 31%, RR 0.79; 95% CI 0.63-1.00), but the differences were not statistically significant. Subgroup analysis showed that with unfavourable cervix, misoprostol may be more effective than vaginal dinoprostone. There was no difference in hyperstimulation syndrome. There were more maternal adverse effects with misoprostol, but no significant differences in maternal and neonatal complications. CONCLUSIONS: Titrated low-dose misoprostol may be a reasonable alternative for IOL in the presence of PROM, particularly in women with an unfavourable cervix. Safety and rare serious adverse events could not be evaluated in a trial of this size.

Naturally processed and presented epitopes of the islet cell autoantigen IA-2 eluted from HLA-DR4.

During immune responses, antigen-presenting cells (APCs) process antigens and present peptide epitopes complexed with human leukocyte antigen (HLA) molecules. CD4 cells recognize these naturally processed and presented epitopes (NPPEs) bound to HLA class II molecules. Epitope identification is important for developing diagnostic and therapeutic tools for immune-mediated diseases and providing insight into their etiology, but current approaches overlook effects of natural processing on epitope selection. We have developed a technique to identify NPPEs using mass spectrometry (MS) after antigen is targeted onto APCs using a lectin-based antigen delivery system (ADS). We applied the technique to identify NPPEs of the intracellular domain of the type 1 diabetes mellitus-associated (type 1 DM-associated) autoantigen insulinoma-associated-2 (IA-2ic), presented by HLA-DR4 (0401). IA-2ic-derived NPPEs eluted from HLA-DR4 constitute 6 sets of peptides nested around distinct core regions. Synthetic peptides based on these regions bind HLA-DR4 and elicit primary T-cell proliferation frequently in HLA-DR4-positive type 1 DM patients, but rarely in non-HLA-DR4 patients, and in none of the HLA-DR4 nondiabetic controls we tested. This flexible, direct approach identifies an HLA allele-specific map of NPPEs for any antigen, presented by any HLA class II molecule. This method should enable a greater understanding of epitope selection and lead to the generation of sensitive and specific reagents for detecting autoreactive T cells.

Metal mediated sterol receptor-DNA complex association and dissociation determined by electrospray ionization mass spectrometry.

The vitamin D receptor (VDR) binds to specific DNA sequences termed vitamin D response elements (VDREs) thereby enhancing or repressing transcription. We have used electrospray ionization mass spectrometry to examine the interaction between the DNA-binding domain of the vitamin D receptor (VDR DBD) with a double-stranded DNA (dsDNA) sequence containing the VDRE from the mouse osteopontin gene. The VDR DBD was shown to bind to the appropriate DNA sequence only when bound to 2 moles of zinc (Zn2+) or cadmium (Cd2+) per mole of protein. Additional binding of Zn2+ or Cd2+ by the protein caused the protein to dissociate from the dsDNA. These results show that the VDR DBD/DNA metal-dependent association occurs when the receptor is occupied by 2 moles of Zn2+ per mole of protein and that further binding of Zn2+ to the protein causes dissociation of the complex.

Analysis of transcription complexes and effects of ligands by microelectrospray ionization mass spectrometry.

The human vitamin D receptor (VDR) and retinoid X receptor-alpha (RXRalpha) modulate gene activity by forming homodimeric or heterodimeric complexes with specific DNA sequences and interaction with other elements of the transcriptional apparatus in the presence of their known endogenous ligands 1alpha,25-dihydroxyvitamin D3 (1, 25-[OH]2D3) and 9-cis-retinoic acid (9-c-RA). We used rapid buffer exchange gel filtration in conjunction with microelectrospray ionization mass spectrometry (microESI-MS) to study the binding of these receptors to the osteopontin vitamin D response element (OP VDRE). In the absence of DNA, both VDR and RXRalpha existed primarily as monomers, but in the presence of OP VDRE, homodimeric RXRalpha and heterodimeric RXRalpha-VDR complexes were shown to bind OP VDRE. Addition of 9-c-RA increased RXRalpha homodimer-OP VDRE complexes, and addition of 1,25-(OH) 2D3 resulted in formation of 1, 25-(OH)2D 3-VDR-RXRalpha-OP VDRE complexes. Addition of low-affinity binding ligands had no detectable effect on the VDR-RXRalpha-OP VDRE transcription complex. These results demonstrate the utility of microESI-MS in analyzing multimeric, high-molecular-weight protein-protein and protein-DNA complexes, and the effects of ligands on these transcriptional complexes.

Umbilical cord plasma concentrations of free morphine following single-dose diamorphine analgesia and their relationship to dose-delivery time interval, Apgar scores and neonatal respiration.

OBJECTIVE: To find the effect of dose-delivery interval on cord-blood levels of diamorphine metabolites and its effect on Apgar sores and neonatal respiration. STUDY DESIGN: Pilot study conducted in labour ward of a district general hospital. One hundred women who had normal delivery and received single dose of 7.5mg of intramuscular injection of diamorphine in labour were recruited in the study. A 2.0 ml sample of umbilical venous blood was collected from the placenta after delivery of the baby. The sample was analysed using RIA method to measure free morphine. Details about the labour and baby's condition at birth were recorded. RESULTS: The concentration of free morphine in the umbilical venous blood was significantly associated with the dose-delivery interval (coefficient (95% CI)=1.08(0.99-1.18), p<0.001). Twenty neonates had low Apgar score (< or =7) at 1 min. The odds of such a low score were raised with higher log free morphine in the cord venous plasma, but not statistically significantly (OR (95% CI)=5.3 (0.84-34), p=0.08). Fourteen neonates required resuscitation. The odds of requiring resuscitation were significantly raised with higher log free morphine: OR (95% CI)=9.3 (1.0-86), p=0.05. CONCLUSION: Concentration of free morphine in the umbilical venous blood after delivery was significantly associated with the dose-delivery interval and this had significant effect on the need for resuscitation.

Detection and quantitation of cellularly derived amyloid beta peptides by immunoprecipitation-HPLC-MS.

A quantitative method for detection of amyloid beta peptides using immunoprecipitation-HPLC-mass spectrometry (IP-LC-MS) is described. Comparison of IP-LC-MS with sandwich ELISA revealed comparable results in the analysis of A beta 1-40 and A beta 1-42 derived from fetal guinea pig cell media and cell lysates. The use of IP-LC-MS not only allows a quantitative method for A beta 1-40 and A beta 1-42 peptides present in Alzheimer's disease (AD), but allows detection of other A beta peptide species that may also play a role in the onset of AD in humans.

Inhibition of human mitochondrial aldehyde dehydrogenase by the disulfiram metabolite S-methyl-N,N-diethylthiocarbamoyl sulfoxide: structural characterization of the enzyme adduct by HPLC-tandem mass spectrometry.

S-Methyl-N,N-diethylthiocarbamoyl sulfoxide (MeDTC-SO) is a known metabolite of the aversion therapy drug disulfiram (DSF). MeDTC-SO is also a potent inhibitor of human mitochondrial aldehyde dehydrogenase (hmALDH) with an IC50 of 1.5 microM. Inhibition of the enzyme by MeDTC-SO resulted in the addition of approximately 100 Da to the molecular mass of the intact protein, as determined by on-line HPLC-electrospray ionization MS (LC-MS). Dialysis of the inhibited protein did not reverse the inhibition, and the molecular mass of 54,533 Da (+/- 0.01%) remained unchanged, indicating that a covalent modification of the protein had occurred. Proteolytic digestion of hmALDH under basic conditions using trypsin at pH 7.8 revealed that the adduct was base labile. However, treating the adducted protein with endopeptidase-Glu-C at pH 3.7 produced a peptide adduct at MH+ = 4924, tentatively attributable to a carbamoylated peptide. This peptide contains three adjacent cysteines, one of which has been implicated as a key amino acid in the highly conserved active site region of ALDH. A pepsin digestion of hmALDH carried out at pH 3.7 and subsequent LC-MS analysis revealed an ion at MH2(2+) = 501.5, corresponding to the carbamoylated peptide FNQGQC1C2C3. This peptide contains the same adjacent active site cysteines. This latter peptide was subjected to LC-MS/MS, which enabled us to determine that the site of carbamoylation was at Cys2. The MS/MS product ion data also confirmed the presence of a carbamoyl group as the adduct species.

One step microelectroelution concentration method for efficient coupling of sodium dodecylsulfate gel electrophoresis and matrix-assisted laser desorption time-of-flight mass spectrometry for protein analysis.

The coupling of the widely used separation technique of conventional sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) with the mass accuracy measurement capability of mass spectrometry (MS) provides a very powerful analytical technique. However, at present, there is no simple, definitive method for coupling the two methods. Typically, separated proteins are extracted from the gel, either as the native protein or as a peptide mixture after in-gel proteolytic digestion, and then analyzed by mass spectrometry. However, the various extraction techniques described previously have been labor intensive and require a large number of steps. The mass spectrometry analysis of very low concentrations of in vivo derived proteins requires minimum sample handling and on-line concentration. Therefore, we have developed an efficient microelectroelution technique that is applied in a single step manner and contains an on-line concentration device. Initial results from this system have shown a high efficiency of analyte elution from the gel and a simple, robust technique for the coupling of SDS-containing gels with MALDI-TOF-MS analysis and a capability of analyzing proteins at the subpicomole level.

Low temperature aqueous electrospray ionization mass spectrometry of noncovalent complexes.

In the present study we describe conditions that permit the characterization of noncovalent protein-substrate complexes in aqueous solution by microspray electrospray ionization-mass spectrometry (ESI-MS), using a heated transfer capillary at low temperature (45 degrees C). Specifically, we examined the binding of calmodulin to two polypeptides; the calmodulin-binding domain of calmodulin-dependent protein kinase II (CamK-II) and melittin. Calmodulin, a well known calcium-binding protein, binds to a number of small amphipathic peptides in a calcium-dependent manner. Our results directly show that both peptides form equimolar complexes with calmodulin only in the presence of calcium. The stoichiometry necessary for the formation of each complex was 1:1:4 for calmodulin:peptide (melittin or CamK-II):Ca2+, respectively. Furthermore, it is demonstrated that the detection of the complex in ESI-MS is source temperature dependent.

Zinc-induced conformational changes in the DNA-binding domain of the vitamin D receptor determined by electrospray ionization mass spectrometry.

Electrospray ionization mass spectrometry (ESI-MS) was used to measure conformational changes within the DNA-binding domain of the vitamin D receptor (VDR DBD) upon binding zinc (Zn2+). As increasing concentrations of Zn2+ were added to the VDR DBD, a gradual shift in the mass envelope to lower charge states was observed in the multiply charged spectrum. The shift in the charge states was correlated to changes observed in the far-ultraviolet circular dichroic (far-UV CD) spectrum of the protein as it was titrated with Zn2+. Both the multiply charged ESI and far-UV CD spectra of the Zn(2+)-titrated protein show that the binding of the first Zn2+ ion to the protein results in very little conformational change in the protein. The binding of a second Zn2+ ion resulted in a significant alteration in the structure of the protein as indicated by changes in both the multiply charged ESI and far-UV CD spectra. Much smaller changes were seen within the multiply charged ESI or far-UV CD spectra upon increasing the Zn2+ concentration beyond 2 mol/mol of protein. The results presented indicate that ESI-MS in combination with CD is a powerful method to measure gross conformational changes induced by the binding of metals to metalloproteins.

Selective increase in cellular A beta 42 is related to apoptosis but not necrosis.

Amyloid beta protein ending at 42 (A beta 42) plays an important role in the pathology of Alzheimer's disease (AD). Here we show an increase in cellular A beta 42 in damaged neurons, with both ELISA and immunocytochemistry. The cellular A beta 42 increase was caused by 3-day treatments with H2O2, etoposide or melphalan, all of which induce genotoxic apoptosis, but not by treatment with sodium azide, which causes necrosis. Secreted A beta was similarly decreased with all these treatments. The cellular A beta 42 increase appeared even with minimal damage (ELISA) and A beta 42-positive cells were TUNEL negative (double staining), indicating that any early apoptosis mechanism may induce the cellular A beta 42 increase. Thus, neuronal apoptosis and cellular A beta 42 increase may be linked in a way that contributes importantly to AD pathology.

Strategy for isolating and sequencing biologically derived MHC class I peptides.

The presentation of MHC class I peptides at cell surfaces and the subsequent cytolytic T-lymphocyte response are critical components of the mammalian immune response. However, the identification and sequencing of such peptides present a considerable analytical challenge since > 10,000 peptides at 10(-15)-10(-18) M concentrations are often present in the mixture. We describe a two-dimensional chromatography approach in conjunction with tandem mass spectrometry to sequence and identify such peptides. After immunoaffinity concentration, and subsequent acetic acid release of MHC class I peptides from MHC protein complex, the peptides are subjected to reversed phase HPLC, where they are separated based on their hydrophilic-hydrophobic character. These coarse fractions are then loaded onto a specially designed membrane preconcentration-capillary electrophoresis cartridge (mPC-CE) and subsequently subjected to on-line mPC-CE-MS analysis. The second dimension of chromatography by CE separation affords resolution of peptides based on their charge/mass (to a first approximation) ratio. Ultimately peptides are sequenced using mPC-CE-tandem mass spectrometry (mPC-CE-MS-MS). We describe the strategy for sequencing < 60 femtomoles of a peptide obtained from 3.10(9) Kb-derived EL-4 cells.

Application of capillary electrophoresis and related techniques to drug metabolism studies.

The use of capillary electrophoresis (CE) for the separation of small organic molecules such as pharmaceutical agents and drug/xenobiotic metabolites has become increasingly popular. This has arisen, at least in part, from the complimentary mode of separation afforded by CE when compared to the more mature technique of HPLC. Other qualities of CE include relative ease of method of development, rapid analysis, and low solvent consumption. The recent introduction of a variety of detector systems (including UV diode array, laser-induced fluorescence, conductivity) and the demonstrated coupling of CE to MS have also aided acceptance of this technology. In the present report, we review the role of CE coupled to various detector systems including a mass spectrometer for the characterization of both in vitro and in vivo derived drug metabolite mixtures. Attributes of CE for this application are demonstrated by discussion of metabolism studies of the neuroleptic agent haloperidol. Various aspects of the development and use of CE and CE-MS for the characterization of haloperidol metabolites, including criteria for selection of parameters such as pH, ionic strength, extent of organic modification, and the use of nonaqueous capillary zone electrophoresis are discussed. We also consider potential limitations of CE and CE-MS for drug metabolism research and describe the introduction of membrane preconcentration-CE (mPC-CE) and mPC-CE-MS as a solution that overcomes the rather poor concentration limits of detection of CE methods without compromising the resolution of analytes or separation efficiency of this technique.