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Background 1,25-Dihydroxy vitamin D3 (DHVD) is the active metabolite of vitamin D, required to maintain blood calcium concentrations. Measurement has proved challenging as it circulates in picomolar concentrations and must be differentiated from other dihydroxyvitamin D species. Clinically, it is essential to be able to determine the cause of hypercalcaemia, which may be due to DHVD excess. Methods The liquid chromatography-mass spectrometry (LCMS) assay which has been developed uses immunoextraction of 0.5 mL serum followed by Amplifex™ derivatization of the dried eluent, with the analysis using the SCIEX 6500+ instrument taking a run time of 11 min. Results The limit of quantitation was determined (15 pmol/L) and the method is linear up to at least 600 pmol/L. Repeatability ranged from 6.1% at 23 pmol/L to 2.5% at 172 pmol/L and intermediate imprecision was 15.6% at 26 pmol/L to 8.3% at 173 pmol/L. The method is unaffected by icterus, haemolysis or lipaemia. Good performance was achieved with the samples from the vitamin D external quality assessment scheme, demonstrating a negative bias compared with the all lab trimmed mean (average –13.8%) and the specific method group (average –7.75%). A negative bias was observed across the concentration range found in 78 patient samples in comparison to a commercial radioimmunoassay (mean –47.8%). This was not unexpected and is likely due to better specificity of the mass spectrometry assay and the lack of a commutable standard reference calibrator. Conclusions We have developed a sensitive and robust LCMS method for the analysis of DHVD in serum, utilizing immunoextraction and derivatization to provide specificity.

Introduction Deaths following diagnosis of venous thromboembolism (VTE) often result from another concurrent illness. The specificity of mortality markers predicting death from pulmonary embolism is unknown. The aim of this analysis was to compare blood predictors of death in patients with confirmed VTE to patients with negative investigations for VTE. Materials and methods Consecutive patients investigated for VTE were prospectively consented from a single hospital over 9 months. VTE was diagnosed and excluded with a standard diagnostic algorithm. Blood was drawn for biomarker analysis and analyzed in batches for NT-proBNP, high sensitivity troponin T, C-reactive protein (CRP), fatty acid binding protein (FABP) and ischemia modified albumin (IMA). Participants were followed for 3 months. The cohort was analyzed in two groups: those diagnosed with VTE and those who had thrombosis excluded. Regression analysis for 3-month mortality was performed for each group. Results 16/153 patients diagnosed with VTE died within three months (10.5%) as did 23/606 patients who had negative investigations for VTE (3.8%). Predictors for death following VTE included cancer, NT-proBNP, troponin T, FABP, and Hb 500 pg/ml in acute cancer associated VTE predicted death with C-statistic of 0.89 (0.80-0.99). Cancer, NT-proBNP and troponin T also predicted death in patients with negative investigations for VTE. Conclusion Several blood markers are not specific for death from PE and may be surrogate markers of global declining health.

Aldosterone is a mineralocorticoid steroid hormone whose measurement in the clinical laboratory is principally performed for the investigation of primary hyperaldosteronism. Traditionally measurement of aldosterone has been performed by radioimmunoassay, however these assays lack specificity as they are prone to interference from structurally related steroid hormones. Herein, we have developed a novel, sensitive and specific method utilising solid phase extraction and quantitation of aldosterone from human plasma by UPLC-MS/MS. Standards, quality controls and samples (250μL) were extracted using Oasis(®) HLB 96-well plates. Extract (30μL) was injected onto a Krudcatcher UPLC In-Line Filter, 0.5μm guard column, coupled to a Kinetex PFP, 100mm×2.1mm, 2.6μm column with methanolic mobile phase gradient elution. Eluant was connected to a Waters(®) Xevo TQS tandem mass spectrometer operating in electrospray negative mode. We detected multiple reaction monitoring (MRM) transitions of m/z 359.0>189.1 for aldosterone and 366.0>194.1 for d7-aldosterone respectively, which co-eluted at 2.65min. Ion suppression was negligible. Mean recovery was 89.6%, limit of detection and lower limit of quantitation were 26pmol/L and 30pmol/L respectively. The assay was linear up to 3200pmol/L (r(2)=0.9999). Mean intra- and inter-assay imprecision and bias were all

Background Measurement of vitamin A (retinol) and E (alpha-tocopherol) in UK clinical laboratories is currently performed exclusively by high-performance liquid chromatography with ultraviolet detection. We investigated whether retinol and alpha-tocopherol could be measured simultaneously by liquid chromatography tandem mass spectrometry. Methods Serum samples (100 μL) were extracted using Isolute + Supported Liquid Extraction plates. Chromatography was performed on a Phenomenex Kinetex Biphenyl 2.6 μm, 50 × 2.1 mm column, and liquid chromatography tandem mass spectrometry on a Waters Acquity TQD. Injection-to-injection time was 4.3 min. The assay was validated according to published guidelines. Patient samples were used to compare liquid chromatography tandem mass spectrometry and high-performance liquid chromatography with ultraviolet detection methods. Results For retinol and alpha-tocopherol, respectively, the assay was linear up to 6.0 and 80.0 μmol/L, and lower limit of quantification was 0.07 and 0.26 μmol/L. Intra and interassay imprecision were within desirable analytical specifications. Analysis of quality control material aligned to NIST SRM 968e, and relative spiked recovery from human serum, both yielded results within 15% of target values. Method comparison with high-performance liquid chromatography with ultraviolet detection methodology demonstrated a negative bias for retinol and alpha-tocopherol by the liquid chromatography tandem mass spectrometry method. Analysis of United Kingdom National External Quality Assurance Scheme samples yielded mean bias from the target value of +3.0% for retinol and −11.2% for alpha-tocopherol. Conclusions We have developed a novel, high-throughput method for extraction of retinol and alpha-tocopherol from human serum followed by simultaneous quantitation by liquid chromatography tandem mass spectrometry. The method offers a rapid, sensitive, specific and cost-effective alternative to high-performance liquid chromatography with ultraviolet detection methodology, and is suitable for routine clinical monitoring of patients predisposed to fat-soluble vitamin malabsorption.

Background Therapeutic drug monitoring of ciclosporin A (CsA) and tacrolimus is traditionally performed using venous whole blood sampling. A number of reports have described development of ultra high-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) methods for the quantitation of CsA and tacrolimus from dried blood spots (DBS), which may offer a convenient alternative. As yet, no such reports have validated this methodology using fingerprick capillary DBS samples collected from transplant patients. Methods Capillary fingerprick DBS were collected from heart and lung transplant patients in a specialist cardiothoracic transplant centre. We utilized our previously published method for the extraction and simultaneous quantitation of CsA and tacrolimus from DBS using UPLC-MS/MS. Drug concentrations measured from DBS were compared to concentrations measured in venous whole blood by our routine clinical UPLC-MS/MS assay. Results In total, 91 heart or lung transplant patients were enrolled onto the study; 46 patients were on CsA therapy and 45 on tacrolimus therapy. Passing–Bablock analysis demonstrated excellent agreement between capillary fingerprick DBS samples and venous whole blood samples. There was a mean positive bias of 2.6 µg/L (95% confidence interval (CI) −2.2 to 7.5 µg/L) for CsA ( n = 45) and mean negative bias of −0.7 µg/L (95% CI −1.1 to −0.3 µg/L) for tacrolimus ( n = 42). Conclusions We demonstrate utility of DBS for serial monitoring of CsA and tacrolimus using UPLC-MS/MS in heart and lung transplant patients. This may offer significant advantages for these patients including the ability to take capillary DBS samples in the community prior to clinic visits.

Objective Patients with symptoms of deep vein thrombosis (DVT) and pulmonary embolism (PE) commonly present to the emergency department (ED). The aim of this study was to assess the role of ischaemia-modified albumin (IMA) testing in the diagnosis of venous thromboembolism (VTE). Methods This was a prospective diagnostic cohort study. Inpatients and ED patients >16 years of age investigated for PE or DVT at a single hospital were eligible for study consent. Blinded IMA analysis was performed on the first blood sample taken from each patient. Patients underwent reference standard investigation for PE or DVT, including 3-month follow-up. Receiver operating characteristic (ROC) curves were constructed for IMA and the IMA:albumin ratio in the diagnosis of all VTE, PE and DVT. A sensitivity analysis was performed. Results 452 patients were consented and investigated for DVT, and 354 patients were consented and investigated for PE (806 in total). 348 patients investigated for PE had IMA testing as did 195 of the first 199 DVT patients. VTE prevalence was 19.7%. The IMA:albumin ratio performed better than IMA alone. The area under the ROC curve (AUC) for IMA:albumin in all VTE was 0.60 (95% CI 0.54 to 0.66), in DVT 0.56 (95% CI 0.46 to 0.65) and in PE 0.63 (95% CI 0.56 to 0.71). In ED patients with symptoms of PE, the AUC for IMA:albumin was 0.69 (95% CI 0.60 to 0.78). Conclusions IMA testing cannot be used alone to diagnose DVT or PE, although there is a moderate association with PE in ED patients.

Cyclosporin A (CsA) and tacrolimus are immunosuppressant drugs principally used in solid organ transplant recipients. Therapeutic drug monitoring (TDM) of both drugs is essential to avoid toxicity related to overdosage, and transplant rejection from underdosage. This necessitates frequent hospital visits to phlebotomy services. Capillary blood sampling onto dried blood spots (DBS) provides numerous advantages to venous whole blood sampling, including the ability for patients to send DBS to the laboratory by post, significantly reducing the number of unnecessary hospital visits. We have developed a novel, simple and rapid method for the extraction and simultaneous UPLC-MS/MS measurement of both CsA and tacrolimus from DBS. The extraction method involved a simple 30 min hot solvent extraction with ultrasonication. Extract (10 μL) was injected onto a Waters Acquity UPLC column filter unit security frit, coupled to a Waters Acquity BEH C18 UPLC column, with methanolic mobile phase gradient elution. Eluant was connected to a Waters Quattro Premier XE tandem mass spectrometer operating in ES+ mode. We detected multiple reaction monitoring (MRM) transitions of m/z 1220>1203 and 1231.9>1215.1 for CsA and d12 CsA respectively which co-eluted at 1.30min, and 821.6>768.5 and 809.6>756.5 for tacrolimus and ascomycin respectively which co-eluted at 1.17 min. Ion suppression was negligible. Mean recovery was 95.5% for CsA and 92.8% for tacrolimus. Limit of detection and limit of quantitation were both 8.5 μg/L for CsA, and 0.5 and 2.3 μg/L respectively for tacrolimus. The assay was linear up to 1500μg/L for CsA (r(2)=0.9999), and up to 50 μg/L for tacrolimus (r(2)=0.9994). Mean intra assay imprecision, inter assay imprecision and bias were all

Genetic engineering or recombinant DNA technology is now routinely applied to the construction and development of new strains of brewing yeast. This is a direct consequence of the power of the technology which facilitates the modification, introduction and stable maintenance of specific genes in brewing yeast, without compromising the intrinsic brewing properties of the yeast itself. The way in which gene technology has been applied to the development of new strains of Bass yeast is briefly illustrated by the provision of plasmid-based systems for ensuring the stable maintenance of recombinant genes, the construction of amylolytic and β-glucanolytic yeast and the design and development of genetic systems for enhancing the value of waste brewers yeast. Commercial and regulatory issues are discussed.

"Golden Rice" is a variety of rice engineered to produce beta-carotene (pro-vitamin A) to help combat vitamin A deficiency, and it has been predicted that its contribution to alleviating vitamin A deficiency would be substantially improved through even higher beta-carotene content. We hypothesized that the daffodil gene encoding phytoene synthase (psy), one of the two genes used to develop Golden Rice, was the limiting step in beta-carotene accumulation. Through systematic testing of other plant psys, we identified a psy from maize that substantially increased carotenoid accumulation in a model plant system. We went on to develop "Golden Rice 2" introducing this psy in combination with the Erwinia uredovora carotene desaturase (crtI) used to generate the original Golden Rice. We observed an increase in total carotenoids of up to 23-fold (maximum 37 microg/g) compared to the original Golden Rice and a preferential accumulation of beta-carotene.

Isoamylases are debranching enzymes that hydrolyze α-1,6 linkages in α-1,4/α-1,6–linked glucan polymers. In plants, they have been shown to be required for the normal synthesis of amylopectin, although the precise manner in which they influence starch synthesis is still debated. cDNA clones encoding three distinct isoamylase isoforms (Stisa1, Stisa2, and Stisa3) have been identified from potato. The expression patterns of the genes are consistent with the possibility that they all play roles in starch synthesis. Analysis of the predicted sequences of the proteins suggested that only Stisa1 and Stisa3 are likely to have hydrolytic activity and that there probably are differences in substrate specificity between these two isoforms. This was confirmed by the expression of each isoamylase in Escherichia coli and characterization of its activity. Partial purification of isoamylase activity from potato tubers showed that Stisa1 and Stisa2 are associated as a multimeric enzyme but that Stisa3 is not associated with this enzyme complex. Our data suggest that Stisa1 and Stisa2 act together to debranch soluble glucan during starch synthesis. The catalytic specificity of Stisa3 is distinct from that of the multimeric enzyme, indicating that it may play a different role in starch metabolism.

The secondary conditioning of cask and bottled beer with live yeast is normally facilitated by ensuring the sedimentation of the yeast to the base of the package prior to dispense. Here we examine the resistance of yeast sediment to disruption in bottled conditioned beer by investigating the attachment of brewing yeast to glass. An attachment assay is described which, when applied to brewing yeast, demonstrated that these yeasts are not naturally adhesive. However, yeast may be encouraged to form an attached mat by the physiological manipulation of starvation. The significance of this observation is discussed in relation to the effect of beer composition on yeast attachment.

Venous thromboembolism (VTE) including deep vein thrombosis (DVT) and pulmonary embolism (PE) is responsible for 32 000 deaths in hospitalised patients every year in the UK. Initial investigation of patients with suspected VTE recommends clinical probability score and D-dimer blood test. The Thromboembolism Assessment and Diagnosis (THREAD) study is a prospective diagnostic VTE study which aims to assess new blood biomarkers which may …

The presence of 2-μm plasmid DNA was demonstrated in each of seven different strains of brewing yeast investigated. These yeasts were used as recipients for 2-μm-based recombinant DNA plasmids carr...

Acinetobacter calcoaceticus strain EBF65/65 harbours a cryptic plasmid, pAV2, which has been shown by electrophoretic separation on agarose gels to have a molecular mass of approximately 13.5 megadaltons (Md). Transfer of the previously described sex factor pAV1 (Hinchliffe & Vivian, 1980a, b) from the hospital strain JC17 into strains possessing pAV2 occurs only at a low frequency, whereas transfer to similar strains lacking pAV2 occurs at a much higher frequency. In EBF65/65, pAV1 may be present in strains possessing or lacking pAV2; pAV1 strains lacking pAV2 correspond to strains previously described as pAV1a (Hinchliffe & Vivian, 1980b) whereas pAV1 strains which also possess pAV2 correspond to pAV1b strains. The genetic evidence presented here is consistent with the hypothesis that pAV2 specifies a host restriction and modification system that is active against pAV1. Physical evidence from agarose gel electrophoresis indicates that pAV1 corresponds to a band of approximately 85 Md in strain JC17. The corresponding band in strains of EBF65/65 is difficult to distinguish because of the presence of a further cryptic plasmid band of approximately 88 Md, designated pAV3. A small cryptic plasmid of approximately 6 Md, designated pAV4, is reported for EBF65/65.

The now well established principles of genetic engineering are described in relation to the solution of problems associated with β-glucans in beer. The application of these techniques has enabled the isolation of a Bacillus subtilis endo-1, 3–1, 4-β-D-glucanase gene which expresses a biologically active enzyme in yeast.15,16 Although this enzyme is capable of hydrolysing beer β-glucans during fermentation, thereby enhancing beer filtration, insufficient β-glucanase is produced in yeast to enable successful commercial implementation. The requirements for the efficient production of β-glucanase in genetically manipulated brewing yeast are described.

The naturally occurring transmissible plasmid pAV1 mediates chromosome transfer and can exhibit two distinct levels of transmissibility in Acinetobacter calcoaceticus strain EBF65/65. The two states of pAV1 have been arbitrarily designated pAV1a (low frequency variant) and pAV1b (high frequency variant). Both variants have the same incompatibility and host range properties and each mobilizes two non-transmissible resistance determinants for tetracycline and neomycin. Sex factor activity has been shown to be stable: however, pAV1b fertility variants can be derived from pAV1a donors following conjugal transfer of pAV1 into new recipient strains of EBF65/65.

The par region of pSC101, required in cis to promote its stable inheritance, was joined, in combination with the tet r determinant of pBR325, to large and small minichromosomes. These hybrid minichromosomes were examined for stability and found to be no more stable than their parent minichromosomes. Indeed, one recombinant plasmid, pEH21, showed reduced stability, which was not attributable to a reduced copy number. Neither pEH21 nor pEH22, a plasmid composed of the same DNA arranged differently, was stabilized by the presence of a Par + pSC101 derived replicon in the same cell. We conclude that the par region of pSC101 does not stabilize minichromosomes.

The naturally occurring plasmid pAV2 restricts the entry of the P class plasmid RP4 and the W class plasmids R388 and S-a into Acinetobacter calcoaceticus strain EBF65/65 from Escherichia coli. The W class plasmids only transfer from E. coli into pAV2-strains. Plasmid RP4 is modified in the presence of pAV2 such that it is no longer restricted on entry into pAV2 recipients of strain EBF65/65.

SummaryA non-transmissible plasmid, pAV5, was isolated from a hospital strain ofAcinetobacter calcoaceticus, JC17. pAV5 confers resistance to the antibiotics tetracycline and neomycin in the genetically characterized strain of A.calcoaceticusEBF 65/65. Transfer of pAV5 can be mediated by the sex factors pAV1 and R751; transfer is occasionally associated with the segregation of the resistance determinants amongst the transconjugants. Phenotypic dissociation of pAV5 corresponds with the formation of two independent plasmids designated pAV51 and pAV52 mediating resistance to neomycin and tetracycline respectively.

We have constructed a set of hybrid yeast Escherichia coli vectors which utilise the site specific recombination function of the Saccharomyces cerevisiae 2 microns plasmid to completely eliminate the bacterial moiety upon introduction into yeast. A number of these plasmids have been shown to exhibit high inheritable stability in both laboratory and industrial strains during non-selective growth. These plasmids are beneficial for the genetic modification of industrial yeast, particularly those used in the production of food and beverages, and are of benefit in the study of plasmid maintenance and heterologous gene expression.

Summary: A naturally occurring transmissible plasmid, designated pAVl, has been isolated in Acinetobacter calcoaceticus. It specifies resistance to sulphonamides and is capable of mobilizing two non-transmissible resistance determinants for tetracycline and neomycin, respectively, within strains of A. calcoaceticus. It is incompatible with the P class R factors RP4 and R751 in A. calcoaceticus. On this basis we conclude that pAVl is a member of the P incompatibility group. However, unlike most other P group R factors, pAVl is not transmissible to strains of Escherichia coli, Pseudomonas aeruginosa, Klebsiella or Proteus mirabilis.

A cloned endo-1,3-1,4-β-glucanase gene from the Gram-positive bacterium B. subtilis has been located by deletion analysis on a 1.4 kb PvuI-ClaI DNA fragment. This gene has been sub-cloned in the yeast LEU2 vector pJDB207 to produce a hybrid plasmid designated pEHB9. pEHB9 has been transformed to S. cerevisiae and shown to direct the synthesis of an endo-1,3-1,4-β-glucanase in yeast. The β-glucanase activity was low and could only be detected in crude cell extracts of yeast harbouring pEHB9.