Prerequisite Delivery Method and Academic Outcomes in a Physician Assistant Education Program.

PURPOSE: The Interservice Physician Assistant Program (IPAP) educates up to 169 matriculants per year. Each service branch sets the admission criteria, including all prerequisites, for their applicants. We hypothesized that prerequisites obtained online/virtual are less rigorous than coursework completed in-person. The purpose of this investigation was to evaluate whether online/virtual prerequisite courses were associated with academic deceleration or attrition at any point. METHODS: Student self-reported data were retrospectively analyzed to evaluate program scores of students who took prerequisites online/virtual or in-person. RESULTS: There were no statistically significant differences in foundational course performance between online/virtual and in-person coursework. In addition, students who took anatomy online performed better than students who completed the coursework in-person (140.6 ± 15.6 vs. 145.6 ± 14.7, P = .05). CONCLUSIONS: This analysis indicates that using the prerequisite source to predict academic difficulty may not be possible in IPAP students. Faculty will need to continue to search for other predictors of academic difficulty.

The role of sleep and heart rate variability in metabolic syndrome: evidence from the Midlife in the United States study.

STUDY OBJECTIVES: Poor sleep and autonomic dysregulation can both disrupt metabolic processes. This study examined the individual and combined effects of poor sleep and reduced heart rate variability (HRV) on metabolic syndrome among 966 participants in the Midlife in the United States II (MIDUS II) study. METHODS: Self-reported sleep was assessed using the Pittsburgh Sleep Quality Index (PSQI). HRV was acquired from 11-minute resting heart rate recordings. Spearman correlations, general linear regression, and logistic regression models were used to examine the study hypotheses. RESULTS: Poor sleep quality was associated with metabolic syndrome when global PSQI scores were evaluated as a continuous (odds ratio [OR]: 1.07, 95% confidence interval [CI]: 1.03 to 1.11) or categorical measure (cutoff > 5, OR: 1.58, 95% CI: 1.19 to 2.10), after adjustment for confounding. There also was an association between reduced HRV and metabolic syndrome (ln [HF-HRV] OR: 0.89, 95% CI: 0.80 to 0.99; ln [LF-HRV] OR: 0.82, 95% CI: 0.72 to 0.92; ln [SDRR] OR: 0.59, 95% CI: 0.43 to 0.79; ln [RMSSD] OR: 0.75, 95% CI: 0.60 to 0.94). When the combined effects of poor sleep and low HRV were examined, the association with metabolic syndrome was further strengthened relative to those with normal sleep and HRV. CONCLUSIONS: To the best of the author's knowledge, this is the first study to suggest a combined effect of poor sleep and low HRV on the odds of metabolic syndrome.

Shift Work Adaptation Among Police Officers: The BCOPS Study.

Few studies have examined shiftwork adaptation among police officers or potential differences in disease biomarkers among adapted and maladapted shiftworkers. This study characterized shiftwork adaptation among 430 police officers from the Buffalo Cardio-Metabolic Occupational Police Stress (BCOPS) study. Police officers working fixed night shifts with symptoms characteristic of adaptation and maladaptation were identified using latent class analysis (n = 242). Two approaches were applied, one with police-specific symptoms and another using more general symptoms as shiftwork adaptation indicators. Biomarkers of inflammation, heart rate variability, and cardiometabolic risk were then compared between shiftwork adaptation groups, and with officers working day shifts, after adjusting for confounding. When analyses included police-specific symptoms, maladapted shiftworkers (n = 73) had more self-reported stress, sleep disturbances, fatigue, and less social support than adapted shiftworkers (n = 169). Using more general symptoms, maladapted officers (n = 56) reported more stress and depression, and less social support than adapted officers (n = 186). In police-specific models, adjusted (least-squares) means (± standard error) of circulating interleukin-6 (IL-6) concentrations in maladapted officers (0.8 ± 0.1 ln[pg/ml]) were modestly elevated relative to adapted shiftworkers (0.7 ± 0.1 ln[pg/ml], p = .09) and relative to permanent day workers (0.5 ± 0.1 ln[pg/ml], p ≤ 0.01), and leptin levels in maladapted officers (9.6 ± 0.1 ln[pg/ml]) exceeded those in the adapted (9.4 ± 0.1 ln[pg/ml], p ≤ 0.01) and day shift groups (9.4 ± 0.1 ln[pg/ml], p = .03). In the general model, adjusted mean tumor necrosis factor-alpha (TNF-α) concentrations among maladapted officers (5.6 ± 0.23 pg/ml) exceeded the adapted (4.8 ± 0.2 pg/ml, p ≤ 0.01) and day workers (5.0 ± 0.2 pg/ml, p = .04), and insulin among maladapted officers was higher (2.4 ± 0.1 ln[uu/ml]) than the adapted group (1.8 ± 0.1 ln[uu/ml], p = .03). No differences were observed for the other biomarkers. The results suggest that maladaptation among police officers working fixed night shifts may lead to increases in leptin, insulin, IL-6, and TNF-α; however, the cross-sectional design and possible residual confounding preclude interpretation of cause and effect. Prospective studies are planned to further characterize the relationship between shiftwork maladaptation and biomarkers of chronic disease risk in this police officer cohort.

Sensory trait variation contributes to biased dispersal of threespine stickleback in flowing water.

Gene flow is widely thought to homogenize spatially separate populations, eroding effects of divergent selection. The resulting theory of 'migration-selection balance' is predicated on a common assumption that all genotypes are equally prone to dispersal. If instead certain genotypes are disproportionately likely to disperse, then migration can actually promote population divergence. For example, previous work has shown that threespine stickleback (Gasterosteus aculeatus) differ in their propensity to move up- or downstream ('rheotactic response'), which may facilitate genetic divergence between adjoining lake and stream populations of stickleback. Here, we demonstrate that intraspecific variation in a sensory system (superficial neuromast lines) contributes to this variation in swimming behaviour in stickleback. First, we show that intact neuromasts are necessary for a typical rheotactic response. Next, we showed that there is heritable variation in the number of neuromasts and that stickleback with more neuromasts are more likely to move downstream. Variation in pectoral fin shape contributes to additional variation in rheotactic response. These results illustrate how within-population quantitative variation in sensory and locomotor traits can influence dispersal behaviour, thereby biasing dispersal between habitats and favouring population divergence.

Status and prospects of plant virus control through interference with vector transmission.

Most plant viruses rely on vector organisms for their plant-to-plant spread. Although there are many different natural vectors, few plant virus-vector systems have been well studied. This review describes our current understanding of virus transmission by aphids, thrips, whiteflies, leafhoppers, planthoppers, treehoppers, mites, nematodes, and zoosporic endoparasites. Strategies for control of vectors by host resistance, chemicals, and integrated pest management are reviewed. Many gaps in the knowledge of the transmission mechanisms and a lack of available host resistance to vectors are evident. Advances in genome sequencing and molecular technologies will help to address these problems and will allow innovative control methods through interference with vector transmission. Improved knowledge of factors affecting pest and disease spread in different ecosystems for predictive modeling is also needed. Innovative control measures are urgently required because of the increased risks from vector-borne infections that arise from environmental change.

Plasmodesmal targeting and intercellular movement of potato mop-top pomovirus is mediated by a membrane anchored tyrosine-based motif on the lumenal side of the endoplasmic reticulum and the C-terminal transmembrane domain in the TGB3 movement protein.

Live-cell fluorescence microscopy was used to investigate the third triple gene block protein (TGB3) of potato mop-top pomovirus and its role in assisted targeting of TGB2 to plasmodesmata (PD). Wild-type and mutant TGB3 proteins were expressed under the control of the 35S promoter or from a virus reporter clone. Assisted targeting of TGB2 to PD was optimal when the proteins were expressed from a bicistronic plasmid in the relative ratios expected in a virus infection, suggesting that excess TGB3 inhibited PD localisation. Contrary to the generally accepted view, bimolecular fluorescence complementation showed that the TGB3 N terminus is located in the cytosol. Mutational analysis to dissect TGB3 sub domain functions showed that PD targeting was mediated by a composite signal comprising an ER-lumenal tyrosine-based motif and the C-terminal transmembrane domain. Mutation of either of these domains also abolished cell-to-cell movement of the virus. The results are discussed in the context of TGB3 membrane topology.

Barley stripe mosaic virus-encoded proteins triple-gene block 2 and gammab localize to chloroplasts in virus-infected monocot and dicot plants, revealing hitherto-unknown roles in virus replication.

Replication of Barley stripe mosaic virus (BSMV), genus Hordeivirus, is thought to be associated with vesicles in proplastids and chloroplasts, but the molecular details of the process and identity of virus proteins involved in establishing the virus replication complexes are unknown. In addition, BSMV encodes a triple-gene block of movement proteins (TGBs) that putatively share functional roles with their counterparts in other hordei-, pomo- and pecluviruses, but detailed information on the intracellular locations of the individual TGBs is lacking. Here, the subcellular localizations of BSMV-encoded proteins TGB2 and gammab fused to green or red fluorescent proteins were examined in epidermal cells of Nicotiana benthamiana and barley (Hordeum vulgare 'Black Hulless'). The fusion proteins were expressed from a BSMV vector or under the control of the cauliflower mosaic virus 35S promoter. The subcellular localizations were studied by confocal laser-scanning microscopy (CLSM). CLSM studies showed that both proteins were recruited to chloroplasts in the presence of viral RNA and that virus RNA, coat protein and gammab protein were detected in plastid preparations from infected leaves. Electron microscope images of thin sections of virus-infected leaves revealed abnormal chloroplasts with cytoplasmic inclusions containing virus-like particles. In addition, cellular localizations of BSMV TGB2 suggest subtle differences in function between the hordei-like TGB2 proteins. The results indicate that TGB2 and gammab proteins play a previously unknown functional role at the site of virus replication.

Oriented immobilisation of engineered single-chain antibodies to develop biosensors for virus detection.

Single chain variable fragment (scFv) molecules were selected from a synthetic phage display library then cloned into a generic vector for expression of the scFv fused to the light chain constant domain of human immunoglobulin with a C-terminal cysteine residue (scFvC(L)cys). A heterobifunctional maleimide linker was synthesised and a strategy for functionalization of gold with the scFvC(L)cys fusion proteins elaborated. Successful covalent attachment of functional scFvC(L)cys was demonstrated using a surface plasmon resonance-based sensor. The results showed that the immobilised scFvC(L)cys molecules were functional and specific binding curves (with response relative to the concentration of virus antigen) were obtained over more than 25 cycles of binding and dissociation. ScFv molecules lacking the C-terminal cysteine performed poorly in similar experiments. The work demonstrates the feasibility of using simple scFv selection and cloning procedures combined with oriented immobilisation of scFvC(L)cys fusion proteins for robust antigen sensing surfaces in immunosensor or other biotechnological applications.

Subcellular localisation, protein interactions, and RNA binding of Potato mop-top virus triple gene block proteins.

Subcellular localisation, protein interactions, and RNA binding of the triple gene block proteins (TGBp) of Potato mop-top virus (PMTV) were studied. The 13-kDa (TGBp2) and 21-kDa (TGBp3) proteins with or without green fluorescent protein fused to their N-terminus, and the 51-kDa protein (TGBp1) were expressed individually from a recombinant Tobacco mosaic virus (TMV) vector. Fluorescent images and Western immunoblotting experiments of recombinant TMV-infected Nicotiana benthamiana cells suggested that TGBp2 and TGBp3 were associated with cellular endomembranes and that TGBp3 was associated with the cell wall, possibly located close to plasmodesmata. In Western blots, TGBp1 was detected in fractions containing the cell wall and those enriched for organelles and membranous structures. Self-interactions were demonstrated with all three proteins in yeast two-hybrid experiments, and a heterologous interaction was found between TGBp2 and TGBp3. No additional heterologous interactions were discovered between the different TGBp and none were detected in an in vitro binding assay. TGBp1 and TGBp2 but not TGBp3 were shown to bind ssRNA in a sequence nonspecific manner. The results support the model where TGBp2 and TGBp3 facilitate delivery and localisation of the ribonucleoprotein complex to the plasmodesmata. However, the process is facilitated by RNA-protein rather than protein:protein interactions between the TGBp1 in complex with viral RNA and membrane-localised TGBp2.

In situ spatial organization of Potato virus A coat protein subunits as assessed by tritium bombardment.

Potato virus A (PVA) particles were bombarded with thermally activated tritium atoms, and the intramolecular distribution of the label in the amino acids of the coat protein was determined to assess their in situ steric accessibility. This method revealed that the N-terminal 15 amino acids of the PVA coat protein and a region comprising amino acids 27 to 50 are the most accessible at the particle surface to labeling with tritium atoms. A model of the spatial arrangement of the PVA coat protein polypeptide chain within the virus particle was derived from the experimental data obtained by tritium bombardment combined with predictions of secondary-structure elements and the principles of packing alpha-helices and beta-structures in proteins. The model predicts three regions of tertiary structure: (i) the surface-exposed N-terminal region, comprising an unstructured N terminus of 8 amino acids and two beta-strands, (ii) a C-terminal region including two alpha-helices, as well as three beta-strands that form a two-layer structure called an abCd unit, and (iii) a central region comprising a bundle of four alpha-helices in a fold similar to that found in tobacco mosaic virus coat protein. This is the first model of the three-dimensional structure of a potyvirus coat protein.

Aphid transmission studies using helper component proteins of potato virus Y expressed from a vector derived from potato virus X.

The genes encoding the helper component (HC) proteins of two strains of Potato virus Y (PVY) were cloned and the proteins expressed from a vector derived from Potato virus X (PVX). The expressed HC contained six N-terminal histidine residues to facilitate purification by metal affinity chromatography. Approximately 2-4 microg/g of purified HC was obtained from leaves of Nicotiana benthamiana plants systemically infected by recombinant PVX. Preparations of the HC protein derived from PVY ordinary strain (PVY(o)) assisted aphid transmission of purified particles of PVY(o) and PVY strain C (PVY(c); a naturally occurring non-aphid transmissible strain of PVY which contains a defective HC), as well as Potato aucuba mosaic virus. The HC derived from PVY(c) contained the Glu-Ile-Thr-Cys (EITC) motif, and mutation of Glu (E) to Lys (K) enabled the mutant PVX-expressed preparations to assist virus transmission by aphids. Expression of HC protein from the PVX vector produced biologically active protein. This approach should facilitate further studies to elucidate more precisely the molecular mechanism of virus transmission by aphids.

Expression of functional recombinant antibody molecules in insect cell expression systems.

Recombinant single-chain variable-fragment molecules (scFv) were constructed from a cell line expressing a monoclonal antibody against African cassava mosaic virus (ACMV) and expressed in Escherichia coli. DNA sequences that encoded the scFv were manipulated to allow scFv expression in insect cell lines. A recombinant baculovirus containing the scFv cDNA was constructed and large amounts of scFv were produced in each of three insect cell lines infected with the baculovirus. However, the scFv were not secreted into the medium by any of the cell lines despite the scFv having been linked to a honeybee melittin leader sequence. The same scFv cDNA construct was introduced into Drosophila DS2 cells and a stable recombinant cell line was obtained that produced scFv that was secreted into the medium. Culture medium containing the scFv was used directly in enzyme-linked immunosorbent assay (ELISA) tests to detect ACMV in plant tissues. Another construct that encoded the Ckappa domain of human IgG was fused to the C-terminus of the scFv that was produced and expressed in Drosophila cells. This scFv derivative also accumulated in the medium and was more active in ELISA than scFv lacking the Ckappa domain.

Properties of a panel of single chain variable fragments against Potato leafroll virus obtained from two phage display libraries.

Twelve single chain variable fragment (scFv) antibodies that bind to particles of Potato leafroll virus (PLRV) were obtained from two naive phage display libraries. Phages were selected against PLRV particles or dissociated PLRV particles immobilised onto tubes. Individual PLRV-binding scFv were identified by ELISA, after their expression either fused to the surface of phage particles, or as soluble scFv (scFv-c-myc), or as scFv-alkaline phosphatase fusion proteins (scFv-AP), obtained by subcloning into pSKAP/S. These procedures resulted in the isolation of scFv with different properties. For example, some of the scFv reacted strongly with virus particles but not with dissociated capsid protein, which suggests that they had reacted with discontinuous epitopes. Others reacted with dissociated capsid proteins and SDS-denatured protein, which suggests that they had reacted with continuous epitopes. ScFv were also subcloned into pC(L) for expression as fusion proteins with human kappa constant region (scFv-C(L)). Expression of these constructs in Escherichia coli yielded 0.2-1 mg protein per litre of bacterial culture. The different scFv fusion proteins were evaluated in ELISA to detect PLRV in leaf extracts of Physalis floridana. Absorbance values obtained with the fusion proteins were greater than those obtained with the scFv-c-myc, and were similar to those obtained in assays done using monoclonal or polyclonal antibodies.

pSKAP/S: An expression vector for the production of single-chain Fv alkaline phosphatase fusion proteins.

The vector pSKAP/S was constructed to enable overexpression of single-chain variable fragment antibody (scFv)-alkaline phosphatase fusion proteins. In pSKAP/S, the scFv were genetically fused to the mutated Escherichia coli PhoA/S gene that encodes an alkaline phosphatase with increased specific activity. The restriction sites incorporated into pSKAP/S allowed the scFv genes to be easily transferred from pUC119-derived phagemid vectors that are used frequently in phage display antibody library technology. Strong transcriptional control of expression was achieved using the tetracycline promoter, and induction of different individual clones with anhydrotetracycline resulted in secretion of most of the scFv-alkaline phosphatase fusion proteins into the culture medium. Although some of the clones secreted fusion proteins that were retained in the periplasm, these proteins could be isolated with a simple extraction procedure. Increased amounts of a scFv-alkaline phosphatase fusion protein were obtained when expressed in the pSKAP/S vector compared with expression in a vector incorporating the lac promoter. Testing for binding of the scFv-alkaline phosphatase fusion proteins to antigen was possible in an ELISA without the need for additional enzyme-conjugated antibodies. The pSKAP/S vector was successfully used to obtain scFv fragments from a preparation of phage-antibody clones after subcloning and expression of individual clones as scFv-alkaline phosphatase fusions, whereas fewer clones (and clones with different properties) were obtained from the same phage-antibody preparations when expressed as soluble scFv fragments. Therefore, the pSKAP/S vector was shown to be useful in extending the range of scFv obtained from phage display libraries.

Rapid production of single-chain Fv fragments in plants using a potato virus X episomal vector.

We have used a plant virus episomal vector, based on potato virus X (PVX) to transiently express a single-chain Fv (scFv) and its diabody derivative in plants. The scFv was directed against a continuous epitope (cryptotope) on the coat protein of potato virus V. A cloned, full-length PVX vector sequence, containing the scFv gene, was used to direct in vitro transcription and the resulting RNA was used to inoculate Nicotiana clevelandii plants. Within a few days, plants developed characteristic symptoms and immunoblot analysis showed that accumulation of scFv protein coincided with accumulation of PVX. Targeting of the scFv to the apoplast greatly increased protein accumulation compared with cytosolic scFv and produced more severe symptoms on infected plants. ELISA demonstrated that the scFv and diabody extracted from infected plants showed the same antigen-binding specificity as that of the parental monoclonal antibody. The PVX vector is a convenient, rapid, low-cost in planta expression system that can also be used for assessment of scFv production and function prior to stable plant transformation.

Fusion proteins of single-chain variable fragments derived from phage display libraries are effective reagents for routine diagnosis of potato leafroll virus infection in potato.

ABSTRACT A panel of 11 different single-chain variable fragment antibodies (scFv) that bind to potato leafroll virus (PLRV) has been studied to assess each one's suitability as practical diagnostic tools. The scFv, previously obtained from naive phage display libraries, were expressed in Escherichia coli as fusion proteins. The fusion proteins comprised scFv joined to either the human light chain kappa constant domain (C(L)), an amphipathic helix (Zip), a combination of C(L) and Zip, or alkaline phosphatase (AP/S). The fusion proteins were tested for their ability to detect, or trap on enzymelinked immunosorbent assay (ELISA) plates, PLRV in extracts of infected potato leaves. The tests done with the different scFv fusion proteins were compared with a standard triple-antibody sandwich (TAS)-ELISA that employs a rabbit polyclonal antibody preparation to coat microtiter plates and a monoclonal antibody, SCR3, to detect PLRV. Of 11 scFvC(L) fusion proteins, 7 detected PLRV as readily as SCR3 when used as detecting antibodies in TAS-ELISA. The limit of detection of purified PLRV for the different scFvC(L) fusion proteins ranged from 250 to 5 ng/ml; that for SCR3 is 5 ng/ml. Of the 11 scFv, 4 cross-reacted with some other luteoviruses. Several scFvC(L) and scFvC(L)Zip fusion proteins trapped PLRV from extracts of infected potato leaves as effectively as the polyclonal antibody preparation. Four scFv fusion proteins were used in a stem print assay to detect PLRV, and the results were similar to those obtained in tests using SCR3. The scFvC(L) fusion proteins retained activity for at least 6 months at 4 degrees C, and all scFv fusion proteins were fully active on reconstitution after lyophilization. A fully recombinant ELISA was devised that detected PLRV in extracts of infected potato, with results comparable to those obtained using the standard TAS-ELISA. The advantages of using scFv fusion proteins for the routine detection of plant viruses include the ability to produce large quantities of reagents cheaply in bacterial fermenters and to incorporate them into standardized tests.

Association of sequences in the coat protein/readthrough domain of potato mop-top virus with transmission by Spongospora subterranea.

A monofungal culture of Spongospora subterranea was unable to acquire and transmit the T isolate of potato mop-top pomovirus (PMTV-T), which has been maintained by manual transmission in the laboratory for 30 years. A recently obtained field isolate (PMTV-S) was efficiently acquired and transmitted by the same fungus culture. Sequence analysis of the readthrough (RT) protein-coding region of PMTV-S showed the presence of an additional 543 nt in the 3' half of the coding region relative to that of PMTV-T. These additional nucleotides preserved the reading frame of the RT protein and inserted 181 amino acids into the RT protein. This was confirmed by a comparison by immunoblotting of the sizes of the RT protein of PMTV-T and other recent isolates of PMTV.

Synthetic antigen from a Peptide library can be an effective positive control in immunoassays for the detection and identification of two geminiviruses.

ABSTRACT Phage-displayed peptides were selected from the Cys 1 random phage display peptide library that bound strongly to the monoclonal antibody (MAb) SCR 20. The binding peptides were fused to the N-terminus of the phage protein pVIII. Preparations of the phage were shown to be effective as controls for the functionality of the SCR 20 MAb in both enzyme-linked immunosorbent assays and dot blot immunoassays. UV irradiation that eliminated phage infectivity did not greatly alter the antigenicity. Peptides displayed on phage are quick and cheap to prepare, and preparations can be standardized to ensure comparability among different assays. The peptide library approach can be readily extended for use with other MAbs to obtain inexpensive and safe standardized positive control reagents for use in immunoassays to diagnose plant disease.

Selection of single-chain variable fragment antibodies to black currant reversion associated virus from a synthetic phage display library.

ABSTRACT Single-chain variable fragment (scFv) antibodies that bind to black currant reversion associated virus (BRAV) were obtained from a synthetic phage display antibody gene library without recourse to animal immunizations. Several different BRAV-specific phage scFv were obtained quickly, after only three rounds of selection against immobilized virus antigen. The phage scFv gave enzyme-linked immunosorbent assay (ELISA) absorbance values that were greater than seven times the control healthy plant extracts. In contrast, comparative tests using a rabbit antiserum failed, because unacceptably high background values were obtained with healthy plant extracts. Two of the scFv were subcloned into the pDAP2 vector for the rapid and efficient production of scFv-alkaline phosphatase fusion proteins. Functional fusion proteins were obtained after expression in Escherichia coli, and preparations from periplasmic extracts detected BRAV in ELISA. The results demonstrate that antibody fragments obtained from a synthetic phage display library are useful research tools, and they proved to be a viable practical alternative when traditional antisera failed to detect BRAV, a weak immunogen. Furthermore, the genetic fusion of antibody fragments to alkaline phosphatase obviates the need for further chemical coupling procedures, and the fusion proteins can be obtained cheaply.

Recombinant antibody fragments that detect enoyl acyl carrier protein reductase in Brassica napus.

Purified Brassica napus enoyl acyl carrier protein reductase (ENR) was used to select specific antibodies from a library of antibody fragments, single-chain Fv (scFv), displayed on filamentous phage. Analysis of the selected clones by BstNI fingerprinting and nucleotide sequencing showed that the scFv were derived from three different human VH germline genes. The binding specificities were confirmed by Western blots and ELISA. The scFv preparations reacted with B. napus ENR, but not with beta-keto reductase, nor enoyl reductase from Escherichia coli. Analysis of fragments generated by CNBr treatment indicates that the scFv 3.13 recognized an epitope located within the N-terminal 80 amino acids of the enzyme molecule. The scFv were used to detect ENR directly in extracts of B. napus seeds.