Viva la VOSCE?

BACKGROUND: The COVID-19 pandemic lockdown precluded face-to-face final Objective Structured Clinical Examinations (OSCE) in the UK. RESULTS: In response, we rapidly developed and then successfully implemented a novel Virtual Objective Structured Clinical Examination (VOSCE). CONCLUSIONS: In this article we both describe and reflect on our experience as well as discuss the implications for future undergraduate assessment as the situation evolves.

Authentication of beef versus horse meat using 60 MHz 1H NMR spectroscopy.

This work reports a candidate screening protocol to distinguish beef from horse meat based upon comparison of triglyceride signatures obtained by 60 MHz (1)H NMR spectroscopy. Using a simple chloroform-based extraction, we obtained classic low-field triglyceride spectra from typically a 10 min acquisition time. Peak integration was sufficient to differentiate samples of fresh beef (76 extractions) and horse (62 extractions) using Naïve Bayes classification. Principal component analysis gave a two-dimensional "authentic" beef region (p=0.001) against which further spectra could be compared. This model was challenged using a subset of 23 freeze-thawed training samples. The outcomes indicated that storing samples by freezing does not adversely affect the analysis. Of a further collection of extractions from previously unseen samples, 90/91 beef spectra were classified as authentic, and 16/16 horse spectra as non-authentic. We conclude that 60 MHz (1)H NMR represents a feasible high-throughput approach for screening raw meat.

Temporal and spatial changes in cell wall composition in developing grains of wheat cv. Hereward.

A combination of enzyme mapping, FT-IR microscopy and NMR spectroscopy was used to study temporal and spatial aspects of endosperm cell wall synthesis and deposition in developing grain of bread wheat cv. Hereward. This confirmed previous reports that changes in the proportions of the two major groups of cell wall polysaccharides occur, with beta-glucan accumulating earlier in development than arabinoxylan. Changes in the structure of the arabinoxylan occurred, with decreased proportions of disubstituted xylose residues and increased proportions of monosubstituted xylose residues. These are likely to result, at least in part, from arabinoxylan restructuring catalysed by enzymes such as arabinoxylan arabinofurano hydrolase and lead to changes in cell wall mechanical properties which may be required to withstand stresses during grain maturation and desiccation.

Remodelling of arabinoxylan in wheat (Triticum aestivum) endosperm cell walls during grain filling.

Previous studies using spectroscopic imaging have allowed the spatial distribution of structural components in wheat endosperm cell walls to be determined. FT-IR microspectroscopy showed differing changes in arabinoxylan (AX) structure, during grain development under cool/wet and hot/dry growing conditions, for differing cultivars (Toole et al. in Planta 225:1393-1403, 2007). These studies have been extended using Raman microspectroscopy, providing more details of the impact of environment on the polysaccharide and phenolic components of the cell walls. NMR studies provide complementary information on the types and levels of AX branching both early in development and at maturity. Raman microspectroscopy has allowed the arabinose:xylose (A/X) ratio in the cell wall AX to be determined, and the addition of ferulic acid and related phenolic acids to be followed. The changes in the A/X ratio during grain development were affected by the environmental conditions, with the A/X ratio generally being slightly lower for samples grown under cool/wet conditions than for those from hot/dry conditions. The degree of esterification of the endosperm cell walls with ferulic acid was also affected by the environment, being lower under hot/dry conditions. The results support earlier suggestions that AX is either delivered to the cell wall in a highly substituted form and is remodelled through the action of arabinoxylan arabinofuranohydrolases or arabinofuranosidases, or that low level substituted AX are incorporated into the wall late in cell wall development, reducing the average degree of substitution, and that the rate of this remodelling is influenced by the environment. (1)H NMR provided a unique insight into the chemical structure of intact wheat endosperm cell walls, providing qualitative information on the proportions of mono- and disubstituted AX and the levels of branching of adjacent units. The A/X ratio did not change greatly with either the development stage or the growth conditions, but the ratio of mono- to disubstituted Xylp residues increased markedly (by about fourfold) in the more mature samples, confirming the changes in branching levels determined using FT-IR. To the best of our knowledge, this is the first time that intact endosperm cell walls have been studied by (1)H NMR.

Fruit juice authentication by 1H NMR spectroscopy in combination with different chemometrics tools.

To discriminate orange juice from grapefruit juice in a context of fraud prevention, (1)H NMR data were submitted to different treatments to extract informative variables which were then analysed using multivariate techniques. Averaging contiguous data points of the spectrum followed by logarithmic transformation improved the results of the data analysis. Moreover, supervised variable selection methods gave better rates of classification of the juices into the correct groups. Last, independent-component analysis gave better classification results than principal-component analysis. Hence, ICA may be an efficient chemometric tool to detect differences in the (1)H NMR spectra of similar samples, and so may be useful for authentication of foods.

Evolving window zone selection method followed by independent component analysis as useful chemometric tools to discriminate between grapefruit juice, orange juice and blends.

This study investigates the use of high resolution 1H NMR as a suitable alternative to the standard chromatographic method for the determination of adulteration of orange juice (Citrus sinensis) with grapefruit juice (Citrus paradisi) based on flavonoid glycoside content. Fifty-nine orange juices (OJ), 23 grapefruit juices (GJ) and 10 blends (OG), obtained from local retail outlets were used to assess the performance of the 1H NMR method. The work presented here introduces the Evolving Window Zone Selection (EWZS) function that holds promise for the automatic detection of spectral regions tailored to discriminate predefined groups. This technique was applied on the pre-processed 1H NMR spectra of the 92 juices. Independent Component Analysis (ICA) is a good alternative to Principal Component Analysis (PCA) for recovering linearly-mixed unobserved multidimensional independent signals and has been used in this study to build supervised models that classify the samples into three categories, OJ, GJ, OG. The regions containing the known flavonoid glycoside markers were selected as well as another zone containing the signals of sucrose, alpha-glucose and other components that were tentatively attributed. ICA was applied on three different groups of selected variables and showed good results for both discrimination and interpretation of the signals. Up to 97.8% of the juices were correctly attributed. This method gave better results than the commonly used PCA method. In addition, the time required to carry out the 1H NMR analysis was less than half the time of the standard chromatographic method.

Comparison of survival following coronary artery bypass grafting vs. percutaneous coronary intervention in diabetic and non-diabetic patients: retrospective cohort study of 6320 procedures.

AIM: To determine whether mortality following percutaneous coronary intervention vs. coronary bypass grafting varies according to whether or not patients have diabetes. METHODS: We used the Scottish Coronary Revascularization Register to identify all patients undergoing revascularization in Scottish NHS hospitals since 1997. We excluded single-vessel disease, left main stem stenosis, and bypass grafting performed at the same time as other operations. We used death certificate data from the Registrar General to identify all subsequent deaths. RESULTS: Of the 6320 eligible procedures, 5042 (80%) were bypass grafts and 1278 (20%) angioplasties. Overall 831 (13%) patients had diabetes with no significant difference by procedure (13% vs. 12%). A total of 382 deaths occurred over a mean follow-up of 2.3 years. Diabetic patients had a poorer prognosis following both surgery (adjusted hazards ratio (HR) 1.43, 95% confidence interval (CI) 1.08, 1.89) and percutaneous intervention (adjusted HR 2.58, 95% CI 1.43, 4.63). Among non-diabetic patients, no significant differences in mortality were detected between the two procedures. Among diabetic patients, no significant difference was detected in those with two-vessel disease. In those with impaired left ventricular function and triple-vessel disease, angioplasty was associated with a significantly higher risk of death (adjusted HR 3.58, 95% CI 1.40, 9.19). CONCLUSIONS: This is the first study to demonstrate statistically significant results that support the BARI trial findings. Our study demonstrated a significant difference for triple-vessel disease but not two-vessel disease. The former may be due to incomplete revascularization using percutaneous intervention. Our results require corroboration from randomized trials.

Unintended effects and their detection in genetically modified crops.

The commercialisation of GM crops in Europe is practically non-existent at the present time. The European Commission has instigated changes to the regulatory process to address the concerns of consumers and member states and to pave the way for removing the current moratorium. With regard to the safety of GM crops and products, the current risk assessment process pays particular attention to potential adverse effects on human and animal health and the environment. This document deals with the concept of unintended effects in GM crops and products, i.e. effects that go beyond that of the original modification and that might impact primarily on health. The document first deals with the potential for unintended effects caused by the processes of transgene insertion (DNA rearrangements) and makes comparisons with genetic recombination events and DNA rearrangements in traditional breeding. The document then focuses on the potential value of evolving "profiling" or "omics" technologies as non-targeted, unbiased approaches, to detect unintended effects. These technologies include metabolomics (parallel analysis of a range of primary and secondary metabolites), proteomics (analysis of polypeptide complement) and transcriptomics (parallel analysis of gene expression). The technologies are described, together with their current limitations. Importantly, the significance of unintended effects on consumer health are discussed and conclusions and recommendations presented on the various approaches outlined.

Discrimination between orange juice and pulp wash by (1)H Nuclear Magnetic Resonance spectroscopy: identification of marker compounds.

The potential of NMR spectroscopy and multivariate analysis methods to detect the adulteration of orange juice with pulp wash is demonstrated. Principal component analysis has been applied to (1)H NMR spectra of >300 orange and pulp wash juices, and stepwise linear discriminant analysis was used to classify the samples. A model with six principal components gave a high success rate of classification (94%) for both training and validation sets. An important principal component loading showed that dimethylproline played a key role in the discrimination between the two types of juice, with higher levels in pulp wash. Dimethylproline was not previously known as a marker compound for orange juice adulteration. An ANOVA test revealed at least 21 other NMR signals that differed significantly between the authentic and pulp wash groups. The compounds they represent could be seen as potential marker compounds in addition to dimethylproline. This makes NMR with chemometrics an attractive screening tool with advantages in terms of rapidity, simplicity, and diversity of information provided.

Structure and conformation of a novel genetically engineered polysaccharide P2.

A new exocellular polysaccharide (P2) has been produced by the manipulation of a glycosyl transferase gene (aceP) involved in the biosynthesis of the polysaccharide acetan by the bacterium Acetobacter xylinum strain CKE5. The P2 polysaccharide has been studied by methylation analysis, reductive cleavage, and 1H and 13C NMR spectroscopy. The data are consistent with the structure predicted when the aceP gene is deactivated: [Molecular structure: see text]. The effect of cooling on proton NMR line width indicates a coil-helix transition in P2 at about 70 degrees C.

Apparent paradoxical vault changes with middle cranial fossa arachnoid cysts--implication for aetiology.

Three cases of middle cranial fossa arachnoid cyst with paradoxical bone changes in the adjacent vault are described, namely, a small middle cranial fossa and pneumosinus dilatans. This association is unusual and unique. The existing literature is reviewed and the probable aetiological factors discussed.

Direct cervical vertebro-venous fistula with radiculopathy and MRI changes resolving after successful endovascular embolisation: a report of two cases.

We report two cases of rare direct cervical vertebro-venous fistula (VVF) presenting with chronic radiculopathy as part of the symptom complex. We describe the MRI and intra-arterial angiography findings before and after successful embolisation. These demonstrate occlusion of the fistula with collapse and thrombosis of the draining extradural venous plexus and, in one case, resolution of MRI signal abnormality in the cervical spinal cord.

Study of the compositional changes of mango during ripening by use of nuclear magnetic resonance spectroscopy.

Liquid-state NMR spectroscopy was used to follow the compositional changes in mango juice during ripening, whereas MAS and HR-MAS techniques enabled resolved (13)C and (1)H NMR spectra of mango pulps to be recorded. Spectral assignment enabled the identification of several organic acids, amino acids, and other minor components, and the compositional changes upon ripening were followed through the changes in the spectra. In pulps, sucrose was found to predominate over fructose and glucose at most ripening stages, and citric acid content decreased markedly after the initial ripening stages while alanine increased significantly. Other spectral changes reflect the complex biochemistry of mango ripening and enabled the role played by some compounds to be discussed. Some differences observed between the composition of juices and pulps are discussed. This work shows that NMR spectroscopy enables the direct characterization of intact mango pulps, thus allowing the noninvasive study of the overall biochemistry in the whole fruit.

A cinnamoyl esterase from Aspergillus niger can break plant cell wall cross-links without release of free diferulic acids.

A cinnamoyl esterase, ferulic acid esterase A, from Aspergillus niger releases ferulic acid and 5-5- and 8-O-4-dehydrodiferulic acids from plant cell walls. The breakage of one or both ester bonds from dehydrodimer cross-links between plant cell wall polymers is essential for optimal action of carbohydrases on these substrates, but it is not known if cinnamoyl esterases can break these cross-links by cleaving one of the ester linkages which would not release the free dimer. It is difficult to determine the mechanism of the reaction on complex substrates, and so we have examined the catalytic properties of ferulic acid esterase A from Aspergillus niger using a range of synthetic ethyl esterified dehydrodimers (5-5-, 8-5-benzofuran and 8-O-4-) and two 5-5-diferulate oligosaccharides. Our results show that the esterase is able to cleave the three major dehydrodiferulate cross-links present in plant cell walls. The enzyme is highly specific at hydrolysing the 5-5- and the 8-5-benzofuran diferulates but the 8-O-4-is a poorer substrate. The hydrolysis of dehydrodiferulates to free acids occurs in two discrete steps, one involving dissociation of a monoesterified intermediate which is negatively charged at the pH of the reaction. Although ferulic acid esterase A was able to release monoesters as products of reactions with all three forms of diesters, only the 5-5- and the 8-O-4-monoesters were substrates for the enzyme, forming the corresponding free diferulic acids. The esterase cannot hydrolyse the second ester bond from the 8-5-benzofuran monoester and therefore, ferulic acid esterase A does not form 8-5-benzofuran diferulic acid. Therefore, ferulic acid esterase A from Aspergillus niger contributes to total plant cell wall degradation by cleaving at least one ester bond from the diferulate cross-links that exist between wall polymers but does not always release the free acid product.

Generation of a novel polysaccharide by inactivation of the aceP gene from the acetan biosynthetic pathway in Acetobacter xylinum.

The acetan biosynthetic pathway in Acetobacter xylinum is an ideal model system for engineering novel bacterial polysaccharides. To genetically manipulate this pathway, an Acetobacter strain (CKE5), more susceptible to gene-transfer methodologies, was developed. A new gene, aceP, involved in acetan biosynthesis was identified, sequenced and shown to have homology at the amino acid level with beta-D-glucosyl transferases from a number of different organisms. Disruption of aceP in strain CKE5 confirmed the function assigned above and was used to engineer a novel polysaccharide with a pentasaccharide repeat unit.

Post-translational modification of nisin. The involvement of NisB in the dehydration process.

The lantibiotic nisin is an antimicrobial peptide produced by Lactococcus lactis. As with all lantibiotics, nisin contains a number of dehydro-residues and thioether amino acids that introduce five lanthionine rings into the target peptide. These atypical amino acids are introduced by post-translational modification of a ribosomally synthesized precursor peptide. In certain cases, the serine residue, at position 33 of nisin, does not undergo dehydration to Dha33. With native nisin this partially processed form represents about 10% of the total peptide, whereas with the engineered variants, [Trp30]nisin A and [Lys27,Lys31]nisin A, the proportion of peptide that escapes full processing was found to be to approximately 50%. This feature of nisin biosynthesis was exploited in an investigation of the role of the NisB protein in pre-nisin maturation. Manipulation of the level of NisB was achieved by cloning and overexpressing the plasmid-encoded nisB gene in a range of different nisin-producing strains. The resulting fourfold increase in the level of NisB significantly increased the efficiency of the dehydration reaction at Ser33. The final secreted product of biosynthesis by these strains was the homogenous form of the fully processed nisin (or nisin variant) molecule. The results presented represent the first experimental evidence for the direct involvement of the NisB protein in the maturation process of nisin.