Increased expression and phosphorylation of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase isoforms in urinary exosomes in pre-eclampsia.

BACKGROUND: Glycolysis is altered in various kidney diseases, but little is known about glycolysis in pre-eclampsia, a multi-system disorder with major pathological effects on the kidney. Urinary exosomes provide a non-invasive alternative for studying changes in kidney metabolism. This study aims to characterise the expression and phosphorylation of isozymes of the key glycolytic regulatory protein, 6-phosphofructokinase-2-kinase/fructose-2,6-bisphosphatase (PFK-2/FBPase-2), in urinary exosomes of subjects with pre-eclampsia (PE), compared to normotensive non-pregnant (NC) and normotensive pregnant (NP) controls. METHODS: A cross-sectional study of NC (n = 19), NP (n = 23) and PE (n = 29) subjects was performed. Exosomes were isolated from urine samples by differential ultracentrifugation, and then analyzed by Western blot and densitometry for expression of PFK-2/FBPase-2 isozymes (PFKFB2, PFKFB3 and PFKFB4) and phosphorylation of PFKFB2 at residues Ser483 and Ser466 and PFKFB3 at Ser461. RESULTS: PFKFB2 expression was increased 4.7-fold in PE compared to NP (p < 0.001). PFKFB2 phosphorylation at Ser483 was increased 2.6-fold in PE compared to NP (p = 0.002). Expression of phosphorylated PFKFB2/PFKFB3 at Ser466/Ser461 was increased in PE, being present in 77.4% (95% CI 59.9-88.9%) of PE and 8.3% (95% CI 1.2-27.0%) of NP samples (p < 0.001). PFKFB3 was more commonly expressed in PE, detected in 90.3% (95% CI 74.3-97.4%) of PE and 8.3% (95% CI 1.2-27.0%) of NP samples (p < 0.001). PFKFB4 had a 7.2-fold increase in expression in PE compared to NP (p < 0.001). No significant differences between NP and NC groups were observed. CONCLUSION: Regulatory proteins that increase glycolysis are increased in the urinary exosomes of subjects with pre-eclampsia, suggesting that renal glycolysis may be increased in this condition.

Profiling undergraduates' generic learning skills on entry to medical school; an international study.

BACKGROUND: Medical education faces challenges posed by widening access to training, a demand for globally competent healthcare workers and progress towards harmonisation of standards. AIM: To explore potential challenges arising from variation in diversity and educational background of medical school entrants. METHOD: This study investigated the reported experience and confidence, in a range of 31 generic skills underpinning learning, of 2606 medical undergraduates entering 14 medical schools in England and South Africa, using a validated questionnaire. RESULTS: Responses suggest that there is considerable similarity in prior educational experience and confidence skills profiles on entry to South African and English medical schools. South African entrants reported significantly more experience in 'Technical skills', 'Managing their own Learning', and 'Presentation', while English students reported increased experience in 'IT' skills. South African undergraduates reported more confidence in 'Information Handling', while English students were more confident in 'IT' skills. The most noticeable difference, in 'IT' skills, is probably due to documented differences in access to computer facilities at high school level. Differences between individual schools within each country are noticeable. CONCLUSIONS: Educators need to acquire a good understanding of their incoming cohorts, and ensure necessary tailored support for skills development.

Is short-term remediation after OSCE failure sustained? A retrospective analysis of the longitudinal attainment of underperforming students in OSCE assessments.

BACKGROUND: Significant improvements in the delivery of criterion-based assessment techniques have improved confidence in standard setting and assessment quality. However, for underperforming students, a lack of evidence about longitudinal performance of this group poses dilemmas to educators when making decisions about the timing and nature of remediation. AIM: To investigate the longitudinal performance of the UK undergraduate medical degree students, with a particular focus on comparing the poorly performing students (i.e. those with borderline or failing grades) with the main cohort of students. METHOD: Over a 5-year period, 3200-student objective structured clinical examination (OSCE) assessments from a single medical school were investigated. A poorly performing subgroup of 125 students was identified and their longitudinal performance in the final 3 years of the undergraduate medical degree analysed. RESULT: The relative performance of this student group declines across serial OSCEs, despite current methods of 'remediation and retest'. CONCLUSIONS: This analysis demonstrates that typically students in the poorly performing subgroup achieve only short-term success with traditional remediation and retest models, and critically show an absence of longitudinal improvement. There is a clear need for institutions to develop profiling models that can help identify this student group and develop effective, research led models of remediation.

Changes in brain morphology in patients with obstructive sleep apnoea.

BACKGROUND: Obstructive sleep apnoea (OSA) is a common disease that leads to daytime sleepiness and cognitive impairment. Attempts to investigate changes in brain morphology that may underlie these impairments have led to conflicting conclusions. This study was undertaken to aim to resolve this confusion, and determine whether OSA is associated with changes in brain morphology in a large group of patients with OSA, using improved voxel-based morphometry analysis, an automated unbiased method of detecting local changes in brain structure. METHODS: 60 patients with OSA (mean apnoea hypopnoea index 55 (95% CI 48 to 62) events/h, 3 women) and 60 non-apnoeic controls (mean apnoea hypopnoea index 4 (95% CI 3 to 5) events/h, 5 women) were studied. Subjects were imaged using T1-weighted 3-D structural MRI (69 subjects at 1.5 T, 51 subjects at 3 T). Differences in grey matter were investigated in the two groups, controlling for age, sex, site and intracranial volume. Dedicated cerebellar analysis was performed on a subset of 108 scans using a spatially unbiased infratentorial template. RESULTS: Patients with OSA had a reduction in grey matter volume in the right middle temporal gyrus compared with non-apnoeic controls (p<0.05, corrected for topological false discovery rate across the entire brain). A reduction in grey matter was also seen within the cerebellum, maximal in the left lobe VIIIb close to XI, extending across the midline into the right lobe. CONCLUSION: These data show that OSA is associated with focal loss of grey matter that could contribute to cognitive decline. Specifically, lesions in the cerebellum may result in both motor dysfunction and working memory deficits, with downstream negative consequences on tasks such as driving.

A technique for the measurement of renal ATP in a large animal model of septic shock.

BACKGROUND: The mechanisms responsible for acute renal failure in sepsis are not understood. Measurement of tissue ATP might help to understand this process but, in the large animal, it is hampered by major technical difficulties. OBJECTIVE: To develop a technique to monitor ATP in the kidney of a large mammal during the induction of septic shock and then circulatory arrest. METHODS: Implantation of a custom-made phosphorus coil around the left kidney. Induction of septic shock by intravenous E. coli administration. Acquisition of 31 P magnetic resonance (MR) spectroscopic data at 3-tesla before and during septic shock over several hours. Induction of euthanasia and measurement of the same 31 P signal immediately and thirty minutes after circulatory arrest. RESULTS: Clear reproducible 31 P MR spectra were obtained before and after the induction of septic shock and euthanasia. They indicated limited changes in ATP during septic shock. An expected rapid and dramatic decrease in ATP occurred with euthanasia. CONCLUSIONS: It is possible to sequentially monitor renal bioenergetics in a large mammal during septic shock using an implanted custom-made phosphorus coil and 3-tesla MR technology. This technique offers a novel approach to the investigation of septic renal failure.

Structural abnormalities remote from the seizure focus: a study using T2 relaxometry at 3 T.

OBJECTIVE: To determine the extent and severity of mesial temporal and subcortical signal abnormalities in patients with partial epilepsy. METHODS: T2 relaxation time maps were acquired in 50 consecutive patients and 55 control subjects on a 3 T MRI scanner. Twenty-two patients had hippocampal sclerosis (HS), 16 had malformations of cortical development (MCD), and 12 had no obvious MR abnormalities (normal MR). The following eight regions were measured bilaterally: hippocampus, anterior temporal lobe (ATL) white matter, amygdala, frontal lobe white matter, caudate, putamen, pallidum, and thalamus. RESULTS: In patients with HS, increased T2 relaxation times were found in the ipsilateral hippocampus and ATL but not in subcortical nuclei. In patients with MCD, increased T2 relaxation times were found in the temporal lobe (hippocampus, ATL) and in subcortical areas (caudate, putamen, and pallidum); in patients with normal MR, increased T2 relaxation times were found in the hippocampus and putamen. The degree of abnormality did not correlate with the duration of epilepsy or the estimated seizure load. CONCLUSIONS: Mesial temporal structures show increased T2 relaxation times not only in patients with hippocampal sclerosis but also in patients with a seizure focus remote from the hippocampus. Patients with normal MR and focal malformations of cortical development have increased T2 relaxation times in subcortical structures. Therefore, abnormalities in T2 relaxation time can be found remote from the seizure focus. They cannot be simply attributed to secondary seizure effects.

Pseudomonas cellulosa expresses a single membrane-bound glycoside hydrolase family 51 arabinofuranosidase.

In the accompanying paper [Beylot, McKie, Voragen, Doeswijk-Voragen and Gilbert (2001) Biochem. J. 358, 607-614] the chromosome of Pseudomonas cellulosa was shown to contain two genes, abf51A and abf62A, that encode arabinofuranosidases belonging to glycoside hydrolase families 51 and 62, respectively. In this report we show that expression of Abf51A is induced by arabinose and arabinose-containing polysaccharides. Northern-blot analysis showed that abf51A was efficiently transcribed, whereas no transcript derived from abf62A was detected in the presence of arabinose-containing polysaccharides. Zymogram and Western-blot analyses revealed that Abf51A was located on the outer membrane of P. cellulosa. To investigate the importance of Abf51A in the release of arabinose from poly- and oligosaccharides, transposon mutagenesis was used to construct an abf51A-inactive mutant of P. cellulosa (Deltaabf51A). The mutant did not grow on linear arabinan or sugar beet arabinan, and utilized arabinoxylan much more slowly than the wild-type bacterium. Arabinofuranosidase activity in Deltaabf51A against aryl-alpha-arabinofuranosides, arabinan and alpha1,5-linked arabino-oligosaccharides was approx. 1% of the wild-type bacterium. The mutant bacterium did not exhibit arabinofuranosidase activity against arabinoxylan, supporting the view that abf62A is not expressed in P. cellulosa. These data indicate that P. cellulosa expresses a membrane-bound glycoside hydrolase family 51 arabinofuranosidase that plays a pivotal role in releasing arabinose from polysaccharides and arabino-oligosaccharides.

Secondary bone grafting of alveolar clefts: a densitometric comparison of iliac crest and tibial bone grafts.

OBJECTIVE: To evaluate changes in the optical density of secondary alveolar cleft bone grafts obtained from two different donor sites over time and to determine whether one donor site gives a higher recipient bone density than the other. METHODS: A prospective study was performed evaluating 40 healthy patients with congenital cleft lip and palate undergoing secondary alveolar bone grafting, 20 (14 boys and 6 girls) having iliac crest and 20 (12 boys and 8 girls) receiving tibial bone grafts. Bone harvest and grafting was carried out by one operator (G.P.). Optical density of iliac and tibial grafts measured using a computerized densitometer, was compared at 6 days, 6 weeks, and 3 months. Due to interference from orthodontic appliances, optical density measurements for 16 subjects were not possible, and these patients were excluded from the study. The length of hospital stay postoperatively for both grafting procedures were recorded. RESULTS: A significant decrease in relative bone density was demonstrated during the 3-month postoperative period in both iliac and tibial bone graft groups (p < .05). The difference in densities between iliac crest and tibial groups were not significantly different at any of the time points (paired t test, p > .05). Subjects undergoing iliac crest grafts stayed an average of 5 days in the hospital postoperatively, compared with subjects with tibial grafts who stayed an average of 3 days postoperatively. CONCLUSION: Optical density measurements of bone grafted into alveolar clefts, reported here for the first time, provide a valuable objective assessment of graft progress. Tibial and iliac crest grafts gave similar optical densities at recipient sites over the first 3 months. Iliac crest grafts required significantly longer postoperative stay; an important consideration in selecting donor sites for secondary bone grafting.

Acute changes in MRI diffusion, perfusion, T(1), and T(2) in a rat model of oligemia produced by partial occlusion of the middle cerebral artery.

Oligemic regions, in which the cerebral blood flow is reduced without impaired energy metabolism, have the potential to evolve toward infarction and remain a target for therapy. The aim of this study was to investigate this oligemic region using various MRI parameters in a rat model of focal oligemia. This model has been designed specifically for remote-controlled occlusion from outside an MRI scanner. Wistar rats underwent remote partial MCAO using an undersize 0.2 mm nylon monofilament with a bullet-shaped tip. Cerebral blood flow (CBF(ASL)), using an arterial spin labeling technique, the apparent diffusion coefficient of water (ADC), and the relaxation times T(1) and T(2) were acquired using an 8.5 T vertical magnet. Following occlusion there was a decrease in CBF(ASL) to 35 +/- 5% of baseline throughout the middle cerebral artery territory. During the entire period of the study there were no observed changes in the ADC. On occlusion, T(2) rapidly decreased in both cortex and basal ganglia and then normalized to the preocclusion values. T(1) values rapidly increased (within approximately 7 min) on occlusion. In conclusion, this study demonstrates the feasibility of partially occluding the middle cerebral artery to produce a large area of oligemia within the MRI scanner. In this region of oligemic flow we detect a rapid increase in T(1) and decrease in T(2). These changes occur before the onset of vasogenic edema. We attribute the acute change in T(2) to increased amounts of deoxyhemoglobin; the mechanisms underlying the change in T(1) require further investigation.