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Cancer immunotherapy holds great promise and has shown durable responses in many patients; however, these responses are not uniform in all patients or all tumour streams. There is an ongoing clinical need for objective diagnostic biomarkers to identify patients that will respond to immunotherapies. Tumour mutational burden (TMB) is a diagnostic biomarker that can stratify cancer patients for response to immune checkpoint inhibitor therapies. It is commonly defined as the average number of somatic mutations per megabase in a tumour exome. Here we describe the TMB biomarker, how it is determined, its underlying molecular basis, the relationship to neoantigens and the issues around its clinical use. This overview is directed toward practising pathologists wishing to be informed of this predictive biomarker.

Anti-myelin oligodendrocyte glycoprotein antibodies (anti-MOG), directed against a component of the myelin sheath, are sometimes detected in the blood or cerebrospinal fluid (CSF) of patients with acute demyelinating conditions. Cortical encephalitic presentations in anti-MOG-antibody-positive patients are recognized but rare, and few pediatric cases have been described. We describe clinical, biochemical, and MRI findings in two children presenting with generalized seizures due to cortical encephalitis, and review potential underlying immunological processes. In both patients, anti-MOG antibodies were detected. Both underwent MRI scans which demonstrated bilateral cortical swelling and T2/fluid-attenuated inversion recovery (FLAIR) hyperintensity with corresponding regions of reduced diffusion. Early detection of anti-MOG antibodies in patients with a similar presentation and imaging features would enable rapid institution of appropriate treatment, and potentially reduce the need for invasive diagnostic procedures such as brain biopsy.

Sharing genomic variant interpretations across laboratories promotes consistency in variant assertions. A landscape analysis of Australian clinical genetic-testing laboratories in 2017 identified that, despite the national-accreditation-body recommendations encouraging laboratories to submit genotypic data to clinical databases, fewer than 300 variants had been shared to the ClinVar public database. Consultations with Australian laboratories identified resource constraints limiting routine application of manual processes, consent issues, and differences in interpretation systems as barriers to sharing. This information was used to define key needs and solutions required to enable national sharing of variant interpretations. The Shariant platform, using both the GRCh37 and GRCh38 genome builds, was developed to enable ongoing sharing of variant interpretations and associated evidence between Australian clinical genetic-testing laboratories. Where possible, two-way automated sharing was implemented so that disruption to laboratory workflows would be minimized. Terms of use were developed through consultation and currently restrict access to Australian clinical genetic-testing laboratories. Shariant was designed to store and compare structured evidence, to promote and record resolution of inter-laboratory classification discrepancies, and to streamline the submission of variant assertions to ClinVar. As of December 2021, more than 14,000 largely prospectively curated variant records from 11 participating laboratories have been shared. Discrepant classifications have been identified for 11% (28/260) of variants submitted by more than one laboratory. We have demonstrated that co-design with clinical laboratories is vital to developing and implementing a national variant-interpretation sharing effort. This approach has improved inter-laboratory concordance and enabled opportunities to standardize interpretation practices.

Sickle cell disease is the most common hereditary hemoglobinopathy, which results in abnormally shaped and rigid red blood cells. These sickle-shaped red blood cells cause vaso-occlusion and ischemic phenomena that can affect any organ in the body. As a common cause of disability, the neurological manifestations of sickle cell disease are particularly important. Neuroimaging has a crucial role in the diagnosis, management, and prevention of the complications of sickle cell disease. These complications can affect the brain parenchyma, vasculature, and skull and can be ascribed directly or indirectly to a vasculopathy of small and large vessels. Vaso-occlusion can cause ischemic stroke. Ischemic damage in the absence of an acute neurological deficit, and therefore only apparent on neuroimaging, is termed silent cerebral ischemia. Weakening of the arterial walls can cause aneurysms. In its most severe form, a vasculopathy of the terminal internal carotid arteries can progress to moyamoya syndrome, characterized by steno-occlusive disease and the formation of friable collateral arteries. Rupture of aneurysms or friable collateral arteries is a potential cause of intracranial hemorrhage. The skull and vertebrae may be affected by extra-medullary hematopoiesis, due to severe anemia, or iron deposition, due to chronic red blood cell transfusion. Impaired blood supply to bone is associated with osteomyelitis and osteonecrosis. Fat embolization syndrome is a rare complication of osteonecrosis, which may cause devastating neurological impairment. Awareness and early recognition of the diverse manifestations of sickle cell disease on neuroimaging is crucial to ensure optimal treatment in a complex patient cohort.

AIM To identify imaging predictors of molecular subtype and tumour grade in patients with isocitrate dehydrogenase (IDH) gene mutant (IDHmut) World Health Organization (WHO) grade 2 or 3 gliomas. MATERIALS AND METHODS Patients with histologically confirmed WHO grade 2 or 3 IDHmut gliomas between 2016 and 2019 were included in the study. Magnetic resonance imaging (MRI) images were evaluated for the presence or absence of potential imaging predictors of tumour subtype, such as T2/fluid attenuated inversion recovery (FLAIR) signal match, and these factors were examined using regression analysis. On perfusion imaging, the maximum relative cerebral blood volume (rCBVmax) was evaluated as a potential predictor of tumour grade. The performance of two experienced neuroradiologists in correctly predicting tumour type on MRI was evaluated. RESULTS Eighty-five patients were included in the study. The presence of T2/FLAIR signal match >50% of tumour volume (p CONCLUSION The presence of matched T2/FLAIR signal could be used to identify tumour subtype when biopsy is inconclusive or genetic analysis is unavailable. rCBVmax predicted astrocytoma grade. Experienced neuroradiologists predict tumour subtype with good sensitivity and specificity.

Background and Purpose— Complex vascular anatomy might increase the risk of procedural stroke during carotid artery stenting (CAS). Randomized controlled trial evidence that vascular anatomy should inform the choice between CAS and carotid endarterectomy (CEA) has been lacking. Methods— One-hundred eighty-four patients with symptomatic internal carotid artery stenosis who were randomly assigned to CAS or CEA in the ICSS (International Carotid Stenting Study) underwent magnetic resonance (n=126) or computed tomographic angiography (n=58) at baseline and brain magnetic resonance imaging before and after treatment. We investigated the association between aortic arch configuration, angles of supra-aortic arteries, degree, length of stenosis, and plaque ulceration with the presence of ≥1 new ischemic brain lesion on diffusion-weighted magnetic resonance imaging (DWI+) after treatment. Results— Forty-nine of 97 patients in the CAS group (51%) and 14 of 87 in the CEA group (16%) were DWI+ (odds ratio [OR], 6.0; 95% confidence interval [CI], 2.9–12.4; P P =0.027) and the degree of the largest internal carotid artery angle (≥60° versus P =0.002) were both associated with DWI+, also after correction for age. No predictors for DWI+ were identified in the CEA group. The DWI+ risk in CAS increased further over CEA if the largest internal carotid artery angle was ≥60° (OR, 11.8; 95% CI, 4.1–34.1) than if it was P =0.035). Conclusions— Complex configuration of the aortic arch and internal carotid artery tortuosity increase the risk of cerebral ischemia during CAS, but not during CEA. Vascular anatomy should be taken into account when selecting patients for stenting. Clinical Trial Registration— URL: http://www.isrctn.com/ISRCTN25337470 . Unique identifier: ISRCTN25337470.

Objectives: Carotid endarterectomy (CEA) is standard treatment for symptomatic carotid artery stenosis but carries a risk of stroke, myocardial infarction (MI), or death. This study investigated risk factors for these procedural complications occurring within 30 days of endarterectomy in the International Carotid Stenting Study (ICSS). Methods: Patients with recently symptomatic carotid stenosis >50% were randomly allocated to endarterectomy or stenting. Analysis is reported of patients in ICSS assigned to endarterectomy and limited to those in whom CEA was initiated. The occurrence of stroke, MI, or death within 30 days of the procedure was reported by investigators and adjudicated. Demographic and technical risk factors for these complications were analysed sequentially in a binomial regression analysis and subsequently in a multivariable model. Results: Eight-hundred and twenty-one patients were included in the analysis. The risk of stroke, MI, or death within 30 days of CEA was 4.0%. The risk was higher in female patients (risk ratio [RR] 1.98, 95% CI 1.02-3.87, p = .05) and with increasing baseline diastolic blood pressure (dBP) (RR 1.30 per +10 mmHg, 95% Cl 1.02-1.66, p = .04). Mean baseline dBP, obtained at the time of randomization in the trial, was 78 mmHg (SD 13 mmHg). In a multivariable model, only dBP remained a significant predictor. The risk was not related to the type of surgical reconstruction, anaesthetic technique, or perioperative medication regimen. Patients undergoing CEA stayed a median of 4 days before discharge, and 21.2% of events occurred on or after the day of discharge. Conclusions: Increasing diastolic blood pressure was the only independent risk factor for stroke, MI, or death following CEA. Cautious attention to blood pressure control following symptoms attributable to carotid stenosis could reduce the risks associated with subsequent CEA. (C) 2015 The Authors. Published by Elsevier Ltd on behalf of European Society for Vascular Surgery. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)

OBJECTIVE: Procedural characteristics, including stent design, may influence the outcome of carotid artery stenting (CAS). A thorough comparison of the effect of stent design on outcome of CAS is thus warranted to allow for optimal evidence-based clinical decision making. This study sought to evaluate the effect of stent design on clinical and radiologic outcomes of CAS. METHODS: A systematic search was conducted in MEDLINE, Embase, and Cochrane databases in May 2018. Included were articles reporting on the occurrence of clinical short- and intermediate-term major adverse events (MAEs; any stroke or death) or radiologic adverse events (new ischemic lesions on postprocedural magnetic resonance diffusion-weighted imaging [MR-DWI], restenosis, or stent fracture) in different stent designs used to treat carotid artery stenosis. Random effects models were used to calculate combined overall effect sizes. Metaregression was performed to identify the effect of specific stents on MAE rates. RESULTS: From 2654 unique identified articles, two randomized, controlled trials and 66 cohort studies were eligible for analysis (including 46,728 procedures). Short-term clinical MAE rates were similar for patients treated with open cell vs closed cell or hybrid stents. Use of an Acculink stent was associated with a higher risk of short-term MAE compared with a Wallstent (risk ratio [RR], 1.51; P = .03), as was true for use of Precise stent vs Xact stent (RR, 1.55; P < .001). Intermediate-term clinical MAE rates were similar for open vs closed cell stents. Use of open cell stents predisposed to a 25% higher chance (RR, 1.25; P = .03) of developing postprocedural new ischemic lesions on MR-DWI. No differences were observed in the incidence of restenosis, stent fracture, or intraprocedural hemodynamic depression with respect to different stent design. CONCLUSIONS: Stent design is not associated with short- or intermediate-term clinical MAE rates in patients undergoing CAS. Furthermore, the division in open and closed cell stent design might conceal true differences in single stent efficacy. Nevertheless, open cell stenting resulted in a significantly higher number of subclinical postprocedural new ischemic lesions detected on MR-DWI compared with closed cell stenting. An individualized patient data meta-analysis, including future studies with prospective homogenous study design, is required to adequately correct for known risk factors and to provide definite conclusions with respect to carotid stent design for specific subgroups.

The travelling check valve (TCV) pump is based on a new class of valve, and is designed for use in pneumatic or hydraulic tube transport. In this article, inventor Ian Doig describes the basic function, design details, advantages and potential applications of the TCV pump. Among its claimed advantages are transport distances and elevations unlimited by pump pressure, and a gentle mode of action suitable for handling delicate materials.

Since the landmark NASCET and ECST trials demonstrated the superiority of carotid endarterectomy over medical therapy in the prevention of stroke for patients with symptomatic carotid artery stenosis, surgical intervention as a part of secondary prevention of stroke has become widespread. However, the newer technology of carotid artery angioplasty and stenting challenges this mode of intervention, promising the benefits of a procedure under local anesthesia and potentially avoiding the surgical complications of cranial nerve palsy and hematoma. Pooled evidence from randomized controlled trials of endarterectomy versus stenting shows a higher rate of stroke or death in the stenting groups—but this finding is mitigated to an extent by the lower incidence of myocardial infarction and cranial nerve palsy in patients undergoing stenting. At present, carotid endarterectomy combined with optimal drug therapy remains the standard of care for symptomatic 70%–99% stenosis of the internal carotid artery, but stenting might be an option in younger patients and in those not suitable for endarterectomy.

BackgroundCarotid artery stenting (CAS) is an alternative to carotid endarterectomy (CEA) for the treatment of carotid stenosis, but safety and long-term efficacy were uncertain.ObjectiveTo compare the risks, benefits and cost-effectiveness of CAS versus CEA for symptomatic carotid stenosis.DesignInternational, multicentre, randomised controlled, open, prospective clinical trial.SettingHospitals at 50 centres worldwide.ParticipantsPatients older than 40 years of age with symptomatic atheromatous carotid artery stenosis.InterventionsPatients were randomly allocated stenting or endarterectomy using a computerised service and followed for up to 10 years.Main outcome measuresThe primary outcome measure was the long-term rate of fatal or disabling stroke, analysed by intention to treat (ITT). Disability was assessed using the modified Rankin Scale (mRS). A cost–utility analysis estimating mean costs and quality-adjusted life-years (QALYs) was calculated over a 5-year time horizon.ResultsA total of 1713 patients were randomised but three withdrew consent immediately, leaving 1710 for ITT analysis (853 were assigned to stenting and 857 were assigned to endarterectomy). The incidence of stroke, death or procedural myocardial infarction (MI) within 120 days of treatment was 8.5% in the CAS group versus 5.2% in the CEA group (72 vs. 44 events) [hazard ratio (HR) 1.69, 95% confidence interval (CI) 1.16 to 2.45;p = 0.006]. In the analysis restricted to patients who completed stenting, age independently predicted the risk of stroke, death or MI within 30 days of CAS (relative risk increase 1.17% per 5 years of age, 95% CI 1.01% to 1.37%). Use of an open-cell stent conferred higher risk than a closed-cell stent (relative risk 1.92, 95% CI 1.11 to 3.33), but use of a cerebral protection device did not modify the risk. CAS was associated with a higher risk of stroke in patients with an age-related white-matter changes score of 7 or more (HR 2.98, 95% CI 1.29 to 6.93;p = 0.011). After completion of follow-up with a median of 4.2 years, the number of patients with fatal or disabling stroke in the CAS and CEA groups (52 vs. 49), and the cumulative 5-year risk did not differ significantly (6.4% vs. 6.5%) (HR 1.06, 95% CI 0.72 to 1.57;p = 0.776). Stroke of any severity was more frequent in the CAS group (15.2% vs. 9.4% in the CEA group) (HR 1.712, 95% CI 1.280 to 2.300;p p = 0.20). There was no difference in the distribution of mRS scores at 1-year, 5-year or final follow-up. There were no differences in costs or QALYs between the treatments.LimitationsPatients and investigators were not blinded to treatment allocation. Interventionists’ experience of stenting was less than that of surgeons with endarterectomy. Data on costs of managing strokes were not collected.ConclusionsThe functional outcome after stenting is similar to endarterectomy, but stenting is associated with a small increase in the risk of non-disabling stroke. The choice between stenting and endarterectomy should take into account the procedural risks related to individual patient characteristics. Future studies should include measurement of cognitive function, assessment of carotid plaque morphology and identification of clinical characteristics that determine benefit from revascularisation.Trial registrationCurrent Controlled Trials ISRCTN25337470.FundingThis project was funded by the National Institute for Health Research Health Technology Assessment programme and will be published in full inHealth Technology Assessment; Vol. 20, No. 20. See the NIHR Journal Library website for further project information. Further funding was provided by the Medical Research Council, Stroke Association, Sanofi-Synthélabo and the European Union.

The aim of this study was to characterize the engineering environment of an instrumented 10 mL miniature stirred-tank bioreactor and evaluate its potential as a scale-down device for microbial fermentation processes. Miniature bioreactors such as the one detailed in this work have been developed by several research groups and companies and seek to address the current bottleneck at the screening stage of bioprocess development. The miniature bioreactor was characterized in terms of overall volumetric oxygen transfer coefficient and mixing time over a wide range of impeller speeds. Power input to the miniature bioreactor was directly measured, and from this the power number of each impeller was calculated and specific power input estimated, allowing the performance of the miniature bioreactor to be directly compared with that of a conventional 7 L bioreactor. The capability of the miniature bioreactor to carry out microbial fermentations was also investigated. Replicate batch fermentations of Escherichia coli DH5alpha producing plasmid DNA were performed at equal specific power input, under fully aerobic and oxygen-limiting conditions. The results showed a high degree of equivalence between the two scales with regard to growth and product kinetics. This was underlined by the equal maximum specific growth rate and equal specific DNA product yield on biomass obtained at the two scales of operation, demonstrating the feasibility of scaling down to 10 mL on the basis of equivalent specific power input.

Pressures on pharmaceutical companies to speed bioprocess development have led to significant interest in small scale, parallel experimentation. A particular focus is cell cultivation and the optimisation of protein synthesis because of the number of biological and engineering variables involved. In this work, we briefly review the current understanding of mixing and mass transfer phenomena in shaken bioreactors with a view to defining criteria for the scale-up of results obtained in shaken microwell systems to conventional laboratory scale. Scale-up approaches are illustrated for two different cell cultures. The first involves an automated microscale process ( 1000 μ l ) for the aerobic fermentation of E. coli JM107:pQR706 overexpressing transketolase (TK) which is subsequently used for asymmetric carbon–carbon bond formation. The kinetics of both the fermentation and bioconversion stages are first quantified as a function of fermentation medium composition (LB or LB-glycerol) and shaking frequency with oxygen transfer rates being identified as rate limiting in certain cases. Successful scale-up of the microwell process (in terms of maximum cell growth rate, biomass yield and specific TK activity) to a 1.4 l scale mechanically stirred bioreactor is then demonstrated based on experiments performed at constant k L a values. The second process investigated involved antibody production in suspension cultures of VPM8 hybridoma cells. Initial results suggest that experiments performed at constant mean energy dissipation rates provide a satisfactory basis for scale translation from shaken microwells ( 800 μ l ) to conical flasks (100 ml) and are indicative of results obtained in a mechanically stirred bioreactor (3.5 l). Overall this work provides an initial insight into the engineering characterisation of shaken bioreactors and how key parameters may be used to define suitable scale-up criteria for different cell cultures.

The risk of a pandemic with a virulent form of influenza is acknowledged by the World Health Organization (WHO) and other agencies. Current vaccine production facilities would be unable to meet the global requirement for vaccine. As a possible supplement a DNA vaccine may be appropriate, and bioprocess engineering factors bearing on the use of existing biopharmaceutical and antibiotics plants to produce it are described. This approach addresses the uncertainty of timing of a pandemic that precludes purpose‐built facilities. The strengths and weaknesses of alternative downstream processing routes are analyzed, and several gaps in public domain information are addressed. The conclusion is that such processing would be challenging but feasible.

A novel method for the measurement of the volumetric oxygen transfer coefficient, kLa, using the catechol-2,3-dioxygenase (XylE) bio-oxidation of catechol yielding 2-hydroxymuconic semialdehyde (2-HS), has been developed for small-scale systems. This method was kinetically characterized and validated by comparison to other established techniques. The bio-oxidation rate was found to be zero order over a catechol concentration from 2 to 7 mM l−1. When the enzyme concentration was in excess and the bio-oxidation rate was mass transfer limited, indicated by a percent DOT of zero, the oxygen transfer rate was determined from the linear increase in product concentration. The method was validated in a 2 l stirred tank vessel equipped with a DOT probe and connected to a gas mass spectrometer. The novel method yielded similar kLa values when compared to the dynamic gassing out method under the same conditions. The applicability of this novel method for small-scale devices was demonstrated by measuring kLa values of up to 150 h−1 in shaken microplates with a working volume of only 200 μl.

The volumetric oxygen transfer coefficient (k(L)a) in shaken microplate microbial cultivations has been modelled using dimensionless groups. The k(L)a correlation was derived from experimental measurements of air-liquid specific surface area and k(L)a values for three microwell geometries operated over a range of shaking frequencies and diameters. The air-liquid surface area was determined from the rate of evaporation and also from high-speed video photography. k(L)a values were calculated from the mass transfer limited growth rate of a strict aerobe, Bacillus subtilis, and were also directly measured using the dynamic gassing out technique. For both surface area and k(L)a measurements there was good agreement between each of the two methods used. The overall correlation for k(L)a values comprised two separate correlations based on different dimensionless groups. The first described the increase in specific air-liquid surface area (a(f)/a(i)) as a function of Froude (Fr) and Bond (Bo) numbers and is represented as: a(f)/a(i) = c(1)Fr(c2) Bo(c3) where c(1) is a geometry-dependent constant, c(2) ranged from 0.51 to 0.86 and c(3) ranged from 0.03 to 0.18. As expected, when the diameter of a microwell decreases, the significance of surface forces increases and the importance of inertial forces decrease. The second correlation related the Sherwood (Sh) number in terms of Reynolds (Re) and Schmidt (Sc) number and is represented as Sh = 0.19 Re-0.68 Sc-0.36. This correlation was independent of microwell geometry. By combining the two correlations it was possible to predict k(L)a values over a wide range of operating conditions. These models provide a rigorous engineering basis for more reproducible and rational selection of operating conditions for microplate scale cell cultivation. (c) 2005 Elsevier Ltd. All rights reserved.

A miniature (2 ml) bioreactor has been developed for the high throughput cultivation of microorganisms. The microplate bubble column bioreactor (MBCB) consists of a static deep well microtiter plate with polyethylene frits (pore size 20 μm) inserted into the base of each of the 48 wells. Air was supplied at a controlled flow rate to the underside of each well and in this way the air passing through the pores of the frit formed bubbles in the liquid biomedium. A volumetric mass transfer coefficient for oxygen (kLa) of up to 220 h−1 could be achieved in this novel system. Since the system was not shaken, as is usually required with cell cultivations in microtiter plates, its performance was simpler to model and predict. A miniature optical oxygen probe was used to measure the dissolved oxygen concentration and it was found that the derived kLa values were proportional to the superficial gas velocity over the range 0–0.02 m s−1. The usefulness of this new device was demonstrated by carrying out 25 parallel experiments to optimise the growth conditions of Bacillus subtilis ATCC6633, a strict aerobe, over a range of pH values and C/N ratios.

A new filter uses flexible elements to enlarge apertures during backwashing. Backwashing needs less energy and is effective. A vertical fixed drum filter can be used to obtain filtration with simultaneous filtering and backwashing. Dr Ian Doig explains.

Since the compositional variety of phospholipids in native lecithins is limited, and structurally diverse synthetic phospholipids are expensive, the aim of this work was to develop a lecithin modification toolbox capable of modifying the fatty acid composition of native lecithins at a convenient lab-scale of 10-100 g. Starting with the native phospholipids of soybean lecithin, two types of fatty acid modification were carried out using either phospholipase A2 from porcine pancreas or a lipase from Rhizopus oryzae. The former was immobilised onto celite and used to selectively hydrolyse the sn-2 positioned fatty acid and the latter, commercially available in an immobilised form, was used to transesterify novel fatty acids onto the sn-1 position. The degree of phospholipid hydrolysis could be controlled between 5 and 95% by varying the contact time with the biocatalyst. A key parameter was the water concentration. By avoiding the presence of a bulk phase of water, emulsion formation was prevented and so simple product recovery was possible. However, sufficient water was required in order to maintain the water activity above 0.2, and because phospholipids increased the polarity of the solvent (hexane), it was necessary to add water in proportion to the lecithin concentration. During transesterification with methyllaurate, up to 43% lauric acid could be incorporated and although the recovery of fully acylated phospholipids was only 28%, due to the formation of hydrolysis products, they were isolated using solvent partitioning in hexane/isopropanol/water. The temperature was important in determining the relative rates of hydrolysis and transesterification and a lower temperature (40 °C) was favourable as the rate of hydrolysis was lowered without affecting the rate of transesterification.

In an effort to produce structurally divergent lecithins for testing of their functional properties and use in food products, several tools have been developed to carry out modifications in the polar head group distribution of the native phospholipids in soybean lecithin. These tools include physical, chemical and enzymatic techniques. Using a combination of acetone de-oiling, ethanol fractionation, N-acetylation and enzymatic hydrolysis and transphosphatidylation with a phospholipase D from Streptomyces sp., a set of lecithins with modified head group distributions were produced. The kinetics of the enzymatic head group hydrolysis and transphosphatidylation was studied in detail. Reaction rates and selectivity (transphosphatidylation / hydrolysis) were affected by both lecithin concentration and donor alcohol concentration. Hydrolysis, forming phosphatidic acid, was strongly dependent on both concentrations, whilst transphosphatidylation, forming phosphatidyl glycerol (or phosphatidyl ethanolamine or phosphatidyl serine), was only influenced by the donor alcohol. This resulted in a reduction in selectivity at high initial lecithin concentrations and suggested the use of a reactant feeding strategy. Enrichment of the phosphatidyl choline content of native soybean lecithin was achieved by ethanol fractionation and phosphatidyl inositol enrichment was by N-acetylation with acetic anhydride followed by de-oiling. The application of these tools, together with others designed to modify the fatty acid composition of phospholipids, was used to produce 10-100 g quantities of divergent lecithins and can be routinely used at lab-scale.

This paper describes the kinetic characterization of a recombinant whole-cell biocatalyst for the stereoselective Baeyer–Villiger type oxidation of bicyclo[3.2.0]hept-2-en-6-one to its corresponding regio-isomeric lactones (−)-(1 S ,5 R )-2-oxabicyclo[3.3.0]oct-6-en-3-one and (−)-(1 R ,5 S )-3-oxabicyclo[3.3.0]oct-6-en-2-one. Escherichia coli TOP10 [pQR239], expressing cyclohexanone monooxygenase (CHMO) from Acinetobacter calcoaceticus (NCIMB 9871), was shown to be suitable for this biotransformation since it expressed CHMO at a high level, was simple to produce, contained no contaminating lactone hydrolase activity and allowed the intracellular recycle of NAD(P)H necessary for the biotransformation. A small-scale biotransformation reactor (20 ml) was developed to allow rapid collection of intrinsic kinetic data. In this system, the optimized whole-cell biocatalyst exhibited a significantly lower specific lactone production activity (55–60 μmol min −1 g −1 dry weight) than that of sonicated cells (500 μmol min −1 g −1 dry weight). It was shown that this shortfall was comprised of a difference in the pH optima of the two biocatalyst forms and mass transfer limitations of the reactant and/or product across the cell barrier. Both reactant and product inhibition were evident. The optimum ketone concentration was between 0.2 and 0.4 g l −1 and at product concentrations above 4.5–5 g l −1 the specific activity of the whole cells was zero. These results suggest that a reactant feeding strategy and in situ product removal should be considered in subsequent process design.

This case demonstrates that the diagnosis of carotid occlusion is important to make as it confers a high future risk of stroke. Advanced neuroimaging that consists of SPECT, transcranial Doppler ultrasound or CT or MR perfusion studies may be able to demonstrate reduction, or inducible reduction, in cerebral blood flow in the appropriate carotid vascular territory. Medical therapy consists of management of vascular risk factors including hypertension and hypercholesterolaemia and the initiation of antiplatelets. This may be supplemented with revascularization of a contralateral carotid stenosis or extracranial-intracranial bypass surgery to alleviate symptoms. However, this technique is not proven to reduce the incidence of ispilateral stroke and is associated with considerable periprocedural mortality.

A 49-year-old patient presents with central retinal artery occlusion. Subsequent imaging reveals thrombus in the carotid artery. Free-floating thrombus (FFT) in the common or internal carotid artery is an uncommon but important diagnosis in the patient who presents with cerebral or retinal ischaemia or infarction. FFT may be idiopathic, secondary to either an underlying arterial abnormality (carotid dissection or atherosclerotic plaque), or a procedural complication following carotid endarterectomy or stenting. The diagnosis is made through non-invasive imaging (carotid Doppler ultrasound, CT angiography or MR angiography) or at catheter angiogram. A characteristic “doughnut sign” of intraluminal filling defect surrounded by contrast is described on axial imaging. Optimal management is debated, and may differ according to aetiology. Treatment options include antithrombotic and anticoagulant drugs, carotid endarterectomy or carotid artery stenting.

The recombinant whole cell biocatalyst Escherichia coli TOP10 [pQR239], expressing cyclohexanone monooxygenase from Acinetobacter calcoaceticus NCIMB 9871, was used in 1.5- and 55-L fed-batch processes to oxidize bicyclo[3.2.0]hept-2-en-6-one to its corresponding regioisomeric lactones, (-)-(1S,5R)-2-oxabicyclo[3.3.0]oct-6-en-3-one and (-)-(1R,5S)-3-oxabicyclo[3.3.0]oct-6-en-2-one. By employing a bicyclo[3.2.0]hept-2-en-6-one feed rate below that of the theoretical volumetric biocatalyst activity (275 micromol x min(-1) x L(-1)), the reactant concentration in the bioreactor was successfully maintained below the inhibitory concentration of 0.2-0.4 g x L(-1). In this way approximately 3.5 g x L(-1) of the combined regioisomeric lactones was produced with a yield of product on reactant of 85-90%. The key limitation to the process was shown to be product inhibition. This process was scaled up to 55 L, producing over 200 g of combined lactone product. Using a simple downstream process (centrifugation, adsorption to activated charcoal, 5-fold concentration with ethyl acetate elution, and silica gel chromatography), we have shown that the two regioisomeric lactone products could be isolated and purified at this scale.

The travelling wave diaphragm pump is a new type of pump invented and patented by Dr Ian Doig, an independent consultant engineer in Australia. In this article he takes us through the details of the design, and the advantages of its non-pulsing operation and the gentle action provided by its special deformable lines. A fully operational demonstration pump has been built and Dr Doig is seeking partners to commercialize the technology.

While considerable progress has been made on the implementation of biocatalytic hydrolyses, there are still a number of engineering issues to address regarding the implementation of biocatalytic redox and carbon-carbon bond forming reactions. The use of process models linked to graphical tools (process maps) to describe the parameter boundaries for effective reactor operation have found particular use for these classes. Such process maps help to describe the properties of the biocatalyst and also the properties of the reactants and products which are frequently labile in the reactor environment. Data to form and verify such models can be rapidly collected at the microwell-scale. The models themselves can either be mechanistic or, as used here, based on experimental data. The output of such models can then be incorporated in graphical process maps and used as a predictive tool to carry out 'what if' studies. These may be, for example, on possible changes to the biocatalyst (via directed evolution) or the implementation of innovative process solutions (such as in-situ product removal). In this way, alternative process flow sheets and operating strategies can be evaluated more rapidly. In this paper, the utility of these numerical and graphical techniques using the Baeyer-Villiger oxidation of bicyclo[3.2.0]hept-2-en-6-one by a whole cell biocatalyst expressing cyclohexanone monooxygenase, is illustrated.

The International Carotid Stenting Study (ICSS, ISRCTN25337470) randomized patients with recently symptomatic carotid artery stenosis50% to carotid artery stenting (CAS) or endarterectomy. CAS increased the risk of new brain lesions visible on diffusion-weighted magnetic resonance imaging (DWI-MRI) more than endarterectomy in the ICSS-MRI Substudy. The predictors of new post-stenting DWI lesions were assessed in these patients.ICSS-MRI Substudy patients allocated to CAS were studied. Baseline or pre-stenting catheter angiograms were rated to determine carotid anatomy. Baseline patient demographics and the influence of plaque length, plaque morphology, internal carotid angulation, and external or common carotid atheroma were examined in negative binomial regression models.A total of 115 patients (70% male, average age 70.4) were included; 50.4% had at least one new DWI-MRI-positive lesion following CAS. Independent risk factors increasing the number of new lesions were a left-sided stenosis (incidence risk ratio [IRR] 1.59, 95% CI 1.04-2.44, p = .03), age (IRR 2.10 per 10-year increase in age, 95% CI 1.61-2.74, p.01), male sex (IRR 2.83, 95% CI 1.72-4.67, p.01), hypertension (IRR 2.04, 95% CI 1.25-3.33, p.01) and absence of cardiac failure (IRR 6.58, 95% CI 1.23-35.07, p = .03). None of the carotid anatomical features significantly influenced the number of post-procedure lesions.Carotid anatomy seen on pre-stenting catheter angiography did not predict of the number of ischaemic brain lesions following CAS.

A growing cell culture of Pseudomonas oleovorans was used to biotransform 1,7-octadiene to 1,2-epoxy-7,8-octene in a continuous-flow bioreactor with an external membrane module. A dense silicone rubber membrane was used to contact an organic phase, containing both the reactant (1,7-octadiene) and the growth substrate (heptane), with an aqueous biomedium phase containing the biocatalyst. Heptane and octadiene delivery to the aqueous phase, and epoxide extraction into the solvent, occurred by diffusion across the dense membrane under a concentration-driving force. In addition, a liquid feed of heptane and octadiene was pumped directly into the bioreactor to increase the rate of delivery of these compounds to the aqueous phase. In this system 1,2-epoxy-7,8-octene accumulated in a pure solvent phase, thus, product recovery problems associated with emulsion formation were avoided. Furthermore, no phase breakthrough of either liquid across the membrane was observed. In this system, the highest volumetric productivity obtained was 30 U.L-1, and this was achieved at a dilution rate of 0.07 h-1, 70 m2. m-3 of membrane area, and a steady-state biomass concentration of 2. 5 g.L-1. The system was stable for over 1250 h. Decreasing the dilution rate led to an increased biomass concentration, however, the specific activity was significantly reduced, and therefore, an optimal dilution rate was determined at 0.055 h-1. Copyright 1999 John Wiley & Sons, Inc.

The mass transfer characteristics of a non-porous silicone rubber membrane contacting an aqueous and an organic phase were determined using a shell and tube mass exchanger. Firstly, the stability of the liquid/membrane interfaces was examined, and when positive aqueous phase transmembrane pressure differentials of up to 2 bar were applied, no bulk transmembrane flow of either liquid was observed. This result was not affected by the addition of surfactants or biomass to the aqueous phase, and therefore it seems that phase breakthrough, common with porous membranes, is avoided. Secondly, the mass transfer characteristics of a range of model solutes were investigated and explained with a resistances in series model. A high membrane/aqueous partition coefficient ( P mem aq ) (approximately>25) resulted in the aqueous phase film resistance limiting, whilst a low P mem aq (approximately P mem aq and the membrane diffusion coefficient. These two parameters could be increased by using a highly swelling solvent (solvent constituting>50% of the swollen membrane volume) with a high organic/aqueous partition coefficient ( P org aq ) for the solute. In this way the membrane resistance for some solutes was decreased, and therefore the overall mass transfer coefficient increased. Finally, the effect of the presence of other hydrophobic solutes on the rate of mass transfer was investigated, and in the case of geraniol and citronellol (two chemically similar solutes with low water solubilities, 0.7 and 0.35 g l −1 , respectively) the effect was similar to a “salting out” phenomena. This resulted in an increase of P org aq for both geraniol and citronellol, and therefore a decrease in flux of the solute transferring to the aqueous phase.

The Membrane Bioreactor for Biotransformations (MBB) is based on the aqueous/organic two-phase system, and uses a tubular silicone rubber membrane to separate the two liquid phases. This avoids the key problem associated with direct contact two-phase processes, specifically, product emulsification. The baker's yeast mediated reduction of geraniol to citronellol was used as a model biotransformation to demonstrate MBB operation. Values for the overall mass transfer coefficient were determined for geraniol, (2.0 x 10(-5) ms-1), and for citronellol, (2.1 x 10(-5) ms-1) diffusion across the silicone rubber membrane. Using these values, and the specific activity of the biocatalyst (5 nmols-1g biomass-1), a suitable membrane surface area: biomass ratio was determined as 2.4 x 10(-3) m2g biomass-1. The bioreactor was operated at this surface area: biomass ratio and achieved a product accumulation rate 90-95% that of a conventional direct contact two-phase system. The slight reduction in product accumulation rate was shown not to be due to mass transfer limitations with respect to reactant delivery or product extraction. Copyright 1998 John Wiley & Sons, Inc.

Kinetics of the stereoselective reduction of geraniol to citronellol by baker's yeast (Saccharomyces cerevisiae) were studied by examining the influence of system parameters on the reaction rate in an aqueous/organic two phase system. This system was used due to the hydrophobic nature of the reactant and product, and enabled simple and accurate measurement of the kinetics, and hence optimisation. This reaction system provided “pseudo steady state” aqueous conditions in batch experiments over short time periods, and therefore allowed direct determination of the biotransformation rate. The maximum biotransformation activity was determined to be 300 nmol min-1 g biomass-1. Of the parameters tested those which most significantly affected the biocatalytic activity were; biomedium pH, reactant concentration, solvent type, and aeration. The optimum pH was found to be 9.5, and this was associated with increased intra-cellular NADPH concentrations. Slight substrate inhibition with geraniol occurred at concentratio...

The Aging in Manitoba (AIM) project includes nearly 9,000 seniors who have been interviewed. Three independent cross-sectional samples (i.e. 1971, 1976, 1983) were subsequently followed (1983–1984, 1990), producing cross-sectional and longitudinal data. A major goal of this paper was to examine non-response over time and representativeness of the panel of AIM survivors. This involved analysis of non-response, calculations of completion/response rates, and comparisons of demographic variables across the AIM sample and the broader Manitoba and Canadian populations. Non-response was very low (4.6% in 1983–1984, 5.0% in 1990) and did not appear to be dependent on such factors as location, gender, education level, or nationality descent. The 95.0 per cent completion rate compares favourably with other longitudinal studies, demonstrating the value of employing rigorous tracking procedures. These findings suggest that attempts to minimize bias and selective attrition in the AIM study have been successful.

Stroke is a common cause of disability and death, but prompt recognition and appropriate initial treatment may save lives or mitigate the morbidity of stroke. Selected patients at specialist centres may benefit from emergency intravenous thrombolysis or decompressive craniectomy, but all patients stand to benefit from stroke unit care and should have their risk factors addressed with consideration of the institution of secondary prevention measures, aimed at reducing the risk of recurrence. A stroke may be the consequence of a number of heterogeneous conditions, and further investigation will aid diagnosis. Structural imaging of the carotid arteries will enable detection of those with a carotid stenosis likely to cause recurrent symptoms. In these patients, revascularization by carotid endarterectomy should be offered as an urgent procedure. Intracerebral haemorrhage and subarachnoid haemorrhage are also important causes of neurodisability. Neurosurgical or neuroradiological intervention to prevent recurrence of haemorrhage in the presence of an underlying vascular abnormality should be considered.

Introduction The biological production of active compounds, ranging from small molecules, such as organic acids, vitamins or antibiotics, through to macromolecules, such as therapeutic proteins or plasmid gene therapy vectors, is of great commercial and social value. The cornerstone of any such bioprocess is the cell cultivation step where a highly selected, and usually engineered, cell-line is grown under carefully controlled conditions. The term cell-line is used here to represent both microbial and mammalian cells. The aim of the cultivation step is to yield the product in as an efficient and cost-effective manner as possible. However, the design and implementation of a cell cultivation process is often a complex, lengthy and costly task. The development of a cell cultivation process typically involves four stages, as shown in Fig. 12.1. Stage 1 involves the initial identification of a native, or wild-type, cell-line that produces the compound of interest, though usually slowly and at low levels. This is followed by Stage 2 in which the productivity of the chosen cell-line [g product (g cells) −1 h −1 ] is enhanced using a variety of microbiological and molecular biology techniques. Stage 3 involves optimisation of the growth media composition and culture conditions; while in Stage 4 , scale-up of the process from laboratory, through pilot plant to manufacturing scale occurs. High-throughput experimentation Traditionally cell culture development has been carried out following a sequential series of experiments, using conventional apparatus with a high labour requirement. Conventional experimentation usually involves carrying out one or only a few experiments at a time.

This work describes the engineering characterization of miniature (2 mL) and laboratory-scale (100 mL) bubble column bioreactors useful for the cultivation of microbial cells. These bioreactors were constructed of glass and used a range of sintered glass gas diffusers with differently sized pores to disperse humidified air within the liquid biomedium. The effect of the pressure of this supplied air on the breakthrough point for gas diffusers with different pore sizes was examined and could be predicted using the Laplace-Young equation. The influence of the superficial gas velocity (u(g)) on the volumetric mass transfer coefficient (k(L)a) was determined, and values of up to 0.09 s(-1) were observed in this work. Two modeling approaches were considered in order to predict and provide comparison criteria. The first related the volumetric power consumption (P/V) to the k(L)a and a good correlation was obtained for differently sized reactors with a given pore size, but this correlation was not satisfactory for bubble columns with different gas diffusers. Values for P/V ranged from about 10 to 400 W center dot m(-3). Second, a model was developed predicting bubble size (d(b)), bubble rising velocity (u(b)), gas hold-up (phi), liquid side mass transfer coefficient (k(L)), and thus the kLa using established theory and empirical correlations. Good agreement was found with our experimental data at different scales and pore sizes. Values for db varied from 0.1 to 0.6 mm, and k(L) values between 1.7 and 9.8 x 10(-4) m center dot s(-1) were determined. Several E. coli cultivations were performed in the miniature bubble column at low and high k(L)a values, and the results were compared to those from a conventional stirred tank operated under identical k(L)a values. Results from the two systems were similar in terms of biomass growth rate and carbon source utilization.

Microscale processing techniques would be a useful tool for the rapid and efficient collection of biotransformation kinetic data as a basis for bioprocess design. Automated liquid handling systems can reduce labor intensity while the small scale reduces the demand for scarce materials such as substrate, product, and biocatalyst. Here we illustrate this concept by establishing the use of several microwell formats (96-round, 96-deep square and 24-round well microtiter plates) for quantification of the kinetics of the E. coli TOP10 [pQR239] resting cell catalyzed Baeyer-Villiger oxidation of bicyclo[3.2.0]hept-2en-6-one using glycerol as a source of reducing power. By increasing the biocatalyst concentration until the biotransformation rate was oxygen mass-transfer limited we can ensure that kinetic data collected are in the region away from oxygen limitation. Using a 96-round well plate the effect of substrate (bicyclo[3.2.0]hept-2en-6-one) concentration on the volumetric CHMO activity was examined and compared to data collected from 1.5-L stirred-tank experiments. The phenomenon and magnitude of substrate inhibition, observed at the larger scale, was accurately reproduced in the microwell format. We have used this as an illustrative example to demonstrate that under adequately defined conditions, automated microscale processing technologies can be used for the collection of quantitative kinetic data. Additionally, by using the experimentally determined stoichiometry for product formation and glycerol oxidation, we have estimated the maximum oxygen transfer rates as a function of well geometry and agitation rate. Oxygen-transfer rates with an upper limit of between 33 mmol.L-1.h(-1) (based solely on product formation) and 390 mmol.L-1.h(-1) (based on product formation and glycerol oxidation) were achieved using a 96-square well format plate shaken at 1300 rpm operated with a static surface area to volume ratio of 320 m(2).m(-3). (C) 2002 Wiley Periodicals, Inc.

Background ICSS showed a significantly higher rate of stroke, myocardial infarction (MI) or death after carotid artery stenting (CAS) compared with endarterectomy. We therefore examined risk factors for CAS. Methods Per-protocol analysis was performed investigating baseline characteristics influencing the risk of stroke, MI or death 30 days after CAS. Logarithmic binomial regression was used to compare this risk in each subgroup. Results CAS was initiated in 828/853 patients allocated stenting. 61 patients suffered stroke, death or MI within 30 days. In univariate analysis, the rate of stroke, MI or death was higher in those patients undergoing a left compared to right-sided procedure (9.4% vs 5.1%, p=0.019), in those who never smoked compared to current smokers (10.2% vs 2.5%, p=0.004), in those with atrial fibrillation (15.4% vs 6.7%, p=0.017), in those with Rankin Score of 4 compared to 0 (17.6% vs 5.9%, p=0.051), in those with stroke compared to amaurosis fugax as the index event (8.4% vs 2.1%, p=0.019), and in those randomised in centres who recruited Conclusion Knowledge of risk factors might be useful in selecting and advising patients on the risk of carotid stenting.

Objectives: Stroke, myocardial infarction (MI), and death are complications of carotid artery stenting (CAS). The effect of baseline patient demographic factors, processes of care, and technical factors during CAS on the risk of stroke, MI, or death within 30 days of CAS in the International Carotid Stenting Study (ICSS) were investigated. Methods: In ICSS, suitable patients with recently symptomatic carotid stenosis > 50% were randomly allocated to CAS or endarterectomy. Factors influencing the risk of stroke, MI, or death within 30 days of CAS were examined in a regression model for the 828 patients randomized to CAS in whom the procedure was initiated. Results: Of the patients, 7.4% suffered stroke, MI, or death within 30 days of CAS. Independent predictors of risk were age (risk ratio [RR] 1.17 per 5 years of age, 95% CI 1.01-1.37), a right-sided procedure (RR 0.54, 95% CI 0.32-0.91), aspirin and clopidogrel in combination prior to CAS (compared with any other antiplatelet regimen, RR 0.59, 95% CI 0.36-0.98), smoking status, and the severity of index event. In patients in whom a stent was deployed, use of an open-cell stent conferred higher risk than use of a closed-cell stent (RR 1.92, 95% CI 1.113.33). Cerebral protection device (CPD) use did not modify the risk. Conclusions: Selection of patients for CAS should take into account symptoms, age, and side of the procedure. The results favour the use of closed-cell stents. CPDs in ICSS did not protect against stroke. (C) 2015 The Authors. Published by Elsevier Ltd on behalf of European Society for Vascular Surgery.

Objective: Cranial nerve palsy (CNP) and neck haematoma are complications of carotid endarterectomy (CEA). The effects of patient factors and surgical technique were analysed on the risk, and impact on disability, of CNP or haematoma in the surgical arm of the International Carotid Stenting Study (ICSS), a randomized controlled clinical trial of stenting versus CEA in patients with symptomatic carotid stenosis. Materials and methods: A per-protocol analysis of early outcome in patients receiving CEA in ICSS is reported. Haematoma was defined by the surgeon. CNP was confirmed by an independent neurologist. Factors associated with the risk of CNP and haematoma were investigated in a binomial regression analysis. Results: Of the patients undergoing CEA, 45/821 (5.5%) developed CNP, one of which was disabling (modified Rankin score = 3 at 1 month). Twenty-eight (3.4%) developed severe haematoma. Twelve patients with haematoma also had CNP, a significant association (p < .01). Independent risk factors modifying the risk of CNP were cardiac failure (risk ratio [RR] 2.66, 95% CI 1.11 to 6.40), female sex (RR 1.80, 95% CI 1.02 to 3.20), the degree of contralateral carotid stenosis, and time from randomization to treatment >14 days (RR 3.33, 95% CI 1.05 to 10.57). The risk of haematoma was increased in women, by the prescription of anticoagulant drugs pre-procedure and in patients with atrial fibrillation, and was decreased in patients in whom a shunt was used and in those with a higher baseline cholesterol level. Conclusions: CNP remains relatively common after CEA, but is rarely disabling. Women should be warned about an increased risk. Attention to haemostasis might reduce the incidence of CNP. ICSS is a registered clinical trial: ISRCTN 25337470.