Process limitations of a whole-cell P450 catalyzed reaction using a CYP153A-CPR fusion construct expressed in Escherichia coli.

Cytochrome P450s are interesting biocatalysts due to their ability to hydroxylate non-activated hydrocarbons in a selective manner. However, to date only a few P450-catalyzed processes have been implemented in industry due to the difficulty of developing economically feasible processes. In this study, we have used the CYP153A heme domain from Marinobacter aquaeolei fused to the reductase domain of CYP102A1 from Bacillus megaterium (BM3) expressed in Escherichia coli. This self-sufficient protein chimera CYP153A-CPRBM3 G307A mutant is able to selectively hydroxylate medium and long chain length fatty acids at the terminal position. ω-Hydroxylated fatty acids can be used in the field of high-end polymers and in the cosmetic and fragrance industry. Here, we have identified the limitations for implementation of a whole-cell P450-catalyzed reaction by characterizing the chosen biocatalyst as well as the reaction system. Despite a well-studied whole-cell P450 catalyst, low activity and poor stability of the artificial fusion construct are the main identified limitations to reach sufficient biocatalyst yield (mass of product/mass of biocatalyst) and space-time yield (volumetric productivity) essential for an economically feasible process. Substrate and product inhibition are also challenges that need to be addressed, and the application of solid substrate is shown to be a promising option to improve the process.

Bilateral lower limb examinations for deep vein thrombosis: A single-centre pilot study comparing request and report parameters for bilateral and unilateral referrals.

INTRODUCTION: Deep vein thrombosis (DVT) is a common pathology with significant morbidity and mortality, often occurring in the lower limb. Ultrasound is the modality of choice for diagnosis of DVT, but all guidance in the United Kingdom assumes a single leg referral. Few studies have addressed the question of bilateral referrals and bilateral DVTs, and it is not known how these should be included in ultrasound protocols. This study aims to compare the request and report parameters of bilateral examinations with those for single leg examinations over a six-month period. METHODS: A single-centre feasibility study collected data on all single and bilateral leg examinations performed by sonographers, over a six-month period at a small general hospital. Data collected for each examination included the referral and report findings. These were compared using basic statistical methods to compare DVT yield by laterality, referrer, DVT site, and patient factors. RESULTS: Six hundred and thirty examinations were included, eighteen of which were bilateral examinations. Although the bilateral leg cohort was small, there were significant differences in DVT yield between the groups, with single leg referrals more than eight times more likely to demonstrate a DVT at ultrasound. CONCLUSION: In a context of limited ultrasound resource, the low DVT yield of bilateral examinations has implications for service design. Further data collection will be needed to validate initial results, and to establish suitable criteria for acceptance of bilateral DVT referrals. IMPLICATIONS FOR PRACTICE: Low yield of DVTs in bilateral examinations can be validated via further research. Bilateral examinations can be explicitly included in DVT service design.

Research ethics systems, processes, and awareness across Europe: Radiography research ethics standards for Europe (RRESFE).

INTRODUCTION: The Radiography Research Ethics Standards for Europe (RRESFE) project aims to provide a cross-sectional snapshot of current research ethics systems, processes, and awareness of such, across Europe together with identifying the associated challenges, education, and training needs. METHODS: A cross-sectional online survey targeting radiography researchers in Europe was conducted. Data collection took place between April 26 and July 12, 2021, using a snowball sampling approach. Descriptive and analytical statistics were used to identify trends in research ethics frameworks across Europe. RESULTS: 285 responses were received across 33 European and 23 non-European countries. Most (n = 221; 95%) European respondents stated ethics approval is required before commencing research in their country. Requirements around research ethics approval and awareness of such requirements varied by European region (X2 (2, n = 129) = 7.234, p = 0.013) and were found to differ depending on the type of research participant and study design. Additionally, European respondents reported ethics approval is a national requirement more often than their non-European counterparts (X2 (1, n = 282) = 4.316, p = 0.049). Requirements for ethics approval were also associated with the undergraduate programme duration (2-year vs. 3-year vs. 3.5 year vs. 4-year vs. multiple programme durations; X2 (4, n = 231) = 10.075, p = 0.016) and availability of postgraduate training (postgraduate training available vs. postgraduate training not available; X2 (1, n = 231) = 15.448, p = <0.001) within respondents' country. CONCLUSION: Respondents from countries with longer programme durations/availability of multiple programme lengths, availability of postgraduate training, and establishment of European Qualifications Framework Level 6 were generally associated with less uncertainty and more comprehensive research ethics requirements. IMPLICATIONS FOR PRACTICE: Results are informative of the current status of research ethics within evidence-based radiography.

Research ethics training, challenges, and suggested improvements across Europe: Radiography research ethics standards for Europe (RRESFE).

INTRODUCTION: The Radiography Research Ethics Standards for Europe (RRESFE) project aimed to provide a cross-sectional view of the current state of radiography research ethics across Europe. This included investigating education and training in research ethics, and identifying the key challenges and potential improvements associated with using existing research ethics frameworks. METHODS: This cross-sectional online survey targeting radiography researchers in Europe was conducted between April 26 and July 12, 2021. Descriptive and analytical statistics were used to identify research ethics education and training trends. Content analysis of qualitative responses was employed to identify significant challenges and proposed improvements in research ethics frameworks of practice. RESULTS: There were 232 responses received across 33 European countries. Most (n = 132; 57%) respondents had received some research ethics training; however, fewer participants had received training on safeguarding vulnerable patients (n = 72; 38%), diversity and inclusivity (n = 62; 33%), or research with healthy volunteers (n = 60; 32%). Training was associated with a greater perceived importance of the need for research ethics review (p = 0.031) and with the establishment of EQF Level 6 training (p = 0.038). The proportion of formally trained researchers also varied by region (p = <0.001). Time-to-ethics-approval was noted as the biggest challenge for professionals making research ethics applications. CONCLUSION: Early and universal integration of research-oriented teaching within the radiography education framework which emphasises research ethics is recommended. Additionally, study findings suggest research ethics committee application and approval processes could be further simplified and streamlined. IMPLICATIONS FOR PRACTICE: The survey contributes to a growing body of knowledge surrounding the importance of education and training in research ethics for assuring a high standard of research outputs in Radiography and has identified hurdles to obtaining research ethics approval for further investigation and address.

A systematic review of non-pharmacologic interventions to reduce anxiety in adults in advance of diagnostic imaging procedures.

OBJECTIVES: Anticipation of a diagnostic imaging (DI) procedure, particularly one involving advanced technology, can provoke feelings of anxiety in patients. Anxiolytics (anxiety reducing drugs) can be used to reduce pre-procedural anxiety in patients, however there are several known disadvantages to this approach. The aim of this systematic review was to identify and evaluate any preparatory non-pharmacological interventions used to reduce patient anxiety in advance of DI procedures. KEY FINDINGS: Database searches revealed twelve studies met the eligibility criteria and were included in the review. A narrative synthesis identified three intervention categories: patient information/education, cognitive strategies (i.e. guided imagery, breathing techniques, imaginative visualisation) and music therapy. CONCLUSION: The current review demonstrates that despite the existence of a number of studies providing some evidence for the effectiveness of a range of anxiety reducing interventions for patients prior to DI, the small number and overall low quality of studies identified makes it difficult to draw firm conclusions regarding the application of a specific intervention in clinical practice. IMPLICATIONS FOR PRACTICE: The majority of interventions included in this review were shown to be practical for inclusion in the clinical setting and did have some positive effect on patient anxiety levels. As a result those professionals working with adults undergoing advanced technology DI procedures may consider implementing some of the strategies that have been discussed within their practice.

The College of Radiographers Research Strategy for the next five years.

This article outlines the updated College of Radiographers (CoR) Research Strategy. This new research strategy will shape the approach to research from the radiography profession over the next five years. This will apply to all the profession and is aspirational and future thinking. The updated research strategy is the fifth research strategy presented by the CoR. Over the last five years, there have been considerable developments within healthcare and healthcare research. As this article is being written we are still in the middle of a global pandemic (Covid-19) which has influenced all our lives. However, despite the challenges of the last year, we are in a stronger position as a profession with more radiographers working towards and gaining masters and doctoral level qualifications. There are more radiographers working in clinical academic roles and there has been further development of radiographers coordinating and delivering research as well as becoming research leaders. This updated research strategy supports the radiography profession in delivering research-based practice over the next five years offering a framework within which radiographers can develop.

Hurricane Allen's Impact on Jamaican Coral Reefs.

Coral reefs of north Jamaica, normally sheltered, were severely damaged by Hurricane Allen, the strongest Caribbean hurricane of this century. Immediate studies were made at Discovery Bay, where reef populations were already known in some detail. Data are presented to show how damage varied with the position and orientation of the substraturn and with the shape, size, and mechanical properties of exposed organisms. Data collected over succeeding weeks showed striking differences in the ability of organisms to heal and survive.

The stability of liposomes in vitro to pH, bile salts and pancreatic lipase.

If liposomes are to be effective as carriers for the oral administration of insulin they must be able to withstand passage through the stomach and small intestine. Multilamellar liposomes, some identical in composition to those used in reported in vivo studies on the uptake of orally administered insulin, were tested in vitro for their stability in the presence of bile salts, pancreatic lipase, and variations in pH. While low or high pH had little effect on most liposomes, 10 mM bile salts caused the release of over 80% of entrapped marker from all liposomes tested except those composed of distearoyl phosphatidylcholine/cholesterol and those composed of dipalmitoyl phosphatidylethanolamine/cholesterol/dicetylphosphate. However, the latter were unstable at low pH. The distearoyl phosphatidylcholine/cholesterol liposomes were also resistant to pancreatic lipase, and therefore may be suitable for use in the oral administration of therapeutic agents.

The uptake of distearoylphosphatidylcholine/cholesterol liposomes by rat intestinal sacs in vitro.

The uptake of free and liposome-entrapped 125I-labelled polyvinylpyrrolidone was measured in an intestinal sac preparation from adult rats. At an equal concentration of 125I-labelled polyvinylpyrrolidone, the rate of uptake of the liposome-entrapped macromolecule by the tissue was over 4-times that of the free macromolecule. The quantity of 125I-labelled polyvinylpyrrolidone present in the serosal fluid of gut sacs, cultured for 2 h, was 1.8-times greater when the macromolecule was entrapped in liposomes than when it was free in the culture medium. When gut sacs were cultured with liposome-entrapped macromolecule, approx. 50% of the total 125I-labelled polyvinylpyrrolidone present in the serosal fluid was associated with a 100 000 X g liposomal pellet.

The uptake of liposome-entrapped 125I-labelled poly(vinylpyrrolidone) by rat jejunum in vitro.

The uptake of free and liposome-entrapped 125I-labelled poly(vinylpyrrolidone) was measured in an intestinal sac preparation from adult rats. At an equal concentration of 125I-labelled poly(vinylpyrrolidone), the rate of uptake of the liposome-entrapped material was four times that of the free macromolecule.

Validation of a liquid chromatography-mass spectrometry method to assess the metabolism of bupropion in rat everted gut sacs.

We have developed a rapid, sensitive and selective LC-MS method for the simultaneous assay of bupropion and its metabolite hydroxybupropion during its intestinal absorption, studied with the rat everted gut sac model. The method was validated in the concentration range of 1-15 microM (0.024-3.58 microg/mL) for bupropion and 0.005-1 microM (0.00127-0.25 microg/mL) for hydroxybupropion with 10 microL injected. Bupropion is used as a probe for the activity of the CYP2B6 isoenzyme of the P450 family of enzymes in man. Its major metabolite hydroxybupropion was found in the serosal media of the gut sac showing that the isoenzyme of the 2B group was active in the intestinal mucosa and metabolized bupropion during its passage across the mucosa. The metabolite was also quantified in the mucosal media indicating its ability to cross the apical membrane of the epithelial cells.

Validation of a liquid chromatography-mass spectrometry method to assess the metabolism of dextromethorphan in rat everted gut sacs.

A rapid, sensitive and selective liquid chromatography-mass spectrometry (LC-MS) method was developed for the simultaneous assay of dextromethorphan and its metabolites in tissue culture medium and its intestinal metabolism studied with the rat everted gut sac model. The method was validated in the concentration range of 0.1-2.5 microM (27.1 ng/mL-0.677 microg/mL) for dextromethorphan and 0.005-0.5 microM for dextrorphan and 3-methoxymorphinan (1.28 ng/mL-0.128 microg/mL) and 3-hydroxymorphinan (1.22 ng/mL-0.122 microg/mL). The limits of quantification (LOQ) were 0.0025 microM (12.5 fmoles, 3.4 pg, 5 microL injected) for dextromethorphan; 0.0025 microM for dextrorphan, 3-methoxymorphinan (24.9 fmoles, 6.4 pg injected), and 3-hydroxymorphinan (25.1 fmoles, 6.1 pg injected) with 10 microL injected. The detection of dextrorphan and 3-methoxymorphinan showed that both the P450 isoforms CYP3A and 2D were active in the intestinal mucosa and metabolised dextromethorphan during its passage across the mucosa.

Left-handed and right-handed metamaterials composed of split ring resonators and strip wires.

The behavior of two structures composed of split ring resonators (SRRs) and strip wires (SWs) is examined through full wave simulations. It is shown that both structures exhibit a transmission peak in the region where the real parts of the electric permittivity and magnetic permeability are presumed to be negative, a property which is usually assumed to imply a negative index of refraction. However, an analysis of the dispersion characteristics and insertion phase of the two structures shows that the first structure, in which the SRRs and SWs are printed on opposite sides of a dielectric substrate, is a left-handed medium in the passband, whereas the second structure, in which SRRs and SWs are printed on the same side, is a right-handed medium in the passband. Hence the transmission magnitude alone does not provide sufficient evidence of a negative index of refraction. To determine the sign of the index correctly, the insertion phase for propagation through several lengths of the structure or calculations of dispersion diagrams are necessary. The impact of the unit cell size on the "handedness" of the structure is also examined.

Application of in situ product-removal techniques to biocatalytic processes.

Biocatalytic processes for the manufacture of small, highly functionalized molecules frequently have limited productivity. A common reason for this is the presence of the reaction products that can cause inhibitory or toxic effects (making poor use of the enzyme) or promote unfavourable equilibria (giving low conversions). In each case, the product needs to be removed as soon as it is formed in order to overcome these constraints and hence increase the productivity of the biocatalytic process. Here, we review the need for in situ product removal and the process research required for its implementation.

Putative immunolocalization of the mechanoelectrical transduction channels in mammalian cochlear hair cells.

Hair cells bear an apical bundle of stereocilia arranged in serried rows. Deflection of the bundle controls the opening and closing of mechanoelectrical transduction channels, thereby altering the conductance across the apical plasma membrane. Two locations for these channels have been proposed in the bundle, either near the bases of the stereocilia or towards their tips. One hypothesis that is consistent with the latter possibility suggests that fine extracellular filaments, which run between the tips of the shorter stereocilia and the sides of the taller stereocilia behind, operate the channels. Determining the precise position of the channels is essential to test this hypothesis. We have therefore attempted to localize them immunocytochemically. Because hair-cell transduction is amiloride sensitive, the channels may have an amiloride-binding site associated with them. We have therefore used a polyclonal antibody raised against another amiloride-sensitive ion channel to hunt for them. This antibody recognizes a 62-64 kDa band in immunoblots of cochlear tissue, and produces discrete labelling in the hair bundle. This is most concentrated just below the tips of the shorter stereocilia, coinciding with a region of specialization in the closely apposed membranes of the short and tall stereocilia but not with either end of the tip link.

Bioadhesion: new possibilities for drug administration?

Bioadhesion (and mucoadhesion) is the process whereby synthetic and natural macromolecules adhere to mucosal surfaces in the body. If these materials are then incorporated into pharmaceutical formulations, drug absorption by mucosal cells may be enhanced or the drug released at the site for an extended period of time. For synthetic polymers, such as the chitosans, carbopols and carbomers, the mechanism of bio/mucoadhesion is the result of a number of different physicochemical interactions. Biological bio/mucoadhesives, such as plant lectins, show specific interactions with cell surfaces and mucin and are seen as the 'second generation' bioadhesives. Bioadhesive systems for drug administration via the buccal and nasal cavities are nearing the market; in the case of nasal bioadhesion, bioadhesive microparticles are used. A bioadhesive formulation for drug administration to the vagina is in use. The gastrointestinal tract is proving a more difficult site because of the rapid turnover of mucus, and relatively constant transit time, but intensive research is in progress. Micro- and nano-particles, coated with either bio/mucoadhesive polymers or specific biological bioadhesives, are showing some promise, but will require considerable research and development before reaching the market.

Liposomes for oral administration of drugs.

In the late 1970s liposome-entrapped insulin was administered by the oral route to both normal and diabetic animals. Results showed that small but significant amounts of insulin could reach the circulation. However, different liposome compositions gave varied results and no mechanism of absorption was elucidated. Subsequent in vitro studies suggested that many liposome compositions used were unstable in the conditions prevailing in the gastrointestinal tract. Using more stable liposomes in an everted gut system, it has been demonstrated that liposomes were pinocytosed by intestinal epithelial cells and transferred to the serosal side of the gut. Recent studies both in vitro and in vivo show that there may be the possibility of enhancing the uptake process to deliver a range of drugs by the oral route.

Enzymatic barriers for GI peptide and protein delivery.

The oral delivery of therapeutic peptides and proteins is a major challenge to pharmaceutical science. The gastrointestinal (GI) tract contains many endo- and exopeptidases, enzymes that hydrolyze peptide bonds and act synergistically to degrade proteins and peptides. It is important to have both qualitative and quantitative data on these peptidases when devising strategies for oral peptide and protein delivery. The greatest threat to therapeutic peptides lies in the lumen of the small intestine, which contains gram quantities of peptidases secreted from the pancreas, as well as cellular peptidases from the mucosal cells, which are constantly sloughed off from the villi. The second major enzymatic barrier is the brush border membrane of the epithelial cells, which contains at least 15 peptidases that together have a broad specificity and can degrade both proteins and peptides. Lysosomal peptidases will also present a barrier to any peptides or proteins endocytosed by the epithelial cells. Although the colon has received some attention as a possible site for peptide delivery, evidence shows that the lumen of the colon contains substantial amounts of peptidase activity, largely because of enzyme production by microorganisms. From a knowledge of the enzymatic barrier, the strategies for oral peptide delivery of enzyme inhibition and the synthesis of enzyme-resistant peptide analogues are logical developments. The latter approach is the most promising.

Enzyme-catalysed carbon-carbon bond formation: large-scale production of Escherichia coli transketolase.

Escherichia coli strain JM107/pQR700 possesses the vector pBGS18, a high copy number plasmid carrying kanamycin resistance, into which a 4.4 kb fragment containing the transketolase gene had been cloned. The bacterium was grown at 20 and 1000 1 scale for the production of transketolase. The specific growth rate was maintained at 0.15 h-1 until the bacterial concentration reached 20 g dry wt per litre at which point the culture was harvested. The clarified cell extract obtained after disruption of the bacteria in a high-pressure homogeniser contained about 230 U ml-1 of the enzyme, which represented about 40% of the total protein released. No further purification was done at large scale as the clarified cell extract could be used satisfactorily for biotransformations.

An accurate fluorometric method to measure the breakdown of gliadin and gliadin peptides.

A simple and accurate method is described to measure the breakdown of gliadin and gliadin peptides. It involves measuring the release of the predominant amino acids glutamine and glutamic acid using a fluorometric double enzyme assay and contains none of the problems normally associated with previously used techniques. The assay is highly accurate in that small concentrations of the free amino acids can be measured with no interference from the peptide bound amino acids. The assay system was used to study the breakdown of: whole gliadin, a peptic/tryptic digest of gliadin (PT gliadin), and a peptic/tryptic/chymotryptic digest of gliadin (PTC gliadin) using a rat intestinal brush border fraction. Both PT and PTC gliadin are hydrolysed at higher rates than is whole gliadin. Exhaustive hydrolysis shows that a brush border fraction can totally break down PT gliadin while an initial linear rate of breakdown is observed (up to 60 min).