Comparing the in vivo function of α-carboxysomes and ß-carboxysomes in two model cyanobacteria.

The carbon dioxide (CO2)-concentrating mechanism of cyanobacteria is characterized by the occurrence of Rubisco-containing microcompartments called carboxysomes within cells. The encapsulation of Rubisco allows for high-CO2 concentrations at the site of fixation, providing an advantage in low-CO2 environments. Cyanobacteria with Form-IA Rubisco contain α-carboxysomes, and cyanobacteria with Form-IB Rubisco contain ß-carboxysomes. The two carboxysome types have arisen through convergent evolution, and α-cyanobacteria and ß-cyanobacteria occupy different ecological niches. Here, we present, to our knowledge, the first direct comparison of the carboxysome function from α-cyanobacteria (Cyanobium spp. PCC7001) and ß-cyanobacteria (Synechococcus spp. PCC7942) with similar inorganic carbon (Ci; as CO2 and HCO3-) transporter systems. Despite evolutionary and structural differences between α-carboxysomes and ß-carboxysomes, we found that the two strains are remarkably similar in many physiological parameters, particularly the response of photosynthesis to light and external Ci and their modulation of internal ribulose-1,5-bisphosphate, phosphoglycerate, and Ci pools when grown under comparable conditions. In addition, the different Rubisco forms present in each carboxysome had almost identical kinetic parameters. The conclusions indicate that the possession of different carboxysome types does not significantly influence the physiological function of these species and that similar carboxysome function may be possessed by each carboxysome type. Interestingly, both carboxysome types showed a response to cytosolic Ci, which is of higher affinity than predicted by current models, being saturated by 5 to 15 mm Ci. This finding has bearing on the viability of transplanting functional carboxysomes into the C3 chloroplast.

A cytosolic acyltransferase contributes to triacylglycerol synthesis in sucrose-rescued Arabidopsis seed oil catabolism mutants.

Triacylglycerol (TAG) levels and oil bodies persist in sucrose (Suc)-rescued Arabidopsis (Arabidopsis thaliana) seedlings disrupted in seed oil catabolism. This study set out to establish if TAG levels persist as a metabolically inert pool when downstream catabolism is disrupted, or if other mechanisms, such as fatty acid (FA) recycling into TAG are operating. We show that TAG composition changes significantly in Suc-rescued seedlings compared with that found in dry seeds, with 18:2 and 18:3 accumulating. However, 20:1 FA is not efficiently recycled back into TAG in young seedlings, instead partitioning into the membrane lipid fraction and diacylglycerol. In the lipolysis mutant sugar dependent1and the ß-oxidation double mutant acx1acx2 (for acyl-Coenzyme A oxidase), levels of TAG actually increased in seedlings growing on Suc. We performed a transcriptomic study and identified up-regulation of an acyltransferase gene, DIACYLGLYCEROL ACYLTRANSFERASE3 (DGAT3), with homology to a peanut (Arachis hypogaea) cytosolic acyltransferase. The acyl-Coenzyme A substrate for this acyltransferase accumulates in mutants that are blocked in oil breakdown postlipolysis. Transient expression in Nicotiana benthamiana confirmed involvement in TAG synthesis and specificity toward 18:3 and 18:2 FAs. Double-mutant analysis with the peroxisomal ATP-binding cassette transporter mutant peroxisomal ABC transporter1 indicated involvement of DGAT3 in the partitioning of 18:3 into TAG in mutant seedlings growing on Suc. Fusion of the DGAT3 protein with green fluorescent protein confirmed localization to the cytosol of N. benthamiana. This work has demonstrated active recycling of 18:2 and 18:3 FAs into TAG when seed oil breakdown is blocked in a process involving a soluble cytosolic acyltransferase.

Targeting patient recovery priorities in degenerative cervical myelopathy: design and rationale for the RECEDE-Myelopathy trial-study protocol.

INTRODUCTION: Degenerative cervical myelopathy (DCM) is a common and disabling condition of symptomatic cervical spinal cord compression secondary to degenerative changes in spinal structures leading to a mechanical stress injury of the spinal cord. RECEDE-Myelopathy aims to test the disease-modulating activity of the phosphodiesterase 3/phosphodiesterase 4 inhibitor Ibudilast as an adjuvant to surgical decompression in DCM. METHODS AND ANALYSIS: RECEDE-Myelopathy is a multicentre, double-blind, randomised, placebo-controlled trial. Participants will be randomised to receive either 60-100 mg Ibudilast or placebo starting within 10 weeks prior to surgery and continuing for 24 weeks after surgery for a maximum of 34 weeks. Adults with DCM, who have a modified Japanese Orthopaedic Association (mJOA) score 8-14 inclusive and are scheduled for their first decompressive surgery are eligible for inclusion. The coprimary endpoints are pain measured on a visual analogue scale and physical function measured by the mJOA score at 6 months after surgery. Clinical assessments will be undertaken preoperatively, postoperatively and 3, 6 and 12 months after surgery. We hypothesise that adjuvant therapy with Ibudilast leads to a meaningful and additional improvement in either pain or function, as compared with standard routine care. STUDY DESIGN: Clinical trial protocol V.2.2 October 2020. ETHICS AND DISSEMINATION: Ethical approval has been obtained from HRA-Wales.The results will be presented at an international and national scientific conferences and in a peer-reviewed journals. TRIAL REGISTRATION NUMBER: ISRCTN Number: ISRCTN16682024.

Enhancement of plant metabolite fingerprinting by machine learning.

Metabolite fingerprinting of Arabidopsis (Arabidopsis thaliana) mutants with known or predicted metabolic lesions was performed by (1)H-nuclear magnetic resonance, Fourier transform infrared, and flow injection electrospray-mass spectrometry. Fingerprinting enabled processing of five times more plants than conventional chromatographic profiling and was competitive for discriminating mutants, other than those affected in only low-abundance metabolites. Despite their rapidity and complexity, fingerprints yielded metabolomic insights (e.g. that effects of single lesions were usually not confined to individual pathways). Among fingerprint techniques, (1)H-nuclear magnetic resonance discriminated the most mutant phenotypes from the wild type and Fourier transform infrared discriminated the fewest. To maximize information from fingerprints, data analysis was crucial. One-third of distinctive phenotypes might have been overlooked had data models been confined to principal component analysis score plots. Among several methods tested, machine learning (ML) algorithms, namely support vector machine or random forest (RF) classifiers, were unsurpassed for phenotype discrimination. Support vector machines were often the best performing classifiers, but RFs yielded some particularly informative measures. First, RFs estimated margins between mutant phenotypes, whose relations could then be visualized by Sammon mapping or hierarchical clustering. Second, RFs provided importance scores for the features within fingerprints that discriminated mutants. These scores correlated with analysis of variance F values (as did Kruskal-Wallis tests, true- and false-positive measures, mutual information, and the Relief feature selection algorithm). ML classifiers, as models trained on one data set to predict another, were ideal for focused metabolomic queries, such as the distinctiveness and consistency of mutant phenotypes. Accessible software for use of ML in plant physiology is highlighted.

Trial summary and protocol for a phase II randomised placebo-controlled double-blinded trial of Interleukin 1 blockade in Acute Severe Colitis: the IASO trial.

INTRODUCTION: Acute severe ulcerative colitis (ASUC) is a severe manifestation of ulcerative colitis (UC) that warrants hospitalisation. Despite significant advances in therapeutic options for UC and in the medical management of steroid-refractory ASUC, the initial treatment paradigm has not changed since 1955 and is based on the use of intravenous corticosteroids. This treatment is successful in approximately 50% of patients but failure of this and subsequent medical therapy still occurs, with colectomy rates of up to 40% reported. The Interleukin 1 (IL-1) blockade in Acute Severe Colitis (IASO) trial aims to investigate whether antagonism of IL-1 signalling using anakinra in addition to intravenous corticosteroid treatment can improve outcomes in patients with ASUC. METHODS AND ANALYSIS: IASO is a phase II, multicentre, two-arm (parallel group), randomised (1:1), placebo-controlled, double-blinded trial of short-duration anakinra in ASUC. Its primary outcome will be the incidence of medical (eg, infliximab/ciclosporin) or surgical rescue therapy (colectomy) within 10 days following the commencement of intravenous corticosteroid therapy. Secondary outcomes will include disease activity, time to clinical response, time to rescue therapy, colectomy incidence by day 98 post intravenous corticosteroids and safety. The trial aims to recruit 214 patients across 20 sites in the UK. ETHICS AND DISSEMINATION: The trial has received approval from the Cambridge Central Research Ethics Committee (Ref: 17/EE/0347), the Health Research Authority (Ref: 201505) and Clinical Trials Authorisation from the Medicines and Healthcare products Regulatory Agency. We plan to present trial findings at scientific conferences and publish in high-impact peer-reviewed journals. TRIAL REGISTRATION NUMBER: ISRCTN43717130; EudraCT 2017-001389-10.

Correction to: Dexamethasone for adult patients with a symptomatic chronic subdural haematoma (Dex-CSDH) trial: study protocol for a randomised controlled trial.

After publication of the original article [1], the authors notified that that one of the BNTRC institutional collaborator names was misspelled.

Dex-CSDH randomised, placebo-controlled trial of dexamethasone for chronic subdural haematoma: report of the internal pilot phase.

The Dex-CSDH trial is a randomised, double-blind, placebo-controlled trial of dexamethasone for patients with a symptomatic chronic subdural haematoma. The trial commenced with an internal pilot, whose primary objective was to assess the feasibility of multi-centre recruitment. Primary outcome data collection and safety were also assessed, whilst maintaining blinding. We aimed to recruit 100 patients from United Kingdom Neurosurgical Units within 12 months. Trial participants were randomised to a 2-week course of dexamethasone or placebo in addition to receiving standard care (which could include surgery). The primary outcome measure of the trial is the modified Rankin Scale at 6 months. This pilot recruited ahead of target; 100 patients were recruited within nine months of commencement. 47% of screened patients consented to recruitment. The primary outcome measure was collected in 98% of patients. No safety concerns were raised by the independent data monitoring and ethics committee and only five patients were withdrawn from drug treatment. Pilot trial data can inform on the design and resource provision for substantive trials. This internal pilot was successful in determining recruitment feasibility. Excellent follow-up rates were achieved and exploratory outcome measures were added to increase the scientific value of the trial.

PRedicting Outcomes For Crohn's dIsease using a moLecular biomarkEr (PROFILE): protocol for a multicentre, randomised, biomarker-stratified trial.

BACKGROUND: The course of Crohn's disease (CD) varies substantially between individuals, but reliable prognostic markers do not exist. This hinders disease management because patients with aggressive disease are undertreated by conventional 'step-up' therapy (in which treatment is gradually escalated in response to refractory or relapsing disease) while those with more indolent disease would be exposed to unnecessary treatment-related toxicity if a more aggressive 'top-down' approach was indiscriminately used. The Predicting outcomes for Crohn's disease using a molecular biomarker trial will assess whether a prognostic transcriptional biomarker, that we have developed and validated, can improve clinical outcomes by facilitating personalised therapy in CD. This represents the first the biomarker-stratified trial in inflammatory bowel disease. METHODS AND ANALYSIS: This biomarker-stratified trial will compare the relative efficacy of 'top-down' and 'accelerated step-up' therapy between biomarker-defined subgroups of patients with newly diagnosed CD. 400 participants from ~50 UK centres will be recruited. Subjects within each biomarker subgroup (IBDhi or IBDlo) will be randomised (1:1) to receive one of the treatment strategies until trial completion (48 weeks). The primary outcome is the incidence of sustained surgery and steroid-free remission from the completion of induction treatment through to week 48. Secondary outcomes include mucosal healing, quality-of-life assessments and surrogate measures of disease burden including number of flares, cumulative steroid exposure, number of hospital admissions and number of Crohn's-related surgeries (assessed hierarchically). Analyses will compare the relative benefit of the treatment strategies in each biomarker-defined subgroup, powered as an interaction analysis, to determine whether the biomarker can accurately match patients to the most appropriate therapy. ETHICS AND DISSEMINATION: Ethical approval has been obtained and recruitment is under way at sites around the UK. Following trial completion and data analysis, the results of the trial will be submitted for publication in peer-reviewed journals and presented at international conferences. TRIAL REGISTRATION NUMBER: ISRCTN11808228; Pre-results.

Dexamethasone for adult patients with a symptomatic chronic subdural haematoma (Dex-CSDH) trial: study protocol for a randomised controlled trial.

BACKGROUND: Chronic subdural haematoma (CSDH) is a common neurosurgical condition, typically treated with surgical drainage of the haematoma. However, surgery is associated with mortality and morbidity, including up to 20% recurrence of the CSDH. Steroids, such as dexamethasone, have been identified as a potential therapy for reducing recurrence risk in surgically treated CSDHs. They have also been used as a conservative treatment option, thereby avoiding surgery altogether. The hypothesis of the Dex-CSDH trial is that a two-week course of dexamethasone in symptomatic patients with CSDH will lead to better functional outcome at six months. This is anticipated to occur through reduced number of hospital admissions and surgical interventions. METHODS: Dex-CSDH is a UK multi-centre, double-blind randomised controlled trial of dexamethasone versus placebo for symptomatic adult patients diagnosed with CSDH. A sample size of 750 patients has been determined, including an initial internal pilot phase of 100 patients to confirm recruitment feasibility. Patients must be recruited within 72 h of admission to a neurosurgical unit and exclusions include patients already on steroids or with steroid contraindications, patients who have a cerebrospinal fluid shunt and those with a history of psychosis. The decision regarding surgical intervention will be made by the clinical team and patients can be included in the trial regardless of whether operative treatment is planned or has been performed. The primary outcome measure is the modified Rankin Scale (mRS) at six months. Secondary outcomes include the number of CSDH-related surgical interventions during follow-up, length of hospital stay, mRS at three months, EQ-5D at three and six months, adverse events, mortality and a health-economic analysis. DISCUSSION: This multi-centre trial will provide high-quality evidence as to the effectiveness of dexamethasone in the treatment of CSDH. This has implications for patient morbidity and mortality as well as a potential economic impact on the overall health service burden from this condition. TRIAL REGISTRATION: ISRCTN, ISRCTN80782810 . Registered on 7 November 2014. EudraCT, 2014-004948-35 . Registered on 20 March 2015. Dex-CSDH trial protocol version 3, 27 Apr 2017. This protocol was developed in accordance with the SPIRIT checklist. Available as a separate document on request.

On-line diffusion profile of a lipophilic model dye in different depths of a hair follicle in human scalp skin.

In skin and hair research, drug targeting to the hair follicle is of great interest in the treatment of skin diseases. The aim of this study is to visualize on-line the diffusion processes of a model fluorophore into the hair follicle at different depths using fresh human scalp skin and confocal laser scanning microscopy. Up to a depth of 500 microm in the skin, a fast increase of fluorescence is observed in the gap followed by accumulation of the dye in the hair cuticle. Penetration was also observed via the stratum corneum and the epidermis. Little label reached depths greater than 2000 microm. Fat cells accumulated the label fastest, followed by the cuticular area and the outer root sheath of the hair follicle. Sweat glands revealed very low staining, whereas the bulb at a depth of 4000 microm was visualized only by autofluorescence. From this study, we conclude that on-line visualization is a promising technique to access diffusion processes in deep skin layers even on a cellular level. Furthermore, we conclude that the gap and the cuticle play an important role in the initial diffusion period with the label in the cuticle originating from the gap.

PhenoMeter: A Metabolome Database Search Tool Using Statistical Similarity Matching of Metabolic Phenotypes for High-Confidence Detection of Functional Links.

This article describes PhenoMeter (PM), a new type of metabolomics database search that accepts metabolite response patterns as queries and searches the MetaPhen database of reference patterns for responses that are statistically significantly similar or inverse for the purposes of detecting functional links. To identify a similarity measure that would detect functional links as reliably as possible, we compared the performance of four statistics in correctly top-matching metabolic phenotypes of Arabidopsis thaliana metabolism mutants affected in different steps of the photorespiration metabolic pathway to reference phenotypes of mutants affected in the same enzymes by independent mutations. The best performing statistic, the PM score, was a function of both Pearson correlation and Fisher's Exact Test of directional overlap. This statistic outperformed Pearson correlation, biweight midcorrelation and Fisher's Exact Test used alone. To demonstrate general applicability, we show that the PM reliably retrieved the most closely functionally linked response in the database when queried with responses to a wide variety of environmental and genetic perturbations. Attempts to match metabolic phenotypes between independent studies were met with varying success and possible reasons for this are discussed. Overall, our results suggest that integration of pattern-based search tools into metabolomics databases will aid functional annotation of newly recorded metabolic phenotypes analogously to the way sequence similarity search algorithms have aided the functional annotation of genes and proteins. PM is freely available at MetabolomeExpress (https://www.metabolome-express.org/phenometer.php).

Time and depth resolved visualisation of the diffusion of a lipophilic dye into the hair follicle of fresh unfixed human scalp skin.

Visualising the penetration pathway of a lipophilic model dye into the hair follicle of fresh unfixed human skin would facilitate optimisation of drug formulations for local delivery to the pilosebaceous unit. A block of fresh human scalp skin was mechanically fixed in a newly designed combination of cutting device/on-line diffusion cell, manual cross-sectioned perpendicular to the skin surface and sealed to create the donor and acceptor compartment. The donor phase consisted of a saturated solution of Bodipy FL C(5) in a citric acid buffer solution. Images were obtained on-line by confocal laser scanning microscopy (CLSM) every 30 min for 16 h. For each time point and each skin region relative intensity values were calculated. The on-line visualisation showed a fast diffusion of the label into the gap of the hair follicle followed by a fluorescent staining in the gap itself. The data strongly indicate that the fluorescence in the cuticle originates mainly from the dye of the gap and not from the surrounding epidermis. The on-line visualisation provides a new and excellent tool to monitor simultaneous changes in distribution profiles in the various skin layers including the hair follicle. This information can be used to determine penetration pathways in the skin.

Permeant lipophilicity and vehicle composition influence accumulation of dyes in hair follicles of human skin.

In skin and hair research drug targeting to the hair follicle is of great interest. Therefore the influence of permeant lipophilicity and vehicle composition on local accumulation has been examined using confocal laser scanning microscopy (CLSM). Formulations saturated with either Oregon Green 488, Bodipy FL C(5) or Bodipy 564/570 C(5) were prepared. The dyes were applied in citric acid buffer, 8% (w/v) surfactants in citric acid buffer or 8% (w/v) surfactants/20% (w/v) propylene glycol in citric acid buffer. Flow-through diffusion experiments were performed with fresh human scalp skin, after which the skin was imaged using CLSM. Diffusion studies showed for Oregon Green 488 (low lipophilicity) a higher flux when applied in citric acid buffer compared to surfactants. In contrast the fluxes of the more lipophilic dyes (Bodipy FL C(5) and Bodipy 564/570 C(5)) are highest when applied in surfactants/propylene glycol. CLSM studies revealed that follicular accumulation increased with (i) a lipophilic dye and (ii) application of lipophilic dyes in surfactants-propylene glycol. Therefore we conclude that targeting to the hair follicle can be increased by the use of lipophilic drugs in combination with surfactant solutions and propylene glycol.

Effect of occlusion on the percutaneous penetration of linoleic acid and glycerol.

The effect of occlusion on the in vitro percutaneous absorption of linoleic acid was investigated. A greater skin concentration of linoleic acid from an ethanolic vehicle was observed in non-occluded experiments compared with occluded experiments (P<0.05). Such changes were not observed as consistently when ethanol was replaced with a less volatile organic solvent (cyclomethicone). These observations were attributed to the increase in the concentration gradient due to the unimpeded evaporation of volatile solvents, which provided a greater driving force and enhanced non-occluded delivery in these systems, compared with occluded systems. Conversely, the percutaneous absorption of a polar material (glycerol) from an aqueous solution did not yield any such differences. While more conclusive comparisons between volatile and non-volatile solvents and penetrants would be required to substantiate fully these comparisons, it is apparent that non-occlusion of volatile solvents may enhance percutaneous absorption. The physicochemical properties of the penetrant, for example its natural state at skin temperature (i.e. solid or liquid) may further determine the degree of enhanced percutaneous absorption compared with occluded environments.

Cyanobacterial carboxysomes: microcompartments that facilitate CO2 fixation.

Carboxysomes are extraordinarily efficient proteinaceous microcompartments that encapsulate the primary CO2-fixing enzyme (ribulose-1,5-bisphosphate carboxylase/oxygenase, RuBisCO) in cyanobacteria and some proteobacteria. These microbodies form part of a CO2-concentrating mechanism (CCM), operating together with active CO2 and HCO3(-) uptake transporters which accumulate HCO3(-) in the cytoplasm of the cell. Cyanobacteria (also known as blue-green algae) are highly productive on a global scale, especially those species from open-ocean niches, which collectively contribute nearly 30% of global net primary fixation. This productivity would not be possible without a CCM which is dependent on carboxysomes. Two evolutionarily distinct forms of carboxysome are evident that encapsulate proteobacterial RuBisCO form-1A or higher-plant RuBisCO form- 1B, respectively. Based partly on RuBisCO phylogeny, the two carboxysome types are known either as α-carboxysomes, found in predominantly oceanic cyanobacteria (α-cyanobacteria) and some proteobacteria, or as ß-carboxysomes, found mainly in freshwater/estuarine cyanobacteria (ß-cyanobacteria). Both carboxysome types are believed to have evolved in parallel as a consequence of fluctuating atmospheric CO2 levels and evolutionary pressure acting via the poor enzymatic kinetics of RuBisCO. The three-dimensional structures and protein components of each carboxysome type reflect distinct evolutionarily strategies to the same major functions: subcellular compartmentalization and RuBisCO encapsulation, oxygen exclusion, and CO2 concentration and fixation.

The coenzyme a biosynthetic enzyme phosphopantetheine adenylyltransferase plays a crucial role in plant growth, salt/osmotic stress resistance, and seed lipid storage.

Coenzyme A (CoA) is an essential cofactor in the metabolism of both prokaryotic and eukaryotic organisms and a universal five-step pathway is utilized to synthesize CoA from pantothenate. Null mutations in two of the five steps of this pathway led to embryo lethality and therefore viable reduction-of-function mutations are required to further study its role in plant biology. In this article, we have characterized a viable Arabidopsis (Arabidopsis thaliana) T-DNA mutant affected in the penultimate step of the CoA biosynthesis pathway, which is catalyzed by the enzyme phosphopantetheine adenylyltransferase (PPAT). This ppat-1 knockdown mutation showed an approximately 90% reduction in PPAT transcript levels and was severely impaired in plant growth and seed production. The sum of CoA and acetyl-CoA levels was severely reduced (60%-80%) in ppat-1 seedlings compared to wild type, and catabolism of storage lipids during seedling establishment was delayed. Conversely, PPAT overexpressing lines showed, on average, approximately 1.6-fold higher levels of CoA + acetyl-CoA levels, as well as enhanced vegetative and reproductive growth and salt/osmotic stress resistance. Interestingly, dry seeds of overexpressing lines contained between 35% to 50% more fatty acids than wild type, which suggests that CoA biosynthesis plays a crucial role in storage oil accumulation. Finally, biochemical analysis of the recombinant PPAT enzyme revealed an inhibitory effect of CoA on PPAT activity. Taken together, these results suggest that the reaction catalyzed by PPAT is a regulatory step in the CoA biosynthetic pathway that plays a key role for plant growth, stress resistance, and seed lipid storage.

Blood transfusion requirements for patients with sarcomas undergoing combined radio- and chemotherapy.

Patients with bony and soft tissue sarcomas may require intensive treatment with chemotherapy and radiotherapy, which often leads to a fall in haemoglobin levels, requiring blood transfusion. There may be advantages in predicting which patients will require transfusion, partly because anaemia and hypoxia may worsen the response of tumours to chemotherapy and radiotherapy. Between 1997 and 2003, a total of 26 patients who received intensive treatment with curative intent were identified. Transfusions were given to maintain the haemoglobin at 10g/dl or above during chemotherapy, and at 12 g/dl or above during radiotherapy. Eighteen (69%) required a transfusion, the majority as a result of both the chemotherapy and RT criteria. There were 78 transfusion episodes, and 181 units of blood given. In the 18 patients who required transfusion, the average number of units was 10.1, but seven patients required more blood than this. The most significant factor influencing blood transfusion was choice of intensive chemotherapy. Intensive chemotherapy and presenting Hb less than 11.6 g/dl identified 13 out of 18 patients who needed transfusion. Adding a drop in haemoglobin of greater than 1.7 g/dl after one cycle of chemotherapy identified 16 out of 18 patients who required transfusion. The seven patients who had heavy transfusion requirements were identified by age 32 or less, intensive chemotherapy and a presenting Hb of 12 g/dl or less. Erythropoietin might be a useful alternative to transfusion in selected patient groups, especially those with heavy transfusion requirements.

On-line visualization of dye diffusion in fresh unfixed human skin.

PURPOSE: The purpose of the current study was to develop a new method to examine the diffusion in fresh unfixed human skin on-line. METHODS: Full thickness skin samples were cut perpendicular to the skin surface (cutting plane facing upwards) with a new cutting device forming part of the final diffusion cell. The donor solution contained 0.1 mg/ml Bodipy FL C5 (moderately lipophilic) dissolved in citric acid buffer, pH 5.0, and the acceptor phase consisted of phosphate-buffered saline, pH 7.4. Images were taken with confocal laser scanning microscopy (CLSM) every 10 min for 8 h. RESULTS: This new method enabled for the first time visualization of concentration profiles in different skin layers simultaneously as a function of time. For this model penetrant, Bodipy FL C5 showed that the lower stratum corneum layer constitutes the greatest barrier to diffusion. Furthermore, there is preferred partitioning of this probe in epidermis vs. either stratum corneum or dermis. CONCLUSIONS: The on-line diffusion cell in combination with CLSM is a promising tool to study diffusion processes of dyes in fresh unfixed skin on-line. The method has the potential to access deeper skin layers as well as to visualize diffusion processes in cells.