Morphology of the ferritin iron core by aberration corrected scanning transmission electron microscopy

As the major iron storage protein, ferritin stores and releases iron for maintaining the balance of iron in fauna, flora, and bacteria. We present an investigation of the morphology and iron loading of ferritin (from equine spleen) using aberration-corrected high angle annular dark field scanning transmission electron microscopy. Atom counting method, with size selected Au clusters as mass standards, was employed to determine the number of iron atoms in the nanoparticle core of each ferritin protein. Quantitative analysis shows that the nuclearity of iron atoms in the mineral core varies from a few hundred iron atoms to around 5000 atoms. Moreover, a relationship between the iron loading and iron core morphology is established, in which mineral core nucleates from a single nanoparticle, then grows along the protein shell before finally forming either a solid or hollow core structure

Quantification of fluid volume and distribution in the paediatric colon via Magnetic Resonance Imaging

Previous studies have used magnetic resonance imaging (MRI) to quantify the fluid in the stomach and small intestine of children, and the stomach, small intestine and colon of adults. This is the first study to quantify fluid volumes and distribution using MRI in the paediatric colon. MRI datasets from 28 fasted (aged 0–15 years) and 18 fluid-fed (aged 10–16 years) paediatric participants were acquired during routine clinical care. A series of 2D- and 3D-based software protocols were used to measure colonic fluid volume and localisation. The paediatric colon contained a mean volume of 22.5 mL ± 41.3 mL fluid, (range 0–167.5 mL, median volume 0.80 mL) in 15.5 ± 17.5 discreet fluid pockets (median 12). The proportion of the fluid pockets larger than 1 mL was 9.6%, which contributed to 94.5% of the total fluid volume observed. No correlation was detected between all-ages and colonic fluid volume, nor was a difference in colonic fluid volumes observed based on sex, fed state or age group based on ICH-classifications. This study quantified fluid volumes within the paediatric colon, and these data will aid and accelerate the development of biorelevant tools to progress paediatric drug development for colon-targeting formulations

Quantification of drug metabolising enzymes and transporter proteins in the paediatric duodenum via LC-MS/MS proteomics using a QconCAT technique

Characterising the small intestine absorptive membrane is essential to enable prediction of the systemic exposure of oral formulations. In particular, the ontogeny of key intestinal Drug Metabolising Enzymes and Transporter (DMET) proteins involved in drug disposition needs to be elucidated to allow for accurate prediction of the PK profile of drugs in the paediatric cohort. Using pinch biopsies from the paediatric duodenum (n = 36; aged 11 months to 15 years), the abundance of 21 DMET proteins and two enterocyte markers were quantified via LC-MS/MS. An established LCMS nanoflow method was translated to enable analysis on a microflow LC system, and a new stable-isotope-labelled QconCAT standard developed to enable quantification of these proteins. Villin-1 was used to standardise abundancy values. The observed abundancies and ontogeny profiles, agreed with adult LC-MS/MS-based data, and historic paediatric data obtained via western blotting. A linear trend with age was observed for duodenal CYP3A4 and CES2 only. As this work quantified peptides on a pinch biopsy coupled with a microflow method, future studies using a wider population range are very feasible. Furthermore, this DMET ontogeny data can be used to inform paediatric PBPK modelling and to enhance the understanding of oral drug absorption and gut bioavailability in paediatric populations

Alginate-iron speciation and its effect on in vitro cellular iron metabolism

Alginates are a class of biopolymers with known iron binding properties which are routinely used in the fabrication of iron-oxide nanoparticles. In addition, alginates have been implicated in influencing human iron absorption. However, the synthesis of iron oxide nanoparticles employs non-physiological pH conditions and whether nanoparticle formation in vivo is responsible for influencing cellular iron metabolism is unclear. Thus the aims of this study were to determine how alginate and iron interact at gastric-comparable pH conditions and how this influences iron metabolism. Employing a range of spectroscopic techniques under physiological conditions alginate-iron complexation was confirmed and, in conjunction with aberration corrected scanning transmission electron microscopy, nanoparticles were observed. The results infer a nucleation-type model of iron binding whereby alginate is templating the condensation of iron-hydroxide complexes to form iron oxide centred nanoparticles. The interaction of alginate and iron at a cellular level was found to decrease cellular iron acquisition by 37% (p < 0.05) and in combination with confocal microscopy the alginate inhibits cellular iron transport through extracellular iron chelation with the resulting complexes not internalised. These results infer alginate as being useful in the chelation of excess iron, especially in the context of inflammatory bowel disease and colorectal cancer where excess unabsorbed luminal iron is thought to be a driver of disease

Systematic review of donor and recipient predictive biomarkers of response to faecal microbiota transplantation in patients with ulcerative colitis.

BACKGROUND Faecal microbiota transplantation (FMT) has previously been explored as a treatment for ulcerative colitis (UC) however, biomarkers that predict and / or are associated with clinical response are poorly defined. The aim of this systematic review was to identify donor and recipient clinical, microbial and metabolomic predictive biomarkers of response to FMT in UC. METHODS A systematic search of the relevant literature of studies exploring FMT in UC was conducted. Data on microbial diversity, taxonomic changes, metabolic changes, donor and recipient microbiota relationship and baseline predictors was examined. FINDINGS 2852 studies were screened, and 25 papers were included in this systematic review. Following FMT, alpha diversity was seen to increase in responders along with increases in the abundance of Clostridiales clusters (order) and Bacteroides genus. Metabolomic analysis revealed short chain fatty acid (SCFA) production as a marker of FMT success. Donors or FMT batches with higher microbial alpha diversity and a greater abundance of taxa belonging to certain Bacteroides and Clostridia clusters were associated with clinical response to FMT. Baseline clinical predictors of response in patients with UC included younger age, less severe disease and possibly shorter disease duration. Baseline recipient microbial predictors at response consisted of higher faecal species richness, greater abundance of Candida and donor microbial profile similarity. INTERPRETATION Distinct changes in gut microbiota profiles post-FMT indicate that certain baseline characteristics along with specific microbial and metabolomic alterations may predispose patients towards a successful therapeutic outcome. Opportunities towards a biomarker led precision medicine approach with FMT should be explored in future clinical studies. FUNDING There no specific funding to declare

Synthesis of FITC alginate.

<p>(Ai) Reaction coupling scheme of FITC onto alginate under peptide coupling conditions. (Aii) Image of fluorescent alginate in normal light (left) and exposed to λ = 365 nm UV light (right). (B) Absorption and emission (red and blue lines respectively) spectra of the fluorescent alginate (FlAlg) product. The native alginate reactant has no absorption or emission profile, however, upon conjugation with FITC a highly absorption and emission peaks are observed.</p

Cellular localisation of alginate with confocal microscopy.

<p>Cells were treated with iron alone (control) or iron and FITC alginate with or without cell-membrane permeabilisation. (A) Cells treated with iron alone as expected showed no FITC signal. (B) Cells treated with iron and FITC alginate showed negligible punctate FITC staining on the cell periphery (C) Cells permeabilised with Saponin and then cultured with iron and FITC alginate showed an abundance of intracellular FITC signal which was mostly cytoplasmic in localisation.</p