Christopher Parmenter

Combined hydrogels that switch human pluripotent stem cells from self-renewal to differentiation

Significance Stem-cell microenvironment has been identified as an important modulator of plasticity, self-renewal, and differentiation. This work details the development of a hydrogel system tailored to promote human pluripotent stem cell (HPSC) self-renewal with a simple chemical microenvironmental switch to direct differentiation. Furthermore, the timing of switching post hydrogel fabrication can promote specific lineage differentiation as in vivo. This system highlights the role of microenvironment on fate choices of pluripotent cells and demonstrates that it may be tailored to control differentiation in vitro. Importantly, this approach may improve the generation of fully differentiated tissues, as demonstrated for cardiogenic differentiation. Our combination of hydrogels allows dense tissue structures to be produced from HPSCs by using a single-step process inaccessible to any current methodology. The ability of materials to define the architecture and microenvironment experienced by cells provides new opportunities to direct the fate of human pluripotent stem cells (HPSCs) [Robinton DA, Daley GQ (2012) Nature 481(7381):295–305]. However, the conditions required for self-renewal vs. differentiation of HPSCs are different, and a single system that efficiently achieves both outcomes is not available [Giobbe GG, et al. (2012) Biotechnol Bioeng 109(12):3119–3132]. We have addressed this dual need by developing a hydrogel-based material that uses ionic de-cross-linking to remove a self-renewal permissive hydrogel (alginate) and switch to a differentiation-permissive microenvironment (collagen). Adjusting the timing of this switch can preferentially steer the HPSC differentiation to mimic lineage commitment during gastrulation to ectoderm (early switch) or mesoderm/endoderm (late switch). As an exemplar differentiated cell type, we showed that directing early lineage specification using this single system can promote cardiogenesis with increased gene expression in high-density cell populations. This work will facilitate regenerative medicine by allowing in situ HPSC expansion to be coupled with early lineage specification within defined tissue geometries.

Biomaterial modification of urinary catheters with antimicrobials to give long-term broadspectrum antibiofilm activity

Catheter-associated urinary tract infection (CAUTI) is the commonest hospital-acquired infection, accounting for over 100,000 hospital admissions within the USA annually. Biomaterials and processes intended to reduce the risk of bacterial colonization of the catheters for long-term users have not been successful, mainly because of the need for long duration of activity in flow conditions. Here we report the results of impregnation of urinary catheters with a combination of rifampicin, sparfloxacin and triclosan. In flow experiments, the antimicrobial catheters were able to prevent colonization by common uropathogens Proteus mirabilis, Staphylococcus aureus and Escherichia coli for 7 to 12weeks in vitro compared with 1-3days for other, commercially available antimicrobial catheters currently used clinically. Resistance development was minimized by careful choice of antimicrobial combinations. Drug release profiles and distribution in the polymer, and surface analysis were also carried out and the process had no deleterious effect on the mechanical performance of the catheter or its balloon. The antimicrobial catheter therefore offers for the first time a means of reducing infection and its complications in long-term urinary catheter users.

Stability of Lactobacillus rhamnosus GG in prebiotic edible films.

Highlights • The concept of prebiotic gelatine based edible films containing probiotics is presented.• Prebiotic edible films effectively protected L. rhamnosus GG.• Inulin and wheat fibre improved the storage stability of L. rhamnosus GG.• Glucose-oligosaccharides and polydextrose reduced lethality during air drying.• Prebiotics resulted in a more compact, less porous and reticular film structure.

Defining the structure of membrane-bound human blood coagulation factor Va

Summary.  Background: Blood coagulation factor (F) Va is the essential protein cofactor to the serine protease FXa. Factor Va stimulates the thrombin‐to‐prothrombin conversion by the prothrombinase complex, by at least five orders of magnitude. Factor Va binds with very high affinity to phosphatidylserine containing phospholipid membranes, which allows the visualization of its membrane‐bound state by transmission electron microscopy (EM).

Stability of Lactobacillus rhamnosus GG incorporated in edible films: Impact of anionic biopolymers and whey protein concentrate

The incorporation of probiotics and bioactive compounds, via plasticised thin-layered hydrocolloids, within food products has recently shown potential to functionalise and improve the health credentials of processed food. In this study, choice of polymer and the inclusion of whey protein isolate was evaluated for their ability to stabalise live probiotic organisms. Edible films based on low (LSA) and high (HSA) viscosity sodium alginate, low esterified amidated pectin (PEC), kappa-carrageenan/locust bean gum (κ-CAR/LBG) and gelatine (GEL) in the presence or absence of whey protein concentrate (WPC) were shown to be feasible carriers for the delivery of L. rhamnosus GG. Losses of L. rhamnosus GG throughout the drying process ranged from 0.87 to 3.06 log CFU/g for the systems without WPC, losses were significantly reduced to 0 to 1.17 log CFU/g in the presence of WPC. Storage stability (over 25d) of L. rhamnosus GG at both tested temperatures (4 and 25 °C), in descending order, was κ-CAR/LBG > HSA > GEL > LSA = PEC. In addition, supplementation of film forming agents with WPC led to a 1.8- to 6.5-fold increase in shelf-life at 4 °C (calculated on the WHO/FAO minimum requirements of 6 logCFU/g), and 1.6–4.3-fold increase at 25 °C. Furthermore probiotic films based on HSA/WPC and κ-CAR/LBG/WPC blends had both acceptable mechanical and barrier properties.

Toward Mid-Infrared, Subdiffraction, Spectral-Mapping of Human Cells and Tissue: SNIM (Scanning Near-Field Infrared Microscopy) Tip Fabrication

Scanning near-field infrared microscopy (SNIM) potentially enables subdiffraction, broadband mid-infrared (MIR: 3-25-μm wavelength range) spectral-mapping of human cells and tissue for real-time molecular sensing, with prospective use in disease diagnosis. SNIM requires an MIR-transmitting tip of small aperture for photon collection. Here, chalcogenide-glass optical-fibers are reproducibly tapered at one end to form a MIR-transmitting tip for SNIM. A wet-etching method is used to form the tip. The tapering sides of the tip are Al-coated. These Al-coated tapered-tips exhibit near-field power-confinement when acting either as the launch-end or exit-end of the MIR optical fiber. We report first time optimal cleaving of the end of the tapered tip using focused ion beam milling. A flat aperture is produced at the end of the tip, which is orthogonal to the fiber-axis and of controlled diameter. A FIB-cleaved aperture is used to collect MIR spectra of cells mounted on a transflection plate, under illumination of a synchrotron-generated wideband MIR beam.

Optimisation of octinyl succinic anhydride starch stablised w1/o/w2 emulsions for oral destablisation of encapsulated salt and enhanced saltiness

Sodium (salt) was encapsulated within the inner water phase of w1/o/w2 food emulsions externally stabilised by starch particles with the ultimate aim of enhancing saltiness perception. The physical properties of the starch particles were modified by octenyl succinic anhydride (OSA) treatment (0–3%) to vary the degree of hydrophobicity of the emulsifying starch. During oral processing native salivary amylase hydrolysed the starch and destabilised the o/w emulsion releasing the inner w/o phase and subsequently sodium into the oral cavity, resulting in a salty taste. Whilst increasing OSA treatment levels increased the stability of the emulsion, intermediate or low levels of starch modification resulted in enhanced saltiness. It is therefore proposed that 1.5% OSA modified starch is optimal for sodium delivery and 2% OSA modified starch is optimal for sodium delivery in systems that require greater process stability. It is also shown that sodium release was further enhanced by oral processing and was positively correlated with native amylase activity. The results demonstrate a promising new approach for the reduction of salt or sugar in emulsion based foods.

Making the practically impossible “Merely difficult”—Cryogenic FIB lift-out for “Damage free” soft matter imaging

The preparation of thinned lamellae from bulk samples for transmission electron microscopy (TEM) analysis has been possible in the focussed ion beam scanning electron microscope (FIB‐SEM) for over 20 years via the in situ lift‐out method. Lift‐out offers a fast and site specific preparation method for TEM analysis, typically in the field of materials science. More recently it has been applied to a low‐water content biological sample (Rubino 2012). This work presents the successful lift‐out of high‐water content lamellae, under cryogenic conditions (cryo‐FIB lift‐out) and using a nanomanipulator retaining its full range of motion, which are advances on the work previously done by Rubino (2012). Strategies are explored for maintaining cryogenic conditions, grid attachment using cryo‐condensation of water and protection of the lamella when transferring to the TEM. Microsc. Res. Tech. 79:298–303, 2016.

Cryogenic FIB Lift-out as a Preparation Method for Damage-Free Soft Matter TEM Imaging

The removal of a thinned lamella from a bulk sample for Transmission Electron Microscopy (TEM) analysis has been possible in the Focused Ion Beam Scanning Electron Microscope (FIB-SEM) for over 20 years either via in situ (by use of a micromanipulator) or ex situ lift-out approaches [1]. Both offer swift, site specific preparation for TEM analysis, particularly in light of advancements in corrected TEM. These techniques, however, are currently only applied to samples at room temperature, typically from the materials sector. The majority of biological samples contain a high degree of water, which will be removed under vacuum, leading to the shrinking and rearrangement of the sample. To overcome this, samples can be prepared by either critical point drying, fixation and resin impregnation (often combined with heavy metal staining) or cryogenic fixation. For both fixed and cryo-preserved samples, the preparation of thin-sections has always typically been prepared with a microtome, which yield samples of 60100 nm. However, these commonly suffer from compression artifacts and/ or knife marks, which distort data. There is also a desire to move away from staining and methods which dehydrate or allow / permit structural or chemical re-arrangement.

Additive manufacture of complex 3D Au-containing nanocomposites by simultaneous two-photon polymerisation and photoreduction

The fabrication of complex three-dimensional gold-containing nanocomposite structures by simultaneous two-photon polymerisation and photoreduction is demonstrated. Increased salt delivers reduced feature sizes down to line widths as small as 78 nm, a level of structural intricacy that represents a significant advance in fabrication complexity. The development of a general methodology to efficiently mix pentaerythritol triacrylate (PETA) with gold chloride hydrate (HAuCl4∙3H2O) is reported, where the gold salt concentration is adjustable on demand from zero to 20 wt%. For the first-time 7-Diethylamino-3-thenoylcoumarin (DETC) is used as the photoinitiator. Only 0.5 wt% of DETC was required to promote both polymerisation and photoreduction of up to 20 wt% of gold salt. This efficiency is the highest reported for Au-containing composite fabrication by two-photon lithography. Transmission Electron Microscopy (TEM) analysis confirmed the presence of small metallic nanoparticles (5.4 ± 1.4 nm for long axis / 3.7 ± 0.9 nm for short axis) embedded within the polymer matrix, whilst X-ray Photoelectron Spectroscopy (XPS) confirmed that they exist in the zero valent oxidation state. UV-vis spectroscopy defined that they exhibit the property of localised surface plasmon resonance (LSPR). The capability demonstrated in this study opens up new avenues for a range of applications, including plasmonics, metamaterials, flexible electronics and biosensors.