The O-antigen of Salmonella enterica serotype Enteritidis PT4: a significant factor in gastrointestinal colonisation of young but not newly hatched chicks

The lipopolysaccharide of Salmonella and other Gram negative pathogenic species has been implicated as a major virulence determinant and in this study we report the role of LPS of S. Enteritidis in the colonisation and persistent gastrointestinal infection of young poultry. The gene encoding the unique O-antigen ligase, waaL, was mutated by insertional inactivation in a well characterised S. Enteritidis strain, S1400/94. The waaL mutant, designated PCP, produced rough colonies on agar medium, did not agglutinate O9 antiserum, did not produce an LPS ladder on silver stained gels and was serum sensitive. PCP and a nalidixic acid marked derivative of S1400/94 (S1400/94 Nal(r)) were used to orally challenge young chicks, separately and together in competitive index experiments. At post-mortem examination of 1-day-old chicks challenged S1400/94 Nal(r) and PCP separately there were no significant differences in the numbers of S1400/94 Nal(r) and PCP bacteria in tissues sampled on days 1, 2. and 5. By day 42 after challenge S1400/94 Nal(r) bacteria were recovered in significantly higher numbers than PCP from the caecal contents (P < 0.001). In competitive index studies in the 1-day-old chick PCP colonised, invaded and persisted in lower numbers than S1400/94 Nal(r). In 4-week-old chicks challenged separately, PCP bacteria were recovered from all tissues examined in significantly lower numbers than S1400/94 Nal(r). In competitive index experiments in 4-week-old chicks, PCP was not detected at any site and at any time point. Therefore, the O-antigen of S. Enteritidis plays art important role in poultry infections although this role is less important in the newly hatched chick. Crown Copyright (C) 2004 Published by Elsevier B.V. All rights reserved.

CLSM method for the dynamic observation of pH change within polymer matrices for oral delivery

If acid-sensitive drugs or cells are administered orally, there is often a reduction in efficacy associated with gastric passage. Formulation into a polymer matrix is a potential method to improve their stability. The visualization of pH within these materials may help better understand the action of these polymer systems and allow comparison of different formulations. We herein describe the development of a novel confocal laser-scanning microscopy (CLSM) method for visualizing pH changes within polymer matrices and demonstrate its applicability to an enteric formulation based on chitosan-coated alginate gels. The system in question is first shown to protect an acid-sensitive bacterial strain to low pH, before being studied by our technique. Prior to this study, it has been claimed that protection by these materials is a result of buffering, but this has not been demonstrated. The visualization of pH within these matrices during exposure to a pH 2.0 simulated gastric solution showed an encroachment of acid from the periphery of the capsule, and a persistence of pHs above 2.0 within the matrix. This implies that the protective effect of the alginate-chitosan matrices is most likely due to a combination of buffering of acid as it enters the polymer matrix and the slowing of acid penetration.

Healable supramolecular polymeric materials

This chapter details the design, synthesis and evaluation techniques required to produce healable supramolecular materials. Key developments in supramolecular polymer chemistry that laid down the design concepts necessary to produce responsive materials are summarized. Subsequently, select examples from the literature concerning the synthesis and analysis of healable materials containing hydrogen bonding, π−π stacking and metal–ligand interactions are evaluated. The last section describes the most recent efforts to produce healable gels for niche applications, including electrolytes and tissue engineering scaffolds. The chapter also describes the design criteria and production of nano-composite materials that exhibit dramatically increased strength compared to previous generations of supramolecular materials, whilst still retaining the key healing characteristics.

An improved methodology for the recovery of Zea mays and other large crop pollen, with implications for environmental archaeology in the Neotropics

We present a simple sieving methodology to aid the recovery of large cultigen pollen grains, such as maize (Zea mays L.), manioc (Manihot esculenta Crantz), and sweet potato (Ipomoea batatas L.), among others, for the detection of food production using fossil pollen analysis of lake sediments in the tropical Americas. The new methodology was tested on three large study lakes located next to known and/or excavated pre-Columbian archaeological sites in South and Central America. Five paired samples, one treated by sieving, the other prepared using standard methodology, were compared for each of the three sites. Using the new methodology, chemically digested sediment samples were passed through a 53 µm sieve, and the residue was retained, mounted in silicone oil, and counted for large cultigen pollen grains. The filtrate was mounted and analysed for pollen according to standard palynological procedures. Zea mays (L.) was recovered from the sediments of all three study lakes using the sieving technique, where no cultigen pollen had been previously recorded using the standard methodology. Confidence intervals demonstrate there is no significant difference in pollen assemblages between the sieved versus unsieved samples. Equal numbers of exotic Lycopodium spores added to both the filtrate and residue of the sieved samples allow for direct comparison of cultigen pollen abundance with the standard terrestrial pollen count. Our technique enables the isolation and rapid scanning for maize and other cultigen pollen in lake sediments, which, in conjunction with charcoal and pollen records, is key to determining land-use patterns and the environmental impact of pre-Columbian societies.

Concurrent recordings of bladder afferents from multiple nerves using a microfabricated PDMS microchannel electrode array

In this paper we present a compliant neural interface designed to record bladder afferent activity. We developed the implant's microfabrication process using multiple layers of silicone rubber and thin metal so that a gold microelectrode array is embedded within four parallel polydimethylsiloxane (PDMS) microchannels (5 mm long, 100 μm wide, 100 μm deep). Electrode impedance at 1 kHz was optimized using a reactive ion etching (RIE) step, which increased the porosity of the electrode surface. The electrodes did not deteriorate after a 3 month immersion in phosphate buffered saline (PBS) at 37 °C. Due to the unique microscopic topography of the metal film on PDMS, the electrodes are extremely compliant and can withstand handling during implantation (twisting and bending) without electrical failure. The device was transplanted acutely to anaesthetized rats, and strands of the dorsal branch of roots L6 and S1 were surgically teased and inserted in three microchannels under saline immersion to allow for simultaneous in vivo recordings in an acute setting. We utilized a tripole electrode configuration to maintain background noise low and improve the signal to noise ratio. The device could distinguish two types of afferent nerve activity related to increasing bladder filling and contraction. To our knowledge, this is the first report of multichannel recordings of bladder afferent activity.

Oxidized alginate hydrogels as niche environments for corneal epithelial cells

Chemical and biochemical modification of hydrogels is one strategy to create physiological constructs that maintain cell function. The aim of this study was to apply oxidised alginate hydrogels as a basis for development of a biomimetic niche for limbal epithelial stem cells that may be applied to treating corneal dysfunction. The stem phenotype of bovine limbal epithelial cells (LEC) and the viability of corneal epithelial cells (CEC) were examined in oxidised alginate gels containing collagen IV over a 3-day culture period. Oxidation increased cell viability (P gels presented larger internal pores (diameter: 0.2 - 0.8 microm) than unmodified gels (pore diameter: 0.05 - 0.1 microm) and were significantly less stiff (P gels was similar to that of unmodified gels suggesting that oxidation may not affect the retention of extracellular matrix proteins in alginate gels. These data demonstrate that oxidised alginate gels containing corneal extracellular matrix proteins can influence corneal epithelial cell function in a manner that may impact beneficially on corneal wound healing therapy.

The secretome of alginate-encapsulated limbal epithelial stem cells modulates corneal epithelial cell proliferation

Limbal epithelial stem cells may ameliorate limbal stem cell deficiency through secretion of therapeutic proteins, delivered to the cornea in a controlled manner using hydrogels. In the present study the secretome of alginate-encapsulated limbal epithelial stem cells is investigated. Conditioned medium was generated from limbal epithelial stem cells encapsulated in 1.2% (w/v) calcium alginate gels. Conditioned medium proteins separated by 1-D gel electrophoresis were visualized by silver staining. Proteins of interest including secreted protein acidic and rich in cysteine, profilin-1, and galectin-1 were identified by immunoblotting. The effect of conditioned medium (from alginate-encapsulated limbal epithelial stem cells) on corneal epithelial cell proliferation was quantified and shown to significantly inhibit (Pgels may regulate corneal epithelialisation through secretion of inhibitory proteins.

Control and analysis of oriented thin films of lipid inverse bicontinuous cubic phases using grazing incidence small-angle X‑ray scattering

Lipid cubic phases are complex nanostructures that form naturally in a variety of biological systems, with applications including drug delivery and nanotemplating. Most X-ray scattering studies on lipid cubic phases have used unoriented polydomain samples as either bulk gels or suspensions of micrometer-sized cubosomes. We present a method of investigating cubic phases in a new form, as supported thin films that can be analyzed using grazing incidence small-angle X-ray scattering (GISAXS). We present GISAXS data on three lipid systems: phytantriol and two grades of monoolein (research and industrial). The use of thin films brings a number of advantages. First, the samples exhibit a high degree of uniaxial orientation about the substrate normal. Second, the new morphology allows precise control of the substrate mesophase geometry and lattice parameter using a controlled temperature and humidity environment, and we demonstrate the controllable formation of oriented diamond and gyroid inverse bicontinuous cubic along with lamellar phases. Finally, the thin film morphology allows the induction of reversible phase transitions between these mesophase structures by changes in humidity on subminute time scales, and we present timeresolved GISAXS data monitoring these transformations.

Instantaneous gelation in Smoluchowski’s coagulation equation revisited

We study the solutions of the Smoluchowski coagulation equation with a regularization term which removes clusters from the system when their mass exceeds a specified cutoff size, M. We focus primarily on collision kernels which would exhibit an instantaneous gelation transition in the absence of any regularization. Numerical simulations demonstrate that for such kernels with monodisperse initial data, the regularized gelation time decreasesas M increases, consistent with the expectation that the gelation time is zero in the unregularized system. This decrease appears to be a logarithmically slow function of M, indicating that instantaneously gelling kernels may still be justifiable as physical models despite the fact that they are highly singular in the absence of a cutoff. We also study the case when a source of monomers is introduced in the regularized system. In this case a stationary state is reached. We present a complete analytic description of this regularized stationary state for the model kernel, K(m1,m2)=max{m1,m2}ν, which gels instantaneously when M→∞ if ν>1. The stationary cluster size distribution decays as a stretched exponential for small cluster sizes and crosses over to a power law decay with exponent ν for large cluster sizes. The total particle density in the stationary state slowly vanishes as [(ν−1)logM]−1/2 when M→∞. The approach to the stationary state is nontrivial: Oscillations about the stationary state emerge from the interplay between the monomer injection and the cutoff, M, which decay very slowly when M is large. A quantitative analysis of these oscillations is provided for the addition model which describes the situation in which clusters can only grow by absorbing monomers.

Time-dependent gel to gel transformation of a peptide based supramolecular gelator

A dipeptide with a long fatty acid chain at its N-terminus gives hydrogels in phosphate buffer in the pH range 7.0–8.5. The hydrogel with a gelator concentration of 0.45% (w/v) at pH 7.46 (physiological pH) provides a very good platform to study dynamic changes within a supramolecular framework as it exhibits remarkable change in its appearance with time. Interestingly, the first formed transparent hydrogel gradually transforms into a turbid gel within 2 days. These two forms of the hydrogel have been thoroughly investigated by using small angle X-ray scattering (SAXS), powder X-ray diffraction (PXRD), field emission scanning electron microscopic (FE-SEM) and high-resolution transmission electron microscopic (HR-TEM) imaging, FT-IR and rheometric analyses. The SAXS and low angle PXRD studies substantiate different packing arrangements for the gelator molecules for these two different gel states (the freshly prepared and the aged hydrogel). Moreover, rheological studies of these two gels reveal that the aged gel is stiffer than the freshly prepared gel.

Development of a wearable assistive soft robotic device for elbow rehabilitation

The loss of motor function at the elbow joint can result as a consequence of stroke. Stroke is a clinical illness resulting in long lasting neurological deficits often affecting somatosensory and motor cortices. More than half of those that recover from a stroke survive with disability in their upper arm and need rehabilitation therapy to help in regaining functions of daily living. In this paper, we demonstrated a prototype of a low-cost, ultra-light and wearable soft robotic assistive device that could aid administration of elbow motion therapies to stroke patients. In order to assist the rotation of the elbow joint, the soft modules which consist of soft wedge-like cellular units was inflated by air to produce torque at the elbow joint. Highly compliant rotation can be naturally realised by the elastic property of soft silicone and pneumatic control of air. Based on the direct visual-actuation control, a higher control loop utilised visual processing to apply positional control, the lower control loop was implemented by an electronic circuit to achieve the desired pressure of the soft modules by Pulse Width Modulation. To examine the functionality of the proposed soft modular system, we used an anatomical model of the upper limb and performed the experiments with healthy participants.

Donor-acceptor π−π stacking interactions: from small molecule complexes to healable supramolecular polymer networks

This chapter presents selected literature examples to review the development of the use of donor–acceptor π–π stacking interactions as transient cross-links in supramolecular polymer networks. The chapter examines notable examples of these highly specific and directional interactions and illustrates how they can be utilised to reliably produce functional supramolecular, self-assembled systems. Knowledge gained from these fundamental studies has enabled the design, synthesis and application of donor–acceptor stacked supramolecular motifs in non-covalent polymer networks, which is exemplified through detailing the production, physical properties and optimisation of healable materials.

The efficacy of potassium sorbate-coated packaging to control postharvest gray mold in raspberries, blackberries, and blueberries

The aim of this work is to build on the success of in vitro studies of an active packaging, produced by coating the surface of post-consumer recycled polyethylene terephthalate (PCRPET) package with an aqueous silicone solution (2%, v/v) containing an antifungal agent (potassium sorbate, KS). Antifungal efficacy was evaluated, in vivo, during the storage of raspberries, blackberries and blueberries by examining their shelf life extension. The packaging effectively delayed the growth of Botrytis by extending its lag-phase, which, in turn, extended the shelf life of the berries by up to 3d. Among the three berries tested, the packaging proved to be more advantageous in the case of raspberries, due to their physiological characteristics and shorter shelf life. Based on sensory panel evaluations, it was shown that the coating, containing KS, did not influence the packaging appearance and transparency, and the fruit did not suffer from any off-flavor development.

Dehydrodipeptide hydrogelators containing naproxen N‑Capped tryptophan: self-assembly, hydrogel characterization, and evaluation as potential drug nanocarriers

In this work, we introduce dipeptides containing tryptophan N-capped with the nonsteroidal anti-inflammatory drug naproxen and C-terminal dehydroamino acids, dehydrophenylalanine (ΔPhe), dehydroaminobutyric acid (ΔAbu), and dehydroalanine (ΔAla) as efficacious protease resistant hydrogelators. Optimized conditions for gel formation are reported. Transmission electron microscopy experiments revealed that the hydrogels consist of networks of micro/nanosized fibers formed by peptide self-assembly. Fluorescence and circular dichroism spectroscopy indicate that the self-assembly process is driven by stacking interactions of the aromatic groups. The naphthalene groups of the naproxen moieties are highly organized in the fibers through chiral stacking. Rheological experiments demonstrated that the most hydrophobic peptide (containing C-terminal ΔPhe) formed more elastic gels at lower critical gelation concentrations. This gel revealed irreversible breakup, while the C-terminal ΔAbu and ΔAla gels, although less elastic, exhibited structural recovery and partial healing of the elastic properties. A potential antitumor thieno[3,2-b]pyridine derivative was incorporated (noncovalently) into the gel formed by the hydrogelator containing C-terminal ΔPhe residue. Fluorescence and Förster resonance energy transfer measurements indicate that the drug is located in a hydrophobic environment, near/associated with the peptide fibers, establishing this type of hydrogel as a good drug-nanocarrier candidate.

In situ gelling systems based on Pluronic F127/Pluronic F68 formulations for ocular drug delivery

This study evaluated the use of Pluronic F127 and Pluronic F68 as excipients for formulating in situ gelling systems for ocular drug delivery. Thermal transitions have been studied in aqueous solutions of Pluronic F127, Pluronic F68 as well as their binary mixtures using differential scanning calorimetry, rheological measurements, and dynamic light scattering. It was established that the formation of transparent gels at physiologically relevant temperatures is observed only in the case of 20 wt % of Pluronic F127. The addition of Pluronic F68 to Pluronic F127 solutions increases the gelation temperature of binary formulation to above physiological range of temperatures. The biocompatibility evaluation of these formulations using slug mucosa irritation assay and bovine corneal erosion studies revealed that these polymers and their combinations do not cause significant irritation. In vitro drug retention study on glass surfaces and freshly excised bovine cornea showed superior performance of 20 wt % Pluronic F127 compared to other formulations. In addition, in vivo studies in rabbits demonstrated better retention performance of 20 wt % Pluronic F127 compared to Pluronic F68. These results confirmed that 20 wt % Pluronic F127 offers an attractive ocular formulation that can form a transparent gel in situ under physiological conditions with minimal irritation.