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The rehydration properties of original whey protein isolate (WPIC) powder and spray-dried WPI prepared from either unheated (WPIUH) or nanoparticulated WPI solutions were investigated. Nanoparticulation of whey proteins was achieved by subjecting reconstituted WPIC solutions (10% protein, w/w, pH 7.0) to heat treatment at 90 °C for 30 s with no added calcium (WPIH) or with 2.5 mM added calcium (WPIHCa). Powder surface nanostructure and elemental composition were investigated using atomic force microscopy and X-ray photoelectron spectroscopy, followed by dynamic visualisation of wetting and dissolution characteristics using environmental scanning electron microscopy. The surface of powder particles for both WPIUH and WPIC samples generally appeared smooth, while WPIH and WPIHCa displayed micro-wrinkles with more significant deposition of nitrogen and calcium elements. WPIH and WPIHCa exhibited lower wettability and solubility performance than WPIUH and WPIC during microscopic observation. This study demonstrated that heat-induced aggregation of whey proteins, in the presence or absence of added calcium, before drying increases aggregate size, alters the powder surface properties, consequently impairing their wetting characteristics. This study also developed a fundamental understanding of WPI powder obtained from nanoparticulated whey proteins, which could be applied for the development of functional whey-based ingredients in food formulations, such as nanospacers to modulate protein–protein interactions in dairy concentrates.

Sonodynamic therapy (SDT) is an emerging stimulus-responsive approach for the targeted treatment of solid tumours. However, its ability to generate stimulus-responsive cytotoxic reactive oxygen species (ROS), is compromised by tumour hypoxia. Here we describe a robust means of preparing a pH-sensitive polymethacrylate-coated CaO2 nanoparticle that is capable of transiently alleviating tumour hypoxia. Systemic administration of particles to animals bearing human xenograft BxPC3 pancreatic tumours increases oxygen partial pressures (PO2) to 20–50 mmHg for over 40 min. RT-qPCR analysis of expression of selected tumour marker genes in treated animals suggests that the transient production of oxygen is sufficient to elicit effects at a molecular genetic level. Using particles labelled with the near infra-red (nIR) fluorescent dye, indocyanine green, selective uptake of particles by tumours was observed. Systemic administration of particles containing Rose Bengal (RB) at concentrations of 0.1 mg/mg of particles are capable of eliciting nanoparticle-induced, SDT-mediated antitumour effects using the BxPC3 human pancreatic tumour model in immuno-compromised mice. Additionally, a potent abscopal effect was observed in off-target tumours in a syngeneic murine bilateral tumour model for pancreatic cancer and an increase in tumour cytotoxic T cells (CD8+) and a decrease in immunosuppressive tumour regulatory T cells [Treg (CD4+, FoxP3+)] was observed in both target and off-target tumours in SDT treated animals. We suggest that this approach offers significant potential in the treatment of both focal and disseminated (metastatic) pancreatic cancer.

Although widely accepted as a water sterilisation technique, chlorination results in the production of potentially harmful by-products, mainly Trihalomethanes. Furthermore, the chlorination process requires specialised infrastructure, management and high costs. In this research paper a potential alternative sterilisation technique was investigated. This rapid three-step process utilized Goethite Nanoparticles and the photosensitising capabilities of a xanthene dye. Rose Bengal (RB) a compound primarily used as a stain to diagnose damaged tissue in the eye was utilized under visible light excitation to sterilise water containing gram-positive Staphylococcus aureus and Bacillus cereus. Bacterial reductions (cfu/ml) of up to 6log10 are reported at RB concentrations of 0.5 mg/L and 10 mg/L for S. aureus and B. cereus, respectively. Goethite Nanoparticles (GNP's), an iron oxyhydroxide, were synthesised by co-precipitation of iron salts and used to adsorb RB post-sterilisation. Poly-vinyl Alcohol (PVA) functionalised GNP's were synthesised to highlight the adsorbent capabilities of the GNP surface. The adsorption capacity for uncoated GNPs was 476.19 mg/g, this reduced to 170.4 mg/g for PVA-GNP's, highlighting the highly porous nature of the synthesised GNP surface. Adsorption was optimal in slightly acidic conditions (pH5-6). The adsorption parameters best followed Lagergens Pseudo-second order kinetics with correlation coefficients close to unity. At the highest envisaged RB concentration (10 mg/L) approximately 20 mg/L GNP's was required to remove the dye from solution post-treatment. Flame Atomic Absorption analysis of the water post-removal revealed Iron concentrations of 0.058 mg/L. This correlates to removal efficacy of 99.71% with residual iron levels below the EU recommended limit of 0.2 mg/L.

Spray-dried whey protein isolate (WPI) powders were prepared at pilot-scale from solutions without heat (WPIUH), heated (WPIH) or heated with calcium (WPIHCa), which were analysed and compared with a control sample (WPIC). WPIC, WPIUH, WPIH and WPIHCa solutions had whey protein denaturation levels of 0.0, 3.2, 64.4 and 74.4%, respectively. Computerised tomography scanning showed that 52.6, 84.0, 74.5 and 41.9% of WPIC, WPIUH, WPIH and WPIHCa powder particles had diameters of ≤30 µm. WPIHCa and WPIH powders were cohesive, while WPIC and WPIUH powders were easy flowing. Marked differences in microstructure were observed between WPIH and WPIHCa. There were no measured differences in wall friction, bulk density or colour.

One current strategy to deal with the serious issue of antibiotic resistance is to use biosurfactants, weak antimicrobials in their own right, with antibiotics in order to extend the efficacy of antibiotics. Although an adjuvant effect has been observed, the underlying mechanisms are poorly understood. To investigate the nature of the antibiotic and biosurfactant interaction, we undertook a scanning electron microscopy (SEM) and atomic force microscopy (AFM) microscopic study of the effects of the tetracycline antibiotic, combined with sophorolipid and rhamnolipid biosurfactants, on Methicillin-resistant Staphylococcus aureus using tetracycline concentrations below and above the minimum inhibitory concentration (MIC). Control and treated bacterial samples were prepared with an immersion technique by adsorbing the bacteria onto glass substrates grafted with the poly-cationic polymer polyethyleneimine. Bacterial surface morphology, hydrophobic and hydrophilic surface characters as well as the local bacterial cell stiffness were measured following combined antibiotic and biosurfactant treatment. The sophorolipid biosurfactant stands alone insofar as, when used with the antibiotic at sub-MIC concentration, it resulted in bacterial morphological changes, larger diameters (from 758 ± 75 to 1276 ± 220 nm, p-value = 10-4) as well as increased bacterial core stiffness (from 205 ± 46 to 396 ± 66 mN/m, p-value = 5 × 10-5). This investigation demonstrates that such combination of microscopic analysis can give useful information which could complement biological assays to understand the mechanisms of synergy between antibiotics and bioactive molecules such as biosurfactants.

A PLGA-based multifunctional biodegradable nanoparticle platform co-harboring hematoporphyrin and indocyanine green has been developed. In vitro studies demonstrate ultrasound and light stimulated generation of cytotoxic reactive oxygen species. In vivo studies show that the ICG component facilitates nIR fluorescence imaging that demonstrates accumulation of IV- administered nanoparticles in tumours. In vivo studies also demonstrate ultrasound- and light-mediated inhibition of tumour growth in animals treated with the platform. Since the platform consists entirely of clinically-approved agents it could find use in sonodynamic- and photodynamic-based therapies for cancer. Statement of Significance We describe a biocompatible and biodegradable nanoparticle-based platform for use in sonodynamic and photodynamic therapeutic approaches for the treatment of cancer. The non-toxic nanoparticles produce cytotoxic reactive oxygen species when exposed to ultrasound and/or light at levels that have no impact on tissues. The system is unique in that it is accumulated by tumours within six hours and has the ability to release its sensitising capability while retaining its imaging capability within a therapeutic time frame. The former could enhance dispersion and sensitising capabilities in less permeable tumour tissues and the latter permits the design of therapeutic approaches that minimize collateral damage to normal tissues.

Composition and relative humidity (RH) can have a profound impact on the physical (flowability, stickiness) and functional (reconstitution) properties of milk powder (MP) and therefore its quality, storage stability and shelf-life. Conventional microscopic techniques are not capable of dynamically imaging the effect of RH on MP at high magnification. The aim of this study was to develop a novel method to characterise in-situ and in real time the hydration and reconstitution of five spray-dried milk protein concentrates (MPCs) using an Environmental Scanning Electron Microscope (ESEM). ESEM was employed to observe the surface microstructure of MPC powders with varying protein content (38.63%–80.94%, w/w), at various RH values ranging from 35% to over 100%. MPC powders were imaged by an ESEM without any prior preparation, and with minimal physical sample alteration, thus providing fundamental insights into MPC hydration and reconstitution. ESEM surface analysis showed particle swelling in all MPCs, and that with increasing protein content, hydration and reconstitution efficiency decreased. For the first time, dynamic particle surface fusion was observed. Such fusion can result in stickiness and caking over time. ESEM methods developed here may provide mechanistic insights into the effects of RH during storage. Surface re-arrangement was also observed in all MPCs, but was impeded in MPC70 and MPC80 thus indicating that this is the rate limiting step for MPC reconstitution. This work validates the use of an ESEM to dynamically characterise MPC powder hydration and reconstitution in-situ and in real-time, at both high magnification and spatial resolution.

Pterygium is a pathological proliferative condition of the ocular surface, characterised by formation of a highly vascularised, fibrous tissue arising from the limbus that invades the central cornea leading to visual disturbance and, if untreated, blindness. Whilst chronic ultraviolet (UV) light exposure plays a major role in its pathogenesis, higher susceptibility to pterygium is observed in some families, suggesting a genetic component. In this study, a Northern Irish family affected by pterygium but reporting little direct exposure to UV was identified carrying a missense variant in CRIM1 NM_016441.2: c.1235 A > C (H412P) through whole-exome sequencing and subsequent analysis. CRIM1 is expressed in the developing eye, adult cornea and conjunctiva, having a role in cell differentiation and migration but also in angiogenesis, all processes involved in pterygium formation. We demonstrate elevated CRIM1 expression in pterygium tissue from additional individual Northern Irish patients compared to unaffected conjunctival controls. UV irradiation of HCE-S cells resulted in an increase in ERK phosphorylation and CRIM1 expression, the latter further elevated by the addition of the MEK1/2 inhibitor, U0126. Conversely, siRNA knockdown of CRIM1 led to decreased UV-induced ERK phosphorylation and increased BCL2 expression. Transient expression of the mutant H412P CRIM1 in corneal epithelial HCE-S cells showed that, unlike wild-type CRIM1, it was unable to reduce the cell proliferation, increased ERK phosphorylation and apoptosis induced through a decrease of BCL2 expression levels. We propose here a series of intracellular events where CRIM1 regulation of the ERK pathway prevents UV-induced cell proliferation and may play an important role in the in the pathogenesis of pterygium.

Clostridium difficile infection is a growing problem in healthcare settings worldwide and results in a considerable socioeconomic impact. New hypervirulent strains and acquisition of antibiotic resistance exacerbates pathogenesis; however, the survival strategy of C. difficile in the challenging gut environment still remains incompletely understood. We previously reported that clinically relevant heat-stress (37–41 °C) resulted in a classical heat-stress response with up-regulation of cellular chaperones. We used ClosTron to construct an insertional mutation in the dnaK gene of C. difficile 630 Δerm. The dnaK mutant exhibited temperature sensitivity, grew more slowly than C. difficile 630 Δerm and was less thermotolerant. Furthermore, the mutant was non-motile, had 4-fold lower expression of the fliC gene and lacked flagella on the cell surface. Mutant cells were some 50% longer than parental strain cells, and at optimal growth temperatures, they exhibited a 4-fold increase in the expression of class I chaperone genes including GroEL and GroES. Increased chaperone expression, in addition to the non-flagellated phenotype of the mutant, may account for the increased biofilm formation observed. Overall, the phenotype resulting from dnaK disruption is more akin to that observed in Escherichia coli dnaK mutants, rather than those in the Gram-positive model organism Bacillus subtilis.

Size, encapsulation efficiency and stability affect the sustained release from nanoparticles containing protein-type drugs.Insulin was used to evaluate effects of formulation parameters on minimizing diameter, maximizing encapsulation efficiency and preserving blood glucose control following intraperitoneal (IP) administration.Homogenization or sonication was used to incorporate insulin into poly(D,L-lactic-co-glycolic acid) (PLGA) nanoparticles with increasing poly(ethylene glycol) (PEG) content. Effects of polymer type, insulin/polymer loading ratio and stabilizer in the internal aqueous phase on physicochemical characteristics of NP, in vitro release and stability of encapsulated insulin were investigated. Entrapment efficiency and release were assessed by radioimmunoassay and bicinconnic acid protein assay, and stability was evaluated using SDS-PAGE. Bioactivity of insulin was assessed in streptozotocin-induced, insulin-deficient Type I diabetic mice.Increasing polymeric PEG increased encapsulation efficiency, while the absence of internal stabilizer improved encapsulation and minimized burst release kinetics. Homogenization was shown to be superior to sonication, with NP fabricated from 10% PEG-PLGA having higher insulin encapsulation, lower burst release and better stability. Insulin-loaded NP maintained normoglycaemia for 24 h in diabetic mice following a single bolus, with no evidence of hypoglycemia.Insulin-loaded NP prepared from 10% PEG-PLGA possessed therapeutically useful encapsulation and release kinetics when delivered by the IP route.

Drug delivery to the brain is still a major challenge in the field of therapeutics, especially for large and hydrophilic compounds. In order to achieve drug delivery of therapeutic concentration in the central nervous system, the problematic blood brain barrier (BBB) must be overcome. This work presents the formulation of a targeted nanoparticle-based drug delivery system using a specific neural cell targeting ligand, rabies virus derived peptide (RDP). Characterization studies revealed that RDP could be conjugated to drug-loaded PLGA nanoparticles of average diameter 257.10 ± 22.39 nm and zeta potential of −5.51 ± 0.73 mV. In vitro studies showed that addition of RDP to nanoparticles enhanced drug accumulation in a neural cell line specifically as opposed to non-neural cell lines. It was revealed that this drug delivery system is reliant upon nicotinic acetylcholine receptor (nAChR) function for RDP-facilitated effects, supporting a cellular uptake mechanism of action. The specific neural cell targeting capabilities of RDP via the nAChR offers a non-toxic, non-invasive and promising approach to the delivery of therapeutics to the brain.

With the advance of nanotechnology in biomaterials science and tissue engineering, it is essential that new techniques become available to observe processes that take place at the direct interface between tissue and scaffold materials. Here, Cryo DualBeam focused ion beam-scanning electron microscopy (FIB-SEM) was used as a novel approach to observe the interactions between frozen hydrated cells and nanometric structures in high detail. Through a comparison of images acquired with transmission electron microscopy (TEM), conventional FIB-SEM operated at ambient temperature, and Cryo DualBeam FIB-SEM, the advantages and disadvantages of each technique were evaluated. Ultrastructural details of both (extra)cellular components and cell organelles were best observe with TEM. However, processing artifacts such as shrinkage of cells at the substrate interface were introduced in both TEM and conventional FIB-SEM. In addition, the cellular contrast in conventional FIB-SEM was low; consequently, cells were difficult to distinguish from the adjoining substrate. Cryo DualBeam FIB-SEM did preserve (extra)cellular details like the contour, cell membrane, and mineralized matrix. The three described techniques have proven to be complementary for the evaluation of processes that take place at the interface between tissue and substrate.

A combined scanning electron microscope and focussed ion beam instrument is suitable for micro- and nanopatterning, cross-sectioning and subsequent imaging, of specimens at room temperature as well as under cryo conditions. In order to reveal internal details, samples are conventionally milled with the ion beam positioned perpendicular to the sample surface. Using this approach certain limitations are frequently encountered, e.g. accumulation of redeposited material, shadowing effects, image distortion and a limited imaging area. Here we show an approach in which samples are pre-trimmed using a microtome to obtain a sample block face that is parallel to the ion beam. This new grazing incidence geometry eliminates the need for removal of bulk material with the ion beam and enables immediate fine polishing of a pre-selected area of interest. Many of the limitations previously described are avoided and in addition milling time is reduced, whilst creating larger cross-sectional areas. Another advantage is that electron imaging can be accomplished by tilting the sample surface perpendicular to the electron beam, providing a geometrically undistorted image. The proposed approach is suitable for materials that can be microtomed, both in ambient and cryogenic conditions, and proves to be of particular benefit for biological and food samples.

Direct pharmacological targeting of the anti-apoptotic B-cell lymphoma-2 (BCL-2) family is an attractive therapeutic strategy for treating cancer. Obatoclax is a pan-BCL-2 family inhibitor currently in clinical development. Here we show that, although obatoclax can induce mitochondrial apoptosis dependent on BCL-2 associated x protein/BCL-2 antagonist killer (BAX/BAK) consistent with its on-target pharmacodynamics, simultaneous silencing of both BAX and BAK did not abolish acute toxicity or loss of clonogenicity. This is despite complete inhibition of apoptosis. Obatoclax dramatically reduced viability without inducing loss of plasma membrane integrity. This was associated with rapid processing of light chain-3 (LC3) and reduction of S6 kinase phosphorylation, consistent with autophagy. Dramatic ultrastructural vacuolation, not typical of autophagy, was also induced. Silencing of beclin-1 failed to prevent LC3 processing, whereas knockout of autophagy-related (Atg)7 abolished LC3 processing but failed to prevent obatoclax-induced loss of clonogenicity or ultrastructural changes. siRNA silencing of Atg7 in BAX/BAK knockout mouse embryonic fibroblasts did not prevent obatoclax-induced loss of viability. Cells selected for obatoclax resistance evaded apoptosis independent of changes in BCL-2 family expression and displayed reduced LC3 processing. In summary, obatoclax exhibits BAX- and BAK-dependent and -independent mechanisms of toxicity and activation of autophagy. Mechanisms other than autophagy and apoptosis are blocked in obatoclax resistant cells and contribute significantly to obatoclax's anticancer efficacy.

The prudent use of the atomic force microscope as a supra-vital live cell imaging tool requires that cell viability must be determined before and after scanning. Complementary optical techniques in conjunction with the fluorescent dyes rhodamine-123 and ethidium homodimer have been used within this study to determine cell viability after increasing loads are applied in contact mode. Guideline force ranges for five commonly cultured cell lines, human squamous carcinoma (A431), fibroblast, HeLa, Potorous tridactylis (PtK2) and rat intestinal epithelial (RIE) cells are given.

This work describes a system for precise re-location of cells within a monolayer after atomic force imaging. As we know little about probe interaction with soft biological surfaces any corroborative evidence is of great importance. For example, it is of paramount importance in living cell force microscopy that interrogated cells can be re-located and imaged by other corroborative technologies. Methodologies expressed here have shown that non-invasive force parameters can be established for specific cell types. Additionally, we show that the same sample can be transferred reliably to an SEM. Results here indicate that further work with live cells should initially establish appropriate prevailing force parameters and that cell damage should be checked for before and after an imaging experiment.

A hysteretic spin transition is induced in a solution assembly of a mononuclear Fe(III) amphiphilic complex which exhibits only gradual spin crossover in the solid state. The hysteretic behavior is dependent on dynamic solution assembly and removal of solvent causes reversion to the original bulk solid magnetic response.

Background: Failure to induce apoptosis is a major limitation of conventional chemotherapy for lung cancer. Resistance to apoptosis contributes to this chemoresistant phenotype. New therapeutic strategies are therefore required to bypass block in apoptosis. Obatoclax (obx) is a small molecule inhibitor of pro-survival BCL-2 family proteins currently being clinically evaluated in both small-cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). However, little is known regarding the molecular mechanisms that might underlie sensitivity and resistance to obx. We are addressing this important question by exploring the pharmacodynamics of obx in NSCLC. Methods: To isolate mitochondria harvested cells were homogenized in ice-cold isolation buffer and the resulting homogenate subjected to differential centrifugation to isolate the mitochondrial fraction. To conduct shRNA transfections, shRNA plasmids targeted against BAX, BAK, BIM and beclin1 were purchased from SA Biosciences (Frederick, MD, USA). H460 cells were transfected with each plasmid and cells were subjected to at least two rounds of selection to create stable clones. For transmission electron microscopy, cells were fixed in double strength TEM fixative (4% paraformaldehyde, 2.5% glutaraldehyde) in 0.1M sodium cacodylate buffer. Samples were analyzed using standard EM procedures. Specific hVps34 Activity was measured in cells deprived of amino acids for 2 hrs in EBSS. Cells were lysed and subjected to immunoprecipitation with anti-hVps34 antibody, and immunocomplexes were assayed for hVps34 lipid kinase activity. Results: shRNA downregulation of BAX/BAK (H460shBAX/BAK) inhibited obx induced caspase 9/3 cleavage, PARP activation, and mitochondrial outer membrane permeabilization. However obx was equally effective in reducing clonogenic survival, and short term viability (48–72 hrs). Obx induced prolific cytoplasmic vacuolation evident by TEM, and processing of LC3I to LC3II was evident in NSCLC H460 and H1975 cells, and SCLC H196 and H146 cells suggesting induction of autophagy. H460shBAX/BAK clones also displayed unaltered LC3 processing. To determine whether LC3 processing following obx was regulated by beclin1, stable knockdown was acomplished using shRNA. 3 clones were selected and exhibited reduced hVpS34 activity, however obatoclax induced equal clonogenic survival and LC3 processing compared to NT shRNA controls. Neither LC3 nor PARP processing was inhibited by 3MA or wortmannin. Also we observed loss of beclin1 expression following obx. Activation of JNK and ERK have been implicated in beclin1 indpendent mitophagy, while JNK is also a known activator of BAX and BIM. Obx induced phosphorylation of JNK but not ERK, and inhibition of pJNK blocked PARP cleavage. Also, stable knockdown of BIM by shRNA did not significantly alter obx EC50 values. In H727 NSCLC cells resistant to obx induced apoptosis, JNK phosphorylation and LC3II formation was observed but to a significantly lesser extent than sensitive cells. Summary: JNK phosphorylation regulates obatoclax induced apoptosis, and this activity is reduced in NSCLC cells resistant to obx. LC3 processing was independent of beclin1, thus the roles of Atg5 and 7 in regulating obx induced LC3 processing and cell fate are ongoing and will be presented. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B30.