Exploring the microstructure of hydrated collagen hydrogels under scanning electron microscopy.

Collagen hydrogels are a rapidly expanding platform in bioengineering and soft materials engineering for novel applications focused on medical therapeutics, medical devices and biosensors. Observations linking microstructure to material properties and function enables rational design strategies to control this space. Visualisation of the microscale organisation of these soft hydrated materials presents unique technical challenges due to the relationship between hydration and the molecular organisation of a collagen gel. Scanning electron microscopy is a robust tool widely employed to visualise and explore materials on the microscale. However, investigation of collagen gel microstructure is difficult without imparting structural changes during preparation and/or observation. Electrons are poorly propagated within liquid-phase materials, limiting the ability of electron microscopy to interrogate hydrated gels. Sample preparation techniques to remove water induce artefactual changes in material microstructure particularly in complex materials such as collagen, highlighting a critical need to develop robust material handling protocols for the imaging of collagen hydrogels. Here a collagen hydrogel is fabricated, and the gel state explored under high-vacuum (10-6  Pa) and low-vacuum (80-120 Pa) conditions, and in an environmental SEM chamber. Visualisation of collagen fibres is found to be dependent on the degree of sample hydration, with higher imaging chamber pressures and humidity resulting in decreased feature fidelity. Reduction of imaging chamber pressure is used to induce evaporation of gel water content, revealing collagen fibres of significantly larger diameter than observed in samples dehydrated prior to imaging. Rapid freezing and cryogenic handling of the gel material is found to retain a porous 3D structure following sublimation of the gel water content. Comparative analysis of collagen hydrogel materials demonstrates the care needed when preparing hydrogel samples for electron microscopy.

Delivery of Temozolomide and N3-Propargyl Analog to Brain Tumors Using an Apoferritin Nanocage.

Glioblastoma multiforme (GBM) is a grade IV astrocytoma, which is the most aggressive form of brain tumor. The standard of care for this disease includes surgery, radiotherapy and temozolomide (TMZ) chemotherapy. Poor accumulation of TMZ at the tumor site, tumor resistance to drug, and dose-limiting bone marrow toxicity eventually reduce the success of this treatment. Herein, we have encapsulated >500 drug molecules of TMZ into the biocompatible protein nanocage, apoferritin (AFt), using a "nanoreactor" method (AFt-TMZ). AFt is internalized by transferrin receptor 1-mediated endocytosis and is therefore able to facilitate cancer cell uptake and enhance drug efficacy. Following encapsulation, the protein cage retained its morphological integrity and surface charge; hence, its cellular recognition and uptake are not affected by the presence of this cargo. Additional benefits of AFt include maintenance of TMZ stability at pH 5.5 and drug release under acidic pH conditions, encountered in lysosomal compartments. MTT assays revealed that the encapsulated agents displayed significantly increased antitumor activity in U373V (vector control) and, remarkably, the isogenic U373M (MGMT expressing TMZ-resistant) GBM cell lines, with GI50 values <1.5 µM for AFt-TMZ, compared to 35 and 376 µM for unencapsulated TMZ against U373V and U373M, respectively. The enhanced potency of AFt-TMZ was further substantiated by clonogenic assays. Potentiated G2/M cell cycle arrest following exposure of cells to AFt-TMZ indicated an enhanced DNA damage burden. Indeed, increased O6-methylguanine (O6-MeG) adducts in cells exposed to AFt-TMZ and subsequent generation of γH2AX foci support the hypothesis that AFt significantly enhances the delivery of TMZ to cancer cells in vitro, overwhelming the direct O6-MeG repair conferred by MGMT. We have additionally encapsulated >500 molecules of the N3-propargyl imidazotetrazine analog (N3P), developed to combat TMZ resistance, and demonstrated significantly enhanced activity of AFt-N3P against GBM and colorectal carcinoma cell lines. These studies support the use of AFt as a promising nanodelivery system for targeted delivery, lysosomal drug release, and enhanced imidazotetrazine potency for treatment of GBM and wider-spectrum malignancies.

The antibiotic vancomycin induces complexation and aggregation of gastrointestinal and submaxillary mucins.

Vancomycin, a branched tricyclic glycosylated peptide antibiotic, is a last-line defence against serious infections caused by staphylococci, enterococci and other Gram-positive bacteria. Orally-administered vancomycin is the drug of choice to treat pseudomembranous enterocolitis in the gastrointestinal tract. However, the risk of vancomycin-resistant enterococcal infection or colonization is significantly associated with oral vancomycin. Using the powerful matrix-free assay of co-sedimentation analytical ultracentrifugation, reinforced by dynamic light scattering and environmental scanning electron microscopy, and with porcine mucin as the model mucin system, this is the first study to demonstrate strong interactions between vancomycin and gastric and intestinal mucins, resulting in very large aggregates and depletion of macromolecular mucin and occurring at concentrations relevant to oral dosing. In the case of another mucin which has a much lower degree of glycosylation (~60%) - bovine submaxillary mucin - a weaker but still demonstrable interaction is observed. Our demonstration - for the first time - of complexation/depletion interactions for model mucin systems with vancomycin provides the basis for further study on the implications of complexation on glycopeptide transit in humans, antibiotic bioavailability for target inhibition, in situ generation of resistance and future development strategies for absorption of the antibiotic across the mucus barrier.

An integrated approach to determine interactive genotoxic and global gene expression effects of multiwalled carbon nanotubes (MWCNTs) and benzo[a]pyrene (BaP) on marine mussels: evidence of reverse 'Trojan Horse' effects.

The interactions between carbon-based engineered nanoparticles (ENPs) and organic pollutants might enhance the uptake of contaminants into biota. The present integrated study aimed to assess this potential 'Trojan Horse', probing the interactive effects of purpose-made multi-walled carbon nanotubes (MWCNTs), a representative ENP, and benzo[a]pyrene (BaP), a ubiquitous polycyclic aromatic hydrocarbon (PAH) pollutant, on the marine mussel Mytilus galloprovincialis. Mussels were exposed to MWCNTs and BaP either alone or in various combinations. The co-exposure of BaP with MWCNTs revealed that the presence of MWCNTs enhanced the aqueous concentrations of BaP, thereby reducing the uptake of this pollutant by mussels as evidenced by lowering BaP concentrations in the tissues. Determination of DNA damage (comet assay) showed a concentration-dependent response for BaP alone which was absent when MWCNTs were present. Global gene expression using microarray analyses indicated that BaP and MWCNTs, in combination, differentially activated those genes which are involved in DNA metabolism compared to the exposures of BaP or MWCNTs alone, and the gene expression response was tissue-specific. Mechanisms to explain these results are discussed and relate primarily to the adsorption of BaP on MWCNTs, mediated potentially by van der Waals interactions. The use of a novel approach based on gold-labeled MWCNTs to track their uptake in tissues improved the traceability of nanotubes in biological samples. Overall, our results did not indicate the 'Trojan Horse' effects following co-exposure to the contaminants and clearly showed that the adsorption of BaP to MWCNTs modified the uptake of the pollutant in marine mussels.

Facilitating treatment of HCV in primary care in regional Australia: closing the access gap.

BACKGROUND: Australia has unrestricted access to direct-acting antivirals (DAA) for hepatitis C virus (HCV) treatment. In order to increase access to treatment, primary care providers are able to prescribe DAA after fibrosis assessment and specialist consultation. Transient elastography (TE) is recommended prior to commencement of HCV treatment; however, TE is rarely available outside secondary care centres in Australia and therefore a requirement for TE could represent a barrier to access to HCV treatment in primary care. OBJECTIVES: In order to bridge this access gap, we developed a community-based TE service across the Sunshine Coast and Wide Bay areas of Queensland. DESIGN: Retrospective analysis of a prospectively recorded HCV treatment database. INTERVENTIONS: A nurse-led service equipped with two mobile Fibroscan units assesses patients in eight locations across regional Queensland. Patients are referred into the service via primary care and undergo nurse-led TE at a location convenient to the patient. Patients are discussed at a weekly multidisciplinary team meeting and a treatment recommendation made to the referring GP. Treatment is initiated and monitored in primary care. Patients with cirrhosis are offered follow-up in secondary care. RESULTS: 327 patients have undergone assessment and commenced treatment in primary care. Median age 48 years (IQR 38-56), 66% male. 57% genotype 1, 40% genotype 3; 82% treatment naïve; 10% had cirrhosis (liver stiffness >12.5 kPa). The majority were treated with sofosbuvir-based regimens. 26% treated with 8-week regimens. All patients had treatment prescribed and monitored in primary care. Telephone follow-up to confirm sustained virological response (SVR) was performed by clinic nurses. 147 patients remain on treatment. 180 patients have completed treatment. SVR data were not available for 19 patients (lost to follow-up). Intention-to-treat SVR rate was 85.5%. In patients with complete data SVR rate was 95.6%. CONCLUSION: Community-based TE assessment facilitates access to HCV treatment in primary care with excellent SVR rates.

Antagonistic Interactions between Benzo[a]pyrene and Fullerene (C60) in Toxicological Response of Marine Mussels.

This study aimed to assess the ecotoxicological effects of the interaction of fullerene (C60) and benzo[a]pyrene (B[a]P) on the marine mussel, Mytilus galloprovincialis. The uptake of nC60, B[a]P and mixtures of nC60 and B[a]P into tissues was confirmed by Gas Chromatography-Mass Spectrometry (GC-MS), Liquid Chromatography-High Resolution Mass Spectrometry (LC-HRMS) and Inductively Coupled Plasma Mass Spectrometer (ICP-MS). Biomarkers of DNA damage as well as proteomics analysis were applied to unravel the interactive effect of B[a]P and C60. Antagonistic responses were observed at the genotoxic and proteomic level. Differentially expressed proteins (DEPs) were only identified in the B[a]P single exposure and the B[a]P mixture exposure groups containing 1 mg/L of C60, the majority of which were downregulated (~52%). No DEPs were identified at any of the concentrations of nC60 (p < 0.05, 1% FDR). Using DEPs identified at a threshold of (p < 0.05; B[a]P and B[a]P mixture with nC60), gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis indicated that these proteins were enriched with a broad spectrum of biological processes and pathways, including those broadly associated with protein processing, cellular processes and environmental information processing. Among those significantly enriched pathways, the ribosome was consistently the top enriched term irrespective of treatment or concentration and plays an important role as the site of biological protein synthesis and translation. Our results demonstrate the complex multi-modal response to environmental stressors in M. galloprovincialis.

Immobilized Enzymes on Gold Nanoparticles: From Enhanced Stability to Cleaning of Heritage Textiles.

Enzyme-based treatments are used in heritage conservation for the effective removal of glues and other damaging organic layers from the surfaces of historic and artistic works. Despite their potential, however, the application of enzymatic treatments is currently limited because of their poor efficiency and low operational and environmental stability. We demonstrate the use of α-amylase immobilized on gold nanoparticles to improve the efficacy of enzymatic treatments enhancing both the reactivity and the stability of the formulations. Gold nanoparticles coated with α-amylase exhibit significant advantages compared to free enzymes. We report up to 5× greater resistance to environmental changes, up to 2× higher efficacy toward removal of starch-based glues from textiles and deeper penetration through the fibers, without causing damage or inducing salt precipitation. These results offer exciting prospects for the development of enzymatic formulations, both for heritage conservation and the wider application of enzymes, such as in medicine, the detergent industry, and green chemistry.

Revealing the nature of morphological changes in carbon nanotube-polymer saturable absorber under high-power laser irradiation.

Composites of single-walled carbon nanotubes (SWNTs) and water-soluble polymers (WSP) are the focus of significant worldwide research due to a number of applications in biotechnology and photonics, particularly for ultrashort pulse generation. Despite the unique possibility of constructing non-linear optical SWNT-WSP composites with controlled optical properties, their thermal degradation threshold and limit of operational power remain unexplored. In this study, we discover the nature of the SWNT-polyvinyl alcohol (PVA) film thermal degradation and evaluate the modification of the composite properties under continuous high-power ultrashort pulse laser operation. Using high-precision optical microscopy and micro-Raman spectroscopy, we have examined SWNT-PVA films before and after continuous laser radiation exposure (up to 40 hours) with a maximum optical fluence of 2.3 mJ·cm-2. We demonstrate that high-intensity laser radiation results in measurable changes in the composition and morphology of the SWNT-PVA film due to efficient heat transfer from SWNTs to the polymer matrix. The saturable absorber modification does not affect the laser operational performance. We anticipate our work to be a starting point for more sophisticated research aimed at the enhancement of SWNT-PVA films fabrication for their operation as reliable saturable absorbers in high-power ultrafast lasers.

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

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.

Navon letters affect face learning and face retrieval.

Identifying the local letters of a Navon letter (a large letter made up of smaller different letters) prior to recognition causes impairment in accuracy, while identifying the global letters of a Navon letter causes an enhancement in recognition accuracy (Macrae & Lewis, 2002). This effect may result from a transfer-inappropriate processing shift (TIPS) (Schooler, 2002). The present experiment extends research on the underlying mechanism of this effect by exploring this Navon effect on face learning as well as face recognition. The results of the two experiments revealed that when the Navon task used at retrieval was the same as that used at encoding then the performance accuracy is enhanced, whereas when the processing operations mismatch at retrieval and at encoding, this impairs recognition accuracy. These results provide support for the TIPS explanation of the Navon effect.

The effectiveness of feedback in multiple-cue probability learning.

How effective are different types of feedback in helping us to learn multiple contingencies? This article attempts to resolve a paradox whereby, in comparison to simple outcome feedback, additional feedback either fails to enhance or is actually detrimental to performance in nonmetric multiple-cue probability learning (MCPL), while in contrast the majority of studies of metric MCPL reveal improvements at least with some forms of feedback. In three experiments we demonstrate that if feedback assists participants to infer cue polarity then it can in fact be effective in nonmetric MCPL. Participants appeared to use cue polarity information to adopt a linear judgement strategy, even though the environment was nonlinear. The results reconcile the paradoxical contrast between metric and nonmetric MCPL and support previous findings of people's tendency to assume linearity and additivity in probabilistic cue learning.

The effects of precedence on Navon-induced processing bias in face recognition.

Macrae and Lewis (2002) showed that repeated reporting of the global dimension of Navon stimuli improved performance in a subsequent face identification task, whilst reporting the features of the Navon stimuli impaired performance. Using a face composite task, which is assumed to require featural processing, Weston and Perfect (2005) showed the complementary pattern: Featural responding to Navon letters speeded performance. However, both studies used Navon stimuli with global precedence, in which the overall configuration is easier to report than the features. Here we replicate the two studies above, whilst manipulating the precedence (global or featural) of the letter stimuli in the orientation task. Both studies replicated the previously reported findings with global precedence stimuli, but showed the reverse pattern with local precedence stimuli. These data raise important questions as to what is transferred between the Navon orientation task and the face-processing tasks that follow.

Effects of processing bias on the recognition of composite face halves.

Research has shown that face recognition accuracy can be improved by prior global processing and impaired by prior local processing (Macrae & Lewis, 2002). The aim of this study was to test the processing bias account of face recognition, using the composite face task (Young, Hellawell, & Hay, 1987), a test of featural recognition. Undergraduate volunteers (N = 75) participated in a between-subjects design that tested their ability to recognize face halves within a composite, following either global or local Navon processing or a control task. Results showed that, as compared with the control task, local processing speeded ability to recognize face halves. These results provide support for the processing bias account of face recognition.