Surface grafting of electrospun fibers using ATRP and RAFT for the control of biointerfacial interactions

BACKGROUND The ability to present signalling molecules within a low fouling 3D environment that mimics the extracellular matrix is an important goal for a range of biomedical applications, both in vitro and in vivo. Cell responses can be triggered by non-specific protein interactions occurring on the surface of a biomaterial, which is an undesirable process when studying specific receptor-ligand interactions. It is therefore useful to present specific ligands of interest to cell surface receptors in a 3D environment that minimizes non-specific interactions with biomolecules, such as proteins. METHOD In this study, surface-initiated atom transfer radical polymerization (SI-ATRP) of poly(ethylene glycol)-based monomers was carried out from the surface of electrospun fibers composed of a styrene/vinylbenzyl chloride copolymer. Surface initiated radical addition-fragmentation chain transfer (SI-RAFT) polymerisation was also carried out to generate bottle brush copolymer coatings consisting of poly(acrylic acid) and poly(acrylamide). These were grown from surface trithiocarbonate groups generated from the chloromethyl styrene moieties existing in the original synthesised polymer. XPS was used to characterise the surface composition of the fibers after grafting and after coupling with fluorine functional XPS labels. RESULTS Bottle brush type coatings were able to be produced by ATRP which consisted of poly(ethylene glycol) methacrylate and a terminal alkyne-functionalised monomer. The ATRP coatings showed reduced non-specific protein adsorption, as a result of effective PEG incorporation and pendant alkynes groups existing as part of the brushes allowed for further conjugation of via azide-alkyne Huisgen 1,3-dipolar cycloaddition. In the case of RAFT, carboxylic acid moieties were effectively coupled to an amine label via amide bond formation. In each case XPS analysis demonstrated that covalent immobilisation had effectively taken place. CONCLUSION Overall, the studies presented an effective platform for the preparation of 3D scaffolds which contain effective conjugation sites for attachment of specific bioactive signals of interest, as well as actively reducing non-specific protein interactions.This research was supported by the Cooperative Research Centre for Polymers (CRCP)

Automated Fourier space region-recognition filtering for off-axis digital holographic microscopy

Automated label-free quantitative imaging of biological samples can greatly benefit high throughput diseases diagnosis. Digital holographic microscopy (DHM) is a powerful quantitative label-free imaging tool that retrieves structural details of cellular samples non-invasively. In off-axis DHM, a proper spatial filtering window in Fourier space is crucial to the quality of reconstructed phase image. Here we describe a region-recognition approach that combines shape recognition with an iterative thresholding to extracts the optimal shape of frequency components. The region recognition technique offers fully automated adaptive filtering that can operate with a variety of samples and imaging conditions. When imaging through optically scattering biological hydrogel matrix, the technique surpasses previous histogram thresholding techniques without requiring any manual intervention. Finally, we automate the extraction of the statistical difference of optical height between malaria parasite infected and uninfected red blood cells. The method described here pave way to greater autonomy in automated DHM imaging for imaging live cell in thick cell cultures

Optimizing interfacial features to regulate neural progenitor cells using polyelectrolyte multilayers and brain derived neurotrophic factor

The development of biomaterials with controllable interfacial features which have the capability to instruct cellular behavior are required to produce functional scaffolds for the treatment of spinal cord injury (SCI). Here, poly-ɛ-caprolactone surfaces were biofunctionalized via layer-by-layer (LbL) deposition. The polyelectrolytes employed in this LbL technique were heparin and poly-L-lysine (PLL), the latter being chosen to improve cell adhesion and the subsequent cellular function of in vitrocultured neural progenitor cells. Material characterization results confirmed the deposition of well structured multilayers. Cell culture studies revealed significant differences in the cellular response to these adhesive/nonadhesive (PLL/heparin) polyelectrolyte multilayer (PEM)surfaces, with neurite outgrowth being significantly promoted on the PLL terminating layers. In addition, brain derived neurotrophic factor (BDNF) was adsorbed onto the LbL surfaces. This combined chemical and biological effect was then characterized in terms of neurite length along with the full length/truncated isoform 1 tyrosine kinase receptor (TrkB-FL/TrkB-T1) and growth associated protein-43 mRNA levels. Here, the authors report the differential effect of adsorbed and soluble BDNF of different concentrations. Adsorbed BDNF promoted neurite outgrowth and led to elevated, sustained TrkB mRNA levels. These findings highlight the potential of PEM biofunctionalized surfaces with integrated chemical and neurotrophin supportive cues to overcome SCI inhibitory environments and to promote regeneration

The effect of Psoroptes ovis infestation on ovine epidermal barrier function

Sheep scab is an intensively pruritic, exudative and allergic dermatitis of sheep caused by the ectoparasitic mite Psoroptes ovis. The purpose of the present study was to investigate the effect of P. ovis infestation on different components of the ovine epidermal barrier within the first 24 hours post-infestation (hpi). To achieve this, the expression of epidermal differentiation complex (EDC) genes and epidermal barrier proteins, the nature and severity of epidermal pathology and transepidermal water loss (TEWL) were evaluated. By 1 hpi a significant dermal polymorphonuclear infiltrate and a significant increase in TEWL with maximal mean TEWL (598.67 g/m(2)h) were observed. Epidermal pathology involving intra-epidermal pustulation, loss of epidermal architecture and damage to the basement membrane was seen by 3 hpi. Filaggrin and loricrin protein levels in the stratum corneum declined significantly in the first 24 hpi and qPCR validation confirmed the decrease in expression of the key EDC genes involucrin, filaggrin and loricrin observed by microarray analysis, with 5.8-fold, 4.5-fold and 80-fold decreases, respectively by 24 hpi. The present study has demonstrated that early P. ovis infestation disrupts the ovine epidermal barrier causing significant alterations in the expression of critical barrier components, epidermal pathology, and TEWL. Many of these features have also been documented in human and canine atopic dermatitis suggesting that sheep scab may provide a model for the elucidation of events occurring in the early phases of atopic sensitisation

Effects of antibiotic-suppelmented media on recovery of enterobacteria

The frequency at which Salmonella typhimurium (ST) and Escherichia coli were recovered from tryptic soy agar (TSA), brilliant green agar (BGA) and MacConkey agar (MAC) alone or supplemented with 2 and 16 µg kanamycin and 0.25 and 2 µg enrofloxacin was investigated

Scission of electrospun polymer fibres by ultrasonication

In this work we show that sonication alone can be used to scission bulk electrospun membranes into short fibres. The mechanism of such scission events is bubble cavitation stimulated by the ultrasonic probe, followed by bubble implosion. The tendency of polymer nanofibres to undergo failure by such a scission process appears to primarily depend on the ductility of the polymer, with brittle, electrospun polymer membranes such as poly(styrene) and poly(methyl methacrylate) readily producing short fibres of approximately 10 μm length. More ductile polymers such as poly(l-lactide) or poly(acrylonitrile) require additional processing after electrospinning and before sonication, to make them conducive to such sonication-based scission. Both the initial diameter of the fibres and the degree of nanofibre alignment of the electrospun membrane influence the final length of the resultant short fibres. It was found that the chemical and physical properties of the short nanofibres were unaltered by the sonication process. We are thus able to demonstrate that sonication is a promising method to produce significant quantities of short fibres of nanometre diameter and microns in length

Development of bovine abomasal organoids as a novel in-vitro model to study host-parasite interactions in gastrointestinal nematode infections

Gastro-intestinal nematode (GIN) parasites are a major cause of production losses in grazing cattle, primarily through reduced growth rates in young animals. Control of these parasites relies heavily on anthelmintic drugs; however, with growing reports of resistance to currently available anthelmintics, alternative methods of control are required. A major hurdle in this work has been the lack of physiologically relevant in vitro infection models that has made studying precise interactions between the host and the GINs difficult. Such mechanistic insights into the infection process will be valuable for the development of novel targets for drugs, vaccines, or other interventions. Here we created bovine gastric epithelial organoids from abomasal gastric tissue and studied their application as in vitro models for understanding host invasion by GIN parasites. Transcriptomic analysis of gastric organoids across multiple passages and the corresponding abomasal tissue showed conserved expression of tissue-specific genes across samples, demonstrating that the organoids are representative of bovine gastric tissue from which they were derived. We also show that self-renewing and self-organising three-dimensional organoids can also be serially passaged, cryopreserved, and resuscitated. Using Ostertagia ostertagi, the most pathogenic gastric parasite in cattle in temperate regions, we show that cattle gastric organoids are biologically relevant models for studying GIN invasion in the bovine abomasum. Within 24 h of exposure, exsheathed larvae rapidly and repeatedly infiltrated the lumen of the organoids. Prior to invasion by the parasites, the abomasal organoids rapidly expanded, developing a ‘ballooning’ phenotype. Ballooning of the organoids could also be induced in response to exposure to parasite excretory/secretory products. In summary, we demonstrate the power of using abomasal organoids as a physiologically relevant in vitro model system to study interactions of O. ostertagi and other GIN with bovine gastrointestinal epithelium

Effects of nitroethane and 2-nitropropanol against Campylobacter jejuni

Campylobacter jejuni is an important foodborne pathogen that colonizes the gut of swine. In this study, the effects of nitroethane and 2-nitropropanol (0, 10 and 20 mM) on growth of C. jejuni were tested during culture in Bolton broth adjusted to pH of 5.6, 7.0 or 8.2. Viable cell counts of samples taken at intervals during incubation revealed main effects (P\u3c0.0001) of nitroethane or 2-nitropropanol on mean specific growth rates thus demonstrating that these were inhibitory to C. jejuni. By 48 h of incubation, C. jejuni concentrations had increased by 1.9 log10 CFU/ml or higher in cultures containing no added nitrocompound

Isolation of Salmonella spp. and bacteriophage active against Salmonella spp. from commercial swine

Bacteriophage are viruses that prey on bacteria and may be a potential strategy to reduce foodborne pathogemc bactena in the gastromtestlnal tract of food animals Phages are fairly common in the gastrointestinal microbial ecosystem of mammals, but the incidence is unknown. If phage are to be an intervention strategy, we must understand their role in the microbial ecology of the gut. From a regulatory perspective, knowing incidence of phage is crucial. Therefore the current study was designed to determine the incidence of phage active against Salmonella spp in the feces of commercial finishing swine in the United States. Fecal samples (n=60) were collected from each of six commercial swine finishing operations. Samples were collected from 10 randomly selected pens throughout each operation. Total number of fecal samples collected in this study was n=360 Salmonella spp were found in 66% of the fecal samples Salmonella spp. were isolated from only 2 farms and the serotypes represented were Schwarzengrund, Anatum, Ohio and Heidelberg Bacteriophages were isolated from fecal sample through 2 parallel methods, 1) initlal enrichment in Salmonella Typhimunum, or 2) initial ennchment in E. colt B (a strain very sensitive to phages), followed by direct spot-testing against Salmonella Typhimurium Bacteriophages active against Salmonella Typhimunum were isolated from 1.1% 4/360) of the individual fecal samples when initially enriched in Salmonella Typhimurium, but E coli S-killing phages were 1solated from 43.8% (158/360) of the fecal samples but only 2 of these Isolates were capable of k1ll1ng Salmonella Typhimunum. Our results mdicate that bactenophage capable of killing Salmonella Typh1murium are fairly w1despread across commercial swine production facilities but may be present at relatively low populat1ons These results md1cate that phage (predator) populations may vary along w1th Salmonella (prey) populations and that phage could potentially be used as a food safety pathogen reduction strategy