A fluorescence in situ staining method for investigating spores and vegetative cells of Clostridia by confocal laser scanning microscopy and structured illuminated microscopy

Non-pathogenic spore-forming Clostridia are of increasing interest due to their application in biogas production and their capability to spoil different food products. The life cycle for Clostridium includes a spore stage that can assist in survival under environmentally stressful conditions, such as extremes of temperature or pH. Due to their size, spores can be investigated by a range of microscopic techniques, many of which involve sample pretreatment. We have developed a quick, simple and non-destructive fluorescent staining procedure that allows a clear differentiation between spores and vegetative cells and effectively stains spores, allowing recovery and tracking in subsequent experiments. Hoechst 34580, Propidium iodide and wheat germ agglutinin WGA 488 were used in combination to stain four strains of Clostridia at different life cycle stages. Staining was conducted without drying the sample, preventing changes induced by dehydration and cells observed by confocal laser scanner microscopy or using a super-resolution microscope equipped with a 3D-structured illumination module. Dual staining with Hoechst/Propidium iodide differentiated spores from vegetative cells, provided information on the viability of cells and was successfully applied to follow spore production induced by heating. Superresolution microscopy of spores probed by Hoechst 34580 also allowed chromatin to be visualised. Direct staining of a cheese specimen using Nile Red and Fast Green allowed in situ observation of spores within the cheese and their position within the cheese matrix. The proposed staining method has broad applicability and can potentially be applied to follow Clostridium spore behaviour in a range of different environments

Measurements of a single lateral jet injected into swirling crossflow

Experiments have been conducted to document the time-mean and turbulent flowfield of a deflected turbulent jet in a confined swirling crossflow. The jet-to-crossflow velocity ratio of 4 was investigated with swirler vane angles of 45 and 70 degrees. A six-orientation single hot-wire technique was used to measure the velocities and turbulence properties of the flow. In addition, a five-hole pitot probe technique was used to measure the time-mean velocities or verification purposes. The results are presented in the form of r-x plots to aid visualization of the fully three-dimensional flowfield. The swirl in the crossflow intensified the local velocity at the location of the injected jet, which effectively reduced the jet-to-crossflow velocity ratio. This caused the trajectory of the injected jet to follow the path of the local flow direction of the crossflow, and reduce its penetration into the crossflow. The time-mean velocity measurements using the hot-wire corresponded to pitot-probe data obtained in identical flow conditions. Turbulence stress data show the same trends as previous swirl flow data without lateral injection. The lateral jet was found to deflect the axis of the precessing vortex core

Multiple closed loop recycling of carbon fibre composites with the HiPerDiF (High Performance Discontinuous Fibre) method

AbstractThe aim of this article is to apply the concept of Circular Economy, where end-of-life products and production wastes are recycled into reusable materials, to carbon fibre reinforced plastics. This not only reduces the amount of material disposed into landfills, but also enables manufacturers to achieve significant savings. While current research focuses on the performance of recycled carbon fibre reinforced composites after one recycling process, this paper aims to investigate the performance of composites remanufactured from short carbon fibres that have undergone multiple recycling loops with the High Performance Discontinuous Fibre (HiPerDiF) method. The HiPerDiF method enables the production of aligned short fibre composites with exceptional mechanical properties. In addition, using short fibres makes the composite material intrinsically easy to recycle. Short virgin carbon fibres underwent two loops of fibre reclamation and remanufacturing. A correlation between the composites’ mechanical properties and the nature of the fibres, i.e. reduction in fibre lengths, as well as the residual matrix accumulation from the reclaiming process over a number of recycling loops, was established

Empires and Percolation: Stochastic Merging of Adjacent Regions

We introduce a stochastic model in which adjacent planar regions $A, B$ merge stochastically at some rate $\lambda(A,B)$, and observe analogies with the well-studied topics of mean-field coagulation and of bond percolation. Do infinite regions appear in finite time? We give a simple condition on $\lambda$ for this {\em hegemony} property to hold, and another simple condition for it to not hold, but there is a large gap between these conditions, which includes the case $\lambda(A,B) \equiv 1$. For this case, a non-rigorous analytic argument and simulations suggest hegemony.Comment: 13 page

Effectiveness of Gaming in Creating Cultural Awareness

Despite the emphasis on cultural competency education in the United States for the past three decades, inequities and disparities in healthcare continue to persist, particularly among minority populations. With the current growing gap in provider and patient cultural congruence, how effectively we train students to work with diverse populations in healthcare settings warrants attention. This article presents the results of a qualitative study on the effectiveness of experiential learning, in the form of the game BaFa’ BaFa,’ in raising cultural awareness among students of health professions. Using thematic analysis, the authors analyzed written reflections from student participants. The findings support earlier studies, conducted mainly outside of healthcare, on the effectiveness of gaming in nurturing cultural awareness

Effect of temperature on the microstructure of fat globules and the immunoglobulin-mediated interactions between fat and bacteria in natural raw milk creaming

Natural creaming of raw milk is the first step in production of Grana Padano and Parmigiano Reggiano Protected Denomination of Origin cheeses. This process decreases the fat content and plays an important role in the removal of clostridia species that may cause late-blowing defects in ripened cheeses. Partial coalescence of fat globules-that may influence fat behavior in cheese making and affect the microstructure of fat in the final cheese product-was observed at creaming temperatures higher than 22\uc2\ub0C by confocal laser scanning microscopy. The widespread practice of heating of milk at 37\uc2\ub0C before creaming at 8\uc2\ub0C resulted in important changes in the size distribution of fat globules in raw milk, potentially altering the ability of fat to entrap clostridia spores. We investigated the role of immunoglobulin classes in both the clustering of fat globules and the agglutination of Clostridium tyrobutyricum to fat globules during creaming. Immunogold labeling and transmission electron microscopy showed that IgA and IgM but not IgG were involved in both clustering and agglutination. Both vegetative cells and spores were clearly shown to agglutinate to fat droplets, a process that was suppressed by thermal denaturation of the immunoglobulins. The debacterization of raw milk through natural creaming was improved by the addition of purified immunoglobulins. Overall, these findings provide not only a better understanding of the phenomena occurring during the natural creaming but also practical insights into how the process of creaming may be optimized in cheese production plants

Plasma surface modification of poly(D,L-lactic acid) as a tool to enhance protein adsorption and the attachment of different cell types

We have studied the influence of oxygen radio frequency glow discharge (RfGD) on the surface and bulk properties of poly(D,L-lactic acid) (PDLLA) and the effect of this surface modification on both protein adsorption and bone cell behavior. PDLLA films were characterized before and after plasma surface modification by water contact angle, surface energy, and adhesion tension of water as well as by scanning electron microscopy (SEM), X-ray electron spectroscopy (XPS), and Fourier transform infra-red (FTIR) spectroscopy. RfGD-films showed an increase in hydrophilicity and surface energy when compared with untreated films. Surface morphological changes were observed by SEM. Chemical analysis indicated significant differences in both atomic percentages and oxygen functional group. Protein adsorption was evaluated by combining solute depletion and spectroscopic techniques. Bovine serum albumin (BSA), fibronectin (FN), vitronectin (VN), and fetal bovine serum (FBS) were used in this study. RfGD-treated surfaces adsorbed more BSA and FN from single specie solutions than FBS that is a more complex, multi-specie solution. MG63 osteoblast-like cells and primary cultures of fetal rat calvarial (FRC) cells were used to assess both the effect of RfGD treatment and protein adsorption on cell attachment and proliferation. In the absence of preadsorbed proteins, cells could not distinguish between treated and untreated surfaces, with the exception of MG63 cells cultured for longer periods of time. In contrast, the adsorption of proteins increased the cells' preference for treated surfaces, thus indicating a crucial role for adsorbed proteins in mediating the response of osteogenic cells to the RfGD-treated PDLLA surface.Contract grant sponsors: Presidential Research Enhancement Fund (PREF), UTHSCSA, San Antonio, TX, USAContract grant sponsor: Foundation lot Science kind Technology (FCT), Portugal; contract grant number: SFRH/BD/11188/2002 and partial funding through FEDER and POCTI programsContract grant sponsor: EU funded Project HIPPOCRATES; contract grant number: NMP3-CT-2003-505758 Contract grant sponsor: European NoE EXPERTISSUES;Contract grant number: NMP3-CT-2004-500283

Modulating bone cells response onto starch-based biomaterials by surface plasma treatment and protein adsorption

The effect of oxygen-based radio frequency glow discharge (rfGD) on the surface of different starch-based biomaterials (SBB) and the influence of proteins adsorption on modulating bone–cells behavior was studied. Bovine serum albumin, fibronectin and vitronectin were used in single and complex protein systems. RfGD-treated surfaces showed to increase in hydrophilicity and surface energy when compared to non-modified SBB. Biodegradable polymeric blends of cornstarch with cellulose acetate (SCA; 50/50 wt%), ethylene vinyl alcohol (SEVA-C; 50/50 wt%) and polycaprolactone (SPCL; 30/70 wt%) were studied. SCA and SCA reinforced with 10% hydroxyapatite (HA) showed the highest degree of modification as result of the rfGD treatment. Protein and control solutions were used to incubate with the characterized SBB and, following this, MG63 osteoblast-like osteosarcoma cells were seeded over the surfaces. Cell adhesion and proliferation onto SCA was found to be enhanced for non-treated surfaces and on SCA+10%HA no alteration was brought up by the plasma modification. Onto SCA surfaces, BSA, FN and VN single solutions improved cell adhesion, and this same effect was found upscaled for ternary systems. In addition, plasma treated SEVA-C directed an increase in both adhesion and proliferation comparing to non-treated surfaces. Even though adhesion onto treated and untreated SPCL was quite similar, plasma modification clearly promoted MG63 cells proliferation. Regarding MG63 cells morphology it was shown that onto SEVA-C surfaces the variation of cell shape was primarily defined by the protein system, while onto SPCL it was mainly affected by the plasma treatment