The physical nature of weak shock reflection

Student Number : 9900131F - MSc (Eng) dissertation - School of Mechanical Engineering - Faculty of EngineeringRecent high-resolution numerical studies of weak shock reflections have shown that a complex flow structure exists behind the triple point which consists of multiple shocks, expansion fans and triple points. This region had not been detected earlier in experimental observations or numerical studies of weak shock reflections due to the small size of this region. New components were designed and built to modify an existing large-scale shock tube in order to obtain experimental observations to validate the numerical results. The shock tube produced a large, expanding cylindrical incident wave which was reflected off a 15° corner on the roof of the section to produce a weak shock Mach reflection with a large Mach stem in the test section. The shock tube was equipped with PCB high-speed pressure transducers and digital scope for data acquisition, and a schlieren optical system to visualise the region behind the triple point. The tests were conducted over a range of incident wave Mach numbers (M12 = 1.060-1.094) and produced Mach stems of between 694 mm and 850 mm in length. The schlieren photographs clearly show an expansion fan centered on the triple point in all the successful tests conducted. In some of the more resolved images, a shocklet can be seen terminating the expansion fan, and in others a second expansion fan and/or shocklet can be seen. A ‘von Neumann reflection’ was not visualised experimentally, and hence it has been proposed that the four-wave reflection found in these tests be named a ‘Guderley reflection’. The experimental validation of Hunter & Tesdall’s (2002) work resolves the ‘von Neumann Paradox’

Characterization of Nitric Oxide Modulatory Activities of Alkaline-Extracted and Enzymatic-Modified Arabinoxylans from Corn Bran in Cultured Human Monocytes

This document is the Accepted Manuscript version of a Published Work that appeared in final form in Journal of Agricultural and Food Chemistry, copyright © American Chemical Society after peer review and technical editing by the publisher. To access the final edited and published work see https://pubs.acs.org/doi/pdf/10.1021/acs.jafc.6b02896The ingestion of foods and food-derived substances that may mediate the immune system is widely studied. Evidence suggests cereal arabinoxylans (AXs) have immunomodulatory activities that may impart health benefits in terms of immune enhancement. This study extracted AXs from corn bran using alkali and developed a modification process using three endoxylanases to obtain fractions of lower molecular weight ranges. In vitro studies showed extracted and modified AXs significantly (P < 0.05) elevated nitric oxide (NO) synthesis by the human U937 monocytic cell line (ranging from 53.7 ± 1.1 to 62.9 ± 1.2 μM per million viable cells) at all concentrations tested (5–1000 μg/mL), indicative of immune enhancement compared to an untreated control (43.7 ± 1.9 μM per million viable cells). The study suggested the dose range and Mw distribution of AXs are key determinants of immune-modulatory activity. AXs in the low Mw range (0.1–10 KDa) were the most effective at inducing NO secretion by U937 macrophages at low AX concentration ranges (5–50 μg/mL), with NO production peaking at 62.9 ± 1.2 μM per million viable cells with 5 μg/mL of AX (P = 0.0009). In contrast, AXs in the high Mw range (100–794 kDa) were most effective at inducing NO at high AX concentration ranges (500–1000 μg/mL) with NO production reaching a maximum of 62.7 ± 1.3 μM per million viable cells at 1000 μg/mL of AX (P = 0.0011). The findings suggest that dietary AXs from corn bran may heighten innate immune responses in the absence of infection or disease

Poloxamer-Mediated Control of Biofilms on Polycarbonate Surfaces

Background: Polycarbonate (PC) is a type of plastic widely used in the food industry as components of food processing equipment and resusable food/water storage containers and bottles. Biofilm formation on food contact surfaces (FCS) such as reusable plastics like PC can pose an potential health risk if insufficiently controlled. Poloxamers have surfactant properties that make them useful as dispersants, detergents, emulsifiers or wetting agents. Poloxamers have been utilised as an emulsifing agent and food additive in the food industry but their application in biofilm control on FCS has not been exploited to date. In particular, the soaking of PC items in poloxamers prior to routine washing processes may present a novel biofilm control measure for reusable products made from PC. Thus, this study investigated the potential use of poloxamers to control biofilm formation on nutrient-exposed PC surfaces. Methods: Poloxamer surfactants (P188 and P407: 0.1, 1.0 and 10.0 mg/ml) were investigated to assess their ability to disrupt biofilms of Gram-positive and Gram-negative bacteria. Staphylococcus aureus (Sa) & Pseudomonas aeruginosa (Pa) biofilms were cultured in nutrient-rich medium on PC surfaces (13mm PC discs; n=6) prior to exposure to poloxamer for 24 hours. The crystal violet (CV) assay was used to determine biofilm mass following treatment of Sa and Pa biofilms with poloxamers. The recovery of Sa or Pa from PC surfaces (n=6) following treatment of biofilms with poloxamer was assessed and biofilm disruption was visualised by confocal and scanning electron microscopy (SEM). Results: Both poloxamer P188 and P407 significantly (P<0.05) reduced Sa and Pa biofilm mass on nutrient-exposed PC surfaces in a dose-dependent manner. In concordance with this finding, treatment of established biofilms of Sa or Pa with poloxamer (P188 or P407) for 24 hours significantly (P<0.05) reduced bacterial recovery from nutrient-exposed PC surfaces. Confocal microscopy and SEM showed extensive biofilm disruption on poloxamer-treated PC surfaces compared to untreated control PC surfaces. Conclusions: This study showed poloxamer surfactants significantly disrupt established biofilms on PC surfaces. These findings suggest treatment of FCS with poloxamers prior to routine washing may be a novel biofilm control measure in the food industry for reusable items made from PC

Using MGN-3 to mediate innate immunity in a diabetic (hyperglycaemic) model of an infected chronic wound

Background The diabetic foot ulcer (DFU) is a type of chronic wound presenting in type 2 diabetics that frequently becomes infected by polymicrobial communities, leading to significant morbidity and mortality. Antibiotics are used as the first line of defence against DFU infections but over-usage has led to widespread antibiotic resistance. To reduce the reliance on antibiotics, novel therapies are desired that can promote infection clearance by stimulating innate host immune responses, thereby either replacing or working alongside antibiotic intervention. This study investigated the use of Biobran (MGN-3) to mediate innate host clearance of typical wound pathogens in a diabetic (hyperglycaemic) model of an infected DFU. Methods Host-pathogen interaction assays (n = 12) were used to assess the effect of MGN-3 treatments on M1 (classically activated macrophage)-mediated phagocytosis of Gram-positive Methicillin-Resistant Staphylococcus aureus (MRSA) and Gram-negative Pseudomonas aeruginosa (PA01) under euglycemic (11mM) and hyperglycaemic (15mM, 20mM and 30mM) culture conditions. The phagocytic ability of M1 macrophage exposed to MGN-3 (0.5, 1.0, 2.0 mg/ml) was compared against bacterial clearance observed in the absence of MGN-3 (untreated control), following treatment with rice starch (0.5, 1.0, 2.0 mg/ml; negative control) and following treatment with bacterial lipopolysaccharide (LPS 5g/ml; positive control). Results Increasing levels of hyperglycaemia significantly (p<0.05) increased the bacterial recovery by impairing M1-mediated phagocytosis. However, MGN-3 and LPS supplementation reversed the detrimental effect of glucose by significantly increasing (p<0.05) M1-mediated phagocytosis of both MRSA and PAO1 in a dose dependent manner compared to the untreated and rice starch-treated controls. Conclusion MGN-3 significantly reversed the detrimental impact of increasing hyperglycaemia on M1-mediated phagocytosis, highlighting the beneficial effect of MGN-3 on promoting bacterial clearance in a dose-dependent manner under hyperglycaemic conditions. These findings suggest the use of MGN-3 in local wound dressings as a potential cost-effective therapeutic strategy to resolve clinical DFU infections warrants further investigation

Pre-treatment with poloxamer surfactant augments estrogen-mediated bacterial clearance by phagocytes in an in vitro model of chronic wound biofilms

Chronic wounds are common in the elderly and lead to substantial morbidity and mortality. Evidence suggests that age-related impaired healing may result in part from a decline in estrogen-mediated signalling with increasing age. Biofilm formation in chronic wounds affecting the elderly can often impede delivery of antibiotic therapy and enable bacteria to evade innate host immune responses. Unresolved biofilms typically lead to pronounced bacterial survival and spread, increased risk of infection by wound pathogens and delayed wound closure. Moreover, given antibiotic resistance is commonplace there is an urgent need to develop new therapeutic strategies to resolve wound infections with reduced reliance on antibiotics. Topical use of surfactants to disrupt established or developing biofilms alongside steroid hormone therapy to stimulate host immunity represents one such emergent strategy to combat biofilms in impaired wound healing states. The aim of this project was to investigate the combined use of poloxamer surfactants with targeted stimulation of innate immune responses by local estrogen replacement therapy as a potential novel dual strategy to resolve wound biofilms in the elderly. Poloxamer surfactants (P188 and P407: 0.1, 1.0 and 10.0 mg/ml) were investigated alone and in combination with hormone supplementation (estradiol: 1x107M) to assess their ability to promote biofilm disruption and inflammatory cell clearance of two key wound bacterial pathogens, Staphylococcus aureus (Sa) & Pseudomonas aeruginosa (Pa). The crystal violet (CV) assay was used to determine biofilm mass following treatment of Sa and Pa biofilms (n=6) with poloxamers, with or without combined estradiol supplementation for 24 hours. Host phagocytes (U937-derived monocytes and macrophages) stimulated with estradiol for 24 hours were assessed in vitro (n=6) in terms of host cell viability and clearance of Sa or Pa following pre-treatment of biofilms with poloxamer. Host-pathogen interactions were visualised by fluorescence and scanning electron microscopy (SEM). Findings indicated estradiol (1x107M) had no detrimental effects on host phagocyte proliferation or viability. Both poloxamers were tolerable to host inflammatory cells (with no significant effect on cell proliferation or viability) when applied at the lower dosage of 0.1 mg/ml, so this concentration was selected for subsequent investigation in the study. Both poloxamer P188 and P407 significantly (P<0.05) reduced biofilm mass when applied directly to Sa and Pa biofilms for 24 hours at a concentration of 0.1 mg/ml. Estradiol had no effect on biofilm mass when applied directly to biofilms but hampered the poloxamer-mediated reduction in biofilm mass when applied concomitantly as a combined treatment with P188 or P407. This interaction between poloxamers and estradiol confirmed sequential (rather than concurrent) dual therapy was more effective at biofilm reduction. Pre-treatment of established biofilms of Sa or Pa with 0.1 mg/ml poloxamer (P188 or P407) for 24 hours significantly (P<0.05) increased subsequent phagocyte-mediated bacterial clearance compared to regimens lacking poloxamer pre-treatment. Similarly, a single therapy approach involving treatment of phagocytes with estradiol alone significantly (P<0.01) reduced bacterial recovery from Sa and Pa biofilms compared with untreated control phagocytes. More importantly however, sequential dual therapy involving pre-treatment of biofilms with 0.1 mg/ml poloxamer (P188 or P407) for 24 hours significantly (P<0.05) enhanced subsequent estradiol-mediated clearance of Sa and Pa by phagocytes compared to corresponding findings observed with single therapy approaches involving either estradiol or poloxamer pre-treatment alone. Overall, this study shows that initial disruption of biofilms with poloxamer surfactants enhances subsequent host-mediated clearance of both Gram-positive and Gram-negative bacteria in vitro, particularly when phagocytes have been exposed to estradiol. These findings suggest that sequential treatment of biofilms with poloxamer followed by stimulation of phagocytes with estradiol may be an effective dual strategy to resolve wound biofilms in the elderly that warrants further investigation

Determining the cellular and molecular mechanisms by which Biobran (MGN-3) mediates innate immunity in a hyperglycaemic model of a diabetic foot ulcer infection

Background Diabetic foot ulcer (DFU) infections frequently become infected by polymicrobial communities, leading to significant morbidity and mortality. Antibiotics are the first line of defence against DFU infections, but over-usage has led to widespread antibiotic resistance. To reduce the reliance on antibiotics, novel therapies are desired that promote infection clearance by stimulating innate host immune responses, thereby either replacing or working alongside antibiotic intervention. This study investigated the use of Biobran (MGN-3) to mediate innate host clearance of typical wound pathogens in a diabetic (hyperglycaemic) model of an infected DFU. Methods Host-pathogen interaction assays (n=12) were used to assess the effect of Biobran (MGN-3: 0.5, 1.0, 2.0 mg/ml) on M1 macrophage-mediated phagocytosis of Methicillin-Resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa (PA01) under increasing glycaemic (glucose: 11, 15, 20 and 30mM) culture conditions. Treatment of M1 macrophages with rice starch (RS: 2.0 mg/ml) or bacterial lipopolysaccharide (LPS: 5g/ml) were used as negative and positive controls respectively, and bacterial clearance was compared against untreated M1 macrophages (untreated control). Furthermore, gentamycin assays were conducted to elucidate whether M1 cells were successfully killing or sequestering bacteria. Host pathogen interactions were visualised by a combination of scanning electron and confocal microscopy. Inflammatory cytokine, nitric oxide and reactive oxygen species (ROS) production were determined by enzyme-linked immunosorbent assay (ELISA), the Griess assay and a membrane permeable fluorescent immunoassay respectively. Results Increasing levels of hyperglycaemia significantly (p<0.05) impaired M1-mediated phagocytosis. MGN-3 and LPS supplementation reversed the detrimental effect of glucose by significantly increasing (p<0.05) M1-mediated phagocytosis of both MRSA and PA01 in a dose dependent manner compared to untreated and RS-treated controls. MGN-3 and LPS supplementation significantly (p<0.05) induced tumour necrosis factor–alpha (TNF) and Interferon beta (IFN-) secretion by M1 macrophage and reversed the glucose-induced reduction in nitric oxide production. Elevated levels of glucose lead to pronounced ROS production by M1 macrophages, which was significantly (p<0.05) alleviated by MGN-3 in a dose-dependent manner. Conclusion MGN-3 significantly reversed the detrimental impact of hyperglycaemia on M1-mediated effector functions including phagocytosis, cytokine secretion and NO/ROS production. These findings highlight the beneficial dose-dependent effects of MGN-3 on promoting bacterial clearance under hyperglycaemic conditions, warranting further investigations to evaluate the use of MGN-3 in local wound dressings as a potential cost-effective therapeutic strategy to resolve clinical DFU infections

Biobran (MGN-3) enhances host-pathogen interaction in an in vitro hyperglycaemic model of an infected diabetic foot ulcer via the CD14/TLR-4 pathway

Background Low grade prolonged inflammation is a key feature of chronic hyperglycaemia (CH) and a pathophysiological characteristic attribute of chronic wounds. CH affects multiple inflammatory pathways leading to dysregulated signalling. Pathogen recognition receptors (PRRs) are a vital component of the innate immune system that recognise Pathogen associated molecular patterns (PAMPs) and Danger associated molecular pathogens (DAMPS), thus enabling an immune response to be elicited. Toll-like receptors (TLRs), particularly TLR-4, is upregulated in patients with an infected diabetic foot ulcer (DFU). Lipopolysaccharide (LPS), a primary ligand for CD14 and its co-receptor TLR-4, has structural similarity to Biobran (MGN-3) in terms of molecular weight and chemical structure, suggesting TLR-4 and/or CD14 may be key regulators of innate immune responses mediated by MGN-3. Thus, this study investigated whether the TLR-4 pathway mediates the effects of MGN-3 on phagocyte function in a diabetic (hyperglycaemic) model of an infected DFU. Methods Host-pathogen interaction assays (n=12) were used to assess the effect of Biobran (MGN-3: 0.5, 1.0, 2.0 mg/ml) on U937-derived M1 macrophage-mediated phagocytosis of methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa (PA01) under increasing glycaemic (glucose: 11, 15, 20 and 30mM) culture conditions. Treatment of M1 macrophages with rice starch (RS: 2.0 mg/ml) or bacterial endotoxin (LPS: 5ug/ml) were used as negative and positive controls respectively, and bacterial clearance was compared against untreated M1 macrophages (untreated control). The TLR-4 pathway was interrogated using TLR-4, myeloid differentiation primary response protein (MYD88) and Toll/IL-1 receptor domain-containing adaptor inducing IFN-beta (TRIF) inhibitors. CD14 was analysed using both flow cytometry to measure membrane-associated CD14 (mCD14) levels and enzyme-linked immunosorbent assay (ELISA) to measure soluble CD14 (sCD14) levels. Host-pathogen interactions were visualised by a combination of scanning electron and confocal microscopy. Results Increasing levels of hyperglycaemia significantly (P<0.05) impaired M1-mediated phagocytosis in a dose-dependent manner. MGN-3 and LPS supplementation reversed the detrimental effect of hyperglycaemia by significantly (P<0.01) increasing M1-mediated phagocytosis of both MRSA and PA01 in a dose dependent manner compared to untreated controls. Inhibition of TLR-4, MYD88 or TRIF significantly (P<0.05) blocked the beneficial effects of MGN-3 on the clearance of both MRSA and PAO1, regardless of the glycaemic state. Flow cytometry showed neither LPS nor MGN-3 affected mCD14 levels under euglycemic conditions. However, hyperglycaemia resulted in a significant increase in mCD14 when M1 macrophages were stimulated with LPS, which was confirmed by immunofluorescence staining. Concomitant treatment of M1 macrophages with MGN-3 and LPS significantly (P<0.01) reversed the effects of hyperglycaemia on LPS-mediated stimulation of mCD14 expression. A significant (P<0.01) increase in sCD14 levels was detected from LPS-activated M1 macrophages at all glucose concentrations. In contrast, MGN-3 treatment (significantly (P<0.05) decreased M1 macrophage-derived sCD14 levels at lower glucose concentrations (11 and 15mM), with sCD14 levels only increasing compared to the untreated control at higher glucose concentrations (20 and 30mM). Conclusion MGN-3 significantly reversed the detrimental impact of hyperglycaemia on M1-mediated phagocytosis of both MRSA and PA01. Inhibition of TLR-4, MYD88 or TRIF blocked the stimulation of phagocytosis observed with MGN-3, confirming the TLR-4 pathway is (at least in part) fundamental for the beneficial activity of MGN-3 on bacterial clearance. MGN-3 was able to reverse the LPS-induced increase in mCD14 expression under hyperglycaemic conditions. Unlike LPS which increased sCD14 at all glucose concentrations, MGN-3 treatment reduced sCD14 levels at lower glucose (11 and 15mM) concentrations. Collectively, these findings suggest TLR-4 and CD14 may be key regulators of innate immune responses mediated by MGN-3, warranting further investigation of MGN-3 as a potential therapeutic strategy for the treatment of patients with an infected DFU

Analysis and characterization of differential gene expression during rapid trophoblastic elongation in the pig using suppression subtractive hybridization

During late peri-implantation development, porcine conceptuses undergo a rapid (2–3 hrs) morphological transformation from a 10 mm sphere to a thin filamentous form greater than 150 mm in length. Elongation of the conceptus is important for establishing adequate placental surface area needed for embryo and fetal survival throughout gestation. Genes involved with triggering this unique transition in conceptus development are not well defined. Objective of the present study was to utilize suppression subtractive hybridization (SSH) to characterize the change in gene expression during conceptus transformation from spherical (8–9 mm) to tubular (15–40 mm) to early filamentous (>150 mm) morphology. Spherical, tubular, and filamentous conceptuses were collected from pregnant gilts and subjected to SSH. Forward and reverse subtractions were performed to identify candidate genes differentially expressed during spherical to tubular and tubular to filamentous transition. A total of 384 transcripts were differentially screened to ensure unique expression. Of the transcripts screened, sequences were obtained for 142 that were confirmed to be differentially expressed among the various morphologies. Gene expression profiles during rapid trophoblastic elongation were generated for selected mRNAs using quantitative real-time PCR. During the transition from tubular to early filamentous conceptuses, s-adenosylhomocysteine hydrolase and heat shock cognate 70 kDa expression were significantly enhanced. A novel unknown gene was isolated and shown to be significantly up-regulated at the onset of rapid trophoblastic elongation and further enhanced in filamentous conceptuses

Modulate your immune response using cereals

Institute of Food Science and Technology (IFST) Young Scientist Competition (2018

Biobran (MGN-3) concurrently reverses lipopolysaccharide-induced elevation of CD14 and impairment of macrophage-mediated bacterial clearance in a model of diabetic wound biofilms

Hyperglycaemia found in type 2 diabetic patients can lead to several complications including a type of chronic wound called a diabetic foot ulcer (DFU). Diabetic foot ulcers are associated with elevated inflammatory markers but defective immune responses that can lead to microbial infection, often characterised by biofilm formation. Cereal-derived fibres called arabinoxylans have shown increased immune function in both the adaptive and innate immune responses in animal models. A cereal-derived fibre called Biobran (MGN-3) has been shown to stimulate immune cells including macrophages, T cell and dendritic cells suggesting it may be of therapeutic benefit to fight infections. This study investigated whether MGN-3 can stimulate the clearance of wound-associated bacterial biofilms under hyperglycaemic conditions and modulate levels of the pattern recognition receptor CD14 on the cell surface of M1 macrophages. Host-pathogen biofilm investigations (n=12) were performed to assess the effect of MGN-3 (0.5, 1.0 and 2.0 mg/ml for 24 hours) on the phagocytosis of both Gram-positive Methicillin resistant Staphylococcus aureus (MRSA) and Gram-negative Pseudomonas aeruginosa (PA01) biofilms by U937-derived M1 macrophages cultured in glucose-supplemented (11, 15, 20, 30mM) medium. CD14 on the cell surface of M1 macrophages was assessed by confocal microscopy and flow cytometry (n=4). Findings showed glucose-supplementation significantly (P<0.05) inhibits the M1 macrophage-mediated phagocytosis of both MRSA and PA01 biofilms in a dose-dependent manner. Moreover, hyperglycaemia significantly (P<0.05) enhanced lipopolysaccharide (LPS)-induced M1 macrophage CD14 surface levels. Treatment of M1 macrophages with MGN-3 (0.5, 1.0 and 2.0 mg/ml) for 24 hours significantly (P<0.05; n=12) promoted the clearance of both MRSA and PA01 biofilms in a dose-dependent manner. Moreover, this is the first study to demonstrate MGN-3 reverses the detrimental effects of hyperglycaemia in a dose-dependent manner, significantly (P<0.05) increasing M1-mediated clearance of bacterial biofilms and reducing LPS-induced CD14 levels (n=4). CD14 levels significantly (P < 0.05; n=4) decreased after dual treatment with LPS and MGN-3 compared to just LPS treatment alone, suggesting competition was taking place between MGN-3 and LPS at the CD14 receptor on M1 macrophages. The findings of this project suggest MGN-3 may be of potential therapeutic benefit for the treatment of DFU patients with wound biofilm infections