Inhibition of myeloperoxidase-mediated hypochlorous acid production by nitroxides

Tissue damage resulting from the extracellular production of HOCl (hypochlorous acid) by the MPO (myeloperoxidase)-hydrogen peroxide-chloride system of activated phagocytes is implicated as a key event in the progression of a number of human inflammatory diseases. Consequently, there is considerable interest in the development of therapeutically useful MPO inhibitors. Nitroxides are well established antioxidant compounds of low toxicity that can attenuate oxidative damage in animal models of inflammatory disease. They are believed to exert protective effects principally by acting as superoxide dismutase mimetics or radical scavengers. However, we show here that nitroxides can also potently inhibit MPO-mediated HOCl production, with the nitroxide 4-aminoTEMPO inhibiting HOCl production by MPO and by neutrophils with IC50 values of approx. 1 and 6 μM respectively. Structure–activity relationships were determined for a range of aliphatic and aromatic nitroxides, and inhibition of oxidative damage to two biologically-important protein targets (albumin and perlecan) are demonstrated. Inhibition was shown to involve one-electron oxidation of the nitroxides by the compound I form of MPO and accumulation of compound II. Haem destruction was also observed with some nitroxides. Inhibition of neutrophil HOCl production by nitroxides was antagonized by neutrophil-derived superoxide, with this attributed to superoxide-mediated reduction of compound II. This effect was marginal with 4-aminoTEMPO, probably due to the efficient superoxide dismutase-mimetic activity of this nitroxide. Overall, these data indicate that nitroxides have considerable promise as therapeutic agents for the inhibition of MPO-mediated damage in inflammatory diseases.

Utilization and optimization of a waste stream cellulose culture medium for pigment production by Penicillium spp

Aims This research sought to determine optimal corn waste stream–based fermentation medium C and N sources and incubation time to maximize pigment production by an indigenous Indonesian Penicillium spp., as well as to assess pigment pH stability. Methods and Results A Penicillium spp. was isolated from Indonesian soil, identified as Penicillium resticulosum, and used to test the effects of carbon and nitrogen type and concentrations, medium pH, incubation period and furfural on biomass and pigment yield (PY) in a waste corncob hydrolysate basal medium. Maximum red PY (497·03 ± 55·13 mg l−1) was obtained with a 21 : 1 C : N ratio, pH 5·5–6·0; yeast extract-, NH4NO3-, NaNO3-, MgSO4·7H2O-, xylose- or carboxymethylcellulose (CMC)-supplemented medium and 12 days (25°C, 60–70% relative humidity, dark) incubation. C source, C, N and furfural concentration, medium pH and incubation period all influenced biomass and PY. Pigment was pH 2–9 stable. Conclusions Penicillium resticulosum demonstrated microbial pH-stable-pigment production potential using a xylose or CMC and N source, supplemented waste stream cellulose culture medium. Significance and Impact of the Study Corn derived, waste stream cellulose can be used as a culture medium for fungal pigment production. Such application provides a process for agricultural waste stream resource reuse for production of compounds in increasing demand.

Interleukin-1β stimulates human renal fibroblast proliferation and matrix protein production by means of a transforming growth factor-β-dependent mechanism

One of the hallmarks of progressive renal disease is the development of tubulointerstitial fibrosis. This is frequently preceded by macrophage infiltration, raising the possibility that macrophages relay fibrogenic signals to resident tubulointerstitial cells. The aim of this study was to investigate the potentially fibrogenic role of interleukin-1beta (IL-1beta), a macrophage-derived inflammatory cytokine, on cortical fibroblasts (CFs). Primary cultures of human renal CFs were established and incubated for 24 hours in the presence or absence of IL-1beta. We found that IL-1beta significantly stimulated DNA synthesis (356.7% +/- 39% of control, P <.003), fibronectin secretion (261.8 +/- 11% of control, P <.005), collagen type 1 production, (release of procollagen type 1 C-terminal-peptide, 152.4% +/- 26% of control, P <.005), transforming growth factor-beta (TGF-beta) secretion (211% +/- 37% of control, P <.01), and nitric oxide (NO) production (342.8% +/- 69% of control, P <.002). TGF-beta (1 ng/mL) and the phorbol ester phorbol 12-myristate 13-acetate (PMA, 25 nmol/L) produced fibrogenic effects similar to those of IL-1beta. Neither a NO synthase inhibitor (N(G)-methyl-l-arginine, 1 mmol/L) nor a protein kinase C (PKC) inhibitor (bis-indolylmaleimide 1, 1 micromol/L) altered the enhanced level of fibronectin secretion or DNA synthesis seen in response to IL-1beta treatment. However, addition of a TGF-beta-neutralizing antibody significantly reduced IL-1beta-induced fibronectin secretion (IL-1beta + IgG, 262% +/- 72% vs IL-1beta + alphaTGF-beta 156% +/- 14%, P <.02), collagen type 1 production (IL-1beta + IgG, 176% +/- 28% vs IL-1beta + alphaTGF-beta, 120% +/- 14%, P <.005) and abrogated IL-1beta-induced DNA synthesis (245% +/- 49% vs 105% +/- 21%, P <.005). IL-1beta significantly stimulated CF DNA synthesis and production of fibronectin, collagen type 1, TGFbeta, and NO. The fibrogenic and proliferative action of IL-1beta on CF appears not to involve activation of PKC or production of NO but is at least partly TGFbeta-dependent.

Enhancing itaconic acid production by Aspergillus terreus

Aspergillus terreus is successfully used for industrial production of itaconic acid. The acid is formed from cis-aconitate, an intermediate of the tricarboxylic (TCA) cycle, by catalytic action of cis-aconitate decarboxylase. It could be assumed that strong anaplerotic reactions that replenish the pool of the TCA cycle intermediates would enhance the synthesis and excretion rate of itaconic acid. In the phylogenetic close relative Aspergillus niger, upregulated metabolic flux through glycolysis has been described that acted as a strong anaplerotic reaction. Deregulated glycolytic flux was caused by posttranslational modification of 6-phosphofructo-1-kinase (PFK1) that resulted in formation of a highly active, citrate inhibition-resistant shorter form of the enzyme. In order to avoid complex posttranslational modification, the native A. niger pfkA gene has been modified to encode for an active shorter PFK1 fragment. By the insertion of the modified A. niger pfkA genes into the A. terreus strain, increased specific productivities of itaconic acid and final yields were documented by transformants in respect to the parental strain. On the other hand, growth rate of all transformants remained suppressed which is due to the low initial pH value of the medium, one of the prerequisites for the accumulation of itaconic acid by A. terreus mycelium. © 2010 Springer-Verlag.

Inhibition of myeloperoxidase- and neutrophil-mediated oxidant production by tetraethyl and tetramethyl nitroxides

The powerful oxidant HOCl (hypochlorous acid and its corresponding anion, −OCl) generated by the myeloperoxidase (MPO)–H2O2–Cl− system of activated leukocytes is strongly associated with multiple human inflammatory diseases; consequently there is considerable interest in inhibition of this enzyme. Nitroxides are established antioxidants of low toxicity that can attenuate oxidation in animal models, with this ascribed to superoxide dismutase or radical-scavenging activities. We have shown (M.D. Rees et al., Biochem. J. 421, 79–86, 2009) that nitroxides, including 4-amino-TEMPO (4-amino-2,2,6,6-tetramethylpiperidin-1-yloxyl radical), are potent inhibitors of HOCl formation by isolated MPO and activated neutrophils, with IC50 values of ~1 and ~6 µM respectively. The utility of tetramethyl-substituted nitroxides is, however, limited by their rapid reduction by biological reductants. The corresponding tetraethyl-substituted nitroxides have, however, been reported to be less susceptible to reduction. In this study we show that the tetraethyl species were reduced less rapidly than the tetramethyl species by both human plasma (89–99% decreased rate of reduction) and activated human neutrophils (62–75% decreased rate). The tetraethyl-substituted nitroxides retained their ability to inhibit HOCl production by MPO and activated neutrophils with IC50 values in the low-micromolar range; in some cases inhibition was enhanced compared to tetramethyl substitution. Nitroxides with rigid structures (fused oxaspiro rings) were, however, inactive. Overall, these data indicate that tetraethyl-substituted nitroxides are potent inhibitors of oxidant formation by MPO, with longer plasma and cellular half-lives compared to the tetramethyl species, potentially allowing lower doses to be employed.

The duration of Chlamydia muridarum genital tract infection and associated chronic pathological changes are reduced in IL-17 knockout mice but protection is not increased further by immunization

IL-17 is believed to be important for protection against extracellular pathogens, where clearance is dependent on neutrophil recruitment and local activation of epithelial cell defences. However, the role of IL-17 in protection against intracellular pathogens such as Chlamydia is less clear. We have compared (i) the course of natural genital tract C. muridarum infection, (ii) the development of oviduct pathology and (iii) the development of vaccine-induced immunity against infection in wild type (WT) BALB/c and IL-17 knockout mice (IL-17-/-) to determine if IL-17-mediated immunity is implicated in the development of infection-induced pathology and/or protection. Both the magnitude and duration of genital infection was significantly reduced in IL-17-/- mice compared to BALB/c. Similarly, hydrosalpinx was also greatly reduced in IL-17-/- mice and this correlated with reduced neutrophil and macrophage infiltration of oviduct tissues. Matrix metalloproteinase (MMP) 9 and MMP2 were increased in WT oviducts compared to IL-17-/- animals at day 7 post-infection. In contrast, oviducts from IL-17-/- mice contained higher MMP9 and MMP2 at day 21. Infection also elicited higher levels of Chlamydia-neutralizing antibody in serum of IL-17-/- mice than WT mice. Following intranasal immunization with C. muridarum Major Outer Membrane Protein (MOMP) and cholera toxin plus CpG adjuvants, significantly higher levels of chlamydial MOMP-specific IgG and IgA were found in serum and vaginal washes of IL-17-/- mice. T cell proliferation and IFNγ production by splenocytes was greater in WT animals following in vitro re-stimulation, however vaccination was only effective at reducing infection in WT, not IL-17-/- mice. Intranasal or transcutaneous immunization protected WT but not IL-17-/- mice against hydrosalpinx development. Our data show that in the absence of IL-17, the severity of C. muridarum genital infection and associated oviduct pathology are significantly attenuated, however neither infection or pathology can be reduced further by vaccination protocols that effectively protect WT mice.

Ananyi (Going) by Tjupi Band

Research background: Ananyi (Going) is an intercultural music project with lyrics sung in Luritja and English, undertaken in collaboration with the Tjupi Band and producer Jeffrey McLaughlin. The project contributes to cultural maintenance for Australian First Nations peoples, and is informed by prior work in this area by scholars including Peter Dunbar-Hall, Chris Gibson and Karl Neuenfeldt. These existing studies have discussed the complexities of intercultural collaboration, and the types of cultural politics that are involved when Indigenous and non-Indigenous musicians and scholars work together on projects of cultural significance. Critical race theory has also informed the creative work, as a means of interpreting the implicit and explicit discourses of race that arise through intercultural creative practice. The project asked the research question, how can collaborative music making contribute to intercultural understanding and the maintenance of Australian First Nations languages and cultures? Research contribution: The project has identified that recorded popular music is important in the maintenance of Luritja language and culture, and that intercultural collaboration in the areas of digital sound production and distribution can assist with cultural maintenance in both local and national contexts. Research significance: The compact disc was released on the CAAMA Music label, and supported through competitive grants from the Australian Government’s Contemporary Music Touring Grant and the Arnhem Land Progress Association (ALPA). The research context of the work is detailed in Brydie-Leigh Bartleet and Gavin Carfoot 2013. "Desert harmony: Stories of collaboration between Indigenous musicians and university students." International Education Journal: Comparative Perspectives 12 (1): 180-196.

Microbial oil production from sugarcane bagasse hydrolysates by oleaginous yeast and filamentous fungi

This study investigated the potential use of sugarcane bagasse as a feedstock for oil production through microbial cultivation. Bagasse was subjected to dilute acid pretreatment with 0.4 wt% H2SO4 (in liquid) at a solid/liquid ratio of 1:6 (wt/wt) at 170 °C for 15 min, followed by enzymatic hydrolysis of solid residue. The liquid fractions of the pretreatment process and the enzymatic hydrolysis process were detoxified and used as liquid hydrolysate (SCBLH) and enzymatic hydrolysate (SCBEH) for the microbial oil production by oleaginous yeast (Rhodotorula mucilaginosa) and filamentous fungi (Aspergillus oryzae and Mucor plumbeus). The results showed that all strains were able to grow and produce oil from bagasse hydrolysates. The highest oil concentrations produced from bagasse hydrolysates were by M. plumbeus at 1.59 g/L (SCBLH) and 4.74 g/L (SCBEH). The microbial oils obtained have similar fatty acid compositions to vegetable oils, indicating that the oil can be used for the production of second generation biodiesel. On the basis of oil yields obtained by M. plumbeus, from 10 million t (wet weight) of bagasse generated annually from sugar mills in Australia, it is estimated that the total biodiesel that could be produced would be equivalent to about 9% of Queensland’s diesel consumption.

Towards improved speech recognition for resource poor languages

In recent times, the improved levels of accuracy obtained by Automatic Speech Recognition (ASR) technology has made it viable for use in a number of commercial products. Unfortunately, these types of applications are limited to only a few of the world’s languages, primarily because ASR development is reliant on the availability of large amounts of language specific resources. This motivates the need for techniques which reduce this language-specific, resource dependency. Ideally, these approaches should generalise across languages, thereby providing scope for rapid creation of ASR capabilities for resource poor languages. Cross Lingual ASR emerges as a means for addressing this need. Underpinning this approach is the observation that sound production is largely influenced by the physiological construction of the vocal tract, and accordingly, is human, and not language specific. As a result, a common inventory of sounds exists across languages; a property which is exploitable, as sounds from a resource poor, target language can be recognised using models trained on resource rich, source languages. One of the initial impediments to the commercial uptake of ASR technology was its fragility in more challenging environments, such as conversational telephone speech. Subsequent improvements in these environments has gained consumer confidence. Pragmatically, if cross lingual techniques are to considered a viable alternative when resources are limited, they need to perform under the same types of conditions. Accordingly, this thesis evaluates cross lingual techniques using two speech environments; clean read speech and conversational telephone speech. Languages used in evaluations are German, Mandarin, Japanese and Spanish. Results highlight that previously proposed approaches provide respectable results for simpler environments such as read speech, but degrade significantly when in the more taxing conversational environment. Two separate approaches for addressing this degradation are proposed. The first is based on deriving better target language lexical representation, in terms of the source language model set. The second, and ultimately more successful approach, focuses on improving the classification accuracy of context-dependent (CD) models, by catering for the adverse influence of languages specific phonotactic properties. Whilst the primary research goal in this thesis is directed towards improving cross lingual techniques, the catalyst for investigating its use was based on expressed interest from several organisations for an Indonesian ASR capability. In Indonesia alone, there are over 200 million speakers of some Malay variant, provides further impetus and commercial justification for speech related research on this language. Unfortunately, at the beginning of the candidature, limited research had been conducted on the Indonesian language in the field of speech science, and virtually no resources existed. This thesis details the investigative and development work dedicated towards obtaining an ASR system with a 10000 word recognition vocabulary for the Indonesian language.

Comparison of intranasal and transcutaneous immunization for induction of protective immunity against chlamydia muridarum respiratory tract infection

Chlamydia pneumoniae causes a range of respiratory infections including bronchitis, pharyngitis and pneumonia. Infection has also been implicated in exacerbation/initiation of asthma and chronic obstructive pulmonary disease (COPD) and may play a role in atherosclerosis and Alzheimer's disease. We have used a mouse model of Chlamydia respiratory infection to determine the effectiveness of intranasal (IN) and transcutaneous immunization (TCI) to prevent Chlamydia lung infection. Female BALB/c mice were immunized with chlamydial major outer membrane protein (MOMP) mixed with cholera toxin and CpG oligodeoxynucleotide adjuvants by either the IN or TCI routes. Serum and bronchoalveolar lavage (BAL) were collected for antibody analysis. Mononuclear cells from lung-draining lymph nodes were stimulated in vitro with MOMP and cytokine mRNA production determined by real time PCR. Animals were challenged with live Chlamydia and weighed daily following challenge. At day 10 (the peak of infection) animals were sacrificed and the numbers of recoverable Chlamydia in lungs determined by real time PCR. MOMP-specific antibody-secreting cells in lung tissues were also determined at day 10 post-infection. Both IN and TCI protected animals against weight loss compared to non-immunized controls with both immunized groups gaining weight by day 10-post challenge while controls had lost 6% of body weight. Both immunization protocols induced MOMP-specific IgG in serum and BAL while only IN immunization induced MOMP-specific IgA in BAL. Both immunization routes resulted in high numbers of MOMP-specific antibody-secreting cells in lung tissues (IN > TCI). Following in vitro re-stimulation of lung-draining lymph node cells with MOMP; IFNγ mRNA increased 20-fold in cells from IN immunized animals (compared to non-immunized controls) while IFNγ levels increased 6- to 7-fold in TCI animals. Ten days post challenge non-immunized animals had >7000 IFU in their lungs, IN immunized animals <50 IFU and TCI immunized animals <1500 IFU. Thus, both intranasal and transcutaneous immunization protected mice against respiratory challenge with Chlamydia. The best protection was obtained following IN immunization and correlated with IFNγ production by mononuclear cells in lung-draining LN and MOMP-specific IgA in BAL.

Characteristics of modal sound radiation of finite cylindrical shells

Characteristics of modal sound radiation of finite cylindrical shells are studied using finite element and boundary element methods in this paper. In the low frequency range, modal radiation efficiencies of finite cylindrical shells are found to asymptotically approach those of the corresponding infinite cylindrical shell when structural trace wavelengths of the cylindrical shells are greater than the acoustic wavelength. Modal radiation efficiencies for each group of modes having the same circumferential modal index decrease as the axial modal index increases. They converge to each other when the axial trace wavelength is much greater than the circumferential trace wavelength. The mechanism leading to lower radiation efficiency of modes with higher circumferential modal index of short cylinders is explained. Similar to those of flat plate panels, change in slope or waviness is observed in modal radiation efficiency curves of modes with higher order axial modal index at medium frequencies. This is attributed to the interference of sound radiated by neighbouring vibrating cells when the distance between nodal lines of a vibrating mode is in the same order or smaller than the acoustic wavelength. Effects of the internal sound field on modal radiation efficiencies of a finite open-end cylinder are discussed.

Bovine colostrum modulates cytokine production in human peripheral blood mononuclear cells stimulated with lipopolysaccharide and phytohemagglutinin

Bovine colostrum has been shown to influence the cytokine production of bovine leukocytes. However, it remains unknown whether processed bovine colostrum, a supplement popular among athletes to enhance immune function, is able to modulate cytokine secretion of human lymphocytes and monocytes. The aim of this investigation was to determine the influence of a commercially available bovine colostrum protein concentrate (CPC) to stimulate cytokine production by human peripheral blood mononuclear cells (PBMCs). Blood was sampled from four healthy male endurance athletes who had abstained from exercise for 48 h. PBMCs were separated and cultured with bovine CPC concentrations of 0 (control), 1.25, 2.5, and 5% with and without lipopolysaccharide (LPS) (3 microg/mL) and phytohemagglutinin (PHA) (2.5 microg/mL). Cell supernatants were collected at 6 and 24 h of culture for the determination of tumor necrosis factor (TNF), interferon (IFN)-gamma, interleukin (IL)-10, IL-6, IL-4, and IL-2 concentrations. Bovine CPC significantly stimulated the release of IFN-gamma, IL-10, and IL-2 (p < 0.03). The addition of LPS to PBMCs cocultured with bovine CPC significantly stimulated the release of IL-2 and inhibited the early release of TNF, IL-6, and IL-4 (p < 0.02). Phytohemagglutinin stimulation in combination with bovine CPC significantly increased the secretion of IL-10 and IL-2 at 6 h of culture and inhibited IFN-gamma and TNF (p < 0.05). This data show that a commercial bovine CPC is able to modulate in vitro cytokine production of human PBMCs. Alterations in cytokine secretion may be a potential mechanism for reported benefits associated with supplementation.