Adenylate cyclase activity during modulation of Bordetella pertussis

During the phenotypic and genotypic changes which occur during the processes respectively known as C modulation and phase degradation, several virulence-associated properties of Bordetella pertussis and adenylate cyclase (AC) are lost (Parton and Durham, 197B; Wardlaw and Parton, 1979). The main aim of the present investigation was to assess whether AC plays a causal role in these two distinct processes as this enzyme is known to play a key regulatory role in the Enterobacteriaceas. Growth of B. pertussis in Stainer and Scholte medium (SS-X) containing high levels of MgSO4, Na2 504, butyrate, Na caprylate, Na succinate or nicotinic acid resulted in C modulation as shown by marked reductions in AC activity, histamine-sensitizing activity (HSA) and the 28k and 3Dk cell-envelope polypeptides (X polypeptides). Although there was some variation between the susceptibility of strains to pro-C-mode salts, there was no instance of components being lost independently of each other. The level of cAMP in the supernate of C-mode cultures was less than 5x of that of X-inode cultures. Magnesium chloride, present at four times the molar concentration of MgSO4 required to induce C modulation, was ineffective at inducing loss of AC activity HSA, or the X polypeptides. During MgSO4-induced modulation, AC activity in three compartments (viz. culture supernate, cell-associated but extracytoplasmic, and cytoplasmic) was reduced to the same extent. Time-course studies on the rate of loss of AC activity, HSA, and the X-polypeptides during ngSO4-induced modulation indicated that these properties were lost simultaneously. Furthermore, losses could be accounted for by complete repression of the synthesis of the components when cells were introduced into C-medium. The low levels of cAMP in the supernate of C-mode cultures could not be accounted for by the ability of pro-C-mode salts to inhibit vitro AC activity, or by inhibition of cAMP excretion by C medium. Loss of AC activity during C modulation required growth and was not due to prolonged exposure to C medium, as chloramphenicol added to C medium prevented loss of AC activity. Loss of HSA and the X polypeptides also required growth or protein synthesis. C-mode cell lysate did not inhibit AC activity of X-mode cell lysate suggesting that loss of AC activity during C modulation is not due to production of an inactivating or inhibitory factor. Similarly, C-mode cell lysate did not destroy HSA present in X-mode cell lysate. Sodium fluoride caused marked inhibition of vitro AC activity, but, at the same concentration, had little effect on the synthesis of cAMP during growth. Growth in X medium containing B. pertussis AC activator (calmodulin) resulted in a four fold-increase in culture supernate cAMP levels. The critical concentration of MgSO4 required to induce loss of the X polypeptides was 10 - 11 mH. In one culture, containing 10 mH MgSD4 and AC activator, partial loss of the X polypeptides occurred yet cAMP levels in the supernate were twice that which normally occurred in X-mode cultures without activator. Respiration rates of amino acids by B. pertussis variants was investigated to determine whether cARP played a role in amino acid catabolism in the organism. The ability of washed suspensions of X- and C-mode and phase IV B. pertussis to respire L-glutamate, L-aspartate, L-proline, L-alanine and L-serine was demonstrated. While differences were found between the respiration rates of different amino acids, there were no significant differences between the ability of variants of B. pertussis to respire any particular amino acid. Phosphonomycin resistance has been used as a convenient method to isolate AC mutants of Escherichia coli. However, eight independently isolated phosphonomycin resistant mutants of B. pertussis possessed the same AC activity as the original strain. Exogenous cAMP and dibutyryl cAnP, in X and C media, had no effect on the production of AC, the X polypeptides or haeraagglutinin. Attempts were made to determine if a cyclic AnP-receptor protein (CRP) analogous to that in E. coli exists in B, pertussis. [3h] cAMP binding activity was demonstrated in several strains of B. pertussis and was about half that obtained for E. coli. Anti-E. coli CRP gave two precipitin lines with E. coli cell extract but none with B. pertussis cell extract. In conclusion, the results of this study suggest that AC does not play a causal role in modulation, and that the mechanism responsible for repressing synthesis of X-mode specific components (such as pertussigen) during modulation, also represses synthesis of AC

An exploration of the role of ethics in leadership decision-making in change initiatives in Queensland government owned corporations

The context for this thesis is a perceived convergence of two themes: high failure rates of change initiatives and growing concern about ethics in leadership decision-making (LDM). Change initiatives have become pervasive in business and government and are increasing in volume, speed and size (Burnes 1996; Keller & Aiken 2008). Change processes however have a high failure rate with significant financial, social and political consequences. One of the consistently identified factors contributing to these failure rates is the ‘Lack of Leadership/Executive Support’ (Chaos, The Standish Group Report 1995; A guide to ERP Success 2001; Challenge of Change: Australia 2010; Cooke et al. 2001; Eser et al. 2007; Keller & Aiken 2008; KPMG 1997; OASIG 1995), although there is limited research of what this comprises. Decision-making is however a core leadership competency (Woiceshyn 2011) increasing concern and research has focused attention on the ethical context in which leaders make decisions (Coldwell 2010; Dassah 2010; Fykse Tveit 2010; Schwab et al. 2010) particularly in the light of recent business scandals. Compounding this situation is the rate of change in business (Coldwell 2010) coupled with the perceived and actual role of leadership decision making (LDM). Consequently, there is increasing pressure on organisations to improve the quality of LDM processes in change initiatives. To date, the interrelationships and behaviours of leaders and the ethics they employ often appear as separate fields of ontological inquiry; in terms of LDM however, these interrelationships form the essence of improving LDM within organisational contexts and their impact on society. This thesis is a qualitative analysis of two case studies of change initiatives in Queensland Government owned corporations focussing on the processes, consequences and outcomes of LDM. The research findings indicate that the quality of LDM may be improved by addressing the two issues of equivocality and uncertainty through the inclusion of ethics and logic in a prescriptive and iterative LDM process. The proposed Q.L.D. model for improving the quality of LDM in change initiatives combines the elements of ethics, logic, organisational learning and change leadership to decrease levels of equivocality and uncertainty. The Q.L.D. model presents a disciplined and focused process for leadership decision making in change initiatives by decreasing the impact of the factors affecting failure rates

Using LiDAR to assess the effect of fire and floods on upland peat bogs, Waterfall Gully, Mount Lofty Ranges, South Australia

A flood exceeding the 100 year average recurrence interval in November 2005 led to the failure of an upland peat bog in Waterfall Gully. The area is prone to severe bushfire and flood events and the control dam at the base of First Falls was filled with sediment sourced from Wilson Bog. A resistant quartzite bar at Fourth Falls has formed a natural constriction point against which burnt logs and debris have collected following previous fire events forming a natural dam resulting in sediment/peat accumulation upstream. The failure of the bog was inevitable as the vegetative material in the log-jam progressively weakened and rotted. Intense flooding triggered the failure but it was augmented by the build up of a critical mass of sediment upstream of the restriction point. The downstream force of the flood waters and the weight of the saturated bog sediments was enough to overcome the basal frictional forces resulting in slumping and headward erosion. LiDAR data clearly shows an erosion channel scoured out by the flood. Approximately 5000 m3 of sediment (-10,100 tonnes) was washed downstream. LiDAR coupled with a tri-spectral scanner has the capacity to identify other upland peat bogs due to their high NDVI value and assess their stability on steep slopes or narrow valleys. Fire is another risk to the stability of these bogs as it has the potential to remove binding vegetation and expose unconsolidated sediments to erosion during subsequent rain events. Groundsurface and vegetation surface DEM\u27s generated from LiDAR combined with NDVI maps derived from a tri-spectral scanner provide an ideal tool to monitor and assess the risk of slumping in other upland peat bogs

Mars oxygen production system design

The design and construction phase is summarized of the Mars oxygen demonstration project. The basic hardware required to produce oxygen from simulated Mars atmosphere was assembled and tested. Some design problems still remain with the sample collection and storage system. In addition, design and development of computer compatible data acquisition and control instrumentation is ongoing

The atypical chemokine receptor Ackr2 constrains NK cell migratory activity and promotes metastasis

Chemokines have been shown to be essential players in a range of cancer contexts. In this study, we demonstrate that mice deficient in the atypical chemokine receptor Ackr2 display impaired development of metastasis in vivo in both cell line and spontaneous models. Further analysis reveals that this relates to increased expression of the chemokine receptor CCR2, specifically by KLRG1+ NK cells from the Ackr2−/− mice. This leads to increased recruitment of KLRG1+ NK cells to CCL2-expressing tumors and enhanced tumor killing. Together, these data indicate that Ackr2 limits the expression of CCR2 on NK cells and restricts their tumoricidal activity. Our data have important implications for our understanding of the roles for chemokines in the metastatic process and highlight Ackr2 and CCR2 as potentially manipulable therapeutic targets in metastasis

Deletion of the protein tyrosine phosphatase PTPN22 for adoptive T cell therapy facilitates CTL effector function but promotes T cell exhaustion

Background Adoptive cell therapy (ACT) is a promising strategy for treating cancer, yet it faces several challenges such as lack of long term protection due to T cell exhaustion induced by chronic TCR stimulation in the tumor microenvironment. One benefit of ACT, however, is that it allows for cellular manipulations, such as deletion of the phosphotyrosine phosphatase non-receptor type 22 (PTPN22), which improves CD8+ T cell anti-tumor efficacy in ACT. We tested whether Ptpn22KO cytolytic T cells (CTL) were also more effective than Ptpn22WT CTL in controlling tumors in scenarios that favor T cell exhaustion. Methods Tumor control by Ptpn22WT and Ptpn22KO CTL was assessed following adoptive transfer of low numbers of CTL to mice with subcutaneously implanted MC38 tumors. Tumor infiltrating lymphocytes were isolated for analysis of effector functions. An in vitro assay was established to compare CTL function in response to acute and chronic re-stimulation with antigen-pulsed tumor cells. The expression of effector and exhaustion-associated proteins by Ptpn22WT and Ptpn22KO T cells was followed over time in vitro and in vivo using the ID8 tumor model. Finally, the effect of PD-1 and TIM-3 blockade on Ptpn22KO CTL tumor control was assessed using monoclonal antibodies and CRISPR/Cas9-mediated knockout. Results Despite having improved effector function at the time of transfer, Ptpn22KO CTL became more exhausted than Ptpn22WT CTL, characterized by more rapid loss of effector functions, and earlier and higher expression of inhibitory receptors (IRs), particularly the terminal exhaustion marker TIM-3. TIM-3 expression, under the control of the transcription factor NFIL3, was induced by IL-2 signaling which was enhanced in Ptpn22KO cells. Anti-tumor responses of Ptpn22KO CTL were improved following PD-1 blockade in vivo, yet knockout or antibody-mediated blockade of TIM-3 did not improve but further impaired tumor control, indicating TIM-3 signaling itself did not drive the diminished function seen in Ptpn22KO CTL. Conclusions This study questions whether TIM-3 plays a role as an IR and highlights that genetic manipulation of T cells for ACT needs to balance short term augmented effector function against the risk of T cell exhaustion in order to achieve longer term protection. What is already known on this topic • T cell exhaustion in the tumor microenvironment is a major factor limiting the potential success of adoptive cell therapy (ACT) in the treatment of solid tumors. • Deletion of the phosphatase PTPN22 in CD8+ T cells improves their response to tumors, but it is not known whether this influences development of exhaustion. What this study adds • Under conditions which promote exhaustion, CTL lacking PTPN22 exhaust more rapidly than WT cells, despite displaying enhanced effector function in their initial response to antigen. • Ptpn22KO CTL express high levels of the inhibitory receptor TIM-3, but TIM-3 signaling does not directly contribute to Ptpn22KO CTL dysfunction. • Ptpn22KO T cells are more responsive to IL-2 through JAK-STAT signaling, which induces TIM-3 expression via the transcription factor NFIL3. How this study might affect research, practice or policy • Strategies aimed at augmenting T cell effector function for ACT should balance improved responses against an increased risk of T cell exhaustion

Mechanistic Target of Rapamycin Complex 1/S6 Kinase 1 Signals Influence T Cell Activation Independently of Ribosomal Protein S6 Phosphorylation

Ag-dependent activation of naive T cells induces dramatic changes in cellular metabolism that are essential for cell growth, division, and differentiation. In recent years, the serine/threonine kinase mechanistic target of rapamycin (mTOR) has emerged as a key integrator of signaling pathways that regulate these metabolic processes. However, the role of specific downstream effectors of mTOR function in T cells is poorly understood. Ribosomal protein S6 (rpS6) is an essential component of the ribosome and is inducibly phosphorylated following mTOR activation in eukaryotic cells. In the current work, we addressed the role of phosphorylation of rpS6 as an effector of mTOR function in T cell development, growth, proliferation, and differentiation using knockin and TCR transgenic mice. Surprisingly, we demonstrate that rpS6 phosphorylation is not required for any of these processes either in vitro or in vivo. Indeed, rpS6 knockin mice are completely sensitive to the inhibitory effects of rapamycin and an S6 kinase 1 (S6K1)–specific inhibitor on T cell activation and proliferation. These results place the mTOR complex 1-S6K1 axis as a crucial determinant of T cell activation independently of its ability to regulate rpS6 phosphorylation

Full Genome Characterization of the Culicoides-Borne Marsupial Orbiviruses: Wallal Virus, Mudjinbarry Virus and Warrego Viruses

Viruses belonging to the species Wallal virus and Warrego virus of the genus Orbivirus were identified as causative agents of blindness in marsupials in Australia during 1994/5. Recent comparisons of nucleotide (nt) and amino acid (aa) sequences have provided a basis for the grouping and classification of orbivirus isolates. However, full-genome sequence data are not available for representatives of all Orbivirus species. We report full-genome sequence data for three additional orbiviruses: Wallal virus (WALV); Mudjinabarry virus (MUDV) and Warrego virus (WARV). Comparisons of conserved polymerase (Pol), sub-core-shell 'T2' and core-surface 'T13' proteins show that these viruses group with other Culicoides borne orbiviruses, clustering with Eubenangee virus (EUBV), another orbivirus infecting marsupials. WARV shares <70% aa identity in all three conserved proteins (Pol, T2 and T13) with other orbiviruses, consistent with its classification within a distinct Orbivirus species. Although WALV and MUDV share <72.86%/67.93% aa/nt identity with other orbiviruses in Pol, T2 and T13, they share >99%/90% aa/nt identities with each other (consistent with membership of the same virus species - Wallal virus). However, WALV and MUDV share <68% aa identity in their larger outer capsid protein VP2(OC1), consistent with membership of different serotypes within the species - WALV-1 and WALV-2 respectively