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Bacterial flagella are rotary nanomachines that contribute to bacterial fitness in many settings, including host colonization. The flagellar motor relies on the multiprotein flagellar motor-switch complex to govern flagellum formation and rotational direction. Different bacteria exhibit great diversity in their flagellar motors. One such variation is exemplified by the motor-switch apparatus of the gastric pathogen Helicobacter pylori, which carries an extra switch protein, FliY, along with the more typical FliG, FliM, and FliN proteins. All switch proteins are needed for normal flagellation and motility in H. pylori, but the molecular mechanism of their assembly is unknown. To fill this gap, we examined the interactions among these proteins. We found that the C-terminal SpoA domain of FliY (FliYC) is critical to flagellation and forms heterodimeric complexes with the FliN and FliM SpoA domains, which are β-sheet domains of type III secretion system proteins. Surprisingly, unlike in other flagellar switch systems, neither FliY nor FliN self-associated. The crystal structure of the FliYC-FliNC complex revealed a saddle-shaped structure homologous to the FliN-FliN dimer of Thermotoga maritima, consistent with a FliY-FliN heterodimer forming the functional unit. Analysis of the FliYC-FliNC interface indicated that oppositely charged residues specific to each protein drive heterodimer formation. Moreover, both FliYC-FliMC and FliYC-FliNC associated with the flagellar regulatory protein FliH, explaining their important roles in flagellation. We conclude that H. pylori uses a FliY-FliN heterodimer instead of a homodimer and creates a switch complex with SpoA domains derived from three distinct proteins.

Natural Killer (NK) cell cytotoxicity and interferon-gamma (IFNγ) production are profoundly suppressed postoperatively. This dysfunction is associated with increased morbidity and cancer recurrence. NK activity depends on the integration of activating and inhibitory signals, which may be modulated by transforming growth factor-beta (TGF-β). We hypothesized that impaired postoperative NK cell IFNγ production is due to altered signaling pathways caused by postoperative TGF-β. NK cell receptor expression, downstream phosphorylated targets, and IFNγ production were assessed using peripheral blood mononuclear cells (PBMCs) from patients undergoing cancer surgery. Healthy NK cells were incubated in the presence of healthy/baseline/postoperative day (POD) 1 plasma and in the presence/absence of a TGF-β-blocking monoclonal antibody (mAb) or the small molecule inhibitor (smi) SB525334. Single-cell RNA sequencing (scRNA-seq) was performed on PBMCs from six patients with colorectal cancer having surgery at baseline/on POD1. Intracellular IFNγ, activating receptors (CD132, CD212, NKG2D, DNAM-1), and downstream target (STAT5, STAT4, p38 MAPK, S6) phosphorylation were significantly reduced on POD1. Furthermore, this dysfunction was phenocopied in healthy NK cells through incubation with rTGF-β1 or POD1 plasma and was prevented by the addition of anti-TGF-β immunotherapeutics (anti-TGF-β mAb or TGF-βR smi). Targeted gene analysis revealed significant decreases in S6 and FKBP12, an increase in Shp-2, and a reduction in NK metabolism-associated transcripts on POD1. pSmad2/3 was increased and pS6 was reduced in response to rTGF-β1 on POD1, changes that were prevented by anti-TGF-β immunotherapeutics. Together, these results suggest that both canonical and mTOR pathways downstream of TGF-β mediate phenotypic changes that result in postoperative NK cell dysfunction. [ABSTRACT FROM AUTHOR]

Supply chain reporting has gained increased scrutiny as companies expand their global supplier networks, address climate change risks/opportunities and with the recent introduction of modern slavery legislation. The question remains as to whether supply chain disclosures address stakeholder requirements. We compare the supply chain disclosure preferences of 20 stakeholders with current disclosure practice for a sample of large listed companies in Australia. We contribute to the emerging supply chain disclosure literature by documenting the factors driving and hindering supply chain disclosure. Further, we offer theoretical and policy implications arising from analysis of stakeholder preferences, drivers and challenges to supply chain reporting. [ABSTRACT FROM AUTHOR]

Increased [alpha]-synuclein gene (SNCA) dosage due to locus multiplication causes autosomal dominant Parkinson's disease (PD). Variation in SNCA expression may be critical in common, genetically complex PD but the underlying regulatory mechanism is unknown. We show that SNCA and the heme metabolism genes ALAS2, FECH, and BLVRB form a block of tightly correlated gene expression in 113 samples of human blood, where SNCA naturally abounds (validated P = 1.6 x [10.sup.-11], 1.8 x [10.sup.-10], and 6.6 x [10.sup.-5]). Genetic complementation analysis revealed that these four genes are co-induced by the transcription factor GATA-1. GATA-1 specifically occupies a conserved region within SNCA intron-1 and directly induces a 6.9-fold increase in [alpha]-synuclein. Endogenous GATA-2 is highly expressed in substantia nigra vulnerable to PD, occupies intron-1, and modulates SNCA expression in dopaminergic cells. This critical link between GATA factors and SNCA may enable therapies designed to lower [alpha]-synuclein production. [alpha]-synuclein dosage | GATA-1 | GATA-2 | gene expression | microarray

Abstract Background Elevated SNCA gene expression and intracellular accumulation of the encoded α-synuclein (aSyn) protein are associated with the development of Parkinson disease (PD). To date, few enzymes have been examined for their ability to degrade aSyn. Here, we explore the effects of CTSD gene expression, which encodes the lysosomal protease cathepsin D (CathD), on aSyn processing. Results Over-expression of human CTSD cDNA in dopaminergic MES23.5 cell cultures induced the marked proteolysis of exogenously expressed aSyn proteins in a dose-dependent manner. Unexpectedly, brain extractions, Western blotting and ELISA quantification revealed evidence for reduced levels of soluble endogenous aSyn in ctsd knock-out mice. However, these CathD-deficient mice also contained elevated levels of insoluble, oligomeric aSyn species, as detected by formic acid extraction. In accordance, immunohistochemical studies of ctsd-mutant brain from mice, sheep and humans revealed selective synucleinopathy-like changes that varied slightly among the three species. These changes included intracellular aSyn accumulation and formation of ubiquitin-positive inclusions. Furthermore, using an established Drosophila model of human synucleinopathy, we observed markedly enhanced retinal toxicity in ctsd-null flies. Conclusion We conclude from these complementary investigations that: one, CathD can effectively degrade excess aSyn in dopaminergic cells; two, ctsd gene mutations result in a lysosomal storage disorder that includes microscopic and biochemical evidence of aSyn misprocessing; and three, CathD deficiency facilitates aSyn toxicity. We therefore postulate that CathD promotes 'synucleinase' activity, and that enhancing its function may lower aSyn concentrations in vivo.

In this paper, we examine the conceptual framework, accounting standards and accounting information relevant to the not‐for‐profit (NFP) sector. Based on the responses of 242 Australian NFP managers, we find support for the inclusion of accountability in the conceptual framework, and for a common set of accounting standards across NFP and for‐profit sectors with additional standards or paragraphs to recognise NFP specific issues. Respondents also rated information within general‐purpose financial reports to be useful for decision making within their organisations. We offer suggestions as to what our findings mean for the development of accounting standards for the NFP sector. [ABSTRACT FROM AUTHOR]

Natural Killer (NK) cells are innate immune responders critical for viral clearance and immunomodulation. Despite their vital role in viral infection, the contribution of NK cells in fighting SARS-CoV-2 has not yet been directly investigated. Insights into pathophysiology and therapeutic opportunities can therefore be inferred from studies assessing NK cell phenotype and function during SARS, MERS, and COVID-19. These studies suggest a reduction in circulating NK cell numbers and/or an exhausted phenotype following infection and hint toward the dampening of NK cell responses by coronaviruses. Reduced circulating NK cell levels and exhaustion may be directly responsible for the progression and severity of COVID-19. Conversely, in light of data linking inflammation with coronavirus disease severity, it is necessary to examine NK cell potential in mediating immunopathology. A common feature of coronavirus infections is that significant morbidity and mortality is associated with lung injury and acute respiratory distress syndrome resulting from an exaggerated immune response, of which NK cells are an important component. In this review, we summarize the current understanding of how NK cells respond in both early and late coronavirus infections, and the implication for ongoing COVID-19 clinical trials. Using this immunological lens, we outline recommendations for therapeutic strategies against COVID-19 in clearing the virus while preventing the harm of immunopathological responses. [ABSTRACT FROM AUTHOR]

The majority of data on human Natural Killer (NK) cell phenotype and function has been generated using cryopreserved peripheral blood mononuclear cells (PBMCs). However, cryopreservation can have adverse effects on PBMCs. In contrast, investigating immune cells in whole blood can reduce the time, volume of blood required, and potential artefacts associated with manipulation of the cells. Whole blood collected from healthy donors and cancer patients was processed by three separate protocols that can be used independently or in parallel to assess extracellular receptors, intracellular signaling protein phosphorylation, and intracellular and extracellular cytokine production in human NK cells. To assess extracellular receptor expression, 200 μL of whole blood was incubated with an extracellular staining (ECS) mix and cells were subsequently fixed and RBCs lysed prior to analysis. The phosphorylation status of signaling proteins was assessed in 500 μL of whole blood following co-incubation with interleukin (IL)-2/12 and an ECS mix for 20 min prior to cell fixation, RBC lysis, and subsequent permeabilization for staining with an intracellular staining (ICS) mix. Cytokine production (IFNγ) was similarly assessed by incubating 1 mL of whole blood with PMA-ionomycin or IL-2/12 prior to incubation with ECS and subsequent ICS antibodies. In addition, plasma was collected from stimulated samples prior to ECS for quantification of secreted IFNγ by ELISA. Results were consistent, despite inherent inter-patient variability. Although we did not investigate an exhaustive list of targets, this approach enabled quantification of representative ECS surface markers including activating (NKG2D and DNAM-1) and inhibitory (NKG2A, PD-1, TIGIT, and TIM-3) receptors, cytokine receptors (CD25, CD122, CD132, and CD212) and ICS markers associated with NK cell activation following stimulation, including signaling protein phosphorylation (p-STAT4, p-STAT5, p-p38 MAPK, p-S6) and IFNγ in both healthy donors and cancer patients. In addition, we compared extracellular receptor expression using whole blood vs. cryopreserved PBMCs and observed a significant difference in the expression of almost all receptors. The methods presented permit a relatively rapid parallel assessment of immune cell receptor expression, signaling protein activity, and cytokine production in a minimal volume of whole blood from both healthy donors and cancer patients. [ABSTRACT FROM AUTHOR]

Efforts by Australian standard-setters to harmonise public sector financial reporting resulted in AASB 1049, which sought to bridge the divide between generally accepted accounting principles (GAAP)-based and government finance statistics (GFS)-based financial statements. However, whether AASB 1049 has resulted in information that is considered appropriate for the public sector has not been examined. We explore this issue by comparing the requirements of AASB 1049 with the responses from a survey of public sector stakeholders on the appropriate accounting treatment and presentation of selected financial items. The analysis suggests consensus with AASB 1049 on presentation issues but less consensus on accounting treatments. [ABSTRACT FROM AUTHOR]

Recent international developments have refuelled the debate on public sector conceptual framework issues. Drawing on the Australian experience, this study surveys stakeholders of public sector financial reports to examine issues of concern in the development of concepts, definitions and principles pertinent to a public sector conceptual framework. The empirical evidence reveals varying degrees of consensus to questions relating to the objectives of financial reporting, the boundaries of financial reporting and financial statement elements. Respondents are generally not supportive of a single conceptual framework for both private and public sectors. The study also draws on the practices from other countries to provide a more insightful analysis. The study informs the progress of the development of a public sector conceptual framework by highlighting areas that need attention and identifying challenges that exist for standard setters in the further development of a conceptual framework that meets the needs of the public sector. [ABSTRACT FROM AUTHOR]

In addition to widespread use in reducing the symptoms of colds and flu, Echinacea is traditionally employed to treat fungal and bacterial infections. However, to date the mechanism of antimicrobial activity of Echinacea extracts remains unclear. We utilized a set of ∼4,600 viable gene deletion mutants of Saccharomyces cerevisiae to identify mutations that increase sensitivity to Echinacea. Thus, a set of chemical-genetic profiles for 16 different Echinacea treatments was generated, from which a consensus set of 23 Echinacea-sensitive mutants was identified. Of the 23 mutants, only 16 have a reported function. Ten of these 16 are involved in cell wall integrity/structure suggesting that a target for Echinacea is the fungal cell wall. Follow-up analyses revealed an increase in sonication-associated cell death in the yeasts S. cerevisiae and Cryptococcus neoformans after Echinacea extract treatments. Furthermore, fluorescence microscopy showed that Echinacea-treated S. cerevisiae was significantly more prone to cell wall damage than non-treated cells. This study further demonstrates the potential of gene deletion arrays to understand natural product antifungal mode of action and provides compelling evidence that the fungal cell wall is a target of Echinacea extracts and may thus explain the utility of this phytomedicine in treating mycoses. [ABSTRACT FROM AUTHOR]

This study examines the usefulness of three accounting systems (cash, Generally Accepted Accounting Principles (GAAP) accrual, and Government Finance Statistics (GFS) accrual) for public sector decision-making. From a survey of internal users, external users, and preparers in Australia, we find that GAAP accrual information is perceived to be relatively more useful and understandable than the other two systems for most decisions examined. The relatively higher ratings for GAAP accrual information differ from earlier studies and may reflect an experience or familiarity effect whereby perceptions of usefulness are enhanced because respondents have become more used to the system. This effect might also explain the lower ratings for GFS accrual. [ABSTRACT FROM AUTHOR]

This study examines the usefulness of accrual accounting information for internal decision-making contexts in the Western Australian public sector. Based on questionnaire responses of public sector managers, it was found that accrual accounting is perceived to be more useful than cash accounting in 16 of the 19 decision situations. These results suggest that the perceived usefulness of the accrual accounting system has improved with the passage of time. It may well be that perceptions of the usefulness of information derived from an accounting system will change over time as users gain familiarity and experience with a system. [ABSTRACT FROM AUTHOR]

While recency effects have been reported in a variety of audit tasks, recent studies suggest that these effects may be mitigated under certain conditions. The importance of investigating order effects in auditors' judgments rests with its potential to impact on the efficiency and effectiveness of audits. Since current studies suggest that recency effects may not impact on all audit situations, it is necessary to identify conditions or variables in the task environment that either induce or mitigate recency. This study examines the occurrence of order effects in auditors' inherent risk assessments, a task not previously examined. Using a case study administered to 70 auditors, this study found that auditors' judgments were not influenced by the order in which audit evidence was evaluated. Rather, the results suggest that judgments of inherent risk may be biased towards conservatism. This may not be surprising given the negative consequences associated with failing to adequately plan an audit. This may cause auditors to act cautiously and thus mitigate recency effects. [ABSTRACT FROM AUTHOR]

This teaching note reports and evaluates an initiative to enhance first-year accounting students' written communication skills in a large class setting. The procedures adopted are outlined and empirical evidence of the effectiveness of these procedures in enhancing writing skills is provided. Overall, the results were positive and encouraging. [ABSTRACT FROM AUTHOR]

This article focuses on reduced audit quality (RAQ).While prior studies have found that auditors engage in higher levels of reduced audit quality (RAQ) behavior when faced with time budget pressure, such studies have not considered the impact of other variables on this relationship. Understanding the different conditions under which time budget pressure can impact on audit work is important because time budget pressure has been shown to be a chronic and pervasive type of pressure faced by professional accountants. The research study in this article, examines the joint effects of time budget pressure and level of risk of misstatement on auditors' propensity to commit RAQ acts in the execution of an audit. Under time budget pressure, a higher risk of misstatement reduces the propensity of auditors to commit RAQ for truncating a selected sample, but not for accepting doubtful audit evidence. Specifically, auditors appear to accept doubtful audit evidence in the presence of high time budget pressure regardless of the level of risk, but appear to fail to test all items in a selected sample in the presence of high time budget pressure only when the level of risk is low. The results suggest that these RAQ acts are not seen as homogeneous by auditors, and that it is perceived by auditors as more tolerable to accept doubtful audit evidence than to truncate a sample.

One obstacle for human solid tumor immunotherapy research is the lack of clinically relevant animal models. In this study, we sought to establish a chimeric antigen receptor (CAR) T-cell treatment model for naturally occurring canine sarcomas as a model for human CAR T-cell therapy. Canine CARs specific for B7-H3 were constructed using a single-chain variable fragment derived from the human B7-H3-specific antibody MGA271, which we confirmed to be cross-reactive with canine B7-H3. After refining activation, transduction, and expansion methods, we confirmed target killing in a tumor spheroid three-dimensional assay. We designed a B7-H3 canine CAR T-cell and achieved consistently high levels of transduction efficacy, expansion, and in vitro tumor killing. Safety of the CAR T cells were confirmed in two purposely bred healthy canine subjects following lymphodepletion by cyclophosphamide and fludarabine. Immune response, clinical parameters, and manifestation were closely monitored after treatments and were shown to resemble that of humans. No severe adverse events were observed. In summary, we demonstrated that similar to human cancers, B7-H3 can serve as a target for canine solid tumors. We successfully generated highly functional canine B7-H3-specific CAR T-cell products using a production protocol that closely models human CAR T-cell production procedure. The treatment regimen that we designed was confirmed to be safe in vivo. Our research provides a promising direction to establish in vitro and in vivo models for immunotherapy for canine and human solid tumor treatment., (©2022 The Authors; Published by the American Association for Cancer Research.)