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"O'Leary, Patrick"
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A single inactivating amino acid change in the SARS-CoV-2 NSP3 Mac1 domain attenuates viral replication in vivo
Despite unprecedented efforts, our therapeutic arsenal against SARS-CoV-2 remains limited. The conserved macrodomain 1 (Mac1) in NSP3 is an enzyme exhibiting ADP-ribosylhydrolase activity and a possible drug target. To determine the role of Mac1 catalytic activity in viral replication, we generated recombinant viruses and replicons encoding a catalytically inactive NSP3 Mac1 domain by mutating a critical asparagine in the active site. While substitution to alanine (N40A) reduced catalytic activity by ~10-fold, mutations to aspartic acid (N40D) reduced activity by ~100-fold relative to wild-type. Importantly, the N40A mutation rendered Mac1 unstable in vitro and lowered expression levels in bacterial and mammalian cells. When incorporated into SARS-CoV-2 molecular clones, the N40D mutant only modestly affected viral fitness in immortalized cell lines, but reduced viral replication in human airway organoids by 10-fold. In mice, the N40D mutant replicated at >1000-fold lower levels compared to the wild-type virus while inducing a robust interferon response; all animals infected with the mutant virus survived infection. Our data validate the critical role of SARS-CoV-2 NSP3 Mac1 catalytic activity in viral replication and as a promising therapeutic target to develop antivirals.
Journal Article
Theories of the state : the politics of liberal democracy
Discusses Pluralist, New Right, Elitist, Marxist and Neo-pluralist theories. Provides summary of intellectual origins, methodology and key strengths and weaknesses with examples from United States, Western Europe, etc.
Book
Targeting peroxiredoxin 1 impairs growth of breast cancer cells and potently sensitises these cells to prooxidant agents
BackgroundOur previous work has shown peroxiredoxin-1 (PRDX1), one of major antioxidant enzymes, to be a biomarker in human breast cancer. Hereby, we further investigate the role of PRDX1, compared to its close homolog PRDX2, in mammary malignant cells.MethodsCRISPR/Cas9- or RNAi-based methods were used for genetic targeting PRDX1/2. Cell growth was assessed by crystal violet, EdU incorporation or colony formation assays. In vivo growth was assessed by a xenotransplantation model. Adenanthin was used to inhibit the thioredoxin-dependent antioxidant defense system. The prooxidant agents used were hydrogen peroxide, glucose oxidase and sodium L-ascorbate. A PY1 probe or HyPer-3 biosensor were used to detect hydrogen peroxide content in samples.ResultsPRDX1 downregulation significantly impaired the growth rate of MCF-7 and ZR-75-1 breast cancer cells. Likewise, xenotransplanted PRDX1-deficient MCF-7 cells presented a retarded tumour growth. Furthermore, genetic targeting of PRDX1 or adenanthin, but not PRDX2, potently sensitised all six cancer cell lines studied, but not the non-cancerous cells, to glucose oxidase and ascorbate.ConclusionsOur study pinpoints the dominant role for PRDX1 in management of exogeneous oxidative stress by breast cancer cells and substantiates further exploration of PRDX1 as a target in this disease, especially when combined with prooxidant agents.
Journal Article
High-Risk Cases at the Intersection of Domestic/Family Violence and Child Protection: Learning from Practice
Domestic/family violence (DFV) and child protection co-occurrence of risk is common, with related homicides remaining a grave public concern. Recognising and prioritising high-risk cases that pose a lethal risk remains a complex challenge for practitioners across a range of services and legal jurisdictions. There are significant gaps in practice knowledge about how to assess and respond to high-risk cases involving child protection and DFV concerns, while working safely and effectively. This paper reports on qualitative research conducted with practitioners from a range of justice and service delivery organisations in Queensland, Australia. A community of practice, supported by the U.S. based Safe & Together Institute, provided the forum for data to be collected on participants’ reflections and observations. An action research framework was employed to connect practice learning into research data through an iterative cycle of reflection and review. Ethnographic note taking was used to document arising policy and practice issues. Discussions in the CoP were also recorded and transcribed verbatim. Thematic analysis was undertaken combining inductive and deductive techniques. Key findings include: learning from and partnering with women; applying a perpetrator pattern-based approach in high-risk cases; engaging with men as fathers; and improving the role of the judicial system. Practice needs to be informed by centring the victim/survivor perspective, which requires a collaborative approach while maintaining system integrity to hold the perpetrator within system responses to ensure accountability. There are important learnings for developing service sector responses and future research on high-risk cases.
Journal Article
RASA2 ablation in T cells boosts antigen sensitivity and long-term function
The efficacy of adoptive T cell therapies for cancer treatment can be limited by suppressive signals from both extrinsic factors and intrinsic inhibitory checkpoints
1
,
2
. Targeted gene editing has the potential to overcome these limitations and enhance T cell therapeutic function
3
–
10
. Here we performed multiple genome-wide CRISPR knock-out screens under different immunosuppressive conditions to identify genes that can be targeted to prevent T cell dysfunction. These screens converged on RASA2, a RAS GTPase-activating protein (RasGAP) that we identify as a signalling checkpoint in human T cells, which is downregulated upon acute T cell receptor stimulation and can increase gradually with chronic antigen exposure. RASA2 ablation enhanced MAPK signalling and chimeric antigen receptor (CAR) T cell cytolytic activity in response to target antigen. Repeated tumour antigen stimulations in vitro revealed that RASA2-deficient T cells show increased activation, cytokine production and metabolic activity compared with control cells, and show a marked advantage in persistent cancer cell killing. RASA2-knockout CAR T cells had a competitive fitness advantage over control cells in the bone marrow in a mouse model of leukaemia. Ablation of RASA2 in multiple preclinical models of T cell receptor and CAR T cell therapies prolonged survival in mice xenografted with either liquid or solid tumours. Together, our findings highlight RASA2 as a promising target to enhance both persistence and effector function in T cell therapies for cancer treatment.
Genome-wide CRISPR screens, biochemical studies and animal models show that
RASA2
has a key role in regulating T cell function and has potential as a genetic target for enhancing anti-tumour immunity.
Journal Article
Worker Safety in High-risk Child Protection and Domestic Violence Cases
PurposeFew studies have investigated how high-risk domestic and family violence perpetrators threaten staff, how organizations support staff, and whether communities of practice improve service coordination and the safety of workers and the families they serve. The purpose of this article is to explore worker safety among practitioners involved in high-risk domestic and family violence and child protection cases.MethodsParticipatory action research methodology was used to investigate and develop cross-sectoral workforce capacity at the intersection of domestic and family violence and child protection practice. This study, based in Queensland, Australia, drew on a subset of data from a larger study of communities of practice. Participants were 15 senior practitioners and team leaders from child protection, women’s and men’s domestic violence services, family support, and justice services. Data were collected in 2018. Data sources included ethnographic notes and transcripts from communities of practice and focus groups. Data were qualitatively analyzed.ResultsFive key themes emerged in the findings: risks to workers associated with the physical environment; advances in perpetrators’ use of technology; failings in police responses and the judicial system; a parallel process between workers’ and women’s responses to threats to their physical and psychological safety; and strategies for improving safety when working with high risk perpetrators of domestic and family violence.ConclusionsImproving the physical and psychological safety of workers in high-risk domestic and family violence and child protection cases requires moving beyond intra-organizational policies and practices and addressing the inter-sectoral and systemic factors that increase risk and reduce safety for child and adult victim/survivors and workers.
Journal Article
Promoting men-inclusive maternity services: exploring the expectations, experiences and needs of men as fathers
Aim
This study aimed to explore the ‘real time’ expectations, experiences and needs of men who attend maternity services to inform the development of strategies to enhance men’s inclusion.
Methods
A qualitative descriptive design was adopted for the study. Semi-structured face-to-face or telephone interviews were conducted with 48 men attending the Royal Brisbane and Women’s Hospital before and after their partner gave birth. Data were coded and analysed thematically.
Results
Most respondents identified their role as a support person rather than a direct beneficiary of maternity services. They expressed the view that if their partner and baby’s needs were met, their needs were met. Factors that contributed to a positive experience included the responsiveness of staff and meeting information needs. Factors promoting feelings of inclusion were being directly addressed by staff, having the opportunity to ask questions, and performing practical tasks associated with the birth.
Conclusion
Adopting an inclusive communication style promotes men’s feelings of inclusion in maternity services. However, the participants’ tendency to conflate their needs with those of their partner suggests the ongoing salience of traditional gender role beliefs, which view childbirth primarily as the domain of women.
Journal Article
Magnetic Resonance Elastography: A Novel Technique for the Detection of Hepatic Fibrosis and Hepatocellular Carcinoma After the Fontan Operation
To evaluate the utility of magnetic resonance elastography (MRE) in screening patients for hepatic fibrosis, cirrhosis, and hepatocellular carcinoma after the Fontan operation.
Hepatic MRE was performed in conjunction with cardiac magnetic resonance imaging in patients who had undergone a Fontan operation between 2010 and 2014. Liver stiffness was calculated using previously reported techniques. Comparisons to available clinical, laboratory, imaging, and histopathologic data were made.
Overall, 50 patients at a median age of 25 years (range, 21-33 years) who had undergone a Fontan operation were evaluated. The median interval between Fontan operation and MRE was 22 years (range, 16-26 years). The mean liver stiffness values were increased: 5.5 ± 1.4 kPa relative to normal participants. Liver stiffness directly correlated with liver biopsy-derived total fibrosis score, time since operation, mean Fontan pressure, γ-glutamyltransferase level, Model for End-Stage Liver Disease score, creatinine level, and pulmonary vascular resistance index. Liver stiffness was inversely correlated with cardiac index. All 3 participants with hepatic nodules exhibiting decreased contrast uptake on delayed postcontrast imaging and increased nodule stiffness had biopsy-proven hepatocellular carcinoma.
The association between hepatic stiffness and fibrosis scores, Model for End-Stage Liver Disease scores, and γ-glutamyltransferase level suggests that MRE may be useful in detecting (and possibly quantifying) hepatic cirrhosis in patients after the Fontan operation. The correlation between stiffness and post-Fontan time interval, mean Fontan pressure, pulmonary vascular resistance index, and reduced cardiac index suggests a role for long-term hepatic congestion in creating these hepatic abnormalities. Magnetic resonance elastography was useful in detecting abnormal nodules ultimately diagnosed as hepatocellular carcinoma. The relationship between stiffness with advanced fibrosis and hepatocellular carcinoma provides a strong argument for additional study and broader application of MRE in these patients.
Journal Article
Transcription and DNA replication collisions lead to large tandem duplications and expose targetable therapeutic vulnerabilities in cancer
Despite the abundance of somatic structural variations (SVs) in cancer, the underlying molecular mechanisms of their formation remain unclear. In the present study, we used 6,193 whole-genome sequenced tumors to study the contributions of transcription and DNA replication collisions to genome instability. After deconvoluting robust SV signatures in three independent pan-cancer cohorts, we detected transcription-dependent, replicated-strand bias, the expected footprint of transcription–replication collision (TRC), in large tandem duplications (TDs). Large TDs are abundant in female-enriched, upper gastrointestinal tract and prostate cancers. They are associated with poor patient survival and mutations in
TP53
,
CDK12
and
SPOP
. Upon inactivating CDK12, cells display significantly more TRCs, R-loops and large TDs. Inhibition of WEE1, CHK1 and ATR selectively inhibits the growth of cells deficient in CDK12. Our data suggest that large TDs in cancer form as a result of TRCs and their presence can be used as a biomarker for prognosis and treatment.
Yang et al. show that transcription–replication collisions lead to large tandem duplications, which are frequent in female-enriched, upper gastrointestinal tract and prostate cancers and are associated with poor survival and mutations in specific genes, such as
CDK12
.
Journal Article