Weakened APC/C activity at mitotic exit drives cancer vulnerability to KIF18A inhibition.

The efficacy of current antimitotic cancer drugs is limited by toxicity in highly proliferative healthy tissues. A cancer-specific dependency on the microtubule motor protein KIF18A therefore makes it an attractive therapeutic target. Not all cancers require KIF18A, however, and the determinants underlying this distinction remain unclear. Here, we show that KIF18A inhibition drives a modest and widespread increase in spindle assembly checkpoint (SAC) signaling from kinetochores which can result in lethal mitotic delays. Whether cells arrest in mitosis depends on the robustness of the metaphase-to-anaphase transition, and cells predisposed with weak basal anaphase-promoting complex/cyclosome (APC/C) activity and/or persistent SAC signaling through metaphase are uniquely sensitive to KIF18A inhibition. KIF18A-dependent cancer cells exhibit hallmarks of this SAC:APC/C imbalance, including a long metaphase-to-anaphase transition, and slow mitosis overall. Together, our data reveal vulnerabilities in the cell division apparatus of cancer cells that can be exploited for therapeutic benefit.

Spermatocytes have the capacity to segregate chromosomes despite centriole duplication failure.

Centrosomes are the canonical microtubule organizing centers (MTOCs) of most mammalian cells, including spermatocytes. Centrosomes comprise a centriole pair within a structurally ordered and dynamic pericentriolar matrix (PCM). Unlike in mitosis, where centrioles duplicate once per cycle, centrioles undergo two rounds of duplication during spermatogenesis. The first duplication is during early meiotic prophase I, and the second is during interkinesis. Using mouse mutants and chemical inhibition, we have blocked centriole duplication during spermatogenesis and determined that non-centrosomal MTOCs (ncMTOCs) can mediate chromosome segregation. This mechanism is different from the acentriolar MTOCs that form bipolar spindles in oocytes, which require PCM components, including gamma-tubulin and CEP192. From an in-depth analysis, we identified six microtubule-associated proteins, TPX2, KIF11, NuMA, and CAMSAP1-3, that localized to the non-centrosomal MTOC. These factors contribute to a mechanism that ensures bipolar MTOC formation and chromosome segregation during spermatogenesis when centriole duplication fails. However, despite the successful completion of meiosis and round spermatid formation, centriole inheritance and PLK4 function are required for normal spermiogenesis and flagella assembly, which are critical to ensure fertility.

Barriers and co-designed strategies for the implementation of negative pressure wound therapy in acute pediatric burn care in Australia: A mixed method study.

PURPOSE: Pediatric burn injuries are a global clinical issue causing significant morbidity. Early adjunctive negative pressure wound therapy improves re-epithelialization rates in children with burns, yet adoption in acute burn care is inconsistent. This investigation aimed to determine barriers to the implementation of adjunctive negative pressure wound therapy for the acute management of pediatric burns and co-design targeted implementation strategies. METHODS: A sequential mixed methods design was used explore barriers to adjunctive negative pressure wound therapy implementation in acute pediatric burn care. An online questionnaire was disseminated to healthcare professionals within four major Australian pediatric hospitals, each with a dedicated burns service. Barriers were coded according to the Consolidated Framework for Implementation Research (CFIR). Semi-structured interviews with senior clinicians tailored implementation strategies to local contexts. A stakeholder consensus meeting consolidated implementation strategies and local processes. RESULTS: Sixty-three healthcare professionals participated in the questionnaire, and semi-structured interviews involved nine senior burn clinicians. We identified eight implementation barriers across all five CFIR domains then co-designed targeted strategies to address identified barriers. Barriers included lack of available resources, limited access to knowledge and information, individual stage of change, patient needs and resources, limited knowledge and beliefs about the intervention, lack of external policies, intervention complexity, and poor implementation planning. CONCLUSION: Multiple contextual factors affect negative pressure wound therapy uptake in acute pediatric burn settings. Results will inform a multi-state stepped-wedge cluster randomized controlled trial. Additional resources, education, training, updated policies, and guidelines are required for successful implementation. It is anticipated that adjunctive negative pressure wound therapy, in conjunction with tailored implementation strategies, will enhance adoption and sustainability. TRIAL REGISTRATION: Australian and New Zealand Clinical Trials Registry: ACTRN12622000166774. Registered 1 February 2022.

1,25-Dihydroxyvitamin D3 Suppresses UV-Induced Poly(ADP-Ribose) Levels in Primary Human Keratinocytes, as Detected by a Novel Whole-Cell ELISA.

Photoprotective properties of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) to reduce UV-induced DNA damage have been established in several studies. UV-induced DNA damage in skin such as single or double strand breaks is known to initiate several cellular mechanisms including activation of poly(ADP-ribose) (pADPr) polymerase-1 (PARP-1). DNA damage from UV also increases extracellular signal-related kinase (ERK) phosphorylation, which further increases PARP activity. PARP-1 functions by using cellular nicotinamide adenine dinucleotide (NAD+) to synthesise pADPr moieties and attach these to target proteins involved in DNA repair. Excessive PARP-1 activation following cellular stress such as UV irradiation may result in excessive levels of cellular pADPr. This can also have deleterious effects on cellular energy levels due to depletion of NAD+ to suboptimal levels. Since our previous work indicated that 1,25(OH)2D3 reduced UV-induced DNA damage in part through increased repair via increased energy availability, the current study investigated the effect of 1,25(OH)2D3 on UV-induced PARP-1 activity using a novel whole-cell enzyme- linked immunosorbent assay (ELISA) which quantified levels of the enzymatic product of PARP-1, pADPr. This whole cell assay used around 5000 cells per replicate measurement, which represents a 200-400-fold decrease in cell requirement compared to current commercial assays that measure in vitro pADPr levels. Using our assay, we observed that UV exposure significantly increased pADPr levels in human keratinocytes, while 1,25(OH)2D3 significantly reduced levels of UV-induced pADPr in primary human keratinocytes to a similar extent as a known PARP-1 inhibitor, 3-aminobenzamide (3AB). Further, both 1,25(OH)2D3 and 3AB as well as a peptide inhibitor of ERK-phosphorylation significantly reduced DNA damage in UV-exposed keratinocytes. The current findings support the proposal that reduction in pADPr levels may be critical for the function of 1,25(OH)2D3 in skin to reduce UV-induced DNA damage.

The current pattern of pediatric burn injuries in an Australian major burns center.

Burns are a common mechanism of pediatric injury worldwide and are a notable cause of disability-adjusted life-years. Burns in children represent a unique challenge, due to the differences from adults regarding physical characteristics, physiology and psychology. This retrospective cohort study examined trends of pediatric burns in New South Wales (NSW), Australia from 2010-22. It specifically focused on the changes in burn etiology and patient characteristics, body area affected, total body surface area, first aid, location and management. It also compared a 'Pre-COVID-19' and 'Peri-COVID-19' era to analyze the impact of COVID-19 on the pattern of pediatric burns, as children are at higher risk of injury during times of social disruption. The study found that burns in children continue to be concentrated in the toddler and preschooler age group and the main mechanisms of injury remain as scald and contact burns. In recent years, there has been a rising trend of friction burns, alongside a fall in flame burns and severe burns. Management of pediatric burns has also evolved, with predominant use of ambulatory care and low rates of admission and operative intervention. Trends in burn injury continue to evolve with time and over the last decade in NSW, key changes in the pattern of pediatric burns have been observed, with evolving mechanisms of injury, reduced severity of burns and a shift towards ambulatory care.

Haspin kinase binds to a nucleosomal DNA supergroove.

Phosphorylation of histone H3 threonine 3 (H3T3) by Haspin recruits the chromosomal passenger complex to the inner centromere and ensures proper cell cycle progression through mitosis. The mechanism by which Haspin binds to nucleosomes to phosphorylate H3T3 is not known. We report here cryo-EM structures of the Haspin kinase domain bound to a nucleosome. In contrast with previous structures of histone-modifying enzymes, Haspin solely contacts the nucleosomal DNA, inserting into a supergroove formed by apposing major grooves of two DNA gyres. This unique binding mode provides a plausible mechanism by which Haspin can bind to nucleosomes in a condensed chromatin environment to phosphorylate H3T3. We identify key basic residues in the Haspin kinase domain that are essential for phosphorylation of nucleosomal histone H3 and binding to mitotic chromatin. Our structure is the first of a kinase domain bound to a nucleosome and is the first example of a histone-modifying enzyme that binds to nucleosomes solely through DNA contacts.

Unremitting pro-inflammatory T-cell phenotypes, and macrophage activity, following paediatric burn injury.

Objectives: The aim of this study was to characterise the dynamic immune profile of paediatric burn patients for up to 18 months post-burn. Methods: Flow cytometry was used to measure 25 cell markers, chemokines and cytokines which reflected both pro-inflammatory and anti-inflammatory immune profiles. Peripheral blood mononuclear cells from 6 paediatric burn patients who had returned for repeated burn and scar treatments for > 4 timepoints within 12 months post-burn were compared to four age-matched healthy controls. Results: While overall proportions of T cells, NK cells and macrophages remained relatively constant, over time percentages of these immune cells differentiated into effector and proinflammatory cell phenotypes including Th17 and activated γδ T cells. Circulating proportions of γδ T cells increased their expression of pro-inflammatory mediators throughout the burn recovery, with a 3-6 fold increase of IL-17 at 1-3 weeks, and NFκß 9-18 months post-burn. T-regulatory cell plasticity was also observed, and Treg phenotype proportions changed from systemically reduced skin-homing T-regs (CCR4+) and increased inflammatory (CCR6+) at 1-month post-burn, to double-positive cell types (CCR4+CCR6+) elevated in circulation for 18 months post-burn. Furthermore, Tregs were observed to proportionally express less IL-10 but increased TNF-α over 18 months. Conclusion: Overall, these results indicate the circulating percentages of immune cells do not increase or decrease over time post-burn, instead they become highly specialised, inflammatory and skin-homing. In this patient population, these changes persisted for at least 18 months post-burn, this 'immune distraction' may limit the ability of immune cells to prioritise other threats post-burn, such as respiratory infections.

Centriole structural integrity defects are a crucial feature of Hydrolethalus Syndrome.

Hydrolethalus Syndrome (HLS) is a lethal, autosomal recessive ciliopathy caused by the mutation of the conserved centriole protein HYLS1. However, how HYLS1 facilitates the centriole-based templating of cilia is poorly understood. Here, we show that mice harboring the HYLS1 disease mutation die shortly after birth and exhibit developmental defects that recapitulate several manifestations of the human disease. These phenotypes arise from tissue-specific defects in cilia assembly and function caused by a loss of centriole integrity. We show that HYLS1 is recruited to the centriole by CEP120 and functions to recruit centriole inner scaffold proteins that stabilize the centriolar microtubule wall. The HLS mutation disrupts the interaction of HYLS1 with CEP120 leading to HYLS1 displacement and degeneration of the centriole distal end. We propose that tissue-specific defects in centriole integrity caused by the HYLS1 mutation prevent ciliogenesis and drive HLS phenotypes.

Reduction of Urinary Tract Infection in Pediatric Surgical Patients Using NSQIP-P and Quality Improvement Methodology.

BACKGROUND: Hospital-acquired urinary tract infections (UTIs) have a detrimental effect on patients, families, and hospital resources. The Sydney Children's Hospital Network (SCHN) participates in the NSQIP-Pediatric (NSQIP-P) to monitor postoperative complications. NSQIP-P data revealed that the median UTI rate at SCHN was 1.75% in 2019, 3.5 times higher than the NSQIP-P target rate of 0.5%. Over three quarters of the NSQIP-P identified patients with UTI also had a urinary catheterization performed intraoperatively. A quality improvement project was conducted between mid-2018 and 2021 to minimize catheter-associated UTIs (CAUTIs) at SCHN. STUDY DESIGN: NSQIP-P samples include pediatric (younger than 18 years) surgical patients from an 8-day cycle operative log. NSQIP-P data are statistically analyzed by the American College of Surgeons and provide biannual internationally benchmarked reports. The project used clinical redesign methodology with a 6-phase process for quality improvement projects. RESULTS: The objectives of the project were to reduce urinary catheter duration of use, educate parents or carers, and improve catheter care and insertion technique by health staff. The duration of a urinary catheter in situ reduced from a median of 4.5 to 3 days from 2017 to 2021. The median NSQIP-P UTI rate at SCHN was reduced by 47.4% from 1.75% in 2019 to 0.9% in 2022. CONCLUSIONS: A multifactorial approach in quality improvement has been shown to be an effective strategy to reduce UTI rates at SCHN, and patient outcomes were improved within a 3-year timeframe. Although this project has reduced UTI rates at SCHN, there remain opportunities for further improvement.

The AID2 system offers a potent tool for rapid, reversible, or sustained degradation of essential proteins in live mice.

Studying essential genes required for dynamic processes in live mice is challenging as genetic perturbations are irreversible and limited by slow protein depletion kinetics. The first-generation auxin-inducible-degron (AID) system is a powerful tool for analyzing inducible protein loss in cultured cells. However, auxin administration is toxic to mice, preventing its long-term use in animals. Here, we use an optimized second-generation AID system to achieve the conditional and reversible loss of the essential centrosomal protein CEP192 in live mice. We show that the auxin derivative 5-Ph-IAA is well tolerated over two weeks and drives near-complete CEP192-mAID degradation in less than one hour in vivo. Prolonged CEP192 loss led to cell division failure and cell death in proliferative tissues. Thus, the second-generation AID system is well suited for rapid and/or sustained protein depletion in live mice, offering a valuable new tool for interrogating protein function in vivo.

Inflammatory proteins and neutrophil extracellular traps increase in burn blister fluid 24h after burn.

Burn wound blister fluid is a valuable matrix for understanding the biological pathways associated with burn injury. In this study, 152 blister fluid samples collected from paediatric burn wounds at three different hospitals were analysed using mass spectrometry proteomic techniques. The protein abundance profile at different days after burn indicated more proteins were associated with cellular damage/repair in the first 24 h, whereas after this point more proteins were associated with antimicrobial defence. The inflammatory proteins persisted at a high level in the blister fluid for more than 7 days. This may indicate that removal of burn blisters prior to two days after burn is optimal to prevent excessive or prolonged inflammation in the wound environment. Additionally, many proteins associated with the neutrophil extracellular trap (NET) pathway were increased after burn, further implicating NETs in the post-burn inflammatory response. NET inhibitors may therefore be a potential treatment to reduce post-burn inflammation and coagulation pathology and enhance burn wound healing outcomes.

Understanding burn injury among Aboriginal and Torres Strait Islander children - results of a two-year cohort study.

BACKGROUND: Despite known inequalities, little is understood about the burden and healthcare experiences of Aboriginal and Torres Strait Islander children who sustain a burn injury and their families. METHODS: The Coolamon Study recruited parents and carers whose children (aged <16 years) were Aboriginal and / or Torres Strait Islander children and had presented to burn units across four Australian states, New South Wales (Sydney), Northern Territory (Darwin), Queensland (Brisbane, Townsville) and South Australia (Adelaide), between 2015 and 2018. Consent was obtained and carers completed baseline and subsequent interviews at 3, 6, 12 and 24 months. Data were collected on the injury event, patient care and safety, sociodemographic factors, health related quality of life (PedsQual), and psychological distress (Kessler K-5). RESULTS: Of the 208 participants, 64 % were male; 26 % were aged less than 2 years and 37 % aged 2-4 years. The most common burn mechanisms were scalds (37 %), contact (33 %) and flame burns (21 %), with more severe burns and flame burns occurring in rural and remote settings. Most carers rated their child's care as either excellent or very good (82 %). Family distress, measured by the K-5, lessened over the 24 months, however the changes were not statistically significant. While 77 % of carers reported that they received enough information, 18 % reported they would have liked more, and 3 % reported no information was provided before treatment. Parents described mixed access to information about the types of support available to them, such as accommodation, meals, travel or cultural support. CONCLUSION: Data from this cohort provide rich new information about risk factors and care received from point of injury through to rehabilitation for Aboriginal and Torres Strait Islander children with burns, providing unique insights into what is needed for appropriate, culturally safe care.

The Australian Traumatic Brain Injury Initiative: Systematic Review and Consensus Process to Determine the Predictive Value of Demographic, Injury Event, and Social Characteristics on Outcomes for People With Moderate-Severe Traumatic Brain Injury.

The objective of the Australian Traumatic Brain Injury (AUS-TBI) Initiative is to develop a data dictionary to inform data collection and facilitate prediction of outcomes of people who experience moderate-severe TBI in Australia. The aim of this systematic review was to summarize the evidence of the association between demographic, injury event, and social characteristics with outcomes, in people with moderate-severe TBI, to identify potentially predictive indicators. Standardized searches were implemented across bibliographic databases to March 31, 2022. English-language reports, excluding case series, which evaluated the association between demographic, injury event, and social characteristics, and any clinical outcome in at least 10 patients with moderate-severe TBI were included. Abstracts and full text records were independently screened by at least two reviewers in Covidence. A pre-defined algorithm was used to assign a judgement of predictive value to each observed association. The review findings were discussed with an expert panel to determine the feasibility of incorporation of routine measurement into standard care. The search strategy retrieved 16,685 records; 867 full-length records were screened, and 111 studies included. Twenty-two predictors of 32 different outcomes were identified; 7 were classified as high-level (age, sex, ethnicity, employment, insurance, education, and living situation at the time of injury). After discussion with an expert consensus group, 15 were recommended for inclusion in the data dictionary. This review identified numerous predictors capable of enabling early identification of those at risk for poor outcomes and improved personalization of care through inclusion in routine data collection.

The Australian Traumatic Brain Injury Initiative: Systematic Review of Clinical Factors Associated with Outcomes in People with Moderate-Severe Traumatic Brain Injury.

The aim of the Australian Traumatic Brain Injury Initiative (AUS-TBI) is to design a data dictionary to inform data collection and facilitate prediction of outcomes for moderate-severe traumatic brain injury (TBI) across Australia. The process has engaged diverse stakeholders across six areas: social, health, clinical, biological, acute interventions, and long-term outcomes. Here, we report the results of the clinical review. Standardized searches were implemented across databases to April 2022. English-language reports of studies evaluating an association between a clinical factor and any clinical outcome in at least 100 patients with moderate-severe TBI were included. Abstracts, and full-text records, were independently screened by at least two reviewers in Covidence. The findings were assessed through a consensus process to determine inclusion in the AUS-TBI data resource. The searches retrieved 22,441 records, of which 1137 were screened at full text and 313 papers were included. The clinical outcomes identified were predominantly measures of survival and disability. The clinical predictors most frequently associated with these outcomes were the Glasgow Coma Scale, pupil reactivity, and blood pressure measures. Following discussion with an expert consensus group, 15 were recommended for inclusion in the data dictionary. This review identified numerous studies evaluating associations between clinical factors and outcomes in patients with moderate-severe TBI. A small number of factors were reported consistently, however, how and when these factors were assessed varied. The findings of this review and the subsequent consensus process have informed the development of an evidence-informed data dictionary for moderate-severe TBI in Australia.

Corrigendum to: Developing economic measures for Aboriginal and Torres Strait Islander families on out-of-pocket healthcare expenditure.

Objective Out-of-pocket healthcare expenditure (OOPHE) has a significant impact on marginalised households. The purpose of this study was to modify a pre-existing OOPHE survey for Aboriginal and Torres Strait Islander households with children. Methods The OOPHE survey was derived through a scoping review, face and content validity, including judgement quantification with content experts. Exploratory factor analyses determined factor numbers for construct validity. Repeatability through test-retest processes and reliability was assessed through internal consistency. Results The OOPHE survey had 168 items and was piloted on 67 Aboriginal and Torres Strait Islander parents. Construct validity assessment generated a 62-item correlation matrix with a three-factor model. Across these factors, item loadings varied, 10 items with high correlations (>0.70) and 20 with low correlations (Conclusion The low level of item loadings to factors in the OOPHE survey indicates interconnectedness across the three-factor model, and reliability results suggest systemic differences. Impeding factors may include cohort homogeneity and survey length. It is unknown how cultural and social nuances specific to Aboriginal and Torres Strait Islander households impacts on results. Further work is warranted. What is known about the topic? Out-of-pocket healthcare expenditure (OOPHE) are expenses not covered by universal taxpayer-funded health insurance. In elderly Australians or those with chronic conditions, OOPHE can cause substantial burden and financial hardship and, in the most extreme cases, induce bankruptcy. Despite higher hospital admissions and disease burden, little is known about how OOPHE impacts Aboriginal and Torres Strait Islander families. Additionally, in Australia, no OOPHE survey tools have been appropriately assessed; this includes for use with Aboriginal and Torres Strait Islander families. What does this paper add? This pilot study modified a pre-existing Australian OOPHE survey for use with Aboriginal and Torres Strait Islander households with children. Knowledge interface methodology was used to bring together Indigenous knowledges with quantitative survey methods. This was critical to ensuring Indigenous knowledges were central to the overall pilot study across item creation, participant focus, outcome contextualisation, interpretation, and resetting dominant norms. Outcomes have demonstrated pertinent points for future work in this area, such as the complexities in developing robust, culturally safe and specific surveys, which reach ideal psychometric levels of validity and reliability for Aboriginal and Torres Strait Islander communities. Certainly, it raises questions for current and future research using surveys in Aboriginal and Torres Strait Islander communities, which are generic and not purpose-built. What are the implications for practitioners? We recommend that OOPHE surveys should be developed with Aboriginal and Torres Strait Islander families from the outset, so they can include important contextual factors for Aboriginal and Torres Strait Islander households.