Poglut2/3 double knockout in mice results in neonatal lethality with reduced levels of fibrillin in lung tissues.

Fibrillin microfibrils play a critical role in the formation of elastic fibers, tissue/organ development, and cardiopulmonary function. These microfibrils not only provide structural support and flexibility to tissues, but they also regulate growth factor signaling through a plethora of microfibril-binding proteins in the extracellular space. Mutations in fibrillins are associated with human diseases affecting cardiovascular, pulmonary, skeletal, and ocular systems. Fibrillins consist of up to 47 epidermal growth factor-like repeats, of which more than half are modified by protein O-glucosyltransferase 2 (POGLUT2) and/or POGLUT3. Loss of these modifications reduces secretion of N-terminal fibrillin constructs overexpressed in vitro. Here, we investigated the role of POGLUT2 and POGLUT3 in vivo using a Poglut2/3 double knockout (DKO) mouse model. Blocking O-glucosylation caused neonatal death with skeletal, pulmonary, and eye defects reminiscent of fibrillin/elastin mutations. Proteomic analyses of DKO dermal fibroblast medium and extracellular matrix provided evidence that fibrillins were more sensitive to loss of O-glucose compared to other POGLUT2/3 substrates. This conclusion was supported by immunofluorescent analyses of late gestation DKO lungs where FBN levels were reduced and microfibrils appeared fragmented in the pulmonary arteries and veins, bronchioles, and developing saccules. Defects in fibrillin microfibrils likely contributed to impaired elastic fiber formation and histological changes observed in DKO lung blood vessels, bronchioles, and saccules. Collectively, these results highlight the importance of POGLUT2/3-mediated O-glucosylation in vivo and open the possibility that O-glucose modifications on fibrillin influence microfibril assembly and or protein interactions in the ECM environment.

Iron and risk of dementia: Mendelian randomisation analysis in UK Biobank.

BACKGROUND: Brain iron deposition is common in dementia, but whether serum iron is a causal risk factor is unknown. We aimed to determine whether genetic predisposition to higher serum iron status biomarkers increased risk of dementia and atrophy of grey matter. METHODS: We analysed UK Biobank participants clustered into European (N=451284), African (N=7477) and South Asian (N=9570) groups by genetic similarity to the 1000 genomes project. Using Mendelian randomisation methods, we estimated the association between genetically predicted serum iron (transferrin saturation [TSAT] and ferritin), grey matter volume and genetic liability to clinically defined dementia (including Alzheimer's disease [AD], non-AD dementia, and vascular dementia) from hospital and primary care records. We also performed time-to-event (competing risks) analysis of the TSAT polygenic score on risk of clinically defined non-AD dementia. RESULTS: In Europeans, higher genetically predicted TSAT increased genetic liability to dementia (Odds Ratio [OR]: 1.15, 95% Confidence Intervals [CI] 1.04 to 1.26, p=0.0051), non-AD dementia (OR: 1.27, 95% CI 1.12 to 1.45, p=0.00018) and vascular dementia (OR: 1.37, 95% CI 1.12 to 1.69, p=0.0023), but not AD (OR: 1.00, 95% CI 0.86 to 1.15, p=0.97). Higher TSAT was also associated with increased risk of non-AD dementia in participants of African, but not South Asian groups. In survival analysis using a TSAT polygenic score, the effect was independent of apolipoprotein-E ε4 genotype (with adjustment subdistribution Hazard Ratio: 1.74, 95% CI 1.33 to 2.28, p=0.00006). Genetically predicted TSAT was associated with lower grey matter volume in caudate, putamen and thalamus, and not in other areas of interest. DISCUSSION: Genetic evidence supports a causal relationship between higher TSAT and risk of clinically defined non-AD and vascular dementia, in European and African groups. This association appears to be independent of apolipoprotein-E ε4.

TAp73 is a marker of glutamine addiction in medulloblastoma.

Medulloblastoma is the most common solid primary brain tumor in children. Remarkable advancements in the understanding of the genetic and epigenetic basis of these tumors have informed their recent molecular classification. However, the genotype/phenotype correlation of the subgroups remains largely uncharacterized. In particular, the metabolic phenotype is of great interest because of its druggability, which could lead to the development of novel and more tailored therapies for a subset of medulloblastoma. p73 plays a critical role in a range of cellular metabolic processes. We show overexpression of p73 in a proportion of non-WNT medulloblastoma. In these tumors, p73 sustains cell growth and proliferation via regulation of glutamine metabolism. We validated our results in a xenograft model in which we observed an increase in survival time in mice on a glutamine restriction diet. Notably, glutamine starvation has a synergistic effect with cisplatin, a component of the current medulloblastoma chemotherapy. These findings raise the possibility that glutamine depletion can be used as an adjuvant treatment for p73-expressing medulloblastoma.

MRI-derived brain iron, grey matter volume, and risk of dementia and Parkinson's disease: Observational and genetic analysis in the UK Biobank cohort.

BACKGROUND: Iron overload is observed in neurodegenerative diseases, especially Alzheimer's disease (AD) and Parkinson's disease (PD). Homozygotes for the iron-overload (haemochromatosis) causing HFE p.C282Y variant have increased risk of dementia and PD. Whether brain iron deposition is causal or secondary to the neurodegenerative processes in the general population is unclear. METHODS: We analysed 39,533 UK Biobank participants of European genetic ancestry with brain MRI data. We studied brain iron estimated by R2* and quantitative susceptibility mapping (QSM) in 8 subcortical regions: accumbens, amygdala, caudate, hippocampus, pallidum, putamen, substantia nigra, and thalamus. We performed genome-wide associations studies (GWAS) and used Mendelian Randomization (MR) methods to estimate the causal effect of brain iron on grey matter volume, and risk of AD, non-AD and PD. We also used MR to test whether genetic liability to AD or PD causally increased brain iron (R2* and QSM). FINDINGS: In GWAS of R2* and QSM we replicated 83% of previously reported genetic loci and identified 174 further loci across all eight brain regions. Higher genetically predicted brain iron, using both R2* and QSM, was associated with lower grey matter volumes in the caudate, putamen and thalamus (e.g., Beta-putamenQSM: -0.37, p = 2*10-46). Higher genetically predicted thalamus R2* was associated with increased risk of non-AD dementia (OR 1.36(1.16;1.60), p = 2*10-4) but not AD (p > 0.05). In males, genetically predicted putamen R2* increased non-AD dementia risk, but not in females. Higher genetically predicted iron in the caudate, putamen, and substantia nigra was associated with an increased risk of PD (Odds Ratio QSM âˆ¼ substantia-nigra 1.21(1.07;1.37), p = 0.003). Genetic liability to AD or PD was not associated with R2* or QSM in the dementia or PD-associated regions. INTERPRETATION: Our genetic analysis supports a causal effect of higher iron deposition in specific subcortical brain regions for Parkinson's disease, grey matter volume, and non-Alzheimer's dementia.

Epigenetic regulator genes direct lineage switching in MLL/AF4 leukemia.

The fusion gene MLL/AF4 defines a high-risk subtype of pro-B acute lymphoblastic leukemia. Relapse can be associated with a lineage switch from acute lymphoblastic to acute myeloid leukemia, resulting in poor clinical outcomes caused by resistance to chemotherapies and immunotherapies. In this study, the myeloid relapses shared oncogene fusion breakpoints with their matched lymphoid presentations and originated from various differentiation stages from immature progenitors through to committed B-cell precursors. Lineage switching is linked to substantial changes in chromatin accessibility and rewiring of transcriptional programs, including alternative splicing. These findings indicate that the execution and maintenance of lymphoid lineage differentiation is impaired. The relapsed myeloid phenotype is recurrently associated with the altered expression, splicing, or mutation of chromatin modifiers, including CHD4 coding for the ATPase/helicase of the nucleosome remodelling and deacetylation complex. Perturbation of CHD4 alone or in combination with other mutated epigenetic modifiers induces myeloid gene expression in MLL/AF4+ cell models, indicating that lineage switching in MLL/AF4 leukemia is driven and maintained by disrupted epigenetic regulation.

Modelling how plant cell-cycle progression leads to cell size regulation.

Populations of cells typically maintain a consistent size, despite cell division rarely being precisely symmetrical. Therefore, cells must possess a mechanism of "size control", whereby the cell volume at birth affects cell-cycle progression. While size control mechanisms have been elucidated in a number of other organisms, it is not yet clear how this mechanism functions in plants. Here, we present a mathematical model of the key interactions in the plant cell cycle. Model simulations reveal that the network of interactions exhibits limit-cycle solutions, with biological switches underpinning both the G1/S and G2/M cell-cycle transitions. Embedding this network model within growing cells, we test hypotheses as to how cell-cycle progression can depend on cell size. We investigate two different mechanisms at both the G1/S and G2/M transitions: (i) differential expression of cell-cycle activator and inhibitor proteins (with synthesis of inhibitor proteins being independent of cell size), and (ii) equal inheritance of inhibitor proteins after cell division. The model demonstrates that both these mechanisms can lead to larger daughter cells progressing through the cell cycle more rapidly, and can thus contribute to cell-size control. To test how these features enable size homeostasis over multiple generations, we then simulated these mechanisms in a cell-population model with multiple rounds of cell division. These simulations suggested that integration of size-control mechanisms at both G1/S and G2/M provides long-term cell-size homeostasis. We concluded that while both size independence and equal inheritance of inhibitor proteins can reduce variations in cell size across individual cell-cycle phases, combining size-control mechanisms at both G1/S and G2/M is essential to maintain size homeostasis over multiple generations. Thus, our study reveals how features of the cell-cycle network enable cell-cycle progression to depend on cell size, and provides a mechanistic understanding of how plant cell populations maintain consistent size over generations.

Quantitative N- or C-Terminal Labelling of Proteins with Unactivated Peptides by Use of Sortases and a d-Aminopeptidase.

Quantitative and selective labelling of proteins is widely used in both academic and industrial laboratories, and catalytic labelling of proteins using transpeptidases, such as sortases, has proved to be a popular strategy for such selective modification. A major challenge for this class of enzymes is that the majority of procedures require an excess of the labelling reagent or, alternatively, activated substrates rather than simple commercially sourced peptides. We report the use of a coupled enzyme strategy which enables quantitative N- and C-terminal labelling of proteins using unactivated labelling peptides. The use of an aminopeptidase in conjunction with a transpeptidase allows sequence-specific degradation of the peptide by-product, shifting the equilibrium to favor product formation, which greatly enhances the reaction efficiency. Subsequent optimisation of the reaction allows N-terminal labelling of proteins using essentially equimolar ratios of peptide label to protein and C-terminal labelling with only a small excess. Minimizing the amount of substrate required for quantitative labelling has the potential to improve industrial processes and facilitate the use of transpeptidation as a method for protein labelling.

An optogenetic method for interrogating YAP1 and TAZ nuclear-cytoplasmic shuttling.

The shuttling of transcription factors and transcriptional regulators into and out of the nucleus is central to the regulation of many biological processes. Here we describe a new method for studying the rates of nuclear entry and exit of transcriptional regulators. A photo-responsive LOV (light-oxygen-voltage) domain from Avena sativa is used to sequester fluorescently labelled transcriptional regulators YAP1 and TAZ (also known as WWTR1) on the surface of mitochondria and to reversibly release them upon blue light illumination. After dissociation, fluorescent signals from the mitochondria, cytoplasm and nucleus are extracted by a bespoke app and used to generate rates of nuclear entry and exit. Using this method, we demonstrate that phosphorylation of YAP1 on canonical sites enhances its rate of nuclear export. Moreover, we provide evidence that, despite high intercellular variability, YAP1 import and export rates correlate within the same cell. By simultaneously releasing YAP1 and TAZ from sequestration, we show that their rates of entry and exit are correlated. Furthermore, combining the optogenetic release of YAP1 with lattice light-sheet microscopy reveals high heterogeneity of YAP1 dynamics within different cytoplasmic regions, demonstrating the utility and versatility of our tool to study protein dynamics. This article has an associated First Person interview with Anna M. Dowbaj, joint first author of the paper.

Timing is everything: A connection between medulloblastoma prognosis and foetal cerebellar development.

The childhood brain tumour medulloblastoma is typically classified into multiple discrete molecular subgroups with characteristic DNA methylation and expression patterns. Several of these subgroups are used as, or proposed to be, an effective basis for treatment stratification. Here, we highlight the close connection between the findings described in a recent series of studies which, together, strongly imply a continuous association between survival outcome, the transcriptional profile of a Group3/Group4 (i.e. non-WNT/non-SHH) medulloblastoma and the specific point during early foetal cerebellar development at which initial pathogenic disruption took place. This has important implications for future efforts to model the disease by incorporating driving molecular features into their specific developmental context. This further suggests that instead of relying upon discrete DNA methylation subgroups, using expression biomarkers as the basis of a continuous risk predictor may produce a more effective risk stratification of patients with Group3/Group4 medulloblastoma.

Molecular characterisation defines clinically-actionable heterogeneity within Group 4 medulloblastoma and improves disease risk-stratification.

Group 4 tumours (MBGrp4) represent the majority of non-WNT/non-SHH medulloblastomas. Their clinical course is poorly predicted by current risk-factors. MBGrp4 molecular substructures have been identified (e.g. subgroups/cytogenetics/mutations), however their inter-relationships and potential to improve clinical sub-classification and risk-stratification remain undefined. We comprehensively characterised the paediatric MBGrp4 molecular landscape and determined its utility to improve clinical management. A clinically-annotated discovery cohort (n = 362 MBGrp4) was assembled from UK-CCLG institutions and SIOP-UKCCSG-PNET3, HIT-SIOP-PNET4 and PNET HR + 5 clinical trials. Molecular profiling was undertaken, integrating driver mutations, second-generation non-WNT/non-SHH subgroups (1-8) and whole-chromosome aberrations (WCAs). Survival models were derived for patients ≥ 3 years of age who received contemporary multi-modal therapies (n = 323). We first independently derived and validated a favourable-risk WCA group (WCA-FR) characterised by ≥ 2 features from chromosome 7 gain, 8 loss, and 11 loss. Remaining patients were high-risk (WCA-HR). Subgroups 6 and 7 were enriched for WCA-FR (p < 0·0001) and aneuploidy. Subgroup 8 was defined by predominantly balanced genomes with isolated isochromosome 17q (p < 0·0001). While no mutations were associated with outcome and overall mutational burden was low, WCA-HR harboured recurrent chromatin remodelling mutations (p = 0·007). Integration of methylation and WCA groups improved risk-stratification models and outperformed established prognostication schemes. Our MBGrp4 risk-stratification scheme defines: favourable-risk (non-metastatic disease and (i) subgroup 7 or (ii) WCA-FR (21% of patients, 5-year PFS 97%)), very-high-risk (metastatic disease with WCA-HR (36%, 5-year PFS 49%)) and high-risk (remaining patients; 43%, 5-year PFS 67%). These findings validated in an independent MBGrp4 cohort (n = 668). Importantly, our findings demonstrate that previously established disease-wide risk-features (i.e. LCA histology and MYC(N) amplification) have little prognostic relevance in MBGrp4 disease. Novel validated survival models, integrating clinical features, methylation and WCA groups, improve outcome prediction and re-define risk-status for ~ 80% of MBGrp4. Our MBGrp4 favourable-risk group has MBWNT-like excellent outcomes, thereby doubling the proportion of medulloblastoma patients who could benefit from therapy de-escalation approaches, aimed at reducing treatment induced late-effects while sustaining survival outcomes. Novel approaches are urgently required for the very-high-risk patients.

POGLUT2 and POGLUT3 O-glucosylate multiple EGF repeats in fibrillin-1, -2, and LTBP1 and promote secretion of fibrillin-1.

Fibrillin-1 (FBN1) is the major component of extracellular matrix microfibrils, which are required for proper development of elastic tissues, including the heart and lungs. Through protein-protein interactions with latent transforming growth factor (TGF) ß-binding protein 1 (LTBP1), microfibrils regulate TGF-ß signaling. Mutations within the 47 epidermal growth factor-like (EGF) repeats of FBN1 cause autosomal dominant disorders including Marfan Syndrome, which is characterized by disrupted TGF-ß signaling. We recently identified two novel protein O-glucosyltransferases, Protein O-glucosyltransferase 2 (POGLUT2) and 3 (POGLUT3), that modify a small fraction of EGF repeats on Notch. Here, using mass spectral analysis, we show that POGLUT2 and POGLUT3 also modify over half of the EGF repeats on FBN1, fibrillin-2 (FBN2), and LTBP1. While most sites are modified by both enzymes, some sites show a preference for either POGLUT2 or POGLUT3. POGLUT2 and POGLUT3 are homologs of POGLUT1, which stabilizes Notch proteins by addition of O-glucose to Notch EGF repeats. Like POGLUT1, POGLUT2 and 3 can discern a folded versus unfolded EGF repeat, suggesting POGLUT2 and 3 are involved in a protein folding pathway. In vitro secretion assays using the N-terminal portion of recombinant FBN1 revealed reduced FBN1 secretion in POGLUT2 knockout, POGLUT3 knockout, and POGLUT2 and 3 double-knockout HEK293T cells compared with wild type. These results illustrate that POGLUT2 and 3 function together to O-glucosylate protein substrates and that these modifications play a role in the secretion of substrate proteins. It will be interesting to see how disease variants in these proteins affect their O-glucosylation.

Identification, function, and biological relevance of POGLUT2 and POGLUT3.

O-glycosylation of Epidermal Growth Factor-like (EGF) repeats plays crucial roles in protein folding, trafficking and function. The Notch extracellular domain has been used as a model to study these mechanisms due to its many O-glycosylated EGF repeats. Three enzymes were previously known to O-glycosylate Notch EGF repeats: Protein O-Glucosyltransferase 1 (POGLUT1), Protein O-Fucosyltransferase 1 (POFUT1), and EGF Domain Specific O-Linked N-Acetylglucosamine Transferase (EOGT). All of these modifications affect Notch activity. Recently, POGLUT2 and POGLUT3 were identified as two novel O-glucosyltransferases that modify a few Notch EGF repeats at sites distinct from those modified by POGLUT1. Comparison of these modification sites revealed a putative consensus sequence which predicted modification of many extracellular matrix proteins including fibrillins (FBNs) and Latent TGFß-binding proteins (LTBPs). Glycoproteomic analysis revealed that approximately half of the 47 EGF repeats in FBN1 and FBN2, and half of the 18 EGF repeats in LTBP1, are modified by POGLUT2 and/or POGLUT3. Cellular assays showed that loss of modifications by POGLUT2 and/or POGLUT3 significantly reduces FBN1 secretion. There is precedent for EGF modifications to affect protein-protein interactions, as has been demonstrated by research of POGLUT1 and POFUT1 modifications on Notch. Here we discuss the identification and characterization of POGLUT2 and POGLUT3 and the ongoing research that continues to elucidate the biological significance of these novel enzymes.

Single-cell DNA sequencing identifies risk-associated clonal complexity and evolutionary trajectories in childhood medulloblastoma development.

We reconstructed the natural history and temporal evolution of the most common childhood brain malignancy, medulloblastoma, by single-cell whole-genome sequencing (sc-WGS) of tumours representing its major molecular sub-classes and clinical risk groups. Favourable-risk disease sub-types assessed (MBWNT and infant desmoplastic/nodular MBSHH) typically comprised a single clone with no evidence of further evolution. In contrast, highest risk sub-classes (MYC-amplified MBGroup3 and TP53-mutated MBSHH) were most clonally diverse and displayed gradual evolutionary trajectories. Clinically adopted biomarkers (e.g. chromosome 6/17 aberrations; CTNNB1/TP53 mutations) were typically early-clonal/initiating events, exploitable as targets for early-disease detection; in analyses of spatially distinct tumour regions, a single biopsy was sufficient to assess their status. Importantly, sc-WGS revealed novel events which arise later and/or sub-clonally and more commonly display spatial diversity; their clinical significance and role in disease evolution post-diagnosis now require establishment. These findings reveal diverse modes of tumour initiation and evolution in the major medulloblastoma sub-classes, with pathogenic relevance and clinical potential.

Translational genomics of malignant rhabdoid tumours: Current impact and future possibilities.

Malignant Rhabdoid Tumours (MRT) are the quintessential example of an epigenetic cancer. Mutation of a single gene, SMARCB1 or more rarely SMARCA4, is capable of causing one of the most aggressive and lethal cancers of early childhood and infancy. SMARCB1 encodes a core subunit of the SWI/SNF complex and its mutation evokes genome-wide downstream effects which may be counteracted therapeutically. Here we review and discuss the use of translational genomics in the study of MRT biology and the ways in which this has impacted clinical practice or may do so in the future. First, the diagnosis and definition of MRT and the transition from a histopathological to a molecular definition. Second, epigenetic and transcriptomic subgroups within MRT, their defining features and potential prognostic or therapeutic significance. Third, functional genomic studies of MRT by mouse modelling and forced re-expression of SMARCB1 in MRT cells. Fourth, studies of underlying epigenetic mechanisms (e.g. EZH2, HDACs) or deregulated kinases (e.g. PDGFR, FGFR1) and the potential therapeutic opportunities these provide. Finally, we discuss likely future directions and proffer opinion on how future translational genomics should be integrated into future biological/clinical studies to select and evaluate the best anti-MRT therapeutic agents.

Advanced molecular pathology for rare tumours: A national feasibility study and model for centralised medulloblastoma diagnostics.

AIMS: Application of advanced molecular pathology in rare tumours is hindered by low sample numbers, access to specialised expertise/technologies and tissue/assay QC and rapid reporting requirements. We assessed the feasibility of co-ordinated real-time centralised pathology review (CPR), encompassing molecular diagnostics and contemporary genomics (RNA-seq/DNA methylation-array). METHODS: This nationwide trial in medulloblastoma (<80 UK diagnoses/year) introduced a national reference centre (NRC) and assessed its performance and reporting to World Health Organisation standards. Paired frozen/formalin-fixed, paraffin-embedded tumour material were co-submitted from 135 patients (16 referral centres). RESULTS: Complete CPR diagnostics were successful for 88% (120/135). Inadequate sampling was the most common cause of failure; biomaterials were typically suitable for methylation-array (129/135, 94%), but frozen tissues commonly fell below RNA-seq QC requirements (53/135, 39%). Late reporting was most often due to delayed submission. CPR assigned or altered histological variant (vs local diagnosis) for 40/135 tumours (30%). Benchmarking/QC of specific biomarker assays impacted test results; fluorescent in-situ hybridisation most accurately identified high-risk MYC/MYCN amplification (20/135, 15%), while combined methods (CTNNB1/chr6 status, methylation-array subgrouping) best defined favourable-risk WNT tumours (14/135; 10%). Engagement of a specialist pathologist panel was essential for consensus assessment of histological variants and immunohistochemistry. Overall, CPR altered clinical risk-status for 29% of patients. CONCLUSION: National real-time CPR is feasible, delivering robust diagnostics to WHO criteria and assignment of clinical risk-status, significantly altering clinical management. Recommendations and experience from our study are applicable to advanced molecular diagnostics systems, both local and centralised, across rare tumour types, enabling their application in biomarker-driven routine diagnostics and clinical/research studies.

Parameter estimation in fluorescence recovery after photobleaching: quantitative analysis of protein binding reactions and diffusion.

Fluorescence recovery after photobleaching (FRAP) is a common experimental method for investigating rates of molecular redistribution in biological systems. Many mathematical models of FRAP have been developed, the purpose of which is usually the estimation of certain biological parameters such as the diffusivity and chemical reaction rates of a protein, this being accomplished by fitting the model to experimental data. In this article, we consider a two species reaction-diffusion FRAP model. Using asymptotic analysis, we derive new FRAP recovery curve approximation formulae, and formally re-derive existing ones. On the basis of these formulae, invoking the concept of Fisher information, we predict, in terms of biological and experimental parameters, sufficient conditions to ensure that the values all model parameters can be estimated from data. We verify our predictions with extensive computational simulations. We also use computational methods to investigate cases in which some or all biological parameters are theoretically inestimable. In these cases, we propose methods which can be used to extract the maximum possible amount of information from the FRAP data.

"I'm outta here!": a qualitative investigation into why Aboriginal and non-Aboriginal people self-discharge from hospital.

BACKGROUND: Occasions of self-discharge from health services before being seen by a health profession or against medical advice are often used by health systems as an indicator of quality care. People self-discharge because of factors such as dissatisfaction with care, poor communication, long waiting times, and feeling better in addition to external factors such as family and employment responsibilities. These factors, plus a lack of cultural safety, and interpersonal and institutional racism contribute to the disproportionately higher rates of Indigenous people self-discharging from hospital. This qualitative study aimed to increase understanding about the causative and contextual factors that culminate in people self-discharging and identify opportunities to improve the hospital experience for all. METHODS: Semi-structured interviews with five Aboriginal and/or Torres Strait Islander (hereafter, respectfully, Indigenous) people and six non-Indigenous people who had self-discharged from a major tertiary hospital in Brisbane, Australia, were audio-recorded, transcribed and thematically analysed. RESULTS: Study participants all respected hospitals' vital role of caring for the sick, but the cumulative impact of unmet needs created a tipping point whereby they concluded that remaining in hospital would compromise their health and wellbeing. Five key categories of unmet needs were identified - the need for information; confidence in the quality of care; respectful treatment; basic comforts; and peace of mind. Although Indigenous and non-Indigenous participants had similar unmet needs, for the former, the deleterious impact of unmet needs was compounded by racist and discriminatory behaviours they experienced while in hospital. CONCLUSIONS: Respectful, empathetic, person-centred care is likely to result in patients' needs being met, improve the hospital experience and reduce the risk of people self-discharging. For Indigenous people, the ongoing legacy of white colonisation is embodied in everyday lived experiences of interpersonal and institutional racism. Racist and discriminatory behaviours experienced whilst hospitalised are thus rendered both more visible and more traumatic, and exacerbate the deleterious effect of unmet needs. Decreasing self-discharge events requires a shift of thinking away from perceiving this as the behaviour of a deviant individual, but rather as a quality improvement opportunity to ensure that all patients are cared for in a respectful and person-centred manner.

Two novel protein O-glucosyltransferases that modify sites distinct from POGLUT1 and affect Notch trafficking and signaling.

The Notch-signaling pathway is normally activated by Notch-ligand interactions. A recent structural analysis suggested that a novel O-linked hexose modification on serine 435 of the mammalian NOTCH1 core ligand-binding domain lies at the interface with its ligands. This serine occurs between conserved cysteines 3 and 4 of Epidermal Growth Factor-like (EGF) repeat 11 of NOTCH1, a site distinct from those modified by protein O-glucosyltransferase 1 (POGLUT1), suggesting that a different enzyme is responsible. Here, we identify two novel protein O-glucosyltransferases, POGLUT2 and POGLUT3 (formerly KDELC1 and KDELC2, respectively), which transfer O-glucose (O-Glc) from UDP-Glc to serine 435. Mass spectrometric analysis of NOTCH1 produced in HEK293T cells lacking POGLUT2, POGLUT3, or both genes showed that either POGLUT2 or POGLUT3 can add this novel O-Glc modification. EGF11 of NOTCH2 does not have a serine residue in the same location for this O-glucosylation, but EGF10 of NOTCH3 (homologous to EGF11 in NOTCH1 and -2) is also modified at the same position. Comparison of the sites suggests a consensus sequence for modification. In vitro assays with POGLUT2 and POGLUT3 showed that both enzymes modified only properly folded EGF repeats and displayed distinct acceptor specificities toward NOTCH1 EGF11 and NOTCH3 EGF10. Mutation of the O-Glc modification site on EGF11 (serine 435) in combination with sensitizing O-fucose mutations in EGF8 or EGF12 affected cell-surface presentation of NOTCH1 or reduced activation of NOTCH1 by Delta-like1, respectively. This study identifies a previously undescribed mechanism for fine-tuning the Notch-signaling pathway in mammals.

Patterns of primary health care service use of Indigenous Australians diagnosed with cancer.

PURPOSE: The role of general practitioners in cancer care has expanded in recent years. However, little is known about utilization of primary health care (PHC) services by patients with cancer, particularly among socio-economically disadvantaged groups. We describe utilization of PHC services by patients with cancer, and the nature of the care provided. The study focuses on a disadvantaged group in Australia, namely Indigenous Australians. METHODS: A retrospective audit of clinical records in ten PHC services in Queensland, Australia. Demographic and clinical data of Indigenous Australians diagnosed with cancer during 2010-2016 were abstracted from patient's medical records at the PHC services. The rates of cancer-related visits were calculated using person years at risk as a denominator. RESULTS: A total of 138 patients' records were audited. During 12 months following the cancer diagnosis, patients visited the PHC service on average 5.95 times per year. Frequency of visits were relatively high in remote areas and among socioeconomic disadvantaged patients (IRR = 1.87, 95%CI 1.61-2.17; IRR = 1.79, 95%CI 1.45-2.21, respectively). Over 80% of visits were for seeking attention for symptoms, wound care, and emotional or social support. Patients who did not undergo surgery, had greater comorbidity, received chemotherapy and/or radiotherapy, and male gender had significantly greater rate of visits than their counterparts. CONCLUSION: The frequency of utilization of PHC services, especially by patients with comorbidities, and the range of reasons for attendance highlights the important role of PHC services in providing cancer care. The reliance on PHC services, particularly by patients in remote and disadvantaged communities, has important implications for appropriate resourcing and support for services in these locations.

Are general practitioners getting the information they need from hospitals and specialists to provide quality cancer care for Indigenous Australians?

BACKGROUND: Cancer care involves many different healthcare providers. Delayed or inaccurate communication between specialists and general practitioners (GP) may negatively affect care. AIM: To describe the pattern and variation of communication between primary healthcare (PHC) services and hospitals and specialists in relation to the patient's cancer care. METHODS: A retrospective audit of clinical records of Indigenous Australians diagnosed with cancer during 2010-2016 identified through 10 PHC services in Queensland is described. Poisson regression was used to model the dichotomous outcome availability of hospital discharge summary versus not. RESULTS: A total of 138 patient records was audited; 115 of those patients visited the PHC service for cancer-related care after cancer diagnosis; 40.0% visited the service before a discharge summary was available, and 36.5% of the patients had no discharge summary in their medical notes. While most discharge summaries noted important information about the patient's cancer, 42.4% lacked details regarding the discharge medications regimen. CONCLUSIONS: Deficits in communication and information transfer between specialists and GP may adversely affect patient care. Indigenous Australians are a relatively disadvantaged group that experience poor health outcomes and relatively poor access to care. The low proportion of discharge summaries noting discharge medication regimen is of concern among Indigenous Australians with cancer who have high comorbidity burden and low health literacy. Our findings provide an insight into some of the factors associated with quality of cancer care, and may provide guidance for focus areas for further research and improvement efforts.