A stagewise response to mitochondrial dysfunction in mitochondrial DNA maintenance disorders.

Mitochondrial DNA (mtDNA) deletions which clonally expand in skeletal muscle of patients with mtDNA maintenance disorders, impair mitochondrial oxidative phosphorylation dysfunction. Previously we have shown that these mtDNA deletions arise and accumulate in perinuclear mitochondria causing localised mitochondrial dysfunction before spreading through the muscle fibre. We believe that mito-nuclear signalling is a key contributor in the accumulation and spread of mtDNA deletions, and that knowledge of how muscle fibres respond to mitochondrial dysfunction is key to our understanding of disease mechanisms. To understand the contribution of mito-nuclear signalling to the spread of mitochondrial dysfunction, we use imaging mass cytometry. We characterise the levels of mitochondrial Oxidative Phosphorylation proteins alongside a mitochondrial mass marker, in a cohort of patients with mtDNA maintenance disorders. Our expanded panel included protein markers of key signalling pathways, allowing us to investigate cellular responses to different combinations of oxidative phosphorylation dysfunction and ragged red fibres. We find combined Complex I and IV deficiency to be most common. Interestingly, in fibres deficient for one or more complexes, the remaining complexes are often upregulated beyond the increase of mitochondrial mass typically observed in ragged red fibres. We further find that oxidative phosphorylation deficient fibres exhibit an increase in the abundance of proteins involved in proteostasis, e.g. HSP60 and LONP1, and regulation of mitochondrial metabolism (including oxidative phosphorylation and proteolysis, e.g. PHB1). Our analysis suggests that the cellular response to mitochondrial dysfunction changes depending on the combination of deficient oxidative phosphorylation complexes in each fibre.

Imaging mass cytometry analysis of Becker muscular dystrophy muscle samples reveals different stages of muscle degeneration.

Becker muscular dystrophy (BMD) is characterised by fiber loss and expansion of fibrotic and adipose tissue. Several cells interact locally in what is known as the degenerative niche. We analysed muscle biopsies of controls and BMD patients at early, moderate and advanced stages of progression using Hyperion imaging mass cytometry (IMC) by labelling single sections with 17 markers identifying different components of the muscle. We developed a software for analysing IMC images and studied changes in the muscle composition and spatial correlations between markers across disease progression. We found a strong correlation between collagen-I and the area of stroma, collagen-VI, adipose tissue, and M2-macrophages number. There was a negative correlation between the area of collagen-I and the number of satellite cells (SCs), fibres and blood vessels. The comparison between fibrotic and non-fibrotic areas allowed to study the disease process in detail. We found structural differences among non-fibrotic areas from control and patients, being these latter characterized by increase in CTGF and in M2-macrophages and decrease in fibers and blood vessels. IMC enables to study of changes in tissue structure along disease progression, spatio-temporal correlations and opening the door to better understand new potential pathogenic pathways in human samples.

Single-cell insights into immune dysregulation in rheumatoid arthritis flare versus drug-free remission.

Immune-mediated inflammatory diseases (IMIDs) are typically characterised by relapsing and remitting flares of inflammation. However, the unpredictability of disease flares impedes their study. Addressing this critical knowledge gap, we use the experimental medicine approach of immunomodulatory drug withdrawal in rheumatoid arthritis (RA) remission to synchronise flare processes allowing detailed characterisation. Exploratory mass cytometry analyses reveal three circulating cellular subsets heralding the onset of arthritis flare - CD45RO+PD1hi CD4+ and CD8+ T cells, and CD27+CD86+CD21- B cells - further characterised by single-cell sequencing. Distinct lymphocyte subsets including cytotoxic and exhausted CD4+ memory T cells, memory CD8+CXCR5+ T cells, and IGHA1+ plasma cells are primed for activation in flare patients. Regulatory memory CD4+ T cells (Treg cells) increase at flare onset, but with dysfunctional regulatory marker expression compared to drug-free remission. Significant clonal expansion is observed in T cells, but not B cells, after drug cessation; this is widespread throughout memory CD8+ T cell subsets but limited to the granzyme-expressing cytotoxic subset within CD4+ memory T cells. Based on our observations, we suggest a model of immune dysregulation for understanding RA flare, with potential for further translational research towards novel avenues for its treatment and prevention.

Replicative fitness and pathogenicity of primate lentiviruses in lymphoid tissue, primary human and chimpanzee cells: relation to possible jumps to humans.

BACKGROUND: Simian immunodeficiency viruses (SIV) have been jumping between non-human primates in West/Central Africa for thousands of years and yet, the HIV-1 epidemic only originated from a primate lentivirus over 100 years ago. METHODS: This study examined the replicative fitness, transmission, restriction, and cytopathogenicity of 22 primate lentiviruses in primary human lymphoid tissue and both primary human and chimpanzee peripheral blood mononuclear cells. FINDINGS: Pairwise competitions revealed that SIV from chimpanzees (cpz) had the highest replicative fitness in human or chimpanzee peripheral blood mononuclear cells, even higher fitness than HIV-1 group M strains responsible for worldwide epidemic. The SIV strains belonging to the "HIV-2 lineage" (including SIVsmm, SIVmac, SIVagm) had the lowest replicative fitness. SIVcpz strains were less inhibited by human restriction factors than the "HIV-2 lineage" strains. SIVcpz efficiently replicated in human tonsillar tissue but did not deplete CD4+ T-cells, consistent with the slow or nonpathogenic disease observed in most chimpanzees. In contrast, HIV-1 isolates and SIV of the HIV-2 lineage were pathogenic to the human tonsillar tissue, almost independent of the level of virus replication. INTERPRETATION: Of all primate lentiviruses, SIV from chimpanzees appears most capable of infecting and replicating in humans, establishing HIV-1. SIV from other Old World monkeys, e.g. the progenitor of HIV-2, replicate slowly in humans due in part to restriction factors. Nonetheless, many of these SIV strains were more pathogenic than SIVcpz. Either SIVcpz evolved into a more pathogenic virus while in humans or a rare SIVcpz, possibly extinct in chimpanzees, was pathogenic immediately following the jump into human. FUNDING: Support for this study to E.J.A. was provided by the NIH/NIAID R01 AI49170 and CIHR project grant 385787. Infrastructure support was provided by the NIH CFAR AI36219 and Canadian CFI/Ontario ORF 36287. Efforts of J.A.B. and N.J.H. was provided by NIH AI099473 and for D.H.C., by VA and NIH AI AI080313.

Cobdock: an accurate and practical machine learning-based consensus blind docking method.

Probing the surface of proteins to predict the binding site and binding affinity for a given small molecule is a critical but challenging task in drug discovery. Blind docking addresses this issue by performing docking on binding regions randomly sampled from the entire protein surface. However, compared with local docking, blind docking is less accurate and reliable because the docking space is too largetly sampled. Cavity detection-guided blind docking methods improved the accuracy by using cavity detection (also known as binding site detection) tools to guide the docking procedure. However, it is worth noting that the performance of these methods heavily relies on the quality of the cavity detection tool. This constraint, namely the dependence on a single cavity detection tool, significantly impacts the overall performance of cavity detection-guided methods. To overcome this limitation, we proposed Consensus Blind Dock (CoBDock), a novel blind, parallel docking method that uses machine learning algorithms to integrate docking and cavity detection results to improve not only binding site identification but also pose prediction accuracy. Our experiments on several datasets, including PDBBind 2020, ADS, MTi, DUD-E, and CASF-2016, showed that CoBDock has better binding site and binding mode performance than other state-of-the-art cavity detector tools and blind docking methods.

OPTIMAL: An OPTimized Imaging Mass cytometry AnaLysis framework for benchmarking segmentation and data exploration.

Analysis of imaging mass cytometry (IMC) data and other low-resolution multiplexed tissue imaging technologies is often confounded by poor single-cell segmentation and suboptimal approaches for data visualization and exploration. This can lead to inaccurate identification of cell phenotypes, states, or spatial relationships compared to reference data from single-cell suspension technologies. To this end we have developed the "OPTimized Imaging Mass cytometry AnaLysis (OPTIMAL)" framework to benchmark any approaches for cell segmentation, parameter transformation, batch effect correction, data visualization/clustering, and spatial neighborhood analysis. Using a panel of 27 metal-tagged antibodies recognizing well-characterized phenotypic and functional markers to stain the same Formalin-Fixed Paraffin Embedded (FFPE) human tonsil sample tissue microarray over 12 temporally distinct batches we tested several cell segmentation models, a range of different arcsinh cofactor parameter transformation values, 5 different dimensionality reduction algorithms, and 2 clustering methods. Finally, we assessed the optimal approach for performing neighborhood analysis. We found that single-cell segmentation was improved by the use of an Ilastik-derived probability map but that issues with poor segmentation were only really evident after clustering and cell type/state identification and not always evident when using "classical" bivariate data display techniques. The optimal arcsinh cofactor for parameter transformation was 1 as it maximized the statistical separation between negative and positive signal distributions and a simple Z-score normalization step after arcsinh transformation eliminated batch effects. Of the five different dimensionality reduction approaches tested, PacMap gave the best data structure with FLOWSOM clustering out-performing phenograph in terms of cell type identification. We also found that neighborhood analysis was influenced by the method used for finding neighboring cells with a "disc" pixel expansion outperforming a "bounding box" approach combined with the need for filtering objects based on size and image-edge location. Importantly, OPTIMAL can be used to assess and integrate with any existing approach to IMC data analysis and, as it creates .FCS files from the segmentation output and allows for single-cell exploration to be conducted using a wide variety of accessible software and algorithms familiar to conventional flow cytometrists.

Distinct lung cell signatures define the temporal evolution of diffuse alveolar damage in fatal COVID-19.

BACKGROUND: Lung damage in severe COVID-19 is highly heterogeneous however studies with dedicated spatial distinction of discrete temporal phases of diffuse alveolar damage (DAD) and alternate lung injury patterns are lacking. Existing studies have also not accounted for progressive airspace obliteration in cellularity estimates. We used an imaging mass cytometry (IMC) analysis with an airspace correction step to more accurately identify the cellular immune response that underpins the heterogeneity of severe COVID-19 lung disease. METHODS: Lung tissue was obtained at post-mortem from severe COVID-19 deaths. Pathologist-selected regions of interest (ROIs) were chosen by light microscopy representing the patho-evolutionary spectrum of DAD and alternate disease phenotypes were selected for comparison. Architecturally normal SARS-CoV-2-positive lung tissue and tissue from SARS-CoV-2-negative donors served as controls. ROIs were stained for 40 cellular protein markers and ablated using IMC before segmented cells were classified. Cell populations corrected by ROI airspace and their spatial relationships were compared across lung injury patterns. FINDINGS: Forty patients (32M:8F, age: 22-98), 345 ROIs and >900k single cells were analysed. DAD progression was marked by airspace obliteration and significant increases in mononuclear phagocytes (MnPs), T and B lymphocytes and significant decreases in alveolar epithelial and endothelial cells. Neutrophil populations proved stable overall although several interferon-responding subsets demonstrated expansion. Spatial analysis revealed immune cell interactions occur prior to microscopically appreciable tissue injury. INTERPRETATION: The immunopathogenesis of severe DAD in COVID-19 lung disease is characterised by sustained increases in MnPs and lymphocytes with key interactions occurring even prior to lung injury is established. FUNDING: UK Research and Innovation/Medical Research Council through the UK Coronavirus Immunology Consortium, Barbour Foundation, General Sir John Monash Foundation, Newcastle University, JGW Patterson Foundation, Wellcome Trust.

Parkinson's disease neurons exhibit alterations in mitochondrial quality control proteins.

Mitochondrial dysfunction has been suggested to contribute to Parkinson's disease pathogenesis, though an understanding of the extent or exact mechanism of this contribution remains elusive. This has been complicated by challenging nature of pathway-based analysis and an inability simultaneously study multiple related proteins within human brain tissue. We used imaging mass cytometry (IMC) to overcome these challenges, measuring multiple protein targets, whilst retaining the spatial relationship between targets in post-mortem midbrain sections. We used IMC to simultaneously interrogate subunits of the mitochondrial oxidative phosphorylation complexes, and several key signalling pathways important for mitochondrial homoeostasis, in a large cohort of PD patient and control cases. We revealed a generalised and synergistic reduction in mitochondrial quality control proteins in dopaminergic neurons from Parkinson's patients. Further, protein-protein abundance relationships appeared significantly different between PD and disease control tissue. Our data showed a significant reduction in the abundance of PINK1, Parkin and phosphorylated ubiquitinSer65, integral to the mitophagy machinery; two mitochondrial chaperones, HSP60 and PHB1; and regulators of mitochondrial protein synthesis and the unfolded protein response, SIRT3 and TFAM. Further, SIRT3 and PINK1 did not show an adaptive response to an ATP synthase defect in the Parkinson's neurons. We also observed intraneuronal aggregates of phosphorylated ubiquitinSer65, alongside increased abundance of mitochondrial proteases, LONP1 and HTRA2, within the Parkinson's neurons with Lewy body pathology, compared to those without. Taken together, these findings suggest an inability to turnover mitochondria and maintain mitochondrial proteostasis in Parkinson's neurons. This may exacerbate the impact of oxidative phosphorylation defects and ageing related oxidative stress, leading to neuronal degeneration. Our data also suggest that that Lewy pathology may affect mitochondrial quality control regulation through the disturbance of mitophagy and intramitochondrial proteostasis.

Poor outcomes in patients with sepsis undergoing emergency laparotomy and laparoscopy are attenuated by faster time to care measures.

AIM: Emergency laparotomy and laparoscopy (EmLap) are amongst the commonest surgical procedures, with high prevalence of sepsis and hence poorer outcomes. However, whether time taken to receive care influences outcomes in patients requiring antibiotics for suspected infection remains largely unexplored. The aim of this work was to determine whether (1) time to care contributes to outcome differences between patients with and without suspected infection and (2) its impact on outcomes only amongst those with suspected infection. METHOD: Clinical information was retrospectively obtained from the 2017-2018 Emergency Laparotomy and Laparoscopic Scottish Audit (ELLSA). Time to care referred to six temporal variables describing radiological investigation, anaesthetic triage and surgical management. Outcome measures [mortality, readmission, hospital death, postoperative destination and length of stay (LoS)] were compared using adjusted and unadjusted regression analyses to determine whether the outcome differences could be explained by faster or slower time to care. RESULTS: Amongst 2243 EmLap patients [median age 65 years (interquartile range 51-75 years), 51.1% female], 892 (39.77%) received antibiotics for suspected infection. Although patients with suspected infection had faster time to care (all p ≤ 0.001) and worse outcomes compared with those who did not, outcome differences were not statistically significant when accounted for time (all p > 0.050). Amongst those who received antibiotics, faster time to care was also associated with decreased risk of postoperative intensive care unit (ICU) stay and shorter LoS (all p < 0.050). CONCLUSION: Worse outcomes associated with infection in EmLap patients were attenuated by faster time to care, which additionally reduced the LoS and ICU stay risk amongst those with suspected infection.

Association between SARS-CoV-2 Symptoms, Ct Values, and Serological Response in Vaccinated and Unvaccinated Healthcare Personnel.

BACKGROUND: SARS-CoV-2 vaccines are effective at reducing symptomatic and asymptomatic COVID-19. Limited studies have compared symptoms, threshold cycle (Ct) values from reverse transcription (RT)-PCR testing, and serological testing results between previously vaccinated vs unvaccinated populations with SARS-CoV-2 infection. METHODS: Healthcare personnel (HCP) with a positive SARS-CoV-2 RT-PCR test within the previous 14 to 28 days completed surveys including questions about demographics, medical conditions, social factors, and symptoms of COVID-19. Ct values were observed, and serological testing was performed for anti-nucleocapsid (anti-N) and anti-Spike (anti-S) antibodies at enrollment and 40 to 90 days later. Serological results were compared to HCP with no known SARS-CoV-2 infection and negative anti-N testing. RESULTS: There were 104 unvaccinated/not fully vaccinated and 77 vaccinated HCP with 2 doses of an mRNA vaccine at time of infection. No differences in type or duration of symptoms were reported (P = 0.45). The median (interquartile range [IQR]) Ct was 21.4 (17.6-24.6) and 21.5 (18.1-24.6) for the unvaccinated and vaccinated HCP, respectively. Higher anti-N IgG was observed in unvaccinated HCP (5.08 S/CO, 3.08-6.92) than vaccinated (3.61 signal to cutoff ratio [S/CO], 2.16-5.05). Anti-S IgG was highest among vaccinated HCP with infection (34 285 aribitrary units [AU]/mL, 17 672-61 775), followed by vaccinated HCP with no prior infection (1452 AU/mL, 791-2943), then unvaccinated HCP with infection (829 AU/mL, 290-1555). Anti-S IgG decreased 1.56% (0.9%-1.79%) per day in unvaccinated and 0.38% (0.03%-0.94%) in vaccinated HCP. CONCLUSIONS: Vaccinated HCP infected with SARS-CoV-2 reported comparable symptoms and had similar Ct values relative to unvaccinated. However, vaccinated HCP had increased and prolonged anti-S and decreased anti-N response relative to unvaccinated.

Tissue factor expression in monocyte subsets during human immunothrombosis, endotoxemia and sepsis.

INTRODUCTION: Tissue factor expression on monocytes is implicated in the pathophysiology of sepsis-induced coagulopathy. How tissue factor is expressed by monocyte subsets (classical, intermediate and non-classical) is unknown. METHODS: Monocytic tissue factor surface expression was investigated during three conditions. Primary human monocytes and microvascular endothelial cell co-cultures were used for in vitro studies. Volunteers received a bolus of lipopolysaccharide (2 ng/kg) to induce endotoxemia. Patients with sepsis, or controls with critical illness unrelated to sepsis, were recruited from four intensive care units. RESULTS: Contact with endothelium and stimulation with lipopolysaccharide reduced the proportion of intermediate monocytes. Lipopolysaccharide increased tissue factor surface expression on classical and non-classical monocytes. Endotoxemia induced profound, transient monocytopenia, along with activation of coagulation pathways. In the remaining circulating monocytes, tissue factor was up-regulated in intermediate monocytes, though approximately 60 % of individuals (responders) up-regulated tissue factor across all monocyte subsets. In critically ill patients, tissue factor expression on intermediate and non-classical monocytes was significantly higher in patients with established sepsis than among non-septic patients. Upon recovery of sepsis, expression of tissue factor increased significantly in classical monocytes. CONCLUSION: Tissue factor expression in monocyte subsets varies significantly during health, endotoxemia and sepsis.

FLONE: fully Lorentz network embedding for inferring novel drug targets.

Motivation: To predict drug targets, graph-based machine-learning methods have been widely used to capture the relationships between drug, target and disease entities in drug-disease-target (DDT) networks. However, many methods cannot explicitly consider disease types at inference time and so will predict the same target for a given drug under any disease condition. Meanwhile, DDT networks are usually organized hierarchically carrying interactive relationships between involved entities, but these methods, especially those based on Euclidean embedding cannot fully utilize such topological information, which might lead to sub-optimal results. We hypothesized that, by importing hyperbolic embedding specifically for modeling hierarchical DDT networks, graph-based algorithms could better capture relationships between aforementioned entities, which ultimately improves target prediction performance. Results: We formulated the target prediction problem as a knowledge graph completion task explicitly considering disease types. We proposed FLONE, a hyperbolic embedding-based method based on capturing hierarchical topological information in DDT networks. The experimental results on two DDT networks showed that by introducing hyperbolic space, FLONE generates more accurate target predictions than its Euclidean counterparts, which supports our hypothesis. We also devised hyperbolic encoders to fuse external domain knowledge, to make FLONE enable handling samples corresponding to previously unseen drugs and targets for more practical scenarios. Availability and implementation: Source code and dataset information are at: https://github.com/arantir123/DDT_triple_prediction. Supplementary information: Supplementary data are available at Bioinformatics Advances online.

Programmed cell death-1 receptor-mediated regulation of Tbet+NK1.1- innate lymphoid cells within the tumor microenvironment.

Innate lymphoid cells (ILCs) play a key role in tissue-mediated immunity and can be controlled by coreceptor signaling. Here, we define a subset of ILCs that are Tbet+NK1.1- and are present within the tumor microenvironment (TME). We show programmed death-1 receptor (PD-1) expression on ILCs within TME is found in Tbet+NK1.1- ILCs. PD-1 significantly controlled the proliferation and function of Tbet+NK1.1- ILCs in multiple murine and human tumors. We found tumor-derived lactate enhanced PD-1 expression on Tbet+NK1.1- ILCs within the TME, which resulted in dampened the mammalian target of rapamycin (mTOR) signaling along with increased fatty acid uptake. In line with these metabolic changes, PD-1-deficient Tbet+NK1.1- ILCs expressed significantly increased IFNγ and granzyme B and K. Furthermore, PD-1-deficient Tbet+NK1.1- ILCs contributed toward diminished tumor growth in an experimental murine model of melanoma. These data demonstrate that PD-1 can regulate antitumor responses of Tbet+NK1.1- ILCs within the TME.

Knowledge, beliefs, and practices related to coronavirus disease 2019 (COVID-19) infection and vaccination in healthcare personnel working at nonacute care facilities.

OBJECTIVE: To characterize experiences, beliefs, and perceptions of risk related to coronavirus disease 2019 (COVID-19), infection prevention practices, and COVID-19 vaccination among healthcare personnel (HCP) at nonacute care facilities. DESIGN: Anonymous survey. SETTING: Three non-acute-care facilities in St. Louis, Missouri. PARTICIPANTS: In total, 156 HCP responded to the survey, for a 25.6% participation rate). Among them, 32% had direct patient-care roles. METHODS: Anonymous surveys were distributed between April-May 2021. Data were collected on demographics, work experience, COVID-19 exposure, knowledge, and beliefs about infection prevention, personal protective equipment (PPE) use, COVID-19 vaccination, and the impact of COVID-19. RESULTS: Nearly all respondents reported adequate knowledge of how to protect oneself from COVID-19 at work (97%) and had access to adequate PPE supplies (95%). Many HCP reported that wearing a mask or face shield made communication difficult (59%), that they had taken on additional responsibilities due to staff shortages (56%), and that their job became more stressful because of COVID-19 (53%). Moreover, 28% had considered quitting their job. Most respondents (78%) had received at least 1 dose of COVID-19 vaccine. Common reasons for vaccination were a desire to protect family and friends (84%) and a desire to stop the spread of COVID-19 (82%). Potential side effects and/or inadequate vaccine testing were cited as the most common concerns by unvaccinated HCP. CONCLUSIONS: A significant proportion of HCP reported increased stress and responsibilities at work due to COVID-19. The majority were vaccinated. Improving workplace policies related to mental health resources and sick leave, maintaining access to PPE, and ensuring clear communication of PPE requirements may improve workplace stress and burnout.

GBMdeconvoluteR accurately infers proportions of neoplastic and immune cell populations from bulk glioblastoma transcriptomics data.

BACKGROUND: Characterizing and quantifying cell types within glioblastoma (GBM) tumors at scale will facilitate a better understanding of the association between the cellular landscape and tumor phenotypes or clinical correlates. We aimed to develop a tool that deconvolutes immune and neoplastic cells within the GBM tumor microenvironment from bulk RNA sequencing data. METHODS: We developed an IDH wild-type (IDHwt) GBM-specific single immune cell reference consisting of B cells, T-cells, NK-cells, microglia, tumor associated macrophages, monocytes, mast and DC cells. We used this alongside an existing neoplastic single cell-type reference for astrocyte-like, oligodendrocyte- and neuronal progenitor-like and mesenchymal GBM cancer cells to create both marker and gene signature matrix-based deconvolution tools. We applied single-cell resolution imaging mass cytometry (IMC) to ten IDHwt GBM samples, five paired primary and recurrent tumors, to determine which deconvolution approach performed best. RESULTS: Marker-based deconvolution using GBM-tissue specific markers was most accurate for both immune cells and cancer cells, so we packaged this approach as GBMdeconvoluteR. We applied GBMdeconvoluteR to bulk GBM RNAseq data from The Cancer Genome Atlas and recapitulated recent findings from multi-omics single cell studies with regards associations between mesenchymal GBM cancer cells and both lymphoid and myeloid cells. Furthermore, we expanded upon this to show that these associations are stronger in patients with worse prognosis. CONCLUSIONS: GBMdeconvoluteR accurately quantifies immune and neoplastic cell proportions in IDHwt GBM bulk RNA sequencing data and is accessible here: https://gbmdeconvoluter.leeds.ac.uk.

Hyperactive CREB subpopulations increase during therapy in pediatric B-lineage acute lymphoblastic leukemia.

Persistence of residual disease in acute lymphoblastic leukemia (ALL) during the initial stages of chemotherapy is associated with inferior survival. To better understand clonal evolution and mechanisms of chemoresistance, we used multiparameter mass cytometry, at single-cell resolution, to functionally characterize pediatric B-ALL cells at disease presentation and those persisting during induction therapy. Analysis of ALL cells from presentation samples (n=42) showed that the most abundant phosphosignals were pCREB, pH2AX and pHH3 and we identified JAK-STAT and RAS pathway activation in five of six patients with JAK or RAS genetic aberrations. The clonal composition of ALL was heterogeneous and dynamic during treatment but all viable cell clusters showed pCREB activation. Levels of pCREB in ALL cells were increased or maintained during therapy and high dimensional analysis revealed a subpopulation of ALL cells at presentation that was positive for pCREB/pHH3/pS6 which increased during treatment in some patients, implicating this signaling node in conferring a survival advantage to multi-agent induction therapy. The small molecule CREB inhibitor, 666-15, was shown to reduce CREB transcriptional activity and induce apoptosis in ALL patient-derived xenograft cells of varying cytogenetic subtypes in vitro, both in the presence and absence of stromal support. Together, these data suggest that the cAMP signaling pathway may provide an opportunity for minimal residual disease-directed therapy for many patients at high risk of relapse.

Comparison of two anti-histones antibodies commercial assays in an immunology laboratory and systemic lupus erythematosus.

BACKGROUND: Anti-histones antibodies (AHAs) are antibodies directed against histone proteins - structural proteins that provides scaffolding for DNA to be wrapped around. AHAs, measured in the serum, are diagnostically helpful in cases of systemic lupus erythematosus (SLE) and drug-induced lupus erythematosus (DILE). In the diagnostic laboratory, they may be measured by enzyme-linked immune-sorbent assay (ELISA) or line immunoassays (LIA); however, the performance of these have never been directly compared in the literature. METHODS: We evaluated a commonly used commercial ELISA and LIA to compare the performance in our immunology laboratory. Retrospective and prospective analyses were undertaken over a 5.5-year period. We also examined their performance in the model disease of SLE and compared their performance to disease activity and the main clinical features. RESULTS: One hundred and thirty-five patients were evaluated including 65 SLE patients. Based on the quantitative cut-offs, there was only moderate agreement between the two assays (κ = 0.444). Both assays only had modest agreement with the clinical situation of the evaluated patients. When considered within the SLE context, both assays were moderately correlated with disease activity. Positive AHAs by ELISA were associated with SLE cytopaenias, and both ELISA and LIA correlated with positive anti-double stranded DNA. CONCLUSIONS: Moderate agreement analytically were seen between ELISA and LIA methods in a general laboratory cohort. Both assays were comparable in their diagnostic performance. ELISA detection of AHAs appears to have some clinical use in our cohort of SLE patients. Future studies are required to explore the clinical utility of AHAs in SLE and other disorders.

Music festival drug checking: evaluation of an Australian pilot program.

BACKGROUND: This paper explores the feasibility of delivering a music festival-based drug checking service in Australia, evaluating service design and stakeholder acceptability. METHODS: Questionnaire and interview data were collected from adult service users and key stakeholders. A mixed methods approach was used to analyse the data on implementation, impact and acceptability. RESULTS: The trial service tested 170 substances with more than 230 patrons (including individuals who attended in groups). Adult service users had an average age of 21 years. Voluntary participation in the evaluation resulted in 158 participants completing the pre-service questionnaire, most of whom also completed the post-service (147 participants). Eleven in-depth qualitative interviews were conducted with patrons in the weeks following the drug checking. Concordance between what the patron expected the drug to be and drug checking results occurred in 88 per cent (n = 139) of the sample. Evaluation results show that the experience of testing and the accompanying harm reduction brief interventions positively impacted on patrons' self-reported drug harm reduction knowledge, trust of health providers and stated drug use intentions. The service was received positively by service users. CONCLUSION: This is the first independent evaluation of a pilot drug checking service in Australia. Consideration of operational feasibility and self-reported behavioural change suggests that the program was successful, although communication about the interpretation of drug checking results could be improved. Future studies should develop strategies for follow-up and consider the applicability of behavioural change theory.

Pentosinane, a Post-Translational Modification of Human Proteins with Underappreciated Stability.

Pentosinane is a structurally complex nonenzymatic post-translational modification of proteins believed to be present in all living things. It falls into the category of advanced glycation end products (AGEs) and is structurally related to the other AGEs pentosidine and glucosepane. Although pentosidine and glucosepane have been widely studied for their role in wide-ranging conditions (e.g., diabetes mellitus, Alzheimer's disease, and human aging), relatively little is known about pentosinane. Interestingly, previous reports have suggested that pentosidine may derive from pentosinane. The (patho)physiological significance of pentosinane in humans is largely unexplored. As a first step to address this knowledge gap, we report herein the first total synthesis of pentosinane. Our synthesis is high yielding (1.7% over seven steps), concise, and enantioselective, and it leverages a strategy for synthesizing 2,5-diaminoimidazoles previously developed by our lab. Access to synthetic pentosinane has allowed us to perform additional studies showing that its oxidation to pentosidine is both pH and oxygen dependent and is substantially slower under physiological conditions than previously believed. Additionally, pentosinane rapidly decomposes under harshly acidic conditions typically employed for pentosidine isolation. Taken together, these results suggest that pentosinane is likely to be more abundant in vivo than previously appreciated. We believe these results represent a critical step toward illuminating the role(s) of pentosinane in human biology.

Development of an Internet of Things Technology Platform (the NEX System) to Support Older Adults to Live Independently: Protocol for a Development and Usability Study.

BACKGROUND: In a rapidly aging population, new and efficient ways of providing health and social support to older adults are required that not only preserve independence but also maintain quality of life and safety. OBJECTIVE: The NEX project aims to develop an integrated Internet of Things system coupled with artificial intelligence to offer unobtrusive health and wellness monitoring to support older adults living independently in their home environment. The primary objective of this study is to develop and evaluate the technical performance and user acceptability of the NEX system. The secondary objective is to apply machine learning algorithms to the data collected via the NEX system to identify and eventually predict changes in the routines of older adults in their own home environment. METHODS: The NEX project commenced in December 2019 and is expected to be completed by August 2022. Mixed methods research (web-based surveys and focus groups) was conducted with 426 participants, including older adults (aged ≥60 years), family caregivers, health care professionals, and home care workers, to inform the development of the NEX system (phase 1). The primary outcome will be evaluated in 2 successive trials (the Friendly trial [phase 2] and the Action Research Cycle trial [phase 3]). The secondary objective will be explored in the Action Research Cycle trial (phase 3). For the Friendly trial, 7 older adult participants aged ≥60 years and living alone in their own homes for a 10-week period were enrolled. A total of 30 older adult participants aged ≥60 years and living alone in their own homes will be recruited for a 10-week data collection period (phase 3). RESULTS: Phase 1 of the project (n=426) was completed in December 2020, and phase 2 (n=7 participants for a 10-week pilot study) was completed in September 2021. The expected completion date for the third project phase (30 participants for the 10-week usability study) is June 2022. CONCLUSIONS: The NEX project has considered the specific everyday needs of older adults and other stakeholders, which have contributed to the design of the integrated system. The innovation of the NEX system lies in the use of Internet of Things technologies and artificial intelligence to identify and predict changes in the routines of older adults. The findings of this project will contribute to the eHealth research agenda, focusing on the improvement of health care provision and patient support in home and community environments. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): DERR1-10.2196/35277.