Essential thrombocythemia: challenges in clinical practice and future prospects.

Essential thrombocythemia (ET) was first described in 1934, and subsequently, progress has been made in better understanding the molecular pathogenesis and which patients may have greatest risk of progression or vascular events. However, it has been more than a decade since a new therapy has been approved for ET. We are beginning to understand more comprehensively both the heterogeneity of this disease, which is largely driven by driver mutation status, as well as the effect of disease-related symptoms, such as fatigue, on patients. In this review we provide a practical overview of diagnosis and management of ET with focus on challenging patient scenarios and some consideration of what comprehensive care might entail. Finally, we also discuss newer therapies and how these might be assessed.

Analysis of Genome-Wide Mutational Dependence in Naturally Evolving Mycobacterium tuberculosis Populations.

Pathogenic microorganisms are in a perpetual struggle for survival in changing host environments, where host pressures necessitate changes in pathogen virulence, antibiotic resistance, or transmissibility. The genetic basis of phenotypic adaptation by pathogens is difficult to study in vivo. In this work, we develop a phylogenetic method to detect genetic dependencies that promote pathogen adaptation using 31,428 in vivo sampled Mycobacterium tuberculosis genomes, a globally prevalent bacterial pathogen with increasing levels of antibiotic resistance. We find that dependencies between mutations are enriched in antigenic and antibiotic resistance functions and discover 23 mutations that potentiate the development of antibiotic resistance. Between 11% and 92% of resistant strains harbor a dependent mutation acquired after a resistance-conferring variant. We demonstrate the pervasiveness of genetic dependency in adaptation of naturally evolving populations and the utility of the proposed computational approach.

Transcriptomic responses to antibiotic exposure in Mycobacterium tuberculosis.

Transcriptional responses in bacteria following antibiotic exposure offer insights into antibiotic mechanism of action, bacterial responses, and characterization of antimicrobial resistance. We aimed to define the transcriptional antibiotic response (TAR) in Mycobacterium tuberculosis (Mtb) isolates for clinically relevant drugs by pooling and analyzing Mtb microarray and RNA-seq data sets. We generated 99 antibiotic transcription profiles across 17 antibiotics, with 76% of profiles generated using 3-24 hours of antibiotic exposure and 49% within one doubling of the WHO antibiotic critical concentration. TAR genes were time-dependent, and largely specific to the antibiotic mechanism of action. TAR signatures performed well at predicting antibiotic exposure, with the area under the receiver operating curve (AUC) ranging from 0.84-1.00 (TAR <6 hours of antibiotic exposure) and 0.76-1.00 (>6 hours of antibiotic exposure) for upregulated genes and 0.57-0.90 and 0.87-1.00, respectfully, for downregulated genes. This work desmonstrates that transcriptomics allows for the assessment of antibiotic activity in Mtb within 6 hours of exposure.

Clinical utility of investigations in triple-negative thrombocytosis: A real-world, multicentre evaluation of UK practice.

Diagnosis of essential thrombocythaemia (ET) is challenging in patients lacking JAK2/CALR/MPL mutations. In a retrospective evaluation of 320 patients with 'triple-negative thrombocytosis', we assessed utility of bone marrow histology (90.9% of patients) and myeloid gene panel (MGP, 55.6%). Supportive histology ('myeloproliferative neoplasm-definite/probable', 36.8%) was associated with higher platelet counts and varied between centres. 14.6% MGP revealed significant variants: 3.4% JAK2/CALR/MPL and 11.2% other myeloid genes. Final clinical diagnosis was strongly predicted by histology, not MGP. 23.7% received cytoreduction (17.6% under 60 years). Real-world 'triple-negative' ET diagnosis currently depends heavily on histology; we advocate caution in MGP-negative cases and that specific guidelines are needed.

Structure of the peptidoglycan polymerase RodA resolved by evolutionary coupling analysis.

The shape, elongation, division and sporulation (SEDS) proteins are a large family of ubiquitous and essential transmembrane enzymes with critical roles in bacterial cell wall biology. The exact function of SEDS proteins was for a long time poorly understood, but recent work has revealed that the prototypical SEDS family member RodA is a peptidoglycan polymerase-a role previously attributed exclusively to members of the penicillin-binding protein family. This discovery has made RodA and other SEDS proteins promising targets for the development of next-generation antibiotics. However, little is known regarding the molecular basis of SEDS activity, and no structural data are available for RodA or any homologue thereof. Here we report the crystal structure of Thermus thermophilus RodA at a resolution of 2.9 Å, determined using evolutionary covariance-based fold prediction to enable molecular replacement. The structure reveals a ten-pass transmembrane fold with large extracellular loops, one of which is partially disordered. The protein contains a highly conserved cavity in the transmembrane domain, reminiscent of ligand-binding sites in transmembrane receptors. Mutagenesis experiments in Bacillus subtilis and Escherichia coli show that perturbation of this cavity abolishes RodA function both in vitro and in vivo, indicating that this cavity is catalytically essential. These results provide a framework for understanding bacterial cell wall synthesis and SEDS protein function.

Differential inhibition of T-cell receptor and STAT5 signaling pathways determines the immunomodulatory effects of dasatinib in chronic phase chronic myeloid leukemia.

Dasatinib is a multi-kinase inhibitor with activity against the SRC kinase LCK, which plays a critical role in T-cell receptor signaling. Dasatinib, initially developed as an immunosuppressive agent, is by contrast, also noted to result in enhanced tumor immunity in a subset of patients. We studied the impact of dasatinib in chronic myeloid leukemia patients and compared it with patients taking other tyrosine kinase inhibitors (TKI) and healthy controls. We found that patients on dasatinib showed inhibition of both T-cell receptor (TCR) and STAT5 signaling pathways, and reduced expression of Teffector pro-inflammatory cytokines. In addition, dasatinib induced selective depletion of regulatory T cells (Tregs) and effector Tregs, particularly in patients with clonal expansion of effector CD8+ T cells, who demonstrated greater and preferential inhibition of Treg TCR intracellular signaling. In addition, we show that dasatinib selectively reduces Treg STAT5 phosphorylation via reduction of IL-2, in relation with the marked reduction of plasma IL-2 levels in patients taking dasatinib. Finally, patients on other TKI had significantly increased TCR signaling in TIM3+ cells compared to patients taking dasatinib, suggesting that chronic SRC kinase inhibition by dasatinib may play a role in preventing TIM-3-mediated T-cell exhaustion and preserve anti-tumor immunity. These data provide further insight into the selective immunomodulatory effects of dasatinib and its potential use for pharmacologic control of immunotherapies.

Whole exome sequencing of FFPE samples-expanding the horizon of forensic molecular autopsies.

Forensic molecular autopsies have emerged as a tool for medical examiners to establish the cause of death. It is particularly useful in sudden unexplained deaths where the cause of death cannot be determined with a regular medical autopsy. We provide the first study of exome data from formalin-fixed paraffin-embedded samples (FFPE) paired with data from high-quality blood samples in forensic applications. The approach allows exploration of the potential to use FFPE samples for molecular autopsies and identify variants in extensive exome data. We leverage the high uniformity of the hybridization capture approach provided by Twist Bioscience to target the complete exome and sequence the libraries on a NextSeq 550. Our findings suggest that exome sequencing is feasible for 24 out of a total of 35 included FFPE samples. When successful, the coverage across the exome is comparatively high (> 90% covered to 20X) and uniform (fold80 below 1.5). Detailed variant comparisons for matched FFPE and blood samples show high concordance with few false variants (positive predictive value of 0.98 and a sensitivity of 0.97) with no distinct FFPE artefacts. Ultimately, we apply carefully constructed forensic gene panels in a stepwise manner to find genetic variants associated with the clinical phenotype and with relevance to the sudden unexplained death.

Evaluation of two temperature storage conditions for full blood count samples from Lifeblood's donors.

BACKGROUND AND OBJECTIVES: Lifeblood completes full blood count samples for selected donors to assess their suitability for future donations. Removing the current practice for refrigerated (2-8°C) storage and aligning with room temperature (20-24°C) storage of other donor blood samples would produce significant efficiencies in blood donor centres. This study aimed to compare full blood count results under two temperature conditions. MATERIALS AND METHODS: Paired full blood count samples were collected from 250 whole blood or plasma donors. These were stored either refrigerated or room temperature for testing on arrival at the processing centre and the following day. The primary outcomes of interest included differences between mean cell volume, haematocrit, platelet count, white cell and differential counts, and the need to produce blood films, based on existing Lifeblood criteria. RESULTS: A statistically significant (p < 0.05) difference for most full blood count parameters results was found between the two temperature conditions. The number of blood films required was similar under each temperature condition. CONCLUSION: The clinical significance of the small numerical differences in results is considered minimal. Furthermore, the number of blood films required remained similar under either temperature condition. Given the significant reductions in time, processing and costs associated with room temperature over refrigerated processing, we recommend a further pilot study to monitor the broader impacts, with the intent to implement national storage of full blood count samples at room temperature within Lifeblood.

Stakeholder perspectives of a pilot multicomponent delirium prevention intervention for adult patients with advanced cancer in palliative care units: A behaviour change theory-based qualitative study.

BACKGROUND: Theory-based and qualitative evaluations in pilot trials of complex clinical interventions help to understand quantitative results, as well as inform the feasibility and design of subsequent effectiveness and implementation trials. AIM: To explore patient, family, clinician and volunteer ('stakeholder') perspectives of the feasibility and acceptability of a multicomponent non-pharmacological delirium prevention intervention for adult patients with advanced cancer in four Australian palliative care units that participated in a phase II trial, the 'PRESERVE pilot study'. DESIGN: A trial-embedded qualitative study via semi-structured interviews and directed content analysis using Michie's Behaviour Change Wheel and the Theoretical Domains Framework. SETTING/PARTICIPANTS: Thirty-nine people involved in the trial: nurses (n = 17), physicians (n = 6), patients (n = 6), family caregivers (n = 4), physiotherapists (n = 3), a social worker, a pastoral care worker and a volunteer. RESULTS: Participants' perspectives aligned with the 'capability', 'opportunity' and 'motivation' domains of the applied frameworks. Of seven themes, three were around the alignment of the delirium prevention intervention with palliative care (intervention was considered routine care; intervention aligned with the compassionate and collaborative culture of palliative care; and differing views of palliative care priorities influenced perspectives of the intervention) and four were about study processes more directly related to adherence to the intervention (shared knowledge increased engagement with the intervention; impact of the intervention checklist on attention, delivery and documentation of the delirium prevention strategies; clinical roles and responsibilities; and addressing environmental barriers to delirium prevention). CONCLUSION: This theory-informed qualitative study identified multiple influences on the delivery and documentation of a pilot multicomponent non-pharmacological delirium prevention intervention in four palliative care units. Findings inform future definitive studies of delirium prevention in palliative care.Australian New Zealand Clinical Trials Registry, ACTRN12617001070325; https://www.anzctr.org.au/Trial/Registration/TrialReview.aspx?id=373168.

Interaction specificity of clustered protocadherins inferred from sequence covariation and structural analysis.

Clustered protocadherins, a large family of paralogous proteins that play important roles in neuronal development, provide an important case study of interaction specificity in a large eukaryotic protein family. A mammalian genome has more than 50 clustered protocadherin isoforms, which have remarkable homophilic specificity for interactions between cellular surfaces. A large antiparallel dimer interface formed by the first 4 extracellular cadherin (EC) domains controls this interaction. To understand how specificity is achieved between the numerous paralogs, we used a combination of structural and computational approaches. Molecular dynamics simulations revealed that individual EC interactions are weak and undergo binding and unbinding events, but together they form a stable complex through polyvalency. Strongly evolutionarily coupled residue pairs interacted more frequently in our simulations, suggesting that sequence coevolution can inform the frequency of interaction and biochemical nature of a residue interaction. With these simulations and sequence coevolution, we generated a statistical model of interaction energy for the clustered protocadherin family that measures the contributions of all amino acid pairs at the interface. Our interaction energy model assesses specificity for all possible pairs of isoforms, recapitulating known pairings and predicting the effects of experimental changes in isoform specificity that are consistent with literature results. Our results show that sequence coevolution can be used to understand specificity determinants in a protein family and prioritize interface amino acid substitutions to reprogram specific protein-protein interactions.

Power coupling losses for misaligned and mode-mismatched higher-order Hermite-Gauss modes.

This paper analytically and numerically investigates misalignment and mode-mismatch-induced power coupling coefficients and losses as a function of Hermite-Gauss (HG) mode order. We show that higher-order HG modes are more susceptible to beam perturbations when, for example, coupling into optical cavities: the misalignment and mode-mismatch-induced power coupling losses scale linearly and quadratically with respect to the mode indices, respectively. As a result, the mode-mismatch tolerance for the ${{\rm HG}_{3,3}}$ mode is reduced to a factor of 0.28 relative to the currently used ${{\rm HG}_{0,0}}$ mode. This is a potential hurdle to using higher-order modes to reduce thermal noise in future gravitational-wave detectors.

Prostate cancer survivorship essentials framework: guidelines for practitioners.

OBJECTIVE: To develop contemporary and inclusive prostate cancer survivorship guidelines for the Australian setting. PARTICIPANTS AND METHODS: A four-round iterative policy Delphi was used, with a 47-member expert panel that included leaders from key Australian and New Zealand clinical and community groups and consumers from diverse backgrounds, including LGBTQIA people and those from regional, rural and urban settings. The first three rounds were undertaken using an online survey (94-96% response) followed by a fourth final face-to-face panel meeting. Descriptors for men's current prostate cancer survivorship experience were generated, along with survivorship elements that were assessed for importance and feasibility. From these, survivorship domains were generated for consideration. RESULTS: Six key descriptors for men's current prostate cancer survivorship experience that emerged were: dealing with side effects; challenging; medically focused; uncoordinated; unmet needs; and anxious. In all, 26 survivorship elements were identified within six domains: health promotion and advocacy; shared management; vigilance; personal agency; care coordination; and evidence-based survivorship interventions. Consensus was high for all domains as being essential. All elements were rated high on importance but consensus was mixed for feasibility. Seven priorities were derived for immediate action. CONCLUSION: The policy Delphi allowed a uniquely inclusive expert clinical and community group to develop prostate cancer survivorship domains that extend beyond traditional healthcare parameters. These domains provide guidance for policymakers, clinicians, community and consumers on what is essential for step change in prostate cancer survivorship outcomes.

Genetic Variant Score and Arrhythmogenic Right Ventricular Cardiomyopathy Phenotype in Plakophilin-2 Mutation Carriers.

INTRODUCTION: Whether detailed genetic information contributes to risk stratification of patients with arrhythmogenic right ventricular cardiomyopathy (ARVC) remains uncertain. Pathogenic genetic variants in some genes seem to carry a higher risk for arrhythmia and earlier disease onset than others, but comparisons between variants in the same gene have not been done. Combined Annotation Dependent Depletion (CADD) score is a bioinformatics tool that measures the pathogenicity of each genetic variant. We hypothesized that a higher CADD score is associated with arrhythmic events and earlier age at ARVC manifestations in individuals carrying pathogenic or likely pathogenic genetic variants in plakophilin-2 (PKP2). METHODS: CADD scores were calculated using the data from pooled Scandinavian and North American ARVC cohorts, and their association with cardiac events defined as ventricular tachycardia/ventricular fibrillation (VT/VF) or syncope and age at definite ARVC diagnosis were assessed. RESULTS: In total, 33 unique genetic variants were reported in 179 patients (90 males, 71 probands, 96 with definite ARVC diagnosis at a median age of 35 years). Cardiac events were reported in 76 individuals (43%), of whom 53 had sustained VT/VF (35%). The CADD score was neither associated with age at cardiac events (HR 1.002, 95% CI: 0.953-1.054, p = 0.933) nor with age at definite ARVC diagnosis (HR 0.992, 95% CI: 0.947-1.039, p = 0.731). CONCLUSION: No correlation was found between CADD scores and clinical manifestations of ARVC, indicating that the score has no additional risk stratification value among carriers of pathogenic or likely pathogenic PKP2 genetic variants.

The clinical and pathological panoply of systemic mastocytosis.

Mastocytosis is a rare disease with varied presentation, myriad symptomatology and variable prognosis. Most patients present with cutaneous disease and mediator-related symptomatology with a small subset having systemic disease (systemic mastocytosis, SM). A subset of the latter develops synchronous or metachronous haematologic neoplasms (SM-AHN), most commonly chronic myelomonocytic leukaemia (CMML). Advanced systemic mastocytosis (ASM) is seen in a relatively small number of patients and is usually associated with organ dysfunction, and may present with hepatosplenomegaly, lymphadenopathy and ascites with progression to leukaemic transformation (mast cell leukaemia/acute myeloid leukaemia) occurring in a few patients. This paper discusses the clinical and pathologic features of the entire spectrum of SM in adults.

The EVcouplings Python framework for coevolutionary sequence analysis.

SUMMARY: Coevolutionary sequence analysis has become a commonly used technique for de novo prediction of the structure and function of proteins, RNA, and protein complexes. We present the EVcouplings framework, a fully integrated open-source application and Python package for coevolutionary analysis. The framework enables generation of sequence alignments, calculation and evaluation of evolutionary couplings (ECs), and de novo prediction of structure and mutation effects. The combination of an easy to use, flexible command line interface and an underlying modular Python package makes the full power of coevolutionary analyses available to entry-level and advanced users. AVAILABILITY AND IMPLEMENTATION: https://github.com/debbiemarkslab/evcouplings.

Contemporary consumer perspectives on prostate cancer survivorship: Fifty voices.

OBJECTIVE: To describe contemporary consumer experiences and priorities of prostate cancer survivorship to inform the development of survivorship guidelines. METHOD: In a cross-sectional qualitative design, semi-structured interviews were undertaken with fifty prostate cancer support group leaders (50% response) across urban and rural/regional Australia. An interpretative phenomenological approach was used to identify participants' experiences and priorities of survivorship. RESULTS: Three themes were identified for informing the development of prostate cancer survivorship guidelines: (a) the Experience of Diagnosis and Treatment, (b) Priorities for Survivorship and (c) Mechanisms for Support. Four priorities for prostate cancer survivorship were: delivering person and men-centred care; improving communication; improving care coordination; and facilitating access to care. Mechanisms for support were as follows: advocacy; peer support; prostate cancer specialist nurses; and communication training for health professionals. CONCLUSIONS: The lack of progress in changing prostate cancer survivorship outcomes for men will become increasingly problematic as this patient population group grows. Co-production provides a way forward to ensure relevance and accessibility for future survivorship initiatives.

Prostate cancer survivorship priorities for men and their partners: Delphi consensus from a nurse specialist cohort.

AIMS & OBJECTIVES: To describe the prostate cancer survivorship experience and priorities from the perspective of prostate cancer specialist nurses. BACKGROUND: Specialist nurses are providing long-term survivorship care to men and their partners however, few prostate cancer survivorship interventions are effective and priorities for nurse-led survivorship care are poorly understood. DESIGN: A three-round modified Delphi approach. METHODS: The study was conducted between 1 December 2018 and 28 February 2019 to develop a consensus view from an expert nurse cohort (43 prostate cancer specialist nurses: 90% response). First, participants described men's prostate cancer survivorship experience and priorities for improving care for men and partners. In subsequent rounds, participants identified key descriptors of the survivorship experience; rated priorities for importance and feasibility; and identified a top priority action for men and for partners. Thematic analysis and descriptive statistics were applied. Guidelines for Reporting Reliability and Agreement Studies informed the conduct of the study. RESULTS: Prostate cancer specialist nurses characterised the prostate cancer survivorship experience of men as under-resourced, disjointed and distressing. In all, 11 survivorship priorities for men and three for partners were identified within five broad areas: capacity building; care coordination; physical and psychosocial care; community awareness and early detection; and palliative care. However, feasibility for individual items was frequently described as low. CONCLUSION: Internationally, prostate cancer survivorship care for men and their partners requires urgent action to meet future need and address gaps in capacity and care coordination. Low feasibility of survivorship priorities may reflect translational challenges related to capacity. Prostate cancer survivorship care guidelines connected to practice priorities are urgently needed. RELEVANCE TO CLINICAL PRACTICE: These findings address key gaps in the evidence for developing national nurse-led prostate cancer survivorship priorities. These priorities can be used to inform survivorship guidelines including nursing care for men with prostate cancer and their partners.

Collaborating across sectors to provide early intervention for Aboriginal and Torres Strait Islander children with disability and their families: a qualitative study of provider perspectives.

Aboriginal and Torres Strait Islander children experience a higher prevalence of disability than other Australian children. Early intervention from across the health, education, and social service sectors is vital for improving outcomes, but families face lack of coordination between services. This study aimed to inform improvements in service access for families of urban-dwelling Aboriginal children with disability through exploring providers' perceptions of factors that influenced working together across sectors. Semi-structured interviews were conducted. Data analysis was informed by the general inductive approach and the Collaborative Practice to Enhance Patient Care Outcomes framework. Twenty-four providers participated. Interprofessional collaborative practice was influenced by interdependent interactional and organizational factors. Interactional factors fit within one of two dimensions: the ability of providers to share common goals and vision within a complex cross-sector service landscape, and influence of interpersonal relationships on their sense of belonging working in a cross-cultural space. Organizational factors also fit within one of two dimensions: the influence of governance in relation to its role in coordination and unlocking the strength of schools as service settings, and the need to formalize processes for effective interprofessional communication. Interprofessional collaborative practice was managed within the context of systemic factors relating to policy and funding. These findings demonstrate the complex interplay of factors related to the cross-sector involvement of providers in early intervention service provision. Consideration of these factors is required to facilitate collaborative cross-sector responses to improve service access for Aboriginal families.Abbreviations: WHO: world health organization; ACCHS: aboriginal community controlled health service; GP: general practitioner; NDIS: national disability insurance scheme.