Structure of the thrombopoietin-MPL receptor complex is a blueprint for biasing hematopoiesis.

Thrombopoietin (THPO or TPO) is an essential cytokine for hematopoietic stem cell (HSC) maintenance and megakaryocyte differentiation. Here, we report the 3.4 Å resolution cryoelectron microscopy structure of the extracellular TPO-TPO receptor (TpoR or MPL) signaling complex, revealing the basis for homodimeric MPL activation and providing a structural rationalization for genetic loss-of-function thrombocytopenia mutations. The structure guided the engineering of TPO variants (TPOmod) with a spectrum of signaling activities, from neutral antagonists to partial- and super-agonists. Partial agonist TPOmod decoupled JAK/STAT from ERK/AKT/CREB activation, driving a bias for megakaryopoiesis and platelet production without causing significant HSC expansion in mice and showing superior maintenance of human HSCs in vitro. These data demonstrate the functional uncoupling of the two primary roles of TPO, highlighting the potential utility of TPOmod in hematology research and clinical HSC transplantation.

Prevalence of moderate or greater permanent childhood hearing impairment and effectiveness of targeted surveillance for babies who pass newborn hearing screening.

OBJECTIVE: To assess the utility of targeted surveillance for the identification of moderate to profound PCHI in babies who pass newborn hearing screening in England and have risk factors. DESIGN: Retrospective analysis. STUDY SAMPLE: A total of 3,957,891 children born 01/04/2012-31/03/2018 in England. RESULTS: A total of 7148 PCHI cases were identified (1.81 per 1,000 babies). 6,707 followed an immediate referral from the screen (1 per 16 referrals), 51 followed targeted surveillance referral (1 per 540 referrals) and 390 without a referral. Audiology uptake was higher following an immediate referral (96.7% overall, 77.2% within NHSP-defined timescales) than following targeted surveillance (63.8% overall, 51.1% within 52 weeks of birth). The screening was 94.5% sensitive overall, with similar sensitivities for each of the risk factors. General linearised logistic regression models identified syndrome as the risk factor with the highest odds ratio (14.08 for all babies, 22.19 for babies without immediate referral). Close family history of hearing loss was the next highest (10.93 for all babies, 12.29 for babies without immediate referral). CONCLUSION: The evidence for a targeted surveillance programme, based on risk factors, for babies in England who pass the newborn screen is not strong.

Development and Validation of a Tool to Identify Anal Incontinence in Pregnant and Postnatal Women.

BACKGROUND: Neuromuscular and mechanical damage to the pelvic floor because of pregnancy and birth can result in anal incontinence. Pregnant and postnatal women are rarely screened for anal incontinence by clinicians who specialize in the care of these women, and no screening tool has been developed for routine use in these women. OBJECTIVE: To develop and validate a tool for use in everyday clinical practice in the care of pregnant and postnatal women. DATA SOURCES: The study includes 2 test phases with separate data sources. Phase I included test and retest phases of the Bowel-Screening Questionnaire in health professionals and women who were pregnant or had recently birthed (n = 45). Phase II included a pilot of the tool compared to 2 current scoring systems (n = 358). SETTING: Large tertiary hospital in South Australia. PATIENTS: Phase II: prospective recruitment of 358 prenatal parous women attending a first antenatal appointment. MAIN OUTCOMES MEASURES: To evaluate the reliability and validity of the developed tool. RESULTS: Test-retest agreement in phase I was excellent for each of the 6 items, with each κ statistic being between 0.83 and 1.0. In phase II, agreement between new and existing tools was fair to good for the detection of anal incontinence symptoms addressed as a composite question (κ between 0.41 and 0.71). Anal incontinence was detected in 191 women (53%) using the new tool, and there was a lower prevalence reported using the Vaizey score (n = 118) and Wexner score (n = 129). Completion rates of the new tool were 99%, higher than both the Vaizey score (33%) and Wexner score (36%). LIMITATIONS: Sample size limits the generalization of findings. CONCLUSION: The questionnaire is reliable and valid, reporting a high incidence of bowel incontinence, with predominant symptoms of rectal urgency and flatus as precursors for worsening function. DESARROLLO Y VALIDACIN DE UNA HERRAMIENTA PARA IDENTIFICAR LA INCONTINENCIA ANAL EN MUJERES EMBARAZADAS Y PURPERAS: ANTECEDENTES:El daño neuromuscular y mecánico del piso pélvico debido al embarazo y al parto puede resultar en incontinencia anal. Las mujeres embarazadas y puérperas rara vez son examinadas para la incontinencia anal por médicos que se especializan en el cuidado de estas mujeres, y no se ha desarrollado ninguna herramienta de detección para uso rutinario en estas mujeres.OBJETIVO:Desarrollar y validar una herramienta para uso en la práctica clínica diaria en el cuidado de las mujeres embarazadas y puérperas.FUENTES DE DATOS:El estudio incluye 2 fases de prueba con fuentes de datos separadas. Fase 1, fase test y retest del Bowel Screening Questionnaire en profesionales sanitarios y mujeres embarazadas o recién paridas (n = 45). La Fase 2 incluyó una prueba piloto de la herramienta en comparación con dos sistemas de puntuación actuales (n = 358).ENTORNO CLINICO:Gran hospital terciario en el sur de Australia.PACIENTES:Fase 2: reclutamiento prospectivo de 358 mujeres con parto prenatal que asisten a una primera cita prenatal.PRINCIPALES MEDIDAS DE VALORACIÓN:Evaluar la confiabilidad y validez de la herramienta desarrollada.RESULTADOS:La concordancia test-retest en la fase 1 fue excelente para cada uno de los 6 ítems con cada estadística kappa entre 0,83 y 1,0. En la fase 2, el acuerdo entre las herramientas nuevas y las existentes fue regular a bueno para la detección de síntomas de incontinencia anal abordados como una pregunta compuesta (kappa entre 0,41 y 0,71). Se detectó incontinencia anal en 191 (53%) de las mujeres que utilizaban la nueva herramienta, y se notificó una prevalencia más baja utilizando la puntuación de Vaizey (n = 118) y la puntuación de Wexner (n = 129). Las tasas de finalización de la nueva herramienta fueron del 99%, más altas que la puntuación de Vaizey (33%) y las puntuaciones de Wexner (36%).LIMITACIONES:El tamaño de la muestra limita la generalización de los hallazgos.CONCLUSIONES:El cuestionario es confiable y válido reportando una alta incidencia de incontinencia intestinal, con síntomas predominantes de urgencia rectal y flatos como precursores del empeoramiento de la función. (Traducción-Dr. Ingrid Melo ).

Are we using best practice to guide laxative use in post-partum patients with obstetric anal sphincter injuries - A retrospective review.

OBJECTIVE: Obstetric anal sphincter injury (OASI) is the leading cause of anal incontinence (AI) in young women. Laxatives are recommended to enhance recovery, however there are no consistent guidelines to guide best practice on the type, frequency, and dose of laxative should be used. This study aimed to evaluate the current use of laxatives following repair of OASIs, and to determine any association with AI. Study design A retrospective cohort study of 356 women who sustained OASIs between January 2016 and June 2020, at a single tertiary centre in Adelaide. Data regarding the type, dose and frequency of laxatives prescribed was extracted from each patient. The degree of OASIs was determined by clinical examination and endoanal ultrasound, and AI was measured by the St Marks incontinence score. RESULTS: Multiple combinations and classes of laxatives were prescribed including bulking agent (Metamucil and Fybogel), emollients (Coloxyl), and osmotic laxatives (lactulose and Movicol). Bulking agents were prescribed for 245 women (68.8%), which is contrary to the current recommendations based on two previous randomised controlled trials. AI reported by 51 (14.3%) women. There were no statistical differences between AI and laxative type, dose, or frequency. CONCLUSION: Considerable variation existed in laxatives prescription. Bulking agents was not associated with higher rates of AI. Further research is required to improve post-partum care in women following repair of OASIs.

Creating change with families: Reflections and recommendations for the care of gender diverse and LGBTQIA+ individuals and their families throughout pregnancy and birth.

OBJECTIVE: To derive a deeper understanding of transgender and non-binary people's experience of pregnancy and birth, and ways to modify practice to provide inclusive care. DESIGN: Case study reports describe the experiences of two transgender and non-binary people who received pregnancy and birth care through a Midwifery Group Practice program. SETTING: A tertiary hospital in metropolitan South Australia with approximately 3800 births per annum. METHODS: Qualitative methodology, utilising open-ended, semi-structured, face-to-face interviews were undertaken postnatally. Interviews were audio recorded and transcribed verbatim to analyse and identify themes. FINDINGS: Both clients feared being misgendered within pregnancy care services. They appreciated the constancy of the Midwifery Group Practice midwife, which meant they did not have to repeat their history to multiple health care providers. They appreciated their pronouns being documented on case notes and welcomed staff attempts to use their preferred terms. Both felt the pregnancy care environment was focussed on cisgender females and found this alienating. They appreciated the midwife's suggestion that the cot card for their baby did not have to be pink or blue. They both suggested staff use more gender-neutral language, and resources, when providing pregnancy care. KEY CONCLUSION: Staff attempted to support these parents, and this was appreciated by them, but the continuity provided by the Midwifery Group Practice model was highly valued by both, regardless of risk status. It was identified that further education for staff was required to facilitate provision of more inclusive care. IMPLICATIONS FOR PRACTICE: The case studies identified a need for greater awareness and education for staff regarding care provision for transgender and non-binary people. Simple adjustments had a big impact. Further research is needed to identify how best to meet the needs of gender-diverse people and address the educational needs of staff.

Post-repair laxative management in obstetric anal sphincter injury guidelines: A narrative review.

BACKGROUND: Childbirth is a common factor which increases the risk of obstetric anal sphincter injuries (OASIS). Damage to the anal sphincters increases the risk of anal incontinence, which has a debilitating impact on the quality of life. Post-repair laxatives are prescribed in this group of women. However, there is no consensus regarding the type or frequency with which they are used, and available guidelines lack consistency and evidence to support the recommendations. AIM: The aim was to review and compare the international, national and local Australian management guidelines for recommendations regarding laxative use in women after OASIS. METHOD: An online literature search of medical and nursing databases such as PubMed, Embase, MEDLINE, CINAHL, Web of Science, Scopus and Cochrane was performed between January 2000 and October 2020. Full-text articles with MeSH headings and Text Words [TW] identified guidelines in the prevention, management and care of OASIS. The search terms included 'obstetric anal sphincter injury', 'OASIS', 'perineal tear', 'postpartum continence', 'bowel injury', 'aperient', 'laxative use' and 'bulking agents'. RESULTS: Thirteen guidelines were included. Laxatives were recommended in most guidelines; however, there was a lack of consistency regarding the type of laxative used, frequency, dose and duration of use. Guidelines were based on historical evidence, with paucity of recently acquired data identified. CONCLUSIONS: There is no consensus regarding an optimal laxative regime for women who sustain an anal sphincter injury after childbirth. Further research is required to develop evidence-based robust clinical guidelines regarding laxative use in women who sustain OASIS.

Parallel Optimization of Potency and Pharmacokinetics Leading to the Discovery of a Pyrrole Carboxamide ERK5 Kinase Domain Inhibitor.

The nonclassical extracellular signal-related kinase 5 (ERK5) mitogen-activated protein kinase pathway has been implicated in increased cellular proliferation, migration, survival, and angiogenesis; hence, ERK5 inhibition may be an attractive approach for cancer treatment. However, the development of selective ERK5 inhibitors has been challenging. Previously, we described the development of a pyrrole carboxamide high-throughput screening hit into a selective, submicromolar inhibitor of ERK5 kinase activity. Improvement in the ERK5 potency was necessary for the identification of a tool ERK5 inhibitor for target validation studies. Herein, we describe the optimization of this series to identify nanomolar pyrrole carboxamide inhibitors of ERK5 incorporating a basic center, which suffered from poor oral bioavailability. Parallel optimization of potency and in vitro pharmacokinetic parameters led to the identification of a nonbasic pyrazole analogue with an optimal balance of ERK5 inhibition and oral exposure.

Recent Advances in Kinase Drug Discovery Part I: The Editors' Take.

This special issue on Advances in Kinase Drug Discovery provides a selection of research articles and topical reviews covering all aspects of drug discovery targeting the phosphotransferase enzyme family [...].

An Alkynylpyrimidine-Based Covalent Inhibitor That Targets a Unique Cysteine in NF-κB-Inducing Kinase.

NF-κB-inducing kinase (NIK) is a key enzyme in the noncanonical NF-κB pathway, of interest in the treatment of a variety of diseases including cancer. Validation of NIK as a drug target requires potent and selective inhibitors. The protein contains a cysteine residue at position 444 in the back pocket of the active site, unique within the kinome. Analysis of existing inhibitor scaffolds and early structure-activity relationships (SARs) led to the design of C444-targeting covalent inhibitors based on alkynyl heterocycle warheads. Mass spectrometry provided proof of the covalent mechanism, and the SAR was rationalized by computational modeling. Profiling of more potent analogues in tumor cell lines with constitutively activated NIK signaling induced a weak antiproliferative effect, suggesting that kinase inhibition may have limited impact on cancer cell growth. This study shows that alkynyl heterocycles are potential cysteine traps, which may be employed where common Michael acceptors, such as acrylamides, are not tolerated.

The thrombopoietin receptor: revisiting the master regulator of platelet production.

Thrombopoietin (TPO) and its receptor, MPL, are the primary regulators of platelet production and critical for hematopoietic stem cell (HSC) maintenance. Since TPO was first cloned in 1994, the physiological and pathological roles of TPO and MPL have been well characterized, culminating in the first MPL agonists being approved for the treatment of chronic immune thrombocytopenia in 2008. Dysregulation of the TPO-MPL signaling axis contributes to the pathogenesis of hematological disorders: decreased expression or function results in severe thrombocytopenia progressing to bone marrow failure, while hyperactivation of MPL signaling, either by mutations in the receptor or associated Janus kinase 2 (JAK2), results in pathological myeloproliferation. Despite its importance, it was only recently that the long-running debate over the mechanism by which TPO binding activates MPL has been resolved. This review will cover key aspects of TPO and MPL structure and function and their importance in receptor activation, discuss how these are altered in hematological disorders and consider how a greater understanding could lead to the development of better-targeted and more efficacious therapies.

Validation of ion mobility spectrometry - mass spectrometry as a screening tool to identify type II kinase inhibitors of FGFR1 kinase.

RATIONALE: The protein kinase FGFR1 regulates cellular processes in human development. As over-activity of FGFR1 is implicated with cancer, effective inhibitors are in demand. Type I inhibitors, which bind to the active form of FGFR1, are less effective than type II inhibitors, which bind to the inactive form. Screening to distinguish between type I and type II inhibitors is required. METHODS: X-ray crystallography was used to indicate whether a range of potential inhibitors bind to the active or inactive FGFR1 kinase conformation. The binding affinity of each ligand to FGFR1 was measured using biochemical methods. Electrospray ionisation - ion mobility spectrometry - mass spectrometry (ESI-IMS-MS) in conjunction with collision-induced protein unfolding generated a conformational profile of each FGFR1-ligand complex. The results indicate that the protein's conformational profile depends on whether the inhibitor is type I or type II. RESULTS: X-ray crystallography confirmed which of the kinase inhibitors bind to the active or inactive form of FGFR1 kinase. Collision-induced unfolding combined with ESI-IMS-MS showed distinct differences in the FGFR1 folding landscape for type I and type II inhibitors. Biochemical studies indicated a similar range of FGFR1 affinities for both types of inhibitors, thus providing confidence that the conformational variations detected using ESI-IMS-MS can be interpretated unequivocally and that this is an effective screening method. CONCLUSIONS: A robust ESI-IMS-MS method has been implemented to distinguish between the binding mode of type I and type II inhibitors by monitoring the conformational unfolding profile of FGFR1. This rapid method requires low sample concentrations and could be used as a high-throughput screening technique for the characterisation of novel kinase inhibitors.

Failure of rapid diagnostic tests in Plasmodium falciparum malaria cases among travelers to the UK and Ireland: Identification and characterisation of the parasites.

OBJECTIVES: Our objective was to systematically investigate false-negative histidine-rich protein 2 rapid diagnostic tests (HRP2-RDT) in imported Plasmodium falciparum malaria cases from travelers to the UK and the Republic of Ireland (RoI). METHODS: Five imported malaria cases in travellers returning to the UK and RoI from East Africa were reported to the PHE Malaria Reference Laboratory as negative according to histidine-rich protein (HRP2)-RDT. The cases were systematically investigated using microscopic, RDT, molecular, genomic, and in in vitro approaches. RESULTS: In each case, HRP2-RDT was negative, whereas microscopy confirmed the presence of P. falciparum. Further analysis revealed that the genes encoding HRP2 and HRP3 were deleted in three of the five cases. Whole-genome sequencing in one of these isolates confirmed deletions in P. falciparum chromosomes 8 and 13. Our study produced evidence that the fourth case, which had high parasitemia at clinical presentation, was a rare example of antigen saturation ('prozone-like effect'), leading to a false negative in the HRP2-RDT, while the fifth case was due to low parasitemia. CONCLUSIONS: False-negative HRP2-RDT results with P. falciparum are concerning. Our findings emphasise the necessity of supporting the interpretation of RDT results with microscopy, in conjunction with clinical observations, and sets out a systematic approach to identifying parasites carrying pfhrp2 and pfhrp3 deletions.

Enhancing the identification of anal incontinence in women of reproductive age.

Anal incontinence is an unpredictable and debilitating condition that can significantly reduce quality of life. Symptoms include the involuntary loss of solid and/or liquid stool, flatus incontinence and rectal urgency. Pregnancy and childbirth are two major factors that increase the risk of anal incontinence in women of reproductive age. Women at high risk of anal incontinence include those with a known history of the condition and those who have experienced severe perineal trauma, particularly after injury to the anal sphincters (third-degree and fourth-degree tears). Routine screening for anal incontinence of women in high-risk groups during pregnancy and after childbirth appears to be limited in clinical practice. This article discusses the potential benefits of screening for anal incontinence, outlines the factors that inhibit and enable screening, describes current bowel screening tools and their limitations, and explores how the identification of anal incontinence in women of reproductive age could be improved.

Small molecule ERK5 kinase inhibitors paradoxically activate ERK5 signalling: be careful what you wish for .

ERK5 is a protein kinase that also contains a nuclear localisation signal and a transcriptional transactivation domain. Inhibition of ERK5 has therapeutic potential in cancer and inflammation and this has prompted the development of ERK5 kinase inhibitors (ERK5i). However, few ERK5i programmes have taken account of the ERK5 transactivation domain. We have recently shown that the binding of small molecule ERK5i to the ERK5 kinase domain stimulates nuclear localisation and paradoxical activation of its transactivation domain. Other kinase inhibitors paradoxically activate their intended kinase target, in some cases leading to severe physiological consequences highlighting the importance of mitigating these effects. Here, we review the assays used to monitor ERK5 activities (kinase and transcriptional) in cells, the challenges faced in development of small molecule inhibitors to the ERK5 pathway, and classify the molecular mechanisms of paradoxical activation of protein kinases by kinase inhibitors.

Paradoxical activation of the protein kinase-transcription factor ERK5 by ERK5 kinase inhibitors.

The dual protein kinase-transcription factor, ERK5, is an emerging drug target in cancer and inflammation, and small-molecule ERK5 kinase inhibitors have been developed. However, selective ERK5 kinase inhibitors fail to recapitulate ERK5 genetic ablation phenotypes, suggesting kinase-independent functions for ERK5. Here we show that ERK5 kinase inhibitors cause paradoxical activation of ERK5 transcriptional activity mediated through its unique C-terminal transcriptional activation domain (TAD). Using the ERK5 kinase inhibitor, Compound 26 (ERK5-IN-1), as a paradigm, we have developed kinase-active, drug-resistant mutants of ERK5. With these mutants, we show that induction of ERK5 transcriptional activity requires direct binding of the inhibitor to the kinase domain. This in turn promotes conformational changes in the kinase domain that result in nuclear translocation of ERK5 and stimulation of gene transcription. This shows that both the ERK5 kinase and TAD must be considered when assessing the role of ERK5 and the effectiveness of anti-ERK5 therapeutics.

Mechanism of homodimeric cytokine receptor activation and dysregulation by oncogenic mutations.

Homodimeric class I cytokine receptors are assumed to exist as preformed dimers that are activated by ligand-induced conformational changes. We quantified the dimerization of three prototypic class I cytokine receptors in the plasma membrane of living cells by single-molecule fluorescence microscopy. Spatial and spatiotemporal correlation of individual receptor subunits showed ligand-induced dimerization and revealed that the associated Janus kinase 2 (JAK2) dimerizes through its pseudokinase domain. Oncogenic receptor and hyperactive JAK2 mutants promoted ligand-independent dimerization, highlighting the formation of receptor dimers as the switch responsible for signal activation. Atomistic modeling and molecular dynamics simulations based on a detailed energetic analysis of the interactions involved in dimerization yielded a mechanistic blueprint for homodimeric class I cytokine receptor activation and its dysregulation by individual mutations.

Hot deformation data for Haynes 214, Haynes 230 and Inconel 740H.

This article presents the datasets gathered for the hot processing of three Ni-based superalloys intended for A-USC application, Haynes 214, Haynes 230 and Inconel 740H. Isothermal compression tests were conducted with a Gleeble 3500 at temperatures between 1000 °C and 1200 °C and strain rates between 0.01/s and 1/s to a full true strain of 0.7. The obtained true stress-true strain curves were used as basis for hot processing maps, linking temperature, stress and strain rate. Subsequently, all samples were sectioned through the geometric centre to provide microstructural information, captured using EBSD, as well as EDX for the evolution of secondary phases. Thermodynamic modelling was performed to validate compositions and mass fraction data from EDX measurements. These combined datasets help in understanding the deformation behaviour of a selected range of superalloys, under commercial processing conditions, aiding in process design optimizations and improvements. For complete interpretation of the data the reader should refer to the related publication "Comparative study of the hot processing behavior in advanced Ni-based superalloys for use in A-USC applications".

Understanding what impacts on disclosing anal incontinence for women when comparing bowel-screening tools: a phenomenological study.

BACKGROUND: There is limited research defining the true prevalence of anal incontinence (AI) in women of childbearing age. Understanding the limitations of the current assessment tools in the identification of AI is paramount for identifying the prevalence of AI and improving the care and management for women of childbearing age. The aim of this research was to explore and develop an understanding of women's experiences in disclosing AI when completing a new bowel-screening questionnaire when compared to two established AI tools. METHODS: A phenomenological qualitative research study was undertaken in a maternity setting in a large tertiary hospital. Parous women in the first trimester of a subsequent pregnancy were recruited to complete a specifically designed screening tool (BSQ), St Marks Faecal incontinence score (Vaizey) and Cleveland (Wexner) score. Qualitative semi-structured interviews were utilised to identify experiences in disclosing AI. RESULTS: Women (n = 16, 22-42 years) with a history of anal incontinence either following the first birth (n = 12) or the second (n = 4) provided differing responses between the three assessment tools. All women answered the BSQ while the Vaizey and Wexner scores were more difficult to complete due to clinical language and participants level of comprehension. Women identified three major themes that were barriers for disclosing incontinence, which included social expectations, trusted space and confusion. CONCLUSION: There are barriers for disclosing AI in the pregnant and post-natal population, which can be improved with the use of an easy assessment tool. The BSQ may facilitate discussion on AI between the patient and health professional leading to earlier identification and improvement in short and long-term health outcomes.

Implementing reasonable adjustments for -disabled people in healthcare services.

Disabled people are one of the groups in society with the greatest health needs, yet they experience some of the most significant barriers to accessing healthcare services. This article describes examples of how three healthcare services have met the Equality Act 2010 duty to make reasonable adjustments for disabled people, so that they are not disadvantaged in accessing these services. Each of these services identified disabled patients, and considered and recorded the specific reasonable adjustments that were required. In doing so, they took time to fully understand the needs of the individual from their perspective. The services collaborated and coordinated the provision of reasonably adjusted care by communicating effectively with other health and social care providers, working together as a team, and treating disabled people as individuals.