Publications by authors named "Martin Howard"

120 Publications

Cell size controlled in plants using DNA content as an internal scale.

Science 2021 06;372(6547):1176-1181

Cell and Developmental Biology, John Innes Centre, Norwich NR4 7UH, UK.

How eukaryotic cells assess and maintain sizes specific for their species and cell type remains unclear. We show that in the shoot stem cell niche, cell size variability caused by asymmetric divisions is corrected by adjusting the growth period before DNA synthesis. KIP-related protein 4 (KRP4) inhibits progression to DNA synthesis and associates with mitotic chromosomes. The F BOX-LIKE 17 (FBL17) protein removes excess KRP4. Consequently, daughter cells are born with comparable amounts of KRP4. Inhibitor dilution models predicted that KRP4 inherited through chromatin would robustly regulate size, whereas inheritance of excess free KRP4 would disrupt size homeostasis, as confirmed by mutant analyses. We propose that a cell cycle regulator, stabilized by association with mitotic chromosomes, reads DNA content as a cell size-independent scale.
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http://dx.doi.org/10.1126/science.abb4348DOI Listing
June 2021

Stigma as a Barrier to Participant Recruitment of Minority Populations in Diabetes Research: Development of a Community-Centered Recruitment Approach.

JMIR Diabetes 2021 May 3;6(2):e26965. Epub 2021 May 3.

Department of Family Medicine and Community Health, University of Massachusetts Medical School, Worcester, MA, United States.

Background: The development of evidence-based care geared towards Black and Latina women living with uncontrolled type 2 diabetes is contingent upon their active recruitment into clinical interventions. Well-documented impediments to recruitment include a historical mistrust of the research community and socioeconomic factors that limit awareness and access to research studies. Although sociocultural and socioeconomic factors deter minorities from participating in clinical research, it is equally important to consider the role of stigma in chronic disease intervention studies.

Objective: We aim to share our discovery of diabetes-related stigma as an underrecognized impediment to recruitment for the Women in Control 2.0 virtual diabetes self-management education study.

Methods: Our initial recruitment plan used traditional strategies to recruit minority women with uncontrolled type 2 diabetes, which included letters and phone calls to targeted patients, referrals from clinicians, and posted flyers. After engaging a patient advisory group and consulting with experts in community advocacy, diabetes-related stigma emerged as a prominent barrier to recruitment. The study team reviewed and revised recruitment scripts and outreach material in order to better align with the lived experience and needs of potential enrollees.

Results: Using a more nuanced, community-centered recruitment approach, we achieved our target recruitment goal, enrolling 309 participants into the study, exceeding our target of 212.

Conclusions: There is a need for updated recruitment methods that can increase research participation of patients who experience internalized diabetes stigma. To address disparities in minority health, further research is needed to better understand diabetes-related stigma and devise strategies to avert or address it.
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http://dx.doi.org/10.2196/26965DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8129881PMC
May 2021

Digital paradigm for Polycomb epigenetic switching and memory.

Curr Opin Plant Biol 2021 06 1;61:102012. Epub 2021 Mar 1.

Department of Computational and Systems Biology, John Innes Centre, Norwich NR4 7UH, UK. Electronic address:

How epigenetic memory states are established and maintained is a central question in gene regulation. A major epigenetic process important for developmental biology involves Polycomb-mediated chromatin silencing. Significant progress has recently been made on elucidating Polycomb silencing in plant systems through analysis of Arabidopsis FLOWERING LOCUS C (FLC). Quantitative silencing of FLC by prolonged cold exposure was shown to represent an ON to OFF switch in an increasing proportion of cells. Here, we review the underlying all-or-nothing, digital paradigm for Polycomb epigenetic silencing. We then examine other Arabidopsis Polycomb-regulated targets where digital regulation may also be relevant.
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http://dx.doi.org/10.1016/j.pbi.2021.102012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8250048PMC
June 2021

Rehabilitation to enable recovery from COVID-19: a rapid systematic review.

Physiotherapy 2021 06 24;111:4-22. Epub 2021 Feb 24.

University of Exeter, UK.

Objectives: To establish the evidence for rehabilitation interventions tested in populations of patients admitted to ICU and critical care with severe respiratory illness, and consider whether the evidence is generalizable to patients with COVID-19.

Methods: The authors undertook a rapid systematic review. Medline (via OvidSP), CINAHL Complete (via EBSCOhost), Cochrane Library, Cochrane Database of Systematic Reviews and CENTRAL (via Wiley), Epistemonikos (via Epistemonikos.org), PEDro (via pedro.org.au) and OTseeker (via otseeker.com) searched to 7 May 2020. The authors included systematic reviews, RCTs and qualitative studies involving adults with respiratory illness requiring intensive care who received rehabilitation to enhance or restore resulting physical impairments or function. Data were extracted by one author and checked by a second. TIDier was used to guide intervention descriptions. Study quality was assessed using Critical Skills Appraisal Programme (CASP) tools.

Results: Six thousand nine hundred and three titles and abstracts were screened; 24 systematic reviews, 11 RCTs and eight qualitative studies were included. Progressive exercise programmes, early mobilisation and multicomponent interventions delivered in ICU can improve functional independence. Nutritional supplementation in addition to rehabilitation in post-ICU hospital settings may improve performance of activities of daily living. The evidence for rehabilitation after discharge from hospital following an ICU admission is inconclusive. Those receiving rehabilitation valued it, engendering hope and confidence.

Conclusions: Exercise, early mobilisation and multicomponent programmes may improve recovery following ICU admission for severe respiratory illness that could be generalizable to those with COVID-19. Rehabilitation interventions can bring hope and confidence to individuals but there is a need for an individualised approach and the use of behaviour change strategies. Further research is needed in post-ICU settings and with those who have COVID-19. Registration: Open Science Framework https://osf.io/prc2y.
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http://dx.doi.org/10.1016/j.physio.2021.01.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7902208PMC
June 2021

Using computational modelling to reveal mechanisms of epigenetic Polycomb control.

Biochem Soc Trans 2021 Feb;49(1):71-77

Computational and Systems Biology, John Innes Centre, Norwich Research Park NR4 7UH, U.K.

The Polycomb system is essential for stable gene silencing in many organisms. This regulation is achieved in part through addition of the histone modifications H3K27me2/me3 by Polycomb Repressive Complex 2 (PRC2). These modifications are believed to be the causative epigenetic memory elements of PRC2-mediated silencing. As these marks are stored locally in the chromatin, PRC2-based memory is a cis-acting system. A key feature of stable epigenetic memory in cis is PRC2-mediated, self-reinforcing feedback from K27-methylated histones onto nearby histones in a read-write paradigm. However, it was not clear under what conditions such feedback can lead to stable memory, able, for example, to survive the perturbation of histone dilution at DNA replication. In this context, computational modelling has allowed a rigorous exploration of possible underlying memory mechanisms and has also greatly accelerated our understanding of switching between active and silenced states. Specifically, modelling has predicted that switching and memory at Polycomb loci is digital, with a locus being either active or inactive, rather than possessing intermediate, smoothly varying levels of activation. Here, we review recent advances in models of Polycomb control, focusing on models of epigenetic switching through nucleation and spreading of H3K27me2/me3. We also examine models that incorporate transcriptional feedback antagonism and those including bivalent chromatin states. With more quantitative experimental data on histone modification kinetics, as well as single-cell resolution data on transcription and protein levels for PRC2 targets, we anticipate an expanded need for modelling to help dissect increasingly interconnected and complex memory mechanisms.
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http://dx.doi.org/10.1042/BST20190955DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7925002PMC
February 2021

Feeling Every Bit of Winter - Distributed Temperature Sensitivity in Vernalization.

Front Plant Sci 2021 27;12:628726. Epub 2021 Jan 27.

Computational and Systems Biology, John Innes Centre, Norwich Research Park, Norwich, United Kingdom.

Temperature intrinsically influences all aspects of biochemical and biophysical processes. Organisms have therefore evolved strategies to buffer themselves against thermal perturbations. Many organisms also use temperature signals as cues to align behavior and development with certain seasons. These developmentally important thermosensory mechanisms have generally been studied in constant temperature conditions. However, environmental temperature is an inherently noisy signal, and it has been unclear how organisms reliably extract specific temperature cues from fluctuating temperature profiles. In this context, we discuss plant thermosensory responses, focusing on temperature sensing throughout vernalization in Arabidopsis. We highlight many different timescales of sensing, which has led to the proposal of a distributed thermosensing paradigm. Within this paradigm, we suggest a classification system for thermosensors. Finally, we focus on the longest timescale, which is most important for sensing winter, and examine the different mechanisms in which memory of cold exposure can be achieved.
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http://dx.doi.org/10.3389/fpls.2021.628726DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7873433PMC
January 2021

Using Health Information Technology to Engage African American Women on Nutrition and Supplement Use During the Preconception Period.

Front Endocrinol (Lausanne) 2020 19;11:571705. Epub 2021 Jan 19.

Department of Family Medicine, Boston University School of Medicine and Boston Medical Center, Boston, MA, United States.

Importance: Healthy nutrition and appropriate supplementation during preconception have important implications for the health of the mother and newborn. The best way to deliver preconception care to address health risks related to nutrition is unknown.

Methods: We conducted a secondary analysis of data from a randomized controlled trial designed to study the impact of conversational agent technology in 13 domains of preconception care among 528 non-pregnant African American and Black women. This analysis is restricted to those 480 women who reported at least one of the ten risks related to nutrition and dietary supplement use.

Interventions: An online conversational agent, called "Gabby", assesses health risks and delivers 12 months of tailored dialogue for over 100 preconception health risks, including ten nutrition and supplement risks, using behavioral change techniques like shared decision making and motivational interviewing. The control group received a letter listing their preconception risks and encouraging them to talk to a health care provider.

Results: After 6 months, women using Gabby (a) reported progressing forward on the stage of change scale for, on average, 52.9% (SD, 35.1%) of nutrition and supplement risks compared to 42.9% (SD, 35.4) in the control group (IRR 1.22, 95% CI 1.03-1.45, P = 0.019); and (b) reported achieving the action and maintenance stage of change for, on average, 52.8% (SD 37.1) of the nutrition and supplement risks compared to 42.8% (SD, 37.9) in the control group (IRR 1.26, 96% CI 1.08-1.48, P = 0.004). For subjects beginning the study at the contemplation stage of change, intervention subjects reported progressing forward on the stage of change scale for 75.0% (SD, 36.3%) of their health risks compared to 52.1% (SD, 47.1%) in the control group (P = 0.006).

Conclusion: The scalability of Gabby has the potential to improve women's nutritional health as an adjunct to clinical care or at the population health level. Further studies are needed to determine if improving nutrition and supplement risks can impact clinical outcomes including optimization of weight.

Clinical Trial Registration: ClinicalTrials.gov, identifier NCT01827215.
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http://dx.doi.org/10.3389/fendo.2020.571705DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7874041PMC
May 2021

Improving the health of young African American women in the preconception period using health information technology: a randomised controlled trial.

Lancet Digit Health 2020 09;2(9):e475-e485

Department of Biostatistics, School of Public Health, Boston University, Boston, MA, USA.

Background: Preconception care focuses on improving women's health before pregnancy as a means to improve their health and future pregnancy outcomes. How to effectively deliver such care is unknown. The aim of this research was to assess the impact of an embodied conversational agent system on preconception risks among African American and Black women.

Methods: We did an open-label, randomised controlled trial of women aged 18-34 years, self-identified as African American or Black, or both, and not pregnant, recruited from 35 states in the USA. Sealed allocation envelopes (in permuted blocks of six and eight, prepared using a random number generator) were opened after enrolment. Intervention participants received an online conversational agent called Gabby that assessed 102 preconception risks and delivered 12 months of tailored dialogue using synthesised speech, non-verbal behaviour, visual aids, and health behaviour change techniques such as motivational interviewing. The control group received a letter listing their preconception risks and encouraging them to talk with a clinician. The primary outcome was the proportion of identified risks at the action or maintenance stage of change at months 6 and 12. The study is registered with ClinicalTrials.gov, NCT01827215.

Findings: From March 11, 2014, through July 8, 2018, 528 women recruited from 35 states and 242 cities across the USA received the Gabby intervention (n=262) or were assigned to the control group (n=266). Participants identified a mean of 21 preconception risks per woman (SD 9·9). In the intention-to-treat analysis, at 6 months, intervention women reported reaching the action or maintenance stage of change for 50·0% (SD 28·9) of those preconception risks identified compared with 42·7% (28·3) in the control group (incidence rate ratio 1·16, 95% CI 1·07-1·26; p=0·0004). This result persisted at 12 months.

Interpretation: The Gabby system has the potential to improve women's preconception health. Further research is needed to determine if improving preconception risks impacts outcomes such as preterm delivery.

Funding: National Institute for Minority Health and Health Disparities.
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http://dx.doi.org/10.1016/S2589-7500(20)30189-8DOI Listing
September 2020

Histiocyte-rich rhabdomyoblastic tumor of the trunk.

JAAD Case Rep 2020 Oct 8;6(10):1001-1002. Epub 2020 Jul 8.

Houston Methodist, DermSurgery Associates, Houston, Texas.

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http://dx.doi.org/10.1016/j.jdcr.2020.06.038DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7508715PMC
October 2020

A theoretical model of Polycomb/Trithorax action unites stable epigenetic memory and dynamic regulation.

Nat Commun 2020 09 22;11(1):4782. Epub 2020 Sep 22.

Humboldt Universität zu Berlin, IRI- Lifesciences, Philippstr. 13, 10115, Berlin, Germany.

Polycomb and Trithorax group proteins maintain stable epigenetic memory of gene expression states for some genes, but many targets show highly dynamic regulation. Here we combine experiment and theory to examine the mechanistic basis of these different modes of regulation. We present a mathematical model comprising a Polycomb/Trithorax response element (PRE/TRE) coupled to a promoter and including Drosophila developmental timing. The model accurately recapitulates published studies of PRE/TRE mediated epigenetic memory of both silencing and activation. With minimal parameter changes, the same model can also recapitulate experimental data for a different PRE/TRE that allows dynamic regulation of its target gene. The model predicts that both cell cycle length and PRE/TRE identity are critical for determining whether the system gives stable memory or dynamic regulation. Our work provides a simple unifying framework for a rich repertoire of PRE/TRE functions, and thus provides insights into  genome-wide Polycomb/Trithorax regulation.
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http://dx.doi.org/10.1038/s41467-020-18507-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7508846PMC
September 2020

Natural variation in autumn expression is the major adaptive determinant distinguishing Arabidopsis FLC haplotypes.

Elife 2020 09 9;9. Epub 2020 Sep 9.

Cell and Developmental Biology, John Innes Centre, Norwich, United Kingdom.

In winter is registered during vernalization through the temperature-dependent repression and epigenetic silencing of floral repressor . Natural Arabidopsis accessions show considerable variation in vernalization. However, which aspect of the repression mechanism is most important for adaptation to different environments is unclear. By analysing dynamics in natural variants and mutants throughout winter in three field sites, we find that autumnal expression, rather than epigenetic silencing, is the major variable conferred by the distinct Arabidopsis haplotypes. This variation influences flowering responses of Arabidopsis accessions resulting in an interplay between promotion and delay of flowering in different climates to balance survival and, through a post-vernalization effect, reproductive output. These data reveal how expression variation through non-coding cis variation at has enabled Arabidopsis accessions to adapt to different climatic conditions and year-on-year fluctuations.
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http://dx.doi.org/10.7554/eLife.57671DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7518893PMC
September 2020

Publisher Correction: Temperature-dependent growth contributes to long-term cold sensing.

Nature 2020 Sep;585(7824):E8

John Innes Centre, Norwich Research Park, Norwich, UK.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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http://dx.doi.org/10.1038/s41586-020-2694-xDOI Listing
September 2020

Framing a Needed Discourse on Health Disparities and Social Inequities: Drawing Lessons from a Pandemic.

Public Adm Rev 2020 Jun 22. Epub 2020 Jun 22.

School of Public Affairs and Administration Rutgers University-Newark 111 Washington Street Newark New Jersey 07102.

COVID-19 provides numerous opportunities for policymakers to consider matters of social equity in relation to the field of public health. Specifically, by reflecting on health disparities in relation to the disproportionate impact of COVID-19 on minority and historically underserved populations, we can leverage a needed discourse on health outcomes for many communities. Grounded in the social determinants of health conceptual framework, this article explores the application of the disproportionate impact of COVID-19 on vulnerable populations and communities of color for a discussion on strategies for minimizing health disparities. This article is protected by copyright. All rights reserved.
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http://dx.doi.org/10.1111/puar.13265DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7361609PMC
June 2020

Temperature-dependent growth contributes to long-term cold sensing.

Nature 2020 07 15;583(7818):825-829. Epub 2020 Jul 15.

John Innes Centre, Norwich Research Park, Norwich, UK.

Temperature is a key factor in the growth and development of all organisms. Plants have to interpret temperature fluctuations, over hourly to monthly timescales, to align their growth and development with the seasons. Much is known about how plants respond to acute thermal stresses, but the mechanisms that integrate long-term temperature exposure remain unknown. The slow, winter-long upregulation of VERNALIZATION INSENSITIVE 3 (VIN3), a PHD protein that functions with Polycomb repressive complex 2 to epigenetically silence FLOWERING LOCUS C (FLC) during vernalization, is central to plants interpreting winter progression. Here, by a forward genetic screen, we identify two dominant mutations of the transcription factor NTL8 that constitutively activate VIN3 expression and alter the slow VIN3 cold induction profile. In the wild type, the NTL8 protein accumulates slowly in the cold, and directly upregulates VIN3 transcription. Through combining computational simulation and experimental validation, we show that a major contributor to this slow accumulation is reduced NTL8 dilution due to slow growth at low temperatures. Temperature-dependent growth is thus exploited through protein dilution to provide the long-term thermosensory information for VIN3 upregulation. Indirect mechanisms involving temperature-dependent growth, in addition to direct thermosensing, may be widely relevant in long-term biological sensing of naturally fluctuating temperatures.
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http://dx.doi.org/10.1038/s41586-020-2485-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7116785PMC
July 2020

The 3' processing of antisense RNAs physically links to chromatin-based transcriptional control.

Proc Natl Acad Sci U S A 2020 06 15;117(26):15316-15321. Epub 2020 Jun 15.

Department of Cell and Developmental Biology, John Innes Centre, NR4 7UH Norwich, United Kingdom;

Noncoding RNA plays essential roles in transcriptional control and chromatin silencing. At antisense transcription quantitatively influences transcriptional output, but the mechanism by which this occurs is still unclear. Proximal polyadenylation of the antisense transcripts by FCA, an RNA-binding protein that physically interacts with RNA 3' processing factors, reduces transcription. This process genetically requires FLD, a homolog of the H3K4 demethylase LSD1. However, the mechanism linking RNA processing to FLD function had not been established. Here, we show that FLD tightly associates with LUMINIDEPENDENS (LD) and SET DOMAIN GROUP 26 (SDG26) in vivo, and, together, they prevent accumulation of monomethylated H3K4 (H3K4me1) over the gene body. SDG26 interacts with the RNA 3' processing factor FY (WDR33), thus linking activities for proximal polyadenylation of the antisense transcripts to FLD/LD/SDG26-associated H3K4 demethylation. We propose this demethylation antagonizes an active transcription module, thus reducing H3K36me3 accumulation and increasing H3K27me3. Consistent with this view, we show that Polycomb Repressive Complex 2 (PRC2) silencing is genetically required by FCA to repress Overall, our work provides insights into RNA-mediated chromatin silencing.
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http://dx.doi.org/10.1073/pnas.2007268117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7334503PMC
June 2020

Preferred and actual place of death in haematological malignancies: a report from the UK haematological malignancy research network.

BMJ Support Palliat Care 2021 Mar 11;11(1):7-16. Epub 2020 May 11.

Epidemiology & Cancer Statistics Group, Department of Health Sciences, University of York, York, North Yorkshire, UK.

Objectives: Hospital death is comparatively common in people with haematological cancers, but little is known about patient preferences. This study investigated actual and preferred place of death, concurrence between these and characteristics of preferred place discussions.

Methods: Set within a population-based haematological malignancy patient cohort, adults (≥18 years) diagnosed 2004-2012 who died 2011-2012 were included (n=963). Data were obtained via routine linkages (date, place and cause of death) and abstraction of hospital records (diagnosis, demographics, preferred place discussions). Logistic regression investigated associations between patient and clinical factors and place of death, and factors associated with the likelihood of having a preferred place discussion.

Results: Of 892 patients (92.6%) alive 2 weeks after diagnosis, 58.0% subsequently died in hospital (home, 20.0%; care home, 11.9%; hospice, 10.2%). A preferred place discussion was documented for 453 patients (50.8%). Discussions were more likely in women (p=0.003), those referred to specialist palliative care (p0.001), and where cause of death was haematological cancer (p0.001); and less likely in those living in deprived areas (p=0.005). Patients with a discussion were significantly (p<0.05) less likely to die in hospital. Last recorded preferences were: home (40.6%), hospice (18.1%), hospital (17.7%) and care home (14.1%); two-thirds died in their final preferred place. Multiple discussions occurred for 58.3% of the 453, with preferences varying by proximity to death and participants in the discussion.

Conclusion: Challenges remain in ensuring that patients are supported to have meaningful end-of-life discussions, with healthcare services that are able to respond to changing decisions over time.
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http://dx.doi.org/10.1136/bmjspcare-2019-002097DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7907576PMC
March 2021

Structure-Based Design and Pharmacokinetic Optimization of Covalent Allosteric Inhibitors of the Mutant GTPase KRAS.

J Med Chem 2020 05 13;63(9):4468-4483. Epub 2020 Feb 13.

Discovery Sciences, R&D, AstraZeneca, Cambridge CB4 0WG, U.K.

Attempts to directly drug the important oncogene KRAS have met with limited success despite numerous efforts across industry and academia. The KRAS mutant represents an "Achilles heel" and has recently yielded to covalent targeting with small molecules that bind the mutant cysteine and create an allosteric pocket on GDP-bound RAS, locking it in an inactive state. A weak inhibitor at this site was optimized through conformational locking of a piperazine-quinazoline motif and linker modification. Subsequent introduction of a key methyl group to the piperazine resulted in enhancements in potency, permeability, clearance, and reactivity, leading to identification of a potent KRAS inhibitor with high selectivity and excellent cross-species pharmacokinetic parameters and in vivo efficacy.
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http://dx.doi.org/10.1021/acs.jmedchem.9b01720DOI Listing
May 2020

Noncoding SNPs influence a distinct phase of Polycomb silencing to destabilize long-term epigenetic memory at .

Genes Dev 2020 03 30;34(5-6):446-461. Epub 2020 Jan 30.

John Innes Centre, Norwich Research Park, Norwich NR4 7UH, United Kingdom.

In , the cold-induced epigenetic regulation of () involves distinct phases of Polycomb repressive complex 2 (PRC2) silencing. During cold, a PHD-PRC2 complex metastably and digitally nucleates H3K27me3 within On return to warm, PHD-PRC2 spreads across the locus delivering H3K27me3 to maintain long-term silencing. Here, we studied natural variation in this process in accessions, exploring Lov-1, which shows reactivation on return to warm, a feature characteristic of in perennial This analysis identifies an additional phase in this Polycomb silencing mechanism downstream from H3K27me3 spreading. In this long-term silencing (perpetuated) phase, the PHD proteins are lost from the nucleation region and silencing is likely maintained by the read-write feedbacks associated with H3K27me3. A combination of noncoding SNPs in the nucleation region mediates instability in this long-term silencing phase with the result that Lov-1 frequently digitally reactivates in individual cells, with a probability that diminishes with increasing cold duration. We propose that this decrease in reactivation probability is due to reduced DNA replication after flowering. Overall, this work defines an additional phase in the Polycomb mechanism instrumental in natural variation of silencing, and provides avenues to dissect broader evolutionary changes at .
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http://dx.doi.org/10.1101/gad.333245.119DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7050481PMC
March 2020

The Discovery of 7-Methyl-2-[(7-methyl[1,2,4]triazolo[1,5-]pyridin-6-yl)amino]-9-(tetrahydro-2-pyran-4-yl)-7,9-dihydro-8-purin-8-one (AZD7648), a Potent and Selective DNA-Dependent Protein Kinase (DNA-PK) Inhibitor.

J Med Chem 2020 04 15;63(7):3461-3471. Epub 2020 Jan 15.

Oncology R&D, AstraZeneca, Cambridge CB4 0FZ, U.K.

DNA-PK is a key component within the DNA damage response, as it is responsible for recognizing and repairing double-strand DNA breaks (DSBs) via non-homologous end joining. Historically it has been challenging to identify inhibitors of the DNA-PK catalytic subunit (DNA-PKcs) with good selectivity versus the structurally related PI3 (lipid) and PI3K-related protein kinases. We screened our corporate collection for DNA-PKcs inhibitors with good PI3 kinase selectivity, identifying compound . Optimization focused on further improving selectivity while improving physical and pharmacokinetic properties, notably co-optimization of permeability and metabolic stability, to identify compound (AZD7648). Compound had no significant off-target activity in the protein kinome and only weak activity versus PI3Kα/γ lipid kinases. Monotherapy activity in murine xenograft models was observed, and regressions were observed when combined with inducers of DSBs (doxorubicin or irradiation) or PARP inhibition (olaparib). These data support progression into clinical studies (NCT03907969).
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http://dx.doi.org/10.1021/acs.jmedchem.9b01684DOI Listing
April 2020

Reassessment of the Basis of Cell Size Control Based on Analysis of Cell-to-Cell Variability.

Biophys J 2019 11 1;117(9):1728-1738. Epub 2019 Oct 1.

Department of Computational and Systems Biology, John Innes Centre, Norwich, United Kingdom. Electronic address:

Fundamental mechanisms governing cell size control and homeostasis are still poorly understood. The relationship between sizes at division and birth in single cells is used as a metric to categorize the basis of size homeostasis. Cells dividing at a fixed size regardless of birth size (sizer) are expected to show a division-birth slope of zero, whereas cells dividing after growing for a fixed size increment (adder) have an expected slope of +1. These two theoretical values are, however, rarely experimentally observed. For example, rod-shaped fission yeast Schizosaccharomyces pombe cells, which divide at a fixed surface area, exhibit a division-birth slope for cell lengths of 0.25 ± 0.02, significantly different from the expected sizer value of zero. Here, we investigate possible reasons for this discrepancy by developing a mathematical model of sizer control including the relevant sources of variation. Our results support pure sizer control and show that deviation from zero slope is exaggerated by measurement of an inappropriate geometrical quantity (e.g., length instead of area), combined with cell-to-cell radius variability. The model predicts that mutants with greater errors in size sensing or septum positioning paradoxically appear to behave as better sizers. Furthermore, accounting for cell width variability, we show that pure sizer control can in some circumstances reproduce the apparent adder behavior observed in Escherichia coli. These findings demonstrate that analysis of geometric variation can lead to new insights into cell size control.
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http://dx.doi.org/10.1016/j.bpj.2019.09.031DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6838950PMC
November 2019

Diagnostic high-throughput sequencing of 2396 patients with bleeding, thrombotic, and platelet disorders.

Blood 2019 12;134(23):2082-2091

East Midlands and East of England Genomic Laboratory Hub, Cambridge University Hospitals NHS Foundation Trust, Cambridge Biomedical Campus, Cambridge, United Kingdom.

A targeted high-throughput sequencing (HTS) panel test for clinical diagnostics requires careful consideration of the inclusion of appropriate diagnostic-grade genes, the ability to detect multiple types of genomic variation with high levels of analytic sensitivity and reproducibility, and variant interpretation by a multidisciplinary team (MDT) in the context of the clinical phenotype. We have sequenced 2396 index patients using the ThromboGenomics HTS panel test of diagnostic-grade genes known to harbor variants associated with rare bleeding, thrombotic, or platelet disorders (BTPDs). The molecular diagnostic rate was determined by the clinical phenotype, with an overall rate of 49.2% for all thrombotic, coagulation, platelet count, and function disorder patients and a rate of 3.2% for patients with unexplained bleeding disorders characterized by normal hemostasis test results. The MDT classified 745 unique variants, including copy number variants (CNVs) and intronic variants, as pathogenic, likely pathogenic, or variants of uncertain significance. Half of these variants (50.9%) are novel and 41 unique variants were identified in 7 genes recently found to be implicated in BTPDs. Inspection of canonical hemostasis pathways identified 29 patients with evidence of oligogenic inheritance. A molecular diagnosis has been reported for 894 index patients providing evidence that introducing an HTS genetic test is a valuable addition to laboratory diagnostics in patients with a high likelihood of having an inherited BTPD.
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http://dx.doi.org/10.1182/blood.2018891192DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6993014PMC
December 2019

Haematology nurses' perspectives of their patients' places of care and death: A UK qualitative interview study.

Eur J Oncol Nurs 2019 Apr 7;39:70-80. Epub 2019 Feb 7.

Epidemiology & Cancer Statistics Group, University of York, York, YO10 5DD, UK. Electronic address:

Purpose: Patients with haematological malignancies are more likely to die in hospital, and less likely to access palliative care than people with other cancers, though the reasons for this are not well understood. The purpose of our study was to explore haematology nurses' perspectives of their patients' places of care and death.

Method: Qualitative description, based on thematic content analysis. Eight haematology nurses working in secondary and tertiary hospital settings were purposively selected and interviewed. Transcriptions were coded and analysed for themes using a mainly inductive, cross-comparative approach.

Results: Five inter-related factors were identified as contributing to the likelihood of patients' receiving end of life care/dying in hospital: the complex nature of haematological diseases and their treatment; close clinician-patient bonds; delays to end of life discussions; lack of integration between haematology and palliative care services; and barriers to death at home.

Conclusions: Hospital death is often determined by the characteristics of the cancer and type of treatment. Prognostication is complex across subtypes and hospital death perceived as unavoidable, and sometimes the preferred option. Earlier, frank conversations that focus on realistic outcomes, closer integration of palliative care and haematology services, better communication across the secondary/primary care interface, and an increase in out-of-hours nursing support could improve end of life care and facilitate death at home or in hospice, when preferred.
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http://dx.doi.org/10.1016/j.ejon.2019.02.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6417764PMC
April 2019

Perspectives of bereaved relatives of patients with haematological malignancies concerning preferred place of care and death: A qualitative study.

Palliat Med 2019 05 30;33(5):518-530. Epub 2019 Jan 30.

1 Epidemiology and Cancer Statistics Group, University of York, York, UK.

Background: People with haematological malignancies have different end-of-life care patterns from those with other cancers and are more likely to die in hospital. Little is known about patient and relative preferences at this time and whether these are achieved.

Aim: To explore the experiences and reflections of bereaved relatives of patients with leukaemia, lymphoma or myeloma, and examine (1) preferred place of care and death; (2) perceptions of factors influencing attainment of preferences; and (3) changes that could promote achievement of preferences.

Design: Qualitative interview study incorporating 'Framework' analysis.

Setting/participants: A total of 10 in-depth interviews with bereaved relatives.

Results: Although most people expressed a preference for home death, not all attained this. The influencing factors include disease characteristics (potential for sudden deterioration and death), the occurrence and timing of discussions (treatment cessation, prognosis, place of care/death), family networks (willingness/ability of relatives to provide care, knowledge about services, confidence to advocate) and resource availability (clinical care, hospice beds/policies). Preferences were described as changing over time and some family members retrospectively came to consider hospital as the 'right' place for the patient to have died. Others shared strong preferences with patients for home death and acted to ensure this was achieved. No patients died in a hospice, and relatives identified barriers to death in this setting.

Conclusion: Preferences were not always achieved due to a series of complex, interrelated factors, some amenable to change and others less so. Death in hospital may be preferred and appropriate, or considered the best option in hindsight.
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http://dx.doi.org/10.1177/0269216318824525DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6507303PMC
May 2019

Center Finding in E. coli and the Role of Mathematical Modeling: Past, Present and Future.

J Mol Biol 2019 03 18;431(5):928-938. Epub 2019 Jan 18.

Computational and Systems Biology, John Innes Centre, Norwich Research Park, Norwich, NR4 7UH, United Kingdom. Electronic address:

We review the key role played by mathematical modeling in elucidating two center-finding patterning systems in Escherichia coli: midcell division positioning by the MinCDE system and DNA partitioning by the ParABS system. We focus particularly on how, despite much experimental effort, these systems were simply too complex to unravel by experiments alone, and instead required key injections of quantitative, mathematical thinking. We conclude the review by analyzing the frequency of modeling approaches in microbiology over time. We find that while such methods are increasing in popularity, they are still probably heavily under-utilized for optimal progress on complex biological questions.
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http://dx.doi.org/10.1016/j.jmb.2019.01.017DOI Listing
March 2019

Reprogramming Cdr2-Dependent Geometry-Based Cell Size Control in Fission Yeast.

Curr Biol 2019 01 10;29(2):350-358.e4. Epub 2019 Jan 10.

Computational and Systems Biology, John Innes Centre, Norwich, UK. Electronic address:

How cell size is determined and maintained remains unclear, even in simple model organisms. In proliferating cells, cell size is regulated by coordinating growth and division through sizer, adder, or timer mechanisms or through some combination [1, 2]. Currently, the best-characterized example of sizer behavior is in fission yeast, Schizosaccharomyces pombe, which enters mitosis at a minimal cell size threshold. The peripheral membrane kinase Cdr2 localizes in clusters (nodes) on the medial plasma membrane and promotes mitotic entry [3]. Here, we show that the Cdr2 nodal density, which scales with cell size, is used by the cell to sense and control its size. By analyzing cells of different widths, we first show that cdr2 cells divide at a fixed cell surface area. However, division in the cdr2Δ mutant is more closely specified by cell volume, suggesting that Cdr2 is essential for area sensing and supporting the existence of a Cdr2-independent secondary sizer mechanism more closely based on volume. To investigate how Cdr2 nodes may sense area, we derive a minimal mathematical model that incorporates the cytoplasmic kinase Ssp1 as a Cdr2 activator. The model predicts that a cdr2 mutant in an Ssp1 phosphorylation site (cdr2-T166A) [4] should form nodes whose density registers cell length. We confirm this prediction experimentally and find that thin cells now follow this new scaling by dividing at constant length instead of area. This work supports the role of Cdr2 as a sizer factor and highlights the importance of studying geometrical aspects of size control.
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http://dx.doi.org/10.1016/j.cub.2018.12.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6345630PMC
January 2019

Temperature Sensing Is Distributed throughout the Regulatory Network that Controls FLC Epigenetic Silencing in Vernalization.

Cell Syst 2018 12 28;7(6):643-655.e9. Epub 2018 Nov 28.

John Innes Centre, Norwich Research Park, Norwich NR4 7UH, UK. Electronic address:

Many organisms need to respond to complex, noisy environmental signals for developmental decision making. Here, we dissect how Arabidopsis plants integrate widely fluctuating field temperatures over month-long timescales to progressively upregulate VERNALIZATION INSENSITIVE3 (VIN3) and silence FLOWERING LOCUS C (FLC), aligning flowering with spring. We develop a mathematical model for vernalization that operates on multiple timescales-long term (month), short term (day), and current (hour)-and is constrained by experimental data. Our analysis demonstrates that temperature sensing is not localized to specific nodes within the FLC network. Instead, temperature sensing is broadly distributed, with each thermosensory process responding to specific features of the plants' history of exposure to warm and cold. The model accurately predicts FLC silencing in new field data, allowing us to forecast FLC expression in changing climates. We suggest that distributed thermosensing may be a general property of thermoresponsive regulatory networks in complex natural environments.
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http://dx.doi.org/10.1016/j.cels.2018.10.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6310686PMC
December 2018

Development of a Novel B-Cell Lymphoma 6 (BCL6) PROTAC To Provide Insight into Small Molecule Targeting of BCL6.

ACS Chem Biol 2018 11 17;13(11):3131-3141. Epub 2018 Oct 17.

Oncology and Discovery Sciences, IMED Biotech Unit , AstraZeneca , 310 Cambridge Science Park, Milton Road , Cambridge CB4 0WG , U.K.

B-cell lymphoma 6 (BCL6) inhibition is a promising mechanism for treating hematological cancers but high quality chemical probes are necessary to evaluate its therapeutic potential. Here we report potent BCL6 inhibitors that demonstrate cellular target engagement and exhibit exquisite selectivity for BCL6 based on mass spectrometry analyses following chemical proteomic pull down. Importantly, a proteolysis-targeting chimera (PROTAC) was also developed and shown to significantly degrade BCL6 in a number of diffuse large B-cell lymphoma (DLBCL) cell lines, but neither BCL6 inhibition nor degradation selectively induced marked phenotypic response. To investigate, we monitored PROTAC directed BCL6 degradation in DLBCL OCI-Ly1 cells by immunofluorescence and discovered a residual BCL6 population. Analysis of subcellular fractions also showed incomplete BCL6 degradation in all fractions despite having measurable PROTAC concentrations, together providing a rationale for the weak antiproliferative response seen with both BCL6 inhibitor and degrader. In summary, we have developed potent and selective BCL6 inhibitors and a BCL6 PROTAC that effectively degraded BCL6, but both modalities failed to induce a significant phenotypic response in DLBCL despite achieving cellular concentrations.
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http://dx.doi.org/10.1021/acschembio.8b00698DOI Listing
November 2018

A gated relaxation oscillator mediated by FrzX controls morphogenetic movements in Myxococcus xanthus.

Nat Microbiol 2018 08 16;3(8):948-959. Epub 2018 Jul 16.

Laboratoire de Chimie Bactérienne, CNRS-Aix Marseille University UMR 7283, Institut de Microbiologie de la Méditerranée, Marseille, France.

Dynamic control of cell polarity is of critical importance for many aspects of cellular development and motility. In Myxococcus xanthus, MglA, a G protein, and MglB, its cognate GTPase-activating protein, establish a polarity axis that defines the direction of movement of the cell and that can be rapidly inverted by the Frz chemosensory system. Although vital for collective cell behaviours, how Frz triggers this switch has remained unknown. Here, we use genetics, imaging and mathematical modelling to show that Frz controls polarity reversals via a gated relaxation oscillator. FrzX, which we identify as a target of the Frz kinase, provides the gating and thus acts as the trigger for reversals. Slow relocalization of the polarity protein RomR then creates a refractory period during which another switch cannot be triggered. A secondary Frz output, FrzZ, decreases this delay, allowing rapid reversals when required. Thus, this architecture results in a highly tuneable switch that allows a wide range of reversal frequencies.
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http://dx.doi.org/10.1038/s41564-018-0203-xDOI Listing
August 2018

Palliative care specialists' perceptions concerning referral of haematology patients to their services: findings from a qualitative study.

BMC Palliat Care 2018 Feb 21;17(1):33. Epub 2018 Feb 21.

Epidemiology & Cancer Statistics Group, University of York, York, YO10 5DD, UK.

Background: Haematological malignancies (leukaemias, lymphomas and myeloma) are complex cancers that are relatively common, affect all ages and have divergent outcomes. Although the symptom burden of these diseases is comparable to other cancers, patients do not access specialist palliative care (SPC) services as often as those with other cancers. To determine the reasons for this, we asked SPC practitioners about their perspectives regarding the barriers and facilitators influencing haematology patient referrals.

Methods: We conducted a qualitative study, set within the United Kingdom's (UK's) Haematological Malignancy Research Network (HMRN: www.hmrn.org ), a population-based cohort in the North of England. In-depth, semi-structured interviews were conducted with 20 SPC doctors and nurses working in hospital, community and hospice settings between 2012 and 2014. Interviews were digitally audio-recorded, transcribed and analysed for thematic content using the 'Framework' method.

Results: Study participants identified a range of barriers and facilitators influencing the referral of patients with haematological malignancies to SPC services. Barriers included: the characteristics and pathways of haematological malignancies; the close patient/haematology team relationship; lack of role clarity; late end of life discussions and SPC referrals; policy issues; and organisational issues. The main facilitators identified were: establishment of inter-disciplinary working patterns (co-working) and enhanced understanding of roles; timely discussions with patients and early SPC referral; access to information platforms able to support information sharing; and use of indicators to 'flag' patients' needs for SPC. Collaboration between haematology and SPC was perceived as beneficial and desirable, and was said to be increasing over time.

Conclusions: This is the first UK study to explore SPC practitioners' perceptions concerning haematology patient referrals. Numerous factors were found to influence the likelihood of referral, some of which related to the organisation and delivery of SPC services, so were amenable to change, and others relating to the complex and unique characteristics and pathways of haematological cancers. Further research is needed to assess the extent to which palliative care is provided by haematology doctors and nurses and other generalists and ways in which clinical uncertainty could be used as a trigger, rather than a barrier, to referral.
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http://dx.doi.org/10.1186/s12904-018-0289-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5822662PMC
February 2018

Absence of warmth permits epigenetic memory of winter in Arabidopsis.

Nat Commun 2018 02 12;9(1):639. Epub 2018 Feb 12.

John Innes Centre, Norwich Research Park, Norwich, NR4 7UH, UK.

Plants integrate widely fluctuating temperatures to monitor seasonal progression. Here, we investigate the temperature signals in field conditions that result in vernalisation, the mechanism by which flowering is aligned with spring. We find that multiple, distinct aspects of the temperature profile contribute to vernalisation. In autumn, transient cold temperatures promote transcriptional shutdown of Arabidopsis FLOWERING LOCUS C (FLC), independently of factors conferring epigenetic memory. As winter continues, expression of VERNALIZATION INSENSITIVE3 (VIN3), a factor needed for epigenetic silencing, is upregulated by at least two independent thermosensory processes. One integrates long-term cold temperatures, while the other requires the absence of daily temperatures above 15 °C. The lack of spikes of high temperature, not just prolonged cold, is thus the major driver for vernalisation. Monitoring of peak daily temperature is an effective mechanism to judge seasonal progression, but is likely to have deleterious consequences for vernalisation as the climate becomes more variable.
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http://dx.doi.org/10.1038/s41467-018-03065-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5809604PMC
February 2018