77 research outputs found
Non-muscle myosins 2A and 2B drive changes in cell morphology that occur as myoblasts align and fuse
The interaction of non-muscle myosins 2A and 2B with
actin may drive changes in cell movement, shape and
adhesion. To investigate this, we used cultured myoblasts as
a model system. These cells characteristically change shape
from triangular to bipolar when they form groups of
aligned cells. Antisense oligonucleotide knockdown of nonmuscle
myosin 2A, but not non-muscle myosin 2B, inhibited
this shape change, interfered with cell-cell adhesion, had a
minor effect on tail retraction and prevented myoblast
fusion. By contrast, non-muscle myosin 2B knockdown
markedly inhibited tail retraction, increasing cell length by
over 200% by 72 hours compared with controls. In addition
it interfered with nuclei redistribution in myotubes. Nonmuscle
myosin 2C is not involved as western analysis
showed that it is not expressed in myoblasts, but only in
myotubes. To understand why non-muscle myosins 2A and
2B have such different roles, we analysed their distributions by immuno-electron microscopy, and found that nonmuscle myosin 2A was more tightly associated with the plasma membrane than non-muscle myosin 2B. This
suggests that non-muscle myosin 2A is more important for
bipolar shape formation and adhesion owing to its
preferential interaction with membrane-associated actin,
whereas the role of non-muscle myosin 2B in retraction
prevents over-elongation of myoblasts
Segmenting Cardiac Muscle Z-disks with Deep Neural Networks
Z-disks are complex structures that delineate repeating sarcomeres in
striated muscle. They play significant roles in cardiomyocytes such as
providing mechanical stability for the contracting sarcomere, cell signalling
and autophagy. Changes in Z-disk architecture have been associated with
impaired cardiac function. Hence, there is a strong need to create tools to
segment Z-disks from microscopy images, that overcome traditional limitations
such as variability in image brightness and staining technique. In this study,
we apply deep learning based segmentation models to extract Z-disks in images
of striated muscle tissue. We leverage a novel Airyscan confocal dataset, which
comprises high resolution images of Z-disks of healthy heart tissue, stained
with Affimers for specific Z-disk proteins. We employed an interactive
labelling tool, Ilastik to obtain ground truth segmentation masks and use the
resulting data set to train and evaluate the performance of several
state-of-the-art segmentation networks. On the test set, UNet++ achieves best
segmentation performance for Z-disks in cardiomyocytes, with an average Dice
score of 0.91 and outperforms other established segmentation methods including
UNet, FPN, DeepLabv3+ and pix2pix. However, pix2pix demonstrates improved
generalisation, when tested on an additional dataset of cardiomyocytes with a
titin mutation. This is the first study to demonstrate that automated machine
learning-based segmentation approaches may be used effectively to segment
Z-disks in confocal microscopy images. Automated segmentation approaches and
predicted segmentation masks could be used to derive morphological features of
Z-disks (e.g. width and orientation), and subsequently, to quantify
disease-related changes to cardiac microstructure
Health system resilience and health workforce capacities: Comparing health system responses during the COVID-19 pandemic in six European countries
Background: The health workforce is a key component of any health system and the present crisis offers a unique opportunity to better understand its specific contribution to health system resilience. The literature acknowledges the importance of the health workforce, but there is little systematic knowledge about how the health workforce matters across different countries. Aims: We aim to analyse the adaptive, absorptive and transformative capacities of the health workforce during the first wave of the COVID-19 pandemic in Europe (January-May/June 2020), and to assess how health systems prerequisites influence these capacities. Materials and Methods: We selected countries according to different types of health systems and pandemic burdens. The analysis is based on short, descriptive country case studies, using written secondary and primary sources and expert information. Results and Discussion: Our analysis shows that in our countries, the health workforce drew on a wide range of capacities during the first wave of the pandemic. However, health systems prerequisites seemed to have little influence on the health workforce's specific combinations of capacities. Conclusion: This calls for a reconceptualisation of the institutional perquisites of health system resilience to fully grasp the health workforce contribution. Here, strengthening governance emerges as key to effective health system responses to the COVID-19 crisis, as it integrates health professions as frontline workers and collective actors
Understanding the experience of initiating community-based group physical activity by people with serious mental illness: a systematic review using a meta-ethnographic approach
Background
People living with serious mental illness (SMI) experience debilitating symptoms that worsen their physical health and quality of life. Regular physical activity (PA) may bring symptomatic improvements and enhance wellbeing. When undertaken in community-based group settings, PA may yield additional benefits such as reduced isolation. Initiating PA can be difficult for people with SMI and so PA engagement is commonly low. Designing acceptable and effective PA programmes requires a better understanding of the lived experiences of PA initiation among people with SMI.
Methods
This systematic review of qualitative studies used the meta-ethnography approach by Noblit and Hare (1988). Electronic databases were searched from inception to November 2017. Eligible studies used qualitative methodology; involved adults (≥18 years) with schizophrenia, bipolar affective disorder, major depressive disorder or psychosis; reported community-based group PA; and captured the experience of PA initiation, including key features of social support. Study selection and quality assessment was performed by four reviewers.
Results
Sixteen studies were included in the review. We identified a ‘journey' that depicted a long sequence of phases involved in initiating PA. The journey demonstrated the thought processes, expectations, barriers and support needs of people with SMI. In particular, social support from a trusted source played an important role in getting people to the activity, both physically and emotionally.
Discussion
The journey illustrated that initiation of PA for people with SMI is a long complex transition. This complex process needs to be understood before ongoing participation in PA can be addressed
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Co-producing a physical activity intervention with and for people with severe mental ill health – the spaces story
SPACES (Supporting Physical Activity through Co-production in people with Severe Mental Illness) is a study which aims to develop an intervention to increase physical activity created with and for people with severe mental ill health (SMI), their carers and professionals involved in physical activity and/or severe mental ill health. People with SMI are less physically active than the general population and have an increased likelihood of experiencing long-term physical health conditions such as cardiovascular and respiratory diseases, diabetes and obesity. The SPACES team employed a comprehensive process of Patient and Public Involvement and Engagement (PPIE) work embedded within a co-production strategy. Researchers worked together from the point of inception with people with lived experience, two of whom became co-applicants, to design and carry out the intervention development stage of the study. This included PPIE work and an iterative process of focus groups and interviews with various stakeholders and a consensus group made up of multiple stakeholders with lived, caring and professional experience. Here, we describe the co-production model we used, the benefits, challenges, achievements and areas for learning and improvement. We offer co-production principles and practical strategy, which we hope will be used, modified, personalised and built on by others. We also offer the idea that laying out the co-production strategy to be employed prior to a study commencing and then comparing how that strategy was or was not met could be a step towards creating more accountability and academic rigour in co-production
The Inner Centromere Protein (INCENP) Coil Is a Single α-Helix (SAH) Domain That Binds Directly to Microtubules and Is Important for Chromosome Passenger Complex (CPC) Localization and Function in Mitosis
The chromosome passenger complex (CPC) is a master regulator of mitosis. INCENP acts as a scaffold regulating CPC localisation and activity. During early mitosis the N-terminal region of INCENP forms a three-helix bundle with Survivin and Borealin, directing the CPC to the inner centromere where it plays essential roles in chromosome alignment and the spindle assembly checkpoint. The C-terminal IN-box region of INCENP is responsible for binding and activating Aurora B kinase. The central region of INCENP has been proposed to comprise a coiled-coil domain acting as a spacer between the N and C terminal domains that is involved in microtubule binding and regulation of the spindle checkpoint. Here we show that the central region (213 residues) of chicken INCENP is not a coiled coil but a ~32 nm long single alpha helical (SAH) domain. The N-terminal half of this domain directly binds to microtubules in vitro. By analogy with previous studies of myosin 10, our data suggest that the INCENP SAH might stretch up to ~80 nm under physiological forces. Thus, the INCENP SAH could act as a flexible dog-leash allowing Aurora B to phosphorylate dynamic substrates localized in the outer kinetochore while at the same time being stably anchored to the stable chromatin of the inner centromere. Furthermore, by achieving this flexibility via a SAH domain, the CPC avoids a need for dimerization (required for coiled-coil formation), which would greatly complicate regulation of the proximity-induced trans-phosphorylation that is critical for Aurora B activation
Human Skeletal myopathy myosin mutations disrupt myosin head sequestration
Myosin heavy chains encoded by MYH7 and MYH2 are abundant in human skeletal muscle, and important for muscle contraction. However, it is unclear how mutations in these genes disrupt myosin structure and function leading to skeletal muscle myopathies termed myosinopathies. Here, we used multiple approaches to analyse the effects of common MYH7 and MYH2 mutations in the light meromyosin region of myosin (LMM). Analyses of expressed and purified MYH7 and MYH2 LMM mutant proteins combined with in-silico modelling showed that myosin coiled-coil structure and packing of filaments in vitro are commonly disrupted. Using muscle biopsies from patients, and Mant-ATP chase protocols to estimate the proportion of myosin heads that were super-relaxed, together with X-ray diffraction measurements to estimate myosin head order we found that basal myosin ATP consumption was increased and the myosin super-relaxed state was decreased in vivo. In addition, myofibre mechanics experiments to investigate contractile function showed myofibre contractility was not affected. These findings indicate that the structural remodelling associated with LMM mutations induces a pathogenic state in which formation of shutdown heads is impaired, thus increasing myosin head ATP demand in the filaments, rather than affecting contractility. These key findings will help design future therapies for myosinopathies
Hypertrophic cardiomyopathy mutations in the calponin-homology domain of ACTN2 affect actin binding and cardiomyocyte Z-disc incorporation
α-Actinin-2 (ACTN2) is the only muscle isoform of α-actinin expressed in cardiac muscle. Mutations in this protein have been implicated in mild to moderate forms of hypertrophic cardiomyopathy (HCM). We have investigated the effects of two mutations identified from HCM patients, A119T and G111V, on the secondary and tertiary structure of a purified actin binding domain (ABD) of ACTN2 by circular dichroism and X-ray crystallography, and show small but distinct changes for both mutations. We also find that both mutants have reduced F-actin binding affinity, although the differences are not significant. The full length mEos2 tagged protein expressed in adult cardiomyocytes shows that both mutations additionally affect Z-disc localization and dynamic behaviour. Overall, these two mutations have small effects on structure, function and behaviour, which may contribute to a mild phenotype for this disease
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