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Background Heart failure and sleep-disordered breathing have been increasingly recognized as co-occurring conditions. Their bidirectional relationship warrants investigation into whether heart failure therapy improves sleep and sleep-disordered breathing. We sought to explore the effect of treatment with sacubitril/valsartan on sleep-related endpoints from the AWAKE-HF study. Methods and Results AWAKE-HF was a randomized, double-blind study conducted in 23 centers in the United States. Study participants with heart failure with reduced rejection fraction and New York Heart Association class II or III symptoms were randomly assigned to receive treatment with either sacubitril/valsartan or enalapril. All endpoints were assessed at baseline and after 8 weeks of treatment. Portable sleep-monitoring equipment was used to measure the apnea-hypopnea index, including obstructive and central events. Total sleep time, wake after sleep onset and sleep efficiency were exploratory measures assessed using wrist actigraphy. The results were as follows 140 patients received treatment in the double-blind phase (sacubitril/valsartan, n = 70; enalapril, n = 70). At baseline, 39% and 40% of patients randomly assigned to receive sacubitril/valsartan or enalapril, respectively, presented with undiagnosed, untreated, moderate-to-severe sleep-disordered breathing (≥ 15 events/h), and nearly all had obstructive sleep apnea. After 8 weeks of treatment, the mean 4% apnea-hypopnea index changed minimally from 16.3/h to 15.2/h in the sacubitril/valsartan group and from 16.8/h to 17.6/h in the enalapril group. Mean total sleep time was long at baseline and decreased only slightly in both treatment groups at week 8 (–14 and –11 minutes for sacubitril/valsartan and enalapril, respectively), with small changes in wake after sleep onset and sleep efficiency in both groups. Conclusions In a cohort of patients with heart failure with reduced rejection fraction who met prescribing guidelines for sacubitril/valsartan, one-third had undiagnosed moderate-to-severe obstructive sleep apnea. The addition of sacubitril/valsartan therapy did not significantly improve sleep-disordered breathing or sleep duration or efficiency. Patients who meet indications for treatment with sacubitril/valsartan should be evaluated for sleep-disordered breathing.

Background: Data derived from wearable activity trackers may provide important clinical insights into disease progression and response to intervention, but only if clinicians can interpret it in a meaningful manner. Longitudinal activity data can be visually presented in multiple ways, but research has failed to explore how clinicians interact with and interpret these visualisations. In response, this study developed a variety of visualisations to understand whether alternative data presentation strategies can provide clinicians with meaningful insights into patient’s physical activity patterns. Objective: To explore clinicians’ opinions on different visualisations of actigraphy data. Methods: Four visualisations (stacked bar chart, clustered bar chart, linear heatmap and radial heatmap) were created using Matplotlib and Seaborn Python libraries. A focus group was conducted with 14 clinicians across 2 hospitals. Focus groups were audio-recorded, transcribed and analysed using inductive thematic analysis. Results: Three major themes were identified: (1) the importance of context, (2) interpreting the visualisations and (3) applying visualisations to clinical practice. Although clinicians saw the potential value in the visualisations, they expressed a need for further contextual information to gain clinical benefits from them. Allied health professionals preferred more granular, temporal information compared to doctors. Specifically, physiotherapists favoured heatmaps, whereas the remaining members of the team favoured stacked bar charts. Overall, heatmaps were considered more difficult to interpret. Conclusion: The current lack of contextual data provided by wearables hampers their use in clinical practice. Clinicians favour data presented in a familiar format and yet desire multi-faceted filtering. Future research should implement user-centred design processes to identify ways in which all clinical needs can be met, potentially using an interactive system that caters for multiple levels of granularity. Irrespective of how data is displayed, unless clinicians can apply it in a manner that best supports their role, the potential of this data cannot be fully realised.

Purpose: Clinical ordinal rating scales of movements, e.g., the Expanded Disability Status Scale, have poor intra- and interrater reliability, are insensitive to subtle differences and result in coarse-grained ratings compared to relative comparative rating methods. We therefore established video-based setwise comparison as a fine-grained, reliable and efficient rating method of motor dysfunction using algorithmic support. Materials and methods: Eight neurologists rated a set of 40 multiple sclerosis patient videos of the Finger-to-Nose-Test using both the newly developed setwise comparison and the established pairwise comparison techniques, which result in a continuous rating scale. Reliability was assessed by the intra-class correlation coefficient. Construct validity was estimated as Pearson’s correlation between the continuous scale and severity ratings according to the Neurostatus scale for upper-extremity tremor/dysmetria and the Nine-hole-peg-test. Comparing the time needed for ratings assessed efficiency. Results: Intra-class correlation coefficient was 0.83 for setwise and 0.7 for pairwise comparison. Correlation to the tremor/dysmetria score of the Neurostatus was 0.86 for both rating procedures and correlation to the Nine-hole-peg-test was 0.64 (setwise) and 0.66 (pairwise). The time needed to rate 40 videos was 22.9 ± 6.9 minutes (setwise) and 77.8 ± 14.5 minutes (pairwise). Conclusions: Setwise comparison is an efficient, valid and reliable method for fine-grained rating of motor dysfunction that can be applied to larger datasets. It is substantially more efficient than pairwise comparison.Implications for rehabilitation Disability rating is crucial in clinical neurorehabilitation and in clinical trials. Humans are naturally inconsistent in rating items on ordinal scales leading to poor intra- and interrater reliability, insensitivity to subtle differences and coarse-grained ratings. Video-based setwise comparison is a new rating method enabling fine-grained, reliable and efficient ratings of motor dysfunction using algorithmic support.

Background In chronic neurological diseases, especially in multiple sclerosis (MS), clinical assessment of motor dysfunction is crucial to monitor the disease in patients. Traditional scales are not sensitive enough to detect slight changes. Video recordings of patient performance are more accurate and increase the reliability of severity ratings. When these recordings are automated, quantitative disability assessments by machine learning algorithms can be created. Creation of these algorithms involves non–health care professionals, which is a challenge for maintaining data privacy. However, autoencoders can address this issue. Objective The aim of this proof-of-concept study was to test whether coded frame vectors of autoencoders contain relevant information for analyzing videos of the motor performance of patients with MS. Methods In this study, 20 pre-rated videos of patients performing the finger-to-nose test were recorded. An autoencoder created encoded frame vectors from the original videos and decoded the videos again. The original and decoded videos were shown to 10 neurologists at an academic MS center in Basel, Switzerland. The neurologists tested whether the 200 videos were human-readable after decoding and rated the severity grade of each original and decoded video according to the Neurostatus-Expanded Disability Status Scale definitions of limb ataxia. Furthermore, the neurologists tested whether ratings were equivalent between the original and decoded videos. Results In total, 172 of 200 (86.0%) videos were of sufficient quality to be ratable. The intrarater agreement between the original and decoded videos was 0.317 (Cohen weighted kappa). The average difference in the ratings between the original and decoded videos was 0.26, in which the original videos were rated as more severe. The interrater agreement between the original videos was 0.459 and that between the decoded videos was 0.302. The agreement was higher when no deficits or very severe deficits were present. Conclusions The vast majority of videos (172/200, 86.0%) decoded by the autoencoder contained clinically relevant information and had fair intrarater agreement with the original videos. Autoencoders are a potential method for enabling the use of patient videos while preserving data privacy, especially when non–health-care professionals are involved.

Background: Wearable sensors allow researchers to remotely capture digital health data, including physical activity, which may identify digital biomarkers to differentiate healthy and clinical cohorts. To date, research has focused on high-level data (e.g., overall step counts) which may limit our insights to whether people move differently, rather than how they move differently. Objective: This study therefore aimed to use actigraphy data to thoroughly examine activity patterns during the first hours following waking in arthritis patients (n = 45) and healthy controls (n = 30). Methods: Participants wore an Actigraph GT9X Link for 28 days. Activity counts were analysed and compared over varying epochs, ranging from 15 min to 4 h, starting with waking in the morning. The sum, and a measure of rate of change of cumulative activity in the period immediately after waking (area under the curve [AUC]) for each time period, was calculated for each participant, each day, and individual and group means were calculated. Two-tailed independent t tests determined differences between the groups. Results: No differences were seen for summed activity counts across any time period studied. However, differences were noted in the AUC analysis for the discrete measures of relative activity. Specifically, within the first 15, 30, 45, and 60 min following waking, the AUC for activity counts was significantly higher in arthritis patients compared to controls, particularly at the 30 min period (t = –4.24, p = 0.0002). Thus, while both cohorts moved the same amount, the way in which they moved was different. Conclusion: This study is the first to show that a detailed analysis of actigraphy variables could identify activity pattern changes associated with arthritis, where the high-level daily summaries did not. Results suggest discrete variables derived from raw data may be useful to help identify clinical cohorts and should be explored further to determine if they may be effective clinical biomarkers.

Background Wearable devices are valuable assessment tools for patient outcomes in contexts such as clinical trials. To be successfully deployed, however, participants must be willing to wear them. Another concern is that usability studies are rarely published, often fail to test devices beyond 24 hours, and need to be repeated frequently to ensure that contemporary devices are assessed. Objective This study aimed to compare multiple wearable sensors in a real-world context to establish their usability within an older adult (>50 years) population. Methods Eight older adults wore seven devices for a minimum of 1 week each: Actigraph GT9x, Actibelt, Actiwatch, Biovotion, Hexoskin, Mc10 Biostamp_RC, and Wavelet. Usability was established through mixed methods using semistructured interviews and three questionnaires, namely, the Intrinsic Motivation Inventory (IMI), the System Usability Scale (SUS), and an acceptability questionnaire. Quantitative data were reported descriptively and qualitative data were analyzed using deductive content analysis. Data were then integrated using triangulation. Results Results demonstrated that no device was considered optimal as all scored below average in the SUS (median, IQR; min-max=57.5, 12.5; 47.5-63.8). Hexoskin was the lowest scored device based on the IMI (3.6; 3.4-4.5), while Biovotion, Actibelt, and Mc10 Biostamp_RC achieved the highest median results on the acceptability questionnaire (3.6 on a 6-point Likert scale). Qualitatively, participants were willing to accept less comfort, less device discretion, and high charging burdens if the devices were perceived as useful, namely through the provision of feedback for the user. Participants agreed that the purpose of use is a key enabler for long-term compliance. These views were particularly noted by those not currently wearing an activity-tracking device. Participants believed that wrist-worn sensors were the most versatile and easy to use, and therefore, the most suitable for long-term use. In particular, Actiwatch and Wavelet stood out for their comfort. The convergence of quantitative and qualitative data was demonstrated in the study. Conclusions Based on the results, the following context-specific recommendations can be made: (1) researchers should consider their device selection in relation to both individual and environmental factors, and not simply the primary outcome of the research study; (2) if researchers do not wish their participants to have access to feedback from the devices, then a simple, wrist-worn device that acts as a watch is preferable; (3) if feedback is allowed, then it should be made available to help participants remain engaged; this is likely to apply only to people without cognitive impairments; (4) battery life of 1 week should be considered as a necessary feature to enhance data capture; (5) researchers should consider providing additional information about the purpose of devices to participants to support their continued use.

Objectives: To examine the feasibility, reliability, granularity, and convergent validity of a video-based pairwise comparison technique that uses algorithmic support to enable automated rating of motor dysfunction in patients with multiple sclerosis (MS). Design: Feasibility and larger cross-sectional cohort study. Setting: The outpatient clinic of 2 specialist university medical centers. Participants: Selected sample from a cohort of patients with MS participating in the Assess MS study (N=42). Videos were randomly drawn from each strata of the ataxia severity-degrees as defined in the Expanded Disability Status Scale (EDSS). In Basel: 19 videos of 17 patients (mean age, 43.4±11.6y; 10 women). In Amsterdam: 50 videos of 25 patients (mean age, 50.0±10.0y; 15 women). Interventions: Not applicable. Main Outcome Measures: In each center, neurologists (n=13; n=10) viewed pairs of videos of patients performing standardized movements (eg, finger-to-nose test) to assess relative performance. A comparative assessment score was calculated for each video using the TrueSkill algorithm and analyzed for intrarater (test-retest; ratio of agreement) and interrater reliability (intraclass correlation coefficient [ICC] for absolute agreement) and convergent validity (Spearman ρ). Granularity was estimated from the average difference in comparative assessment scores at which 80% of neurologists considered performance to be different. Results: Intrarater reliability was excellent (median ratio of agreement≥0.87). The comparative assessment scores calculated from individual neurologists demonstrated good-excellent ICCs for interrater reliability (0.89; 0.71). The comparative assessment scores correlated (very) highly with their Neurostatus-EDSS equivalent (ρ=0.78, P

Background: Evaluation of pain and stiffness in patients with arthritis is largely based on participants retrospectively reporting their self-perceived pain/stiffness. This is subjective and may not accurately reflect the true impact of therapeutic interventions. We now have access to sensor-based systems to continuously capture objective information regarding movement and activity. Objectives: We present an observational study aimed to collect sensor data from participants monitored while performing an unsupervised version of a standard motor task, known as the Five Times Sit to Stand (5×STS) test. The first objective was to explore whether the participants would perform the test regularly in their home environment, and do so in a correct and consistent manner. The second objective was to demonstrate that the measurements collected would enable us to derive an objective signal related to morning pain and stiffness. Methods: We recruited a total of 45 participants, of whom 30 participants fulfilled pre-defined criteria for osteoarthritis, rheumatoid arthritis, or psoriatic arthritis and 15 participants were healthy volunteers. All participants wore accelerometers on their wrists, day and night for about 4 weeks. The participants were asked to perform the 5×STS test in their own home environment at the same time in the morning 3 times per week. We investigated the relationship between pain/stiffness and measurements collected during the 5×STS test by comparing the 5×STS test duration with the patient-reported outcome (PRO) questionnaires, filled in via a smartphone. Results: During the study, we successfully captured accelerometer data from each participant for a period of 4 weeks. The participants performed 56% of the prescribed 5×STS tests. We observed that different tests made by the same participants were performed with subject-specific characteristics that remained consistent throughout the study. We showed that 5×STS test duration (the time taken to complete the 5×STS test) was significantly and robustly associated with the pain and stiffness intensity reported via the PROs, particularly the questions asked in the morning. Conclusions: This study demonstrates the feasibility and usefulness of regular, sensor-based, monitored, unsupervised physical tests to objectively assess the impact of disease on function in the home environment. This approach may permit remote disease monitoring in clinical trials and support the development of novel endpoints from passively collected actigraphy data.

BACKGROUND In chronic neurological diseases, especially in multiple sclerosis (MS), clinical assessment of motor dysfunction is crucial to monitor the disease in patients. Traditional scales are not sensitive enough to detect slight changes. Video recordings of patient performance are more accurate and increase the reliability of severity ratings. When these recordings are automated, quantitative disability assessments by machine learning algorithms can be created. Creation of these algorithms involves non–health care professionals, which is a challenge for maintaining data privacy. However, autoencoders can address this issue. OBJECTIVE The aim of this proof-of-concept study was to test whether coded frame vectors of autoencoders contain relevant information for analyzing videos of the motor performance of patients with MS. METHODS In this study, 20 pre-rated videos of patients performing the finger-to-nose test were recorded. An autoencoder created encoded frame vectors from the original videos and decoded the videos again. The original and decoded videos were shown to 10 neurologists at an academic MS center in Basel, Switzerland. The neurologists tested whether the 200 videos were human-readable after decoding and rated the severity grade of each original and decoded video according to the Neurostatus-Expanded Disability Status Scale definitions of limb ataxia. Furthermore, the neurologists tested whether ratings were equivalent between the original and decoded videos. RESULTS In total, 172 of 200 (86.0%) videos were of sufficient quality to be ratable. The intrarater agreement between the original and decoded videos was 0.317 (Cohen weighted kappa). The average difference in the ratings between the original and decoded videos was 0.26, in which the original videos were rated as more severe. The interrater agreement between the original videos was 0.459 and that between the decoded videos was 0.302. The agreement was higher when no deficits or very severe deficits were present. CONCLUSIONS The vast majority of videos (172/200, 86.0%) decoded by the autoencoder contained clinically relevant information and had fair intrarater agreement with the original videos. Autoencoders are a potential method for enabling the use of patient videos while preserving data privacy, especially when non–health-care professionals are involved.

Background: Accurate clinical assessment in multiple sclerosis (MS) is challenging. The Assess MS system is being developed to automatically quantify motor dysfunction in MS, including upper extremity function (UEF) and mobility. Objective: To determine to what extent combinations of standardized movements included in the Assess MS system explain accepted measures of UEF and mobility. Methods: MS patients were recruited at four European MS centres. Eight movements were selected, including tasks of activities of daily living (ADL) and classical neurological tests. Movements were recorded on video and rated by experienced neurologists ( n = 5). Subsequently, multivariate linear regression models were performed to explain the variance of the Nine-Hole Peg Test (9HPT), Arm Function in Multiple Sclerosis Questionnaire (AMSQ) and Timed-25 Foot Walk test (T25WT). Results: In total, 257 patients were included. The movements explained 62.9% to 80.1% of the variance of the 9HPT models, 43.3% and 44.3% of the AMSQ models and 70.8% of the T25WT. In all models, tasks of ADL contributed most to the variance. Conclusion: Combinations of movements are valuable to assess UEF and mobility. Incorporating ADL tasks into daily clinical practice and clinical trials may be more valuable than the classical neurological examination of UEF and mobility.

Physical activity (PA) is a key component in the treatment of a range of chronic health conditions. It is therefore important for researchers and clinicians to accurately assess and monitor PA. Although advances in wearable technology have improved this, there is a need to investigate PA in greater depth than the sum of its total parts. Specifically, linking deep PA data to patient outcomes offers a valuable, and unexplored use for wearable devices. As a result, this paper extracts useful features from accelerometer data (Actigraph GT3X Link), and applies machine learning algorithms to predict daily pain and stiffness. This was applied to a population of 30 arthritis patients and 15 healthy volunteers. Participants were provided with an Actigraph and asked to wear it continuously for 28 days. Results demonstrate that it is possible to predict both pain and stiffness of patients using the extracted accelerometer features.

Objective. We evaluate the stride segmentation performance of the Adaptive Empirical Pattern Transformation (ADEPT) for subsecond-level accelerometry data collected in the free-living environment using a wrist-worn sensor.Approach. We substantially expand the scope of the existing ADEPT pattern-matching algorithm. Methods are applied to subsecond-level accelerometry data collected continuously for 4 weeks in 45 participants, including 30 arthritis and 15 control patients. We estimate the daily walking cadence for each participant and quantify its association with SF-36 quality of life measures.Main results. We provide free, open-source software to segment individual walking strides in subsecond-level accelerometry data. Walking cadence is significantly associated with the role physical score reported via SF-36 after adjusting for age, gender, weight and height.Significance. Methods provide automatic, precise walking stride segmentation, which allows estimation of walking cadence from free-living wrist-worn accelerometry data. Results provide new evidence of associations between free-living walking parameters and health outcomes.

Furrow ingression is asymmetric in cytokinesis in the Caenorhabditis elegans zygote. A combination of quantitative high-resolution live-cell microscopy and theoretical modeling revealed a mechanistic basis for asymmetry: feedback among membrane curvature, cytoskeletal alignment, and contractility. The model also suggests that asymmetry promotes energy efficiency., During cytokinesis, the cell undergoes a dramatic shape change as it divides into two daughter cells. Cell shape changes in cytokinesis are driven by a cortical ring rich in actin filaments and nonmuscle myosin II. The ring closes via actomyosin contraction coupled with actin depolymerization. Of interest, ring closure and hence the furrow ingression are nonconcentric (asymmetric) within the division plane across Metazoa. This nonconcentricity can occur and persist even without preexisting asymmetric cues, such as spindle placement or cellular adhesions. Cell-autonomous asymmetry is not explained by current models. We combined quantitative high-resolution live-cell microscopy with theoretical modeling to explore the mechanistic basis for asymmetric cytokinesis in the Caenorhabditis elegans zygote, with the goal of uncovering basic principles of ring closure. Our theoretical model suggests that feedback among membrane curvature, cytoskeletal alignment, and contractility is responsible for asymmetric cytokinetic furrowing. It also accurately predicts experimental perturbations of conserved ring proteins. The model further suggests that curvature-mediated filament alignment speeds up furrow closure while promoting energy efficiency. Collectively our work underscores the importance of membrane–cytoskeletal anchoring and suggests conserved molecular mechanisms for this activity.

Aneuploidy is a common feature of human solid tumors and is often associated with poor prognosis. There is growing evidence that oncogenic signaling pathways, which are universally dysregulated in cancer, contribute to the promotion of aneuploidy. However, the mechanisms connecting signaling pathways to the execution of mitosis and cytokinesis are not well understood. Here, we show that hyperactivation of the ERK1/2 MAP kinase pathway in epithelial cells impairs cytokinesis, leading to polyploidization and aneuploidy. Mechanistically, deregulated ERK1/2 signaling specifically downregulates expression of the F-box protein Fbxw7β, a substrate-binding subunit of the SCFFbxw7 ubiquitin ligase, resulting in the accumulation of the mitotic kinase Aurora A. Reduction of Aurora A levels by RNA interference or pharmacological inhibition of MEK1/2 reverts the defect in cytokinesis and decreases the frequency of abnormal cell divisions induced by oncogenic H-RasV12. Reciprocally, overexpression of Aurora A or silencing of Fbxw7β phenocopies the effect of H-RasV12 on cell division. In vivo, conditional activation of MEK2 in the mouse intestine lowers Fbxw7β expression, resulting in the accumulation of cells with enlarged nuclei. We propose that the ERK1/2/ Fbxw7β/Aurora A axis identified in this study contributes to genomic instability and tumor progression.

Motor dysfunction, particularly ataxia, is one of the predominant clinical manifestations in patients with multiple sclerosis (MS). Assessment of motor dysfunction suffers from a high variability. We investigated whether the clinical rating of ataxia can be improved through the use of reference videos, covering the spectrum of severity degrees as defined in the Neurostatus-Expanded Disability Status Scale. Twenty-five neurologists participated. The variability of their assessments was significantly lower when reference videos were used (SD = 0.12; range = 0.40 vs SD = 0.26; range = 0.88 without reference videos; p = 0.013). Reference videos reduced the variability of clinical assessments and may be useful tools to improve the precision and consistency in the clinical assessment of motor functions in MS.

Quantitative analysis relating cell, nuclear, and chromosome size in C. elegans embryos predicts two levels of chromosome length regulation in response to both cell and nuclear size., Multicellular development requires that cells reduce in size as a result of consecutive cell divisions without increase in embryo volume. To maintain cellular integrity, organelle size adapts to cell size throughout development. During mitosis, the longest chromosome arm must be shorter than half of the mitotic spindle for proper chromosome segregation. Using high-resolution time-lapse microscopy of living Caenorhabditis elegans embryos, we have quantified the relation between cell size and chromosome length. In control embryos, chromosome length scaled to cell size. Artificial reduction of cell size resulted in a shortening of chromosome length, following a trend predicted by measurements from control embryos. Disturbing the RAN (Ras-related nuclear protein)-GTP gradient decoupled nuclear size from cell size and resulted in chromosome scaling to nuclear size rather than cell size; smaller nuclei contained shorter chromosomes independent of cell size. In sum, quantitative analysis relating cell, nuclear, and chromosome size predicts two levels of chromosome length regulation: one through cell size and a second in response to nuclear size.

SummaryGenome stability relies upon efficacious chromosome congression and regulation by the spindle assembly checkpoint (SAC). The study of these fundamental mitotic processes in adult stem and progenitor cells has been limited by the technical challenge of imaging mitosis in these cells in situ. Notably, how broader physiological changes, such as dietary intake or age, affect mitotic progression in stem and/or progenitor cells is largely unknown. Using in situ imaging of C. elegans adult germlines, we describe the mitotic parameters of an adult stem and progenitor cell population in an intact animal. We find that SAC regulation in germline stem and progenitor cells is distinct from that found in early embryonic divisions and is more similar to that of classical tissue culture models. We further show that changes in organismal physiology affect mitotic progression in germline stem and progenitor cells. Reducing dietary intake produces a checkpoint-dependent delay in anaphase onset, and inducing dietary restriction when the checkpoint is impaired increases the incidence of segregation errors in mitotic and meiotic cells. Similarly, developmental aging of the germline stem and progenitor cell population correlates with a decline in the rate of several mitotic processes. These results provide the first in vivo validation of models for SAC regulation developed in tissue culture systems and demonstrate that several fundamental features of mitotic progression in adult stem and progenitor cells are highly sensitive to organismal physiological changes.

Despite its functional importance and well known clinical impact in Multiple Sclerosis (MS), the cerebellum has only received significant attention over the past few years. It is now established that the cerebellum plays a key role not only in various sensory-motor networks, but also in cognitive-behavioural processes, domains primarily affected in patients with MS. Evidence from histopathological and magnetic resonance imaging (MRI) studies on cerebellar involvement in MS is increasingly available, however linking these pathological findings with clinical dysfunction remains challenging. There are promising advances in technology that are likely to improve the detection of pathological changes within the cerebellum, which may elucidate how pathology relates to disability.

The black-and-white patterning of tendon fascicles when visualized by light microscopy, also known as crimp, is a well-known feature of fiber-forming collagens. However, not much is known about its development, function and response to strain. The objective of this study is to investigate the interaction of tenocyte and crimp morphology as well as their changes with increasing age and acute strain. In contrast to previous studies, which used indirect measures, such as polarized light, to investigate the crimp structure, this study visualizes internal crimp structure in three dimensions without freezing, sectioning, staining or fixing the tissue, via two-photon imaging of green fluorescent protein expressing cells within mouse tail tendon fascicles. This technique further allows straining of the live tissue while visualizing changes in crimp morphology and cell shape with increasing specimen length. Combining this novel microscopy technique with computational image and data analysis revealed a complex relationship between tenocytes and the extracellular matrix that evolves with increasing age. While the reduction of crimping with strain was observed as expected, most of the crimps were gone at 0–1% strain already. Even relatively low strains of 3% led to pronounced changes in the crimp structure after relaxation, particularly in the young animals, which could not be seen with bright-field imaging. Cell length and gap width increased with strain. However, while the cells were able to return to their original length even after high strains of 6%, the gaps between the cells widened, which may imply modified cell–cell communication after overstretching.

Summary Microtubules (MTs) are cytoskeletal polymers that undergo dynamic instability, the stochastic transition between growth and shrinkage phases. MT dynamics are required for diverse cellular processes and, while intrinsic to tubulin, are highly regulated. However, little is known about how MT dynamics facilitate or are regulated by tissue biogenesis and differentiation. We imaged MT dynamics in a smooth muscle-like lineage in intact developing Caenorhabditis elegans. All aspects of MT dynamics change significantly as stem-like precursors exit mitosis and, secondarily, as they differentiate. We found that suppression, but not enhancement, of dynamics perturbs differentiated muscle function in vivo. Distinct ensembles of MT-associated proteins are specifically required for tissue biogenesis versus tissue function. A CLASP family MT stabilizer and the depolymerizing kinesin MCAK are differentially required for MT dynamics in the precursor or differentiated cells, respectively. All of these multidimensional phenotypic comparisons were facilitated by a data display method called the diamond graph.

The AWAKE-HF study evaluated the effect of sacubitril/valsartan (S/V) versus enalapril on sleep using actigraphy and a home sleep study in patients (pts) with heart failure with reduced ejection fraction (HFrEF). Pts 18 to 80 years of age with New York Heart Association (NYHA) class II/III HFrEF

SummaryThe presence of a single centromere on each chromosome that signals formation of a mitotic kinetochore is central to accurate chromosome segregation [1]. The histone H3 variant centromere protein-A (CENP-A) is critical for centromere identity and function; CENP-A chromatin acts as an epigenetic mark to direct both centromere and kinetochore assembly [2–4]. Interpreting the centromere epigenetic mark ensures propagation of a single centromere per chromosome to maintain ploidy. Thus, understanding the nature of CENP-A chromatin is crucial for all cell divisions. However, there are ongoing debates over the fundamental composition of centromeric chromatin. Here we show that natively assembled human CENP-A nucleosomes are octameric throughout the cell cycle. Using total internal reflection fluorescence (TIRF)-coupled photobleaching-assisted copy-number counting of single nucleosomes obtained from cultured cells, we find that the majority of CENP-A nucleosomes contain CENP-A dimers. In addition, we detect the presence of H2B and H4 in these nucleosomes. Surprisingly, CENP-A associated with the chaperone HJURP can exist as either monomer or dimer, indicating possible assembly intermediates. Thus, our findings indicate that octameric CENP-A nucleosomes mark the centromeric region to ensure proper epigenetic inheritance and kinetochore assembly.

A growing number of domains, including affect recognition and movement analysis, require a single, real number ground truth label capturing some property of a video clip. We term this the provision of continuum labels. Unfortunately, there is often an uncacceptable trade-off between label consistency and the efficiency of the labelling process with current tools. We present a novel interaction technique, setwise comparison, which leverages the intrinsic human capability for consistent relative judgements and the TrueSkill algorithm to solve this problem. We describe SorTable, a system demonstrating this technique. We conducted a real-world study where clinicians labelled videos of patients with multiple sclerosis for the ASSESS MS computer vision system. In assessing the efficiency-consistency trade-off of setwise versus pairwise comparison, we demonstrated that not only is setwise comparison more efficient, but it also elicits more consistent labels. We further consider how our findings relate to the interactive machine learning literature.

Processing bodies (PBs, or P bodies) are cytoplasmic granules involved in mRNA storage and degradation that participate in the regulation of gene expression. PBs concentrate nontranslated mRNAs and several factors involved in mRNA decay and translational repression, including the eukaryotic translation initiation factor 4E-transporter (4E-T). 4E-T is required for PB assembly, but little is known about the molecular mechanisms that regulate its function. Here, we demonstrate that oxidative stress promotes multisite 4E-T phosphorylation. We show that the c-Jun N-terminal kinase (JNK) is targeted to PBs in response to oxidative stress and promotes the phosphorylation of 4E-T. Quantitative mass spectrometry analysis reveals that JNK phosphorylates 4E-T on six proline-directed sites that are required for the formation of the 4E-T complex upon stress. We have developed an image-based computational method to quantify the size, number, and density of PBs in cells, and we find that while 4E-T is required for steady-state PB assembly, its phosphorylation facilitates the formation of larger PBs upon oxidative stress. Using polysomal mRNA profiling, we assessed global and specific mRNA translation but did not find that 4E-T phosphorylation impacts translational control. Collectively, these data support a model whereby PB assembly is regulated by a two-step mechanism involving a 4E-T-dependent assembly stage in unstressed cells and a 4E-T phosphorylation-dependent aggregation stage in response to stress stimuli.

Drosophila Evi5 is a Rab-GAP that acts on the membrane-trafficking mediator Rab11 to promote guidance receptor polarization during border cell migration., Membrane trafficking has well-defined roles during cell migration. However, its regulation is poorly characterized. In this paper, we describe the first screen for putative Rab–GTPase-activating proteins (GAPs) during collective cell migration of Drosophila melanogaster border cells (BCs), identify the uncharacterized Drosophila protein Evi5 as an essential membrane trafficking regulator, and describe the molecular mechanism by which Evi5 regulates BC migration. Evi5 requires its Rab-GAP activity to fulfill its functions during migration and acts as a GAP protein for Rab11. Both loss and gain of Evi5 function blocked BC migration by disrupting the Rab11-dependent polarization of active guidance receptors. Altogether, our findings deepen our understanding of the molecular machinery regulating endocytosis and subsequently cell signaling during migration.

Epigenetic mechanisms regulate genome activation in diverse events, including normal development and cancerous transformation. Centromeres are epigenetically designated chromosomal regions that maintain genomic stability by directing chromosome segregation during cell division. The histone H3 variant CENP-A resides specifically at centromeres, is fundamental to centromere function and is thought to act as the epigenetic mark defining centromere loci. Mechanisms directing assembly of CENP-A nucleosomes have recently emerged, but how CENP-A is maintained after assembly is unknown. Here, we show that a small GTPase switch functions to maintain newly assembled CENP-A nucleosomes. Using functional proteomics, we found that MgcRacGAP (a Rho family GTPase activating protein) interacts with the CENP-A licensing factor HsKNL2. High-resolution live-cell imaging assays, designed in this study, demonstrated that MgcRacGAP, the Rho family guanine nucleotide exchange factor (GEF) Ect2, and the small GTPases Cdc42 and Rac, are required for stability of newly incorporated CENP-A at centromeres. Thus, a small GTPase switch ensures epigenetic centromere maintenance after loading of new CENP-A.

SummaryPolar body extrusion (PBE) is the specialized asymmetric division by which oocytes accomplish reduction in ploidy and retention of cytoplasm. During maternal gametogenesis, as in male meiosis and mitosis, cytokinesis is accomplished by a ring rich in active Rho, myosin, and formin-nucleated F-actin [1–7]. However, unlike mitosis, wherein the contractile ring encircles the cell equator, the polar body ring assembles as a discoid cortical washer. Here we show that in Caenorhabditis elegans, the meiotic contractile ring transforms during closure from a disc above the spindle to a cylinder around the spindle midzone. The meiotic midbody tube comprises stacked cytoskeletal rings. This topological transition suggests a novel mechanism for constriction of an initially discoid cytokinetic ring. Analysis of mouse PBE indicates that midbody tube formation is a conserved process. Depletion of the scaffold protein anillin (ANI-1) from C. elegans results in large and unstable polar bodies that often fuse with the oocyte. Anillin is dispensable for contractile ring assembly, initiation, and closure but is required for the meiotic contractile ring to transform from a disc into a tube. We propose that cytoskeletal bundling by anillin promotes formation of the midbody tube, which ensures the fidelity of PBE.

An automated, quantitative 4D image analysis method is used to track kinetochore dynamics in metaphase cells., During mitosis in most eukaryotic cells, chromosomes align and form a metaphase plate halfway between the spindle poles, about which they exhibit oscillatory movement. These movements are accompanied by changes in the distance between sister kinetochores, commonly referred to as breathing. We developed a live cell imaging assay combined with computational image analysis to quantify the properties and dynamics of sister kinetochores in three dimensions. We show that baseline oscillation and breathing speeds in late prometaphase and metaphase are set by microtubule depolymerases, whereas oscillation and breathing periods depend on the stiffness of the mechanical linkage between sisters. Metaphase plates become thinner as cells progress toward anaphase as a result of reduced oscillation speed at a relatively constant oscillation period. The progressive slowdown of oscillation speed and its coupling to plate thickness depend nonlinearly on the stiffness of the mechanical linkage between sisters. We propose that metaphase plate formation and thinning require tight control of the state of the mechanical linkage between sisters mediated by centromeric chromatin and cohesion.

Formation of cancerous translocations requires the illegitimate joining of chromosomes containing double-strand breaks (DSBs). It is unknown how broken chromosome ends find their translocation partners within the cell nucleus. Here, we have visualized and quantitatively analysed the dynamics of single DSBs in living mammalian cells. We demonstrate that broken ends are positionally stable and unable to roam the cell nucleus. Immobilization of broken chromosome ends requires the DNA-end binding protein Ku80, but is independent of DNA repair factors, H2AX, the MRN complex and the cohesion complex. DSBs preferentially undergo translocations with neighbouring chromosomes and loss of local positional constraint correlates with elevated genomic instability. These results support a contact-first model in which chromosome translocations predominantly form among spatially proximal DSBs.

BackgroundSensor-based recordings of human movements are becoming increasingly important for the assessment of motor symptoms in neurological disorders beyond rehabilitative purposes. ASSESS MS is a movement recording and analysis system being developed to automate the classification of motor dysfunction in patients with multiple sclerosis (MS) using depth-sensing computer vision. It aims to provide a more consistent and finer-grained measurement of motor dysfunction than currently possible. ObjectiveTo test the usability and acceptability of ASSESS MS with health professionals and patients with MS. MethodsA prospective, mixed-methods study was carried out at 3 centers. After a 1-hour training session, a convenience sample of 12 health professionals (6 neurologists and 6 nurses) used ASSESS MS to capture recordings of standardized movements performed by 51 volunteer patients. Metrics for effectiveness, efficiency, and acceptability were defined and used to analyze data captured by ASSESS MS, video recordings of each examination, feedback questionnaires, and follow-up interviews. ResultsAll health professionals were able to complete recordings using ASSESS MS, achieving high levels of standardization on 3 of 4 metrics (movement performance, lateral positioning, and clear camera view but not distance positioning). Results were unaffected by patients’ level of physical or cognitive disability. ASSESS MS was perceived as easy to use by both patients and health professionals with high scores on the Likert-scale questions and positive interview commentary. ASSESS MS was highly acceptable to patients on all dimensions considered, including attitudes to future use, interaction (with health professionals), and overall perceptions of ASSESS MS. Health professionals also accepted ASSESS MS, but with greater ambivalence arising from the need to alter patient interaction styles. There was little variation in results across participating centers, and no differences between neurologists and nurses. ConclusionsIn typical clinical settings, ASSESS MS is usable and acceptable to both patients and health professionals, generating data of a quality suitable for clinical analysis. An iterative design process appears to have been successful in accounting for factors that permit ASSESS MS to be used by a range of health professionals in new settings with minimal training. The study shows the potential of shifting ubiquitous sensing technologies from research into the clinic through a design approach that gives appropriate attention to the clinic environment.

To elucidate the regulation of kinetochore microtubules (kMTs) by kinetochore proteins in Saccharomyces cerevisiae, we need tools to characterize and compare stochastic kMT dynamics. Here we show that autoregressive moving average (ARMA) models, combined with a statistical framework for testing the significance of differences between ARMA model parameters, provide a sensitive method for identifying the subtle changes in kMT dynamics associated with kinetochore protein mutations. Applying ARMA analysis to G1 kMT dynamics, we found that 1), kMT dynamics in the kinetochore protein mutants okp1-5 and kip3Delta are different from those in wild-type, demonstrating the regulation of kMTs by kinetochore proteins; 2), the kinase Ipl1p regulates kMT dynamics also in G1; and 3), the mutant dam1-1 exhibits three different phenotypes, indicating the central role of Dam1p in maintaining the attachment of kMTs and regulating their dynamics. We also confirmed that kMT dynamics vary with temperature, and are most likely differentially regulated at 37 degrees C. Therefore, when elucidating the role of a protein in kMT regulation using a temperature-sensitive mutant, dynamics in the mutant at its nonpermissive temperature must be compared to those in wild-type at the same temperature, not to those in the mutant at its permissive temperature.

We present an algorithm for the three-dimensional (3D) tracking of multiple fluorescent subresolution tags with super-resolution in images of living cells. Recently, we described an algorithm for the automatic detection of such tags in single frames and demonstrated its potential in a biological system. The algorithm presented here adds to the tag detector a module for relative tracking of the signals between frames. As with tag detection, the main problem in relative tracking arises when signals of multiple tags interfere. We propose a novel multitemplate matching framework that exploits knowledge of the microscope point spread function to separate the intensity contribution of each tag in image regions with signal interferences. We use this intensity splitting to reconstruct a template for each tag in the source frame and a patch in the target frame, which are both free of intensity contributions from other tag signals. Tag movements between frames are then tracked by seeking, for each template-patch pair, the displacement vector providing the best signal match in terms of the sum of squared intensity differences. Because template and patch generation of tags with overlapping signals are interdependent, the matching is carried out simultaneously for all tags, and in an iterative manner. We have examined the performance of our approach using synthetic 3D data and observed a significant increase in resolution and robustness as compared with our previously described detector. It is now possible to localize and track tags separated by a distance three times smaller than the Rayleigh limit with a relative positional accuracy of better than 50 nm. We have applied the new tracking system to extract metaphase trajectories of fluorescently tagged chromosomes relative to the spindle poles in budding yeast.

SummaryAn elegant quantitative model to explain cellular oscillations during cytokinesis reveals a novel function for polar blebbing and raises the question as to why cells live right on the edge.

This paper presents new learning-based techniques for measuring disease progression in Multiple Sclerosis (MS) patients. Our system aims to augment conventional neurological examinations by adding quantitative evidence of disease progression. An off-the-shelf depth camera is used to image the patient at the examination, during which he/she is asked to perform carefully selected movements. Our algorithms then automatically analyze the videos, assessing the quality of each movement and classifying them as healthy or non-healthy. Our contribution is three-fold: We i) introduce ensembles of randomized SVM classifiers and compare them with decision forests on the task of depth video classification; ii) demonstrate automatic selection of discriminative landmarks in the depth videos, showing their clinical relevance; iii) validate our classification algorithms quantitatively on a new dataset of 1041 videos of both MS patients and healthy volunteers. We achieve average Dice scores well in excess of the 80% mark, confirming the validity of our approach in practical applications. Our results suggest that this technique could be fruitful for depth-camera supported clinical assessments for a range of conditions.

The physical separation of a cell into two daughter cells during cytokinesis requires cell-intrinsic shape changes driven by a contractile ring. However, in vivo, cells interact with their environment, which includes other cells. How cytokinesis occurs in tissues is not well understood. Here, we studied cytokinesis in an intact animal during tissue biogenesis. We used high-resolution microscopy and quantitative analysis to study the three rounds of division of the C. elegans vulval precursor cells (VPCs). The VPCs are cut in half longitudinally with each division. Contractile ring breadth, but not the speed of ring closure, scales with cell length. Furrowing speed instead scales with division plane dimensions, and scaling is consistent between the VPCs and C. elegans blastomeres. We compared our VPC cytokinesis kinetics data with measurements from the C. elegans zygote and HeLa and Drosophila S2 cells. Both the speed dynamics and asymmetry of ring closure are qualitatively conserved among cell types. Unlike in the C. elegans zygote but similar to other epithelial cells, Anillin is required for proper ring closure speed but not asymmetry in the VPCs. We present evidence that tissue organization impacts the dynamics of cytokinesis by comparing our results on the VPCs with the cells of the somatic gonad. In sum, this work establishes somatic lineages in post-embryonic C. elegans development as cell biological models for the study of cytokinesis in situ.

SummaryFollowing DNA replication, eukaryotic cells must biorient all sister chromatids prior to cohesion cleavage at anaphase. In animal cells, sister chromatids gradually biorient during prometaphase, but current models of mitosis in S. cerevisiae assume that biorientation is established shortly after S phase. This assumption is based on the observation of a bilobed distribution of yeast kinetochores early in mitosis and suggests fundamental differences between yeast mitosis and mitosis in animal cells. By applying super-resolution imaging methods, we show that yeast and animal cells share the key property of gradual and stochastic chromosome biorientation. The characteristic bilobed distribution of yeast kinetochores, hitherto considered synonymous for biorientation, arises from kinetochores in mixed attachment states to microtubules, the length of which discriminates bioriented from syntelic attachments. Our results offer a revised view of mitotic progression in S. cerevisiae that augments the relevance of mechanistic information obtained in this powerful genetic system for mammalian mitosis.

In Saccharomyces cerevisiae, histone H3 lysine 56 acetylation (H3K56ac) occurs in newly synthesized histones that are deposited throughout the genome during DNA replication. Defects in H3K56ac sensitize cells to genotoxic agents, suggesting that this modification plays an important role in the DNA damage response. However, the links between histone acetylation, the nascent chromatin structure, and the DNA damage response are poorly understood. Here we report that cells devoid of H3K56ac are sensitive to DNA damage sustained during transient exposure to methyl methanesulfonate (MMS) or camptothecin but are only mildly affected by hydroxyurea. We demonstrate that, after exposure to MMS, H3K56ac-deficient cells cannot complete DNA replication and eventually segregate chromosomes with intranuclear foci containing the recombination protein Rad52. In addition, we provide evidence that these phenotypes are not due to defects in base excision repair, defects in DNA damage tolerance, or a lack of Rad51 loading at sites of DNA damage. Our results argue that the acute sensitivity of H3K56ac-deficient cells to MMS and camptothecin stems from a failure to complete the repair of specific types of DNA lesions by recombination and/or from defects in the completion of DNA replication.

The mitotic spindle, due to its striking form, has been imaged for well over 100 years. Composed largely of microtubules and chromosomes, the spindle also contains numerous proteins whose roles include biochemical and biophysical regulation of mitosis. Given the transient, dynamic nature of the spindle, the light microscope continues to be the main tool employed to unlock its mysteries. In this chapter, we will discuss modern light microscopy techniques commonly used for imaging this intricate cellular machine as well as provide examples and protocols. We will also describe some biological preparations and experimental regimes for investigation of the mitotic spindle.

INTRODUCTIONThe development of cloning vectors for green fluorescent protein (GFP) and the simplicity of yeast reverse genetics allow straightforward labeling of yeast proteins in living cells. Budding and fission yeast are therefore attractive organisms in which to study dynamic cellular processes such as growth, cell division, and morphogenesis using live cell fluorescence microscopy. This article focuses on methods to culture, mount, and observe budding yeast cells using three-dimensional (3D) microscopy, but the methods are broadly applicable to other types of cells and other imaging techniques. The emphasis is on 3D imaging, because yeast cells are roughly spherical, and most organelles in yeast move in three dimensions. Three-dimensional imaging also makes it possible to apply image restoration methods (e.g., deconvolution) to obtain sharper images with better definition. This is important, because yeast cells are small (haploid Saccharomyces cerevisiae cells have a diameter of ~4–5 µm) relative to the resolution of even the best optical microscope (~0.25 µm).

Protein dynamics generate adaptive cellular architecture. This concept is exemplified by kinetochores, organelles that orchestrate chromosome segregation during mitosis. In this review, we will focus on protein dynamics at kinetochores and discuss how these dynamics impact chromosome motility during mitosis.