Kinematics-based tracking of cells and fluorescent beads using feature vectors

Tracking of cells or fluorescent beads from images of deforming or developing biological systems is a central challenge in biomechanics. In the former case, the objective is often to find the same cell in a tissue or on a Petri dish that has been imaged before and after time in an incubator. In the latter case, the objective is often to estimate mechanical tractions based upon displacement of fluorescent beads embedded in a defined extracellular matrix. A great number of techniques exist for this purpose, and all face challenges in matching cells and beads from one image to the next and in identifying mismatches. Here, we present a simple, fast, and effective technique for matching cells and beads using “feature vectors” that connect a cell or bead to a set of its nearest neighbors. A generalized feature vector deformation gradient tensor is defined that enables the use of standard kinematics to estimate the maximum likelihood matches between cells or beads in image pairs. We describe the strengths and limitations of the approach and present examples of its application

Understanding Cellular Noise with Optical Perturbation and Deep Learning

Noise plays a crucial role in the regulation of cellular and organismal function and behavior. Exploring noise's impact is key to understanding fundamental biological processes, such as gene expression, signal transduction, and the mechanisms of development and evolution. Currently, a comprehensive method to quantify dynamical behavior of cellular noise within these biochemical systems is lacking. In this study, we introduce an optically-controlled perturbation system utilizing the light-sensitive Phytochrome B (PhyB) from \textit{Arabidopsis thaliana}, which enables precise noise modulation with high spatial-temporal resolution. Our system exhibits exceptional sensitivity to light, reacting consistently to pulsed light signals, distinguishing it from other photoreceptor-based promoter systems that respond to a single light wavelength. To characterize our system, we developed a stochastic model for phytochromes that accounts for photoactivation/deactivation, thermal reversion, and the dynamics of the light-activated gene promoter system. To precisely control our system, we determined the rate constants for this model using an omniscient deep neural network that can directly map rate constant combinations to time-dependent state joint distributions. By adjusting the activation rates through light intensity and degradation rates via N-terminal mutagenesis, we illustrate that out optical-controlled perturbation can effectively modulate molecular expression level as well as noise. Our results highlight the potential of employing an optically-controlled gene perturbation system as a noise-controlled stimulus source. This approach, when combined with the analytical capabilities of a sophisticated deep neural network, enables the accurate estimation of rate constants from observational data in a broad range of biochemical reaction networks.Comment: 33 pages, 4 figure

Viscoelastic spectrum analysis and the identification of a fung viscoelastic material

Despite its many limitations, the Fung “quasi-linear viscoelastic” constitutive model continues to serve as a workhorse of the biomechanics community. A central challenge in applying the model is that it requires a specific form for the relaxation spectrum that is difficult to relate to easily obtained experimental spectra such as a generalized Maxwell relaxation spectrum. Here, we present a simple and general technique for obtaining a from relaxation data a viscoelastic spectrum appropriate to the Fung model. We apply the model to identify several biomaterials that are modeled reasonably by a Fung model, and many more that are not

An approach to quantifying 3D responses of cells to extreme strain

The tissues of hollow organs can routinely stretch up to 2.5 times their length. Although significant pathology can arise if relatively large stretches are sustained, the responses of cells are not known at these levels of sustained strain. A key challenge is presenting cells with a realistic and well-defined three-dimensional (3D) culture environment that can sustain such strains. Here, we describe an in vitro system called microscale, magnetically-actuated synthetic tissues (micro-MASTs) to quantify these responses for cells within a 3D hydrogel matrix. Cellular strain-threshold and saturation behaviors were observed in hydrogel matrix, including strain-dependent proliferation, spreading, polarization, and differentiation, and matrix adhesion retained at strains sufficient for apoptosis. More broadly, the system shows promise for defining and controlling the effects of mechanical environment upon a broad range of cells

Virtual blebbistatin: A robust and rapid software approach to motion artifact removal in optical mapping of cardiomyocytes

Fluorescent reporters of cardiac electrophysiology provide valuable information on heart cell and tissue function. However, motion artifacts caused by cardiac muscle contraction interfere with accurate measurement of fluorescence signals. Although drugs such as blebbistatin can be applied to stop cardiac tissue from contracting by uncoupling calcium-contraction, their usage prevents the study of excitation-contraction coupling and, as we show, impacts cellular structure. We therefore developed a robust method to remove motion computationally from images of contracting cardiac muscle and to map fluorescent reporters of cardiac electrophysiological activity onto images of undeformed tissue. When validated on cardiomyocytes derived from human induced pluripotent stem cells (iPSCs), in both monolayers and engineered tissues, the method enabled efficient and robust reduction of motion artifact. As with pharmacologic approaches using blebbistatin for motion removal, our algorithm improved the accuracy of optical mapping, as demonstrated by spatial maps of calcium transient decay. However, unlike pharmacologic motion removal, our computational approach allowed direct analysis of calcium-contraction coupling. Results revealed calcium-contraction coupling to be more uniform across cells within engineered tissues than across cells in monolayer culture. The algorithm shows promise as a robust and accurate tool for optical mapping studies of excitation-contraction coupling in heart tissue

Confidence Intervals for Concentration and Brightness from Fluorescence Fluctuation Measurements

AbstractThe theory of photon count histogram (PCH) analysis describes the distribution of fluorescence fluctuation amplitudes due to populations of fluorophores diffusing through a focused laser beam and provides a rigorous framework through which the brightnesses and concentrations of the fluorophores can be determined. In practice, however, the brightnesses and concentrations of only a few components can be identified. Brightnesses and concentrations are determined by a nonlinear least-squares fit of a theoretical model to the experimental PCH derived from a record of fluorescence intensity fluctuations. The χ2 hypersurface in the neighborhood of the optimum parameter set can have varying degrees of curvature, due to the intrinsic curvature of the model, the specific parameter values of the system under study, and the relative noise in the data. Because of this varying curvature, parameters estimated from the least-squares analysis have varying degrees of uncertainty associated with them. There are several methods for assigning confidence intervals to the parameters, but these methods have different efficacies for PCH data. Here, we evaluate several approaches to confidence interval estimation for PCH data, including asymptotic standard error, likelihood joint-confidence region, likelihood confidence intervals, skew-corrected and accelerated bootstrap (BCa), and Monte Carlo residual resampling methods. We study these with a model two-dimensional membrane system for simplicity, but the principles are applicable as well to fluorophores diffusing in three-dimensional solution. Using simulated fluorescence fluctuation data, we find the BCa method to be particularly well-suited for estimating confidence intervals in PCH analysis, and several other methods to be less so. Using the BCa method and additional simulated fluctuation data, we find that confidence intervals can be reduced dramatically for a specific non-Gaussian beam profile

The epidemiology of chickenpox in England, 2016-2022: an observational study using general practitioner consultations

Chickenpox is a common childhood disease caused by varicella-zoster virus (VZV). VZV vaccination is not part of the UK childhood immunisation programme, but its potential inclusion is regularly assessed. It is therefore important to understand the ongoing burden of VZV in the community to inform vaccine policy decisions. General practitioner (GP) chickenpox consultations were studied from 1 September 2016 to 9 December 2022. Over the study period, the mean weekly chickenpox consultation rate per 100,000 population in England was 3.4, with a regular peak occurring between weeks 13 and 15. Overall, rates decreased over time, from a mean weekly rate of 5.5 in 2017 to 4.2 in 2019. The highest mean weekly rates were among children aged 1-4 years. There was no typical epidemic peak during the COVID-19 pandemic, but in 2022, rates were proportionally higher among children aged < 1 year old compared to pre-pandemic years. Chickenpox GP consultation rates decreased in England, continuing a longer-term decline in the community. The COVID-19 pandemic impacted rates, likely caused by the introduction of non-pharmaceutical interventions to prevent SARS-CoV-2 transmission. The lasting impact of the interruption of typical disease transmission remains to be seen, but it is important to monitor the chickenpox burden to inform decisions on vaccine programmes

Report of the Regional Coordination Meeting for the North Atlantic (RCM NA) 2015

The 12th RCM North Atlantic was held in Hamburg (Germany) 14-18 September 2015. The main purpose of the RCM is to coordinate the National Programmes (NP) of the Member States (MS) in the North Atlantic region. National Programmes for 2011-2013 have been rolled over for the period 2014-2016. Therefore, the main focus at this year was to improve regional data collection, analysis and storage and the evolution towards Regional Coordination Groups (RCG).The impact of the introduction of the landing obligation and preparations for its implementation was also discussed taking into account possible changes in scientific sampling schemes. The participation of four National Correspondents make possible to address National administration issues related to the oncoming EU MAP. A data call was launched by the chairs of the RCM NA, RCM Baltic and RCM NS&EA where MS were requested to upload data for 2014 in the regional database (RDB Fishframe) hosted by ICES. All MS except France and Northern Ireland complied with this request on landings and effort data. All MS except France uploaded sample data for 2014. French data were available for the meeting using a web base interface. Evaluation of the data call for submission data to the RDB revealed the numbers of species in landings and sample data and the numbers of metiers in effort data are in general data stable. RCM NA see big improvements in the work MS are doing regarding data calls coming from a situation where some countries didn´t provide any data to a new scenario where everyone is providing data; at the same time the overall quality has significantly improved, which is a large step forward. Regional data collection, analysis, storage and the evolution towards Regional Coordination Groups (RCG). Optimizing and harmonizing fisheries management across MS is dependent on improving regional coordination. The group discussed various needs and aspects relevant for facilitating future work of the RCM. Future tasks for the RCM don’t differ much from the current tasks. The discussion was focused on the structure of the RCGs, funding and short term needs to address tasks in an efficient way in the future. Regional coordination encompasses many different aspects, ranging from regional cooperation, sampling design, quality control procedures, data storage and analysis to the actual coordination, reporting and accountancy. Current task sharing and coordination procedures as well as future mechanisms are partially covered under the current MARE study 2014/19 (FISHPI). The project and its progress were presented to the group. The outcomes of this study will demonstrate future procedures based on case studies. As substantial effort and costs are involved to facilitate the process of regional coordination, the group highlighted the importance to access to budgets to cover these costs. Development of the RDB is also crucial for future work of the RCGs; funds are needed for the development. Additionally, RCM NA identified 4 supra regional topics where work can be done intersesionally in cooperation with the rest of RCMs: (1) Cost sharing of funding surveys; (2) Impact of landing obligation; (3) reviewing the ICES list of data needs ; and (4) review and follow up on RDB upload logs. Due to the importance to moving to a regional catch sampling scheme, an exercise was realised using the distribution of landings by harbour and fleet segment as a proxy of sampling frames that could hypothetically operate in a regional probability based design. The exercise was based on landing weight, for the simple reason that this was the only complete variable that was available for all the various national data sets. A regional sampling design can however be optimized in any number of ways (e.g. by landings value, by métier diversity, by species diversity, by number of fishing trips). The aims and aspirations of the end users need to be defined to ascertain which is most appropriate. It is one of the overriding advantages of a regional sampling design (as opposed to the aggregation of national designs) that the overall coverage can be set out to achieve regional goals. The RCM NA analyzed and discussed the main achievements of WKISCON2. It was clear that concurrent sampling at-sea is a long-established practice in most MS and that, where it was applied, concurrent sampling of fishing trips on-shore resulted in substantial increases in species collected without jeopardizing the main uses of data. Stock assessment and discard estimation and management are the major current uses of concurrent sampling data. Concurrent sampling has also been providing other benefits than its initial reason, such as advice to local, national and international authorities, research on MSFD descriptors, mixed fisheries and gear interactions and on mortality of rare species, data-poor stocks and PETS. It was clear that concurrent sampling being a statistically valid method for species selection which has proven to fulfil different end-users needs, implementation constraints hinder concurrent sampling on-shore. Thus, in order to meet end-users needs and to overcome the constraints that may arise from the implementation of concurrent sampling in some countries, particularly on-shore, RCM NA considers that different statistically sound approaches other than concurrent sampling must be developed to be tested in the field, so they may provide useful alternatives. Introduction of the landing obligation and its impact in the implementation in scientific sampling schemes. In terms of evaluating the impact of the introduction of the Landing Obligation (LO) regulation on data collection, there is only limited experience as the current implementation only covers Pelagic and Industrial fisheries in this region but MS have or are preparing for the implementation where they can. It is currently perceived that this year is a transition period for the pelagic fisheries and that these fisheries and control agencies are not fully implementing the LO (managing but not enforcing). As a result MS did not have a lot of comments on the current year and are in general preparing for next year. During the meeting it was decided to gather further information to address this issue by getting member states who were present to fill in a table on “Monitoring the impact of the landing obligation on data collection in the North Atlantic region” outlining the current state of play. This table could be considered as a live document which should be filled in year by year as the Landing Obligation is phased in. This table will then serve to provide an historical record as countries can document the changes year by year and will also provide guidance and act as a learning tool to all member states on how other countries are implementing the LO. National administrations The group discussed the proposal for task sharing and criteria for joint surveys. RCM NS&EA and RCM NA 2014 discussed a cost model for the present joint MS financed surveys and for future joint surveys. In addition to this model, the RCM NA 2015 highlighted that four categories of surveys should be considered in relation to task sharing and criteria for joint surveys. In the light of cost sharing, the group commented that the current DCF recast proposal refers to ‘exploitation of stocks’ rather than EU TAC or landings. Given the relative stability, EU TAC shares are the preferred basis for sharing costs. The exploitation of stocks shall be interpreted as EU-TAC share as a default. In specific cases, RCGs can in the future agree on different interpretation where needed and feasible. Fully agreement among the group was concerning to the engagement and participation of National Correspondent (NC) in this meeting. The future role of the NCs in the RCG context was discussed, indicating a formal role for the NCs in the RCG process to approve and agree on regional arrangements. However, the current recast of the DCF doesn’t include the formal involvement of the NCs in the coordination procedures and meetings. RCM NA highlights this as potentially problematic for the foreseen formal role of the NCs. Other items on the agenda were the consideration of the follow up of relevant recommendations made last years by Liaison Meeting and presentations and relevant development from ICES, EC and SC-RDB

The Epidemiology of Chickenpox in England, 2016-2022: An Observational Study Using General Practitioner Consultations.

Chickenpox is a common childhood disease caused by varicella-zoster virus (VZV). VZV vaccination is not part of the UK childhood immunisation programme, but its potential inclusion is regularly assessed. It is therefore important to understand the ongoing burden of VZV in the community to inform vaccine policy decisions. General practitioner (GP) chickenpox consultations were studied from 1 September 2016 to 9 December 2022. Over the study period, the mean weekly chickenpox consultation rate per 100,000 population in England was 3.4, with a regular peak occurring between weeks 13 and 15. Overall, rates decreased over time, from a mean weekly rate of 5.5 in 2017 to 4.2 in 2019. The highest mean weekly rates were among children aged 1-4 years. There was no typical epidemic peak during the COVID-19 pandemic, but in 2022, rates were proportionally higher among children aged < 1 year old compared to pre-pandemic years. Chickenpox GP consultation rates decreased in England, continuing a longer-term decline in the community. The COVID-19 pandemic impacted rates, likely caused by the introduction of non-pharmaceutical interventions to prevent SARS-CoV-2 transmission. The lasting impact of the interruption of typical disease transmission remains to be seen, but it is important to monitor the chickenpox burden to inform decisions on vaccine programmes

Cellular and Matrix Mechanics of Bioartificial Tissues During Continuous Cyclic Stretch

Bioartificial tissues are useful model systems for studying cell and extra-cellular matrix mechanics. These tissues provide a 3D environment for cells and allow tissue components to be easily modified and quantified. In this study, we fabricated bioartificial tissue rings from a 1 ml solution containing one million cardiac fibroblasts and 1 mg collagen. After 8 days, rings compacted to <1% of original volume and cell number increased 2.4 fold. We initiated continuous cyclic stretching of the rings after 2, 4, or 8 days of incubation, while monitoring the tissue forces. Peak tissue force during each cycle decreased rapidly after initiating stretch, followed by further slow decline. We added 2 μM Cytochalasin-D to some rings prior to initiation of stretch to determine the force contributed by the matrix. Cell force was estimated by subtracting matrix force from tissue force. After 12 h, matrix force-strain curves were highly nonlinear. Cell force-strain curves were linear during loading and showed hysteresis indicating viscoelastic behavior. Cell stiffness increased with stretching frequency from 0.001–0.25 Hz. Cell stiffness decreased with stretch amplitude (5–25%) at 0.1 Hz. The trends in cell stiffness do not fit simple viscoelastic models previously proposed, and suggest possible strain-amplitude related changes during cyclic stretch