Advances in computation and neuronal modeling have enabled the study of entire neural tissue systems with an impressive degree of biological realism. These efforts have focused largely on modeling dendrites and somas while largely neglecting axons. The need for biologically realistic explicit axonal models is particularly clear for applications involving clinical and therapeutic electrical stimulation because axons are generally more excitable than other neuroanatomical subunits. While many modeling efforts can rely on existing repositories of reconstructed dendritic/somatic morphologies to study real cells or to estimate parameters for a generative model, such datasets for axons are scarce and incomplete. Those that do exist may still be insufficient to build accurate models because the increased geometric variability of axons demands a proportional increase in data. To address this need, a Ruled-Optimum Ordered Tree System (ROOTS) was developed that extends the capability of neuronal morphology generative methods to include highly branched cortical axon terminal arbors. Further, this study presents and explores a clear use-case for such models in the prediction of cortical tissue response to externally applied electric fields. The results presented herein comprise (i) a quantitative and qualitative analysis of the generative algorithm proposed, (ii) a comparison of generated fibers with those observed in histological studies, (iii) a study of the requisite spatial and morphological complexity of axonal arbors for accurate prediction of neuronal response to extracellular electrical stimulation, and (iv) an extracellular electrical stimulation strength–duration analysis to explore probable thresholds of excitation of the dentate perforant path under controlled conditions. ROOTS demonstrates a superior ability to capture biological realism in model fibers, allowing improved accuracy in predicting the impact that microscale structures and branching patterns have on spatiotemporal patterns of activity in the presence of extracellular electric fields. In the study of extracellular electrical stimulation of neural systems, spatial and temporal patterns of activity are strongly influenced by tissue geometry. One established approach to studying this relationship is through morphologically detailed equivalent circuit models of neurons, including axons. While these models are invaluable for many different applications, they are especially useful for prediction of tissue response to extracellular stimulation, where explicit morphologies aid the prediction of activation thresholds under varying biological and stimulating conditions , 1998. FDespite an understanding that geometry and topology influence activity, much of the biological realism in these studies is reserved for dendritic rather than axonal arbors. This lack of realism in axonal fibers becomes especially disconcerting when considering both that (i) central nervous system axon terminal arbors are often highly branched and tortuous relative to the mostly straight and long nerves of the periphery, and (ii) under most typical stimulating conditions, axons have shorter chronaxies than somas and dendrites . It follin situ cell density at tissue scale. This becomes particularly apparent when considering that the unique geometry of the dentate gyrus, which changes from septal to temporal poles, requires dramatic inter-region variety in the volume, orientation, and contour of afferent EC axons.Despite the long record of hippocampal observation, axonal morphology is not as scrupulously described as the somas and dendrites of many cell types. With a few exceptions, studies yielding explicit reconstructions through morphometric analysis of neuronal branching have focused on dendritic arbors and overlooked their axons . The deaDespite the general absence of morphometrics for axons, the shape of terminal fields, distribution of synaptic spines, and a rough measure of anatomic domains of axon terminal fields from either histology or other imaging methods provide data from which minimally functional axons can be grown. Explicit dendritic reconstructions, spine counting, and anterograde and retrograde staining experiments provide information regarding synaptic targets. Distributions of synaptic targets combined with a knowledge of the general path and origin of a fiber is sufficient to generate a functional structure that captures the tertiary conformation and local divergence of the axon terminal arbor. When representing a neural process as a graph, nodes placed at synapses and edges to connect between them and the soma can effectively reconstruct a functional dendritic arbor . LikewisBeyond minimally functional morphologies, sophisticated models of realistic neuronal branching have been proposed with primary application to either generating unique and artificial dendritic trees or reconstructing them from a series of images. There are two chief types of generative models: (i) stochastic  or (ii) Consequently, we present in this paper a new graph-based algorithm for generating biologically realistic tree representations of axon terminal arbors. The proposed model is inspected for its utility in studying extracellular electrical stimulation of cortical tissue through analysis of the impact of arbor topography and morphometry on activation thresholds and, by extension, spatiotemporal patterns of activity in the hippocampus. The results of this work comprise (i) a quantitative analysis of the generative algorithm proposed, (ii) presentation and quantitative/qualitative description of generated fibers, (iii) comparison to leading alternative methods, (iv) demonstration of a method to reduce spatial complexity of axonal arbors while maintaining accurate prediction of neuronal response to extracellular electrical stimulation, and (v) an extracellular electrical stimulation strength–duration study. The value of these studies is twofold: (i) establishing the novelty and utility that this modeling system yields and (ii) determining if stimulation–response recruitment order  for straight, long, large-diameter, and myelinated peripheral fibers is similarly true of small, highly branched, and unmyelinated cortical fibers.en passant connective schema, where most pre-synaptic boutons are non-terminal. Many more features and their sources are detailed in The study presented here focuses on accurately capturing the emergent spatial features of spiny stellate EC axons within the dentate gyrus in Sprague-Dawley rats. The model is designed to be flexible to the inclusion of novel morphometric criteria as new experimental data become available but, at present, it is clearly important that (i) fibers are constrained to 1–1.5 mm within the septotemporal axis , (ii) laIn the construction of a spatial ordered tree, the number and topography of target nodes strongly determines the emergent features of the resulting graph. Therefore, the selection of nodes is an important step in the successful generation of biologically appropriate axonal trees. This process was executed using the Kjoenigsen rat hippocampal atlas slice at −3.34 from Bregma to segment model boundaries . Past efTo summarize: the number of synaptic spines in the outer and middle third of hippocampal granule cell dendritic arbors the size, number, and density of granule cells, and the number/density of EC cells contributing to the perforant path provide the necessary arithmetic for deducing the number of synapses made within each axon terminal field . When conn−2)( trees that span a set of targets (n), few of which are biologically plausible  or 5,900 (three synapses per target cell) target cells rather than 17,700 target cells and used a single node from each to guide arbor growth. Perforant path fibers were generated using each of these node counts and then each was also line simplified. The Ramer–Douglas–Peucker (RDP) algorithm for line simplification was used on each of these trees to determine the minimum node count required to approximate full complexity fibers  according to Eq. 1.The TREES toolbox, like ROOTS, depends upon carefully selected nodes or points to grow a graph; therefore, spanning trees generated by this method can use the same set of synaptic or cellular targets as those utilized by the proposed system. Unlike ROOTS, however, the only parameter that can be adjusted to improve the performance of the resulting morphology is the balancing factor (BF). This BF represents the weighting of priority for path length versus conductive delay in the spanning tree that is generated. To study this approach, a new topology grown via TREES utilizing the same control points as a ROOTS fiber. The BF was calibrated by minimizing a multi-objective function (MOF), formulated as an unweighted sum of independently normalized mean root square error for each of the following morphometrics: Euclidean distance/path-length (BF), branch order, bifurcation angle, and total path length. The BF of the arbor generated by TREES was set at the value that minimized the difference between the MOF scores of the arbors generated by each system. To maximize similarity between these two morphologies through calibration of the TREES BF, direct (Euclidean) vs. path length ratios were calculated for each carrier node, then a histogram of these data was fit with a kernel density estimate (KDE via Gaussian smoothing). This process was repeated for path length, branching order, branch length, and branching angle. The KDEs for direct vs. path length and branching angle for the TREES axon were subtracted from those for the ROOTS fiber. These differences were independently normalized and the RMSE was calculated. Each of these metrics were summed without any weighting. The TREES BF was then calibrated through minimizing the summed normalized root mean-squared error of these differences , branching order (dBO), total path length (dPL), branch length (dBL), and bifurcation angle (dBA). Unlike TREES and the proposed algorithm, L-NEURON doesn’t rely on a preselection of target nodes to construct a topology, but rather depends upon measures of branching structure of a tree, or morphometrics. Fitted distributions to these measurements are then stochastically sampled to grow a tree. This approach is intended to capture branching patterns without much regard for the emergent spatial features of a tree. Because of the lack of an extensive database of EC axons from which to take branching measurements, we resorted to using the companion tool L-MEASURE to extract morphometrics from one of our own generated arbors to gauge the feasibility of using stochastic methods to generate axonal arbors when such a dataset does become available . ExtractVirtual arbors were ultimately evaluated based on their ability to capture known spatial features of EC axon terminal fields, including a complex geometry which conforms to the contours of the dentate gyrus.en passant fibers. Because fiber diameter has been shown to influence both conduction velocity and excitability, it was important to explore the response characteristics of fibers with this sub-micron variation in diameter  with widths between 0.025 and 1.4 ms were delivered at each of the two distances. The extracellular voltage throughout the model space was estimated using an analog of Coulomb’s law with material resistivity of 3.8 Ω-m (Eqs 2 and 3) .I. The conductance, σ, is the inverse of resistivity. Radial distance, r, is found by Eq. 3:where Φ is the field potential resulting from a current source, By adding new voltage sources in series with the circuital elements representing each section of membrane, extracellular potentials calculated via Eqs 2 and 3 were applied to neuronal compartments within the NEURON model using the “extracellular” mechanism. Stimulation was delivered over a range of current amplitudes designed to cover, at a minimum, rheobase to twice rheobase for each set of stimulating conditions.The second analysis examined the same models and stimulating setup but focused on the temporal distribution of the response at rheobase. The time it took each compartment in the fiber to reach action potentiation was recorded, used to populate a histogram, and then Gaussian smoothed to present a KDE. These plots were used to identify the impact of boutons on conduction velocity throughout the complex arbor.In order to demonstrate the differences between the relatively sophisticated axonal models grown via ROOTS and more simple axon representations, a simple distance-based delay was estimated based on the average conductance velocities of the ROOTS fibers and visualized using KDEs after the manner previously described. The distance-based conduction delay mechanism just explained is described in figures and relevant results sections as a “pure-delay” mechanism because no explicit cable model is used to approximate the delay. To clearly demonstrate the differences that are not readily seen in the KDE, pair-wise differences  of compartmental APs using ROOTS and the pure-delay mechanism were measured and plotted.Neural Roots (Roots)1,2 along with concise documentation. Neural Roots was written with Python 3.6.9 and has a few non-standard dependencies, including: SciPy, NetworkX  a qualitative and quantitative assessment of the proposed algorithm, (ii) a comparative analysis with alternative methods, and (iii) the presentation of strength–duration curves and an analysis of EC fiber recruitment order and temporal distribution of the response to extracellular stimulation.There are two chief loops within the core ROOTS algorithm: branch extension and bifurcation. As the algorithm proceeds, the complexity of branch extension decreases while that of bifurcation increases, this can be seen in Consideration of the complexity of the resulting fiber is essential because simulation of just a few of these fibers at full complexity could be highly taxing on a single-processor computer. Ramer–Douglas–Peucker line simplification was performed on axons with two or three synapses per target granule cell . en passant synapsing; presentation of both DG and CA3 terminal fields with the preservation of a fissure between the two fields; and finally, the tree structure passed through a plausible topography of DG and CA3 arbor domains which enables in situ levels of connectivity.Example graph models of EC axon terminal fields which incorporate these simplifications generated by the proposed algorithm are presented in A morphology generated via our own graph-based algorithm  was seleen passant connection schema which typifies EC axons of the perforant path, where terminal branchlets are reportedly rare. Attempts were also made to generate accurate morphologies using the TREES MatLab toolbox. These efforts yielded trees superior to those generated by the L-Neuron method, though still deficient in important ways. An axon generated by TREES was selected for quantitative and qualitative comparison with that yielded by earlier analysis using the ROOTS system . Each tree was constructed using the same set of target nodes; therefore, all differences in patterns of branching arise from differences between the two algorithms. The TREES BF was then calibrated through minimizing a MOF, attempting to find the best possible match between TREES and ROOTS arbors. This process yielded a functional axon from the TREES algorithm, though differences remained that could not be resolved through manipulation of the BF alone . DespiteAn additional challenge not satisfied by the TREES approach is that of performing acute turns. A portion of the perforant path innervates the CA3/CA2 region of the hippocampus and therefore must be able to extend from the end of the enclosed blade of the dentate into these other domains. The algorithm presented in To determine the importance of including boutons when simulating extracellular electrical stimulation of axon models, strength–duration curves for three diameter patterns of a ROOTS arbor  of this modeling approach and the study presented in this paper is the lack of an extensive dataset describing EC axons in the perforant path, including rich morphometrics. Existing explicit reconstructions have readily identifiable errors and, therefore, should not be used as solitary sources of branching morphometrics or be virtualized and used in stimulation models without sophisticated automated and/or manual repair . The issWhile the present study espouses a ROOTS, there are many algorithms that might achieve topological characteristics reminiscent of neuronal branching.Although existing stochastic models may be effective for dendritic arbor generation, they are not naturally adapted to the construction of axonal morphologies because they fail to reliably conform to pre-determined geometries or volumes that are irregular or highly non-symmetrical . The abiWhen guided by serial histological sections, spanning-tree algorithms provide an efficient approach to reconstructing spatial trees. However, without some modification, traditional minimum-spanning-tree algorithms can result in spatial error and distorted branching patterns . Using hIt is plausible that established stochastic methods, known to be highly proficient at recapitulating tree morphometrics, could be modified to allow directedness and improved conformation to terminal field volumes. Despite these feasible and time-worthy alternatives, the model presented in this study has demonstrated sufficient performance in terms of accuracy, flexibility, and computational complexity.While many alternatives to RDP for tree simplification exist, this method was preferable to other node cluster-and-merge methods  because these alternatives have the potential to introduce large differences in branching patterns, creating additional challenges in evaluating the equivalence of simplified and unsimplified fibers. Evaluation of the RDP simplification approach could be done in a very straightforward fashion because line simplification doesn’t result in changes in branching patterns—simplified arbors were compared to the reference case on the basis of path length and subjective evaluation of an acceptable RDP-epsilon, or the maximum distance between original and simplified contours.Analysis in this paper involving extracellular field estimations used analytical methods that fail to completely account for anisotropy and heterogeneous resistivity of the tissue volume or full-wave propagation of electric fields. More sophisticated field estimation techniques  that account for complex impedance or by filtering point-source estimations to account for amplitude dampening and phase-shift of stimuli could have been used but were deemed unnecessarily complex for the principle questions under examination in this article . It shouThe study of strength versus duration of stimulus in the measurement of activation thresholds as performed in this paper does not provide conclusive data which concretely establishes the connection between fiber size and excitability for hippocampal networks. This is due to the realistic elements still missing from the models used, including non-uniform diameters of fibers, irregular bouton geometry and volumes, varying bouton topography, and an electrophysiological study of any differences in channel density and dynamics between boutoned and non-boutoned axon regions. Despite these limitations, it seems important to consider what impact boutons may have on fiber excitability within the terminal fields of small, highly branched, and unmyelinated axon fibers. Importantly, these results imply that action potentials in extracellular anodally stimulated axon terminal fields are initiated in boutons with lower input resistance than nearby portions of the fiber and that failing to consider the impact of boutons on conduction velocity will likely result in meaningful temporal errors. However, thorough experimental work is needed to confirm this possibility. It is further valuable to recognize that for long pulses delivered at more distant locations uniformly large diameter fibers approximate the vastly more complex fully boutoned fibers quite well and may provide a viable approach to reducing the computational demands of models that include detailed axon terminal arbors.While much remains to be done, this study represents a step forward for detailed computational modeling of complex neuronal systems. Where previous models were either focused on peripheral axons with less complex arbors or used sophisticated methods to generate dendritic trees but neglected axons altogether, the model presented here demonstrates an approach to constructing functional axonal morphologies that can be used for diverse applications, including extracellular electrical stimulation of the cortex. It should be noted that while ROOTS is itself general, expert knowledge of the tissue system being virtually recapitulated is required: branching criteria and synaptic or cellular targets must be provided to the tool. Critical morphometric parameters include bifurcation angles, branch extension angle, and internode length; these must be independently determined by users. Despite these requirements, ROOTS presents many opportunities to increase the sophistication of model-based studies of neural tissue system dynamics and central nervous system neuromodulating devices such as deep brain stimulation or potential hippocampal memory prostheses . ROOTS cAs computer technology matures, constructing virtual models of brain parts has become an increasingly valuable approach to understanding how patterns of activity emerge in different neuronal structures. Many efforts to model populations of neurons have emphasized the implementation of biological realism for cell bodies and dendrites while settling for simplistic representations of axons. This neglect leads to potentially large errors in predictions of when and where synaptically driven activity in a neural circuit might occur. To address this concern, we have developed a novel algorithm called ROOTS to generate biologically realistic axon models for use in computer simulations of the brain. With realistic axons in place, such models can be used to predict how different regions of the brain respond to stimulation from implanted electrodes as part of a prosthetic device. This improvement in the realism of tissue models of the brain will provide superior support to on-going work to reveal the mechanisms of brain disorders and to optimize devices or drugs that are used to treat them.All datasets generated for this study are included in the article/CB designed the models and algorithms, conducted the analysis, and prepared the manuscript. AM and J-MB provided support for the design, analysis, and manuscript preparation. J-MB, DS, GL, and TB oversaw the project scope, project conception, and worked to maintain funding to support the project.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Studies in high-income countries have documented a consistent gradient between socio-economic status (SES) and high blood pressure (HBP), a key risk factor for cardiovascular disease (CVD). However, evidence from Latin American countries (LA) remains comparatively scarce and inconclusive.Encuesta de Salud, Bienestar y Envejecimiento) (SABE) conducted in 2015. SES was measured by educational achievement and household assets. CVD risk factors included objectively measured HBP and body mass index (BMI), as well as behaviors .Data for 3,984 individuals came from a nationally representative survey of individuals aged 60 years or above in Colombia . Being in the highest asset quartile (most affluent) is associated with a 44% lower risk  of HBP compared to those in the lowest asset quartile (most deprived). Individuals with lower SES are more likely to smoke, not engage in regular physical activity and not regularly consume fruits or vegetables. In contrast, individuals with higher SES are more likely to consume alcohol and, those with more assets, more likely to be obese.Among older Colombians there exists a marked SES gradient in HBP as well as several additional risk factors for CVD. The results highlight the importance of a public health approach towards HBP and additional CVD risk factors that takes into account the specific conditions of older individuals, especially among disadvantaged groups. The prevalence of chronic non-communicable diseases (NCD) is increasing around the world, representing a substantial share of health care expenditures as well as morbidity and mortality in low and middle-income countries (LMICs).  Besides A critical feature of the rising epidemic of NCDs is the presence of growing socio-economic inequalities by which the disease burden is becoming increasingly concentrated in disadvantaged groups. ,11 The eIn contrast, evidence on SES inequalities in HBP and additional risk factors for CVD in LA remains comparatively scarce and conflicting. Existing studies among older individuals in LA suggest that SES is either associated with HBP,  only incEncuesta de Salud, Bienestar y Envejecimiento  this study systematically assesses the relation between SES , objectively measured HBP and additional risk factors for CVD including obesity, physical activity, fruits and vegetable intake, smoking and alcohol consumption.Using representative data for individuals aged 60 years and above from the Data from this study came from the SABE 2015 survey.  The latthttps://github.com/PhilippHessel/Colombia_SES_CVD_OlderIndividuals).The SABE data can be requested from the Colombian Ministry of Health. Although the user agreement does not permit sharing the data directly, under the following link we include the data request form, detailed instructions on how the data can be requested, as well as complete replication materials  none, 2) primary (incomplete or complete), 3) secondary or post-secondary (complete or incomplete).Assets were measured based on a summative index indicating whether individuals’ households possessed a functioning version of each of the following  items orFor assessing prevalence of HBP we relied on assessments of both systolic blood pressure (SBP) as well as diastolic blood pressure (DBP). Both were assessed using an electronic manometer  and recorded in millimeter of mercury (mm Hg). The obtained values were recorded after five minutes of resting in the sitting position, with three consecutive measures being obtained, while waiting at least 30 seconds between each measure. Following the manual of the Colombian Ministry of Health,  individu2). Individuals were classified as being overweight if their corresponding BMI was > 30 kg/m2. Additional risk factors for CVD included in this study include excessive alcohol consumption, low consumption of fruits and vegetables, lack of physical activity, smoking as well as being overweight. Alcohol consumption was operationalized in the form of a binary variable distinguishing between respondents not consuming any alcohol and those drinking alcohol at least once a week or more often. Consumption of fruits or vegetables was measured in the form of a binary variable distinguishing individuals that consume fruits or vegetables at least twice a day from those that consume fruits or vegetables less frequently. Lack of physical activity was measured by a binary variable capturing whether or not a person participated at least three times per week in any sports or physical activity. Smoking was operationalized by distinguishing individuals who currently smoked with those that either never smoked or those that had smoked in the past.Prevalence of obesity was measured based on anthropometric measures of body height and weight using a portable stadiometer (SECA 213) and an electronic scale . Based on the measures of height and weight we calculated individuals’ body mass index (BMI) in units of kilograms (kg) by the square of body height in meters (mCovariates include age (in years), gender as well as region of residence. The latter represent the five census regions of Colombia .± standard deviations (SD) or the distribution of its sub-categories in percent (%) , separately by gender  are shown in As The aim of this study was to systematically assess associations between SES and HBP as well as additional risk factors for CVD among older individuals in Colombia, drawing on nationally representative data from the SABE survey. The results show that there exists a marked SES gradient in HBP among older individuals. Thus, older individuals with higher SES have a substantially lower risk of suffering from HPB compared to those with lower SES. Having higher SES also is associated with lower likelihood of unhealthy behaviors in terms of smoking, fruits or vegetable consumption as well as physical activity. In contrast, individuals with higher SES are more likely to consume alcohol and, those with more assets, more likely to be obese.The results of this study—showing a consistent and marked SES gradient in HBP across all SES categories—are consistent with studies in high-income countries and a study among older individuals in Brazil . HoweverThe absence of a clear SES gradient in HBP in various LA countries may be the result of several factors. On the one hand, selection effects at higher ages due to which individuals with lower SES are less likely to survive to higher ages, may explain a lower SES gradient among older individuals. ,30 Such With regard to the results for additional CVD risk factors the results of this study are in line with a systematic review on the relation between SES and risk factors for NCDs in LMICs showing that individuals with low SES are more likely to consume less fruits and vegetables, engage in less physical activity and are more likely to smoke compared to their counterparts with higher SES.  The findThe existence of a substantial SES gradient in HBP and several additional risk factors for CVD among older individuals in Colombia is highly relevant in the context of the Rio Political Declaration on Social Determinants of Health. ,37 AmongSome limitations should be considered. First, due to its cross-sectional nature, the results of this study only reveal associations between SES and CVD, as well as associated risk factors. In consequence, the results have no causal interpretation, i.e. they do not suggest that there would exist a causal relation between SES and health or related behaviors. Secondly, although the data from the SABE study are nationally representative, with a comparatively large sample size, information on blood pressure was only collected for a sub-sample of respondents. Respondents were randomly invited to participate in the blood pressure assessment. However, supplementary analyses reveal that the sub-sample for which data on blood pressure is available differs slightly in terms of observed characteristics from the remaining sample. With regard to average educational achievement and number of disposable household assets, the sub-sample that includes data on blood pressure is slightly more educated  and has a slightly larger number of disposable assets . Although both differences are statistically significant, overall, they are relatively small. With regard to the main research question of this study, the circumstance that individuals with higher SES are slightly over-represented in the sub-sample used in this study, in tendency, should result in an under-estimation of the SES gradient in terms of health and health behaviors, rather than an over-estimation.This study is the first to assess inequality in key risk factors for CVD according to SES among a representative sample of older individuals in Colombia. In contrast to results from other LA countries, inequalities among older individuals in Colombia are substantial, revealing that individuals with lower SES bear a significantly larger burden of HBP than their counterparts with higher SES. The results emphasize that a comprehensive public health approach for CVD that aims to reduce SES inequality among older individuals should take into account specific risk factors and needs for care of disadvantaged groups.S1 Appendix(DOCX)Click here for additional data file.


Propensity score techniques can reduce confounding and bias in observational studies. Such analyses are able to measure and balance pre-determined covariates between treated and untreated groups, leading to results that can approximate those generated by randomized prospective studies when such trials are not feasible. The most commonly used propensity score -based analytic technique is propensity score matching (PSM). Although PSM popularity has continued to increase in medical literature, improper methodology or methodological reporting may lead to biased interpretation of treatment effects or limited scientific reproducibility and generalizability. In this study, we aim to characterize and assess the quality of PSM methodology reporting in high-impact otolaryngologic literature.PubMed and Embase based systematic review of the top 20 journals in otolaryngology, as measured by impact factor from the Journal Citations Reports from 2012 to 2018, for articles using PSM analysis throughout their publication history. Eligible articles were reviewed and assessed for quality and reporting of PSM methodology.Our search yielded 101 studies, of which 92 were eligible for final analysis and review. The proportion of studies utilizing PSM increased significantly over time (p < 0.001). Nearly all studies  specified the covariates used to calculate propensity scores. Covariate balance was illustrated in 67.4% (n = 62) of studies, most frequently through p-values. A minority  of studies were found to be fully reproducible according to previously established criteria.While PSM analysis is becoming increasingly prevalent in otolaryngologic literature, the quality of PSM methodology reporting can be improved. We provide potential recommendations for authors regarding optimal reporting for analyses using PSM. Randomized controlled trials (RCTs) provide the highest level of evidence when examining the effects of particular exposures or interventions on an outcome of interest. However, despite their ability to minimize selection bias and confounding, surgical RCTs are often viewed as expensive, challenging to conduct, limited in scope, and subject to extensive ethical considerations or debate –3. TheseIn an attempt to improve comparisons between cohorts in observational studies, statistical methodologies have been developed in order to reduce confounding when randomization is not possible . The mospropensity scores as a way to measure and balance baseline characteristics between two groups. A propensity score is defined as the probability (0 to 1) of receiving a treatment based on recorded baseline characteristics of an individual ) (Table 5).Employing univariate logistic regression to assess associations between study PSM reporting characteristics and findings of significant results, we found studies that were fully reproducible were 78% less likely to report significant results compared to studies that were not fully reproducible . Additionally, studies that reported the regression model used to create the PSM model were 72% less likely to find significant results . Following mixed effect multivariable logistic regression analysis, there were no statistically significant associations between covariates and the presence of statistically significant results  in our mixed-effects multivariable logistic regression, albeit by slim margins, it nevertheless may indicate that reproducibility may represent a proxy for appropriate experimental methodology. In other words, studies neglecting to report core components of reproducibility may have been less fastidious, be it intentionally or not, in ensuring the robustness of other components of their methodology. This is especially pertinent in study designs using propensity score matching, given the numerous steps where errors can occur, which can be further compounded by PSM’s iterative nature.Fig 2 and Table 2. We propose several recommendations for authors to consider. First, covariates used to generate propensity scores and rationale for these selections should be specified. Second, in order to ensure matched pairs do not differ substantially, balance between covariates may be assessed through use of standardized mean differences, thereby minimizing biased treatment effects. Third, authors may also consider conducting a sensitivity analysis to further ensure robust matching. Fourth, all four criteria for reproducibility of a propensity-matched analysis should be reported, as discussed above. Fifth, in order to assess the external validity of a study, readers may find it useful if the matched sample size is specified and baseline characteristics or summary statistics for pre- and post-matched populations are provided [Table 1. Additionally, although no significant difference in reproducibility was found between studies with or without statistician coauthors, it is advisable for individuals with strong statistical knowledge and background to be involved in the study and be listed as a co-author if appropriate, as implementation of PSM can be a complex process. However, as a reminder to readers, PSM is still limited by its ability to only control for confounders that are known and measurable. Well-designed RCTs, by contrast, are able to control for all confounders by virtue of treatment randomization and thus remain the gold standard in clinical research.The results of the data presented herein demonstrate that the reporting of PSM methodology in otolaryngology has room for improvement. This is especially important given that the prevalence of PSM utilization is increasing by year as shown in provided . A summaThe results of this study should be interpreted in the context of its study design and in consideration of several important limitations. For one, we were unable to identify the presence of a statistician co-author in eleven studies, and our analysis of this variable may therefore be incomplete. In addition, our multivariable logistic analysis of the effect of PSM methodology reporting on statistical significance was based solely on the primary outcome as stated by the authors. In certain situations, studies with non-significant primary outcomes may have had statistically significant secondary outcomes which may be of clinical importance which would have been overlooked by our model. We also acknowledge that some of the included studies may have hypothesized equivalence between two groups or treatments, in which case lack of statistical significance may have been a positive result which would have been misclassified in our analysis. Furthermore, we acknowledge that this investigation is likely subject to a degree of publication bias in that studies with positive results compose the majority of published literature. This must be considered a potential confounder in our analysis of statistically significant results . Lastly,PSM represents a valuable tool for researchers to minimize bias and confounding in analyses of observational studies. In this systematic review of high-impact otolaryngology journals, the prevelance of PSM analysis was found to be increasing by year. Despite this, the quality of PSM methodological reporting in otolaryngology can be improved in order to ensure unbiased interpretation of results. We provide authors with recommendations to maximize scientific rigor in such studies. Authors, reviewers, and readers alike should be cognizant of such considerations when designing or interpreting studies utilizing PSM methodology.S1 TableCompleted PRISMA checklist for our literature review.(DOC)Click here for additional data file.


Naturally occurring, or spontaneous diseases in large animals remain a largely untapped resource, and given the similarities in pathophysiology to humans they not only allow for studying new treatment strategies but also disease etiology and prevention. Naturally occurring disease models, particularly for longer lived large animal species, allow for studying disorders at an age when the disease is most prevalent. As these diseases are usually also a concern in the chosen veterinary species they would be beneficiaries of newly developed therapies. Improved awareness of the progress in animal models is mutually beneficial for animals, researchers, human and veterinary patients. In this overview we describe advantages and disadvantages of various animal models including domesticated and companion animals used in regenerative medicine and tissue engineering to provide an informed choice of disease-relevant animal models.Rapid developments in Regenerative Medicine and Tissue Engineering has witnessed an increasing drive toward clinical translation of breakthrough technologies. However, the progression of promising preclinical data to achieve successful clinical market authorisation remains a bottleneck. One hurdle for progress to the clinic is the transition from small animal research to advanced preclinical studies in large animals to test safety and efficacy of products. Notwithstanding this, to draw meaningful and reliable conclusions from animal experiments it is critical that the species and disease model of choice is relevant to answer the research question as well as the clinical problem. Selecting the most appropriate animal model requires in-depth knowledge of specific species and breeds to ascertain the adequacy of the model and outcome measures that closely mirror the clinical situation. Traditional reductionist approaches in animal experiments, which often do not sufficiently reflect the studied disease, are still the norm and can result in a disconnect in outcomes observed between animal studies and clinical trials. To address these concerns a reconsideration in approach will be required. This should include a stepwise approach using  The use of sentient animals for research purposes is a controversial topic, which has raised public and ethical concerns and is criticized by opponents claiming that animal models often do not generate appropriate benefit with regards to their potential risks and harm and as a consequence, are often ethically not permissible. The increasing status of pets as family members and corresponding high level of veterinary care for privately owned pets further amplifies the controversy over the use of animals for research purposes.in vitro experiments. Although in vitro models allow for systematic, standardized analysis of various cellular, biophysical and biochemical cues in a controlled environment, without the natural variability inherent to in vivo animal models, they can only offer an abstract insight into the pathophysiology of diseases and disorders. Therefore, while animal models cannot yet be replaced, the number of animals used should be reduced to a minimum and experiments involving animals should be optimized with regard to their translatability and the welfare of the animals.However, animal models are still an important and, at a regulatory level, a compulsory component of translational research, which cannot yet be replaced by However, to date a reductionist approach often using immature laboratory species is commonly employed . Small r1,2. For successful and timely translation from animal models to regulatory approval and clinical application, a step-wise development using laboratory animals for screening and early development work, followed by a large animal model such as the pig, sheep or horse which offers a more realistic approach for late development and pivotal studies would be more appropriate , the USA Federal Food and Drug Administration (FDA) and the International Society for Stem Cell Research (ISSCR) recommend the use of large animal models to evaluate efficacy, durability, dose response, degradation and safety of advanced therapeutic medicinal products (ATMPs)ropriate .Moreover, animals develop many naturally occurring (or spontaneous) diseases that are equivalent to human disease leading to the development of the “One Health One Medicine” concept which presumes that diseases in men and animals  have similar aetiologies and pathophysiologies and require analogous therapeutic approaches. Hence, human and veterinary medicine can mutually benefit from research that applies a one health approach. Using large animal models with naturally occurring disease with a similar pathophysiology as in humans, allows study of not only new treatment strategies but also disease development and prevention at a relevant age. However, although using naturally occurring disease models best reflect disease complexity, standardization of disease grade and availability of sufficient clinical case numbers for recruitment into studies can be challenging.reduce, refine and replace animal models) of using the smallest possible number of animals, animal models need to be optimized to the greatest possible extent  is a heterogeneous disease for which no single animal model perfectly recapitulates the complex etiology and clinical manifestations of the human disease . CurrentSurgical models have the advantage of repeatability and reproducibility as well as rapid onset and progression , but forThe validity of chemically induced models for OA has been questioned  due to tAnimal models are further widely used in osteoporosis research. They include, among others, models for disuse induced osteoporosis, glucocorticoid-induced osteoporosis and postmenopausal osteoporosis. The most popular animal models of postmenopausal osteoporosis are those generated in the mouse, rat, sheep, and nonhuman primates by ovariectomy .The choice of the animal models differ markedly, depending on the objectives of the study. It has to be noted that rodents for example are of limited value for investigating intra−cortical bone remodeling, because they lack true Haversian cortical bone remodeling under physiological conditions due to their small weight . Larger Ascaris antigens, house dust mite, cockroach extracts), used alone or in combination, are difficult to compare and to interpret  are a well-accepted, well-established and clinically relevant animal model particularly for musculoskeletal disease, which is of major interest in regenerative medicine.Horses  , ultrasoAlso the lack of traceability of cells injected for cell therapies could be overcome to a certain extent by using either super paramagnetic iron oxide particle (SPIO) for MRI  or nucleSome disadvantages of using the horse as a model include high costs of animal of animal purchase, maintenance/handling as well as ethical concerns and lower acceptance of the horse as an experimental animal compared to small animal studies by the lay public. In addition, some key parameters building the framework used in studies applying Omics approaches are not well enough researched in horses yet. A restrictive annotation status and availability of equine specific antibodies, molecular tools and markers are limiting factors. A major challenge when using horses is that their weight precludes non-weight-bearing investigations postoperatively. Significant limitations may arise regarding biomechanical strains, which far exceed those considered physiologic in humans and other animal models, which could render the stabilization of injured structures, transplants and/or sutures ineffective. Therefore, horses are a less amenable model for meniscus or bone repair. Nonetheless, these are major challenges in equine patients and several different attempts have been made or are envisaged to support healing of these structures by regenerative medicine approaches  which maHorses commonly suffer from naturally occurring tendon injuries (tendinopathy) and degenerative joint disease (osteoarthritis − OA) with similar pathophysiology to the human in terms of etiology and risk factors, which include over-exercise, age and genetic factors . As athlAn example is the equine superficial digital flexor tendon (SDFT) which performs a similar function to the human Achilles tendon during high-speed locomotion. In both species, their respective tendons are one of the most frequently injured  with ageAcute and chronic Achilles tendon pathology is estimated to be responsible for as many as 50% of all sports-related injuries in humans . The incHowever, due to the challenges of standardization of disease grade and availability of sufficient clinical case numbers for recruitment of horses with naturally occurring disease, a number of induced equine models have been developed to investigate both tendon and joint disease.Several surgically induced tendon injury models have been developed to try to achieve a standard lesion size, anatomical location and the ensuing inflammatory response as well as time to treatment . While mAnother example to illustrate “what the horse can tell the human” is Osteoarthritis, a degenerative joint disease characterized by progressive loss of articular cartilage. Adult articular cartilage has limited capacity for repair and regeneration . Any disA number of surgically induced equine models of articular cartilage degeneration and healing have been developed which were reviewed by Horses, analogous to humans, commonly suffer from asthma. Asthma is a chronic inflammatory disease characterized by airway hyper-responsiveness and airway remodeling due to increased mucus production, epithelial fibrosis, hypertrophy and hyperplasia of airways smooth muscles, and gland enlargement . This re5. The standard therapy is based on corticosteroid administration to reduce airway obstruction thus improving quality of life. However, about 20% of people are corticosteroid resistant and do not respond to therapy (Around 300 million people worldwide (both adults and children) suffer from asthma and hence the societal impact is high therapy . CorticoThe gold standard species for studies into human asthma would be human patients, but such studies are ethically impossible, because of the large number of patients requiring repeated biopsies to understand the causes of remodeling. Therefore, although requiring ethical authorizations, animal models are essential to advance understanding of the disease.Severe equine asthma (SEA), which occurs spontaneously in horses , shares IL2, IL23, and IL17, compared to the mouse , imbalance in collagen homeostasis, profibrotic environment, tissue hypoxia and inappropriate cell apoptosis are discussed as contributing factors .Interestingly ponies heal better and faster than horses, with ponies yielding a quicker and more intense inflammatory response and an improved resistance to infection as compared to horses . Some ofAnother challenge of using horses as animal models, particularly for orthopedic disease, is so called supporting limb laminitis (SLL). Laminitis is a disorder of the tissue suspensory apparatus which suspends the distal phalanx to the inside of the horse’s hoof wall. SLL of the contralateral or supporting limb occurs when horses are forced to bear weight predominantly unilaterally (with the supporting limb) for prolonged periods, due to a severe, unilateral lameness. Mechanical loading or overloading of the supporting limb is the primary factor in its pathogenesis .Ovis aries) provide unique opportunities in research as an experimental and pre-clinical animal model  which allows for good simulation of healing and remodeling processes of bone or cartilage tissue . In addiSheep have been involved in studies for treating critical-sized bone defects using scaffolds with or without Mesenchymal Stem cells (MSCs). These treatments were shown to enhance bone formation and improve mechanical properties if compared to gold standard reparative methodologies like bone grafts .Although the ovine knee cartilage differs in thickness to human cartilage , it provides a close match regarding mechanical properties for preclinical studies . Tissue Furthermore, regenerative strategies for osteoarthritis  have been investigated in sheep . Of partSpontaneous cartilage lesions (including osteoarthritis) have been observed in the sheep without experimental induction . These aSheep, like horses, are ideal candidates for tendinopathy modeling, but cheaper and easier to handle and house. The ovine species also serves as an adequate and effective model to study peripheral nerve regeneration, because of the similar nerve size  and simiApart from conventional autografts and allografts for repairing peripheral nerve injuries in sheep , tissue Using sheep models, MSCs were shown to play a reparative role in intervertebral disc regeneration. Injection of MSCs led to a reduction of degeneration of the discs compared to the control group .in-situ heart valve replacement for the pulmonary valve using a resorbable synthetic graft. 12 months post-implantation the tissue-engineered valve was shown to be colonized by host cells and replaced by newly formed tissue with a mature organization of the extracellular matrix without any sign of valve calcification :MSCs led to a reduction of inflammation and oedema and an improved oxygenation in sheep models of acute respiratory distress . In an iRecently, The sheep model has also been described for wound healing studies because it allows for the creation of relatively large and deep wounds to mimic the typical scenario of traumatic injuries like burn injuries or decubitus ulcers. In a similar ovine wound model, Recently, A different tissue engineering approach to treat myelomeningocele was used by Watanabe et al. to treat spina bifida wounds with a gelatin/collagen sponge hybrid scaffold .in vivo as well as in vitro models are required. Fetal sheep share many important physiological and developmental characteristics with humans and have hence proven themselves invaluable models for mammalian physiology  provides sufficient temporal resolution to translate findings obtained in sheep into human parameters .Fetal sheep have a fully functioning immune system by 75 days of gestation (gd) . They prFor all these reasons, results obtained in the fetal lamb have been directly applicable to the understanding of human fetal growth and development and are highly predictive of clinical outcome in a variety of applications including in utero stem cell transplantation .Due to their special stomach system  bio-availability and efficacy of drugs administered orally is questionable for the human GI tract. Moreover, prolonged inappetence and application of non-steroidal anti-inflammatory drugs, antibiotics or both resulting in sustained high acidity in the abomasum may cause abomasal ulceration. Also stress, high dietary fiber and inadequate dietary fiber are believed to play a role .Therefore, pain management and anti-microbial management have to be planned carefully and adapted to meet the special requirements of sheep . Sheep gSome of the most important advantages and disadvantages of using sheep as model animals are summarized in Porcine models present the advantage of having similarities with the human in terms of gastrointestinal anatomy, metabolism and physiology . When coTraditionally, animal models used for preclinical testing of new drugs and toxicology studies have been rodents, mainly mice and rats, for the primary screening studies. Nonetheless, because translation from rodents into humans is often not fully realized, regulatory agencies also demand the use of non-rodent models. Pigs are increasingly being used as an alternative to dogs or primates, the previous nonrodent species of choice . HoweverWith the advent of DNA recombination and gene editing technologies, modifying the pigs genome has enabled its use as a genetic model of numerous human diseases . This isThe pig has been used as a teaching and research animal model in surgery in the past decades. Starting in the 1990s, it became so prominent in academic and surgical training that it can be regarded as default model for non-survival surgical teaching classes, substituting the dog . Its ubiThe minipig has been used as a model in the development of dermatological products , and morIn this particular area of biomedicine, the pig is experiencing a higher increase in adoption when compared to other large animal models  and sevePigs have also been used recently as a model of amyotrophic lateral sclerosis (ALS). This research has been based on the use of transgenic pigs with a mutated human copper/zinc superoxide dismutase 1 gene that mimics the human neurodegenerative disease in these pigs . SimilarPigs suffer from porcine malignant hyperthermia also known as porcine stress syndrome which is characterized by hyperthermia triggered by stress, certain anesthetic agents or intense exercise and may lead to sudden death . Some ofThe importance of companion animals to serve as models for human disease has received significant attention through the One Health initiative which aims to “break through the species barrier” in a drive toward a better link between medical and veterinary research for the benefit of both the human and veterinary patient .While the definition of companion animals covers a range of animals this article extends only to the dog and cat as models, as they share remarkable similarities with the human and provide unique opportunities for developing advanced therapeutics.One of the main reasons why dogs returned as a focus of genetic research is related to the specific population structure that has been created over the past 150−200 years.To fully appreciate and exploit the biomedical potential of dogs , some insight into the unique canine population structure is necessary. Domesticated dogs were subjected to rigorous breeding selection, for instance for behavioral traits and/or specific morphological features such as excessive muscle formation, short limbs or a specific coat color . Illustr2+) its Jekyll and Hyde character becomes evident in the involvement in chemical reactions leading to the production of reactive oxygen species. In a Fenton reaction, Cu+ catalyzes the formation of the highly reactive hydroxyl radical (OH.). In the converse Haber-Weiss reaction Cu2+ inactivates the damaging superoxide radical O2. Therefore, regulation of its intracellular free concentrations is of utmost importance and needs to be controlled within very narrow limits  or superoxide dismutase  . Since cw limits . Severalw limits , endemicw limits , and idiw limits . These aCopper disorders also affect sheep and dogs . DeleterThe discovery of the COMMD1 mutation and subsequent investigations into functions of COMMD1 is an intriguing example for a useful exploitation of inbred dog strains to reveal novel molecular and genetic pathways. Genetically speaking the big advantage of canine genetics to benefit human genetics is the ease to discover modifier genes. This is a needle-in-a-haystack technology in men even today, but the specific genetic population structure in inbred dogs clearly facilitates this approach.Labrador retrievers are among the most popular breeds in the Western world.It was already known for a long time that approximately one in every three first-line relatives of Labradors retrievers with copper toxicosis had elevated copper levels . This puThis genetic study clearly illustrates the power of the canine model. Explaining 12% of the phenotypic variation with an ample 250 dogs doesn’t even remotely resemble the number of human patients used to explain similar percentage for age at menarche, Inflammatory Bowel Disease (IBD) and Rheumatoid arthritis (RA) for which over 100,000 individuals were included .The examples prove that due to the specific population structure of inbred dog breeds, genetic studies can be successfully performed even for rare and/or complex genetic diseases.In order to investigate COMMD1-deficient dogs as a preclinical model for liver stem cell transplantations, a breeding colony of five COMMD1 deficient dogs was created on a Beagle background and followed for over 4 years . This moThe most prevalent non ischaemic cardiomyopathies in humans are hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy (DCM), reported to affect 1 in 500 and 35 in 100,000 people, respectively 2017, . ArrhythThe development of cell-based approaches in the feline and canine clinic will have significant benefits for translation in human cardiomyopathy treatment.Hypertrophic cardiomyopathy is the most common cardiomyopathy in both humans and cats with a prevalence of approximately 0.1−0.2% and 16%, respectively . There iThe underlying molecular pathogenesis driving HCM remains to be elucidated although a common pathway is thought to exist in both humans and cats in which altered calcium handling within the myofilaments enhances calcium sensitivity, causing maximal force production and energy deficiency promoting mitochondrial dysfunction, cell death, fibrosis and cardiomyocyte hypertrophy .Studies using myocardial tissue from a cat homozygous for the MYBPC3/R820W mutation suggest that increased myofilament calcium sensitivity can occur in the absence of haploinsufficiency, which is common feature in human MYBPC3 mutations . IncreasSuch studies highlight the need to identify detailed molecular mechanisms for precise drug targeting. However, there are practical limitations with obtaining sufficient heart tissue and the survival of isolated primary cardiomyocytes is poor. Induced pluripotent stem cells (iPSC) or embryonic stem cells (ESCs) represent an alternative and robust source for preparing cardiomyocytes. The development and use of human ESCs represents an ethical dilemma and while less of an issue in veterinary species, there are only two reports of ES-like cells from cats, but these do not replicate indefinitely in culture unlike true ES cells. iPSCs on the other hand do not have the concerns associated with ESCs and can be relatively readily prepared from somatic cells.Feline iPSCs have recently been reported for the first time by our group, the development of which represents a significant step in the generation of iPSC derived cardiomyocytes from a veterinary species . It paveiPSCs derived from patients with HCM or iPSCs with a genetic mutation inserted using CRISPR to model HCM, display characteristics of hypertrophic cardiomyocytes in culture  suggesti6. It is the second most common cardiac disease in dogs and accounts for 10% of canine cardiac diagnosis  and chondrodystrophic (CD) dogs. The last group presents with disproportionally short limbs, caused by aberrant endochondral ossification of long bones. Dachshunds are typical examples. The molecular mechanisms of this short limb phenotype is associated with a retrogene insertion of the FGF4 fibroblast growth factor 4 gene. This leads to elevated levels of FGF3 signaling. Interestingly, whereas CD dogs are more prone to intravertebral disc degeneration (IVDD), the insertion of the retrogene renders short-limb dogs less likely to develop OA in comparison with NC-dog . These eDespite walking on four legs in contrast to men walking on two only, both species develop intervertebral disc degeneration with great similarities and similar prevalence. Link-N is a protein involved in proteoglycan stabilization  and is highly homologous between men and dogs. However, neither human link-N nor canine link-N can protect cultured canine intervertebral disc cells form degeneration, whereas human link-N improved glycosaminoglycn deposition in human and bovine chondrocyte-like cell cultures .In a classcial pre-clinical study a controlled release system for the COX-2 inhibitor celecoxib (cyclooxygenase-2) was tested in a dog model for IVDD . Since cNaturally occurring cranial cruciate disease has been studied extensively in veterinary medicine . It can ;Dogs also suffer from naturally occurring meniscal pathologies and hence lend themselves as potential translational models to study mechanisms of degeneration or for testing new treatment strategies . The canSweigart et al., 2004;Bélanger et al., 1996;Abelew et al., 2000;Cats often serve as models to study spinal cord healing and comparative aspects in neurosurgery  allow optimal engraftment of injected cells for different diseases. It also needs to be determined whether multiple injections will be more beneficial and if so the question arises whether there is an associated increase in risk of an adverse immune reaction. Cell therapies likely function via a paracrine mechanism and as such alternative approaches such as cell-free extracellular vesicle fractions or soluble factors, need to be explored that may reduce some risks posed by cell administration particularly of allogeneic cells.For tissue engineered constructs implantation studies using animals with similar size and weight as human patients are crucial to test the implants under relevant biomechanical conditions.To answer these questions pre-clinical trials with patient cohorts of sufficient size are required which need to be designed robustly to measure appropriate safety and efficacy readouts. Equivalent diseases in animals makes them not only relevant models which offer a more accurate evaluation of safety and efficacy of new treatments, but at the same time are potential beneficiaries of new treatment approaches. Hence, human and veterinary medicine can mutually benefit if one appreciates the similarities.IR contributed to conceptualization, writing the manuscript, merging the parts contributed by other authors, and revision and editing of the manuscript. PB, AL-C, LM, MP, ES-F, LD, DC, and FS wrote the manuscript. FJ revised and edited the manuscript. JD wrote, revised, and edited the manuscript. LP conceived the idea and wrote, revised, and edited the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The organization of microtubule arrays in immune cells is critically important for a properly operating immune system. Leukocytes are white blood cells of hematopoietic origin, which exert effector functions of innate and adaptive immune responses. During these processes the microtubule cytoskeleton plays a crucial role for establishing cell polarization and directed migration, targeted secretion of vesicles for T cell activation and cellular cytotoxicity as well as the maintenance of cell integrity. Considering this large spectrum of distinct effector functions, leukocytes require flexible microtubule arrays, which timely and spatially reorganize allowing the cells to accommodate their specific tasks. In contrast to other specialized cell types, which typically nucleate microtubule filaments from non-centrosomal microtubule organizing centers (MTOCs), leukocytes mainly utilize centrosomes for sites of microtubule nucleation. Yet, MTOC localization as well as microtubule organization and dynamics are highly plastic in leukocytes thus allowing the cells to adapt to different environmental constraints. Here we summarize our current knowledge on microtubule organization and dynamics during immune processes and how these microtubule arrays affect immune cell effector functions. We particularly highlight emerging concepts of microtubule involvement during maintenance of cell shape and physical coherence. The cytoskeleton plays a major role for accomplishing numerous immune cell effector functions. Microtubule filaments are crucially important for directing migratory cells to their final destination and for mediating specific cell-cell interactions between an antigen-presenting cell and a T cell. Microtubules are composed of α-/β-tubulin heterodimers which polymerize in a head-to-tail fashion to form a polar protofilament with the α-tubulin subunit exposed to one end – designated as minus-end – and β-tubulin exposed to the other end, which is commonly referred to as the plus-end . In mostLeukocytes are commonly known as white blood cells, which constitute the major component of the body’s defensive unit against diseases. In contrast to erythrocytes, many types of leukocytes exist, which mainly originate from hematopoietic stem cells in the bone marrow. They are classified either by structure into granulocytes and agranulocytes or by cell lineage into myeloid or lymphoid cells. In contrast to other highly specialized cell types, which often reorganize microtubules into non-centrosomal arrays during differentiation, leukocytes primarily nucleate microtubules from the centrally located centrosome with individual filaments extending radially toward the cell periphery . One excOne of the most characteristic features of all leukocytes is their highly motile behavior. Lymphocytes constantly patrol the body for foreign antigen by recirculating from blood, through tissue, into lymph and back into the blood stream. Myeloid cells such as neutrophils and monocytes emigrate from the blood stream into tissues when they detect changes on the surface of blood vessels that transmit signals of injury or infection . A critiamoibe; Greek for “change”). By contrast, adhesion-dependent migration occurs as a consecutive sequence of leading-edge protrusion, transmembrane force coupling by adhesion receptors and contraction of the cell rear to promote de-adhesion, closely resembling Abercrombie’s three-step mesenchymal migration mode  cells. Skin TRM cells exhibit highly dynamic shape changes when migrating within the constrained epidermal layer with extension of multiple competing protrusion similar to dendritic cells. Treatment of TRM cells in the skin with microtubule destabilization agents induces cell elongation and fragmentation of extending dendrites suggesting that an intact microtubule network is also required to maintain cellular coherence of TRM cells within the epidermis  at the peripheral SMAC. Similarly, microtubule filaments project to the immune synapse and associate with ADAP into a ring-like structure . Loss ofProvided that microtubule minus-ends are anchored at the MTOC these data support a model, in which the dynein-dynactin complex localizes to the plasma membrane at the immune synapse and exerts the force, which is required to recruit the MTOC close to the contact site . In thisin vitro reconstitution experiments showed that barrier-attached dynein is indeed able to trigger microtubule shrinkage and to generate pulling forces, which are sufficient to center microtubule asters in confining geometries  in the cell membrane . DAG is Glued . Both coGlued . Knockdo protein . More re protein . Depleti protein . When dy protein .Independently of the mechanism of force generation and transmission, microtubules need to be anchored at the immune synapse to efficiently allow MTOC reorientation. Based on the “search and capture” model, which was first proposed in the context of microtubule-chromosome interaction during mitotic spindle assembly , a matheMicrotubule organizing center polarization toward the immune synapse was also reported in antigen-presenting cells such as dendritic cells and B cells . In bothThe mechanisms of MTOC translocation in B cells rely on the interplay of actin and microtubule filaments at the centrosome: in addition to its role as MTOC, the centrosome functions as an organizer of a local network of actin filaments, which can impose physical constraints on the microtubule cytoskeleton, specifically affecting the growth and shape of microtubule filaments as well as the dynamics of the centrosome . CentrosRecently, the mechanistic link between local lysosome fusion at the B cell immune synapse and microtubule dynamics has been addressed : lysosomTogether, in antigen-presenting cells the direct actin-microtubule crosstalk determines the capacity of the MTOC to repolarize and nucleate microtubules in response to external stimuli and enables local lysosome delivery at the immune synapse.Initially, association of lytic granules with the MTOC and its repositioning toward the immune synapse have been identified as the only prerequisite for targeted secretion while plus-end directed motor transport along microtubule filaments was dispensable for specific killing . Lytic gper se is not essential for the spatial specificity of fusion events (Whether centrosome polarization is a prerequisite for efficient cytotoxic T lymphocytes and natural killer cell-mediated killing is still a matter of debate. While in several studies impaired or delayed MTOC translocation as well as abrogation of transient centrosome docking with the plasma membrane is associated with reduced cytotoxicity , other sn events .Here we have summarized the multiple faces of microtubule involvement during immunological processes ranging from leukocyte migration to immune synapse formation and vesicle polarization. We have learned from recent studies that leukocyte microtubules are essential for the induction of tissue immunity by maintaining cell coherence of migrating leukocytes and regulating polarized secretion of granules and lysosomes. As such, microtubule arrays deserve precise attention and exact description in future studies of immunological processes.Migrating dendritic cells exhibit differences in the dynamics of front- versus back-directed microtubule filaments. How this polarization is achieved and what are the key regulators of microtubule dynamics in migrating leukocytes still remain open questions. Advanced light sheet microscopy in combination with recently developed photo-switchable compounds and optogenetic constructs that target microtubule stability and dynamics are potent tools to investigate subcellular microtubule dynamics with high spatiotemporal resolution allowing to understand the functional principles of microtubule dynamics in tissue navigating cells . SimilarAdaptive immunity is to a large extent influenced by mechanical forces. Microtubules accumulate a great diversity of PTMs, which can alter microtubule dynamics and thus control the mechanical properties and function of microtubule filaments. Acetylation of α-tubulin at the luminal surface of microtubules affects filament stability and is associated with long lived microtubule subpopulations . FunctioDuring dendritic cell migration acetylation occurs predominantly on front directed microtubules . In partIn the future, it will be interesting to employ life cell sensors to analyze the role of PTMs in a detailed spatiotemporal manner during processes such as leukocyte migration and immune synapse formation. The tools are currently limited; however, recent advances now enable monitoring of tyrosinated microtubules in living cells .Moreover, T cell activation and effector function is a mechanosensitive process, in which force transduction across the T cell receptor complex generates significant traction stresses at the cell-cell interface (reviewed by Another interesting feature of leukocytes that has received limited attention during the past years is the centrosome-nucleus connection. It is well established that stationary or slow-moving cells such as neurons or cells of mesenchymal origin exhibit a stable front-back polarization . HoweverThe immune synapse is a well-known site of polarized membrane transport and localized vesicle release. While this process is fairly well understood in T cells and cytotoxic cells, it is less clear how polarized secretion is regulated in antigen-presenting cells. MTOC relocalization in dendritic cells seems to be dispensable for the specific delivery of cytokines and T cell activation. As dendritic cells are able to interact and activate several T cells simultaneously  it seemsIn contrast to mesenchymal cells, many of the core cell biological characteristics of the leukocyte microtubule cytoskeleton are currently poorly understood. Given that chemokines and other external factors, such as the mechanochemical composition of the microenvironment, act upstream of cytoskeletal regulation  it will Both authors contributed to the conceptual development of the work, literature research, and writing of the manuscript. AK composed the figures.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Despite increases in STIs among those over 40, little is known about the social context of STI transmission among people experiencing relationship transition in midlife, and few sexual health promotion initiatives are targeted at this group. This study sought to identify factors shaping STI risk perceptions and practices among midlife individuals either contemplating or having sex with new partners following the end of a long-term relationship.Participants were purposively selected from respondents to Britain’s third National Survey of Sexual Attitudes and Lifestyles, using three eligibility criteria: aged 40–59, reported experience of the end of a marital or cohabiting relationship with an opposite-sex partner in the past 5 years, and willingness to participate in a qualitative interview. Qualitative data were generated via face-to-face interviews with 10 women and 9 men and analysed inductively using thematic analysis, with themes then organised using a socioecological framework.Participants’ accounts of new sexual partnerships in midlife indicate that STI risk perceptions and practices are shaped by factors operating at multiple levels across the socioecological arena . Constraints on, and resources for, the navigation of sexual safety include self-perceived STI risk rooted in past rather than present circumstances; legacies of mistrust within former relationships; intersecting gender-age dynamics in negotiation of risk prevention strategies with new partners; peers and younger relatives’ influences on understandings of sexual risk and safety; postrelationship change in social networks that increase or mitigate vulnerability to sexual risk; age-related barriers to accessing condoms; and disconnection from safer sex messaging and services culturally coded as for the young.Improving sexual health among midlife adults requires age-sensitive interventions designed to address multilevel constraints, and harness positive influences, on the navigation of sexual safety at this stage of life. While younger people continue to account for the majority of STI diagnoses, increases in rates of STIs among people over 40 have been observed in high-income countries in recent years.Despite prominent calls for greater attention to sexual health among those over 40,Drawing on qualitative data, this paper uses a socioecological framework to conceptualise the social context of STI risk perceptions and practices among people who have experienced the breakdown of a cohabiting/marital relationship with an opposite-sex partner in midlife see . ModifieIndepth interviews were conducted with a subsample of respondents to the third British National Survey of Sexual Attitudes and Lifestyles . Full survey methods are described elsewhere.Participants eligible for this study were 323 women and 205 men aged 40–59 years (defined here as midlife) who reported having experienced the end of a cohabiting/marital opposite-sex relationship in the last 5 years. The social context of STI risk among individuals ending same-sex relationships was deemed to warrant a dedicated, culturally focused study. All Natsal-3 respondents were asked in the questionnaire to indicate willingness to participate in a follow-up interview (no topic defined); among those eligible for this study, 279 women and 176 men agreed. We drew a purposive sample guided by recency of participating in the Natsal-3 survey, roughly equal gender distribution and geographical spread across Britain.Interviews were conducted with 19 individuals  see . Our samInterviews were in participants’ homes, with written consent obtained from all participants. The topic guide explored perceived norms and experiences regarding new sexual relationships at midlife, conceptualisation and prioritisation of sexual health, safer sex strategies, and help-seeking for sexual health information and advice. Interviews were audio-recorded and professionally transcribed.We conducted thematic analysis, guided by Braun and Clarke’s six-stage framework.new opposite-sex partner in the past year. Among this subgroup, around half considered themselves to be not at all at risk of STIs or HIV, almost three-quarters reported condomless sex with new sexual partner(s) in the past year, while only around 1 in 20 reported having attended a sexual health clinic in the past year , lack of STI testing, lack of knowledge of partner’s STI status). Accounts of self-perceived low STI risk were strongly grounded in self-identity regarding one’s orientation to relationships , which appeared to foster a sense of perceived insulation from STI risk, especially when combined with a belief that the new partner(s) was similarly oriented. Loss of fertility  appeared to also strongly affect motivation to use condoms, with several women and men expressing low prioritisation of ‘safer sex’ in the context of removed risk of pregnancy.Many participants described their willingness and ability to initially trust new sexual partner(s) as severely reduced due to the legacy of struggles and trauma in former relationships . Scepticism about former partners’ monogamy was often cited as a motivation for a midlife sexual health check in theory, although not all who expressed doubt sought STI testing—a situation participants sometimes spontaneously accounted for by expressions of residual faith that their (now ex) partner would only have had sex with ‘low risk’ partners. Among those who had sought testing during midlife , timing between doubt of a partner’s monogamy and testing varied; while some reported seeking testing immediately on discovery (suspected or confirmed) of a partner’s non-monogamy, others described a lag between doubt and testing as they waited until later points, such as when the relationship had definitively ended, when planning to start dating or at the start of a new relationship.Negotiation of sexual safety with new partners was clearly constrained by intersecting gender-age dynamics. Prior strategies for avoiding unwanted pregnancy were sometimes no longer relevant  and required renegotiation. Condoms were commonly described by women and men as embarrassing to discuss and use with new midlife partners for various reasons, including perceived association with youth, lack of familiarity after decades of condomless sexual activity and their perceived exacerbation of erectile dysfunction in new partnerships. Middle-aged men’s particular resistance to condom use was described by both men and women, presenting challenges for women’s insistence, and expressed resentment at having to assume sole responsibility for initiating their use. Accounts of STI testing in the early stages of a new sexual partnership were mostly attributed to requests (and sometimes insistence) by female partners, rather than at men’s own initiation. Where testing had occurred, results were sometimes characterised as static , even after condomless sex with new partners in the interim. In the absence of normalised expectations of mutual STI testing, evaluation of the STI status of potential partners was often based on inadequate indicators, such as appearance, demeanour, perceived wealth, assumptions (rather than discussions) about their sexual history, duration between meeting and sexual activity, and among men, women’s willingness to engage in specific sexual practices .known others within their personal networks , as well as perceived norms within wider local communities. Some participants described pressure from friends and family to repartner, while simultaneously being warned that sexual cultures had shifted since they were last ‘single’ and required new strategies to keep themselves ‘safe’. In some cases, friends and family were recruited to help assess the sexual risk associated with new  partners, although sometimes using unreliable indicators, such as the individual’s apparent interest and ease in integrating into new social networks. Exposure to younger relatives’  own sexual health practices were an important context for updating knowledge and validating safer sex strategies, such as negotiating mutual STI testing in the early stages of a new partnership.Peers and social networks were important informal systems shaping understandings of norms relating to sexual risk and safety at midlife, with participants commonly comparing their own experiences of dating, sex and risk negotiation with new partners with those of similarly aged, needing additional support at times of relationship transition and being needed by others . Women especially described prioritising children’s emotional needs following their separation/divorce  over their own.Change in social networks following relationship transition both increased and mitigated sexual risk among interviewees. For some, total loss of contact or growing emotional distance from formerly close friends and family members  fostered feelings of social isolation and reduced opportunities for social support regarding their personal lives. Some men described new patterns of socialising postdivorce/separation that increased vulnerability to sexual risk, including more time spent with casual acquaintances in social environments with community norms that encouraged high alcohol consumption, frequent partner change, age mixing, paying for sex, and ‘othering’ of those at risk of STIs and HIV . By contrast, others (mostly women) described feeling more socially integrated after relationship transition, within both existing and newly forged social networks, and having more freedom to discuss intimate matters  with friends once they no longer felt bound by loyalties of discretion to former partners. Yet, in the context of competing midlife demands, several participants conveyed limited capacity to prioritise new personal and sexual relationships, or their own health ; there was an evident tension between Several features of the wider social and structural context appeared to contribute to understandings and experiences of navigating sexual safety at midlife. Greater public discussion about sex and sexual health information than in previous eras was broadly described as positive, as were increasing representations in mainstream and social media of sexuality among midlife and older adults, although not all portrayals were seen as helpful . News stories were also credited with raising generalised awareness of rising rates of STIs among those over 40, yet some participants noted they did not relate to the extreme case examples commonly included in this coverage. In terms of healthcare, participants’ belief that ‘sexual health’ is coded as relating to young people extended to a perception of specialist sexual health services as youth-focused and therefore embarrassing to access as a midlife adult . Only one participant reported sexual health being discussed in general practice settings, although valued discussions about contraception and sexual function , lubrication) had occurred during clinical encounters focused on treatment and management of other conditions . Barriers to accessing condoms in non-sexual health settings  included fear of ageist judgement, especially among women.Despite awareness of rising prevalence of STIs among midlife adults, few participants in this study consistently used condoms with new partners, routinely sought STI testing or perceived themselves at risk of STI. A tendency to characterise one’s exposure to STI risk in relation to a self-identity grounded in former relationship status, rather than current circumstances, may shed some light on specific age-related factors contributing to the common underestimation of one’s STI risk.The strengths of this study include rich description and prioritisation of subjective meanings; use of a socioecological framework to highlight the multilevel social context shaping perceptions and practices; and a sample that included non-users of sexual health services. The limitations include potential social desirability bias within face-to-face interviews and exclusion of those acquiring new sexual partners concurrent with an ongoing marital/cohabiting relationship—a likely subgroup of midlife people experiencing new STI diagnoses. A parallel investigation with individuals who have ended a same-sex relationship in midlife would more fully expand understanding of factors shaping STI risk perceptions and practices among those over 40.Given the compounding pressure on sexual health services in the UK, including increases in STIs and dramatic cuts to funding,In terms of services, participants’ accounts indicated virtual absence of GP-initiated discussion of sexual matters and a perception of specialist sexual health services as youth-focused. Previous research suggests preference for STI diagnosis and treatment in general practice among at-risk individuals not attending sexual health clinics.No matter what age you are […] if you have sex with a new or casual partner, make sure you use condoms and get regularly tested’.anyone experiencing sex with a new partner, regardless of their age.In the context of an ageing population, with many people sexually active into later midlife, maintaining momentum for sexual health protection throughout the life course is crucial. Public Health England recently urged: ‘Midlife men and women with new sexual partners experience multiple constraints on their navigation of STI risk and prevention.STI prevention services and campaigns geared towards younger people may not adequately address these age-specific factors.Efforts to reduce and prevent STI transmission in midlife should extend beyond increasing individual knowledge and address sexual risk and vulnerability among midlife men and women in the broader context of their lives and social networks.


Individuals with Mild Cognitive Impairment (MCI) may have limited access to intervention programs that support their mental and physical health. The COVID-19 pandemic has put them at an even greater risk of not having access to such programs. While there is currently no cure, there is growing evidence that intervention programs may attenuate the progression from MCI to dementia, particularly those which 1) have potential to reduce the level of cardiovascular risk factors, 2) employ cognitively stimulating activities, and 3) create opportunities for social interaction . Many mind-body interventions, such as tai chi, yoga, and mindfulness classes, contain these three elements and have been shown to benefit individuals diagnosed with MCI, including improving cognition . Tele-technology  can aid in overcoming the logistical barriers by bringing instructors and interventions to these individuals to help them stay engaged and attend activities more frequently from the comfort and convenience of their home. We will present recent findings from a user study with 8 stakeholders  to assess barriers and facilitators to using tele-technology to bring instruction of mind-body interventions to individuals diagnosed with MCI. This poster will present guidelines for delivering such interventions based on our findings from the user study, including safety and training protocols.


In a recent study, Robinson and colleagues  applied Prediction of chronological age itself is of limited use beyond specialised fields such as forensic science. However, the new field of Geroscience proposes that biological aging, a set of interrelated molecular and cellular changes associated with aging, drive the physiological deterioration that is the root of multiple age-related health conditions . RobinsoSomewhat surprisingly, age acceleration measured through metabolomics was uncorrelated with assessments based on established epigenetic clocks. One may speculate that epigenetic clocks specifically target the hallmark of epigenetic stability which was not captured through metabolomic analysis. Negligible correlations between different biological age markers have been reported in other cross-sectional studies possibly suggesting that the different hallmarks of aging are independent rather that multifaceted expressions of the same core process . HoweverMetabolomic age acceleration showed similar or somewhat stronger associations with most of the assessed risk factors as epigenetic clocks that were similarly trained on chronological age. However greater sensitivity to risk factors was observed with an epigenetic clock trained on “phenotypic age” a composite of clinical markers selected based on prediction of time-to-death. This raises important questions regarding the optimum approach to derive biological age markers using untargeted approaches such as “omics”. Aging is driven by both intrinsic  aging and disease specific processes and it is practically difficult to separate these processes. Identifying molecular markers associated with chronological age, particularly in populations including younger, healthy individuals, presents one route to identifying intrinsic biological age markers, distinct from early effect markers of specific disease processes, that may precede subsequent changes in phenotype and health . It is oMetabolomic analysis in blood can provide a more complete picture of biological processes involved in aging than DNA methylation analysis, as metabolites represent the final products of cellular metabolism including in organs and tissues throughout the body, rather than in just blood cells themselves. All the hallmarks of aging may be expected to have detectable effects on the metabolome and overlap significantly with the effects of metabolic disorders . The metAge-related disease including cancers, cardiovascular disease and dementia, provide the greatest health burden in developed countries and it is hoped that targeting the biological aging process, particularly earlier in life, will have greater success in reducing the burden of disease than the current approach of disease-specific treatments in later life . Assessm


Age related hearing loss, or presbycusis, is a global condition that is increasing in its prevalence. Despite being one of the most common chronic conditions among the older population, there is much more to understand about its association with other aspects of physical and emotional health and well-being. Current research is suggesting that hearing loss is more prevalent in those with cognitive impairment compared to those without cognitive impairment. This study analyzed the incidence of hearing loss and its linkage to mild cognitive impairment in a community-dwelling geriatric population. With the increasing prevalence of this condition in both rural and urban communities of Alabama, it becomes a more pressing matter to understand comorbidities and risk factors for future decline in functioning. This study was conducted in an interdisciplinary geriatrics primary care outpatient clinic in a Family, Internal, and Rural Medicine department affiliated with a university medical center in the Deep South. Ninety-one participants completed the Montreal Cognitive Assessment (MoCA) and a hearing screening. Hearing screenings were conducted in quiet rooms in the medical center using Phonak hearing screening cards. Detection of 500, 1000, 2000, and 4000 Hz tones was assessed. Pearson correlation analyses demonstrated an association between hearing loss mild cognitive impairment. Poorer hearing was significantly associated with lower scores on the MoCA. Conducting behavioral health screenings like this in other primary geriatrics clinics and community settings could improve care and identification of patient needs by integrating important data regarding comorbidities and independent living.