Methamphetamine (METH) is an illicit psychostimulant that is subject to abuse worldwide. While the modulatory effects of METH on dopamine neurotransmission and its neurotoxicity in the central nervous system are well studied, METH’s effects on modulating microglial neuroimmune functions and on eliciting neuroinflammation to affect dopaminergic neurotoxicity has attracted considerable attention in recent years. The current review illuminates METH-induced neurotoxicity from a neuropathological perspective by summarizing studies reporting microglial activation after METH administration in rodents. Assessing microglial reactivity in terms of the cells’ morphology and immunophenotype offers an opportunity for comprehensive and objective assessment of the severity and nature of METH-induced neuronal perturbations in the CNS and can thus contribute to a better understanding of the nature of METH toxicity. We reach the conclusion here that the intensity of microglial activation reported in the majority of animal models after METH administration is quite modest, indicating that the extent of dopaminergic neuron damage directly caused by this neurotoxicant is relatively minor. Our conclusion stands in contrast to claims of excessive and detrimental neuroinflammation believed to contribute and exacerbate METH neurotoxicity. Thus, our analysis of published studies does not support the idea that suppression of microglial activity with anti-inflammatory agents could yield beneficial effects in terms of treating addiction disorders. The effects of METH in the central nervous system (CNS) are well studied, and for some time now, METH has been reported to increase dopamine (DA) neurotransmission through regulation of dopamine transporter activity –3, and tWe view microglia as “sensors of pathology,” cells that continuously monitor neuronal well-being and become alerted (activated) when neuronal activity is disturbed or compromised by injury, disease, or neurotoxic agents. Microglia are thus the first line of defense against toxic insults and most sensitive sentinels of neurotoxic effects and neuronal damage in general. Their responses to disturbed neuronal homeostasis likely result in neuroprotective and/or reparative mechanisms in order to minimize neuronal damage. As we discuss here, the experimental evidence available does indeed support this notion by showing relatively mild activation of microglia after METH and little, if any, evidence of frank degeneration of DA neurons.+ 2 and Na+ ions, dysregulation of mitochondrial function, neuronal energetic imbalance, and overproduction of reactive oxygen species. The primary goal of the current review is to re-evaluate this neurotoxicity from the perspective of reactive microglial cell changes, as neuroinflammatory reactions have been reported to occur following METH administration and are believed to causally contribute to METH-induced neurotoxicity [For a variety of reasons methamphetamine’s neurotoxic effects, although well-studied, are not well-defined . In expetoxicity –7.Fig. 1Rather than viewing microglia as potentially harmful cells that attack neurons, we view microglia as sensitive biological indicators of neuronal perturbations and perhaps the most reliable cellular sentinels of neurotoxic effects and neuronal damage in general. Microglia have long been recognized as “sensors of pathology”  because We understand the term “neurotoxicity” to mean that METH exposure is directly toxic to neurons, but not necessarily in the sense that METH kills neurons. Dopaminergic neurons are of particular interest because METH is known to directly target dopamine neurons; it competes with the uptake of released dopamine via the dopamine transporter (DAT); it disrupts vesicular storage of dopamine; and induces reverse transport of dopamine through the transporter termed “dopamine efflux” –15. ThesMicroglial reactions visualized histopathologically are more telling than biochemical measures of GFAP in that the microscopic observation of microglial responses better inform about the regional specificity, nature, and severity of METH-induced neurotoxicity. Not only do microglia display different morphological forms after a damaging event, their surface immunophenotype is marked by presence of multiple membrane-bound receptors that undergo characteristic changes during activation, which are detectable with antibodies directed against a variety of immunological important molecules, such as major histocompatibility (MHC) antigens, complement and Fc receptors, integrins, surface immunoglobulins, and toll-like receptors, in addition to presence of intracellular microglial antigens, like the ionized calcium-binding adapter molecule 1 recognized by the Iba1 antibody . Neverththe critical morphological feature for identifying activated microglia. Studies show that in the striatum microglia remain ramified, but upregulate isolectin B4 binding relative to controls where microglia are unstained [In mice exposed to METH, there are noticeable differences in the nature and intensity of microglial activation that occurs in the nigrostriatal dopaminergic system versus how microglia respond in limbic structures, such as hippocampus and amygdala. While the effects of METH on microglial biology in the ventral tegmental area (VTA) are unclear and there are no reports of microglial activation occurring around VTA dopaminergic neurons, in the striatum where dopaminergic terminal degeneration has been suggested by positive silver staining and decreased levels of dopamine and TH , 23, 24,nstained –27. In anstained . These onstained . Togethenstained . Interesnstained .The direct effects of METH are not limited to neurons; there is also vascular toxicity and blood-brain barrier (BBB) damage, as well as hyperthermia and seizures that contribute to neurotoxicity and to microglial activation. Not surprisingly, hyperthermia and neuroinflammation are much exacerbated if animals receive bacterial lipopolysaccharide after METH . While tMETH is reported to affect immune function in multiple and generally suppressive ways, which were reviewed recently . In termThe claim that METH-induced neuroinflammation perpetuates neurodegeneration is speculative and unproven, and it is unclear whether it contributes to METH-induced neurotoxicity . We are Additional evidence against the idea that neuroinflammation causes neurodegeneration stem from observations that the microglial response in the striatum after METH administration is quite mild and can be characterized as minimal microglial activation, if it occurs at all Fig. . This slIn summary, immune responses are not required to elicit METH neurotoxicity, rather the immune reaction is a response to the damaging effects of this toxic substance on neurons. Whether or not microglia play an important role in cellular mechanisms of addiction remains an interesting question at this time that will require further investigation Fig. .Fig. 2a


Animal models remain invaluable for study of respiratory diseases, however, translation of data generated in genetically homogeneous animals housed in a clean and well-controlled environment does not necessarily provide insight to the human disease situation. In vitro human systems such as air liquid interface (ALI) cultures and organ-on-a-chip models have attempted to bridge the divide between animal models and human patients. However, although 3D in nature, these models struggle to recreate the architecture and complex cellularity of the airways and parenchyma, and therefore cannot mimic the complex cell-cell interactions in the lung. To address this issue, lung slices have emerged as a useful ex vivo tool for studying the respiratory responses to inflammatory stimuli, infection, and novel drug compounds. This review covers the practicality of precision cut lung slice (PCLS) generation and benefits of this ex vivo culture system in modeling human lung biology and disease pathogenesis. Respiratory disease is a leading cause of death, closely following heart disease, stroke and cancer according to the World Health Organization . DespiteOne factor for the limited success in identifying new drug therapies in many disease areas is the lack of translatable animal models used to evaluate efficacy and safety . MammaliHistorically, basic studies in respiratory research have primarily relied on transformed human cell lines, stimulated in culture and in isolation. More recently, culture systems comprising airway epithelial cells grown as a 2D monolayer or as a more complex 3D differentiated air liquid interface (ALI) culture including goblet, club and basal cells, have been mainstays of in vitro modeling –9. Use oModel systems such as those described above have pushed the boundaries of in vitro modeling capabilities, however, they still have drawbacks. Reliance on artificial scaffolds and the difficulty of acquiring primary cells from patients prevents such systems from completely recapitulating the complexities of respiratory disease. For structural relevance tracheal pieces in organ baths have been traditionally utilized for monitoring for smooth muscle function and constriction , 19. ForThe concept of using tissue slices to study organ metabolism and toxicology was initiated in the 1920s, with substantial improvements in slicing technology since. Initially, liver slices were usually cut manually resulting in high variability of thickness and limited viability . The useFor soft tissue such as lung, which is closely inter-networked by extracellular matrix and honeycombed to enable maximal gas exchange, it is technically challenging to obtain PCLS. However, a major breakthrough was achieved by Placke and Fisher in 1980s  by infus2 and 95–100% air humidity under tissue culture conditions, and medium is changed daily  cells, IgE increase, and airway hyperresponsiveness, bronchoconstriction and remodeling [Animals have been successfully utilized to mimic various components in the pathophysiology of human asthma. Mice and rats are commonly used to replicate aspects of allergen induced asthma. The mouse develops relevant features of human allergic asthma upon allergic sensitization and challenge, resulting in allergen-specific response of T helper type 2  is a chronic lung disease pathologically characterized by progressive interstitial fibrosis, with an incidence of estimated 2.8–18 cases per 100000 people per year in Europe and North America , 76. IPFPCLS have been successfully used to study the early onset of lung fibrosis in IPF. By exposure to TGF-β1 and cadmium chloride, PCLS from human or rat have shown relevant pathohistological changes observed in early lung fibrosis, including upregulation of important pro-fibrotic genes, increased thickness of alveolar septa, and aberrant activation of pulmonary cells , 85, 86.The PCLS system has been used with some success to study innate responses to viral challenge, and to a lesser extent bacterial or bacterial component challenge. Use of the PCLS system has enabled a better understanding of which cells are infected in the intact lung as compared with in vitro air liquid interface cultures . Goris eRegulatory toxicology testing is highly reliant on in vivo experiments in animals where it is key to identify organ toxicities that represent responses in a complex physiological environment. At present this is an essential step to identify and exclude risk in humans. In order to reduce candidate drug attrition due to toxicology or lack of clinical safety, predictive toxicology strategies have been implemented in earlier stages of the drug discovery process . Such a In vitro systems such as ALI cultures have been applied to detect adverse impact of drugs in these more complex in vitro system, where the models comprise human primary epithelial cells on a basement membrane with a focus on predicting the toxic effects of inhaled drugs . However2-receptor agonists have been shown to relax airway smooth muscle of contracted PCLS, mimicking their performance in animal models or in clinical trials [The more complex mix of cells and architecture in lung slice models is an attractive proposition for efficacy and toxicology testing, representing a translatable system between cell line or primary cell culture systems and the in vivo situation Fig. . Indeed,l trials –105.Fig.2-receptor agonists necessitates development of novel therapeutic strategies. Targets relevant to asthma pathogenesis have therefore attracted more attention in recent years and investigations in PCLS have benefitted the evaluation of these targets as potential therapeutics. For example, inhibition of histone deacetylase by trichostatin A abrogates airway constriction in human PCLS and simultaneously inhibits airway hyperresponsiveness in antigen-challenged mice [PCLS have also been utilized to evaluate alternative targets for asthma treatment as increasing tolerance towards glucocorticoids and βged mice . Furtherged mice . Indeed ged mice , 106–109With regards to safety of inhaled therapeutics, key functional aspects and damage are assessed using well-validated pathological readouts on standardized safety studies intended for safety pharmacology assessments . HoweverAlthough a lung slice comprises a section of lung tissue with the spatial arrangement and context preserved, the fact remains that tissue removed from the living organisms is to some extent fixed with regards to the cell populations contained within. Thus, a cultured PCLS represents a snapshot of cell populations currently resident in the lung tissue at time of excision but does not provide access to the wider, recruitable immune system, which is so critical in the whole organism. Additional consideration needs to be taken for heterogeneity in the lung. Differences in epithelial integrity, resident immune cell populations and responses to stimulation will be found between different locations within a single lobe. Furthermore, when studying diseased lung, heterogeneity between samples will further complicate data interpretation. Therefor appropriate power analysis should be used to account for slice to slice variability.It is well documented that upon pathogenic invasion, the resident immune and tissue cells in the lung can release messenger molecules  that attract further immune cells to the site to deal with the invading organism –113. TheThe inability to recruit non-resident immune cells, and viability of only 2 weeks, limits the extent to which the immune response in a cultured PCLS system can replicate the in vivo situation. However, the initiation and early signals induced by bacterial and viral pathogens can be studied in the PCLS model. This affords a wealth of opportunities to investigate the signals of the innate immune system and resident lung cells such as alveolar macrophages, alveolar type II cells and club cells. The lung slice model also provides an opportunity to investigate modification, by immunomodulatory treatments under investigation for inflammatory lung disease, of the initial innate immune signals released by resident lung cells upon exposure to pathogens. A challenge with any treatment of the PCLS system is the route of administration. Because the entire slice is bathed in the compound or stimulant of interest, one cannot directly translate treatment of the slice to inhaled or systemic application in vivo. Furthermore, translation of dosing from PCLS to human will prove difficult for the same reason. Despite this limitation, use of PCLS could provide great utility in both testing efficacy, but also in assessment of the potential adverse immunosuppressive impact of these drugs on host defense.The PCLS model is typically used as a static system, not “breathing” as the lung typically would. This becomes important when modeling diseases such as ventilator induced lung injury, which rely on the mechanical stress applied by ventilation to damage the epithelial barrier . A limitIn vitro models of respiratory disease have matured over the past decades, changing from single cell systems to complex multidimensional, multicellular systems Table . They arMethods for high-throughput processing and culture, improved drug delivery techniques to enable application of compounds to either the airway or blood stream, and incorporation of additional immune components will enhance this already powerful model. Despite their current drawbacks, PCLS are well situated to be a powerful model system for both basic research and drug screening workflows.


Stroke is a leading cause of long-term disability worldwide and often impairs walking ability. To improve recovery of walking function post-stroke, researchers have investigated the use of treatments such as fast functional electrical stimulation (FastFES). During FastFES treatments, individuals post-stroke walk on a treadmill at their fastest comfortable speed while electrical stimulation is delivered to two muscles of the paretic ankle, ideally to improve paretic leg propulsion and toe clearance. However, muscle selection and stimulation timing are currently standardized based on clinical intuition and a one-size-fits-all approach, which may explain in part why some patients respond to FastFES training while others do not. This study explores how personalized neuromusculoskeletal models could potentially be used to enable individual-specific selection of target muscles and stimulation timing to address unique functional limitations of individual patients post-stroke. Treadmill gait data, including EMG, surface marker positions, and ground reactions, were collected from an individual post-stroke who was a non-responder to FastFES treatment. The patient's gait data were used to personalize key aspects of a full-body neuromusculoskeletal walking model, including lower-body joint functional axes, lower-body muscle force generating properties, deformable foot-ground contact properties, and paretic and non-paretic leg neural control properties. The personalized model was utilized within a direct collocation optimal control framework to reproduce the patient's unstimulated treadmill gait data (verification problem) and to generate three stimulated walking predictions that sought to minimize inter-limb propulsive force asymmetry (prediction problems). The three predictions used: (1) Standard muscle selection  with standard stimulation timing, (2) Standard muscle selection with optimized stimulation timing, and (3) Optimized muscle selection (soleus and semimembranosus) with optimized stimulation timing. Relative to unstimulated walking, the optimal control problems predicted a 41% reduction in propulsive force asymmetry for scenario (1), a 45% reduction for scenario (2), and a 64% reduction for scenario (3), suggesting that non-standard muscle selection may be superior for this patient. Despite these predicted improvements, kinematic symmetry was not noticeably improved for any of the walking predictions. These results suggest that personalized neuromusculoskeletal models may be able to predict personalized FastFES training prescriptions that could improve propulsive force symmetry, though inclusion of kinematic requirements would be necessary to improve kinematic symmetry as well. Approximately 15 million people experience a stroke each year  is a promising treatment for improving walking ability post-stroke. However, little data currently exist for determining the best way to customize the treatment to individual-specific gait deficits. The rationale for FastFES training is that fast speed walking by itself can improve gait biomechanics in individuals post-stroke, while task-specific electrical stimulation of selected muscles can provide feedback to the nervous system to promote motor learning of the appropriate timing and activation of the stimulated muscles  who was a non-responder to the standard FastFES treatment protocol. The subject gave written informed consent, and the study was approved by the institutional review boards of Emory University and the University of Florida. Collected data included full-body video motion capture data , bilateral force plate data from a split-belt instrumented treadmill with belts tied , and surface EMG data from 14 muscles per leg , althougExperimental data were collected for several types of trials. Static trial data were collected in which the subject stood upright with an anatomically neutral joint alignment for several seconds with feet pointing forward. Data from this static trial were used for scaling an initial generic musculoskeletal model and determining the locations of reflective surface markers on the model (see below). Six different isolated joint motion trials were performed to facilitate calibration of lower-body joint positions and orientations in the body segments. One isolated joint motion trial was performed for each hip, knee, and ankle, where each trial exercised all functional axes for the selected joint  in their respective body segments as well as parameters defining the positions and orientations of marker triads placed on the pelvis, thighs, shanks, and feet of the model. The cost function minimized the sum of squares of errors between experimental and model-predicted marker positions using all motion trials together. Each function evaluation performed an OpenSim “Inverse Kinematics” analysis to calculate the current marker location errors. Matlab's “lsqnonlin” non-linear least squares algorithm was used to perform the optimization. The calibrated joint and marker triad positions and orientations were applied to the model and used in a final OpenSim “Inverse Kinematics” analysis to determine joint position, velocity, and acceleration time histories for subsequent steps of the model personalization process.The second step involved personalization of the model's EMG-driven muscle-tendon force and moment generating properties using data from 40 gait cycles and OpenSim “Inverse Dynamics” and “Muscle” analyses. The data needed for this step included joint kinematics found as in the first step along with ground reaction and EMG data. Three tasks were performed in preparation for this model personalization step. First, EMG data from 40 gait cycles were processed  as described in Meyer et al. , resultiOnce these preparatory tasks were completed, we formulated an optimization problem that sought to calibrate Hill-type muscle-tendon model parameters  along with parameters defining the surrogate musculoskeletal geometry. The cost function minimized the weighted sum of squares of errors between three types of lower-body quantities: (1) inverse dynamic and model-predicted total joint moments, (2) published experimentally measured , we incorporated muscle synergy techniques into the optimization process to estimate the activations for the 4 muscles in each leg with missing EMG signals  force symmetry for three FastFES treatment scenarios: (1) Standard muscle selection with standard stimulation timing, (2) Standard muscle selection with optimized stimulation timing, and (3) Optimized muscle selection with optimized stimulation timing. These three treatment scenarios built upon a baseline treatment optimization that predicted the theoretically achievable improvement in AP force symmetry under unstimulated conditions so that the effects of electrical stimulation could be isolated.ton, off-time toff, and amplitude A. Since our optimal control prediction problems used controls related to muscle activation  rather than muscle excitation , we developed a closed-form equation that approximated the amplitude and shape of the activation output produced by activation dynamics  by adding simulated electrical stimulation to two selected paretic leg muscles. For each treatment optimization, simulated electrical stimulation was added on top of the subject's simulated unstimulated muscle activations. Electrical stimulation waveforms were assumed to be simple step functions defined by an on-time astim(t) is the amplitude of activation produced by electrical stimulation at the current time t, tend is the final time of the gait cycle, and c1, c2, toffset1, and toffset2 are adjustable parameters. For any stimulated muscle, we used non-linear least squares optimization to calibrate the four parameters c1, c2, toffset1, and toffset2 to match the output of the muscle's activation dynamics as closely as possible given a step function input of amplitude one. Thus, given A, ton, and toff for any muscle, Equation (1) with calibrated parameters was used to define the muscle's time-varying activation from electrical stimulation. The form of Equation (1) also allows for electrical stimulation to extend beyond the end of the gait cycle and wrap around into the start of the same gait cycle. The total activation of a stimulated muscle was assumed to be the sum of its activation from electrical stimulation and its activation from muscle synergies, where the sum was constrained to be less than one. Furthermore, the maximum activation from electrical stimulation was bounded to be ≤ 0.7 so that total activation would never exceed one.In this equation, As a starting point for treatment optimization, we formulated and solved a baseline optimal control problem to quantify AP force asymmetry in the absence of electrical stimulation but with minimization of AP force asymmetry added to the cost function  and medial gastrocnemius (GasMed)—with standard stimulation timing  ranging from 2.5 to 6.4 Nm and mean absolute errors (MAE) between 2.0 and 4.7 Nm . Ground The three FastFES treatment optimizations predicted progressively lower AP force asymmetry relative to the baseline optimization with no muscle stimulation  along wiPredicted reductions in AP force asymmetry were accompanied by notable changes in predicted propulsive as well as braking force ; Table 8While the predicted joint angles for the four treatment optimizations were similar to the experimental gait motion, some differences were still evident . For parPredicted lower body joint moments also exhibited notable changes in response to the different simulated stimulation conditions . For allThis study used a personalized neuromusculoskeletal walking model coupled with direct collocation optimal control to predict how FastFES treatments should be implemented to maximize propulsive force symmetry for an individual post-stroke who was a non-responder to the standard FastFES training protocol. Though FastFES is a promising treatment for post-stroke gait neurorehabilitation, methods for customizing FastFES prescriptions to the unique needs of individual patients have yet to be developed. Using treadmill gait data collected from a non-responder to FastFES training, we personalized a full-body neuromusculoskeletal model and then used it to predict improvement in the subject's AP force symmetry for three FastFES treatment scenarios: (1) Standard muscle selection with standard stimulation timing, (2) Standard muscle selection with optimized stimulation timing, and (3) Optimized muscle selection with optimized stimulation timing. Overall, the more flexibility a FastFES treatment optimization was given, the more the subject's predicted AP force asymmetry was reduced. Our results suggest that for this particular subject, (1) Stimulation of standard muscles  with standard timing should produce an acute improvement in the subject's propulsive force symmetry between the two legs, (2) A comparable improvement in propulsive force symmetry could potentially be achieved for this subject by stimulating TibAnt with standard settings and GasMed with decreased amplitude but increased duration, (3) A larger improvement in the subject's propulsive force symmetry could potentially be achieved by stimulating Sol and Semimem in place of TibAnt and GasMed, and (4) Large improvements in propulsive force symmetry may not guarantee large improvements in joint motion symmetry. Thus, future optimal control studies should explore adding kinematic symmetry terms to the optimization cost function so that improvements in both types of symmetry can be predicted simultaneously. The methodology developed in this study therefore provides only a first step toward computational design of personalized FastFES prescriptions that are customized to the unique functional limitations of the patient.with stimulation (the situation predicted by the model), AP force symmetry is improved due to an acute response to the electrical stimulation, often termed an orthotic effect. This orthotic effect demonstrates that the FES is able to augment force generation in the ankle muscles and generate greater paretic leg propulsion while the FES is on. In contrast, after multiple sessions of FastFES training, when gait performance is evaluated in the community or measured in the lab without stimulation, AP force symmetry is determined by the therapeutic or long-term retention effect of the treatment. The therapeutic effect may be influenced by multiple factors including the magnitude of favorable neuroplasticity induced through repeated training as well as improvements in muscle strength, cardiovascular endurance, and psychosocial factors. EMG data published in the previous FastFES clinical study  selected by our third treatment optimization would be logical choices based on interpretation of their biomechanical roles. While paretic (right) propulsive force generation was clearly inhibited in the subject's experimental gait pattern, braking force was also larger on the paretic side than on the non-paretic side, likely due to poor coordination. Thus, muscle stimulation that acts to decrease early-stance braking and/or increase late-stance propulsion would improve propulsion symmetry in this subject. Indeed, the optimization that selected the best two muscles to stimulate chose one muscle to increase propulsive force in late stance (Sol) and another muscle to decrease braking force in early stance (Semimem), consistent with minimization of AP force impulse asymmetry. Several studies have reported that Sol and GasMed contribute to forward acceleration of the trunk in mid to late stance , more synergies are required than indicated by synergy analysis of muscle activation data alone. Because our walking predictions are dynamically consistent, the muscle activations controlling the model must be of high enough fidelity to reproduce the subject's experimental data closely. Based on our previous work  might be more effective for this subject than stimulation of the two standard muscles (GasMed and TibAnt), we examined the structure of the subject's synergy vectors (SVs) for 2 and 3 paretic leg synergies . We chosInterestingly, the three FastFES treatment optimizations predicted comparable values for peak paretic propulsive force during stance phase  as well Our study possesses several important limitations that inform interpretation of our current results and suggest directions for future investigation. First, only a single subject was studied. We specifically selected a non-responder to FastFES training so as to maximize our chances of identifying alternate stimulation protocols that could potentially improve the subject's walking ability. Whether or not the same modeling approach would work for other subjects will require further investigation. Second, no measurements were available for the forces and torques exerted by the handlebar on each hand, plus the hand loads estimated during the model personalization process were applied to the model during the treatment optimization process. Experimental measurement of hand loads would greatly simplify model personalization. Allowing the hand loads to vary during treatment optimization could alter our propulsive force predictions. Third, experimental stimulation does not target individual muscles as directly as modeled in this study. In practice, medial and lateral gastrocnemius are often stimulated together by a single electrode. Even if GasMed and TibAnt were well-targeted for electrical stimulation, some stimulation would likely “bleed” into other plantarflexor and dorsiflexor muscles. Modeling of this “bleeding” phenomenon would impact our treatment predictions, though the extent to which the predictions would be changed is unknown. Fourth, no measure of kinematic asymmetry was included in the optimal control cost function. When initiating this study, we expected that improved propulsive force symmetry would naturally result in improved joint motion symmetry. This expectation proved to be incorrect, suggesting that some measure of kinematic asymmetry should be included in future optimal control studies of FastFES treatment design. However, no published study to date has presented an optimal control problem formulation that is capable of turning an asymmetric walking motion into a symmetric one. Thus, development of a problem formulation that enforces kinematic symmetry remains an important challenge for the neuromusculoskeletal modeling research community. Fifth, simulated electrical stimulation was explored for only two paretic leg muscles due to current technical limitations in the FastFES hardware and software. If stimulation of three or even four muscles was investigated, it is possible that substantial improvements in both propulsive force symmetry and kinematic symmetry could be predicted. However, stimulation of more than two muscles is not feasible with the electrical stimulation system used in this study. Sixth, our FastFES treatment optimizations assumed that the subject's neural control strategy remained largely unchanged by the application of FES. In reality, if a subject responds to FastFES training, one would expect his or her neural control strategy to change over the course of treatment as favorable neuroplasticity occurred , all model personalization optimizations could be completed in roughly 5 h of CPU time, while each treatment optimization required between 3 and 15 min of CPU time. Thus, the primary bottleneck was not computation time but rather the time required to learn the entire computational workflow, process the experimental data to get them into the correct format, and identify appropriate optimization problem formulations to get each step to work properly the first time. In the present study, the most challenging problem formulation issues were related to how to minimize propulsive force asymmetry, model the hands grasping the handlebar, and predict the best two muscles to stimulate. None of these issues had been explored in previous optimal control studies of human walking, and all of them required running hundreds of optimal control problems before appropriate problem formulations could be identified. We are continuing to refine our model personalization and treatment optimization workflow so that the steep learning curve currently required to become proficient in the entire process can be eliminated as a bottleneck.One of the biggest limitations of this study was our inability to evaluate experimentally our optimal FastFES treatment prediction. Such an evaluation would have required completion of an additional training study for this subject, which unfortunately we were unable to perform. We hope to be able to apply our optimal treatment prediction to this subject, as well as explore the use of model-based optimal treatment predictions for other subjects, as part of a future FastFES training study.In conclusion, this study explored the feasibility of using subject-specific neuromusculoskeletal models combined with direct collocation optimal control to predict novel FastFES treatment prescriptions that may improve a specified treatment target—in this case, inter-leg propulsive force symmetry. The ability to tailor neurorehabilitation treatments to the unique needs of individual patients would be an important next step in modern healthcare. In the case of stroke, walking deficits vary widely from patient to patient, highlighting the need for an objective and effective treatment customization process. In the current study, a computational approach to FastFES treatment customization predicted that changing the stimulation amplitude and timing of a typically stimulated muscle, or changing which two muscles are stimulated, may improve a specific subject's paretic propulsion significantly. While this computational approach is still too difficult and time consuming to be feasible on a large scale, future improvements in computational methodology and technology may eventually make it possible to perform this approach on a routine basis, potentially allowing treatment decisions to be based on objective predictions of a patient's post-treatment function.Simtk.org at https://simtk.org/projects/predictfastfes.The Matlab code and experimental data used to perform this study can be found on The human subject was recruited at Emory University and gave informed written consent. The study was approved by the institutional review boards of Emory University where the data were collected and the University of Florida where the data were modeled.NS performed all model personalization and treatment optimization tasks, wrote the first draft of the manuscript, and assisted with preparation of figures and tables. AM assisted with development of the personalized neuromusculoskeletal model, modification of the optimal control problem formulations to accommodate the goals of the present study, and review of the manuscript draft. JA oversaw experimental data collection for the project, processed all experimental data, and provided feedback on the manuscript draft. LT and TK recruited the experimental subject, organized the experimental data collection session, helped identify treatment scenarios to optimize, and provided feedback on the manuscript draft. BF coordinated the entire project, oversaw model personalization and treatment optimization tasks, edited the manuscript draft, and prepared the final figures and tables.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.


Adding biomarker information to real world datasets (e.g. biomarker data collected into disease/drug registries) can enhance mechanistic understanding of intra-patient differences in disease trajectories and differences in important clinical outcomes. Biomarkers can detect pathologies present early in disease potentially paving the way for preventative intervention strategies, which may help patients to avoid disability, poor treatment outcome, disease sequelae and premature mortality. However, adding biomarker data to real world datasets comes with a number of important challenges including sample collection and storage, study design and data analysis and interpretation. In this narrative review we will consider the benefits and challenges of adding biomarker data to real world datasets and discuss how biomarker data have added to our understanding of complex diseases, focusing on rheumatoid arthritis. Addition of biomarker information to real-world datasets is challenging but could capture clinically useful information.Biomarkers can detect pathologies present early in disease potentially paving the way for preventative interventions.Biomarkers can quantify important exposures underpinning intra-patient differences in disease outcomes to facilitate precision medicine.Real world datasets include information collected from individuals in routine clinical care with a particular condition or diagnosis or receiving a particular drug, as opposed to patients recruited to trials. Real world data can enhance patient management because the length of follow-up can be much longer than randomized control trials (RCTs); they include patients with complex issues, of the type seen in routine clinical settings, who are often excluded from RCTs due to the exclusion criteria; and cohort sizes tend to be larger than conventional RCTs. They provide health care professionals and researchers with information on people with diseases, at the individual and group level, often over time enabling analysis of trends in treatment, risks of adverse events (e.g. infection) and disability.Insight into the outcomes that are linked to particular disease trajectories can be obtained from clinical and demographic factors, disease and treatment history, and co-morbidity and risk exposures that are recorded in real world databases. However, these factors often only partially explain intra-patient differences in important clinical outcomes, suggesting that other factors contribute. Such factors could be biological, many of which can be measured directly or through a proxy; such biomarkers can correlate with stage of disease, response to treatment or adverse events . BiomarkAdvantages of biomarkers over routinely collected clinical data are that the markers are measured objectively and therefore not subject to observer bias, they can be measured reliably and precisely, and they are closer to the pathology and may provide mechanistic insight. Some biomarkers become so accepted that they become part of routine data collection; for example, anti-citrullinated peptide antibodies (ACPA) detected by anti-cyclic citrullinated peptide (anti-CCP) antibodies are now routinely tested because they define a subset of cases with RA with a more severe disease course .In this review we will consider the advantages that addition of biomarker data to real world datasets brings, discuss how biomarker data have improved our understanding of disease outcomes in RA, and consider the challenges faced when interpreting biomarker discoveries or when planning to add biomarker data to real world data collections.Once a patient presents with inflammatory arthritis, it is now widely accepted that the primary goal should be to control disease activity as quickly as possible. Indeed, real world data have shown that control of disease activity within the first 6 months of presentation of inflammatory arthritis can influence outcome 20 years later . Over thHLA-DRB1 and RA development  and subjective  measures, the latter scores receiving higher weightings . We havePsychological factors, including high pre-treatment anxiety scores, have been reported to be associated with non-response to methotrexate, with the definition of non-response including patients who stopped their treatment before 6 months . HoweverEven with an optimized treatment response outcome that is reflective of synovial inflammation, the identification of biomarkers predictive of response would still not be straightforward as a number of factors can confound response prediction. We have previously reported that self-reported adherence to TNFi therapies was sub-optimal (27% of patients self-reporting non-adherence) and correlated with future response . TherefoRA is a relapsing–remitting disease in which patients experience moments of high and low disease activity as part of the natural disease course. Therefore a distinction that is difficult to make in real world datasets is whether disease activity has improved because of successful treatment, or because disease activity was initially very high and has more room to improve before the subsequent clinical visit. In a trial setting the issue is resolved by randomizing patients to one of two (or more) treatment arms but that is not possible in the real world. In statistics the phenomenon is referred to as regression towards the mean and must be considered when designing biomarker studies or when interpreting results from real world datasets. Ensuring matching of the baseline characteristics of the two comparator groups can be used for within-cohort studies or propensity score matching  can be uOnce biomarkers of clinical response to treatment have been identified, the next challenge will be to test if they are predictive of response to a specific treatment or if they are specific to a particular disease. Here numerous datasets are required, including patients exposed to alternative treatments, across different stages of disease and across different inflammatory diseases. In the UK, these datasets have already been established in highly successful national stratified medicine programmes in RA, psoriasis, systemic lupus erythematosus, Sjogren's syndrome, autoimmune hepatitis and primary biliary cirrhosis. The Immune-Mediated Inflammatory Disease Biobanks in the UK (IMID-Bio-UK)  is bringAnalysing real world databases, with or without biomarker data, exposes a number of potential biases that require consideration. A recent editorial published in Nature has highlighted statistical challenges that should be considered in designing and analysing studies. These include regression to the mean, natural variation, the selection of an appropriate outcome measure and using a continuous rather than a dichotomous outcome. Some of these issues are discussed in more detail in the review by Prof. Til Sturmer as part of this supplementary edition.A large number of biomarker studies have been published yet few markers have reached the clinic. When interpreting the findings of biomarker studies, therefore, a degree of scepticism is healthy. Validation of findings is a pre-requisite but even if replicated in independent datasets, a biomarker may not add sufficient information to make it clinically useful. How predictive a biomarker needs to be to be clinically useful will depend very much on the context in which it is used. For example, diagnostic accuracy is very important for tests that will determine whether treatment is given or withheld but a test with less stringent performance characteristics may be adequate where choices are being made between treatments, i.e. preferential prescribing (complementary diagnostics). Several studies are underway currently to establish recommended cut-offs for tests aimed at guiding selection of the first biologic therapy for RA, for example. Demonstration of clinical utility may need to be followed by health economic assessment to show that the cost of the test does not outweigh the benefit accrued. Helpfully, the OMERACT Consortium has developed guidelines for the clinical validation of biomarkers .In summary, sceptics would argue that, despite huge investment in biomarker research, few successes have emerged. We would argue that biomarker studies have added value to real world data collections in inflammatory arthritis and have already identified biomarkers that are being used or have the potential to be used in routine clinical practice. These include anti-CCP testing, which is already in use; drug level and antidrug antibody testing in patients being treated with TNFi, which is currently being reviewed by NICE; and anti-CarP antibody testing, which requires further assessment of clinical utility. A major goal for biomarker research is to help inform and personalize the choice of treatment recommended to patients to maximize clinical benefit. Before this goal can be realized in RA, a quantitative disease activity measure(s), which is easy to measure for all patients and is reflective of synovial inflammation, is first needed and will likely be discovered by well-designed biomarker experiments. For example, assessing blood-based biomarkers for correlation with MRI-observed synovitis might identify a biological surrogate for active joint-specific inflammation that is better than CRP alone and can be objectively measured in all patients. Other major obstacles to overcome include the inadequate control of confounding, which can partly be addressed by testing for drug adherence and anti-drug antibody titres and by more sophisticated methods of matching patient groups for comparison. Reproducibility of biomarker discoveries to date has been hampered by a lack of statistical power that can partly be addressed by increased funding for establishing and maintaining real world datasets and collection of biological samples as part of expected practice. Discovery of biomarkers of clinical importance will also likely require large collaborative efforts involving many academic groups and industry partners. The emergence of high dimensional datasets and advanced data-driven statistical methods, such as machine learning, offer the potential to develop accurate, robust and discriminative statistical classifiers of important clinical outcomes in RA. It is possible that many small differences can be combined to develop scores that will, together, allow better prediction of clinically important outcomes; for example, genotypic risk scores have been proposed to aid identification of patients at the highest risk of developing cardiovascular disease . HoweverFunding: D.P. is funded by the NIHR Manchester BRC. We acknowledge support from Arthritis Research UK (grant ref 21754) and MRC/Arthritis Research UK stratified medicine award, MATURA (grant ref MR/K015346/1).Disclosure statement: The authors have declared no conflicts of interest.


Carcinus maenas) shows strong robustness of transient dynamics in keeping not only the activation sequences but also specific cycle-by-cycle temporal relationships in the form of strong linear correlations between pivotal time intervals, i.e. dynamical invariants. The level of variability and coordination was characterized using intrinsic time references and intervals in long recordings of both regular and irregular rhythms. Out of the many possible combinations of time intervals studied, only two cycle-by-cycle dynamical invariants were identified, existing even outside steady states. While executing a neural sequence, dynamical invariants reflect constraints to optimize functionality by shaping the actual intervals in which activity emerges to build the sequence. Our results indicate that such boundaries to the adaptability arise from the interaction between the rich dynamics of neurons and connections. We suggest that invariant temporal sequence relationships could be present in other networks, including those shaping sequences of functional brain rhythms, and underlie rhythm programming and functionality.By studying different sources of temporal variability in central pattern generator (CPG) circuits, we unveil fundamental aspects of the instantaneous balance between flexibility and robustness in sequential dynamics -a property that characterizes many systems that display neural rhythms. Our analysis of the triphasic rhythm of the pyloric CPG ( As experimental and theoretical studies show, sequence generation is a key computational phenomenon to encode, control and execute information in sensory, central and motor networks19. In many cases, robust sequences underlie what is usually simply viewed and termed as a rhythm. Unveiling general principles in the generation and coordination of neural sequences, particularly in transient regimes, is an important step in relating neural activity to function and has potential impact in other fields such as rehabilitation technology, robotics and control theory23.Robust sequences of neural activations can be found in any nervous system, from simple invertebrate circuits25. Their robust and highly coordinated neuron activation sequences arise from the combination of intrinsic cell and synaptic dynamics27. Invertebrate CPGs are key neural circuits to understand sequence generation and coordination, as their cells and connections have been identified and mapped, like in the crustacean pyloric CPG that we use in this study30. Most CPGs have what is called a non-open topology31, i.e., all neurons in the CPG receive input from other cells in the circuit for transient closed-loop computation. An essential component of these sequence generator networks is reciprocal inhibition between pairs of neurons. Mutual inhibition together with electrical coupling and other non-reciprocal interactions32 underly the timing of neuron activations that shape each cycle35. CPGs activate muscles that produce motor rhythms and their proper function is crucial for animal survival.Central pattern generators (CPGs) are neural circuits that produce flexible rhythmic motor patterns36. On the other hand, in vivo recordings of CPG activity display further temporal characteristics and a larger degree of irregularity than in vitro recordings38. Recordings of isolated cells show that most of them are highly irregular42. Rich intrinsic cell and synaptic dynamics, arising from different time scales27, enable neurons comprising the CPG to readily establish a network rhythm in concert26. Recent work has proposed that variability in cycle period can be controlled by synaptic feedback44. External and intrinsic factors continuously affect the system inducing transients and therefore making them an important element of the system functionality, which can be better observed in recordings of irregular activity  neuron, a group of six to eight pyloric neurons (PY), two electrically coupled Pyloric Dilator (PD) neurons and the Anterior Burster (AB), also electrically coupled to the PDs57). Pyloric rhythm under ethanol is characterized by a remarkably flexible and long PD burst duration, and variability in hyperpolarization in both neurons. At the same time, LP burst duration presents much less variability as compared to the PD neuron. Despite the large irregularity induced by ethanol, the sequence LP-PY-PD in the rhythm is still preserved. After washing or ethanol evaporation, regular activity is recovered.Irregular activity can also be seen in cases of intrinsic variability in the preparation, which may be a result of external neural modulation, severed modulator nerves, etc. Panel B in Fig. The analysis of variability of CPG rhythms in all conditions was assessed by defining specific intervals with precise time references using the first and last spike of bursts from intracellular recordings. We chose seven intervals , i.e., a Departing from well-defined time references at the burst beginning and end in the LP and PD neurons, we analyzed entclass1pt{minimaApproximate phase maintenance observed in previous studies was revealed by averaging intervals across preparations (see Introduction). Following the same procedure by calculating interval averages for each preparation, correlation is found between all intervals and the averaged period and even among them in control and in most cases in ethanol conditions  entclass1pt{minimaFigure mentclass2pt{minimThe presence of both invariants A major contribution of our work is the demonstration of cycle-by-cycle adjustments that give rise to the invariants, which can be indirectly be seen in Figs Panel A in Fig. mentclass2pt{minim62. Some of these sequences are often very robust and directly related to the execution of motor commands, cognitive decisions and behavioral actions. When generating robust sequential activations, cycle-by-cycle flexibility and fine tuning of instantaneous periods, phases and event timings can be crucial for the optimization and achievement of effective functions. In this paper, we have addressed this issue in a well-known experimental model where these questions can be more easily examined.Although typically characterized by frequency and synchronization, most brain rhythms throughout the nervous system are built from sequential activations of groups of neurons36. However, the analysis of irregular CPG rhythms, rich in transient dynamics and not only in steady state activity, can unveil important properties of the robust neuron and network dynamics underlying sequence programming and coordination. Irregular rhythms were obtained in this study by two means: intrinsic irregularity, and biophysical disruption with ethanol. Moderate ethanol application did not disrupt the anti-phase relationship between LP and PD neurons, thus the robustness of the sequence was kept, but evoked variable burst duration and hyperpolarization intervals. The effect of ethanol was reversible and in most cases the neurons returned to their original rhythm after ethanol was washed out or evaporated.Most experimental and computational studies on CPGs focus their analysis on regular regimes, frequently discarding non-regular transient activityAs opposed to traditional regular activity recordings, irregular rhythms caused by intrinsic factors in the preparation presented high variability in hyperpolarization intervals and waveforms in both LP and PD neurons. LP presented larger plateaus and higher variability in burst duration while PD activity remained less variable. Irregularity induced by ethanol, however, presented remarkably flexible and long PD burst durations, while LP burst duration was more restricted. Ethanol also induced variability in the hyperpolarization intervals in both neurons.Our results show that the CPG tends to preserve specific cycle-by-cycle temporal relationships between neurons even under extreme conditions, which points out to the circuit’s highly effective negotiating properties and the dynamical arrangement of the motor rhythm to balance robustness and flexibility. Using an adequate time reference frame and experimental conditions to expose transient dynamics, our characterization of cycle-by-cycle variability in CPG circuits has revealed the presence of dynamical invariants in neural sequences. Cycle-by-cycle analysis allowed to center the study of dynamical invariants in transient regimes, without losing the temporal relationship between pivotal time intervals building the sequence. Results show that 59. When connectivity is asymmetric, the selection of the time references to define the intervals is crucial for exposing potential dynamical invariants. LP neuron receives less connections than other neurons in the pyloric circuit. Therefore, it has more flexibility to adapt and coordinate its activity with the rest of the circuit elements in a cycle-by-cycle basis, making this neuron a better candidate as a time reference . Also note that the two invariants observed during cycle-by-cycle transients are different than the approximate phase maintenance reported in previous works that used cross-preparations analysis, steady activity recordings, and other time references59. Approximate phase maintenance, obtained by averaging phase and periods in different preparations or in the same preparation under different treatments59, might reflect some aspects of the unveiled invariants, but not their presence in cycle-by-cycle analysis, in particular during transients. In our analysis, averaging intervals within preparations .Most neural functions are supported by neuronal oscillatory activity, often simply referred to as a rhythm71, but most importantly, the sequential activation of its constituent elements. Inhibition based mechanisms offer specific time windows where neurons can express their excitability, balancing the robustness of the sequence and the flexibility to tune activation timings. The actual execution of a sequential neural command, e.g., in the performance of a movement, is determined not only by the serial order of individual participants but also by their timing. This is the case for the pyloric CPG, as most likely fine timing adaptations are required to optimize the function of the motor plant beyond keeping the sequence needed to move food from one side to the other.Most neural rhythms, as pyloric neural oscillations, are based on inhibition as the main mechanism shaping not only the rhythmic activity21 can provide autonomous solutions to different situations informed by sensory feedback.The unveiling of dynamical invariants in the spatio-temporal patterns of neural activity may have an important impact in robotics. Traditional robotic locomotion control paradigms are based on ad-hoc rules to deal with different scenarios . The concept of dynamical invariants provides an alternative way to autonomously build constraints to drive behavior in all situations. In this context, a dynamical invariant based CPG control arising from the connectivity and rich intrinsic neuron dynamics66. We foresee that the study of specific time references and dynamical invariants in different neural systems will provide novel views on the functional role of brain rhythms and their constituent sequences.Beyond spiking-bursting activity and CPG function, dynamical invariants in other brain rhythms can underlie the creation of cyclic windows within oscillations when synaptic input can be most efficiently integrated for the effective execution of sequences generated in a given informational contextCarcinus maenas) were purchased locally and maintained in a tank with 13–15 °C artificial seawater. Crabs were anesthetized by ice for 15 min before the dissection. The procedures followed the European Commission and Universidad Autónoma de Madrid animal treatment guidelines. The stomatogastric nervous system was dissected following standard procedures and pinned in a Sylgard-coated dish containing Carcinus maenas saline (in https://www.st-andrews.ac.uk/wjh/dataview/), first applying a FIR filter and then a threshold-crossing criterion to detect the beginning of each spike. Since we used a high threshold and worked with intervals, calculated by subtracting consecutive time references, the error introduced by using the beginning of the spike is mostly cancel out. In each recording, the distribution of the spikes was used to select the time windows that defined the intra and inter burst intervals, and particularly the first and last spike of each burst (see Matlab scripts in Adult male and female shore crabs (To analyze and quantify regular and irregular recordings, we considered several interval measures based on precise time references at the beginning and at the end of the bursts (see Fig. The coefficient of variation defined as entclass1pt{minimaThe significance level In this case, the number of hypothesis is the correlations between different combination of the defined time intervals (A t-test was also performed to tested the significance of the correlation coefficients among 16 experiments for each hypothesis Standardized cycle intervals where Experimental data analysis was implemented with Matlab. In the Supplementary informationSupplementary VideoSupplementary scriptsDataset


Hospital-acquired malnutrition is a significant issue with complex aetiology, hence nutrition interventions must be multifaceted and context-specific. This paper describes the development, implementation and process evaluation of a complex intervention for improving nutrition among medical patients in an Australian hospital. An integrated knowledge translation (iKT) approach was used for intervention development, informed by previous research. Intervention strategies targeted patients (via a nutrition intake monitoring system); staff (discipline-specific training targeting identified barriers); and the organisation (foodservice system changes). A process evaluation was conducted parallel to implementation assessing reach, dose, fidelity and staff responses to the intervention using a mixed-methods design . Staff-level interventions had high fidelity and broad reach . Patient and organisation interventions were implemented effectively, but due to staffing issues, only reached around 60% of patients. Staff found all intervention strategies acceptable with benefits to practice. This study found an iKT approach useful for designing a nutrition intervention that was context-specific, feasible and acceptable to staff. This was likely due to engagement of multiple disciplines, identifying and targeting specific areas in need of improvement, and giving staff frequent opportunities to contribute to intervention development/implementation. Malnutrition is a significant issue that health organisations, clinicians and researchers have attempted to address for decades ,2. DespiDeveloping and implementing health interventions that are feasible, acceptable and effective in the hospital setting is challenging. This is especially the case with nutrition interventions, where previous research has shown to be either effective but not practical or sustainable ; or feaOur team used this approach to develop a multifaceted, tailored intervention to improve nutrition care, delivery and intakes among patients in an acute medical setting. Understanding the processes underpinning development and implementation of interventions is important when evaluating its outcomes to give insight into what worked, for whom and under what conditions. The medical research council (MRC) of the United Kingdom recommends conducting process evaluations for complex interventions for more meaningful understanding of its effects ,13. The This program of research used an iKT approach  to develA process evaluation was conducted parallel to the implementation of the intervention, following MRC guidance for process evaluation of complex interventions . The proThe study was conducted in the acute medical unit (AMU) at a 364-bed public metropolitan teaching hospital in southeast Queensland. The AMU is a 28-bed ward with a 1:4 to 1:6 nurse-to-patient ratio. Participants were patients and family members receiving care on the ward and staff providing care/services to the ward , and foodservice staff).The research team engaged knowledge end-users in three ways: (1) including end-users on the study team; (2) consulting with a nutrition reference group developed specifically for the study; and (3) engaging in regular group discussions with staff on the study ward see . The resFindings from Phase 1  and Phase 2 (stakeholder interviews) informed the intervention design, in conjunction with theory/evidence from the literature. Baseline meal audits highlighted that nutritional intakes among patients on the AMU were poor and that several factors affected nutrition at patient, staff and organisational levels. Interviews with patients, families and staff provided more detail on why and how these complex factors occurred and interacted, and how they might be addressed. Interview findings are reported in detail elsewhere  but in sIn conjunction with the nutrition reference group, the research team brainstormed possible intervention strategies targeting the identified problem areas at patient, staff and organisation levels. Focus groups were held with nursing, foodservice, and allied health staff on the AMU to share the findings from Phases 1 and 2 and to seek feedback. In these sessions, a member of the research team presented findings from previous study phases and asked staff if the findings resonated with their experiences with nutrition care on the ward. Next, the researcher facilitated discussions around possible strategies to address the problem areas identified, allowing staff to present their ideas first and then seeking feedback on the strategies brainstormed by the research team and nutrition reference group. Notes taken in these sessions were collated and used by the study team and nutrition reference group to finalise the intervention plan. The intervention components are detailed in Quantitative data were collected on intervention implementation  during Phases 3 (implementation) and 4 . For patient-level interventions, researchers recorded use of nutrition intake magnets on patient whiteboards. Staff training logs were used to evaluate intervention reach (number of staff present at each training session), dose (time spent delivering each session) and fidelity (content covered in each session) of the discipline-specific staff training. Data on organisation-level interventions (earlier breakfast delivery and use of new ‘full + hot breakfast’ diet) were collected during the post-intervention audit period. Qualitative data on end-user  responses to and interactions with the intervention were collected by taking notes during informal, semi-structured individual and focus group interviews. Questions were asked using a semi-structured interview guide with questions informed by the theoretical domains framework (TDF), which is commonly used to assess barriers and facilitators to change in complex health care settings ,19.Quantitative data  were analysed descriptively using frequencies (number and percent) using IBM SPSS Statistics for Windows version 23.0 . Qualitative data were analysed using content analysis of notes taken during interviews and focus groups. Patient-level implementation: nutrition intake magnets were present on patients’ whiteboards on 228 of 418 meal occasions observed (55%). They were most commonly in place at dinner (60%) and lunch (59%), whilst at breakfast only 47% of patients had magnets present. The majority of patients (64%) had green magnets, indicating adequate nutritional intake; 12% were orange (moderate risk) and 24% were red (high risk). The implementation record noted that the AMU nutrition assistant (responsible for updating magnets) was on leave for two out of the four weeks of data collection without position backfill. Staff-level implementation: delivery of the staff-level intervention (discipline specific staff training) in terms of fidelity, dose and reach is described in Organisational-level implementation: the purpose-developed ‘full + hot breakfast’ diet was implemented by the foodservice dietitian prior to the Phase 4 audit. This diet was prescribed to 59% of eligible patients  by nursing staff. The remaining 41% of patients were prescribed the standard ‘full diet’.Breakfast delivery times improved from pre-intervention  to post-intervention (consistently delivered between 0745–0805 h).Nurses, dietitians, nutrition assistants and foodservice staff said they found the intervention acceptable; they thought it was useful to staff and helpful to patients. Their detailed feedback provides insights into possible causal mechanisms for why the intervention may have worked.Nutrition intake magnets: the nutrition intake magnets were spoken about frequently. Staff described how the magnets prompted them to change their practice in some way, and noted that they helped indicate patients who were at risk or needed help. For example, foodservice staff described how they spent extra time assisting patients with meal set up, made an effort to communicate poor intakes to nursing staff, or left items for patients to eat later when collecting trays if they noticed a red or orange magnet. Nutrition staff thought the magnets were useful to doctors as a reminder of how they could engage in patients’ nutrition. The dietitian thought the magnets could feasibly be continued as a part of usual practice on the ward. Nurses agreed, but suggested some changes to the magnet system, such as using a single magnet with two sides (red for inadequate intake and green for adequate intake), rather than having three separate magnets  to simplify the process.‘Full + hot breakfast diet’: access to this new diet was considered by most staff to be an acceptable intervention. Foodservice and nutrition staff thought it would result in benefits such as reduced food wastage and a more patient-centred approach to nutrition. This diet was seen to reduce menu fatigue, with four hot options available each day; and patients had the ability to order hot items only if they wanted to , thus limiting food wastage. The nutrition assistant indicated this diet was so useful it was since being applied to patients all around the hospital  even after the study finished.Nursing staff engagement: when asked why they thought the intervention worked, nutrition staff highlighted the engagement of nursing staff on the AMU played an integral role in the implementation and uptake of the intervention. The leadership from nurses in authoritative positions  and support from nurses who were key influencers were thought to positively affect the responses and behaviour of all nurses on the ward. The dietitian said AMU nurses already had a good knowledge and understanding of the importance of nutrition and described them to be nutrition-focused, which she thought was another reason as to why they responded well to the intervention. The nutrition assistant described high nurse engagement in the intervention; for example, some nurses helped to update patients’ nutrition intake magnets even though this was proposed as the nutrition assistant’s role. Interestingly, whilst other professions highlighted that nurses played a central role in the success of the intervention, nurses themselves did not acknowledge this. Even after being shown the study findings, some nurses showed apprehension in believing the changes in nutrition intakes were a result of the intervention and sought alternate explanations. Several nurses proposed other factors that they thought might have affected the results; however, the researcher facilitating focus groups was able to explain that these factors were not at play or had already been accounted for. For example, some nurses thought the post-intervention data collection may have occurred when more students were present on the ward, so more patients may have received feeding assistance. However, pre- and post-intervention data collection periods were done at the same time of the year (February–March) one year apart, with similar student numbers on the ward in both time periods. After these explanations, nurses were more accepting of the results. Some nurses suggested the regular presence of researchers on the ward  helped to raise the profile of nutrition in practice. Medical staff engagement: the dietitian found that having a medical consultant supportive of the intervention from the outset had a large impact on the receptiveness of the rest of the medical team to the intervention. She described how one consultant, who already prioritised nutrition as an important part of patient care, made an effort to communicate the medical staff training aspect of the intervention  to the rest of their team. Some nurses reported that they had heard doctors asking patients about nutrition  during ward rounds, and said one team in particular incorporated this into their rounds and their medical notes in the electronic medical record. An unexpected outcome of the intervention was that tensions between foodservice and nursing staff described in Phase 2 during pre-intervention staff interviews were not apparent in post-intervention focus groups. When asked what (if anything) had changed in this respect , most staff said nothing had changed. When asked/prompted about the issues raised previously, both nursing and foodservice staff said these were not really an issue.Clinical effectiveness of the intervention  is reported elsewhere . In this mixed method study, the development, implementation and process evaluation of a tailored, multifaceted intervention for improving nutrition care, delivery and intakes among patients in an acute medical setting was described. This paper highlights the importance of an iKT approach  in the development, implementation and evaluation of complex interventions. Changing practice is widely acknowledged to be a difficult process. Knowledge translation approaches make practice change more achievable by partnering with knowledge end-users throughout the entire research process and co-developing interventions and implementation strategies . The useIntervention reach for staff training was high , likely due to influential staff being on the study team  or supportive of the study . Fidelity was 100% across all staff groups  and dose was similar across staff in each group. This was reflected in other process outcomes, such as patient-level interventions; 59% of patients eligible for the ‘full + hot breakfast diet’ were allocated it by nurses, and 55% of patients had nutrition intake magnets in place during the post-intervention audit. It was noted that the nutrition assistant, who was responsible for updating patients’ nutrition intake magnets, was on leave for two of the four weeks that the audit took place, and her position was not backfilled. Hence, the finding that only about half the patients had their magnets updated daily is not surprising. We did note that some nurses took on this responsibility in some instances; however, temporary/casual nursing staff likely did not receive the training so they would not know what the magnets meant. Several nurses suggested using a single magnet with two sides rather than having three separate magnets, which they thought would simplify the process in several ways. Firstly, they highlighted that the orange magnet was redundant as patients either ate well or ate poorly, and the system should reflect this. Secondly, by using just one double-sided magnet , nurses could keep whiteboards updated more easily as they would not have to go looking for the correctly coloured magnet. The nutrition assistant kept the coloured magnets in a box, and not all nurses knew where it was kept; whereas a double-sided magnet would always be present on each patient’s whiteboard. These suggestions were important for future tailoring of the intervention. Finally, the organisational-wide strategy (moving breakfast to an earlier time) was effective, with breakfast consistently being delivered at 0800 h post-intervention, compared to inconsistent delivery between 0800–0900 h pre-intervention. This change was important, as it enabled the ward to meet one of Queensland Health Statewide Foodservices Network’s key performance indicators of no longer than 13 h between the last meal service and the next day’s first meal service . HoweverResponses by staff to the intervention were mostly positive, and several discussed how they thought it worked to improve nutrition care, delivery and intakes . Many of their perceptions aligned with the TDF, which is often used to explain human behaviour in complex clinical environments . For exaNurses’ high engagement in the intervention correlated with several theoretical domains. Firstly, nutrition staff thought nurses responded well to the intervention as they generally had a good knowledge and understanding of the importance of nutrition and described the ward to be ‘nutrition-focused’; aligning with domains ‘knowledge’, ‘skills’ and ‘environmental context and resources’ . Also, nurses seemed to generally take ownership for nutrition and saw it as their role, which translated into some nurses taking responsibility for intervention strategies ; consistent with the ‘social/professional role and identity’ domain. Finally, the leadership, support and influence of nurses in authoritative roles  were seen to play a large role in engaging all ward nurses and ultimately the intervention’s success, consistent with previous literature . This alSeveral staff said the nutrition intake magnet system prompted them to change their practice or behaviour. For example, nurses said it helped them identify at-risk patients, foodservice staff said they spent more time assisting patients with meal set-up and communicate with nurses, and the dietitian thought it helped to remind doctors to discuss nutrition. This aligns with the ‘memory, attention and decision processes’ domain, which involves retaining information, focusing selectively on aspects of the environment and making clinical decisions . This maProcesses relating to the iKT approach used may have had indirect effects on the success of the intervention. For example, the regular presence of nutrition researchers on the ward and regular contact between researchers and ward staff could have increased staff awareness and increased the profile of nutrition. The fact that the ward dietitian, nurse unit manager and nurse clinical facilitator were involved in the study may have increased nurses’ acceptance of it. Regular updates provided to ward staff (such as presenting findings from each study phase and facilitating discussions around these) may have acted as an audit and feedback type mechanism, whereby staff were provided with data specific to their ward and patients, highlighting areas in need of improvement . FinallyThis study has several limitations. Firstly, the intervention was evaluated using a pre/post design (findings will be reported elsewhere), which was most feasible for the co-development approach. Secondly, this study was conducted in one medical ward, at one Australian hospital, so the findings cannot be generalised. However, this detailed example of how to use an iKT approach to develop, implement and evaluate a multifaceted intervention targeting improved nutrition among hospitalised patients provides an implementation model for testing in other sites. Finally, due to the limited study timeframe, we were unable to assess intervention sustainability. This study used an iKT approach to develop and implement a complex, tailored intervention aiming to improve nutrition care, delivery and intakes among patients in an acute medical ward. This paper described the intervention co-development process (with end-users) and presented an evaluation of the processes underpinning implementation in a real-world clinical setting. Quality of intervention delivery  was high, which is most likely attributed to the strong stakeholder engagement. In particular, involvement of nurses in authoritative positions on the ward  seemed to increase acceptance and use of intervention strategies by ward nurses. The engagement of multiple disciplines, the identification and targeting of specific areas in need of improvement in that ward, and providing staff with frequent opportunities to contribute to intervention development and implementation were also important. Whilst developing the intervention was time and resource intensive, it ultimately resulted in positive outcomes. Hence, sharing our learnings is important for advancing implementation science in the field of nutrition by providing an example of how an iKT approach can be used to develop and implement a malnutrition prevention intervention in an acute medical setting.


Porphyromonas gingivalis produce a large amount of butyric acid as a metabolite. Though butyric acid has been reported to have an anti-inflammatory effect on inflammatory diseases in the gastrointestinal tract, it has been suggested to contribute to the progression of periodontal disease in the oral cavity. The concentration of butyric acid in periodontal tissue of patients with periodontitis patients is reported to increase with the progress of the periodontal disease state. However, the influence of butyric acid on periodontal disease progression is not well known.Periodontopathic bacteria such as In this review, we have considered the relationship between butyric acid and periodontal disease with respect to the findings reported till date and the knowledge we newly obtained . We have studied the relationship between butyric acid and periodontal disease by analyzing the effect of butyric acid on normal human gingival fibroblasts, which are a major component of periodontal tissue. We observed that gingival fibroblasts underwent cytostasis and apoptosis via extrinsic and intrinsic pathways upon long-term exposure to butyric acid. In addition, we showed that TNF-α produced by gingival fibroblasts treated with butyric acid plays an important role in inducing exogenous apoptosis.Butyric acid produced by periodontopathic bacteria may promote progress of the periodontal disease state. Butyric acid is known to act as an HDAC inhibitor. Thus, we believe that advanced epigenetic analysis of the effects of butyric acid on gingival fibroblasts will help elucidate the periodontal disease pathology and facilitate discovery of new targets for periodontal disease treatment. Porphyromonas gingivalis, Treponema denticola, Tannerella forsythensis). However, little attention has been paid to the effect of metabolites produced by periodontal pathogens on the progression of periodontal diseases. The bacterial flora differs considerably at the supragingival and subgingival level and the metabolites produced are also different. The cariogenic bacteria in supragingival plaques, such as Streptococcus mutans, produce lactic acid, which induces dental caries by lowering the pH of dental enamel [Porphyromonas gingivalis and Fusobacterium nucleatum in subgingival plaques produce a large amount of short chain fatty acids including butyric acid as metabolites [Periodontal disease is a lifestyle-related disease . Periodol enamel . Periodoabolites . Howeverabolites . As a reabolites –16. TherPorphyromonas gingivalis are known to differ between healthy and inflamed gingival fibroblasts [Porphyromonas gingivalis than HGFs collected from healthy subjects. The pro-inflammatory cytokine mRNA expression in inflamed subjects is reportedly upregulated by lower LPS and shorter stimulation time compared to that in healthy subjects [High concentrations of butyric acid are reported to induce apoptosis in immune cells –19. Thisroblasts . HGFs cosubjects . Therefosubjects , the expsubjects .Normal human gingival fibroblasts were reported to be less sensitive to butyric acid . The numPeriodontal disease is also an inflammatory disease. The concentration of pro-inflammatory cytokines such as IL-6 and TNF-α in gingival crevicular fluid (GCF) increases with the progress of periodontal disease . Pro-infButyric acid is known to act as an HDAC inhibitor , 26. HDAButyric acid produced by periodontal pathogens may collapse the homeostasis in periodontal tissues. Considering the metabolites of periodontal pathogens, which have not been noticed so far, may lead to the discovery of novel targets for treatment. It is thus important to further clarify the effect of butyric acid on periodontal tissues in order to elucidate the mechanism of progression of periodontal disease pathology and develop breakthrough treatments.


ASBO is a common cause of emergency surgery and the use of laparoscopy for the treatment of these patients is still under debate and conflicting results have been published, in particular regarding the high risk of iatrogenic bowel injury. In fact, although over the last few years there has been an increasing enthusiasm in the surgical community about the advantages and potential better outcomes of laparoscopic management of adhesive small bowel obstruction (ASBO), recently published studies have introduced a significant word of caution. From 2011 in our centre, we have started to systematically approach ASBO in carefully selected patients with a step-by-step standardized laparoscopic procedure, developed and performed by a single operator experienced in emergency laparoscopy, collecting data in a prospective database. Inclusion criteria were: stable patients (without diffuse peritonitis and/or septic shock with suspicion of bowel perforation), CT scan findings consistent with a clear transition point and therefore suspected to have a single obstructing adhesive band. Patients with diffuse SB distension in the absence of a well-defined transition point and suspected to have diffuse matted adhesions  should be initially managed conservatively, including gastrografin challenge. Up to date, 83 patients were enrolled in the study. The rate of iatrogenic full-thickness bowel injury was 4/83 (4.8%); two of these cases were managed with simple repair and the other two required bowel resection and anastomosis. Conversion to open was performed in 3/4 of these cases, whereas in one a repair of the full-thickness injury was completed laparoscopically. All the iatrogenic injuries were detected intraoperatively and none of the reoperations that occurred in this series were due to missed bowel injuries. At 30 days follow-up, none reported incisional hernias or SSI or death. With the described accurate selection of patients, the use of such standardized step-by-step technique and in the presence of dedicated operating surgeons with advanced emergency surgery laparoscopic expertise, such procedure can be safe and feasible with multiple advantages in terms of morbidity and LOS. A careful preoperative selection of those patients who might be best candidates for laparoscopic adhesiolysis is needed. The level of laparoscopic expertise can also be highly variable, and not having advanced surgical expertise in the specific subspecialty of emergency laparoscopy, ultimately resulting in performing standardized procedures with proper careful and safe step-by-step technique, is highly recommended. Dear Editor,We read with great interest the study by Behman et al. on laparoscopic adhesiolysis . DespiteFirstly, careful preoperative selection of those patients who might be best suited for laparoscopic adhesiolysis is needed; the level of laparoscopic expertise may be highly variable, and having advanced surgical expertise in the specific subspecialty of emergency laparoscopy , leadingWe have started to systematically approach ASBO in carefully selected patients from 2011 with a step-by-step standardized laparoscopic procedure, developed and performed by a single operator experienced in emergency laparoscopy, collecting data in a prospective database . Up to MIn detail, the inclusion and exclusion criteria in our experience were:adult patients;informed consent;initial CT diagnosis of complete ASBO with an identifiable transition point and an anticipated single obstructing band with completely collapsed distal small bowel loops;and/or with a radiological/clinical evidence of failure of a NOM trial with hyperosmolar WSCM via NGT.Inclusion criteria:hemodynamic instability and preoperative shock;diffuse peritonitis and/or evidence of severe intra-abdominal sepsis;high suspicion of gangrenous/perforated bowel;high probability of diffuse and dense matted adhesions (e.g. multiple previous laparotomies ≥ 3 with intraoperative findings of diffuse dense and matted adhesions);preoperative diagnosis of any other cause of complete mechanical SBO than adhesions .Exclusion criteria:previous midline laparotomy was not considered an absolute contraindication;suspicion of bowel strangulation and/or volvulus and/or bowel ischaemia without gangrene or perforation;localized clinical peritonitis;CT findings of free abdominal fluid;CT or AXR findings of small bowel distension with a size > 4 cm;CT additional findings such as SB faeces sign, SB thickening, and mesenteric oedema/vascular engorgement.The following were NOT exclusion criteria from diagnostic laparoscopy in our experience, although these are risk factors for conversion after an initial diagnostic laparoscopy:Careful interpretation of the CT scan findings may be useful in identifying good candidates for a laparoscopic approach. We also recommend that only fully trained and experienced laparoscopic surgeons should attempt this technique. The correct surgical technique is of paramount importance to avoid bowel injuries. From our experience, once a laparoscopic approach is decided, we recommend not to use Veress needles or blind insertion of the first port in close proximity to the previous scars. We believe that a safe entry to the abdomen can be best obtained by either inserting the first Hasson trocar in the left flank with open access or by using a blunt dilating tip, optical trocar entering the abdominal wall at the level of the Palmer point, under direct vision step 1—Fig. —Fig. 1 . Once thFurther tips and tricks derived from our experience are not to immediately search for the transition point and for the strangulating band by manipulating the distended and fragile bowel loops, but rather to start with identification of the caecum, then starting to explore from the collapsed distal ileal loop in a distal-to-proximal fashion step 2—Fig. —Fig. 2. Any bleeding during lysis of adhesions is generally minor and often self-limiting. Gauze compression may help and any persistent bleeding can be dealt with at the end of the operation, after releasing the obstruction and before finishing the procedure. Finally, adhesiolysis should be limited to only the obstructing band or to the adhesions that need to be divided to reach the transition point. Further extensive adhesiolysis is unnecessary, potentially harmful and should therefore be avoided. Careful observation for any serosal tears over the loops which were manipulated may allow intraoperative laparoscopic simple repair of these, thus preventing postoperative fistula or leak. Full-thickness injury usually mandates conversion. In our previously mentioned prospective single-operator series (unpublished data), the current rate of iatrogenic full-thickness bowel injury is 4/83 (4.8%); two of these cases were managed with simple repair and the other two required bowel resection and anastomosis. Conversion to open was performed in 3/4 of these cases, whereas in one a repair of the full-thickness injury was completed laparoscopically. All the iatrogenic injuries were detected intraoperatively and none of the reoperations that occurred in this series were due to missed bowel injuries.With the described accurate selection of patients, the use of such standardized step-by-step technique  and in tIn the study by Behman, patient selection criteria are not specified since the study is a population-based cohort study where data have been collected from health administrative records. However, some degree of patient selection emerges from the study, since patients undergoing laparoscopic procedures were younger, had fewer comorbidities and were cared for at larger hospitals.We therefore agree with the authors’ conclusions that surgeons should approach laparoscopic adhesiolysis with a high level of awareness and use strategies to mitigate the risks. We suggest that laparoscopic adhesiolysis is not for all patients and may not be for all surgeons. Ideally, laparoscopic adhesiolysis should be only performed in high volume centres with specific expertise in emergency laparoscopy, especially in tertiary referral centres with availability of operating room and laparoscopic equipment at all times, and with highly trained surgical and OR staff.As investigators of the current Finnish–Italian LASSO trial4, we eagerly await the results and will hopefully clarify further details about safety and outcomes of the laparoscopic treatment of ASBO.