Synaptic plasticity, the experience-induced change in connections between neurons, underlies learning and memory in the brain. Most of our understanding of synaptic plasticity derives from in vitro experiments with precisely repeated stimulus patterns; however, neurons exhibit significant variability in vivo during repeated experiences. Further, the spatial pattern of synaptic inputs to the dendritic tree influences synaptic plasticity, yet is not considered in most synaptic plasticity rules. Here, we investigate how spatiotemporal synaptic input patterns produce plasticity with in vivo-like conditions using a data-driven computational model with a plasticity rule based on calcium dynamics. Using in vivo spike train recordings as inputs to different size clusters of spines, we show that plasticity is strongly robust to trial-to-trial variability of spike timing. In addition, we derive general synaptic plasticity rules describing how spatiotemporal patterns of synaptic inputs control the magnitude and direction of plasticity. Synapses that strongly potentiated have greater firing rates and calcium concentration later in the trial, whereas strongly depressing synapses have hiring firing rates early in the trial. The neighboring synaptic activity influences the direction and magnitude of synaptic plasticity, with small clusters of spines producing the greatest increase in synaptic strength. Together, our results reveal that calcium dynamics can unify diverse plasticity rules and reveal how spatiotemporal firing rate patterns control synaptic plasticity. Synaptic plasticity—the activity-dependent modification of synaptic strength—is widely hypothesized as the neural substrate of learning and memory throughout the brain . For insCortical neurons exhibit significant trial-to-trial variability in vivo in response to the same external sensory stimulus ,17. TriaExperiments suggest that spatial organization of synaptic inputs on the dendritic tree are important for plasticity. For instance, in vitro, cortical synaptic plasticity has been shown to depend not only on rate and timing, but also on cooperativity of inputs , which iIn spiny projection neurons (SPNs) of the striatum, the input nucleus of the basal ganglia and the focus of this paper, dopamine and calcium activated signaling pathways interact to produce synaptic plasticity. In vitro, a dopamine signal occurring within 1–2 s of synaptic activation produces long term potentiation . In vivoOne approach to investigate how in vivo variability influences synaptic plasticity is to evaluate calcium signals in response to in vivo spike trains. The spatiotemporal dynamics of the calcium signal, and not just peak calcium, determine the occurrence and direction (potentiation or depression) of plasticity ,30,31. SCRCNS.org (accessed on 23 March 2019) repository. The anterior lateral motor cortex was selected because those pyramidal neurons project to the striatum, and the spike trains were collected during a learning task known to involve the striatum [We developed a biologically-constrained computational SPN model with multiple ion channels identified in SPNs, real dendritic morphology, explicit dendritic spines, sophisticated calcium dynamics, and a calcium-based plasticity rule. Model parameters were determined using our parameter optimization software to fit the model to electrophysiological current injection data . Synaptistriatum ,42. The https://moose.ncbs.res.in, accessed on 6 June 2022 (version 3.2.0.dev20190806)). We used a D1 SPN morphology obtained from the Luebke repository [neuromorpho.org, accessed on 6 June 2022 (ID: Apr29IR2b) [A validated, biophysical SPN model we previously published  was updapository  on neuror29IR2b) . Dendrite values  using a e values . Values As previously described , the mod3.3/α1I) . We newl3.3/α1I) ,63. ChanModel optimization was done using ajustador software ,64 that Intracellular calcium concentration was modeled with diffusion, calcium-binding proteins (buffers), and transmembrane calcium pumps. One-dimensional radial diffusion of buffers and calcium was modeled with concentric shells in dendrites, while spine heads and necks implemented one-dimensional axial diffusion in cylindrical slabs connected from the spine neck to the submembrane shell of the dendritic shaft . TransmeThe sources of calcium influx in the model included the voltage gated calcium channels and the NMDAR synaptic channel. Calcium concentration in the submembrane shell was used for calcium-dependent channel activation  or inactivation .A channels were included on the dendritic shaft with a density of 4 synapses per dendritic compartment. Activation of these synapses is described in Excitatory NMDAR and AMPAR synaptic channels were included on spine heads, and each cortical synaptic input activated both the NMDAR and AMPAR. Inhibitory GABAThe calcium-based plasticity rule used dual amplitude and duration thresholds for spine calcium concentration to determine LTP and LTD, as we previously described . For LTDCRCNS.org (accessed on 23 March 2019) repository, consisting of recordings from 25 simultaneously recorded anterior lateral motor cortex pyramidal neurons with 90 repeated trials [In vivo cortical spike trains were obtained from a d trials . A singlTo generate controlled trial-to-trial variability of spike timing, the initial trial was repeated 10 times with random jitter of each spike on each repetition. Trial-to-trial variability was limited to standard deviation of spike timing while constraining the same total number of spikes per spike train within each trial. The random jitter was generated from a truncated normal distribution using a standard deviation of 1, 10, 100, or 200 ms . Experiments consisted of 10 trials with a single standard deviation. Trials were separated by a 1 s intertrial interval, during which time membrane potential returned to resting potential and spiking activity ceased.We also implemented a different type of trial-to-trial variability that allows variability of spike rate for individual synapses, but maintains the same spike timing to the neuron as a whole. Variable spike rate was implemented by randomly moving individual spikes from one pre-synaptic input train to another, with the probability of each spike being moved between 10 and 100%. To maintain the distribution of spike counts per trial, the randomly selected target train probability was weighted by the number of spikes of the target trains. This ensured both the overall spike timing pattern to the whole neuron was maintained as well as the distribution of spike counts to synapses, while allowing small variations in spike rate for each synapse from trial to trial.To evaluate the role of spatial pattern, a single trial was repeated using different mappings of spike trains to clusters of synapses. Each simulation used a different number of spines per cluster and a different cluster length (dendritic distance between the most separated pair of spines in the cluster). In addition to excitatory inputs, the neuron also received two types of inhibitory inputs. All inhibitory inputs were constructed from Poisson processes with mean firing rates consistent with both striatal fast spiking interneurons (FSIs) and low threshold spiking interneurons (LTSIs). FSI inputs were generated with a mean firing rate of 12 Hz  and densAnalysis of simulations used Python3 and the following python packages: Numpy, Scipy, Pandas, Scikit-learn, Statsmodels, and Matplotlib. For analysis relating spatiotemporal input patterns to magnitude and direction of synaptic weight change, we introduce a “weight-change triggered average,” which is constructed by grouping synapses per trial into bins based on the value of the weight change at the end of a trial, computing the instantaneous firing rate of the spike train input to each given synapse, and averaging the instantaneous firing rates within each bin of synaptic weight change. This is analogous to a spike-triggered average , but usiRandom forest regression was applied to the instantaneous firing rate to a synapse, instantaneous firing rate to neighboring synapses, cluster length, and the distance from synapse to soma. Random forest regression applies a non-linear method for predicting the weight change from a set of features, such as pre-synaptic firing rate. The prediction is determined from a set of hierarchical rules, where each rule partitions the weight change based on a single feature. A non-linear method was required because of the non-linear relationship between weight change and pre-synaptic firing. The instantaneous firing rate of the input was discretized into 1-5 features (time samples), where 1 time sample calculates the mean firing rate for the entire trial, and 5 time samples calculates mean firing frequency for subsequent 200 ms time intervals. To determine the optimal features for predicting the weight change, a random forest regression was performed for each combination of features. For each set of simulations, the trial-level weight changes were randomly subdivided into a testing set (1/N of the data) and a training set (the remainder of the data); subdividing the data and performing the random forest regression was repeated N times. Then analysis of variance was used to determine which combination of features best predicted the weight change.https://github.com/neurord/moose_nerp/releases/tag/v2.2 (accessed on 6 June 2022) and modelDB  Code used for parameter optimization is available on github (https://github.com/neurord/ajustador/releases/tag/v2.1 (accessed on 6 June 2022).All code for simulation and analysis is available on github . We compared ending weight of every synapse to its total pre-synaptic spike count across all 10 trials for experiments at different levels of trial-to-trial variability A. We fouThough spike count per synapse was consistent for experiments, the trial-to-trial variability introduced changes in spike timing that altered the time-varying instantaneous pre-synaptic firing rate for each synapse. Thus, we next investigated the effect of the temporal pattern of pre-synaptic firing rate on synaptic plasticity. We computed a weight-change triggered average pre-synaptic firing rate by binning trials and synapses based on the magnitude of synaptic weight change following an individual trial, computing the instantaneous pre-synaptic firing rate vs. time for each synapse and trial, and averaging across synapse-trials within each bin.Our results show that synapses that strongly potentiate exhibit a weight-change-triggered average pre-synaptic firing rate with a high peak firing rate late in the trial. In contrast, synapses that strongly depress exhibit an earlier peak firing rate or a moderate sustained pre-synaptic firing rate. Synapses with little or no weight change exhibit a low pre-synaptic firing rate A. This pPrior work has shown that synaptic plasticity can be affected by spatiotemporally cooperative synaptic activity—that is, multiple synapses on the same dendritic branch active within a limited time window ,80,81,82We repeated simulations using 125 different mappings of spike trains to clusters of synapses . However, the large number of observations and non-linear relationships may impede discovering spatiotemporal predictors of synaptic weight change. Thus, we used machine learning techniques to identify features that were predictive of synaptic weight change.We evaluated several spatial properties that may influence change in synaptic weight, including cluster size, mean firing rate of neighbors and maximum firing rate of neighbors. To calculate mean firing rate of neighbors, for every synapse, its 19 nearest neighbors’ spike trains were combined and a single time series for instantaneous firing rate was calculated from the spike train representing all 19 nearest neighbors. Neither the mean firing rate of the neighbors C, nor thTo quantitatively assess how spatiotemporal pattern of synaptic input controls synaptic plasticity, we used random forest regression to predict synaptic weight change using several features. First, we used the calcium dynamics to determine an upper bound on the regression score (a measure of predictive ability), since the learning rule directly depends on calcium. Then, we used instantaneous firing rate to the synapse, together with either firing rate of neighboring synapses, cluster length, spines per cluster or distance of synapse to the soma. Both the instantaneous firing rate and the calcium concentration were discretized into a small number of time samples to coarsely represent the mean over time: e.g., 1 sample measures the mean firing rate for the entire trial.To evaluate the role of within-trial correlations between neurons, we repeated simulations using spike trains in which interspike intervals (ISIs) were shuffled. Using the same number of synaptic inputs resulted in predominantly synaptic depression  showing relationships between spatial synaptic input patterns and plasticity. For instance, in SPNs in vitro, spatially clustered synaptic inputs can produce supralinear depolarization and calcium transients ,71,76,83μm , which inot be sufficient for formation of synaptic clusters. Our data-driven model of calcium dynamics accounts for global processes (produced by somatic spiking) and local processes (such as NMDA spikes), and the calcium-based synaptic plasticity rule can produce spatial clustering.Functional synaptic clustering is a key component in plasticity and learning; however, it is unclear whether functional clustering arises from synaptic connectivity in development, or if activity dependent plasticity can generate functional clusters starting from random connectivity. By using randomly distributed inputs, our results suggest that functional synaptic clusters may emerge from potentiating neighboring synapses that each receive higher than average input. Our results are consistent with another modeling study investigating the impact of synaptic clustering on somatic membrane potential with in vivo-like conditions , which sOur work predicts that synapse specific potentiation will occur for spatially clustered synapses during the induction phase of plasticity. However, a second critical component for synapse-specificity involves the maintenance phase of long term synaptic plasticity. Recent theoretical work  has addrA common concern about data-driven computational models is that they are underconstrained, and thus it is important to show that model results are robust to parameter variation. Our model of a direct pathway striatal SPN is directly based on our prior one  which diOur results suggest a mechanism for spatially balanced potentiation and depression, which could provide homeostatic balance and prevent runaway potentiation. Synapses with small increases in calcium, produced by nearby depolarization and/or weak synaptic inputs, exhibited synaptic depression. These observations are consistent with in vivo experiments that reveal spine shrinkage in inactive spines accompanying structural potentiation of nearby spines, suggesting heterosynaptic depression as an important compensatory plasticity mechanism ,96. Our This study has important implications for plasticity in variable in vivo conditions by showing that precise spike timing is not required. It is clear from numerous in vivo studies that neither the frequency based protocols nor the spike timing dependent protocols used in brain slices exactly replicate the patterns of synaptic inputs that occur during behavioral learning. Instead, these protocols are tools that are useful for understanding the molecular mechanisms underlying synaptic plasticity.Similar to a non-spatial model of pyramidal neurons , our worOur research is a major advance over prior work which implemented a plasticity rule based on simplified calcium dynamics in a non-spatial model  or derivThe distinct temporal patterns that were associated with potentiation or depression have implications for striatal and wider basal ganglia circuit function. Though corticostriatal LTP requires dopamine in addition to calcium elevation , prior eAnother consequence of our research is to provide a mechanism for the observed decreasing trial-to-trial variability in cortical and striatal neurons in vivo during learning . CorticoOur results show that the spatial aspects of synaptic integration may contribute to synaptic plasticity. However, spiking network models that incorporate spike-timing dependent plasticity rules to investigate the effect of plasticity on network activity neglect spatial patterns of synaptic inputs to a single neuron, reducing neurons to point processes ,105,106.


Thousands of long noncoding RNAs (lncRNAs) are actively transcribed in mammalian genomes. This class of RNAs has important regulatory functions in a broad range of cellular processes and diseases. Numerous lncRNAs have been demonstrated to mediate gene regulation through RNA-based mechanisms. Simultaneously, non-functional lncRNA transcripts derived from the activity of lncRNA loci have been identified, which underpin the notion that a considerable fraction of lncRNA loci exert regulatory functions through mechanisms associated with the production or the activity of lncRNA loci beyond the synthesized transcripts. We particularly distinguish two main RNA-independent components associated with regulatory effects; the act of transcription and the activity of DNA regulatory elements. We describe the experimental approaches to distinguish and understand the functional mechanisms derived from lncRNA loci. These scenarios reveal emerging mechanisms important to understanding the lncRNA implications in genome biology. The development of high-throughput sequencing technologies has shed light on transcriptome knowledge leading to the identification of thousands of transcriptionally active genomic regions that do not derive from protein-coding genes but produce long noncoding RNAs (lncRNAs) ,2,3. In Typical regulatory DNA elements such as promoters and enhancers drive lncRNA transcription or are embedded into lncRNA loci ,10. SurpIn this review, we discuss the underlying mechanistic principles associated with the activity of lncRNA loci beyond the RNA-dependent function. In particular, we discuss recent insights into the regulation originated from the activity of transcription and the regulatory elements of hosted lncRNA loci. We also provide an updated overview of the available methods for interrogating the mechanistic scenarios derived from these observations to distinguish the functional components of given lncRNA loci.cis-acting mechanism) [Cis-acting lncRNAs regulate gene expression at the local level by remaining tethered to their transcription site to modulate gene regulation through chromatin looping, modulate the binding of transcription factors, and the establishment of histone modifications to their target regions [In general, lncRNAs are coexpressed with adjacent or neighboring protein-coding genes ,15. Althchanism) ,17. Cis- regions ,19,20. AGene regulation may be influenced by neighboring transcription, where the act of transcription of a given locus per se can regulate nearby genes . This neS. pombe, the phosphate-responsive permease gene (tgp1+) is regulated by the transcription factor Pho7 in response to phosphate stimulus. The lncRNA nc-tqp1 is transcribed upstream of tgp1+ upon phosphate starvation. The activity of noncoding transcription originated from nc-tqp1 inducing tgp1+ silencing by an increase in nucleosome occupation over the promoter region, which physically prevents Pho7 binding [nc-tqp1, noncoding transcription originated from serine-induced lncRNA SRG1 silences SER3 by increasing nucleosome density over SER3 promoter in response to serine stimulus. The increased nucleosome compactness interferes with the binding of transcription factors on the SER3 promoter [nc-tqp1 and SRG1 are upstream lncRNAs whose transcription act interferes with the binding of transcription factors by modulating nucleosome displacement over promoter regions.Noncoding RNA transcription can be originated not only from intragenic but also from intergenic genomic regions . This ob binding . Similarpromoter . Thus, nIn addition to the gene silencing induced by the physical displacement of nucleosomes, lncRNA transcription may influence the chromatin state through modifying the profile and deposition of histone marks, binding of TF, and the activity of RNAPII. During the progression of transcription, the RNAPII associates with chromatin remodeling complexes, transcription factors, and histone chaperones, contributing to the chromatin state dynamics during transcription . There aTcra locus is involved in V(D)J recombination during thymocyte development [In mammals, the elopment . This loelopment . IntriguD. melanogaster, the Polycomb Response Elements (PRE) silence genes by targeting Polycomb Group (PcG) complexes, which are necessary for homeotic gene regulation [Fab-7 PRE in the bithorax locus preventing the repressive effect by the PcG complex [LYZ) is transcribed under lipopolysaccharide (LPS) stimulation [LYZ by the enhancer. Upon LPS stimulation, the upstream enhancer becomes active and transcribed into the lncRNA LINoCR leading to CTCF eviction from the enhancer and subsequent LYZ activation [Uph) is transcribed upstream to Hand2. The Uph transcription is necessary to maintain the binding of GATA-4 and the activation of two overlapped enhancer elements-driven Hand2 gene expression [In gulation . Noncodi complex . Thus, lmulation . The instivation . Oppositpression . In suchThymoD is transcribed from an upstream intergenic region from the Bcl11b gene, which harbors an enhancer element. The act of transcription repositions the enhancer element from the nuclear periphery to the nuclear interior, triggering the enhancer activation. Moreover, ThymoD transcription facilitates CTCF/Cohesin complex occupancy over the enhancer and the Bcl11b promoter, which juxtaposes enhancer–promoter communication into a loop structure [Although architectural proteins such as CTCF and cohesin complex mediates chromatin structuration through facilitating a loop formation between the enhancers and promoters , lncRNA tructure   embedded into lncRNA loci can exert cis-regulation separately from the lncRNA transcript or the act of transcription ,52. PromIn parallel, examining available high throughput sequencing data such as chromatin accessibility (DNase I and ATAC-seq), histone modifications , and TF binding from ChIP-seq can help to clarify the context in which a lncRNA locus can exert its function. Moreover, chromosome conformation capture  data can inspect to elucidate the possible interacting pattern of the putative regulatory element with potential target genes .The process of transcription by itself can drive local gene expression regardless of the synthesized transcript as described above. To unravel whether the transcription process coming from a locus of interest exerts cis-regulation, transcription termination is the frequently used method. Inserting polyA-terminator sequences immediately from the lncRNA TSS allows premature transcription termination ,14. UsinCollectively, all the experimental approaches described before should be applied to unraveling the complex molecular mechanism of a lncRNA locus, which in turn led us to identify if the candidate lncRNA locus is, in fact, a functional RNA transcript. However, it could be experimentally complex, especially in investigating the dynamic regulation of lncRNA loci during cell differentiation systems or organism development. Finally, these elegant and carefully experimental approaches can lead us to dissection of all the possible regulatory scenarios coming from lncRNA loci. Examples of lncRNA locus where one or more mechanistic principles may act together to regulate gene expression in a particular context have been reported ,49,59.Our knowledge about gene regulation has evolved with the discovery of the lncRNA genes. Even though evidence demonstrates that some lncRNA transcripts are byproducts of transcription or transcriptional noise, others are functional RNA molecules involved in gene regulation. Even more, the DNA sequences where lncRNA emerges and the process of transcription can influence local gene expression. This underlines the importance of applying genetic tools to study the RNA and DNA as regulatory modalities emerging from lncRNA loci, in which several possible modes of action can arise from disentangling the molecular basis of lncRNA loci. The examples described here highlight the need to apply rigorous experimental approaches to identify functional lncRNA transcripts as well as putative regulatory DNA elements associated with gene regulation. These emerging mechanisms of action by themselves can exert gene regulation with relevant biological functions such as cell differentiation, imprinting, development, and diseases. Fortunately, emerging exciting genome editing tools like CRISPR-Cas9 coupled together with information about the epigenetic landscape have enabled distinguish the lncRNA mechanisms of action. Although these efforts, it remains to clarify the molecular mechanisms and the functional roles of lncRNA which are complex loci given the existence of processes associated with their production and the activity of regulatory DNA elements that originated it. Future research will allow characterizing on genome-wide scale potential non-functional lncRNA transcripts as well as new putative functional regulatory mechanisms associated with lncRNA loci. It will be also interesting to unveil if these regulatory mechanisms observed in lncRNA loci also take place in other noncoding loci such as miRNAs, piRNAs, or cirRNAs and their contribution to cell biology and disorders.


Many older adults are sexually active, but ageism fuels the neglect of older adults' sexuality in research and sexual education. The purpose of this study was to (1) assess older adults’ willingness to adopt technology tools to receive innovative, tailored sexual health education via the internet, (2) investigate their sexual health needs, and (3) contribute to development of an assessment tool and tailoring algorithm. Tailoring is a precision approach that matches educational content to users’ needs instead of offering the same suboptimal one-size-fits-all content to everyone. We conducted an online survey using Qualtrics research panels of older adults . We found that 30% reported they were likely to try tailored internet-based education for older adults; an additional 34% were unsure. The results establish priority areas in sexual health needs, including behavioral patterns, knowledge gaps, health-related constraints, and attitudinal and communicational issues. These components provide the basis for the tailoring assessment questionnaire. Respondents also rated the personal relevance of 28 educational modules covering sex in later life . Statistical relationships between sexual health needs and personally relevant modules contribute to the development of a tailoring algorithm for creating educational bundles matched to older adults’ unique needs.


Salmonella Typhi. Typhoid fever is prevalent in developing countries where diets can be less well-balanced. Here, using a murine model, we investigated the role of the macronutrient composition of the diet in maternal vaccination efficacies of two subunit vaccines targeting typhoid toxin: ToxoidVac and PltBVac. We found that maternal vaccinations protected all offspring against a lethal-dose typhoid toxin challenge in a balanced, normal diet (ND) condition, but the declined protection in a malnourished diet (MD) condition was observed in the PltBVac group. Despite the comparable antibody titers in both MD and ND mothers, MD offspring had a significantly lower level of typhoid toxin neutralizing antibodies than their ND counterparts. We observed a lower expression of the neonatal Fc receptor on the yolk sac of MD mothers than in ND mothers, agreeing with the observed lower antibody titers in MD offspring. Protein supplementation to MD diets, but not fat supplementation, increased FcRn expression and protected all MD offspring from the toxin challenge. Similarly, providing additional typhoid toxin-neutralizing antibodies to MD offspring was sufficient to protect all MD offspring from the toxin challenge. These results emphasize the significance of balanced/normal diets for a more effective maternal vaccination transfer to their offspring.Children are particularly susceptible to typhoid fever caused by the bacterial pathogen  Salmonella Typhi, which is prevalent in developing countries where diets can be less well-balanced. Here, we used mice to study the role of nutrition in maternal vaccination efficacies of two subunit vaccines targeting Salmonella’s typhoid toxin. We found maternal vaccinations protected all offspring from a lethal-dose typhoid toxin challenge in a balanced/normal diet (ND) condition, but the lack of protection in a malnourished diet (MD) condition was observed in the PltBVac group. Our data indicate that the difference in maternal vaccination outcomes between ND and MD offspring was due to the less effective maternal antibody transfer from MD mothers to their offspring. Providing additional proteins to MD mothers or additional toxin-neutralizing antibodies to MD offspring saved all malnourished offspring from a lethal-dose typhoid toxin challenge, highlighting the importance of balanced/normal diets for effective maternal vaccination outcomes.Typhoid fever is a life-threatening systemic infectious disease caused by  Salmonella enterica serovar Typhi (S. Typhi) is the cause of the life-threatening systemic disease typhoid fever, resulting in ~10.9 million reported cases annually and affecting billions of individuals estimated to be exposed to this pathogen  using ImageJ software. For each group, a minimum of 10–20 images were taken and the results of two independent experiments were plotted as bar graphs. GraphPad Prism software was used to test the statistical significance of the data.For metagenomics, we isolated and sequenced DNAs of the small intestine contents via a nanopore sequencing and analysis pipeline previously established with modifications . BrieflyData were tested for statistical significance with the GraphPad Prism software. The number of replicates for each experiment and the statistical test performed are indicated in the figure legends. Image analysis and quantification were performed using ImageJ.


Disruption of circadian rhythmicity distorts physiological and psychological processes and has major consequences on health and well-being. A chronic misalignment within the internal time-keeping system modulates alcohol consumption and contributes to stress-related psychiatric disorders which are known to trigger alcohol misuse and relapse. While there is growing evidence of the deleterious impact of circadian disruption on male physiology and behavior, knowledge about the effect in females remains limited. The present study aims to fill the gap by assessing the relationship between internal desynchronization and alcohol intake behavior in female rats. Female Wistar rats kept under standard 24-h, 22-h light-dark conditions, or chronic 6-h advanced phase shifts, were given intermittent access to 20% alcohol followed by an extended alcohol deprivation period. Alcohol consumption under altered light-dark (LD) conditions was assessed and emotional behavior during alcohol abstinence was evaluated. Internally desynchronization in female rats does not affect alcohol consumption but alters scores of emotionality during alcohol abstinence. Changes in affective-like behaviors were accompanied by reduced body weight gain and estrous irregularities under aberrant LD conditions. Our data suggest that internal desynchronization caused by environmental factors is not a major factor contributing to the onset and progression of alcohol abuse, but highlights the need of maintaining circadian hygiene as a supportive remedy during alcohol rehabilitation. Alcohol abuse is a widespread public health concern with adverse social and economic ramifications. Individuals with alcohol use disorder (stocktickerAUD) have impaired control over drinking and continue to drink despite deleterious consequences to their health and professional lives . stocktiAlthough the causes of excessive alcohol consumption are multifactorial, including genetic, socio-economical, and psychological factors, growing evidence point to the important role of the internal time-keeping system in the development and progression of alcohol abuse . The cirNevertheless, circadian rhythm disruption affects not only the reward-related neural circuitry but also contributes to stress-related mood disorders. Clinical studies have linked clock gene variants with depressive and anxiety-like behavior in humans  schedule for 3 weeks. Following acclimatization, all animals were randomly assigned to be kept under standard (CTRL) or desynchronizing lighting conditions. We used two different light-dark (LD) paradigms to induce circadian desynchrony: A chronic phase shift schedule (SHIFT), consisting of 6-h advanced shifts in the onset of the light phase every second day, by shortening the dark phase  were kept individually in transparent cages equipped with running wheels  in light  and temperature-and humidity controlled  rooms. All animals had access to food and water rk phase , and a srk phase .To induce voluntary alcohol consumption in rats, the intermittent alcohol exposure (IAE20%) paradigm was used . The IAEReward-based anhedonia-like behavior was assessed by monitoring sucrose intake . First, The OFT was performed to evaluate exploratory and anxiety-like behavior . Prior tThe EPM was used to assess anxiety-related behavior . The mazThe MBT was used to evaluate impulsiveness, irritability, and compulsive behavior. Burying behavior of harmless objects such as marbles is suggested as a form of impulsive behavior  or indicn = 12), SHIFT (n = 6), and LD22 (n = 4) independently and in combination with alcohol consumption, deprivation, and re-introduction of alcohol . The severity of chronodisruption was assessed using the following circadian parameters: daily fragmentation (intradaily variability–IV), day-to-day stability (interdaily stability–IS), and the difference between day and night activity levels (relative amplitude–RA).In the first experiment, we evaluated the impact of various light conditions and alcohol consumption on circadian rhythmicity . InternaThis experiment determines if rats kept under artificial LD conditions will voluntarily consume more alcohol in a two-bottle choice paradigm compared to animals under standard LD24 conditions. Animals were kept either under LD24, SHIFT, or LD22 conditions and given intermittent access to alcohol for 20 sessions . We asseFollowing alcohol access, alcohol was withheld to determine if high alcohol consumption levels would be maintained following a prolonged abstinence period. Thereafter, the animals were given again intermittent access to 20% alcohol for 10 alcohol-drinking sessions as described above. Comparisons were made between the mean alcohol consumption and alcohol preference, respectively, during the 10 alcohol-drinking sessions immediately before and after the abstinence period.We next compared the behavioral performance of animals kept under LD alterations alone and LD alterations during alcohol abstinence in a variety of behavioral tests . In the We determined the effect of chronic LD alterations on body weight without and with alcohol access and deprivation. Relative changes in body weight were calculated based on the individual body weight at the beginning of the experiment. As stress and artificial light conditions influence the female estrous regularity, we characterized the estrus cycle over 24 days (24 days as a multiplicand of a 4-day estrous cycle) of each LD paradigm and alcohol condition.We quantified circadian rhythmicity by continuous registration of locomotor activity using running wheels. Summed wheel revolutions were recorded and stored over 10 min intervals using the VitalView system . Locomotor activity data from the last 14 days of each LD condition and phase of alcohol exposure were used for the Chi-square-periodogram and circadian parameter analysis. Chi-square-periodograms  were comp < 0.05. A one-way ANOVA (LD effect) was used to compare the rhythm parameters IV, IS and RA between the LD regimens, followed by a Dunnett’s post-hoc test if a significant main effect was found. Further evaluation of the effect of the light regimen or alcohol exposure on circadian parameters during periods of alcohol access or abstinence was conducted by a two-way ANOVA  and Dunnett’s post hoc comparison.Statistical analysis was performed using the GraphPad Prism 9 software package . All results were presented as mean ± standard error of the mean (SEM) and were examined for normality and homogeneity of variance. The significance level was set at Alcohol consumption and preference, as well as water consumption between the animals kept under different LD conditions over the 20 exposure sessions, were assessed by a two-way repeated measures ANOVA (LD and time effect). The effect of LD conditions and progression of alcohol exposure on alcohol intake and preference before and after alcohol abstinence was assessed in a two-way repeated measure ANOVA.post hoc analysis when a significant overall effect was found.Behavioral tests were analyzed using two-way ANOVA , followed by Sidak’s 1  = 0.04154, p = 0.9594, one-way ANOVA], a significant main effect of the LD conditions on IS  and RA  was found. Both parameters were significantly reduced in animals exposed to non-24-h light cycles  and artificial LD paradigms  during alcohol abstinence .post hoc analysis, however, did not reveal significant differences between groups.A significant main effect of the LD conditions was found , Table 3Post hoc analysis revealed a significant decrease in locomotion during alcohol abstinence in all three experimental groups  = 12.95, p = 0.0179, two-way ANOVA]. In contrast, chronic intermittent alcohol consumption alone was not altering the estrous cyclicity under standard LD conditions .All rats showed a progressive increase in body weight over time . Body weThe link between the disruptions of the circadian system and alcohol consumption has been recognized for decades. While most of the research focused on male cohorts, the effect of internal rhythm disruption on alcohol consumption in females is less known. We show that the induced internal desynchrony through chronic advanced shifts and LD22 persists throughout the course of alcohol intake and abstinence, but is unaffected by the respective alcohol condition. Although aberrant light schedules, which simulate shiftwork and jet lag, tend to increase alcohol consumption and preference, we observe no significant differences between our experimental groups in female rats. Importantly, we demonstrated that internal desynchrony affects performance in tests of anxiety-like behavior during periods of alcohol absence. However, these behavioral alterations do not seem to predict or interact with alcohol consumption before abstinence or during re-exposure (data not shown). Taken together, our results demonstrate that internal circadian desynchronization strongly affects female physiology, but has only minor effects on mood-related behavior and alcohol consumption if the two factors were considered separately. When combining the effects of alcohol consumption and circadian disruption, changes in mood-related behaviors became more apparent, which emphasizes the important contribution of dysregulation of circadian functioning in the etiology of mood-related behavior in females under alcohol abstinence.Experimental conditions of jet lag or constant, abnormal light conditions on alcohol consumption led to disparate results, depending on heterogeneity and breadth in approaches and concepts associated with circadian disruption. In our experiment, we observed similar alcohol intake (g/kg/24 h) levels as previously reported in female Fischer, Lewis rats, and P rats , 2014 unNumerous factors including species, sex, duration of aberrant light exposure, the direction of phase shift, and the time between shifts affect the independent and combined effects on behavioral and physiological variables . HoweverSeveral studies indicate that molecular components of the circadian clock play a crucial role in the control of alcohol-drinking behavior  and suggLastly, peripheral oscillators may impact the reward circuit in addition to the central clock. The release of sex hormones like estradiol is controlled by the circadian system  and the Extensive literature supports the association between circadian rhythm disruption and the emergence of anxiety and depression-like symptoms . Since nReduced exploratory behavior and increased anxiety-like behavior in rats under alcohol withdrawal have been demonstrated in the past . In lineIncreased reactive behavior during alcohol abstinence was not evident in the marble burying task. In our experiment, alcohol-experienced rats exhibited decreased burying behavior, whereas previous studies have shown that female rodents enhance marble-burying behavior during alcohol abstinence . As chroReduced sucrose consumption is often used as a measure of anhedonia to assess depressive-like traits in rodents . StudiesOur study provides a foundation to understand how circadian desynchronization affects emotional behavior in female rats under alcohol abstinence. The female organism consists of a unique sex-dependent neurobiological, chronobiological, and hormonal setting, that distinctly reacts to chronic light-dark changes. In future studies, it will be important to distinguish between operational and acute sex-hormonal factors mediating the consequences of internal desynchronization and the effects of alcohol intake in females. A more comprehensive understanding of the role of sex-dependent circadian risk factors will be needed to provide sex-specific alcohol rehabilitation treatments incorporating affective disorders as an important factor in individuals performing under shiftwork conditions.The original contributions presented in this study are included in the article/supplementary material, further inquiries can be directed to the corresponding authors.The animal study was reviewed and approved by the Canadian Council on Animal Care and the Concordia University Animal Care Committee (certificate number: 30000256).CM and SA conceived and designed the experiments. CM conducted the experiments and assisted by KS. CM analyzed and interpreted the data with input from KS and SA. CM did the data visualization. CM wrote the manuscript and revised by SA and KS. SA supervised the project. All authors contributed to the article and approved the submitted version.


With 2176 participants recruited, D-CARE is the largest pragmatic clinical trial of dementia care strategies, to date. At four clinical trial sites (CTS), D-CARE will compare the effectiveness of three dementia care strategies over 18 months: 1) by nurse practitioners or physician assistants within a health care system, 2) by social workers or nurses at community-based service organizations (CBO), or 3) usual care. Primary outcomes include person with dementia (PWD) behavioral symptoms and caregiver strain. Other outcomes include the PWD quality of life and ability to reach personal goals, and caregiver self-efficacy, distress, and depressive symptoms. Recruitment began in June 2019 with a basic protocol in which participating providers reviewed lists generated from the electronic health records (EHR) of patients who had a diagnosis of dementia, allowing the removal of patients who should not be contacted and giving an opportunity to provide information about the family caregiver. Some practices gave “blanket” referral allowing research staff to recruit participants directly. Other practices provided direct referrals via EHR communications to the research team. Self-referrals triggered by public postings in clinics and CBOs, social media, and media coverage were also accepted if a dementia diagnosis was confirmed in the EHR. By March 16, 2020, all in-person recruitment visits were suspended due to COVID-19. In response, informed consent was switched to telephone with verbal consent as permitted by State and Institutional regulations. This symposium describes the creative approaches employed by CTS’ to respond to these challenges and reach the recruitment goal in January 2022.


We illustrate a notable case of an elderly male presenting to a community hospital with six out of 10 substernal chest discomfort and electrocardiogram changes consistent with an anterolateral myocardial infarction. Percutaneous Coronary Intervention (PCI) was initiated following aspirin and anticoagulation administration, which further revealed a critical distal left main spontaneous coronary artery dissection (SCAD). Cardiothoracic and Vascular Surgery consults led to the recommendation of emergent two-vessel Coronary Artery Bypass Grafting (CABG). The patient’s clinical status resolved to full recovery and was discharged on postoperative day five. The incidence of SCAD in older men has not been well documented in current literature. Prevalence in older males is 0.02%. However, it rises to 10.8% in females less than 50 years of age and with acute coronary syndromes (ACS) and ST-segment elevation. Our aim is to incorporate this case report into the current literature and help improve early diagnosis and treatment based on current recommendations. Spontaneous coronary artery dissection (SCAD) is a rare condition characterized by a non-iatrogenic, non-traumatic separation of the coronary vessel wall layers. A false lumen gets created between either the tunica intima and media or the tunica media and externa ,2. BloodSCAD currently has a prevalence rate of 0.2% in a population of 11,605 patients from an interventional cardiology laboratory database. Of those with SCAD, about 0.07% is found in men and 0.6% in women . Remarka2.A 61-year-old Caucasian male was transferred to the emergency department via ambulance with a chief complaint of “aching” six out of ten substernal chest discomfort. His past medical history is significant for controlled hypertension, hyperlipidemia, osteoarthritis, and gastroesophageal reflux disease (GERD). His pertinent home medications included valsartan-hydrochlorothiazide and hydrocodone-ibuprofen. Previous surgical history included a spinal stimulator placement for chronic back pain and a Nissen fundoplication for GERD. He is a former cigarette smoker but stated his last tobacco product use was in 1986. He infrequently consumes alcohol and denies any previous illicit substance use. He lives a physically active lifestyle due to his highly demanding job in landscaping despite having a BMI of 28 kg/mUpon initial presentation, the electrocardiogram (EKG) revealed ST-segment elevations in leads I, aVL, V2, V3, and V4 along with reciprocal J-point depressions in leads II, III, and aVF. The EKG changes were consistent with an anterolateral myocardial infarction and warranted further emergent intervention via cardiac catheterization. The patient was immediately started on aspirin and heparin therapy in the emergency room and was then transferred to the catheterization lab for emergent investigation. With the modified Seldinger technique, right femoral access was obtained as the guidewire and catheter proceeded proximally. An ectatic aortic root was noted on ascension to the coronary vessels, and the Q catheter  engaged the left coronary artery (LCA) while the JR4 catheter  engaged the right coronary artery (RCA). Diagnostic studies revealed a two-vessel disease with a critical distal left main spontaneous coronary artery dissection occluding approximately 90% of the vessel and a critical proximal left anterior descending (LAD) artery stenosis Figure . IncidenPrior to cardiothoracic surgery, the Society of Thoracic Surgeons' (STS) Risk Calculator from the STS Adult Cardiac Surgery Database was computed, and the results were discussed with the patient. Risk scores showed a 3.606% risk of operative mortality, 21.057% risk of prolonged ventilation, 3.040% risk of re-operation, and an overall morbidity/mortality risk of 28.804%. After the isoelectric arrest was accomplished, a two-vessel CABG was successfully anastomosed using the Left Internal Mammary Artery (LIMA) to LAD and a saphenous vein graft to the mid-Obtuse Marginal (MOM) artery. After anastomoses, the patient was given protamine sulfate for reversal of the heparin drip and return of preoperative hemodynamic status was achieved with no complications. The pericardium was reconstructed, and the sternum was closed with the usual Figure-of-8 wires. The patient tolerated the procedure well without any complications and was discharged to the intensive care unit.Post-operatively, the patient had an uneventful recovery with only minimal lower extremity edema and peri-operative anemia. The plan of action was to slowly begin weaning the patient off the IABP. By postoperative day two, the IABP frequency was 50% augmentation with stable hemodynamics and eventually was discontinued later that same day. The patient was started on in-patient aspirin, clopidogrel, and enoxaparin. In-patient physical therapy was consulted on postoperative day three for evaluation and treatment in order to guide the patient to return to the prior level of functioning safely. By postoperative day four, the patient was ambulating close to 100% without any assisted device or difficulty while maintaining appropriate sternal precautions. He achieved complete stability by postoperative day five and was prepared for discharge with education regarding lifestyle modification for risk factors. He was additionally begun on amiodarone, aspirin, atorvastatin, metoprolol tartrate, and hydrocodone-acetaminophen prior to discharge. Future plans for outpatient follow-up will include discussions regarding the addition of angiotensin-converting enzyme (ACE) inhibitors or angiotensin II receptor blockers (ARBs) to improve long-term patient outcomes.The low incidence and prevalence of SCAD events make recognition and medical management two of the most important aspects of achieving positive patient outcomes. The case presented outlines typical features of the acute coronary syndrome (ACS) accompanied by atypical patient demographics and the ultimate outcome followed by a suggested treatment algorithm Figure  5]..5].Since symptoms of SCAD may be indistinguishable from other causes of ACS, a physician should have increased levels of suspicion when patients present with sudden onset of symptoms during isometric exercise or under emotional distress; however, it still remains a challenge to make a clear-cut diagnosis . CurrentAs mentioned above, the demographic impacted by SCAD is “typically” women of reproductive age with symptoms of ACS related to predisposing factors such as fibromuscular dysplasia (FMD), pregnancy, multiparity, connective tissue disease, and chronic inflammatory conditions which create increased wall tension in the coronary artery leading to progressive tunica intima or media degeneration ,8. Out oAn alternative diagnosis to consider in ACS presentations similar to SCAD may be Takotsubo Cardiomyopathy (TTC). TTC is a condition that is believed to occur due to a sudden catecholamine surge from physical or emotional stress, more often seen in females. Despite the intense physical demands of our patient’s occupation, the cardiac angiogram showed no evidence of left ventricular apical ballooning in the absence of significant coronary stenosis. Buccheri and Zambelli explored a link between SCAD and TTC via analysis of published case reports, ultimately hypothesizing a possible causative relationship between SCAD leading to TTC or vice versa . HoweverThere are several treatment modalities for SCAD such as conservative measurements, percutaneous coronary intervention (PCI), and CABG. A Howard University Hospital meta-analysis of 440 patients with single lesion SCAD in the LCA or RCA systems from 1931 to 2008 showed that patients had better outcomes with early aggressive therapy using stent placement or CABG compared to conservative therapy  . HoweverPatients that develop SCAD are expected to manifest a wide variety of symptoms during multiple follow-ups in their lives. Cardiovascular-related events that occur include chest pain, anxiety, depression, recurrent myocardial infarctions, congestive heart failure, stroke, and recurrent SCAD. Major adverse cardiac events were reported in 15 to 37% between a five-to-seven-year interval . The recWe present an interesting case of an elderly-aged male with minimal risk factors developing SCAD in the left main coronary artery requiring aggressive therapy with CABG. On presentation at the emergency room, the patient experienced typical ACS symptoms along with ST-segment elevations correlating to an anterolateral myocardial infarction, and upon further investigation using angiography SCAD was diagnosed. SCAD is often an underdiagnosed subcategory of ACS which requires immediate recognition and initiation of therapy to improve long-term patient success. Limited case reports due to low disease incidence allow this case to boost current literature in order to improve early recognition and outcomes of SCAD among physicians with high levels of clinical suspicion.


Randomized controlled trials (RCTs) have demonstrated a survival benefit for adjuvant platinum-based chemotherapy after resection of locoregional non-small cell lung cancer (NSCLC). The relative benefits and harms and optimal approach to treatment for NSCLC patients who have major comorbidities  are unclear, however.We used a simulation model to run in-silico comparative trials of adjuvant chemotherapy versus observation in locoregional NSCLC in patients with comorbidities. The model estimated quality-adjusted life years (QALYs) gained by each treatment strategy stratified by age, comorbidity, and stage. The model was parameterized using outcomes and quality-of-life data from RCTs and primary analyses from large cancer databases.Adjuvant chemotherapy was associated with clinically significant QALY gains for all patient age/stage combinations with COPD except for patients >80 years old with Stage IB and IIA cancers. For patients with CHF and Stage IB and IIA disease, adjuvant chemotherapy was not advantageous; in contrast, it was associated with QALY gains for more advanced stages for younger patients with CHF. For stages IIB and IIIA NSCLC, most patient groups benefited from adjuvant chemotherapy. However, In general, patients with multiple comorbidities benefited less from adjuvant chemotherapy than those with single comorbidities and women with comorbidities in older age categories benefited more from adjuvant chemotherapy than their male counterparts.Older, multimorbid patients may derive QALY gains from adjuvant chemotherapy after NSCLC surgery. These results help extend existing clinical trial data to specific unstudied, high-risk populations and may reduce the uncertainty regarding adjuvant chemotherapy use in these patients. Lung cancer is the leading cause of cancer death in the United States, with non-small cell lung cancer (NSCLC) representing >85% of lung cancer cases , 2. LocoA substantial proportion of patients with locoregional NSCLC have serious comorbidities that are related to smoking. The comorbidities most commonly observed in patients with NSCLC include cardiovascular disease (20–25%) and chronic obstructive pulmonary disease  –10. PatiSurgery is the standard treatment for locoregional NSCLC, but a large proportion of patients remain at risk of cancer recurrence after resection , 12. SevThe risk/benefit ratio of adjuvant chemotherapy may substantially differ in patients with comorbidities due to differences in adverse events from cancer treatment, competing mortality risks (non-lung cancer deaths), and poorer quality of life –22. PatiSimulation modeling is a comparative effectiveness technique that incorporates existing data into a mathematical model allowing the conduct of computer-generated “in silico clinical trials,” in which a simulated cohort of patients are “randomized” to treatments and have different health outcomes according to pre-specified health state transition probabilities. Compared to using retrospective databases analyses alone, simulation modeling has the advantage of integrating data from many valid sources including randomized clinical trials, can generate personalized treatment recommendations for a large variety of patient profiles, and can consider quality of life parameters, frequently missing in observational cancer databases. In this study, we aim to determine the indications for adjuvant chemotherapy in patients with locoregional NSCLC and smoking-related comorbidities (COPD and cardiovascular diseases) by developing a simulation model to run in-silico comparative trials of platinum-based adjuvant chemotherapy versus observation.We comprehensively reviewed the literature and conducted primary analyses of large cancer datasets to assess factors unique to patients with NSCLC and comorbidities. We then incorporated this information into a novel adjuvant chemotherapy-focused treatment extension of the Lung Cancer Policy Model, a validated, comprehensive micro-simulation model of lung cancer detection, development, progression, treatment, and survival . This trth criteria) NSCLC and comorbidities populate the model and transition through different health states. Monthly transition probabilities were ascertained from primary analyses of large clinical datasets, clinical trial results, and a review of the literature  with a waiver of consent in compliance with the Code for Federal Regulations Title 45 Part 46.116.COLT-M parameters include mortality (both from NSCLC and competing risks), complications from treatment, and quality of life utilities. Parameters and data sources are listed in To estimate the benefits of adjuvant platinum-based chemotherapy on NSCLC-specific mortality, we used the results of the Lung Adjuvant Cisplatin Evaluation (LACE) pooled analysis . The month edition criteria) from 2000–2013 who underwent lobectomy but did not receive adjuvant chemotherapy. Survival time was calculated from lobectomy date to date of death from NSCLC or other causes. Surviving patients were censored at the time of last known contact. Cause of death was determined from SEER data. The Fine-Gray competing risk model was used to estimate sub-hazards of death from NSCLC vs. other causes; cumulative incidence functions were generated and used to calculate cause-specific survival curves and monthly death rates. The monthly death rates were then incorporated into COLT-M as transition probabilities for each cycle. With this data, we also calculated 5-year overall survival rates, an outcome that is reported commonly in cancer clinical trials. We then modeled until age 100 or death and outputed 10-year overall survival rates for patients with comorbidities who underwent lobectomy followed by adjuvant chemotherapy or observation.We used population-based data from Surveillance, Epidemiology, and End-Results database linked to Medicare (SEER-Medicare) to estimate mortality from both NSCLC and competing risks  in patients who underwent lobectomy and did not receive adjuvant chemotherapy. SEER-Medicare links SEER registry data with Medicare claims records and has information on NSCLC characteristics and outcomes, as well as allows to assess for comorbidities and complications. We identified 7,852 patients from SEER-Medicare diagnosed with locoregional NSCLC , a HR of 2.41 (95% CI: 1.64–3.55) was used in our model to increase the non-NSCLC mortality rate in the first 6 months after adjuvant chemotherapy, after which non-NSCLC mortality returned to background levels . We usedCOLT-M also accounted for quality-adjusted life years (QALYs), an outcome which considers both quality and quantity of life . We deriGains in QALYs were used to compare patients with comorbidities who received adjuvant chemotherapy vs. observation. Adjuvant chemotherapy was considered the optimal treatment strategy when it was associated with least a 0.25 (3 month) QALY increase. Otherwise, observation was considered the favored strategy. This threshold was selected based on the American Society of Clinical Oncology benchmarks for clinical significance for NSCLC .th edition Stage IB-IIIA NSCLC and comorbidities according to age category , comorbidity type , and NSCLC AJCC 8th edition stage . We conducted analyses for lone comorbidities  and combinations of comorbidities . We conducted one-way sensitivity analyses, evaluating model output when varying treatment effectiveness and toxicity (from RCTs) parameters over their 95% CIs.For the base case analyses, we compared QALY gains and 5-year survival rates of adjuvant chemotherapy vs. observation alone. In these analyses, we used COLT-M to calculate survival and QALYs in patients with AJCC 8SEER-Medicare analyses found that each increasing age category was associated with greater risk of non-NSCLC cancer death and female sex was associated with a decreased risk of death from non-NSCLC causes .One-way sensitivity analyses evaluated the robustness of model outputs to changes in key parameters  Tables.We conducted simulated clinical trials using the COLT-M, an extension of an established lung cancer microsimulation model, to assess the indications for adjuvant platinum-based chemotherapy in locoregional NSCLC patients with comorbidities. Our results showed that the optimal treatment strategy (adjuvant chemotherapy vs. observation) differed by age, sex, and comorbidities. While some patients benefited from adjuvant therapy, some older patients, particularly with earlier stages of disease, had better outcomes with observation after undergoing lobectomy. This study shows that while comorbidities should not prevent the use of adjuvant chemotherapy in many patients, a less aggressive treatment strategy may be the preferred approach for a select group of patients who are older or have major cardiac comorbidities.Meta-analyses of RCTs and retrospective observational studies have shown that adjuvant chemotherapy for early-stage NSCLC after lobectomy leads to improvements in overall survival, with absolute increases ranging from 4 to 9% , 34, 35.Simulation modeling is a way of leveraging existing data to assess the risks and benefits of adjuvant chemotherapy and can provide guidance for a wide variety of patient profiles. In our study, simulated patients in COLT-M received treatment with either adjuvant chemotherapy or observation. Our simulated clinical trials considered both quantity and quality of life and compared QALYs between the treatment groups according to patients’ NSCLC stage, sex, age category, and comorbidity profile. In general, patients were more likely to have a greater benefit from adjuvant therapy if NSCLC stage was more advanced. This is consistent with adjuvant therapy clinical trial data and previous observational studies . Our stuWe also found that many patients with COPD benefit from receiving adjuvant chemotherapy. Lung cancer patients with COPD have worse outcomes and are less likely to receive adjuvant therapy , 40. AddCardiovascular comorbidities were most frequently associated with lack of clinically significant superiority for adjuvant chemotherapy, particularly in older patients and earlier stage tumors. Our findings were likely driven by substantial increases in non-lung cancer death with these conditions (which attenuates the benefits of chemotherapy), as well as higher treatment toxicity and a negative influence on quality of life. While there is currently very limited guidance on use of adjuvant chemotherapy, patients with lung cancer and comorbid cardiovascular diseases are known to have worse overall survival largely due to competing risks of death , 45. OurOur analyses showed that female sex was associated with improved survival, worse chemotherapy toxicity, and worse quality of life, findings which are consistent with the existing literature –48. In oA strength of our study is that we used a robust modeling approach to assess the indications for adjuvant chemotherapy in older patients with locoregional NSCLC and comorbidities. This simulation modeling approach is well-established in evaluating lung cancer screening and treatment and has been used to evaluate treatment benefits of surgery in patients with comorbidities , 49–51. In summary, we used a simulation model to evaluate QALY increases from adjuvant chemotherapy for patients with resected locoregional NSCLC and comorbidities. We found that many older patients with multiple comorbidities can benefit from adjuvant therapy; however, older individuals with cardiac comorbidities may be better managed with observation. Our simulation model results help to extend existing clinical trial data to unstudied, high-risk populations for whom limited primary data exist and may reduce clinical uncertainty regarding the use of adjuvant chemotherapy. Our study findings can be used to guide future research on treatment in patients with early-stage NSCLC and comorbidities, such as studies on adjuvant targeted therapies and immunotherapy.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.


Targeted therapies have significantly improved survival rates and quality of life for many cancer patients. However, on- and off-target side toxicities in normal tissues, and precocious activation of the immune response remain significant issues that limit the efficacy of molecular targeted agents. Extracellular vesicles (EVs) hold great promise as the mediators of next-generation therapeutic payloads. Derived from cellular membranes, EVs can be engineered to carry specific therapeutic agents in a targeted manner to tumor cells. This review highlights the progress in our understanding of basic EV biology, and discusses how EVs are being chemically and genetically modified for use in clinical and preclinical studies. They ca systems . The proEV secretion has been observed in virtually all kingdoms of life. EV biogenesis and cargo loading are tightly linked processes, and these steps are strictly regulated to ensure that the subsequent interaction with target cells produces the desired biological effects . Cargos Small non-coding RNAs have been found within EVs, and may control molecular events in recipient cells by regulating the activity of target mRNAs, for example . These RThe lipid composition of EVs is very similar to that of their parental cells; this provides an ideal physical barrier to protect internal cargo from premature degradation. In addition to providing EV integrity, the large amounts of sphingomyelin and cholesterol prevent EV cargos from degradation by both nucleases and proteases . Such liA suite of fatty acids in EVs provide the building blocks for the generation of signaling intermediates that regulate multiple physiological processes . ConsistDespite their advantageous small size and ability to cross the blood brain barrier, natural EVs require further optimization before they can be used for drug delivery. Major limitations include the lack of target cell specificity and an inability to store sufficient payload quantities. Great strides have been made with regard to improving the loading of heterogenous cargo . EV membCellular nanoporation (CNP) is the method of choice for delivery of nucleic acids, including large mRNAs, to EVs . The mRNThe single-chain transmembrane glycoprotein PDGFR has been exploited for breast cancer immunotherapy by Cheng et al., who designed nanoscale controllers termed synthetic multivalent antibody retargeted exosomes (SMART-Ex) . Using tInterestingly, EV-type structures from non-mammalian organisms are also being evaluated for their potential as drug carriers. A case in point are bacterial outer membrane vesicles that have been engineered to express a HER2-specific antibody fused to the transmembrane region of the bacteria pore-forming protein, cytolysin A .The therapeutic efficacy of EVs can be increased by bioconjugation of nanobodies or bispecific antibodies that bind to specific molecules and receptors on target cells. The ExoMAb approach exemplifies this strategy of engineering EVs to express non-natural antigens . BrieflyCirculating EVs play a continuous ‘cat and mouse’ game with patrolling macrophages, which can phagocytose EVs upon recognition. This clearly reduces the effective half-life of EVs in the body. CD47 (mentioned above) can again be exploited here: by binding to signal regulatory protein alpha (SIRPα) on the surface of macrophages, CD47 sets up a ‘don’t eat me’ signal , 30. Thuvia enzymatic removal of sialic acid moieties also changes their biodistribution  conjugation to the surface of EVs increases their half-life  Figure  have been used for targeted delivery of chemotherapeutic drugs  , active methods of loading such as mechanical or chemical opening of the EV membrane via sonication, electroporation, saponin treatment or freeze-thaw cycles can be employed  preferentially home to and target the tumor cells from which they were derived  Table . FurtherGlioma is a devastating malignant brain tumor with a high mortality rate due to low penetration and efficacy of the available chemotherapeutics. TEX are already having an impact in this tumor type. For example, TEX-mediated delivery of IMV-001  to patients with recurrent malignant glioma was more effective than other delivery methods due as it was associated with an increased number of tumor-infiltrating lymphocytes and enhanced anti-tumor immunity , 58. In In the liver cancer setting, administration of functionalized TEX loaded with an adjuvant, high mobility group nucleosome-binding protein 1 (HMGN1), reduced tumor size and potentiated immunogenicity in a mouse model of hepatocellular carcinoma  Table .Pleural effusions and ascites are abnormal fluid collections within the thoracic and peritoneal cavity, respectively. They are frequent in terminal stage malignancies, and require aspiration and paracentesis to manage disease conditions. TEX may be particularly suited to drug delivery for pleural effusions. For example, A549 tumor cell-derived EVs loaded with methotrexate (MTX) induced neutrophil recruitment in the effusion fluid and improved primary malignant pleural effusion (MPE) in non-small cell lung cancer patients Table 1.In addition to tumor cells themselves, TEX are also present in ascites. These ascites-derived TEX can also be exploited for cancer treatment, as exemplified in a clinical trial  -derived EVs (STEX) arise from different tissues in normal and disease conditions and have beneficial effects in wound healing , myocardin vitro (in vivo by decreasing cyclooxygenase (COX)-2 expression  are one of the most studied STEX sources because they are easier to obtain and harvest through subcutaneous lipoaspiration; this is much less painful and less ethically sensitive than collecting bone marrow or embryonic stem cells , 81. In se model   significantly reduced tumor burden in a C6 glioma rat model  Table  , which express MHC-I- and MHC-II-bound antigen peptides as well as other adhesion molecules . PioneerDEX can activate CD4+ T cells by inducing Th1 and Th2 immune responses, irrespective of the maturity of the DCs . HoweverNK cell-derived EVs (NEX) can carry cargo such as cytotoxic proteins, miRNAs, and cytokines that employ multiple mechanisms to kill tumor cells . They alMacrophages can be categorized into anti-tumor M1 and protumor M2 subtypes, and reprogramming between these phenotypes has been exploited for anticancer therapy . MacrophFruits and vegetables have been used historically as medicines to treat numerous diseases. Following the discovery of plant-derived EVs (PEX), which can be isolated by extracting apoptotic vesicles from leaf, sunflower seeds, and roots , it was The consumption of grapes may reduce the impact of risk factors associated with multiple diseases, including cancer . This haGinger contains gingerol, which has powerful antioxidant and anti-inflammatory properties ; togethein vitro study showed that curcumin significantly arrested the growth of human colon cancer cells  have great potential utility as magnetic nanoplatforms for targeted drug delivery , as theyin vivo using prostate-specific membrane antigen (PSMA) aptamers . EVs may offer a natural solution to this problem because they are nanosized membrane vesicles that can easily pass through the BBB. The combination of BBB penetration and tumor cell-selective targeting using EVs has been compellingly demonstrated in glioma. Specifically, EVs decorated with RGERPPR, a peptide ligand for neuropilin-1 which is highly expressed on glioma cells but not other neuronal cells , 131, wevia the delivery of DOX to TNBC xenografts using MEX coated with the c-Met binding peptide, CBP (Triple negative breast cancer (TNBC) has the worst prognosis of all breast cancers, primarily because it lacks estrogen and progesterone receptors, and has extremely low levels of HER2 , 135. Hoide, CBP , 138.2C QD). This compound has higher photothermal conversion efficiency than AuNRs, and V2C QD-loaded EVs have superior biocompatibility and long circulation times combined with advanced antitumor activity  have unique properties that make them attractive for applications in bioimaging, drug delivery, and photothermal therapy in cancer . Irradiaactivity  (Table Photodynamic therapy (PDT) requires light and a photosensitizing chemical substance that produces an oxygen radical to elicit cell death . EVs conThere is great demand for safe, efficient, and versatile drug delivery systems for cancer therapy. Engineered EVs hold great promise in this regard, given their ability to specifically target and efficiently transfer therapeutic agents into cancer cells. However, more evaluation is required to produce and incorporate engineered EVs into clinically relevant systems, weighing the potential risks and benefits of this new approach. While the use of allogenic EVs appears feasible, the selection of parental cells, assessment of immunologic and oncogenic effects, and risk of viral contamination need to be carefully considered.in vivo (Successful clinical translation of EVs depends on the availability of reliable methods for large-scale production, isolation, and characterization to minimize lot-to-lot variations in drug-loaded EVs . A potenin vivo . AlthougThe author confirms being the sole contributor of this work and has approved it for publication.


The concurrent additional tasking impacts the walking performance, and such impact is even greater in individuals with mild cognitive impairment (MCI) than in healthy elders. However, effective training program to improve dual task walking ability for the people with MCI is not immediately provided. Therefore, this study aimed to determine the effects of cognitive and motor dual task walking training on dual task walking performance and the responding brain changes in older people with MCI. Thirty older adults with MCI were randomly allocated to receive 24 sessions of 45-min cognitive dual task training , motor dual task training , or conventional physical therapy . Gait performance and brain activation during single and dual task walking, and cognitive function assessed by trail-making test  and digit span test were measured at pre-, post-test, and 1-month follow-up. Both CDTT and MDTT improved dual task walking with responding activation changes in specific brain areas. The improvements in motor dual task walking performance after both dual task trainings were significantly better than after CPT in the older adults with MCI. Both cognitive and motor dual task training were feasible and beneficial to improve dual task walking ability in older adults with MCI.Trial Registration: The trial was registered to Thai Clinical Trial Registry and the registration number is TCTR20180510002 (first registration date: 10/05/2018). The impaired gait performance may indicate a risk of fall6, which is an important issue for the aged populations. As dual task walking may be affected by motor ability7, growing evidence demonstrated that the gait performances highly correlated with general cognitive function in the healthy older population3. Prefrontal cortex (PFC) plays a crucial role in cognitive function, and was evident to be involved in dual tasking9. Increased activation in PFC was observed during dual task walking in healthy adults, and older adults tended to activate more compared to younger participants10. Other motor brain areas such as premotor cortex (PMC) and supplemental motor area (SMA) were also found to activate during dual task walking, which were responsible for stabilizing gait and adapting walking speed14.Dual task walking, such as talking or carrying a bag while walking, is essential in daily life. However, both cognitive and motor dual task impact the walking performance, including decreased speed, increased stride time and stride time variability15. In people with MCI, decreased speed, increased stride time, variability were observed during cognitive dual task walking as compared with single walking, with inconclusive results in PFC activation21. The influence of additional task on walking was even greater in individuals with MCI than in healthy elders21, which may further impact their daily activities and increase fall risks. Thus, dual task walking ability should be addressed especially in cognitive impaired population. However, the effective training program to improve dual task walking ability is not immediately provided.Cognitive impairment in aging population has drawn growing attention in the recent decade. Mild cognitive impairment (MCI) is a brain function syndrome involving the onset and evolution of cognitive impairments beyond those expected based on the age and education of the individual22. The beneficial effects of dual task training have been demonstrated in several populations with different clinical conditions23. Specifically, cognitive dual task training has been reported to exert beneficial effects on cognitive and physical functions in older adults with and without cognitive impairment25. The overview of systematic reviews has further suggested that such training program is preferred over single task training as targeting motor performance under dual task situation in healthy older adults24. However, the effect of dual task training on dual task walking performance in older adults with MCI is not known. Furthermore, whether different dual task training would exert different effects in the MCI population needs further elucidation for possible clinical applications. Therefore, this study aimed to determine the effects of cognitive and motor dual task walking training on dual task walking performance and responding brain changes in older adults with MCI. We hypothesized that cognitive and motor dual task walking training would provide task-specific effects on dual task walking performance in older adults with MCI, and the brain changes would correlate to the improvement of walking performance after training.Dual task training is designed in accordance with the principles of motor learning and task specificity, to improve the dual task performanceForty-five older adults with MCI who met the eligibility criteria were recruited, and 14 of them declined to participate due to personal reasons. Thirty-one participants were randomly assigned to the conventional physical therapy group , cognitive dual task training group , or motor dual task training group . One in the CDTT group dropped out before the pre-test assessment. Therefore, total of 30 participants completed the training sessions and post-test measurements. One in the CPT group and one in the CDTT group missed the follow-up assessment because of transportation issue. The study flow chart was shown in Fig. The median age for all participants was 79.5  years old. The median mini-mental state examination (MMSE) scores were 28 (IQR = 27–29) and the median Montreal cognitive assessment (MoCA) scores were 22 (IQR = 20–23.8). No significant differences among groups were found in basic data and measures at pre-test Table .Table 1BTable The motor dual task gait performance in three groups are presented in Table The speed, cadence, stride length, and stride time during single walking were improved after CDTT. Significant within-group improvements in speed, cadence, stride length, spatial variability, and stride time were found in the MDTT group. No significant change was found after CPT. Furthermore, the training effects on cadence and stride time after CDTT was significantly better than CPT at follow-up Table . HoweverThere were no significant changes in cognitive function after CDTT, MDTT or CPT, except for the trail-making test part B (TMT-B). The time needed to complete TMT-B was significantly less after 24 sessions of MDTT, and such effect remained at the 4-week follow-up Table .Table 5C28, few have explored the effect of different training on gait performance29. This study demonstrated both cognitive and motor dual task trainings improved dual task walking and single walking performance. Furthermore, the effects of both dual task trainings on the motor dual task walking performance were superior to CPT in the older adults with MCI. Nevertheless, only MDTT resulted in significantly superior effect at 1-month on both dual task walking performances as compared with CPT. In addition, improvements in dual task gait performance correlated with the activations in the specific brain areas.Although many studies have examined the interventions on cognitive function in individuals with MCI30. However, the virtual reality-based dual task balance training did not benefit the Timed Up-and-Go test under dual task condition in the institutionalized older persons with MCI31. In present study, both cognitive and motor dual task trainings improved walking performance under dual task conditions, but the CPT, as practicing single tasks consecutively, did not. Present results were consistent with results of other studies in different populations33, suggesting the specific dual task training effect over single task training. Moreover, the improvements in gait speed  were higher than the minimum detectable change value (16 cm/s) during dual task walking34, which may indicate the clinical importance of the favorable results.As dual task walking ability is essential for daily activities, previous studies have shown that simultaneous cognitive and aerobic training improved cognitive dual task walking ability more than physical training in people with MCI35. The reasons for inconsistent results between participants with MCI and stroke are not known. We further noted the lasting effects (at 1-month follow-up) on dual task walking performance seemed to be evident after MDTT, while the lasting effects on single walking performance was significant after CDTT. The concept of task priority may lend some explanations for such results after motor and cognitive dual task training. Previous studies proposed the prioritization of dual task performance depended on the complexity of the posture-related task as well as the additional tasks38. In present study, the additional cognitive tasks may be too challenging for our participants to maintain the posture control and gait stability, thus they needed to allocate attentional resources on posture and gait control during CDTT. As a result, we observed the significant improvement in single walking performance after CDTT with little changes in cognitive function tested by TMT and digit span test. The insignificant improvements in cognitive function as demonstrated by executive and memory function were also noted in the meta-analysis study after combined cognitive-physical intervention39.It draws our attention that motor and cognitive dual task training did not result in distinct effects in accordance with the dual task types in the MCI population. Previously we found that CDTT improved cognitive dual task gait performance and MDTT improved motor dual task gait performance in people with chronic stroke40. Therefore, the participants could have ample opportunity to practice the integration of two simultaneous tasks, which resulted in superior beneficial effects on dual task walking performance even after 1-month post training. In addition, it should be mentioned the implications of changes in gait variability after MDTT. Studies showed that the gait variability was able to predict fall risk, and was related to executive function in the aging populations with and without cognitive impairment41. In line with the improvement in dual task walking performance, we noticed the beneficial effects on TMT-B after MDTT. As mentioned earlier, cognitive flexibility plays an important role in dual tasking in the aged population42, and TMT-B is used to assess executive function, specifically as the ability of cognitive flexibility43. Taking together, our MDTT program improved dual task walking ability by the adaptation of dual tasking and possibly decreased fall risks in older adults with MCI.On the other hand, the additional motor tasks did not seem very complex or difficult, so the participants may not need to prioritize the additional task over the walking during MDTT. In other words, MDTT might benefit from minimizing the crosstalk, which reduced the requirement for attention on the additional motor tasks during training44. By contrast, CPT did not improve any walking performance measured in present study. A meta-analysis demonstrated multi-modal training, similar to CPT in present study, could improve single walking performance with a relatively long period of training, e.g. 90 min per session for 40 sessions in older adults with MCI or mild dementia45. Our CPT programs were 45 min per session, including 15 min of gait training, for a total of 24 sessions. On the other hand, our dual task training combined either cognitive or motor task concurrently with gait training throughout the 45-min session, and resulted in single walking improvement. Therefore, to improve single walking performance may need relatively long duration of walking activity.It is worth mentioning that our two dual task training programs were also able to exert clinically important improvements in single walking speed, as the median change values  were in the range of minimal clinical important differences (10–20 cm/s)46. According to our results, positive correlations between the changes in gait performance and brain activation after CDTT, suggesting that CDTT enhanced the brain activations in prefrontal and motor-associated brain areas extensively to provide positive effects. In line with our findings, previous study proposed that older adults increased the activation in SMA and frontal cortex as a compensatory strategy to ensure a better task performance47. PMC and SMA played important roles in gait control, and prioritizing the walking tasks during CDTT might specifically induce activations of these brain areas. Regarding the PFC activation, although it may reach the resource ceiling for cognitive tasks in the aged population, Logan et al. demonstrated that providing external instructions potentially helped to reverse the deactivation of frontal cortex48. Thus, we speculate that CDTT may increase the recruitment of PFC resource by verbal cues from the therapist, which may partially contribute to the improvements in walking performance. As for the MDTT, the reduction in spatial variability correlated with the decreased activation in PFC during motor dual task walking after training, which may be explained by the cortical adaptation for the processing of dual-tasking49. Previous studies demonstrated decreased PFC activation at late stage of motor learning, accompanied by the shifting of neural networks51 to promote neural efficiency even after the learning process46. We thus think the beneficial effects on dual task walking of MDTT may be related to the enhancement of neural efficiency, and that of CDTT may be related to the recruitment of neural resources.Although both cognitive and motor dual task training exerted beneficial effects on dual task walking ability, the underlying brain changes in response to different types of dual task training seemed different, which may indicate different strategies for the improvementsThere were several limitations in this study should be noted. First, the sample size was relatively small in each training group, and there were drop-outs at the 1-month follow-up. To address this issue, we applied the non-parametric statistical methods with Bonferroni adjustment and intention-to-treat in order to draw unbiased conclusion. The significances in the between-group comparisons were with large effect size, which may help to validate the training effects. Second, the task priority was not specifically emphasized during our dual task training programs, and the instructions were given according to the performance for each participant during training. Further studies are needed to investigate the influences of task prioritization and task complexity on training effects to establish a comprehensive guideline for the application of dual task training.Our study provides the first evidence about different dual task trainings on dual task walking performance and related brain changes in individuals with MCI using three parallel groups with randomized controlled design. Both cognitive and motor dual task training for 24 sessions improved walking performance under single and dual task condition, while the CPT did not exert significant change in gait performance. Furthermore, the changes in responding brain activation during walking were related to dual task walking improvement after CDTT and MDTT. The possible mechanisms for the different dual task training should be investigated and validated in future studies.This study was a three-arm parallel single blinded (assessor blinded), randomized controlled trial. The Block of 6 randomization was used, and the random allocation sequence was generated via sealed envelope drawn by a person who was not involved in the study. Participants were enrolled and randomly assigned by the researchers to one of the three groups: cognitive dual-task training (CDTT), motor dual-task training (MDTT) or conventional physical therapy group (CPT). A sample of 30 participants was suggested to be sufficient with effect size of 0.4 (medium effect size), power of 0.80 and a two-tailed alpha level of 0.05 using G*Power version 3.1. The basic data including age, gender, educational level, mini-mental state examination (MMSE) scores and Montreal cognitive assessment (MoCA) scores were obtained and assessed before the interventions. All participants received 45 min of training according to their group assignment, 3 sessions per week for a total of 24 sessions instructed by a physical therapist. All outcomes were measured before (pre) and after completing 24 sessions of interventions (post), and at 1-month follow-up assessment (follow-up). The study protocol was approved by the Institutional Review Board of National Yang-Ming University . The trial was prospectively registered to Thai Clinical Trial Registry (TCTR20180510002) on 10/05/2018 and conformed to the CONSORT checklist. All experiments were performed in accordance with relevant guidelines and regulations.54, and (3) the ability to walk for 60 s independently without any assistive device. The exclusion criteria included (1) unstable medical condition and (2) any neurological disease, psychological disorder, or learning disability known to interfere with participation in the study. Informed consent was obtained from all participants.Older adults with MCI were recruited from community centers, nursing homes and long-term care facilities in Taiwan. The inclusion criteria were (1) age ≥ 65 years old, (2) MCI was determined by MMSE ≥ 24 and MoCA < 26Single walking (SW): Participants were asked to walk at their self-selected speed.Walking while performing a cognitive task: Participants were asked to walk at their self-selected speed while subtracting three from a random three-digit number serially.Walking while performing a motor task: Participants were asked to walk at their self-selected speed while carrying a tray, with a cup of water on it, with both hands.The primary outcome was gait performance under dual task conditions, and the secondary outcomes included single walking performance, brain activation during walking, and cognitive function. Data for gait performance and brain activation were obtained simultaneously during 3 walking conditions described below. Participants were asked to sit on a chair for 60 s, and then to stand still for at least 15 s to stabilize the hemodynamic response before each walking trial. The participants were asked to walk back and forth on the hallway for 60 s in each walking trial. Each condition repeated twice with a total of 6 walking trials in a random order.55. The kinematic data was recorded and synchronized wirelessly for two sensors, and transferred to a tablet computer. Gait parameters included speed, cadence, stride length, stride time, and gait variability. The spatial and temporal gait variability were calculated as the coefficient of variation (= standard deviation/mean × 100%) of the stride length and stride time, respectively. To increase the measurement reliability, the gait parameters recorded in two trials of each walking condition were averaged for statistical analysis. In addition, to quantify the interference of additional task on walking, the dual task cost of gait speed (DTC) was calculated according to the following formula. The higher DTC value indicates the more interference caused by the additional task to walking speed.ST: walking speed during single walking, SDT: walking speed during cognitive (motor) dual task walking.The gait performance was assessed by two wearable Physilog sensors  attached to the lateral side of each shoe. The sensor was validated and had fair reliabilities (0.71–0.87) for measuring single and dual task walking performance1. The data was band-filtered , and wavelet filtering was used for the motion artifact correction in each channel. The preprocessed signals were converted to concentration in oxygenated (HbO) and deoxygenated hemoglobin (HbR) using the modified Beer–Lambert law58. The relative changes in HbO and HbR concentrations for each walking condition were obtained using a 5-s baseline collected before each session. The HbO and HbR changes were averaged over two repetitions for each walking condition (5–40 s after the onset) to improve signal-to-noise ratio. The index of hemoglobin differential was calculated by (Hbdiff = HbO − HbR) for further analysis. The Hbdiff values in multiple channels corresponding to each brain area were averaged to provide a better indication of hemodynamic response in the specific area  imaging system  was used to detect the cortical hemodynamic response to indicate the brain activation during walking. A total of 14 source-detector channels were arranged over the bilateral prefrontal cortex (PFC), premotor cortex (PMC), and supplementary motor cortex (SMA). The procedure of signal processing referred to our previous studyThe cognitive function was assessed by the modified trail making test (TMT) and digit span test.59. The modified version of TMT-A and B for the Chinese speakers were used in this study60. The time needed to complete each task was recorded for statistical analysis.The TMT are widely used to evaluate executive function in different populations61. Participants were asked to repeat series of numbers forward and backward instructed by the assessor. The total scores ranged from 0 to 28, and the higher scores indicated the better performance.Digit span test was used to assess sustained attention and working memoryParticipants received 24 sessions of 45-min one-on-one training program according to their group assignment.Participants in CDTT group practiced cognitive tasks during walking on level surface. The cognitive tasks included repeating phrases, counting number forward or backward, playing phonemic word chain games, reciting a poem, having conversations, and reciting a sentence backward.Participants in MDTT group practiced motor tasks during walking on level surface. The motor tasks consisted of holding balls, raising an umbrella by both hands, waving a rattle, beating a castanet, bouncing a basketball, and holding one ball and concurrently kicking a basketball.The complexity of walking for both dual-task training groups was modified by walking forward, backward and on an S-shaped route. The difficulty in tasks and walking complexity was progressively adjusted to challenge participants. The 8-week programs for the cognitive and motor dual-task training groups are shown in Supplementary Table Participants in CPT group received a multicomponent exercise program consisting of muscle strengthening, balance, and gait training for 15 min each. The strengthening exercise focused on the lower extremity muscles, including gluteal muscles, quadriceps, hamstrings, calf muscles and ankle dorsiflexors. The balance training included weight shifting to different directions while standing, tandem standing, standing on a foam, and one-leg standing. Participants were asked to walk forward, backward and on an S-shaped route during the gait training. The resistance for muscle strengthening, and the difficulty of the balance and gait training were adjusted accordingly as the participants progressed.ε2) of effect size was calculated for the between-group comparisons 62. Change values were calculated by subtracting the pre-test data from the post-test or follow-up data. Spearman’s rank correlation coefficient was used to determine the correlation between the post-intervention changes in gait and brain activation during walking. All the data was analyzed by SPSS 24.0. The statistically significant level was set at 0.05.Non-parametric test was used for comparisons due to heterogeneity of the study sample tested by Shapiro–Wilk test. Intention-to-treat analysis was used for the missing data at follow-up assessment. Descriptive statistics were generated and presented in median  for all variables. The between-group differences in basic data and outcome measures at pre-test were examined using Kruskal–Wallis test and Chi-square test. The within-group training effect on gait performance and cognitive function was evaluated by Friedman test in each group. The between-group differences were analyzed by Kruskal–Wallis test using change values, and post hoc analysis with Dunn–Bonferroni test. Epsilon-square estimate (Supplementary Information.