According to an influential account of aging effects on reading, older adults (65+ years) employ a more “risky” reading strategy compared to young adults (18–30 years), in which they attempt to compensate for slower processing by using lexical and contextual knowledge to guess upcoming  words more often. Consequently, while older adults may read more slowly, they might also skip words more often (by moving their gaze past words without fixating them), especially when these are of higher lexical frequency or more predictable from context. However, this characterization of aging effects on reading has been challenged recently following several failures to replicate key aspects of the risky reading hypothesis, as well as evidence that key effects predicted by the hypothesis are not observed in Chinese reading. To resolve this controversy, we conducted a meta-analysis of 102 eye movement experiments comparing the reading performance of young and older adults. We focused on the reading of sentences displayed normally , conducted using an alphabetic script or Chinese, and including experiments manipulating the frequency or predictability of a specific target word. Meta-analysis confirmed that slower reading by older compared to younger adults is accompanied by increased word-skipping, although only for alphabetic scripts. Meta-analysis additionally showed that word-skipping probabilities are unaffected by age differences in word frequency or predictability effects, casting doubt on a central component of the risky reading hypothesis. We consider implications for future research on aging effects on reading. Eye movement research has helped build detailed accounts of visual, cognitive, and oculomotor control processes underlying skilled reading , 2009, eSlower and more disrupted reading by older adults generally is attributed to visual and cognitive declines in older age, including slower processing of visual and linguistic information  which may lack the statistical power required to detect effects reliably. Second, there is lack of uniformity in the age ranges investigated across studies, and in whether visual and cognitive assessments are administered to exclude participants with abilities outside the normal range. Consequently, differences may exist in participant profiles for studies showing and not showing specific patterns of aging effects. Third, evidence suggests that task demand characteristics can influence the likelihood of older adults engaging in different reading behaviors. Research in this area has nevertheless reached a level of maturity at which it may be possible to gain insights into aging effects using meta-analysis techniques. This was the approach taken in the present research, which used meta-analysis to examine aging effects across 102 separate eye movement studies conducted in either an alphabetic script or Chinese. Meta-analysis has the advantage of synthesizing findings from multiple studies to calculate an overall effect e.g., , providiOur aim was to describe the current state of play regarding aging effects on eye movements in reading. We also aimed to provide a systematic assessment of the risky reading hypothesis, which seems timely considering recent studies that appear to contradict its predictions. Accordingly, we conducted a meta-analysis that focused on whether, compared to young adults, older adults (a) produce slower and more disrupted reading, that is, with longer reading times, more and longer fixations and more regressions across the whole of sentences, longer reading times on individual words and regions, and larger effects of word frequency and predictability in reading times for words; (b) make longer forward saccades and skip words more frequently, as characterized in the original The full data set and analytic code used are available (see Author Note). No materials were used. The number of studies and participants that contributed to each analysis are given in Online Supplement.Relevant articles were identified using the search terms “eye movement*,” “reading,” and “age OR aging OR ageing OR older adult*” in Web of Science and the China Academic Journals database in August 2021. The same search terms were used to find unpublished studies in doctoral/master’s theses using major online thesis databases . Screening identified articles meeting the following inclusion criteria: (a) the article was published in English or Chinese; (b) the study examined eye movements during sentence reading in either an alphabetic language or Chinese; (c) both young (16–35 years) and older (60+ years) age groups were included, with descriptive statistics provided for each group; (d) participants were native readers of the studied language and data were available for first-language (L1) reading; (e) participants were reported to be free of eye or neurological disease; (f) at least one normal display condition was included, that is, with no unusual formatting, gaze-contingent display changes, highly unusual sentence structures , or concurrent task demands ; (g) descriptive statistics were available for sentences displayed normally for a least one measure examined in the meta-analysis. Individual experiments in multi-experiment articles/theses were considered separately unless using the same participants. Duplicates  were removed.Adult age differences were examined across sentences , multiword regions , and individual words in sentences (using measures incorporating eye movements for a single word). At the sentence level, we analyzed the following variables: words per minute reading rates for alphabetic scripts/characters per minute reading rates in Chinese, sentence reading time (ms), average fixation duration (ms), number of fixations, number of regressions, and forward saccade length (in letters/characters). For multiword regions, we analyzed the following variables: first-pass reading time , regression-path duration , and total time . At the word level, we analyzed the following variables: first-fixation duration , gaze duration , total reading time , and word-skipping probability (%). Additionally, adult age differences in word frequency and word predictability effects were examined using word-level measures. Only data from normal reading conditions were included (see the inclusion criteria above), averaged across different normal reading conditions  where these were used.Online Supplement.Word-level analyses were restricted to single-word regions in sentences and averaged across words if multiple single-word regions were reported . For analyses of word frequency and word predictability, only studies which employed experimental manipulations were included, as corpus-based studies typically do not report descriptive data. For studies where more than two word frequency or predictability conditions were included , we included the highest and lowest frequency conditions. WebPlotDigitizer  was usedlonger reading times than words that are high in word frequency or predictability), the size of the frequency effect was calculated as the mean of the low-frequency condition minus the mean of the high-frequency condition, and the predictability effect was calculated as the mean of the low predictability condition minus the mean of the high predictability condition. For word-skipping probability , the size of the frequency effect was calculated as the mean of the high-frequency condition minus the mean of the low-frequency condition, and the predictability effect was calculated as the mean of the high predictability condition minus the mean of the low predictability condition. This meant that all measures could be interpreted similarly, that is, with all frequency and predictability effects expected to be positive. The standard error of the mean difference was used as the measure of variance for these analyses. Forest plots were created using the forest function within the meta package . Finally, to establish whether effects obtained in alphabetic scripts might generalize to Chinese, we then present analyses examining all studies, with script type  deviation coded  and included as a moderator variable. Analyses of script type were entirely between-study, as no studies examined the reading of both alphabetic languages and Chinese.Meta-analysis results for alphabetic languages are shown in Older adults had longer sentence reading times, longer average fixations, more fixations, and more regressions, compared to young adults.Forest plots for multiword region measures are shown in Forest plots for word-level measures are shown in Overall, the meta-analysis showed that older adults read more slowly than young adults by making more and longer fixations and more regressions, including spending longer reading individual words and multiword regions. However, forward saccades were longer and words were skipped more often by the older adults.Older adults had larger word frequency effects in gaze duration and total reading time. However, the age difference was not reliable for first-fixation durations or skipping probability. Age differences in the size of word predictability effects were not reliable for any measures.n = 3 for predictability effects on word-skipping to n = 8 for word frequency effects for the reading time measures).Overall, there was evidence that, in comparison to young adults, older adults made disproportionately longer gaze durations and total reading times on words of low frequency compared to words of high frequency. However, there were no reliable age differences in the size of the predictability effects for reading times. Moreover, there was no indication of an age difference in the size of word frequency or word predictability effects in word-skipping. Accordingly, the meta-analysis presents no evidence that age differences in word-skipping are driven by older adults making greater use of lexical or contextual knowledge to guess (and so skip) upcoming words more frequently. We note, however, that this interpretation is based on only a small number of experiments .As with findings for alphabetic scripts, word frequency effects were larger for older than younger adults in reading time measures. Unlike the data for alphabetic scripts, word predictability effects were larger for older than younger adults across all reading time measures. Critically, there was no age difference in the effects of word frequency or predictability on word-skipping, and so no evidence that older adults made greater use of lexical or contextual knowledge to skip upcoming words. As for the analyses of word frequency and predictability effects in alphabetic languages, we note that this interpretation is based on only a small number of experiments . For each experiment (shown as filled circles), the age difference (older adults’ mean minus young adults’ mean) is plotted against the standard error. In the absence of bias, a symmetrical funnel-shaped pattern of filled circles should be observed, such that experiments with smaller standard errors  cluster toward the top of the graph and experiments with larger standard errors  scatter along the bottom. A visual inspection indicates lack of symmetry in several plots. There are many reasons why funnel plots are not symmetrical, including chance , and there were no longer significant age differences in the size of the word frequency effect for first-fixation duration . However, all other measures produced the same direction and similar statistical significance as the original analyses. Notably, almost all unpublished studies showed the same direction of effects as published studies. Publication bias therefore did not appear to affect our findings or interpretations to any meaningful extent.To assess the likelihood of publication and other biases, funnel plots were created using the funnel function in the meta package . These ag chance . Neverthg chance  to trim g chance . We thenThe present meta-analysis pooled data from 102 experiments to determine age differences in eye movement behavior. We found a clear pattern of aging effects across multiple studies, even though such effects were not observed in some individual studies. In addition, data from both alphabetic scripts and Chinese confirm that older adults read more slowly, by making more fixations and regressions, and spending longer fixating words compared to young adults. Older readers of alphabetic languages make longer forward saccades and skip words more often than young adults; however, older Chinese adults make shorter forward saccades and skip words less often. There is some evidence that older adults have larger word frequency and word predictability effects in reading times than young adults, however, neither script type showed evidence of an age group difference in the effects of these variables on word-skipping.For alphabetic scripts, including English, older adults read more slowly, made more and longer fixations and more regressions, and spent longer reading individual words. Our meta-analysis also confirms that this slowdown in reading in older age is coupled with increased word-skipping and generally longer forward saccades. This aspect of our findings is consistent with the risky reading hypothesis as described by This leaves the question of why older adults exhibit higher word-skipping probabilities and longer forward saccades when reading alphabetic scripts. The meta-analysis suggests these effects are not moderated by a word’s frequency or predictability and so cannot be explained in terms of older adults making greater use of lexical or contextual knowledge to guess upcoming words (and so skip them more often). However, the effects might be compatible with an account in which older adults attempt to compensate for slower processing by guessing words identities based on only visual and orthographic information about upcoming words . Such an account may resonate with a simulation of aging effects by The meta-analysis provided clear evidence for cross-script differences in aging effects, indicating that findings for alphabetic languages may not be generalizable to other scripts. We found that older adults read Chinese more slowly than young adults, by making more and longer fixations and more regressions. However, whereas aging effects for alphabetic scripts were characterized by increased word-skipping and longer forward saccades, for Chinese they involved skipping words infrequently and making shorter forward saccades compared to young adults.Various explanations of these cross-script differences are possible. These include participant sampling differences across experiments with different scripts and also a specific age-related cost for Chinese. However, an explanation based on sampling differences seems unlikely given the care taken in matching young and older adult participants in most experiments, including in terms of participants’ years of education, reading experience, and vocabulary knowledge. There is therefore no indication from these studies that the young and older adult participants differ in terms of literacy . Moreover, the finding from the present meta-analysis that age differences in effects of word frequency are broadly similar across alphabetic scripts and Chinese suggests that the young and older adults who participated in these experiments have similar language processing capabilities. One strong possibility is that the visual and cognitive demands associated with the Chinese script are a specific source of difficulty for older adult readers. Indeed, our analyses which included script type as a moderator indicated that age differences in sentence reading times were larger in Chinese than alphabetic languages, which is consistent with the notion that the reading of Chinese may be particularly challenging for older readers. These demands are likely to include visual processing difficulties associated with identifying complex characters e.g., , as wellWe note, however, that we did not find any studies that directly examined cross-linguistic age differences, and so presently our understanding of such effects is entirely based on comparisons of different studies. Such comparisons are limited by methodological differences across studies . While direct cross-linguistic comparisons of eye movements in reading are inherently difficult , future The meta-analysis provided clear evidence for age group differences in the effects of word frequency on the processing of fixated words. This was because, compared to young adults, older adults made disproportionately longer reading times for low-frequency words relative to high-frequency words. This larger word frequency effect in older compared to young adults occurred for both the reading of alphabetic languages and Chinese. Coupled with the finding that older readers make generally longer fixations on words, these effects might be consistent with slower lexical processing in older age. This slower processing might be a consequence of cognitive decline. Alternatively, it may be because growth in the mental lexicon from a lifetime of exposure to written language can slow access to specific lexical items e.g.,  or becauThe meta-analysis also provided some evidence of age group differences in the effects of word predictability. Older readers of Chinese had disproportionately longer reading times for low predictable compared to high predictable words in comparison to their younger counterparts. This indicates that young and older Chinese readers differ in their use of context when processing words. Older readers of alphabetic languages also showed numerically larger predictability effects than young adults in gaze durations and total reading times, although these findings did not reach conventional standards of statistical significance. Intriguingly, studies using event-related potentials (ERPs) have typically found that an ERP component (the N400) known to be sensitive to the contextual fit of a stimulus is smaller or delayed for older relative to younger adult participants, indicating that older adults might be less able to use context predictively e.g., . This coIn this final section, we turn to a discussion of how our experience in performing the present meta-analysis might inform best practice in conducting and reporting eye movement studies of aging effects and inform directions future research. First, while the meta-analysis produced generally clear results, some discrepancies among studies were observed, particularly for alphabetic scripts. Some of this variability will be attributable to variations in stimulus material characteristics, for example, sentences that vary in length and/or complexity, multiword regions that differ in the number of words, or critical words that differ in length, frequency, or predictability. As noted in the Introduction, some between-study variance might be because of small sample sizes in some studies, differences in the profile of participant groups across studies, or use of experimental methods that promote more careful reading . We note that studies that have recently been cited as counterevidence for the risky reading strategy e.g.,  had slowBenefits may also be obtained from fuller assessment and reporting of the visual and cognitive capabilities of participant groups. Many studies (including from our laboratory) have not reported these variables comprehensively and so it is unclear whether some discrepancies between studies might be attributable to the inclusion of participants with visual or cognitive capabilities outside the normal range. Including better information about participant profiles will improve the comparability of study findings and advance understanding of how individual differences in visual and cognitive capabilities might affect reading performance. Improvements to current assessment methods should include more rigorous assessment of visual abilities, which decline markedly in older age and are affected by eye disease common to older adults  do not fully report all eye movement measures that are useful for assessing aging effects. In some cases, this was sufficiently serious that experiments, including several considered to be landmark studies in the field, violated our inclusion criteria. These included studies which either did not report raw means or variance or did not provide descriptive statistics separately for normal and nonnormal text displays e.g., , 2006. FFinally, we consider it important to note that investigations of aging effects on eye movements in reading conducted to date tend to be limited to the reading of isolated sentences by young (18–35 years) or older adults (65–80 years). Few studies have investigated reading multi-sentence texts but see , 2019 al10.1037/pag0000522.supp


This review serves as an introduction to a special issue of Frontiers in Physiology, focused on the importance of physiological performance curves across phylogenetic and functional boundaries. Biologists have used performance curves to describe the effects of changing environmental conditions on animal physiology since the late 1800s (at least). Animal physiologists have studied performance curves extensively over the past decades, and there is a good foundation to understanding how the environment affects physiological functions of individuals. Our goal here was to build upon this research and address outstanding questions regarding the mutability and applicability of performance curves across taxonomic groups and levels of biological organization. Performance curves are not fixed at a taxonomic, population, or individual level – rather they are dynamic and can shift in response to evolutionary pressures  and epigenetic programming . The mechanisms underlying these shifts are being increasingly used to predict the efficacy with which plasticity and heritability of performance curves can render individuals and populations less vulnerable to climate change. Individual differences in physiological performance curves (and plasticity of performance curves) can also have cascading effects at higher levels of biological organization. For instance, individual physiology likely influences group behaviors in non-additive ways. There is a need therefore to extend the concept of performance curves to social interactions and sociality. Collectively, this special issue emphasizes the power of how within- and between-individual shifts in performance curves might scale up to the population-, species-, and community-level dynamics that inform conservation management strategies. Physiological performance curves describe changes in physiological rates with respect to an environmental gradient. The y-axis can represent any level of physiological performance, from single-molecule  to whole-animal measures . Similarly, the x-axis can describe a wide range of environmental gradients, including those associated with abiotic  and biotic factors . The shapes of physiological performance curves thereby describe physiological rates as a function of the acute environment. The precise shapes can vary within and between taxonomic and functional boundaries. Determining the mechanisms that drive these changes is important for understanding the diverse strategies and limitations that shape animal responses to their environments.A comparative approach has been particularly powerful to help trace the cascading effects of environmental change across levels of biological organization. Reductionist approaches focusing on single-enzyme thermal performance curves, for instance, indicate that breakdowns in animal performance at high temperatures cannot simply be ascribed to protein denaturation, as was once thought. Rather, the shapes of these single-enzyme curves suggest that decreases in whole-animal performance likely reflect more nuanced shifts in enzyme microstates and activation energies see . Thus, ubetween populations and species, for instance, differences in shape can point to adaptations that promote fitness in local environments. Comparing how performance curves shift as a function of the environment within individuals and populations, on the other hand, can indicate whether plasticity allows animals to defend performance against environmental change. These comparative approaches can also help to assess tradeoffs associated with specific physiological strategies.In an even broader sense, a comparative approach has also helped identify the adaptive and plastic  responses that animals use to overcome environmental variability. By comparing performance curves In this Special Issue, we focus on molecular and physiological mechanisms underlying performance curves and how performance curves may shift in response to an environmental change, by both genetic and epigenetic mechanisms. Responses to the environment are likely to differ between phylogenetic groups, and we are particularly interested in differences between ectotherms and endotherms. Along these lines, we also aim to extend the concept of performance curves from individuals to groups of animals to ask how the physiology of performance curves of individuals can impact emergent properties, like social behavior.In the simplest terms, physiological performance curves effectively represent dose-response relationships – an increasing “dose” of some environmental factor alters the response of a physiological system. Many ancient societies clearly recognized the phenomenon of dose-dependence. Ancient Greeks, for instance, recognized that wine in excess would dull the senses  in a frog warmed from 2 to 32°C, then cooled back down to 2°C again the following day (2-production) between these various taxonomic groups to investigate the metabolic properties that correspond to these distinct evolutionary strategies , for instance, represents the lowest PO2 at which an oxyregulator can regulate some rate of metabolic oxygen consumption (MO2), below which it oxyconforms , but also understanding the physiological strategies and limitations that bound these responses. The critical oxygen tension  may promote higher fitness by implying increased rates of growth and development . In otheinverted U-shape arguably represents the most common shape for a physiological performance curve, where performance is maximized at some intermediate environmental state and decreases as the environment changes in either direction. Thermodynamic effects on enzymatic rate processes, for instance, mean that catalytic activity is often optimized at some temperature that may be reflective of local environmental conditions. Cooler temperatures reduce performance by limiting the free energy available for activation, whereas warm temperatures ultimately limit performance by shifting the distribution of active enzyme microstates and potentially raising activation energies  typically between two and three for biological systems. In other words, physiological reaction rates double to triple for every 10°C increase, and are halved or third for every 10°C decrease . Another common type of performance curve in physiology is dose-response curves that are classically sigmoidal in shape  suggested that shifts in thermal performance curves for growth represented local adaptation to seasonal growth periods (Performance curves that differ between populations (intraspecific) and species (interspecific) can reflect adaptive differences in environmental tolerance e.g., –C. This  periods . Specifi periods .Galeolaria caespitosa) is optimized to the temperatures experienced during embryogenesis. Specifically, there was a warmward skew in thermal survival curves with increasing embryonic temperature. Together, these findings demonstrate that specific aspects of the thermal performance curve  are shaped differently by developmental conditions.Performance curves that differ as a function of the developmental environment can reflect developmental plasticity e.g., –F. Here,Poecilia formosa) adjusts the thermal sensitivity of open-field swimming behavior, but not maximal swimming performance, as a function of developmental temperature. Interestingly, the authors find the opposite pattern in the closely related but sexually reproducing Atlantic molly (Poecilia mexicana), where performance curves for maximal swimming performance, but not open-field swimming behavior, shift as a factor of developmental temperature. This divergence between species suggests that genetic mechanisms are important in mediating the strength of epigenetic responses.Clonal organisms represent a particularly powerful tool for studies in developmental plasticity because genetic backgrounds are easily controlled. In this special issue, Melanoplus boulderensis) from higher elevations have greater capacity for developmental plasticity in response to seasonal changes  in the environment. Here, shifts in thermal performance curves for hopping distance and feeding rates revealed that different populations (and sexes) responded to developmental changes in day-length and thermal variability in complex ways. Together this work suggests that the interaction between genotype (populations) and epigenetics  shape ecologically important responses.Developmental plasticity can itself represent an adaptive trait. That is, the capacity for developmental plasticity can vary between individuals and populations e.g., ,E. In thBokermannohyla ibitiguara) to determine their capacity for reversible acclimation. Here, a within-individual approach would likely not be possible because temporal responses to the changing thermal environment would be confounded with developmental stage. While the authors found that thermal performance curves for aerobic scope shifted with acclimation temperature, the effect is likely to reflect a pathology of high acclimation temperature rather than a regulated compensatory response. That is, aerobic scope collapsed in tadpoles acclimated to the upper boundary of temperatures naturally experienced in their microhabitat . Thus, not all curve shifts within and between individuals represent adaptive plastic responses – a comprehensive understanding of the performance parameter in question, the life history of the focal species, and the acute effects of the environmental input(s) used represent important points of interpretation.Performance curves that shift temporally within individuals as a function of the environment can reflect acclimation e.g., –I. WhileGambusia holbrooki) that were each reversibly acclimated to both a cool and warm temperature to show that focusing on mean values can mask underlying variation and obscure bet-hedging dynamics – particularly when populations are undersampled. Further, by focusing on plasticity at an individual level, An increasing body of work has also revealed that there can be substantial inter-individual variation in the capacity for acclimation; that is, some individuals have phenotypes that are particularly plastic, and others have phenotypes that are more fixed . While some of this variation is likely to be genetically determined, the capacity for reversible acclimation itself also appears to be a plastic trait that can be programmed in response to the developmental environment. An implication of individual variation in the capacity for acclimation is that mean acclimation responses cannot predict population responses to environmental change in meaningful ways. With a bet-hedging strategy, for instance, only a subsection of the population may persist under conditions that favor plastic over fixed phenotypes. In this special issue, Danio rerio) embryos at thermal extremes. Specifically, the authors used a targeted pharmacological approach to generate thermal reaction norms for mitochondrial  and cardiac (heart rate) performance in embryos pre-exposed to a range of five temperatures. Collectively, their work demonstrates that comparing performance curves down levels of biological organization can help elucidate mechanisms driving whole-animal responses. A note of particular interest is that the mechanisms that modulate mitochondrial performance with changing temperatures are non-linear – mitochondrial oxygen consumption is constrained by reduced rates of ATP production at low temperatures and amplified by increasing rates of proton leak at high temperatures.Shapes of performance curves across functional boundaries and levels of biological organization can be particularly useful to determine potential mechanisms underlying higher-level traits. In this special issue, In the same way performance curves can help uncover mechanisms underlying physiological responses to the environment, they can also be used to make predictions about emergent properties of a system at higher levels of organization. In this special issue, Performance curves represent a powerful tool to understand the implications of dynamic traits in changing environments. While the approach itself has been around for more than a century, this special issue showcases underused or emerging ways in which performance curves can be applied in comparative fields. We partitioned curve shapes, curve shifts, and functional boundaries into distinct subsections for simplicity, but we acknowledge that biological systems are rarely so defined. The complexity of natural systems is reflected by the studies included in this special issue, where comparisons within and between individuals, populations, endotherms and ectotherms , and phyAL and FS conceived the ideas. AL wrote the manuscript. FS edited the manuscript. All authors contributed to the article and approved the submitted version.AL was supported by a Discovery Grant from the Natural Sciences and Engineering Research Council of Canada . FS was supported by an Australian Research Council Discovery Grant (DP190101168).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


It is known that the cell environment such as biomechanical properties and extracellular matrix (ECM) composition dictate cell behaviour including migration, proliferation, and differentiation. Important constituents of the microenvironment, including ECM molecules such as proteoglycans and glycosaminoglycans (GAGs), determine events in both embryogenesis and repair of the adult lung. Mesenchymal stromal/stem cells (MSC) have been shown to have immunomodulatory properties and may be potent actors regulating tissue remodelling and regenerative cell responses upon lung injury. Using MSC in cell-based therapy holds promise for treatment of chronic lung diseases such as idiopathic pulmonary fibrosis (IPF) and chronic obstructive pulmonary disease (COPD). However, so far clinical trials with MSCs in COPD have not had a significant impact on disease amelioration nor on IPF, where low cell survival rate and pulmonary retention time are major hurdles to overcome. Research shows that the microenvironment has a profound impact on transplanted MSCs. In our studies on acellular lung tissue slices (lung scaffolds) from IPF patients versus healthy individuals, we see a profound effect on cellular activity, where healthy cells cultured in diseased lung scaffolds adapt and produce proteins further promoting a diseased environment, whereas cells on healthy scaffolds sustain a healthy proteomic profile. Therefore, modulating the environmental context for cell-based therapy may be a potent way to improve treatment using MSCs. In this review, we will describe the importance of the microenvironment for cell-based therapy in chronic lung diseases, how MSC-ECM interactions can affect therapeutic output and describe current progress in the field of cell-based therapy. COPD alone is the 3g cancer . COPD pag cancer . The comg cancer . IPF, afg cancer  is also g cancer  and sharg cancer . No treag cancer . IPF is g cancer . Therefog cancer . The lacg cancer , and theg cancer .in situ environment of the injected MSC.Common to both diseases is the structural remodeling of the lung tissue, resulting in impaired gas exchange that leads to decline in lung function. To date, the knowledge of the inherent capacity of the lung to repair is limited, but mesenchymal stromal cells (MSC) have been proposed to have a therapeutic potential due to their regenerative and immunomodulatory abilities in combination with their low immunogenicity attributed to low or absent human leukocyte antigen (HLA) expression . The str6–108 cells per dose, single dose or in some cases, up to 4 doses. Completed trials using primarily one dosage regimen show in COPD trials a safe profile and ameliorated clinical parameters in up to 50% of patients  or intratracheal routes of administration of bone marrow, umbilical cord or adipose tissue derived MSC in a dosage range of 10patients . In a sm, alleviating fibroblast activation and ECM deposition along with anti-inflammatory outcomes . Pre-cliMSCs are extensively explored as cell therapy candidates and are known for their potential to differentiate into multiple cell lineages, however their major therapeutic mechanism is from interactions via paracrine signaling and cell-cell contact . MSCs haIn vitro, HGF inhibited epithelial-mesenchymal-transition (EMT) through inhibition of transforming growth factor β (TGF-β) signaling . EVs arKGF/FGF7). Interessed MSCs . These din vitro and in vivo  chondroitin/dermatan sulfate (CS/DS) and keratan sulfate (KS) . A fourtArguably one of the most important interactions between cells is mediated through growth factors and cytokines. These factors travel large distances in the body to recruit and direct cells where they are needed. A largely overlooked process is how growth factors interact with the ECM via GAGs and how the ECM can increase or decrease the cell response to growth factors. Thus, the ECM serves as a reservoir for growth factors, which are slowly released over time. At the same time the growth factors bound to the ECM via the GAGs become protected from circulating proteases. Because of these properties, if the ECM is injured the release of growth factors will be instant, thus triggering an immediate cell response. For example, latent TGF-β  in the Ein vitro  and influence cell behavior . To mainin vitro studies showing how MSC are differently affected by the environment the cells encounter. Increasing research is focused on how to control the therapeutic effect of MSC, where one option being explored is by modulating the microenviroment in situ or by injectable biomaterials.There are several in situ (Encapsulating MSC in injectable hydrogels is extensively explored to increase retention time and to modulate the cellular activity in situ  by proviin situ . Effortsin situ . So far,in situ . Apart fin situ . The advin situ . De Santin situ . Transplin situ . Hydrogein situ . As discin situ .In contrast to solid hydrogels, macroporous biomaterials or scaffolds introduces a more complex 3D environment, where the surface properties of the scaffold can be tuned to functionalize cell attachment, differentiation and migration, as well as to enhance cell diffusion to the scaffold.In a recently published article, a collagen-based scaffold was developed with macroporous structures to mimic the distal lung tissue. The scaffold was functionalized with collagen binding HGF to enhance the regeneration of alveolar-like structures . When maThere are increasing evidence that MSC have a cellular memory, with sustained responses to preceding stimuli, which may influence the desired effect of MSC-based therapies . MSC culin situ may hold difficulties in its approach in retaining tuned levels of cell mediators or in adapting specific ECM components in the location of transplanted MSC. Identifying key elements or biological properties of the ECM warrants further investigation as this would allow for more precise and directed manipulation of the pathological tissue in situ. The environmental responsiveness of the MSCs creates both challenges and opportunities in applying transplanted MSC as a cell-based therapy in lung diseases that are marked with massive structural and compositional tissue remodeling. Thus, pre-conditioning of MSC may hold a more transient approach in overcoming the powerful influential impact of surrounding tissue on cellular behavior, providing a more instant regenerative response.For MSC therapy to become an efficient alternative to current medicine in IPF and COPD, it is essential to ensure the functional outcome of the cells in the diseased tissue. One way is to control the microenvironment in which the cells are delivered. Recent advances have demonstrated that MSC actively interact and modify their pericellular environment, that there is an active interplay between the surrounding properties, both in terms of structural properties and chemical composition, thus will play a role in directing MSC function . Severalin situ cell guidance over time as the scaffold is degraded and replaced with functional lung tissue. By providing structural support, we anticipate that both the MSC retention time will be greatly improved with remained activity and allowing endogenous cells to repopulate lost tissue area.The artificial scaffolds hold great promise in future therapeutic purposes as it can combine cell-instructive cues together with a supporting microenvironment, delicately designed in both structure and biomechanics, that together yield an optimal curative setting for MSC. The purpose of a controlled microenvironment is to create an interim support as well as Altogether, harnessing the microenvironment holds promise to ameliorate future cell therapeutic regimens to be able to induce lung regeneration and healing of lung tissue in chronic lung diseases marked with massive structural remodeling like IPF and COPD.


Intravascular imaging was introduced 30 years ago to assess coronary artery morphology and facilitate percutaneous coronary intervention (PCI). Today, invasive imaging has an established role in clinical practice and research . CumulatKuku et al. published in this issue describes the evolution in catheter design and presents the evidence supporting the value of this modality in guiding PCI and detecting vulnerable plaques. Muller—the inventor of NIRS imaging—and Madder, also highlight, in their review, the potential of hybrid NIRS-based imaging in optimizing PCI results and identifying high-risk lesions and patients who would benefit from aggressive treatments targeting atherosclerosis, and introduce a combined NIRS-OCT system that is expected to have clinical applications in the near future (Muller and Madder). Another hybrid intravascular imaging modality that appears superior to standalone IVUS or OCT imaging in assessing plaque phenotype is the combined IVUS-OCT imaging, which has also reached clinical translation. Several prototypes have been presented over the last years and one of them the Novasight HybridTM System  had recently first-in-man studies (Near-infrared spectroscopy (NIRS)-IVUS imaging is the first hybrid intravascular imaging modality that had clinical applications. The catheter incorporates a NIRS and IVUS integrated probe that acquire simultaneous imaging data which are co-registered to generate hybrid NIRS-IVUS images. The review article of  studies . Ono et  studies .Khraishah and Jaffer).The above developments in intravascular imaging may enable more precise evaluation of plaque composition but cannot detect vascular inflammation which is an important instigator of plaque evolution. Near-infrared fluorescence (NIRF) imaging was introduced to address this unmet need. This modality can be combined with IVUS or OCT and appears capable to identify the presence of macrophages, cathepsin protease activity, oxidized low-density lipoprotein and thrombus. NIR autofluorescence (NIRAF)-OCT intracoronary imaging has been performed in patients and may identify high-risk plaques with intraplaque hemorrhage or oxidized lipid, without the need for a contrast agent. The developments in the field are described in detail by the inventors of NIRF in a comprehensive review that also discusses the potential value of hybrid NIRF-IVUS and OCT-NIRF imaging in secondary prevention . The study of Otsuka et al.). The inventors of this approach present a thorough review which describes the basic principles of PS-OCT and highlights its value in characterizing atheroma. Summarizing their research in the field the authors suggest that PS-OCT is capable to assess collagen and smooth muscle cell content in fibrous caps, and improve the efficacy of OCT in identifying necrotic cores, macrophage accumulations and cholesterol crystals—plaque features that are seen in high-risk lesions. Finally, PS-OCT offers a unique potential to automatically quantify tissue types allowing fast processing of large imaging data.In parallel to the design of novel hybrid imaging catheters for more detailed assessment of the atheroma, efforts have been made to optimize standalone intravascular imaging catheters. Toward this direction polarization sensitive (PS)-OCT imaging was introduced. This approach relies on the analysis of the polarization properties of the reflected OCT signal . This approach offers increase signal penetration (>20 mm) and is capable to acquire images with a resolution of >50 μm. Preliminary data has shown that it is able to characterize plaque composition. OpUS is still in its infancy and its full potential has not been evaluated yet; moreover further developments are needed before this modality has applications in interventional cardiology .Despite the high resolution of OCT there are plaque micro-features that cannot be visualized by this imaging technique. To address this limitation micro-(μ)OCT has been developed that allows plaque imaging with a resolution of 1–2 μm. Kilic et al.)—and thus more precise identification of vulnerable lesions and high-risks patients that will benefit from emerging therapies targeting atherosclerosis  and positron emission tomography (PET)-CT/CTCA. In contrast to intravascular imaging that is associated with a risk of complications and do not allow complete assessment of the coronary artery tree, non-invasive imaging appears as the ideal approach for stratifying cardiovascular risk as it can be used in asymptomatic individuals, requires a lower dose of radiation and enables complete study of all the major coronary arteries . Yet CTCCB drafted the manuscript while FJ, YO, and PS reviewed, edited, and approved the final draft. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. 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Pain is managed using a biopsychosocial approach and pharmacological and non-pharmacological treatments. Transcutaneous electrical nerve stimulation (TENS) is a technique whereby pulsed electrical currents are administered through the intact surface of the skin with the intention of alleviating pain, akin to ‘electrically rubbing pain away’. Despite over 50 years of published research, uncertainty about the clinical efficacy of TENS remains. The purpose of this comprehensive review is to critically appraise clinical research on TENS to inform future strategies to resolve the ‘efficacy-impasse’. The principles and practices of TENS are described to provide context for readers unfamiliar with TENS treatment. The findings of systematic reviews evaluating TENS are described from a historical perspective to provide context for a critical evaluation of factors influencing the outcomes of randomized controlled trials (RCTs); including sample populations, outcome measures, TENS techniques, and comparator interventions. Three possibilities are offered to resolve the impasse. Firstly, to conduct large multi-centered RCTs using an enriched enrolment with randomized withdrawal design, that incorporates a ‘run-in phase’ to screen for potential TENS responders and to optimise TENS treatment according to individual need. Secondly, to meta-analyze published RCT data, irrespective of type of pain, to determine whether TENS reduces the intensity of pain during stimulation, and to include a detailed assessment of levels of certainty and precision. Thirdly, to concede that it may be impossible to determine efficacy due to insurmountable methodological, logistical and financial challenges. The consequences to clinicians, policy makers and funders of this third scenario are discussed. I argue that patients will continue to use TENS irrespective of the views of clinicians, policy makers, funders or guideline panel recommendations, because TENS is readily available without prescription; TENS generates a pleasant sensory experience that is similar to easing pain using warming and cooling techniques; and technological developments such as smart wearable TENS devices will improve usability in the future. Thus, research is needed on how best to integrate TENS into existing pain management strategies by analyzing data of TENS usage by expert-patients in real-world settings. Pain is managed using a biopsychosocial approach, and pharmacological and non-pharmacological methods. Therapeutic neuromodulation techniques deliver thermal, mechanical, chemical and electrical stimuli to the body, and are recommended in medicine, physical therapy and nursing for relief of pain. Electrotherapeutic neuromodulation techniques are categorized as; invasive, such as percutaneous electrical nerve stimulation, electroacupuncture, spinal cord stimulation and deep brain stimulation; or non-invasive, such as transcutaneous electrical nerve stimulation (TENS) and TENS-like techniques.Pain mechanisms: a new theory by Melzack and Wall which suggested that electrical stimulation of low threshold peripheral nerves in the skin could inhibit activity of centrally located nociceptive transmission neurons, thus relieving pain  . NICE diThe debate about the efficacy of TENS has remained unresolved despite over 50 years of published research. Analyses of the costs, risks and benefits suggests that TENS rates favourably with standard care and NICE thresholds for Quality-Adjusted Life Year ,13,14. TThe purpose of this comprehensive review is to critically appraise previous clinical research on TENS to inform future strategies to resolve the ‘efficacy-impasse’. A brief overview of the principles and practices of TENS is provided for readers unfamiliar with TENS treatment. This is followed by a description of systematic reviews of RCTs, and a critical appraisal of factors influencing findings; including sample populations, outcome measures, TENS techniques, and comparator interventions. Issues arising are used to optimise the design of future RCTs, and systematic review and meta-analysis. It is suggested that the ‘efficacy-impasse’ would be resolved by large multicentred RCTs using an enriched enrolment with randomized withdrawal design, and a meta-analysis of the effect of TENS on pain intensity during treatment, irrespective of pain condition . FinallyThe review is narrative in style and draws on three decades of experience of conducting reviews and meta-analyses of TENS for various types of pain. Narrative reviews can be vulnerable to selection and evaluation biases and opinion-oriented arguments, so readers are directed to key references for comprehensive coverage of topics of further interest. The intention is to challenge dogma where necessary and to catalyse scholarly debate about future directions for research, practice and health care policy.This section provides a brief overview of the principles and practice of TENS as a foundation for issues discussed in later parts of the review. Readers familiar with TENS may wish to proceed directly to In health care, transcutaneous electrical nerve stimulation (TENS) refers to the use of a portable device that generates pulsed electrical currents that are delivered across the intact surface of the skin via conducting electrodes to stimulate peripheral nerves . TENS isThe safety profile of TENS compares favourably against medication. Adverse events associated with TENS are minor and include erythema and itchiness beneath or around the electrodes, and a vasovagal response in some individuals, manifesting as nausea and dizziness. Contraindications are few. From a legal perspective, manufacturers recommend not to use TENS for patients with epilepsy, malignancy, deep vein thrombosis or an active electrical device implant . It is possible to use TENS in some of these patients following careful assessment and providing electrodes are placed at sites distant to the hazard  within, or close to, the site of pain  without concurrent activation of higher threshold small-diameter nociceptive nerve fibres  of pulsed currents determines the rate of neuronal impulses and is limited by the absolute and relative refractory periods for the axon. For conventional TENS, pulse frequencies below 250 pulses per second (pps) are used as they produce a pleasant tingling sensation. In principle, pulses of current between 50 and 500 microseconds in duration ) are optimal to activate low threshold large-diameter fibres (A-beta) without concurrently activating high threshold small-diameter nociceptive fibres (A-delta and C). Commonly, TENS devices deliver biphasic pulse waveforms with zero net current flow  as this Conventional TENS is the most common technique used in clinical practice. Other techniques such as acupuncture-like TENS are used in specific situations or for patients who have not responded to conventional TENS .There is a raft of ‘TENS-like techniques’ described in the literature, with manufacturers and advocates claiming specific roles and indications. Whether such techniques show superiority in efficacy, safety or utility over placebo, conventional TENS and/or other treatments is a matter for debate and lies outside the scope of this review (for review see ).TENS modulates nociceptive input at peripheral , segmental  and extrasegmental (‘descending inhibition’) sites. Stimulation of low threshold large-diameter non-noxious cutaneous afferents reduces activity and excitability in sensitised or non-sensitised central nociceptive transmission cells, in segments of somatic receptive fields related to the location of the electrodes ,31,32,33Delivery of higher amplitude currents stimulates high threshold cutaneous (A-delta) afferents within deeper tissue and produces long-term depression of central nociceptor cell activity persisting up to 2 h post-stimulation ,35,36. ITENS also extinguishes ‘incoming’ orthodromic impulses conducted in peripheral afferents . Low intensity  TENS extinguishes impulses conducted in low threshold cutaneous afferents arising from non-noxious somatosensory receptors such as mechanoreceptors ,40. At hThe central inhibitory effects during TENS occur through a complex interplay between excitatory and inhibitory neurotransmitters and neuromodulators, influenced in part by the frequency of the pulsed current (for review see ). These Recently, evidence has emerged that the analgesic effects of TENS for knee pain associated with osteoarthritis is influenced by genetic variation within the catechol-O-methyltransferase (COMT) and endothelin receptor type A (EDNRA) genes . In futuIt is possible that TENS indirectly reduces pain by changing other physiological processes including; increasing microperfusion which is beneficial for ischaemic tissue, arterial insufficiency and claudication due to arterial and neuropathic diseases; reducing heart rate and systolic and diastolic blood pressure; and influencing local reflex control of visceral functions to counteract detrimental effects associated with constipation and urinary retention (for review see ).Decisions to offer treatments to patients involves the integration of clinical experience and patient values, with the best available research information about efficacy and effectiveness . Clinical experience suggests that TENS may provide short-term relief of pain during or immediately after treatment for any type of acute or chronic pain. Pains with a restricted distribution may be easier to treat because it is possible to permeate TENS sensation throughout the painful area. However, clinical experience alone cannot distinguish effects attributed to the active ingredient of a treatment from non-specific effects associated with the act of administering a treatment including patient expectation of a treatment having effects, natural fluctuations in symptoms, reporting bias to please the practitioner, and contamination from concurrent treatment.active ingredient of a treatment, evaluated under ideal circumstances and controlled conditions. In contrast, evaluations of ‘effectiveness’ involve measurement of beneficial and harmful effects of the whole treatment package when given in real-life healthcare practice, and whether this treatment package matters to the patient. Efficacy studies tend to be ‘explanatory’ in design, whereas effectiveness studies tend to be ‘pragmatic’ in design  used doses of TENS which approached this. Duration of treatment was less than 4 weeks in 83% of the trials, and in 85% of the trials stimulation occurred less than 10 h per week, with 67% of the patients having less than ten sessions of TENS.” [In 1997, McQuay et al.  publisheleagues,  who demof TENS.”  p. 48.Two decades later, there continues to be an absence of RCTs evaluating long-term TENS treatment. Most RCTs evaluate TENS treatment over a period of days  or weeks  rather than months. Many RCTs to date prescribe a fixed number of treatments of certain duration, whereas in clinical practice patients use TENS as often as needed. There remains a necessity to match treatment schedules with measurement protocols in RCTs, including distinguishing during treatment effects from cumulative effects and long-term outcomes (discussed previously). The need for pragmatic trials where participants are trained to personalize TENS treatment according to their needs is discussed later.Repeated TENS treatment may result in a decline in pain relief in some individuals (TENS tolerance). A variety of biopsychosocial factors have been implicated including; dead batteries, perished leads, a worsening pain problem and waning of the initial enthusiasm for a new treatment. Repeated TENS has been shown to cause tolerance in mu and delta opioid receptors ,151 withParadoxically, repeated TENS treatment may intensify pain relief in others (cumulative benefit) with a variety of biopsychosocial factors involved including; increasing familiarity and competence at using equipment, improved skills at optimising treatment on an as needed basis, re-calibrating treatment expectations, and TENS indirectly improving functions of daily living. Repeated TENS also reduces neuronal excitability and sensitization at peripheral and/or central sites over time and may reset physiological sensitization to normal ,31. RCTsNon-invasive electrical stimulation of the skin with the intention of stimulating peripheral nerves to alleviate pain(a)… a portable, battery-powered generator of monophasic or biphasic pulsed electrical current delivered in a repetitive manner, with a maximum peak-to-peak amplitude of approximately 60 milliamperes (mA) into a 1 kilohm load.” [‘standard TENS device’ defined as “m load.”  p. 12, a(b)electrodes attached to the surface of the skin that would also include electrodes integrated into garments such as knee braces, cuffs, gloves and/or socksEquipment consisting ofTechniques that produce strong but comfortable TENS sensations using any type of pulse pattern, pulse frequencies no more than 250 pulses per second (pps), and pulse durations no more than 1 millisecond (1000 µs). Intensities above motor threshold are acceptable providing TENS is administered with the intention of alleviating pain.Techniques that administer TENS at the site of pain or over nerve bundles near to the site of pain. TENS at remote acupuncture points would not be in-scope.Any TENS treatment schedule providing it matches with intended symptomatic relief of pain, and in RCTs matches measurement schedules.Investigators face major challenges in defining the scope of TENS in relation to type of devices, electrical characteristics, electrode placement sites, and adequacy of TENS technique. The following operational criteria have been published to define the scope of TENS by Johnson et al. :Non-invaRCTs have evaluated TENS versus placebo TENS ; versus no treatment or waiting list control; versus treatments partly or wholly as standard of care ; and versus other treatments, both pharmacological and non-pharmacological, not considered to be used in routine practice. Debates about the efficacy of TENS tend to concentrate on evaluations using placebo comparators.Sham TENS devices with no current outputSham TENS devices with current above sensory detection threshold that fades to zero current output usually within 45 sActive TENS below sensory detection threshold, so the participant cannot sense itActive TENS above sensory detection threshold with infrequent pulses using interpulse intervals beyond that expected to produce physiological effectsPlacebo controls are used to isolate effects associated with the active ingredient of a real treatment from biases associated with receiving a treatment. In drug trials placebos are designed to be identical in appearance, taste and odour to the ‘real’ drug but contain an inert substance rather than an active chemical ingredient. It is important that placebo controls are indistinguishable from test interventions to conceal (blind) which intervention is placebo and which is ‘real’ from trial participants, practitioners and assessors. A variety of placebo comparisons have been used in RCTs of TENS including:Sham TENS devices with no current output are most commonly used as they isolate effects of pulsed electrical currents per se, i.e., the active ingredient of TENS.Electrical currents are only a means (stimulus) to selectively activate low threshold afferents. Thus, from a physiological perspective any modality that selectively stimulates low threshold peripheral nerves, such as vibration, warmth and cold may generate similar effects as TENS, although the quality of the sensory experience may differ. The role of TENS sensation in outcome has been neglected. Historically, electrical paraesthesia was considered critical to the success of TENS and spinal cord stimulation. More recently the role of electrical paraesthesia in spinal cord stimulation has been revisited due to the development of spinal cord stimulators that do not generate electrical paraesthesia . Moreover, a variety of non-invasive electroanalgesic (TENS-like) techniques that do not generate sensations during stimulation are available on the market . The current consensus is that for conventional TENS, a strong comfortable TENS sensation is a criterion for adequacy of TENS. This raises challenges in blinding TENS interventions in RCTs.Blinding involves concealing the nature of interventions, including whether an intervention is a placebo. This is to reduce bias associated with expecting treatments to be beneficial and/or harmful from study participants and/or investigators and/or therapists. Double-blinding whereby participants and assessors are unaware of the nature of an intervention, is considered gold-standard in drug trials, although technique-based treatments that are administered by a practitioner may need to be triple blind, i.e., participant, practitioner and assessor. Leakage of blinding is reduced by isolating these individuals from each other, although this is particularly challenging especially between the practitioners administering and participants receiving treatments.‘Some types of electrical stimulating devices produce tingling sensations during treatment, and some do not, such as microcurrent therapy’;‘During the study you may or may not experience sensations from the stimulating device’; and‘During the study you may or may not receive a placebo intervention’.There is a longstanding debate about the fidelity of blinding participants and practitioners in studies of TENS. Active and placebo drugs can be made identical in appearance, with neither providing sensory cues. It is not possible to blind participants to TENS sensation, raising concern about trial participants guessing which intervention is the placebo . It is possible to create uncertainty about which intervention is ‘real’ using pre-study briefings. For example, by informing participants that:These types of statements meet ethical standards for informed consent because they are truthful whilst creating uncertainty about which intervention is a placebo.Transient sham TENS devices that deliver current for 45 s that then fades to zero milliamps  have been developed to reinforce blinding of participants, practitioners and assessors. Rakel et al.  found noThere is inconsistency in judgements of the risk of performance and assessor bias (blinding) between systematic reviews. Some reviewers assign high risk of performance bias to all RCTs on the premise that it is impossible to blind the sensory experience of active TENS, whereas others assign low risk of bias on the premise that sham TENS devices coupled with participant briefing information create sufficient uncertainty. The majority of reviewers assign unclear risk of performance and assessor bias because RCT reports provide insufficient operational detail about blinding.Critical appraisals of literature undertaken to develop safety guidelines suggest that TENS is a safe intervention and that adverse events are predominantly skin irritation and post TENS tenderness that are infrequent, mild in severity, and of minor consequence ,21. TherIt is clear from TENS is of no value in acute pain.” and “The use of TENS in chronic pain may well be justified but it has not been seen.” [There is a requirement for a randomised trial to address the issue. It will be difficult to design and organise, it will need to be multicentred in the UK and other European countries may need to be included, it will require large numbers of patients and simple outcome measures. Without it, a potentially valuable intervention may be underused, or a useless intervention may continue in use.” [In 1997, McQuay et al. published a Health Technology Assessment of outpatient services for pain control in the U.K. . McQuay n seen.”  p. 49. Min use.”  p. 49.Issues with quality, study size and lack of data meant we were unable to draw any conclusion on TENS-associated harms or side-effects or the effect of TENS on disability, health-related quality of life, use of pain-relieving medicines or people’s impression of how much TENS changed their condition.” [There have been hundreds of clinical trials and over 100 systematic reviews published on TENS since McQuay et al.’s statement. In 2019, Gibson et al.  publishedition.”  p. 3.A large multicentered randomized controlled trial that enables participants to tailor TENS treatment according to their needs, including skills to optimise response and troubleshoot issues. Ideally, data should be gathered in a ‘real-world setting’ providing an ecologically valid insight to the value or otherwise of TENS in clinical practice. Enriched enrolment with randomized withdrawal studies are ideally suited for such needs.A comprehensive meta-analysis that estimates the magnitude of during treatment effects irrespective of the type of pain, and includes an evaluation of precision, consistency and certainty of the effect size estimate based on study quality and risk of bias.To overcome the ‘efficacy-impasse’ there needs to be improvements in the quality of RCTs and a re-appraisal of approaches to systematic review. I offer two solutions:Greater investment in large multicentred RCTs for precise estimates of effect size, and for scrutiny of large data sets not published in scientific literature such as those residing with device manufacturers.Better control of biases in RCTs, especially bias associated with blinding interventions and isolating individuals with different roles from each other.Improved clarity and detail in trial reports to enable replicability of methodology and evaluation of reproducibility of findings, in line with the Template for Intervention Description and Replication (TIDieR) checklist , includiEvaluation of TENS in ecologically valid settings  using outcome measures meaningful to individual patients  with long-term follow-up outcome.In 2020, Travers et al.  recommenGladwell et al. argues that evaluations of TENS should capture what patients use TENS for rather than what practitioners or investigators prescribe, i.e., participants should not be passive recipients of treatment schedules ,115,116.Gladwell et al. argues that TENS is a complex intervention and that long-term users of TENS optimise benefits and minimise problems by learning, through trial and error, personalised treatment strategies appropriate for their personal needs ,115,116.RCTs need to be developed to enable participants to tailor TENS treatment according to their personal situation. Operational considerations have been incorporated into criteria published by Bennett et al.  to optimScreen volunteers for factors influencing treatment failureDeliver TENS training on how to use TENS safely and how to optimise treatmentInclude a ‘skills development’ period where participants practice using TENS, personalise the positioning of electrodes and the TENS settings, and refine a pattern of TENS usage through a systematic process of trial and error. This will develop skills to adapt treatment according to needDetermine, through a process of shared decision-making, outcomes that participants find meaningful and realistically achievableIdentify participants who have unresolvable adverse events, are uncompliant or decide that TENS is not appropriate treatment for their needsMany logistical challenges of tailoring and optimising TENS treatment within RCTs could be overcome by including a ‘run-in’ phase that could:Enriched enrolment with randomised withdrawal trials have been used to evaluate the efficacy of drug medication by identifying likely responders in a clinical population prior to randomisation into treatment arms for assessment of efficacy. Enriched enrolment with randomised withdrawal trials involve an observational ‘run-in’ phase (often of two weeks) to optimise dosage and assess adverse effects, and an RCT that evaluates efficacy using participants most likely to respond . There have been no attempts to deliver an enriched enrolment with randomised withdrawal trial of TENS for any painful condition.Phase one of an enriched enrolment with randomised withdrawal study on TENS would optimise TENS treatment and identify participants most likely to respond. Phase two would determine whether electrical currents are responsible for benefits or harms associated with treatment, as represented in Phase one utilises an open-label design with all participants receiving TENS treatment. Participants would be trained to use TENS and would learn how to self-administer treatment and tailor TENS technique according to personal need. Participants unable to tolerate adverse effects, are non-compliant with instructions for TENS technique, or opt not to use the treatment in the future are identified as ‘non-responders’ and withdrawn during or at the end of the phase. Only, potential treatment responders are permitted to enroll into the randomised controlled trial in phase two.Phase one would be used to develop skills to be able to optimise electrode positioning and electrical characteristics which may be awkward, inconvenient and time-consuming. Recent technological advances that may help to optimise treatment include; electrodes woven into clothing, smart TENS electrodes with algorithms for precise targeting of currents, interfacing of the TENS device with mobile technology, and data capture systems that can monitor TENS usage ,164,165.Phase two uses an enriched sample of ‘responders’ to assess whether effects observed in phase one were due to the active ingredient of TENS  rather than expectation or natural improvement of the condition. The enriched sample of participants would be free from unresolvable adverse events, and competent to self-administer and optimise TENS treatment. This should reduce treatment heterogeneity.The ability to conceal (blind) real and placebo interventions is a challenge because participants would have had prior exposure to TENS sensation in phase one. Strategies to overcome risk of performance bias were discussed previously.One criticism of enhancement enriched randomized withdrawal trial is that findings have limited external validity because populations arriving at clinics consist of non-responders. However, data gathered in phase one of the trial provides valuable ‘real-world’ data including, estimates of the incidence of responders, adverse events, compliance, and treatment satisfaction. This will provide a wholistic picture of efficacy and adverse events. The financial cost of delivering a large fully powered multicentred-placebo controlled enhancement enriched randomized withdrawal study would be high.Patients, clinicians, policy makers and funders urgently need an estimate of the magnitude of symptomatic relief of pain during treatment irrespective of the type of pain. This would answer the question ‘Does TENS sensation alleviate (soothe) pain in the moment?’ i.e., immediate short-term pain relief. An evaluation of precision, consistency and certainty of the answer needs to be based on study quality and risk of bias.In 2019, Johnson et al. published a protocol for a meta-analysis to calculate effect size estimates for both benefit (pain intensity) and for harm (adverse events) from RCT data versus placebo (Meta-TENS study) . The proThroughout the decades, calls for large multicentred RCTs have been unmet. The largest RCTs to date were delivered on modest funding  restricting the samples within trial arms to no more than approximately 100 participants , with placebo TENS (n = 99) and no treatment (n = 99) for women with fibromyalgia [, with plLimited evidence for TENS showed no clinically important difference compared with sham TENS and usual care across several outcomes at less than 3 months, and no longer term evidence was identified.” [NICE judged these findings to be insufficient evidence to support a recommendation to offer TENS for chronic primary pain, concluding that “tified.”  p. 29. THence the ‘efficacy-impasse’ is likely to remain for the foreseeable future, resulting in reduced availability of TENS within public health systems  and a reluctance of private healthcare insurance systems to cover treatment costs . Researchers place great credence on efficacy, whereas evidence-based health care practice also considers clinical experience, including patient values underpinned by physiological plausibility. Observational research spanning half a century demonstrates that some patients rely on TENS and use it for many years, and research from basic sciences supports physiological plausibility see . In addiNowadays, electrodes are available that are woven into garments and interfaced with smart phone technology for more precise targeting of currents without the need to reposition electrodes. TENS usage data can be uploaded to the Cloud and machine learning software used to create personalized TENS treatment schedules. Analysis of large data sets of usage and outcomes from real-world settings may be a relatively cost-effective means of gathering ecologically valid data from new patients to resolve doubts about the utility and potential efficacy of TENS. Therefore, it is unlikely that uncertainty about efficacy will discourage patients from purchasing their own TENS equipment or discourage practitioners from indicating TENS in the future .This comprehensive review appraises reasons for longstanding uncertainty about the efficacy of TENS that has persisted for over half a century. Inconsistent and imprecise effect size estimates found by RCTs result from the use of inadequate sample sizes, heterogeneous populations, muddled measurements of outcome, inappropriate TENS technique, dosage and regimen, and difficulties blinding placebo interventions. The review reveals tensions between using currents to create pleasant non-painful sensations for symptomatic relief of pain, and using specific combinations of electrical characteristics of currents to target pathology and mechanisms associated with different pain conditions. Enriched enrolment with randomised withdrawal studies and a comprehensive meta-analysis to determine whether strong nonpainful TENS alleviates (‘soothes’) pain ‘in the moment’ are offered to resolve uncertainty about efficacy.However, it may be necessary to concede that it is impossible to generate sufficient evidence about efficacy because operational challenges are insurmountable. This situation is not unique to TENS. There is longstanding uncertainty about analgesic efficacy for most nonpharmacological technique-based analgesic interventions including acupuncture, electrophysical agents  and a wide variety of manual therapies. Consequently, practice remains based on local policy and dogma. Perhaps clinicians, policy makers, funders and researchers need to reconsider the value of persisting with the production and publication of so many RCTs that fail to answer questions about efficacy.


Organoids well recapitulate organ-specific functions from their tissue of origin and remain fundamental aspects of organogenesis. Organoids are widely applied in biomedical research, drug discovery, and regenerative medicine. There are various cultivated organoid systems induced by adult stem cells and pluripotent stem cells, or directly derived from primary tissues. Researchers have drawn inspiration by combination of organoid technology and tissue engineering to produce organoids with more physiological relevance and suitable for translational medicine. This review describes the value of applying organoids for tumorigenesis modeling and tumor vaccination. We summarize the application of organoids in tumor precision medicine. Extant challenges that need to be conquered to make this technology be more feasible and precise are discussed. It hin vitro construction , used epigenetic Infinium MethylationEPIC BeadChip (EPIC), a microarray chip from Illumina that interrogates more than 850,000 CpG sites to analyze DNA methylation status of 25 human cancer organoids . This daKRAS, CDKN2A, TP53, and SMAD4 in normal pancreatic organoids eventually developed to a state recapitulating pancreatic ductal adenocarcinoma  in the presence of T cell-stimulating growth factors, antigen-specific cytotoxic T cells were selected and amplified in about half of total samples  secrete miscellaneous cytokines, chemokines, and growth factors to create a conducive microenvironment for tumor progression . The proThe deficiency of blood vessels in the organoid system brings challenges . One stuin vitro operation  is conducive to oncotherapy, which is combined with clinical treatment to form complementarity –124. NGSOrganoid culture along with NGS and single-cell sequencing (scRNA-seq) is good to hunt for therapeutic targets and discover mutation-associated neoantigens (MANAs) for fresh targeted remedy or tumor vaccination Figure 3in vitro from surgery tissues and added coronavirus disease 2019 (COVID-19) candidate vaccines into the culture system, a predominant tool to verify vaccine efficacy, to observe whether tonsil organoids incite immune responses, specific immunocytes, and antibodies against viral surface spike proteins  and the Shanghai Municipal Committee of Science and Technology (21140901600 to DX).ZM and YW served as lab technicians for Shanghai OneTar Biomedicine Co. Ltd.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Bushfires substantially increase the environmental health risks for people living in affected areas, especially the disadvantaged  and those with pre-existing health conditions. Pregnant women exposed to bushfire smoke are at a greater risk of adverse pregnancy and foetal outcomes, especially if they smoke tobacco, which may compound the toxic impacts. Bushfires may also exacerbate mental stress, leading to an increase in smoking. There are gaps in the evidence and more research is required on the combined effect of bushfire smoke and tobacco smoke on pregnant populations. Bushfires are a type of wildfire that occur in “the bush”, which is a collective term for forest, scrub, woodland, or grassland in Australia and New Zealand. Bushfire smoke, which contains noxious gases and particulate matter  and other toxic gases), are a substantial health risk and may trigger cardiovascular, respiratory, and inflammatory health events [ectively .Pregnant women may be especially susceptible to bushfire smoke due to physiological changes during pregnancy and likelihood of adverse effects on the foetus, especially if they smoke or live in a smoking household. Since tobacco smoke and bushfire smoke have similar constituents, we explored the risks related to concomitant exposures. The article aims to highlight the adverse health effects experienced by pregnant women living in bushfire-affected areas, especially the effects on their foetus and smoking behaviours, and comment on the research needed to minimise the previously mentioned adverse effects.3 increase in mean daily PM2.5 exposure in pregnancy can increase the likelihood of children developing wheeze by 56%  and cold or flu by 35%  in early childhood [Very few published studies have explored the direct effect of bushfire or wildfire smoke inhalation on pregnancy and birth outcomes. Due to the highly toxic nature of the exposures, evidence has relied on natural experiments and cohort studies, with or without a control or comparator group ,6. Pregnhildhood . While mhildhood . This inhildhood .Adverse birth outcomes  have been long associated with active and passive tobacco smoking among pregnant women. These adverse effects in pregnant women are very similar to the exposure to air pollutants from bushfire smoke ,9,12. SmBushfires are also associated with adverse mental health effects. A longitudinal survey of 2063 young adults found an association between the experience of traumatic bushfire-related incidents  and increased levels of tobacco smoking , as well as uptake of smoking (OR 1.11 95% CI 1.01–1.23) by non-smokers . As far Bushfire smoke may be problematic for a wide range of health research, especially smoking related trials that are evaluating smoking cessation or relapse prevention interventions. Exhaled CO measurement is a frequently used method for biochemically verifying smoking abstinence in smoking cessation clinical trials. However, the large bushfires in a vicinity may affect the validity and reliability of CO monitoring due to those enrolled in the study inhaling CO from the fires and as a result exhaling more CO than what can be attributed to their smoking alone. In such cases, if adequate guidance is not provided to on-ground research personnel and the participants, results of the study may overestimate smoking and incorrectly label non-smokers or ex-smokers as smokers.High quality evidence on the effects of bushfires on human pregnancy outcomes is lacking. We also do not know the best ways to mitigate these adverse effects. The 2019–2020 Australian bushfires provided an opportunity  to understand more about the impact of smoke pollution on the natural course of pregnancy for exposed women. Observational studies (cohorts or case-controls) would help investigate the impacts of bushfires on pregnancy outcomes and smoking behaviours. Additionally, qualitative research is required to understand the relationships between exposure to bushfires and their effect on women’s mental health and smoking behaviours.Although there is no fail-safe way to eliminate the health effects of bushfire smoke, changing individuals’ behaviours may reduce harm. Various health agencies recommend protective measures for pregnant women; however, the evidence base of many recommendations is uncertain as they are based on expert opinion. However, recommendations related to smoking cessation advice are derived from high quality evidence, generated through systematic reviews and meta-analyses of RCTs . As withWildfires or bushfires are becoming a global problem and Australian bushfires could help inform wildfire research in general. The 2019–2020 bushfire season presented Australia with unique challenges for its high-priority populations, especially those pregnant women who were concurrently exposed to bushfire and tobacco smoke. More research is needed to generate high quality evidence about the effects of bushfire smoke pollution in pregnancy, how these exposures can be mitigated, and build clinical guidelines for health practitioners with pregnant clients. The cohort of 2019–2020 pregnant women should be followed up to better understand the combined impacts of bushfires on the physical and mental health of pregnant women, especially those who are smokers or exposed to second-hand smoke.