Asthma is now recognized as a heterogeneous disease, encompassing different phenotypes driven by distinct pathophysiological mechanisms called endotypes. Common phenotypes of asthma, referred to as eosinophilic asthma, are characterized by the presence of eosinophilia. Eosinophils are usually considered invariant, terminally differentiated effector cells and have become a primary therapeutic target in severe eosinophilic asthma (SEA) and other eosinophil-associated diseases (EADs). Biological treatments that target eosinophils reveal an unexpectedly complex role of eosinophils in asthma, including in SEA, suggesting that “not all eosinophils are equal”. In this review, we address our current understanding of the role of eosinophils in asthma with regard to asthma phenotypes and endotypes. We further address the possibility that different SEA phenotypes may involve differences in eosinophil biology. We discuss how these differences could arise through eosinophil “endotyping”, viz. adaptations of eosinophil function imprinted during their development, or through tissue-induced plasticity, viz. local adaptations of eosinophil function through interaction with their lung tissue niches. In doing so, we also discuss opportunities, technical challenges, and open questions that, if addressed, might provide considerable benefits in guiding the choice of the most efficient precision therapies of SEA and, by extension, other EADs. Asthma is a highly prevalent chronic respiratory disease responsible for a considerable health burden worldwide . As geneA very important component of the latest GINA definition is the recognition of asthma as a heterogeneous disease . In spitEosinophils currently occupy the central stage in asthma management. The realization that asthma and especially its most difficult cases are highly heterogeneous in terms of phenotype and mechanisms of disease has drastically switched therapeutic approaches from a “one fits all” strategy toward precision medicine ,8,17. AsNonetheless, anti-eosinophil biological treatments mostly provide clear benefits specifically in SEA, but not in non-T2 asthma , indicatIn this review, we address our current understanding of the role of eosinophils in asthma. We further address the possibility that different SEA phenotypes may be driven by differences in eosinophil biology through eosinophil “endotyping” or plasticity. Finally, we discuss the technical challenges and open questions that, if addressed, could provide considerable benefits in guiding the choice of the most efficient precision therapies of SEA and, by extension, of other EADs.Eosinophils are a specialized type of granulocytes most often characterized in different species by their distinctive red or pink specific granule staining under the action of acidic dyes in bright field microscopy . EosinopNotwithstanding the protective or beneficial roles eosinophils may have played in our long ancestry—and are likely still playing today—eosinophils are mostly studied in the context of EADs . EADs haThe study of eosinophils has long suffered from prejudice due to their apparent proximity with neutrophils. Like neutrophils, they are classically viewed as terminally differentiated and short-lived once they exit the bone marrow. Eosinophils are therefore mostly envisioned as circulating “sentinels” that invade tissues following immune detection of pathogenic threats. This view is, however, to be mitigated, as significant populations of eosinophils are present in homeostatic conditions in various human organs, including the lungs, and eosinophils may be involved in tissue morphogenesis . In respTissue recruitment of eosinophils involves the activation of their surface integrins in response to chemotactic cues including eotaxins and lipid mediators, inducing their extravasation in target tissues . Once inIn line with their revised role as regulators of tissue morphogenesis and repair, several lines of evidence suggest that eosinophils actively contribute to the airway remodeling process in asthma. Airway remodeling refers to structural changes of the airway walls as a result of repeated injury and repair processes. In asthma, airway remodeling is characterized by increased airway smooth muscle cell mass, epithelial cell hyperplasia, goblet cell metaplasia, and thickening of the reticular basement membrane (RBM) by deposition of collagen, tenascin, and other matrix proteins, leading to a progressive loss of lung function . NotablyAltogether, the “built-in” activities of eosinophils have contributed to making them strong suspects in asthma pathophysiology, prompting the development of specific “anti-eosinophil” biologicals.While eosinophils have long been “usual suspects” in asthma, anti-eosinophil biologicals offer unique opportunities for directly establishing their level of involvement in disease manifestations. In this regard, clinical trials of anti-eosinophil biological therapies revealed that the role of eosinophils in asthma is more complex than anticipated . PuzzlinIt therefore appeared from early trials that eosinophils were not systematic direct drivers of BHR and airflow limitation, raising the question of their actual contribution, if any, to asthma symptoms. At about the same time, other trials established a strong correlation between eosinophilia and poor asthma control and exacerbation rates ,46,47. TIn spite of the progress allowed in SEA management, not all patients respond equally well to anti-eosinophil therapeutics. Further studies therefore aimed at determining which factors may influence the response to anti-eosinophil treatments . PerhapsAltogether, current clinical data suggest a generally stronger response to anti-eosinophil therapy in T2-high, possibly non-atopic, adult-onset SEA with degraded lung function. Part of the explanation to the above is probably that patients with adult-onset SEA or with FAO start “worse off” or have more room for improvement, as they tend to experience more severe exacerbations and more degraded lung function before the initiation of anti-eosinophil therapy . NonetheIt is to be expected that a significant part of the benefit of anti-eosinophil therapeutics simply arises from the decrease in (or full depletion of) circulating eosinophils, dampening their subsequent accumulation in the airways. Nonetheless, eosinophilia alone does not seem sufficient to fully explain how eosinophils cause more frequent, more severe exacerbations and negatively affect lung function in specific SEA phenotypes. An intriguing observation of early randomized clinical trials with mepolizumab is that similar reductions in exacerbations are obtained with a range of mepolizumab doses, even though a significant population of residual sputum eosinophils persists in patients receiving lower doses of therapy . In addiThis raises a series of interconnected questions. First, what are the particular processes in SEA patients driving eosinophil recruitment and activation of their detrimental activities? Second, what are the eosinophil activities most relevant to their pathogenicity in SEA? Third, are these differences in eosinophil pathogenicity induced at the level of the lung tissue through local “plasticity” of eosinophils, or do they rather  arise systemically within eosinophils themselves through what we refer to below as eosinophil “endotyping” ? AnsweriHere, we introduce the concept of eosinophil “endotypes”, a term we derive from the adaptation of the asthma endotype concept. Like how endotypes of asthma refer to built-in differences in pathophysiological mechanisms of disease, eosinophil endotypes would refer to specific functional programs imparted to eosinophils (or subsets thereof) as part of their developmental process in specific contexts. In this model, (subsets of) eosinophils with specific activities different from those of “classical” (viz. steady state) eosinophils would arise as a consequence of pathology-associated signals perceived by the developing progenitors of eosinophils . Differe+ Ly6G+ murine myeloid progenitors that normally give rise to neutrophils may express eosinophil genes in response to IL-5 ex vivo [Eosinophil endotyping would occur during eosinophil development from their hematopoietic progenitors . Classic ex vivo . While t+ CD101lo “regulatory” and Siglec-Fhi CD101+ “inflammatory” eosinophils is not specific to asthma. In transgenic mice overexpressing IL-5, Siglec-Fhi and Siglec-F+ eosinophil subsets with distinct gene expression profiles were detected in the bone marrow and blood [Clues of eosinophil endotyping in asthma have been reported in both humans and mice. In mice, only one subset of eosinophils is commonly detected in the blood and lungs in homeostasis. However, following the development of airway allergy to house dust mite antigens, two subsets of eosinophils become detectable in the blood and lungs of mice . One of nd blood . “Reguland blood , suggestnd blood ,97,98.The above observations in mice raise the question of the mechanisms leading to endotyping of inflammatory eosinophils during eosinophilopoiesis. IL-5 is expected to be an essential player in the process. Indeed, neutralization of IL-5 potently antagonizes inflammatory eosinophil development in mice . In addiEvidence of eosinophil endotyping in humans is scarcer yet much more ancient. Studies in the 1980s reported the presence of hypodense eosinophils in asthmatic patients. Hypodense eosinophils segregate with mononuclear cells in density gradients, away from “classical” normodense eosinophils. Increased numbers of hypodense eosinophils are present in the blood, bronchoalveolar lavage, and lung tissue of SEA patients . Even thAs a cautionary note, we need to stress that the reciprocal translation of murine and human observations of eosinophil endotyping may not be entirely straightforward in our current state of knowledge . We acknIn this work, we compared the gene expression profiles of circulating blood eosinophils in healthy patients and allergic SEA patients receiving either mepolizumab (anti-IL-5) or omalizumab (anti-IgE) therapy. Mepolizumab treatment in SEA patients consistently leaves a residual population of eosinophils, representing approximately half the normal eosinophil blood counts in the general population . We aimeWe must also consider a second common blind spot: only normodense eosinophils are usually recovered as part of human eosinophil purification procedures. Because the patients in our study had only mild eosinophilia and were well-controlled, we do not expect them to have harbored prominent populations of hypodense eosinophils. Yet, we argue that hypodense eosinophils may be important in our understanding of the pathogenic roles of eosinophils in SEA and EADs.Third, our study only included allergic SEA patients for matching purposes with omalizumab-treated control patients. Yet, we speculated that asthma endotypes could also influence eosinophil endotyping. We expected that different SEA phenotypes could result in different signals being sent from the diseased lung to developing eosinophil progenitors, influencing their downstream pathogenic potential in tissues. As noted above, eosinophils appear to be particularly detrimental in adult-onset, possibly non-allergic, highly remodeling SEA. We therefore advocate that future studies should assess and compare the heterogeneity of blood (and possibly bone marrow) eosinophils of asthma patients with different phenotypes of SEA.Finally, and most importantly, molecular analyses on eosinophils are still essentially performed on bulk eosinophils. Eosinophils are notably difficult cells to work with using current gold-standard molecular single-cell profiling techniques such as single-cell RNA sequencing. Yet, endotypes that would develop as subsets of total eosinophils (such as hypodense eosinophils) could easily go unnoticed using bulk analyses. Advanced molecular and phenotyping techniques such as scATAC-seq (a transposase-based accessibility assay) or single-cell DNASE1 hypersensitivity assays could provide alternative solutions if they prove applicable to eosinophils ,107.hi eosinophils derive from Siglec-F+ eosinophils in airway allergies through plasticity [hi eosinophils seem to readily develop in the bone marrow and circulate in allergic mice and in other models of eosinophilia [+ eosinophils may acquire a Siglec-Fhi phenotype through local plasticity in the lungs or if endotyping is solely responsible for the existence of the two murine eosinophil subsets. With this cautionary note given, here, we will consider a complementary source of human eosinophil diversity: local plasticity (Eosinophil “endotypes” remain little-studied but need to be considered a possible explanatory factor with regard to subsequent eosinophil phenotypes in tissues. For instance, it has been proposed that lung Siglec-Fasticity . Howevernophilia ,107. Theasticity . Taking The long-held view of eosinophils as invariant cells is already being extensively revised with the realization, initiated in mouse models, that eosinophils can be more diverse than initially anticipated ,107,109.Eosinophils are relatively easy to identify in mouse and human tissue using histological staining or flow cytometry, but assessing their relative adaptations to different tissue compartments or niches remains a challenge. As we discussed above, density and candidate cell surface marker approaches have revealed different eosinophil phenotypes. Yet, it is likely these different phenotypes themselves encompass eosinophils with different local adaptations. For instance, eosinophils have strong tropism toward lung nerve endings and can modulate nerve function and influence lung innervation during development and in asthma ,112,113.We expect that relating eosinophil plasticity to its pathological roles in SEA and EADs will require paying attention to some of the same “blind spots” discussed for eosinophil endotypes above, mainly timing and asthma phenotypes. Given the apparently more detrimental role of eosinophils in highly remodeling adult-onset SEA, understanding how eosinophils respond to the particular bronchial microenvironment during exacerbations in this asthma phenotype should also be highly prioritized. Toward this goal, and to generally decipher the role of eosinophils in EADs, we expect that understanding local plastic adaptations of eosinophils will require the power of novel techniques such as spatial transcriptomics or spatial epigenomics. In addition, special attention should be dedicated to different modes of eosinophil activation and cytolysis, which could also be influenced by endotyping and plasticity and be critical determinants of eosinophil function in SEA.Eosinophils should no longer be considered short-lived, invariant, terminally programmed effector cells in asthma or EADs. As suggested by preclinical models, through observations in severe asthma and by the use of anti-eosinophil biologicals in SEA treatment, eosinophils are certainly more labile and adaptable than is envisioned in classical paradigms. Irrespective of the origin of the differences in the involvement of eosinophils in disease manifestations, be it endotyping or plasticity, we advocate that in-depth scrutiny of systemic and local eosinophil responses will provide insights critical for the understanding and management of eosinophilic asthma phenotypes and, by extension, of other EADs.


Parent-child similarities and discrepancies at multiple levels provide a window to understand the cultural transmission process. Although prior research has examined parent-child similarities at the belief, behavioral, and physiological levels across cultures, little is known about parent-child similarities at the neural level. The current review introduces an interdisciplinary computational cultural neuroscience approach, which utilizes computational methods to understand neural and psychological processes being involved during parent-child interactions at intra- and inter-personal level. This review provides three examples, including the application of intersubject representational similarity analysis to analyze naturalistic neuroimaging data, the usage of computer vision to capture non-verbal social signals during parent-child interactions, and unraveling the psychological complexities involved during real-time parent-child interactions based on their simultaneous recorded brain response patterns. We hope that this computational cultural neuroscience approach can provide researchers an alternative way to examine parent-child similarities and discrepancies across different cultural contexts and gain a better understanding of cultural transmission processes. Cultural values, beliefs, and practices are transmitted across generations . ParentsPast research has examined parent-child similarities and discrepancies in beliefs, behavior, and physiological adjustment across diverse cultural contexts. Guided by the value transmission model , parent-Decades of research in developmental psychology has studied parent-child similarity or synchrony at the behavioral level , a commonly used method in neuroimaging, has enabled researchers to explore shared responses across different brains . For exaIntersubject representational similarity analysis (IS-RSA), a newly developed computational method in neuroimaging, is implemented to search the mapping between individual variations in experiences during naturalistic viewing and variations in preferences or other differences . For exaSocial communications between parents and children not only rely on verbal content, but also depend on non-verbal behavior because some social signals contain cultural-specific meanings and are only expressed through non-verbal communications . Thus, aTo understand the cultural differences in parent-child similarities, researchers could ask whether specific facial behavioral patterns may be shown during parent-child interaction in some cultural groups, and whether these behavioral patterns signal any particular psychological meaning. For example, previous cross-cultural facial expression research found that, although some expressions  were similarly represented, some expressions  were distinctly represented across different cultures, suggesting that the communication purpose of expressions might differ across cultures . This reAlthough analyzing non-verbal behaviors during dyadic interaction could help researchers to examine what kinds of social signals might occur during interaction, it is still difficult to disentangle what kinds of psychological processes might occur within each individual during their interactions. Thus, in this section, we suggest that researchers could take a hybrid approach to decode the complexity of psychological processes during dyadic interactions by collecting dyadic brain data with hyperscanning techniques, and analyzing these data with brain signatures established via computational methods . By takiHyperscanning aims to measure brain responses for the two individuals simultaneously during their social interactions. Hyperscanning has been used to examine diverse research topics, including dyadic trust decision-making , social Since multiple psychological processes might be simultaneously involved during dyadic interactions, brain signatures established via computational methods are particularly useful to decode what psychological processes might occur during parent-child interaction . In geneThe above approach has been successfully used in predicting not only basic sensations, such as pain intensity  or sustaComputational cultural neuroscience approach provides powerful tools that can make important and unique contributions to the field. First, this approach can inform the understanding of cultural transmission, providing insights into how cultural values and practices are passed on across generations not only at belief and behavioral level, but also at the neural level. For example, researchers can examine cultural similarities and differences in neural synchrony within parent-child dyads when they engage in interactions or communications. As parenting practices or behaviors are shaped, in part, by their culture, computational cultural neuroscience provides techniques to capture how children’s brains are wired to their parents in the dynamic process of cultural learning.Second, in addition to examining between-culture variability in parent-child dyads at the neural level, this approach allows researchers to explore individual differences across families within the same culture, which can elucidate the cultural transmission process at the individual level. Within each culture, there is likely to be variability in the extent to which parents adopt cultural values and norms, thereby creating variability in parent-child interactions and ultimately children’s development. Therefore, parent-child neural similarities may represent meaningful differences across families within the same culture, and it is important to examine what factors contribute to such differences. For example, using representational similarity analysis, recent research suggests that on average, American mothers and their children do not show similar neural patterns during stress . HoweverThird, this approach will also shed light on the links between beliefs, brain, and behavior. For example, it is informative to investigate how belief similarities in parent-child dyads are related to brain similarities, and how brain similarities in parent-child dyads are linked to behavioral similarities. More importantly, future research can explore the meaning and developmental consequences when parent-child similarity at one level does not match with the similarity at another level. This line of research will not only extend prior research that links beliefs, brain, and behavior at the intrapersonal level , but also explore the complex relations among beliefs, brain, and behavior at the interpersonal level .Although this computational cultural neuroscience approach provides a novel perspective to examine parent-child similarities in diverse cultural contexts, limitations of this approach should be noted. First, although using pre-trained brain signatures to analyze hyperscanning data can help researchers to examine what psychological processes might occur within interacting individuals, it is still possible that some important psychological processes are not covered by pre-trained brain signatures. Thus, more future studies are needed to establish brain signatures representing most relevant psychological processes during social interactions. Second, most of the methods described in this review are designed for cross-sectional studies, but meaningful cultural changes in attitudes or behavior can provide important information in longitudinal studies. For instance, a previous longitudinal study showed that even in the same cohort of immigrants, changes in self-construal during the process of acculturation were reflected in the relative engagement of brain areas implicated in self-referential processing . Thus, fTaken together, this integrative review highlights the importance of understanding parent-child similarities and discrepancies at the neural level across cultures. We also introduce several cutting-edge computational methods to explore intersubject neural similarities, decode social signals during communications, and unravel the complexities of real-time parent-child interactions based on their simultaneous recorded brain response patterns. This computational cultural neuroscience approach aims to promote the use of computational methods to get a deeper understanding of cultural transmission processes, and hope to provide theoretical accounts and innovative tools for this promising line of research.Both authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.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.


Approximately 10% of all COVID patients develop long COVID symptoms, which may persist from 1 month up to longer than 1 year. Long COVID may affect any organ/system and manifest in a broad range of symptoms such as shortness of breath, post-exercise malaise, cognitive decline, chronic fatigue, gastrointestinal disorders, musculoskeletal pain and deterioration of mental health. In this context, health institutions struggle with resources to keep up with the prolonged rehabilitation for the increasing number of individuals affected by long COVID. Tai Chi is a multicomponent rehabilitation approach comprising correct breathing technique, balance and neuromuscular training as well as stress- and emotional management. In addition, practicing Tai Chi elicits the relaxation response and balances the autonomic nervous system thus regulating respiration, heart rate, blood pressure and vitality in general. Moreover, Tai Chi has been shown to increase lung capacity, improve cognitive status and mental health, and thereby even the quality of life in diseases such as chronic obstructive pulmonary disease (COPD). Hence, we advocate Tai Chi as potent and suitable rehabilitation tool for post-COVID-19-affected individuals. Tai Chi is a multicomponent exercise that promotes self-efficacy and empowers affected individuals to actively contribute to their own disease management.Tai Chi has been shown to improve life quality and clinical parameters of chronic obstructive pulmonary disease (COPD) and other complex diseases.Tai Chi builds strength, improves mobility and balance, and elicits activation of the relaxation response.Tai Chi is suited for rehabilitation after COVID-19 and treatment of long COVID symptoms.Tai Chi potentially improves lung function by counteracting the fibrotic scar formation and may decrease chronic fatigue syndrome by balancing the autonomous nervous system and thereby the risk for development of mental disorders.Tai Chi may reduce the economic burden associated with COVID-19 rehabilitation.We recommend to implement Tai Chi for acute COVID-19 rehabilitation directly after discharge from the hospital and for long COVID.The Tai Chi rehabilitation program should last for a minimum of 3 months with twice training for 1 h per week.Training by oneself in between the weekly classes should be encouraged.Physical inactivity increases the risk for modern society diseases such as diabetes and cardiovascular diseases, and likely even susceptibility for infectious diseases such as COVID-19. Recent data have shown that the risk for hospitalization and death due to COVID-19 increases more than 2-fold in physically inactive persons compared to persons performing at least 150 min/week moderate to vigorous physical activity  . MoreoveA severe COVID-19 disease course can lead to fibrotic changes in the lung  that couRecently it has been shown that relaxation response training upregulates genes associated with energy metabolism and mitochondrial function while downregulating genes linked to inflammatory response and stress-related pathways . Such boThe inflammatory response during COVID-19 is tightly connected with the oxidative stress response leading to accumulation of reactive oxygen species (ROS) and reactive nitrogen species (RNS), by inducing mitochondrial dysfunction and secretion of proinflammatory cytokines. Moreover, ROS also activate transforming growth factor b (TGFb), a key factor for developing lung fibrosis.2max) decreases the ROS load and DNA damage, respectively, and stimulates key antioxidant enzymes (While occasional high-intensity physical activity has been shown to promote oxidative stress, regular exercise with moderate intensity (40–59% VO enzymes . The samOne of the most debilitating long COVID signs is fatigue, which is often occurring in attacks of severe physical and mental tiredness that might result in mental health deterioration such as development of anxiety or depression . It has In addition, a recent study has shown that insomnia, anxiety and depression are very prevalent among the general population during the COVID-19 pandemics with twice as high prevalence as compared to non-pandemic periods and even higher among patients with COVID-19 . These oInitiation of Tai Chi practice soon after recovering from acute COVID-19 could potentially decrease the risk for developing long-term COVID. Tai Chi improves both blood and energy flow, activates the relaxation response and thus balances the autonomic nervous system  and can Tai Chi potentially improves lung function by counteracting the fibrotic scar formation and decreasing long-term COVID fatigue and thereby the risk for development of mental disorders. Moreover, the practice is at the same time likely to increase muscular strength, mobility and vitality, which should in turn empower the individuals affected by COVID-19 to actively contribute to their recovery. Hence, implementation of Tai Chi in rehabilitation of COVID-19-affected individuals, for both short- and long-term disease courses, can be highly recommended.MZ and JC designed the manuscript. MZ wrote the manuscript together with JC and MK. All authors contributed to the article and approved the submitted version.JC is holding rehabilitation classes at Fundación Neumológica Colombiana for COPD and post COVID patients. In addition, he has his own Tai Chi classes. 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.


The nutritional hallmark of chronic critical illness (CCI) after sepsis is persistent inflammation, immunosuppression, and catabolism syndrome (PICS), which results in global resistance to the anabolic effect of nutritional supplements. This ultimately leaves these patients in a downward phenotypic spiral characterized by cachexia with profound weakness, decreased capacity for rehabilitation, and immunosuppression with the propensity for sepsis recidivism. The persistent catabolism is driven by a pathologic low-grade inflammation with the inability to return to homeostasis and by ongoing increased energy expenditure. Better critical care support systems and advances in technology have led to increased intensive care unit (ICU) survival, but CCI due to PICS with poor long-term outcomes has emerged as a frequent phenotype among ICU sepsis survivors. Unfortunately, therapies to mitigate or reverse PICS-CCI are limited, and recent evidence supports that these patients fail to respond to early ICU evidence-based nutrition protocols. A lack of randomized controlled trials has limited strong recommendations for nutrition adjuncts in these patients. However, based on experience in other conditions characterized by a similar phenotype, immunonutrients aimed at counteracting inflammation, immunosuppression, and catabolism may be important for improving outcomes in PICS-CCI patients. This manuscript intends to review several immunonutrients as adjunctive therapies in treating PICS-CCI. Advances in intensive care unit (ICU) technology, support systems, care bundles, and evidence-based protocols have improved ICU survival by largely preventing early deaths . Girard In 2020, Rosenthal et al., published an analysis of CCI patients from the UF SCIRC sepsis database in which it was found that, despite receiving adequate macronutrients starting early in the ICU, CCI patients did not respond the way RAP patients did. Rather, they experienced a persistent acute phase response  and failed to become anabolic . These dOver the past few decades, guideline recommendations for ICU protein supplementation have increased from 0.8 g/kg/day up to as high as 2 g/kg/day  in an attempt to offset catabolism and anabolic resistance . The empAdding to the rationale for higher protein supplementation in PICS-CCI was another recent publication from a Protein Summit that recommended protein prescription in the range of 1.2–2.5 g/kg/day in the ICU setting to optimize nutrition, to preserve muscle mass, and to decrease mortality . FinallyArginine is a conditional amino acid with a wide range of bioactive impact but under states of physiologic stress and ongoing inflammation becomes depleted. Barbul and colleagues were early champions of arginine metabolism and reported its importance in wound healing and as an immunologic regulator ,50,51,52Branched-chain amino acids  are another group of amino acids that, when supplemented, have demonstrated decreased muscle catabolism and hypertrophic muscle growth through increased protein synthesis . Frank CGlutamine supplementation may prove to be important as its pluripotent ability to serve as an antioxidant, in gluconeogenesis, and to enhance immune function is lost during times of great stress. Skeletal muscle generates the majority of endogenous glutamine, but during major catabolic insults, demand for glutamine becomes far greater than supply. This phenomenon results in glutamine becoming a conditional amino acid. Glutamine serves as the primary oxidative fuel for rapidly dividing tissues, such as small bowel mucosa, proliferating lymphocytes, and macrophages . GlutamiLipids remain a crucial macronutrient and mainstay nutritional therapy for the critically ill and surgical populations, but controversy still exists regarding the digestion, absorption, and oxidation of lipids in hyperdynamic states . While uAn advanced understanding of the bioactive nature of omega 3 FAs (EPA/DHA) has shown us that there is an even greater endogenous byproduct of lipid metabolism called Specialized Pro-Resolving Mediator (SPMs) . These hICU care, as advanced as it may seem, has limitations, which has generated research interests for a better understanding of what a healthy, diverse microbiome can provide a host during critical illness. Since 1907, when the Nobel Laureate Elie Metchnikoff first described the concept of probiotics, the overarching opinion has been that probiotics are safe and have a role in treating gastrointestinal diseases, but the use has not been supported by the literature . InsightSupplementing the critically ill and post-surgical patients with both pre- and probiotics has proven beneficial in many settings, but evidence lacks as to which specific species are superior to improve the dysbiosis and can they be beneficial to PICS-CCI? Despite the amounting research that has shown benefit, supplementing probiotics is still not considered the standard of care in all ICU settings. Nevertheless, the idea is that ‘bioecological control’ has blossomed in critically illness and polytrauma patients: to supply viable beneficial bacteria or a substrate that enhances these specific beneficial bacteria instead of trying to eliminate the pathogen ,109. CouAnton et al. performed a meta-analysis to identify nutritional and pharmacologic interventions targeting chronic low-grade inflammation in older adults. In this analysis, he identified probiotics as having the largest impact on decreasing the serum biomarkers of inflammation (IL-6 and CRP). Angiotensin II receptor blockers and omega 3 fatty acids also significantly reduced these biomarkers but to a lesser extent compared to probiotics . Other cAnabolic and anti-catabolic agents may be beneficial in mitigating persistent muscle breakdown, a pillar of PICS-CCI. Herndon and colleagues described numerous adjuncts to mitigate catabolism in the pediatric burn population that may be of value in the PICS-CCI population, including (a) growth hormone , (b) intPICS-CCI is the high cost of ICU survival and the new, dominant chronic MOF phenotype in ICUs and LTACs. The underlying pathobiology contributing to this increasingly common CCI phenotype are being unraveled. We now know that evidence-based, gold standard nutritional protocols fail to have much of an effect on the PICS-CCI phenotype as this patient population continues to exhibit a cachexia-like response to our nutritional strategies. Commencing therapeutic strategies early in critical illness may mitigate progression into CCI. When PICS-CCI is established, combatting the pillars will likely require multiple modalities. Nutritional strategies such as the use of various immunonutrients are forthcoming and are one modality to hopefully alter the clinical trajectory of PICS-CCI. Among these strategies, the ones that hold the highest potential are protein concentration ≈2 g/kg/d, specific immuno-amino acids, omega 3 fatty acids, specialized pro-resolving mediators, probiotics, and anabolic agents.


Bone, as a physiological and anatomical construct, displays remarkable intrinsic healing capacity. The overwhelming majority of fractures will heal satisfactorily, if aligned anatomically, compressed and immobilised appropriately. Of the 10% of fractures that do not heal, even under ideal mechanical and biological conditions, further consideration must be given to augment bone healing. Management strategies for non-union pose a significant clinical challenge to the practicing orthopaedic surgeon. Stem cell therapy is beginning to demonstrate significant potential for augmented bone repair in the context of non-union. This review attempts to contextualise the function of stem cells within this clinical setting, reviewing the relevant cellular mechanisms and clinical applications. From evaluating the literature base, there is a lack of high-quality evidence examining the role of mesenchymal stem cells (MSCs) within this research focus. Appropriately designed randomised controlled trials are required to evaluate this research area further, with a view to guiding future treatment options for the practicing orthopaedic surgeon. Bone, as a physiological and anatomical construct, displays remarkable intrinsic healing capacity . The oveStem cell therapy is beginning to demonstrate significant potential for translational clinical application, across a variety of surgical and medical specialties. Both in vitro and in vivo experimental models have demonstrated promising results; positive therapeutic effects relating to augmented bone repair in the context of disease have been produced , 3.This review aims to explain what a stem cell is, relating this to their physiological function and highlighting the variety of stem cells present, whilst discussing methods of stem cell isolation for use in bone repair models. This review will also aim to evaluate the fundamentals of bone repair at the cellular level, focusing on the context of fracture non-union. An insight into future stem cell application in bone repair and disease will also be included, with limitations of the current evidence base also explored. We also hope this paper serves as a useful primer and aide-memoire for the practicing orthopaedic surgeon on the evolving concepts of bone healing and stem cell therapy in the context of non-union.MethodologyA focused literature search was performed using the Pubmed/MEDLINE index. Inclusion criteria included the use of human studies, papers written in the English language and full papers. Published abstracts, wholly non-human studies and non-English language papers were excluded. All relevant literature was screened for inclusion, with relevant papers proceeding to further full screen evaluation.Stem cell biologyTo evaluate the advances in stem cell therapy relating to bone repair and disease, the fundamentals of stem cells must be explained to provide clinical context. 'Stemness' is a term used to describe a cell with stem-cell qualities compared to a non-stem cell . A succiConcerning stem cell potency, a 'true' stem cell is multipotent, differentiating into more than one cell type. In contrast, a progenitor cell derives from a multipotent stem cell but is unipotent, dividing into a single progeny . DependeSelf-renewal describes the mode of proliferation specific to stem cells. For a cell to be self-renewing, it must produce a daughter cell with the same replicative potential of the mother cell. An example of this type of cell lineage are neural stem cells, where both daughter and mother cells are multipotent .Types of stem cellStem cells can be broadly divided into two categories: those of embryonic origin and those of non-embryonic origin. Human embryonic stem cells (ESCs) are considered the ‘gold standard’ for developing understanding of stem cell pluripotency . ESCs arThe focus of this review concerns adult stem cells (ASCs). An example of these are haematopoietic stem cells (HSCs), which have the capacity to derive all types of mature blood cell . There aOne stem cell population that derives the aforementioned cell types are mesenchymal stem cells (MSCs), cells found primarily in adult bone marrow . MSCs arConcerning clinical musculoskeletal application, the scope of use is far-reaching , 13, 21.Mesenchymal stem cells are introduced into the clinical site either by direct injection into the clinical site, or along bioengineered scaffolds which seek to promote proliferation, MSC survival and are considered more osteoconductive surfaces in comparison to the host surface .Principles of normal bone repair and maintenance: fracture healingConsolidation of a fracture site occurs through a combination or primary (direct) bone healing, secondary (indirect) bone healing, or a combination of both mechanisms. This can be augmented by micro motion at the fracture site, however an overload of motion can lead to non-union of the fracture . A widelDirect bone healing  occurs when fracture fragments are compressed and are directly opposed. Compression is produced by surgical implantation, for example dynamic compression plating, or by immobilisation (for example splinting or cast immobilisation). Implants directly compress the fracture fragment, which on mechanical loading stimulates bone formation allowing the joint to bear load as time progresses. Initially the implant bears mechanical load, but as healing progresses and more bone is laid down, load bearing is transferred gradually to newly formed bone. Osteoclasts and osteoblasts produce bone by forming constructs known as ‘cutting cones’, whereby osteoclasts resorb bone and osteoblasts deposit bone following this. Only bone present at the implant-bone interface is restored with this method. Repair needs to occur outside of the interface to ensure mechanical integrity of the construct, thus a process known as gap healing occurs, whereby pockets of fractured bone not at the interface are repaired by periosteal osteoblastic activity. Approximately 12 weeks after implantation the newly formed bone acquires the tensile strength of normal bone . If the Within the quoted literature, fracture healing is dependent principally upon five conditions at the fracture site. These include there being a sufficient blood supply, a fracture-haematoma, a population of cells diverse enough to enhance healing and provide nutrition to the site, a constant mechanical environment, and an osteoinductive scaffold for repair to occur on -3, 26.Fracture non-union: definition and aetiologyFor the purposes of the practicing orthopaedic surgeon, a non-union is a fracture that will not heal without further intervention . The cauDistinguishing between different types of non-union is important for clinical practice and guides surgical management. The classification of morphological type of non-union allows the clinician to target treatment appropriately. Non-union can be classically divided into hypertrophic union, whereby the fracture ends are viable and capable of mounting biologic reaction, and atrophic, whereby fracture ends are inert and incapable of mounting a biologic reaction . The accManagement of non-union is dependent on complex decision making relating to surgical, implant and patient factors. Broadly speaking, hypertrophic non-union is treated by ensuring adequacy of mechanical stability at the fracture site. As the fracture site is considered biologically active, callus formation will be considered sufficient without external modification , 36. AtrPreparation and conditioning of MSCsThe successful differentiation of MSCs to osteoblasts is the basis of potential treatment options in non-union; autologous stem cells could serve to provide a native osteoblast population for the recipient, stimulating bone growth at the fracture site.The preparation and conditioning of MSCs is fundamental in facilitating differentiation into osteoblasts. Historically, the vital factors governing successful differentiation were considered scaffold composition, the surface scaffold topography and the interaction between the stem cell and the scaffold.Two approaches to conditioning stem cells specifically down an osteoblastic lineage have been demonstrated in the literature: the ‘top-down’ and ‘bottoms-up’ approaches . The firAs discussed by Kim and Mikos, the use of the ‘top-down’ approach invariably incorporates the use of external factors such as BMP-2 at supra-physiological levels, leading to possible complications reported as over-activation of osteoclasts, heterotopic ossification and autoimmune dysregulation . To bypaA further growth-area focuses on the isolation of specific MSC sub-populations . MSCs caClinical applicationsThe first reported clinical use of stem cell engineering in fracture repair was carried out by Vacanti et al. in 2001 . A patieClinical studies have highlighted that patients injected with bone marrow of which the MSC concentration was less than 1000 MSCs/ml did not experience augmented bone growth, whereas patients injected with bone marrow aspirate of which the MSC concentration was around 1500 MSCs/ml experienced rapid healing , 41. TheMultiple innovative surgical strategies have been developed for bridging bone defects, such as vascularised bone grafting, employing the Ilizarov technique and the Masquelet technique . One sucAnother potential therapeutic option involving a different population of human stem cell has also been explored clinically. The use of human multipotent adipose-derived stem cells (hMADSCs) could potentially counteract some of the problems associated with isolation of MSCs. Obtaining hMADSCs from adipose tissue is non-invasive, and adipose tissue is generally abundant in-patient populations. Adipose-derived stem cells are also considered to have a high degree of cellular plasticity; meaning they can differentiate into a variety of cell populations . One stuPercutaneous autologous bone-marrow grafting has now been used in several studies with success, illustrating that MSCs present in extracted bone marrow are playing a useful therapeutic role in aiding the repair of a fracture. A correlation has also been uncovered relating to the efficacy of the treatment and the number of osteoprogenitor cells present in the graft, merely underlining the importance of the initial stem cells in the treatment process . FurtherStrengths and limitationsSignificant clinical limitations exist relating to the long-term efficacy of MSCs in the treatment of non-union. Whilst a short-term positive clinical efficacy has been demonstrated by recent work, long-term 5-10 year follow-up of patients has not been evaluated within the current literature base , 31, 38.Further, undertaking MSC research in this context appears to require translational academic expertise in musculoskeletal tissue engineering and orthopaedic surgery. Barriers to access are reflected in the paucity of robust translational literature, with most research produced in tertiary academic centres currently. Further to this end, there appears to be distinct heterogeneity relating to method of obtaining stem cells, isolation, and subsequent introduction into the defect. Such heterogeneity makes comparison between methods challenging.Critically, there does not appear to be an internationally agreed definition for ‘non-union’, either defined through radiological or clinical parameters. Thus, production of robust meta-analyses and quantitative analysis of stem cell use within this setting would appear unfeasible, given seemingly equivalent studies would have inherent methodological heterogeneity. Heterogeneity in the assessment of fracture consolidation makes discerning whether MSCs are clinically beneficial difficult, with direct comparison between study outcomes poor due to this lack of uniformity in outcome measurement. Outcome measurement should focus on not just radiological outcome, but patient reported outcomes, combining holistic measurement of pain and functional status .Further, prospective cohort analysis appears to be the prevailing study methodology, with no control subjects appearing within the scope of any such trial of MSC application in non-union. Without control subjects, examining the true effect of MSCs is challenging both from a clinical and methodological standpoint.This review references and appraises the key literature relating to stem cell usage in the non-union setting, contextualising the key research advances and areas of research focus. We have also summarised the salient clinical and basic science literature relating to bone repair at the cellular level. In summary, the role of MSCs in the treatment of non-union does not appear to be fully understood. Understanding surrounding isolation, culture, and implantation of stem cells in the context of non-union is based on principally heterogenous case studies, lacking randomisation and homogenous research techniques. There is a lack of high-quality evidence examining the role of MSCs within this research focus. Appropriately designed randomised controlled trials are required to evaluate this research area further, with a view to guiding future treatment options for the practicing orthopaedic surgeon.


A cornerstone of HIV prevention clinical trials is providing a combination prevention package to all trial participants. The elements included in that standard of care (SoC) package evolve as new prevention modalities are developed. Pre-exposure prophylaxis (PrEP) was recommended by the World Health Organization for persons at high risk of acquiring HIV, but not all countries immediately adopted those recommendations. The South African Medical Research Council (SAMRC) convened a summit to discuss issues relating to SoC and PrEP in HIV prevention clinical trials taking place in lower- to middle-income countries (LMIC). Policymakers, regulators, ethicists, experts in law, researchers, representatives of advocacy groups, and the HIV Vaccine Trials Network (HVTN) presented a framework within which SoC principles could be articulated. A group of subject matter experts presented on the regulatory, ethical, scientific, and historic framework of SoC in clinical trials, focusing on PrEP in South Africa. Summit participants discussed how and when to include new HIV treatment and prevention practices into existing clinical guidelines and trial protocols, as well as the opportunities for and challenges to scaling up interventions. The summit addressed challenges to PrEP provision, such as inconsistent efficacy amongst different populations and various biological, virological, and immunological explanations for this heterogeneity. Advocates and community members propagated the urgent need for accessible interventions that could avert HIV infection. The meeting recommended supporting access to PrEP in HIV prevention trials by (1) developing PrEP access plans for HIV vaccine trials, (2) creating a PrEP fund that would supply PrEP to sites conducting HIV prevention trials via a central procurement mechanism, and (3) supporting the safety monitoring of PrEP. This report summarizes the presentations and discussions from the summit in order to highlight the importance of SoC in HIV prevention clinical trials.The online version contains supplementary material available at 10.1186/s13063-021-05754-z. HIV prevention has evolved tremendously over the last decade. Traditionally, antiretroviral therapy (ART) was prescribed to people living with HIV (PLWH) to slow the progression of AIDS when CD4+ T-cell counts dipped below 200 cells per microliter. As ART reduces viral load, it was hypothesized to work as ‘treatment as prevention’ (TasP) to inhibit sexual transmission. In a trial of serodiscordant couples that compared early ART administration (irrespective of CD4 counts) to delayed, the early treatment reduced the risk of sexual HIV transmission to their uninfected partner by over 90% , 2. ThisThe ‘inverse’ of HPTN 052 was investigating the efficacy of ART when administered to the HIV-uninfected partner in a serodiscordant relationship, a concept of pre-exposure prophylaxis (PrEP). Several randomized controlled trials (RCTs) comparing different combinations of oral tenofovir disoproxil fumarate (TDF) and TDF/emtricitabine  in serodiscordant couples have shown that PrEP can reduce HIV acquisition, although to different extents based on the population . PreventA cornerstone of HIV prevention clinical trials is providing a combination prevention package  to all trial participants. The package provision is part of an obligation to minimize the participants’ risk of acquiring HIV, as assumption of risk can be underestimated in RCT participants , 11. As In 2015, the World Health Organization (WHO) made a recommendation for the inclusion of oral PrEP as part of a combination HIV prevention package for people at substantial risk of HIV infection . In 2017As a result of the lack of PrEP licensure for those at high risk of HIV acquisition in South Africa, the SAMRC reached out to stakeholders in communities, government, academia, and pharmaceutical companies to discuss issues concerning PrEP provision as SoC in HIV prevention trials. The 2-day summit began with presentations from experts on ethics, regulatory processes, clinical studies, basic science, and community engagement, followed by a day of panel discussions with the goal of providing a comprehensive and thought-out plan on PrEP provision for HIV prevention trials in-country. The overall objective of the meeting was to engage regulatory, legal, and ethical frameworks, whilst considering perspectives of government, community organizations and advocates, and the funders of research to review the principles behind setting SoC for prevention clinical trials in LMIC to address the PrEP gap. There were 18 presentations on the first day  project, which is a multi-partner, 5-goal project to develop and implement primary care guidelines in South Africa , and HVTN 705/HPX2008 [ClinicalTrials.gov: NCT03060629]). PrEP was offered at each instance of risk reduction counselling along with other prevention methods provided during participant study visits and during the informed consent process. Where possible, sites could offer PrEP from their own pharmacies and staff were trained to prescribe and manage PrEP initiation and follow-up. Each clinical site received an up-to-date list of pharmacies and clinics that stocked and prescribed PrEP in their areas. PrEP training for site staff was offered periodically to enable PrEP prescribing and management. The more recent HPX3002/HVTN 706 (Mosaico) trial conducted amongst MSM and transgender individuals in the Americas and Europe offered a slightly different approach to PrEP administration to ensure all participants could receive access to the highest standard of prevention. The trial was designed to give volunteers three options regarding PrEP: (1) participants already on PrEP or who wished to take PrEP will not be included in the study, but referred to PrEP resources in their community if requested; (2) participants who decide to take PrEP after receiving vaccination can remain in the study; and (3) participants not wanting to take PrEP can be included in the study [Following the summit, the SAMRC, with additional support from the Fred Hutchinson Cancer Research Center (parent institution of the HVTN) and industry partners, set up a PrEP fund for drug acquisition and laboratory monitoring for HIV prevention study participants across sub-Saharan Africa. This enabled the SAMRC to purchase PrEP at state tender prices and utilize pre-existing negotiated laboratory contracts and within-study systems to extend available PrEP funds. PrEP drug provision and related essential laboratory monitoring support were subsequently initiated for interested and eligible HVTN efficacy trial participants at the 24 clinical trial sites conducting trials in sub-Saharan Africa (HVTN 702 [he study .Additional file 1: Supplement Table 1. Summit presentations.


Integrin-linked kinase (ILK) has been implicated as a molecular driver and mediator in both inflammation and tumorigenesis of the colon. ILK functions as an adaptor and mediator protein linking the extracellular matrix with downstream signaling pathways. ILK is broadly expressed in many human tissues and cells. It is also overexpressed in many cancers, including colorectal cancer (CRC). Inflammation, as evidenced by inflammatory bowel disease (IBD), is one of the highest risk factors for initiating CRC. This has led to the hypothesis that targeting ILK therapeutically could have potential in CRC, as it regulates different cellular processes associated with CRC development and progression as well as inflammation in the colon. A number of studies have indicated an ILK function in senescence, a cellular process that arrests the cell cycle while maintaining active metabolism and transcription. Senescent cells produce different secretions collectively known as the senescence-associated secretory phenotype (SASP). The SASP secretions influence infiltration of different immune cells, either positively for clearing senescent cells or negatively for promoting tumor growth, reflecting the dual role of senescence in cancer. However, a role for ILK in senescence and immunity in CRC remains to be determined. In this review, we discuss the possible role for ILK in senescence and immunity, paying particular attention to the relevance of ILK in CRC. We also examine how activating Toll-like receptors (TLRs) and their agonists in CRC could trigger immune responses against cancer, as a combination therapy with ILK inhibition. The development of colorectal cancer (CRC) is a multistage process during which mutations in epithelial cells of the intestinal inner layer accumulate. In the early stages of CRC, benign polyps are formed; however, an accumulation of specific mutations in these polyps result in the formation of adenomas, which have a potential to develop to a more advanced stage of cancer. Subsequently, tumors can spread and metastasize to different areas of the body via the lymphatic system and blood vessels .According to the International Agency for Research on Cancer, in 2018 there were about 1.85 million new cases of CRC and 900,000 deaths in both sexes globally . The CRCThe classification of CRC depends on etiology and genetics. There are three main groups: sporadic, familial and hereditary. The sporadic group represents the majority (75%) of CRC cases . This grMany studies have shown that tumor-infiltrating immune cells are associated with prognosis and metastasis of CRC. IBD is a chronic inflammation of the gastrointestinal tract (GIT) and is an important risk factor for developing and promoting colon cancer, i.e., colitis-associated cancer (CAC). IBD presents as two types: ulcerative colitis (UC) and Crohn’s disease (CD). CD affects the small intestine and large intestine, in addition to the mouth, esophagus, stomach and the anus, while UC mainly affects the colon and the rectum . InflammCellular senescence was identified more than five decades ago as an arrest of the cell cycle in cultured fibroblasts associated with limited proliferative capacity . It is cIntegrin-linked kinase (ILK) has been implicated as a molecular driver and mediator in both inflammation and tumorigenesis of the colon . ILK funMore importantly, although there is less understanding of the role for ILK in cellular senescence in the cancer context, some reports that have suggested an involvement for ILK in cellular senescence regulation. One study has shown that cellular senescence is induced and suppressed skin tumors and benign colon adenomas by peroxisome proliferator-activated receptor-β/δ (PPARβ/δ) via repressing ILK expression and Akt phosphorylation (pAkt) . AnotherIn this review, we aim to focus on a possible association of ILK with cellular senescence regulation and cancer immunity in CRC. This review summarizes up-to-date studies of ILK and its crosstalks with signaling pathways involved in cancer growth and progression. Also, we analyze the possible role of ILK in the inflammatory response of senescence which is considered the deleterious factor in tumor progression and cancer immunity. We also examine how activating Toll-like receptors (TLRs) could trigger anti-tumor immune responses and be used as a combination therapy with ILK inhibition in CRC.ILK was first discovered in 1996 as an intracellular serine/threonine kinase and adaptor protein interacting with β1 integrin cytoplasmic domain . ILK is The ankyrin domain of ILK binds adaptor proteins such as particularly interesting new cysteine-histidine-rich protein (PINCH)  and ILK-The PH-like domain is located in the center of the ILK protein and binds phosphatidylinositol 3,4,5-triphosphate (PIP3), which is essential for activation of ILK via phosphoinositide 3-kinase (PI3K) . PIP3 isThe ILK pseudokinase domain interacts with cytoplasmic domains of β1 and β3-integrin subunits and different actin-binding adaptor proteins, including α-Parvin and Paxillin . The maiThe dynamic communication between the extracellular matrix (ECM) and actin is mediated by ILK in its role as an adaptor and mediator protein to support cell adhesion, spread and migration. This is mediated when integrin binds to the ILK/PINCH/Parvin complex, termed IPP . A tightHelicobacter pylori /NF-κB/tumor necrosis factor alpha (TNF-α) signaling pathway by lipopolysaccharide (LPS), a major component of the outer membrane of Gram-negative bacteria such as r pylori . Since Hpathways , it remaXenopus laevis, mice and zebrafish) is lethal, affecting adhesion and migration processes as well as vasculature development , ILK knockdown (KD) does not affect the adhesion rate. However, it reduces the spread of cells, as they remained rounded for a longer time (∼18 h) in comparison with ILK wild-type (WT) cells . In addiILK has been revealed to contribute to different non-cancer diseases. The most important related to cancer development is inflammation. ILK KO in epithelial cells of the mouse intestine displays a reduction in inflammation of the colon (colitis) and inflammation-induced cancer (CAC) . MoreoveFurthermore, ILK contributes to other diseases. For example, ILK inhibition in polycystic kidney disease in mouse models has shown a reduction in fibrosis cyst growth with improved renal function and survival . ILK alsILK has been proposed to play a critical role in cancers. It has been established for about two decades that ILK overexpression and dysregulation are associated with development and progression of different cancers, including CRC . ILK is ILK is involved in regulation of epithelial-mesenchymal transition (EMT), an important cellular process regulating migration, invasion and chemoresistance in CRC . ArtificFurthermore, ILK overexpression has a central role in apoptotic resistance. ILK knockout in HT29 CRC cells under hypoxia conditions induces apoptosis, as well as decreases invasive activity . In gastTherefore, while the function of ILK is diverse in different contexts, nonetheless, based on the above studies, ILK is implicated in normal and disease contexts via utilizing its critical function to mediate or regulate several cellular processes including cell shape, adhesion and tissue integrity, as well as cell growth, invasion and migration. Such cellular processes are regulated by dynamic actin organization which is well known to be regulated by ILK. In addition, ILK is implicated as an important molecule in different compartments such as epithelial, endothelial and inflammatory cells to mediate cellular processes regulation and inflammatory response.ILK interacts with different critical signaling pathways  regulatiin vitro ILK upregulation in SW480 colorectal cancer cells promotes upregulation of Snail, slug, vimentin and MMP9, subsequently increasing proliferative, migratory and invasive activities , VEGF and VEGFR gene activation  may be regulated by ILK or Akt in inflammatory and cancer contexts. Our laboratory has shown that myeloid-ILK-deficient cells in an experimental colitis model exhibited reduced activation of NF-κB and PI3K signaling pathway, but elevated STAT3 activation and proliferation of intestinal epithelium . Consequin vitro and in vivo  signaling in colon cancer, some studies have demonstrated such an interaction in different cell types. For example, in gastric cancer cells, ILK silencing inhibited drug resistance via pAkt and pErk suppression . FurtherTherefore, ILK has numerous interactions with different cell signaling pathways and also mediates communications among these pathways, indicating its unique function and potential to be a therapeutic target and modulate the TME and suppress tumor growth.The tumor microenvironment is an area surrounding a tumor that has different cellular components and factors that support tumor growth and progression . TME comSenescence was identified more than five decades ago as an arrest of the cell cycle in cultured fibroblasts due to limited proliferative capacity . It is cThere are different morphological and molecular phenotypes for induced senescent cells. Microscopically, senescent cells exhibit a size enlargement and flattened shape . The nucIn early studies, senescence was thought to have an anti-tumor impact. Besides arresting cell growth, SASPs secreted by senescent cells recruit immune cells eliminating the senescent cells suppressing tumorigenesis . This ceHowever, during last two decades it has been recognized that hallmarks of senescence are more complex and not only involve growth arrest as described above but also have an ability to drive cancer development and progression . SenesceSeveral reports have revealed that a subset of senescent cancer cells can escape from senescence . For insin vitro and in vivo, and could be responsible for cells escaping from senescence and supporting tumor growth  or on endosomal membranes . The cell membrane TLRs bind proteins or lipids, whereas endosomal membrane TLRs bind nucleic acids . TLR sigin vivo the TLR agonists suppressed tumor growth and the effect was improved by using a combination of the agonists or together with chemotherapy (As reviewed recently, stimulating TLRs combined with chemotherapeutic agents, which are known induce senescence, may suppress tumor growth and stimulate anti-tumor immunity . The TLRotherapy . Our labotherapy .In contrast, TLR activation induces inflammation and this is implicated in carcinogenesis and cancer progression . The natChronic inflammation as evidenced by IBD is a risk factor initiating CAC or CRC. Inflammatory response recruits inflammatory cell infiltration and that leads to tumor initiation. Inflammation is also involved in all stages of cancer including growth, invasion and metastasis. Chemotherapy or radiotherapy treatment of tumors may prevent growth temporarily; however, after a period, the tumors will regrow. Different bodies of evidence show that arresting tumor growth is mediated by TIS. However, because the induced senescent cells are still active in metabolism and transcription, they will produce SASPs. There are pronounced heterogeneous inflammatory molecules in SASP, the SIR, that have an impact on rescuing tumor growth again and promote progression via different mechanisms including growth arrest escape, immunosuppressive cell infiltration and immune evasion. ILK has been implicated as a mediator in both inflammation and tumor growth. ILK also has been suggested to prevent senescence induction in the cancer context. Nonetheless, the knowledge about the role of ILK in cellular senescence and inflammatory response mediated by SASPs remains incomplete. Targeting ILK in solid tumors such as CRC could be effective in suppressing tumor growth via promoting TIS, enabling recruitment of anti-tumor immune cells into the TME thereby regulating SASP secretion . What isSA and BW planned the review. SA drafted. AA, RB, and BW edited the review. All authors contributed to the article and approved the submitted version.RB was Chief Scientific Officer and shareholder of Catherics Pty Ltd. BW was Director and shareholder of this company. 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.


Strategies to increase the bio-functionality of staple food, such as bread, by incorporating whole-grain wheat flour or flour from other, non-wheat grains instead of refined wheat flour are often constrained with the lack of their techno-functionality, despite the associated beneficial effect on consumers' health and well-being. Most of the available studies investigating the possibilities to improve technological and sensory quality of bread prepared using whole-grain wheat and non-wheat flours still rely on formulation approaches in which different additives and novel ingredients are used as structuring agents. Less attention has been given to technological approaches which could be applied to induce structural changes on biopolymer level and thus increase the breadmaking potential of whole grains such as: modification of grain and biopolymers structure by germination, flour particle size reduction, dry-heat or hydrothermal treatment, atmospheric cold plasma, high-pressure processing or ultrasound treatment. Strategies to modify processing variables during breadmaking like dough kneading and hydration modification, sourdough fermentation or non-conventional baking techniques application are also poorly exploited for bread preparation from non-wheat grains. In this paper, the challenges and opportunities of abovementioned processing strategies for the development of bread with whole-wheat flours and non-wheat flours from underutilised gluten-containing or gluten-free cereals and pseudocereals will be reviewed throughout the whole breadmaking chain: from grain to bread and from milling to baking. Feasibility of different strategies to increase the technological performance and sensory quality of bread based on whole-grain wheat flours or flours from other, non-wheat grains will be addressed considering both the environmental, safety and nutritive advantages. Triticum aestivum) flour. However, in recent years, there has been renewed interest in fortifying or replacing refined wheat flour with whole-grain wheat flour, or flour from gluten-free cereals , pseudocereals  and ancient cereals [Bread, regardless of the type, production process and geographical origin, is traditionally produced from refined common wheat  that could act as structure forming agents instead of diluted or absent gluten , 10. In However, relatively little research has been conducted on technological approaches for improving breadmaking potential of whole-grain and non-wheat cereals. As noted by Parenti et al.  instead Therefore, the aim of this review is to provide a critical opinion on current and future-looking sustainable technological innovations and strategies utilized to increase the technological performance and sensory quality of bread based on whole-grain and non-wheat cereals. Improvement strategies discussed in this paper encompassed the whole bread production chain Fig. : from ra2+, Fe2+/3+, Ca2+, Mg2+, Mn2+ and Cu2+ [Modification of grain and biopolymers structure by germination is mostly performed to initiate nutrient compositional changes which are associated to health benefits. During the germination process degradation of macromolecules occurs due to increased enzyme activities: (i) starch is hydrolysed by amylolytic enzymes to maltose, glucose, dextrins and oligosaccharides, resulting in its higher digestibility –15; (ii)and Cu2+ . Moreoveand Cu2+ , 19. Gerand Cu2+ , 18, recand Cu2+ .Although increase in enzymatic activity produced by germination has mostly a detrimental effect on the breadmaking potential of cereals, with proper adjustment of the germination parameters it can be a promising tool to improve both the nutritional and technological properties of cereal-based food. In general, germination leads to softer and more fragile grain as a consequence of enzyme action which results in lower damaged starch content upon milling . This, aHowever, shorter germination times, low substitution levels or addition of some improvers  to germinated wheat flour could increase technological performance of whole-grain cereals , 17, 25.In addition, optimized α -amylase activity can improve the bread shelf-life and sensory attributes . It was Flour particle size can significantly alter bread functionality and technological quality. If a micronization, such as jet milling, is applied to produce fine wheat flour with extremely low particle size, flour with increased digestible starch content is obtained . When usHowever, it seems that pulverization of flour is not promising technology concerning bread technological quality since whole-grain wheat jet milled breads (flour volume median diameter = 17–53 μm) were characterized with reduced specific volume and moisture content and increased crumb hardness in comparison to breads with flour having volume median diameter of 84 μm . The samDifferent flour heat treatments such as dry-heat treatment or hydrothermal treatments (below or above starch gelatinization temperature) are being increasingly applied to improve the functionality of alternative cereals flour. It was shown that dry-heat treated sorghum flour produced breads with increased specific volume and more cells per slice area. This was ascribed to increased viscosity of sorghum flour dough as a consequence of starch granule swelling due to heat induced partial gelatinization as well as denaturation of both proteins and enzymes . In addiIt was also revealed that gluten-free flours (maize or rice) blanching results in doughs with higher consistency, adhesiveness, springiness and stickiness due to the partial gelatinisation of the starch, which further led to improved bread quality , 39.When flour/starch heating is carried out in the presence of water without fostering a complete starch gelatinization, as it is the case with annealing (treatments in excess or at intermediate water contents below the gelatinisation temperature) and heat-moisture treatment (exposure of starch to higher temperatures at very restricted moisture content), increase in the starch gelatinization temperature, water binding capacity and granule susceptibility to enzyme hydrolysis occurs , 41. TheIt was shown that application of hydrothermally treated rice and maize flour to manufacture rice and maize semolina-based breads increased the specific volume and decreased the hardness and chewiness of the gluten-free breads, due to higher initial viscosity imparted by treated flours enabling the entrapment of air bubbles in the dough .When hydrothermal treatments are performed above gelatinization temperature starch granules are irreversibly losing their integrity, a process known as pre-gelatinization . ParentiIf pre-gelatinization is achieved with the aid of extrusion cooking  besides amylose and amylopectin leaching from disrupter starch granule, breakage of the amylose and amylopectin chains, denaturation of proteins, enzyme (in)activation and Maillard reactions occurred . ExtrusiAtmospheric cold plasma (ACP) is a non-thermal processing technology that so far was applied at different stages of the cereal processing chain for a range of applications including improved germination, microbial decontamination, toxin degradation and biopolymer structural changes for improved functionality . The modHowever, most of the studies investigating plasma-induced changes in grain/flour/dough structure are based on breadmaking potential of refined wheat flour, biopolymer changes in whole grain wheat or the safety aspects of plasma application for alternative grains decontamination. The studies concerning plasma application to enhance breadmaking performance of whole-grain or non-wheat cereals are scarce. Since some preliminary studies have shown that ACP treatment is effective just in increasing breadmaking potential of weak flours , some fuHigh-pressure processing (HPP) represents novel processing technology which is mainly used for non-thermal treatment for fruit juices preservation . GeneralUltrasound treatment, as a non-thermal processing tool, has been intensively utilized for microbial and enzyme inactivation, bioactive component extraction and food components modification for increased functionality . HoweverWhile it was shown that ultrasound modulation of flour functionality depends on the treatment time , 63, theIn general, ultrasound treatment of whole-grain flour significantly increases water solubility, water absorption and swelling power of quinoa, buckwheat and rice flour –64. It aJalali et al.  have shoFlour transformation to dough is performed by hydration and mixing operations, where different processing variables can be modified in order to achieve optimum dough and bread quality. Appropriate water content and temperature ensure optimal dough rheology and consistency, avoiding undesired softening or hardening. Proper choice of mixing speed and temperature will avoid dough warming and excessive weakening, while kneading time management prevents both over- and under-mixing and allows dough aeration and its capacity to retain gases .Water content influences dough quality in the following manner: adding too much water during kneading generates soft and sticky dough, while dough with water content below the optimal water absorption of the flour will be harder to knead . IncreasGomez et al.  have alsAlthough being an ancient biotechnology, sourdough fermentation has gained renewed interest as a tool for better exploitation of non-wheat cereals in breadmaking . SourdouBeside the fact that sourdough fermentation contributes to enhanced nutritional properties of bread  and phytic acid reduction, leading to increased mineral, protein and free amino acids bioavailability; it has significant impact on bread techno-functionality , 72.Taking advantage of LAB ability to produce certain polymers and modify the main structure-building components of flour such as starch, arabinoxylans and proteins, sourdough fermentation was used to improve dough and bread technological properties such as loaf volume, water absorption of the dough, dough rheology and machinability . CertainS. cerevisiae [On the contrary, reports on the fate of starch during sourdough fermentation are contradictory. In the case of the wholegrain wheat flour, sourdough fermented bread exhibited higher resistant starch content and lower glycaemic response than the corresponding products leavened with revisiae . Howeverrevisiae .Saccharomyces cerevisiae.The effect of sourdough fermentation on techno-functionality of bread prepared with alternative cereals is summarized in Table 2, ethanol, diacetyl, hydrogen peroxide, fatty acids, reuterin, fungicin, etc.) also contribute to bread preservation thus prolonging its shelf life [Besides bread technological quality, organic acids together with other LAB metabolites .In comparison to conventional, partial-vacuum baking of gluten-free bread did not have significant impact on bread volume and texture; however, it resulted in product which became stale more slowly than the control .Microwave and infrared baking are considered as time- and cost-efficient processes. Although microwave and microwave-assisted hot air baking increase gluten-free bread crumb hardness and result in pale bread crust compared with the hot air baking, it was shown that these techniques can reduce the digestibility of starch and glycaemic index of the bread and increase loaf volume .Application of single infrared radiation  results mostly in products of inferior quality, due to the high rate of heating which influence sudden and thick crust formation and the prevention of the product expansion thus leading to lower specific volume and higher firmness values than conventional baking , 92. HowAnother novel baking technique, jet impinging, based on forced convection heating, increases the heat transfer efficiency during the baking process , but resOhmic heating is an innovative technology in which an alternating electrical current is passed through a material, generating heat by dissipation of the electrical energy due to material's own electrical resistance, allowing rapid and uniform heat distribution .2 is released during heating enabling bread expansion.Bender et al.  have shoIn order to increase the potential of non-conventional baking techniques while minimizing the disadvantages a combination of them (hybrid heating) can be applied. Combination of infrared lamps and electric heating coils enables 28% reduction in baking time, while resulting in breads comparable with breads baked in conventional electrical heating in terms of crumb firmness, volume, moisture content and colour . HoweverImpact of abovementioned processing strategies on breadmaking potential of whole-grain wheat and non-wheat flours is summarized in Fig. This review has highlighted that different technological strategies can be used to increase techno-functionality of whole-grain wheat and non-wheat flours and sensory properties of final product—bread. They are mostly performed with the aim to alter biopolymer structure and thus increase its functionality and encompass the ones used to provoke starch pre-gelatinization , reduce starch retrogradation , induce gluten strengthening through oxidation (atmospheric cold plasma) or gluten hydrolysis . It was elucidated that despite the opportunities offered by different conventional and emerging technologies and approaches, the gaps between technological and nutritional strategies for improving breadmaking potential of whole-grains still exist, especially when other, non-wheat grains are used. Namely, effectiveness of reviewed technological approaches largely depends on initial flour composition and quality. Therefore, further investigations are needed, particularly with respect to the ones including combined technologies  to further increase technological and sensory quality of bread from whole-grain non-wheat cereals while preserving health beneficial properties.