Stroke is a life-threatening condition in which accurate diagnoses and timely treatment are critical for successful neurological recovery. The current acute treatment strategies, particularly non-invasive interventions, are limited, thus urging the need for novel therapeutical targets. Arginine vasopressin (AVP) receptor antagonists are emerging as potential targets to treat edema formation and subsequent elevation in intracranial pressure, both significant causes of mortality in acute stroke. Here, we summarize the current knowledge on the mechanisms leading to AVP hyperexcretion in acute stroke and the subsequent secondary neuropathological responses. Furthermore, we discuss the work supporting the predictive value of measuring copeptin, a surrogate marker of AVP in stroke patients, followed by a review of the experimental evidence suggesting AVP receptor antagonists in stroke therapy. As we highlight throughout the narrative, critical gaps in the literature exist and indicate the need for further research to understand better AVP mechanisms in stroke. Likewise, there are advantages and limitations in using copeptin as a prognostic tool, and the translation of findings from experimental animal models to clinical settings has its challenges. Still, monitoring AVP levels and using AVP receptor antagonists as an add-on therapeutic intervention are potential promises in clinical applications to alleviate stroke neurological consequences. Stroke results from an acute central nervous system injury caused by the disruption of cerebral blood flow or bleeding within or around the brain, with consequent neurological damage and loss of function. Ischemic stroke due to cerebral blood vessel occlusion comprises most of the cases , followed by intracerebral hemorrhage stroke (28%), and subarachnoid hemorrhage stroke (10%) . Un. Un11]. Regarding ischemic stroke, in one study, treatment with a V1R antagonist increased AQP4 expression . ContrarAVP influences ion balance in astrocytes and endothelial cells. The ion imbalance caused by Na/K pump dysfunction is aggravated by V1aR-mediated luminal ion transporters’ activity enhancement ,136. AVPStimulation of V1aRs found on cerebral vasculature leads to endothelin-1 overexpression and protein kinase C (PKC) pathway activation, which translates into oxidative stress exacerbation and BBB disruption. A study conducted by Faraco et al. has shown that water deprivation-induced AVP hypersecretion elicits AVP-mediated oxidative stress leading to cerebrovascular dysregulation in murine brains . Moreover, AVP may damage BBB by stimulating matrix metallopeptidase 9 (MMP9) expression in the brain endothelium . The actAVP elicits the vasoconstriction of cerebral blood vessels via V1aR, causing an increase in cerebral perfusion pressure (CPP) . In the In the rodent TBI model, AVP aggravated injury-elicited inflammatory response. Mechanistically, AVP amplifies CXC and CC chemokines synthesis in the endothelium and astrocytes. In line with this, AVP stimulates the expression of high-mobility group box1, a well-known inflammatory cytokine, in astrocytes during hypoxia/reoxygenation ,151. A rAs highlighted in As summarized above, increases in AVP are detrimental to stroke and have a considerable potential of being used as a therapeutic target as the mechanisms are directly associated with AVP receptors. Furthermore, although AVP is an unstable peptide, it is released in equimolar concentrations with copeptin and validated as a surrogate marker of AVP.Clinically, copeptin measurements in acute ischemic stroke serve as a tool to predict functional outcomes ,166. A mAnother promising application of copeptin measurement is to predict stroke after an episode of TIA. Previous studies have shown that patients with a higher copeptin level measured immediately after the TIA event are at a greater risk of developing a stroke or any cerebrovascular re-event ,169. In In general, copeptin seems to be an effective biomarker in risk stratification after ischemic stroke and in predicting vascular events after TIA. However, copeptin is also associated with low specificity or low discrimination as a biomarker in other conditions. For instance, a few studies assessed the risk of stroke-associated infections using copeptin ,171,172.Likewise, applying copeptin as a diagnostic tool to distinguish stroke patients from stroke-free subjects has been tested with inconsistent results ,22,173. In summary, copeptin seems to be an effective biomarker opening an opportunity to improve stroke prognosis. It should be considered that the field of novel biomarkers regarding cerebrovascular disease management is growing rapidly. The combination of both circulating blood protein biomarkers and genetic testing can be used for cerebrovascular risk assessment and outcome stratification after an ischemic event ,175,176.The mechanisms mentioned above, especially brain edema exacerbation, prompted the rationale for AVP receptor antagonism in stroke. The use of a selective V1aR antagonist (SR49059) in MCAO rodent stroke models improved neurological outcomes and reduced infarct area ,135,178.OPC-31260, a V2R antagonist, prevented water and sodium accumulation in the brain in both general cerebral hypoxia and SAH rat models. Moreover, in both studies, V2R antagonism enhanced plasma AVP increase in subjected rodents ,28.Tolvaptan is a clinically approved V2R antagonist, successfully used in hyponatremia treatment . HoweverConivaptan is an FDA-approved V1aR and V2R antagonist . The maiOverall, antagonism of just V1R or both V1R and V2R provides a better outcome in rodent stroke models than using V2R antagonists alone. Recent literature regards Conivaptan as a safe, mixed AVP receptor antagonist that has the potential to supplement stroke treatment. Nevertheless, phase II studies are needed to test the effectiveness of Conivaptan in stroke.During the acute phase of stroke, AVP release is uncontrolled and larger than observed in physiological conditions. The mechanisms for AVP release in stroke are independent of plasma osmolality and involve a complex interplay and synergism between regulatory systems, systemic factors, and local components. Among them, AVP secretion underlying mechanisms include an interplay between the sympathetic and renin-angiotensin system; systemic changes in glycemia; and regional increase in glutamate excitability, as well as maladaptation of the neurovascular unit (reviewed in ). The reTargeting the AVP receptor with pharmacological receptor antagonists, either individually or in combination, aims to prevent pathological events spurred by V1R and V2R activation. A mixed AVP antagonist, Conivaptan, achieved the best safety and neurological outcomes, providing the most significant degree of potential in stroke treatment. Of note, most of the beneficial effects of AVP receptor antagonists were achieved in ischemia-reperfusion models. Thus, it is reasonable to infer that AVP receptor antagonism-oriented therapy is more efficient when applied together with the recanalization of the occluded vessel. In line with this, we believe that future studies involving AVP receptor antagonists should focus on using AVP antagonists as an add-on therapy for standard recanalization (thrombolysis/thrombectomy) treatment. It is crucial to recognize the potential of the anti-edematous effects of AVP receptor antagonists in post-stroke secondary brain damage alleviation. We strongly feel that treatment of post-stroke brain edema using AVP receptor antagonists in concert with standard brain edema treatment could be a viable means of stroke outcome improvement and mortality rate reduction, especially in patients with refractory brain edema . As demoA limited number of studies have focused on the possibility of using AVP receptor antagonists in animal models of hemorrhagic stroke. Although ischemic stroke is epidemiologically more frequent than hemorrhagic stroke, we suggest that the efficacy of vasopressin antagonists in the latter should also be investigated . Thus, wIn this review, we pointed out the involvement of AVP in some stroke-related pathophysiological events. The multiplicity of mechanisms involved in primary and secondary brain damage in stroke points out the still underexplored areas of possible AVP influence. We propose that further research should be undertaken in the following areas: (1) reperfusion injury, (2) neuroinflammation, (3) vasospasm, (4) oxidative stress, and (5) BBB disruption.This review provides an update on understanding AVP involvement in ischemic stroke. Moreover, it highlights viable future research areas to explore. Our work would lend itself well to clinicians conducting clinical trials involving AVP receptor antagonists and researchers exploring the molecular mechanisms behind other neurological conditions. The ultimate goal of this review is to provide a springboard for further studies on the involvement of AVP in stroke settings and the implication thereof in modern medicine. In our view, the biggest challenge would be the time-consuming and cost-intensive process of clinical trials, particularly considering other competing research areas regarding stroke therapy, including neuroprotective drugs, safer/cheaper thrombolytic agents, and extended time windows protocols for thrombolysis/thrombectomy. Moreover, since the animal model for human ischemic stroke does not adequately reflect the human brain conditions in stroke, the discussed results of animal studies could be delusive, thus undermining the rationale for large-scale studies. To overcome these challenges, further experimental investigations are needed to establish the complete basis of the AVP mechanism of action in terms of stroke. For instance, the current animal stroke models should be improved by considering the age, metabolic state, and common comorbidities of stroke patients, as well as the anatomical and molecular differences between experimental animals and humans. Due to the significant burden of stroke in the world’s rapidly growing and aging population, the scientific fields studying novel stroke therapeutics and stroke pathomechanism are one of the central issues currently being researched. The line of research regarding the involvement of AVP in stroke is especially significant, given that some drugs that could putatively diminish the detrimental effect of AVP are widely available for treatment in other medical scenarios, thus making them easily accessible in case of a positive translation process. In conclusion, overlapping detrimental events occurring both in stroke and AVP hypersecretion models and the effectiveness of AVP receptor antagonists provide evidence to infer that AVP plays a vital role in stroke development. However, the indirect nature of the gathered data does not provide us with the essential details to determine the overall involvement of AVP-mediated mechanisms in stroke. Moreover, the differences between ischemic and hemorrhagic stroke regarding AVP release and the mechanism of action remain largely uncovered. Nevertheless, monitoring AVP levels and targeting AVP receptors are of paramount potential in clinical applications. Undoubtedly, molecular and prospective clinical studies are needed to discover the precise mechanisms behind AVP effects and integrate the measuring of copeptin or/and the use of AVP receptor antagonists in clinical practice.A stroke is associated with the release of AVP into the blood in response to various endogenous stimuli;Overproduction of AVP is most likely detrimental to the brain, primarily due to the AVP-elicited brain edema;Copeptin is a surrogate marker of AVP and can be used as a prognostic biomarker of stroke outcome;AVP receptor antagonists can potentially be used as an add-on therapeutic intervention in stroke.Based on the data included in the sections of the manuscript, we can summarize that:


How do proteins interact in the cellular environment? Which interactions stabilize liquid–liquid phase separated condensates? Are the concepts, which have been developed for specific protein complexes also applicable to higher-order assemblies? Recent discoveries prompt for a universal framework for protein interactions, which can be applied across the scales of protein communities. Here, we discuss how our views on protein interactions have evolved from rigid structures to conformational ensembles of proteins and discuss the open problems, in particular related to biomolecular condensates. Protein interactions have evolved to follow changes in the cellular environment, which manifests in multiple modes of interactions between the same partners. Such cellular context-dependence requires multiplicity of binding modes (MBM) by sampling multiple minima of the interaction energy landscape. We demonstrate that the energy landscape framework of protein folding can be applied to explain this phenomenon, opening a perspective toward a physics-based, universal model for cellular protein behaviors. Biological processes are achieved through different protein communities that range from binary complexes to cellular bodies. Specific rules governing the formation of these organizations also regulate the spatial and temporal characteristics of the underlying biological activities. How are these rules encoded in the amino acid sequence enabling protein function under a variety of cellular conditions? Recent discoveries highlight that, in addition to specific protein complexes, higher-order assemblies, ranging from ordered amyloids to dense liquid droplets, contribute to a wide range of biological activities . In partThe article is organized as follows: First, we give a historical overview of protein interactions, introducing ordered and disordered binding modes. Second, we describe the phenomenon of context-dependent interactions. Third, we introduce the landscape model to describe interaction behaviors. Fourth, we propose the energy landscape framework as a universal model for protein folding and higher-order assembly.The binding mode in stoichiometric protein complexes is usually classified based on the structural changes upon interactions. The binding mode is classified as ‘ordered’ if the resulted complex has a well-defined structure in the bound state, while ‘disordered’ if the complex samples an ensemble of conformations . In the Alternatively, disordered proteins may remain disordered in their bound states forming conformationally heterogeneous complexes ,14 with These two scenarios are associated with distinct sequence features. High complexity sequence motifs can form a well-defined interaction pattern, while low complexity motifs form ambiguous interaction patterns  Figure B. This cTaken together, proteins may interact via a wide range of binding modes, from ordered binding mode with a well-defined contact pattern to disordered binding modes with heterogeneous contact patterns, which are encoded in the local complexity of the protein sequence.Kip1 cell-cycle kinase inhibitor, for example, increased stability of the α-helix motif compromises the inhibitory function [The experimentally observed binding characteristics reflect the binding mode of the interaction. The dissociation constant of disordered proteins with ordered binding modes strongly depends on the flanking sequences, which can modulate the affinity by orders of magnitude ,23. Flanfunction . In addifunction .In disordered binding modes, the affinity linearly depends on the length and the number of binding motifs involved , indicatWhy does Nature use different binding modes for interactions? Evolvability requires sequence flexibility to avoid accumulation of lethal variations . Thus, cTaken together, different biological processes require proteins with different local sequence complexities, resulting in different binding modes for interactions.2+ concentration, which modulates the binding affinity of Ets-1 to DNA by orders of magnitude through multisite phosphorylation of Ets-1, which has an autoinhibitory effect via regulating the stability of the interaction element [2+-induced multisite phosphorylation, which modulates the mode of interaction between the transcription factor and its cognate sequence [The cell responds to millions of stimuli. Nevertheless, biological processes are achieved with high accuracy under a wide variety of cellular conditions. For example, the Ets-1 transcription factor functions under a variable control to regulate stem cell differentiation . Its gen element . Thus, csequence .Overall, cellular conditions translate into changes in the binding modes of the interactions, with an impact on specificity, structural order or biological pathway . In the Liquid–liquid phase separation of proteins in particular depends on the cellular context . DropletDPp), which is proportional to the probability of sampling disordered binding modes [To illustrate this point, we have analyzed mutations in droplet-forming proteins, FUS , TDP-43ng modes  as well ng modes . We founTaken together, protein interactions follow changes in the cellular environment and context-dependence is linked with multiplicity of binding modes.Protein structures are usually interpreted within the energy landscape framework. The polypeptide chain aims to achieve an energy optimum during folding, which is often associated with the minimum of a funnel. Because of the many-body problem, not all interactions can be optimized simultaneously, leading to suboptimal contacts and energetic frustration . It was Structures formed through templated folding of disordered proteins upon binding their partners comprise high density of suboptimal contacts . This inThe analogy between the folding and interaction energy landscapes  suggestsThe energy landscape framework, for example, has been successfully applied to explain different mechanisms of amyloid aggregation . DiffereAs the same physical principles drive organization of the polypeptide chain at different levels, the energy landscape framework is universal, from stoichiometric specific complexes to nonstoichiometric higher-order assemblies.Cellular organizations of proteins present a challenge to relate sequence and biological activity. Depending on the associated processes, protein sequences have evolved to high or low complexity. While structure–function relationships of high complexity sequences can be described by deterministic models, these cannot be readily applied to low-complexity sequences ,64. At lExperimental results highlight the complexity of the protein interactions, in particular in the cellular environment, where assembly/disassembly is regulated by a wide variety of conditions. Therefore, proteins sample a wide range of binding modes, from ordered to disordered binding. Many proteins are also capable of interacting through a multiplicity of binding modes (MBM), resulting in variable behaviors according to the cellular context . ProteinIt is challenging to decipher how the interaction behavior, quantified by MBM, is encoded in the protein sequence. In contrast to the traditional process of identifying specific binding motifs, one needs to investigate the local sequence bias of the potential interaction sites. The local sequence bias determines the ordered or disordered binding modes of interactions. Because of context-dependence, the interaction motifs will change with the cellular conditions. Thus, if the local sequence bias can be modulated considerably by the minor alterations in the flanking regions, for example, by post-translational modifications or by masking a few interaction sites, the binding element will be prone to sample high MBM. In contrast, if the local sequence bias is robust to alterations in the flanking regions, the interaction will be unimodal with low MBM. These principles are represented by binding  and dropThe common origin of interaction characteristics in higher-order assemblies, for example, liquid–liquid phase-separated droplets and stoichiometric protein complexes, will facilitate deciphering the sequence codes for physiological functions and aberrant states. Identifying non-native interactions, based on energetic considerations, is more straightforward than identifying universal-binding motifs applicable to most proteins. Analysis of heterogeneous conformational ensembles can be helpful in this endeavor ,65.In summary, protein interaction complexity in the cellular environment is manifested in the assembly through multiplicity of binding modes. The physical origin of MBM is energetic frustration, and thus can be described within the energy landscape framework similarly to protein folding. Recognizing the common origin of protein folding and higher-order assembly will facilitate the development of physics-based, quantitative models for biomolecular condensates.Proteins interact via a wide range of binding modes from ordered to disordered binding, from well-defined to ambiguous contact patterns.Protein interactions sample a multiplicity of binding modes (MBM) depending on the cellular context.MBM is critical for aggregation of liquid–liquid phase-separated droplets to solid-like amyloid fibers.The energy landscape framework is a universal, physics-based framework linking folding to assembly across all scales of protein communities.


Hematopoietic stem cell transplantation (HSCT) is a common method for patients such as hematologic malignancies. However, HSCT generally has a higher risk of secondary solid cancer development. The aim of this study was to emphasize the need for lifelong follow-up of oral secondary solid cancer. The patient was a male who underwent HSCT for chronic myelogenous leukaemia at the age of 31 years. He underwent ten onsets on oral secondary solid cancers during his subsequent follow-up of more than 20 years. In conclusion, patients after HSCT require lifelong observation of oral secondary solid cancer, which may be accompanied by repeated new and recurrent occurrences. Hematopoietic stem cell transplantation (HSCT) is a vital therapeutic method for most patients who have hematologic malignancies such as leukaemia and non-malignant disease such as aplastic anaemia. HSCT has increased steadily over the past three decades worldwide –3, and hAlthough a relatively large number of oral secondary solid cancers have been reported, including statistical observations, most were merely reporting of first onset and have a short follow-up period , 7. OralIn this study, we report a case of oral secondary solid cancers with ten times repeated occurrence and/or recurrence, which has been followed up for more than 20 years after undergoing HSCT at the age of 31 years in order to emphasize the need for long-term observation of the onset of oral secondary solid cancer.In April 2019, a 51-year-old man visited the oral and maxillofacial surgery clinic to continue follow-up of oral examination after HSCT. The patient was previously diagnosed with chronic myelogenous leukaemia in 2000. In September 2000, the patient underwent an allogeneic HSCT from his human leukocyte antigen–matched sister. Immediately after the transplantation, graft-versus-host disease (GVHD) of oral mucosa appeared. However, no prominent symptoms of GVHD other than the oral cavity were observed, and immunosuppressive therapy associated with GVHD was not performed. In addition, no local therapy was given.After that, the patient could continue to follow up at the previous oral surgery clinic for oral GVHD and underwent surgery for secondary solid cancers of right buccal mucosa and left mandibular gingiva, both of which were diagnosed as squamous cell carcinoma (SCC) in February 2012; left mandibular gingiva in July 2012 (SCC); left mandibular gingiva in September 2012 (SCC); and right upper lip (SCC) in July 2013. Subsequently, biopsies were performed on the left upper lip in November 2017 and on the right side of the dorsum of the tongue in December 2017, both of which were negative for malignancy. After that, regular observations were continued, but due to work, our department took over the flow-up.His medical history other than that associated with chronic myelogenous leukaemia included hypertension, hyperuricemia, chronic kidney disease, diabetes, and dyslipidemia. Current medications included omega-3-acid ethyl esters (hyperlipidemia), atorvastatin calcium hydrate (hypercholesterolaemia), amlodipine besilate (hypertension), perindopril erbumine (hypertension), febuxostat (hyperuricemia), and insulin aspart (diabetes).On examination, erythema was observed on the right side and left margin of the tongue, while on the dorsal surface, exophytic masses were found. The left maxillary anterior gingiva showed whitish change . The rigAfter the biopsies, in June, surgical resections were performed. The diagnosis of the left dorsal surface of the tongue and the right buccal mucosa was severe dysplasia, and lower lip was SCC, and the left maxillary palatal gingiva was carcinoma in situ Figures . To dateWith improved clinical outcomes of hematologic diseases performed by HSCT, there is rising interest in secondary solid cancers after HSCT. Among them, oropharyngeal region is the most prominent high-risk organ of development of secondary solid cancers , 7. In tSkin and oropharyngeal secondary solid cancer is predominantly squamous cell carcinoma . VariousThe symptoms of oral acute GVHD and chronic GVHD may overlap. Diagnostic features of oral chronic GVHD include lichen planus-like changes, characterized by hyperkeratotic white lines and lacy-appearing lesions on the oral mucosa, and distinctive features of chronic GVHD include xerostomia (dryness), mucoceles, mucosal atrophy, ulcers, and pseudomembranes . SymptomThe clinical and histological features of chronic GVHD are very similar to those of oral lichen planus. In both diseases, the immune system plays a primary role in the pathogenesis, and the same mechanisms are considered to be associated with malignant transformation. Clinically, it may be difficult to distinguish the secondary solid cancers from chronic GVHD with lichen planus-like changes. The most frequent clinical features presented at diagnosis of the solid cancers were described to be plaques 50%), exophytic masses (39%), and ulcers (28%) [0%, exophOther risk factors of the oropharyngeal cancers included reduced-intensity conditioning regimens associated with HSCT, autologous HSCT and hereditary disorders such as Fanconi anaemia in paediatric patients. Limited to oral cancer, long-term immunosuppressive therapy associated with chronic GVHD ≥24 months, conditioning regimens with limited field irradiation, and male gender were risk factors . In immuSecondary oral solid cancers can occur at any site in the oral cavity, but Mawardi et al. stated that the occurrence at the tongue was 56%, being the most predominant site, followed by buccal mucosa, 39% –10. In tAs for follow-up, it is predicted that the secondary oral solid cancers will occur for a lifetime, so regular and uninterrupted scrutiny is necessary. Screening every six months may be considered for patients at high risk for developing oropharyngeal cancer . EvaluatThis study reported a patient with repeated new and recurrent secondary oral solid cancers who were followed up for more than 20 years after HSCT. The number of leukaemia survivors with HSCT is expected to increase in the future. In particular, there has been a dramatic increase in the number of transplant recipients who are more than 50 years of age at transplant over the past decade . Dentist


In recent decades, extracellular vesicles (EVs), as bioactive cell-secreted nanoparticles which are involved in various physiological and pathological processes including cell proliferation, immune regulation, angiogenesis and tissue repair, have emerged as one of the most attractive nanotherapeutics for regenerative medicine. Herein we provide a systematic review of the latest progress of EVs for regenerative applications. Firstly, we will briefly introduce the biogenesis, function and isolation technology of EVs. Then, the underlying therapeutic mechanisms of the native unmodified EVs and engineering strategies of the modified EVs as regenerative entities will be discussed. Subsequently, the main focus will be placed on the tissue repair and regeneration applications of EVs on various organs including brain, heart, bone and cartilage, liver and kidney, as well as skin. More importantly, current clinical trials of EVs for regenerative medicine will also be briefly highlighted. Finally, the future challenges and insightful perspectives of the currently developed EV-based nanotherapeutics in biomedicine will be discussed. In short, the bioactive EV-based nanotherapeutics have opened new horizons for biologists, chemists, nanoscientists, pharmacists, as well as clinicians, making possible powerful tools and therapies for regenerative medicine. Regenerative medicine aims at the structural restoration and functional reestablishment of impaired or missing tissues by replenishing, replacing or repairing cells, tissues and organs, holding great promise for human healthcare in vivo stem cell proliferation and targeted delivery to the injured sites Although effective, there are considerable limitations restricting the wider clinical application of stem cell therapy, including 1) biosafety concerns deriving from abnormal differentiation, immune rejection and tumorigenic risk e.g., microRNA (miRNA), messenger RNA (mRNA), and DNA), as well as soluble small molecules inherited from their parental cells Figure 1). Indeed, EVs exhibit great superiority over the conventional stem cell therapy for regenerative medicine, such as, multiple biologically therapeutic effects, exogenous cargo delivery functions, enhanced biocompatibility and biosafety, as well as improved reproducibility and stability In general, EVs, typically including exosomes, microvesicles (MVs), and apoptotic bodies based on their size and biogenesis, have originally been considered as a disposition apparatus for waste materials from cells, but are now recognized as biological cell-secreted nanoparticles (30-2000 nm) enclosed by double phospholipid membranes, which play important roles in intercellular communication Despite EVs having shown considerable potential in regenerative medicine application, their development is still at an early stage, where more attention should be devoted to both fundamental research and clinical practices. Herein, we provide a systematic review of the latest progress of EVs for regenerative medicine. The biogenesis, function and isolation technology of EVs will be introduced firstly. Then, the underlying therapeutic mechanisms of the native unmodified EVs as well as engineering strategies of the modified EVs as regenerative entities will be discussed. Afterwards, a specific focus will be placed on the tissue repair and regeneration applications of EVs on various organs including brain, heart, bone and cartilage, liver and kidney, as well as skin. More importantly, current clinical trials of EVs for regenerative medicine will be briefly presented. Finally, the remaining challenges and some insightful perspectives of the currently developed EV-based nanotherapeutics in biomedicine will be briefly highlighted, which will be beneficial to the future development of the EVs or other bioinspired nanotherapeutics. We believe that this review will appeal to different researchers of biologists, chemists, nanoscientists, pharmacists, clinicians as well as scientists from interdisciplinary fields.More than half a century has passed since EVs were discovered as “platelet-dust” in 1967 Figure 2): 1) Exosomes are small vesicles (30-150 nm) secreted from the plasma membrane which go through invagination and inward budding, ultimately exhibiting a typical cup-shaped structure; 2) Microvesicles  are formed by directly budding from the cell membrane; 3) Apoptotic bodies  are budded from the plasma membrane of apoptotic cells with shrinkage and fragmentation. The biogenesis mechanisms of EVs involve a series of endocytosis and exocytosis. During the formation of exosomes, twice invagination of the plasma membrane occurred. For the first time, membrane proteins and extracellular soluble molecules are enclosed in the cup-shaped structure, which further forms the early-sorting endosomes (ESEs) and late-sorting endosomes (LSEs) in vivo and in vitro. Moreover, the EV biogenesis may also be regulated by different cell types, culture conditions, and cell sources As mentioned above, the EVs are normally divided into three major subtypes (http://www.microvesicles.org/), which is one of the most predominant EV databases generally available.EVs contain a variety of bioactive substances such as proteins, lipids, nucleotides and some soluble metabolites. Extensive research has revealed that the proteins of EVs usually include membrane proteins, cytoplasmic and nuclear proteins. Some specific proteins present on EVs such as Alix, TSG101 and tetraspanins (transmembrane proteins) CD9, CD63 and CD81 can serve as characteristic biomarkers for the identification of EVs. It is noteworthy that some organelles and their associated proteins may also be enriched in EVs under certain specific conditions. For example, some EVs selectively package mitochondria as well as mitochondria-related proteins or nucleic acids and subsequently become mitochondria-rich EVs Figure 3). Through binding of the specific ligand from EVs  and their corresponding affinity receptors on the target cell surface , EVs can dock on the target cell surface and further trigger intracellular signaling pathways via specific receptor-ligand interaction EVs typically interact with the membrane of target cells in three ways once released extracellularly: receptor-ligand interaction, endocytosis, or membrane fusion to internalize EVs  EVs are nano-to-micro sized extracellular vesicles distributed in various complex fluid environments e.g., cell debris and organelles) from cell culture media or biological fluids containing exosomes by numerous cycles of centrifugation with speed from 300 ×g to 100,000 ×g at 4 °C Among these separation and purification techniques, differential ultracentrifugation and density-gradient centrifugation are the two most widely used traditional approaches and are considered the “gold standard” for EV isolation. Differential ultracentrifugation stepwise removes different components , physicochemical or biological variations of the EVs , and even additional field forces , which typically require low sample consumption and fast processing time. Attractively, the microfluidic technique is more suitable for the quantitative detection of EVs, holding great promise for clinical disease diagnosis due to a handful of starting materials and high sensitivity Apart from the above-mentioned common EV isolation techniques based on different sedimentation coefficients, densities or molecular weights, some new techniques for efficiently and specifically obtaining EVs have emerged. Immunoaffinity capture is mainly based on specific binding between specific biomarkers such as surface antibodies of EVs and antibody-recognized ligands immobilized on beads or filters, in which additional elution procedure is required to discard unbound fraction and collect the desired bound EVs. Such method is characterized by high specificity, purity and high cost at the same time Of note, on the basis of the inherent pros and cons of each EV isolation technology, there is currently no specific EV separation technique that is considered to be suitable for all research Figure 5), eventually ameliorating the adverse effects of diseases and promoting regenerative functions As key molecules for intercellular communication, EVs extensively mediate the exchange of information in both physiological and pathological states of cells, which influence the function of recipient cells et al., EVs derived from human bone marrow mesenchymal stem cells (BM-MSCs) can effectively alleviate inflammation in a mouse acute lung injury model by down-regulating the expression of macrophage inflammatory protein-2, which may be related to the expression of keratinocyte growth factor (KGF)-mRNA in the injured site  et al. have demonstrated that the levels of transforming growth factor (TGF) β1, pentraxin 3, let-7b-5p within EVs greatly affected the immune response in T cell receptor (TCR)- or Toll-like receptor 4 (TLR4)-stimulated splenocytes, and mediating the therapeutic effects of MSC-EVs for the treatment of ocular Sjög-ren's syndrome Until now, numerous research groups have disclosed the immunomodulatory effects of EVs in various disease models in vitro and in vivoAs is well-documented, apoptosis plays a resistance role in tissue regeneration following injury such as ischemia et al. reported that exosomes derived from MSCs can positively activate AKT, Erk, and sata3 signaling pathways in wound healing and induce the expression of growth factors, such as HGF, NGF, IGF-1, and stromal-derived growth factor-1 (SDF1), thereby promoting the proliferation and migration of vascular endothelial cells to form new blood vessels, eventually accelerating skin healing via miR-210 in the mouse myocardial infarction model, which might be associated with the inhibition of the Efna3 signaling pathways et al. have demonstrated the potential of exosomes derived from macrophages could inhibit inflammation and accelerate the healing of diabetic wounds in the skin defect model rats by synergistically inhibiting the secretion of IL-6 and TNF-α, inducing the proliferation and migration of endothelial cells, as well as promoting angiogenesis of diabetic wounds It has been well-documented that angiogenesis is a crucial process in promoting tissue repair via adaptive regulation of the Nrf2 defense system et al. demonstrated that EVs derived from young human donor fibroblasts exhibited a glutathione-S-transferase activity, which can ameliorate senescence-induced tissue damage of old mice by increasing the old cells' antioxidative capacity, further promoting physical recovery In addition to the above-mentioned potential therapeutic mechanisms related to native EVs as regenerative entities, EVs can also affect oxidative stress. Overproduction of reactive oxygen species (ROS) and resultant oxidative stress are closely associated with the pathogenesis of various diseases by breaking the redox homeostasis Table 2). Among these cargos, miR-21, IL-10 and VEGF-mRNA within EVs have been considered to play a major role in tissue regeneration Collectively, tissue regeneration is a cascading and progressive process, which is a comprehensive result of multiple elements rather than relying on the promotion or suppression of a single pathologic factor. In general, the regenerative performance of EVs is superimposed, which may be involved in immunomodulatory, cell proliferation and anti-apoptosis, tissue angiogenesis, antioxidation, and anti-inflammation at the same time. In this regard, we summarized the regenerative mechanisms of specific components in EVs from the perspective of EVs-containing cargos  in different disease models  on the surface of EV-secreting donor cells via metabolic glycoengineering by metabolic glycoengineering (MGE) using unnatural sialic acid tetraacetylated N-azidoacetyl-D-mannosamine (Ac4ManNAz). Combined with the bioorthogonal copper-free click chemistry, dibenzocyclooctyne (DBCO)-terminated hyaluronic acid was covalently edited on the resultant EV surface to specifically target the CD44-overexpressing cells, which enables a prolonged blood circulation and favorable targeting ability of the modified EVs in both rheumatoid arthritis and tumor mouse models Besides genetic engineering, EV donor cells can also be modified by a chemical engineering strategy, which commonly combines metabolic engineering and clicks chemistry A final endogenous strategy for straightforward EV functionalization is the physical engineering method where the content of specific cargo in EVs could be changed by direct physical force to the donor cells or regulation of cell culture conditions. The common physical methods include incubation, electroporation, and extrusion via co-incubation at room temperature Co-incubation by directly co-incubating EVs with various chemical compounds under different conditions, is a simple and inexpensive EV engineering strategy that will not destroy the integrity of the EV membranes and show little effect on their natural functions. Although co-incubation is widely used in EV research, this approach is more suitable for hydrophobic compounds, such as small molecule drugs and specific RNA Electroporation is the most promising strategy for exogenous cargo loading into EVs, in which a high-intensity short-duration voltage-current disturbs the phospholipid bilayer of the EVs and creates small-transit pores on the membrane of EVs. Thus, small molecule drugs or nucleotides can subsequently diffuse into the interior of EVs. The integrity of the membrane is then recovered automatically. Specifically, the electroporation approach is widely used for loading different nucleotides  Apart from co-incubation and electroporation, sonication is the third most wildly used exogenous engineering strategy for EV modification. In this method, the mechanical shear force produced by the sonicator probe breaks the membrane integrity of the EVs and promotes the exogenous cargo to diffuse into the EVs during membrane deformation. There are studies demonstrating that the loading efficiency of sonication is much higher than that of co-incubation and electroporation Mechanical extrusion can be used to encapsulate various exogenous cargos particularly synthetic nanoparticles (NPs) into EVs by extruding the mixture of the cargos and EVs through a syringe-based mini-extruder with polycarbonate membranes of pore size from 200 nm to 400 nm. Briefly, mechanical extrusion deforms the membrane of EVs, further promoting EVs vigorously mixed with the cargo. Currently, the mechanical extrusion method has always been applied to facilitate the encapsulation of synthetic NPs such as IONPs, AuNPs Freeze/thaw cycles are a relatively simple but not very commonly used approach for loading different cargos in EV such as chemotherapeutic drugs and enzymes via either covalent or noncovalent methods In addition to the chemical engineering strategy towards the source cell membrane prior to EV isolation, direct EV membrane modification after EV secretion is another important exogenous approach for EV functionalization Table  To achieve the most satisfying surface modification and additional functionalization outcomes of the modified EVs, several crucial factors should be considered before the selection of the most suitable EV engineering method, such as the type of cargo being engineered, different EV sources, the purity of the modified EVs, as well as the structural integrity and intrinsic bioactivity of the EV itself.In summary, proper engineering methods will result in optimal cargo loading or superb conjugating efficiency in modified EVs and accordingly enhance their targeting capabilities and therapeutic efficacies. The principle, advantages, disadvantages, and applicable molecular types of different engineering strategies for modification of the EVs for biomedicine have been summarized in In light of the above introduction on native and modified EVs, currently, there are two main strategies for the utilization of EVs in regenerative medicine. On one hand, native or natural EVs produced from certain source cells such as MSCs or immune cells can be directly served as potential candidates for regeneration therapies. On the other hand, outside of the inherent therapeutic effects, EV itself can be simultaneously acted as a delivery carrier through engineering various therapeutic agents in EVs to achieve a cascading progressive regenerative effect. Herein, we will briefly review the therapeutic application of different EVs on various organs in regenerative medicine, based on the strategies mentioned above.et al. proved the neurological recovery function of the embryonic stem cell derived small EVs (ESC-sEVs) which were collected by the classical differential centrifugation method in ischemic stroke mouse models  via the TGF-β/Smad signaling pathway, which resulted in reduced neuronal death as well as nerve regeneration after ischemic stroke. This study suggested that the nature EVs could be utilized as a powerful therapeutic tool for brain injuries and other autoimmune diseases.The brain is one of the most important and most complex organs, serving as the center of the nervous system in the human body. Neurological impairments caused by traumatic brain injury (TBI) and different types of neurodegenerative diseases such as ischemia stroke, Alzheimer's disease, and Parkinson's disease in brain organs, are the major reasons for the long-term physical disability of adults, especially the increasing aged people, posing a considerable threat to public health Figure ) In addition to being widely used as native therapeutic agents for neuroregeneration, EVs have also been engineered to transport various cargo for neurological repair via proliferation of the cardiomyocytes, inhibition of cardiac cell apoptosis, pro-angiogenesis, and reduction of infarct size via the enriched miR-486-5p in mice and nonhuman primate MI models high monocyte recruitment through the contained miRNA-24-3p The human heart is a primary organ in the circulatory system, playing a central role in delivering oxygen and nutrients to the tissues through the pumped blood. In recent decades, cardiovascular diseases (CVD) are the top leading cause of death all over the world according to the World Health Organization, causing both health and economic burdens for patients and families et al. have designed a dually-targetable grafted magnetic nanoparticle (GMNP) as the in vivo vesicle shuttle to selectively capture, transport and release circulating therapeutic exosomes with proangiogenic and cardiac repair functions by intravenous injection for ameliorating angiogenesis and heart function . Particularly, the GMNP surface engineered two types of antibody (GMNPEC) via an acid-cleavable hydrazone bonds, in which one antibody anti-CD63 can bind to CD63 antigens on the surface marker of EVs and the other antibody anti-MLC can target to the myosin-light-chain surface markers on the injured cardiomyocytes. By taking advantages of an external magnetic field and the conjugated anti-MLC antibody, the endogenous circulating therapeutic exosomes captured by GMNPEC would be actively accumulated and responsively released in the acidic infarcted heart areas. In both rabbit and rat MI models, the released exosomes in injured cardiac tissue significantly led to reduction in infarct size, promotion of angiogenesis, and improvement of heart functions  in situ cardiac patch formation with intrapericardial injection of biocompatible hydrogels containing EVs to achieve minimally invasive delivery of therapeutics into the pericardial cavity for cardiac repair . The intrapericardial injection-based cardiac patch showed robust cardiovascular repair performance and improved cardiac functions in both mouse MI model and a clinically relevant porcine model The delivery methods of EVs for MI therapy include intravenous, intramyocardial, intracoronary, and intrapericardial injections et al. used exosomes derived from human mesenchymal stem cells (hMSCs) which pre-differentiated for different durations ranging from 4 to 20 days to decorate 3D-printed titanium alloy scaffolds (Ti-scaffolds) for bone regeneration  in vitro osteogenic differentiation of hMSCs and subsequently regenerated bone tissue in the rat radial bone defect model by simultaneously upregulating the osteogenic miRNAs and downregulating the anti-osteogenic miRNAs within the osteogenic exosomes to trigger the PI3K/Akt and MAPK signal pathways.The injuries and defects of bone or cartilage caused by trauma, degenerative diseases, aging, and other factors are extremely common in daily life, such as fractures, bone defects and osteonecrosis et al. applied an extrusion approach to amass the exosome mimetics (EMs) from hMSCs for skeletal repair  via inhibition of miR-29a to enhanced osteogenesis. In addition to being widely used for bone regeneration, numerous studies have recently reported that EVs can also be effective in promoting the cartilage repair and regeneration. Zhang et al. demonstrated that exosomes derived from MSCs could display an efficient osteochondral regeneration outcome by promoting the proliferation of chondrocytes, enhancing matrix synthesis, and modulating a regenerative immune phenotype via exosomal CD73-activated AKT and ERK signal pathways To further improve the regenerative ability and production yield of the hMSCs-derived exosomes, Fan et al. demonstrated that EVs derived from HUC-MSCs can protect liver injury in mice ischemia/reperfusion injury (IRI) model through downregulating CD154 expression on intrahepatic CD4+T cells because the CD154 highly expressed CD4+T cells would trigger the inflammatory response in liver and deteriorate liver IRI. Further experiments proved that Chaperonin Containing TCP1 Subunit 2 (CCT2) within HUC-MSCs-EVs could modulate the calcium channels to reduce the CD154 synthesis on CD4+ T cells, leading to a robust therapeutic potential of UC-MSC-EVs in alleviating IRI in liver  The liver and the kidney are two of the most pivotal organs in human body carrying out various essential functions, including detoxification, excretion of waste, protein synthesis, electrolyte balance, and metabolites production. Currently, acute liver failure has been considered as one of the leading causes of death globally Figure ) Figure ). Based on the above examples, both the native EVs themselves and EVs-engineered scaffolds can be utilized as an advanced regenerative entity for liver or kidney tissue regeneration.To achieve a combined tissue regeneration effect for CKD therapy, Ko's group developed a multifunctional PME/PDRN/TI-EV scaffold consisting of poly (lactic-co-glycolic acid) (P), magnesium hydroxide (M), and porcine kidney extracellular matrix (E), which further engineered with therapeutic components polydeoxyribonucleotide (PDRN) as well as EVs derived from tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ)-loaded MSCs (TI-EVs)  Figure ).Skin injuries are common acute or chronic epithelial tissue injuries often caused by trauma, burns or diabetes. Cutaneous wound healing is a highly programmed process generally involving four steps: hemostasis, inflammation, proliferation, and remodeling phases https://www.clinicaltrials.gov/ct2/, NCT02565264). Another study aims at assessing the safety and efficacy of AGLE-102 in the treatment of lesions in subjects with epidermolysis bullosa, where the AGLE-102 is an allogeneic EV product derived from MSCs of a healthy person. This study is expected to begin in April 2022 (NCT04173650).In recent few decades, with more in-depth research on the biogenesis and functions of EVs, either nature or modified EVs have become promising bioactive nanotherapeutics for the diagnosis and treatment of various diseases et al. have also found that lung spheroid cell-derived exosome can alleviate the symptoms of pulmonary fibrosis caused by bleomycin and silica through reestablishing normal alveolar structure, reducing collagen accumulation, and decreasing myofibroblast proliferation, ultimately promoting lung regeneration Of note, since the novel coronavirus (COVID-19) outbreak began at the end of 2019, the discovery of fatal cytokine storms in patients with moderate to severe COVID-19 diseases has spawned some clinical trials to use EV products for COVID-19 treatment because the EVs can regulate immunity, restore oxygenation, and suppress inflammation. Recently, a clinical trial of MSC-derived exosomes has been carried out to reduce inflammation in critically ill patients with COVID-19, in which the MSC-derived exosomes can greatly inhibit the pulmonary fibrosis pathways (NCT05191381). Actually, as early as in 2020, Dinh Figure 11).The past few decades have shown that the natural cell-derived EVs with fascinating merits including inherent biological functions, low immunogenicity, desirable biocompatibility, as well as ability to realize disease-specific drug delivery, play significant roles in a wide range of physiological or pathological processes as well as a rich variety of diseases treatments, especially in regenerative medicine, which not only affording very powerful tools for diagnostics and therapeutics but also providing new opportunities for clinical translation of the nanotherapeutics. Hence, a large amount of native or engineered EVs have been extensively studied and fabricated for the treatment of various diseases. Although several achievements have been obtained in preclinical trials of EV-based nanotherapeutics, there are still several ongoing scientific challenges and key technical issues that need to be addressed before their further widespread applications and practical clinical utilization , thus leading to their distinct biological functions and subsequent therapeutic outcomes. Particularly, some certain subpopulations with unique morphological and physicochemical features may be more suitable than other subpopulations for specific biomedical applications. Besides, cell passage number as well as cell culture environment can also affect the actual functions of EVs, which substantially determines whether EVs perform a therapeutic or pathogenic effect. For example, EVs generated from early passaged MSCs may have better therapeutic efficacy than EVs generated from late passaged MSCs, while EVs generated from 3D cultured MSCs may be more effective than 2D EVs There is increasing evidence that EVs from different donor cells show distinct physicochemical indexes  EVs cannot respond to the pathological environmental stimuli as stem cells do, leading to a lack of plasticity for EVs in the treatment of various diseases; 2) current immature large-scale production processes, additional isolation procedures and optimal quality control of EVs make them much more expensive than stem cell therapies, greatly hindering the clinical translation of EVs; 3) the clinical studies of stem cells cannot be simply transferred to the stem cell-secreted EVs because the therapeutic mechanisms of EVs in some specific diseases may be completely different from their source stem cells. Although challengeable in principle, with continued advances in expanding knowledge of EV biology and therapeutic mechanism as well as with strong cooperation in multidisciplinary fields of bioengineering, chemistry, material science, nanotechnology, clinical medicine, and industry, we believe that not only more potential in tissue engineering and regenerative medicine of EVs will be discovered, but also it may ignite further explorations and inspiration for clinical applications of safe and efficient nanotherapeutics in various biomedical fields in the near future.


Epigenomics encompasses a broad field of study, including the investigation of chromatin states, chromatin modifications and their impact on gene regulation; as well as the phenomena of epigenetic inheritance. The epigenome is a multi-modal layer of information superimposed on DNA sequences, instructing their usage in gene expression. As such, it is an emerging focus of efforts to improve crop performance. Broadly, this might be divided into avenues that leverage chromatin information to better annotate and decode plant genomes, and into complementary strategies that aim to identify and select for heritable epialleles that control crop traits independent of underlying genotype. In this review, we focus on the first approach, which we term ‘epigenome guided’ improvement. This encompasses the use of chromatin profiles to enhance our understanding of the composition and structure of complex crop genomes. We discuss the current progress and future prospects towards integrating this epigenomic information into crop improvement strategies; in particular for CRISPR/Cas9 gene editing and precision genome engineering. We also highlight some specific opportunities and challenges for grain and horticultural crops. Epigenomics, through its exploration of the combinatorial code of chromatin modifications, can play a role in crop improvement strategies by enriching our understanding of crop genomes and as the molecular basis of traits. The investigation of heritable epigenetic variation that underpins traits in crops is an accelerating area of research with many exciting challenges on the horizon, including better understanding the epigenetic basis of traits, the stability and heritability of epialleles, and the sources of epigenetic variation in crops. These aspects have been reviewed extensively in recent years . In thisteosinte branched 1 (tb1) [VEGETATIVE TO GENERATIVE (VGT1) enhancer of ZmRap2.7 [Crop genomes vary dramatically in size , ranging 1 (tb1)  and the ZmRap2.7 , where gde novo annotate or refine existing genome annotations [Different parts of the genome, such as expressed genes, silent genes, TEs and CREs are marked by different chromatin states. These are defined by the presence or absence of specific covalent histone or DNA modifications. Studying the chromatin states of different genomic elements has greatly advanced our understanding of the roles of various chromatin modifications. However, researchers have noted the power of reversing this approach and using chromatin states to otations ,20,21 orotations . There aThe specificity and quantity of transcription are regulated via the interactions between sequence-specific DNA-binding transcription factors (TFs) and the CREs in promoters and distal enhancers . TranscrMedicago truncatula, rice, tomato [ATAC-Seq (the assay for transposase-accessible chromatin with high-throughput sequencing) is a powerful approach for the identification of CREs. It takes advantage of an engineered Tn5 transposase's preference to insert into accessible chromatin regions . This me, tomato , sorghum, tomato . Signifi, tomato . Additio, tomato , includi, tomato ,28 and M, tomato . These cHistone modifications are an important chromatin modification that can be used to guide genome annotation. Histones are primarily defined by their role in the packing of DNA into chromatin fibers. Nucleosomes represent a barrier to transcription, accordingly, their placement and modifications affect transcription or silencing of particular DNA regions; some modifications are more favorable to transcription. To facilitate these processes, histones are subject to a wide variety of post-translational modifications including methylation, acetylation and phosphorylation . The golThese patterns of chromatin modifications, especially histone modifications, can give insight into the regions of the genome that may be fruitful targets for gene editing. In particular, this information is useful not only to identify functional genes but also to refine gene boundaries and resolve inconsistencies in gene annotations. Mendieta et al. , appliedaccessible gene distal plant CREs are likely characterized by three types of chromatin signatures /CRISPR-associated protein 9 (Cas9) have paved the way for a new era of genome engineering . These tWUSCHEL (WUS) and CLAVATA3 (CLV3) in meristems is to maintain the balance between cell differentiation and self-renewal. In tomato, natural and induced mutations that affect the expression of WUS and CLV3 can positively affect the size of tomato floral meristems that leads to enlarged fruits compared with the wild progenitors [CLV3 has generated a range of new alleles that in turn produce a continuum of phenotypic variation for tomato fruit size [CLE genes [CLE knockout alleles had a deleterious effect on yield, while cis-regulatory editing to change gene expression boosted yield. Other recent work in both rice [In contrast with targeting a coding sequence, there is great potential in using CRISPR/Cas to generate expression variation by targeting gene-regulatory sequences ,57. For genitors . In factuit size  and indeuit size . EngineeLE genes . In thisoth rice  and tomaoth rice  has usedoth rice ; thus, wFWA leading to highly heritable changes in gene expression and flowering time [Epigenome editing might also help address some of the shortcomings of gene knockout. Promoter DNA methylation is strongly associated with decreased gene expression ,65. Receing time . TherefoVERNALIZATION1 (VRN1) affect both flowering, shoot architecture and root architecture in wheat and barley [TEOSINTE BRANCHED1 (TB1) affects both lateral branching and inflorescence architecture in maize and wheat [CLV3–WUS stem cell homeostasis network [Editing gene promoters and CREs may offer the possibility to introduce novel alleles in ways that cannot be achieved by knockout mutations or conventional breeding. This may be extremely useful in some specific situations as outlined in d barley ; likewisnd wheat ,74. Targnd wheat  and the  network , or to gDNA methylation is highly stable throughout most of the vegetative development of a plant, this means that the profile of a single tissue, for example, a leaf could potentially be extrapolated to predict genes and CREs relevant in other tissues or organs ,77–79. IDEMETER-like DNA demethylases mutant, SlDML2, where the resulting phenotype was a non-ripening tomato [In several species, DNA methylation patterns have been observed to change during fruit ripening, including tomato , pepper g tomato . The mecg tomato . In orang tomato , presentDNA methylation patterns in grains also need careful consideration. In developing rice grains DNA methylation profiles fluctuate through grain development as part of the regulatory mechanism for seed maturation . DNA metInstances in which DNA methylation is more dynamic could present issues when using UMRs from vegetative tissues as predictions for CREs in reproductive tissues. One possibility is that predicted CREs might become methylated and no longer function in these tissues. Yet, these regions could still be edited to alter expression patterns in non-reproductive tissues, for example, by removing a repressor element. More likely, the more dynamic nature of DNA methylation in fruits and grains raises the possibility that cryptic CREs could be uncovered in these tissues due to the removal of DNA methylation. Indeed, this is the case in tomatoes, where cryptic binding sites for the transcription factor RIN are uncovered during ripening . The samEpigenome guided improvement is already being deployed for effective modification of traits in crops.The presence, absence and combination of chromatin modifications can be used to locate genes and gene-regulatory regions in crop genomes.The fruits and grains of crop species may represent special cases that require further investigation because patterns of DNA methylation can change in the cells that comprise these reproductive organs.We foresee great potential for epigenome guided improvement in the future, most notably to pinpoint targets for genome engineering to fine-tune gene expression.


Clinicians monitor mechanical ventilatory support using airway pressures—primarily the plateau and driving pressure, which are considered by many to determine the safety of the applied tidal volume. These airway pressures are influenced not only by the ventilator prescription, but also by the mechanical properties of the respiratory system, which consists of the series-coupled lung and chest wall. Actively limiting chest wall expansion through external compression of the rib cage or abdomen is seldom performed in the ICU. Recent literature describing the respiratory mechanics of patients with late-stage, unresolving, ARDS, however, has raised awareness of the potential diagnostic (and perhaps therapeutic) value of this unfamiliar and somewhat counterintuitive practice. In these patients, interventions that reduce resting lung volume, such as loading the chest wall through application of external weights or manual pressure, or placing the torso in a more horizontal position, have unexpectedly improved tidal compliance of the lung and integrated respiratory system by reducing previously undetected end-tidal hyperinflation. In this interpretive review, we first describe underappreciated lung and chest wall interactions that are clinically relevant to both normal individuals and to the acutely ill who receive ventilatory support. We then apply these physiologic principles, in addition to published clinical observation, to illustrate the utility of chest wall modification for the purposes of detecting end-tidal hyperinflation in everyday practice. VT) [PL) that distends the lung but also the airway pressures used to guide ventilation, the important influence of the chest wall is often discounted or ignored altogether outside such clinical extremes as morbid obesity, severe skeletal deformity, or abdominal compartment syndrome.Clinicians usually monitor mechanical ventilatory support using airway pressures—primarily the plateau and driving pressure, recorded during passive inflation. Indeed, the focus of lung-protective ventilation centers on the numerical values of these airway pressures, which are considered by many to determine the risk or safety of the applied tidal volume (VT) . Althougrestricts lung expansion. Doing so for diagnostic or therapeutic purposes is seldom performed in the clinical setting, with several exceptions. Scattered reports, for example, indicate that external compression of the chest wall to alleviate hyperinflation may be a temporizing and life-saving measure for status asthmaticus [Actively limiting chest wall expansion through chest wall loading (applying weights or pressures to its surface) simultaneously hmaticus , 3. Whilhmaticus –6. This reducing PEEP, positioning the torso more horizontally, and loading the chest wall through application of external weights or manual pressures to the body surface [improved compliance of lungs and integrated respiratory system , as mechanical ‘paradox’. Detecting its presence may hold potential for therapeutic interventions.Yet, recent literature describing the mechanics of severe ARDS, primarily in patients with late-stage ARDS secondary to COVID-19, has raised awareness of the potential diagnostic (and perhaps therapeutic) value of doing the polar opposite, i.e.,  surface –14. Even surface . We refeOur purpose in this interpretive review is twofold. We first describe underappreciated lung and chest wall interactions that are clinically relevant to both normal individuals and the acutely ill who receive ventilatory support. We then apply these physiologic principles in addition to published clinical observations to illustrate the utility of chest wall modification for the purposes of detecting end-tidal hyperinflation in everyday practice.Anatomically, the chest wall is formed by two interacting compartments above and below the diaphragm that jointly influence pressures within the pleural cavity. The forces of inflation can be viewed as expanding the ribs while simultaneously depressing the diaphragm against the opposing pressure of the abdomen. Under passive conditions, these compartments unavoidably interact with one another, distending or contracting in relative proportion to their individual compliances, which differ with pathology, body habitus, age, lung volume, body position, and gravitational gradients. Because externally imposed loads on the supine chest wall are applied selectively to only one of these diaphragm-separated compartments (either the rib cage or abdomen), it is helpful to consider the characteristics of each, and how the distribution of compartmental compliances varies with weights, manual pressure and body positioning.CRC) is several times greater than that of the abdomen (CAB) [CCW). Unlike the gas-filled supradiaphragmatic compartment, the volume of the abdomen itself is essentially fixed but may change its shape, diaphragm-bounded position, and flexibility. Chest wall compliance deteriorates with advancing age, due primarily (but not exclusively) to stiffening of the rib cage [CCW changes relatively little for face forward (supine) postures over the range of 0° to 90°, even as the resting lung volume  and partitioned components (CRC and CAB) of CCW change markedly [In healthy normal subjects, the inflation compliance of the rib cage (en (CAB) ; togetherib cage . Interesmarkedly . The posmarkedly .CRC  falls progressively in response increasing compression, whereas CAB does not until the diaphragm is pushed to its tethered limit. Neither extreme of low chest wall compliance is entered under usual clinical conditions when managing ARDS using lung protective strategies. Notably, the aerated volume of the lung in ARDS (the ‘baby lung’) is much lower than that occupied by the infiltrated, edematous entire lung, which tends to remain normally distended at FRC, as does the chest wall. It follows, therefore, that the aerated baby lung and chest wall operate from different points on their respective pressure–volume curves. The inherent compliance of the chest wall in ARDS also depends on the causative disease process, tending to be lower in extrapulmonary lung injury than in pneumonia [The inflation compliance of the normal chest wall remains relatively high and changes little throughout much of the volume range that extends from FRC to total lung capacity . Howeverneumonia .Fig. 1PrCCW) during tidal breathing [By contrast with ARDS, the hyperinflated airspaces of exacerbated asthma and COPD are surrounded by an equally distended chest wall. Estimates made in such patients suggest low inflation compliance of a quasi-normal chest wall that often encroaches on its upper inflection zone , 5 to 8 cmH2O PEEP may be needed simply to offset volume losses of upright FRC associated with this positional change.Gravitational forces exert an important influence on lung volumes, ventilation distribution, and iastinum . FRC deciastinum . If one 2 efficiently [Patients with severe airflow obstruction generally lose much less total volume than do normal subjects of similar age when assuming supine recumbency, in part due to air-trapping that occurs as a result of extensive airway closure through part or all of the breathing cycle ; in suchiciently .2), the expiratory reserve volume  may nearly disappear even when fully upright due to loading of the chest wall by adipose tissue [PL falls below a critical threshold during the tidal deflation phase [In massive obesity  accentuates the tendency for dorsal and peri-diaphragmatic atelectasis and consolidation [Recumbency redistributes lung volume by altering the geometry of the thoracic shell and its contents. When supine and horizontal, the heart and mediastinal contents more directly compress the left than the right lower lobe bronchi. This asymmetric anatomy helps explain the tendency for atelectasis to develop more commonly in the left lower lobe in post-operative and bedridden patients—especially in those with cardiomegaly . Because regions . Consequlidation , 32.Ppl is lower when prone than supine, largely due to positional reshaping of the thoracic cavity and offloading the weight of the heart and mediastinal contents [CCW to decline. Although conversion from the supine to prone position in ARDS is usually accompanied by marginal net changes of total resting lung volume (< 15%), the distribution of compressive regional atelectasis changes significantly. As the larger, well-perfused, and previously compressed dorsal regions become better aerated, oxygenation in ARDS usually improves [The gradient of contents . The supimproves . While limproves  and seveimproves .Ppl increases minimally as intra-abdominal pressures (IAPs) rise over a range from 0 to 10 cmH2O. However, experimental studies conducted in healthy anesthetized pigs indicate that once IAP rise above its upper baseline value of ≈ 10 cmH2O, approximately half of any further incremental increase of IAP transmits to the pleural space at the end of inflation, raising the plateau and driving pressures associated with a fixed VT [Transmission of extrapulmonary pressure increments between the rib cage and abdomen that occur naturally or during chest wall loading is a complex function of body position, lung volume, and the relative distensibility of these compartments. Selective compartmental pressure increases are reflected to a varying extent on the other side of the diaphragmatic barrier . For exafixed VT . Transmifixed VT .Ppl, even at rather high levels of IAP [Ppl are limited by a domed diaphragm stretched to its rib-tethered limits [As IAP rises, contraction of the resting lung volume occurs via the open circuit during the deflation phase of tidal breathing, preventing major increases of end-expiratory s of IAP . Factorsd limits .Ppl increases that originate within the supra-diaphragmatic compartment arise naturally from expansion of the lungs or from large pleural effusions. These generally exert only modest effects on IAP. The rising Ppl that develops during passive inflation, for example, causes IAP to elevate by a smaller amount that varies inversely with the ratio of CRC to CAB.Ppl that originate from loads above the diaphragm exert two actions that may influence cardiac performance:Ppl raises right atrial pressure and tends to decrease venous return as higher intraluminal pressures upstream simultaneously fill the extensive capacitance vessels of the splanchnic bed and lower extremities [Venous return depends on the pressure gradient between the ‘upstream’ mean systemic pressure, influenced heavily by the venous reservoir below the diaphragm, and the central venous (or ‘downstream’) pressure located within the thoracic cavity. Increased remities , 41. Unlremities . Lackingremities .Ppl above the diaphragm also reduce left ventricular afterload, although assuming cardiac contractility is not markedly impaired, this potential benefit is of low magnitude and relatively inconsequential.Selective increases of The effect of chest wall loading on hemodynamics depends on the site—above or below the diaphragm—at which loading occurs. Selective increases of In contrast, the effect of selectively increasing IAP is variable. Modest increases in IAP tend to increase venous return due to a transfer of blood volume from splanchnic capacitance vessels to the central veins , a shiftIn non-acute care settings, binding of the abdomen or chest wall may help improve the efficiency of breathing efforts in patients with certain neuromuscular diseases, for example, diaphragmatic paralysis , 47. BinFor most ICU providers, deliberate reduction of FRC by external compression of the rib cage or abdomen is an unfamiliar and somewhat counterintuitive practice. In specific clinical settings, understanding the physiological principles just reviewed should help the clinician to apply and modulate external loads of the chest wall compartments while monitoring central airway pressure with the intent of improving diagnosis and ventilatory management.CRC, CCW, and CRS [Body positioning, while not commonly perceived as a form of chest wall modification does, in fact, do so, and thereby acts as a powerful complement to regulation of airway pressure for modifying lung mechanics. The fundamentals of prone positioning and its value for ARDS management are well-established . Elevati and CRS .decreased lung and respiratory system compliances  in a specific subset of patients with severely reduced aerating capacity, including those with severe and unresolving ARDS [Although more upright positioning often improves these same compliance components in disease, such positioning may result in ing ARDS –51. Thising ARDS . In thisCRS following placement into a more upright position only occurs if a simultaneous positional decrease in CL outweighs the predictable increase in CCW that arises from less cephalad diaphragmatic pressure , elevations of plateau and driving pressure that occur with upright repositioning are the result of increased end-tidal overdistension prevailing over recruitment. In such patients, more horizontal (flatter) torso positioning will result in decreased plateau and driving pressures as end-tidal overdistension becomes alleviated [Assuming that the abdomen itself is not constricted in the process, any decrease in ure Fig. . In otheleviated .Fig. 2EfCCW, i.e., the slope of its tidal pressure–volume curve). Described methods of weighting include placing saline or sand bags over the sternum or abdomen, usually at supine angles approximating 0° to horizontal [Dominance of end-tidal overdistension may also be revealed by maneuvers that impose external pressure on the chest wall, applied upright or supine. The thorax can be compressed along the ventral-dorsal  plane by weights that externally load the chest wall to displace its pressure–volume relationship rightward  than selective compression applied over the rib cage. Therefore, discounting the local effects of a ‘sternal push’, which might favor decompression of disproportionately overdistended ventral lung units , the ‘be posture . IndepenVT, and degree of lung inflation, loading sufficient to reduce lung volume during tidal ventilation can be easily confirmed by demonstrating a significant rise in the plateau airway pressure during an end-inspiratory breath hold. With total lung and chest wall volumes unchanged, an upward deflection of the plateau pressure during an end-inspiratory breath hold of ≥ 2 cmH2O caused by compressive loading that reverts immediately to baseline following compressive release, indicates the potential of that loading force to reduce end-tidal chest volume when tidal breathing resumes [VT with the first breath following a loading maneuver  during tidal-volume controlled ventilation also confirms the impact of the compression.Precisely how best to conduct these compressive maneuvers is an unsettled question being pursued in ongoing research . Yet, wh resumes , 51, 56,PL and resting lung volume. While their underlying physiologic mechanisms are relatively well-understood, they have not been subjected to rigorous clinical studies that confirm or refute their diagnostic value for guiding safer ventilator adjustments or worth as sustained therapeutic interventions. Nonetheless, solid data regarding compressive mechanics are available that allow prediction of the impact of PL alterations on common problem types encountered in intensive care practice. Patients with ARDS, in particular, are proposed as potential candidates for chest wall modification.All methods of chest wall loading share the unifying characteristic of altering PL may encourage collapse of unstable units in ‘recruitable’ lungs; indeed, when unstable lung units are prevalent, encouraging their collapse by horizontal positioning or chest wall restriction is illogical and contraindicated. Illustrating this point, success in simulating the gas exchanging benefits of prone positioning by sustained ventral chest compression has generally been limited or elusive during the early stages of acute lung injury.In ARDS, tidal closure and re-opening of vulnerable lung units has been identified as a mechanical stimulus for VILI . Indeed,CL by reducing VT and/or PEEP simultaneously attenuates barotrauma risk, ventilating stress, strain, and power [VT strategies are employed [CL (and CRS) but also redistribute the VT and intrapulmonary perfusion. What benefit—if any—accrues to gas exchanging efficiency by relief of end-tidal hyperinflation remains to be defined.In a different micromechanical environment, however , the ‘recruitment first’ priority—and, therefore, the treatment perspective—may change. As injury progresses and worsens over time, lung units become less recruitable, the viable baby lung shrinks in capacity, and the tendency for end-tidal hyperinflation poses greater hazard . Improvind power . In factemployed . When suVT and PEEP presumed to be ‘lung protective’ without it [More horizontal positioning, placement of sternal weights, and active compression of the upper abdomen have been shown to elicit mechanical paradox suggestive of end-tidal overdistension in patients with late-stage ARDS despite the use of thout it , 51, 56.VT affect the entire respiratory system—both thoracic and abdominal compartments in unison. In contrast, all described methods of chest wall modification alter properties of one compartment disproportionately. Although informative reports have detailed specific clinical responses and physiologic observations in selected populations, numerous questions regarding chest wall modification—many with immediate clinical relevance—remain little researched. For example, although best described in severe ARDS (primarily due to COVID-19), the true prevalence of paradoxical responses to chest wall modification among a general population of patients, including those with and without pre-existing chest wall or lung pathology of any type and severity, is unknown. Randomized trials of chest wall modification that test the clinical value and consequences of detection and reversal of end-tidal overdistension are currently lacking.Reductions of PEEP and/or PL in zones of end-tidal hyperinflation, an objective that for an unchanging VT may be met by reducing FRC or by regional stiffening the chest wall. For exploratory diagnostic maneuvers of brief duration, safety concerns regarding chest wall modifications are few. However, when sustained, such interventions with therapeutic intent have the potential to influence lung architecture, distributions of perfusion and ventilation, hemodynamics, and gas exchange.Whether for diagnostic or therapeutic purposes, effective chest wall modification depends on reducing end-inspiratory VT, and body positioning? Even within the same diaphragm-delineated compartment, external compression is applied over a confined surface footprint; just how widely shared that localized volume restraint may be among contiguous and non-contiguous lung sectors is unknown.The relative efficiency of these compression methods, their optimal execution (‘standardization’), as well as safety, patient tolerance, and durations of any resulting benefit need to be determined. Even fundamental physiologic questions remain only partially answered. For example, how does each method of chest wall modification  affect global and regional lung mechanics, hemodynamics, gas exchanging efficacy with variations of PEEP, VT, or body positioning.Because the lung is surrounded by a regionally diverse and multi-compartmental chest wall, reliance on the plateau and driving pressures that accurately characterize the passive respiratory system may not always reflect the internal environment of the lung. Important physiologic and clinical outcome investigations wait to be conducted to standardize, compare, and prove the value  of various chest wall modifications for decision-making and intervention in conditions such as ARDS. Nonetheless, it seems reasonable to propose that detection of unanticipated lung mechanics by a loading maneuver may usefully alert the clinician to reconsider ongoing therapeutic choices for PEEP,


Idiosyncratic drug‐induced liver injury (DILI), although an infrequent entity, was the cause of acute liver failure in 13%–15% of cases in reports from the United States and Sweden , three prospective studies  and one retrospective study evaluating UDCA in DILI treatment were identified. Additionally, a case report, a pilot study, two randomized clinical trials (RCT) and one retrospective study were found evaluating UDCA in DILI prevention. Twenty-four DILI cases and 6 clinical studies reported benefits of UDCA therapy in time required for total bilirubin (TBL) decrease, normalization or reduction of liver test parameters, or avoiding increase in aminotransferases when used in prevention. Unfortunately, we cannot reach a firm conclusion about the role of UDCA in DILI management and prevention due to the limitations in the design of the published studies.In the systematic review of Bjornsson et al. analyzed the literature with regards to the potential role of corticosteroids in DILI management, with most studies analyzing the effects in moderate/severe DILI and suggesting beneficial effects, except for in drug-induced fulminant ALF. The best response was found in patients with drug-induced autoimmune-like hepatitis (DI-AILH). Patients with liver injury due to checkpoint inhibitors (CPIs) also seem to respond to corticosteroids. However, some of these patients recovered spontaneously and some were steroid unresponsive. Similar to what occurs for UDCA, the lack of well-designed controlled RCT does not allow firm conclusions to be drawn on the efficacy of corticosteroids in DILI.Sanabria-Cabrera et al. This systematic review included eleven studies, of which eight were RCT. Two out of three RCT and one observational study concluded effectiveness of NAC in prevention of anti-tuberculosis drug-related DILI. NAC for treatment of non-acetaminophen DILI was part of studies evaluating NAC in ALF of different aetiologies. Among the benefits of NAC were improvement of liver transplant (LT)-free survival, improved liver parameters and reduced length of hospital stays, however, the limitations detected across studies once again prevent clear conclusions.N-Acetylcysteine in non-acetaminophen (non-APAP) DILI prevention and treatment was reviewed by Licata et al. found improved prognosis and reduced mortality when administered within 8–24 h from APAP overdose in 34 studies with almost 20,000 patients. Although NAC dose regimen and treatment length varied among the studies, all showed benefits.With regards to NAC in APAP DILI however, Li et al. They classified hepatoprotective drugs , drugs for cholestasis , immunosuppressants (Glucocorticoids) and specific treatment agents .A general review of agents used for treatment or prevention of DILI was carried out by Randomized clinical trials were only found for evaluations of hepatoprotective drugs. Treatment with magnesium isoglycyrrhizinate (MgIG) in DILI seems to increase normalization rate of ALT and AST when comparing with tiopronin treatment. Bicyclol showed higher reduction of ALT than polyene phosphatidylcholine and was superior to silybin and diammonium glycyrrhizinate in DILI treatment. Sylimarin was observed to improve ALT alterations better than UDCA in DILI in one RCT, however, these results were not confirmed in other RCTs compared with placebo. Therefore, the definite therapeutic effects of these drugs are still to be confirmed by prospective randomized and controlled studies.byBenic et al., twenty-eight articles were included, and among them eight RCT. They found the following agents: bicyclol, Paeoniae radix rubra, calmangafodipir, livina-polyherbal preparation, cytisin amido phospate, picroliv, fomepizole, magnesium isoglycyrrhizinate, plasma exchange, and S-adenosylmethionine. Among them, only bicyclol and MgIG showed low to moderate evidence of liver profile improvement compared to phosphatidylcholine and placebo, respectively.In the systematic review of novel therapies in DILI, Delire et al. reviewed the management of DILI due to Immunotherapy (CPIs). Temporary CPI withdrawal has been proposed in case of grade 2 and 3 hepatitis and permanent discontinuation for grade 4 hepatitis. Corticosteroids can be administered to patients with grade 2 or more severe liver injury. For those corticosteroid refractory cases, second-line immunosuppressive drugs have been proposed (mycophenolate mofetil).There is only limited information about safety in CPIs rechallenge after temporary withdrawal, although it seems an appropriate option in many cases. As a matter of fact, the guidelines for management of liver toxicity due to CPIs are mainly based on expert opinions and case series.Delire et al. (9). Corticosteroids and other immunosuppressants have been used for treatment as well as prophylaxis. The benefit of corticosteroid treatment seems clear, but is also associated with reduced gene therapy activity. Data in this field is very limited and the majority remains investigational.In the context of gene therapy, in clinical trials for haematological diseases incidences of liver toxicity may occur in up to 60% Treatment in DILI remains a challenge. DILI with different phenotypes and different culprit drugs could benefit from a different treatment strategy. Except for NAC in APA-induced liver injury with demonstrated efficacy in the improvement of liver damage, only moderate or very limited evidence of benefits has been shown for other treatment options that have been tried . Further well-designed controlled RCT are needed in order to find therapies that might alter the natural history of DILI.