Real-time transmission electron microscopy of electrochemical reactions is one of the most significant breakthroughs poised to enable radical shift in our knowledge on how materials behave in the electrochemical environment. This review, therefore, summarizes the scientific discoveries enabled by in situ transmission electron microscopy, and specifically emphasizes the applicability of this technique to address the critical challenges in the rechargeable ion battery electrodes, electrolyte and their interfaces. New electrochemical systems such as lithium–oxygen, lithium–sulfur and sodium ion batteries are included, considering the rapidly increasing application of in situ transmission electron microscopy in these areas. A systematic comparison between lithium ion-based electrochemistry and sodium ion-based electrochemistry is also given in terms of their thermodynamic and kinetic differences. The effect of the electron beam on the validity of in situ observation is also covered. This review concludes by providing a renewed perspective for the future directions of in situ transmission electron microscopy in rechargeable ion batteries.An in-depth understanding of material behaviours under complex electrochemical environment is critical for the development of advanced materials for the next-generation rechargeable ion batteries. The dynamic conditions inside a working battery had not been intensively explored until the advent of various  In situ TEM is a powerful tool that helps to understand energy storage behaviors of various materials. This review summarizes the critical discoveries, enabled by in situ TEM, in rechargeable ion batteries, and foresees its bright future for extensive applications. These problems are complicated because of the evolution of battery electrodes, electrolytes and solid electrolyte interphases (SEIs) during battery operation, which cannot be easily investigated under the required sealed working environment. Various methods such as X-ray and neutron scattering, Raman, cathodoluminescence and electrochemical impedance spectroscopy have been developed to characterize the material properties and behaviours in a battery. Among these techniques, transmission electron microscopy (TEM) stands out for high spatial/temporal resolution, superior sensitivity to inhomogeneity and versatile capabilities for a systematic study of material phases, compositions and electronic structures. Therefore, real-time TEM techniques plating and why repetitive cycling leads to capacity fading. Charge storage dynamics and kinetics. Intercalation, alloying and conversion reactions are generally recognized as three mechanisms dominating Li+ storage in battery electrodes+ transport without significant structural change+ and the host element A  with formation of Li–A alloys0 and formation of LiX charge processes in LiMn2O4 cathode were found to exhibit a ‘fringe’ region featuring cubic–tetragonal transition (2O4 nanowires4 (LFP) nanoparticles using in situ liquid TEM alloying process alloying behaviours are discovered. These inspiring findings have greatly improved the development of alloying electrodes, particularly Si-based anode. As such, their structural integrity and capacity retention are enhanced via surface and nanoscale engineering21222324Among various alloying-based electrodes, silicon (Si) is of great interest because of its abundance in nature and high theoretical capacity of 4,200 mAh g process . To date process , Ge  during the conversion of various MX phases2627280 particles were found to be structurally connected with each other inside the LiX matrix, as illustrated in 2  and Cu–Cu1+2O  in CuO  show reversible LiLiX ref. . Despiten 2 ref. . The higref. 3O4 30, MoO3   in CuO 34 and ot2O3 ref.  and Co3O  affect the (de)lithiation behaviours were well explored 36. Additomposite 41, wherein situ TEM has revealed positive roles of defects in enhancing battery reaction kinetics. For example, lithiation featuring stress-driven dislocation plasticity+ transport  could be reasonably generated and controlled during material synthesis.Crystal defects are conventionally expected to degrade battery performance, while ransport 42 were bThermal stability analysis. With the increasing demand for densely packed lithium ion batteries for high-energy density applications, safety has become one of the major concerns. Localized thermal failure could result in chain reactions for large-scale thermal release and even explosion particularly in Co -rich oxides at their charged states. Understanding the mechanisms of thermal failure is thus the prerequisite for effective improvement of battery safety.in situ heating stage in TEM, the thermal decomposition of LixNi0.8Co0.15Al0.05O2 particles up to 450 °C was systematically studied and a layer to rock salt phase transition as well as surface porosity evolution associated with O release are dynamically recorded2, the well-known commercialized cathode material for lithium ion battery, where the O evolution shows facet-dependent feature and is correlated with local-phase transition  upon heatingWith an SEI and Li dendrite growth. Direct observation of the evolution of SEI and Li (de)plating on electrode/electrolyte interface with high spatial resolution is the prerequisite for understanding and improving interface-limited electrochemistry in batteries. Using in situ TEM, the substrate-sensitive Li plating behaviour (ehaviour 46 and th6/ethylene carbonate (EC)/diethyl carbonate (DEC) electrolyte was dynamically recorded at nanoscale resolution plating typically contains liquid-based electrolytes, a sealed liquid cell has been increasingly utilized to mimic the authentic battery reaction environment10solution 49, whereSolid-state electrolyte. Compared to the conventional flammable liquid electrolyte in lithium ion batteries, solid-state electrolyte (SSE) is safe, nonflammable and compatible with metallic Li, and even allows more flexible configurations such as thin film and miniature batteries+ transport property around the interface region. In situ TEM with analytical capability has been intensively applied to dynamically track interfacial Li+ diffusion/migration kinetics, providing in-depth understanding of the ionic transport mechanisms.in situ TEM study, that is, the single nanowire-based battery (2/LiPON and c-LLZO/Li) captured the existence of structurally disordered domains, unit cell-level phase transition and subsequent electronic structure change of transitional metals in the formed interphases  and Li+ (0.76 Å). For example, the lithiation front in SnO2 nanowires features a dislocation-rich zone, making the lithiated SnO2 ductile and free of fracture, while Na+ interact with the host much stronger and results in a softened structure of poor plasticity, high Na+ diffusion barrier and disconnected Sn network, which largely decrease the sodiation kinetics  prior to conversion of WO3 into W and CaO was explicitly revealed at atomic scale3O4 nanocubes, Mg2+ insertion shows no sign of conversion reaction other than Mg plating, while Al3+ insertion is even more sluggish2O3) covering the surface of metallic anodes, as well as the large repulsive forces encountered by the inserted cations.To date, there are few in situ observation should be taken into serious consideration. These effects also vary based on different in situ TEM designs as categorized below.The energy of an incident electron during TEM operation ranges from tens to hundreds of KeVs, which can trigger side reactions in targeted materials and adversely affect the imaging process. The typical negative effects include atomic displacement and e-beam sputtering caused by elastic (electron–nucleus) scattering, and specimen heating/contamination/damage due to inelastic scatteringBeam effect on solid-state open cell design. Within the solid-state open cell, the main concern is the stability of Li2O under electron beam and the subsequent effect on the observed battery electrochemistry. The electron beam was reported to either slow down the electrochemical lithiation , dimethyl carbonate (DMC) and DMC+EC, as well as LiTf in dimethylsulphoxide and LiPF6 in EC/DMC. A per-frame dose of 13.7 e− nm−2 f is sufficient to cause breakdown of LiAsF6-based electrolytes with precipitation of LiF nanoparticles in the solution  under electron beam, other researchers also captured SEI nucleation and growth on Li deposit charge, CV, choronoamperometry and electrochemical impedance spectroscopy inside TEM. Recent reports have shown the possibility of measuring pA-level current flowing through a single nanowire-based batteryThe quantification of nanoscale electrochemistry inside TEM is critical to correlate structural changes with the electrical output at various states of (dis)charge. While most With recent developments in the design of microfluidic and electromechanical chips and TEM column configurations, microscopists now can study electrochemical reactions in more complex environments. For instance, with the introduction of a thin-film heater within liquid cells, it would be quite interesting to study electrochemical reactions at various temperatures to understand the nature and composition of SEI growth as a function of temperature. These studies can also provide new understanding about the origins of thermal failure in rechargeable ion batteries. Another interesting study would be to introduce oxygen or water vapour in environmental TEM equipped with open cell battery holders to track the structural and chemical evolution of SSEs. This can help electrochemists to explore the degradation mechanisms accounting for the failure of solid-state batteries in ambient atmosphere. Including force measurement features in the electrochemical holders can be used to quantitatively explore the mechanical degradation in flexible batteries. Ion irradiation in TEM during electrochemical reactions can help us gain a better understanding of the working environment of delicate batteries widely used in aerospace and nuclear environments. These environments are rich in intensive ion flux, which can potentially result in ionization, atomic displacement, impurity production and energy releasein situ TEM to image the charge distribution in Ge nanowire during normal (de)lithiation91With the advent of new detection and imaging probes in TEMs, innovative windows of opportunities can be opened towards understanding the underlying mechanisms of complex electrochemical reactions. A direct observation of charge distribution under battery-operating conditions is important to assess interior potential distribution. Off-axis electron holography has been recently incorporated with in situ liquid-cell TEM, which can provide systematic information regarding interfacial dynamics and kinetics. However, the introduction of multistimuli and multiprobes into the narrow gap between the two pole pieces within TEM can potentially degrade the imaging resolution and increase the chance of cross-contamination, which will demand a careful design of the hardware configuration.In addition to electrons, signals such as photons and secondary ions can also be generated in TEM. The capability of introducing light (laser) and simultaneously collecting the excited photons from targeted materials has been demonstrated previously for both Raman and cathodoluminescence spectroscopies93in situ TEM community is the reduced spatial/energy resolution due to presence of membranes for microfluidic devices, thick liquid electrolytes or various stimuli along the beam path, which largely decrease the accuracy of imaging or spectroscopy analyses. In particular, the spatial resolution of the liquid cell falls far behind that of traditional imaging, not to mention the elemental and electronic structure sensitivityin situ TEM by reducing the liquid thickness along the electron path, replacing the thick SiN windows with thinner layersin situ observation100One problem plaguing the in situ TEM, it is becoming important to tackle some of the scientific challenges that new battery chemistries are facing. For example, the study of Li–O2 batteries using in situ liquid TEM is very difficult because of the practical difficulty of introducing O2 in the TEM chamber. This complicated system, however, was simplified by flowing an O2-rich electrolyte into the liquid cell in TEM2 and Zn–O2 batteries that have high chance of major breakthroughs. Another interesting chemistry would be the high-voltage aqueous electrolyte Li–ion batteries that have been realized by the ‘water-in-salt’ conceptIn situ liquid TEM experiments can probably replace the currently used organic electrolyte with aqueous electrolytes. This can ease the operational challenges of organic-based liquid electrolytes such as carbon contamination, liquid flow clogging and H2O-sensitive organic electrolyte. The study of multivalent batteries can be pursued with a liquid-cell TEM technique and with a proper choice of electrolytes and electrodes.With everyday progress in the field of in situ TEM, one should realize the limitations of this technique. These include the requirement of delicate specimens with extremely thin thicknesses, minimized contamination and maximized stability, as well as the effect of confined volumes on the electrochemical reactions. With these considerations, it is necessary to combine other complementary methods  with in situ TEM to enable a more comprehensive evaluation of dynamic electrochemical reactions.In addition to several promising directions to be further explored by 2, Li–S and Na–ion batteries) that have been realized by in situ TEM. Compared to other in situ techniques, TEM enables direct visualization and spectroscopy of the electrochemical processes at subnanoscales. The sensitivity of TEM in capturing localized information and inhomogeneity also complements other in situ methods sensitive to bulk information. Significant findings include, but are not limited to, the positive role of crystal defects in charge transport, anisotropic lithiation, phase boundary migration kinetics during cycling, SEI and metallic dendrite evolution in liquid electrolytes, and the key factors limiting the ORR and OER kinetics in metal–air batteries. We foresee that in situ TEM holds great potential to further advance our fundamental knowledge of nanoscale electrochemistry events in rechargeable ion batteries. Along these lines, multidimensional and cross-disciplinary efforts from electrochemists, microscopists and nanofabrication scientists are needed to address the current battery challenges and pave the roadmap for new discoveries in this field. Some of the future directions can encompass (1) performing quantitative electrochemistry at nanoscale; (2) advancing multiprobe analysis during nanoscale electrochemistry; (3) developing new stimuli to study batteries under nonequilibrium environments; (4) enhancing the image resolutions; and (5) exploring the challenges of new battery chemistries beyond Li–ion batteries.To conclude, we have summarized the current scientific discoveries in rechargeable batteries .Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


In recent years, some new processes have been proposed to explain how alcohol may influence behavior, psychological symptoms and alcohol seeking in alcohol-dependent subjects. In addition to its important effect on brain and neurotransmitters equilibrium, alcohol abuse also affects peripheral organs including the gut. By yet incompletely understood mechanisms, chronic alcohol abuse increases intestinal permeability and alters the composition of the gut microbiota, allowing bacterial components from the gut lumen to reach the systemic circulation. These gut-derived bacterial products are recognized by immune cells circulating in the blood or residing in target organs, which consequently synthesize and release pro-inflammatory cytokines. Circulating cytokines are considered important mediators of the gut–brain communication, as they can reach the central nervous system and induce neuroinflammation that is associated with change in mood, cognition and drinking behavior. These observations support the possibility that targeting the gut microbiota, by the use of probiotics or prebiotics, could restore the gut barrier function, reduce systemic inflammation and may have beneficial effect in treating alcohol dependence and in reducing alcohol relapse. Indeed, the anti-inflammatory effect may be one of the many mechanisms by which antidepressants exert their therapeutic effect.17 A meta-analysis has shown that systemic tumor necrosis factor (TNF)α levels decrease over time in treatment-responsive patients, but remained elevated in treatment non-responders.4Over the past few years, numerous studies have supported that inflammation might be important for the development of psychological disorders such as depression,19 Recent evidence suggests the gut microbiota as a new important factor in health and disease, including neuropsychiatric disorders.20 Complex pathways, involving endocrine, immune and neural signaling, mediate the communication between the intestinal bacteria and the central nervous system (CNS), thereby influencing brain function, mood and behavior.21 Changes in the composition of the gut microbiota have been observed in various psychiatric disorders such as autism,23 depression,24 Parkinson’s disease25 and alcohol dependence,27 and interested readers may refer to excellent reviews describing the gut–brain axis and its potent role for mental illnesses.29The origin of inflammation in alcohol dependence and other psychiatric diseases is however not yet clear. Several factors such as psychological stress, early life stress, obesity, diet , oxidative stress linked to ethanol metabolism and alcohol-induced liver steatosis could be involved. Another potential source of inflammation is the gut microbiota, a huge community of microorganisms colonizing the intestine, that interacts with the host and influence many aspects of physiological processes such as body homeostasis, metabolism and immunity.This review will focus on the role of gut microbiota—as an important regulator of the immune response—in the pathophysiology of alcohol addiction. New potential strategies targeting the gut, and not directly the brain, will be suggested to improve health and psychological symptoms of alcohol-dependent patients.31 Overall, animals have a natural aversion for alcohol and do not develop a true addiction as observed in humans. Moreover, the rate of ethanol metabolism and elimination is five times greater in mice than in humans.32 Alcohol administration to rodents  requires artifices that do not directly mimic human drinking patterns, and the damages alcohol causes to the organs in animals represent only a part of the spectrum of that observed in humans . In addition, numerous differences exist in the characteristics of the murine and human immune system, with, for instance, the balance of circulating lymphocytes and neutrophils,34 the numbers of Toll-like receptors (TLRs) expressed at the immune cell surface (13 in rodents and 10 in humans)35 and the species-specific differences in TLR regulation following, for example, stimulation with lipopolysaccharide (LPS),36 a component of Gram-negative bacteria considered a key factor in the development of alcoholic liver disease in rodent models by stimulating the TLR4–CD14 pathway.37 However, to our knowledge, it remains unknown whether differences in gut microbiota composition between mice and humans are consistent with a differential pattern of TLR expression and stimulation. Although imperfect, animal models are still useful if their limitations and weaknesses are understood and taken into consideration. We will hence present, in this review, data obtained in both animal and human studies.Animal models have proved very useful in addressing mechanistic questions on the role of inflammation in alcohol dependence and studies on germ-free (GF) mice have been extremely instrumental in introducing the concept that the gut microbiota may largely influence behavior. However, data obtained in animals should be interpreted with caution especially with regard to the changes in human behavior and the possibility to extrapolate them to human disease should carefully be checked in well-designed clinical studies.38 However, the effects of alcohol on the immune response are largely influenced by the dose and the length of exposure (acute vs binge vs chronic). Although numerous studies have shown that acute alcohol exposure inhibits the pro-inflammatory response in vitro and in vivo,39 chronic alcohol abuse results in the activation of the immune response. Alcoholic patients show a general dysregulation of the immune system, which make them more susceptible to bacterial (pneumonia and tuberculosis) or viral (HIV and hepatitis C) infections.40 They exhibit elevated levels of circulating pro-inflammatory cytokines41 as well as signs of inflammation in various peripheral organs  and in the brain.39Alcohol consumption is known to alter both innate and adaptive immune responses.42 These receptors are located on the cell surface or within the cells and can sense, recognize and bind pathogen-associated molecular patterns that mainly consist of bacterial or viral products such as LPS from Gram-negative bacteria, peptidoglycans (PGN) and lipoteichoic acid from Gram-positive bacteria, flagellin, nucleic acids (DNA and RNA) or damage-associated molecular patterns . The interaction of pattern recognition receptors with their specific ligands activates intracellular signaling pathways resulting in the production of a plethora of cytokines, chemokines and type 1 interferon,44  and humoral immunity (B cells and plasma cells that produce immunoglobulins).45Innate immune cells, comprising monocytes/macrophages, dendritic cells, neutrophils, eosinophils, mast cells and the natural killer lymphocytes, express pattern recognition receptors, which include TLRs and Nod-like receptors.feron,44 . Innate 41 However, the results reported in the literature are highly heterogeneous and even contradictory. To date, there is no consensus on what inflammatory cytokines are actually changed by alcoholism itself. Discrepancy is likely due to coexisting comorbidities, including the stage of liver disease, the nutritional status, obesity, metabolic disorders, age, drug use, methodology used (in vivo vs ex vivo), the status of ethanol intake at the moment of the study (active drinkers vs sobers) all of which could influence the production of cytokines. For instance, monocytes from alcoholic hepatitis patients show a higher spontaneous NFκB activity associated with greater production of cytokines and chemokines compared to healthy controls,48 whereas another study showed spontaneous increased production of pro-inflammatory cytokines in monocytes of alcoholic patients without liver disease but not in alcoholics with liver disease.49 Peripheral blood T lymphocytes are slightly increased and express activation antigens , which correlate positively with alcohol intake in patients that do not suffer hepatitis.50 Despite a lower number of B lymphocytes, these patients usually have higher blood levels of circulating immunoglobulins  possibly reflecting an abnormal regulation of antibody production and/or a manifestation of auto-immunity or molecular mimicry.52 All these data demonstrate an altered immunity in alcoholics with a persistent activation of T cells that may result in an inappropriate immune response to pathogens and impaired host defense. The gastrointestinal tract is the primary site of interaction between the microorganisms and the immune system, and recent evidence supports that disturbances in the bacterial community result in dysregulation of the immune cells.20Numerous studies demonstrated systemic inflammatory changes in alcoholics.21 In line with this assumption, GF mice exhibit multiple spontaneous brain changes including hyperactivity of the hypothalamus–pituitary–adrenal axis,53 leaky blood–brain barrier (BBB) permeability,54 axon hypermyelination55 as well as behavioral changes such as reduced anxiety,56 impaired social interactions57 and cognition.58 The mechanisms underlying the bidirectional communication between the gut and CNS are multiple and highly complex involving immune, neural and endocrine pathways. Below we summarize those for which experimental and human data suggest their potential role in alcohol addiction with a particular emphasis on immune pathways.In addition to influence host physiology, metabolism and immunity, accumulating data indicate that the gut microbiota also communicates with CNS, and thereby influence brain function and behavior.The gut, and more particularly the gut microbiota, is a major source of pro-inflammatory agents that have the capability to stimulate immune cells of target organs.62 and in alcohol-dependent subjects.65 The intestinal barrier is mainly composed of enterocytes, tightly bound to their neighboring cells owing to apical junctional proteins  known as tight junctions and adherens junctions.66 The barrier function is reinforced by a protective mucus layer elaborated by the goblet cells and antimicrobial substances, such as regenerating islet derived (Reg)3b and Reg3g secreted by the Paneth cells, which shape the composition of the intestinal microbiome.67 In addition, numerous immune cells in the lamina propria play an essential role in defending the intestinal mucosa against invading bacteria.66By using different probes and markers, several independent studies have shown an alteration of the gut barrier function, also referred to as ‘leaky gut’, in rodents exposed to alcohol68 NFκB activation,69 upregulation of intestinal circadian clock gene expression,70 overexpression of miRNAs that inhibit tight junctions translation71 and reactive oxygen species production.30 In addition, pro-inflammatory cytokines such as TNFα have been demonstrated to downregulate tight junctions expression and cause disruption of the gut barrier.73 High expression of TNFα in macrophages of the lamina propria has been found in duodenal biopsies of alcoholics.74 Those patients were also characterized by alteration of the thickness of the duodenal mucus layer75 as well as by decreased Reg3g protein expression.76How alcohol, and its primary metabolite acetaldehyde, causes gut leakiness is not yet well-established but multiple mechanisms have been proposed. They include myosin light-chain kinase activation,59 dietary fibers60 or probiotics61 that all modify the composition of the gut microbiota. This indicates that therapeutic strategies targeting the gut microbiome may be effective in the treatment of alcohol use disorders.Accumulating evidence demonstrates that a new factor, the gut microbiota, is involved in ethanol-induced leaky gut. Animal studies have shown that improvement of ethanol-induced gut barrier dysfunction can produce beneficial effects on distant organs. Although data are scarce with regard to the gut–brain interaction, this has been clearly demonstrated for the gut–liver axis where both improvement of intestinal barrier integrity as well as liver injury can be achieved by using antibiotics,77 make possible the identification of qualitative and quantitative microbial changes induced by chronic alcohol abuse. Gut dysbiosis  has been reported in rodents chronically exposed to ethanol80 as well as in the colonic mucosa and fecal samples of alcoholics81  secreted by the bacteria that could also participate in gut barrier dysfunction.Culture-independent next-generation sequencing techniquesholics81 . DysbiosBifidobacterium—that reinforce the intestinal barrier function), or whether alcohol induces gut barrier alteration, which in turn results in enteric dysbiosis .62 whereas ethanol-induced gut dysbiosis occurs later, after 8 or 10 weeks.80 Data in humans suggest a more complex interplay between microbes and gut permeability. Intriguingly, two independent studies have shown that only some, but not all, actively drinking alcoholic subjects, presented with alteration of the gut microbiota,27 which correlated with increased intestinal permeability.27 Indeed, patients with dysbiosis had higher intestinal permeability, whereas patients without microbial alterations did not, despite heavy alcohol consumption.27 In addition, both studies have shown that sober alcoholics still exhibited gut dysbiosis,27 despite a total restoration of intestinal permeability after >2 weeks of abstinence.63 Therefore, it seems that (1) alcohol consumption alone is not sufficient to increase intestinal permeability or to induce gut dysbiosis and (2) dysbiosis without alcohol exposure  can co-exist with a normal gut barrier function. These findings suggest that both alcohol consumption and gut dysbiosis are necessary to induce leaky gut. In line with this hypothesis, recent experiments of fecal transplantation from human alcohol-dependent subjects to GF mice fed with alcohol revealed that the gut microbiota might play a causative role in the modulation of intestinal permeability and in the development of alcoholic liver disease.84 Mice harboring the gut microbiota from a patient with severe alcoholic hepatitis developed greater intestinal permeability, higher bacterial translocation and more severe liver inflammation than mice harboring the gut microbiota from a patient without alcoholic hepatitis, despite the same amount of alcohol consumed. These observations support the hypothesis that the gut microbiota contains pro-inflammatory signals, which likely derive from pathobionts. Although this study brings crucial information in gut–liver axis research, further studies using fecal transplantation are urgently needed not only to confirm a causal relationship between dysbiosis and gut permeability in alcohol dependence but also to better define the role of the gut microbiota in the modulation of neurological processes that ultimately influence behavior of patients.Animal models of chronic alcohol exposure have shown that ethanol-induced leaky gut is a very early event that occurs within 2 weeks of alcohol feeding,Lactobacillus and Bifidobacterium has been shown in animal exposed to alcohol and in alcohol-dependent subjects. Consequently, restoration of these bacteria could represent a potential target to improve alcohol-related diseases. In experimental models of alcoholic liver disease, modulation of the gut microbiota by the use of probiotic Lactobacillus GG or dietary fibers reduces gut leakiness, endotoxemia, inflammation and improves liver function.85 In humans, a 5-day supplementation with probiotics Bifidobacterium bifidum and Lactobacillus plantarum 8PA3 during alcohol detoxification had greater effect on the reduction of liver enzymes than abstinence alone81 and a 4-week administration of Lactobacillus casei Shirota to alcoholic cirrhosis patients improved the neutrophil phagocytic capacity.86 Although these studies tempt to show a benefit in term of liver disease, no data are currently available sustaining a potential benefit of probiotics for brain alterations and psychological symptoms in alcoholics. However, in otherwise healthy subjects, previous interventional studies have demonstrated beneficial psychotropic effects of probiotics with improvement of anxiety and depression,87 cognitive reactivity to sad mood (due to reduced rumination and aggressive thoughts),88 stress-associated abdominal symptoms89 and brain activity.90 In major depressive disorder patients receiving probiotics, improvement of depression scores was associated with a reduction of inflammatory biomarker hsCRP.91 The mechanisms underlying the psychotropic effect of probiotics in humans has not yet been elucidated, whereas in rodents, a strain of Lactobacillus has been shown to decrease depression and anxiety-like behaviors through the activation of the vagus nerve.92Decrease in beneficial bacteria such as 93 They exert their health effects through the production of beneficial metabolites such as short-chain fatty acids  with antimicrobial activity, lower intestinal pH to inhibit pathogen growth such as Proteobacteria, reinforce the colonic defense barrier and exhibit anti-inflammatory properties.94 Prebiotics might be safer and more efficient than probiotics since they have a broad effect on the gut microbial ecosystem and can change the abundance of >100 bacterial taxa.97Faecalibacterium prausnitzii and Bifidobacterium, which are drastically decreased in alcoholics,81 exhibit anti-inflammatory properties99 and their abundance increased after consumption of prebiotics galacto-oligosaccharides or inulin-type fructans in healthy volunteers100 and in obese patients.96 In rats, consumption of prebiotics has been associated with neurochemical changes in the CNS with increased hippocampal brain derived neurotrophic factor and glutamate receptor expression,101 which are involved in the regulation of numerous behaviors including anxiety/depression, cognitive performance and addiction.102Prebiotics are selectively fermented ingredients that result in specific changes in the composition and/or activity of the gastrointestinal microbiota, thus conferring benefit(s) upon host health.103 In rodents, depletion of the gut microbiota by antibiotics induced changes in brain neurochemistry and cognitive impairment,105 and, importantly, modified the behavioral response to a psychostimulant drug.106 In this latter study, oral, but not intraperitoneal, administration of antibiotics resulted in increased sensitivity to the behavioral effects of cocaine, which could be reversed by the administration of short-chain fatty acids. Intraperitoneal injection of minocycline, known to alter neuroimmune and cytokines expression in the brain, was found to reduce ethanol intake but no link with the gut microbiota has been examined in this study.107 Finally, in humans, the use of antibiotics has been associated with an increased risk of depression and anxiety.108Antibiotics are another way to modulate the gut microbiota but they have been associated mainly with negative outcomes. No improvement of endotoxemia or liver function has been shown in alcoholics receiving the broad-spectrum antibiotic paromomycin for 4 weeks.Prospective, randomized, placebo-controlled, well-designed clinical trials are definitely needed to evaluate the effect of modulation of the gut microbiota (by pro- or/and prebiotics) on alcohol dependence and, more particularly, on the psychological symptoms  as well as on the different behavioral aspects of alcohol addiction, such as impulsivity, compulsivity, alcohol-seeking behavior, stress, cognitive and executive functions.41In vivo, ethanol is likely not sufficient to induce the peripheral inflammatory response observed in alcoholics, as elevated plasma pro-inflammatory cytokines are still found after a period of sobriety,9 suggesting that stimuli other than ethanol might challenge the immune system. Furthermore, inflammation is thought to be involved in the development of other psychiatric disorders where alcohol does not play a role.111 The origin and mechanisms contributing to systemic inflammation in alcohol dependence as well as in other neuropsychiatric diseases are not yet fully understood but increasing evidence suggests that the gut microbiota might take part to this process.Alcohol-dependent subjects present with chronic low-grade systemic inflammation as witnessed by elevated plasma levels of TNFα, interleukin (IL)-1β, IL-6, IL-8, IL-10 and hsCRP even in the absence of actual bacterial or viral infection.8 Indeed, LPS receptors TLR4 and CD14 as well as PGN receptor TLR2 expression and activation were found to be higher in PBMCs of alcoholics compared to healthy subjects. In addition to elevated plasma PGN levels, the PBMCs expression of NOD2 (an intracellular receptor that binds the bioactive structure of PGN), was found to be increased in alcoholics. Activation of the transcription factor AP-1 and Nod-like receptor protein 3 inflammasome in PBMCs was suggested to contribute to the elevated plasma levels of IL-1β and IL-8. By contrast, downregulation of TNFα and IL-6 in PBMCs suggests that these two latter cytokines, which are actually increased in the plasma of alcoholics,9 might originate from other sources, like different circulating immune cells  or peripheral organs such as the liver or the gut wall itself that are also directly in contact with ethanol and bacterial components. Detailed analysis of the inflammatory pathways in these two organs and whether they release pro-inflammatory cytokines into the blood in response to alcohol abuse would further enhance our knowledge concerning the origin of systemic inflammation in alcoholics.Peripheral blood mononuclear cells (PBMCs) represent an essential defense barrier against gut-derived bacterial products entering the bloodstream and may therefore contribute to the chronic low-grade systemic inflammation in alcohol-dependent subjects. Mechanistic analyses performed in naturalistic conditions have revealed that LPS and to a higher extent PGN, derived from Gram-negative and Gram-positive bacteria, respectively, can contribute to the activation of PBMCs.112 In brief, this theory supports that peripheral infections lead to the activation of the innate immune system, through the recognition of bacterial or viral compounds by TLRs, and consequently the production of pro-inflammatory cytokines.43 The cytokines are able to reach the brain113 and subsequently induce the three components generally observed during sickness, that is, fever, a neuroendocrine response and behavioral changes such as fatigue, lassitude, inability to concentrate, irritability, loss of appetite and withdrawal from normal social activities. When inflammation persists, as occurs in chronic inflammatory diseases, sickness behavior may transform into depressive symptoms.115 A large body of experimental and clinical evidence supports a causal role for inflammation in the development of various psychiatric disorders. For instance, a substantial proportion of cancer or hepatitis C patients treated with IFN-α and IL-2 develop psychiatric symptoms including depression.117 Patients suffering major depression, who present with high baseline levels of circulating pro-inflammatory markers, showed improvement of depressive symptoms when treated with infliximab, a monoclonal antibody that binds and blocks the actions of TNFα.118 Modulation of the immune system and induction of inflammation during gestation in rodents and primates has also been implicated in the development of autism- and schizophrenia-related behavior.120 Finally, intravenous injection of LPS to healthy humans induced increased serum levels of TNFα, IL-6 and cortisol, which were associated with depressed mood, increased anxiety and decreased memory performance.121The induction of behavioral symptoms by peripheral inflammation is a pivotal element of the sickness behavior theory.122 Very often, affective disorders precede the onset of alcohol addiction. Importantly, even if a proportion of alcoholic patients are not diagnosed for mood or anxiety disorders, their levels of depressive and anxious symptoms are significantly higher than in healthy subjects and that is why anxious and depressive symptoms, craving, as well as cognitive dysfunction are considered important (neuro)psychological markers of addiction severity. Pro-inflammatory circulating cytokines were found to positively correlate with scores of depression, anxiety and alcohol craving9 in alcoholics. The association between inflammation and alcohol craving was confirmed in another study,8 where the improvement of craving scores during short-term alcohol withdrawal correlated with the decrease in IL-1β and IL-8 expression in PBMCs of patients—with IL-8 being considered the best predictor of craving.9 By contrast, the anti-inflammatory cytokine IL-10 was negatively correlated with psychological scores and craving at the end of a 3-week detoxification.9Alcohol use disorders frequently occur with other psychiatric conditions. Almost one-third of alcoholics present with mood disorders  and 37% of the patients do have an anxiety disorder.123 Nevertheless, as negative emotional states and alcohol craving play a crucial role in negative reinforcement, a major factor favoring drug-seeking behavior and relapse,124 these observations highlight the possibility that reducing inflammation could help patients to improve their psychological well-being and subsequently reduce the probability of relapse. Modulation of the gut microbiota by the use of pro- or prebiotics is one potential way to reduce inflammation125 and there is now clinical evidence that probiotic supplementation reduces psychological symptoms, stress and changes brain connectivity in humans. So far, the effect of pro- or prebiotics on addictive behavior has not been investigated.The limitation of these correlational data in humans is that they do not address causality between changes in peripheral inflammation and modifications in craving or alcohol consumption. By contrast, data obtained in rodent models argue in favor of a cause–effect relationship showing, for instance, that genetic deletion of inflammatory genes is associated with a change in alcohol preference and consumption.127 Afferent vagus nerve endings express receptors to IL-1 and prostaglandins,128 and consequently appear to be important for relaying information about the immune status to the brain. Pro-inflammatory cytokines produced or released in blood or peripheral organs  activate vagal transmission and induce de novo synthesis of cytokines in projection regions of the vagus nerve, particularly the nucleus of the tractus solitarius129  by perivascular macrophages and brain endothelial cells that express TLRs and IL-1 receptors. Microglia and astrocytes are part of the brain innate immune system, as they express pattern recognition receptors and can consequently respond to pathogen-associated molecular patterns and damage-associated molecular patterns by producing pro-inflammatory cytokines.134 Repeated exposure to alcohol leads to a long-term activation of microglia and astrocytes that secrete pro-inflammatory cytokines resulting in neuronal damage, cell death and behavioral changes such as anxiety-like behavior and impaired cognitive function.136 By using knockout mice and small interfering RNA, researchers found that activation of TLR4 in microglia and astrocytes following ethanol exposure is crucial to induce neuroinflammation138 and BBB impairment.139 Moreover, peripheral injection of LPS induces long-lasting increase in ethanol drinking,140 suggesting a major role of TLR4 in alcoholic disease. By contrast, a recent comprehensive study across multiple laboratories, using different animal species and different models of drinking patterns, has shown that TLR4 was not a critical determinant of excessive drinking.141Chronic alcohol abuse also activates brain immune cells that results in neuroinflammation and epigenetic changes, which could favor addictive behavior.145 as well as by studies using genetic deletion of immune genes. Indeed, mutant mice lacking chemokine (Ccl2/MCP-1) or cytokine (IL-6) genes or their receptors displayed reduced ethanol preference and consumption.146 By contrast, transgenic mice overexpressing IL-6 showed increased alcohol preference.147 In humans, increased expression of innate immune genes , a danger signal exerting cytokine-like effects) has been shown in the brain of alcoholics collected post-mortem.149The involvement of the brain immune system in the modulation of alcohol consumption and addictive behavior has also been shown by studies reporting upregulation of TNFα, IL-1β, IL-6 and MCP-1 expression in several brain areas of rodents chronically exposed to ethanol,132 In brain slice cultures, TNFα has been shown to reduce glutamate transport,150 thereby increasing extracellular glutamate levels151 that lead to an hyperexcitability state which could inactivate the frontal cortex, with possible influences on mood and cognition.Ethanol exposure could also contribute to the neurobiology of addiction by altering the glutamate signaling through immune mechanisms.152 such as GABA, serotonin and dopamine, which are important regulators of the brain reward circuit. Gut bacteria can also release short-chain fatty acids following the fermentation of dietary fibers. These compounds have neuroactive properties that could directly influence brain function and behavior.153 The tryptophan/kynurenine pathway is regulated by several enzymes tightly controlled by the immune system.154 Under inflammatory conditions, this pathway is activated and tryptophan, the precursor of serotonin, is converted into kynurenine, which in turn is converted into other neuroactive metabolites. Depletion of serotonin and production of kynurenine metabolites that could cross the BBB and exert neurotoxic actions is also one potential means of communication between the periphery and the brain. A study has suggested that the anti-depressant effect of probiotic Bifidobacterium infantis could be due to its modulation of the tryptophan/kynurenine pathway.155 Finally, some bacteria  display molecular homology with neuropeptides , which may result in the production of auto-antibodies that bind both the bacterial proteins and neuropeptides. Auto-antibodies display a dual function depending on their affinity for the peptide and can therefore serve as a peptide carrier or by contrast result in peptide neutralization.52 The potential role of molecular mimicry and auto-antibodies has been suggested in some psychiatric disorders  and correlations have been found between levels of auto-antibodies against neuropeptides and anxiety scores156Additional mechanism, yet incompletely understood, might also be taken into consideration when exploring gut–brain communication . For ins29 Although tremendous amount of experimental and clinical work on ethanol-induced leaky gut, endotoxemia and gut dysbiosis has been performed in the gut–liver axis, very few studies have been dedicated to analyze the effect of gut microbiota on brain and behavior in alcohol dependence, although being a psychiatric disease.Increasing evidence attributes a role for the gut microbiota and gut-derived microbial components as immune modulators that could contribute to the development of gut, liver, systemic and brain inflammation in alcohol dependence. Although this review particularly focuses on inflammation as a way for the gut to communicate with the brain, one has to keep in mind that other pathways involving, for example, the vagus nerve, neurotransmitters and metabolites definitely participate to the complex bidirectional gut–brain interactions.8 However, other important cytokines such as TNFα and IL-6 likely originate from other type cells or other target organs such as the gut or the liver. Analysis of the inflammatory pathways in organs affected by alcohol abuse would help to clarify the potential sources of systemic inflammation in alcohol dependence and finally enhance our mechanistic comprehension of the disease.Circulating blood cytokines have the capacity to convey the peripheral inflammatory message to the CNS eventually resulting in the alteration of mood and behavior including drinking behavior in alcoholics. Recent data obtained in a large cohort of alcohol-dependent patients implicated PBMCs in the release of IL-1β and IL-8 into the blood.157 using human stool sample, from alcoholics or from major depressive disorder patients, transferred to GF mice found that specific dysbiosis contributes to the development of alcoholic liver disease or of depressive-like behavior, also emphasizing the feasibility of using fecal transplantation from human to mouse. As members of the gut microbiota exerting protective function such as F. praustnizii and some strains of Bifidobacterium are found exclusively in human gut microbiota, the use of humanized mice (GF mice receiving human microbiota) might be a more appropriate model to investigate the involvement of the gut microbiota in health and disease, despite several limitations linked to this procedure.158 One is tempted to speculate that the use of humanized mice could better mimic human behavior in relationship to alcohol abuse and microbial changes, especially in the case of addictive behavior such as alcohol-seeking behavior. Nevertheless, we are still convinced that more studies generated directly in humans and well-designed clinical trials need to be carried out to elucidate the complex interactions between the gut, the immune system and the brain and to better reflect the combined effects of excessive alcohol consumption together with all other parameters affected by alcohol dependence. To date, how alcohol induces a leaky gut and intestinal dysbiosis in some, but not all alcoholics, despite similar amounts of alcohol consumed, remains largely unknown. One could hypothesize that initial difference in the microbial composition may exist before the development of alcoholism in some susceptible patients. Only longitudinal prospective studies could answer this question.Bacterial components originating from the intestinal microbiota, concurrently with alcohol exposure, seem to be strong inducers of the immune response. In addition to its modulatory effect of the immune system, the gut microbiota has also the ability to influence brain function and behavior. However, to date, studies almost exclusively base their conclusions on correlations between gut modifications, inflammation and behavioral changes. A formal proof of a direct cause and effect relationships is still lacking in humans. Two recent elegant studies159 will definitely help to explore the mechanisms underlying peripheral immune-to-brain communication in humans.The modulation of gut microbiota—by the use of pro- or prebiotics—and its effect on gut barrier, inflammation and the different behavioral aspects of addiction should be tested in rigorously conducted, placebo-controlled clinical trial. As diet is a major factor influencing the gut microbiota composition, dietary assessments should be carefully reported when performing clinical studies. Potential confounding factors such as obesity, diabetes and inflammatory bowel disease should be excluded. Finally, future studies should be targeted to better analyze the brain inflammatory response in relation with functional changes in the various brain circuits implicated in addiction in humans. Due to obvious ethical reasons, the effect of chronic alcohol consumption on brain inflammation has been exclusively studied in animal models. Therefore, the use of newly developed, inflammatory-specific tracers for functional magnetic resonance imaging or PET scan


Preponderance of proinflammatory signals is a characteristic feature of all acute and resulting long-term morbidities of the preterm infant. The proinflammatory actions are best characterized for bronchopulmonary dysplasia (BPD) which is the chronic lung disease of the preterm infant with lifelong restrictions of pulmonary function and severe consequences for psychomotor development and quality of life. Besides BPD, the immature brain, eye, and gut are also exposed to inflammatory injuries provoked by infection, mechanical ventilation, and oxygen toxicity. Despite the tremendous progress in the understanding of disease pathologies, therapeutic interventions with proven efficiency remain restricted to a few drug therapies with restricted therapeutic benefit, partially considerable side effects, and missing option of applicability to the inflamed brain. The therapeutic potential of mesenchymal stromal cells (MSCs)—also known as mesenchymal stem cells—has attracted much attention during the recent years due to their anti-inflammatory activities and their secretion of growth and development-promoting factors. Based on a molecular understanding, this review summarizes the positive actions of exogenous umbilical cord-derived MSCs on the immature lung and brain and the therapeutic potential of reprogramming resident MSCs. The pathomechanistic understanding of MSC actions from the animal model is complemented by the promising results from the first phase I clinical trials testing allogenic MSC transplantation from umbilical cord blood. Despite all the enthusiasm towards this new therapeutic option, the caveats and outstanding issues have to be critically evaluated before a broad introduction of MSC-based therapies. Inflammatory diseases represent the biggest threat to the preterm infant. They affect all organs including the immature lung, brain, eye, and gastrointestinal tract with extensive and lifelong consequences for the patient's health. So far, efficient therapeutic interventions are restricted to a limited number of drugs and most pathomechanistic insights are available for the inflammatory damage to the immature lung. Therefore, this review is focused on the disease pathology of lung injury and on the therapeutic concepts to protect the immature lung from inflammatory damage. Exogenous mesenchymal stromal cells (MSCs) exert many positive effects on organ development and regeneration  and atteBronchopulmonary dysplasia is the chronic lung disease of the preterm infant which affects more than 60% with a gestational age < 28 weeks in the US and more than 30% of infants < 30 weeks in Europe , 5. BPD The pathogenesis of BPD is caused by the distortion of physiologic lung development in the critical period of the saccular stage. BPD constitutes a multifactorial disease which is caused by the interaction of a plenty of pre-, peri-, and postnatal factors. Being small for gestational age with intrauterine lung growth restrictions caused by placental insufficiency, smoke-induced injury, or diseases emerging during pregnancy, genetic predisposition and the immaturity of the lung with its insufficiency of anti-inflammatory surfactant production represent important prenatal conditions. Pre- and postnatal infections, mechanical ventilation, and oxygen supply are the central triggers of disease pathology with an overwhelming inflammatory reaction in the immature lung which induces or further aggravates lung injury , 12.β, IL-6, IL-8, and TNF-α and the absence or downregulation of anti-inflammatory cytokines including IL-4, IL-10, and IL-13 and of lung growth factors including growth factors such as FGFs, VEGFA, and PDGF-A which are required for further physiologic development of the epithelial, mesenchymal, and endothelial compartments of the lung [α, MIP-1β, and TGF-β1 that augment and sustain the inflammatory reaction [κB signaling which boosts inflammation and inflammatory cell survival and aggravates the distortion of the central pathways of lung development. PDGF receptor α, bone morphogenetic protein, fibroblast growth factors, PDGFR-α, Wnt/β-catenin, retinoic acid, VEGFA, and HIF signaling constitute critical pathways affected [α, HIF-2α, and VEGFA activity [β1-mediated TTG2, PLOD2, and FGF signaling, PDGFR-α-dependent VEGFA action, and myofibroblast and mesenchymal progenitor cell function [β1 [α, and NFκB add further levels of complexity but can explain why most promising preclinical therapies failed to prove superiority in the clinical setting [Characteristic features of lung damage initiation are the overweight of classical proinflammatory cytokines including IL-1reaction . The infaffected , 16. Earaffected . The chaaffected . Furtheractivity . In the function –21. Not function . Histopafunction –26. Of ifunction  and impation [β1 , 29, PDG setting , 28–33  the selection of the most effective cell preparation fulfilling quality criteria which yet have to be defined, (2) the optimal number, time point, and route of MSC cell administration, and (3) the need for validation of clinical criteria to predict the infant at risk benefiting from such a therapy. At present, MSCs represent the most promising candidates for prevention or treatment of BPD and other inflammatory diseases of the preterm infant as MSCs proved therapeutic efficacy in several animal models mimicking BPD, intraventricular hemorrhage, or periventricular white matter injury. In contrast, data on alveolar and endothelial progenitor cells and on epithelial cells from the amniotic fluid  display Experimental data on exogenous and resident pulmonary MSCs suggest that both the application of exogenous physiologic cell preparation and the reprogramming of resident pulmonary MSCs represent highly promising therapeutic interventions. While the therapeutic potential of reprogramming resident pulmonary MSCs is still at the very beginning and limited to in vitro investigations, the application of exogenous MSCs requires thorough studies addressing the concerns raised above. One often ignored argument against exogenous MSC preparation is that these cells do not display all properties inherent to resident pulmonary MSCs and that safety issues must be taken into account concerning potential long-term side effects which require decades of research before definitive answers can be given for the preterm situation. It is clearly established that exogenous MSCs do not possess the capability to produce lung growth factors and cytokines and lung-specific proteins including that of the extracellular matrix and do not have the characteristic profile of upregulated transcriptions factors specific for the lung , 150. SpTaken together, MSC-based therapies represent a highly promising research field to ameliorate or overcome the deleterious effects of overwhelming inflammation in the preterm infant. Despite the tremendous progress in pathomechanistic understanding obtained from animal trials during the recent years and the first promising phase I clinical trials, a lot of research effort is needed to bring the optimized MSC product into the upcoming clinical trials. As we have learned from the multicenter studies from the past, the look at the long-term aspects is of central importance for the overall and sustained success of stem cell-based therapies in the preterm infant.


There are four major types of EB, including EB simplex (EBS), junctional EB (JEB), dystrophic EB (DEB) and Kindler’s Syndrome based on ultrastructural mutation level in tissue of skin and mucosa.2 Patients with EB suffer from many complications of this disease such as infection, deformities of upper and lower extremities, gastrointestinal stricture, dysphasia and odynophagia due to narrowing of the esophagus, widespread skin ulceration that makes them susceptible to skin cancer, urinary tract dysfunction and kidney fibrosis. Itch is another main problem of these patients that can disrupt their normal function.3 Fibrosis of both small and large intestines of these patients can cause malabsorbtion, constipation, failure to thrive and weakness. Fibrosis due to severe skin ulcers and inflammation leads to joint contractures in EB patient and makes them defective and decreases the quality of life in these patients.5Epidermolysis bullosa (EB) is a hereditary genetic disease characterized by varying degrees of skin and mucosa fragility.6 Suppressing the TGF-β via reducing fibrosis can reduce the problems of patents with EB.7Recent in-vivo and in-vitro studies confirm the pivotal role TGF-β in fibroblast proliferation and collagen synthesis leading to fibrosis.9 Because of these effects, this drug is used for the treatment of atopic dermatitis and scleroderma.10 Anti-fibrotic effect of tranilast is shown in myocardial fibrosis in mice with viral myocarditis.11 Anti-cancerogenic effect of this medicine is also determined in-vivo.12Several studies showed that tranilast, N--anthranilic acid, is a mast-cell stabilizing antihistamine and anti-inflammatory and anti-oxidant drug capable of suppressing the TGF-β.Therefore, we conclude that administering this safe drug for patients with EB, specially in DEB and JEB, can mitigate the fibrotic complications of this disease, such as fibrosis of the skin, kidney, esophagus and bowel as well as alleviating the pruritus and preventing the cutaneous cancers. It introduces a novel and very safe medication for a very debilitating disease and can encourage researchers, especially those who have access to this drug, to conduct further trials on this topic.Not applicable.The authors declare that there is no conflict of interests regarding the publication of this paper.


NAC (NAM/ATAF/CUC) proteins constitute one of the biggest plant-specific transcription factor (TF) families and have crucial roles in diverse developmental programs during plant growth. Phylogenetic analyses have revealed both conserved and lineage-specific NAC subfamilies, among which various origins and distinct features were observed. It is reasonable to hypothesize that there should be divergent evolutionary patterns of NAC TFs both between dicots and monocots, and among NAC subfamilies. In this study, we compared the gene duplication and loss, evolutionary rate, and selective pattern among non-lineage specific NAC subfamilies, as well as those between dicots and monocots, through genome-wide analyses of sequence and functional data in six dicot and five grass lineages. The number of genes gained in the dicot lineages was much larger than that in the grass lineages, while fewer gene losses were observed in the grass than that in the dicots. We revealed (1) uneven constitution of Clusters of Orthologous Groups (COGs) and contrasting birth/death rates among subfamilies, and (2) two distinct evolutionary scenarios of NAC TFs between dicots and grasses. Our results demonstrated that relaxed selection, resulting from concerted gene duplications, may have permitted substitutions responsible for functional divergence of NAC genes into new lineages. The underlying mechanism of distinct evolutionary fates of NAC TFs shed lights on how evolutionary divergence contributes to differences in establishing NAC gene subfamilies and thus impacts the distinct features between dicots and grasses. Arabidopsis thaliana genome account for over 90% of the duplications in transcription factors (TFs) and signal transducer gene families , is now recognized as an important evolutionary force in the origin and diversification of plants , ATAF1 and -2, and CUC2 (cupshaped cotyledon) . Compared to the NAC domains, the C-terminal regions of NAC TFs are rather variable  and result in the diversity of the transcriptional activities showed by NAC proteins . The NAC proteins form one of the largest families of plant-specific TFs, with <100 members discovered in Arabidopsis, rice, and other major angiosperm lineages, which probably expanded via WGD and other gene duplication events , whereas only approximate 30 NAC TFs are present in early land plants  , Brassica rapa (release v1.1)   , Sorghum bicolor (release v1.0)  were downloaded from JGI (http://www.phytozome.net/) or EnsemblePlants (http://plants.ensembl.org/index.html) to construct a local BLAST database by using BLAST v2.2.27.Protein-coding transcripts of http://www.ncbi.nlm.nih.gov/sites/cdd; Zhu et al., Multiple query NAC sequences from different lineages were used for each subfamily to obtain comprehensive samples through BLAST searches of our local database. Sequences collected from the same species with DNA identity> 95% and without indels were regarded as possible alleles  when necessary. In order to objectively assess alignment quality, the column scores of each amino acid site were estimated in ClustalX v2.1  and at least six and five members from dicot and grass species, respectively . The ratio of non-synonymous substitutions per non-synonymous site (dN) to synonymous substitutions per synonymous site (dS)  of homologous gene pairs , and equilibrium frequencies for sites in the three rate ratio classes . Comparison between models of rate heterogeneity variation across sites without vs. with switching among substitution rate ratio (M3 vs. M3 + S1) was conducted, and the chi-squared test was employed to estimate the significance of difference.In order to reveal the molecular evolutionary patterns of the COGs within NAC subfamilies, we further conducted likelihood analysis on the same data sets used in Our previous analysis based on 837 NAC sequences of land plants demonstrates a 21-subfamily configuration of NAC proteins, some of which are dicot-/monocot-specific  the ancient duplications which occurred in the MRCA of dicot (dicot-specific duplication) or grass (grass-specific duplication) before their divergence, and (b) the recent duplications which are specific to a particular organismal lineage (one or few closely related species) of dicot (dicot species-specific duplication) or grass (grass species-specific duplication). We separately examined these two types of duplications in each one of the 31 COGs, and summarized the results in Table Glycine max, having the most recent event occurred 13 Mya  have undergone one or more ancient duplications, of which more occurred in grass  than in dicot   and the other involved in the relatively specific processes (category 2)  are significantly higher than that of the corresponding subfamilies composed of the homologs from the dicot; and 11 grass subgroups  show the opposite result Figure .P < 0.05) test in all the phylogenetic trees  as parameters were added to the nested substitution models (Table p1 + p2) are identified to be under purifying selection with ω1 < 0.023 and ω2 < 0.724 in all the COGs (Table p3) of sites within the genes in COGs are identified to be under positive selection with ω3 values considerably larger than 1.0. Among these positive selection sites, the number of sites for genes in COGs of Ib_1, II_2, Va_1, Va_2, and Vb is significantly asymmetric across dicot and grass families, suggesting that the selection mainly occurred either in the dicot or in the grass subclade of these phylogenetic trees. This result provides evidence of divergent selection between dicots and grasses  was observed  uneven constitution of COGs and contrasting birth/death rates among subfamilies, (2) distinct patterns of gene duplication and loss between dicots and grasses, (3) a relatively higher evolution rate in grass than in dicot in most subfamilies analyzed, and (4) asymmetrical selection pattern between dicots and grasses. Clearly, with these findings, a general picture has emerged for the mode, tempo, and consequence of the evolution of NAC TFs during the angiosperm evolution, and several relevant conclusions can now be addressed.B. rapa after its divergence from A. thaliana: one triplication that arose approximately 13 Mya  through 41 populations of wild emmer wheat revealed great diversity in NAM-B1 locus both between and within populations. Meanwhile, the polymorphism of NAM-B1 gene was significantly correlated with multiple eco-environmental factors (unpublished data). Thus, it is possible that the NAM-B1 divergence may contribute to fitness as an adaptive evolution strategy to novel ecological challenges.The above observations guaranteed dicots recruited more members of NAC TFs than monocots but usually with a lower evolution rate within certain COGs/subfamilies. Therefore, two diverse evolutionary scenarios can be proposed, the relatively more copies of NAC TFs in dicots might interact with each other as part of a network through sub-functionalization, on the other hand, with high evolution rate, grass NAC TFs were always accompanied by diversifying selection (positive selection). In fact, the poplar PtrWND TFs (Ic subfamily) interact with each other and perform a diverse combination of heterodimers together with their downstream TFs to form a transcriptional network, thus activating the secondary wall biosynthetic program during wood formation  and seed plants, including gymnosperms and angiosperms (Zhong et al., Arabidopsis, and woody poplar; and the number of SWNs in poplar is nearly twice of Arabidopsis. This increase in the number of SWNs in poplar was probably due to a salicoid duplication event, a WGD event that occurred near the emergence of Salix and Populus lineages 60-65 Mya (Tuskan et al., Arabidopsis counterparts (Zhong et al., Coincidentally, the NAC TFs from Ic subfamily were found to function as master switches in the secondary wall biosynthesis, one of the pivotal features that vascular plants have evolved to enable themselves to conquer dry land around 430 Mya (Raven and Johnson, However, several studies suggest that the number of genes in a family initially depends on their functional requirement. However, after the number reached a sufficient level, the number can fluctuate occasionally Nei, . In our Arabidopsis and rice have revealed NAC genes to be induced by at least one type of abiotic stress such as salinity, drought, cold, or abscisic acid (Fang et al., The whole-genome expression profiling studies in Among the stress-related NAC subfamilies, the Vb subfamily possessed the smallest ω value, with >9 sites evolving under relaxed selection, suggesting that members of this subfamily are more conserved than others. It was shown that members of Vb subfamily tend to function at the convergence point of abiotic stress signaling and pathogen defense that was induced by multiple stresses (Fujita et al., CUCs, all of which belong to Ia subfamily. After the CUC3 gave birth to CUC1/2 through gene duplication event at the base of extant seed plants (Vialette-Guiraud et al., CUCs evolved differentially since the initial duplication. CUC2 did not diverge much from the ancestral gene, while CUC1 evolved more profoundly from its ancestor through neo-functionalization (Hasson et al., Another example emerges from the evolutionary process of In this study, we reported divergent evolutionary patterns of NAC TFs, both among subfamilies and between dicots and grasses. Observations also showed that the distinct fates of NAC TFs are associated with plant lineage diversification and gene duplication events. Considering our results along with functional data, we hypothesize that rapidly born NAC TFs and relaxed selection in certain NAC COGs/subfamilies may have permitted substitutions that are responsible for functional divergence into new lineages. Meanwhile, the high evolution rates have increased complexity of interaction of NAC TFs following the concerted duplications from a MRCA of all the extant angiosperms.XJ and JR were in charge of data collection, analyses, and manuscript drafting and finalizing. EN and XY worked on revising and finalizing the manuscript. JP took care of the whole project, i.e., applying for grants, designing the experiments, outlining and finalizing the manuscript. All relevant authors and institutions have approved the submission for publication of this manuscript; and all persons entitled to authorship have been so named and have agreed to the submitted version of the manuscript.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.