Inflammatory bowel disease (IBD) coincides with profound shifts in microbiota and host metabolic energy supply and demand. The gastrointestinal epithelium is anatomically positioned to provide a selective barrier between the anaerobic luminal microbiota and host lamina propria, with the microbiota and epithelium participating in an intricate energy exchange necessary for homeostasis. Maintenance and restoration of the barrier requires high energy flux and places significant demands on available substrates to generate ATP. It is recently appreciated that components of the microbiota contribute significantly to a multitude of biochemical pathways within and outside of the mucosa. Decades-old studies have appreciated that byproducts of the microbiota provide essential sources of energy to the intestinal epithelium, especially the colon. More recent work has unveiled the existence of numerous microbial-derived metabolites that support energy procurement within the mucosa. It is now appreciated that disease-associated shifts in the microbiota, termed dysbiosis, places significant demands on energy acquisition within the mucosa. Here, we review the topic of host- and microbial-derived components that influence tissue energetics in health and during disease. Numerous studies over the last two decades have revealed these communities of microorganisms as essential for health. Found on both external and internal surfaces, including the skin, gastrointestinal tract, and oral mucosa, the microbiota is intimately integrated with the host and is as unique as our fingerprint. Although the human microbiota comprises of bacteria, fungi, archaea, and protozoa, bacteria vastly outnumber the other microorganisms by orders of magnitude, with the majority residing in the colon. A commonly referenced, initial estimation of the bacterial to human cell ratio in a body was 10:1, although the ratio has since been revisited and updated to ~1:1.2 For instance, an ancestor of modern eukaryotes engulfed an α-proteobacterium capable of oxidative phosphorylation, establishing an endosymbiotic relationship that led to the mitochondria as essential and defining organelles.3 The gut microbiota functions as another organ that resides in the host, albeit of microbial origin. This microbiota consists of organisms of different lineages that communicate with each other and the host, manipulates and redistributes energy, and mediates physiologically vital chemical transformations, providing us with essential functionalities upon which we depend.4 It has long been known that inflammatory bowel disease (IBD) is marked by a state of energy deficiency that involves dysbiosis of the composition and function of the gut microbiota concurrent with a loss of colonic epithelial barrier function.5–7 In this review, we will discuss newer and older literature regarding host- and microbial-derived components that constitute interdependent energy exchanges vital for colonic homeostasis and disease remission.Eukaryotes and microbes have long existed in mutually beneficial, symbiotic relationships with one another, relationships thought as fundamental to the development and evolution of multicellular life.8 Subsequently, the microbiota diversifies and stabilizes under the influence of environment, diet, genetics, and gut physiology into an adult-like composition by the age of three, growing to the highest cell density of any recorded ecosystem.4 Upon community stabilization, gut microbiota differences between individuals are shown to associate with many environmental and lifestyle aspects such as body mass index (BMI), exercise frequency, and, notably, diet.8 Gut microbes presumably have a tight co-evolutionary history with the host, while undergoing selective pressure from microbial competitors and the host alike.9 This environment is one that commonly produces a microbial community in which few groups occur in high abundance with several in low abundance.11 The vastly dominant gut microbial phyla are Firmicutes, Bacteroidetes, and Actinobacteria;12 and although the gut is home to great diversity at the species level, the microbiota is essentially comprised of divergent lineages of closely related bacteria from eight divisions.11The human microbiota develops after birth and is shaped by a number of variables, including gestational age, delivery type, source of milk, weaning period, and exposure to antibiotics.13 By determining what substrates are available for fermentation, diet fundamentally influences microbiota composition based on differential microbial fermentative capabilities, with dietary-induced shifts in bacterial diversity and microbiota-derived end products observed as quickly as 24 h.14 For instance, it is well demonstrated that Prevotella is enriched in individuals with a plant-based diet rich in fiber and simple sugars, whereas Bacteroides dominates in those consuming a Western diet high in animal proteins and saturated fat.15 These dietary-linked microbiota compositions highlight the functional differences between Prevotella and Bacteroides, as Prevotella is more specialized for the degradation of plant material and relatively lacks lipolytic and proteolytic fermentative capabilities, whereas Bacteroides show more specialization for animal carbohydrates and proteolytic fermentative capacity.17 Symbiotic relationships such as that shared between the host and gut microbiota are dependent on the environment, with diet being a substantial environmental variable in this system that facilitates the expansion or restriction of certain microbial populations through selective competition for ingested nutrients.The composition of the gut microbiota is unique to each individual, much like a fingerprint. Numerous microbiome analyses over the years have provided useful insight into the functional capability of the microbiota. One such analysis revealed that the human distal gut microbiota is uniquely enriched in carbohydrate, amino acid, nucleotide, and coenzyme metabolic capability relative to other microbes. Further analyses identified that carbohydrate, nucleotide, amino acid, and energy metabolic pathways were among those enriched in the gut microbiome relative to the human genome, offering insight into the vast functional capability the microbiota can provide the host.18 In this, our microbiota offers us access to nutrients we otherwise could not. In addition to the resistant starch and non-starch polysaccharides mentioned above, a consistent carbohydrate source is provided by host epithelial goblet cells that secrete large amounts of highly glycosylated mucin proteins. The diverse capacity of gut microbes to utilize a wide variety of carbohydrates benefits the microbial community as a whole through cross-feeding, whereby the differential degradation abilities provide mono/oligosaccharides to the entire microbiota, affording a degree of flexibility in maintaining symbiotic function in a perpetually fluctuating nutrient environment.Despite broad fermentative ability, the physiology of the gut microbiota is exceptionally suited for polysaccharide metabolism, reflecting the available resistant starch and non-starch polysaccharides (dietary fiber) gut microbes evolved around and are accustomed to harvesting for fuel in the gut microbiota-occupied niche. Humans produce ~17 gastrointestinal enzymes to mainly digest starch, whereas the gut microbiota produces thousands of complementary enzymes that depolymerize the xylan-, pectin-, and arabinose-containing carbohydrates remaining from consumed grains, fruits, vegetables, nuts, and legumes.19 Current hypotheses on protective mechanisms of prebiotics include changes in the intestinal microbiota and improving the intestinal barrier.19 Several studies showed that prebiotics shift the intestinal microbiota toward a beneficial composition in both animal models and human studies, as they increase the quantity of beneficial bacteria, such as bifidobacteria and lactobacilli, at the loss of disease-inducing bacteria.21Supplementing diet with non-digestible substrates, called prebiotics, for microbial fermentation have been reported to show beneficial effects in both of the IBDs ulcerative colitis (UC) and Crohn’s disease (CD), as well as chronic pouchitis.22 A beneficially functional gut microbiota requires continuous adaptation to the variable nature of food intake, in that the microbiota is capable of processing a range of molecules into the compounds required by the host. As discussed above, this capability is achieved in part through compositional plasticity and cross-feeding, but also through a high level of functional redundancy.24 This functional redundancy is in part due to horizontal gene transfer occurring 25-times more often in gut bacteria than between other bacteria, stimulating the evolution and selection of specific core functions in the intestinal ecosystem.25 Indeed, one of the most prominent findings of the Human Microbiome Project was that although taxonomic composition significantly differs among individuals, the abundances of metabolic pathways are relatively consistent.27 In this, two microbiotas differing in composition at the species and/or genetic level may produce similar protein and metabolite profiles.24 Over the years, attention has shifted from microbiome-based, associative studies toward the mechanistic determination of the molecular interactions between the microbiota and host. An important insight resulting from these studies is that many microbiota–host interactions are mediated by metabolites secreted or modified by the microbiota and/or host.28 A recent study comparing germ-free and colonized mice revealed that such microbial metabolites affect the chemistry of all organ systems, highlighting how the gut microbiota signals to distant organs and influences whole-body metabolism.29Analogous studies of the microbiome at other levels corresponding to active genes (metatranscriptomes), proteins (metaproteomes), and metabolites (metabolomes) are comparatively lacking to microbiota composition studies, but may be more relevant in understanding how the gut microbiota defines and contributes to host function.30–33 Commonly, these GI diseases present a multifaceted pathophysiology involving dysbiosis of the composition and metabolism of the microbiota and intestinal epithelial barrier loss, with this increased epithelial permeability allowing inappropriate immunological stimulation by luminal antigens. In the chronic disease state of UC, these factors perpetuate each other in a convoluted relationship between the dysbiosis-induced loss of microbiota-derived metabolites, dysfunctional epithelial barrier, and unresolving inflammation.34 This intricate relationship motivates the search for therapeutic approaches that address both host processes and the microbiota.35 As epithelial barrier is responsible for creating a habitat that promotes a healthy microbiota, isolating that microbiota from the host immune system, and coordinating crosstalk between the two,37 targeting epithelial barrier restoration may have such a pleiotropic response. To do so necessitates defining the contribution of microbiota-derived metabolites to gut barrier function and the dysbiosis-induced shifts of those metabolites in disease.The importance of intestinal homeostasis to whole-body health is manifested by numerous gut-organ axes, where disease in other organs often occurs with gastrointestinal (GI) diseases. It is appreciated that dysbiosis of the gut microbiota is not limited to IBD and associates with irritable bowel syndrome (IBS), allergies, asthma, metabolic syndrome, and cardiovascular disease.38–40 The microbiota is also a substantial contributor to host energy procurement. This is exemplified in that germ-free (GF) mice lacking a microbiota are lean in comparison to conventionally-raised (CR) mice, with the colonization of GF mice inducing rapid weight gain and increased adiposity.41 The contribution of the microbiota to the human energy requirement is estimated to be 5–10%,42 with a considerable local impact on the large intestine. For example, analyses of the total available energy (TAE), a metric that accounts for the total available chemical energy in a system as ADP, ATP, and phosphocreatine,43 from colon tissue extracts reveal that GF mice have ~55% (p < .001) of the TAE shown in CR counterparts.44 Much of this microbiota-derived energy is devoted to a monolayer of intestinal epithelial cells (IECs) residing at the frontier of the microbiota–host interface that construct and maintain a mutually beneficial, selective barrier between the gut microbiota and host. In doing so, these IECs establish the first line of defense against pathogen infection, but also provide fuel and habitat for microbial symbionts. Two primary differentiated host epithelial cell types are responsible for constructing and maintaining barrier in the large intestine – the goblet cell and enterocyte – with distinct and essential contributions to this key component of gut homeostasis.The GI tract is tasked with managing nutrient and waste flux in a manner that protects the host from luminal pathogenic microbes and antigenic materials. Intestinal homeostasis requires substantial energy input, with the gut devoted ~20% of total cardiac output while consuming 10–20% of the available oxygen.45 In a healthy colon, mucins are continuously secreted at a rate of ~2 – 4 μm/min, creating a flow that physically repels microbes and turns over the mucus barrier hourly.46 The transition from the inner to the outer layer is in part endogenously controlled to assist mucin clearance, and allow mucin to provide lubrication for the fecal stream.47 The prominent secreted mucin is MUC2, which has a high level of O-glycosylation that accounts for ~80% of the total molecular weight of intestinal mucus.48 This glycosylation helps shield the mucin protein backbone from host and microbe proteases and binds the water necessary for gel formation. The integrity of the mucus layer is critical for health, as it is the first intestinal structure that a pathogen must overcome to establish infection. Genetic ablation of MUC2 expression or loss of O-glycans in mice incites spontaneous colitis and sometimes cancer.50 Correspondingly, structural weakening of the mucus barrier is implicated as an early event in UC pathogenesis,51 while patients with active UC have an abnormally penetrable inner mucus layer and altered O-glycosylation profile.53Goblet cells secrete large amounts of mucin proteins to construct a barrier that physically separates the epithelium and microbiota. A healthy colonic mucus barrier has two layers – a dense, stratified inner layer impenetrable to most luminal microbes and an outer, less dense layer containing microbes and dietary material.54 After which, the translation of secretory proteins on the endoplasmic reticulum (ER) is fueled by GTP hydrolysis, followed by numerous ATP consuming processes like protein translocation, folding, post-translational modifications, and trafficking.55 The extensive nucleotide demanding process of O-glycosylation occurs once the mucin reaches the Golgi, requiring nucleotide sugar precursors to sequentially add single monosaccharides.56 Given the high energy requirement for ER function in secretory cells, energy deprivation disrupts protein folding and glycosylation, causing ER stress. To overcome this imbalance in protein folding capacity, ER stress activates a pathway called the unfolded protein response (UPR) that engages transcription factors and enzymes in an effort to reestablish ER homeostasis.57 Notably, several proteins that sense and respond to ER stress are O-glycosylated, which is thought to function to relay the status of the Golgi and secretory apparatus to regulate the UPR.58 The dependency of the ER on energy balance is underscored by exquisite sensitivity and coordination of responses to energy fluctuations. The UPR transcriptionally regulates glucose synthesis and breakdown genes, in addition to having important roles in fatty acid and cholesterol metabolism.59 Furthermore, the ER directly communicates with mitochondria to increase ATP regeneration during times of energetic demand.60 Prolonged ER stress induces inflammation and apoptosis, as demonstrated by mice aberrant in MUC2 oligomerization leading to MUC2 precursor accumulation-induced ER stress, increased apoptosis, and spontaneous colitis.61 The colitis developed in these mice exhibited the decreased goblet cell number, decreased MUC2 production and secretion, MUC2 precursor accumulation, and increased ER stress phenotypes characteristic of UC.62 Interestingly, colonic epithelial defects in the key UPR component X-box binding protein 1 (XBP1) are reported in UC, identifying the ER stress pathway as a common genetic contributor to the disease.63 Given the intimate relationship with and dependence on energy metabolism for ER function and the state of energy deficiency of the colonic mucosa during UC,5–7 it is reasonable that poor energy balance may be a significant contributor to the penetrability of the mucus barrier during disease that must be addressed for healing.The secretion of large amounts of mucin protein is a substantial energy and nucleotide demanding process at multiple levels. Fundamentally, nucleotide templates are required for ribosomal RNA generation and messenger RNA transcription, with the need for nucleotides most prominent during transcription.65 Adherens junctions (AJs) provide the strong adhesive bonds responsible for maintaining cellular proximity and junctional complex stability.66 Loss of AJs disrupts cellular contacts, polarization, and differentiation, while inducing premature apoptosis.67 The AJs are made up of a family of transmembrane proteins called cadherins that form strong, homotypic interactions with molecules from neighboring cells. The cytoplasmic end of cadherins interacts with catenin proteins that link the AJ to the cellular cytoskeleton and a perijunctional actomyosin ring. This AJ system provides the stability necessary for TJ formation and resultant sealing of the paracellular space.Colonic enterocytes contribute to barrier function by forming intercellular adhesion complexes mediated by tight and adherens junctions, termed the apical junction complex (AJC). The most apical tight junctions (TJs) are the primary regulators of paracellular permeability to solutes and macromolecules (gate function), while polarizing the enterocytes into apical and basolateral regions that establish a gradient between the intestinal lumen and basolateral membrane (fence function).66 TJs are multi-protein complexes, with the most important members from the claudin family. Peripheral membrane or scaffolding proteins such as the zona occludins (ZO) are crucial to TJ formation, notably through linking TJ proteins to the actin cytoskeleton. At least two independently regulated means of transport across TJs are identified. The leak pathway allows passage of large solutes, even molecules as large as proteins and proinflammatory bacterial lipopolysaccharides.69 Flux across this pathway is increased by proinflammatory cytokines such as interferon-γ and tumor necrosis factor, which are highly expressed in the chronically inflamed intestine.71 The other pathway represents the canonical association with TJ function, in which small pores formed by claudin proteins exclude molecules larger than 4 angstroms in a charge selective manner.69 Both size and charge selection can be independently or jointly regulated in response to various physiological or pathophysiological stimuli.66 Dysregulation of the TJ and thus paracellular flux is another hallmark of UC.71–73 For instance, a study that measured epithelial resistance as a metric of TJ function revealed an 80% reduction in resistance in samples from UC patients with an inflamed colon.74 Perhaps unsurprisingly, restoration of homeostatic TJ characteristics correlates with quiescent UC and mucosal healing, notably in part through increased expression of the actin associating ZO proteins.75Small molecules, including potential antigens, easily diffuse through the mucus layers. Epithelial paracellular flow is typically more permeable than transcellular flux, conferring TJs as the rate limiters of transepithelial transport and principle determinant of mucosal permeability.76 Much of this energy is devoted to the cytoskeletal components tasked with junctional regulation, which connect to a network that transduces adhesive and mechanical signals from the membrane, into the cell, and back to mediate AJC regulation.78 Accommodating the various cell morphologies and movements that occur in a monolayer requires junctions to be both strong and plastic, a functionality that requires an exceptionally active cytoskeleton rich in actin filaments working to stabilize and cycle junction proteins.79 To this end, the AJC complex is supported by a network of TJ-associating F-actin bundles and the dense circumferential actomyosin ring contiguous with AJs that forms one of the most organized and active actin networks found in nature.80 This ATP-dependent actomyosin ring provides stability and intercellular tension that forces paracellular flux through the TJ,81 while ATP-fueled turnover, or treadmilling, of the F-actin bundles facilitates the extension and contraction of actin filaments to, in part, cycle TJ proteins.82–85 Not only does mucosal healing in UC necessitate TJ reformation and homeostatic regulation, but also epithelial migration for wound closure and cellular polarization. It was demonstrated that T84 model IECs devote nearly 20% of cellular TAE to cytoskeletal activity in such a wound healing scenario.43 Given that these processes require exceptional cytoskeletal capacity and energy supply to drive restoration and maintenance of the AJC, they are likely hindered by the energy-deficient state associated with UC.5–7Studies using ATP depletion models suggest that the AJC commands substantial energy to control paracellular flux.27 Microbes in the gut community compete for nutrients, but also participate in complex cross-feeding relationships in which the excreted product from one strain is the preferred energy source for another.86 Deficiency in one component of this intricate cross-feeding relationship can have extended consequence across the bacterial community, inducing global shifts in microbiota composition and metabolism. Such a deficiency may then deprive the colonic epithelium of metabolites upon which it relies for energy procurement and barrier function. Restoring homeostasis to this multifaceted, interdependent system calls for an equal therapeutic approach. Given that maintenance of energetic relationships is paramount to microbiota and host function in health, understanding and identifying breaks in these metabolite-mediated energy circuits presents a means to address disease.All cells require a source of energy to maintain cellular functions, growth, and reproduction. Fundamentally, the microbiota bioreactor is a system that transforms otherwise-indigestible complex carbohydrates into energy that fuels bacterial communities and the host, producing a remarkable number of metabolites in the process. It is of ongoing interest to define what metabolites are microbial-derived and the responsible taxa.87 This deficit is partially filled by oligosaccharides, but dietary RS are often the single largest contributor to colonic microbial growth.88 These RS are fermented by specialized microorganisms to ultimately produce the SCFAs acetate, propionate, and butyrate. Succinate and lactate are also prominent fermentation products but generally do not accumulate higher than 5 mM in healthy adults, as they are substrates for other bacteria, including propionate and butyrate producers.89–91 Protein fermentation can also occur in the distal colon, as various amino acids can be used to produce SCFAs, but is less favored to carbohydrate substrate and also generates potentially toxic metabolites such as ammonia, indoles, and phenols.89 Such an imbalance between SCFAs and toxic metabolites may contribute to the pathogenesis of IBD and colon cancer.92Humans consume a wide range of complex carbohydrates, many of these dietary polysaccharides endure digestion and pass through the stomach and small intestine. These resistant starches (RS) and non-starch polysaccharides  reach the colon, providing the primary energy source for the system, where they are fermented by the microbiota to short-chain fatty acids (SCFAs). Even though NSP completely resists digestion by intrinsic human intestinal digestive enzymes, its intake may account for only 25% of the calculated production of SCFAs.87 Acetate is a common fermentation product of many gut anaerobes and is also produced by reductive acetogenesis, accounting for the greater accumulation, while propionate and butyrate are produced by distinct bacterial subsets.89 These SCFAs are efficiently taken up by the gut mucosa, with only 5–10% estimated to be excreted in feces, and have significant impact on host physiology as energy substrates, regulators of gene expression, and signaling molecules recognized by specific receptors.93–95 However, these SCFAs are differential in their influences, fate, and tissue distribution. For instance, propionate contributes to gluconeogenesis in the liver while acetate reaches highest concentrations in the blood and primarily transports to muscle.96 This review will center around butyrate production as this SCFA is the most increased by RS consumption,97 the preferential fuel source of the colonic mucosa, and a foundational contributor to colonic microbiota–host energy circuits.Colonic SCFAs exist in molar ratios of approximately 60:20:20 for acetate:propionate:butyrate, with total SCFAs reaching up to 140 mM in the proximal colon.Eubacterium rectale, Eubacterium hallii, and Faecalibacterium prausnitzii as among some of the most abundant and dominant butyrate-producing species, and encompass the primary biosynthetic routes utilized for butyrate production.98 Notably, E. rectale and F. prausnitzii are capable of producing butyrate from the oligosaccharide inulin in an acetate-dependent manner, yet both appear limited in their capacity or are unable to degrade RS.98–100 It is appreciated that the primary degradation of RS is conducted by two currently known species – Ruminococcus bromii and Bifidobacterium adolescentis (and other related Bifidobacterium species).97 As such, these primary degraders are responsible for producing the available mono/oligosaccharides from RS and acetate needed by E. rectale and F. prausnitzii for butyrate production, and therefore are regarded as “keystone species.”101 Accordingly, increases in the abundance of R. bromii by diets supplemented with RS are concomitant with increases in E. rectale and butyrate.99 The human bacterium Ruminococcus champanellensis is a member of this very important keystone group due to an ability to degrade the NSP cellulose.88 Butyrate production from E. hallii is distinguished in that the bacterium utilizes lactate and acetate as substrates.98 Lactate is a common end product of bacterial fermentation produced, among others, by the genera Lactobacillus and Bifidobacterium, which are regarded as key members of the gut microbiota due to their health-promoting capabilities.98 The keystone species B. adolescentis degrades RS to produce lactate in addition to acetate through a unique metabolic pathway to Bifidobacterium named the “bifid shunt,” functioning as a primary degrader for butyrate production by E. hallii.Clostridia are the major butyrate-producing class, and particularly, Bacteroides thetaiotaomicron, Ruminococcus gnavus, Ruminococcus torques, Bifidobacterium bifidum, and Akkermansia muciniphilia are known as capable of partial or full mucin degradation.102 With glycans constituting 80% of the dry weight of mucin and the microbiota able to access this fuel source, it is as though the colonic epithelium itself produces a prebiotic. Since discovery as an abundant member of the microbiota in 2004, A. muciniphilia has gained attention for its therapeutic potential in treating UC.103 The mucin protein backbone has primarily O-glycosylated and some N-glycosylated chains of 2 to 12 monosaccharides of mostly galactose, fucose, N-acetylgalactosamine, N-acetylglucosamine, mannose, and sialic acid.102 Mucin degradation by A. muciniphilia results in the release of oligosaccharides and production of acetate and propionate fueled by an impressive ability to utilize up to 85% of the complex mucin structure as the sole carbon and nitrogen source.102 Because of this capacity to access mucin glycans and cross-feed the microbial community, A. muciniphilia is thought of as a keystone species. Support for this is found through the concomitant enrichment of mucolytic and non-mucolytic butyrogenic bacteria in the mucus environment and was demonstrated by A. muciniphilia supporting the butyrate-producing gut commensal Anaerostipes caccae.104 Furthermore, Clostridium cluster XIVa species, encompassing well-known butyrate producers such as E. rectale and E. hallii, account for nearly 60% of the mucin-adhered microbiota, conferring A. muciniphilia as a local primary degrader with this proximity to the colonic epithelium thought to facilitate butyrate bioavailability.105As mentioned earlier, colonic goblet cells secrete large amounts of highly glycosylated mucin proteins that provide a significant carbohydrate source for the microbiota. Only 106 To maintain focus on microbiota–host energy circuits, the scope of this review will be primarily limited to the role of butyrate in epithelial energy and barrier function.Butyrate has a wide range of influence over cellular processes in the colonic mucosa that include G-protein coupled receptor signaling and histone deacetylase (HDAC) inhibition to regulate gene expression, both of which mitigate chronic inflammatory responses.107 Because most colonic butyrate exists in the dissociated form, apical epithelial transport from the lumen and into the cell is facilitated via several transporters, mainly the SCFA-HCO3− exchange, monocarboxylate transporter isoform 1 (MCT1), and sodium-coupled monocarboxylate (SMCT1) transporters. A steep concentration gradient exists between luminal and systemic butyrate concentrations, with systemic availability of colonic-administered butyrate shown to be 2%.108 This is in part due to different affinities of colonocyte apical and basolateral SCFA-HCO3− exchange transporters for butyrate , through which butyrate is sequestered for energy procurement.108–110 As an energy substrate, butyrate undergoes β-oxidation to form acetyl-CoA, which enters into the TCA cycle to produce the NADH that drives the electron transport chain (ETC) and oxygen consumption to ultimately regenerate ATP. This energetic supply provides critical support to the cytoskeleton and thus barrier, bestowing IECs an unsurpassed capacity to rapidly polarize and form strong AJCs.111 A substantial contribution of butyrate to barrier function stems from ATP provision to the cytoskeleton, but also through upregulating the expression of actin-binding proteins like synaptopodin by HDAC inhibition and activation of other transcription factors like STAT3, SP1, and AMPK that induce genes encoding for TJ components.112Butyrate is the preferential fuel source of the colonic epithelium, with oxidation of this SCFA accounting for over 70% of the cellular oxygen consumption in the distal colon.114 This oxygen depletion stabilizes hypoxia-inducible factor (HIF), a transcription factor that regulates many genes important for intestinal barrier function, such as the TJ protein claudin 1 (CLDN1).115 Oxygen concentrations in colon tissue range from ~1% near the lumen and increase to ~5-10% in the vascularized submucosa and muscle layers.113 The ETC can function at near anoxia and is not limited by intracellular oxygen until levels reach 0.3%.117 Instead, flux through the ETC during hypoxia is attenuated by HIF-dependent and HIF-independent mechanisms, a major benefit being decreased formation of mitochondrial reactive oxygen species that can incur cellular injury.118–121 HIF modulates glucose metabolism by increasing glucose uptake and flux through glycolysis, while shunting the resulting pyruvate from conversion into acetyl-CoA and entrance into the TCA cycle toward lactate production and efflux.122–128 In doing so, butyrate-induced HIF stabilization molds metabolism to confer butyrate as the primary source of acetyl-CoA for the TCA cycle and thus mitochondrial ATP regeneration in the healthy colon. Additionally, the HIF-mediated upregulation of glycolysis also drives ATP regeneration, further promoting epithelial energy balance and function. The energy provided from butyrate oxidation also supports mucin production complemented by the upregulation of MUC2 expression by HIF, providing fuel and habitat for the microbiota and altogether contributing to mucus and AJC barriers  for proliferation and to support the ribosomal RNA generation, messenger RNA transcription, and glycosylation required for sustained mucin secretion.56 This purine nucleotide requisite is met via two pathways – the salvage pathway, which is true to its namesake and utilizes exogenous purine as substrate for nucleotide generation, and the ATP and nutrient-consuming de novo pathway which sequentially constructs purine nucleotides from phosphoribosyl pyrophosphate (PRPP), at the very costly expense of 5 ATP per purine molecule produced. Previous studies have demonstrated that the gut mucosa preferentially salvages purines in lieu of the energy and nutrient-consuming de novo pathway in the presence of available purine substrate.44 This salvage is an efficient, resource-conserving alternative to de novo purine biosynthesis.The purine nucleobase hypoxanthine is a significant product of the gut microbiota that provides a readily available substrate for efficient purine nucleotide biogenesis.44 For example, colonic enterocyte model T84 cells show a substantial decrease in AJC barrier resistance when subjected to hypoxia, an energetically depleting state of oxygen deprivation representative of their natural environment. Hypoxanthine supplementation significantly increased the total available energy (TAE) in hypoxic cells, concomitant with complete recovery of AJC function. Further analyses revealed that the energetic benefit afforded by hypoxanthine supplementation promoted actin polymerization and AJC stability, and increased AJC formation from a depolarized cell state.43 Extended in vivo studies revealed that depletion of microbial purine production through streptomycin treatment significantly decreased colonic tissue purine levels, and that those purine levels could be restored by colonization with streptomycin-resistant, purine-producing bacteria.44 The colonic mucosa was found to be dependent upon this microbiota-sourced purine for energy balance and nucleotide biogenesis during dextran sodium sulfate (DSS)-induced colitis. Colonic tissue depleted of microbiota-sourced purines showed increased ER stress concurrent with loss of inner mucus layer thickness and sterile integrity during the colitic insult, rendering the barrier more penetrable to microbes. Tissue energy balance and the sterile integrity of the mucus barrier was recovered, and ER stress alleviated, by reconstitution of exogenous purine supply through colonization with purine-producing bacterial or oral hypoxanthine supplementation, identifying purines as a limiting substrate in such processes.44 In analogous work, lower amounts of hypoxanthine were recently observed in the stools of IBS patients.131 Notably, the microbiota was found to also use hypoxanthine as a substrate, endowing the metabolite as a cross-feeding substrate. Additionally, IBS patients appeared to have decreased fecal hypoxanthine as a result in part of increased microbial utilization and breakdown, particularly by Lachnospiraceae, and the ensuing purine starvation in the colonic epithelium identified as a potential new mechanism underlying IBS.131 Overall, microbiota-sourced purines appear as substantial contributors to and critical substrates for colonic energy homeostasis, barrier function, and potentially a healthy microbiota, warranting further characterization  and monocarboxylate transporter 12 (MCT12). Tissues with high energy demand are abundant in CrT owing to its unidirectional transport and ability to concentrate intracellular molecules against a gradient, while MCT12 facilitates diffusion.137Creatine supplementation has been long used as means to drive muscle energy production and energetic capacity in athletes and bodybuilders and is the subject of a clinical trial as a potential therapeutic for UC . ATP homeostasis is highly dependent on the efficient action of the creatine kinase (CK) circuit, as a large amount of cellular energy is stored as phosphocreatine. Creatine kinases utilize a large creatine pool (creatine + phosphocreatine) to distribute energy from regions of ATP production to regions of ATP consumption, acting as a temporal and, due to the subcellular compartmentalization of CKs, spatial ATP buffer. Creatine is equally provided physiologically by diet and endogenous production. Biosynthesis of creatine occurs simply through two enzymatic steps in which arginine and glycine are condensed by arginine:glycine aminotransferase (AGAT) to form guanidinoacetic acid (GAA) predominantly in the kidney, then methylation of GAA utilizing S-adenosyl methionine (SAM) by guanidinoacetate methyltransferase (GAMT) to form creatine in the liver, which is released into the bloodstream and taken up in other tissues.134 At this time we are unaware of any studies characterizing a role for MCT12, which may contribute to IEC creatine metabolite exchange with the bloodstream. A murine diet fortified in creatine conferred enhanced AJC barrier resistance and decreased disease susceptibility in experimental models of colitis, with creatine kinases shown to localize to the AJC in support of ATP regeneration and to draw high energy phosphates to the region.132 This work also identified creatine kinases and CrT as HIF targets, further highlighting their importance to epithelial energy homeostasis and function. Moreover, mice deficient in endogenous creatine production due to AGAT mutation show increased AJC dysfunction and disease susceptibility to experimental colitis,135 while an inability to transport and utilize exogenous creatine is demonstrated to impede AJC formation and cells enriched in creatine transport show enhanced barrier formation  is significantly diminished in UC.143 Such a decrease in host butyrate uptake likely results in an under-representation of the extent of fecal butyrate depletion, as decreased MCT1 itself, a target upregulated by butyrate,144 is indicative of decreased microbiota-derived butyrate. This lack of microbiota-derived butyrate coincides with diminished butyrate-producing species, notably F. prausnitzii and Roseburia hominis,145 and members of Clostridium cluster XIVa.146In 1980, Roediger reported that colonocytes obtained from UC patients were deficient in butyrate oxidation and postulated that the disease has a substantial “energy deficiency” component.142 In UC, lactate can accumulate to 100 mM and succinate to 24 mM.90,91 Correspondingly, positive FMT outcomes associate with reinstation of the broken energy circuits contributing to diminished SCFA production and cross-feeding substrate accumulation. For instance, patients who maintain clinical remission show normalized levels of butyrate-producing and lactate-consuming members of Clostridium cluster IV (including F. prausnitzii) and XIVa  with drastic concomitant reduction in fecal lactate levels.147 Furthermore, microbial taxa that discriminated positive therapy outcomes include the keystone species R. bromii and A. muciniphilia that degrade RS and mucin glycans, respectively, to provide mono/oligosaccharides and acetate for the aforementioned butyrate producers, with A. muciniphilia possibly contributing to the remission-associating decrease in fecal succinate.147 Of note, significant fecal metabolite increases in the salvageable purine nucleobases xanthine and adenine were observed upon FMT, indicating shifts in microbial purine metabolism, with sustained remission of UC correlating with decreases in fecal hypoxanthine and xanthine. As reinstatement of mucin production and restoration of the mucus barrier is inherent for the sustained remission of UC and concurrent habitat and fuel for A. muciniphilia and Clostridium cluster XIVa,105 and colonic epithelial utilization of microbiota-sourced purine is instrumental to mucin generation,44 it is intriguing to postulate a fundamental role for microbial purines in positive therapeutic outcomes.Recent fecal microbiota transplantation (FMT) experiments assessing the efficacy of the treatment in UC and associated fecal microbiome and metabolome shifts provide invaluable insights into the compositional and metabolic dysbiosis of the disease state, and the changes from that state accompanying long-term remission, notably in lactate- and succinate-consuming energy circuits. In a healthy colon, lactate is typically found at less than 5 mM and succinate around 1–3 mM because of their function as microbial cross-feeding metabolites for butyrate and propionate formation, respectively.The Prolongation of Life.28 Despite nearly three decades of effort to that end, probiotics have generally failed to live up to their expectations.28 Next-generation probiotics (NGPs) will include bacteria selected for a specific activity and genetically modified microorganisms (GMMs) designed for a specific function to consistently and locally deliver deficient microbiota-derived metabolites in diseases.148 The genetic engineering of probiotic strains offers therapeutic promise by endowing bacteria with beneficial functions to target a specific disease, and such GMMs have been made to treat diseases such as cancer, infections, metabolic disorders, and inflammation.149 Similar GMMs may be generated that are enriched in lactate and/or succinate consumption and SCFA and/or purine production to shift the IBD environment to a more homeostatic state and facilitate healing.Interest in treating human diseases with beneficial microorganisms long predates recent discoveries, originating in a 1910 publication by Nobel laureate Elie Metchnikoff, A lesson derived from the FMT studies is that successful treatment of IBD is multifaceted and variable across individuals. Individually, microbial-derived metabolites such as butyrate and hypoxanthine exhibit specific influences on the gut epithelium, but as a diverse body of bacterial species and influential metabolites coexist in the human intestines, this plethora of microbes and their metabolic functions need to mesh in order to regulate energy metabolism and maintain homeostasis . Success


Although melanoma is the least frequent type of skin cancer, it accounts for the majority of skin cancer-related deaths. Large-scale sequencing efforts have led to the classification of melanoma into four major subtypes . These sequencing studies have also revealed that melanoma genomes are some of the most mutated genomes of all cancers and therefore have a high neoantigen load. These findings have resulted in the development and clinical use of targeted therapies against the oncogenic BRAF→MEK→ERK pathway and immune checkpoint inhibitors for the treatment of metastatic melanoma. Although some patients with metastatic melanoma benefit immensely from these transformative therapies, others either become resistant or do not respond at all. These clinical challenges have intensified the search for new drug targets and drugs that can benefit patients who are either intrinsically resistant or have acquired resistance to targeted therapies and immunotherapies. Numerous signaling pathways and oncogenic drivers can cause changes in mRNA transcription that in turn drive melanoma initiation and progression. Transcriptional regulation of mRNA expression is necessary to maintain cell identity and cellular plasticity via the regulation of transcription factor expression and function, promoter/enhancer activities, chromatin regulators, and three-dimensional genome organization. Transcriptional deregulation can arise due to genetic and/or non-genetic alterations in the genome. Specifically, these deregulated transcriptional programs can become liabilities for melanoma cells due to their acquired dependencies on these programs for survival, which can be harnessed to develop new therapies for melanoma. In this article, we present an overview of the mechanisms that result in the transcriptional deregulation of mRNA expression in melanoma cells and assess how these changes facilitate melanoma initiation and progression. We also describe how these deregulated transcriptional pathways represent new opportunities for the development of unconventional and potentially impactful treatments for metastatic melanoma. Melanoma accounts for over 132,000 cases each year worldwide. The incidence of melanoma is higher in Western counties than in other parts of the world, with the highest incidences occurring in Australia and New Zealand. Basic research to understand the genetics of melanoma and the subsequent translation of fundamental discoveries into clinical applications has resulted in the development and use of several transformative therapies to treat patients with metastatic melanoma. Among those are therapies that target the BRAF→MEK→ERK pathway, and particularly the use of BRAFV600E and MEK inhibitors to treat BRAF-mutant melanoma. The melanoma genome is highly mutagenized and, therefore, has a high neoantigen load, making melanoma significantly more immunogenic compared with other cancers. Therefore, melanoma has emerged as one of the cancer types that is most responsive to immune checkpoint blockage-based immunotherapies.Transcriptional regulation of mRNA expression serves as a key determinant of transcriptome and proteome diversity in mammalian cells. Patterns of mRNA expression determine cell identity and allow cells and tissues to acquire specific functional and phenotypic characteristics, which collectively are necessary for whole-body homeostasis to maintain normal physiology and organismal survival. Genetic or epigenetic alterations in cancer cells can hijack transcriptional networks to attenuate tumor-suppressive transcription and promote oncogenic transcription factor expression and pro-oncogenic gene expression signatures. The deregulation of mRNA expression is one of the characteristics of all cancer cells, and most cancer types can be classified solely on the basis of their mRNA expression patterns , 2. BecaTranscriptome-wide profiling of patient-derived melanoma samples has led to the identification of specific signatures associated with major oncogene subtypes  and prognoses –5. More Melanoma cells can acquire changes in transcriptional pathways and accumulate dysregulated gene expression by a variety of mechanisms. Among these mechanisms are the direct mutations of transcription factors and chromatin regulators or changes in expression that alter their specificity, localization, association with other proteins, and/or promoter occupancy. Similarly, direct mutagenesis of histone proteins and noncoding regulatory genomic regions can create new sites for transcriptional regulators to bind and abolish the old binding sites. In addition, three-dimensional architectural changes can lead to changes in gene expression by altering topologically associated domains (TADs) and long-range DNA/chromatin interactions.In most cases, transcriptional deregulation benefits melanoma cells by allowing them to proliferate under various stressful conditions and thus spread to distant organs. Therefore, deregulated transcription pathway regulators represent unconventional and largely untapped targets for drug-target discovery and drug development. In this review, we provide an account of key drivers of transcriptional alterations of mRNA expression in melanoma, and discuss how the discovery of these drivers provides new opportunities to develop drugs to treat metastatic melanoma.Transcription regulation is a complex biological process that in eukaryotic cells involves a series of regulators, including transcription factors, co-activators, and chromatin regulators. The transcription of coding RNAs (mRNAs) and noncoding RNAs  is deregulated in melanoma. In this review, we will focus on transcriptional deregulation of mRNA expression, which can occur via the following four major mechanisms: 1) genetic or non-genetic modulation of transcription factors; (2) genetic or non-genetic modulation of DNA-modification proteins, chromatin regulatory proteins, or histone proteins; (3) genetic or non-genetic modulation of gene regulatory elements; and (4) chromatin conformation changes  array data were combined with gene expression analysis of the NCI-60 panel of cell lines . In addiTranscription factors can also be regulated as a result of non-genetic changes, such as post-translational modification and/or changes in interacting partners, which in turn govern transcription factor localization, DNA binding, and association with transcriptional co-activator proteins. Consistent with the role of post-translational modification in the modulation of transcription factor function, MITF is post-translationally modified by a number of kinases. For example, c-KIT upregulates MITF expression via MEK/ERK pathway-mediated phosphorylation of MITF at Serine 73 . SimilarAnother transcription factor that is mutated in melanoma is tumor suppressor p53, which is mutated in about 15% of melanomas . MutatioAnother interesting example of a transcription factor with important roles in melanoma is the transcription factor SR-BOX 10 (SOX10), which is characterized by a DNA-binding motif known as the high mobility group (HMG) domain. SOX10 is well known for its role in neural crest and oligodendrocyte development; it becomes upregulated in the pre-migratory cells of the neural crest and is essential for the committed development of neural crest cells into melanocytes , 19. An Another example of the role of SOX10 in melanoma is the finding that ERK phosphorylates and regulates SOX10 sumoylation at lysine 55, which is required for the regulation of its transcriptional activity and target selection in BRAF-mutant melanoma. In particular, this study discovered that SOX10 functions as a regulator of the stemness transcription factor FOXD3. This study also showed that the depletion of SOX10 sensitized BRAF-mutant melanoma cells to BRAF inhibitors . CollectSimilarly, brain-specific homeobox/POU domain protein 2 (BRN2), also known as POU3F2, has been shown to promote melanoma metastasis. Specifically, BRN2 was shown to be a target of the MAPK pathway , which pThe MAPK pathway that is activated in melanoma as a result of mutations in BRAF, NRAS, or NF1 has been shown to regulate the expression, stability, and activity of a number of different melanoma-relevant transcription factors. In particular, sustained MAPK pathway activity can lead to phosphorylation and increased stability of c-FOS . SimilarDNA and chromatin regulators are often mutated or post-translationally modified in melanoma. Two major groups of DNA-modifying proteins have been identified and functionally categorized: DNA methyltransferases (DNMTs) and DNA demethylases. DNMTs catalyze the transfer of a methyl group from S-adenyl methionine to the fifth carbon of cytosine residues to form 5-methylcytosine (5mC) . DNA demAmong the three major DNMTs, DNMT3A and DNMT3B are commonly overexpressed in melanoma , 38. AddLike DNA, histone proteins can undergo post-translational modification. Post-translational changes to histones serve as regulatory signals for transcription. Chromatin regulatory proteins are classified as writers, erasers, or readers based on their activity . Writer Y646) is observed in 3% of human melanomas, and focal amplification of EZH2 was noted in 15 of 262 (5.7%) cases of melanoma in TCGA [Several examples of direct alterations of histone writers, erasers, and readers have been identified in melanoma . For exa in TCGA .A recent example of histone writer deregulation in melanoma is the histone methyltransferase EHMT2 . EHMT2 is a histone methyltransferase that methylates histone H3 on lysine 9 (H3K9me2) . One stuAlthough cancer genome sequencing studies initially focused on genetic mutations in protein-coding genes, recent studies have looked at noncoding regions and discovered intronic mutations and promoter/enhancer mutations that directly impact the transcription of cancer regulatory genes , 54. An In addition to TERT promoter mutations, a less well-described mutation in the succinate dehydrogenase complex subunit D (SDHD) promoter has been observed in melanoma . Unlike One of the prerequisites for mRNA transcription is the ability of transcription factors and activator proteins to access open chromatin. The National Institute of Health (NIH) Roadmap Epigenomics Consortium analyzed 111 reference human epigenomes and an additional 16 samples from ENCODE to uncover how epigenetic processes contribute to human biology and disease . One of The chromatin state is an important predictor of the mutational landscape in cancer cells. The Roadmap Epigenomics Consortium performed a landmark study in which they showed that the cell-of-origin chromatin organization shapes the mutational landscape of cancer. Chromatin accessibility and modification in combination with replication timing explained up to 86% of the spatial variance in mutation rates among cancer genomes . The besMore recently, another large-scale study used the assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) to analyze the chromatin accessibility landscape of 410 tumor samples representing 23 different types of primary human tumors, including 13 melanoma samples . The stuA simplistic view of transcriptional regulation is that it is a locus-specific event with RNA pol II and other transcription factors and machinery assembling to transcribe mRNA. It has become clear, however, that long-range interactions among promoters and enhancers can result in the activation or repression of mRNA expression . RecentlIt is clear that a series of transcriptional regulators and mechanisms are dysregulated in melanoma, resulting in changes in mRNA expression and alteration of the melanoma proteome. However, the identification of alterations in transcription factors or chromatin regulators alone does not provide any information on the functional implications of these alterations in melanoma initiation and/or progression. Therefore, in most cases, further functional validation studies were conducted to establish the importance of discovered alterations in melanoma initiation and/or progression Fig. . One exaIn addition to direct transcription factor action in melanoma progression, DNA methylation-regulating proteins such as DNMTs and the TET family of active DNA demethylases have been shown to play an important role in melanoma initiation and progression , 40, 70.DNMT3B has also been shown to promote melanoma growth. The loss of Dnmt3b in a Braf/Pten mouse model resulted in a dramatic decrease in melanoma formation . The autQ61KInk4a−/− mice [Y641F allele to cooperate with the conditionally activatable alleles BrafV600E and NrasQ61R in the presence of the tamoxifen-inducible tyrosinase-Cre allele (Tyr-CreERT2) in mice [Y641F allele cooperates with BrafV600E alone or in combination with PTEN haploinsufficiency to promote melanoma formation and maintenance. However, the Ezh2Y641F allele did not accelerate NrasQ61R melanomagenesis, with or without the loss of p16Ink4a. Further analysis of previous sequencing results showed that EZH2 mutations in human melanoma co-occur with activating mutations in BRAF (p = 0.006) and are mutually exclusive with NRAS mutations (p = 0.004) [Y641F expression caused global redistribution of H3K27me3. A more recent study showed that EZH2 plays an important role in melanoma genesis and metastasis by silencing genes that are necessary for the integrity of primary cilia [Another example of a functional association between mutation/overexpression and melanoma initiation and progression is the chromatin modifier EZH2. Several studies have shown an important role for EZH2 in melanoma initiation and progression –73. All −/− mice . The eff−/− mice . The sam−/− mice . A secon in mice . This st= 0.004) . In addiry cilia –73. FurtSimilar to EZH2, the histone methyltransferase EHMT2 drives melanoma growth and promotes an immunosuppressive microenvironment by activating the WNT signaling pathway. Melanoma cells with high levels of H3K9me2, a histone modification associated with EHMT2 enzymatic activity, were sensitive to the EHMT2 inhibitor UNC0642, indicating a dependency of EHMT2/G9a-amplified melanoma on EHMT2/G9a . FurtherTERT promoter mutations are present in melanoma; however, it is not yet known how they function to promote melanoma. One possibility is that TERT promoter mutations drive increased TERT expression and increased telomerase activity. The treatment of melanoma cells with BRAF inhibitors reduced TERT expression and telomerase activity, suggesting that the MAPK pathway was necessary for TERT expression in these cells . The samMelanoma is arguably at the top of the list in regard to successful outcomes achieved with next-generation personalized therapies and immunotherapies. However, not all melanoma patients benefit from BRAF/MEK inhibitors or immunotherapies. Therefore, the quest to identify additional druggable targets in melanoma is still ongoing. Chromatin modifiers and transcription factors such as BCL6 have emerged as potential drug targets in various cancers , 76. TheAlthough it is clear that melanoma is highly dependent on transcriptional regulators for initiation and progression, the clinical use of transcriptional regulatory proteins in patients with melanoma is restricted compared with that in patients with other types of cancers, and is thus far largely restricted to HDAC inhibitors and DNMT inhibitors. The substantial activity in the development of clinical drugs targeting transcriptional regulatory proteins in other types of cancers might provide significant clinical opportunities for the treatment of metastatic melanomas that are refractory to currently approved therapies. One major advantage of pursing that line of inquiry is the fact that for several clinical grade inhibitors, such as EZH2 and embryonic ectoderm development (EED) proteins, data on human pharmacokinetics, pharmacodynamics, and toxicity and, in some cases, clinical trial outcomes in other cancer types are already available . Therefo


The current enormous gender disparity in the sciences has drawn considerable attention and sparked widespread debate. Working together, researchers, women scientists who have been discriminated against in scientific recognition and promotions, and supportive journalists and legislators are addressing the question, ‘where are women in the sciences?’ by documenting inequities and exploring root causes. To probe the effectiveness of such efforts in addressing the gender discrepancy, in this paper I analyzed the top 100 Altmetric COVID-19 publications to determine differences between first/last authored male and female papers. The outcomes were striking: the efforts in increasing awareness of the current scientific gender discrepancy among some stakeholders  were initially effective, as there were no significant gender discrepancy in Altmetrics scores, news coverage, discussions on blogs and social media coverage.Unlike conventional scientific metrics , the Altmetrics index is a new way of looking at the impact of scientific papers, providing a wide range of information from various sources, including mainstream media coverage, discussions on blogs and mentions on social media . AnalysiSupplementary Table for details) [I extracted information on the COVID-19 papers with the top 100 Altmetrics scores using Altmetric Explorer  . It is ndetails) . From thThe Altmetric Top 100 papers on COVID-19 included: 65 (65%) men and 35 (35%) women as first authors, 83 (83%) men and 17 (17%) women as last authors; and 58 (85%) men and 10 (15%) women as both first and last authors. These findings strongly support the existence of a gender gap in COVID-19 publications, which agrees with other reports [Working together, researchers and supportive journalists are increasing awareness on the current issues of gender imbalances in science’s backyard . For exaFollowing this seemingly successful initial step in improving gender parity in our scientific backyard, one feasible next step would be to address imbalances in the number of publications across male and female scientists, which requires effective and timely action through the involvement of more influential stakeholders . Gender balance in the number of publications and authorship  is crucial, as women make up a considerable proportion of the scientific, engineering and health workforce  (e.g., ∼Click here for additional data file.


Migration is an energy-intensive, multi-step process involving cell adhesion, protrusion, and detachment. Each of these steps require cells to generate and consume energy, regulating their morphological changes and force generation. Given the need for energy to move, cellular metabolism has emerged as a critical regulator of both single cell and collective migration. Recently, metabolic heterogeneity has been highlighted as a potential determinant of collective cell behavior, as individual cells may play distinct roles in collective migration. Several tools and techniques have been developed and adapted to study cellular energetics during migration including live-cell probes to characterize energy utilization and metabolic state and methodologies to sort cells based on their metabolic profile. Here, we review the recent advances in techniques, parsing the metabolic heterogeneities inherent in cell populations and their contributions to cell migration. Cellular energy utilization and metabolic plasticity can improve cell fitness and regulate disease progression by maximizing energy production and providing the necessary intermediates for biosynthetic processes7. However, it has only recently been reported that cell metabolism may play an important role in migration9, with intracellular energy generation still being explored.Cellular bioenergetics play a significant role in many essential biological processes including growth and proliferation, allowing cells to adapt to a changing environment3. Cells can move both as individual cells and as collective cohorts, adopting a variety of migratory modes, ranging from Rac1-dependent mesenchymal13 to RhoA-mediated amoeboid migration16. In addition to plasticly switching between these migratory modes or adopting intermediate characteristics of each21, migratory cells are also able to switch between two main metabolic pathways: glycolysis or oxidative phosphorylation (OXPHOS)22. Energy requirements depend on cell morphology, physical properties of surrounding environments, and cell–environment interactions and are therefore determining factors in cell migration3.Recent evidence suggests that cells have distinct energetic needs depending on their mode of migration26. It has been reported that both single and collective cell migration may demand that certain energetic requirements be met during migration30. In single cells, the more energy-efficient, mitochondrial ATP generation occurs at the leading edge of cells8, whereas during collective migration, there is no consensus in the current literature on energy production at the leading edge of the cell front compared to the central follower cells32. Cell metabolic pathways and energy sources are of key interest in the context of cancer, as a better understanding of these processes could provide efficient targets for treating cancer and inhibiting cell invasion. Therefore, newly developed tools and techniques available for studying cell metabolism during migration have enabled further investigation of cellular energetic needs and the mechanisms by which energy is generated.Collective migration is characteristic of a number of biological processes, including development, wound healing, and invasive diseases like cancer, where energy utilization and efficiency have recently been shown to play a significant role36. However, as more is learned on the heterogeneity of cell populations and migratory ability, it becomes clear that understanding individual cell energetics can shed light on these complex and intricate processes. Molecular probes that can be stably expressed in multiple cell lines are able to map these minute changes on a cell-by-cell basis to study single cell energetics in the context of larger cell populations. Many fluorescent biosensors thus exist to interrogate cell metabolism in real time, including those measuring the metabolic intermediates lactate37 and pyruvate38 or mitochondrial membrane potential39. Here, we have focused on just a few of the many probes used to investigate cell migration and metabolism.Cell metabolism has primarily been characterized using bulk techniques analyzing metabolic genes and proteins and with tools such as the Seahorse Analyzer XF that reports oxygen consumption and extracellular acidification rates of cell populations40, actin polymerization41, and cytoskeletal remodeling43. ATP:ADP ratios are useful in reporting the availability of these metabolic intermediates. The PercevalHR probe boasts the ability to track real-time ATP:ADP ratios and localization as cells migrate, providing substantial insight about the relationship between cell energy utilization and migration in various cell microenvironments44. PercevalHR combines a fluorescent protein with the bacterial regulatory protein GlnK1 that competitively binds to active ATP and ADP45. The probe has two distinct excitation wavelengths for ATP and ADP binding and has been successfully used to measure a ratio of these values in yeast cells46, HeLa cells47, pancreatic beta cells49, neuronal cells50, fibroblasts51, metastatic breast cancer cells30, and many others to report cell energy status.Adenosine triphosphate (ATP) and adenosine diphosphate (ADP) are two key components of energy transfer in cells, with ATP being essential in multiple migratory mechanisms including actomyosin contractility52, generating interest in how this altered energy dynamic regulates migration and interaction with the extracellular matrix (ECM)53. In dense, more-challenging environments, ATP:ADP ratios in cancer cells are elevated, likely to meet the higher energetic demands of cells navigating the ECM9  reduces to NADH when it accepts an electron, and both are essential cofactors in glycolysis, the citric acid cycle, and OXPHOS61. While NAD+ and NADH levels were both found to decrease in cells lacking Citrin, levels of glycolysis and OXPHOS similarly decreased, suggesting that Citrin aids in providing intermediates for both energetic pathways. However, Peredox is not the only probe that takes advantage of the competitive binding of Rex. Similar variations of the construct have been used to highlight glycolysis as a determinant of cell migration in development. A Rex probe paired with yellow fluorescent protein reported that neural crest migration depends heavily on glycolysis and is abrogated in cells utilizing OXPHOS62. This has been echoed in recent studies, where increased glycolysis and its intermediates were linked to increased migration68. These probes allow visualization of the inherent metabolic plasticity of cells and how migratory phenotypes are altered when cells utilize different energy pathways.Recently, Peredox was used to interrogate the role of Citrin, the mitochondrial transporter, in increasing cancer cell invasion by measuring the relative levels of NAD+ and NADH during energy production in cells where the transporter was silenced69. This probe has been used as an indicator of glycolysis to study cancer cell migration in particular, as cancer cells are known to exhibit increased glucose uptake and glycolysis to support increased proliferation. Increased 2-NBDG fluorescence has been correlated with increased migration in cells62 and directly related to an increase in ATP hydrolysis9. Not only has 2-NBDG been used in ex vivo live tissues successfully70 but also, in future work, it could be used to track glucose uptake and therefore relative glycolytic rates ex vivo and in vivo during migration through highly relevant microenvironments to investigate dynamic metabolic plasticity.Glucose fuels glycolysis, releasing ATP and other factors that feed into OXPHOS. 2-NBDG is a modified, fluorescent glucose analog that is effectively taken up by cells, but not utilized during glycolysis, and thus serves to report glucose uptake in cells2O2)71. H2O2 serves as an important second messenger since it is an oxidizing agent during cell metabolism72. Multiple probes have been developed incorporating a fluorescent protein with the H2O2-sensing protein OxyR to obtain real-time, live-cell tracking of H2O2 localization in the cell to gain insight on the redox state and H2O2 gradient74. The recently developed and most sensitive of these probes is HyPer 7, which can detect very low concentrations of H2O2 while being pH stable72. Using this probe, protrusion formation and cell polarization was shown to rely significantly on the H2O2 concentration at the leading edge of cells and the steepness of the H2O2 gradient from the protrusion to the cell body was found to directly correlate with the stability of the protrusion. This gradient, observed using a HyPer variant, has additionally been reported to change based on mechanical environmental cues, for instance confinement, to alter lamellipodia formation and induce migration toward available oxygen75. Though further work is required to understand exactly how this H2O2 gradient and signaling directs migration and cell polarization, the increased sensitivity of this probe, and others, has provided a unique avenue for further exploration.Cells can respond to various environmental cues to regulate and drive migration. The “redox status” of cells, describing the relative levels of regulators of oxygen, can be determined by these cues and is regulated by the production of intermediates such as reactive oxygen species (ROS) like hydrogen peroxide , a key regulator of ROS production and redox state, was inhibited in breast cancer cells, collective migration was significantly inhibited, suggesting that ROS generation and regulation plays an important role not only during single cell migration but also in collective invasion and migration108. As energy utilization in this mechanism is still not fully understood, molecular probes and fluorescent labeling have been employed to determine how the differences between these two distinct cell groups are established and affected by metabolic requirements. For instance, cells expressing GFP-labeled keratin-14 in primary breast tumor organoids were found not only at the leading edge of collective cells to direct migration but also in follower positions that can engage in leader/follower switching events in response to both microenvironmental cues and intracellular energy needs110. Utilizing RFP-tagged and CycleTrak-labeled MDA-MB-231s, it has been shown that cells guided by environmental cues may compete for a dynamically changing leader cell position to minimize energetic costs during ECM invasion77. Thus, probing individual cell behavior with various fluorescent biomarkers has further clarified the metabolic determinants of different subpopulations during collective migration; moreover, these findings have suggested that the regulation of leader/follower cell metabolic profile by the surrounding microenvironment is critical to collective migration.Observing the metabolic plasticity of collectively migrating cells has led to a recent focus on the distinction between the independent roles of leader/follower cells. The formation of phenotypically unique leader cells from follower cells is reportedly dependent on dynamic collective stresses in follower cells that can in some instances pull leader cells into position111, molecular probes allow for metabolic profiling of individual cells in collective migration, highlighting the spatial and temporal heterogeneities in leader/follower dynamics. The PercevalHR probe has been used to observe available energy in leader cells during collective cancer cell invasion in vitro through dense collagen, which is directly related to leader cell lifetime and leader/follower positions31. Using the 2-NBDG probe, it has been separately reported that leader cells exhibited higher glucose uptake than follower cells, suggesting increased glycolysis31, while another reported the preferential usage of OXPHOS by leader cells32. Though it is clear that collective cell populations maintain complex metabolic heterogeneity, how cells utilize different metabolic pathways during collective migration remains unclear. These real-time cell tracking techniques not only shed light on cell metabolism during migration but also, more importantly, point to the need for further investigation into leader versus follower cell behavior.Molecular probes are a powerful tool for dissecting the processes of collective migration, especially in the context of bioenergetics and cancer. Unlike Seahorse, which requires large cell populations112. These techniques employ the use of photoconvertible tags to identify and mark individual cells in collective populations that can then be sorted and expanded into discrete subpopulations. By creating separate populations characterized by mutations affecting invasive capability113, highly invasive leader cells were shown to rely on focal adhesion kinase–fibronectin signaling to promote invasion, while follower cells instead are recruited to leader cells via irregular VEGF-based vasculogenic signaling112. Further definition of unique subpopulations can also be determined using single-cell sequencing, probing the metabolic heterogeneities in bulk expression profiles to link metabolic gene expression during in vitro cell migration with in vivo metastasis117. By specifically studying these populations, a fuller understanding of their interactions and roles in collective cell migration can be gained, particularly in the context of invasive diseases like cancer.To understand the needs of individual leader and follower cells during migration, researchers have taken advantage of the ability to physically sort heterogeneous subpopulations based on their distinct phenotypic, genotypic, and metabolic profiles using tools such as photoactivation and spatiotemporal genomic and cellular analysis (SaGA)120. High levels of NAD(P)H autofluorescence may serve as a useful biomarker for increased OXPHOS119, though it may be difficult to fully parse the contributions of NADH and NADPH to metabolism and whether these changes specifically point to mitochondrial respiration or glycolysis57. Nonetheless, autofluorescence has provided a probe-free avenue for exploring the metabolic preference in cells that could lend to easier characterization of migratory metabolism in the future.Recently, it has been shown that cells can be sorted without the use of specific probes or photoactivation but rather based on autofluorescence of the metabolic intermediates NAD(P)H and FAD31, while others show a dependence on OXPHOS32, as shown by increased sensitivity of leader cells to mitochondrial-targeting treatment. During glycolysis, the tumor microenvironment is acidified owing to the increase in lactate production by cells121, which has been linked to the activation of matrix metalloproteinases123, important mediators of matrix degradation. This evidence supports the idea that leader cells rely more heavily on glycolysis, potentially to aid in matrix remodeling for migration, and further highlights how leader/follower phenotypic heterogeneity may be sustained or partially altered by the microenvironment. As future studies continue to reveal distinct leader/follower profiles, these opposing reports may be resolved with further investigation into how leader/follower dynamics and complex microenvironmental cues regulate migratory metabolism.As studies continue to investigate the metabolic activity of leader/follower cell populations, conflicting reports have emerged regarding the utilization of glycolysis and/or mitochondrial respiration during collective migration. In cancer, leader cells have been shown in some instances to rely on glycolysis, as demonstrated by increased ATP/ADP ratios and increased glucose uptakein vivo are still lacking and will be essential in understanding cellular energy status of migrating cells in highly complex and relevant environments. Additionally, in primary or patient-derived cell lines where cells are more fragile and last shorter time periods in vitro, molecular probes requiring several passages for transduction and even selection can be technically difficult to utilize. Therefore, the development of a less-invasive tool to visualize and quantify intracellular energy status in these cells will be particularly useful in future cell migration work.In very recent work, cell migration research has focused on understanding the role of metabolic heterogeneities in cell migration. Studies have narrowed from visualizing intracellular energetics in single cell and collective groups of cells to sorting cells into specific populations and characterizing the phenotypic and genotypic differences in these subpopulations. This is made feasible through the use of photoactivation and cell sorting, with characterization made simple using molecular probes that allow visual and quantitative comparison of metabolic processes in migrating cells. However, tools and techniques for monitoring and quantifying energy production and consumption in real time Recent research has revealed that individual cell populations hold key heterogeneities that independently contribute to the migration of collective cell groups as a whole, with metabolic intermediates playing an essential role in determining the function and role of these cells. Whether these heterogeneities are inherent in the cell or dependent on surrounding conditions has yet to be fully explored and is of key interest in future work. As it is still unclear which metabolic pathways are prioritized by specific cell subpopulations, further investigation into these questions is essential to fully understand the mechanisms of both single and collective cell migration, especially in the context of a complicated disease-developing environment.


The tumor vasculature is essential for tumor growth and metastasis, and is a prime target of several anti-cancer agents. Increasing evidence indicates that tumor angiogenesis is stimulated by extracellular vesicles (EVs) that are secreted or shed by cancer cells. These EVs encapsulate a variety of biomolecules with angiogenic properties, and have been largely thought to stimulate vessel formation by transferring this luminal cargo into endothelial cells. However, recent studies have revealed that EVs can also signal to recipient cells via proteins on the vesicular surface. This review discusses and integrates emerging insights into the diverse mechanisms by which proteins associate with the EV membrane, the biological functions of EV membrane-associated proteins in tumor angiogenesis, and the clinical significance of these proteins in anti-angiogenic therapy. Tumor growth and metastasis depend on the development of a tumor vasculature to supply oxygen and nutrients. It is widely recognized that tumors secrete a repertoire of soluble factors that stimulate recruitment of endothelial progenitors, growth and maturation of endothelial cells, and assembly of these cells into vessels . In the EVs are produced by virtually all types of cells, but are often more highly secreted by cancer cells than by normal cells . To dateMass spectrometry-based proteomic analyses of EVs derived from diverse cell types and body fluids have revealed several thousand vesicular proteins ,15. Of tThe MVB pathway plays an essential role in regulating the sorting of integral membrane proteins, and intimately ties protein sorting to exosome biogenesis. The epidermal growth factor receptor (EGFR) is a prototypic cargo that is sorted by the MVB pathway . FollowiWhereas earlier studies showed that ubiquitination of cargo is important for their recognition by the early ESCRT complexes ,25, subsCells that are depleted of key components of all four ESCRT complexes have been found to form MVBs, suggesting that ESCRT-independent mechanisms also regulate the MVB pathway . KajimotIn addition to cell surface receptors, a number of ligands have been detected in EVs isolated from body fluids and from diverse cell types including cancer cells. These ligands include interleukins (IL), chemokines and members of the epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), and transforming growth factor (TGF) families ,49,50,51Following synthesis, most cytokines are trafficked to the endoplasmic reticulum (ER) and then to the Golgi apparatus, a process that is directed by their N-terminal signal peptide sequence  Figure . Upon soWhereas earlier studies assumed that cytokines are encapsulated within EVs , recent VEGFA mRNA [189), but not other VEGF isoforms, is preferentially enriched in small EVs that are secreted by ovarian, colorectal, and renal cancer cells and are present in body fluids of patients with these cancers, and that VEGF189 is bound to the surface of these EVs [189 in small EVs secreted by cancer cells that express VEGF189, and assaying the binding of ‘free’ recombinant VEGF189 to small EVs secreted by VEGF-deficient cancer cells, the authors estimated that post-secretion binding accounts for only ~25% of the VEGF189 in small EVs, implicating that de novo synthesized VEGF189 is largely sorted to small EVs [189 interacts with the surface of small EVs through heparan sulfate binding  on the surface of breast cancer cell-derived microvesicles of 0.5 to 1.0 μm in diameter [90K was generated by crosslinking of VEGF165 by tissue transglutaminase, and associated with microvesicles through binding heat shock protein 90 (Hsp90) [90K was not detected in small EVs and, conversely, other isoforms of VEGF were not detected in microvesicles [165 trafficking has revealed that VEGF165 undergoes unconventional secretion whereby VEGF165 is retained on the outer surface of the plasma membrane and is then released on vesicles that shed from the cell surface [Intriguingly, recent studies of VEGFs indicate that the association of a cytokine with the EV surface might not occur as a post-secretion event. Several isoforms of VEGF are generated through alternative splicing of GFA mRNA . Ko and hese EVs . In thismall EVs . Further binding . Of the  sulfate . Becausend CXCL1 ,61. By cdiameter . VEGF90K (Hsp90)   are essential to enable endothelial cells to invade tissues and form new vessels . One of By contrast, several other EV integral membrane proteins can elicit signaling by interacting with proteins on the plasma membrane of endothelial cells. Sato and colleagues recently identified that the ephrin type B receptor 2 (EPHB2) is present on small EVs secreted by head and neck cancer cells, and that EV-associated EPHB2 stimulates tumor angiogenesis by activating the STAT3 signaling pathway via engagement of ephrin-B2 on the surface of endothelial cells . WhereasGiven that many of the pro-angiogenic integral membrane proteins on EVs retain signaling-competence, these proteins likely retain the same orientation on the EV membrane as on the plasma membrane of the parental cell. This assumption could particularly hold for ectosome-associated membrane proteins because ectosomes form by outward budding of the plasma membrane . The MVB90K has been shown to stimulate VEGFR2 phosphorylation in endothelial cells and tube formation [189 has also been shown to stimulate VEGFR2 phosphorylation, endothelial cell migration, and tube formation in vitro and to increase tumor microvessel density in vivo [189 to stimulate endothelial cell migration and tube formation was not inhibited when EV uptake by endothelial cells was blocked, but was abrogated when endothelial cells were treated with an antibody that blocks ligand binding to VEGFR2 [189 interacts with the extracellular domain of VEGFR2 and elicits VEGFR2 signaling in endothelial cells independently of uptake. VEGF isoforms are biologically active as homodimers [189 has been found to be present on small EVs as a homodimer [In their analysis of protein topology in EVs, Cvjetkovic and colleagues not only identified that many integral membrane proteins are oriented ‘inside-out’ in EVs but also that many cytosolic proteins localize on the surface of EVs . As descormation . Small E in vivo . The abio VEGFR2 . These fmodimers  and VEGFomodimer . By contomodimer ,75. Thesomodimer ,75.189 on the surface of cancer cell-derived small EVs remain almost unchanged at 24 h following incubation in plasma at 37 °C, indicating that the association of this pro-angiogenic cytokine with the EV surface profoundly increases its stability [90K, which comprises crosslinked VEGF165, is two-fold more effective in stimulating endothelial tube formation than an equivalent amount of free VEGF165 [The secretion of EVs was initially described as a means of eliminating unneeded biomolecules from cells . It is ntability . It is a VEGF165 . Amphire VEGF165 . Exosome VEGF165 . In addi VEGF165 . ThroughAgents that inhibit angiogenesis have been among the most extensively studied anti-cancer therapies. Bevacizumab, a humanized antibody that neutralizes VEGF, was the first anti-angiogenic agent approved for the treatment of solid tumors and remains widely used . A more The resistance of tumors to anti-angiogenic therapy has been attributed to several mechanisms such as the utilization by tumors of existing vessels , infiltr189 is signaling-competent but is not neutralized by bevacizumab [189 associates with the surface of small EVs through heparin-binding, and that the engagement of VEGF189 with high molecular weight (HMW) heparin reduces its recognition by bevacizumab [189 might therefore stem from a conformational change in VEGF189 that is induced by its engagement with heparin (90K activates VEGFR signaling and is not neutralized by bevacizumab [90K associates with microvesicles through interacting with Hsp90, and that treatment with an Hsp90 inhibitor releases VEGF90K from microvesicles, restoring sensitivity of the ligand to bevacizumab (In addition to treatment-induced hypoxia and resultant changes in EV release and composition, there are several other mechanisms by which EVs could decrease efficacy of anti-angiogenic therapy. On one hand, EVs might mask the therapeutic agent. Simon and colleagues identified that bevacizumab is internalized by glioblastoma cells, trafficked into endosomes and then displayed on EVs secreted by these cells . This EVacizumab . The autacizumab . It has acizumab , and thaacizumab . The ina heparin . In anotacizumab . The autacizumab . Taken t90K [189 have been found to be approximately five-fold higher in patients with progressing disease than in those with stable or regressing disease in a cohort of patients with newly diagnosed metastatic renal cell carcinoma who were treated pre-surgically with single-agent bevacizumab [How can biological studies of EVs be translated into studies that improve outcomes of cancer patients? One major limitation of anti-angiogenic therapy has been the lack of reliable biomarkers that can predict clinical response . Finding90K . Furtheracizumab . Howeveracizumab . These fIn addition to the potential use of EV-associated proteins as biomarkers, the possibility of using natural EVs as therapeutic agents has been raised by findings that EVs secreted by some types of normal cells can inhibit angiogenesis. The heart is renowned for its immunity to cancer, and EVs isolated from heart progenitor cells have been shown to inhibit tumor growth and angiogenesis in a fibrosarcoma model . MicropaIn summary, there is increasing evidence that EVs released by cancer cells and by stromal cells display a repertoire of integral membrane proteins and membrane-anchored proteins that stimulate tumor angiogenesis and can enable tumors to evade anti-angiogenic therapy. However, there remain several gaps-in-knowledge. Firstly, functional studies to date have focused on a given type of EV, and differences in paracrine signaling mediated by different types of EVs are poorly understood. More comparative analysis of the composition and functional significance of membrane proteins between different types of EVs is needed. Paramount to these efforts is the development of more rigorous methods to isolate and clearly distinguish the different types of EVs. Secondly, further investigation is needed into the mechanisms by which pro-angiogenic proteins interact with the surface of EVs, and how the interactions of these proteins with the EV membrane modulate their topology, signaling competence and stability. As discussed, recent studies of EV membrane-associated VEGF highlight the importance of evaluating differences in the conformation of EV-associated proteins and their cellular or soluble counterparts in designing therapeutic agents that target these proteins. Thirdly, recent studies indicate that EVs are a promising class of therapeutic vehicles. Further investigation of EV membrane proteins will yield important insights for facilitating cell-specific delivery and the targeting of specific subcellular destinations.