In the last decade, amphiphilic cellulose (AC) is emerging as attractive biomaterial for different therapeutic use, due to its unique chemical and physical properties. Using it as alternative to synthetic polymers, AC opens up new avenues to prepare new bio-sustainable materials with low impact in the cellular environment. Herein, most recent methods to synthesize and processing AC materials from different sources—i.e., cellulose nanofibers, bacterial cellulose, cellulose derivatives—will be discussed. By an accurate optimization of morphology and surface chemistry, it is possible to develop innovative amphiphilic platforms, promising for a wide range of biomedical applications, from drug delivery to molecular/particle adsorption. The. The5]. lomorphs . This colomorphs . Celluloeatments .In -linked polysaccharides, anhydroglucose units present in a chair conformation where hydrophilic hydroxyl groups are in equatorial position and the hydrophobic C–H bonds are in an axial position. α--linked polysaccharides form a cavity rich in C–H bonds that is suitable for encapsulating hydrophobic chemicals, including for delivery in living organisms .−3), high tensile strength , and a high Young’s modulus  [Cellulose molecules accumulate to form cellulose microfibrils, which are characterized by the presence of either highly ordered  or less-ordered (amorphous) regions . Cellulo region) .Individual cellulose molecules are brought together into larger units known as microfibrils, which are packed into larger units called micro-fibrillated cellulose. The diameter of these microfibrils is around 20 to 50 nm. The microfibrils are formed during the biosynthesis of cellulose and are several micrometers in length. The ordered regions are cellulose chain packages that resemble nanocrystalline rods.These nanocrystalline parts contain highly ordered  domains which can be separated and extracted from different cellulose sources through acid hydrolysis using sulfuric, hydrochloric, or phosphoric acid. Acid hydrolysis facilitates the cleaving of accessible glycosidic linkages within the cellulose microfibrils resulting in the characteristic rod-shaped nanocrystals . CNCs ha2SO4-hydrolyzed CNCs were measured from sedimentation tests in a wide set of 59 solvents and binary mixtures [Cellulose nanocrystals (CNCs) are usually considered to be isotropically polar nanoparticles with amphiphilic behavior in suspension. The Hansen solubility parameters of wood-based Hmixtures . It was mixtures ,16.Moreover, CNCs have rich functional moieties, such as aldehyde and hydroxyl groups, which offer a variety of modifications to control their physical or chemical properties. The presence of an aldehyde groups at the end of a CNC permits the selective modification of reactive reducing ends to prepare amphiphilic CNCs with special features. Otherwise, some relevant limitations in the use of CNCs for particle entrapment concern the stabilization of the polymers from oil-water emulsions. Different modifications were carried out to increase the hydrophobicity of CNCs. For example, end-group modification by the introduction of hydrophobic polystyrene chains was carried out to increase the viability of modified CNCs in emulsifying toluene and hexadecane compared to unmodified CNCs . MoreoveRecently, nanocellulose has been applied in drug delivery applications . HoweverCellulose nanocrystals, with many hydroxyl and sulfate groups on the surface, can stabilize metal nanoparticles effectively. Moreover, CNCs derived from sulfuric acid hydrolysis are super-hydrophilic and show great dispersibility in water because of the abundant hydroxyl and surface sulfate ester groups, which make great supports for heterogeneous catalysts for hydrodeoxygenation reactions. In the field of amphiphilic cellulose nanocrystals, benzyl-polyethyleneimine modified cellulose nanocrystals were developed via the periodate oxidation of cellulose nanocrystals and reductive amination, which exhibited pH-responsive amphiphilicity due to the existence of hydrophilic amino- and hydrophobic benzyl groups .Cellulose nanofibers (CNFs) have been used as an alternative non-toxic and bioactive material in a broad field of innovative nanostructured materials . CNFs weMoreover, the surface-bound charged CNFs were reported to act as dispersing and structural-forming agents for multifunctional exfoliating. Amphiphilic CNFs attract with graphite through hydrophobic interactions, whereas hydrophilic surfaces assist in the dispersion of calcium phosphates in aqueous media . MoreoveCellulose can be produced by organisms such as vascular plants, algae, certain types of bacteria, and even some animals . BacteriCellulose and its derivatives have been demonstrated to be non-toxic in both animals and humans, which makes it an ideal material for biomedicine . CelluloMany attempts have been carried out to overcome this problem. All commercially available cellulose products are obtained by solid phase reactions, more or less swollen, state (heterogeneous reactions) because of insolubility of cellulose caused by supramolecular structure . ThroughThe most promising strategy to use cellulose is taking in advantage with the natural abundance of different hydroxyl groups to attempt chemical modifications. Cellulose derivatives can be synthesized not only by etherification or esterification of neat cellulose, but also oxidation, silylation and polymer grafting .There are many kinds of cellulose derivatives used frequently, such as cellulose acetate (CA), methylcellulose (MC), ethylcellulose (EC), hydroxyethylcellulose (HEC), hydroxypropylcellulose (HPC), sodium carboxymethylcellulose (CMC) and others.CMC a water-soluble cellulose ether, can be prepared from the reaction of cellulose with sodium monochloroacetic acid. CMC can be characterized by different degrees of substitutions (ranging from 0.4 to 1.3) depending on the average number of carboxymethyl groups per glucose unit. CMC degrades completely at low rates in the environment to form nontoxic intermediates . MoreoveDue to the low processability of neat cellulose, the synthesis of cellulose derivatives such as cellulose esters or ethers generates valuable properties, including suitable hydrophobicity/hydrophilicity, thus enabling the use of cellulose derivatives in various applications. Dong et al., demonstrated new cellulose ether-based amphiphiles, cellulose ω-carboxyalkyl derivatives, which prevented crystallization of supersaturated solutions of poorly water-soluble drugs. The authors prepared cellulose ethers with the required balance of hydrophobicity/hydrophilicity through two techniques: starting from a hydrophilic methyl cellulose or by a one-pot homogeneous reaction. A series of cellulose ethers with an adaptable degree of substitution and proper and terminal functionality were prepared. These derivatives also inhibited nucleation from supersaturated solutions of the poorly water-soluble drug and enhanced their potential use as amorphous solid dispersion .Amphiphilic celluloses (ACs) combine hydrophilic parts and hydrophobic moieties. The hydrophilic parts are responsible for hydration and swelling, while the hydrophobic moieties minimize water contact. Introducing or grafting hydrophobic segments to hydrophilic cellulose represents a promising tool for constructing amphiphilic systems. There are two main strategies for constructing an amphiphilic system: covalent conjugation and non-covalent intermolecular interaction. In covalent conjugation, cellulosic materials containing hydroxyl groups can react with a wide range of hydrophobic moieties via ester bonds to form amphiphilic polymers. The covalent chemical bonds between cellulose and hydrophobic moieties can be used in biomedical applications especially drug release. A non-covalent strategy may refer to electrostatic interactions, host–guest recognition, hydrogen bonding, or charge transfer interaction .Because they have both hydrophobic and hydrophilic parts, amphiphilic cellulose copolymers can self-assemble into micelle structures when in contact with water or a selective solvent . ParticuVarious amphiphilic polymers such as carboxymethyl cellulose acetate butyrate have gained increasing importance in coating technologies and pharmaceutical research. Carboxymethyl cellulose acetate butyrate can be prepared by esterification of CMC with acetic and butyric anhydrides. The main character of carboxymethyl cellulose acetate butyrate is its dispersity in water to form stable colloidal solution. Moreover, its ability to control the release rate of highly water-soluble compounds and enhance the dissolution of poorly water-soluble drug proposes variety of applications especially in pharmaceutical and drug delivery applications .One convenient route for introducing new chemical and physical properties to cellulose is graft modification. In contrast to block copolymers, graft copolymers have special architectures that contain a main chain or backbone and many side-chains, which could cause a unimolecular association and result in the unimolecular micelles . In the In the case of grafted copolymers, however, additional parameters, mainly graft density and graft chain length, should be taken into account to correlate the molecular and micellar characteristics .Amphiphilic graft copolymers are generically composed of a hydrophilic main chain and hydrophobic branches which are spaced comparatively closely along the main chain. After appropriate chemical modifications, amphiphilic cellulose achieves suitable properties of biodegradable polymeric surfactants. The design of amphiphilic polysaccharide systems has been sought through the grafting of macromolecules  with polymer chains such as carboxymethylcellulose and polylactide or poly(ethylene glycol) (PEG) to chitosan and carboxymethyl chitosan .As to cellulose derivatives, hydrophobically modified O-(hydroxyethyl) cellulose (HEC) is known as a cellulosic surfactant that possesses unique properties in aqueous solution . DependiThe most commonly produced chemically modified cellulose derivatives are cellulose ethers and esters made by reactions of hydroxyl groups of cellulose. Basing on the way the hydrophobic branch is anchored to the hydrophilic substrate, the graft copolymerization onto cellulose and its derivatives is generally performed by different approaches including ,51: “gra(a)Conventional free-radical polymerizationFree-radical polymerization still represents the most common method of synthesizing polymers in industrial fields due to its versatility . The app(b)Controlled radical polymerizationThe need to obtain polysaccharide-based hybrids with precise and tailored properties for synthetic graft length, chemical composition and topology, led to development of controlled radical polymerization (CRP). These techniques are tolerant of moisture and compatible with a large range of functional groups. They are based on the addition of species that ensure the reversible trapping of the “active” propagating radical species as “dormant” species through reversible termination or reversible transfer . This stThe recent advent of CRP such as nitroxide-mediated polymerization (NMP), atom transfer radical polymerization (ATRP) or reversible addition-fragmentation chain transfer (RAFT). Among CRP techniques, ATRP is still the most widely used due to its versatility and compatibility with a wide range of vinyl monomers and solvents, and or its solid surface modification . Graftinl-lactide) (PLLA), interesting for their applications in biomedical materials, have been widely grafted onto cellulose via ROP [2), the widely used catalyst in the production of biodegradable polyesters due to its low biologic toxicity. Because it avoids transesterification reactions, Sn(Oct)2 ensures the synthesis of final graft copolymers in high yield, controlled molecular weight, and narrow distribution. A coordination–insertion mechanism is thought to be initiated by a tin–alkoxide species formed prior to ROP [ROP is a well-established technique to polymerize cyclic monomers to prepare aliphatic polyesters of cellulose . The dri via ROP . A schem via ROP . On the  via ROP . For thi via ROP . Particur to ROP .Cellulose graft copolymers with complex architectures, such as a dual-, block-, or centipede-like graft can be synthesized by combing ROP with other polymerization methods, such as ATRP . For exaIt is well known that cellulose is hard to dissolve because of its morphology and high crystallinity . As prevSeveral in the last three decades’ methods have been explored to dissolve the cellulose. Traditionally, these have involved defibrillation by viscous processes aimed at inducing the formation of regenerated cellulose fibers. However, these procedures involved the use of aggressive solvents to chemically modify the macromolecules, which caused problems in waste disposal, health safety and environmental pollution . More reAC has enormous potential for drug delivery applications due to its high surface area-to-volume ratio and high polymerization . Its higRecently, AC has been successfully used to improve the bioavailability of orally delivered drugs. Several experiments confirmed that the peculiar properties of AC may be modulated to design innovative drug-delivery systems with improved stability in the gastrointestinal tract and poor permeability across the intestinal epithelium to minimize the bioavailability of therapeutic molecules . Accordil-lactide) copolymers via the ROP technique. The solubility of the graft copolymers in organic solvents improved. The prepared amphiphilic copolymers were self-assembled into nanoparticles for delivery of anticancer drug oleanolic acid. The copolymer nanoparticles displayed a high drug-loading efficiency and a prolonged drug release. The amphiphilic carboxylated cellulose-graft-poly(l-lactide) copolymer nanoparticles provided a novel platform for drug-delivery applications  than carboxymethyl cellulose acetate (1.78%) which may have been related to the high degree of substitution of the hydrophobic parts .It was reported that up to 80% of drugs suffer from poor bioavailability due to poor aqueous solubility. Various methods have been established to improve drug solubility and bioavailability. Amorphous solid dispersions represent some of the most efficient formulations to improve drug solution concentration. The polymers prepared for amorphous solid dispersions matrices have shown certain structural criteria: (1) a terminal carboxyl group interacts with the drug and can act as a pH trigger to enhance the release process; (2) a degree of hydrophobicity controls miscibility with hydrophobic drugs; (3) a degree of hydrophilicity controls the release of a drug in an aqueous environment, and (4) a sufficiently high glass-transition temperature (Tg) that can immobilize the drug, prevents recrystallization and ensures that polymer dispersion remains in the glassy state .Cellulose ω-carboxyesters have been established as effective matrix polymers for amorphous solid dispersion applications with various drug molecules. The moderate hydrophilicity arising from carboxylic acid groups promotes its amphiphilicity to work effectively as amorphous solid dispersion matrices . Dong etAlso, olefin cross-metathesis was reported as z promising technique for preparing amphiphilic derivatives of hydroxypropyl cellulose. The preparation of olefin-terminated hydroxypropyl cellulose derivatives followed by cross-metathesis with various acrylates and hydrogenation afforded stable, saturated products. The 5-carboxypentyl hydroxypropyl cellulose derivative showed high promise as a crystallization inhibitor of telaprevir from a supersaturated solution. Previous articles showed that amphiphilic cellulose has substantial promise in drug-delivery and amorphous solid-dispersion applications .Zhenzhen Liu et al., reported that cationic amphiphilic cellulose copolymers could be prepared through grafting hydrophobic poly (p-dioxanone) chains onto hydrophilic quaternized cellulose derivatives via a ring-opening polymerization reaction, which was performed in 1-butyl-3-methylimidazolium chloride using 4-dimethylaminopyridine or 1,8-diazabicyclo (5.4.0) undec-7-ene (DBU) as s catalyst, . StudyinThermo-responsive micelles were prepared by reductive amination between hydroxypropyl methyl cellulose containing an amine group  as hydrophobic block. The reaction produced diblock, triblock and three-armed copolymers with different hydrophilic/hydrophobic ratios. The geometrical structure of copolymers strongly affected the micelle size as well as the cloud point of the hydroxypropyl methyl cellulose-JEF copolymers. Spherical nano-micelles were formed by the self-assembly of copolymers in aqueous solution, and the micelle size was tailored by varying the block length of the HPMC and the geometrical structure. Three-armed HPMC–-JEF copolymers presented a lower critical micelle concentration and smaller micelle size compared to linear diblock and triblock ones. MTT present outstanding cytocompatibility, suggesting that these novel HPMC-JEF copolymers can be safely used as a potential drug carrier .Various forms of amphiphilic cellulose were prepared and characterized for different industrial applications. The most promising approach was the use of amphiphilic cellulose materials to develop support for immobilizing nanoparticles. For example, Li et al., prepared cellulose sponges through dual-cross-linking cellulose nanofibers with γ-glycidoxypropyltrimethoxysilane and polydopamine. The highly porous and flexible cellulose sponge was used as a carrier for palladium nanoparticles, which are homogeneously dispersed on the surface of a cellulose sponge with a narrow size distribution. The Pd@cellulose sponge exhibited high catalytic activity and efficient recyclability in Suzuki and Heck cross-coupling reactions .Amphiphilic cellulose was synthesized by grafting hydrophobic groups onto hydrophilic cellulose nanocrystals and used as a support for PdNi alloy nanoparticles. The as-synthesized catalysts exhibited encouragingly high performance in the hydrodeoxygenation of vanillin under mild conditions . AmphiphTajima et al., investigated nanofibrillated bacterial cellulose produced by culturing a cellulose-producing bacterium in a medium supplemented with carboxymethylcellulose will aggregate in organic solvents, -i.e., a cellulose-producing bacterium named Gluconacetobacter intermedius NEDO-01 in a medium supplemented with hydroxypropylcellulose. The prepared hydroxypropyl-nanofibrillated bacterial cellulose was dispersible in various polar organic solvents, and this property has valuable applications in molding and complexation .2 efficiently stabilized the oil/water interface [Recent trials have been carried out to understand the stabilization mechanism as a function of the amphiphilic properties of the nanocrystals through tuning the surface-charge density. Kalashnikova et al., used two sources of cellulose: cotton linters and bacterial cellulose to explore their emulsifying capability. The results showed that the electrostatic interaction plays the main role in the control of the interface. It has been demonstrated that a decrease in the charge density of cellulose nanocrystals below 0.03 e/nmnterface .Another strategy for preparing amphiphilic CNC is by chemical pretreatment of cellulose with periodate oxidation followed by reductive amination. This strategy may suggest the preparation of cellulose nanofibers without the widely used acid hydrolysis. Visanko et al., used three butylamine isomers to fabricate CNCs with amphiphilic features. The characterization tools showed that iso- and n-butylamine attach the highest number of butylamino groups to the cellulose fibers . The preAlternative approaches involve the fabrication of nano-fibrillar aerogels by coupled TEMPO oxidation and mechanical blending assemble into monolithic forms of porous structures . In thisGao et al., prepared amphiphilic cellulose in cellulose NaOH/urea aqueous solution. Amphiphilic cellulose was blended with polysulfone as an additive for preparing porous membranes. The hydrophobization chain segment of amphiphilic cellulose provided compatibility between cellulose and polysulfone, and hydrophilic, and antifouling protection were then created from the hydroxyl surface of amphiphilic cellulose. The water flux and the tensile strength were enhanced after adding nanosized amphiphilic cellulose into the membrane .Films prepared from cellulose nanofibrils are commonly brittle, which may limit its application. Various additives have been proposed to increase the ductility of nanocellulose films . Among tE. coli. The results showed that amphiphilic cellulose materials are promising for inhibition the growth of bacteria.Surface curvatures and surface anti-wetting synergistically act to inhibit bacterial contamination. Several trials were carried to transform the hydrophilic character of cellulose material into an amphiphilic character. Jin et al. prepared titania–PFOTMS composite thin films onto the cellulose nanofiber by chemical etching and coating . The preAmphiphilic cellulose materials are composed of hydrophobic and hydrophilic moieties; the former ones enable polymer solubility in aqueous solvents, while the latter ones enable the formation of polymeric micelles in aqueous solvents due to hydrophobic interactions. These peculiar features in combination with the high flexibility of these materials to be processed allows fabricating cellulose in different forms  with an enormous potential in different applications in different areas, i.e., biomedicine, drug delivery, bio-environment.


COVID-19 has affected long-term residential care (LTRC) disproportionally due to the high-risk population, lack of resources and insufficient preventative measures. Protective measures, including quarantine and strict visitation restrictions have made transitions into LTRC more challenging. Further insight is needed to understand how residents, relatives and staff have experienced this during the COVID-19 pandemic. During four months of fieldwork in a LTRC facility in Switzerland, a rapid ethnography consisting of interviews, observations, informal conversations and document analysis was conducted. This study included a total of 14 residents, 21 healthcare staff from varying departments and 7 relatives of residents. First results indicate that protective measures interfere with a resident’s ability to find meaningful activities and interactions within LTRC as well as the possibility to maintain mobility. This and limited family contact following a move into LTRC prevents a smooth transition from home to LTRC and impacts overall resident quality of life.


Leukocyte recruitment to the site of injury is a crucial event in the regulation of an inflammatory response. Tight regulation of interactions between the endothelium and circulating leukocytes is necessary to ensure a protective response to injury does not result in inflammatory disease. Rising interest in the broad immunoregulatory roles displayed by members of the glycan-binding galectin family suggests that these proteins could be an attractive target for therapeutic intervention, since their expression is significantly altered in disease. The focus of this review is to summarize current knowledge on the role of galectins in leukocyte trafficking during inflammation and the clinical approaches being taken to target these interactions for treatment of inflammatory disease. Glycans are one of the four major components that constitute cells, accompanied by nucleic acids, proteins and lipids. Recognition of specific glycan motifs by glycan-binding proteins (lectins) is crucial for facilitating highly sophisticated cross-communication between leukocytes in the bloodstream and endothelial cells (ECs) lining the blood vessels . Severalvia the MEK5/ERK5  pathway. Consequently, this prevents initiation of the inflammatory response through the downregulation of adhesion molecules and thus reduced leukocyte recruitment to the endothelium , reduce the velocity of leukocytes rolling along the endothelium through binding to heavily glycosylated counter-receptors  involved in the capture and subsequent rolling events of the leukocyte adhesion cascade . This inWhilst many aspects of the leukocyte trafficking cascade have been studied in detail, a lot still remains unknown. Specifically, the galectin family of glycan-binding proteins have recently come to light as functionally important immunoregulatory proteins with involvement in leukocyte activation, apoptosis and in mediating leukocyte adhesion and migration , 14. Enhvia their unique carbohydrate recognition domains (CRDs) (https://www.ncbi.nlm.nih.gov/genbank/) Galectins-1, -3, -8 and -9 are amongst the most studied members since many of their functions have been closely linked to inflammation and disease  . At pres disease . Importa disease , allowin disease Figure 2 disease . Additio disease .Key conserved amino acids in the CRDs across different galectins are responsible for the specific binding to β-galactoside-containing glycans. Despite these similarities, each galectin type reserves specificity for particular branched glycan’s and glycan motifs attached to proteins and lipids alike Figure 2in vitro cultured human macro and microvascular ECs, with expression largely intracellular ], a Toll-like receptor (TLR) 3 ligand  PhD studentship [222392/Z/21/Z]. HM is supported by the MCR [MR/T028025/1]. AI is supported by Birmingham Fellowship and AMS Springboard Award [SBF003\1156].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.


Dynamic changes in circulating and tissue metabolites and lipids occur in response to exercise-induced cellular and whole-body energy demands to maintain metabolic homeostasis. The metabolome and lipidome in a given biological system provides a molecular snapshot of these rapid and complex metabolic perturbations. The application of metabolomics and lipidomics to map the metabolic responses to an acute bout of aerobic/endurance or resistance exercise has dramatically expanded over the past decade thanks to major analytical advancements, with most exercise-related studies to date focused on analyzing human biofluids and tissues. Experimental and analytical considerations, as well as complementary studies using animal model systems, are warranted to help overcome challenges associated with large human interindividual variability and decipher the breadth of molecular mechanisms underlying the metabolic health-promoting effects of exercise. In this review, we provide a guide for exercise researchers regarding analytical techniques and experimental workflows commonly used in metabolomics and lipidomics. Furthermore, we discuss advancements in human and mammalian exercise research utilizing metabolomic and lipidomic approaches in the last decade, as well as highlight key technical considerations and remaining knowledge gaps to continue expanding the molecular landscape of exercise biology. The term metabolism—derived from the Greek word “metabole” meaning “change”—defines all the chemical reactions that change molecules within living systems. Exercise represents a major challenge to whole-body and cellular energy homeostasis, and a multitude of molecular responses to acute exercise  are engaged to combat energy stress at the cellular and whole-body level . During neration . The relize ATP;  “anaerobneration ,7,8. Theneration .Although tremendous progress in the understanding of the cellular and molecular mechanisms involved in the responses to acute exercise has occurred over the past decades through traditional “reductionist” approaches, these approaches are limited to studying a biochemical pathway or molecular target of interest in isolation. As a result, further insight into the complex integrative nature of exercise-regulated molecular metabolic networks has been limited, and critical knowledge gaps remain ,9. FortuMetabolomics is defined as the comprehensive study of metabolites present in a given biological system  ,11. The Metabolites are heterogeneous compounds that possess various physicochemical properties, but are generally classified as either hydrophilic polar molecules  or hydrophobic non-polar molecules  . The hum® consortium has provided a lipid classification system including a comprehensive list of lipid categories , classes and subclasses [® Structure Database contains more than 45,000 unique lipid structures [Lipidomics, considered a subfield of metabolomics ,19, is tbclasses . As of Dructures .Emerging only at the start of the new millennium, metabolomics and lipidomics represent the newest applications among global omics-based approaches . The rapidly growing fields of metabolomics and lipidomics have dramatically expanded over the past 15–20 years, thanks to major advancements in analytical instrument technologies and bioinformatic analysis platforms. As a result, there has been a substantial increase in the application of metabolomics and lipidomics across a wide range of research fields, including health and disease , toxicolStudying the metabolome (including the lipidome) is appealing for expanding our understanding of complex biological systems in the context of exercise, as metabolites lie downstream of all other layers of biological regulation. Therefore, the metabolome reflects the cumulative changes resulting from processes involving the genome, transcriptome and the proteome, as well as their interactions with the environment . The metAnother advantage of studying the metabolome/lipidome is that the central reactions and pathways essential to energy metabolism, growth and nutrient supply are highly conserved across mammalian species, meaning that metabolite measurements obtained from other mammalian species such as rodents may be relevant and translational to humans ,30. In aOmics-based approaches such as metabolomics and lipidomics are generally described as unbiased, global experimental strategies to identify and/or quantify as many compounds as possible within a biological system. However, different approaches to investigating metabolites and/or lipids within a biological system are currently available, as briefly outlined below and reviewed elsewhere in more detail ,32:This approach aims to reproducibly measure as many metabolites/lipids as possible in a given biological sample. Depending on the platform utilized, the untargeted strategy generally yields a metabolite detection coverage in the hundreds to low thousands using a combination of different separation and detection modes (described below). This approach provides semi-quantitative data, meaning that peak areas are reported for each metabolite instead of absolute concentrations. These peak areas allow the assessment of the relative abundance of detected metabolites between experimental groups. Of note, metabolite identities are usually unknown prior to data acquisition and analysis when using this approach. As opposed to the untargeted strategy, the targeted approach aims to provide absolute concentrations of a set of known metabolites  by using authentic chemical standards and calibration curves for each pre-selected metabolite. Recent developments in commercially available targeted metabolomics kits now facilitate the identification of up to 188 metabolites, and up to 1184 different lipids for lipidomics .This third approach is less common. However, it is gaining popularity amongst many metabolomics research groups. This approach represents an intermediate strategy between untargeted and targeted approaches where a higher number of known metabolites  are investigated using a single chemical standard and ad hoc calibration curve for multiple metabolites, thus providing approximate metabolite concentrations.The untargeted approach is primarily used as a hypothesis-generating method where the research question is generally unbiased with respect to metabolite identification. In contrast, the targeted approach can be used as a hypothesis-testing and experimental validation method, as it provides much higher sensitivity and specificity in comparison to the untargeted approach, but with reduced metabolite coverage. Therefore, the untargeted approach permits the identification of new potential biomarkers and pathways, which can be further validated and more accurately quantified via the targeted approach. Although these approaches are complementary, the targeted strategy provides the most quantitative insights into metabolite dynamics in response to stimuli such as exercise .Regardless of the type of metabolomic approach utilized, the main analytical platforms used in metabolomic studies are mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy ,19. We p1H, 13C, 15N, conferring their magnetic properties. The magnetic orientations of these atoms, which have random directions, become aligned when a magnetic field is applied. Then, a pulse of electromagnetic radiation at a specific “resonance” frequency (dependent on the atom and magnetic field) is applied, causing nuclei “excitation” and subsequent “relaxation” when the radiation pulse stops. During relaxation, nuclei emit the radiofrequency waves absorbed during the excitation phase, thus generating radiofrequency peaks in a frequency spectrum  after Fourier’s transformation. Briefly, NMR spectroscopy is a technique based on the interaction of an applied magnetic field with the nuclei of atoms which possess an odd number of protons or neutrons, including NMR spectroscopy is used as a non-destructive technique and allows the measurement of chemical and physical properties of molecules, therefore helping identify and/or quantify molecules of interest. In theory, this can be performed in solid, liquid and gas states from frozen to very high temperature materials, although researchers typically focus on only one aggregation state based on practicality and feasibility. Numerous applications using “pulse sequence” have been developed to expand the capability of NMR techniques. Pulse sequence is analogous to music scores for an orchestra to create the right harmony, which is the spectrum in NMR. Application of the right pulse sequence can enable sample information such as chemical structure, molecular shape, size and molecular aggregation to be obtained. Since NMR is non-destructive, the sample can be reused to gain more information using different pulse sequences, unless the sample condition has changed during the experiment . The major limitation of the NMR technique is low sensitivity compared to MS, resulting in reduced metabolite coverage  with NMR ,38. Compm/z) and peak intensity . Further information can be collected through tandem MS (MS/MS or MS2) in which either intact “precursor” ions are fragmented into “product” ions, or ions already fragmented during MS undergo further fragmentation, providing additional structural information about a given compound detected and thus enhancing compound identification capacity. MS2 is particularly useful for compounds with identical m/z values [In MS-based techniques, the compounds present in a biological sample are converted to ions (with either a positive or negative charge) through the use of an ion source. The formed ions then enter the MS instrument which detects ions and their abundance, providing a mass spectrum displaying mass-to-charge ratio , it is typically but not exclusively combined with chromatographic separation techniques such as gas chromatography (GC) and liquid chromatography (LC). Chromatography columns contain a stationary phase that interacts with the sample and uses the affinity of molecules to separate them within complex matrices. As molecules flow along the column, their different affinities for the stationary phase result in different retention times in the column and thus sequential introduction into the MS instrument, therefore enhancing sensitivity and identification capacity ,37. Due Lipidomics shares similar analytical techniques with metabolomics ,40. Althm/z values can be individually selected to visualize their signal intensity (thus abundance) within the tissue section. By merging the different colour-coded m/z signals, spatial distribution and abundance of different lipid species can be visualized throughout the tissue [Direct analysis from biological matrices is mainly performed by MS imaging. An appealing characteristic of this method is its ability to determine the spatial distribution of thousands of lipid species in tissue sections without any labelling . The prie tissue ,44. Anote tissue .The analysis of lipid extracts without separation is often referred to as “shotgun” lipidomics or direct infusion-based lipidomics, whereby a given lipid extract is continuously injected in the MS instrument, generally after selective ionization by an ion source, which provides some lipid separation . DespiteA typical untargeted metabolomics/lipidomics workflow is composed of several experimental and analysis steps . The firm/z ratio and peak intensity in MS; and chemical shift in NMR, i.e., resonance frequency of a nucleus relative to a standard with a value of 0) of the compounds present in samples through the use of one  of the analytical platforms mentioned above. Once acquired, raw peak intensity data are processed to permit further analysis. In MS, data processing comprises many steps including conversion of raw peaks into data matrices, noise filtering, retention time correction, chromatogram alignment, peak detection, data normalization, and eventually, compound “putative” identification by matching metabolite/lipid spectra against in-house libraries and available databases such as HMDB, METLIN or LIPID MAPS. Putatively identified compounds are then benchmarked by the investigator, and the relative levels of identification confidence are assigned and reported according to the Metabolomics Standard Initiative (MSI). The (MSI), and more recently, the Lipidomics Standards Initiative (LSI), have notably been created to standardize the confidence levels for metabolite and lipid identification [Next, data acquisition refers to the detection and characterization  and partial least square-discriminant analysis (PLS-DA) are common examples of unsupervised and supervised multivariate methods, respectively [Following identification , a broad range of statistical analyses is performed to determine potential differences between samples and/or experimental groups. Commonly used statistical methods include univariate and multivariate analysis, either in an unsupervised or supervised manner. ANOVA and ectively . Brieflyectively ,54. A plThe last step of the workflow is data integration and interpretation, which allows the investigator to link detected compounds with their biological context using publicly available software tools and databases that further enable pathway and enrichment analysis, metabolite/lipid mapping and visualization. Among these available databases, the Kyoto Encyclopedia of Genes and Genomes (KEGG), LIPID MAPS and MetaboAnalyst are widely used in applications of metabolomics and/or lipidomics ,55. HoweFollowing the introduction of metabolomic and lipidomic approaches, analytical platforms and experimental workflows above, we overview in this section metabolomic and lipidomic findings made over the last decade in the context of acute exercise. We selected 25 primary research articles and one systematic review specifically focusing on molecular metabolic responses to a single bout of exercise  within the first minutes/hours and up to 72 h following this single exercise bout, in healthy subjects. Articles that exclusively investigate the effects of acute exercise on the metabolome/lipidome in subjects with disease states , as well as articles investigating the effects of repeated exercise bouts , were not included in this review. We discuss metabolomic/lipidomic findings related to both acute aerobic and acute resistance exercise bouts, with the term “endurance” used to define an aerobic exercise bout of 30 min or longer duration. As opposed to aerobic exercise which typically consists of repetitive physical activity against relatively low loads and requires the use of oxygen for energy conversion, resistance exercise consists of muscle contractions performed against relatively high loads ,58. FindAn acute bout of exercise dynamically impacts the human metabolome in a range of biological fluids including blood, plasma/serum, urine and sweat, amongst others. The high variability between existing human studies in terms of age, sex, BMI, exercise type (resistance versus aerobic/endurance), modes , sample types, collection time points, as well as analytical platforms used, presents challenges in distilling these large datasets into a consensus molecular metabolic signature of exercise. Blood is considered an integrative biofluid given that it contains metabolites exchanged between organs and is therefore suitable for relatively comprehensive metabolic profiling . Blood cFollowing a qualitative systemic review of human exercise metabolomics studies by Sakaguchi et al. , SchrannDespite some metabolite classes such as amino acids and derivatives showed mixed responses  between exercise types  as well as differences between endurance studies amongst the 27 studies analyzed , other mBloodThe mixed responses observed in amino acid levels following an acute exercise bout  may be eOverall findings from this systematic review  are suppIn contrast to some of the above findings, other studies have identified elevated serum levels of amino acids including alanine, tyrosine and phenylalanine in males following marathon running, whereas alterations of cholesterol and steroid metabolism following a marathon were consistently reported in these two studies ,70, as en = 18), average (n = 40) and low (n = 18) performers, based on VO2max  and race completion time . Blood samples from these three groups were examined for potential differences in metabolomic responses to marathon running. Immediately post-race, the low performers exhibited a significant increase in a wide range of acylcarnitines (from short to medium- and long-chain) in plasma compared to the average and top performers groups. One possible explanation for these differences in acylcarnitine levels between top/average and low performers is that low performers may have a reduced capacity to oxidize lipids, which may in turn lead to an accumulation of acylcarnitines. Differences in arginine metabolism and urea cycle related-metabolites between low and top performers have also been reported [Metabolomic responses to exercise in blood have also been shown to be influenced by an individual’s level of fitness. For instance, in a study by Schader and colleagues , male amreported . Argininreported . Furtherreported . These oreported . HoweverAnother recent metabolomics analysis of plasma from young active men performing an acute time-to-exhaustion cycling trial allowed the separation of metabolomic profiles in a 20-min window, and the identification of biomarkers at the onset of fatigue . One keyRecently, Contrepois et al. investigated metabolic responses to acute aerobic exercise  using multi-omics  blood profiling . The stuUrineInterestingly, metabolomics analyses of blood samples have also proven useful in studying the role of liver in exercise metabolism. Indeed, Hu et al. recently highlighted liver-skeletal muscle crosstalk during acute exercise by analyzing arterio-venous differences of metabolites in: (1) the hepato-splanchnic bed; and (2) the exercising and resting leg, in young men . These dSalivaAlthough blood is the most commonly studied biofluid in the research area of exercise metabolomics, an increasing body of work in the field of exercise involves analysis of other biofluids including urine, saliva and sweat. After blood, urine is seemingly the most commonly analyzed biofluid in the context of exercise metabolomics. In humans, metabolite profiling of urine samples is appealing since it has proven to be more stable, under less homeostatic regulation than other biofluids , and colSweatSaliva has gained attention over the past few years in the study of exercise metabolomic biomarkers. Like urine collection, saliva is collected non-invasively and does not require specialized laboratory facilities or skilled healthcare professionals. However, only few metabolomics-based studies investigating the effects of exercise in saliva have been conducted to date  or the rate of perceived fatigue. Several confounding factors and current pitfalls of sweat-based metabolomics studies have been reported, including the absence of localized sweat rate measure; normalization methods of analyte concentrations; sweat collection devices utilized, which can also constitute a great source of interindividual and inter-study variability; and the presence of skin bacteria and cosmetics that may interact with sweat metabolites [Sweat also represents an understudied biofluid in the field of exercise metabolomics, partly due to its relatively low metabolite concentrations. However, sweat metabolomics has proven useful in other contexts such as cancer diagnostics . Among mabolites . This, iOverall, metabolomics studies analyzing human biofluids in the context of exercise are far more common than human studies investigating tissues. One of the main reasons for the currently limited tissue metabolomics studies involves the more invasive nature of human tissue biopsies  compared to routine blood sampling or sweat, urine and saliva collection. However, it has been demonstrated that blood and skeletal muscle metabolomes have very little overlap, thus suggesting an overall limited ability to potentially identify muscle tissue-specific metabolites from blood samples . MammaliAs opposed to human studies, other mammalian studies  assessing the effects of a single bout of exercise on the metabolome have predominantly analyzed tissues relative to biofluids. These biofluids including saliva, sweat and urine are not collected as easily and not available in sufficient volumes in small mammals such as rodents. Several metabolomics studies in rodents have analyzed skeletal muscle following an acute bout of exercise. Building upon the results in human biofluids demonstrating differential responses to exercise depending on fitness level and performance, it has been shown in mouse hindlimb skeletal muscle that metabolic responses to exercise are dependent on the time of the day during which exercise is performed . In a stFurthermore, metabolomic analysis of both plasma and hindlimb skeletal muscle from rats with high and low running capacities has provided insights into substrate utilization during and following an exhaustive running bout . FollowiIn addition to rodents, tissue metabolomic responses to acute exercise have also been studied in other mammalian species. For instance, the skeletal muscle metabolome in horses was recently investigated after a single incremental exercise test to exhaustion, in both an untrained and trained status . Only 31The liver, which is crucial to whole-body energy supply and maintenance of metabolic homeostasis during exercise, has also been studied using metabolomic approaches. In 2010, Huang and colleagues were the first to use metabolomics to investigate changes in liver metabolic profile induced by exhaustive treadmill running in rats . Increasn-3 and n-6 polyunsaturated FA (PUFA) and metabolized by CYP were also released into the bloodstream following an acute maximal treadmill test using the Bruce protocol [The release of vasoactive metabolites from the working skeletal muscle and vascular endothelium during exercise is well documented . These nprotocol  in healtprotocol ; and 12,protocol . Diols sprotocol , potentiprotocol . The phyprotocol . 2max, respectively) in healthy humans, regardless of sex, age and physical activity level [While no significant changes in plasma levels of other detected epoxides and diols were observed following maximal exercise in humans, Stanford and colleagues recently showed substantially increased levels of circulating 12,13-dihydroxyoctadecanoic acid —the downstream product of 12,13-EpOME—after a moderate-intensity exercise bout (cycling and running at 70 and 75% VOty level . In thisty level . These lty level . Howeverty level  and mitoty level . The bio2max) [n-6 LA , supporting the inclusion of 9- and 13-HODE as oxidative stress biomarkers [Increased plasma levels of 12,13-DiHOME and other lipid mediators have also been observed in plasma collected from trained male cyclists. In this study, cyclists completed a 75-km cycling bout at moderate intensity (~70% of VO2max) . Increas [n-6 LA  that havomarkers .Gollash and colleagues have helped expand the understanding of metabolic responses to acute maximal aerobic exercise by investigating changes in lipid profiles from red blood cells (RBC), a constituent of blood largely overlooked in metabolomic/lipidomic studies . RBC rep2 and its metabolites TXB2 and 12-hydroxyheptadecatrienoic acid (12S HHTrE). TXB2 and 12S HHTrE are considered biomarkers of TXA2 biosynthesis and are more easily detected since TXA2 is rapidly degraded into TXB2 or 12S HHTrE [2, PGE2, PGF2α and PGI2. Likewise, PGs are also rapidly converted to primary 6 or 15-keto and secondary 13,14-dihydro-15-keto metabolites. Next, LTs are composed of LTA4 and derivatives including LTB4 and anti-inflammatory/pro-resolving lipoxins LXA4 and LXB4. Efforts to characterize lipid mediators derived from COX, LOX and CYP pathways responding to acute resistance exercise have also been performed . Using t2S HHTrE . PGs com2 and 12S HHTrE; (2) PGD2, PGE2 and its derivative 15-keto PGE2, 15-keto PGF2α, 6-keto PGF1α; (3) LTB4 and derivatives LXA4, LXB4; (4) pro-inflammatory AA-derived 12-hydroxyeicosatetraenoic acid (12-HETE) and its byproduct tetranor 12-HETE, along with the anti-inflammatory 15-HETE. Early responses to resistance exercise were also marked with immediate increases in EPA-derived resolvins (RvE1) and DHA-derived 10(S),17(S)-DiHDHA (protectin D1), which are generated by LOX enzymes and exhibit anti-inflammatory properties. In contrast, LOX-mediated LA derivatives such as 9- and 13-HODE and their degradation products 9- and 13-oxo-octadecadienoic acids (9- and 13-oxo-ODE) tended to decrease in the first half hour following exercise before significantly increasing (compared to 30 min post-exercise) and peaking at 2–3 h of recovery. Similarly, CYP-mediated LA metabolite 9,10-EpOME and downstream product 9,10-DiHOME were also elevated at 2–3 h of recovery. Finally, elevations in 11,12- and 14,15-DHET-CYP-mediated metabolites of AA-were also found in the early recovery phase. Most lipid metabolites returned to basal levels within 24 h post exercise, except for a few metabolites which peaked  or remained significantly elevated (12(S) HHTrE). These findings pinpoint the activation of pro-inflammatory and pro-resolving pathways following acute resistance exercise, both in the early and later stages of exercise [Early resistance exercise responses (0–3 h of recovery following exercise) comprised increased levels of (1) TXBexercise .In addition to FA-derived lipid mediators involved in pro- and anti-inflammatory/pro-resolving pathways induced by acute exercise, lipidomics has also helped recently uncover other lipid species belonging to other categories and classes and playing potential roles in exercise metabolism. Indeed, the lipidomics arm of Contrepois and colleagues’ work  revealedSimilar to the blood analyses previously described, further lipidomics analyses of skeletal muscle have uncovered inflammatory responses to acute resistance exercise. Lipidomic analyses of muscle biopsies from young active men during a single resistance exercise session indicated augmented inflammatory response in the early recovery phase . Two houLipidomic responses to acute resistance exercise are however influenced by an individual’s age. Applying targeted lipidomics to skeletal muscle from both young (~22 years old) and older (~74 years old) healthy men who undertook a single resistance exercise bout, Rivas et al. observed significant differences in skeletal muscle ceramides between the two cohorts . Ceramid2max) treadmill running bout to exhaustion. Of the 933 plasma lipid species detected, 130 were known lipids. Despite the lack of statistical power in this pilot study, 13 lipid species were changed following exercise, including seven FA and six phospholipids; three phosphatidylcholines (PCs), one lysophosphatidylcholine (LPC) and two sphingolipids (SMs). Although not reaching statistical significance, five TAGs decreased by more than 20% following exercise. While six identified phospholipids , (P-36:2), (P-36:4); LPC (18:0); SM (d36:1) and (d42:2)), along with several FA such as LA, LNA (C18:3), and the n-6 11,14-eicosadienoic acid (C20:2), were increased by supramaximal exercise, the remaining FA  were decreased post-exercise. The increase in plasma unsaturated FA may reflect lipolysis in the adipose tissue, reported to contain a higher ratio of unsaturated/saturated TAGs compared to plasma FA [In other mammals, to date very few studies have been performed to investigate the effects of acute exercise on the blood lipidome. To our knowledge, the only study aimed to specifically characterize the lipidome following acute exercise in mammals to date is a pilot study performed in Thoroughbred horses . Four holasma FA . Phospholasma FA . Althouglasma FA .As stated previously, the feasibility of investigating liver biopsy responses to exercise in humans is very low; therefore, utilizing other mammalian species has helped gain a deeper understanding of tissue lipidomic responses to acute exercise. In 2010, Hu and colleagues were one of the first to conduct a lipidomic study to uncover molecular responses to acute treadmill running exercise in mouse liver . Among 1More recently, this same group used a similar endurance exercise protocol to study the lipidomic responses to acute exercise in both liver and hindlimb skeletal muscles (gastrocnemius and soleus) in mice . Their tThe extensive exercise metabolomics and lipidomics literature from the past decade discussed above has helped build a strong foundation to continue expanding the molecular landscape of exercise in the next decade. To continue expanding the exercise molecular landscape in the next decade, metabolite and lipid identification/annotation still represents a main challenge and bottleneck of untargeted metabolomics and lipidomics approaches, in contrast to protein identification in proteomics, for example. Whereas proteins are composed of a finite and more manageable combination of different amino acids that can be sequenced by matching experimental peptides against in silico fragmentation spectra, metabolites (including lipid species) are a highly heterogenous group of small molecules resulting from countless different chemical structures and atomic combinations, although predominantly composed of the elements C, H, N, O, P and S . Despite2 (and sometimes MSn) is required to reach the highest level of confidence, as fragmentation patterns help elucidate molecular structures and distinguish metabolites with similar m/z and retention times by matching them with fragmentation patterns of authentic chemical standards within metabolite libraries. Nevertheless, most libraries are still largely incomplete, therefore the number of authentic chemical standards available represents a current limiting factor to metabolite identification of the broader metabolome. Additionally, compounds can exhibit different levels of confidence in identification/annotation, making data integration and interpretation even more challenging since most commonly used dedicated tools  require metabolite identification  to integrate the data into biological context [Next, metabolites and lipid features (such as mass-to-charge ratios and retention times) that meet quality control criteria can still correspond to numerous molecular structures. Their identification—a term used when the highest level of confidence is reached; level 1—or annotation   notably  context . 2 is however more time- and resource-consuming. In addition, validation of metabolite identification/annotation still requires extensive human intervention, since this step is usually performed manually and requires expertise in chemical structure and biochemistry. This hurdle may become a growing issue as the number of metabolites to manually validate increases with the expansion of metabolite libraries in the years to come. It is also important to note that MS2 is not always sufficient to distinguish structural isomers—compounds with identical molecular formula but different chemical bond arrangements between atoms—and stereoisomers—compounds with identical formula and chemical bond arrangements but different spatial orientation of groups in the molecule [n may be required to validate the identification of a metabolite or lipid species. Of note, NMR represents a quicker and cheaper alternative (in terms of cost per sample) to MSn with regard to structural elucidation [Efforts to expand libraries with authentic standards in the next decade will help exploit the full potential of untargeted metabolomics by yielding a much higher coverage of unequivocally identified metabolites. MSmolecule ,140,141.cidation .One of the main challenges encountered in human exercise studies is the high interindividual variability in genetic background, sex, age, lifestyle, environmental exposure and nutritional and health status , which rhttps://motrpac-data.org (accessed on 15 December 2020) [Human metabolomics studies to investigate the molecular mechanisms of acute exercise are however starting to be performed at a larger scale. Indeed, a recent study investigated blood metabolic profiles of over 400 middle-aged adults, uncovering metabolic signatures associated with cardiometabolic health . In addier 2020) ,146. Metabolomics/lipidomics studies in the fields of sport and exercise physiology to date have mostly been conducted using only male participants, as highlighted in a recent human exercise metabolomics review , with onAnother major challenge in exercise-related metabolomics and lipidomics studies is the ability to directly compare studies between independent studies and research groups. The current lack of reproducibility and the common discrepancies observed within a given research field may in part be attributed to intrinsic and extrinsic/environmental confounding factors described in the previous section, as well as experimental factors see . Study dTo deal with the complexity and heterogeneity of metabolomics and lipidomics datasets  and theUntargeted metabolomics/lipidomics is a hypothesis generating method and as such, future work should also focus on following up on these generated hypotheses, notably by using targeted approaches to validate findings and provide more quantitative insight  into exercise-regulated metabolites/lipids and biochemical pathways. Although metabolite and lipid concentrations provided by these targeted approaches are crucial to enhancing the measurement accuracy of exercise-induced metabolite changes, they alone only provide a snapshot of metabolic reactions that have just occurred. Therefore, complementary analyses such as metabolic flux analysis, also called fluxomics—a method that combines stable isotope tracing of metabolites with MS or NMR spectroscopy—help depict metabolic reaction capacities, therefore allowing more mechanistic insight into the dynamics of molecular metabolic reactions that may explain, at least partially, observed differences in metabolite concentrations between and/or within individuals over a certain time or exercise intervention . In addiWith respect to compound identification, the numerous and constantly evolving technologies in the metabolomics and lipidomics fields also provide tremendous potential for metabolomics and exercise research communities in the next decade. For example, the combination of IMS with chromatography and MS methods will help increase metabolite identification capacity. IMS-MS instruments are now available and implementation of IMS to current analytical platforms used in the field of exercise will help improve coverage of identified metabolites, therefore expanding capacity to map molecular responses to exercise. Likewise, the implementation of multidimensional approaches—where several separation methods are combined into a single analysis  to enhanAdvancement in technologies will also aid how omics research of human exercise and athletic performance will be taken from the “bench and into the community.” Considerable interest in the use of dried blood spots (DBS and miniaturization technologies) in human performance fields may offer several important advantages over conventional whole blood sample collection. First, sample collection is less invasive compared to venepuncture and easy to perform . This is particularly important for maximizing participant recruitment in the context of frequent repeated blood sampling. Second, blood sampling can be performed by an individual and away from a laboratory setting, such as the home of a clinical patient following only minimal training. Third, the use of miniaturized devices allows for low volume of blood to be collected , compared to standard venepuncture sampling which requires a minimum of ~2 mL of whole blood. Finally, as the name implies, samples are air-dried and can be shipped by mail to laboratories at minimal expense and without the need for maintaining stable temperature environments. Such devices will benefit from discovery research, where stable and quantifiable metabolites can be selected and developed for measurement of metabolites collected via miniaturized technologies. Considerable work is already occurring with DBS and dried urine spots, and companies are now developing technologies which can provide users with live measurement of metabolite concentrations which might be useful indicators of health or fitness phenotypes. These approaches will facilitate personalized exercise interventions and provide sport and exercise scientists with data that can inform decision making, health tracking and athlete phenotypes. In other mammalian species, recent efforts to utilize mouse genetic diversity panels and recombinant inbred mouse strains in metabolomics/lipidomics research have proven valuable tools for genetic mapping  and investigating environmental exposure, providing new insights into compound identification, as well as the molecular basis of metabolic health and disease. For example, a recent large-scale genome-lipid associated map and resource termed LipidGenie was generated by analysing liver and plasma samples from diversity outbred mice, which permitted the identification of unknown lipids from MS data by mapping molecular lipid features to genetic loci . In addiIn the next decade the exercise biology and metabolism fields will continue to benefit from these ongoing efforts in the metabolomics and lipidomics research community. Metabolomics/lipidomic analyses may ultimately be capable of being performed as part of routine health checks to assess an individual’s metabolic status  and responses to a given stimuli  as well as predisposition to certain diseases. This will in turn enable highly tailored and personalized exercise, diet, and/or medical interventions to prevent, delay and combat metabolic disease .The unbiased global measurement of metabolomes and lipidomes from different sources such as biological fluids and tissues are constantly improving. They represent a promising avenue to unravel the complex and interconnected metabolite and lipid networks underlying the molecular responses to exercise throughout the body. In the context of exercise biology, the application of metabolomics and lipidomics as a hypothesis-generating approach has dramatically increased the number of biological targets measured simultaneously thanks to the major technological advances over the last decade. This has allowed the measurement of hundreds to thousands of metabolites (including lipid species) in a single run and subsequently revealed biomarkers of exercise intensity, training state and exercise capacity, fatigue, among others. In summary, findings made over the past decade have revealed numerous metabolites regulated by acute aerobic/endurance and resistance exercise in various biofluids and tissues across mammalian species, including: glycolysis end-products and TCA cycle intermediates; FA, acylcarnitines and TAGs; ketone bodies; nucleotides and derivatives; amino acids and derivatives; vitamins; steroid hormones; as well as less characterized complex lipids with various functions, pro- and anti-inflammatory properties and vasoactive properties .Although considered the closest reflection of an organism’s metabolic phenotype, the metabolome and the lipidome alone will not be sufficient to understand to complexity of exercise’s molecular landscape. Therefore, extensive efforts to integrate metabolomics and lipidomics data with other layers of biological regulation such as the genome, proteome and phosphoproteome will also be required. Importantly, expansion of metabolite and lipid databases will convert a much higher proportion of data into useful information and meaningful biological insights. In addition, continued advancements in instrumentation and analytical platforms in the metabolomics and lipidomics field will help standardize and harmonize experimental procedures from study design, sample handling, data acquisition, processing and analysis, as well as reporting. Together these efforts in the next decade will help maximize the utility of metabolomic and lipidomic profiling in exercise biology, metabolic health and disease, and beyond.


This cross-sectional study analyzes trends in out-of-pocket (OOP) health expenses in the United States during the last 2 decades and compares the distribution of services that contribute most to OOP spending. One goal of the 2010 Patient Protection and Affordable Care Act (ACA) was to limit patient out-of-pocket (OOP) health expenses. This cross-sectional study aimed to analyze trends in OOP health expenses in the United States during the last 2 decades and compare the distribution of services that most contribute to OOP spending.STROBE) reporting guideline for cross-sectional studies. Institutional review board approval and informed consent were waived because the data were publicly available. Data from the National Health Expenditures (NHE) Accounts from 2000 to 2018 were analyzed. OOP expenditures represent total payments made in the form of deductibles, coinsurance, and health and flexible savings accounts and by individuals who are uninsured. Expenditure estimates were converted to per capita estimates, and the Consumer Price Index was used to adjust all values to 2018 US dollars. Significance was determined using linear regression with gamma error distribution and log link. Significant changes in trends between pre-ACA (2000-2009) and post-ACA (2010-2018) OOP health expenses were determined using an interaction term between ACA and calendar year within the regression. Statistical analyses were 2-tailed (α < .05) and performed using Stata version 15.0 (StataCorp).This study followed the Strengthening the Reporting of Observational Studies in Epidemiology  (P for trend < .001).From 2000 to 2018, total OOP per capita health expenses increased from $1028 to $1148. The average annual growth rate (AAGR) of OOP spending significantly decreased following the ACA  . Mean (S = .001) . Total p1 The ACA implemented OOP spending maximums and increased access to preventive care services, which may have counteracted high OOP spending in HDHP plans. However, ACA-mandated price ceilings are still significant—$7900 in 2019—which may explain why OOP spending is still increasing annually.2 We speculate that ACA-imposed spending limits for HDHPs account for substantial OOP savings. Furthermore, access to coverage for individuals who were previously uninsured may account for additional OOP savings.Compared with the pre-ACA period, OOP spending increased at a slower rate for almost all health care services during the post-ACA period. Tax legislation in 2003 encouraged employers to provide high deductible health plans (HDHPs) to their employees; however, HDHPs do not reduce OOP spending because of the high upfront cost of care.3 demonstrated that at in-network hospitals, some services provided by anesthesiologists, pathologists, or assistant surgeons were billed as out-of-network services, with patients held responsible for additional costs. These costs may come under control with the passage of recent federal legislation to limit surprise billing.AAGRs for OOP health expenses have increased for physician services since the introduction of the ACA, possibly because of increased use of out-of-network care. Cooper et al6OOP health expenses for prescription medications decreased rapidly from 2010 to 2018. Reasons for these findings include increased prevalence of prescription to nonprescription switches for medications, increased number of clinicians using nonprescription medications as first-line management, loss of patent protection for name-brand drugs, and increased use of prior authorization for prescriptions.Although the ACA may provide a partial explanation for OOP spending trends, we cannot definitively attribute these changes to the ACA alone. Coinciding economic events, such as the Great Recession, likely decreased consumer willingness to pay OOP for health-associated costs. In the current study, we were unable to determine whether these decreases were secondary to disproportionately high rates of Baby Boomers becoming eligible for Medicare. Moreover, savings in OOP health spending may be nullified by increased taxpayer spending on Medicaid. Access to coverage for individuals who were previously uninsured through Medicaid expansion may account for the greatest OOP savings.


Whether or not capillary pericytes contribute to blood flow regulation in the brain and retina has long been debated. This was partly caused by failure of detecting the contractile protein The aim of this review is to summarize recent developments in detecting Evidence suggests that for visualization of the small amounts of With guidance of actin biology, innovative labeling and imaging techniques can reveal the molecular machinery of contraction in pericytes. For a ,in vivo, which typically exhibited dilation or constriction with slow onset but with prolonged duration in response to physiological (a few seconds of odor exposure),in vivo as well, but currently remains technically challenging.First, capillary radius changes induced by downstream pericytes are heterogeneous. As reported by Hall et al.,,,,,–in vivo induced actin polymerization in these pericytes.,,,,cis and trans acting factors determine the outcome of expression in different cells and under various physiological conditions.–,,The second problem is difficulty in detecting small amounts of In summary, before concluding that downstream pericytes do not express 2,,,,Actin, an evolutionarily well-conserved microfilament, is the most abundant protein  in mammalian cells except for a few types.in vivo,,,,,,,,Cytoplasmic and smooth muscle isoforms of actin were detected in cultured pericytes.3Amanita phalloides, binds to monomers in actin filaments by interacting with two neighboring monomers.,–Actin is found in polymerized [polymeric/filamentous(F)] or depolymerized [monomeric/globular(G)] forms. Phalloidin, obtained from the mushroom Immunofluorescent staining with antibodies against actin is the conventional method of labeling actin in fixed tissues.in vivo, impacting stress fiber functioning and cytoskeletal architecture.,,A different set of tools is required for actin imaging in live cells and tissues. GFP tagging of monomeric actin was developed but GFP-actin was not efficiently incorporated into the polymerized form.Drosophila, a concentration-dependent disintegration of actin cytoskeleton has been reported.,Alternative probes are also available. Utrophin (UtrCH), an actin binding protein from humans, can bind to actin globules, hence; serve as a genetically encoded label for F-actin.4,,There is a highly dynamic balance between two actin forms, F-actin and G-actin in live cells, which rapidly shifts toward the polymerized form to promote contractility upon mechanical or chemical stimulation.,,–,F-actin polymerization is one of the universal cellular mechanisms providing contractility, motility, and tonus; therefore, it likely plays a role in pericyte contraction as well, as does it in vSMCsDifferential labeling of F-actin and G-actin in the same cell (see below) allows calculation of F/G actin ratio from fluorescence intensities and this ratio is significantly higher in capillaries with smaller juxtanuclear diameters relative to the segment origin5,,,We can take vSMCs as the proxy cells to gain insight into the contractile machinery and actin cytoskeleton in pericytes. In vSMCs, actin filaments are attached to focal adhesion points (dense plaques) on the plasma membrane to transmit the intracellular forces to the extracellular matrix,,,,,,,,,ex vivo,,In vSMCs, contractile and cytoskeletal actin filaments are differentially organized.6,,A number of pharmacological agents, mostly natural toxins, are commonly used to investigate actin polymerization-depolymerization dynamics . Besides,,Other pharmacological tools with different mechanisms are also available to modulate actin machinery. Latrunculins are derived from sea sponge.7,,,,Myosin is the principal cellular motor protein. Among 35 subclasses,,Actin-myosin cross-bridge cycling is accepted as the main contractile mechanism for muscle cells. Upon actin-myosin interaction, power stroke moves myosin along the actin filament.8Presence of contractile proteins like


This analysis was conducted to compare different systemic treatment options in the therapy of localized and resectable soft-tissue sarcoma. We tested these in a network against the current standard of resection with or without radiotherapy, which allowed direct and indirect comparisons. Chemotherapy given after the local therapy and chemotherapy added to regional hyperthermia before the operation were associated with a longer survival and likeliness of being disease free and alive. Chemotherapy tailored to the specific subtype of sarcoma did not improve outcomes. Adjuvant chemotherapy might be more helpful in male rather than female patients.p = 0.017). Likewise, neoadjuvant chemotherapy combined with regional hyperthermia (naCTx + HTx) also led to superior OS . Both neoadjuvant chemotherapy alone (naCTx) and perioperative chemotherapy (periCTx) did not improve OS . Histology-tailored chemotherapy (htCTx) also did not improve survival compared to surgery ± radiotherapy . The network analysis of DFS, LRFI, and DRFI revealed a similar pattern between the different treatment regimens. Adjuvant chemotherapy significantly improved DFS, LRFI, and DRFI compared to surgery ± radiotherapy. In direct comparison, this advantage of adjuvant chemotherapy was restricted to male patients  with no effect for female patients . Conclusions: Standardized chemotherapy in high-risk soft-tissue sarcoma appears to be of added value irrespective of timing. The benefit of adjuvant chemotherapy seems to be restricted to male patients. The addition of regional hyperthermia to neodjuvant chemotherapy achieved the best effect sizes and might warrant further investigation.Background: The standard treatment of high-risk soft-tissue sarcoma consists of surgical resection followed by risk-adapted radiation therapy. Further treatment options that may improve local and systemic tumor control, including chemotherapy, are not well established. Due to the heterogeneity of the disease, different systemic approaches as well as their application at different time points have been attempted. Methods: We conducted a systematic literature search for randomized clinical trials in the treatment of localized, resectable high-risk adult soft-tissue sarcoma comparing different treatment modalities according to the PRISMA guidelines. We extracted published hazard ratios and number of events for the endpoints overall and disease-free survival  as well as local and distant recurrence-free interval . The different modalities were compared in a network meta-analysis against the defined standard treatment surgery ± radiotherapy using the inverse-variance heterogeneity model. Results: The literature search identified 25 trials including 3453 patients. Five different treatment modalities were compared in the network meta-analysis. The addition of adjuvant chemotherapy significantly improved OS compared to surgery ± radiotherapy alone (HR = 0.86; CI-95%: 0.75–0.97;  Limb-sparing surgery with adjuvant or neoadjuvant radiotherapy has been accepted as the standard treatment for resectable soft-tissue sarcomas located in the extremities, trunk, or head and neck region ,2,3. TheOverall, soft-tissue sarcomas of the extremities and trunk are a very heterogeneous group of tumors consisting of many differently behaving histologically types with varying potential for local and distant recurrence. This is the rationale for a tumor-specific chemotherapeutic approach, consisting of a tailored systemic therapy for each subtype of sarcoma. At present, there is little knowledge about the optimal treatment regimen and sequencing to achieve the best outcome. Therefore, we sought to analyze the different approaches in a network meta-analysis.We conducted a literature search in the electronic databases MEDLINE and EMBASE until June 2021, without restrictions regarding language or publication status, for controlled randomized trials including surgery with or without radiation therapy and different timings or different therapeutic strategies of systemic therapy in resectable soft-tissue sarcoma in accordance with the PRISMA guidelines for network meta-analyses ,12. The We excluded trials comparing different chemotherapy regimens in the same oncological setting, e.g., regimen 1 vs. regimen 2 in the adjuvant setting, as the comparison of different systemic substances and dosages was not subject of this analysis. Patients not amendable for local therapy due to locally advanced disease or metastatic spread were also excluded from the analysis. Studies including GIST or any pediatric soft-tissue sarcoma were not part of this analysis. We extracted the hazard ratios or event numbers from the published randomized trials. In studies where only survival curves were available, we estimated the corresponding hazard ratios using the method published by Parmar et al. and Tierney et al. ,14.The quantitative meta-analysis of data from individual patients of the ‘Sarcoma Meta-analysis Collaboration’ group provided the best estimates of effect sizes for the analyzed endpoints . UnfortuA meta-analysis of effect sizes on overall survival (OS), disease-free survival (DFS), local recurrence-free interval (LRFI), and distant relapse-free interval (DRFI) was performed using the inverse variance heterogeneity model (ivhet). The definition of the analyzed endpoints was derived from the published trials. A correction of the follow-up time in the LRFI and DRFI odds ratios was not possible due to the small number of studies and different median follow-up times. All statistical analyses were carried out using the Microsoft Excel  add-in MetaXL . Due to possible heterogeneity of the study populations, the inverse variances of heterogeneity model (ivhet) by Doi et al. were chosen as a comparison method . This mep-values lower than 0.05.A subgroup analysis of matching endpoints and cohorts was possible for the direct comparison of surgery ± radiotherapy versus surgery ± radiotherapy + adjuvant chemotherapy. The effect of adjuvant chemotherapy with regard to gender of 2 EORTC trials was available from one trial only ,17 and aThe PRISMA flow chart for the literature search is presented in n total = 3.465) which we analyzed in this network analysis. Twenty trials evaluated the effect of adjuvant chemotherapy in addition to local therapy alone. One trial compared neoadjuvant chemotherapy to surgery and/or radiotherapy [Overall, we identified 13 publications that reported the results of 25 trials  comparing neoadjuvant chemotherapy to immediate surgery ± radiotherapy [The adjusted network effect sizes of all indirect comparisons  depended on one relatively small trial (otherapy . Howeverp = 0.017). Neoadjuvant chemotherapy  and perioperative chemotherapy (indirect comparison) had higher effect sizes than adjuvant chemotherapy, but did not reach statistical significance  and ). Tailored neoadjuvant chemotherapy (indirect comparison) had no statistical effect of significance . Regional hyperthermia in combination with neoadjuvant chemotherapy (indirect comparison) resulted in the highest effect size of all tested modalities in the network .The network analysis regarding overall survival is shown in Adjuvant chemotherapy significantly improved DFS, LRFI, and DRFI compared to surgery ± radiotherapy. Neoadjuvant chemotherapy added to regional hyperthermia also resulted in significant longer DFS. The point estimates in the network with the corresponding confidence intervals for the residual treatment modalities did not reach significantly. However, we detected the same trends for the treatment modalities as in the analysis of OS, trials with neoadjuvant chemotherapy reporting favorable effect size estimates.p = 0.004) compared to female patients  receiving adjuvant chemotherapy with a significant test for heterogeneity (p = 0.010). We further detected a trend for longer overall survival for the marginally resected patients  with adjuvant chemotherapy. No differences were found for the other subgroups studied, such as T-stage, grading, radiotherapy, or type of chemotherapy  high-grade soft-tissue sarcomas. The addition of systemic chemotherapy to the standard treatment in different sequences have been tested in several randomized trials with partially inconsistent results and several missing direct comparisons between treatment strategies. n = 134) comparing neoadjuvant chemotherapy followed by surgery ± radiotherapy to immediate surgery ± radiotherapy [Adjuvant chemotherapy and neoadjuvant chemotherapy in combination with regional hyperthermia were the only treatment strategies in the network meta-analysis that lead to statistically longer overall survival in comparison to surgery with or without radiotherapy . When raotherapy . Furtherotherapy ,49.For sole neoadjuvant chemotherapy and perioperative chemotherapy (before and after surgery) the same before mentioned limitation account regarding the estimates in the network meta-analysis. Nevertheless, the effect size indicate that these treatments could be beneficial for high risk soft-tissue sarcoma patients  comparedDisappointingly, histologically tailored neoadjuvant chemotherapy using trabectedin for high-grade myxoid liposarcoma, gemcitabine plus dacarbazine for leiomyosarcoma, high-dose ifosfamide for synovial sarcoma, etoposide plus ifosfamide for malignant peripheral nerve sheath tumors, and gemcitabine plus docetaxel for undifferentiated pleomorphic sarcoma, did not show a benefit in any investigated endpoint in the indirect comparison to surgery ± adjuvant radiotherapy . In the In this analysis, we present an update on the benefit of adjuvant chemotherapy in adult soft-tissue sarcoma  beyond the previous assessments ,17. AddiSystemic therapy in soft-tissue sarcomas remains a grey area where additional factors like localization of the tumor, histological subtype, feasibility of the surgical resection, type, and dosing of the systemic therapy should be considered when making a treatment decision. Further, the data analyses might be limited by the small sample size and the fact that patients from randomized trials might not reflect the real world patient population. However, complementary to the data from the randomized studies, Graham et al. used the National Cancer Database (NCDB) to observe the association between overall survival and chemotherapy in 5436 patients with the five most common subtypes of soft-tissue sarcoma with primary disease localized to the extremity or trunk . Multi-aNumerous attempts have been published predicting the individual patient’s prognosis using validated nomograms ,54. PasqThe comparison of different treatment strategies for the treatment of adult soft-tissue sarcoma in a network meta-analysis demonstrates that adjuvant chemotherapy and neoadjvuant chemotherapy combined the regional hyperthermia improve overall survival. The modest benefit of adjuvant chemotherapy might be restricted to high-risk patients and male gender. Neoadjuvant chemotherapy strategies might be more effective than adjuvant therapy, but the conclusion are limited by the number of included patients and available randomized studies.This work was presented at the Annual ASCO Meeting in 2020.


Eukaryotic genomes are highly complex and divided into linear chromosomes that require end protection from unwarranted fusions, recombination, and degradation in order to maintain genomic stability. This is accomplished through the conserved specialized nucleoprotein structure of telomeres. Due to the repetitive nature of telomeric DNA, and the unusual terminal structure, namely a protruding single stranded 3′ DNA end, completing telomeric DNA replication in a timely and efficient manner is a challenge. For example, the end replication problem causes a progressive shortening of telomeric DNA at each round of DNA replication, thus telomeres eventually lose their protective capacity. This phenomenon is counteracted by the recruitment and the activation at telomeres of the specialized reverse transcriptase telomerase. Despite the importance of telomerase in providing a mechanism for complete replication of telomeric ends, the majority of telomere replication is in fact carried out by the conventional DNA replication machinery. There is significant evidence demonstrating that progression of replication forks is hampered at chromosomal ends due to telomeric sequences prone to form secondary structures, tightly DNA-bound proteins, and the heterochromatic nature of telomeres. The telomeric loop (t-loop) formed by invasion of the 3′-end into telomeric duplex sequences may also impede the passage of replication fork. Replication fork stalling can lead to fork collapse and DNA breaks, a major cause of genomic instability triggered notably by unwanted repair events. Moreover, at chromosomal ends, unreplicated DNA distal to a stalled fork cannot be rescued by a fork coming from the opposite direction. This highlights the importance of the multiple mechanisms involved in overcoming fork progression obstacles at telomeres. Consequently, numerous factors participate in efficient telomeric DNA duplication by preventing replication fork stalling or promoting the restart of a stalled replication fork at telomeres. In this review, we will discuss difficulties associated with the passage of the replication fork through telomeres in both fission and budding yeasts as well as mammals, highlighting conserved mechanisms implicated in maintaining telomere integrity during replication, thus preserving a stable genome. Saccharomyces cerevisiae telomeres are comprised of 300 ± 75 bp of double stranded heterogeneous TG1–3/C1–3A repeats with a 8-15 nt overhang  [reviewed in ]. With foverhang . SimilarC0–1G1–8 .Like the rest of the genome, telomeres must be accurately duplicated during S-phase to ensure proper cell division. DNA replication is initiated at multiple replication origins in a bidirectional way . At eachThis review compares telomeric replication by conventional replicative machineries in humans and two lower eukaryotic model organisms, budding and fission yeasts. We first focus on difficulties encountered by the replisome in reaching the chromosomal ends, followed by a description of possible outcomes of interrupted “conventional” telomeric replication and the main pathways involved in proper telomere replication completion.Replication stress can be defined by the transient slowing or arrest of replication fork progression . At chroin vitro  folds into stable anti-parallel G4s, whereas G4s were unfolded when the complementary strand was present  or other non-B DNA structures in vitro . G-quadrin vitro . Whereas present . Hence,  present . Consequly short .In addition to the specific telomeric DNA structure, transcription from subtelomeric and telomeric areas and the presence of RNA-DNA hybrids could hamper fork progression. Indeed, different species of non-coding RNAs produced from subtelomeric and telomeric areas in yeasts and vertebrates, including humans, have been described . Of the In vitro, DNA-bound TRF1 and TRF2 block replication fork progression  experiments done on metaphase chromosomes [reviewed in   . The FPC humans) ]. The Ti humans) . The req humans) . In addi humans) .tel1Δ mec1Δ cells behave like telomerase-negative cells, exhibiting telomere shortening and senescence  compared to S. cerevisiae (16), as genetically engineered single chromosome budding yeast was able to produce survivors with circularized chromosomes  that arose via a single major loss event of telomeric repeats. It is thought that this event is triggered by stresses encountered during DNA replication and the resulting single critically short telomere is enough to cause growth arrest . Consist1–3 repeats of the G-rich overhang engaging in BIR with interstitial telomeric sequences (ITSs). These sequences are located in the subtelomeric region and can be used in order to repair a broken telomere by non-reciprocal translocation mechanisms   mechanisms, thus leading to an unlimited proliferative potential [reviewed in ]. Telomelomeres) , 1997. Alomeres) ]. Like ilomeres) . RAD52 clomeres) . As thorlomeres) ]. Thus, lomeres) , [reviewlomeres) ].S. pombe by binding to 3′ G-rich ssDNA created by reversed or broken replication forks, thereby recuperating telomere replication and protecting telomeres from inappropriate HDR , and the 3′ ssDNA overhang is bound by Pot1  complex . Howeveromplexes . Furtheromplexes . In addiS. cerevisiae, the CST complex promotes telomere homeostasis via several mechanisms. As the cell cycle progresses into late S phase and semiconservative DNA replication nears completion, removal of RNA primers at the lagging strand and resection at the leading strand produces 8-15 nt telomeric 3′ G-overhangs . The much longer repeat tracts as compared to yeasts could be the reason for an inherently increased potential for replication stress, therefore requiring more means for maintaining fork stability. However, we would like to propose an alternative view. An evolutionary key difference between yeasts and human cells resides in telomerase being constitutively expressed in yeast, whereas it is not expressed in most human cells. Thus, in yeasts, recovery from telomeric replication fork collapse could be achieved by telomerase action, as already observed in fission yeast . This efTherefore, frequent fork stalling at telomeres in human cells, despite the known risks associated with them, may be somewhat beneficial as they allow local and transient action of major DDR kinases (ATM and ATR) at telomeres, required for post-replicative processing of ends and efficient engagement of repair activities. Hence, a deeper understanding of replication stress in somatic cells versus cancerous cells (telomerase-inactive vs -active) could be important in advancing development of new drugs in cancer biology (see section “Alternative Lengthening of Telomeres”).EB and EP wrote the manuscript. RW amended and completed the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.