Further than this though, artificial cell researchhas the aim of combining synthetic materials and nonliving macromoleculesinto ordered assemblies with the ability to carry out more complexand ambitious cell-like functions. These activities can provide insightsinto fundamental cell processes in simplified and idealized systemsbut could also have an applied impact in synthetic biology and biotechnologyin the future. To date, strategies for the bottom-up fabrication ofmicrometer scale life-like artificial cells have included stabilizedwater-in-oil droplets, giant unilamellar vesicles (GUV’s),hydrogels, and complex coacervates. Water-in-oil droplets are a valuableand easy to produce model system for studying cell-like processes;however, the lack of a crowded interior can limit these artificialcells in mimicking life more closely. Similarly membrane stabilizedvesicles, such as GUV’s, have the additional membrane featureof cells but still lack a macromolecularly crowded cytoplasm. Hydrogel-basedartificial cells have a macromolecularly dense interior  that better mimics cells, in addition to mechanicalproperties more similar to the viscoelasticity seen in cells but couldbe seen as being not dynamic in nature and limiting to the diffusionof biomolecules. On the other hand, liquid–liquid phase separatedcomplex coacervates are an ideal platform for artificial cells asthey can most accurately mimic the crowded, viscous, highly chargednature of the eukaryotic cytoplasm. Other important key features thatresearchers in the field target include stabilizing semipermeablemembranes, compartmentalization, information transfer/communication,motility, and metabolism/growth. In this Account, we will brieflycover aspects of coacervation theory and then outline key cases ofsynthetic coacervate materials used as artificial cells , finishing with envisioned opportunities and potentialapplications for coacervate artificial cells moving forward.Cells have evolved to be self-sustainingcompartmentalized systemsthat consist of many thousands of biomolecules and metabolites interactingin complex cycles and reaction networks. Numerous subtle intricaciesof these self-assembled structures are still largely unknown. Theimportance of liquid–liquid phase separation (both membranelessand membrane bound) is, however, recognized as playing an importantrole in achieving biological function that is controlled in time andspace. Reconstituting biochemical reactions  Artificialcell research will improve our fundamental understanding of biologicalsystems, offer insights into how life could have evolved on earlyearth, and open the door to a wide range of possible health technologiesfrom therapeutics to tissue engineering and organoid development.4The development of artificialcells as interactive systems thatmimic features and functions of life is a significant research challengewith the potential for far reaching impact.6 This molecular approach complements top-down artificial cell fabrication,which utilizes parts of deconstructed eukaryotic cells (or even wholeprokaryotes) as functional building blocks, included in cell-sizedsynthetic scaffolds. Irrespective of the fabrication approach taken,synthetic cells aim to mimic several features of biological cells:(i) Compartmentalization has been shown to be essential both for the individual structure of the cell and for many intracellular processeswhich occur exclusively within specific organelles. (ii) Informationprocessing is performed in a number of ways, from biomolecular geneticmaterial reproduction to the application of chemical signals. (iii)Cell adaptability and motility are important traits to mimic withartificial cells. (iv) Finally, the more challenging features to mimicgrowth and division–which are beginning to be investigated.Bottom up assembly of synthetic macromolecules, biologicalcomponentssuch as nucleic acids and proteins, and other small molecules canallow for the production of capsules or droplets with cell-like properties.8 Advantages ofthese systems are ability to include protein synthesis machinery andother delicate supramolecular processes due to the idealized aqueousenvironment. Various groups have shown elegant examples of dynamicnonequilibrium self-assembly inside aqueous droplets as well as processingof genetic information and production of proteins.9 The downsides of GUVs and aqueous droplets as artificialcells are the limited resemblance of the aqueous lumen to the viscoushighly crowded cytosol of natural cells. Other artificial cell systemstake advantage of droplets  to perform compartmentalized reactions and cytoskeleton mimickingself-assembly processes.8 Hydrogel-basedartificial cells can have physical properties more similar to thoseof cells than of droplets,10 and recentexamples have shown in vitro translation transcription(IVTT) expression of proteins inside micrometer-sized hydrogels.11 The final main class of artificial cell materialsare based on liquid–liquid phase separated (LLPS) dropletsor complex coacervates, which mimic the viscous, dynamic, and electrolyte-richnature of the cell interior and are in fact also naturally observedin living cells. These macromolecular condensates are important formany intracellular processes and membraneless organelles, includingthe formation of RNA rich phases like nuclei and stress/p granules.13 The fabrication of artificial cells from macromolecular coacervatesis therefore a highly relevant method for mimicking biology. Indeed,coacervate droplets (both with and without outer membranes) have beenstudied by various groups with recent successes in elucidating RNAlocalization as well as duplex thermodynamics and mechanisms, alongwith incorporation of bacterial systems for dynamic/growing artificialcells.15The field of artificial cell research has seen rapid growth, withresearch groups taking different approaches to mimicking life-likecharacteristics of cells with synthetic materials overview 1. Possib16 Finally, wemention some areas of research where coacervate-based artificial cellscan continue to push the boundaries of biomimetic systems, to achieveincreasingly complex and dynamic life-like materials.In this Account, we will highlight new developmentsof syntheticand semisynthetic macromolecular coacervate materials in mimickingcellular systems. We start with briefly discussing some fundamentalaspects of coacervate formation, including an overview of the syntheticmacromolecules used for coacervate artificial cells to date. We thenconsider the key features of natural cells and how coacervates havemimicked these, referring to key examples from literature and ourgroup’s own research. Excellent recent reviews on hydrogels,vesicles, and aqueous droplets as artificial cells can be found, sothese will not be considered here.217 This phase separationcan also be classified as aggregative or segregative depending onwhether the macromolecules involved predominately exist in one phasetogether or separate phases and also depends heavily on conditionssuch as pH, salt concentration, and temperature. When these condensatesare formed from two oppositely charged polymers, they are termed complexcoacervates, or from one self-condensed ampholytic polymer–simplecoacervates. Complex coacervates were first observed in the early1900s, and their remarkable resemblance to the cytoplasm of cellswas already mentioned. Oparin already in 1953 described coacervatedroplets as an early form of a cell with potential to help explainthe origin of life on earth.18 More recently,the use of complex coacervates to study and model artificial cellshas been not only rediscovered, with particular interest in proteinand RNA function in these liquid condensates, but also extended toother aspects of cellular function such as motility and signaling.Solutions ofmacromolecules in aqueous conditions can form distinctpolymer dense droplets see 2.17 ThisBoth associative and segregative coacervation canbe representedwith phase diagrams, where variation of two specific conditions ofthe coacervate formation is combined with the observation of one/twophases on the plot 2a. The c19 The groupof Stephen Mann, and others, have utilized dextran polysaccharidesto great effect  dextran).20The synthetic polymer materials so far investigatedas complexcoacervate-forming systems include charged macromolecules in threemain categories: modified polysaccharides, polypeptides, and syntheticpolymers 2b.While21 Advantages of syntheticpolypeptides include the use of natural amino acids as building blocks,variability of side chain functional groups, and possibility for growing,including active droplets.25 Synthetic polymers are beginning to be used increasinglyfor artificial cell assembly.26 Their useallows for facile variation of charge density and molecular weight,access to alternative charge functional groups, architectures, andincorporation of aspects of natural systems through bioconjugationstrategies.31 Charge density along the polyelectrolyte backbone has been shownto play a significant role in enzymatic processes inside coacervatedroplets. Synthetic polymer chemistry and peptide chemistry allowdesign of libraries made of structures with varying charge densities.34 Finally, combinations of polysaccharides, polypeptides and syntheticpolymers, with other natural multivalent anions such as DNA/ATP/hyaluronicacid/heparin are also being studied as coacervate artificial cellsystems; however, these will be covered only briefly in this review.Polypeptides have alsobeen extensively studied as complex coacervate-formingmacromolecules.3In orderto mimic living systems, it is useful to break the criteriadown into the minimal necessary features. The following sections coverkey aspects of life-like systems which can be incorporated in coacervate-basedartificial cells: membranes,  compartmentalization,communication, motility, and metabolism and growth.3.135 Necessary biomoleculesare retained while certain small molecules exchange freely with theexterior environment, with more complex systems evolving with membranereceptors and ion channels to allow signaling and other tasks. Membraneson the coacervate droplet cell mimics can be created with a varietyof molecules from lipids, synthetic block copolymers, protein conjugates,synthetic peptides, or even lysed cell membranes. In addition to amphiphilemembrane formation, particle-based membranization and stabilizationof cell-sized droplets is an interesting approach and could allowincorporation of different functionalities at the droplet surface.37 The coupling of membranes with coacervate systems has progressedcoacervates to be more life-like by allowing semipermeable diffusioninto the droplet, while also increasing stability to coalescence.Although coacervate dropletswithout an outer membrane are able to act as micro/macroscale condensatesfor dynamic processes involving facile exchange of molecules, bothcoalescence and nonselective uptake of molecules can sometimes hindertheir use as artificial cell systems. Living cells control the exchangeof nutrients and waste with the environment through a semi permeablelipid bilayer membrane.19 The blockcopolymer consisted of a hydrophilic steric stabilizing poly(ethyleneglycol) block, a hydrophobic poly(caprolactone-gradient-trimethylenecarbonate), and an anionic poly(glutamic acid) block to facilitateanchoring to the net positively charged coacervate (PEG-(PCLgTMC)-PGlu). The particular design of this polymer allowsa balance among electrostatically driven coacervate attraction, hydrophilicsurface stabilization, and hydrophobic membrane self-association.Membranized artificial cells allow for distinct populations to bepresent, and therefore with the addition encapsulated enzymes, theycan enable the study of complex multicomponent processes on extendedtime scales.Our group developed a triblock terpolymer for the interfacial stabilizationof cell-sized coacervate microdroplets 3.19 The 38 in early studies thegroup employed fatty acids as membranes.20 Sodium oleate fatty acid spontaneously self-assembled at the interfaceof poly (PDDA)–adenosine triphosphate(ATP) coacervates, as well as poly(lysine)–oligonucleotidecoacervates.20 The membranes were multilamellarand mediated selective uptake of small molecules and large (bio)molecules.The group has more recently investigated lysed cell membranes as reconstitutedmembranes in artificial cell systems .39 The Keating group assembled phospholipidmembranes around both synthetic polymer-based and protein-based coacervatedroplets through a lipid thin-film resolvation method.40 The membrane composition could be varied dueto formulation from commercial synthetic phospholipids , 1,2-dioleoyl-sn-glycero-3-phosphotidylethanolamine (DOPE), and 1,2-dioleoyl-sn-glycero-3-phosphotidylserine (DOPS), DOPE-PEG 2 kDa),highlighting the modular nature of their system.Mann and colleagues pioneered the use of coacervatecell mimicmaterials in 2011;3.244Living cells rely on compartmentalization to function. For example,the genome is compartmentalized in the eukaryotic cell nuclei throughthe biomolecular condensate of DNA with histone proteins, and alongwith the nuclear envelope and nuclear matrix, maintains the integrityof genes as well as helps to regulate gene expression. This key organellefeature of cell systems is an interesting target for synthetic mimicsand allows the study of complex multicomponent kinetics and cascadeprocesses.15 The peptidematerials, decapeptides of arginine, lysine, and aspartic acid, weresynthesized by solid phase peptide synthesis and, when combined, formedcell-sized droplets with an inner phase of higher peptide densitythan that of the outer phase .Interestingly, this led to differences in RNA partitioning and RNAdissociation in the different phases, with higher amounts of duplexRNA in the outer phase, and offers insights into how membranelessorganelles are able to stabilize or destabilize nucleic acids in specificcompartments without the additional help of particular proteins orother binding molecules.Recently, Choi et al. used short polypeptides to form multiphasecoacervates and investigated the kinetics of RNA duplex formationin these different but neighboring phases 4.15 The 45 Coacervates formed multilayer arrangements ifthe coacervate to coacervate interfacial tension was lower than theinterfacial tension between the surrounding media and the coacervates.The demonstration of this concept in this elegant article highlightshow multiphase coacervates can be rationally designed from their monomercompositions and could be an interesting area for further research.If we consider coacervate systems formed solely from synthetic polymers,then there are fewer examples of hierarchical multiphase compartmentalization.One elegant example from Capasso Palmiero et al. shows how the modularityof synthetic polymers can be used to screen for a wide range of life-likecoacervate properties , hypothesized to mimic aminoacid groups present in specific attractive interactions, e.g., hydrogenbonding, pi–pi aromatic interactions, or electrostatic chargeimbalances, led to simple coacervate droplets that could be tunedto uptake certain molecules of interest. In addition, the authorscombined the discovered polymers to form multiphase droplets as primitivecell mimicking materials.Multiphase coacervate droplets have been shownto form over a widerange of coacervate materials , ssDNA, and ATP, to poly(trimethyllysine), poly,and DEAE-dextran) by, for example, the group of Spruijt.operties 5.26 The 42 The encapsulationof polymersomes or other vesicles can allow for precise separationof components, and therefore enable the study of chemical systemsor enzymatic networks.Further to these excellent examples of compartmentalizationthroughadditional hierarchical phase separation, we have demonstrated thatthis can also be achieved by inclusion of polymersomes in coacervateartificial cell systems.3.346 While artificial cells remain simplified modelsfor intricate living cell systems, they offer an opportunity to conductstudies involving signaling and reaction networks in a biomimeticcompartmentalized fashion. Researchers have made progress in modelingcommunication behavior in both artificial cell–artificial cellsystems and artificial cell–living cell systems. The mechanismsemployed for artificial cell communication are varied and can be triggeredby stimuli either external or internal to the system. Small moleculesignaling chemicals can diffuse into and from coacervates dependingon electrostatic interactions or hydrophobicity, while larger biomolecules,such as nucleic acids or proteins, can have additional physical and/ormultivalent interactions making controlled uptake and excretion fromcoacervates challenging. In such cases, different moieties can beused to tune noncovalent affinities, for example, nucleic acid strands,or histag NTA chemistry can be used to promote coacervate uptake.Future applications of this type of research could include use ofartificial cells to regulate antimicrobial activity, inducing celldifferentiation, rescuing particular cell functions, inducing celldeath, and use in biohybrid synthetic tissues.3Individual unicellularorganisms and populations of cells in tissues rely on biochemicaland mechanical signaling to communicate and drive collective behavior.47 This simplifiedsignaling concept provides a method to engineer life-like communicationpathways involving protein secretion, similarly to pathways in higherorder multicellular constructs. Other systems have been developedfocusing on other important biomolecular signals, such as DNA itself,to achieve information transfer in artificial cell populations. Thegroup of de Greef in particular have utilized aqueous vesicles toachieve DNA strand communication via diffusion,48 while the group of Walther have employed DNA-based coacervatesfor similar DNA strand communication studies.49So far many types of molecules have been investigated forartificial cell communications, including proteins, DNA, and smallmolecules (generated from enzyme-based cascade reactions). Our grouphas recently developed a protein shuttling system, where particularDNA strands are used as triggers to induce uptake and release of fluorescentproteins in two coacervate cell populations 6.47 This51 In polymer coacervateartificial cells, enzymes have been used to sense small moleculesthrough production of fluorescent moieties able to be easily detected.This has been expanded recently by Liu et al., who showed that enzymecontaining artificial cells could be built into tissue-like structuresmaintaining segregation of the coacervate populations .The anticoagulation activity of the produced NO was then investigatedby measuring real time light scattering of blood plasma, which inthe NO producing device was dramatically reduced (t1/2 = 280 min) compared to the control non-NO producingdevice (t1/2 = 32 min).In the realm of small molecule signaling and communication,earlywork was carried out in lipid vesicles, where bacteria would producea bioluminescent signal on sensing of a lipid vesicle synthesizedsugar molecule.ulations 7.50 A tuThis highlighted research in biomolecule and smallmolecule signalingin artificial cell systems represents an exciting direction for biomimeticmaterials and prototissue synthesis from a bottom-up strategy. However,there are still many unexplored research directions. One interestingexample for artificial cell communication studies is the incorporationof further functional genetic material to create systems capable ofcollective behavior based on signaling with more life-like pathways.3.4Cell migration is fundamentalto the development and health of living organisms. Aspects of tissuecharacteristics, such as wound healing, immune response, and developmentalbiology, all require cell movement. This cell motility can be determinedby chemical or mechanical cues. Failure in aspects of these motilesystems can lead to diseases including tumor growth, neurodevelopmentaldisorders, and vasculature diseases, therefore increasing understandingof cell motility could lead to new therapeutic opportunities.52 The PDDA/ATP coacervates were stable to coalescencedue to an interfacial increase in noncovalent interactions when thecoacervates were transferred from buffer to deionized water. Movementof the charged droplets through a maze structure was performed withtwo orthogonal DC fields. Achieving motion through electric fieldof a noncovalent electrostatically assembled structure is noteworthy,as this has only been shown with rigid or cross-linked particles before.Interestingly, when an AC field was applied to a solution of coacervatedroplets they formed aligned chain-like structures of connected yetdistinct droplets, due to the polarizable nature of the phase separateddroplets. This transient polarization of cell-like coacervate dropletswill be useful as a model system for further understanding electrodynamicsand hierarchical assembly of living systems.Engineering the movement of artificial cells can be achieved notonly with chemical fuels but also with magnetism, electric fields,and light. The latter three are interesting options because they canbe triggered externally to potentially achieve some form of spatiotemporalcontrol. Agrawal et al. used defined electric fields to investigatethe movement of coacervate droplets both individually through a microfabricatedpath and collectively as groups of droplets in an oscillating AC field.53 Using bioorthogonal click chemistry, the enzymes urease and catalasewere conjugated to the polymer membrane of the amylose coacervatesystem. Due to the fluid-like nature of the self-assembled polymermembrane, the enzymes were able to laterally diffuse, thus introducingtransient asymmetry to the artificial cell surface configuration ofenzymes. Chemical fuel mediated movement of the coacervate cell-likedroplets was determined by a balance of enzyme density and transientasymmetry with medium densities having a higher probability of displayingasymmetric arrangement of surface enzymes. These recent examples highlightthe use of electric fields and chemical fuels to drive the motionof coacervate artificial cells; other examples of noncoacervate systemshaving induced motion include systems propelled with light irradiationor are due to other physical phenomena such as Marangoni flows.54Our group has alsolooked at the motility of coacervate artificialcells, using chemical fuels and enzymes as motors 8.53 Usin3.555 Such a system wouldrequire a dynamic equilibrium to maintain the appropriate levels ofnutrients as well as waste removal, in order to have continued metabolicprocesses over longer time scales. Linked to metabolism are the functionsof growth and eventually division (in the case of a self-sustainingsystem).56 All of these dynamic functionshave only been shown for polymer-based coacervate artificial cellsin a few cases .58Some of the most important aspects of cellular machinery are alsothe most complex and difficult to replicate in synthetic systems.Metabolism, the process of energy consumption and waste production,is vital to cells and relies on interconnected mechanisms of (macro)moleculeproduction and degradation, in the presence of energy-rich phosphatecompounds ATP/ADP. The reproduction of these complex pathways in artificialcells is challenging.59 Lysine rich protein K72 in the presence of ADP did notform droplets, but in the presence of PyK, added phosphoenol pyruvatewas hydrolyzed to pyruvate and ATP was formed. The produced ATP wasable to form coacervate droplets with K72, and the growthof droplets was controlled by the calculated addition of the fuelmolecule phosphoenol pyruvate. The most advanced example of a dynamicand metabolic coacervate artificial cell to date was recently publishedby the Mann and Li groups ,the protocells could not only grow in volume (2×) but also undergomorphogenesis, transforming from spherical to amoeba-like nonsphericalshapes due to the internal metabolic activity.Nakashima et al. investigatedactive coacervate protocells that were able to grow in size througha pyruvate kinase (PyK) catalyzed mechanism.oups see 9.14 The 4The recent explosionof interest in liquid–liquid phaseseparation and biomolecular condensates in cells is increasing ourunderstanding of the biophysics involved and our recognition of theimportance of phase separation behavior in the origin of early cellstructures. In the future, multidisciplinary teams of chemists, physicist,biologists, and materials scientists will be able to tackle more complexresearch questions involving coacervate-based artificial cells. Themost exciting recent research in the artificial cell field, has involvedintegration of life-like functions such as dynamic metabolism andgrowth in micrometer-sized hierarchical materials. This will continuein the future with further integration of genetic information andIVTT machinery into coacervate droplets, which has to date been morechallenging than their equivalent incorporation into aqueous dropletsor GUVs. Achieving this could allow production of cytoskeletons orcell division machinery in situ, to further extend the lifetime ofartificial cells. IVTT in coacervate artificial cells would also allowmore complex communication based on nucleic acids and signaling proteinsor cytokines. Additionally, the materials used for complex coacervatescan, depending on the charged state, be toxic to cells. Future researchwill further develop these systems to be more biocompatible, whilestill maintaining enough charge density to form stable coacervatedroplets.60 Use of membrane stabilized coacervateartificial cells for the fabrication of tissues could open the doorto a number of new applications in tissue regeneration and organoidformation. The controlled placement of artificial cells in 3D spacecould also allow for higher degrees of spatiotemporal control in themimicking of functions such as communication. We think one of themost exciting advances in the field will come from the interactionof artificial cells with living cells to create hybrid materials withinteractive properties. The assembly of interactive materials thathave the ability to self-organize, can dynamically repair, and canalso sense and respond to their biological surroundings will be aresearch target for the future. The interplay between artificial andliving cells could lead to new applications in biomedicine, includingnew ways to modulate or restore diseased cell behavior and add orrepair cell function. The isolation of particular cell-like characteristics in vitro also allows the study of various pharmaceuticallyrelevant interactions in a life-like environment and could lead toartificial cell screening of drug compounds in the future. From amore industrial viewpoint, it is possible artificial cells could aidin the growing applications of synthetic biology for the advancementof sustainable manufacturing of chemicals, for agricultural applications,or in the food and drink industry.Another particularly exciting avenue of research willbe assemblyof artificial cells into macroscopic materials with higher order structure.Some groups have already made early progress in this regard, for example,several groups have utilized 3D printing and other droplet manipulationtechniques to fabricate artificial cells into 3D synthetic tissuesor prototissues.5In this Account, we havesummarized new developments of syntheticand semisynthetic polymer materials in the mimicking of cellular systemswith coacervate droplets. Fundamental aspects of coacervate formationwere first discussed with a focus on the ionic interactions of complexcoacervates systems. Factors affecting liquid–liquid phaseseparation were also outlined, and phase diagrams briefly highlightedas a convenient visual tool to understand whether a particular materialand conditions will phase separate. The materials so far investigatedas complex coacervate-forming artificial cell systems include chargedsynthetic polymers, modified polysaccharides, and polypeptides.Second, with reference to leading examples from the literatureand our group’s own research, we examined the key featuresof natural cells and how coacervate materials have been successfulin mimicking these. Life-like artificial cell systems should ideallyhave viscoelastic mechanical properties, outer membranes, hierarchicalcompartmentalization, communicative features, motility, and possiblythe ability to have self-sustaining functions like metabolism, growth,and division. Finally, we have mentioned some areas of research wherecoacervate-based artificial cells can have impact and advance thefield of biomimetic systems, to achieve increasingly life-like materials.


It is increasingly clear that Intratumor heterogeneity (ITH) fuels tumor evolution, matching the concept of cancer as a heterogeneous ecosystem of spatially and temporally modulated cell subpopulations, which exploits dynamic strategies to hijack local and systemic resources and tissue(s) space . ITH hasDistinct lncRNA/miRNA/protein regulatory axes have been described in many tumor subtypes, including colorectal cancer (CRC) . As wellAt a microenvironmental level, lncRNA/miRNA axes may affect immunity, as suggested by recent works which identify clinically relevant lncRNA immune-related signatures in tumors. According to this, it has been proposed that a lncRNA signature of tumor immune heterogeneity can predict the risk of distant metastasis in loco-regionally advanced nasopharyngeal carcinomas , or the At an epigenetic level, it should be noted that an intimate relationship between lncRNA and nuclear domains can be confidently traced, with lncRNAs mediating nuclear phase separation thereby affecting spatial gene expression during tissue development . This isBased on these evidence, future research efforts should aim to correlate lncRNA expression and ITH, by conducting a spatiotemporal analysis of lncRNA/miRNAs dynamics within cell subpopulations in the tumor, at a multilevel resolution. According to this, it will be of interest to gain insight on how lncRNAs hubs may fine tune cell states and phenotypes under adaptive stress responses (such as chemo- and radiotherapy).Importantly, we have described specific waves of microRNA clusters from EphA2- and EphB2-expressing CRC human and murine tumors, which have allowed us to define some stemness-related and progression-related functions to EphB2 and EphA2, respectively . The useA key question to pose herein is what mediates the cancer cells–TME crosstalk by cargoing lnc- and mi-RNAs. An obvious and important candidate for such a function is represented by the exosomes ,17. For Another foreseeable area of investigation is about how to exploit such regulatory circuits for therapeutic purposes. The pleiotropic nature and mode of action of lncRNAs may somewhat discourage precision-based approaches. However, this apparent downside may turn out to be useful if one considers using natural compounds, for which the mechanism of action is broad by definition. For example, this is the case of celastrol and butein—both naturally occurring compounds capable of modulating a large fraction of ncRNAs ,19.Finally, due consideration must be devoted to the models. Are we using clinically relevant models to study and dissect lncRNA functions? To date, the vast majority of the published research employs a two-dimensional culturing of the cells. Albeit useful, this may limit the clinical relevance of the gained knowledge., since many modulators of lncRNA behavior, including matrix stiffness , presenc


PhysioKit, an open-source, low-cost physiological computing toolkit. PhysioKit provides a one-stop pipeline consisting of (i) a sensing and data acquisition layer that can be configured in a modular manner per research needs, and (ii) a software application layer that enables data acquisition, real-time visualization and machine learning (ML)-enabled signal quality assessment. This also supports basic visual biofeedback configurations and synchronized acquisition for co-located or remote multi-user settings. In a validation study with 16 participants, PhysioKit shows strong agreement with research-grade sensors on measuring heart rate and heart rate variability metrics data. Furthermore, we report usability survey results from 10 small-project teams  who used PhysioKit for 4–6 weeks, providing insights into its use cases and research benefits. Lastly, we discuss the extensibility and potential impact of the toolkit on the research community.The proliferation of physiological sensors opens new opportunities to explore interactions, conduct experiments and evaluate the user experience with continuous monitoring of bodily functions. Commercial devices, however, can be costly or limit access to raw waveform data, while low-cost sensors are efforts-intensive to setup. To address these challenges, we introduce  Physiological signals have been actively explored in a wide range of research fields, given their usefulness in tracking health, as well as physical and psychological states. Particularly in human–computer interaction (HCI), much attention has recently been paid to physiological computing research, where it focuses on how to evaluate user responses to interventions, enhance user interactions with technology, or investigate how to support interpersonal interactions . Physiolhttps://www.apple.com/uk/watch/, accessed on 28 September 2023, Fitbit, https://www.fitbit.com/, accessed on 28 September 2023, Garmin, https://www.garmin.com/, accessed on 28 September 2023) has boosted the interest in physiological sensing forward, there is a need to pay attention to the challenges of using such devices, including limitations in robustness associated with sensor misplacement, body movements and ambient noise, reduced flexibility in adapting an acquisition interface to the study protocol, and relatively high costs of hardware or software  understand the difference between using this product and using physiological sensors directly.” (P3).When participants were asked what they appreciated about PhysioKit relates to and builds upon existing research.In this section, we discuss our main findings and show how PhysioKit is a fully open source toolkit for physiological computing that implements an Arduino-based sensing and signal acquisition layer, offering researchers flexibility to configure one or more Arduino-compatible physiological sensors . The simple and user-friendly interface of the software application layer is effective in streamlining the physiological data acquisition for a variety of applications, including those involving biofeedback. Its provision to synchronize data acquisition for remotely located users is enabler for conducting remote studies that require acquiring physiological signals. Furthermore, it is cumbersome to manually inspect segments of acquired signals, specifically in case of long acquisition duration and high number of participants. SQA-Phys introduced in this work can be applied both in real-time and as a post-processing step to automate the quality assessment of PPG signals, significantly reducing efforts of researchers. Furthermore, access to raw data not only gives HCI researchers ownership and control over the data, but also enables computational research to develop robust algorithms for handling real-world environments. Taken together, these aspects all contribute to enhancing the usability of the toolkit for researchers.PhysioKit is well below the least expensive of the commercial sensors mentioned in The overall hardware cost of the PhysioKit and the gold standard for HR. The PRV metrics also show good agreement during the baseline condition, while there is acceptable agreement during experimental conditions involving significant movement. This difference between performance related to HR and PRV can be explained by the findings of a recent study that assessed the validity and reliability of PPG derived HRV metrics, and found that PPG sensors are less reliable for HRV measurements [PhysioKit shows better agreement with the gold standard compared to the performance of existing PPG-based commercial devices mentioned in PhysioKit can be largely attributed to its sensing and signal acquisition layer, as well as the processing pipeline that includes signal quality assessment, filtering, and extraction of physiological metrics.The validation study in urements . HoweverRegarding flexible configuration, the validation study results show similar performance of the finger PPG sensor and ear PPG sensor, suggesting that alternative sensor sites for PPG can be explored to achieve different research objectives and support accessibility. The slightly low signal quality (pSQI) observed for finger PPG sensor can be attributed to the possible voluntarily movement of finger leading to more frequent motion artifacts, whereas inability to ambulate ear lobe makes it more promising site for acquiring PPG signals. This interpretation is further supported by the lower pSQI of ear-PPG signals for an experimental condition involving guided face movement A.PhysioKit, demonstrating its versatility in supporting data acquisition in different settings. The usability survey results  ,91. The PhysioKit an open-source toolkit are two-fold: (i) offering a flexible and cost-effective solution for research community, and (ii) leveraging the contributions from research community in addressing the existing limitations of PhysioKit towards introducing support for additional physiological sensors and biofeedback modalities, as well as extending the validation study for range of applications, sensor modalities and higher numbers of participants. The latter objective is expected to be achieved as a future work. Currently, the signal quality assessment module (SQA-Phys) mentioned in this work is limited to assess the quality of PPG signals, which could be extended to signals acquired using other sensors. Additionally, although the existing implementation of SQA-Phys provides high temporal resolution, its outcome is restricted binary classification at a given time-point. By using an appropriate activation function  at the final layer of the 1D-CNN architecture, as well as replacing the classification loss with the regression loss function, it is possible to obtain a continuous score from 0 to 1 to gain better insights into the signal quality, which will be addressed as a future work.The objectives of making PhysioKit to build a dataset to train affective music recommender system, no projects required performing data analysis based on the event-marking labels. In future work, this aspect of the toolkit can be further explored and validated. The hands-on training for groups participating in the usability study was provided owing to unavailability of elaborate installation and usage instructions for the toolkit at early development stage. The published repository of PhysioKit offers easy installation steps, along with detailed usage instructions. Furthermore, as a future work, tutorials for different application types will be made available at the repository homepage.While one project used event-marking function of PhysioKit for its use in real-world scenarios.Our future implementation plan further considers implementing a processing pipeline for contact-less sensing methods, such as an RGB camera-based remote PPG ,96 and aPhysioKit, an open-source and cost effective physiological computing toolkit that streamlines physiological signal acquisition and analysis for various HCI studies and applications. Uniquely, PhysioKit can synchronize signal collection in multi-user studies with both co-located as well as remote users. The evaluation study on heart rate and pulse rate variability measurements demonstrated the performance of PhysioKit compared to the reference system. The comparable signal reliability from the finger and ear PPG channels further indicates the possibility of enhancing accessibility to support participants with certain physical impairments . Further, the usability study highlighted the usefulness, satisfaction and ease of use associated with PHysioKit, emphasizing its positive impact in various application scenarios. PhysioKit provides further useful features for researchers and practitioners: visual biofeedback, which can be extended to other forms of biofeedback such as audio and haptics; and machine-learning-driven signal quality assessment that can significantly reduce efforts and time on manual signal inspection . Also, raw physiological data are stored in an accessible format, which can be organized as per the study protocol and participant ID and can foster opportunities for researchers to easily apply state-of-the-art analysis. PhysioKit thus provides a one-stop approach that supports physiological sensing, data acquisition and computing supporting for a broad spectrum of studies and HCI applications. This paper introduced


Given the enormous and diverse levels of stressors circulating in our environments, living systems, at any given stage of their life cycle are exposed to singular stress factors or a combination of stressors acting on them simultaneously. In this context, stress-homeostatic processes have been central to the field of Stress Response Biology. Among small animals, and as occupants of varied ecological niches, insects exhibit a diverse range of survival tactics. An array of impressive organismal- and cellular-level strategies, and defence machinery enable insect responses against potential stress-induced damages, thereby ensuring their survival and propagation even under challenging conditions.The field of stress response biology has gained rapid attention in the past few decades, with growing evidence and warnings that our environments are changing. Over the years, insects as representative animal models have served as reliable and tractable model systems to better understand and predict the impacts of changing environments e.g., . This ReThere were three original research articles and one review published in this issue that cover representative insects from various orders, namely, Coleoptera, Diptera, Hemiptera, Hymenoptera, and Orthoptera. These contributions provide an engaging overview of the adaptation profiles of various insect species and their survival responses to habitat-specific stress conditions. This Research Topic collates recent trends in insect stress biology at the organismal-, behavioural-, cellular, physiological- and molecular-levels using multidisciplinary approaches and cutting-edge tools and techniques.Khurshid et al. report noteworthy findings on the critical role of antioxidant enzymes and Heat-Shock Protein (HSP) genes in the management of redox homeostasis under heat-inflicted oxidative damage in the green peach aphid, Myzus persicae. Cause-effect relations of thermal sensitivity in insects has been an important research area, and this study unravels the implied potential of insect stress biology research in the management of this highly adaptable and polyphagous insect pest that burdens the agricultural industry.Fudlosid et al. present new insights on the effects of microplastic-based diets administered in the tropical house cricket, Gryllodes sigillatus. This study examined whether supplementing a standard diet with either polyethylene microplastic beads (75–105 μm) or polyethylene terephthalate microfibers (<5 mm) would affect selected life history characteristics. Interestingly, while microplastic beads did not have a significant impact on growth and development of G. sigillatus, polyethylene fibers were detrimental to the cricket. In summary, the plausible physiological mechanisms for sex-specific impacts of microplastics are yet unclear. However, future investigations examining if and how microplastics are transformed in the insect gut will reinforce our foundational knowledge to devise approaches for tackling the global concerns of plastic pollution.Menail et al. outline an excellent comparative account of thermal sensitivity and mitochondrial metabolic flexibility in three flying insect species, Apis mellifera, Drosophila melanogaster and Leptinotarsa decemlineata. By measuring oxygen consumption in permeabilized flight muscles from these three species, the authors established how thermal stress is tolerated under conditions involving different oxidative substrates. In conclusion, differential mitochondrial plasticity across insect species demonstrated in this study considerably expands our understanding of the survival dynamics and resilience to thermal stress in some insects over others.Mayekar et al. offer a comprehensive analysis of the use of clinal variation as a promising tool to monitor population-level correlations between active genomic responses to climate change. Drawing from case studies in Drosophila, a cosmopolitan and genetically amenable model, this review highlights interesting broader-scale comparisons of genetic and plastic traits that are not currently well understood. The authors shed light on clinal shift patterns to explain how organisms across latitudes/altitudes are responding to climate change. Taken together, this review proposes the role of clinal variation studies to complement measures of mitigating climate change.Overall, this Research Topic provides original research data and an in-depth review that enlighten us on comparative stress-induced adaptive frameworks in various insect species. Additionally, we anticipate that findings from these studies will pave the way for translational research as well as help leverage the implied potential of the field of stress response biology in supporting evidence-based policymaking in climate change management and mitigation, and biodiversity conservation.As co-editors, we enjoyed working with the authors and we sincerely thank them for submitting their interesting work to this topic Research Topic.With more exciting discoveries awaited for the future, we envisage that this Research Topic will serve as a foundation to stimulate further interest in gaining cross-axes insights from evolutionary, cellular, eco-physiological and biochemical perspectives on insects.We hope our readers will enjoy this work, as much as we enjoyed putting it together for you!This Research Topic is dedicated to the memory of Prof. Hans-Joachim Pflüger who passed away on 25 January 2022.Prof. Pflüger, whom we dearly called ‘Jochen’ was a Professor of Functional Neuroanatomy/Neurobiology at Freie Universität Berlin, Germany. As one of the founding members and one of the presidents of the German Neuroscience Society, Jochen was a backbone to the Neurosciences community in Germany and Europe.Jochen’s research focused on how sensory feedback mechanisms, neuronal networks, and neuromodulation lead to meaningful behavior in insects and other arthropods. When not in the lab, Jochen was a keen birder, loved nature and enjoyed taking students to field excursions.Apart from his professional credentials, Jochen was a wonderful human being who touched the lives of many! As his co-editors, we thank Jochen for his unwavering support, generous scientific and technical advice and for co-editing the submissions published in this Research Topic Research Topic.


The loss of human uricase activity may have allowed humans to survive environmental stressors, evolution bottlenecks, and life-threatening pathogens. While high urate levels may contribute to developing gout and cardiometabolic disorders such as hypertension and insulin resistance, low urate levels may increase the risk for neurodegenerative diseases. The double-edged sword effect of uric acid has resurrected a growing interest in urate’s antioxidant role and the uricase enzyme’s role in modulating the risk of obesity. Characterizing both the effect of uric acid levels and the uricase enzyme in different animal models may provide new insights into the potential therapeutic benefits of uric acid and novel uricase-based therapy.Uric acid is the final product of purine metabolism and is converted to allantoin in most mammals via the uricase enzyme. The accumulation of loss of function mutations in the  Uric acid is the final product of purine metabolism in humans. Serum urate levels vary significantly between different species. Particularly, humans have the highest urate levels compared to all other mammals. Markedly high uric acid levels in humans have been caused by the loss of the uricase enzymatic activity during the Neogene period ,2.uricase gene [In humans, two-thirds of uric acid is excreted through the kidney and one-third through the gastrointestinal tract . While hase gene ,5,6,7. IIn mammals, uricase is the primary enzyme that converts uric acid to allantoin, a more water-soluble form. This comparative biology approach paved the road for developing the recombinant form of uricase to treat advanced tophaceous gout and prevent uricosuria in treating certain hematological and solid tumor malignancies ,9,10,11.uricase gene rendered humans the only mammal to develop gout, it was also proposed that retaining uric acid provided an adaptive mechanism for human survival [In most mammals, uric acid levels could range between 1 and 2 mg/dL [survival ,14. Noneuricase gene, during the evolution process, rendered the enzyme undetectable in humans and specific monkeys [Uricase is present in nearly all living organisms, from bacteria to mammals, with a common evolutionary origin. The gradual accumulation of genetic mutations, mainly nonsense mutations, in the  monkeys . Specifi monkeys . The occ monkeys ,16.The loss of the uricase enzyme in humans and the extensive reabsorption of uric acid suggest that retaining uric acid is essential to human health; hence, evolutionary physiology has treated uric acid not as a harmful waste. Also, this high conservation of uric acid has rendered humans susceptible to uric acid changes induced by diet and their propensity to develop gout spontaneously. Despite the well-established relationship between high uric acid levels and gout, researchers have been actively trying to elucidate the health benefits of uric acid, its role in adaptation to different environments, and potentially inform the development of less immunogenic uricase-based gout therapy .Proposed theories about the role of uric acid have been previously described . For exaIt is hypothesized that the loss of the L-gulono-γ-lactone oxidase gene, which is involved in vitamin C biosynthesis, was driven by the significant ingestion of fruits and vegetables, a primary source of vitamin C. As a result, uric acid became the primary antioxidant and free radical scavenger when fruit and vegetable consumption dramatically decreased due to substantive climate changes . Even thPopulation dietary patterns and cultural habits have been implicated in the high prevalence of hyperuricemia and gout among specific population groups . For exaThe objective of this review article is to highlight the effects of high and low serum urate levels on human health. A review of the implication of genetics, as it intersects urate levels and various phenotypes associated with serum urates, is critical to fully characterize the contribution of different environmental changes to human health. Additionally, analyzing the ramifications of the loss of the uricase gene could expand our knowledge base to better characterize the various physiological roles that uric acid could play in human health and explain the current public health trends and population risk differences for common and chronic diseases.SLC2A9, SLC16A9, SLC17A1, SLC22A11, SLC22A12, ABCG2, PDZK1, GCKR, LRRC16A1, HNF4A/G, and INHBC are some of the major determinants in uric acid levels and modulators of gout risk  has been well established. Excessive sodium intake has been shown to increase blood pressure and the onset of hypertension. Increased sodium consumption is associated with increased water retention, peripheral vascular resistance, oxidative stress, and microvascular remodeling . FollowiWhile being a naturally occurring antioxidant, uric acid has been strongly implicated in developing the hypertension epidemic and maintaining blood pressure during the early hominoids. Fossil evidence suggests that early hominoids heavily relied on fruits for their dietary needs. With a fruit-based diet, the sodium intake during the Miocene period was expected to be very low to maintain blood pressure. To evaluate this hypothesis, mild hyperuricemia was induced in rats by inhibiting uricase, recreating the pre-historic conditions by feeding the rats a low-sodium diet. Compared with hyperuricemic rats, normal rats receiving a low-sodium diet experienced either no change or a drop in systolic blood pressure. In contrast, systolic blood pressure increased in hyperuricemic rats, proportional to uric acid levels, and could be mitigated using allopurinol ,46. TherUric acid levels may also contribute to the salt sensitivity associated with developing hypertension. A study using rats treated with oxonic acid and a low-salt diet for seven weeks resulted in mild hyperuricemia compared with controls. Following the discontinuation of oxonic acid, uric acid levels did not differ between groups. However, the renal tissue obtained from rats with hyperuricemia showed persistent arteriolopathy with decreased lumen diameter and mild interstitial inflammation. When rats were then randomized to a low- or high-salt diet, an increase in blood pressure from the high-salt diet was observed only in rats that had been previously hyperuricemic . CollectInflammation remains the principal suspect linking gout and cardiovascular diseases. Mechanisms whereby serum urate levels could contribute to the development of hypertension are partly related to the uric acid’s primary effect on the kidney. These mechanisms include activating the renin–angiotensin–aldosterone system (RAAS) and the disposition of urate crystals in the urinary lumen. Additional evidence suggests that elevated serum uric acid can decrease nitric oxide production, causing endothelial injury and dysfunction. It is well established that monosodium urate crystals can activate the NLRP3 inflammasome cascade and the production of inflammatory cytokines such as IL-1 beta and IL-8 . AlthougA reverse causality of hypertension and hyperuricemia is also possible. Patients treated for hypertension could develop hyperuricemia secondary to decreased renal blood flow, increased BMI, and concomitant use of medication that reduces uric acid excretion or increases uric acid production. Another proposed hypothesis suggests that, given the route of elimination, uric acid can directly cause renal injury, which could result in the activation of RAAS, causing hypertension .Hypertension is an epidemic affecting more than 30% of adults worldwide . While gHyperuricemia is an independent risk factor for multiple cardiovascular disease risk factors, which can, collectively, elevate the risk of cerebrovascular events. Nonetheless, the direct role of uric acid levels in developing stroke events is inconclusive ,54. On tThe incidence and prevalence of hypertension and obesity were minimal in specific populations until Westernization and the adoption of a Western diet ,58. AddiThe underlying association mechanism between westernization and the development of hypertension is complex. However, the most likely cause is fueled by the acculturation and assimilation of the Western world, which brings multiple changes in dietary habits and lifestyle factors . For insThe old hypothesis that the chemical structure of uric acid could mimic specific brain stimulants, such as caffeine or methylxanthine, may have provided selective advantages to early hominoids . To thisMultiple epidemiological studies have examined the association between serum urate levels, gout diagnosis, and brain-related diseases ,67. SpecSLC2A9 , cumulative scores of the risk alleles were created based on the total number of minor alleles across the three loci [SLC2A9 minor alleles versus those with ≤2 minor alleles [SLC2A9 score, was not statistically significant (p = 0.056) [To assess the causality between uric acid and PD, a Mendelian randomization approach was used to systematically evaluate the inherent risk of lower urate levels and disability requiring dopaminergic treatment in early PD patients. Genotyping 808 patients for three loci across the ree loci . Serum u alleles . A 5% in= 0.056) .SLC2A9 and PD in the Han Chinese population. Using a case–control study design, the serum urate levels were significantly lower in PD patients than in controls. Individuals with the rs1014290 TT and CT genotypes had significantly higher serum urate levels than the CC genotypes [SLC2A9-encoded transporter (GLUT-9) might be an alternative approach for urate-elevating therapy, compared to urate precursor administration as a potential PD treatment target.Another study evaluated the association of a single nucleotide polymorphism (rs1014290) within enotypes . The lowThe role of uric acid levels in mild cognitive impairment (MCI), a common diagnosis that precedes the development of clinical AD, has been extensively investigated. While asymptomatic hyperuricemia can go undiagnosed and rarely treated, exploring the association between hyperuricemia and neurodegenerative diseases may be confounded by recall bias, disease misdiagnosis, and secondary causes of hyperuricemia. Nonetheless, patients with gout are considered to have the highest uric acid burden and represent the extreme phenotype of hyperuricemia. Therefore, the association between gout diagnosis and the risk of neurodegenerative diseases has been of significant interest in neurodegenerative disease research.Interest has emerged in investigating the association between serum urate levels and mild MCI. Given the transitional stage between declining cognition and a clinical diagnosis of AD, clinical interventions targeting MCI can become a window of opportunity to arrest or slow the progression of the disease . To thisMultiple studies have shown that subjects with AD have lower serum urate levels than matched controls. These findings have ushered in the hypothesis of uric acid being a neuroprotective compound and potentially a therapeutic target in neurodegenerative diseases. However, lower brain urate levels in AD patients than matched controls could be owing to the reduced metabolic activities that might lead to reduced uric acid production. To this extent, urate levels may serve as a proxy for overall brain metabolic activity rather than being the culprit of the disease. Additionally, one of the significant serum urate predictors is the overall nutritional status and BMI. Clinical manifestations of AD are often preceded by substantive weight loss, which may significantly account for the lower serum urate levels in the AD presentation.Generally, patients with hyperuricemia tend to have a higher cardiovascular disease burden, warranting a concomitant use of varying medications that may affect serum urate levels. Also, serum urate levels can be influenced by sex, obesity, genetics, and overall nutrition. Therefore, the measurement of uric acid may not accurately reflect the inherent urate level of the individual to draw a robust association between serum urate levels and the condition of interest, let alone the cross-sectional design of these studies. While uric acid may serve as a potent antioxidant, a robust epidemiological study designed to identify the optimal urate levels to maximize the benefits and minimize the risks is needed.Selective advantages to developing hyperuricemia in distinct population groups may have been driven by natural selection. This natural selection advantage may partly explain the inherently increased predisposition for hyperuricemia or gout among select population groups, especially when challenged by unprecedented environmental threats or culturally altering events. Besides being a naturally occurring antioxidant, urate levels may have enabled humans to survive crucial environmental stressors and pathogenic threats. When crystallized, urate may trigger the innate immune response. For this reason, monosodium urate crystals are considered a critical natural endogenous immune response trigger. With infectious diseases being one of the strongest drivers of natural selection, it is presumed that genes associated with raising urate may have been selected during major depopulation events. This theory may partly explain the genetic predisposition for hyperuricemia/gout and the higher mean uric acid levels in the Pacific and Oceania regions compared with whites and the Western world ,88,89,90Historically, exposure to pathogenic or parasitic infections such as malaria has pressured the human genome of distinct populations to develop disorders like sickle-cell disease and glucose-6-phosphate dehydrogenase deficiency as a survival switch. Therefore, it is plausible that the inherent risk of hyperuricemia could be considered another adaptation to protect against malaria or other pathogens that threatened our ancestors. Urate levels released from erythrocytosis because of malaria infection have been shown to activate the host’s inflammatory response, facilitating the detection of the parasite . Given tUric acid levels are the strongest predictor of developing gout via the deposition of monosodium urate crystals in and around the joints. The chronic suppression of uric acid levels is the hallmark of reducing the MSU crystal burden and preventing future gout flares. While different treatment modalities have been developed to lower serum urate, most treatments are focused on inhibiting uric acid production. In the advanced forms of gout, the recombinant form of uricase has been used to reduce the tophi burden or if the patient is not responsive to available treatments. In addition to gout, high uric acid levels have been incriminated in different chronic disease states, including hypertension, metabolic syndrome, diabetes, non-alcoholic fatty liver disease, and chronic kidney disease ,41,93,94While numerous experimental and clinical studies support the role of uric acid as an independent cardiovascular risk factor, a handful of studies also suggested that lowering serum urate may improve blood pressure. These studies included pre-hypertensive obese , hyperteA growing body of evidence also suggests that urate-lowering therapy does not affect blood pressure. A crossover single-center study enrolled ninety-nine participants randomized to 300 mg of allopurinol or placebo over four weeks . There wn = 149) with serum uric acid ≥5.0 mg/dL were randomly assigned to probenecid, allopurinol, or placebo [In another double-blind placebo-controlled trial, overweight or obese participants  ,76. AmonIn patients with early PD, administering inosine was safe, tolerable, and effective in raising serum and cerebrospinal fluid urate. However, among patients recently diagnosed with PD, treatment with inosine compared with placebo did not result in a significant difference in the rate of clinical disease progression, questioning the use of inosine as a treatment for early PD ,103. AnoDespite the disappointing results of the urate-elevating studies, it is critical to recognize that of the above studies, some lacked the sample size to detect adequate differences. Additionally, inosine use could be problematic in achieving the desired serum urate levels as it can enter the purine salvage pathway. While addressing the sample size issue for future studies is obvious, the mechanism to elevate uric acid requires a robust thought process and potentially targeting uric acid excretion transporters.The loss of activity mutations in the uricase gene may have been protective in situations of famine and food scarcity; thus, it rapidly took over the ancestral population, likely driven by harsh environmental factors during the Miocene era. In modern times, this adaptation is possibly leading to a state of hyperuricemia, increasing the risk of gout and other cardiometabolic disorders. Today, all humans are considered uricase knockouts. Loss of the uricase enzyme resulted in the inability to regulate uric acid effectively. The adaptation to famine and food scarcity in early hominoids dictated crucial changes to obtain and conserve energy for survival. In contrast to animal experiments and observational studies, targeting uric acid levels for therapeutic purposes beyond the purpose of gout remains conflicting.As hyperuricemia and gout animal models continue to be instrumental in advancing the field of gout and uric acid metabolism, it is equally important to recognize that animal models are inherently predisposed to eliminating uric acid. Therefore, the effects of raising uric acid levels in animal models may not reflect the outcomes seen in human studies. Additionally, the sex effect on uric acid levels reinforces the need for adequate and robust analyses by sex. Studies have shown that women are more sensitive to the cardiometabolic effects of increased uric acid after menopause versus men. Uric acid levels may also present as a J-shaped phenomenon. Therefore, careful study design and a robust rationale for classifying or categorizing uric acid levels are needed to minimize the effect of extreme uric acid levels.While genetics play a significant role in developing hyperuricemia or gout, it is equally important to recognize that the same genetics are therapeutic targets to modify uric acid metabolism. Genetic polymorphisms have been implicated in the racial difference in gout prevalence and may also contribute to the heterogeneity in response to urate-lowering or urate-elevating therapies. Therefore, genetic investigations into uric acid metabolism in clinical trials need to be considered in designing the study. Finally, as described earlier, uric acid levels are the culmination of endogenous cellular processes and external dietary sources; therefore, accounting for dietary and lifestyle habits in uric acid levels may minimize the effect of study confounders.


Pancreatic cancer is a leading cause of cancer-related death, with a poor overall survival rate. Although certain risk factors have been identified, the origins of pancreatic cancer are still not fully understood. Surgical resection remains the primary curative treatment, but pancreatic surgery is still associated with high morbidity and mortality rates, and most patients will experience recurrence. The impact of pancreatic cancer on patients’ quality of life is significant, with an important loss of healthy life in affected individuals. Traditional outcome parameters, such as length of hospital stay, do not fully capture what matters to patients during recovery. Patient-centered care is therefore central, and the patient’s perspective should be considered in pre-operative discussions. Patient-reported outcome and experience measures (PROMs and PREMs) could play an important role in assessing patient perspectives, but standardized methodology for evaluating and reporting them is needed. This narrative review aims to provide a comprehensive overview of patient perspectives and different patient-reported measures in pancreatic cancer surgery. Understanding the patient perspective is crucial for delivering patient-centered care and improving outcomes for patients with pancreatic cancer. Pancreatic cancer (PC) represents the fourth leading cause of cancer-related death, and has been estimated to become the second by 2030 ,2. An ovPC therefore has particularities in terms of surgical treatment, complications and poor survival, which inevitably impacts patients’ quality of life. A systematic review of the burden of PC in Europe demonstrated a 98% loss of healthy life in affected individuals . TraditiPatients should thus have the opportunity to express their expectations and experiences for the care that they receive. There has been growing interest in this approach for a decade, and this could be beneficial to patients, families and care providers themselves ,15. ThesThis scoping review aims to provide a comprehensive overview of patient perspectives, and provide a summary of the different patient-reported outcome and experience measures of patients undergoing pancreatic surgery for PC.A patient’s definition of a successful outcome may be different from that of surgeons. Patient-reported outcome measures (PROMs) are defined as any patient’s health condition self-report without interpretation of a response by a caregiver ,17. PROMA prospective qualitative study including patients undergoing abdominal surgery showed that the themes of recovery identified were returning to habits and routines, resolution of symptoms, overcoming mental strains, regaining independence and enjoying life . InteresPROMs usually take the form of questionnaires that can be completed pre- and post-operatively, allowing a better understanding of the recovery process . DiseaseAnother brief, fully patient-derived and disease-specific questionnaire has been developed by a Norvegian team . This PAThe Functional Assessment of Cancer Therapy-Hepatobiliary (FACT-HEP) is a questionnaire measuring general and hepatobiliary disease specific aspects of quality of life, including 27 core items and an additional 18 hepatobiliary-specific items  that assess symptoms and function ,27. ThisThe M.D. Anderson Symptom Inventory for gastrointestinal cancer (MDASI-GI) was developed to assess physical and psychological symptom prevalence and burden among the GI cancer population . It contThe Gastrointestinal Quality of Life Index (GIQLI) is an instrument designed to measure the quality of life in patients with gastrointestinal disease . It measPatients with PC often suffer from disease-related symptoms. The Edmonton Symptom Assessment System (ESAS) measures the severity of 9-symptom domains and has been validated for use in oncology, including PC . A populThe Hospital and Anxiety Depression Scale (HADS) is a self-rating scale, mainly used for rapid assessment of patients’ anxiety and depression, and is one of the tools for the screening of these psychological aspects ,35,36. TThe Acceptance of Illness Scale (AIS) is a questionnaire designed for measuring any disease acceptance . This top = 0.0015) of PC [The Katz Activities of Daily Living (ADL) index is a dichotomous categorization of 6 daily activities: bathing; dressing; toileting; transferring; continence; and feeding. Scores range from 6  to 0  . A study5) of PC .A systematic review confirmed that PROMs currently used in abdominal surgery lack adequate measurement properties, which precludes their use to support value-based surgical care . InvestiFinally, the Pancreatic Cancer Action Network (PanCAN) patient registry, developed by an American team, is an online, voluntary pancreatic cancer-specific registry enabling patients to self-report sociodemographics, disease/management characteristics and PROMs . This tyPatient-reported experience measures (PREMs) are tools for assessing experiences with health care from the patient’s perspective. While PROMs measure health care outcomes as described above , PREMs assess patients’ needs and experiences of their care or health service  ,48. TablA systematic review concluded that PREMs with good psychometric characteristics were lacking in oncology, and had low reliability and construct validity . AnotherThe Picker Patient Experience (PPE) questionnaire is a tool for assessing the patient experience of care, and consists of 15 questions assessing eight key aspects of patient care . These aThe EORTC IN-PATSAT32 questionnaire is a tool evaluating patient satisfaction with care during hospital stays . The queThe Nordic Patient Experiences Questionnaire (NORPEQ) is a short self-completed questionnaire with evidence for data quality, reliability and validity . The NORThe patient experience questionnaire (PEQ) was produced with 18 items on five dimensions  . The valThe majority of PROM and PREM questionnaires are given to patients at different key moments in patient care, typically in pre-operative clinics with the post-operative questionnaire being collected after surgery. The ideal timing to complete the questionnaire may vary depending on the type of procedure and associated additional treatments . There has been an increase in the number of electronic tools developed to support patients during cancer care. Patient portals and groups on social media with various and different means of virtual communications did appear. A systematic review showed that electronic systems have the potential to help patients manage side effects of cancer treatment, though comparison across studies was difficult due to the wide range of available assessment tools . StructuTo avoid patient burden and increase patient compliance, tools and items should be carefully selected. It should also be emphasized that the more often a questionnaire is administered to patients, the shorter it should be. For example, several successful studies have adopted only 10 to 20 items for weekly administration ,67. FurtSeveral limitations need to be considered when collecting and interpreting PROM and PREM data. The patients may not be in a physical or psychological state to give accurate feedback. They may also be anxious about the potential negative impact of their answers on the management of their pathology, thus adjusting their responses accordingly by overrating healthcare quality . Time coTraditional outcome parameters do not fully capture what matters to patients recovering from PC surgery. Patients’ perspectives include an interplay of physical, psychological and social factors that should be addressed in pre-operative discussions when planning surgery for pancreatic cancer. PROMs and PREMs will play an important role in the near future, but there is a crucial need to develop standardized methodology for assessing and reporting them.


We opened the Research Topic regarding biological mechanisms and therapeutics in stress, pain and/or addiction. Cumulating evidence highlights substantial central nervous system (CNS)-mechanism overlaps among stress, pain and addiction. Stress and pain are interacting and influencing each other at multiple levels, including genetic, biological, cognitive, behavioral, and social factors  tractography in male and female rats and found hypersensitivity of female rats and gender differences in pain-associated brain connectivity such as the anterior cingulate cortex (ACC), prefrontal cortex (PFC) and ventral thalamus. They suggest the existence of sexual dimorphism of brain associated with pain sensitivity .First, Kim et al. demonstrated a critical role of hypothalamic orexinergic neurons between stress and psychiatric diseases. While it is well known that stress is an important precipitating factor for the development of psychiatric conditions for major depression  plays an important physiological role in brain function and also plays a critical role in substance abuse. The neuropeptides called orexin (or hypocretins) have been identified as being located exclusively in the cell bodies of the LH. Kim's group has previously demonstrated that mechanical stimulation (MS) of the ulnar nerve produces strong inhibitory effects on cocaine addiction–like behaviors through an LH-lateral habenula (LHb) circuit .Third, Liao et al. endeavored to uncover a novel neural circuit overlapping stress and pain. Many patients with stress-related emotion disorders are comorbid with chronic abdominal pain, indicating that stress-primed emotional status may enhance abdominal pain magnitude . Their research suggests that stress may cause the plastic changes of medial septal-lateral septal-accumbal circuit and thus produces stress-primed abdominal pain.Fourth, Yeo and Roh provided a therapeutic mechanism of rapamycin in an animal pain model and its signaling pathway. Neuropathic pain in the orofacial area is commonly observed in humans and markedly reduces the quality of general life by disrupting food intake, face washing, and tooth brushing . They demonstrated that rapamycin alleviated both mechanical and cold allodynia in orofacial pain mice. Enhanced p-p38 MAPK and p-MKK4 in orofacial pain mice were suppressed following rapamycin treatment. They also showed that the increased p-p38 expression colocalized with microglia but neither neurons nor astrocytes and suggested that suppression of orofacial pain by rapamycin is associated with the inhibition of p-MKK4/p-p38 MAPK-mediated microglial activation in the TNC (Yeo and Roh).Last, The above articles proved the sexual dimorphism of brain associated with pain, a novel neural circuit in stress-primed pain, the role of microglia and associated downstream signaling in pain medication, the critical role of lateral hypothalamic orexinergic neurons in addiction and increasing resilience of stress-associated psychiatric diseases. While the articles revealed the distinct and shared mechanisms between stress and pain or between stress and psychiatric diseases, no articles regarding the shared mechanism underlying pain and addiction were collected. Despite the limitation of our Research Topic, we believe that the above publications contribute to our current understanding in stress and/or pain or addiction and also help for elucidating the underlying mechanisms and discovering new therapeutics against stress, pain and/or addiction. We appreciate all authors and reviewers for their substantial contribution to this Research Topic.HK: Writing — original draft, Writing — review & editing. IS: Writing — original draft, Writing — review & editing.


Studying the genetic makeup of viruses and phages through genome analysis is crucial for comprehending their function in causing diseases, progressing medicine, tracing their evolutionary history, monitoring the environment, and creating innovative biotechnologies. However, accessing the necessary data can be challenging due to a lack of dedicated comparative genomic tools and viral and phage databases, which are often outdated. Moreover, many wet bench experimentalists may not have the computational proficiency required to manipulate large amounts of genomic data.We have developed VAPEX (Virus And Phage EXplorer), a web server which is supported by a database and features a user-friendly web interface. This tool enables users to easily perform various genomic analysis queries on all natural viruses and phages that have been fully sequenced and are listed in the NCBI compendium. VAPEX therefore excels in producing visual depictions of fully resolved synteny maps, which is one of its key strengths. VAPEX has the ability to exhibit a vast array of orthologous gene classes simultaneously through the use of symbolic representation. Additionally, VAPEX can fully analyze user-submitted viral and phage genomes, including those that have not yet been annotated.https://archaea.i2bc.paris-saclay.fr/vapex/.VAPEX can be accessed from all current web browsers such as Chrome, Firefox, Edge, Safari, and Opera. VAPEX is freely accessible at  Viruses are small infectious agents whose replication occurs obligatorily inside the cells of living organisms and relies on cellular metabolism. They are composed of genetic material (either DNA or RNA) surrounded by a protein capsid. Enveloped viruses harbor an extra lipid bilayer membrane surrounding the capsid. Viruses infect virtually any eukaryal or archaeal cell. Bacteria are not immune from viral infection and their viruses are called phages, short for bacteriophages.Viruses cause a number of human diseases such as influenza, AIDS, Ebola, and SARS-CoV2, and pose a constant threat to public health. The occurrence of viral epidemics threatens also all varieties of cultivated plants to sustain human population and its domesticated animals  and recoet al. 2020), VAPEX can create comprehensive and fully resolved synteny maps for both bioinformaticians and wet lab experimentalists.To our knowledge, there is currently a lack of public web services that provide comprehensive genomic comparisons of bacteriophages, eukaryotic viruses, and archaeal viruses especially when user-submitted genomes are involved. As a solution, we introduce VAPEX—a user-friendly and interactive web server aimed at facilitating in-depth examination of natural virus and phage genomes including user-submitted sequences. By utilizing the well-established symbolic representation developed for the WASPS web server  or bitmap (PNG) formats for further analysis.Filoviridae not only in mammals and birds but also in fish and amphibians (Filoviridae (Filoviridae. Surprisingly, the RNA-dependent RNA polymerase and glycoprotein encoded by the Wuhan sharpbelly bornavirus belong to the same orthologous groups as the corresponding proteins in related viruses, suggesting a considerably lower diversity in Filoviridae than anticipated. These findings suggested that VAPEX's sensitivity is comparable to that of molecular phylogenetics while offering a broader and more contrastive analysis of the evolution of related viruses.Recent events have highlighted the significance of tracing the origins of RNA viruses, such as SARS-CoV-2 that has caused a global pandemic or Ebola that has led to highly fatal epidemics. The onset of the Ebola virus disease outbreak in West Africa between 2013 and 2016 signaled the inception of widespread real-time molecular epidemiology (oviridae . As expebtad528_Supplementary_DataClick here for additional data file.


This approach also is used now to produce these mimics of small organs utilizing individual induced pluripotent stem cells (iPSCs). Normal tissue and organ biochemistry and physiology as well as tissue interactions and cross talk are faithfully reproduced in each of these structures. Hence, they have become legitimate models of both physiologic state and development. When iPSC from disease states are used to produce these structures, they can be used to study the details of individuals’ pathologies and possible therapeutics. Papers contributed to this Research Topic describe how stem cells, organoids, assembloids, and organ-on-chip models may soon foster public health through precision medicine and clinical interventions.Soon after pluripotent embryonic stem cells (ESCs) were isolated from preimplantation embryos, the cells were grown individually into 3 dimensional embryoid bodies and, later, organoids, assembloids and “organs-on-chips.” These models recapitulate embryonic and adult tissues in organs ex vivo . When the iPSCs are isolated from humans and other mammals with genetic diseases, they also serve as models of these diseases. For example, in Hirschsprung’s disease, a congenital lack of enteric nerve development can lead to an almost complete absence of GI tract motility and insufficient nutrient absorption. And these abnormalities are recapitulated in organ-like structures formed from stem cells with the Hirschsprung’s disease-causing mutations in PHOX2B . Their results show that cryopreservation of these stem cells is possible although, as of now, with reduced viability. And there was no detected effect of such preservation on the functions of resultant nervous system cell subtypes.Interestingly, such diseases can likely be corrected through transplantation of mutation-free stem cells into affected regions of the GI tract . Such stForrester-Gauntlett et al.). While Grhl-knock out in mouse ESCs apparently led to this diminished epithelial integrity, Grhl-knock out cells otherwise developed normally expressing neural, neuronal, and sensory hair cell markers. These studies are important because mutations in this transcription factor gene, GRHL2, in humans are associated with sensorineural deafness. Moreover, such hereditary hearing loss may soon be treatable through gene therapy including gene replacement, suppression, and editing . Hence, these three-dimensional structures might be an autologous source of cells for cell-based bladder tissue engineering. Nevertheless, the mechanisms of recovery of apparently normal smooth muscle cell contractility in microtissues remains to be determined. Moreover, such muscle cells seem unlikely to benefit patients with neurogenic bladder dysfunction . These smooth muscle stem cells were derived from the rat adipose-derived stem cells these authors used originally to alleviate inflammatory bowel disease (Finally,  disease . Interes disease , so theiThe juxtaposition of new stem cell biology and 3-dimensional growth technologies has led to a revolution in regenerative biology. Based on the ability to produce stem cells from adult tissues of individuals, the ability to recreate functional tissues has burgeoned into a robust approach to disease physiology, therapeutic test beds, and organ tissue replacement. Importantly, the creation of screening systems with tissue arrays provides direct approaches to minimally invasive autologous organ tissue. Ultimately, combined with gene editing technology direct correction of genetic disease states to homeostatic states will be possible. These exciting advances are explored in the suite of papers presented here and promise a new generation of biologic approaches to disease amelioration.


Respiratory syncytial virus (RSV) infections are a constant public health problem, especially in infants and older adults. Virtually all children will have been infected with RSV by the age of two, and reinfections are common throughout life. Since antigenic variation, which is frequently observed among other respiratory viruses such as SARS-CoV-2 or influenza viruses, can only be observed for RSV to a limited extent, reinfections may result from short-term or incomplete immunity. After decades of research, two RSV vaccines were approved to prevent lower respiratory tract infections in older adults. Recently, the FDA approved a vaccine for active vaccination of pregnant women to prevent severe RSV disease in infants during their first RSV season. This review focuses on the host response to RSV infections mediated by epithelial cells as the first physical barrier, followed by responses of the innate and adaptive immune systems. We address possible RSV-mediated immunomodulatory and pathogenic mechanisms during infections and discuss the current vaccine candidates and alternative treatment options. Orthopneumovirus genus of the family Pneumoviridae . Infection . The adonfection . + memory T cells mediate viral clearance in the absence of RSV-specific CD4+ T cells and antibodies in mice, emphasizing the protective role of CD8+ memory T cells in the absence of other adaptive immune cells [+ memory T cells were also involved in excessive weight loss and increased disease severity and immunopathology in infected mice mediated by the increased secretion of IFN-γ compared to control mice [+ T cells and antibodies. In a recent study, peripheral blood mononuclear cells were isolated from healthy children who encountered RSV infection during infancy. Restimulation of these PBMCs showed an impaired memory T-cell response irrespective of the disease severity during RSV infection when compared to children that were not infected during infancy [Because repeated RSV infections are common, an impairment of RSV-specific memory T cell generation may be assumed . In micene cells . Besidesrol mice . This st infancy . The alt infancy . The reduced survival of memory T cells may be due to intrinsic cell programming, as studies in infant mice infected with influenza virus showed an increased expression of T-bet and a reduced expression of survival factor CD127 on the surface of T cells compared to infected adult mice. This altered phenotype persisted even after the adoptive transfer of infant Trms into adult mice, indicating that the host environment is not responsible for the inefficient establishment of memory and that infant Trms are intrinsically programmed for short-term immunity .Generating memory T cells is essential for long-term protection against recurrent infections. Fully functional memory T cells are produced during RSV infections that can mediate protection upon reinfection. The rapid decrease in RSV-specific memory T cells and impaired functionality in infants and older adults indicate that this protection is not long-lasting and predisposes the host to recurrent infections. Further studies will be necessary to better understand the role of memory T cells during RSV infection and immunopathogenesis, and the role of RSV in regulating these processes.After the first isolation of RSV from chimpanzees in 1956 and infants in 1957, the first formalin-inactivated vaccine, FI-RSV, was developed in the 1960s ,312 and Vector-based vaccines have been used for decades for several pathogens, showing their potential to induce protective immunity in recipients after vaccination. They are generated by integrating a viral sequence encoding a target antigen into a non-pathogenic, often replication-deficient viral vector, like modified vaccinia virus or adenoviruses . Vector-In contrast to whole virus vaccines, subunit vaccines are based on one or more purified viral antigens . TherefoE, administered intramuscularly, significantly reduced the risk of ALRI in vaccine recipients compared to the placebo group [A similar subunit vaccine, RSVPreF3, is based on a prefusion F protein antigen and was developed by GSK. Intramuscular vaccination of healthy pregnant women during their late second or third trimester with unadjuvanted RSVPreF3 was safe and induced neutralizing antibodies that were transferred to the fetus . Transplbo group . The genbo group . In May During the SARS-CoV-2 pandemic, mRNA vaccines against COVID-19 attracted much interest. These mRNA-based vaccines, coding for a specific viral antigen, are administered intramuscularly using lipid nanoparticles . After tProphylactic treatment with monoclonal antibodies represents an alternative to vaccination. Palivizumab, a humanized IgG1-type monoclonal antibody, was approved in 1998 to treat high-risk infants during their first RSV season ,331. PalThe stabilization of the prefusion F protein is a critical factor for the development of vaccines and human monoclonal antibodies for the prevention of severe RSV infections in infants, older adults, and the immunocompromised. The progress in vaccine development is best illustrated by the recent approval of Arexvy and Abrysvo, which offer protection for the older population from RSV-associated ALRI. For infants, the vaccination of pregnant women seems an attractive option as Pfizer’s PreF-protein vaccine was approved recently by the FDA based on promising data from phase III clinical trials. Despite decades of research, RSV remains a substantial global burden, mainly affecting infants, older adults, and immunocompromised individuals. After the early failure of FI-RSV-based vaccines in the 1960s, research has focused on understanding the host’s immune response and possible immunomodulatory effects of RSV that may counteract a protective response and may even enhance immunopathological effects. This review emphasizes that effective and well-balanced immune responses are fundamental for reducing RSV-associated disease. As summarized, RSV-altered immune cell activity and differentiation robustly impact the cellular and humoral immune response. This dysregulation is mainly characterized by a Th2-skewed immune response leading to more severe disease and immunopathology. In the end, altered RSV-mediated responses of the innate and adaptive immune system may affect and suppress the generation of a proper immunological memory leading to impaired and short-lived immunity. Comprehending these alterations is thereby of great interest, although further studies will be necessary to address this in more detail.Solving the prefusion conformation of the RSV F protein and its stabilization demonstrated the immense progress made in vaccine development after decades of basic research. As a consequence of this breakthrough, two preF-based vaccines have recently received licensure for use in older adults as both safety and efficacy were demonstrated. Although the recently developed RSV vaccines seem highly efficacious in older adults, their transmission-reducing potential and durability of protection in this age group require further investigation. The risk of waning antibody levels, also seen after RSV infection, may call for annual vaccination to protect this vulnerable age group.A preF-protein-based vaccine for the vaccination of pregnant women was licensed recently by the FDA. This way, infants can be protected by vaccine-induced maternally derived antibodies during their first RSV season. Furthermore, several vaccine candidates are currently in phase III clinical trials showing promising results for the vaccination of older adults and pregnant women. Maternal vaccination seems to be a promising approach for the infant population, at least during their first RSV season, by the transplacental transfer of neutralizing antibodies, as direct vaccination of newborns may result in a suboptimal immune response considering their immature immune system. Maternal vaccination, however, may bear certain risks, taking into account the current developments in relation to the RSVPreF3 vaccine and the higher abundance of premature births after vaccination with one candidate vaccine. Although a significant amount of data has been collected recently, our understanding of the immunological mechanisms during a natural RSV infection is still incomplete. Considering the phenomena of immunosenescence and preexisting immunity of older adults on one side and the immaturity of the infant’s immune system on the other, analyzing the differences between the immune responses of the two main age groups at risk is critical for vaccine development. Further studies in infants and older adults will be necessary to fully comprehend the protective and harmful immune responses upon RSV infection and vaccination and to pave the way toward a better understanding of cell- and antibody-mediated immunity to RSV infection.