Vicia faba L.) is a promising protein crop for temperate climates, owing to its remarkable yield potential (up to 8 tonnes ha−1 in favourable growing conditions) and high protein content (~29% dry matter basis). Nevertheless, the adoption of faba bean protein in plant-based products that aim to resemble animal-derived counterparts is hindered by its distinctive taste and aroma, regarded as “off-flavors”. In this review, we propose to introduce off-flavor as a trait in breeding programs by identifying molecules involved in sensory perception and defining key breeding targets. We discuss the role of lipid oxidation in producing volatile and non-volatile compounds responsible for the beany aroma and bitter taste, respectively. We further investigate the contribution of saponin, tannin, and other polyphenols to bitterness and astringency. To develop faba bean varieties with diminished off-flavors, we suggest targeting genes to reduce lipid oxidation, such as lipoxygenases (lox) and fatty acid desaturases (fad), and genes involved in phenylpropanoid and saponin biosynthesis, such as zero-tannin (zt), chalcone isomerase (chi), chalcone synthase (chs), β-amyrin (bas1). Additionally, we address potential challenges, including the need for high-throughput phenotyping and possible limitations that could arise during the genetic improvement process. The breeding approach can facilitate the use of faba bean protein in plant-based food such as meat and dairy analogues more extensively, fostering a transition toward more sustainable and climate-resilient diets.The growing interest in plant protein sources, such as pulses, is driven by the necessity for sustainable food production and climate change mitigation strategies. Faba bean ( In the Vicia faba L.)  is still minor  and Vicia faba var. minor Beck. (field bean). Broad bean and field bean mainly exhibit differences in the morphology of the seeds , with broad bean possessing larger seeds than field bean , vernalization requirement and photoperiodic induction . As a re® in 2004  . At the  in 2004 . DespiteFaba bean is gaining popularity in several countries as an ingredient for plant-based products. However, faba bean ideotypes for modern food applications  are lacking in the market. The development of improved varieties with good techno-functional properties , together with nutritional improvement, is pivotal to expanding the use of faba bean ingredients in the food industry. Interestingly, new varieties could be bred for protein components and amino acid composition, which are intrinsic components impacting the techno-functionality of the products during processing .2One of the major obstacles to adopting a diet with reduced meat consumption is the lack of familiarity with meat-alternative products and their limited aroma, taste and texture attractiveness .Flavour is a multisensory perception defined by taste and aroma, which are influenced by the food matrix and its complex physico-chemical affinity with flavor-active chemical compounds . Aroma rPrior to starting breeding programs and setting breeding objectives, plant breeders need understanding about off-flavor molecules and which of them highly impact on off-flavor in the final ingredients. In this way, marker compounds for screening for unpleasant flavors can be defined. It is important to understand that not all compounds detected in a food or food ingredient contribute to its flavor, and determining the real impact of the diverse aroma and taste compounds on perception typically involves laborious methods. Furthermore, the sensory impact of compounds is not solely based on their concentration, but it also relies on their specific sensory thresholds. Some compounds are detectable by some consumers at ultra-trace levels . The impIt is worth noting that the identification of key off-flavor compounds depends on several factors. For instance, different varieties of faba exhibit a different level of off-flavors compounds , but theThe dominant flavor of faba bean has been traditionally described as dried pea flavor, bitter aftertaste and unpleasantly fruity . SpecifiIn the following subparagraphs, we discuss a summary of molecules potentially involved in off-flavor development in faba bean and their origins. 2.1The unpleasant aroma of faba bean protein fractions is associated with the release of certain volatile compounds originating from non-volatile precursors like lipids, amino acids, and carbohydrates. The chemical reactions leading to the development of aroma compounds mainly occur during harvesting, postharvest processing, and storage . Even thLipid peroxidation occurs via autoxidation or enzymatic oxidation which requires the presence of lipoxygenase. The susceptibility of fatty acids to lipid peroxidation augments proportionally with the degree of unsaturation . Oleic aLipid oxidation is not the only source of unpleasant aroma development in faba bean. Other compounds, such as terpenes, can also influence the aroma profile of faba bean. For example, 2.2The bitter and astringent unpleasant taste of legumes is associated with non-volatile compounds present in seeds. The information on the role of non-volatile compounds in the taste of faba bean is scarce, but it is widely accepted that polyphenols , saponins, amino acids and peptides are bitter and/or astringent compounds , and alsVicia faba  measured in a spectrometric way e.g., in , or theycia faba . Many ofcia faba , so it ccia faba  and are cia faba . The phecia faba  and havecia faba . Howevercia faba . In factcia faba , but theAn important class of polyphenols in faba bean is represented by the tannins. Tannins are mainly classified into two categories based on their reactions with hydrolytic agents: condensed tannins and hydrolysable tannins. Hydrolysable tannins have a central carbohydrate core esterified to phenolic carboxylic acids such as gallic acid and ellagic acid and are usually referred to as ellagitannins and gallotannins. Condensed tannins are mainly polymerised products of flavan-3-ols. The most occurring classes are procyanidins, formed by (epi)catechins, and prodelphinidins, formed by (epi)gallocatechins . TanninsSaponins are known to contribute to the bitter, astringent, and metallic taste of pea and soybean . They arin vitro taste receptor assays. However, although many toxic compounds  have been tested on receptor level , yet their flavor characteristics have remained unexplored . V-C comor level , vicine or level . Given tor level  will alsOff-taste cannot only be evoked by compounds inherently present in the seeds like described above, but it can also be evoked by non-volatile lipid oxidation compounds. In pea protein isolates, it has been shown that bitterness was mainly caused by four trihydroxy-octadecenoic acid isomers , four hydroxy-octadecadienoic acid isomers -9-hydroxyoctadeca-10,12-dienoic acid), one oxo-octadecadienoic acid, one octacosatetraen, 1-linoleoyl glycerol, 2-hydroxyoleic acid, 2-hydroxypalmitic acid, α-linolenic acid, and linoleic acid . Some of3Various thermal, chemical, or enzymatic strategies have been investigated to bridge the flavor gap in plant-based proteins by eliminating, modifying, or masking the development of undesirable taste and aroma . HoweverIt is important to specify that preventing the formation of off-flavors is a breeding target for the use of faba bean as ingredient in plant-based dairy or meat analogues, but not for other applications where seeds are cooked or consumed freshly. Breeding efforts to improve seed quality in faba bean must consider the significant variation in consumer preferences influenced by cultural and personal factors . Some coThe following paragraphs illustrate the candidate pathways that are critical to major off-flavors development and how they can be targeted with breeding aimed at improving the quality of faba bean for plant-based meat and dairy analogues.3.13.1.1LOX genes are known to regulate the enzymatic oxidation rate of polyunsaturated fatty acids, such as linoleic acid (C18:2) and linolenic acid (C18:3), through the expression of the lipoxygenase enzyme  and alcohol dehydrogenase (ADH) see . The steLOX family genes are conserved among legumes. In fact, lipid-oxidations products,  are considered markers for “beany” flavor in other legumes such us chickpea, lentil, and yellow pea  among three distinct commercially available pea types has been reported . In addilipoxygenase2 inactivation, has shown that LOX-free variants can exhibit diminished off-flavor in soy-milk production   . Based oAlthough some LOX enzymes do not require the prior release of fatty acids from their storage forms, the hydrolysis of acyl lipids to free fatty acids (FFAs) is generally required for their oxidising activity in plants . Lampi e3.1.2Ideally, faba bean cultivars to produce protein fractions for meat and dairy analogues ought to exhibit decreased levels of C18:2 and C18:3, thereby diminishing the available substrate for lipoxygenase activity. Oil stability, characterised by resistance to oxidation, is affected by the ratio between C18:1, C18:2, C18:3 in the total oil composition. Enhancing the level of C18:1 while reducing C18:2 and C18:3 is an important breeding target to reduce fatty acid oxidation.Fatty Acid Desaturase (FAD) genes are responsible for adding double bonds to the oleic acid (C18:1) to form linoleic (C18:2) and linolenic acids (C18:3) (fad2 (GmFAD2–1A and GmFAD2–1B) into the same genetic background can effectively raise oleic acid content up to 80%, by disrupting the desaturase pathway  have led to the development of ultra-low-linolenic soybean oil (less than 1%), positively affecting oil shelf life and reducing off-flavors  . In part (C18:3) , while F (C18:3) . The fab pathway . Crispr- pathway . Likewis-flavors . Additiobean oil . Studiesoduction . This isIt should be noted that both linoleic (ω6) and linolenic (ω3) acids are essential fatty acids for humans, and deficiencies in these fatty acids are linked to various health concerns . Hence, 3.23.2.1Plant breeding efforts have led to the development of several low-tannin or zero-tannin faba bean varieties that are currently sold on the market. Low tannin genotypes present a reduction in condensed tannin content from ~8% to up to 0.01% on the dry matter . Breedinzt1 and zt2 (Medicago ortholog WD40 transcription factor TTG1 (Transparent Testa Glabra 1) was identified as the causal gene for the white flower phenotype (zt-1 locus)  , while tt2 locus . Despitet2 locus , their senotypes . These tzt1 and zt2 are expected to act in the last steps of the pathway between the synthesis of dihydroflavonols and flavan-3-ols  gene accumulate virtually no saponins, paving the way for the development of peas with reduced bitterness and astringency  or cross-pollination . It is important to consider that faba bean demonstrates a mixed-mating system, exhibiting partial outcrossing (cross-pollination) behaviour with an average outcrossing rate of about 30%. The co-existence of selfing and outcrossing in the flowers of the same plant of faba bean requires modifications of traditional breeding methods for self-pollinated crops while also considering that strategies for highly outcrossing species may not be entirely applicable . FlavourTo be able to identify the genetic components of off-flavors and to develop breeding tools in faba bean, there is the need to explore genetic variation. In this view, zero tannin germplasm can serve as a valuable starting material in breeding schemes, as it already has reduced bitterness and astringency. In addition to the zero-tannin germplasm, further investigation of natural variation in off-flavor-related traits within the faba bean germplasm is required. It is worth mentioning that the current genetic diversity of faba bean to exploit in breeding is restricted to the cultivated gene pool , as the To optimise the utilization of the extensively available germplasm and identify potential lines for crossbreeding, creating smaller “core collections” may be beneficial, as these would be more accessible to breeders and easier to increase seed availability from selected accessions . Since t4.1Exploring over 43,000 accessions of faba bean preserved in approximately 40 gene banks  to identFor volatile flavor compounds, Gas Chromatography (GC) in combination with mass spectrometry (MS) is a widely used analysis apparatus to separate a mixture of many volatile compounds to identify  each of the compounds. Before this analysis can take place, the volatiles first need to be extracted from faba bean flour. The extraction technique chosen determines the type and quantity of compounds extracted, as not all compounds can be extracted by each extraction method. Vacuum distillation and solvent extraction are accurate, but very laborious and time-consuming . Faster,For non-volatile flavor compounds, high-performance liquid chromatography (HPLC) or ultra-high-performance liquid chromatography (UPLC) coupled with mass spectrometry (MS) are commonly used. These techniques can detect and quantify saponins, lipid derivatives, tannins, phenolics, and other compounds that influence taste perception and astringent mouthfeel. Extraction steps are crucial for analysing non-volatile compounds, as solvents used to impact the solubility of compounds . As for 4.2zt1 and zt2 loci involved in the biosynthetic pathways of tannins, there has been no attempt to map quantitative trait loci (QTL) or genes controlling off-flavors development in faba bean.The field of faba bean genomics has historically trailed behind that of other legumes, resulting in a slower pace of advancements in trait improvement and breeding programs . MoleculThe lack of a historical focus on off-flavors in faba bean breeding presents a unique opportunity for modern genomics to contribute significantly to this field. The recent release of the giant genome sequence (~13 Gb) of the faba bean cultivar Hedin/2  opens a Mapping major genes and QTL associated with off-flavor development can expedite the discovery of novel genes and the identification of natural variation. The complexity of off-flavor pathways likely involves a combined effect of multiple alleles, requiring quantitative approaches in breeding. Several examples of quantitative approaches have been reported in legume breeding literature to map QTL regulating molecules with taste and aroma properties, including saponin, flavonoid, alkaloid, fatty acid compositions, and hexenal . The curLotus japonicus , Medicago truncatula , cowpea  hold great promise for accelerating progress in faba bean breeding. These advancements will ultimately contribute to the development of improved faba bean varieties that are manufactured into food products that are more appealing to consumers, helping to drive the expansion of meat and dairy analogues.4.3Breeding to remove off-flavor compounds from faba bean seeds has the potential to improve the taste and palatability of meat or dairy analogues. However, this approach also carries some potential drawbacks. Most of the secondary metabolites likely involved in off-flavor  are required by plants for various functions, such as pigmentation, growth, reproduction, and resistance to pathogens. As such, they represent adaptive traits that have undergone natural selection during evolution . The eliPlants augment the production of polyphenols in response to abiotic stressors as a strategy to assist adaptation to drought, heavy metal exposure, salinity, extreme temperatures, and ultraviolet radiation . TanninsSaponin represents a chemical barrier against pathogens . PositivFurthermore, removing secondary metabolites might negatively impact the nutritional value of the beans. For instance, some polyphenols serve as important sources of antioxidants and are considered health-promoting compounds . In addiConsidering the potential drawbacks reported, it would be preferable in breeding to specifically target genes that are expressed within the seeds only. This allows the production of secondary metabolites in other plant tissues, ensuring the production of molecules that are involved in defense mechanisms against biotic or abiotic stresses in some parts of the plant. It is also important to account that the disruption of secondary metabolism may lead to unforeseen effects elsewhere in the plant’s metabolic system, which is complex and highly interconnected. The elimination of bitter and astringent compounds could change the availability of certain nutrients, which may lead to the production of new compounds or changes in the concentration of existing ones. Therefore, careful monitoring of any changes in the bean’s composition is crucial to ensure that no harmful or unwanted compounds are produced.Establishing strong collaborations between plant breeders and the food industry, alongside rigorous monitoring and evaluation of the breeding program outcomes, will be crucial for ensuring that the development of new faba bean varieties aligns with both consumer preferences and the long-term sustainability of the crop.5The plant-based food industry is increasingly using faba bean as a key ingredient in meat and dairy alternatives. Although technological solutions can mitigate the off-flavors present in faba bean ingredients, they are energy-intensive and costly. We firmly believe that the most sustainable solution to eliminate off-flavors involves plant breeding, with a focus on developing varieties that are tailored for these applications and that ensure minimal to no off-flavors. Previous breeding research on other crops has identified genes behind off-flavor synthesis. Moreover, as we deepen our understanding of the molecules responsible for off-flavors, and with the onset of the genomic era in faba bean research, we can uncover additional genetic pathways. Such insights can significantly benefit faba bean breeding. The success of breeding initiatives aiming to improve off-flavor profiles depend on several factors, including the availability or creation of genetic variations, the quantitative nature of the trait, its heritability, and the efficiency of high-throughput screening methods, among others. For improved faba bean varieties with reduced off-flavors to be both competitive in the market and attractive to farmers, they must also exhibit high yield and resistance to both biotic and abiotic stresses. Complementing these traits in new varieties can promote the transition toward a more sustainable and climate-resilient diets.AL: Conceptualization, Formal Analysis, Visualization, Writing – original draft. WR: Formal Analysis, Investigation, Writing – review & editing. OB: Writing – review & editing. LP: Writing – review & editing, Funding acquisition, Project administration. LT: Funding acquisition, Project administration, Writing – review & editing, Conceptualization, Supervision.


Life-like systemsneed to maintain a basal metabolism,which includesimporting a variety of building blocks required for macromoleculesynthesis, exporting dead-end products, and recycling cofactors andmetabolic intermediates, while maintaining steady internal physicaland chemical conditions . A compartment,such as a unilamellar vesicle, functionalized with membrane-embeddedtransport proteins and metabolic enzymes encapsulated in the lumenmeets these requirements. Here, we identify four modules designedfor a minimal metabolism in a synthetic cell with a lipid bilayerboundary: energy provision and conversion, physicochemical homeostasis,metabolite transport, and membrane expansion. We review design strategiesthat can be used to fulfill these functions with a focus on the lipidand membrane protein composition of a cell. We compare our bottom-updesign with the equivalent essential modules of JCVI-syn3a, a top-downgenome-minimized living cell with a size comparable to that of largeunilamellar vesicles. Finally, we discuss the bottlenecks relatedto the insertion of a complex mixture of membrane proteins into lipidbilayers and provide a semiquantitative estimate of the relative surfacearea and lipid-to-protein mass ratios  that are required for the construction of a syntheticcell. Their concerted actionensures an out-of-equilibrium state by importing fuel molecules orbuilding blocks for biosynthetic purposes and by exporting waste productsthat would otherwise become harmful in the lumen.Life exists away fromthermodynamic equilibrium. In fact, the propertiesand behavior of cellular systems are largely governed by the kineticsof fuel and building block supply rather than by their thermodynamicstability. In living organisms, the out-of-equilibrium state is maintainedwithin a confined space bounded by a semipermeable membrane.3 In bacteria, the fraction ofmetabolism-related genes ranges from 35% in Mycoplasma pneumoniae, a pathogen with limited metabolic functions, up to 47% in the modelorganism Escherichia coli. JCVI-syn3a, the (known)living organism with the simplest genetic makeup, also employs one-thirdof its genes for metabolism and physicochemical homeostasis.3Within thecellular boundary, a set of catalyzed chemical reactionscollectively termed metabolism  enables cellsto remain out of equilibrium. It is therefore not surprising thata large portion of the gene products is dedicated to sustaining metabolicactivity.Mycoplasma mycoides capri.3 This top-down approach, also applied to other model organismssuch as Bacillus subtilis can be sourced (and engineered) from differentorganisms, in order to obtain the desired functions. Artificial partsdesigned de novo in the laboratory are also used to reproduce specificaspects of living organisms.16 However, fully autonomoussynthetic cells ultimately rely on templates encoding the instructionsfor their self-reproduction, growth, and division, which are executedby the synthetic cell machinery. While nonbiological components andapproaches (nonenzymatic reactions) are very important, no artificialalternative has to date been created to replicate such componentsand replace the biological system based on nucleic acids. Therefore,we argue that the construction of autonomous synthetic systems aimedto recapitulate the fundamental aspects of living cells will relyon components that can be genetically encoded.The engineering of minimal synthetic cells strippedfrom nonessentialfunctions is currently an active area of research with many scientificand technological challenges.17  from Salmonella enterica and finding applications in the construction of synthetic cells38 but also DNA39 and protein40 sequencing.41 Nonselectivepores allow molecules to diffuse in or out the synthetic cell accordingto their concentration gradients. Toxins with pore diameters of 1.4–4.0nm are availabe30 so that macromolecules  are retained while metabolites can enter or leave thecompartment. A main disadvantage is that small molecules cannot beaccumulated against their concentration gradient, which is an essentialfeature of living systems. enterica and α-heus aureus self-assus aureus3. Next tOpen, selective systems. Reconstituting membrane transportersin lipid vesicles generates selectively open systems that can maintainan out-of-equilibrium state by accumulating specific nutrients andexcreting unwanted end products. Such systems are typically drivenby ATP or electrochemical ion gradients. They allow cells to growunder environmentally changing or low-nutrient conditions, and theyshould ultimately be reproduced in synthetic systems.Beyond open systems: open environment. A selectivelyopen system that is placed in a closed environment will ultimately reach equilibration, i.e., when the substrates runout and the waste products accumulate. This can be avoided by openingthe external environment, that is, by introducing a continuous nutrientflow that simultaneously removes products, whereas the synthetic systemsare retained .3 and contain genomes with an upperlimit in size of ∼6 Mbp 21 but also to some free-living bacteria 22  with a diameterof 1–2 μm.Synthetic cells that are to be built bottom-up frommolecular buildingblocks can be designed by following the principle of compartmentalizingthe metabolic machinery. This raises a question of what the optimalvolume for such a compartment would be. Many bacteria have volumesof less than 1 μmf ∼6 Mbp 2a.20 Thebique)22 2b. These25 the ensemble of ionicfluxes across the bilayer generated by the action of membrane-embedded,ion translocating proteins is called ion motive force .26 The proton and sodium ion gradients are the main sourcesof electrochemical energy known in cells from all domains of life.27 Thus, a minimal cell-like system must efficientlyincorporate simple pathways to utilize and regenerate ATP, NAD(P)H,and IMF, not only to drive the metabolism but also to maintain physicochemicalhomeostasis.To facilitate the construction of a complete syntheticmetabolicnetwork, it is arguably beneficial to work with a simpler set of metabolic modules that can be combined into a more complex system.These modules must be sufficiently simple to allow in-depth characterizationof their kinetic behavior, which subsequently will facilitate theexploration of parameters that lead to emergent properties and functionaldesigns that do not exist in nature. Modules that in our view areessential in any design and upon which a metabolic network can bebuilt relate to the provision of energy and the establishment of ionicgradients along the membrane. Specifically, adenosine triphosphate(ATP) and nicotinamide adenine dinucleotides NAD((P)H)are the hub metabolites fueling life-like systems by provision offree energy and reducing equivalents;Here, we present a perspective and semiquantitativeanalysis ofthe requirements for the bottom-up construction of a basal metabolismsuitable for synthetic cells, with a focus on the functions of thecell boundary. We propose that the compartment of the synthetic systemshould be selectively permeable to nutrients and waste products, andwe give an overview of the lipid and protein components that shouldbe included in a minimal membrane. We use JCVI-syn3a as a model organismto explore the vital functions of the energy supply, physicochemicalhomeostasis, transport of nutrients and waste products, and membranegrowth. Lastly, we examine how different volumes would affect therelative protein surface area and lipid-to-protein mass ratios ofthe boundary and how these properties would reflect on the growthrate of the synthetic cells.246 Membrane transportproteins that undergo large conformational changes in the translocationstep typically make essential interactions with, e.g., lipid headgroups or require specific hydrophobic chains.52 Thus, if metabolism and energy conservation are to rely on multiplemembrane proteins for selective communication with the external environment functionality.Therefore, other vesicle-forming compounds, such as single chain-amphiphilesand block copolymers, generally do not or poorly support membraneprotein function, with a few notable exceptions.ironment1, naturaironment3.4.1. Pe2.152 Complex mixtures extracted from natural sources, such as polar lipidextracts , are alsoused, as their varied composition provides a close-to-native environmentthat meets the structure/activity requirements of many proteins. However,these mixtures do not easily enable us to determine the lipid propertiesminimally required for functionality, an important aspect of the building-to-understandapproach.Selecting a lipid bilayercomposition consisting of a minimal set of defined phospholipids thatenable the functionality of a wide variety of membrane proteins isessential in building a synthetic cell 3. Phosph62 The ratio of a bilayer to nonbilayertype of lipid is important for membrane formation and protein activity.52 We screened different synthetic phospholipidmixtures for the optimization of the activity of several membraneproteins. We have found that a minimal lipid mixture of PG/PE/PC (PC= phosphatidyl choline) at mole fractions of 25:25:50, supplementedwith a sterol or specific lipid  vesicles widely usedin the field of biophysics ;ipid see 2.2 suppo63 Oleic (18:1 cis-9) and palmitic (16:0) acids are the most abundant acyl chains inliving cells.66 We typically use dioleoyl or oleoyl-palmitoyl chainsin our synthetic lipid mixtures.48 Theglobal properties of lipid membranes such as lateral pressure in theheadgroup and acyl chain region are important for the insertion andfolding efficiency of  membrane proteins, and thesefactors also have to be considered in the choice of lipid mixtures70  lead to a randomorientation of the proteins in the membrane. This approach has theadvantages that the quality of the membrane protein can be assessedprior to and after reconstitution, that the system is well-defined,and that complex membrane proteins with multiple transmembrane segmentscan be incorporated. While detergent-mediated protein reconstitutionworks well with minimal modules, increasing the system complexitybrings about bottlenecks. In our attempts to compartmentalize theenzymes for phospholipid biosynthesis in LUVs, we have experiencedthe following technical limitations: (i) a requirement for high proteinyields and purity levels, which are not trivial to obtain for certainmembrane proteins; (ii) the difficulty of encapsulating peripheralmembrane proteins, which tend to bind to the outer surface of thevesicles; and (iii) the low probability of distributing all componentsover the vesicle population, which leads to an increasing number ofinactive vesicles .Membrane protein reconstitution in GUVs. The reconstitutionof membrane proteins in GUVs currently poses a major challenge andrequires the optimization of conventional formation methods or entirelynew approaches. Most commonly used are gel-assisted swelling and electroformation.228 Integrin αIIbβ3, a membrane-anchoredprotein with a soluble active domain, has been reconstituted in GUVsby gel-assisted swelling.229 The voltage-gatedion channel KvaP has been reconstituted by gel-assisted swelling andelectroformation.230 The latter approachhas also been used to reconstitute a Ca2+-ATPase, the ABCtransporter Opp,231 and bacteriorhodopsin.232 Solvent displacement is a less-trivial methodfor proteo-GUVs preparation, due to the use of organic solvents, althoughit was successfully adapted to reconstitute the integrin αIIbβ3via the formation of water-in-oil droplets.233 To date, there is no general or robust protocol that allows thereconstitution of any complex, multispan transmembrane proteins (suchas the membrane transporters) into GUVs of desired lipid composition.Transcription-translation in GUVs. The in vitro synthesisof membrane proteins meets several requirements, including: (i) controlover the orientation of the protein, unless it operates bidirectionallylike solute/product antiporters; (ii) regulation of protein expressionat the genetic level; and (iii) heredity of the molecular machinery,thereby avoiding dilution over daughter cells. Thus, externally fed,luminal synthesis of proteins is the route of choice toward the synthesisof autonomous cell-like systems that grow and divide. Due to the highmolecular complexity of the protein synthesis machinery, transcription-translationin confinement is preferred in GUVs. Transcription-translation inconfinement may be performed either by encapsulating cellular extracts216 or recombinant systems, such as the PURE,218 within vesicles. While cell extracts have amore complete formula and show higher expression yields,217 purified components are minimal yet highlydefined and thus often preferred for the construction of syntheticcells. Both transcription-translation approaches also face technicalchallenges, including the difficulty of expressing functional ribosomes,which to date has only been achieved by coupling rRNA transcriptionto purified ribosomal proteins.234 In addition,the impaired ribosomal processivity poses a limit at the translationallevel.235Membrane insertion. Finally, the in vitro transcription-translationof membrane proteins requires coupling to a mechanism  for the insertion and folding offunctional membrane proteins. Some polytopic membrane proteins havebeen reported to self-insert in lipid bilayers in vitro without theaid of an insertion machinery such as the Sec translocon  and the SRP-receptorFtsY, two components required for SecYEG-dependent targeting and insertion.In our view, there is no compelling evidence for efficient and functionalinsertion in the lipid bilayer of polytopic membrane proteins in theabsence of translocon and foldase components.din ref ). Exampl2.273 proteins derived fromextremophiles may benefit from phosphoglycolipids,74 while the functionality of archaeal membrane proteins maydepend on ether-type phospholipids.75 Nonpolarlipids such as sterols  can be requireddue to specific interactions with the proteins embedded in the membrane76 or their effect on the overall physicochemical properties of membranes for activitysee refs  and 78).Ka).81 Fatty acid vesicles are much more permeable to smallpolar molecules than phospholipid vesicles82 and can spontaneously undergo growth and division,85 which makes these amphiphiles particularly interesting for origin-of-lifestudies, given their prebiotic plausibility.87 The integrity of fatty acid vesicles is extremely sensitive to environmental physicochemical conditions; for example, low concentrationsof divalent cations88 or pH changes89 can be detrimental to the bilayer stability.While their dynamic behavior was likely an advantage for the earlyonset of life on earth, it is undesirable for modern life that needsto maintain physicochemical homeostasis in a variety of environmentalconditions. It is therefore not surprising that no known living organismrelies on fatty acids alone for spatial confinement, although it isnoteworthy that fatty acids and single-tail lipids can constitutea significant fraction of the amphiphiles that form the membrane ofa cell.90Single-chainamphiphiles, e.g., fatty acids, can also self-assemble into bilayerstructures when present above threshold concentration and within acertain pH window synthetic cells as precursors for membrane expansion  are the precursorsof phospholipids. While it is possible to recapitulate isolated featuresof phospholipid vesicles with single-chain amphiphiles,xpansion 3.4.1. InFinally, it is important to note that each lipid type incorporatedin a synthetic cell adds to the complexity of the system. Ultimately,a biogenesis/feeding mechanism is required for each lipid type presentin the synthetic cell. Engineering biosynthetic pathways is a requirementfor the autonomy of cell-like systems. It is thus important to haveinsight into the lipid requirements of the membrane proteins minimallyneeded to build a synthetic cell and, possibly, direct the choiceof proteins toward orthologs with not too diverse lipid dependencies.2.3101 The size and volumeof LUVsare in the same range as those of many microorganisms  to large-unilamellar vesicles  and giant-unilamellar vesicles .rganisms 2, while 2.3.1101 which yields vesicles with a narrow size distribution.102 LUVs are then prepared by fusing preformedSUVs via freeze–thaw cycles, followed by an extrusion stepto obtain more evenly size-distributed LUVs.103 Solvent displacement methods101 (suchas reverse-phase evaporation) are also commonly used to produce LUVs,104 with the advantage of high encapsulation efficiencyof soluble components and the possibility of forming asymmetric lipidbilayers.SUVs can beprepared by the hydration of a lipid film followed by sonication,105 electroformation,107 and gel-assisted swelling.108 Varioussolvent displacement methods have been customized for GUVs formation,e.g., reverse-phase evaporation,109 invertedemulsions,110 and cDICE.111 Microfluidic tools are also widely used for the preparationof GUVs.112 The reconstitution of membraneproteins in GUVs poses a significant challenge.113 Currently, there is no robust technique to produce stable proteo-GUVsfor any membrane protein. Methods are generally optimized for a certainmembrane protein or synthetic cell module .Engineeringan autonomous, functional synthetic cell will ultimately require manyenzymes, each with a copy number of one or higher. From the data availablefor JCVI-syn3a, we argue that ∼500 genes will be needed fora bottom-up constructed cell. Accounting for membrane proteins (∼30%of the proteome) and the presence of multimers, this number reasonablytranslates to ∼250 soluble enzymes. Autonomy dictates thatthe metabolic machinery is eventually self-expressed from a genomictemplate. However, it is likely that initial engineering designs willhave to rely on the encapsulation of pre-existing components due tothe molecular complexity and technical limitations currently facedby in vitro transcription-translation systems 3. In thiFigure S1). These estimations assume an infinitely smalllipid bilayer, encapsulation efficiencies for each enzyme of 100%,and homogeneous size of the vesicle population. In practice, LUVsdisplay a large size distribution,63 leadingto a fraction of vesicles that does not contain all pathway componentsand is thus inactive.42 In addition, multipleproteins are likely to interact together, which will also have aneffect on the encapsulation efficiency. Further, it is technicallychallenging to sufficiently concentrate multiple proteins to the requiredvolume and encapsulate them without enormous loss of the preciouspurified components. Nevertheless, these estimations indicate thatvesicles with a radius of ≥130 nm (within the range that iscommonly employed for membrane reconstitutions) are large enough forthe construction of life-like synthetic cells.Despite their relativelymonodisperse size distribution, SUVs have an internal volume thatis too small to accommodate sufficient amounts of enzymes for theconstruction of a synthetic cell, even at micromolar concentrations;hence, the encapsulation becomes stochastic, and most vesicles willbe empty. A radius of at least 70 nm is required to ensure that eachvesicle contains at least one or more copies of a given enzyme at5 μM concentration, while a radius of 100 nm would ensure that10 copies are minimally present 4a. To en118 and components of the cytoskeleton123 have already been introduced into GUVs.In addition, GUVs also provide a larger membrane surface area to reconstitutemembrane proteins once a suitable generic method has been developed.However, GUVs incur the cost of a less-favorable surface-to-volumeratio. This poses a serious problem for the delivery of large amountsof nutrients and other solutes by membrane transport proteins124  and incorporatedmetabolites (or building blocks). Recyclable cofactors are metabolicintermediates that are responsible for the transfer of a functionalgroup. These compounds transiently bind to the enzyme during the catalyticcycle and are subsequently reloaded with a new functional group. Netimport (or synthesis) of such metabolites is not necessary as longas the cell does not grow. Examples of recyclable cofactors are phosphoryldonor ATP and electron donor NAD(P)H. The formulationof a specific cocktail of these recyclable metabolites is tightlyinterconnected with the ultimate metabolic reaction network design.We stress the importance of engineering recycling mechanisms in orderto avoid systems reaching thermodynamic equilibrium.On the basis ofthe ATP requirements of an autonomous life-likesystem, Sikkema et al. have proposed a list of essential metaboliccomponents.Incorporatedmetabolites are the constituent units of macromolecules and lipids. These metabolitesare sequestered from the cytoplasm and used by the cell to expandor replicate its own components before division. Therefore, buildingblocks should always be sourced from the external milieu. Prostheticgroups that are tightly bound to the enzymes are also regarded asbuilding blocks.6Based on the proposed classification, we identifyseveral lipidbilayer-dependent modules that will be key for any design of a syntheticcell, namely: energy provision, physicochemical homeostasis, metabolitetransport, and membrane expansion 9.We preBox 2Energy and redox power provision The genome of JCVI-syn3aencodes the full glycolytic pathway. Glucose, mannose, and glucosamineare imported by PtsG, which is the membrane component of a PEP-dependentphosphotransferase system.146 The sugarsare phosphorylated concomitantly with their transport and then metabolized. N-Acetylmannosamine is presumably taken up by an unknownABC transporter and also metabolized into fructose-6-phosphate. Thefinal product of glycolysis, pyruvate, is converted to acetate viathe pyruvate dehydrogenase complex, phosphate acetyltransferase, andacetate kinase, yielding extra ATP. The genes for the FoF1-ATPase are present in JCVI-syn3a, allowing the generationof a PMF at the expenses of ATP. JCVI-syn3a synthesizes NAD+ and NADP+ via uptake of the precursor nicotinate (videinfra). NAD+ is reduced in the glycolytic pathway. Alternatively,a side shunt of glycolysis consisting of a nonphosphorylating glyceraldehyde3-phosphate dehydrogenase (GapN) is responsible for NADP+ reduction.Physicochemical homeostasis.Ionand pH homeostasis. The genome of JCVI-syn3aencodes an ATP-consuming sodium/potassium antiporter , a P-type ATPase for magnesium uptake (mgtA), an ABC-importer for phosphate , and a putative magnesium/calciumtransporter (corA). Sodium/proton antiport is assumedin the metabolic reconstruction; however, the corresponding gene(s)are not essential or quasi-essential. Yet, we consider one or moresodium/proton or potassium/proton antiporters essential for pH homeostasis.We note that a variety of transporters  are coupled to the PMF (vide infra).Osmotic pressure control. Dedicated compatiblesolute importers are not annotated in JCVI-syn3a,and the metabolic model does not account for osmotic pressure regulation.In fact, Mycoplasma species do not possess a cellwall and most likely have little or no turgor.147 However, they still need to regulate their internal volume.Although the specific regulatory mechanisms are unknown, we arguethat potassium ions and amino acids  transportplay a role in volume control in JCVI-syn3a.Metabolitetransport.Nucleotides. A putative ABC transporter  mayimport all (deoxy)ribonucleosides, which are then phosphorylatedby the kinases Tdk, Dak1, and Dak2 to yield the corresponding nucleotides.Nucleobase import is also included in the metabolic model with a protonsymport reaction . The nucleobasescan then be converted into nucleotides by several phosphoribosyl-transferases.Amino acids. JCVI-syn3aimports oligopeptides by an ABC importer , which are hydrolyzed by peptidases (ietS and others). A specific l-glutamate/l-aspartatetransporter (gltP) ensures the uptake of l-glutamate, which is the most abundant amino acid in Mycoplasma species148 and possibly important forvolume regulation (vide infra). In the metabolic model, l-glutamate and l-aspartate are taken up via a proton symportmechanism. In addition, two uncharacterized membrane proteins aremodeled as putative unselective proton symporters for other aminoacids.Vitamins and polyamines. JCVI-syn3a encodes one energy-coupling factor (ECF) module149  and several uncharacterized substrate-bindingsubunits  that have been assigned to the transportof folate, riboflavin, coenzyme A, nicotinate, and pyridoxal; an Scomponent (ecfS3) responsible for the uptake of 5-formyl-THFis also included in the metabolic reconstruction. ABC-type importersfor thiamine  and spermine  are quasi-essential in JCVI-syn3a.Waste export. In JCVI-syn3a,several membrane proteins are annotated as ABC-type exporters. However,their functions are unknown.Membrane expansion.Phospholipidbiosynthesis. Free fatty acids, cholesterol,and triacylglycerols are supplied by the hosts of Mycoplasma species,151 and, in turn, JCVI-syn3a relieson an exogenous feed of these components. The free fatty acids arephosphorylated in the cytosol by soluble fatty acid kinases , followed by binding to the acyl-carrier protein (ACP) and transferto glycerol 3-phosphate . Glycerol 3-phosphate is formed by phosphorylation of glycerol(glpK), which may passively permeate through themembrane.144 JCVI-syn3a encodes the pathwayfor phosphatidylglycerol (PG) and cardiolipin (CL) formation  but lacksenzymes for phosphatidylcholine (PC) and phosphatidylethanolamine(PE) biosynthesis. JCVI-syn3a also produces lipogalactan  and Gal-DAG (cps and others).Membrane protein insertion. JCVI-syn3a encodes the machinery for the cotranslationalinsertionof membrane proteins , coupled to signal-recognition-particledocking (ftsY), quality-control (ftsH), and protein excretion (lspA). Othercomponents involved in protein insertion/translocation in E. coli are missing in JCVI-syn3a ,152 but JCVI-syn3amay use other chaperone-like proteins to substitute for SecB, similarto B. subtilis.153Uncharacterized processes. Despite the drastic genomereduction, JCVI-syn3a encodes 38 essential and quasi-essential membraneproteins for which it has not yet been possible to predict a function,that is, one-third of all membrane proteins; the same is true alsofor its soluble proteins. Computational efforts are ongoing to bridgethe gap between these genes and the minimal proteome.1523.126 Exponentiallygrowing cells typically contain cytosolicATP in the millimolar range. Importantly, upon hydrolysis, this cofactoris recycled, in order to continuously restore the energy pool. Theimportance of ATP recycling becomes evident by comparing the numberof ATP equivalents present in a cell and the number of ATP equivalentsrequired to sustain a full growth and division cycle. For example,JCVI-syn3a contains approximately 3.29 × 10–3 mmol/gDW ATP  to sustain its metabolism throughout a cell cycle,3 which implies ∼104 rounds ofATP recycling or a turnover of the cellular ATP pool of ∼1.5s–1.ATP is the main energycurrency of living cells./gDW ATP 1, that i3, comparable tothat of LUV-sized synthetic cells. In agreement, it was calculatedthat 3.6 × 108 ATP equivalents are needed for a vesicleof equal volume to undergo a full cell cycle,62 while E. coli (1 μm3) needs asmuch as 2.4 × 1010 ATP equivalents to sustain a cellcycle.62 A comparable energy requirementwould be sufficient for a small GUV with a diameter of 1–2μm. However, assuming that such a correlation would hold forlarger volumes, it can be estimated that an average GUV with a diameterof 50 μm  would face a much higherenergy demand , which againpoints to small GUVs (or very large LUVs) as preferable compartmentsfor bottom-up synthetic cells.The amount of ATP required to sustain the cellularmetabolism scaleswith the molecular complexity and thus with the cellular volume. JCVI-syn3ahas an internal volume of 0.03 μm125Besides ATP, electrochemicalion gradients  play an essential role in energy provision. Themagnitude and composition of the PMF vary for different cell typesand environmental conditions, but typical PMF values range between−150 and −200 mV (−15.5 to −20.7 kJ/mol).126 These redox cofactors play key roles in ATPand PMF (re)generation,127 protectcells against reactive oxygen species,128 function in cell signaling,129 and regulategene expression and cell division.132 Numerous central metabolicpathways and cellular processes rely on the oxidation and reductionof redox cofactors as well as on the (re)generation of ATP andPMF. Consequently, a minimal out-of-equilibrium metabolic networkshould include modules for ATP, PMF, and redox cofactor (re)generationgeneration have been providedto in vitro systems by introducing organelles or crude membranes suchthylakoids, chromatophores, and inverted membrane vesicles. For example,isolated thylakoid membranes have been encapsulated in microdropletsand used to drive the COThe use of such native microcompartments can provide the energyand redox cofactors for complex metabolic networks. However, theymust be derived as fractions from living systems, have a complex andpartly undefined molecular composition, and are not easily amenablefor further engineering of cell-like systems. Therefore, better-defined,minimal modules are required for building a synthetic cell.3.1.2136 as well as in vitrotranscription-translation.137The interconnectionof ATP, PMF, and redox power provision in livingcells has inspired the development of synthetic metabolic modulesfrom purified and reconstituted components. For instance, an artificialphotosynthetic organelle has been developed to generate a PMF andthe subsequent synthesis of ATP upon illumination. The organelleswere encapsulated within GUVs and used to power carbon fixation andactin polymerization138 In this system, ATP was produced externally and utilized to promotethe cell-free expression of a reporter gene in the medium. The coreconstitutionof a bo3 quinol oxidase and an ATP synthaseled to the production of ATP by basic oxidative phosphorylation.139 These synthetic organelles have reported ATPsynthesis rates139 that in some cases are comparableto that of E. coli FoF1-ATPasein membrane vesicles.140A PMFhas also been generated by NADH oxidation coupled to ubiquinone reductionwith reconstituted mitochondrial complex I.3.1.3l-arginine breakdown pathway has beenreconstituted to generate ATP in LUVs and drive membrane transportto elicit partial volume and pH homeostasis.141l-Arginine breakdown only requiresone membrane protein (ArcD), which imports l-arginine inexchange for l-ornithine (the end product of the pathway),plus three soluble enzymes ; the formed CO2 and NH3 leave the vesicles by passive diffusion.Hence, the pathway runs for hours, away from equilibrium, and producesone ATP per l-arginine when ADP plus inorganic phosphateare supplied. The production of ATP has been used to drive compatiblesolute uptake63 and glycerol 3-phosphatesynthesis.42 The current design of the l-arginine breakdown pathway produces ATP at a rate that wouldallow an LUV-sized synthetic cell62 togrow and undergo a complete cell cycle in about 10 h . We note that a Pi importer is ultimatelyneeded for sustained ATP formation by l-arginine breakdown,as part of phosphate will be incorporated in lipids and nucleic acids.In addition, a net uptake of ATP (or synthesis from precursors thatare taken up as building blocks) is required under growth conditionsto prevent dilution of the internal nucleotide pool over the daughtercells and, most importantly, to provide sufficient building blocksfor the synthesis of nucleic acids . This allows feedingof ATP from the outside to the synthetic cell in exchange for internalADP . Such an exchange ofATP for ADP generates an inside-negative membrane potential and doesnot change the overall pool of available adenine nucleotides insidethe synthetic cell.2– into L-lactate1–, as found in lactic acid bacteria.142 The uptake of L-malate2– occurs in exchangefor L-lactate1–, and the L-malate/L-lactate antiportthus generates a ΔΨ. A proton is consumed in the decarboxylationreaction inside the vesicles, hence the formation of a ΔpH.These pathways are advantageous for the modular design of a syntheticmetabolism because the overall electrochemical gradient is formedindependently of the pathway for ATP synthesis, in contrast to designsfor energy conservation based on oxidative phosphorylation (vide infra).Metabolite decarboxylation can be used to drive the uptake of buildingblocks, such as amino acids and sugars, via secondary transportersand pH gradient  are formed. An exampleis the decarboxylation of L-malateterssee 3.3.1. AnE′0 = −0.43 V)25 compared to nicotinamides(E′0 = −0.32 V) and itsability to permeate lipid membranes.144 In a minimalredox pathway, formate is utilized by a dehydrogenase that concomitantlyreduces NAD+.28 The producedNADH can be coupled to NADP+ transhydrogenation, regeneratingNAD+ and at the same time forming NADPH. This pathway forthe regeneration of redox cofactors is functional in both LUVs andGUVs, and it has been used for NADPH-dependent conversion of glutathionedisulfide into reduced glutathione, a known antioxidant that protectsliving cells against oxidative stress.128Pathways for the regeneration of reducingequivalents have alsobeen designed. Formic acid is a convenient electron donor due to itslow standard reduction potential 155 in order to maintain theirionic strength within physiological limits. It is unclear how JCVI-syn3aoperates the volume homeostasis .l-argininebreakdown pathway from Lactococcus lactis has beenreconstituted in a synthetic system with the aim to develop an ATPgeneration module , sugars, mostamino acids, vitamins, nucleotides, compatible solutes, and variousmetabolic end products.173 In these cases, membranetransport proteins are needed for import or export, either by facilitateddiffusion or by active transport. Membrane transporters enable compartmentalizedsystems to maintain an internal chemical composition different fromthe external environment. They allow the accumulation of nutrientsagainst their concentration gradients and generate electrochemicalgradients for energy conservation and physicochemical homeostasis mayenter the cell by simple diffusion through the lipid bilayer 7. Similaeostasis1. We preeostasis8.3.3.1180 Nucleobase and nucleoside import is retained by JCVI-syn3a  butwould require a reductase187 and a suitableelectron donor to produce the corresponding deoxy forms ribonucleobase and (deoxy)ribonucleosideimport.VI-syn3a 2 and is orms see 3.1.3.189 while oligopeptide import playsa pivotal role in the nutrition and communication of Gram-positivebacteria.190 JCVI-syn3a is also equippedwith a mechanism for oligopeptide import 194 that will rapidly convert the peptides into amino acids, it willbe possible to deliver all 20 amino acids for protein synthesis. Thesignificantly higher demand for certain amino acids  may be satisfied by tuning thepeptide composition of the medium or by introducing additional selectiveamino acid transporters.For amino acids, obligate parasites use transporters for extractionfrom their hosts,e import 2. Di/tri114 We envision that this systemcould be expanded in the future with selective building block uptakestrategies, such as the ones proposed heretofore to keep macromoleculebiosynthesis away from thermodynamic equilibrium.A milestone was set in the bottom-upsynthesis of macromoleculesby coupling self-encoded protein expression to DNA duplication inconfinement.149 vitamins and polyamines, consisting of a single transporter backbone(ECF module) coupled to a variety of highly specific substrate-bindingsubunits (S components)149 2. We arg3.3.2195). However, a minimalcell-like system designed from scratch should also encompass exportstrategies for metabolic waste products that are membrane-impermeable.To this end, membrane antiporters that are selective for both thesubstrates and end products of a certain reaction network , while othersare beyond the focus of this perspective  and to the cellular lumen (soluble components).201 Cargo release by controlled vesicle fusionhas been successfully demonstrated in the presence of a variety offusogenic compounds , but it has not been assessed whetherleakage occurs in the fusion process. Programmable vesicle fusionoffers unique advantages compared to strategies that involve de novosynthesis of lipid. Feeding (phospho)lipids through pre-existingvesicles is fast and yields significant membrane expansion; the dispensabilityof protein expression for lipid biosynthesis and of actual lipid synthesisis an additional advantage. Hence, an overall optimization of thecellular resources, which can be repurposed toward other metabolicmodules, can be envisioned. Next to membrane expansion, controlledfusion also brings the added bonus of feeding cytosolic nutrientsand cellular components that are difficult to produce or recycle internally. However, in the absence of a mechanism to internallyregulate the production and display of fusogenic tags, fusion-mediatedmembrane expansion remains dependent on external triggers, which mayultimately compromise the autonomy of synthetic life-like mimics.In livingcells, phospholipid biosynthesis occurs via complex, multistep reactioncascades.VI-syn3a 2. Analog16 We argue that membrane proteins incorporatedin vesicles prepared from such biomimetic lipids will likely be functional,207 provided the appropriate headgroup compositionis supplied has been explored through various approaches: (i) chemical, (ii) mixedchemo-enzymatic, and (iii) enzymatic. The chemical synthesis of phospholipidanalogues has been achieved by linking an acyl-chain donor to a functionalizedlysophospholipid by diverse reaction mechanisms, including click-chemistry,native chemical ligation, imine chemistry, transacylation, and others(see ref ), yieldisupplied 2.1. Howe99 Chemo-enzymatic phospholipid formation has also been coupled tothe synthesis of the acyl-chain precursors by means of a type I fattyacid synthase.208 Beyond the advantageof eventually linking phospholipid formation to the regulation ofgene expression, this route avoids the intrinsic molecular complexityof its multistep natural biosynthetic counterparts (vide infra). Inaddition, it provides a simple approach for phosphatidylcholine formation,which has not yet been realized in in vitro membrane model systems.A linkto the genome may be established by mixed chemo-enzymaticapproaches, where chemical reactions  are coupled to biochemical ones to demonstrate de novo vesicle generation.For example, Bhattacharya et al. have developed a minimal syntheticpathway for lipid formation consisting of a water-soluble enzyme thatactivates a single chain amphiphile, followed by spontaneous acylationof a functionalized phospholipid precursor.209 Phospholipidbiosynthesis has also been achieved in vitro by expressing PS biosyntheticenzymes within GUVs, albeit with modest yields.38 Genome-encoded phospholipid biosynthesis in confinementhas in later studies been expanded by the inclusion of an acyl-chainformation module.210Finally, phospholipid formation can be achieved in vitro with afully enzymatic approach by using  multistepbiosynthetic routes. With this approach, significant bilayer expansionwas demonstrated by detergent-mediated reconstitution of the purifiedenzymes for PE and PG formation on the outer surface of small-unilamellarvesicles (SUVs). Despite its molecular complexity, this system ishighly versatile. For instance, the PE and PG levels can be variedby changing the relative enzyme concentration, and different acyl-chainmixtures can be obtained by varying the pool of single-chain amphiphilessupplemented as substrates.42 Furthermore, for sustainable lipid synthesisand membrane growth, lipid flip-flop mechanisms are required to movelipids from the cis to the trans side of the membrane .211A common bottleneckof all approaches that rely on long-chain fattyacids is the poor solubility of these and other bilayer-relevant amphiphilesin aqueous solutions. This poses an inherent limitation to phospholipidformation in nonleaky compartments, but it can be circumvented byprovision of a continuous flow of diluted acyl-chain precursors.3.4.2214 for the reconstitution of purified membranecomponents into LUVs . These also ref 3.E. coli)217 or reconstituted cell-free translation systems .218 This approach is ultimately preferred overdetergent-mediated reconstitution for autonomous cell-like systems.The PURE system has the advantage of being composed of well-definedcomponents, and it has thus found wide application in bottom-up syntheticbiology.219 However,membrane proteins synthesized in vitro by transcription-translationwith PURE tend to display low activities,38 partly due to limitations intrinsic to PURE vailable 2. The im224 or expression from a DNA template.225 In vitro expressed SecYEG has been claimedto spontaneously self-embed into lipid bilayers, albeit with extremelypoor efficiency.226 Therefore, we argue that catalyticamounts of Sec/YidC should be initially added to the membrane bilayerof the cell-like system in order to facilitate the transertion of additional Sec/YidC andother membrane proteins.The Sec/YidC cotranslational insertionpathway has been studiedin membrane systems prepared either via detergent-mediated reconstitution3.5227 (Table S2), yet they are required for cellular growth,and their functions need to be unraveled.3 It is likely that these essential proteins will have consequencesfor the design of bottom-up constructed synthetic cells. In earlyengineering stages, a degree of uncertainty (use of components withunknown functions) will likely have to be tolerated in order to achievecomplex functions such as membrane growth and division. Some evidencefor this is already available. For example, certain membrane proteinsdisplay higher activities in membrane vesicles than in reconstitutedvesicles of defined lipid compositions  and cell cycle phases,membrane proteins generally occupy a large fraction of biologicalbilayers. Plasma membranes have a lipid-to-protein mass ratio of about1:1 w/w, which corresponds to a surface occupancy by proteins of ∼25%.243The fluid mosaic model245 In order to obtain optimal proteinfunctionality in LUVs, a lipid-to-protein mass ratio of at least 20:1w/w is required.246 Protein insertion coupled to in vitro transcription-translationis to date also insufficient for creating low lipid-to-protein massratios  proteins decreases at low lipid-to-proteinmass ratios.tios see 3.4.1124 Here, we narrowthe problem down to estimating the smallest membrane surface areathat would enable one to reconstitute membrane modules required fora minimal metabolism at a realistic lipid-to-protein mass ratio. Todo so, we use the JCVI-syn3a annotated membrane functions (Table S2) as a proxy of what is needed in synthetic vesicles.Giventhe challenge to produce synthetic vesicles with physiological levelsof protein crowding, the question arises whether, at relatively highlipid-to-protein mass ratios, such membranes can incorporate enoughproteins to have sufficient capacity to transport all nutrients andother solutes needed for a minimal metabolism. This issue is particularlyimportant for GUVs, as the surface-to-volume ratio is inversely proportionalto the radius of the vesicles and membrane transport may become rate-limitingfor luminal processes.Supporting Information Methods). The abundance of eachprotein was taken from the proteomic data of JCVI-syn3a in the exponentialphase of growth .145 This assumption is reasonable given thatthe total surface area of JCVI-syn3a (r = 200–250nm)247 is comparable to that of LUVs extruded through400 nm polycarbonate filters (raverage ≈ 230 nm).63 The relative proteinsurface area occupancy is the ratio between the total protein surfacearea and the total surface area of a sphere of a given radius.The total surface area occupied by membrane proteins can be estimatedfrom the sum of their horizontal sections, each protein being approximatedto a cylinder embedded perpendicularly in the membrane plane . A higher doubling time may lowerthe required membrane protein abundance to values feasible in LUVs.For example, a doubling time of ∼50 h would lead to intermediateprotein copy numbers that would enable >20:1 w/w lipid-to-proteinmass ratios in LUVs with raverage ≈230 nm. This estimation assumes an inverse correlation between theprotein abundance and the doubling time, which probably will not holdwhen the protein abundance becomes too low. Also, protein copy numberslower than a certain threshold are not desirable due to stochasticityissues.From the protein surface areaoccupancy, lipid-to-proteinmassratios can be estimated  are sufficiently large for the reconstitution of JCVI-syn3amembrane proteins at physiological abundance and doubling time of∼2 h, but vesicles with a radius of 1 μm, akin the sizeof bacterial cells like 5The componentsof the synthetic metabolic (sub)systems giverise to dynamic system-level behaviors, and computational modelingis key in understanding these emergent behaviors. Different typesof modeling are conceivable, and some of them are outlined below andin Box 4Model structure. The model structure will largelyconsist of differential equations for each metabolite. These differentialequations contain terms for each reaction in which a metabolite participates.The majority of these terms are enzymatic rate equations. These rateequations describe the rate of the (transport) reaction in questionand may be a function of multiple metabolites . Knowing the substrates and products of a reaction isnot sufficient to obtain a rate equation, as this also depends onthe mechanism . In addition to the mechanismitself, further assumptions about the kinetics can introduce or removeterms from the rate equation. For instance, it is often assumed thatspecific steps in the enzyme mechanisms are slower than other steps,which generally allows removal of some terms in the equation. Forinformation about enzyme mechanisms and kinetics see refs  using a parameter estimation algorithm.Parameter values listed in a database are apparent parameter values.They are obtained under particular environmental conditions and assuminga particular rate equation. Rate equations in a dynamic model oftendiffer from the rate equation used in characterization (see above).Consequently, a single apparent parameter is often a function of multipleparameters in the dynamic model.260 Therefore,care needs to be taken when incorporating database parameter values.For parameter estimation, both experimental data and a model are required.The result of parameter estimation is the set of parameter valuesthat minimize the difference between the experimentally measured systemvariable and the simulated in silico system variable for the exactsame conditions. For the intricacies of parameter estimation algorithmssee refs  encodegene productsthat catalyze 175 metabolic reactions. These metabolic reactions werefirst modeledequation 4 for eacl-arginine breakdown pathway63 imports l-arginine from the externalenvironment and converts it in three enzymatic steps to ATP and wasteproducts. The waste products  leave the internal environment (by secondary active transportor passive diffusion).63 There are at leasttwo system-level behaviors that emerge when the pathway operates.First, the pH of the internal environment changes due to proton consumptionby the enzymatic reactions and passive diffusion of ammonia.63 A pH change could affect the enzymatic ratesand the passive diffusion of protons across the membrane . Second,an unwanted transport reaction results in the buildup of a membranepotential. In the unwanted reaction, external l-arginineis not exchanged for l-ornithine (as was intended) but forthe intermediate product L-citrulline. The exchange of l-arginineand l-ornithine is electroneutral, but the exchange of l-arginine for L-citrulline imports a net positive charge, consequentlybuilding up a membrane potential. The generated membrane potentialin turn decreases the l-arginine/L-citrulline antiport.63 Thus, even in a simple bottom-up system, severaleffects, often of a physicochemical nature, on the system level canoccur simultaneously. To ultimately understand the full dynamics ofa bottom-up-built system, it is important to considerthese system-level effects when integrating different metabolic modules,as they likely feed back onto other processes. For this reason, computationalmodels are indispensable.A bottom-up constructed minimal metabolismmay eventually be integratedwith other key cellular functions, also resulting in a synthetic cellthat is capable of growth and division. However, the bottom-up approachcurrently works with metabolic systems without any gene regulation.Despite their apparent simplicity, much mechanistic detail needs tobe considered to provide a full dynamic description of such a system.For example, the 251In our view, models of bottom-up systemshave to be developed ina stepwise manner in tandem with the construction of the metabolicnetworks in the lab, starting with individual components and increasingthe complexity of the system until all components are present. Ateach step the in silico simulations are compared to in vitro experimentswith the same level of complexity. If there is a discrepancy betweenthe two, then the model (and consequently our understanding of thesystem) is incomplete. Hypotheses that resolve these discrepanciescan then be tested in silico and afterward verified experimentally.In contrast to modeling a large top-down developed system, modelingof a small bottom-up developed system yields system-level insightswith mechanistic detail.6In this Perspective,JCVI-syn3a has been used as inspiration forthe design of bottom-up constructed life-like synthetic cells. Tooperate a cell away from thermodynamic equilibrium, we reason thata boundary composed of lipids and selective membrane proteins is required.Hence, we provide a comprehensive description of the membrane modulesminimally needed for sustainable metabolism and physicochemical homeostasis.We have evaluated the effect of the compartment size on the encapsulationefficiency and the surface area required for sufficient solute fluxand other membrane protein functions. We conclude that bacteria-sizedvesicles with a diameter of 1–2 μm are the most suited.We argue that the implementation of efficient protocols for the preparationof bacteria-sized vesicles is important. In parallel, robust in vitrotranscription-translation and methods for membrane protein insertionneed to be developed further. Such efforts will significantly advancethe development of sustainable metabolic networks, which may ultimatelylead to autonomous growth of synthetic cells.


Quantum teleportation has been developed to simultaneously realize the Hertz rate and the 64-km distance through fiber channels, which is essential to real-world application of quantum network. Thanks to its recent fast development, teleportation-based quantum information science has become a promising field that inspires many important applications. Quantum teleportation enables the remote transfer of quantum state in quantum communication network6 and the long-range interaction among quantum states in distributed quantum computation7. So far, great efforts have been made in quantum teleportation with a variety of quantum systems. Quantum optics-based teleportation offers a promising avenue towards quantum networks, where the quantum states are key resources of quantum information science, and not only coherent states9 but also nonclassical states10 have been experimentally teleported. For practical application, the high-rate quantum teleportation is demanded for effectively transferring quantum state12. Meanwhile, it is also required to teleport quantum state over remoter users. The transfer distances have been extended over 1400 km with a low-Earth orbit satellite13, and over 100 km through commercial optical-fiber networks14, respectively. Therefore, it is required to simultaneously realize quantum teleportation with the both long distance and high rate in real-world scenario.Quantum teleportation is one of the most important protocols in quantum information science, and enables the transfer of an unknown quantum state over long distances by using quantum entanglement resourceLight: Science & Applications, the team led by Qiang Zhou from the Institute of Fundamental and Frontier Sciences, University of Electronic Science and Technology of China has reported an experimental realization of a Hertz-rate quantum teleportation system through a real-world fiber network15. The techniques of high-performance time-bin entangled source with a periodically poled lithium niobate (PPLN) waveguide and a fully running feedback system for quantum states distribution are employed, thus a weak coherent single photon with decoy state is transferred at a rate of 7.1 ± 0.4 Hz among different real-world buildings connected by 64-km-long fiber channel, as illustrated in Fig. For practical quantum teleportation network, in a newly published paper in 17. On quantum channel, there are still improvement spaces for high performance quantum teleportation. The rate of quantum teleportation can be increased by improving efficiency and repetition rates of generation, manipulation and measurement. Besides improving indistinguishability, the high-quality quantum light source19 and entanglement enhancement21 provide possibility of high-fidelity quantum teleportation. Continuous variable (CV) quantum information processing system benefits from high efficiency generation and detection, as well as unambiguous state discrimination, although its fidelity is limit due to the losses. Meanwhile, discrete variable (DV) system can perform high fidelity quantum information processing as a result of resisting the losses, although it is restricted by probabilistic operations. Thus, the hybrid architecture of both CV and DV approaches may have potential advantages on combination of two approaches11. By combining the complex quantum states, such as multiple degrees of freedom and high-dimensional quantum states, quantum teleportation can increase its capacity23. Furthermore, the distance of teleportation can be improved by integration techniques of free space and fiber channel, and even quantum repeater5. On the quantum node, various platforms, including atomic ensembles24, single atoms25, trapped ions27, solid-state quantum systems28, and nuclear magnetic resonance29, enable quantum teleportation between matter nodes. In the future, the hybrid approach of these above technologies provides possible way to realize a high-performance quantum teleportation network.It is foreseeable that the quantum teleportation can give rise to exciting inspirations for both advanced quantum technology and quantum network applications, as illustrated in Fig. 18. Besides, quantum teleportation is potentially applied to distributed quantum computation7. Quantum teleportation can distribute local gate operations between distant users, and be used to link the distributed quantum computing units. This work establishes an important step from proof-of-principle demonstrations to real-world applications of quantum teleportation.Looking forward, while the quantum teleportation establishes an important foundation of quantum network, it also fosters inspirations to future possible applications. Quantum teleportation will play an essential role to realize quantum communication towards global scale


The clinical spectrum of acute SARS-CoV-2 infection ranges from an asymptomatic to life-threatening disease. Considering the broad spectrum of severity, reliable biomarkers are required for early risk stratification and prediction of clinical outcomes. Despite numerous efforts, no COVID-19-specific biomarker has been established to guide further diagnostic or even therapeutic approaches, most likely due to insufficient validation, methodical complexity, or economic factors. COVID-19-associated coagulopathy is a hallmark of the disease and is mainly attributed to dysregulated immunothrombosis. This process describes an intricate interplay of platelets, innate immune cells, the coagulation cascade, and the vascular endothelium leading to both micro- and macrothrombotic complications. In this context, increased levels of immunothrombotic components, including platelet and platelet-leukocyte aggregates, have been described and linked to COVID-19 severity.Here, we describe a label-free quantitative phase imaging approach, allowing the identification of cell-aggregates and their components at single-cell resolution within 30 min, which prospectively qualifies the method as point-of-care (POC) testing.We find a significant association between the severity of COVID-19 and the amount of platelet and platelet-leukocyte aggregates. Additionally, we observe a linkage between severity, aggregate composition, and size distribution of platelets in aggregates.This study presents a POC-compatible method for rapid quantitative analysis of blood cell aggregates in patients with COVID-19. The human body produces a series of immune responses when it gets infected with SARS-CoV-2, the virus that causes COVID-19. One of these responses involves platelets, the blood clotting factor sticking to immune cells to form cell aggregates in the bloodstream. We aimed to understand the significance of these cell aggregates in COVID-19 disease progression. A quantitative imaging approach was used to investigate the number and components of these cell aggregates in SARS-CoV-2 infected patient blood. We observed blood from severe COVID-19 patients was associated with higher numbers and specific composition of cell aggregates. Our method can potentially support the risk stratification of severe patients to prevent complications in COVID-19 and other medical disorders, where immune cells are shown to aggregate. Klenk, Erber et al. quantitatively detect blood cell aggregates in COVID-19 patients using label-free digital holographic microscopy. They find that COVID-19 severity associates with the amount of platelet and platelet leukocyte aggregates in the peripheral blood. The clinical manifestation of Coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) ranges from asymptomatic infection to a life-threatening disease, which is characterized by acute respiratory distress syndrome (ARDS) and multi-organ failure.4. Vascular endothelial cell dysfunction, a hyperinflammatory immune response, and hypercoagulability result in a systemic prothrombotic phenotype in COVID-19 that has been linked to morbidity and mortality7.A hallmark of severe COVID-19 is COVID-19-associated coagulopathy (CAC) that can manifest in micro- and macrothrombi leading to multi-organ injury and failure. While the underlying cellular and molecular mechanisms have not been completely understood, several lines of evidence suggest that CAC is driven by dysregulated immunothrombosis, a complex interplay of innate immune cells like neutrophils and platelets, the coagulation cascade, fibrinolytic pathways and the vascular endothelium8. Likewise, the C-reactive protein (CRP), another acute-phase protein and inflammatory marker, guides prognosis and disease severity and may also predict the risk of venous thromboembolism10. However, the relevance of both markers for clinical management, including anticoagulation, requires further investigation12. Numerous studies have evaluated platelet function in patients with COVID-19. There is growing evidence of platelet hyperactivation contributing to dysregulated immunothrombosis in COVID-19 via secretion of intracellular granula and surface expression of prothrombotic membrane proteins14. Several other markers of coagulation, fibrinolysis, endothelial dysfunction, extravesicular vesicles, and novel soluble biomarkers have been investigated9. While there is an evident potential for many of the markers to guide risk stratification, hardly any marker or algorithm on multiple markers has been translated into clinical practice. Reasons are diverse and include lack of biomarker accuracy, insufficient prospective validation, complex diagnostic workflow, and economics17. Specialized and not yet automated laboratory techniques are often required, rendering routine measurement in clinical practice unfeasible.The clinical management of COVID-19 patients largely depends on disease severity and complications. Thus, early prediction of the course of the disease and markers for the occurrence of adverse events are highly relevant for patient management. Several attempts to establish predictive markers or prediction models have been made, and the investigated markers include numerous factors involved in CAC: Plasma levels of D-dimers are frequently elevated in COVID-19 patients and have been linked to thromboembolic complications, disease severity, and mortality23. Those aggregates can be detected by several methods. One example is the usage of blood smear analysis, where PP aggregates and macroplatelets have been observed in COVID-19 patients24. However, the amount of blood cells that can be evaluated manually is limited and hampers statistical analysis. Fluorescence flow cytometry overcomes the problem of low statistical power but lacks spatial resolution and workflow automation. Forward and side scatter information combined with fluorescent antibodies staining enables cell detection with high specificity but lacks differentiation between single platelets and PP aggregates. Aggregates consisting of different cell types, such as LP aggregates, can be detected but not quantitatively evaluated. PP aggregates, however, cannot be analysed at all23. Also, the sample preparation steps for the fluorescent staining may interfere with cell biology and sample composition29. Image-based flow cytometer approaches combine the advantages of microscopy and flow cytometry and have shown promising results23. However, the potential negative effects from the time delay due to incubation periods and the possible interferences of other sample preparation steps remain. To our best knowledge, there is no label-free high-throughput methodology allowing the detection of aggregates and the analysis of their composition on a single platelet level suitable for point-of-care (POC) applications.Further, increased levels of platelet aggregates (PP aggregates) as well as platelet-leukocyte aggregates (LP aggregates), including platelet-monocyte and platelet-neutrophil aggregates, have been described in COVID-19 patients, linked with disease severity, and could thus serve as biomarkers−1 , with a microfluidic chip and a customized image analysis, resulting in a label-free high-throughput imaging flow cytometer Fig. . ContrarWHO ordinal scale for clinical improvement for hospitalised patients with COVID-1931, patients were classified into moderate COVID-19 (score 4 or 5) and severe COVID-19 (score 6 to 10). Female and male volunteers (age 29 to 67) served as blood donors for reference measurements. The study was conducted in accordance with the declaration of Helsinki and approved by the ethics committee of the Technical University Hospital of Munich (221/20 S-SR and 620/21 S-KK). Informed consent was obtained from all subjects or their legal representatives.36 patients (age 32 to 83) with PCR-confirmed SARS-CoV-2 infection admitted to the Klinikum München rechts der Isar between November 2020 and 2021 were prospectively enrolled. Blood samples  were collected using 2 ml Blood Gas-Monovettes® from Sarstedt and subjected to DHM analysis within 30 min after the blood draw. To minimize mechanical stimuli, samples were carefully transported to the nearby prototype. Demographic, clinical, and laboratory data were retrospectively compiled by chart review. Based on the W = 4 × 106 Da, Sigma Aldrich).Blood samples were diluted in a measurement buffer with a factor of 1:100  and immediately subjected to analysis. The measurement buffer contains 99.95% phosphate-buffered saline (PBS) and 0.05% polyethylene oxide . Interferograms are generated by off-axis holography combined with a double-shearing interferometric approach. Partially coherent light is emitted by a 528 nm SLED (Osram) in combination with a Koehler illumination unit and passes through the sample toward the objective. The objective  has a 40× magnification and a numeric aperture of 0.55 for a compromise between lateral and axial resolution to ensure a high yield of platelets in focus. A grating is used to generate the different diffraction maxima, which, together with a spatial filter, lay the foundation for the double-shearing approach. Interference of the single beams is recorded with the camera at an off-axis angle between the beams, resulting in the interferogram. The GS3-U3-32S4M camera (Teledyne FLIR LLC) generates 105 images per second at an exposure time of 5 µs. Reconstruction of the phase and amplitude images out of the holograms was performed using the OsOne Software Version 5.12.12 (Ovizio Imaging Systems). Parallel measurement of up to 20 cells per image allows high-throughput of 500−2000 cells/s. For each test, ~50,000 blood cells per captured for analysis.The Image Analysis Pipeline is composed of three distinct steps: preprocessing of the phase images, Mask R-CNN based segmentation, and analysis of detected cells and aggregates.32. Subsequently, these contours were filtered based on size. Each cell or cell aggregate (represented by a contour) was then saved as a smaller image section for further processing. Masking of individual cells was used to remove unwanted noise from fluid, particles, and other cells. This was done based on the earlier calculated threshold mask. Normalization is the last essential preprocessing step. The images were first clipped to limit the value range. Afterward, min–max normalization was applied to transform the image values into the range 0–1 suitable for neural networks.Preprocessing is the first step of the image analysis pipeline, which includes background subtraction, cell detection, masking, and normalization. Background Subtraction is needed to remove background noise and artifacts of the fluid channel. Cell Detection is enabled by thresholding and contour finding in the acquired images. In the first step, a binary threshold was applied to the phase images. Contours were extracted from the resulting binary images based on the algorithm of Suzuki et al. 33 for refining the individual aggregate components and identifying their cell types. Mask R-CNN allows both object detection and object mask computation simultaneously by performing four steps. In the first stage, a convolutional network (backbone) receives the input images and provides a convolutional feature map. Secondly, a region proposal network (RPN) provides candidate regions called regions of interest (RoI). This is done by sliding a small network over the convolutional feature map. In the third stage, a RoI align layer utilizes bilinear interpolation to provide feature maps of the same size as the RoI. These maps are then used in the fourth stage for classification and bounding box regression. A small FCN is applied to each RoI to predict the individual object masks on a pixel resolution. In this work, a ResNet5034 is used as a backbone.Segmentation is the second step of the image analysis, which allows identification of single blood cells, aggregates, and their components. In this work, we employ a combination of classical thresholding for a rough evaluation of the cell locations for preprocessing and, as a second stage Mask R-CNNThe performance and reliability of a neural network depend highly on the quality, amount, and selection of training data. For this purpose, a great amount of ground-truth annotated data is needed. However, manual labeling of cell images is, in this case, not feasible due to the quality and quantity of required ground-truth data. Therefore, we used a method of creating a synthetic dataset of aggregates, made by composing multiple classified single-cell images together to form cell aggregates. This is based on pure blood cell populations extracted from whole blood.Using this synthetic dataset of aggregates, the neural network is trained on 200,000 cell images in total. The performance of the trained network is evaluated on both computer-generated aggregates as well as manually labeled images.Analysis of the detected cells and aggregates is the last step of the processing pipeline. Hereby, based on the segmentation and classification results, image patches are categorized as single cells or cell aggregates. The number of cells in an aggregate as well as their cell classes and their morphological parameters are saved for each patch. This allows an in-depth analysis of both single components and whole aggregates at the same time.35, and the neural network components were built in Tensorflow 2.8.036.All image analysis tools were written in Python 3.8.8 (Python Software Foundation). Computer vision algorithms were mainly adapted from OpenCV 4.5.5p < 0.05; **p < 0.01; ***p < 0.001 and ****p < 0.0001). Correlation values between aggregates and various clinical parameters were obtained by Spearman’s rank correlation. To consider multiple observations per patient, within- and between-subject correlations between platelet aggregates and relevant biochemical parameters were estimated. Linear and exponential growth function fits were executed using OriginPro (2021b). Missing data were not imputed. Graphs were plotted using OriginPro (2021b) and R studio (1.4.1106) and assembled using Inkscape (1.1.2).Statistical analyses were performed using IBM SPSS Statistics 25 (SPSS Inc), the statistical software R version 4.0.2 , Microsoft Excel (V16.51), and OriginPro (2021b). The Shapiro-Wilk test was applied to test for normal distribution. The significance of non-normal distributed data was analysed using the non-parametric Kruskal-Wallis-ANOVA with a subsequent Dunn’s test where significance levels are indicated by asterisks (*n = 36) and healthy references (n = 15) were measured once at each sampling. Longitudinal samples  were intended to be collected, starting as early as possible from admission to the ICU. Each measurement contains approximately 50,000 blood cells, which were recorded in less than two minutes.Patients  and blood from healthy donors as control (n = 15). Based on the WHO ordinal scale for clinical improvement for hospitalized patients with COVID-19, the patient cohort was divided into a moderate (score 4 or 5) and severe group (score 6 to 10)31. The patient characteristics are described in Supplementary Table We then employed our prototype to study cell aggregate formation in COVID-19 using peripheral blood from patients with a positive SARS-CoV-2 polymerase chain reaction test (PCR)  aggregate formation was significantly higher in both moderate and severe COVID-19 patients compared to healthy donors Fig. . Of noteTo test whether cell aggregates qualify to predict severity in COVID-19 patients, we then analysed the cell aggregates detected at the initial measurement (day 0). PP aggregates were significantly higher in severe (9.06%) and moderate COVID-19 (3.29%) patients compared to healthy donors (1.52%). In line with the maximum level, PP aggregates were higher in severe COVID-19 compared to moderate cases at day 0 Fig. . Likewisn = 10) to patients infected with variant SARS-CoV-2 (n = 26), which included the B.1.1.7 , B.1.351  and B.1.617.2  variants . Further, we found a significant within-subject correlation between PP aggregates and procalcitonin (PCT), which is an established marker for hyperinflammation and used to monitor bacterial (super)infections in clinical practice . While there is an association of CRP with severity in our cohort , we did not observe a correlation between PP aggregates and the inflammatory markers CRP and Interleukin-6.We next aimed to correlate our findings with established biochemical parameters. Elevated D-dimer levels have been associated with coagulopathy and suggested as a biomarker to predict mortality in COVID-19tes Fig. . The witice Fig. . This su25, which can lead to artificial platelet aggregation. To investigate these alterations, we obtained blood samples from nine severe COVID-19 patients and three healthy references and assessed PP aggregate percentages in each sample after 0, 60, and 120 min. We found a significant difference between both groups right after sample collection with mean values of 1.69% and 8.91% for healthy and severe, respectively. With increasing storage time, the number of aggregates and standard deviation increases in both groups impeding the differentiation of the two clusters at 120 min  12 days after ICU admission due to a rapid deterioration of the respiratory function. While the total platelet count was within the reference range and no increase of PP aggregates was observed during the first 9 days, there was a significant increase in PP aggregates with a large fraction of aggregates containing three or more platelets on day 12, right before initiation of ECMO therapy. Due to the clinical deterioration on that day, a second CT scan showed dramatic progression compared to the initial scan . It is unlikely that an overlap of the cells solely explains the occurrence, as the percentage of platelets in whole blood compared to erythrocytes is low, and the dilution minimizes spatial coincidences. PP doublets may thus appear physiologically or as a side product of the blood draw  variant in 21 and the B.1.1.7  variant in 4 of 26 patients infected with SARS-CoV-2-variants. Both variants are known to be more virulent compared to wild-type SARS-CoV-28. However, optimal cut-off levels for diagnostic or therapeutic decisions and the value of D-Dimer in disease monitoring remain a matter of debate46. In line with a prognostic value for disease severity, we observed a positive between-subject correlation for D-Dimers and PP aggregates. In contrast and consistent with previous reports, longitudinal measurements of D-Dimer and PP aggregate levels did not correlate23. The prognostic value of PCT in COVID-19 is equally conflicting48. Currently, serial assessment of PCT levels is recommended to identify secondary bacterial infections that often require antibiotic treatment49. Intriguingly, we found a significant within-subject correlation between PP aggregates and corresponding PCT levels , hinting at a possible link of PP aggregates with superinfections that might be interesting to be addressed in future projects.Our cohort size limited the correlation of PP aggregates with specific clinical outcomes, including thromboembolic events. For the same reason, the observed associations of CRP serum concentration and PP aggregates with COVID-19 severity did not translate into a correlation between the two parameters. However, we correlated two routinely assessed laboratory parameters with serial measurements of PP aggregates. D-Dimers result from degradation of fibrin in thrombi, are used as markers for coagulation and fibrinolysis, and have strongly been linked with the severity of COVID-19We finally present two exemplary cases showing a correlation between the serial assessment of PP aggregate levels and the clinical course. While PP aggregates and aggregate composition did not greatly vary in patient 1, we observed a reversed trend of total platelet count and PP aggregates in patient 2. In parallel with the clinical deterioration of the patient 12 days from admission, PP aggregates tremendously increased that day and were even higher on day 20. While the increased platelet consumption within the extracorporeal blood circuit presumably contributes to the observed drop of the total platelet count following initiation of ECMO therapy, the consumption of platelets on day 12 is likely caused by the formation of PP aggregates as the blood sample was collected before the start of the ECMO therapy. The decrease in platelets on day 12 is comparable to the values of day 2, where still no rapid worsening of the patient status occurred. This observation suggests that PP aggregates could be used to differentiate causes of platelet consumption.A few limitations to this study should be mentioned. Our analysis was performed monocentric as we had only one measurement setup available, and the POC intention limited a multicentric design, resulting in a small cohort size. Although gender and age proportions were comparable for the different cohorts (Supplementary Fig. 52. This technology to study cell aggregates with single platelet resolution in low shear conditions is a promising prognostic or diagnostic tool for various systemic diseases.In summary, we present a POC-compatible method that allows rapid, label-free phase image-based quantification of micro-aggregates. The technique was employed to characterize PP aggregates in COVID-19, and a high correlation with disease severity was reported. While future studies are required to validate and substantiate our findings, we demonstrate that this technology yields clinically relevant findings and is not limited to COVID-19. Increased PP and LP aggregates have been observed in several medical disorders, including sepsis, cancer, or cardiovascular diseasesPeer Review FileSupplementary FileDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Reporting Summary


Burn patients are susceptible to infections and are often treated with broad-spectrum antibiotics. Vancomycin is a common antibiotic administered alone, or in combination with piperacillin-tazobactam (Pip/Tazo). This combination therapy was associated with increased rates of acute kidney injury (AKI) in burn patients when compared to Vancomycin alone or in combination with other antibiotics. However, recent studies failed to demonstrate these findings. We sought to determine the degree of nephrotoxicity of Vancomycin, Pip/Tazo, and their combination, with burn severity.A retrospective, multi-institutional study was performed utilizing TriNetX Global Health Research Network that encompasses de-identified patient records from 55 health care organization across the US. We identified the population of burn patients between 2000-2020 and stratified them into 3 groups by TBSA < 10%, 10-39%, or 40%+. Patients were further stratified into cohorts by administration of broad-spectrum antibiotics within 3 months of their burn: Vancomycin, Pip/Tazo alone, in combination (Combo), or neither. Cohorts were propensity score matched for age at index, gender, race, ethnicity, and pre-existing conditions that predispose to AKI. We explored patient distribution and conducted a comparative outcome analysis to determine the risk of developing AKI in 7- and 90-day outcome windows.Vancomycin and Pip/Tazo were compared to control groups and showed increased risk of AKI in all TBSA groups and time outcomes(p< 0.0001). Before stratifying by TBSA, all burn patients administered combination antibiotics showed increased risk of AKI when compared to control, Vancomycin alone, and Pip/Tazo. In patients under < 10% TBSA, combo antibiotics increased AKI incidence in both 7- and 90-days when compared to all other groups. In the 10-39% TBSA group, combination therapy increased the risk of AKI when compared to Pip/Tazo at 7-days. Increased risk of AKI occurred in the combo group when compared to both Vancomycin and Pip/Tazo in 90-days. In the 40%+ TBSA cohort, combination antibiotics did not increase the incidence of AKI in either time period or when compared to Vancomycin and Pip/Tazo. The results of the comparative analysis are included in Table 1.Broad-spectrum antibiotics use in burn patients is associated with increased risk of AKI, this risk is higher with Vancomycin and Piperacillin/Tazobactam combination. Combo therapy increased AKI in < 10% and 10-39% but not 40%+ TBSA. These findings can be used to monitor for early manifestations of AKI when using broad-spectrum antibiotics.The decision to initiate antimicrobial therapy is crucial in burn patients as they are already at risk for AKI. TBSA can be a useful tool for guiding risk stratification.