Reactivity and Transformation ofVesicles

Vesicles as such are only models of the shell of biological membranes. In order to obtain a more meaningful biological model for the cell, one has to have biochemicals inside the membranes as well as reactions that are of biological interest. This has been achieved and here we will review some fundamentals of reactions in vesicles, focusing on fatty acid vesicles.

In particular, two aspects ofthe use offatty acid vesicles as mo del of early cells reveal how such synthetic compartments have been investigated in origins oflife studies. These are: (A) the realization of fundamental biochemical reactions inside fatty acid vesicles and (B) the self-reproduction offatty acid vesicles. These two important features of the vesicles' reactivity and transformation will be discussed at first separately. Later it will be shown how a route can be found to functionally couple them together. It is important to realize that the model systems illustrated here are inspired by the autopoietic theory and represent the first attempts to concretely understand the basic process of living cells by constructing them.

(i) Compartmentalized Reactions

The main idea is that simple and complex reactions ofbiochemi-cal significance can be carried out in the inner cavity of vesicles. A preliminary concept relating to the permeability of vesicles was introduced in the previous section. Lipid vesicles and fatty acid vesicles exhibit selective permeability toward different molecules. Large and charged molecules have low permeability and therefore do not cross the hydrophobic membrane easily. In contrast, small molecules, even if charged, can permeate the bilayer barrier at a measurable rate. Hydrophobic and amphiphilic molecules, on the other hand, accumulate in/on the membrane. This means that it is possible in some cases to feed vesicles through external addition of precursors. The classical application is the entrapment ofan enzyme inside vesicles will external addition of substrates. The substrates permeate into the vesicles, where they react with the entrapped enzyme and the reaction product may be released or not from the vesicle, depending on its chemical nature.

Following this scheme (with various modifications), several enzymes have been studied inside vesicles, such as carbonic anhydrase, a-chymotrypsin, P-galactosidase, etc. (for a recent review, see ref. 17). But how can an enzyme be inserted inside the vesicles? The

Figure 12.5. Two important reactions carried out inside fatty acid vesicles. Polynucleotide phosphorilase (PNPase) is entrapped inside oleate vesicles, then ADP is added externally; it slowly permeates into the vesicles by crossing the membrane and it is polymerized inside (to give poly(A)). As indicated in the arrow, the process is carried out in self-reproducing vesicles (A). A template RNA, Qp-replicase and all low molecular weight compounds are trapped inside oleate vesicles and the replication of RNA is followed, simultaneously to the self-reproduction of vesicles (B).

Figure 12.5. Two important reactions carried out inside fatty acid vesicles. Polynucleotide phosphorilase (PNPase) is entrapped inside oleate vesicles, then ADP is added externally; it slowly permeates into the vesicles by crossing the membrane and it is polymerized inside (to give poly(A)). As indicated in the arrow, the process is carried out in self-reproducing vesicles (A). A template RNA, Qp-replicase and all low molecular weight compounds are trapped inside oleate vesicles and the replication of RNA is followed, simultaneously to the self-reproduction of vesicles (B).

entrapment is generally carried out by preparing the vesicles in the solution where the enzyme is solubilized. Although enzymes are supposed to be passively entrapped during the formation ofthe vesicles, in some cases, e.g., with basic enzymes and negatively charged surfactants, electrostatic forces can play a large role determining the entrapment yield and the location ofthe enzyme in the vesicle. After vesicle formation, the enzyme left in the external medium can be removed by gel filtration, centrifugation, dialysis or ultrafiltration. In the case of very large vesicles (>10 ^m), the enzyme can also be directly injected into the vesicles by microinjection techniques.

One ofthe first important experiments on biochemical reactions inside vesicles, with the aim of constructing processes relating to a minimal cellular compartment, was carried out in as early as 1990 by Schmidli et al:18 the four enzymes responsible for the synthesis of lecithin were entrapped inside lecithin liposomes, with the idea that lecithin would be produced inside the liposomes, leading to the growth and possibly division of the liposomes from the inside—a typical autopoiesis experiment. The experiment was partly successful, but it could not be completed as intended when the enzymes were withdrawn from commercial availability.

In order to simulate two very important processes for the origins of early protocells, the synthesis of poly(adenilic acid)19,20 and the self-replication of RNA21 have been carried out in fatty acid vesicles (Fig. 12.5). In the first case, polynucleotide phosphory-lase (PNPase) was entrapped inside oleate vesicles (at pH 9) and adenoside diphosphate (ADP) was externally added (Fig. 12.5A). Thanks to the nonzero permeability of the nucleotide, poly(A) was isolated at the end of the reaction from inside the vesicles. Once polymerized, the product in fact cannot escape from the inner vesicle cavity. This system shows how a combination of polymerization and semi-permeability produces an "emergent" outcome, i.e., the formation ofprotocells containing a macromolecule. This is a model ofthe early formation of functional vesicles containing a functional biopolymer. In ancient times, a small catalyst might have played the role of PNPase. Notice that, in principle the vesicle membrane may also assist the polymerization reaction, or reaction inside the vesicle may lead to the formation of some molecule that later acts as a catalyst for polymerization.

In the second case (Fig. 12.5B), an RNA strand was replicated by means of the enzyme QP-replicase.21 The enzyme and the substrates for the reaction (RNA and nucleotides) were trapped simultaneously inside oleate vesicles and the replication of RNA was achieved. Enzymatic RNA replication inside vesicles is of course pertinent to the origin of functional protocells: it is a model for early compartmentalized RNA replication catalyzed either by RNA itself, as in the RNA world hypothesis, or by other catalysts such as proto-enzymes.

As it will be discussed later, the relevance of these investigations (Fig. 12.5A,B) is also related to the simultaneous self-reproduction of the vesicles, which was allowed to occur together with the compartmentalized reactions, so that a system ofself-reproducing vesicles with internalized polymerization or internalized self-replication was realized. Therefore these models, even if they do not explain how the first protocells originate, provide a solid experimental approach to the understanding of compartmentalized reactions.

It has also been possible to carry out the PCR reaction in liposomes;22 and later on ribosomes also could be entrapped in the inner core of vesicles, thus permitting for the first time the synthesis of a polypeptide inside a vesicle.23 This last experiment opened the gate to a series of subsrquent experiments aimed at expressing protein synthesis in liposomes.

It is well accepted today that proteins can be expressed inside vesicles, by entrapping simultaneously a gene and the entire biochemical machinery needed to carry out the transcription-translation processes. Green fluorescent proteins, T7 RNA polymerase and a-hemolysin have been successfully expressed inside phospholipid vesicles. Again, the interested reader should consult recent reviews for details.2,24 Some experiments developing the notion of minimal cell are described as follows.

(ii) Self-Reproduction ofVesicles

We have mentioned before the capability of vesicles to undergo "self-reproduction" processes. This is of course very important as it permits a closer modeling of the biological cells. We would like to sketch here the basic principles of such a mechanism and the reader may referr to specialized literature for further details.2

Supramolecular structures such as vesicles, micelles and reverse micelles can undergo a "self-reproduction" process. The terminology here is important, since the term "self-replication" i.e., an exact complementary replica of the template molecule, is properly applicable to small molecules as well as nucleic acids. In contrast to self-replication, self-reproduction deals with the formation of new structures via growth-division processes, which largely do not proceed with stringent molecular-level controls as in the case of nucleic acid replication. Molecules self-replicate, whereas cells self-reproduce. As a typical example, a vesicle can grow and divide into two daughter vesicles having different sizes, so that the initial structure (the vesicle) reproduces to form two new vesicles that are similar but not identical.

Pioneering investigations on self-reproduction of supramolecular structures were carried out by Luisi and coworkers in the 1990s, giving rise to the discoveries—in chronological order—of reverse micelles, micelles and vesicle self-reproduction.25

Vesicle self-reproduction was achieved in 1994, using fatty acid vesicles and a fatty acid precursor (Fig. 12.6). The experimental strategy is clearly inspired by the autopoietic approach, namely the achievement of a dynamical system that works in the following manner: a bounded particle (the vesicle) takes up the "nutrients" needed for its growth, transforms them—within its boundary—

Figure 12.6. Self-reproduction of oleate vesicles. Oleic anhydride, a water-insoluble precursor, is layered above an alkaline solution containing preformed oleate vesicles. Oleate vesicles take up anhydride molecules by dissolving them in the hydrophobic bilayer and increasing the surface where hydrolysis can take place. The products of the reaction, two oleate molecules, increase the vesicle surface and bring about vesicle growth (non necessarily spherical), with a consequent destabilization of the structure that collapses into two or more small vesicles. The overall process leads to an autocatalytic number growth of the vesicles. If preformed oleate vesicles are omitted, they form spontaneously after the initial stage of anhydride hydrolysis and then catalyze the whole process after a lag phase.

Blank Worksheet Mitosis

Figure 12.6. Self-reproduction of oleate vesicles. Oleic anhydride, a water-insoluble precursor, is layered above an alkaline solution containing preformed oleate vesicles. Oleate vesicles take up anhydride molecules by dissolving them in the hydrophobic bilayer and increasing the surface where hydrolysis can take place. The products of the reaction, two oleate molecules, increase the vesicle surface and bring about vesicle growth (non necessarily spherical), with a consequent destabilization of the structure that collapses into two or more small vesicles. The overall process leads to an autocatalytic number growth of the vesicles. If preformed oleate vesicles are omitted, they form spontaneously after the initial stage of anhydride hydrolysis and then catalyze the whole process after a lag phase.

into particle components (membrane-forming molecules) and consequently grows, reaches an unstable state and then divides into two or more new particles. The transformation of precursors into elements of the autopoietic particle must occur within the particle itself, for this is the key interior that distinguishes the autopoietic self-reproduction of vesicles from a spontaneous formation of vesicles.

Experimentally, this process can be realized by stratifying oleic anhydride (the precursor) on a basic solution where oleate vesicles are present. Oleic anhydride is slowly hydrolyzed at the interface with the basic solution. Simultaneously to this spontaneous process, other oleic anhydride molecules are taken up by preformed oleate vesicles, since they can be solubilized within the bilayer membrane. Once in the membrane, oleic anhydride can be hydro-lyzed and therefore transformed into two oleate molecules, with a corresponding increase of membrane surface. The oleate vesicle is therefore growing by incorporating membrane precursors, which are transformed into building-blocks of the membrane itself, with consequent growth and the eventual production of new vesicles (with a mechanism which is still poorly understood).

The stoichiometry ofa growth-division process can be schematically summarized as follows:

where V represent a vesicle and S the membrane-forming compound (for the sake of simplicity, S can represent the precursor as well). Accordingly, the process is autocatalytic, since more vesicles are produced, more precursor can be taken up, more vesicles will be pro -duced and so on. Experimentally, autocatalytic processes are characterized by sigmoidal profiles and this was actually demonstrated in the self-reproduction of oleate vesicles (Fig. 12.7). Notice also that if preformed vesicles are not included in the experimental setup, they can be formed after the initial stage of spontaneous hydrolysis of the anhydride, with the result that the overall process becomes a spontaneous pathway from surfactant precursors to vesicles without the need of preformed structures. The driving force for entry into the self-reproduction cycle comes from the thermodynamics of anhydride hydrolysis and the self-assembly of fatty acids.

The second example of oleate vesicle self-reproduction exploits the pH-dependent assembly properties of fatty acids. In fact, since at high pH oleate molecules self-assemble as micelles, whereas at intermediate pH (8.5) they form bilayers and therefore vesicles, the addition of oleate micelles into a buffered solution of preformed oleate vesicle also represents a way of feeding vesicles with the aim of observing their growth and division, i.e., self-reproduction. Actually this strategy is currently adopted by many researchers, for it does not have the drawback of the oleic anhydride approach in involving a biphasic system.

Oleate micelles and oleate vesicles are both macroscopically single-phase systems and therefore the process of vesicle self-reproduction can be easily followed by spectroscopic techniques. When oleate micelles (in equilibrium with the monomeric form) are added to a pH 8.5 buffered solution they can form vesicles spontaneously by rearranging their packing modes. However, when preformed oleate vesicles are present in such solution, an additional pathway might be the uptake of oleate (in the form of monomers or micelles) from preformed vesicles, exactly as in the previous case of oleate vesicles and oleic anhydride. In this second pathway, the preformed vesicles grow and divide, starting up self-reproduction dynamics. The two competitive processes of de novo vesicle formation vs uptake-growth-division can be distinguished by labeling the preformed vesicles with a water-soluble marker such as ferritin molecules (Fig. 12.8). By measuring the distribution of ferritin molecules inside the vesicles before and after the addition of oleate micelles, it has been shown that the vesicles can actually grow and a small but significant guantity of ferritin-containing vesicles are

Figure 12.7. Kinetic profiles of the system illustrated in Figure 12.6. When preformed oleate vesicles are present in the aqueous phase, oleic anhydride is quickly hydrolyzed (curve A). When preformed oleate vesicles are not present at the start, they form after the initial alkaline hydrolysis of oleic anhydride, soon after the critical aggregation concentration is reached. The S-shaped profile (curve B) clearly indicates an autocatalytic mechanism, i.e., vesicles that catalyze the formation of other vesicles.

Time, h

Figure 12.7. Kinetic profiles of the system illustrated in Figure 12.6. When preformed oleate vesicles are present in the aqueous phase, oleic anhydride is quickly hydrolyzed (curve A). When preformed oleate vesicles are not present at the start, they form after the initial alkaline hydrolysis of oleic anhydride, soon after the critical aggregation concentration is reached. The S-shaped profile (curve B) clearly indicates an autocatalytic mechanism, i.e., vesicles that catalyze the formation of other vesicles.

formed by the division of the grown vesicles, as indicated by the reduced number of ferritin molecules per vesicle caused by the distribution of ferritin molecules into "daughter" vesicles.

Modern studies on fatty acid self-reproduction have revealed a series of interesting features that cannot be discussed in detail here, such as the existence ofa "matrix" effect, a sort of"templating" effect exerted by preformed vesicles, resulting in the formation of a vesicle population with conserved sizes ;26 the competition between vesicles of different sizes for uptake of oleate micelles;27 or the competition between osmotically "stressed" and "relaxed" oleate vesicles.28

An original contribution combining vesicle self-reproduction and inheritable "compositional" information made by Doron Lancet is shown in Box 12.1.

(iii) Strategies for Functional Coupling of Core and Shell Transformations

We have seen that: (i) simple and complex biochemical reactions (including self-replication) can be carried out with vesicles; (ii) vesicles can self-reproduce spontaneously upon the external addition ofa suitable surfactant precursor. Starting from these considerations, which have come from experiments, it might be asked ifit is possible to further couple these two processes to create a simple model of an autopoietic self-reproducing cell.

From the theoretical viewpoint, the solution can easily be envisioned: a metabolic network that reproduces itself should be encapsulated within a vesicle and some of the processes in the network should be capable ofproducing the membrane-forming component from suitable precursors. The system can be simple in construction at the beginning and become more sophisticated at a later stage of development (Fig. 12.9). The vesicle would grow either in terms of internalized molecules based on cyclic networks that produce more and more molecules of the members of the cycles as the reactions proceed, or in terms of membrane surface. As in the case of vesicle self-reproduction, collapse from an unstable state may be expected

Figure 12.8. The use of ferritin as water-soluble probe to investigate the formation of oleate vesicles. In the first case (A), oleate micelles form oleate vesicles independently from the preformed vesicles (the experiment was performed using phosphatidylcholine vesicles as preformed vesicles). The mechanism involves the de novo formation of oleate vesicles, with no interaction between oleate micelles—or oleate monomers— and preformed vesicles; consequently, the label is not diluted. In the second case (B) oleate micelles—or oleate monomers—are taken up by preformed phosphatidylcholine vesicles that grow as new membrane-forming molecules are incorporated into the membrane. Following this initial step, the vesicle becomes unstable (the growth might also be nonspherical) and divide into two or more "daughter" vesicles. Accordingly, the probe may or may not be statistically distributed among the new vesicles. The reduction of the average number of ferritin molecules per vesicle suggests the existence of a growth-division pathway (Vesicles, micelles, fatty acids and ferritin molecules are not drawn to scale).

Figure 12.8. The use of ferritin as water-soluble probe to investigate the formation of oleate vesicles. In the first case (A), oleate micelles form oleate vesicles independently from the preformed vesicles (the experiment was performed using phosphatidylcholine vesicles as preformed vesicles). The mechanism involves the de novo formation of oleate vesicles, with no interaction between oleate micelles—or oleate monomers— and preformed vesicles; consequently, the label is not diluted. In the second case (B) oleate micelles—or oleate monomers—are taken up by preformed phosphatidylcholine vesicles that grow as new membrane-forming molecules are incorporated into the membrane. Following this initial step, the vesicle becomes unstable (the growth might also be nonspherical) and divide into two or more "daughter" vesicles. Accordingly, the probe may or may not be statistically distributed among the new vesicles. The reduction of the average number of ferritin molecules per vesicle suggests the existence of a growth-division pathway (Vesicles, micelles, fatty acids and ferritin molecules are not drawn to scale).

Box 12.1. Lipid "Composomes" and Their Role as Prebiotic Replicators

The role that supramolecular aggregates, as lipid and fatty acid vesicles, can have in the origin of life is not only related to their property of providing a compartment for the development of biochemical reactions. The Graded Autocatalysis Replication Domain (GARD), developed by Doron Lancet and coworkers at the Weizmann Institute, Rehovot, Israel, provides a possible—and unorthodox—scenario for a gene-free propagation ofinformation.29,30 In fact, it is proposed that in the very early stages ofprebiotic evolution, supramolecular assemblies such as vesicles, composed of several kinds of molecular components, could homeostatically grow and split, generating new assemblies that have the same composition as the parent one. The homeostatic growth is favored by selective adsorption and transformation of compounds from the environment. In this way, a particular composition and organization of an assembly represents "information" transmitted through generations of assemblies. The existence of such "composomes" has been demonstrated by detailed computer simulations, that have also shown also the possibility of transition from one composome form to another in a simple evolutionary progression.

Metabolic Network Diagram

Figure Box 12.1.1. The "composomes", specific compositional states capable of homeostatic growth and information-preserving fission.

Figure 12.9. Diagram of a self-reproducing autopoietic protocell. A metabolic network is confined in the inner volume of a vesicle. The internal metabolic network "E" (consisting of one or more enzymes) catalyses the formation of the membrane-forming compound S from the precursor A, which is initially present in the environment and permeates through the membrane. S can decay to Y. The whole metabolic network, here indicated as "E", is also reproduced within the vesicle, fed by the set of precursors "B" and producing the waste molecules "W". This would represent a core-and-shell self-production. Notice the production of the internal components and that of the shell component S are functionally linked, because a product of the internal network catalyses the formation of S. Based on the balance between constructive (anabolic) and destructive (catabolic) processes, the protocell can undergo growth, collapse or—when the processes are balanced—enter into a homeostatic regime. When the protocell constructs its components, it grows and divides and the internal components are statistically distributed among the "daughter" cells. Since no control is exerted on vesicle division, some of the new vesicles may be deprived of key components and therefore enter into "death".

to give rise to two or more "daughter" vesicles, each containing part ofthe internalized components. If the number ofinternalized components is high, the probability ofhaving all the components within each daughter vesicle is also high and the number of"active" vesicles actually increases. Vice versa, since the process is uncontrolled, the division of internalized molecules may proceed unevenly leading to some daughter vesicles not being able to sustain the metabolic network owing to the lack of some of the components.

The system described in Figure 12.9 is autopoietic and according to early considerations by Maturana and Varela,7 it might even be called alive; however recent studies have led to a reassessment of this view.8,31 Going back to experimental approaches, one may ask how the system illustrated in Figure 12.9 can be implemented in the laboratory.

The first attempt in this direction was realized by Zepik et al,32 who model the system represented in Figure 12.9 by simplifying the network of internal reactions, so that only two reactions are operative : the synthesis and the degradation ofS, the membrane-forming molecule. Both reactions, chemical in nature (hydrolysis of anhydride as the S-forming reaction and oxidation ofS as the S-depleting reaction), can be regulated in rate so that one can observe the three different states shown on the right hand side of Figure 12.9. In particular, it has been shown that a homeostatic regime can actually be achieved through a the fine regulation of the anabolic and catabolic processes.

From the origin of life point of view, it would be very important to demonstrate that simple chemical systems can evolve, self-assemble and self-organize in the way depicted in Figure 12.9. At the current level of knowledge, however, this objective seems very difficult to achieve, especially if some degree of complexity and/or sophistication is to be built into the system. Moreover, if one wishes to reproduce the pathway leading from simple molecules to the first functional macromolecules (protoenzymes or ribozymes), to the first "metabolic" cycles, to self-replication or self-reproduction, one is faced with a lack of basic knowledge. It can be said—notwithstanding the great efforts devoted to experimental and theoretical research—that almost all the key transitions have simply not be defined.

A possible scenario involves the spontaneous generation of functional macromolecules (one of the greatest challenges in contemporary studies on origins of life), in/on prebiotically plausible self-assembling vesicles, such as the fatty acid vesicles and to give rise to the initiation of confined metabolism. From this stage onward, the self-maintenance and self-replication ofthe internalized reaction network would have to be attained, followed by the origin of the genetic code and still later by coded self-replication and core-and-shell self-reproduction (Fig. 12.10). In this protocell "evolutionary" pathway, interactions with the environment are fundamental, since the internalized metabolism has to be somehow coupled to the composition of the external medium. Vesicle self-reproduction, mediated by externally supplied or internally synthesized surfactants, would have to fulfill the role ofmultiplying protocells, at first in a stochastic way, generating diversity and competition between protocells and later on under the control of internal metabolism. Other phenomena, such as fusion, growth-division, competition, selective processes and the entire rich reactive landscape ofvesicles could advantageously enable supramolecular systems to escape the world ofinanimate matter. This hypothetical scenario, which yet remains largely speculative, focuses on the concept of individuality and population derived from the compartmentation ofreaction networks, on their possible evolution and on their stepwise increases in complexity.

In order to understand some of the fundamental transitions depicted in Figure 12.10, several experimental and theoretical investigations have been carried out in recent years. In particular, it would be very important to address the numerous questions and aspects concerning the properties and biological functions of very early cells, those that do not possess a full-fledged arsenal of genes, enzymes, fine metabolic regulations, feedback loops and controlled functions. This "historical" aspect ofprimeval cells perfectly complements the theoretical issue of the minimal number of components and functions that would make a system "alive". Can we construct such a system?

Within the framework of feasible, concrete and robust— although difficult—experiments, we can "limit" ourselves to the investigation of semi-synthetic constructs that employ extant enzymes/genes and synthetic compartments to build "minimal cells", i.e., cell-like structures that display living properties based on the scheme in Figure 12.9. Of course, the choice of using evolved enzymes and genes stems from the absence of known primitive (and less specific) "enzymes" that would be perfectly fitting for the design and construction of protocells.

The achievement of such a goal would represent, even more than a historical reconstruction of the early living cells, a proof-of-principle concerning the definition and emergence of life as the coordinated, collective property of a network of compartmentalized interacting components/reactions.

Many recent advances (for a review, see ref. 24) indicate that vesicles with internalized reactions that are as complex as protein expression, occurring in single steps or in cascades and observable for periods of a few hours to several days, have become accessible to the experimentalist.

In essence, the key event remains the coupling between the internal metabolism (intended here as the formation and replication of functional macromolecules) and the reproduction of the whole vesicle. The coupling must be functional, i.e., the two processes must be coupled chemically by a reaction or a network of reactions, or by some molecule that is common to the two pathways. Previously, it has been demonstrated that internalized reactions and vesicle self-reproduction can occur simultaneously, as in the case ofQP-replicase RNA replication and vesicle self-reproduction.

However, the two processes in this instance are just overlaid one on the other, with no functional coupling established between the core- and the shell-reproductions.

A possible approach illustrated in Figure 12.11 consists of a vesicle that synthesizes its membrane components thanks to the presence of a catalyst—an enzyme or a set of enzymes—that is itself produced by an internalized reaction network. In principle this is experimentally feasible by combining in vitro functional protein expression and the above-mentioned processes of vesicle growth-division. Ultimately, all the components required for the production of the enzyme(s) need to be replicated and evenly shared between the daughter vesicles. This construct is designated a semi-synthetic minimal cell.2,24

Such minimal cells, far from being perfect, can enrich our understanding of the biophysics and biology of early cells, their requirements in term of substrate permeability, dynamics of internalized reactions, disposal ofwaste materials, energy requirements, the quest of simultaneous core-and-shell reproduction and interaction with the environment and possibly other cellular individuals. The final goal of realizing the construct depicted in Figures 12.9 and 12.11 has not yet been achieved. We believe, however, insight into several important issues on the origin of cellular life can be gained along the path to this goal, because such "limping" cells, missing in many evolved functions, might represent useful models for the early cells that are open to experimental testing and verification.

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