 Good morning everyone from all over the world. I have this honor just to start this training. I just choose this title, the word of sugars. But you know, I wanted to ask also a subtitle, carbohydrate for dummies, because I don't know exactly, you know, what is your level of knowledge. So I'm sure that our students are very also educated people in, in psycho science. So please, please take, take it, you know, with a grain of salt because, you know, it's going to be a general presentation of what we, what we do, what we plan to do, and the things we are facing in the world of psycho science. So you see the first slide, it's quite, it's quite an amazing slide, because it shows some of the monosaccharide with different orientation, configuration, and the big there is also, you know, sucrose, because whenever we talk, okay, can I have now, I have to, to, to, to, I have to change my presentation. No, no, I have to push. I'm sorry. Now I have. You can choose. No, no, no, I have to, to, to, to make sure that I can change my, okay, okay, good. Very good. So I mean, yeah, I mean, when we, when we talk about sugars, you know, everyone thinks about sucrose. And of course, sucrose is a very important component of our field. It's a fantastic disaccharide is now produced, you know, as a molecularly pure product with about 145 millions tons per year. A little bit of history, sucrose was discovered in New Guinea. And then they went to India. And so in India, this is where the name of the Sakura was, was given to this very nice product. And Sakura mean grain. And from Sakura, we have Sakura's, we have all the things which we know now about sucrose. And then, you know, by several, several travels, he just reached Bersha, then went to, from, from India, then went to Spain, and from Spain, of course, you know, that was a time of discovery. And so people realize that planting sugar cane was most efficient into this part of the world. And of course, this is a tragedy because this is the beginning also of this extreme slavery. And that, you know, took place over several hundred years. And now the good point is that, you know, we are becoming slave, our safe slave of sucrose. And we think that a significant part of the obesity that our world is facing may be related to consumption of sucrose. Okay, I don't want to go much into that. So what do you mean by sugars? We, we are referring to a very vast family of molecules that are homogeneous and very diversified. They are all made of carbon, hydrogen and oxygen. So this is what we refer them as carbohydrates in English. It had the carbon in French. In German, saccharide and so forth. And we have several families, one family. This is the oligosaccharide. This one is polysaccharide. And then with respect to a lot of biological implication, we have the so-called lack of conjugate, lack of peptide, lack of protein, lack of lipids. And that will go more into the details of that. So whenever we have always a difficulty in describing our science, because we can talk about sugars, we can talk about carbohydrates, we can talk about oligo or polysaccharides, and we can talk about black hands. So we will be, I will be using these different words, you know, over my presentation. In terms of molecular diversity, of course, we have the carol molecules, or we have L and B shape of sugars. And then they are formed in the form of carbon chain, going from three to ten carbons at home. And all the carbon atoms are functionalized. So also they're characterized by the coexistence of a carbonate group. And we have also agrocyl groups which are forming aldoses and ketoses. And they display very interesting character, which is electrophile and nucleophile. So that's about all the chemistry that I will be showing. As you see just that we set the scene. Quite interestingly, we don't have only sugars on Earth, but also sugars have been discovered in space, not too long ago. And so there has been a report of this glycoaldehyde molecule, which I've been identifying in not too far away from Earth, only 400 light years. And this is some sort of moiety that we find in ribose, and that we find also, and going also away from ribose to RNA and DNA, and also some part which we find in our monosaccharides. So that's interesting. And we have also other evidence of the existence of sugars in space. Now, in terms of depiction, most of the time whenever the students are taught about carbohydrate, this is what they are taught about. This is a famous Fisher discovery, which of course made a good sense whenever I mean it was a fantastic discovery, but it is wrong in a sense that this linear type of representation of monosaccharide are wrong in terms of the existence. So there have been been following up different type of a presentation. So this is one type of presentation whenever we saw the relative orientation of the aggressive group. This has been also translated into this representation, where we have also a three dimensional vision. And more recently, because of the complexity of these sugars and because also their general occurrence, different kinds of colors have been representing. So this is some sort of historical presentation at the evolution of the depiction of monosaccharide. Quite interestingly, the fact that Amy Fisher gave the configuration was almost he had a choice between L and D, and it took the good choice, because the absolute configuration of sugars of glucose was only established in 1951 by this great crystallographer by food from the Netherlands. So it took about 60 years or so before this configuration was established. Actually, a great progress has been made whenever many scientists involving glycoscience realized that it was required to define to have convention to describe our with a symbol nomenclature to define the sugars. So this is a great paper because there are like like 40 co-authors on this. And this is actually what we call SNFG, which give colors to these different, these eight different monosaccharides and then give different shape to the exos, to the anacetyl hexamine and so forth. This is how we are representing our sugars. And I strongly suggest to anyone starting the field to use this symbol nomenclature to have the rest of the world understanding what they want to describe. A small things now, where do carbohydrates stand in the scheme of the molecular paradigm of the central dogma of life science. They are supposed to always describe a secondary metabolites. So secondary doesn't mean they are just the less important. So actually there's a product, a very complex enzyme. And the enzyme action, wherever enzyme are required nucleotide sugars are required and to give birth to this complex oligosaccharide. This is here a polysaccharide and to give also a birth to this complex glycolic which are also as important. So this is this oligosaccharide, this carbohydrate, another direct product of a gene, and this is quite of course a complicated complication. So in terms of complexity of glycans, so this is just a slide which I borrow from our colleagues here. That shows that we are dealing here with carbohydrates, for example in chloroplasts, that here we have the chromosome, here we have other types of glycolipids here. And these are some sort of information content that is found in genome. Then the transcription leading to transcriptome, there goes there with much information. Then we go to proteome, with a translation to protein, which is also an important set of information. And what we call today the glycom, which shows that an extended amount of information. And this slide here, this part of the slide here shows the differences between the way bases, between the way amino acid are linked together in a very linear fashion. In contrast to what the carbohydrates are, there's a lot of possibility of linking this monosaccharide together. And for the first time for biological molecules, these particular molecules show some sort of possibility of branching, which of course complicated a lot the analysis and then the construction and the description. So this is a complexity of glycan, which we are facing. So we don't say they are difficult molecules that are challenging. Now, since we are dealing with a different community, we have to find a way to describe this molecule. This is an example where this molecule, which is called Lewis X and solid acid on core two. I mean, some people can understand what it means, which can be translated also in the high you pack in a condense I you pack type of presentation, which is correct, of course. And also that everybody speaks I you pack fluently. And if you are a chemist, you better have this type of presentation here at this molecule, which can make good sense and you see the branching over the branching here. So if you are a biologist, you prefer to have this presentation here, wherever following the SNMG presentation, we have these galactose residue, we have these sucrose residue, we have cellic acid residue and so forth, which is fine. But actually, of course, if you are in biology, you will like to know where this particular motif occurs, you know, to which protein is it linked. And if you are doing a cell membrane, you would like to understand where this molecule, maybe part of that quality is located into this membrane. However, none of these representation are available for bioinformatics. So the way to describe bioinformatics are several ways. This is one of the so called like a city, which is a way to describe the different components and the different way this monosaccharides are linked together. This is linked. So there are other types of representation available for bioinformatics. But this one is still human readable. Another one, you know, like the one that Keoko has been developing are less human readable. But this is of course a big issue in the field of bioinformatics, how to encode the complex information about this issue. As I said, we are dealing with a very challenging molecule. So this is what I'm calling the isomer barrier. They have a number of monomers and of course substitution also can occur to these monomers. They have different way of connecting these different monomers. I show here the way of having the so called beta and the so called alpha type of configuration. And they can be linked to this position. So it should be two, three, four, six and so forth. And beside that also have branching points. So these are for the difficulty in terms of isolating and characterizing this molecule that of course a synthesis. In terms of crystallization that only very difficult to crystallize the only sugar which is easy to crystallize is sucrose. So it was complicated to get three dimensional structure of carbohydrate. In terms of chemical synthesis that also quite challenging. And as I said, you know, they are not a direct product of a gene so they are different from proteins and they cannot be amplified by PCR so different from nucleic acid. So these are the different points which we have to face in order to move forward in our elucidation understanding comprehension and to see how this molecule participate in a very important biological effect. So to summarize, so this is my slide whenever you say okay whenever you deal with nucleic acid or with protein you have here a linear sequences we can translate these sequences. And this, for example, can be read saying okay I am a moglobin. Whenever we deal with the freedom with structure of carbohydrate, we have to not only be able to construct this molecule and to understand how they interact with the rest of the world. And this could be for example the signal for this molecule saying I am a cancer cell. As I say many people speak English, not as people for the time being speak Chinese. And again, you know, this is the difficulty of deciphering the meaning of this structure. And as I said before in bioinformatics, the difficulties to encode the structure is quite simple. So a few examples that we want to go too much into that we characterize oligosaccharide and polysaccharide. They are structurally complex molecule and they have a lot of functional diversity. So we found this oligosaccharide and polysaccharide all over in your life. The oligosaccharide by definition, they are composed of four to ten monosaccharide. And sometimes there is a result or the transformation of the degradation of polysaccharide. You know some of them, dextrin, fructose oligosaccharide and galacto oligosaccharide, they are quite used, quite studied in human nutrition. We of course know the cyclo dextrin. And more recently there have been, of course, this discovery of human milk oligosaccharide. And now we have the possibility of going through synthesis and also biosynthesis of this human milk oligosaccharide, which can be synthesized and produced in very high in pure quality and added to formulas. So this is an added value also at this oligosaccharide. For polysaccharides, we have even increased diversity. They are used as energy storage. I will go into that. They are used also as structural support. So this is corn, structural support. These can be cellulose, but they can be chitin here. There is plasticity as well. Plasticity at the component of the cell wall polysaccharide. These are pectin, which are also important for industrial application. And these algis, algis are full of polysaccharide. And then we have this polysaccharide, which are involved in blood cloning and also the many important polysaccharide, which forms the extracellular matrix. So as you can see here, there is a diversity of role and function, which is fantastic. And we still have to be fully discovered. So this is my slide and that talk about starch. If you look at all these fields, I mean, human today, we have been living on these resources for almost 10,000 years. So they are different. We have rice, we have potatoes, we have corn, we have wheat. But if you extract the component of these, you will find here the starch granules. You see, they're all different, different in size, different in morphology. But if we look at this granule under polarized light, they all show a different, this common aspect, which is a multi force that indicates there is a crystalline order into each of these granules. And the name of the game was to understand how these things are put together. So that's again, you know, an illustration of having the granules here and how this granule can produce a very nice use and the microscope. Now I will show you a movie, which is here, which has and okay, which has sound. This is a magnification factor. We go from one. So there's no sound here. Is there a sound? Sorry. Is there a sound? Did you say there is sound? Yes. You don't have a sound? No. Oh, sorry. It's very, very low, in fact. Okay. I'm sorry for the sound. Is that better? No, we can't hear you. Oh, you can hear. Okay. Sorry. Okay. So that was my, my Google search, you know, whether I have to stop these things. I cannot stop it. Maybe you can stop sharing your screen and then go back. Maybe I can stop sharing the screen. How do I do so? And the top of the, of your screen. Yeah, yeah, but okay. Okay. So that's it. Thank you. We are back now. Yeah. So again, you know, so going to start, as I said, only glucose. And this is, this is made, you know, a very simple way in a sense, glucose molecule, which are linked alpha one four. So this is the amylose component. But the most important part is the amylopectin, which shows a lot of energy. And the, the, the challenge was to identify how these things are put together because they are the natural reserve of energy of all plants. And it took us several years to go from the understanding that starting from a glucose residue, they are forming double helical structure, which has parallel stranded. That is double helical structure that is double helical structure are put together in the form of a crystalline plate late, which can contain maybe 500 or so at the double helical structure. This plate late are organized into what we call block lead. And the organization of the block lead follows a very nice principle, which is a so-called phyto tactic principle that govern the most energy of the plant. And these block leads are put together into layers here, which are responsible by the organization to the things which we see under polarized light. So now we have a consistent description of a five or six orders of magnitude of the different ways starch granule is made of. And this is still maintaining the identity of matter because this is a reserve of energy. And by analogy, now I say, this is what I described to you going from the glucose to starch. But I like to draw this analogy because with also the one here, which is of course the one to maintain information. Each of the key things in nature, energy for plants, genetic information, they start from double helical structure made on a polysaccharide backbone. So having the same repeat unit, about 202 nanometers. Here we have anti-parallel things. Here we have a parallel organization, but they still follow the same sort of organization, architecture, overall five orders of magnitude. So to me, this is a very stimulating observation or thinking that we can draw from all this talk. Now we change gear and we go back bacterial polysaccharide. So we heard about the gram-positive bacteria, gram-negative bacteria. They contain a significant amount, a very important amount of carbohydrate. We have the peptidoglycan. The ways they are built together is different between the things. And we have here, for example, a very important lipo-polisaccharide which is found in the gram-negative bacteria and the toxin. And here we have the components of the peptidoglycan. They all serve as protective layers of all these bacteria and understanding how they are put together is still not completely understood. And this would be also a nice step forward to understand that just to have also an intelligent way to fight this bacteria. But these bacteria also produce polysaccharide. So for example, this polysaccharide, they are playing a vital role in the maintenance and functioning of the biofilm. And they contribute also to the pathogenicity and the anti-fungal resistance of the bacteria. They are produced by a lot of bacteria here. I just mentioned lactic acid bacteria, which are so useful for functional properties and industrial application. And these polysaccharide are used, you know most of them because they are used as thickness stabilizers and so forth. And there are, for example, cellulose is also produced by bacteria, chitosan also, hyaluronic acid is also produced by bacteria. But also we can find a new bacteria in extreme marine habitats. And we have also now this isopolysaccharide, which are more and more identified in morphology. So there is all world of new structures to be discovered. The things of proteoglycan, I mentioned that before in term of extracellular matrix, they are forming that we have a cork protein, which one or more covalently attach a glycosaminoglycan. And they occur in the connective tissue of extracellular matrix. The components are known to most of us. Converting sulfate, keratin sulfate. Most of the people know hyaluronic acid because hyaluronic acid is used also in cosmetic. So this is to make you look nice. And eparin as well. Eparin, of course, is a tremendously important product because it's injectable, blue thinner and catalyzed the anti-coagulant activity of anti-crombin. And the bioactive moiety of eparin, which is responsible for this particular expression of functionality, it sees pentasaccharide, which is some sort of an accident in this long chain. And this pentasaccharide now, it took several years, of course, several decades of research to identify the pentasaccharide and to grow for chemistry, chemical synthesis of this pentasaccharide. There was a tremendous, tremendous achievement by our community to make these drugs which is now fully available. But eparin is also involved in a significant amount of interaction with the so-called eparin binding protein, with enzymes, with lipoprotein, that also has a growth factor and so forth and so forth. So we have a list here of all the interactions that eparin is taking place. So a unique and very important component. Now let me see from dream to reality. So this is actually what the people from cosmetic would like you to look at. Most of the time, this is a way that people in biology would like to consider a cell. Quite nice. Actually, this is a dream. The reality is more like this one. Wherever, you know, we have this program here, but here we have also this lack of calyx. This lack of calyx occurs at all the periphery of the cell. It's very essential. But for the time being, there is a lot of an understanding to go, to characterize this different polysaccharide and other tremendous role in terms of the protection of the bacteria, but also in terms of the interaction of the bacteria. So this is all a field of black oak conjugate, where even we realize that complex and there is recognition. So again, this is a growing whereby some glycoprotein, some glycolipids are represented. As you can see, they are just at the periphery of the cell and of course play a tremendous role in the cellular, the social life as a cell, so to speak. And this is an example again, a famous electron microscopy representation of glycocalix. As we got to the cytosol here, that display that shows the role of these conjugates. So these glycoconjugates, they're from a very large family of glycol molecules. We have glycolipids. We have the where we have the lipids like ceramide and so forth, which are linked to glycol. There are the lipopolysaccharide, where these polypsaccharide are anchored to the membrane lipid. As the whole families of glycoproteins, and this also includes the protoglycan. The peptidoglycan are there also, covalently due to peptide. And there were also the glycopeptide, which result from the degradation of oligopeptide linked to oligosaccharide. So this is a very, very important family. And this is like a conjugate. There are key mediators in the cellular social life that we are dealing with. So this is true for bacteria. And again, I show the ground positive or the ground negative bacteria. This is true in human and animals. That's true also in plants, where this interaction are playing a role in the growth of the plant and the protection of the plants. But this is also true in viruses and in fungi. So all these glycan are playing a critical function in the areas of cell signaling, molecular recognition, immunity, and inflammation. And this is also one of our duty to understand that. Key things which occur for these glycocontrogate are key words which are the multi-valency, affinity, and ability. Actually, most of the glycan binding protein, which are important here, all the recognition event I just suggested. These glycan binding proteins are multiple binding side, or they only go, only go, may rise to achieve multi-valency. This is, for example, here, where we have the cell which has a different way to interact with the insectine, or we have also a long chain here. So multi-valency is a way to facilitate cell-cell interaction directly or indirectly. And when we talk about multi-valency, then we have to address two issues. Issues of affinity. The affinity is a well-known, of course, concept in drug design, or so forth. However, you have this here, the growing of this monoclonal antibody, and we know what it means by affinity. And also, we have always to consider the ability, how much of these things are interacting. Because most of the times, the interaction between the glycan and the protein is not very high. But actually, the sum of this interaction, and we call that the velcro type of interaction, makes the recognition process to occur. And this is quite unique to our field, and sometimes people developing drugs see the most of taking, talking about the affinity about the ability, and it's difficult to conceptualize. I mean, the concept is there, but to translate the concept of ability into drug discovery. So recapitulation of all these molecular interactions that involve carbohydrate, viruses, cancer cells, hormones, enzymes, antibodies, glyco-conjugate, lectins, toxins, and bacteria. So I name most of them, and this is, of course, a very popular slide in our field. Whenever we talk about some of them, we have also to consider the biosynthesis. And the biosynthesis of this cell, of this oligosaccharide, that's the glycan, I mean, it's a very complex process. We know the final details of this complex until recently, and this is this complex that takes place in the endoplasmic reticulums that goes through a series of linkage of synthesis, degradation, control of quality, and so forth, to go to the Golgi and to be transferred as a block to the external part. And so this is the case of this end-linked and O-linked glycan, where we have very famous and very well-studied nowadays with the asparagine link here and the important role of fucose. And then the different type of glycan, which are identified as high hybrid complex. And we know also that these are the end glycan, but there are also the O-glycan, which are linked on serine and pheonine, which are smaller. And they're all composed from a series of limited amount of monosaccharide, which are shown here. And this end glycoprotein, they are just here, and they, like for example, these are the so-called decoration, which are more than decoration on a given protein, and you see they're important. Sometimes such a glycan here can cover up to 400 square, Armstrong square, which means they are playing a role not only in terms of recognition, but also in terms of physical chemistry. They are just assuring some stability. They are also solubility. They can go for epitope masking and also they can be involved, or not involved in interaction or recognition. So first of all, I try to identify the nature of this glycan, their location on a given protein, and understand their role in the recognition and protection. It's also a key point, key area of research in the glycoscience. So I'll give you some example about sugars and infection. I mean, this is like a timely, because we are facing a very difficult time. And we should be just through virus and glycan. This slide here shows a summary of the different flu virus episode, which we have. We usually have the seasonal flu, which is essentially affecting humans. But also we have the so-called H1N1, which is referring to the Spanish flu in 1919 that killed over 40 million people in Europe. And then this has been changing. So H means for emagglutinin and for neuraminidase. This one is responsible for recognition, for interaction. And this neuraminidase is responsible for cleaving. And they are acting on a very simple, similar, a sim-like-that type of glycan. And the only difference between the so-called levy and glycan and the human glycan is the nature of the branching here, of the salic acid on the core here, galactose in the back. In one case, we call it, it's referred as being too free linked. And the other one is too things linked. So if you just have a look here on the piece of paper, you don't see too much difference. But actually the three-dimensional differences are quite important, which of course is a relief because we don't like to have too much of a mutation for this viral to interact with this one. So the way that human and alien receptor interact with emagglutinin, and of course this is what essentially happens in China, where there is a closeness between human being, between pigs, and between also hands here, and which are forming reservoirs for propagation of this avian. And again, I show the differences between the two sixes and the two three, which you can see better because if you look at the primary representation, that's salic acid, alpha-1-6, beta-1-4, you don't see too much difference. So the virus frame are just called according to this surface protein. So this would be H1N1. And I show this one. So H1, a hyaluronic emagglutinin here, is responsible for binding the virus to the cell via the salic acid on the membrane. So there is here the insertion, the vagination of the virus. And the neuramididase catalyzes the agrolysis of salic acid and which allows budding and release of the virus. So these are the key events. So I just have to apologize for any colleagues in virology because this is very preliminary type of presentation that gives you the figures. So whenever we go about this lectin binding, so this is what is seen from microscopy, and we have been the three-dimensional structure at this structure, as this has been solved. This occurs as a trimeric protein, and these are the binding sites of this primary protein, surely, onto the salic acid. And these are providing the anchoring point for the virus to enter. Comparison has been made also between the avian and the human emagglutinin, between the so-called H3 avian, the so-called Hong Kong episode in 1968 and the human one, the Spanish one. And the differences here are in the different type of amino acid which are involved into the binding. They are not too much different, as you can see. The tricky point is, of course, with neuraminidase, which cleans the salic acid on our cell, and then in this case, the crystal structure has been solved. So this is a tetrameric protein also, and this is the linkator to the salic acid, and this is responsible for the budding. From these things, conception of neuraminidase has been 20. So the first one, so there are inhibitors. So the first one has been shown here. So N1 with salic acid. The second one is, of course, a famous tamiflu. So this has been devised knowing the three-dimensional structure of all these interactions and doing a lot of very, very subtle, very nice organic synthesis. So that led to tamiflu. And the relenza was developed afterwards from this particular knowledge of three-dimensional structure. So I'm stressing the word of three-dimensional because I will talk about that in the afternoon. But we have to understand, you know, how these things go. And this is, for example, molecular dynamics simulation, the influenza variant. And you see in yellow here, we have neuraminidase. In orange, we have emagglutinine. And there are interactions. So nowadays, we are capable of running molecular dynamics simulation for a long time, trying to understand how these molecules behave. Whether, for example, there is bivalent service spacing between the so-called spike protein to make it compatible with the bivalent antibody association and so forth. So this is a present status of knowledge in terms of structure and also dynamics and interaction with the membrane. I will finish here with oligosaccharide as antigen determinant, giving a list of commercial markers, a cellular addition and so forth. And I will finish by the simple example of blood group antigen. So I will skip two slides, which are important, two series of slides, which are important in terms of xenon antigen, finishing with a sugar infection. I think that the sugars are varying from one species to the other one. And this was a discovery of glycan and blood groups by this Lanzterner. So this gentleman was so famous, they made a stamp in his honor. But actually it was not the only one. There was this also great lady, Dr. Watkin. So we made ourselves a stamp to recognize your contribution. So this is a blood group antigen, however, made of very simple sugars, clupenac, galactose, anesthesiogalactose, amine and frucose. They are making the so-called blood group O, blood group A and blood group B with very, very tiny differences. The tiny differences, which are fantastically important in terms of the blood transfer and so forth. And so this is the example where we have the so-called A, B and O system. So that this A and B system, I show them here, O antigen, which is devoted at the side chain here, here. And there are on glycoproteins and glycolipids in red cell membranes. And that also occurs on most cells and tissues in human animals. So they are here. And of course, we have to understand more about that. But there is a very striking observation that the worldwide distribution of human blood groups is not the same. And we have zones in our planet, whether there is more O zones here, zones whether that A zones with the blood group A and B zones. For example, A is quite prominent in Europe and is less prominent in other parts of the world. And some world has been, work has been made to make a relationship between the occurrence of this zones here and also to the existence of epidemia. So people have been relating the occurrence, the influence of black plague in Europe and though that only the, basically most of the people carrying the A zone survive from this epidemia. In contrast, other cholera has been also addressed here and then a bit, the people surviving cholera would be more likely to display a particular type of blood group and the same also from malaria. So this is also some sort of vision that we may have that we don't know exactly what's the role of these blood group substances. But what we think that our sugars are the results of the co-evolution between animals and microorganisms. And the course is that, so this is my wife, Ani Emberty who prepares this idea. So it's a result of evolution and so we think that the biological role and significance of the black hands on our cells is actually to create and generate diversity. So that's about what I wanted to describe because this is a very broad description, but very light description because I could not do the details of all these things just to show that we have an immense task in front of us of trying to identify, characterize, construct, understand how this mechanism goes together. And that information is important because it's putting all the significant amount of information together from which we can draw conclusion and I wanted to address you to this article that Frédéric and Davidé and myself published in the blog Blackopedia and this article is a description of what are the tools which are available in bioinformatics in the cyberspace which are available if you want to address issues regarding the analysis, regarding the characterization, the representation, the occurrence of all these very fascinating molecules and to move forward in the understanding of the role and function of these. And so that was my presentation which is hopefully finishing time.