 Yeah, I can see well, I can see now I can see that's it. Perfect. Good. So as I said, I will talk about plasmid taxonomy, something we are developing right now but already working have been working on it for a few years right now. So the idea here is if we aim to if if we want to understand plasmid function plasmid biology, we need the first thing we need is to make it clear what are the evolving units in another words. What are is the subject for evolution, so what you know, and in the same way as we don't analyze one giraffe to know what's going on, but the species of giraffe is what should we do about plasmids and in order to do that we know already by some a. work that has been done before that a. For bacterial genomes it has been found that a bacteria of the same species usually share have a core genome as a some said before, which is maybe half of the genome or it depends it depends, but a they are 95% identical. While individuals in different species are much less so there is a valley here of similarity, which makes the. Say the definition of the species as the genomes which share a lot of DNA a quite impressive not as in this paper here so a we did more or less the same for plasmids and. And the result. was this paper that we published a last year or what now over a year ago and in which, as you can see here, each node in this graph is a plasmid a plasmid sequence and some plasmids are a related to each other, there is a net between two nodes, when there is a similarity over. A 70% over 80% of the of the or 70% of the genome or more than half of the genome a and so so you see plasmids cluster and we can analyze these clusters now and that's what we did. No, before that a okay so a what we realize is that plasmids cluster indefinite groups and there is not much in between, so that this will be a similar representation to the representation of bacterial units, so in the same by the same reason that we believe there are species, because there are. bacteria or bacterial isolates which we have we have high similarities, the same happens for for plasmids and they cluster in these groups and we will analyze these clusters. So when we. analyze these clusters in in detail a of I cannot see okay a we see that when we here we look at the plasmids of the order enterobacterial is which contains equalize almonella and everything, as you see on top of the this column here. And here you see that most these are the PT use of plasmid taxonomic units, and you can see that most of them they appear in one or a few general so I, I was surprised to find that most plasmid taxonomic units. are they derived from one species one genus of bacteria, also a so in some cases, they can appear in other a general as well, but not in that many as we will see so each PT you has it's a determined a host range. These are the genomes in which these plasmids have appeared naturally, not the. A bacteria to which we can transfer this plasmid this is a different thing and I will explain probably on Wednesday. But here you have all the PT use in the in the this is the old the old data from the our published paper, so we have here a 37 PT use which are more plus that means that they are transmissible by continuation. That contain most of the plasmids in enterobacterial which are group in five classes, the classes are defined by the relaxed genes, because this is like this lowest clock in a in a plasmid. And, and therefore there are five classes, and each class, we can distribute in several families, which share the whole traffic. So in order to define the taxonomy of a plasmid, we will use the mob type, which is the relaxes class, then the family which somehow defines the trust system and everything that is connected to that and several in many occasions are the partition genes and so on. And then the PT you, which contains a large backbone of that plasmid species. So what are the most important results in our paper. First, that most PT use have a preferred host genus, as I said, and as it is shown in this figure. The second, that the trust system is always conserved in almost well always conserved with the exceptions that I will tell later in in what we call a family. And then one important question or result is that the rep system is not conserved in in many PT use. And that is because as we think now, the red region can or has to change quickly when a plasmid moves from one bacterium to another. And therefore you can have, you have many PT use a that have different replication formulas. And this is shown here in a in a close up zoom to the mob F class, the mobile class has a subclass which is F one, which are a number of classmates that appear in interbacterial is because then there are others in cyanobacteria, arctinomycetes and so on, which contain also the mobile class. But here, we have three families in F one F 11 family, always the transfer system is a ti plus me type transfer system will be one be to be three and so on. In mobile 12 is like a F plus me, which contains like 25 or 30 teams involved in transfer, and each one has their own PT use. And so, as you can see this character hierarchy of classes, families, and PT use a tell us a lot about the properties of our class, the structural properties of this. Okay, so, yeah, I will skip this one. So now we have updated our that our database to a rough set 200, which means we have analyzed over 19,000 plus. That's more than double that we use before. This is the overall picture of the network. And in this network. Now we have 634 pt use, which contain for more members. That is almost about 60% of the plasmids. And of them now we have decided only to name those which have 10 or more plus, but still, this is about half of the total. And that is because they're very small pt use only four or five members. And we have found that they are not so stable, let's say, and in the new classification, sometimes they get attached to another one and so on. So I will explain a little bit more about this. But so this is already a huge atlas, let's say of the pt use in the in all the bacterial kingdom. So, and here you have the names for everything that we have, we find in the in the entero bacterial is older. Here we have a seven over 7000 plus meets that that means almost one third of all plus meets in NCBI will belong to these two hosts in this order. There are 226 pt use more than four and 144 name, which means with 10 or more members. And here you have them names. We try to keep the in C classification when possible for these, but sometimes we have like new names and so on. But here you have all the complexity and of this cluster again. Well, this is color by the mob class. So you have members of five different classes in the entero bacterial disorder. And as just to show one example. This is a PTU I one that can be used and as an example, the PTU contains 259 members. And here. This is a different network. This is an agnet network is a network which is constructed by the relationship of plus meets with the different protein families. So each of the gray node is a protein family. Each of the color node is a is a plus. So, as you can see, all plus meets within the PTU I one share a number of plus meet protein families, which will be so to say the core of this PTU and there, then there are many other protein families which are present in a few or many of the plus meets but not, but no one when you see here, the, the distribution of hosts, you see that Salmonella E coli and Clebsiella and Shigella are more or less distributed more or less randomly. So that's why we think this I one plus meets are able to colonize, let's say, all the enterobacteriaceae family. Here you have the same network, but color by the presence of the relaxation. This is a P P class relaxation. So it is it is blue. There are no others except when this black is two black dots that contain two mobs. And that's probably because they are cointegrates with somebody, something else I we are analyzing all this. There are also a few which have no relaxes as you can see these three, four, five, six or so. We have found this in in many PTUs that you have all plus meets. If they can if the PTU let's say contains a relaxes many plus meets contain that relaxes and no other, but some plus meets have lost it and they may have lost even the whole tradition we have a, we have a paper published with Eduardo Rocha in in bio archives for the moment, and it shows the dynamics of the PTUs and it is shown there that PTUs are, as you can expect for plus meets, relatively unstable, compared to the chromosome so you get variants, quite often, in these cases, they are still part of the PTU but if the losses or gains are higher, they will get expelled from there. So, and then we have for instance if we look here. We have drawn here everything which is related to the I1 PTU by at least one edge. And as you can see that there we have connections with the C, E85, the BOK set, which is this part of the I1, which didn't contain the I pillars or something different, and E84. And as you can see, and I show this picture just to show that there are plus meets which are left outside the PTU because of the rules, the strict rules we put to include a plus meeting a PTU, but they are still connected with some of the plus meets in the PTU. And that may be interesting in this outbreak analysis or so on because you may have, you know, cointegrate for instance this plus meet here seems to be a cointegrate of an I1 and a C plus, and so on. So, there are many plus meets which lie outside the PTUs because they failed to comply with one of the rules and this is good because that keeps the PTU well defined. But you may have one plus meet like this and I will go to this at the end of my talk, which is not too far now. So, when we now look at the different PTUs, where they appear, what we find is that some PTUs like FS or FY, this is typical of Salmonella only. The typical of your senior only are a specific of a species. Some are specific of a given genus. Some are specific to a family, and this is the majority. Like 80 PTUs here. Up to the order, another 30 or so, but very few PTUs can lie outside an order like a class or a film. These ones are new here and we are still analyzing them. So, what this means is that the rearm of a PTU is the family if it's a conjugative element, or maybe less if it's a non transmissible and no mobile. And here the host range of a mob minus or no mob PTUs are narrower than the transmissible ones. And, okay, let's keep this just just to finish. You have here in this address, the complete network of the of a plus meets in the database and you can click on one, like for instance, our 100 if you search any plus meet which is in NCBI, you can find it here, and all your the connections of that plus meets console here. I mean, because this is called by the class, the F class is read. So many of these plus meets will be in the F a cluster but you can see everything you know you can click on each plus meet and see where it lies in the in the company and it gives you all the connections. And in order to classify your your plus meets, we developed this tool called copla in which you can, you are upload the sequence of your plus meet and it gives you an output, which a several files, the PTU prediction that gives you the PTU. But if it's assigned the host range from one to six as I explained before, the score, which is, if it's over point nine is very good. The size of the plus meet and then the mob, which tells you the class and the MPF which tells you, more or less, the family of the plus. And also the replicant formula using a rep TV. Also the antibiotic the antimicrobial resistances that we find, and this is very important, the least of all connections of that plus not only the ones which belong to the same PTU. And these others that I said that are in the periphery of the PTU and therefore can help you in the analysis of your plus meet or the project in which you're involved. And I think that's all I wanted to say. This is my group here and I would like to acknowledge main, mainly to a Santiago redondo who did a lot of almost all of the bioinformatics development and these tools. I think are the young who did also a lot on the analysis and a rancher, a penile, who is also involved in the analysis of all of this, and I shouldn't forget for Fernandez who helps me, helps me in many things which are directly related or indirectly related to this project. And thank you very much for your attention. Thanks very much for another fantastic talk. Oh, it looks like we've got a hand up from James for a question. Oh, sorry, I thought there was a round of applause. Sorry. I don't know how to do zoom. I don't know how to do zoom. I don't know how to get. Okay, fine. Around applause at least and if anyone has any questions. Now's a good time you have a few minutes before we start our next talk. Okay, as you will have seen on the on the schedule there was a chance. So there's a question from Olivia. Sorry, Olivia costlets saying what kind of data do you use to determine the host range of a plasmid. What kind of data do you use to determine the host range of a plasmid. The only thing we do is to find out in what genome genera are the host of the plasmids in NCBI. So for instance for this I one PTU the PTU has 256 genomes. So we, we determine that what we determine the sequence tells you the genome the host genera and the host species. And then we add the different species of the different genera and we grade that from one to six, if the old plasmids appear in one. In one species is one in one genomes is two in one family is three in an order is four in a class is five and in a film is six. We have never found a plasmid which goes over that appears in different pilot. Although we know of course that plasmids can be delivered from one film to another. And we do that in the lab. Any follow up there Olivia is that okay. You could feel sorry. No it's okay I was just checking. I was just asking Olivia to tell us if that was sufficient answer but I think it is anything from anyone else to remove on. So we've got one from Liam Shaw, who's speaking later this week, as is Olivia. I'm just going to read it to you. Read it out. Thanks for great talk Fernando. I've read the papers associated. I think they're fantastic too. I wanted to ask about intra cluster density. Does host range mean that any member of a PTU works in any of its hosts. I don't know the short answer is I don't know because I didn't check it. But I guess I don't know. It's a good question because if plasmids within a PTU can have different replication formula. Maybe they acquire a given replicon. To be able to replicate in one host so we can see one plus meeting one host because it has acquired just a replicon. To make it able to replicate in that host and not changing anything else or not very much. So I don't know this should be checked for each PTU but it's a good question in the sense that now that we have defined what is a member of a PTU, what's the collection, you can analyze those as belonging to the PTU. And then you say if it's each plasmid in the PTU can replicate in all of the available hosts or not. But one thing is that you're able to replicate. And another thing is that you appear there. Because I don't know what ice what we see is that a conjugation apparatus apparatus is are in fact, most of them are very broad host range. They appear to be able to send a plasmid almost everywhere, but that they send the plasmid there doesn't mean that the plasmid is able to be maintained there for a long time. You can replicate, but then there will be some say, interactions with the host, and what what we see is that the plasmids tend to appear mostly in one gene. In most of the cases. That's kind of interesting. There's another question here, maybe since there are like 20 minutes at the end of the these talks, we can discuss more know because he's kind of a time for the next. I don't mind to be answering questions. I, I, let's move on, but the the message from Teresa Cooke is a comment rather than the question. So I'll just read it out. It seems you consider plasmids originated and evolved in different hosts. But databases are biased by clinical and very recent isolates. And it is probable they have another origin moved between bacterial species and selected in different species afterwards so everything is biased by worry. That's a good theory, but I'm afraid is not true, at least by what I see I saw. Let's follow that up in the discussion section then because that's okay okay yeah good. So, Teresa I hope that's okay we will follow up that at the discussion section. And then, so it's my pleasure to introduce. Victor Mathew. He's going to be talking to us about characterization of the pollens and firm acutes shows a close evolutionary relationship between primary independent poll bees coding or PBS I guess coding elements and conjugative mobile genetic elements. Thank you.