 Hello everyone and good morning thank you Ravi for this kind introduction and today I would like to talk a little bit about this unique protein which is called the musin and I will show you how we are trying to develop a specific and selective fluorescent probe for musin. So for the next 15 minutes I'm going to start with the basics. So first of all what is musin? Well musin it's one of the biggest and more complex protein within our body. Think that more than 5,000 amino acids compose a single monomer of musin and to most of these amino acids are bonded sugars which are called glycans and these glycans can be up to 80 percent of the total mass of the protein and because of the presence of the glycans musin can retain a huge amount of water and because of its physicochemical properties musin can assemble into this polymeric glycol conjugates which forms a three-dimensional network around which mucus is organized and you have to know that basically we have mucus all over the wet surfaces of our body starting from the eyes going to the airways and up to the gastrointestinal tract and these three-dimensional metrics formed by mucus it's extremely important for us because it is the first barrier of the organism against the penetration of you see viruses bacteria but also drugs and unfortunately we know really well musin because of the so-called mucus-related disorders like cystic fibrosis and COPD where musin it's overproduced and it generates a viscous mucus but in addition to these mucus-related disorders in the last years we are facing an increased interest on musin and we are gaining more and more scientific evidence about the implications of musin in different kind of tumors and for example it has been proposed that secreted musins could be a diagnostic and prognostic biomarker in pancreatic cancer which you can see it's the third most deadly type of cancer worldwide and today one of the most interesting I would say methods to detect biomarkers at serum level consist indeed of fluorometric detection mediated by fluorescent probes and these techniques are really interesting because they are simple and relatively cheap and most importantly because they are extremely sensitive and we got inspired by this work where the the authors developed a fluorescence probe to detect lipase at serum level and with this probe they were able to discriminate between healthy people and people having increased serum level of lipase and so the idea our idea it's kind of similar because we are trying to develop a fluorescent probe to be used to detect musin at serum level and potentially to use this fluorescent probe to to screen between healthy people and people at risk of pancreatic cancer and today among the most interesting fluorescent probes we have definitely the the polymethane dyes such as quareins, croconins and cyanines. Structurally these dyes are formed by these poly conjugated bonds flunked by two two moieties, an electron donor and an electron acceptor and sorry and they are really interesting because they are quite photo stable in in solutions and they have really high absorption and emission properties and their photo physical properties can be easily tuned by changing the length of this of these poly conjugated bonds or changing the the functional groups and for our purpose we focused on squareins because in the last year squareins have been widely studied and they found many applications spanning from solar cells to light emitting diode and even they have been proposed for biological applications. So as I was saying previously squareins as well the other polymethane dyes are characterized by really high absorption coefficients and the high quantum yields and high fluorescence intensities but this happens only when they are dissolved in organic solvents in fact the main drawback of squareins as well the other polymethane dyes is that as soon as they get in contact with water they form insoluble aggregates and the fluorescence it's completely quenched. But an interesting phenomenon happening with squareins is that when they get in contact with specific proteins they can increase their fluorescence and we call this phenomenon a turn-on of fluorescence and the idea behind this project it's trying to exploit this phenomenon to develop a fluorescent probe and detect musing using indeed the turn-on of fluorescence. So we started by investigating these four squareins you see all of them have a negative charge at pH 7.4 because of the carboxylic group on the lateral moieties and they belong to two different classes one being the indolinine-based and the second the benzindolinine and the other differences between them consists of the length of the lateral chain you see here we have two carbon atoms while he ate carbon atoms. So we started by studying how these molecules interact with the musing by fluorescence spectroscopy and here we got really good results because for all of the four squareins we got an increase of the fluorescence. You see with the squareins with the short alkyl chain we got a mild increase of fluorescence up to fourfold while the squaring with the longer alkyl chain we got a higher increase of fluorescence up to 45-fold and yeah this was quite a good result but the initial joy was quickly dampened because then we repeated the experiment with using instead of musing we used albumin and this time we got even higher increases of fluorescence and for our purpose such a result it's not good at all because as you can imagine at serum level the most abundant protein is albumin and consequently when detected musing in serum it could induce a really strong background signal so again our purpose is to develop a fluorescent probe which responds to musing but not to albumin. So we next try to understand if what is the mechanism behind this phenomenon so we had a look at the structural activity relationship and here we found an interesting behavior for example if you have we have a look at the turnon we see that the squareins with the short alkyl chain can induce a quite mild increase of fluorescence while the other with the longer alkyl chain have really strong increases of fluorescence and we also investigated the kinetics of the interaction by monitoring the time requested to reach an equilibrium and a sort of a plateau and here again we see that the more complex is the squaring and the longer is the time requested to to reach the stability we also calculated the dissociation constant which gives us information about the affinity between the the squareins and and musing and again we see a kind of relationship with the structure of the squaring because the the more complex is the squaring the lower is the affinity for musing and we then put together this experimental data and computed a correlation matrix with some molecular descriptors and here we got quite an interesting result because we found that lipophilicity could could modulate the interaction with musing because for example these molecular descriptors the molecular weight the total surface area the partition and the distribution coefficient all of them are related to the lipophilicity of the of the molecule and basically all of them are strictly correlated and they give us the same information which in other words is that the higher is the lipophilicity of this of the squaring the higher will be the turn on a fluorescence but at the same time the higher the lipophilicity the lower will be the affinity for musing and the longer will be the interaction time and this actually could make sense because when we study the interaction by absorption spectroscopy we see that increasing the concentration of the of musing actually increases the band corresponding to the monomeric form of of of the squaring while at the same time the increase of the protein concentration decreases the band corresponding to the H- aggregates of the squaring so we speculate that the mechanism behind the interaction with the protein could be a kind of desegregation and solubilization on the lipophilic spots of musing because you have to know that in addition to these really hydrophilic domains which are heavily glycosylated musing has also naked portions of the peptide core which are called cysteine rich domains and usually this falls into more hydrophobic domains and so we speculate that the aggregate of squaring could attach and interact on on these hydrophobic spots so according to to these observations then we wondered if we could increase the selectivity for musing by increasing the lipophilicity of the squaring so what we've done was to synthesize new squarings and the first thing we've done was to get rid of the carboxylic group on the lateral moieties because they are quite hydrophilic and then we increase the length of of the lateral chain up to four carbon atoms and we obtain this new squaring which we called it C4 squaring C4 then we recorded the interaction again with the two proteins and this time we see that the signal we record with musing and with albumin it's quite overlapped so this means that on one hand we reduce the selectivity for albumin and on the other hand we increase the the selectivity for musing so it looks like we really modulated the selectivity for musing by modifying the lipophilicity of the squaring so in the next step we wanted to increase even more the lipophilicity of this structure so we introduced two bromide atoms on on the lateral on the indolinine groups and we obtained this bromide C4 squaring and for the first time we were able to get higher responses to musing with respect to albumin and this is really a great result but still we we have a response to albumin and for our purpose it's it's not suitable because again we are we want to have a dye which responds to musing but it's not sensitive to albumin so because of this we wanted to increase even more the the lipophilicity of the bromide C4 and this time we introduced the benzoindolinine moiety instead of the indolinine and unfortunately this new structure this new squaring was so lipophilic that it was completely insoluble in water and it's actually is quite insoluble even in organic solvent and the signal recorded was not reliable so in this case we we had a solubility solubility problem so what to conclude what i've just shown you is just is that squarines could potentially be used as fluorescent probes to detect musing because they can increase their fluorescence in the presence of of musing and among the structures we studied we found that the bromide C4 is the best their former because it was more selective for musing with respect to albumin but we are we plan to to modulate the structure of bromide C4 to increase its response to musing and we also plan to test positively charged squarines because we speculate that cationic squarines could interact with the negatively charged glycans present on musing by electrostatic interaction but we still have to do all of this and let's see and finger crossed so i would like to thank my supervisor professor Sina Vicentine and Nadia Barbero and Carlotta Pontremoli from the Moff lab at the department of chemistry here at University of Turin and last but not least for sure i would like to thank you for your attention and i'll be glad to take your questions thank you thank you cosmon i appreciate you finishing well in time so we have time for questions full five minutes okay let me see if there are any hands hi rally i actually have a question yeah sure so yeah luka and then um hankona after that okay look yeah yeah first very nice presentation thank you um i just had a couple of so i saw that actually you do see a shift in the emission when you when you move it to the in some yeah some cases it's more it's more uh it's stronger like uh when you have this 45 time announcement you also have a shift uh in the in the emission peak so first question is could you use this as a as a way uh beyond the intensity and the other question if is if this protein has a structure that was resolved ever so that you could maybe uh identify better which are the side the position where the dive bind to the protein yes thank you okay thank you luka really interesting questions i will start with the first one uh yeah this could be uh one of the solution but i don't think uh it would be reliable because we observe this kind of shift not only with musin but also sorry also with albumin and even with other kind of proteins because we are testing now for example we tested the trypsin the fibronogen and the different other kind of proteins because uh of course at serum level we the most abundant protein is albumin but it's not the only protein so again we we will have the same problem because the shift will be observed even with the other problem with the other proteins and regarding the structure of musin uh today we don't have the complete structure uh we have some portions for example we have uh resolved the some hydrophobic domain some for example the foam filler brand which usually is at the bottom of the protein but we don't have the complete structure especially because musin as i as i was saying it's highly glycosylated and um we don't know exactly uh how the sugar chains are attached to musin so yeah i hope i i answered your questions okay now ancona you had your question yeah i think my question already got partially answered because i was going to ask whether you have tested with other proteins that would be present in serum and it seems like this probe is also interacting with other proteins yeah uh actually yeah we um actually published that that work and we saw that for example with fibronogen and trypsin we had really high uh signals and uh but usually the most uh reactive protein i would say remain albumin so yeah thank you thank you yeah i don't see any other hands so kosmin i get a chance to ask a question so um is musin a membrane bound protein actually does it have a transmembrane part yeah there are two kinds of musins the secreted and the transmembrane musins the for example the transmembrane musin are attached to the cellular membrane with the an intracellular domain and usually this kind of musin constitutes the first layer of mucus which is actually it's more hydrated but at the same time there are different other kind of musins which are the secreted one which constitutes the upper layer of the mucus with the the reality dimensional network created by by musins yeah is it so then and i assume that you're trying to detect the soluble musins right the one that actually gets secreted out isn't that correct uh well we are using commercial musins in this case we used porcine gastric musin which is extracted from the stomachs of pigs and um yeah the the most part of it is definitely secreted musins but uh we cannot be sure that uh among the the secreted musins are are present even transmembrane musin because of you know of the extraction process and maybe part of the transmembrane musin is also present but it's mostly it's mostly secreted musin right so yeah so then that's so that's what my confusion is actually so how does increasing i thought i saw in your data that the lipophilicity anti correlated with affinity right initially right which makes sense based on what we just discussed but then later on you increase the lipophilicity and you got stronger binding i didn't make sense of that if you why why does that happen which is this this slide you're referring maybe this one yeah this one this one tells me that lipophilicity is oppositely correlated to affinity for example right yeah right and now if you go to when you went to your square ends you try to increase the lipophilicity of the square ends and you got a better yeah because yeah because even even though the the response to musin it's higher with the the lipophilicity of the square end the this is not related to the affinity we saw and also because we are not sure if we could talk about affinity in this case because we are looking at the interaction between an aggregate and a protein and not an interaction between the single molecule and the protein so i'm not sure if we can talk about affinity oh okay okay so so you're just increasing the ability to form the clusters the aggregates so that you can actually interact with them okay exactly yeah yeah okay makes sense okay thank you cost man i think that answers my question for all the all the questions yeah sure okay great so i think that brings us to the end of the day i think i will hand it over to luka at this point to make any final announcements