 I don't see the speaker. Let's see if there is a roundup. He's here. He's here. So Shahi Karamunde alone, can you please share your screen on YouTube yourself? Here you are. Okay, fine, go. Good afternoon, everyone. Should I start the talk? Yes, yes, go. Please. Good afternoon, everyone. My name is Shahi Karamunde alone. I'm going to give a talk on the title named Tracking the Early Events of Aggregation in A-beta switch peptides employing protein charge transfer spectra. So here, here the protein of our interest is the A-beta peptide. So this peptide is responsible for the Alzheimer disease and which happens by forming insoluble amylate fibrils which deposit in the neurofilm. So this is the schematic of the aggregation pathway. So this is the A-beta monomer, which have a fast kinetics initially, which transforms into the partially folded beta sheet structure. And after that, there is a slope transitions which forms the fibrils or the proto fibrils and then the fibrils. So there are various methods to characterize this aggregation pathway. Some of them are using extrinsic fluorophores like 511D, which binds to the fibrils and give fluorescence. Or some other are the congoate assay or the ANS assay. And some other are the DLF dynamic light scattering and all. But here we are mainly focused in the early stages of aggregation. So this is a very fast rapid process. And to characterize this thing is a bit difficult and a bit tricky. So this is the monomer, this is the beta sheet structure, which is formed. So generally, 3D can be useful. Circular dichroism, because there is a transition in the conformational, in the beta sheet structure. But the problem is that since it is fast, it is a bit problematic. So for that, we used some switch peptides. This is the schematic of the switch peptide and going into details. So this is the peptide. This is the peptide, the A beta 140. This is the sequence of the peptide. And this is the amylate core. This part, this amylate core. So I'm using this A beta 16 to 22 of this A beta peptide and doing all the experiments. So what does this switch does is when it is in the switch off mode, this isn't actually a peptide one. So when, so it isn't actually a peptide one. This switch F off is in the pH, aesthetic pH, like pH 2. So when it's in the pH 2, there isn't any peptide backbone. But when it is transferred to pH 7 or say in some basic pH like pH 7, then there is some O2N SL transfer that takes place. And then only it becomes the peptide backbone. And then only it, the aggregation happens. So this part, this switch peptide thing gives us enough time to systematically analyze this kinetics of the early stages of aggregation. So these switch peptides help us to monitor the aggregation in the early stages, in its early stages. And here we use our novel technical approaches to monitor them, which I'm going to discuss in the future slides. Before that, the composition that I'm going to use, this is the peptide, this is the peptide sequence. This is the switch over here. And we did various mutations. We did various mutations like lysine and glutamate in that fragment. But unfortunately, some of them got precipitated and some other didn't show any aggregation because of the charges of the residue switch inserted and created some, which forbid the aggregation assembly. So finally, we ended up with these five peptides. Some of them were acetalates, some were not. And some we used for lower concentration, as low as 50 micromolar, and some were at 200 micromolar. So these are the five peptides which we used for our aggregation assembly. So before doing the pro charts, we first did the conventional techniques like the CD, circular dichrogym along with some other dyes. So here I'm highlighting the CD, the circular dichrogym spectrum. To know more, I am just highlighting this is a sample, the random, not my data, just a sample to make you understand better. So generally, what happens initially in the random coil state of the A-beta peptides, it is in the random coil state. So it gives a peak at around this one, this green line. But when it forms beta sheets, then it becomes the red one. This is for the alpha elix, but it doesn't show like that in the real scenario. It just changes from the random coil to the beta sheet. So from green to red, this transition takes place. So in our case, we did for all the five peptides. And what we found out is that as the time progressed, the increase in the 220, which is a characteristic of the beta sheet, increased for all the cases, for all the peptides, whether it is acetylated or non-cylinder, as well as for lower, as well as higher. The lower concentrations took a bit longer time, whereas the higher concentrations, it was quite rapid. Although there were some switches available, but still some of the irrigation was so fast as around 60 minutes only. We suggest that the conformational changes are happening in that irrigation. Then we did ANSSA. In that ANSSA, what happens is when ANSSA is a fluorescent probe, which when being in the free state, it doesn't give any fluorescence. And it maximizes around 520. But when bound to hydrophobic surfaces, when the monomers are coming closer, it gives higher fluorescence. And MSHA also gets blue shifted. So in this case also, what I found is that as the aggregation proceeded, we see a blue shifted with the increase in fluorescence in the ANSSA. And this is consistent for all the peptides, which suggests that the aggregation is happening, and they're coming closer. Again, we did THTSA also, high-velopment TSA, which is a characteristic for the hybrid formation of the peptides. And its fluorescence is around 490 nanometer, when excited at 450. In free state, it gives very low fluorescence because there are no fibrils. But when bound to fibrils, it gives high fluorescence. So in this case, there are all these five peptides. We found that, yes, as the aggregation proceeded, there is increase in the THT absorbance suggesting there are some fibril formation, whereas in some of the peptides, basically in some of the non-ethic peptides only, there wasn't that much THT increase. As you can see from the graph, this is the kinetics, which we indicated the area over time. So we integrate the area, and then plot it versus time, and we found out these are kinetics for all the cases. And in this case, there wasn't much change, but in this case, there are some changes. For this thing, THT was able to detect the changes, but in only cases of that fibril formation. So till now, we have discussed only the conventional part. So my topic mainly starts from here only. So which is the unconventional intrinsic chrome force. Basically, whatever till now I have discussed, in all the cases, there are some extrinsic chromophores which you are adding, which may alter the kinetics. But so we need to have some intrinsic chromophores that are already present in the peptides. So generally the intrinsic chromophores that are present in the proteins or peptide system are the peptide backbone or the aromatic amino acids. But the absorbance of the aromatic amino acids and the peptide backbone, they absorb only till 320. After that, there isn't, they should not absorb. But what we found from our previous reports, it was reported that some of the proteins which are very highly charged, they show absorbance way beyond 320 nanometer, some of the highly charged like lysosome Hs and all. So they showed much higher absorbance even in the, what is called the higher wavelength region. So this may be because of some, some of the reasons that was not known till then. But in 2017, from a lab, we proposed that this, we took alpha-3CM monomeric protein. And in this case, we saw that the absorbance in this tail was in the absorbance in this region was quite high. Although this protein doesn't have any aromatic amino acid. It was free of phenylene, tyrosine and tryptophan. Although it was showing absorbance in this region. What was, it was postulated was that there was some electronic charge transfer that has been happening from the cation from the cation to the anion. Like there are multiple kinds of charge transfer that may be happening. And some of the charges are from the backbone, the electron-rich backbone to the electron-deficient side terminal of the lysine. And in some case, the electron-rich glutamate to the backbone of the relatively electro positive backbone. And this transitions are happening from the HOMO of the more cationic entity to the LUMO of the relatively electronegative species. So in our case, this is for the monomeric protein. But the basis was that this absorbance was mainly dependent on the 3D proximity of these charged residues. So what we assume was that during aggregation, when the monomers were expected to come closer, they should interact. And this kind of transition should happen. And we should get increased absorbance in this tail region. So what we found is that for all the peptides that we used in this study, we found that with time, it increased with time for all the peptides. So this suggests that this approach of the absorbance is increasing with time as compared to the circular dichrolytic data. This is the kinetics that is the kinetics. We calculated all the, what is it called? We took the absorbance data for all the wavelengths and plotted versus time. And we found out that we are getting an increase for all the peptides. Some of the increase were very steep, whereas some were very flat kind of. But anyway, we are getting some, we are getting appreciable changes over here. So that was the approaches absorbance that I've discussed. So in 2020, it was also reported that in contrast to the approaches absorbance, there is approaches luminescence also, when the excited electron went to the LUMO from the HOMO, it may then come again back to the HOMO and recombine. And then it gives appreciable luminescence. So this is the schematic of this luminescence, this red one, what is called this red one. It is again, when the electrons, when it is being excited, it charges, if the electrons going up and then it is coming back down to the peptide bag one or some other highly electro positive form, the origin from where it was generated. So we get appreciable luminescence, all in this study was based on a monomeric protein only. So similar pattern we expect in this case also, we did the approaches luminescence to monitor the switch-shifted assembly and we found out that as the time increased, we got increased signal of luminescence and it was consistent for all. One case it wasn't, we didn't show much of the changes. We did the excitation of various wavelengths at 295 and took the reading from beyond. And it was consistent for multiple wavelengths possible. And we then plotted it versus time and we found out that, yes, it also shows similar kind of trajectory as the approaches absorbance, suggesting both of them are, both of the phenomena are quite related with each other. Then we tried to see some kinetics. We modeled the, whatever I have shown here, we tried to model the kinetics and we found out what is called one time at which half of the education was complete. We named it T half. And then we plotted this T half for all the various like CD absorbance as well as the luminescence data. And we tried to show it in one slide like for various peptides. And we found out that more or less, they were like quite correlating with each other and this one was less, it was less. And then, but in some cases, it was not that exact, suggesting that the phenomena behind all these methods, although they were able to see the changes, but the phenomena behind everything, every other techniques was a bit different. So that may be the reason behind this, some of the changes observed in some of the cases. Whereas in most of the cases, it was quite consistent. Then the earlier, whatever I have discussed in the luminescence, that was a steady state luminescence. But here we use time-resolved luminescence decay to monitor the phase-referred assembly. In this case, it was, this is the decay plot. We excited the samples and then we collected the fluorescence in various channels and then we get the decay. And then we fitted this decay in this expression. That is the, this is the tau one and then alpha, tau i and then alpha i. The alpha is the respective contribution from the tau i. When fitted in a discrete model, we found that the three exponential models fitted better and we got alpha one, tau one, alpha two, tau two and alpha three, tau three, which has been shown here. So here we can see that these are the alpha, this gray one of the alpha values of the tau alpha one and then alpha two and then alpha three and the dashed one of the aggregates. So we found that the contribution from the lower, the contribution from the lower lifetime were higher for all the peptides. This gravers. But as the, but with aggregation, that even intensified more, that amplitude, it increased even more in most of the cases. Suggesting that with aggregation, the mean lifetime that we calculated decreased and this can also be a parameter to identify the changes in the switch-shifted assembly. The reason of this being as the aggregation process, there was some chemical, there was some entities that may be forming which come down from the LUMO quite quickly as compared to the monomers, which is giving to the more population of the lower lifetime components as observed in this data. So this approaches luminescence lifetime, time-resolved luminescence in Disney intensity can also give us information about this aggregation. And all these methods are based on intrinsic chromophores. We are not labeling or doing anything like that. Then we also did the main analysis of the same lifetime luminescence intensity. This is a model-free approach, this main analysis maximum entropy method. This is a model-free approach. The earlier fit was we did for one order, two order, three order, but in this case, we just did for 100 orders. And then this is a model-free approach. And we found out that, yes, it complements the data of the discrete analysis and we are getting three exponentials, three distributions, which is satisfying, which is satisfying the same data of the discrete model as well. Finally, we tried to compare all the techniques. This is the CD220. This is the absorbance at 340. This is CD220. And we tried to change the percentage change of all the techniques. And we found out that the NSDST, although they were good, they were as compared to 200% kind of change, but it's absorbance and the CD were much more efficient and much more sensitive and much more sensitive than the other data. So in conclusion, we can say that the approaches absorbance as well as luminescence were able to detect the changes in the aggregation, in the early stages of aggregation and the approaches absorbance highly correlated with the CD data, which is a characteristic of the conformational changes in the secondary structure as compared to the THD and ANF. And also this approaches absorbance, if not it is better than the CD, it is as good kind of with the CD. And our switch peptide model can become a template or can become a standard model to study the early stages of aggregation in the switch peptide and can enable us to screen drugs for a bit of aggregation. This is the references that I had and I want to acknowledge IIT Guwahati India for providing you with all the necessary equipment for my work. Thank you. Thank you, Shah. There is a little time for that question and any, please use the chat or raise your hand. Can I ask a question? Sure. Yes. Thank you very much for the talk, it was really interesting. How, so the luminescent spectrum, how the spectrum, how efficient is that signal? I mean, do you need to do a special background cleaning or is the efficiency, the yield of that process is high now? So what I mean is that if you want to use that as a vehicle compatible probe or for luminescent probe, so is that the yield is high, we need to do some background cleaning or something like that? Yeah, actually the yield was a bit lower because they are not what is called the conventional chromophores. The extinction coefficient of this absorbance was low in this region, as well as the quantum yield also. I haven't showed the quantum yield here, but the quantum is for very kind of low kind of thing in this case. But anyway, this gives us a different region apart from the conventional techniques and further improvement needs to be done in this wavelength region because in this, what is called this, in the higher wavelength region, even if we can just increase the quantum yield also, like that we can also use this thing directly for like imaging and also because it won't interfere with the cellular components as well. Yeah, okay. Really helpful if you can at least increase that thing. Thanks. Thank you. Any other question? Can I ask you a question? Please, Umash. Yeah, Shahar Kamal, that was a very nice presentation and interesting observations. I'm just wondering whether, you know, you have tried ionic strength to dependence on the aggregation of your protein. And if at all you do that, any change in fluorescence, would you attribute that to, you know, the structural changes or the effect of ionic strength on fluorescence? Okay, sir. We could have increased or like changed the ionic strength but the one problem that lies with it is that it may affect the aggregation assembly also. So if we just want to see the changes in the fluorescence, like how the interactions are, what is the charge transfer, the degree of charge transfer that has been affected but in other way, the aggregation will also be hampered. So we can't directly say whether it is because of the aggregation that has been happening or like because of the screening of the charges that it's taking place. Because initially when I tried to incorporate some of the charges, while adding two license, we are not getting aggregation happening. So just a single mutation is like hampering the segregation formation. So increasing the salt or something like that, increasing the ionic concentration may help us to get a better insight about the, what is called the better insight about the charge transfer phenomena but it will in a way affect the aggregation and then we cannot have an inclusive result about it. Excellent. Thanks, love. Thanks. Okay. I think it is time to switch to the next talk. So thanks.