In the previous lecture, you obtained a glimpse of the principle of different label-free technologies and their possible applications. In today's lecture, we are going to talk about the recent advancements in the field of label-free techniques and how one could integrate different technology platforms. Imagine that we have talked about mass spectrometers, we have talked about SPR, we have talked about BLI. Can we start comparing these technologies? Can we start integrating these technology platforms? This is what we are going to talk in today's lecture where I am going to talk to you about SPR imaging, one of the latest high throughput platform which can do similar thing like SPR but much more in high throughput manner. Integration of SPR with mass spectrometers for identification of protein interactions and some of the pros and cons and comparison of two major technology platforms for label-free biosensors like SPR and BioLayer Interferometry. So my today's lecture, I hope is going to further unfold the applications of label-free technologies in analyzing different molecular interactions. So let us continue with my lecture which I have delivered in this workshop. So SPR-I, SPR imaging is one of the high-special resolution platform which allows for high throughput analysis of various type of biomolecular binding interactions that you want to measure. Here you can intentionally that you are using imaging platforms. So imagine that you had the gold chip which was you are using for SPR but in SPR gold chips you have the very small region where the gold part is there rather if you have the full chip which is having the gold and then now you are using imaging to image the entire surface. And then that image is going to generate some sort of pseudo image like a microarray which will show you that you know each one of those features which you have printed on the chip will give you an image which looks like a microarray but it is not actually microarray. So that kind of concept was used can we image the whole chip rather than you know exciting on a specific angle only which what you use in the SPR case. So you are radiating by the light imaging on the detector arrays you are using some sort of CCD devices which are not so costly. And then this particular platform showed potential to combine with the microarray based technology because anything now which you can do in high throughput way for the whole chip is very much compatible with the microarrays. So this is where you know one of the image I am showing with one of the experiment I was doing in Josh lab earlier when we were developing some SPR imaging based platform on the early prototypes. So as I mentioned to you you are seeing something here just the pseudo image it is not the actual you know you are not measuring fluorescence these are some of the pseudo image obtained from this kind of platforms which shows you compatibility with SPR imaging. So what happens here conceptually we have this prism we have this gold chip but now here we are exploring the full chip potential to scan the whole surface here and then by generating this SPR image again the concept is same we are still looking for reflection angle we are measuring the percentage reflectivity change when you have too many spots printed on the whole chip surface you need to find out some common angle which can be best angle for doing this SPR experiment. And that is how you have to play with the conditions to find out what can be that best angle which can be fixed for the whole chip surface to do those kind of assays. Once you find that and again that may not be the best and most accurate angle it is a 19 you have decided or 20 you have decided you may have to play with more to find out what can be the best angle to freeze for the whole chip to be scanned. Nevertheless the idea here is to generate the pseudo image of whatever is printed on the chip without adding any kind of fluorescence labels you are just generating the image on the gold chip. I am showing you now one of the workflow which was done several years ago which was on a prototype to give you feel of you know how these kind of experiments can be done. So this is you know prism here and this is a chip full of the microarray kind of chip where you can add all the you know protein feature which you want to analyze and there are certain holes on this you know on both the sides. And these holes you can now combine with the flow cell from which your liquid can come and pass which you want to test out. And then now you can screen the whole chip surface on the SPR kind of setup and it is you know just reminds probably Josh that you know one of the old setups we had the prototypes where now you can test eight antibodies for example or eight different proteins in very much to format like you know you are developing a new instrument setup and these kind of you know solutions are coming in you have the gold chip everything is you know passing from those and then you know is going back in the washing containers. So these kind of platforms could be used and it result into these images which are the pseudo images as I mentioned to you it shows you these kind of spots. Now all these red green what is shown here these are actual spots but along with them whatever you see the gray color is the just background. So software are artificially add some blue color dots these spots could be used for subtracting the background. So in this way now you are you are ready to test a lot of molecules unlike the four what you can do just by using the listening technologies. And these are known as the regions of interest otherwise in any kind of micro experiment any kind of SPR imaging experiment you have to ensure that you know what you are measuring is the right signal and not just kind of you know that particular feature what intensity it has. So you have to take into consideration the intensity of these spots in combination with the background what is there. So therefore for each of these spots we had four of the differences spots. So if you subtract the average of these four from this particular one and same you do for every spot then probably you are only measuring what is printed on each of the features right. Then you have removed all kind of side effects coming from those chips. And then now you can see that how much difference it could make. For example this was the actual spot which is showing you the binding. And now this is something coming from your blue spot because you know buffer is flowing on the whole chip.  Now if you subtract this then your shape got changed your sensor ground looks little different now right. So these are the kind of thing which can make the your curse very different. If you are just not using normalization or if you are using those background subtraction which is very crucial. One should really normalize these features on the chip surface and becomes more critical here because you have many spots printed on the long chip and imagine that you know when the liquid is flowing from one side and reaching to the other side it will take some time. And you are you know you are start seeing binding for some molecule and then eventually you will see for every other molecule on the chip right. So then they will show the different type of time at which the binding started. But ideally you want to compare all of them together. So then you have to normalize again them so that now everything whatever you see so it was coming from you can see the intensity that different looking here for different angles. Now you have normalize the SPR curves and everything is now looking same then now you have a opportunity to compare their signals. So the SPR signals which is we talked about sensor ground they are the one actually if your eyes are pretty expert and tuned to look at a data immediately start telling you a lot of information. The SPR data just you know it shows you the different curves will tell you that you know what is happening for the kinetics how much concentration I can see for those proteins what is the surface capacity, molecular weight etc. And many times you know these kind of curves which are showing you the just the difference in the on rate and off rate can be very powerful because imagine that you know you are working towards developing different drugs. And objective for you know those drug development probably you might have quoted several examples. If you want to really make that drug work very fast right you want immediate effects for you know any accidental condition any kind of you know headache kind of things you want just very quick response. So your on rate you are looking at that time versus your off rate becomes very different right. So you want immediate action to happen something has to go bind very quickly and then if you are looking at you know the sleep response you are looking at long-term response for a given particular drug then you are thinking that okay let it goes off slowly you know you do not want it to just you know show a very quick binding and release you are thinking about it should go very long so that you have more effect for that particular molecule. So both of these conditions probably can give you the same KD value it will show you same kind of response overall. But on rate and off rate could be different and those can be so detrimental or useful for your kind of drug discovery you know portfolio which you want to develop. So therefore these kind of you know curves are very crucial as you are watching and there are many companies who work with us who do the testing in our labs in our facility who are trying to look at many of their you know the biosimilar products and some of these kind of testing they do. We can see from their experiments that you know just looking at the shapes of these curves of sensorogram can make them feel excited or not so excited because immediately it tells them it is working or not working. So all these kind of stuff is just visually you are seeing of course you need to do better fitting of these curves to find out the actual values for concentration or looking at the KD values but you know many times these experiments gives you lot of visual feel of doing those analysis. But as I said you know if you want to really get the proper values you have to do good cropping you have to do good fitting in this case for example as I mentioned your response timing is different because you have different molecules printed on the whole gold surface where you have some starting right now and some starting later part so then your response units are shown differently here but now you are normalizing all of them and then you want to see comparison of their responses throughout the whole experiment. So therefore data processing becomes very crucial but advantage of using SPR imaging is like high density microarrays where you are using less of the analytes which you want to test. In the same kind of sample which you have available especially the drug molecules which are very low in quantity or some of the clinical samples I think you have opportunity over there to not test out on hundreds of features simultaneously right with SPR imaging platforms. So that is something you know time saving cost saving of course and going to be much more reproducible experiment and then within the same type of condition which you provide now you can study the behavior of lot of molecules under the same condition which is very critical. If you are varying the you know chip to chip there will be some variation and the day to day temperature variation lot of things even you are doing under controlled condition but if you split hundred analyte testing on 25 chips versus doing on one will have huge difference. So buffer temperature many variables are there and if they are on the same spot at least you are you can neglect them you can normalize them you have a better ways of having the controls to negate those kind of you know negative effects from coming from those molecules. All right so latest popular technologies one of them is Bayakor T200 is one of them offered by G-Healthcare. There are many type of chips which are coming from these particular type of technologies where you know how you want to immobilize your molecules sometime you have different type of carboxy methyl based surfaces sometime nickel NTA kind of chips can be very useful for you if you have histag based proteins. So you can select the right kind of chips for your use and accordingly you can make use of them. Bayakor T200 is right now one of the industry leading technology which is pretty much GLP approved so whatever data you obtain here can be very much be sent for FDA or other kind of approvals and that's a lot of industries are looking for these kind of technology platforms. Nevertheless you know these kind of technologies are giving you very precise data but they're very costly you know considering the how much cost it takes to do the experiments. So many times if you are in the discovery development mode and you have hundreds of you know compounds or libraries to screen this may not be the best platform to start with because you are limited with the you know how much cost you have to screen that many large number of compounds. So new technology platforms like one which I'm showing you here which is biolayer interferometry based platform can be very powerful for first round of screening. It may not have that sensitive on-date off-rate and those kind of KD values which you want to obtain but it will definitely tell you binding happening yes no in a very quick format using your solutions what you have for different type of molecules to test out and here they're looking at the interference pattern. So on this sensor there are some you know these antibodies which you want to test out they're immobilized and then they go inside the like ELISA plate and then they those sensors are going to go and test out your your binding using the interference change which is going to happen. So this something is we have a follow-up lecture so I'm not talking much detail but I wanted to give you the feel that you know in which way you can move forward from different type of platforms. So a good thought can be you know from the microarray experiments you have got leads let's say you know 200 molecules which is of your interest now you can go to BLI to narrow it down further now you can use probably be a code to test out you know how many of those you want to know whether the exact on it offered right okay so there are many label free platforms which are possible I'm restricting right now the applications of those mainly based on the SPR right now because that's what something which we have currently available at IT Bombay and I'm also running a core facility which is offering services for many type of applications for internal users and external users and the kind of you know this particular distribution but what it shows to you that we are doing all kind of applications here looking at even from protein fiber interactions protein liposomes protein protein various organic molecules and one you know interesting experiment came when somebody wanted to print bacteriophage virus on the chip and test with the E. coli whether those abiding can be seen or not so you know all kind of thoughts you can have what you want to measure on the SPR and of course popular ones includes protein peptides nanoparticles concentration analysis something which we developed a new essay and optimization of many new applications so that's the ongoing thing we always want to see that what more we can obtain from these instrument platforms so I'm now kind of you know flashing it through couple of application slides with just an intention that what all things can be done on these platforms these are not my results just coming from our facility whichever people are running the experiments but what it shows to you that you know variety of things can be done for example one of the chemistry faculty lab was looking at one of the Kellexeran molecule and copper binding and to do that there was no chip which was available for us to do immobilization so we gave them the bare gold chip the small part of the chip and then they did the immobilization themselves of those and after doing those particular this is a step which are shown to you so sometime you may not have ready made ships available for the kind of functionalization which you want to follow and that's when you can use the bare ship where you can try out your own immobilization chemistry which could be then used to further do the SPR experiment on bare core chip so that's what we have some bare gold chips as well so this one showing you two different experiments the KD values obtained are pretty close and that was at least you know interesting experiment for us to try out because it was not the ready made chip available for it and still immobilization was pretty reproducible another lab from electrical engineering their intention is to have some benchmarks so let's say you know bare core technology the benchmark available in the in the field if you can measure you know an interaction and generate some KD from bare core and now if you develop another biosensor and you can show that you know I can generate same kind of KD then probably you are talking you know really robust platform development right so even for any new technology development you need to have some sort of you know benchmark development so therefore what they wanted to do they wanted to just look at the myoglobin protein binding with the anti-myoglobin protein and in fact they're looking at also the serum now so when we try to do this thing we were able to pretty much get good concentration of this myoglobin protein and that's something which made them very happy because now they can test their own biosensors and see the benchmarking how close these values are another experiment is from the bio labs here in the protein carbohydrate interaction which is one of the very common thing which we see many people want to look at local binding protein and different type of carbohydrates binding to the proteins and again you know why I'm showing your duplicates because it's become very crucial to give us the confidence that different experiments are giving the same KD values again I'm not talking the biology of it probably I should not talk is others work but I'm just giving you the feel of variety of applications which are possible by doing SPR kind of technologies now one of the industry sample which of course I cannot disclose which was trying to compare that what data we obtain on beacore how that you know similar or dissimilar when they are running to their parent company somewhere in Switzerland and and they had that kind of numbers and the you know KDs in their mind and they wanted to compare the things here and those molecules show pretty close binding based on these beacore experiments we also have some faculty from chemistry who are looking at protein is small organic compound their interactions in this case one of the unknown protein which is not known to us 27 KD protein was immobilized and then we are looking at how small organic compounds can bind to them so again many of these time the KDs are very close and that is giving us you know good confidence that these experiments are you know in duplicates are working well one interest which we had in our lab was to measure some of the known protein concentration in the serum for example from our discovery approach of using a diage and coitraic based platforms couple of protein we identified which looks good to us and as a potential biomarkers so we thought can we measure the level of those proteins in the serum using SPR and to do that you know some new assays were developed which was you know which we termed as a concentration free calibration analysis which was a CFCA analysis where you know different type of you know patients from this is one of the malaria project when we had patients suffering from either a phalcipin or vivax malaria or dengue fever and we wanted to measure the level of one of the proteins serum iodide A and we wanted to see that you know how that protein concentration is different in different type of conditions and because we already had idea for this protein from our other data set discovery data set so we felt pretty confident that you know we can now measure and this is what you need you know you cannot do validation on on large number of samples using mass pack or other type of technologies so then you have to come down to either ELISA or these kind of label free approaches which could do screening of you know the same molecules in a very high throughput manner so this was a good experiment for us to to show that you know we can actually measure the concentration of this protein of interest to us again you know sometime you have to develop these assays in the beginning so you need pure protein it's good idea to try out something if you are planning to do this kind of experiments with the known proteins for which you have the purified proteins available build the confidence and the say conditions from those then you can apply for some unknown compounds as well all right so i'm not showing you some data actual data from two different type of technology platforms so we compared for a couple of proteins both BLI platform and by a koti 200 platform for a few experiments because you know the cost wise they are very different but sometime the you know looking at the acceptability wise things are different and the kind of GLP compliance wise only one platform is more compatible so can we use this you know at least the BLI platform as the first level of screening with high confidence so in this case we use anti beta microglobulin and this antibody and the protein interaction so using SPR and BLI we tried binding for various time points analysis which we did different concentration series came pretty close actually you know this is showing you 20 the overall kd is almost 3 e to the power minus 9 and it is also 2.11 e to the power minus 0 9 these things just imagine was done just you know in a like this kind of instrument what is being brought here not available in my lab that time so not in the best optimized conditions but still the way i can see there's not huge variation just the measuring these kind of you know the abundant molecule bindings then we looked at some of the you know compounds of interest for a given project when we had some drug protocol to look for their binding and again things are pretty close you know coming from the two platforms available we also tested you know another drug which is shown here curcumin and prumbagan which we are testing for some bacterial protein against those and we are seeing you know the changes that are pretty close coming from both the platforms so you know what i want to convey you that you know many times just don't just rush for one technology platform think about if you have to test out many compounds what can be the cost for doing that what can be the variations of doing those experiments start you know using those platforms which can narrow down your your leads and hits then do the final experiments on something which is really you know acceptable and of course if you have huge amount of you know funds available for doing that project then you can do everything on the same platform but otherwise you can mix and match and choose which technologies can give you what sometime you know a good experiment may involve every technology which we are you know studying in different workshops right now here starting from you know discovery from my my spec based stuff and microarray based stuff you know take those leads forward and then validate using these kind of label free platforms so everything you know has its own utility and you have to pick the technologies in the right context all right so now over the period but you would have realized from various lectures given by Josh that there is so much need for having integration of new technologies and development of new technologies and that's the coupling different existing platform becomes very crucial in Josh lab we thought couple of years ago and many people are still working on developing those technologies of coupling Napa technology with SPRE waging based platforms the initial experiments done by Manuel Funtis and then I joined the lab where actually you know these are you know quite old Napa chips 2006 2008 that kind of timing so the glass lights are showing these kind of signals which I'm sure by now you'll have seen it has improved tremendously and then the same features printed on the gold slide are showing much better much more intense signals so first of all the objective was to to see whether Napa concept can work on gold or not because so far it was all done on the on the glass slides and first time it was you know planned whether we can mimic the same thing on the gold surface but looking at the 3D view then AFM images it was I think you know apparent that probably Napa could work on the gold surface as well but the traditional Napa chemistry which you are aware of having you know many components in the master mix right which included you know BSA BS3 cross linker you had the capture antibody and of course your clone of interest the serine of that having GST tag so all of them were actually making the whole mass too much which you are adding on a given chip surface right so the initial experiment when it was started the baseline when you are printing so much mass on the gold chip and in the SPR kind of platforms you are only measuring that how much binding you can see happening right so to begin with itself for the baseline itself is going off the response unit so here you have literally no room to find out how much binding you can observe later on because with the buffer alone the baseline itself is very high which is passing beyond the response unit of the instrument which is the SPR imaging platform so I talked to you about you know briefly about the technology which is SPR imaging you have been exposed for Napa arrays think about the combining the two to harness the both the power so if you add the entire GST antibody and you want to measure the binding of entire GST with the GST protein then probably you have no room here to see the binding where the binding is happening because it's already passing beyond the response unit of the equipment so the next thing was whether the same Napa chemistry can work here probably not so then there were you know a newer approaches came which was one of them was Coil-Coil chemistry of using E-Coil and K-Coil can our clones may have you know E-Coil and the chip may have the K-Coil and using those particular type of chemistry now if you chip you know print your all the clones using the same Napa concept but now you have the K-Coil peptide the capture peptide and each of the clone contains E-Coil tag so you are not talking about peptide-peptide interactions here and it's a very you know strong interaction not having so much material printed on the chip surface so you have you know no you know the same issue what you have seen earlier probably could be resolved by using the new chemistry of Napa so now if you had the self-re-expression system on the chip itself and imagine everything now you're doing on the gold slide earlier what are you doing on the glass slide but Napa chemistry is modified with the E-Coil and K-Coil peptides so now if the proteins are being formed with the you know this interaction of E-Coil and K-Coil peptides the haptamards being formed now you will have the tight interaction you can see these proteins synthesized on the chip surface and which could be then detected and used for the further interaction studies so now the baseline which was seen here earlier now the same baseline is much lower here with the new chemistry right so the old chemistry what's in the new chemistry and now you can have much more room for measuring any kind of binding responses you can change different concentration and you can still see how much binding you can observe so this case is not only entire GST which was injected for testing how much GST proteins are being synthesized so several experiments were done in this case initially you know various type of proteins were tested for expression first of all whether on the gold chip with the new chemistry can we detect the protein expression so every protein has the GST tags so in this case you know handful of 8 or so protein were tested out and you can see there all of them are showing binding but is there the real binding or the bulk effect you have to negate those possibilities so another experiment was done where sequential antibodies were injected starting from anti-P53 and type 4 and type June antibody on the same chip and now if you can see the triplicate spots are showing binding in response to those particular antibody which you are injecting then probably this binding is not bulk effect you are what you are measuring is protein specific antibodies you are using so protein specific responses you are observing right in case of GST it can it can be something you know everything is showing your response so probably may not be very confident thing that it is proteins are always being expressed but if you are using a specific antibody for P53, 4 or June then probably you have a specific type of you know measurements which you can make here and then you know some protein interactions were tested using June and FOS as a pair FOS was injected and June was you know printed as the cDNA on the chip and there was some binding observed similarly MDM 2 and P53 were tested out and then you know those kind of things showed some binding so you know a next experiment which was planned which was more kind of high throughput experiment and which was very challenging to really do that thing on the prototypes is what we had available where we have you know from one of the companies in Cambridge that time gave lot of clones of P53 mutants and these are all hot spot mutants of you know different P53 regions to test out you know how MDM 2 protein it binds to the wild type of P53 versus a different type of mutant forms of it and our this is as a cartoon images to show you we are trying to look at 144 spots binding how that can be measured simultaneously once you do the experiment it was very you know first time high throughput type of experiment being performed 144 spots binding you are trying to observe simultaneously but it was very complicated because there was no software available that time which could do normalization which could process the data and there was you know lot of issues especially we were also finding that you know probably there was some sort of bulk if it we could observe even in the control spots there is some sort of you know binding which one could be observed so for the next steps of these kind of technology development which is even that time we were seeing that even for Napa chemistry there are you know the halotech chemistry which you are seeing now is showing much robust signal so could we now try your the same thing on the you know SPRI platform using the revised Napa chemistry and that is where now people in the George lab are following up the work which they are building a new type of SPRI platforms where halotech based chemistry could be used with the chloroalkanes and then probably that will have much more robust signals which is what is required for the low abundant proteins for the clinical sample context so this is how the technology you know development takes place this is one of the approach you can appreciate could be used for high throughput screening which I am sure eventually will come to the market another thing is which you can think about coupling the surface plasmon resonance technology with the mass spectrometry based platform because just imagine that you know SPRI having you know its own unique property then mass spec can identify the proteins very effectively so if you are just doing a testing of a known protein with a known potential interactor on SPRI this is what you do usually right you have a known protein which you immobilize and you are floating one of the potential known protein to find out the interaction so then you are not doing much justice to your actual experiment and the technology because you are already knowing the things so much and then you are saying these two are should be binding but now you are I am confident they are actually binding this is what you do on SPRI right what can be actual power of the technology that you have a unknown protein printed and now you are passing your you know a lot of compounds and you know you can see are they binding or not right and then can I discover something new which is binding but what is that new thing which is binding which you have no control which have no idea so then eventually you want to identify that particular binding with the mass spec so but you know in real situation if you have done mass spec you would realize you need enough of the peptides which is required for detection of those peptides and the SPRI experiment you are doing is things that you know just very low concentration that is not sufficient for you to give you enough peptide for doing the experiments so it is of course not a novel thought it is just you know a practical concept which we want to to employ to really find out the unknown interactors but to detect them using mass spec but even the practical concept can become very difficult when you do the actual experiments so the concepts are you know very straightforward we are employing one of the SPRI technology and one of the mass spec you know technology two together are trying to get the best of the two technologies to identify the unknown interactors in this case again we started as a proof of concept which are two micro globulin experiment and then use all the four flow cell because you need lot of analyte to bind which you can recover and then use for the mass spec testing so all the four flow cells were immobilized and now when you are passing your beta micro globulin protein then you are enriching them and we actually you know use it pretty high concentration 30 micro micro per ml flow rate was used 5 micro per minute all the four flow cells were employed after doing you know series of cycles so we did that for maybe almost 20 hours or so several rounds on all the four flow cells with the intention that you know enough of the protein should bind beta micro globulin should bind on the anti beta micro globulin and then can we chop off that particular bound protein beta micro globulin then get peptide is selected out of those proteins and then finally we candidate them using mass spec that was the objective so after doing mass spec sexes are not so great but still we could see eight peptides coming out of the same protein of interest at the top hit beta 2 micro globulin you can see here as a first hit of mass spec these are the kind of chromatogram which we saw so then we tried on another protein which was of the you know physiological interest lipoprotein E and intention was again to you know look for their binding this time we could see four distinct peptides so at least you know good news was and this one we are using QTOF platforms not the latest orbit technologies so we could see the right protein and it detects the right peptides which was good news but of course many times you do not have opportunity to run all four flow cells for so many round of cycles to really enrich your protein so how low we can go is what we have to now optimize using mass spec based platforms but this can be something which is useful for biological experiment when you really want to identify the unknown interactants where you see binding but you do not know the protein of interest so you can employ both SPR and MS platforms all right it's SPR MS you know now we have at least tested with five protein pairs which looks more confident we are kind of evaluating those technology to see limit of detection what could be best used and some of these could be you know heavily useful for the clinical sale especially serum type of samples you have a known protein of interest and you want to see you know where it binds with other interactants so lot of ligand fishing applications can be you know thought of build on this application which is what we are testing out and some of the GE application scientists are also working with this so this particular technology having lot of applications including it could be used for the PTM kind of analysis as well as the inhibitor screening analysis but you know now we have to really use the best of the mass spec power to go to the lowest detection limit with the minimal run cycle with the minimal repeat cycle then only I think this can make some impact in the actual samples of interest all right so in summary I think I have given you probably a broad overview of various interactomic field you know in which way the dynamic molecules to really understand them you need a gamut of technologies you cannot just rely on one that I have studied in this course and I can now start going using for every of my application you have to very open to look for various type of platforms which are available to you I am sure with overall you are probably convinced that you know there is lot of potential of using these technologies label free technologies and the different platforms available starting from you know looking at conductance based principle reflectance based principles interference based principles many of those could be utilized you can choose which platform can be useful for your kind of application but you know I think you have to really open to think about what should be your experimental strategy starting from where you should start from discovery workflow to reaching to the validation phase and then for for you know finally you want to translate those finding to the you know actual products so you have to follow you know a good pipeline which involves very much you know all kind of technologies which you are studying in this course as well which are being taught in the other courses in the proteomics field right now now you are able to understand the utility of label free technologies in the field of interactomics and also integration of novel technology platforms especially SPR imaging SPR with mass spectrometers as well as other technology platforms in the proteomics areas which could be utilized for multiplexing for these interaction studies that can even lead to identification of novel interacting partners or establishing with confidence the high throughput screening for the drugs in a given context the experiments that were discussed in this lecture would have also provided you an insight into wide applications of label free techniques it will definitely encourage you to think about how to plan these experiments using label free biosynthesis especially for your own experiments and I must say whether you are going to work in the areas of proteomics in depth or not but you will at some stage need to study protein-protein interaction or protein drug interactions in the upcoming lectures we will also discuss about coupling of immunoprecipitation with mass spectrometry to detect and identify the interacting partners and the molecules of your interest I hope these novel technology platforms which we are discussing in the last two lectures and in the upcoming lecture will make you much more aware of available technology platforms for your applications thank you


This paper proposes a new methodology to estimate ground-level PM2.5 concentrations using satellite-derived 500M AOD data. The proposed geometrically weighted regression, GWR, model combines the simplified high-resolution MODIS aerosol retrieval algorithm, CERA, AOD product, with meteorological variables such as PBLH, RH, WS, and temp to capture the spatio-temporal dynamics in the PM2.5 AOD relationship. The estimated ground-level PM2.5 concentration has 500M resolution at the MODIS satellites over past moments twice a day, which can be used for air quality monitoring and haze tracking at the urban and regional scale. This article was authored by Yang Bai, Lee Sin Woo, Kai Chen, and others.


dents learn things new and fascinating and relevant to them. I'm very excited. Chris Anglem has entered the waiting room. I have never been more excited in my life to be interrupted by a presentation. Chris, can you hear me? Chris? I think you're muted. You're muted. He can't hear you. Chris, can you hear me? Chris, can you hear me? Chris, I've never been so happy to hear your voice. All right, what can you see? How was his audio for you guys? Cody? Can you hear me okay? It's faint, but I can make out a lot of the most good words. The point is we're going to be talking today about challenges and the words of the day with the American-Tunneled Communities. Yeah, I'm good, but I don't think you'd be to use the next slide, but I was just going to say look, I'm going to reverse the article because the first thing that's different is the one bit, you know, for primary resources to get into there. The inverse is looking at the one bit, it's a, okay, yeah, I'm special. The University of Columbia, the University of Columbia and both the reference library and the art notes, the university art, and the district of Columbia for the seven years. The university, a little bit about the university, but we continue with the district of Columbia with sort of a lot of the university, created from several predecessor institutions, including minor school, wills, and future college, future college, federal city college, Washington, after which I'm a minor, who was an abolitionist of the form that I was in to the school, which I was able to make in 1951. We did that in the previous cities and all sort of nations, probably urban, land, land, university. Since I'm a minor, I formed a university in 1951, a university that played a key role in the education of African-American leadership of Columbia. Okay, next slide. Next slide, please. So before we talk about the rewards and challenges, we'll talk about what are the significance of HBCUs before the civil rights movement, historically black colleges and universities who are HBCUs offered evidence. One of their only pathways to a college is to the HBCUs to provide inclusive and supportive environment for African-Americans to obtain higher education. Okay, next slide, please. And one of their advantages today of HBCUs is that they provide a welcoming inclusive environment, one appealing factor, a feature of HBCUs and all educational institutions because of that provide welcoming environment for people of all backgrounds. HBCUs, a student, aspires to being themselves and being part of an inclusive welcoming culture and HBCUs do this very, very well. HBCUs have always provided a safe and affirming space for African-American students. So they instill a sense of cultural pride. They also show students from Canada who navigate racism, unfortunately, found in many other types of institutions. Okay, next slide, please. Next slide, please. Why itself? Since we're talking about learning about our guys, we talked a bit about HBCUs so we have a bit of background on this. Now we'll shift a little to talking about HBCUs and our guys and what. So we need to first understand why every student should learn how to use archives. So archives are not just places where documents or records are stored. They're witnesses to African-American history using archival materials about students doing things directly with the people and stories from the past. Students can hear about events directly from those experienced students who also relate to the lives and experience of the primary source readers to their own lives and experience. Archival records also serve the strength of the collective memory to protect people's rights, private and identity, children's rights and identity. And again, they help them understand the paths they came from and why things are the way they are today. And it's kind of lost as far as inspiration pride and growth over time. Okay, next slide. Next. Some of the significance, particularly of HBCU archival collections is that HBCUs have had a longstanding tradition of developing collections that document the rich African-American experience with rare unique materials. HBCUs have potential to enrich current narratives with the history of African-American education, scholarship and activism of university presidents back to the students and alumni. Despite their significance, however, HBCUs and archives are not easily accessible or much of the sense to do with the historical underfunding of these archives and the fact that they have not always been well publicized either. Okay, next slide. Next slide. So, some of the challenges that we have in particular in serving our students at HBCUs are that many are first generation college students. Some are not completely ready when they started. Many have re-research goals. Many may not be able to really understand their history. Some may be learning difficulties or other disabilities. Some are family needs that our parents are in need to support their family. So that is an added stressor to the pursuing academic barriers. And many face some serious challenging challenges which hinder their academic progress. Okay, next slide. And further challenges may be are that many are faced with issues of day-to-day means and have not had the opportunity to learn much about history or archives coming to the university and they may not be familiar with archives or understand the relevance or usefulness of archives. And most have no background in use of archives or the yet for any sort of grounding or understanding of archives. So we're starting from very much from the beginning stages and teaching them about the importance and significance of archives and how this can be an enjoyable experience for them. Okay, next slide. In some of our rewards in serving our students that are seeing the students enjoy and coming to their history and culture in ways that they have not before. Seeing students view their history from the excitement of the first person perspective to the stories of real people living real lives and making real contributions to the community. So the stories that can be apparent in telling in the to our students we're not talking about history in terms of merely dates and names and we're seeing actual stories being told for such a measurement of the current and also involved students. Can we go back to the last slide please? Seeing and just that learning that enjoying and owning their history and culture in ways that they have not before seeing the students view history from the excitement of the first person perspective the stories of real people living real lives and making contributions to their community seeing how students can add to the body of knowledge by adding their own stories and experiences or stories of their friends and family members to this whole story and history. So in my experience of as an archivist and teaching students how to use the art archives, primary sources and so on we first started off with primarily focusing on the institutional history and also on the student organization which was done by and the sororities and fraternities that got a great deal of interest in the great deal of and many of the students found this to be very beneficial or very helpful and so on. In recent years I began noticing a much more student interest in the areas of social justice and the universities of role in the subject of social justice and the way committee and the great animal work in terms of our universities' role in social justice or particularly in the black lives matter and events like that in the past four or five years we've been searching for racism and getting lost in violence and activism. And so we looked from campus and created some lesson plans that didn't really work well. We worked with various of our nation's campus that worked on social justice issues. We found this to be very rewarding and the students found it to be very rewarding as well, except with a light bulb or a lot of them, they'd see it themselves and they'd see the use of primary and source materials in a way that they have not been able to realize and quote. So we found this to be very rewarding and enriching experience for them and they also become better researchers and they've also become better in terms of better animals, better animals in these sort of events. So with that well included my presentation and with that I'll ask for any questions or comments or if you might have a problem with getting problems with my audio so if you might want us to get your questions invited you can use the iPhone and I will answer you with this iPhone. I'm going to do and I appreciate your attention and your coming to our conference today with interest in how they can do but please submit your questions in the comment section. Yeah, we will take any questions and we'll try and relay them to you or we can certainly try and put them into the chat, Chris if we have any. Is there any questions in the room? Anybody would like to ask Chris a question? Okay, I do have one question that Lee, so I'll pull up the chat window here and I'll see if I can pull that up here, Chris. You want to type it in? Or I don't care. What kinds of contemporary collecting plays here? Is it as simple as analog records? What type of contemporary collecting are you doing? Web, R, cutting, etc. Can you see that question, Chris? The question is what type of contemporary are you doing with R, R, cutting, etc. Primarily as we're doing a long-term campaign launch that doesn't get very influential in the process but the students are not therefore interested in a lot of them about the things you might I mean not the things you might want in terms of licensing but they really should try to work closely with the student organizations and the job that the teachers and the students are in the lead lead lead up we're looking for it and we are getting more and we're going to do a little fun with the last thing physical jobs and also a lot of work. I think we're up off of whatever archiving is much more than digital archiving as well. There's train pudding? In terms of what the student archiving is much more than digital archiving, is there a plot of a leader or an individual? Can you comment on that? When a relic of a leader or a worker is operating on the project, can you comment on that as well? Thank you. Any other questions? Over in the... Oh, I'm sorry? Yes? Can you discuss the new relationship with DCU and DC City Archives? What they're expecting to achieve and also some of the controversies that seem to be arising in the media? Discuss the relationship with DCU and DC Government Archives? Both the benefits that they're expecting and the negative consequences on campus. Consequences, right? Hey, how about that? Alright, another question for you, Chris. Discuss the relationship with DCU and DC Government Archives? The benefits and consequences on campus? I see work actively with DC Archives when I referred to quite a bit often to DC Government Archives when they have a need for those and I think the benefits of handling... I think they supplement each other very well and we don't... There isn't any sort of overlap between the two of them that are Archives and UDC Archives and strictly institutional Archives with some of the Government documents pertaining to specifically UDC but also to education in general and sort of benefits that seem to just be a relationship between the two of them and the students and the faculty and other researchers have benefited greatly from this and they've gotten the information that they needed and I can't think of the consequences that have been mutually beneficial for both the researchers whether they've been academic students or otherwise and for us so we value our relationship with DC Archives and the other Archives in DC will find this to be a very constructive relationship and we hope to broaden this relationship over time. Any more questions? Well, Chris, I really would like to... Chris, I want to thank you very much for making this possible and I apologize that you were unable to get down here in person and what I will do is I will share your email with people that if anybody would like to reach out to you and... If you want to come up to UDC to look at our Archives and visit our Archives and look where your research needs and the delays that we can deal with and sort of how you would like people to approach or build them. Excellent. Thank you very much, Chris. See Hang Lin at UDC, yes. Alright, so if anybody wants his email address, that's it there in the chat so you can reach out to him with any additional questions or comments and as he said there, I'm sure he would be happy to... to host you at UDC and see any of the rich collections that he's got there. But thank you all very much for working through us with this technical issues that we had with his presentation. Thanks to Chris for making himself available wherever he is somewhere in the state of New York. For those of you that are still here, we're going to invite Lee to come on up here. We are just going to roll right into our informal feedback session. Feel free to hit us up with any questions, comments. I'd like to say in any meeting that I'm in, moans of despair, cries of anguish, things that you would have liked to have seen, things that you would want us to do differently in the future. Lee's already said she's not doing it next year. If you, you know, put some money in the tip jar on your way out, that might be persuaded to do it next year. So, and if you're leaving, thank you for coming. There might still be some people downstairs. Yeah, you can go check them with those. Also, check out the rotunda. Please check out all the exhibits. I understand the World Series trophy is in the sports exhibition downstairs. All right, hit us. ASL shift change. Elizabeth? Yes. I'm not below starting just calling on people. I just did. I'm going to start calling on people because people whose names I know. There's only a few people left in the room whose names I know. I will just make a quick comment and say, just thank you all for coming. I think we started to plan this a little later than we would have in the years pre-pandemic. So thank you all for being up for experimenting and going with the flow with us. I think we're really thinking, especially moving into the next few years and seeing where the pandemic takes us. But just, I would be curious to know about whether the existing format that we did this time around, the same as we used to, still works. Whether you thought being in person was a good thing. Those are just some things we're going to be thinking about moving forward in the future with this event. This could be a time to transform it. It could be a time to keep the original structure. Just some food for thought. Well, I've come to that. This is the first time that I have come to a, you put on, like archives or PCRAs. And it's the first, you're coming out of the pandemic, which is supposed to mean that everything's back to normal, but it doesn't work that way at all. It's a great practice. So thank you for starting my good practice again. And I don't know that I'm sort of in a different archived space than most people here, but I would have loved if there was a round table time for a panel to hear about how people would feel like it was different. So kind of like a birds with a feather kind of event where people could choose like a topic, like reference or something and connect on it. Yeah. Or have maybe two or three different people from different institutions address similar ideas. Like ask me anything kind of thing, like just, you know. Or maybe a shorter presentation. But for instance, you had a person who was at about moving. So maybe two or three institutions that had balanced on the game for moving. Or two or three at a Spartan stretch. Or two or three that were working on student game as part of their tour. Thank you. Can I just wonder about this? I think this class section here might be really beneficial to hear about some different institutions. And then maybe even have some more clear examples of maybe some of the successes.  I think you could have benefited from having people involved with presenting. And also having some work. You're not really good at it. Maybe you could have more constituents as well. Like at the Kennedy Center, presentation is marvelous. It could have been even more expansive if you got like somebody not in archives to be using archives in a different way for the first time. For example, some school programs or an alley. The great mechanism in which we figured out how to get the right box onto the right shelf. That's all pretty amazing. And I would have loved to hear more about how that would be. So I will say that when the Archives Fair first started, the DC Archives Fair, it was started by the National Archives Assembly, which is a professional organization of NARA staff. And it was specifically targeted for repositories here in DC. We partnered with the Smithsonian Professional Group. We also used to bring in the Library of Congress Professional Group. And we broadened it to include the Mid-Atlantic Regional Archives Conference. And it grew and grew and grew. And then we had the benefit of being able to have technological advancements that we had originally were going to live stream a lot of these sessions. And we did live stream, I want to say, in 2018. And we recorded all of these sessions today.  And we'll send that link out to everybody when that's all ready. And so what we will do, and what we will continue to do, is that with more lead time, I think what Lee was saying is that when we have more lead time, we can hopefully have more sessions. We can have, you know, because we have this space, there's another conference room here, there's another conference room there. So we would have the ability to have run as many as three or four different session blocks, you know, three or four different sessions per block and have up to 12 sessions over the course of a day.  We could have theater presentations. I think the options are limitless. Now, I would like, actually I would like to ask Jim a question, is for people with where you are in a situation where you have a need, do you have, when you go to conferences that you, I mean, could you contact me to arrange for, to do this presentation, what is your sort of expectation when you go to a professional meeting as far as to be able to present and be presented to? Really, it depends on the expectations to evolve them. The team of interpreters, the flexibility of schedule, often we have a situation where we want to go professionally to a conference or a professional organization and we have a established schedule and we also decide interpreter availability and then often the professional organization has no budget to hire interpreters for the full day. Especially to be able to accommodate us, we all decided to go in different areas. So really this is the ideal setup, what we had today. So this logistically makes sense where they're seated, the camera, everybody moving around and that can be very typical and also it's a very common experience for us. So this has been really good. I was very happy that you were here today and I was happy that we were able to have those accommodations for you and I was in your presentation when I heard that you had gone to SAA because it is not my recollection that SAA has had interpreters in the past. Yeah, that's the point of the conference that we were talking about during COVID. So that was online. Right. And that does make it easier for services if they have online services and they're able to hire an interpreter remotely. But on-site can be a bigger challenge. And that's one of the feedbacks that I was going to share with you. So sometimes it's a good idea to also have hybrid mode for next year, maybe that could be a consideration to make the conference remote as well as in person. Well, we're glad you were able to be here. Me too. Anyone else? But I didn't really know that somebody happened to plug it and then just making sure that on absolutely every list or would I get sent out? I know I shared it with the Merrick Administrator. I shared it with the Merrick Caucus Reps, the DC, Maryland, and Virginia ones. I did put it on the Merrick Facebook page. We sent it out to Syask. I sent it out to the NARA assembly page. Where are you? Yeah. And actually my wife who works at the Smithsonian, she's like, did you send it out to the library schools? And I was like, yeah, I forgot about the library schools. And the thing is I can't put my finger on. There used to be like, you know, there was the old archives list, the Merrick Caucus list and like the SA distribution list. And I was like, I couldn't put my finger on where I needed to send it to make sure that I hit as many people as possible. But again, it's again, we've sort of lost how to get in touch with people over the past three years. Can we bear at work? Yes. What happened to you that it was last week? Yes. Then you know, like my lost thing like this? I'm also not coming in Monday. So I'm certainly hoping that now that we've had an archives fair in this new pandemic era, that more people will be aware of it. And then when we start planning a lot earlier in the next year, this will be a bit less of an issue than it was this year. I wanted to add something also. We got our information through Merrick too. And we wanted to know if we could see with the WR, are the world research library connection? Consortium. Consortium. So we could see more institutions that are similar to ours being able to attend here. Sure. That would be a good idea. Jim, do you have a contact for them? For the WRLC? Yeah, I can definitely put that on the webpage if you want me to. I'd be happy to. Can you just email me? Email me the contact? Absolutely. Thank you. Perfect. So I'm a NAR employee. So I would say even internally, it seems to be very much communication. I think there's one mention in the New Testament, but even the most recent, like we found out some things it wasn't mentioned on that list. Which I know in the past have been widely admitted by NAR employees. I know a number of colleagues who just didn't know. Are you an assembly member? I am an aspiring assembly member. I did send it out to the assembly membership. So it did go out to that. It was on the external NARA facing calendar. I don't know whether it was on the internal facing NARA calendar. And I did have some people post it internally. I don't know whether it was on the ICN though. But again, that was just something that it didn't get pushed out to as many outlets as I would have liked to have it done. And I think that was just a factor of we got the ball rolling late. And there are only so many hours in the day. And I would say also just trying to get a group back too. Yeah. It has been, what, three or four years? Three years. 19 was the last one. 18 is the last one I remember. But I think there was one in 19. Yeah. Sir. Discussing. Yes. Today was our in service day. Yeah. Right. Yeah. Yeah. So when we started looking at dates for the Archives Fair today, the reason we selected the date for today is because we wanted the archivist to be able to speak. And as she said in her remarks, she leaves for Seattle tomorrow. So today was the only day that she could speak this week. And this was the week we wanted. So that's why we scheduled it for today, knowing that she was on travel the rest of this week. And then we were like, OK, we can have the archivist, but we won't have anybody in research services because it was in in service day. Well, again, yes, one more in the back. I just wanted to say thank you again. There's plenty of honey at night. I hope it comes with lots of suggestions. But I just put together a lot of conference for the last week. I really appreciate how hard it is to get going again and to pull back all the little threads and pieces that you used to have in place and not anymore. This is not my first rodeo, but it's also unfortunately not my last rodeo. First pandemic, most pandemic. Yeah. Again, thank you all very much for coming. And we will see you in 2024 if not before. Thanks all. Thanks all. Thank you so much for everything. We appreciate you inviting us. Thank you for providing the interpreter. We very much appreciate it. Right. Yes. Thank you. Yeah, that's it. Perfect. Thank you.


Well, hello, dandies. Welcome to By You and Me. I'm Dr. David Fontana-Colderow, the public relations director for the NFC South. Yes, the NFC South. It's like the NFC North without sleeves. Please welcome my blind jazz band co-host and emergency contact, D.D. DeLune. Who that? He's not just a Saints fan. He's legitimately asking. He's blind. Now, I don't know if y'all done a bud. But we got quite the cage-and-cage match this week. We got Delana Falcons and the New Orleans Saints. For preview, we now go to Scarlett P. Street W. Tante live in Atlanta. Oh, greetings, sweet and avid. Scarlett, my dear, do you like the Falcons chances this week? Oh, yes, indeed. That quarterback's name might be Breeze, but I predict this Sunday he'll be gone with the wind. Pfft. Isn't the Tampa Red Book a mess? See, I noticed you was dressed like our division's top quarterback, Ryan Fitzmagic. Indeed I am, because I like looking like a dad trying to infiltrate a high school party. Usually it's hard to find Harvard boys in Tampa unless they miss their connecting flight on the Spirit Airlines. The only time I got from Harvard winds up in Tampa, is if he's chasing his missing teen daughter. I, for one, would like to welcome him to the NFC South. Yes, the NFC South, every bookie's nightmare. Well, this has been by you and me. Join us next week when our guest will be Steve Starkeesian and the French Vampire. Who lives in my backyard? Timberdome! Now, back to Terry Blueberry and the other Fox guys. Is it me or does Jimmy Johnson's voice sound a little less sexy later in the day? Oh, well that's because that's Tony Gonzalez.


Parents who constantly post their kids online are risking a lot more than just annoying their friends. By 2030, Sharon Ting is expected to play a role in two-thirds of identity fraud cases facing young people. Children's identities can be stolen and used in things like baby role play, in which Instagram users steal images of children, give them a new name and personality, and claim them as their own. Almost a third of parents who were surveyed said they had never thought to get their child's permission before posting them, and 55% said they weren't concerned about repercussion. Posts may seem harmless, but they live online forever and have the potential to be found by the child's future employers, friends, and romantic prospects. So the next time you see someone Sharon Ting, keep in mind that


I'm an everyday woman, woman, woman Welcome to Everyway Woman They're all the major series happening in the hospital I'm living my life, life, life Living day by day Live from Los Angeles, here's Everyway Woman I tend to baby their kids too much But then again, I don't have children It's actually now But if I did have children, I would too But you know what? But it's not just parents, it's the whole institution of anything our kids touch Like for example, the sports teams where everyone gets a trophy Everyone is not awesome Everybody's got that great How long have they been on this? Seven years old, though So? We need to be sad about it They have to learn disappointment Yes, there's always going to be someone on the team Learning to be as gracious as a loser Exactly And if you don't ever lose, then What's that going to teach you guys?  You know what? I feel bad My son knew I was working And he stubbed his fingers in the door And I was like, that's what you get Because I'm working I'm working I'm working I'm working You're not working at your group But that's me Because I know life can be hard But what I do is I try to make sure That they have a balance And I think as a parent You need to give them a balance You need to let them still be a child And still give them that balance of reality There's this new parenting technique That you can't let the child cry Anytime No, no 24-7 Physical touch I'm not trying to carry groceries To the car They have to earn a little dollar Or five dollars that I give them And when they have a birthday party We go to a party together You pick your cake You pick what color you want Because I don't want them to be lukewarm And confused all the time I want them to know Directly what it is that they want So when the time comes They can go after it Everybody else has to make the decision for them I have a point where my daughter She was very picky And we're trying to get her to start to choose clothes Now she's really super picky now But when we took part in that But we would lay stuff out and go Pick something And she had to pick something She had to pick what I put on her Anna has an interesting question What about what's on TV though? What is the TV showing? Our children Oh my gosh The TV show And I won't say the name But it was showing the kids How to lie to their parents See what I mean?  I don't know I don't know Some science I mean that's Because TV is changing a lot of things anymore It is But I was talking Our comedian Who was here And I was talking to a friend Who was telling me That they use television shows For learning moments Like okay this is a prison show And I was like You watch that with your kids And they're like yeah But I tell them If you do this and this and this This is where you end up This is what you want to happen to you So in some of those cases So it's just like that comedian Yeah it's exactly Earlier in their stay I was like Oh yeah I've heard that before But I have to say I have been exposed to The results of what happens When parents don't When kids are not told no I'm a human resource professional And I run an internship program Particularly for high school students And I distinctly remember We have program guidelines And they are very clearly communicated And we had one student Who was selected for the internship And they couldn't fulfill One part of the preliminary requirements Because they were going out of country And you need to be present for it So they had their parent call First the workforce development center That we work through They had the parent call there And ask for an exception And when they said no there The parent called me And asked for an exception And I said First of all If it's the parent calling And not the student I already know Who's driving it That's an automatic turn off But then when I told her no And now mind you I'm a program facilitator The book stops with me When I told her no She politely asked me Well is there anybody else I could talk to I let her know I said You know what It's your guys' decision Whether you take this trip Or you don't But I let you know Your son has a difficult decision In front of him And ultimately He didn't do it That's the outcome No no no I see a lot What's wrong with that Biggest section Well no no I would call anybody Who I need to call To get my child into a program And you know what Understandable But when we have 500 students Who all wanted to Be present And who tried To compete for this And weren't selected And we're willing To fulfill the requirements You know We have to draw the Line somewhere And that is something That when we hear no It's a good thing I remember reading a book And someone told me How your child know And I remember thinking What do you mean Life is about know They need to learn How to hear it How to accept it How to process it Right So I mean Life to be a land Of limitless opportunity So you must Got out of yes Yes And I know I mean I know who Called the producers To get you on We'll be back With more Everyway Woman Stay tuned Coming up next More Everyway Woman Are you in Everyway Woman Over the years I grew up without a father My mother had to play Both roles as a mother And a father With us today Is a single parent Who is not only making A difference in the lives Of his children But also in our community Stay tuned to Everyway Woman First in a voice And today we're Focusing on fathers We'd like to welcome Marcus Ham to the show And I want to know What it's like to be An active participant In your son's life While being separated From his mother I think the concept Of a co-parent Is one that You don't really hear About in society But when things Don't work out Between the parents I think it's always Important for those parents To, you know, Just still work To take care of the child How do you do that? Well, you just have To become a daily Active part Of the child's life You know, it's It's mandatory, you know And you have to be Motivated to do that You know that this Child needs structure They need this, you know Both parents in their life It makes such an impact On every stage of their Development from birth All the way up into adulthood How do you... What if one partner Is not willing to be An active parent? How do you work over that To stay to, as you said Be in part, because it's mandatory How do you work through that? You know, unfortunately You know, everyone has Different circumstances It really does take That individual to kind of Step up and really look At themselves and ask The question, why? Why do I not want To be involved in this child's life? You know, is it because of The mother or the father? Or is it because I'm afraid, you know Maybe it's just I don't know what to do No one knows what to do When they first step into A child's life Were you afraid of that?  You know, I can't Change it, I'm not Going to try it You embrace it It's interesting though Because so many single moms Right now are not letting Fathers participate They're saying I've got this I can do it all I don't need you And I'm so glad that You're really giving a face To the father and saying You know, you do need me Yeah, we all have Our roles in a child's life What does your role look like? My role for my son I think really has a lot to do With giving him a Mirror image to look at You know, he emulates A lot of the things that I do Like what?  Marcus, where did you learn Where did you get this concert from Where did you learn The importance of being a father? Well, thankfully I had both parents in my life And my father played a very strong role And continues to to this day What did he teach you?   Even at a younger age He kind of warned against the potential Of, you know, being in a situation Where the relationship may not work out And how important it is that No matter what, you know You need to be a father to that child And Joseph made a good point That oftentimes single mothers Say it can do it all Talk a little bit about the notion That the only man who can teach a man To be a man is a man What are your thoughts about that? Is the Superman? I do agree to the extent And of course I understand That this isn't always possible In everyone's life And there's nothing to be held against Any child that doesn't have A father figure in their life But I have to say that, you know Being a father and seeing the difference In what my role is in my son's life I know that my role is necessary to him And I know that it's going to make A very big difference in the outcome Of his future And I think that more fathers Need to take on that position And be a daily part of their child's life So what would you say to those Deadbeat dads? I would say, you know It's time to man up You know, it's time to put aside Whatever your personal issues are No matter what they are Whether they're personal From your childhood Or whatever is going on Between you and the person in your life And just focus on that child You know, I love what you said About the roles And traditionally the role of the mother Is the nurturer What would you say is the role Of the father in a child's life The specific role?  I mean, before you were a father You were Marcus Right, I was Marcus I moved to Los Angeles From the East Coast And I came with the dreams Of film and fame Having a son, you know It became more about him Than it did about me And my focus went towards What can I do That's going to work for both of us What's going to guarantee That he has a future And give me the opportunity To grow as well So were you the same man now That you were then? I can say, you know In a lot of ways I'm not And I say that because For the better? Yeah, definitely for the better Because it's not just me anymore I don't make any decisions for myself I make decisions for the both of us You know, Marcus, what's the one thing That you want your son to always Remember about you? What's the one thing? I think That he's grown in I just want him to remember That I was always there And I want him to know that That came with the choice That I made And that he's going to have That choice in his time as well Wow, that's powerful That you are always there Thank you for always being there For being that superhero dad And I hope that serves to An example to the fathers that are watching And the mothers let him be a father Stay tuned with more Everyway Woman when we come back We'll be back with Everyday Fitness Are you an Everyway Woman Are you an Everyway Woman Coming up next We have Carly Who's going to be talking to us About benefits of weightlifting I better see you at the gym Later after this Stay with us here at Everyway Woman We're going to set the record Straight on women lifting weights Our fitness expert is here To explain You know, I've always I'm actually excited Your hair First off, I'm loving your legs Well, this comes from weight training So this is why I'm here I saw you walking I'm like, I wish I had those legs Yeah I've always wondered though Me, you know A bit chunkier So I don't want to turn My fatness into, you know Muscle like I mean So you're trying to Like bulking up Yeah, bulking up That's what it's called I'm glad that you brought that up Because that is a misconception A lot of women are very afraid of Of doing You have to lose weight first, right?  But then you kind of said that Lifting weights You kind of burn calories too Lifting, oh yeah Lifting weight I didn't know that Lifting weight You're adding to your engine Which is your muscles Lean mass Muscles that you have to your body So the more muscles you have That's why guys have a high metabolism Because they have more lean tissue Or muscles What's metabolism? It's when basically Your body is using the food Or anything that you have Storage of what you eat And burning it up That's your metabolism So you have metabolism Just to keep you alive right now To breathe Your body is burning calories Okay And then when you exercise It's even higher And you're burning more calories So what's going to make a difference Is you have more lean tissue Such as men have bigger engines For the fact that they have more muscle They burn more calories Such as like I know women get frustrated When they do like a boyfriend Let's say My boyfriend and I are going to have A little contest And lose body weight And the women are working harder But the men are barely doing anything And they're losing so much faster Right For the fact that they're naturally Natural composition Is that they have more lean tissue And they have more testosterone To make them burn faster So then for example Let's say I go to the gym What kind of weights Would you recommend for me to do? Are you a beginner right? A total beginner So if you were a beginner You want to start maybe like 12 to 15 reps So that's how many times you're going to live Such as like if you're going to do Some ice curls Oh these are reps Right But let's say for women They have problems with like The rear The back of the arm So tricep exercise is very important So you could do maybe 12 to 15 reps Times With the weight Where you feel comfortable That you can do it 12 to 15 times So you kind of have to play a little bit So and you want to make sure That you're always pushing yourself A little bit So you can be stronger And you're building muscle So you can burn calories So do maybe The last few reps of those You should start feeling a little resistance You should feel a little tough What do you do for your legs? This comes from squats Lunges And heavy by the way Heavy lifting So I'm not going to get I think I look okay right? You lift heavy ones Yeah but in order to get these legs I have to push myself Challenge my body Challenge Exactly To get where I need to go So that's from heavy lifting And consistently Now that we're talking about heavy lifting What is too much lifting For anyone Maybe somebody that's watching right now Too much lifting I don't know if there's too much I mean if you're doing improper form So then you don't want to do too much Where you're doing improper form You get heart You will see that in the gym as well Let's say that I don't want to knock the guys But sometimes the guys Their yoga comes with weight training right And then you'll see them doing crazy Like swinging and what not And you know That comes with you know They just have this challenge And they want to be able to lift that weight But if they do that They can also get hurt So proper technique is very important In training How can they get hurt though From weight training You could throw your back out You know If you're swinging If you're doing let's say A heavy dumbbell curl Right Swing your body Right And then you couldn't be like Oh my back Right And you didn't even use The muscles to do the exercise You used your back Is it true that like This is one fear I've always had As you get older You can't exercise as much And I'm thinking like weights Right You Well people it's a little bit harder But actually it's more important That you exercise when you're older Because when you get older Your muscles actually atrophy Or get smaller And that's your metabolism decreases Thank you So much I learned so much Thank you for your advice And we will be right back With EveryWay Woman This has been an EveryWay Woman production Every day woman Woman Woman In every way Yeah Living day by day


How I made millions with Apple devices. Are you wondering what the best operating system is? Is it Android? Is it Apple? In this video, I'm going to explain to you how Apple has actually made me millions of dollars and we're starting right now. Hey, I'm JR Fisher. If you're new to the channel, welcome. Thanks for being here. I really appreciate it. Don't forget to subscribe. There's a big button down there. Don't forget to turn on bell notifications. There's a bell next to it. You need to ring the bell. Turn on all notifications so you don't miss any of my videos so I can let you know. You can be part of the VIP family, the Fisher family, that gets notified as soon as I do one of these videos. Now I've been selling online since 2009 and I've sold millions of dollars of both digital and physical products. And I've got to admit, Apple has had a lot to do with that. In this video, I'm going to talk to you about why the Apple and iOS operating system is the best thing for you to use for digital marketing. Now, do me a favor. If you agree or disagree, put it in the comments below. Have you had experience with Apple or PC operating systems? Let me know what you think of it. Put it in the comments section below. Okay, let's get started. Now, when I first started my business, it was 2009. And I had a PC. I didn't really have a laptop at the time. I had a desktop computer and I liked it. I thought it was great. I'd had it for a few years. I was working on it. And I met my wife at the time and she was using Apple. And I thought, oh my gosh, Apple. That's that quirky company. I don't know anything about them. I watched her work on her computer several times. And man, it just seems so difficult and so foreign to me to screen on there. And I'm not a real technical person. And I kept looking at it thinking, wow, I don't want to get involved with that. That looks too difficult. Now, it came a point that I need to upgrade my phone. And I've had the Android system on my phone. And she liked Apple. She had a Apple computer, which I had watched her work on some, but it looked really confusing. But I decided that time I was going to go ahead and get an Apple phone. Now, this is going back to 2010. And I went ahead and got an Apple phone. Now, I remember talking to the guy at the Verizon store and I asked him, what was more popular? Was it the iPhone or was it all these other phones? And he said, well, it's about equal. I said, what do you mean it's about equal? He says, well, it's about 50% iPhones and then 50% of all these other phones. I said like Motorola and Galaxy and all the others. He goes, yeah, yeah, all those are about 50%. And then Apple's 50%. And I thought to myself, well, if all of those are 50%, then really that's spread out amongst a whole lot of different manufacturers. So actually Apple's way ahead of them. So I went ahead and got an Apple phone and I grew to where I really liked it. I thought, wow, this is a really good phone. But I was still using PCs. My office had PCs. I still had all the desktop computers were all PCs. And the only thing I had was an iPhone. But I did notice I was a little bit more efficient with the iPhone. I liked how the appointments all synced up on the iPhone. And it just seemed a little bit easier to me. So in 2012, I actually got a Mac Air and things changed dramatically from that point on. The Mac Air was so nice. It was so sleek. It was so fast. It never got viruses. It was easy to work on. Everything I did synced up with my phone and a whole new world opened up. I gotta tell you, it was a whole lot easier to work in this operating system that Apple had than I had ever seen before with PCs and Android and all that. And it's not just me that thinks this. There are some profound differences in an Android operating system and an Apple operating system. I wanna go over those so you can kind of compare for yourself and make your own decisions. But I gotta tell you, if you're doing digital marketing, it is so very important that you have a system that is synced up that everything works together with so that you can grow your business. It's gonna save you so much time and hassle. I can't tell you how many hours I spent trying to get viruses off my computer. I don't do that anymore. I haven't done that in over 10 years. It's just not a problem for me. Now, some people will tell you it's just branding or status that has iPhone in front of everybody else, but I think it's a whole lot more than that. Generally, if you're using the iOS operating system, it is faster and it is smoother than Android. I've used them both and I can tell you there's a huge difference. Now, the fastest operating system right now at the time of this video is the iPhone Pro Max, which I have. I have the iPhone Pro Max. I love the phone. It features a six core CPU in it and it is amazing. And sometimes we get lost in specifications and all that stuff and I'm not a technical person, but it has been proven that the operating system on iOS is just plain faster than the one that Qualcomm makes. It's just a fact. And I think it has a lot to do with the fact that the operating system for Apple just goes into one device, the iPhone. That's it. It just goes into one device, whereas Qualcomm's processor has to go in a multitude of different devices from different manufacturers. So there's gonna be some slowdown just because of that because they've got to make it to where it works with all these different devices. Now, the part that I thought was the most confusing was using the system and how it worked and how it differed from PC. But in fact, once you learn the Apple operating system, it is actually much easier to operate than any of the Android devices. Now, it is true that Android has really up their game and they have more and more things they can do, but essentially the thing that Apple users really like is the simplicity of the operating system. It may not do as many different things, but you really don't need it and that makes it cleaner and faster to operate. And what I like is that it's all based on a series of columns and rows so that you see everything you have on an iPhone.  The other thing that I really like about Apple is that the updates you get as soon as they're available. They notify you, you can update them. Now, the downside to that is that if you have an older Apple device, you may run into some difficulties. I generally will upgrade every two years so that my Apple device is the newest out there, but I will tell you, if you've got an older device, this can be something that's gonna work against you a little bit. But if you keep up with the hardware, you're gonna love this because your device is always updated. Now, it has been proven that certain Android devices have failed at getting the updates and they're very difficult to install on some of the devices. The other thing that's really cool is the seamless integration of all the devices. Whether you have an iWatch, whether you have an iPhone, whether you have an iPad, whether you have MacBook Pro, they all interact perfectly and they all update at the same time, which is a seamless integration which makes your work and your plays are a whole lot easier when using these devices. The other thing I like is that the actual Apple Store is policed and curated a whole lot better than the Google Play Store so that you can really kind of rest assured if it's in there, it's probably gonna be safe for you to use. Any developer who has an app has to go through a really lengthy process to get their app into that Apple Store. So you can really be rest assured that it's gonna be easier for you to use, it's gonna be easier for you to update and it's probably gonna work just fine. Also, the thing I like about the App Store is it's less confusing. It doesn't have a bunch of ads that confuse you when you're trying to find the apps. It just works cleaner and nicer. Now, Google has improved, I'm not gonna say they haven't, and their security has improved and that's great, but it still just doesn't stand up to Apple. Additionally, Apple's CarPlay works seamlessly. When you go and use the Android and the Google type of integration with your car, it just doesn't work as well. It just doesn't. Don't get me started on this one, but support, oh my God, support is unbelievable. I did have a PC I was using for another purpose and I remember one night I had to go to the Apple Store and I also had to go to this PC Store and the PC Store was the Microsoft Store in the mall. And when I went into Apple, they're so organized, they're standing there up front, they've got the iPad, they already know you're coming, they get you sitting down at a table there, somebody comes over, they're an expert at what they do, they get the problem solved, you're done. I went over to the Microsoft Store, you didn't know where to walk to, people would say things like, what do you want? What are you looking for? In Apple, they just seem to know. They just seem to know what you wanted and what you needed. I got sat at a table, I waited for about 45 minutes, somebody came over and they said that's not something I work on, the other person that needs to help you is over there, they should be done a little bit. I ended up spending about three hours there. Now I'm not gonna say I didn't get the problem solved, but I can tell you, it was not the same experience. By far it was not the same experience. They had really no customers in the store which surprised me that I couldn't get help, but in addition to that, they had very few employees and the ones that were there acted like they just didn't really care. Now my understanding is the Apple employees get paid a little bit more, but the one thing they all say is that the benefits are unreal. If you have to take off for a personal reason or you're sick or there's some issues with your family, it's not a problem. As far as benefits go, they all get benefits. They love the fact that they get benefits and it just seems like a better run company. Now let's talk about resale value. Oh my gosh, resale value is phenomenal on Apple phones. It just is. I always get a very large credit if I'm gonna trade in my phone or if I wanna sell it, I can normally get good money for the phone whereas with Android phones, that's just not the case. Maybe it's because there's so many manufacturers, there's so many of them out there, there's so many choices that it kinda dilutes the market. Now the iPhone XS Max was introduced in September 2018 and it sold for $1,099. That's a lot of money, don't get me wrong, but a used based iPhone XS Max is currently about $400 to $500 on eBay which means that's only about a 50% loss. That's just not the case with Android phone. The Samsung Galaxy Note 9 was released in August of 2018 which is just a month before for about $1,000. Only $99 difference and that's the 128 gigabyte version. But you can find those on eBay now for 250 to 300 bucks. So that's a 70% loss. So when you look at it that way, the iPhone actually may be cheaper. And keep in mind, this is one example. I know Samsung has Android phones and all out there, but the point is overall across the board, the cost of owning an iPhone, I think is cheaper than owning an Android because of what the value is when down the road. Now I'm not gonna tell you that the iOS operating system is categorically better than all Android. I'm not gonna say that. I'm gonna say I feel like it is. I'm gonna say I think it's a lot better but you've gotta judge that on your own based on the information I've given you in this video. Now I wanna follow up on one story. In the beginning of this video when I told you in 2012, I got a Mac Air. And I loved it. I loved it. I don't have a Mac Air anymore. I've got the MacBook Pro. And you may say, well, if the Mac Air, so great, why don't you still have it? This is 2020. And I will tell you in 2019, I looked at my Mac Air one day and I had so much on it. You know, I didn't have the memory on it that I wanted. But that was seven years later and it worked just fine. But I wanted another computer. So in 2019, I got my MacBook Pro, which is this computer right here. And that computer served me for seven years. And I will tell you all the years prior to that, I had PCs and I had Android phones and they just didn't last. I mean, I had problems with them. They would get too slow and the memory wouldn't work. I would have to constantly reboot them. And it was frustrating. And I think of how many extra computers I would buy in a seven year period. There's no way I would buy a PC and it'd be good for seven years. The MacBook Air that I still have works fine. I don't even need this computer. I just wanted it because I knew it would be faster and have more storage and all that. Now, I really appreciate you listening to this video. I'm sure I'm gonna get some feedback on what your opinions is, especially if you're an Android user and you watch this video. But if you're an Apple user, give me your feedback too. Let me know what you think of Apple. Tell me if you agree with me on this. You know, I love them. I'm sticking with them.  I swayed one time and I got an Android phone. I think it lasted like two weeks and I just couldn't stand it. I had to go right back to Apple again. So that was the only time that I left Apple in the past, what, 10 years, something like that. So guys, I really appreciate you listening. If you haven't subscribed yet, please do so. There's a big button down there. You gotta hit that big button. And next to it, there's a bell. You gotta ring the bell, turn on all notifications so you become part of the Fisher family. The VIP group that gets notified every single time I do one of these videos. I really appreciate you watching and listening. And I want your comments. Put them down there. I really appreciate it. I'll see you in the next video. Hey, thanks for watching my video. Don't forget to subscribe to my channel and click that little bell right here so you can be notified every time I do a new video. Also, click on one of those videos there. Keep watching on my channel.