Welcome to Monet Café. I'm glad you've joined me here today where we're gonna talk about how to create paintings with explosive color. This is part of my dynamic sky series actually on repurposed paper that I'll be talking about soon. I'm calling it fire in the sky. Hello and welcome to Monet Café and this video is proof that Monet Café is not a physical place but it is wherever you are and wherever your painting space is. As and where I am right now I'm not in my home studio but I am bringing lessons to you still from my parents basement. Many of you may know that my mother passed away not long ago and I'm here staying with my dad and we're just all helping each other through this grief and loss but I've set up in the basement here. They have a beautiful home and a beautiful basement but I'll give you a little tour of kind of what the setup is here and I'll be here as long as it takes to help us all get through this. So here is a little tour of my setup. The basement area I'm working in is actually bigger than my small home studio. So I have plenty of room but I did have to pack up a lot of supplies just to make sure I had what I needed. Lots of little things. I did actually video on some of those carry-all bins just a video or so ago from Arteza. I love those and I brought plenty of pastels so I didn't have to worry about not having something. I took their coffee table and put it underneath for a second layer under of papers and things I need. These are some extra supplies I have in the windowsill. Those are my neutrals there. So I definitely have lots of room which is very nice. Now lighting is always a challenge especially not just when you're painting but when you're producing videos. So I have my ring light you saw there. I love my ring light. I have that in my Amazon shop by the way along with a lot of other things in my videos and I have my computer editing setup station on one wall. Of course I have some pastel books for my patrons. I'm trying to get started back with story time that we have on my Patreon page. It's a lot of fun. Now this is the backside of my studio area on the other side of the fireplace. A beautiful cozy room which is definitely comfortable and peaceful. So I'm creating some paintings of skies dramatic skies on some repurposed pastel paper. I have a video where I showed how I repurposed this piece of pastel paper. It's you art paper from a previous painting and now I have already applied the underpainting using acrylic ink and watercolor and you art paper receives that beautifully. And I've also already applied some darks with one one little new pastel for the darks. So I'm gonna get started with this painting. You will see it from start to finish and I'm glad you're here. I hope you'll subscribe if you haven't already. Like this video, make some comments and I look forward to seeing what you have to say and see if you try repurposing paper yourself. All right here we go. Here I am marking off my painting area on the repurposed you art paper like I mentioned. My reference image is one that I took from my own backyard and I really liked that long format. So my paper even though it was wide wasn't quite the same dimension. So I just have a piece of masking tape here even though I use artist tape a lot. Masking tape works well as well and the area will end up being 8 inches by 21 inches. And now let's get started with this underpainting. I love underpaintings because they really give a roadmap for your final painting and in this one I'm using a combination of acrylic ink made by Dayla Rowney. You can use whatever brand you want and watercolor. So this is the golden color. I think this is Indian yellow acrylic ink. This video won't be about the underpainting necessarily like I do in a lot of my videos but you can still see the process. And what I'm doing here is I'm really just, I put some just directly on my brush here because I wanted it a little darker. What I had used before was a little bit diluted with water. But what I'm doing here is I'm really just getting the concept of the painting, the big shapes. I have a video I just shared on that and now I'm moving to watercolor. I'm just looking at this reference photo and I'm trying to create a little bit of the color and the energy. Now at this point you do not want to get too tied up into details. This is about the motion and the energy and just really giving yourself a good painterly very loose start. So this is proof though that you art paper is definitely water friendly. You know it is because I washed this paper off from a previous painting and now I'm using it once again to apply some wet mediums. The watercolor, I don't think I've ever combined watercolor and acrylic ink for an underpainting but I didn't quite have the colors of acrylic ink that I needed for creating some of the colors on this. So once again this is just a big energetic base to begin the painting. I'm continuing to use the watercolor to get some of the darker cloud shapes in the sky and again just creating big shapes. Also when you do this use the biggest brush possible. It really helps to create that painterly feel. And now I'm pretty soon I'm going to be adding some acrylic ink. I know that I can get a little bit of a darker color with acrylic ink and I want to go ahead and establish some of those darker areas. This is Purple Lake acrylic ink and I believe I did use this green one here. I'll have to get the name of that color. And first I just used the Purple Lake. Now I mixed that right from the jar. I didn't add any water to this and I could have used my other big brush but I think I didn't wash it out or something. So I'm using this just to get in these tree shapes. Generalities is all you need at this point. Actually my paper was still wet here. I didn't wait for it to dry. If I had waited for it to dry the Purple Lake acrylic ink would have showed up even darker. So just getting in my shapes and then once I'm done with this I am ready for the pastel. Once my surface is dry I'll be using a new pastel spelled in new pastel by Prismacolor. This one's called Spruce Blue. They're a little bit harder. I do like my Prismacolor new pastels. They're great for initial layers and I find myself using this little Spruce Blue for my darks. I needed to re-establish my darks. Now I'm going to make a point early on in this video that you'll see as I create the painting. You'll see this in action. I am typically going to be working darker to light. Now I know that those background trees are going to be lighter. Oh let me pause here.  It's like the little packing peanuts and it's really great for blending. Being away from home I didn't have all of my supplies. I usually use a piece of pipe foam insulation but I asked my dad could we find anything that has a similar consistency and I found these packing peanuts and I think I'm going to order them. Just keep them in my studio because they're really really great for blending. Okay back to the background trees. They're a little darker now but you'll see me in this painting working dark to light and I'll be talking more about that strategy throughout the video. Now here are the primary pastels I used. I love this Paris collection made by Sennelier. These are gorgeous. Now this is 120 half sticks of pastels and I love buying half stick sets because you get more for your money. You don't have to buy the whole stick. You get double the pastels for the same price typically. Look at these gorgeous oranges and reds. One of the reasons I chose this set for this particular painting and this is a great set with lots of different colors and values. I will be using another set of pastel for a few colors that I will show in a minute. So what I've done here is I have gotten one of the darker colors. This is sort of like a gray very neutral gray purple and it's not so dark like the trees but I'm just doing kind of what I was doing in the under painting. I'm establishing my large shapes.  If you just use one fresh out of the box they have these little tails. You kind of have to grind them down but because of those two aspects it's not going on as smoothly as maybe it would with a brand new flat piece of UART paper. But I don't worry about these things because I know that in time pastels start to layer themselves and usually with initial layers I do a little blending anyway. I'll break out my little packing peanut again. You see that? That's exactly where I'm talking about where it had a little ripple in the paper. So don't stress out if that happens. I do have a lot of comments here on the YouTube channel also in our Facebook group Monet Cafe Art Group and from my patrons from my Patreon page where you're a little bit like oh you feel like things aren't just working out because it doesn't have this smooth application but typically that's just the nature of this medium. Often we get our things smoothed out the more pastels we add. Now here you'll see what I was talking about the Sennelier pastels. This is one I haven't used yet and sometimes you kind of have to sand them off a little bit to get a smooth flat edge. So I just put me a piece of watercolor paper next to me in order to be able to do that with some of these pastels. And because this is such a wide format painting I'm having a little bit of a hard time and finding a place to put the little reference image like I like to insert for you guys to refer to while I'm painting. By the way my patrons from my Patreon page you will get some extra goodies from this video. One will be the actual reference image in your Patreon post and also you're going to get a breakdown of my colors. I'm going to give you a color chart. And by the way if you're not sure what a patron is I have a Patreon page. It's patreon.com slash Susan Jenkins. $5 a month is all it is to just support this channel and it helps keep these free videos coming but you also get not only some extra content but also it's really just kind of neat. I get to see your work more and I have a lot of other little special things that you can unlock by becoming a patron. I have the special groups, special Facebook groups, special Instagram group and a Discord server, all kinds of neat little things that our patrons can experience. So it's awesome and I love my patrons. Thank you all for supporting me. Alright, so back to my point about working dark to light. Some of these color choices that I'm making for the sky are not only darker in value than I may end up with. They are also more brilliant in color. You remember the title of this video? How to create dynamic color. That's one of the most common questions that I get is how do you make those color choices?  And let me brag on the Sennelier Paris set though. The 120 pastels that you saw that I showed earlier. It just is full of dynamic color choices. It has some brilliant oranges, reds, turquoisey colors and I've found it really is a great overall set. If you're looking to invest, Sennelier is a great brand but you're going to see my unison set soon. That's also a great set. Alright, so look at the color choices I'm making. You don't see that brilliant pink or magenta I'm using in the sky. It's really more subtle. But what I'm doing is I'm taking the colors that are already there and I'm intensifying it, making it more dramatic. Now the neat thing is just like with the value, I can tone down the color and the value by my layering choices that I make later. I'm going to talk a little bit right now about focal point and that will tie in to what I'm about to talk about with how you can neutralize certain colors. You want to identify your focal point early in your painting and I think from this reference photo it was pretty easy to see. It was that brilliant contrast between the light bright yellowy color of the sky and that dark cloud. High contrast is really something that our eyes naturally go to for a focal point. We're just that's just how we're created to see things that are high contrast with color and value. And so I knew that was the focal point. So my strategy is to keep that the highest contrast with value and also the most intense with color and mark making our eyes also go to edges and sharp lines more than they do soft edges. So back to my point about laying down some of these colors that are just more brilliant and dynamic. I will choose to keep some of them intensified with color. But as the painting progresses, I will actually neutralize some of the colors that are extending out and above and away from that focal point area. In other words, I don't want the eye to be drawn to the edges of the painting. I want it to come into that focal area and the other elements will be more subdued around the edges. Almost like a little halo or eclipse effect around the edges. So in those outer areas, I will neutralize the colors more, soften the colors more, and and I can do that by right now I'm doing it. Actually, I'm taking this little kind of a mauve pastel and I'm layering it and softening some of the darker colors, making the values a little less intense. Now I know there are going to be some spaces in between these clouds and once again, I'm choosing a darker value that I later will add lighter, bluish, leaning a little bit more towards turquoise values. Those little spaces of the sky that's showing in between the clouds. They make some really interesting little negative shapes.  But it's going to give a nice contrast for the lighter value I lay down on top of it.  They're already in the perfect little shape. This is almost the size that I prefer to use my pastels. You'll see me at some point in the video actually breaking some of the Sennelier pastels into smaller, in half basically. When I show my unison set coming up pretty soon I think. It's the unison 120 half stick set. The unison half sticks are actually just about the size of this little packing peanut that I have in my hand. The Senneliers are a little bit longer.  And so they actually can be for me anyway broken in half again, just to get a little size that's like I said about the size of this packing peanut. So remember when I talked about some of the initial applications not going down so smoothly and I said don't worry about that. The pastels will actually start to blend that. But also we can use these little blending tools. Now I don't blend a lot at final stages of my painting but I do in the initial working part the initial stages of the painting. I'm doing this not only to kind of smooth out some of that paper showing through but also to soften some of the edges of the clouds. You know if I was to give a piece of advice that I would think would be really up there with importance with pastel painting I would say that remember that the painting is a process. Often I think we we think it's supposed to look good before it really will. You may have heard a lot of other pastel artists say and you may have experienced this early on that your painting has to go kind of like through an ugly stage first where you're kind of like what is going on here. And so that might be something that initially might deter some people from pressing forward and pursuing this medium because you kind of just want to get results real quickly in your painting process. And it's a process and when we learn this process and we learn how to apply things and to be patient about it then we start to really embrace it and understand it's just the nature of this medium and really most painting mediums I would say even with oil and acrylic you you do kind of the same processes with blocking in, working big shapes to small shapes, working big values and color and then gradually getting to where you're honing in on the final finish. So don't get frustrated if you feel like your painting is going through an adolescent stage and you just feel like man I'm not really good at this. Oh my gosh my husband can attest to the fact that I did this so much early on a painting. Even when I started to get okay with pastel painting I'd start something and he'd hear me comment oh my gosh look at this is a mess. He says you say that at every painting but once again it's the nature of painting process. But the more you paint the more you start to realize this it's just the way it is and you gain more confidence. Alright here's something I would like to mention I'm creating a little bit of negative shapes in these trees right now. Notice how I'm carving into the trees some spaces rather than trying to draw individual branches but I wanted to make a note now I'm moving on with this pastel now but with regards to some of those brilliant colors that are going to be behind the trees peeking through with they're called sky holes it's the little holes in between things in the trees with things as with sunsets or really with any kind of background behind the sky the colors and values uh well let me talk about value first the values are going to be a little bit darker in those tree shapes than the sky just on the outside of it like say you've got a brilliant yellow sky behind the tree well in those little spaces in the in the negative shapes you're going to go a little bit darker in value it's basically because the branches are acting as a filter and if you put the same light value in those little spaces those negative shapes it's going to look like popcorn it's going to just stand out too much and be very unnatural now with the color in the little sky I'm sorry I'm not still working on the tree while I'm describing this but with color it's going to be a little bit more neutral sometimes you can have a few of them that are really brilliant if you want a good focal point but for the most part the leaves and the things of the trees are going to neutralize the color a little bit so often we go a little darker in value with the sky holes between the tree branches and a little bit more neutral in color so that's just a general rule of thumb so that was me just making some little notes kind of that I want to remember to do that all right you see I've been adding look at these brilliant colors once again all from the Sennelier Paris set and I am punching up this color it's really what is already there to a degree in the photo I often say too sometimes I think I see color in a photo that may just be so subtle and then I punch it up and if you want to get better at seeing color subtle colors I have in my patreon group I've given you guys a lot of if you're one of my patrons you know what I'm talking about I've given you guys a lot of little color tests and color games one's called this an app called I Love Hue HUE and it's a free app I think it's android and iphone but it's a color game and it will start to help you to see the subtleties of color and the better you get at that the more that you will be able to see those subtleties in a reference image or in life and punch them up make them a little more intense I sometimes see green in a sky that you initially wouldn't even think to put that in the sky or purple in a landscape and you know and sometimes too also just learning the rules of color allows you to get more exploratory and creative one is color temperature I have a video that talks about color temperature and if you understand it we know that things get cooler in the shadows right like a tree if you're hot in the sun you go out and you get in the shade of a tree and you're cooler so colors also get cooler well what happens when colors get cooler they get more blue or more purple so often in your shadowy areas you can add a little hint of blue or purple even if you don't see it at all in your reference image so you can learn to not only see the subtleties of color and intensify them if you learn the rules of color and color theory you can exaggerate those as well so those are just a couple of little tips once again i'm trying to stay true to the subject of this video which is creating dynamic color in a painting and you know after all i love the fact that many people do realistic painting and i'm amazed at some of the realism that can be achieved but for me personally as an artist i like to get creative i like to explore things that may not be there and to me that's kind of what art is we're made in the image of god i got a little feedback on my last video or a little negative comments for you know expressing my faith but i can't help but adore god's wonderful creation and we're created in his image to create ourselves now here is the unison 120 half stick set that i have been talking about i love this set as well i just had one of my patrons ask a question in our facebook group just for my patrons he's like which one should i get the 120 senelle a set or the 120 unison set he asked this even before this video and i recommend them both really you can't go wrong with either one they both have some brilliant color but i do think the paris half stick set has a bit more vibrancy in color um i love actually how the unison set is laid out so user friendly it has the colors and values laid out so easy to identify and also it has some great neutrals in the set as well so both very good sets again you can't go wrong with either one and now i'm doing like i said before i'm using one of the senelle a pastels this is one of the ones that i broke in half notice it's a little more neutral it's kind of a mavi color and this is what i was talking about being able to go back lighten values see the clouds getting a little bit lighter a little softer neutralize things a bit it's not quite as purply i think i had applied some purples you can see that purple over to the right in one of those right clouds now so um once you get the bold colors down you have the choice to be able to neutralize them soften them or not and so i always recommend getting your little bit of a darker value in first and perhaps a little bolder color in first and then you have those decisions with layering that you can make later to hone in on your focal point and that means sometimes neutralizing things that are further away from it i'm using the same color here to kind of get in some of those clouds um this is also another focal point strategy that i'm using i believe it's called convergence it's another term i think they use for it is pointing you notice how the clouds are all almost kind of coming in and pointing hey viewer look at that focal point right there where the bright yellow is going to be and so i'm doing that not only with the clouds i'm doing it with the landscape notice the type of strokes that even though they're preliminary strokes in the foreground i made them all kind of sweeping up and over a hill coming in towards that focal point area and so i believe i've really started personally as an artist to focus on focal point a lot more in the last few years i believe it's what uh makes a great painting there's a lot of elements of course that make a great painting but when you have a really really good focal point and the viewer sometimes doesn't even know why they're drawn to the painting but you as the artist have used your artistic license and your knowledge as an artist to lead the viewer's eye to the area that i think usually is what initially drew you to that reference image to begin with that's definitely what was powerful about the reference image to me was that it just looking into the distance now i get a little philosophical and faithful here because i do admire creation i admire our creator and what he's given us and so when i look at an image sometimes i personify things i think these trees are looking at a grand show a beautiful color display that the lord is giving them and it's causing them to look to the future look to the light and look to hope again i mentioned at the beginning of this video i've lost my mother recently it's actually been about a month now and um she saw the lord everywhere and what a believer oh my goodness i'm just so blessed i've had such an amazing mother i miss her so much but i look at things and i can see the hope that god has given us and i'll i'll stop there like i said i i don't want to offend people i did get some not so nice comments really some very nasty comments if you want to know the truth um from my last video not many most of you just don't even care what i go off on a tangent with but it is what it is i gotta be who i am and i know a lot of you like that and appreciate my honesty so notice now how i'm giving some little highlights of that sun is catching up on some of those clouds some of those brilliant colors are on the underneath side of some of those clouds also another rule with color temperature is things are going to obviously be warmer at the source of the sun thus the bright yellows oranges and reds but they're going to get cooler as they get away from the sun so notice how the colors as they're moving away to that right side there they're getting more pink and they're getting more um that magenta color and eventually getting even cooler so that's just the way nature works things get cooler away from the sun so another little color rule that you can keep in mind that will help your art even when the reference photo may not show it very um obviously a reference photo sometimes just inspires you and then you've got to take these rules and this knowledge that you've learned to create a painting that once again like i said lets the viewer see what you see and what you want them to see so i loved creating this painting i put on my um inspirational music i have some music that i listen to while i paint and it's music that i can't share on here youtube will give me a copyright strike if i share the music that has a copyright and by the way if you haven't heard the cello music of hauser oh my goodness it is absolutely beautiful it's one of my mom's more recent favorite artist and he's actually beautiful to look at too he's a handsome man and i can't help but love him because he looks so much like my youngest son or my youngest son looks like him so he's a beautiful person and seems to be a beautiful soul and his music is just gorgeous so if you want to find some good music to listen to while you paint it's usually very inspiring i haven't mentioned yet but i wanted to mention that i have sped this video up just a tad so that it's not so long this painting i think probably took over two hours maybe pushing closer to three i had to stop and start a lot with some stuff i'm helping working with my dad and helping him with a lot of projects like i said while we're working through the loss of our my mama so so anyway but that's fine and so it did take you know a little longer and i knew i wouldn't be able to upload all of that footage so but i think i am going to add some music since i was just talking about music at this point while you watch the rest of the painting process i might speed up the tail end of this video a little bit more to keep it under an hour but you've gotten 30 minutes of commentary here and by the way i'm gonna add a song here let me go see what the name of the song is it's from the youtube audio library that's a place i can go for my youtube videos where i know the music they provide does not have a copyright and that's not always my favorite music so i really have to search to find songs that i think are pleasing to go with the painting but this one is called almost in f and then it has tranquility after it i don't know if that's part of the title but it's by kevin mcleod he has a lot of music in the youtube audio library so i hope you enjoy this all right okay i gotta talk now right here is where i was talking about focal point my highest contrast with lights and darks is going to be in this area my most distinct mark making is going to appear notice right now how your eye is going to go right to that area over and above any other area in the painting and it's because of the intensity of color the mark making and also the contrast between lights and darks all right now listen to the music i hope you'll subscribe to this channel if you haven't already i hope you'll find me in all those little places i have at the end of my video like this video i love it when you comment and become a patron if you'd like it it really does help when you support this channel all right guys enjoy and don't go anywhere i'll be back at the end of the video i think i may have missed a little bit of footage here but i wanted to go ahead and give some commentary here now remember when i mentioned those background trees were a little too dark before i put them darkest value down first i am toning them down a bit now but using warm values typically when it's a bright sunny day i cool off the trees in the distance but because this is a sunset and there's so much light coming back there a lot of times we warm our background trees up by putting warmer tones and it creates that glow the sun is glowing down on these trees also notice the ground i have decided to do what happens in nature a lot of times the colors from the sky will be reflected down upon the ground and i used focal point strategies to also create that pointing feeling which is why everything feels like it's being your eyes just being pulled into that direction also to notice the color and value changes in the foreground they are going well let's go back to the back trees there they're going from the lighter values of the sun because that brightest yellow is way back there and they're gradually getting a little darker notice how they get more orange and then eventually i've even got a little might not can see it in this video a little bit of those pinks and magentas coming forward into the foreground towards the viewer's eye here is the final and actually i didn't have to speed up the video anymore so i hope you learned a lot and enjoyed this i'm going to show you again these are the senelier pastels i used i have a habit if when i use a set to turn them up so you can see or i can remember which colors i used but once again my patrons i'm going to make you a color chart with all of these colors i used and i pray you guys enjoyed this learned a lot and as always happy painting


Doing better and more than you thought you could in less time than you thought it would take while helping others do the same for themselves Excellence is always driving to get better Excellence is never an accident. It is always the result of high intentions to your effort and intelligent execution It represents the wise choice of many alternatives choice not chance determines your destiny Excellence is never an accident. It occurs with hard work dedication devotion higher level of thinking Empathy and the desire to be better Providing your very best effort without hesitance giving of yourself all that you can even if you believe you can Excellence is figuring out what others need in order to realize their goals Being one needs to be done at doing it without them. That's not my job When you put your own agenda and ego aside and focus on the people you can serve Bringing the best of yourself every day because it encourages others to do the same just lead by example Excellence is possible only with joy Excellence is remembering to bring gentleness and humor to work with you and never know when those two traits will come in handy Excellence is going above and beyond what is expected of you both as a part of an entity and as an individual without complaint or reservation Excellence needs no definition. You know it when you see it Excellence is someone who always goes above and beyond the call of duty to help others Excellence isn't taught it is achieved Excellence is knowing which work to do to the absolute highest standard as a showcase and which work to be done without putting fancy touches on Being happy with your life Seeing that excellence comes from a life of hard work and practice at being a good Excellence is preparation and taking the chance to improve Excellence is knowing that you have done your best under the circumstances and then move on to set new goals to achieve Excellence is not being afraid of your own screw-ups apologizing when you're wrong and trying even when it's difficult Excellence is letting go of the old ways to pursue new vision Excellence is establishing work priorities based on mission strategic goals or themes and values Doing the best work possible all the time just because it's what's right Excellence is the continual motion of striving forward building to be better than good exceeding expectations of yourself and others Excellence is doing the very best we can on any given day Excellence is the result of carrying more than others think is wise Risking more than others think is safe dreaming more than others think is practical expecting more than others think is possible Excellence is not perfection. This is an important distinction and the two should not be interchangeable Excellence is putting every effort into what you believe in Excellence is staying calm and accepting human limits


Good afternoon, everyone. And thank you for being here. I'm pleased to have Susanna Davis, our executive director of racial equity here with me today to talk about a new initiative called Ideal Vermont, which is helping municipalities do more to increase racial equity at the local level. Ideal kicked off last month with 14 communities signing on right out of the gate, including Bennington, Burlington, Brattleboro, Essex, Fairfax, Hartford, Hardwick, Hinesburg, Orange, Richmond, South Burlington, St. Albans, Timmouth, and Winooski. And while we want to see more towns join, this is an impressive start covering 18 or eight counties across the state. As we work to increase equity and make sure Vermont is a welcoming, inclusive state, getting communities directly involved is important. Susanna and her team at the Office of Racial Equity have been essential to our efforts. They've added an equity lens as we've developed new policies. So we make sure we're reducing barriers for everyone and rethinking the tools needed to address existing inequity. But this can't be just about a top-down approach. We need to help at the grassroots level as well, driven by communities themselves. And that's what this initiative aims to do. We want to encourage municipalities to commit to this work and to take action that fits the needs of their community. By signing on to Ideal, towns will learn from each other with the guidance of Susanna's team, as well as trusted partners from the Vermont Declaration of Inclusion, Vermont Community Foundation, Abundant Sun, the Vermont League of Cities and Towns, the Vermont Council on Rural Development, and experts from across the state and country. Members will share strategies, data, and information to take concrete steps toward a Vermont that is more equitable, more diverse, more welcoming, and ultimately more inclusive. And this local perspective and collaboration is what makes us think this initiative will have a real impact. It will reflect a community level of commitment to these values and bring local leaders with that kind of thinking to the table. So we're all pulling in the same direction. That's why I was proud to propose the funding to help get Ideal off the ground, though I want to thank the Vermont Community Foundation for its investment in the program as well. The fact is, we need to do this work. Because while Vermont is a great place to live with tight-knit communities who care about their neighbors, not everyone has been welcomed, and we have some work to do to make sure they are. We're not only doing this because everyone deserves to be treated with dignity and respect and have the same chance to build a good life, but it's also the right thing to do to put ourselves on better footing with a stronger economy, more workers, the best schools and prosperous communities in every corner of the state. As I've said many times, one of the biggest challenges we face is our demographics, which impacts everything, from our financial stability to our workforce. To reverse these trends, we need to attract more young and diverse families, which will help grow our economy. To achieve this, Vermont must be more welcoming to families from all walks of life, which means we need to respect and celebrate our differences. This is critical to making sure we have the safe, healthy and bright future we want for all of Vermont, and I think ideal can help. So with that, I'll turn over to Susanna to talk more about this program and how to get involved. Susanna. Gracias, Governor. So as the Governor stated, this is an initiative that brings municipalities together to take meaningful action on equity and inclusion at the community level in Vermont. The communities who are member municipalities are being supported by the Office of Racial Equity through an initiative that has several main components. One is convenings. The next is workshops, followed by technical assistance, some data coaching, access to grant funds, and access to other thought leaders and a resource library and online platform. I need to underscore that this initiative is not another exercise in more and more trainings. Recognizing the importance of education and training broadly, we also know that equity work doesn't get solved just through equity and inclusion trainings. It's an important component, absolutely. And yet after the listening and the learning have to come, the leading, right? We have to do after we've listened and learned. And so that's what this initiative is intended to do, to help municipalities and help municipal leaders who may feel either uncertain about their efforts or who feel maybe very confident about their efforts and want to be able to have the support of their peers around the state. We bring them together and offer them opportunities to work with state agencies and with each other to be able to implement their learning. So, for example, our convenings are going to offer member municipalities ways for them to do that reflective and introspective work to commune with one another, discussing what's working, what's not, challenges, aspirations, et cetera. Workshops will be more in-depth and will provide them an opportunity to hear not just from subject matter experts in the state, but also from those outside the state, perhaps national or international thought leaders on related topics. There is technical assistance provided by our sister agencies so that once municipalities understand the menu of options that they have available to them to implement equity at the local level, we have people in the state agencies who can help them actually do it. Concretely, what could that look like as an example? Well, a municipality might say, hey, we want to have a more equitable housing or zoning or land use policy. Well, we have a lot of land use experts in state agencies and in state government and also in places around the country who are able to help you get into the weeds about what does it mean to change a policy, or to update a policy, or to think of something new. Then we also have data coaching, which is going to help municipalities not just understand the value and the importance of data, both qualitative and quantitative, but also helps the state to be able to see more of it. There's only so much that we have access to and we find that building trust in communities means that you're able to get eyes into qualitative and quantitative data sets at different levels. So the data coaching will help us have more visibility into what's happening in Vermont's communities and it also helps Vermont's communities understand and utilize data in a way that makes sense and is actually helpful. We're also providing a web platform for member municipalities to be able to access that is complete with a resource library that offers everything from model policies to templates to reports, other information that they may not otherwise have known where to find. All of that is brought together in a way that's looked at and vetted by us to make sure that it adheres to our overarching mission and principles on equity and centralizes it in one location so that our member municipalities have access to it in a way that is efficient of their time. Finally, the access to grants piece is going to help even the playing field so that for those municipalities for whom equity work would otherwise have been cost prohibitive, we're at least able to offer them some supplemental support to be able to do the work that they otherwise are very passionate about. So we have about 14 members in the cohort, as the governor stated, and they range between a whole host of municipalities who are a little bit more well resourced to those who may not be. We have some cities who are larger in population and we have some who are a lot smaller in population and that's a good thing. That's what we want. Vermont is an eclectic and unique place and we need to see that reflected in our member municipality roster. Ultimately, this is an initiative that creates a bridge not just between municipalities but also between municipalities and the state. There are a lot of things that people look to state government to do and to fix and we're working hard on equity and we're not going to stop working hard on equity and yet there's a lot of built-in injustice in this country that has manifested over the years at the local level, right? Think redlining and zoning, think education policy, think policing and community safety. These are all issues that could in part be addressed at the state and federal levels but that absolutely have to be tackled at the local level as well if we really want to move the needle. And so what we're hoping to do is to work with municipalities to help them accomplish what they uniquely are situated to do. So we're playing a convening and assistive role in that we are going to continue to serve the member municipalities in the cohort and to support them on their journey and to also gain insight and strength from them in ways that we can make our work at the state level more effective. Thank you, Shazana, and we'll open up the questions at this point. I think it is about Vermont that has maybe held us back historically in this work. I mean, you grew up in a community that has very large immigrant population but yet you say that there's more that needs to be done. Why? You know, it's interesting. I do think back about my upbringing growing up in Barrie and I look at other communities as well, be it Rutland, having a very diverse background in all types of immigrants. But we stalled out, it seems. And I don't know if that was part of the economy that we weren't really growing. We had a spurt of growth and a lot of what we see, whether it's in the trades, particularly in Barrie and in Rutland, the stone trades. And that attracted many of the French Canadian population, those from Scotland and other countries as well. But then that, again, plateaued and then took a nosedive in some respects because of globalization. And much of the manufactured stone was done in China and India and it really did undermine the market. So we haven't had the growth that we, I believe that we should. That's why we've been focused on trying to grow the economy and to fill the jobs we already have but also to grow our diversity. So with growth, I believe that there's opportunity and we'll be working to make sure that we bring a more diverse population into Vermont. Can we learn from in this new chapter? I think that learning from our history is valuable. I mean, as a state, we are uniquely situated in between a lot of other places that are significantly more diverse racially. But I do just want to underscore the importance of recognizing that Vermont has a lot of diversity in other forms that may not necessarily be racial, right? There are already tens of thousands of us people of color in the state, but aside from that, you have diversity across the ability spectrum. You have gender and sexual identity diversity. You have a very, very interesting mix of people of different socioeconomic statuses living very close together, political diversity. So the state is already really well versed in understanding how to work together and live together alongside people who are different from you. But in the United States, race has been a particularly bitter factor that has been difficult for people in dominant groups, I think, to get over. And so, yeah, I think learning from our history is going to be important, but truth is we already know everything we need to know to be able to take action on matters of equity and justice. It really just means that we know that we have certain systems that have been set up intentionally to privilege some and disadvantage others. We know what those systems are. It's not a secret. The question is, are we actually brave enough to do something about it? And for people who have been in positions of privilege for a long time, getting to equity feels like something is being taken from them because they're accustomed to outsized privilege. That's really an experiential journey that people have to be able to go on, but we have the answers. It's just about people being willing to actually do what's needed. And does ideal Vermont contemplate those other forms of diversity, gender identity, sexual orientation, et cetera? Or is it more purely racial? Because it's an outgrowth of the Office of Racial Equity, ideal Vermont leads with race. When you correct for a number of other demographic factors, whether it's socioeconomic status, gender, sexual identity, ability, more often than not, race always is a better predictor of a person's outcomes because it is the factor upon which most of the discrimination happens in this country. So we lead with race because we see that as the most critical of these forms of inequities. However, ideal Vermont absolutely does focus on not just race, but other forms of equity as well because you can't talk about something like, I don't know, you can't talk about the fact that people who are trans or nonconforming are subjected to higher rates of attack or murder, especially considering the events of this past weekend, without acknowledging that within that group trans women of color are particularly at high risk, right? So the intersectional identities are important, and we do plan to highlight those wherever they're appropriate. When looking at the work that's being done through ideal, what types of metrics and data do we look for at the local level to show that it's paying off or to measure success of some of this work? Yeah, I don't know the intricacies of the ideal data points, but we do need to measure it so that we know that we're effective and that we're moving forward. Susanna, do you? Yeah. A lot of the metrics that we would use to measure the success of ideal Vermont really have to do with how government functions, particularly at the local level. Some of those are going to include things like staffing, recruitment, and retention in town government itself. Now, again, in some jurisdictions around the state, that's really easy to track because you have larger numbers of staff. In others, you may have three people who work in that office, and it's hard to really gain statistically significant numbers based on that. But either way, it's an important tool, right? Not just how local government is serving people, but who is part of that local government serving people. It also includes looking at things like the diversity and the inclusion of other parts of local government, right?  We're also going to pan out a little bit and look at more population level outcomes. Things like what are the justice outcomes having to do with local policing, right? Are they learning anything from this program and adapting their policing practices accordingly? We're going to look at things like home ownership rates, right? Things like approvals or denials of certain kinds of land use petitions based on factors related to equity. Things like policy change. How many unjust policies can we identify that exist at the local level that are being modified and at what rate? We're going to look at things like which municipalities are investing tangibly in equity. And that might mean making sure that local programming or youth programming is inclusive of more people. But it also might mean where are we investing? Big picture. We're going to look at things like school policies. Recognizing that the school system in the state is separate from the distinct municipal leadership system. There's still a lot that we can learn. We do have people in the cohort who do represent the school districts. And so being able to see the learnings from Ideal Vermont trickle into not just local governance, but also local education policy, et cetera, is going to be really important. At the end of the day, a lot of the metrics that we're going to be collecting may not appear to be flashy or exciting, but they're important because the small and everyday ways in which inequity shows up at the local level, that's the needle that we're trying to move. And when it moves, it moves in such small increments that we may not necessarily see it in the big picture. But one of the things that's really unique about Vermont is that there's a big focus on community and on civic engagement at the local level, and so those effects really are felt locally. How much of that money comes from the state of Vermont? How much comes from the community? $220,000 was committed from state of Vermont funds over the course of two years, supplemented by the availability of funds from the Vermont Community Foundation available to the municipalities to advance specific equity projects in their jurisdictions. Do you want to give us an update on where the Truth and Reconciliation Commission is at with its duties and what you see ahead as sort of the next step? Truth and Reconciliation Commission. So I am a member of the first of three groups leading up to the formation of the commission. We identified the candidates. We would like to invite to serve on the selection panel, which would then select the members of what will ultimately be the commission. We sent out a number of invitations. We had one-on-one conversations with our prospective selection panel members, and we had secured the correct number of people. We convened them for their first meeting. I think it was last week. And so right now the selection panel is getting up and running and preparing to potentially hire a staff person because that is permitted under statute so that they can have a little bit of administrative support while they work through selecting candidates for the commission seats. And based on your experience with that process today, how are you feeling about, you know, the prospective success of this endeavor? You know, I think that everybody has a really different perspective on truth and reconciliation processes globally, and to say nothing of here in Vermont. And so recognizing that mine is just one opinion. I tend to back load my expectations in this process. That is to say, I think that the greatest marker of our success through the truth and reconciliation process will be with what the state does after the findings are made. I think that truth-seeking and hearing people's stories and having them recount their truths and their histories is extremely important. And I don't want to undercut that. And yet we don't want it to be in vain by asking people to be re-traumatized by recounting painful family histories if we're not going to actually try to remedy it and make it right. So my greatest expectations really are going to be on the back end with what state government does in response to the findings. I said, you're the only person of color in this room, which is probably something that you're accustomed to in this state, which is the reason that ideal is so important. I wonder, when you moved here for this job, and we're so glad you took this job, and you're doing a fantastic job, did you always feel that you were listened to when the state government reflected you? Do you mean by virtue of my race or any other factors? Because I carry a lot of identities. Did I always feel included when I came here? Well, I mean, there are always very few brown people in the room with me, so numerically speaking, no. Although I do think it's important that as the country and the state grows increasingly multicultural that we also keep in mind that sometimes it's hard to know a person's ethnic background. And I have often been surprised in rooms not knowing who else is with me in the room and that there are people in the room who are also people of color. So have I always felt included, like as a professional in my job, by my colleagues, by my bosses? Absolutely. I did feel listened to. I do feel listened to. I think that a small part of that is people being afraid to look like racists, so they're extra, extra nice for reasons they don't need to be. But for the most part, I do think that we're making an impact. And being the lonely only has a lot of challenges to it.  But I'm measuring my feeling included by what we're getting done, how easy it is to get it done and to work with colleagues internal and external. And centering not really my experience, but the experience of people of color across the state because I carry a lot of privilege. And so my experience as the lonely only in a room is not the same as the experience of another person, a woman of color in Vermont in the room. So I'm a little bit less concerned about how that's affecting me and more how it's affecting other people who don't have the level of access that I have had. Governor, for you, obviously the idea of starting racial equity inclusion. But the recent shooting of Colorado Springs, I was wondering how you're feeling Vermont is doing with equity inclusion on all groups including LGBT communities and others as well? Well, we can always do better. But we're moving in the right direction, I believe, here in Vermont.  But again, I think we're ahead of many other states. We always, always have room to grow and more to learn and being more sensitive to the needs of others and being more compassionate, I think, is a big part of that. But listening is the most important thing. And again, being more sensitive to what others are feeling, how they're feeling and making sure that they feel included. And specific initiatives like I-Neal, do you think that's necessary for other groups as well in the state? Well, I think we can learn a lot from what we're doing here, again, just making sure that we're being more accepting, that we are, again, understanding what other people are going through, taking a walk in their shoes and just being more aware. But we'll learn a lot from this. And I do believe that we can utilize this for other initiatives as well. Because it really does have to be a grassroots effort. I mean, you can take a top-down approach, you can proclaim something, you can pass a law, you can pass a resolution, but it's not as meaningful unless it's grown from the grassroots. And everyone then is a part of it. And when you're part of it, you want to be successful. To that question, in the past several days, advocates for the LGBTQ community have said that there is a direct line between language, legislative proposals that minimize or dehumanize, for example, trans people, say that they are not welcome in certain spaces like bathrooms or locker rooms or whatnot with violence. There's a direct line these advocates say. Your reflections on that, especially in the wake of the Colorado violence? Again, yeah, in light of that, but in what we've seen over the last month here in Vermont as well, we just need to step back and tone down the rhetoric and understand what people are going through. And I personally believe that we have the right, the basic right to be whoever we want to be, the basic fundamental human right. And we shall accept that. We celebrate that, and we need to do more to make sure that we practice that. Do you think your own part of national, I mean local parties not here a lot, but nationally, do you think that they need to hear that? Yeah, absolutely. Yeah, I think we need to, I think we're doing maybe as well as any other state. We have a long ways to go as well, but we have a huge divide across the country. Did you open an invitation to all the municipalities in Vermont to join? We had, I think it was 80, was that what it was? Almost 80. 80 that we sent the invitation to. 83. 83. And then we had 14 responses. So, but that's okay. We have to prove ourselves. We believe that the 14 we're starting with will pave the way for others to see what can be done and learn from that as well. Because it's, you know, it's difficult for small communities to, from a staffing perspective, just understanding what's available to them. And I think this is an approach with our help to an helping hand from the state can show the way for them to take advantage of this as well. And are they, are these municipalities committing to anything beyond saying they want to initiate this process and go down this road? Are you looking for commitments from them that demonstrate to you they're ready to buy in in a meaningful way? Yes. So one thing that I do want to note is that this initiative is not invite only, it never was. We did send out some proactive invitations to that, to certain municipalities we thought could be a good fit, but it's always been open to all 251. And we hope that we will be pleasantly surprised by hearing from some municipalities who want to step up. But in so doing, they are committing, yes, to more than just saying, yeah, we like equity, we've checked this box, right? We're looking for demonstrable commitment. We're looking for actual action results and, you know, tangible steps. Now, recognizing that one could make one's best efforts at tangible steps and still not get a policy passed. That doesn't mean that you can't be in the initiative. But we're looking for commitment and we're okay with having a smaller cohort of genuinely dedicated members as opposed to a larger cohort of frequent absentees and those who are just, again, there to check the box. So what we're really going to be doing is drilling down deep with them to give them the information and the assistance and potentially the funding they need to make meaningful change at the local level, which means we want to see meaningful change at the local level. And sure, there are lots of low-cost, no-cost ways to do that, but we're looking for people to do the hard work as well. And are there going to be regularly scheduled check-ins, meetings between member municipalities and your office, for example? Yes. Okay. What were some of the main reasons why municipalities didn't want to step up? We didn't actually hear from anyone who said that they didn't want to be part of it. We did have a lot of people who we hadn't heard back from yet. Some of them are still thinking it over, I think. And I think we did hear from one municipality specifically who said we would love to, but we are short on staff, which is extremely understandable. I think some of the hesitation on the part of some municipalities is that they don't know what they're signing up for. They may want to wait and see based on what the current cohort is experiencing. I also recognize that there's a lot of talk around equity and inclusion, and it may be the case that some municipalities want to wait to see if this is worth it. And I get that, too. So we are committed to making it worth it for everyone who does join. But in exchange, we really want to see real effort. Governor, Burlington's racial equity, inclusion, and belonging office has developed an official policy of limiting access to the city-owned Burlington area community gardens to people who will sign a document declaring personal support for diversity, equity, and inclusion. What do you think of diversity programs and policies that, in effect, deny access to public services for people who won't sign statements of agreement? Yeah, I think we need inclusion on all levels. So we want to make this as easy as possible for everyone to be involved. And that means being inclusive to all walks of life. So I don't know about the policy you're speaking of, but I'm just speaking from my standpoint. I think it's important that we are inclusive across the board, because we need people to step up to join this effort in order to be successful. Ms. Davis, are you aware of that policy? I haven't had the privilege of being able to speak to that office directly about the details of it to be able to come down one way or another. I would want to know more about the policy before publicly opining on it. Governor, we are a week out from the election. Now that the dust has settled on some of these statewide and local races, what's been on your mind recently as you've kind of ruminated on the results? Locally, or here in the state? Oh, here in the state. Well, it's, again, unfortunate that I was a bit disappointed in some of the races. I'd hope that we would have and maintain the numbers to have some more balance in this state, but we lost ground. So we're going to have to, again, play the cards we've dealt. I'm sure we'll get a lot done, but we're going to have to make sure that we're all working together in order to do that. You mentioned Republicans lost ground in the House and the only statewide office that one was yours. What does it say about the direction that you're headed for? Well, I don't think it's just right now. I mean, this has been a trend for a number of years, I think, as I reflect. I think it's been almost, it's been over a decade and I've been the only statewide Republican office holder in the state. So this isn't exactly something that's changed, but it isn't getting any better at this point. And I think that some of the national politics we just need to be we need to do better. Again, we have I've been able to be successful from from my days in the Senate. I've always served in the minority. I've never served in the majority and I've always been able to get something done because I've been willing to work across the aisle. Bipartisanship, I think, is important. But as I've said many times, bipartisanship is a great term but it's a two-way street. It can't be a one-way street. So, need bipartisanship on both sides regardless of the numbers. Who would you like to see a certain minority leader in the next biennium? I don't know. Patty has been been leading but I don't know if she's going to continue. I haven't spoken to her. Have you had any conversations with the presumptive speaker or Senate president? Have not, but we will be I think we have something scheduled with speaker next week. But I haven't spoken to the presumptive President Pro Tem yet either. How do you expect that conversation to go? I mean, what's the Hey, have you been or are you going to be looking at a substantive conversation? We obviously disagree on a lot of stuff. What can we do? I think that's the way we approach almost every conversation on both sides. How we can work together. Where are there areas of common interest? Because there are a lot of areas of common interest here in Vermont. We just may not agree on the path to getting there. So we'll have those conversations and again I have a deep amount of respect here and I've worked with the presumptive Pro Tem Senator Baruth in the past. I served with him as well. So it's not as though I don't know the different personalities and different people. Can you anticipate a similar conversation? We've always had a good relationship and I look forward to continuing that. Since this is Thanksgiving last week, what are you thinking before this? Well, I'm again thankful that we no longer have the pandemic over our head that we've moved forward. We've come a long ways in the last two or three years. We've been through a lot together as a state and I look forward to have a lot of opportunity in front of us with things that we're talking about like today with ideal with some of the opportunities in terms of the economy. We're, and we have a lot of federal money that has been appropriated to us. We have an opportunity to really transform the state in a positive way. I look forward to doing the tough work, working together with others who have the same thought process and the same goals and I'm certain that Vermont, I feel very good about the direction of the state if we can work together because we have a lot of opportunity here. Well, as the Indigenous day of mourning approaches, I would say I am grateful for the fact that Vermont's Indigenous community has been welcoming this work and of me and has permitted me into spaces with them so that we can find better ways to create a meaningful meaningful relationship. Part of that is through things like the Truth and Reconciliation Commission, but a lot of it goes far deeper. It really has to do with day-to-day interactions, not just big splashy policies, so I'm grateful for them for being there for the day-to-day things. Okay, thank you all very much. Have a wonderful Thanksgiving.


Hello students. In the previous lecture, I talked to you about using protein arrays, how one could screen autoantibodies from the brain tumor patients. Ideas to give you the workflow, what is involved of doing the actual biological experiments, actual clinical experiments where one could try to obtain some meaningful insight from these protein chips or different type of proteomic technologies. So, in the last lecture, I essentially talked to you about the workflow involved in doing these experiments. And then I talked to you about one case studies on gliomas, serum sample, how one could probe that on the chip and try to find the potential biomarkers for autoantibody screening. Let me continue a bit more on that because it is important for us to know that same platform could be used for variety of similar diseases and also one could not only use the serum sample but also other type of bio fluids. So, I am going to continue that application further and talk to you about another disease right now which is meningioma. Just to give you brief context to that, the meningiomas they derived from meninges of the brain which is you know the duramatter, arachnoid and parameter just go back little bit basics of your anatomy and think about the geology and look at you know the brain structure and you understand that you know while it looks like broadly they are all brain tumor but they within the different regions of the brain these kind of tumors can arise and actually their aggressiveness and the way of treatment are very different depending on the location as well. So, these tumors which are meningiomas these are most common, most predominant type almost 37% of among the all these CNS tumors. But other tumor which you can see the bottom panel which is the gliomas while they are all you know the not the highest numbers but they are the most aggressive like the glioblastomalty for me they are most aggressive tumor type and they have the origin differently which is from the gliocels. So, meningiomas in general they are slow growing well differentiated and demarcated they are mostly benign but of course as the disease progresses towards the grade 2 and 3 they might invade the brain and then you know one could have even the various malignant effect and potentially the death of the patient. But that is not so common as compared to if you think about the glioma type of tumors which are fibrillary and diffuse in nature and they are very aggressive and very malignant and they migrate and invade the entire normal brain tissues. So, I hope you got some idea that you know there are different type of tumors and when you are obtained the patient serum sample from different type of brain tumors there is a curiosity that is the same type of antibodies are going to be seen or are there going to be different set of antibodies which are going to be showing the signal in response to glioma differently and meningoma differently. So, that is the idea for this experiment where if you see on the screen we use the same workflow as I talked to you in the last lecture you know like a western blot scheme we have the protein arrays we probe the patient serum sample on the chip which is having the primary antibody and now if the antibody antigen reaction happens then we are going to add the secondary antibody which is anti human IgG linked with the Psi 3 or Psi 5 fluorophores and then you can edit those signals. In this case for the meningoma we had the patients from the control and the grade 1 and grade 2 and an idea was can we find out some distinct pattern of the proteins which is unique for the meningoma auto antibodies. So, very briefly when we get these samples from Pratay Memorial Hospital where our collaborators are there from the clinical side before the sample come to us all the you know the patients information is available where each patients immunohistochemistry data is available and based on those information they do the WHO grading. So, the patients belong to the grade 1, grade 2, grade 3 and also their MRI images are there. So, when it is looking at the radiological based features of the tumor as well. So, now in this case we can see that the tumor is in which location is that based on a skull base or supertentorial. So, while now we are going to screen the patient serum sample from the meningoma different grades, but we already have information whether the patients belong to the which grade of the tumor and what are the radiological location of those tumors. So, once we do the screening but as you can understand in the protein arrays we are taking one chip and one patient serum sample. So, we are generating the data which is all distinct. So, one could reanalyze the data in different manner based on the WHO grading scheme or based on the radiological base classification. So, after doing the screening we first wanted to ask the question based on the radiological location of the tumor can we identify the protein signature which could segregate patients into the radiological subgroups. And interestingly some of the proteins like vimentin, myosin 9, fibronectin and ribosome binding protein they showed the different differences even based on the location of the tumor which is a skull base or supertentorial. When we started looking at different grades of the tumor now based on the WHO criteria we found some very interesting significantly dysregulated autentic bodies in meningoma patient. And you know I have shown on the left side image one of the protein cryome which is highly expressing in the healthy individual but in the disease of the brain tumor patients of meningoma you can see no signal is seen. So, there is significant down regulation but other protein like MAPK3 or EFCAB2 proteins they are showing that in most of the meningoma patients the signal is very high very intense. So, of course, some of these are the interesting observation which one would like to follow up validate and look for are there you know the distinct signal from the meningoma patients which one could you start utilizing it for the reduction of these kind of diseases. On the right side what you see those are volcano plots where we are like after doing all the analysis we are looking at which are the proteins emerging from the protein microarray platform which are quite significantly distinct when we compare from the healthy control with the meningoma grades. Further what we are looking at you know how some of these protein which are significantly disintegrated or significantly altered from the healthy individual to the great patients can we also validate these proteins using western blot. So, for some protein which we have antibodies we try to validate those and especially if you see the selenium binding protein 1 and TPD 52 proteins for those we also try to validate their levels using western blots and we found quite similar trends in both array platform as well as the western blot images. Further looking at all the alternative bodies which are changing from the healthy versus meningomas and healthy versus meningoma grade 1 or healthy versus meningoma grade 2 we try to map all those proteins into different type of enrichment analysis and we looked at which are the major pathways which are perturbed. So, we found there are 48 pathways where most of these proteins are mapping which are disintegrated in meningoma grade 1 as compared to the healthy controls and some of these pathways were quite relevant which are shown in this slide here which are based on the map kinase cascade, EGFR signaling, osteopantine mediated events, signaling by NGF or signaling to RAS. Many proteins like NRAS, MAPK3, MAPK1, PTPN11 some of these are already implicated in majority of these pathways and also some of them are known from other independent studies that they are quite relevant proteins in the disease biology of meningomas. Further we try to enrich these various candidates looked at their interaction networks and found that meningoma grade 1 gave us 4 distinct clusters of protein interactions whereas the grade 2 only gave us one cluster for the looking at the protein interaction network analysis. These are shown on the left side and the top right side. Further we have also utilized the geo terms emerging from the healthy control versus meningoma grade 1 and try to map these interaction networks in this figure which you see in the bottom panel. I think it is important to appreciate the different technologies are giving us you know certain clues which are quite relevant to look for any unknown factors for that disease but it is good idea to also see how robust those signals are. Can we measure those proteins from different platforms and still see the same pattern. So we took the same patient samples from the serum of meningoma and then apply those on the mass spectrometry based platform and looked for how many of those auto antibody we can also detect when we look at the serum samples from the mass pack. And this table shows that there are many protein where we could now start comparing our signal from the tissue proteomics versus auto antibody response across different grades of you know the grade 1 and grade 2. And interestingly many protein which showed higher up regulation even from the tissue proteomics using mass spectrometer they were also seen higher from the serum proteomics of the auto antibody response using protein arrays. So these are protein which are differently quite interesting and needs further investigation and validation on the larger patient cohorts. Likewise this slide shows we also had some proteins which were down regulated and several of them also showed common pattern when we measured them from the mass spectrometers or from the protein array platforms. Finally ideas can we also validate these proteins and when it comes to validation then many times we are limited with the antibody approaches. So in our lab and you know there are many scientists working worldwide in the proteomics field they are trying to employ different type of platforms for doing validation. And selected reaction monitoring which is mass spectrometry based triple quadrupole mass pack based assay it is coming very powerful way of doing validation especially in the context of when we very when we do not have antibody then we can do these kind of measurement of civil peptides and try to monitor their progression in the mass pack and look at their intensity and then try to utilize that information to look for the protein quantification. Of course you know this whole slide and content is very complex but the idea is to just simply give you the feel that the same protein we are trying to measure using western blot using the protein arrays and using the you know selected reaction monitoring based targeted assays and can all of them give us the confidence that these proteins are always showing the same trend when we are trying to measure them for the healthy versus disease patients. So from this part of meningioma patient serum sample screening for auto antibody using human proteomerase I hope you are convinced that this is our very robust platform which in a single short analysis can give you idea for how many proteins are dysregulated and in this particular case we found 489 proteins were dysregulated in the grade 1 and 104 proteins in the grade 2 patients of meningiomas. Then further looking at you know the much deeper biology of them different type of pathway analysis when we start narrowing down the candidates and we found several proteins are very interesting biomarker candidates for meningiomas like IJG4, TPD52, selenium binding protein and we try to also do their validation using different type of proteomic technologies. Now this work was published in Onko Targets again the work involved variety of collaborators from Qatar Memorial Hospital the clinicians some technologies from the Johns Hopkins University and we have even within IT Bombay the PhD students post-doc rates and other faculty members who are bringing expertise from the you know the big data analysis disciplines. So the entire team is then able to try to understand the complex problems and in the last lecture I talked to you about the gliomas and this was a study based on the meningioma serum sample screening. But when we talk about auto antibody screening it is always not limited only with the serum sample. So I am now going to talk to you about of the same patients we also had for not as many as I talked for the glioma and meningioma for the serum sample but we also had the cesium sample from the less number of patients and we thought can we start using the same platform of human proteomerase and add the cerebrospinal fluid samples on the chip and see what kind of auto antibody we can see when we have the cesium samples. So just to give you the you know brief idea when we are talking about you know the screening using variety of samples one could look for variety of you know a sample type it could be the tissue biopsies it could be the blood serum of plasma or it could also be cerebrospinal fluid. So the top panel shows you the variety of options available for looking at the biomarker candidates but cerebrospinal fluid that is the most proximal fluid which is present in the CNS tumors and 25% proteins are you know very specific for the brain only. It is less invasive technique although it is not commonly used for the you know any kind of test but in the complications when they are taking the CSF sample this could be a valuable sample for looking at auto antibodies. CSF is very attractive because it is closest from the tumor location and it has a lot of advantages which is shown in the table here. It very much mimics the biochemistry of the brain it is present with the blood brain barrier low complexity and that number of proteins have been reported which are very unique to the brain only in the CSF. So in some way it is very true reflection of the physiology which is happening inside the brain but it is challenging because the protein concentration is very low and the salt concentration is very high. So to get the right signal after removing the salt is one of the major challenge of course availability of the CSF sample is not always. So one would only use that when it is required for clinical conditions and it is invasive technique not non invasive although less invasive as compared to the tumor biopsies. So I will summarize the results in a nutshell in a very brief manner. So when we looked at the GBM patients auto antibody response using cerebrospinal fluid we are now able to see some new proteins emerging which we had missed out from the serum samples and one of them interesting protein is chalurin road GEF kinase. Now this protein promotes the exchange of GDP by GTP regulates neuronal shape growth and plasticity and you know it is actually involved in the actin cytoskeletal remodeling it is also detected as the driver 1 mutation in the other type of which in SCC cancer. Other protein which looks interesting is nuclear protein 4 which is expressed predominantly in the fetal brain and testis and localizes in the nucleolus and has a RNA binding domain. So both of these proteins showed in majority of the grade 4 patients very high and robust signal from the CSF sample whereas in the control we could not see much of the signal from these proteins. And the proteins interesting is the structural maintenance of chromosomal protein 1a and this is the part of a cohesion complex and very important part of the kinetro core it interacts with BRCA1 and is involved in the DNA repair. Other interesting protein was SW1 SNF complex subunit SMARCC2 protein which is a subunit of the large ATP dependent chromatin remodeling complex it actually regulates the transcription of the genes by altering chromatin structures. So again the similar kind of workflow and data analysis strategy was employed here you can see lot more heterogeneity in the different patient samples and the signals and the pre normalization the patterns are very different but only after post normalization when all the signal looks uniform now we can see that which are the proteins differentially expressed with high confidence and the below panel the the volcano plot shows if you are not normalized you will see large number of proteins in the red which are showing the differential expression whereas on the right side now you can see the less number of protein which are showing the differential expression after doing the post normalization. The similar strategy we applied for the CSF of the meningioma patients idea is can we now find out some new proteins which were missed out using the serum sample and of course we did see some new proteins emerging again in the left side you see a heat map the comparison of the control versus you know the meningioma patients which are from different grades and some interesting protein like RBPGA protein which is the DNA binding protein and also principle effector of the Norse signaling pathway showed up regulation in the grade 1 and grade 2 patients which was totally absent in the control. Another protein which is LDL red 4 protein which is involved in the attenuation of canonical TGF beta signaling and shows increased abundance which is also associated with the increased cell proliferation and migration in the hepatic cancer cells not known for the brain tumors but shown in the liver cancer again showed you know very clear signal distinct signal in the grade patients of meningioma grade 1, 2 and 3 and no signal was seen for the control population. Then ideas can we start mapping these proteins in the interaction networks and look at where they are showing the maximal role and it gives at least some idea that you know many of the proteins which we are identifying they are involved in the cytokine cytokine receptor interactions, transmembrane receptor protein also belonging to the serine 3 unit kidney signaling pathways most of them are showing some GTPase activities also involved in the membrane regions. So you start getting some idea for the biological consequence of you know these proteins where they are showing their immunogenic properties. So from this part we can conclude that a few proteins show distinct response when we looked at the cerebrospinal fluid samples both in the gliomas and meningiomas and of course we have the panel of the protein which are quite distinct in the glioma patients as compared to the meningioma patients. Proteins like SMC1A and SMARCC2 were found to be associated with chromatin remodeling and showed immunogenic response in the higher-grade patients of the gliomas. Authentic body response for the NOL4 protein which is a cancer testless antigen protein and chalurin protein was detected in the GBM patients. Few proteins like RBPJ, LDLRAD4, EDIL3, NINJ2 they showed significant immunogenic response in both meningioma grade 1 and grade 2 patients. I hope now you are convinced that protein arrays could be a very powerful way of looking at autentic body responses from the serum and cerebrospinal fluid and one could look at different type of autoimmune diseases one could also look at even different type of cancer and complex diseases where there is a possibility of autentic body production. Now, let us be shift gear and very briefly talk to you about protein-protein interactions using NAPA arrays. So, most of the study which I talked to you was essentially on the purified proteins printed on the chip and 17,000 to 18,000 proteins were printed and those represents the maximum proteome coverage for humans, so human proteome arrays. At the same time the same kind of arrays could also be utilized for studying protein-protein interaction. But protein-protein interaction becomes more you know interesting if you think about how best you are going to look at the protein expression in its you know most native condition possible and that is what I think NAPA arrays provide a much more powerful way because we are expressing the protein from the DNA directly on the chip in situ and then think about an experiment where when you are you know the query protein for which you want to measure the interaction if that protein also is the DNA and going to get expressed along with the protein when you add the in vitro transcription translation mix then that will be very powerful. So, I am just showing you this image which talks to you about studying protein-protein interaction using NAPA arrays using co-expression analysis. Here as I mentioned idea is that on the chip you have let us say 10,000 spots which are all having the cDNA and you are going to express the protein using in vitro transcription translation mix which we talked earlier the cell-free expression based arrays. Now when you are adding your query protein rather than adding the purified protein you are simply adding the DNA of that query protein. So, in this case we are adding the DNA query for the FOS protein but keep in mind that now you have to have some strategy for the measurement of the interactions. So, now idea is if the FOS protein is you know with the DNA is going to come and bind on the chip it is going to show interaction with the June protein which is known in tractor and might be showing interaction with some other protein as well. So, how to measure these protein? So, the FOS in this case was having the flag tag whereas all other proteins on the chip is having the GST tag and based on this if all the proteins are expressing the proteins on the chip and the DNA from the FOS then now we are going to have the proteins expressed on the chip and going to show the interactions where it is going to show the binding. This is an idea for doing the co-expression based experiment. So, a similar experiment we were also teaching some students at the Code Spring Harbor Laboratory in one of the previous courses which I conducted with Dr. Josh LeBair. You have heard Josh talking about different type of applications. I am trying to convey you here that these experiments are not very difficult to perform and these experiments were done with the participants in a workshop which we conducted without having the you know kind of you know big laboratory setup over there. We took these arrays which having the CDNA printed within APA chemistry and the expression was performed in the course by the participant themselves. An idea here was the same experiment can we use you can see the layout of the chip on the left side where we are hoping that you know the FOS is going to bind to the June with the yellow spots which are seen and on the right side you see after doing the actual experiment we could see those signals for the duplicate spots of the June showing the binding with the FOS query protein. We also looked at measurement of anti flag antibody and can we now measure the where the FOS proteins are binding and based on that we could now measure the signal and look for the quantification of that. So this shows another small array which again you know idea is to just show you the crew data the small raw data which even can be obtained in a small setting in a workshop which participant themselves are able to do these experiments and in this case again with the FOS flag query DNA which is after co-expression going to bind to the June protein when could start measuring the signal and you can see these are the duplicate spots which are seen for the June protein. And likewise we had another experiment here we showed that you know now when we are measuring the binding with the FOS not only that you know the FOS protein which is you can see in the red is binding with the June but also we could identify some new interactors and in this case FKBP5 was shown as a potential new interactor where the FOS protein was binding. Additionally now these arrays could be done for use for the other applications as well especially to look for the kinase assays and Josh has talked to you about various type of phosphorylation experiments but I am just again showing you this experiment done in the course by the participants where they took these upper arrays did the phosphorylation experiment dephosphorylation and rephosphorylation. Idea was can we monitor some of these kinase proteins and look at their expression and because we do not want to have any confounding effect coming from the ribate reticuloside lysate or IVT mix that is why we are doing dephosphorylation step in between and then again dephosphorylation to ensure that those phosphorylation what we are measuring to they are actually coming from the actual activity of these kinase proteins. So after doing that then we were able to measure the signal for you know the controls and dephosphorylation, post lysate phosphorylation, rephosphorylation of these proteins and some proteins you know look quite interesting when we start measuring their trends for the ABL protein, BCR ABL mutant we had on the chip and you know just it gives you the idea that lot of interesting biology could be studied very rapidly if you know how to utilize these kind of array platform to address biological questions. So I hope I was able to convey you that you know protein arrays could be utilized for different type of interesting biological applications looking at the clinical context to that one could look at auto antibody screening looking at the you know a protein of interest one could do protein-protein interactions one could also look at you know various type of activity for the PTM modifications like kinase activity and different ways of arrays can be utilized from the human proteome arrays to the NAP arrays. So protein microarrays are definitely a very powerful platform for various applications specifically for measuring the protein protein and protein other biomolecular interactions, post-translation modifications and biomarker discovery. Auto antibody screening definitely shows the power of using protein arrays. Directly from the crude serum or cesar samples we are able to screen this kind of you know the auto antibody response which is very close to the clinical type of testing which is not possible using very robust mass spectrometry based platform. Mass spec card you know advanced instruments but for doing the analysis you need to extract the protein out from serum and cesar samples clean them up digest them get the peptides out and then only analyze the peptide then do a lot of analysis. Now when you are you want to analyze the serum or cesar you have to also do additional step of depletion you want to remove the abundant proteins and then only you will be able to get the right signal. So therefore using arrays and directly by using the crude serum or cesar sample shows you the you know how powerful this platform is you will you are able to measure the the signal which is not possible from other technology platforms. I hope you know you are learning these techniques for in the field of interactomics and big data sciences but also able to appreciate where each of the technology gives you more advantage and where the technologies have limitations. I hope you will start studying more about not specifically only protein arrays but variety of protein technologies and start thinking about which technology can address your belcher questions of interest much more powerful and robust manner. Thank you.


Welcome to what Chelsea eats. Thank you so much for joining me today. I just wanted to come quickly and just talk to you a little bit about making choices. I get emails every single day almost with the call sign. Help me, help me Chelsea, help me. I need help. These fibroids, I need help. It's bleeding, I need help. I have anemia, I need help. And so I want to just say, first off, there are so many resources in the videos that I have on YouTube. So if you haven't taken the time to watch those videos, I know it can be labor-intensive, sometimes boring, whatever. But if you want help, that's the first place to start. One is free. One is good information. And one, there's healing to be had by following the advice that I give you on those videos, you know? So my suggestion is that you take the information that is free and run with it. And really, you know, and gross yourself into learning what it is that I'm teaching you because what I'm teaching you, it really will help you. So the second part of this is the thing that I want to say here today is you have choices to make. You can, and they're your choices. It doesn't matter what the choice is, but they are your choices. And you get to decide whether or not your choices are going to be good for you or if they're going to be not so good for you.  It is a choice whether or not you choose to eat healthy food.  Either way, it's a choice and it is your choice. And that choice is going to lead to either blessings or curses, unfortunately. I don't mean to use the word curses.  So if you choose to take care of yourself, you'll get the fruit of taking care of yourself, health, strength, energy, positivity. But you can also choose death, which is not taking care of yourself, not exercising, not eating well, not doing the things that you know you need to do to take good care of yourself. All of it is a choice. Now, sometimes our choices are like, we can't make the choice, we feel like we can't make the choice. You're so bogged down in how bad we feel mentally, emotionally, spiritually, we're just in a rut and we just feel like we can't make any good choices that we're stuck in our terrible choices, you know? And we don't have to stay in that vein. We can actually make good choices. So if you find that you feel stuck, it means it's time for you to turn to God and ask for some help.  And that's fine. It doesn't matter to me.  And if you choose to follow Christ, great. If you choose to follow another path, that's good too. Because it's the thing that you feel like is the best for you, okay? So, but all I'm saying in that is if you feel like you are stuck and you are in bondage, I am telling you that the way out of it is to turn to God. And when you are in a battle like that, it's time for you to hit your knees and start talking to the one that can get you out of the problem that you're in. So if you find that you can't make good choices, you wanna make good choices and you can't make good choices, ask him to help you.  You know, like knock on the door until you get the answer that you're looking for. But I think what happens is a lot of times we want God to change things in our lives and we want the results of having health and strength and all that stuff, but we don't wanna do what it takes to get there. And that's what we have to evaluate because God is there to help us. We sometimes just don't want to do the thing that's hard. And that's a choice too. That's why I'm saying everything is a choice. If we are in a rut where we feel like we can't get out, we feel trapped and in bondage, well, your choice, a good choice that will get you out of it is to turn to God. Turn to God and ask him to help you get out and follow through with it. Don't just ask one time, keep on asking until you get out of it. And don't like sit there for five minutes and just say, well, he didn't help me. Ask until you get what you need. And if you need help with this, put a comment below in the description box of this video and ask for help. Show up whenever I'm doing the prayer sessions. On Sunday, I will be doing a prayer session at 3 p.m. Eastern Standard Time. I think that's June 5th on Sunday Eastern Standard Time. And I'll be happy to pray for you during that time. But just ask for help. There's help for you. And also if you need to know something about how to help your body to heal naturally from fibroids, start going through these videos on this YouTube channel. Like there's a ton of stuff. If you don't have time to do that, you can set up an appointment with me to do a discovery call to see if my 90-day walk, the Fibroid Freedom Group Coaching Program is the right thing for you. And if it is, then maybe we can have you be a part of the group. The next group starts on July 11th. And you can set an appointment with me, set an appointment sooner rather than later, because I get booked up really quickly because I only coach on certain days for the discovery calls. So if you want to talk to me, get on my calendar and let's talk, let's see if it's a right fit for you. And if you're not interested in doing the 90-day program, schedule a 50-minute session with me so that I can find out what's going on with you and write a protocol for you so that you can get on the right path to healing. I will put links to both of those down in the description box of this video. But I just came impromptu today just to tell you that everything is a choice and you can get out of this situation that you're in.  You do not have to be looking eight months pregnant every day. You don't have to do that. It's a choice. It's a choice. You can choose to get out of this situation or you can choose to stay in it. What is it gonna be? You are able to do anything that you set your mind to and God is able to do exceedingly and abundantly above all that you could ask or think. Some of you want babies. You can have a baby. There's testimonies about women who have had fibroids and the doctor was telling them to remove their womb and they had the baby. A healthy baby, healthy pregnancy, that's possible.  So let me just tell you, I'm gonna stop now, stop talking but I just wanted to encourage you. Whether we talk ever again, I just wanna say you have, you can make a choice to get well, you can. There, right here on this channel is information at your fingertips that can help you on your way to health. All right, ladies, lots of love to you. I'll talk to you later. Take care.