Okay everyone we're going to get started and I want to welcome everyone to our virtual grand rounds today with with Sheila Baker who's on the screen here right now. And I'd also like to welcome I think we have our first cohort of applicants for our residency here and many of them will be online this morning so this is our typical and somewhat atypical as well grand rounds we needed eight o'clock on Wednesdays, usually in person but. And, but today we're having a guest lecturer from University of Iowa, and it's Sheila Baker, and Sheila just to give a little bit of background because I don't think Sheila and I have ever met in person. As far as I know so she's she's from the Midwest, or at least trained did her undergraduate degree at university University of Wisconsin, Stevens point and then got her PhD at the Medical College of Wisconsin. She did a postdoctoral fellowship from 2003 to 2005 at mass I near, and then was a research associate at Duke University. And since then she's been in Iowa at where she became an assistant professor into in 2010, and was promoted to associate professor, and she is in biochemistry so biochemistry of eye disease, and she'll be talking about KCNV to retinopathy a story of mice and men. And for those of the residents here we're doing our retinobole tomorrow so this could have been an it could have been a question on there because this. What's important about KCNV to, and she will. We'll talk about that and what's interesting in terms of electrophysiology today.  But when I saw the title of our talk and said, as a clinician, I do know something about that and, even though it's a rare disease I think it's on the order of a one in a million disease. I'm going to base here of our patients and I do see patients with this and all I had to do was write a quick email to Emily Spoth our genetic counselor, and she came back within 10 minutes and said, These are the three patients that we have with this disease. So, this is translational this can be a translational talk and to start things out. I'm going to have Lydia Sauer, give a just a very brief presentation of our and some pictures of our three patients before we go to Sheila. Thank you very much. I'm just going to share the screen here. I can everybody see this. So I'm going to talk about just the clinical cases of Casey and me to write an opposite that we have seen at the more and I center. And as Dr. Winston mentioned, there are there are three cases that we have seen all three of those have been genetically. Analyzed. The first one is a 23 year old male with a compound hydrozygous Casey and the two mutation with two pathologic alleles. The second one is a 37 year old female that is heterozygous in the US. And the third one is 16 year old female with a compound heterozygous that is likely pathologic. And with the US has a very similar phenotype and we think that she actually has the disease. And here are the three fundus photographs, as well as auto fluorescence fundus auto fluorescence intensity images. And I just want to highlight the clinical features and the general things about this mutation. So it's an autosomal recessive rod cone disease that is not up. One set is in the first to second decade of life, and it has a prevalence of one in 865,000 people with an incidence of five new cases per year. So it's pretty rare. And Casey and me to encodes for a voltage gated potassium channel that sets the photo receptor resting potential and is responsible for the voltage response. Symptoms that the patients will experience are central scatomas for visual acuity variable photophobia and red queen does comatopsia with a relative loose bearing. And then the findings that we see on imaging would be a ring like a wall side change in the macula central atrophy increased for real auto fluorescence and variable OCT findings with like variable auto retinal integrity loss of the ellipsoid zone and all hypo reflective spots. And all three patients showed this in both of the eyes. And the very important passive economic finding is the electro retinogram signature where in the dark adapted state, the a wave is normal to normal with a late negative component. The wave has a relatively high amplitude and it's called or can be super normal. And these are just two of our patients who had EIG imaging. And that's a very classic finding that's very specific to Casey and the two mutations. And I think that Sheila is going to talk more about that. And with that, I'm actually going to have Nana come up because he has some fun facts about her.  Thank you. Awesome. Good morning everyone so we couldn't let her start without a couple of fun facts. So she has two cats we might be able to get a glimpse of during her presentation, named Dungey and monkey girl. And then she has. She's an avid reader, a big fan of the murder bot diaries. So with pleasure, we'll pass it over to Dr Baker. And so you can begin your presentation here. All right. Thank you very much. That was lovely. Let me just share my get my screen set up here. Okay. Hopefully now. We should be good you should be seeing my title slide and able to hear me. Is that all good. All right, thank you. So, first of all, I want to thank you for accommodating my request to have this meeting virtually there are a number of personal reasons why travel is difficult for me. And one upside for me out of the whole chaos that is the pandemic is that we learn how to do virtual meetings better so I really appreciate you, allowing me to do this. I'm going to tell you about work we've been doing in my lab recently investigating the biochemistry molecular biology underlying casey and V2 retinopathy. And I didn't know we'd have such a lovely presentation so I have my real quick intro to the disease. So we commonly call it in the field casey and V2 retinopathy. And it's in that name is very sensible it's a disease of the retina caused by mutations in the gene casey and V2. And I'll be telling you all about how that affects the protein whose name is Katie 8.2. And more formally at least according to OMIM the disease that should be called retinal cone dystrophy 3B, but the only other name I have ever come across is cone dystrophy with supernormal rod responses. This name is very cumbersome but I like it because it helps me understand what's going on. And I also like it because it gives me a short acronym I can put on all my title slides. I will be going back and forth using CDSRR or casey and V2 retinopathy interchangeably. As we just saw the key features of this disease is that it starts early in the first and second decade of life. It's characterized by a macular degeneration that typically can start as early as the second decade of life and it's progressive. Sometimes it starts later. And when you have a macular degeneration you tend to get a lot of phenotypes consistent with cone dystrophy such as loss of visual acuity loss of color vision and photophobia. So this part helps with the name of the disease the first part is cone dystrophy. This is part of genetic explanations for why a person might have a cone dystrophy. What sets this particular disease apart is that it's also has variable amounts of night blindness in patients and most strikingly it's characterized by abnormal ERG. And that's where the part of the disease named supernormal rod responses come from. This is truly unique. And this, because the ERG is a recording of the electrical activity the retina this really gives me some insight into what's going on. And then I have one pitch fundus image from a patient seen at Iowa so as we just learned this disease is incredibly rare, but there doesn't seem to be any particular genetic or ethnic group where the disease shows up it just happens all around the world you'll find cases everywhere. And we have them at Iowa, it was actually, I mean, obviously feel bad for the patients but I'm quite glad to hear that you've seen them and you can help me understand this disease from your perspective. I will also make a point that I think that there's a high likelihood that this disease is under diagnosed. And to help explain that I want to tell you my own experience, sort of with a patient. So I'm a basic fundamental scientist. I'm a lab rat myself, and I don't interact in the clinics but when I first got this grant to study kcmd2 retinopathy I have someone contact me was really, really quite nice they just started by thanking me for actually studying their incredibly rare disease. But through a series of communications this particular patient volunteered their medical history in an effort to help me better understand what was going on and this had a huge impact on me and well as my lab very motivating I want to encourage you that when you do see maybe rare patients that that perhaps the PhDs in your eye center are studying, go ahead and invite them into the clinic and let them see exactly how the disease is affecting the life of their patients it can be really rewarding. So this particular patient, their history starts as young as five years old. They went into clinic they were treated for amblyopia, also reported at that time and he was already struggling with tracking small objects had poor color vision, especially under low contrast conditions. In his early teens he was really struggling with reading in dim light, and he went in for testing he was given a visual field test but he failed it. But there was no diagnosis given at that time that he was just told, yeah you just did a bad test, and they let him go on with his life so a few years later. And he went back for more testing because he was starting to notice progressively failing central vision, which he described as there's a dark blob over people's faces, which is exactly how my grandmother with amd describes her vision. Adaptation for this patient is delayed and he described that as anytime he moves from a bright room to a dark room, or conversely a dark room into a bright room it takes has to pause and it takes quite a bit of time for him to adjust. And he was a little cutie is not too bad at that point. And then, just a few years later, he was diagnosed with something similar to star guards disease. And the main complaints that he had is he continued to have difficulty tracking objects. When he was a young child that was tracking small objects that were hard for him. And now it's becoming harder and harder, even the large objects, including cars. So I was very glad to hear that he had stopped driving, although his excuse for stopping to driving is that he's having a hard time seeing stop lights road signs and potholes. I think you should be more concerned about the other cars more than the potholes, but there you go. He was under dim light, meaning rod media vision was becoming worse in his visual cutie was dropping off a little bit more. Finally, when he was 25 years old, he underwent genetic testing and finally had an ERG done, and that ERG and the genetic testing is what allowed him to move from you have something wrong with your retina that kind of looks like this other disease to an accurate diagnosis cone dystrophy with super normal rod responses, CDSRR or ACMV2 retinopathy. At this point, we really can't do very much for the patient. He's working with various low vision devices he uses a lot of contrast inversion works under very bright light, and on the and can still work on the computer had into use zoom and large fonts. I'm in a position where I'm a photoreceptorologist and I think that I know enough about photoreceptors that I was really intrigued by this disease and I thought I might have some ideas and be able to explain what was going on. And if I could do that, I thought that I could work with people such as yourselves and we might be able to come up with better options for patients like this person as well as your own patients and any others in the future. For those of you who are particularly interested. This patient did when he had his genetic testing done it's found that he was heterozygous for two variants in case you need to. One is pathogenic and I have a good explanation I believe for why that is. But if you're really interested in the molecular details, you'll have to stick around for my research seminar later on today. And then there's a mutation where we really don't know what's going on, which is unfortunate for the patient but good for me because it means there's more work I have to do. All right, so now what exactly is Casey and you to. It is a relatively small gene I'm sorry I think I'm sharing this window there we go with that a little bit easier. So it's a relatively small gene, simple two exons coding sequence is a little bit less than two kilobases. That's good anyone who's thinking about gene therapy like aha that can fit into a a b viruses if this seems like a good idea. And again it's very rare, but in the last 15 years or so I've come through everything in pub bed and I found at least 61 variants that I think it's reasonable say that they are definitely linked to causing the disease. When you look at the types of various show up in the gene, about 50% of them are nonsense mutations or frame shifting mutations that are going to result in the absence of protein expression. And I think that's interesting this is a case where such patients would be the best candidates if anyone were to develop a gene therapy for this disease. The other 50% of the mutations are missense mutations, and I've drawn on to this cartoon showing the topology of the protein Katie 8.2. So this is a membrane protein it has six membrane spanning domains. So what is the organization is that of a typical voltage gated ion channel. So it has a domain that functions, or can function in sensing voltage, and then it has a domain that will form a poor has a third domain that's a little bit unique to this particular ion channel as it has it's called the T one domain, and that's used to interact with other ion channels that are related to it. And I'll be telling you a lot more about that in a second. The missense mutations have been reported for the gene will affect the entire length of the protein they're scattered throughout the different domains, but the bulk of them that we know about today, impact the T one domain. So what is that T one domain. How does it affect the protein and then how does the protein affect our vision. Right. This T one domain is going to be important for assembly. So I have to tell you about now, you have to learn about more than just one item channel today. You have to learn a little bit about the larger family of voltage gated potassium channels. A family that subdivided into different groups you have KB one, two, three, and four. These all form ion channels that are expressed throughout the nervous system in the heart, the pancreas, and in several other places at lower levels. I'm always familiar with these channels because they are responsible for generating a propagating action potentials in the nervous system. Consequently patients with mutations in the genes and coding these channels, typically present with epilepsy. One of the things that I find interesting is that you can sort of fine tune neuronal signaling by adjusting the exact type of ion channels that a neuron will express in diversity in signaling within this family is achieved by mixing and matching different sub units. So it affects the biophysical properties of the channel. You get slightly different. Transmission effects. This family is divided. So this is a family where no kissing cousins are allowed. So for instance, this, the genes within the KB one subfamily can mix and match and assemble assemble with each other, but they don't interact with KB for KB three is alone. So KB two is the interesting exception. KB two has a is closely related to a large subfamily that is called the KBS for silent. And I will explain that in just a moment. I will say that in the retina, even though we have all of these different KB genes and the genome in retina. We only have two to worry about one from this KBS family are 8.2 culprit for the topic today, as well as KB 2.1. I think that they form an ion channel that would look something like this with the transmembrane domains for me to voltage sensor here, pour in the center, and then that T1 domain hanging down like a gondola and cytoplasm. So these ion channels are expressed in photoreceptor inner segments, and just a word of warning here. We all know that culturally we like to put things that are most important to us at the top. When we present slides, and as a photoreceptorologist, all my retina pictures have photoreceptors at the top. So you need to take a just a brief second and flip that image in your in your head to have it make sense.  So what you see here is labeling for KB 8.2 and 2.1. They're found in the inner segments of photoreceptors. Now, what's the deal with having both of these. They're having interdependence and first let me tell you a bit more about KB 8.2 and why it's called the silent channel. The easiest way to explain that is to show you this nice experiment from a paper in 2012. These are heterologous cells have been transfected with protein. You can see if you if you express KB 8.2 all by itself forms this beautiful reticular pattern, but there's none of it out at the plasma membrane. So if an electrophysiologist puts a patch pipette on that cell they will record no current. Hence, it's an electrically silent channel. So there's just no channel actually at the surface. However, when you co express 8.2 with 2.1. Now you do get it released from internal stores and it traffics out to the surface and you record a current. But having 8.2 assembled with 2.1 does affect 2.1 as well. So it's trafficking, but it's activity. So what happens in this is of getting a figure from the literature and so the paper in 2007. And this is a little bit complicated but I don't just take you through the key observation here, at least in my mind. So the upper plot is the normalized current plotted as a function of membrane voltage. What you need to pay attention to is this range down here. So photoreceptors in the dark adaptive condition have a resting numbering potential about minus 35. And then when they hyperpolarize a response so like they go more negative minus 70. So, there's a little bit too much information in this graph but if you'll pay attention to the lines with the square symbols this is the activation of data for this channel. The big squares low are just KV 2.1 in this channel forms a beautiful well functioning homo tetramer channel throughout the nervous system, but its activation kinetics are such that in a full receptor, it would be closed at all times. However, when you add in 8.2. Now the activation curve shifts the left. So that at photoreceptor operating potentials you do have open channels. Now there's not a whole lot, but that's exactly what you want because if you only have a small population of channels that have a high open probability. That means small changes in the number of channels that are open or closed are going to have a big effect on the output of the cell. So adding 8.2 changes the membrane potential at which the channels will open. The other thing that happens is it affects when they will close. So in the lower plot you're looking again at normalized current as a function of time at this time with 8.2 channels are open, they will inactivate with a set kinetics, but when you add in 8.2 that is slowed by nearly 50%. So I have taken you through this in this details, not because I'm an electrophysiologist and therefore you can't be fascinated by this even though it is really cool. But these two features, a channel that opens at more negative potential and has a very slow inactivation is characteristic of at the time this was published a channel for which we did not know the molecular carrier. It's called receptors carrying a current known as IKX. So just a potassium current with the designation of X because the molecular identity of the channel is not known. So the work that I'm describing here led to the proposal that the molecular carrier of this particular current was this heteromeric channel. What I hope I convince you of today is that when we look at mouse models, where we disrupt this channel and this has worked on my lab as well as two other labs in the last three years, hopefully you'll be convinced that this is indeed true. We know this. So the significance of that is more impactful if we go back take a step back and think about how photoreceptors signal. So, I'm glad that there are some younger students in the audience so hopefully this won't be too much review for for everyone. So we've got a cartoon here of a rod photoreceptor and we're zooming in now in the inner outer segment. As we know we have outer segment is responsible for light detection right this is our adoption this is a photo transduction cascade that biochemists love to ask you detailed questions about just to see if you were paying attention. The top shot is if you think about photoreceptor signaling in terms of the currents experienced by the cell is easiest to describe the dark adapted photoreceptor as having a circulating dark current. There's positive ions flowing into the outer segment in the dark. And this is balanced by positive ions flowing out of the cell from channels in the inner segment. So, the bulk of that positive efflux comes from itx or KB 2.18.2. Also need to point out that because the sodium potassium ATPase exchanges an unequal number of sodium for potassium ions. This is actually a net efflux of positive charge from this transporter. So it also contributes in a smaller way to this net circulating current. This is what keeps the photoreceptor depolarized in the dark went too fast that's okay when the lights flash on what's going to happen is activation of the photo transduction cascade. It's closed the channels in the outer segment, breaking that circulating current. There's no immediate response to what's happening in the inner segment. So the membrane potential moves more negative. Now the channel does deactivate it just does so a little bit slower, and while it's open and there's an opportunity for this channel to sort of help shape and filter the ultimate response of the cells. As the light continues or gets brighter, the cell will driven into more and more negative potentials until there's other channels that will help reset the cell until you reset the photo transduction cascade. And I mostly mention this current right here is on this slide, because in the last few years, 10 years we've actually done a lot of work on hdn one. So if you have questions about that, and you want to talk about it and you're one on one, we can do that. But coming back to Casey and the two disease, understanding that this channel is helping to control the resting membrane potential in the dark. And the fact that doesn't immediately switch off when the lights are turned on allows it to filter responses. Make let's us make some predictions about what would happen if you have a non functional channel, which is what we think is the case in our disease of interest. It should reduce or delay the photo response. So, in order to test this.  But I want to address big elephant room first of all. This disease is characterized by a macular degeneration and mice don't have a macula. So some of you might be thinking, why are you wasting my time with this. Well, I don't think I'm wasting your time I think we can learn a lot from mice, even though they don't have a macula. The only other argument is this in the mice, we know that they have a very beautiful array of rods that's actually quite similar to the human distribution and structure and function distribution. So whatever's going wrong in case in me to retinopathy that the rods would hopefully be phenocopy. In terms of the cones, we have roughly the same number of cones to rod ratio mice is in humans, we're just missing the macula and that high density for central vision. But that means that if what's going wrong in the diseases intrinsic to cone physiology, or is either driven by coupling to dysfunction rods, we should still be able to recapitulate the basic mechanism in mice. On the other hand, if all of your assumptions that it's silly to study a macular disease in mice is correct that actually by testing that in mice, I can confirm that. And we can say if we don't see a few copy of the disease, it tells us that there is indeed something unique by the structure or function of the macula that's contributing to the disease. And that will help us focus in on future efforts to understand the disease and treat it. So I hope I made my case. With that, we made a knockout mouse. This is a time when our mouse genome editing facility was fairly new so it's very fun to play with designing the, the CRISPR guides and making our mouse but we set this up to cause a deletion that would result in truncation of the protein very, very early on. We should get no protein. We do Western blots indeed KV 8.2 is gone. We looked at a few related channels and there were some minor changes we don't really know what those mean yet. So next I'll draw your attention over here this is our immunohistochemistry again photoreceptor site up in the wild type we have labeling in the inner segment. And in the knockout the labeling is gone. You'll also notice that in the knockout the outer nuclear layer is thinner so we have retinal degeneration. I don't know about that, but first I want to address the ERG phenotype because that's the diagnostic characteristic of this disease. And if this mouse model is going to be useful at all it has to copy that aspect. So here we go you saw some ERGs really quickly. Thanks to Lydia really appreciate that. These are some on the left hand side you have sample ERGs again taken from the literature. Here you have a stimulus condition that will evoke a rod dominated response. You have hyperpolarization of the photoreceptors reflected in the downward a wave. And then you have the upward reflection of the B wave that comes from the response of the deep polarizing or on bipolar cells in the inner random. So what you see here is that the a wave response is fairly similar all of that varies across patient. But what's unique and definitive is that the B wave is larger. This is the super normal rod response part of the name of the disease. If you use a stimulus condition that evokes more cone dominated responses that get normal patients give you a robust B wave here's the a wave beautiful B wave. But the B wave is now reduced when it's elicited by cone signaling. And if you use a flickering light stimulus to isolate cones, you see that again the cone responses reduce. What we have on the left hand side are ERGs from the mouse. And the simple description is they're the same as humans, where you see differences, they're mostly just mouse human. So for instance, on the rising B wave of the mouse ERG you see a lot more of these oscillatory potentials. That's a mouse human difference. But what we have is we have the super normal B wave response for the rods, and we have reduced signaling coming from cones. Now what you can do with the mice that difficult to do with the patient is you can put them through more extensive ERG testing, and try to learn more so we did quite a lot of that. I'm going to show you just two sets of experiments. The first is asking more detail about the broad response. So here's what we're doing is we're using dark adapted mice we give them a flash of light that elicits a response. And then we repeat that with increasing intensity of light so you get that what's called this flash family. And the really amazing thing here is when you compare the wild type traces which are black to the knockout traces which are in red, you see that the effect is different depending on the lighting. So here early on in response to very low light, you'll see that the red traces are delayed for when you see the B wave and then that B wave is small at the very most dim light. Just a slight increase in light that will start moving us into that super normal B wave response. And what you can also see here is I'm going to focus in on the a wave because the a wave is reflecting hyper polarization is that closure of channels and outer segments, where KV 2.18.2 is really contributing to regulation. And so the way it's easiest to see what's happening here in these lighting conditions and they're very dim light. So this is very slow it's delayed to respond, you do get a full amplitude response just takes a long time to get there. However, when you go to brighter light, it speeds up, and you now start seeing the onset of this wave matching for wild type and knockout. But if you're going to go fast, you can't get as big of a response so the a wave plateaus out. Here's the summary, under dim, very dim response, lighting conditions, the a wave is delayed, and a brighter flash and keep in mind this is bright for a rod, the amplitude is reduced. Okay, how does that make sense. Let me give that a try. I'm going to come back to my cartoon of signaling that's happening. I'm skipping the dark adaptive condition we're just looking at the light in the light the channel in the outer segment is close. And the IKX channel should be there and slowly inactivating, but in this disease where you don't have 8.2, you're left with just 2.1. And remember that means it's voltage of activation is shifted so it should not be open. Effectively you have zero channel here for IKX. And the only thing supporting the light response here in this knockout would be the activity of sodium potassium ATKs. And forgive me for being maybe a little bit too simple, but one way I like to describe this is, you're really messing up the amount of potassium you have in the extracellular space around the photoreceptor really don't have enough go juice to get the response you need. I don't know why under some conditions that photoreceptor sacrifices speed for amplitude and vice versa. But the effect is the same if you don't have enough positive charge in the extracellular space. One of those two things is going to happen and we happen to see both of them. Okay, I'm going to throw in here just a quick note now about the B wave mostly because that's just a characteristic of the name right it's the supernormal rod responses, which is actually a little bit deceptive because under the most dim lighting conditions for rods. The B wave is actually underperforming. And it's only after this you make this switch between sacrificing time versus amplitude that you start to see that supernormal response. So, this is a little bit hard to think about, but the way that it makes sense to me is I want to come back to one of these traces over here, because the onset of the B wave is always delayed, even though this a wave is not reaching its, its full amplitude you don't have any wave response. And it's, and it sort of square off it keeps going, you don't, and that means you can't really drive the cell all the way to its full hyper polarized condition you don't have the other mechanisms to help return the cell to its resting normal timing. So in some ways, even though you don't have a large amplitude you have more continuous drive for the signal and I think that's why you get the larger B wave, not because there's an effect specifically in the bipolar styles, but just because the information coming from rods is not stopping. So you get an accumulated large or supernormal B wave response. So the first set of more set of ERGs I want to show you here we're using a different stimulus assign you soil to flicker stimulus. So the first set of experiments were designed to be more similar to what you might see in the clinic. This one is a little bit more helpful for me. So it'll flicker we're just taking a step closer to a more normal lighting experience. And we can, we can analyze the waveforms you get from this and one of the things that we've learned is that across various frequencies. The response is always reduced in the knockout mice. And because this is a flickering stimulus, you know this is a large part driven by cones so this is absolutely be producible see that across the board. When you're when we use this particular ERG protocol under very low frequencies we can actually isolate information from the rods. And here this is what the raw waveforms look like below this is that light stimulus. So the white would, when the lights go on we elicit the positive B wave. Again black is our wild type and red is our knockout. And then because the signal continuously flickers we actually isolate. So we are able to see another component of the ERG that we didn't see in our first set of experiments and that is a second positive going wave that is called the C wave. Now this is important in this disease because the C wave is generated by the Mueller cells, and they do this in response to the light induced drop in exercise or potassium. So without KB 2.1 KB 8.2 that extra cellular potassium amount should be lower. Therefore the change between light and dark should be smaller, and indeed the C wave is smaller. So we can see that, and this then conforms to our hypothesis. Right, we did more ERG but I'm going to spare you. We're going to now move on to the degeneration. Now, here in mice. We used OCT again retinas upside down to the way most of you guys look at it. But here in the yellow bars this is the, the outer nuclear layer for receptor layer so we just measured that over time and wild type and knockout. And you can focus in here on panel C. What we see is that at two months of age we have something in the retina, not to submit, not too big video, about 10% thinner at this point, but that progresses fairly slowly so that by about 10 months of age we've lost about 30%. Now, this is a little bit different because the majority of patients that I've read about for KCMD 2 retinopathy don't have any obvious thinning of the peripheral retina. So this might be a mouse specific thing here. The other thing is an information I have on this slide is labeling for GFAP this is a marker for reactive gliosis. And indeed you can see that that is upregulated in the retina and you can see that the mule cells are starting to express the GFAP and it's starting to fill some of the processes in the retina. This is very commonly seen anytime you have a photoreceptor degeneration, but this is happening very early on in this particular disease that at two to three months when we're just starting to see photoreceptor drop out. We're seeing the mule cells respond, which might be sensible because they're electrically part of the problem as we saw with the C wave and the ERGs. And here's a really important question. What about the cones in cones are only 3% of the photoreceptors so for losing 30% of the retina know a lot of this is coming from the rods. What's happening to the cones. The answer that we did retinal flat mouth and we labeled for code arrested. Now this is obviously very low magnification picture you can't see individual cells. But what we did is we collected regions of interest at different eccentricities. And that's because even though the mouse right now does not have a macula there's a very slight increase in density of cones as you go more towards the center. So we did get these different eccentricities, comparing the cone density. And what we saw is absolutely no difference between wild type and neck out. We first did this experiment with six months old mice. And then my colleague slash competitor from Australia who had a different line of KBA point to mice published her own analysis. You can see a slight loss of cone density. So we repeated this experiment our mice, but this time aging them out to one year of age. And this is the data from the one year old mice, no difference, no loss of cones. At this point, though, I wanted to push a little bit harder on what would be happening with cones. And we have in the lab, a strain of mice, which you may or may not have heard of before. So if you've heard of the NRL mouse, take a moment today dream I'm just going to explain this to those of you haven't. NRL is a transcription factor expressed in the retinas required for specification of cones. So if you have a knockout mouse, all of the photoreceptors are cone like photoreceptors instead of being rods. The NRL mouse is used throughout our field to study cone dominant biology in the retina, but it's a difficult retina to work with because as you can see from this picture, it's morphologically abnormal, the cone and nuclei, instead of being a nice smooth layer form of polypholdings and rosettes. So instead I got a variation of the NRL knockout from Christian Grimm, where he crossed it with another line of mice and obtained a normal beautiful morphology. Boyer the outer segments, or where the outer nuclear layer happens me just all cones. So to make sure I'm very clear that this is not the pure NRL knockout line. We call this the cone full mouse. Here's our own histology. The retina looks pretty good. The outer nuclear layer is not as well organized. It's not as compact, but that's because it's cones not rods, and the outer segments are obviously much shorter. This is not a degenerated retina. It's just missing long rod outer segments. So we took this mouse, we crossed it to our KV 8.2 knockouts. And what did we get? Well, when we did ERGs now targeting the cones, you can see again black as wild type, red as knockout, the responses reduced exactly by 50%. So one of my questions, which you may remember early on was, is it possible that the defect in cone driven signaling, that reduction in the B wave is actually driven by the dysfunctional rods? And the answer from this experiment is no. It's intrinsic to cone signaling. B and C are just quantitations of the A wave. The A wave in this protocol is very, very small. There's a lot of variability. We don't see anything that statistically significant, but we do see this reliable reduction in the B wave. So this experiment was done at one month of age on the mice, because we know that in the anteroom knockout in the cone full mice, there is a degeneration that occurs. So this experiment was done at one month of age to check and make sure everything was okay. We did histology at two months, and then we followed it up with it four months. In two months, the cone full mice with a normal copy of KB 8.2 looked as fine, as well as the animals that are missing 8.2. But when we looked at four months here, the outer nuclear layer, it's a little bit more disorganized in this particular image, but it's actually within statistical normal range. However, by four months, the animals missing 8.2 have a striking degeneration. So in this particular environment, cones do degenerate in the absence of KB 8.2. This brings me to my summary of the mouse work, and this is work that we've all published. So I'm going to do a quick summary and then move on to unpublished data so you can learn what I'm thinking about for the future. We have a mouse model where the ERG phenotype is common to mice and humans. Those features I've described in the name of the disease, and it supports the hypothesis that the problem is insufficient extracellular potassium. Can't support the dark current required for resting membrane potential and they can't filter responses in response that come from light. In the mice, we see clear degeneration of rods. I don't really understand if this is consistent with the human disease as it's described in literature right now. Most patients do not have a rod degeneration. That said, the macula, even though we tend to think of that as being an area of really high cone density is also an area of really high rod density. And if we ever have access to histological samples from donor patients, maybe we will see that there are indeed rods dying more frequently. But it could also just be a mouse specific thing. This is something we just have to be mindful of as we continue. When we looked at this unusual mouse model with the all cones, we were able to learn is that the cones will be generate, but only in the absence of rods. And I want you to be patient with me, but this is one idea that this sparks in me and it requires further testing but perhaps the way to think about translating this to the humans, perhaps the fragility of the foveal cones right so in the center of the foveal is pure cones. Maybe the absence of rods there is what makes that particular population cells more sensitive and they degenerate which then propagates out into the progressive macular dystrophy we see. And again, could be mouse only so we need to test that, because as you saw from those retina pictures, even though this cone foe mouse has an all cone retina, it is still not the morphology of a foveal. So we still have to figure out exactly what is making cone so sensitive to degeneration in this disease. With that caveat clearly stated and clearly in mind, I think that as I go forward if I use both of these models normal mice and our cone foe mice, and I pay attention to these caveats think we can still use them and make progress and understanding the basic biology what's happening. Okay. See I'm starting to run short on time so I'm going to speed up but feel free to interrupt me. Oh, we need to stop and ask questions. One thing I'm doing to go forward in the future is, I was wondering what else is changing in the retina. One experiment we did is we looked at, we're measuring the various metabolites in the retina. The vast majority of things did not change but we saw changes in two metabolites. One is an AA or an acetyl aspartic acid, and the other is in Tori. What these two. These are free amino acids, meaning they look like amino acids but they don't actually get incorporated into proteins. And the common is that they both function as osmolites. So, changing levels of these molecules can reflect water stress or stress to your protein folding. What's strange to me is that in one case, the NA levels are going up, but in the other case Tori and the levels are going down. And this is an effect due to genotype and not age. Our young mice are one and a half months old. Our old mice are a little over a year old, in this case, and you see the effect at both ages. So I was going to tell you just a bit more about what we think we know about NA and Tori. I'm going to go really really quick through that. We really don't know very much about NA in the retina per se, but we do know that increased levels are hallmark of the disease called Canavan disease. Canavan disease is caused by mutation in an enzyme that breaks down NA, releasing acetate that is used to build lipids in all the dendrocytes. So you have a defect in myelin synthesis in terms of the retina that's obviously going to affect the optic nerve, but we don't know if it has any effect on the retinal neurons themselves. Tori is more fun. Sorry. We think we know a lot about Tori. If you look in the literature does a whole bunch of things regulate cell volume, forms bile salts in the digestive system. It's been purported to act as an antioxidant at the inflammatory blah blah blah everything. So criminals will sell it to you until you eat it. This is a very dangerous thing. So beware. However, when you look at the scientific literature. There's some interesting things. Every few years as a paper on Tori and retinal health. Tori depletion causes rapid retinal degeneration effects cone for receptors ganglion cells rods. If you mutate the transporter required for bringing Tori into photoreceptors you get a rapid retinal degeneration. So as I go forward, I want to study more about the biochemistry of the channel and I'll be telling people more about that in my research talk. And from this talk, I want to say we've learned more about the basic biology of this channel. In some patients gene therapy might be a viable option. The big hurdle there is going to be the very rarity of this disease gene therapy is incredibly expensive to develop and deploy. Maybe what we learn in mice about various metabolites will give us an alternate or complimentary tool though to help patients sooner. So stay tuned for that. And if I have time I have just one more slide. I wanted to show to you guys because I want feedback on this. When you think about the future we always have to ask what don't we know. So Casey and be to so causes a retinal disease. But Casey and be to is not only expressing photoreceptors. There's some evidence that at least at lower levels it's expressed for instance in the hippocampus.  Don't know the significance of this but in a paper from about 10 years ago, it was reported that within a group of pediatric epileptic patients. There were Casey and be to polymorphisms. So we know that from this standpoint of retinal disease, you have to do ERG to accurately diagnose the disease. But if a patient is subject to his prone to seizures, you don't want to put them under extensive ERG testing. So we don't know if if epileptic patients with mutations and Casey and be to might have a vision defects, for sure. Conversely, we don't know do our Casey and be to read out the few patients are they prone to perhaps having seizures or other cognitive impairments. I don't know. It's pretty rare but I'm really curious and feedback on that to determine if it is worth taking the time and effort to look more deeply using our mouse models at what's going on in other parts of the brain. With that, I will stop and take any questions I have time to answer and just stay on my. Thank you slide this is my, my group and my collaborators might flood the course. Very nice. And a translational talk of how we can go back and forth between human and mouse disease. I know we have other things that other people have to go to the next minute or two are there any quick questions for anyone here. And if not, I would encourage you at noon she will be speaking on online. Yeah, so there's even more detail and research people will be having it. Anything more that you want to say, Jim. Okay, all right, well thank you very much. Okay, thank you for your attention.


The Special English Division began in 1959 as an experiment to communicate with non-native English speakers slowly and simply. And that's the VOA, Special English. The experiment was a success, and in 2013, Special English became Learning English. Today, we have loyal teachers and students from all over the world. Our interview show, Talk to Us, brings some of them together to practice their English-speaking skills. Voice of America helped me to keep my mind busy. Our video series, Let's Learn English, is a popular learning tool. Le Van Cong and his family from Vietnam have their own special way of using it to learn English. Most city politicians in big town are using sticky money to have a party on the cruise ships. So taking money from a gentleman, oh no, oh no, you can't say, what I can I do now is we will put them here on the new day. I was looking for many ways to learn English, so when I found a VOA, I said this is the page I was looking for, a web page not having ads, this is a huge benefit. VOA content, particularly its articles, has been helping me not only to improve my English, but to increase my knowledge in many areas. I love VOA. I started listening to VOA back in 1997, Special English. I did learning, then after a few years, I was a little busy. I could not join, thanks to lockdown and COVID, I rejoined the VOA. And in this way, I'm connected and still listening for more years to come. And right now, I can, as big, be more confident. And I love you so much. I listen to every podcast, believe me. I have finished American presidency four times. I have a brain now to speak English. I can speak English everywhere. I need to speak English when the pandemic began. Well, she made me to know you, to enjoy your VOA. And it was very, very good for me. It was a new life for me. Now, VOA is important to us, teaching us English and, in my opinion, fighting misinformation. Faking news, especially at this time of the pandemic, with trust and information. So, thanks, VOA. All the best. We have it there. It is up to you if you take it or not. Thank you, VOA. And you're happy it's your anniversary? Yeah, it's everybody's anniversary.


Hello, I'm Dan Cunningham in Washington. Accepting an invitation from Senate Judiciary Committee Chairman Patrick Leahy, Justice Anthony Kennedy testified before the committee on February 14. For more than 90 minutes, the Justice commented on a wide range of topics with the Senators, including judicial independence, security for judges and their families, televised coverage of Supreme Court oral arguments, and judges' pay. On that last topic, Judges' Pay, Justice Kennedy urged prompt action by Congress. But as you mentioned, Mr. Chairman, the condition of our salaries is something that requires discussion. It's a subject that is frankly most awkward for me to talk about, and yet it's a sensitive subject that I think we should discuss in a candid and frank way. The raw fact is that the congressional policy with reference to judicial compensation is threatening the excellence of our judiciary. Judicial independence presumes an excellent judiciary. I've got some graphs in the statement, and it shows that the real income of the average American worker has risen by 15 percent, and the real earnings of the judiciary and the Congress has decreased in the same period of time by 25 percent. That's a 43 percent differential, and if and when this committee recognizes the urgent action is required, just as urgent action is required for the security measures that you're considering in the bill today. It's very important that you keep in mind the objective of restoring the judiciary to its preeminent place. $160,000 for a district judge, the present salaries. Sounds like a lot of money the average American and it is, but it's insufficient for us to attract the finest members of the practicing bar to the bench. The justice set a major problem has been the tradition of linking the pay of federal judges to congressional salaries. I think it's been a failed policy for both sides of the bargain, and I understand that Congress maintains two residences. They have tremendous travel expenses that we don't. So that's a problem that should be addressed. On the other hand, we simply can't wait another 20 years, and we have benchmarks. I think you should set judicial salaries and then in due course, whenever in your discretion you decide to do so, set congressional salaries as well. But the so-called linkage has been unfortunate and it has hurt the judiciary very, very badly, and I think it's quite unprincipled and quite unfair. Some of the senators said they agreed with points made by Justice Kennedy. The compensation issues are critically important and we need to address the concerns that you have brought out. Reporting from Washington, I'm Dan Cunningham.


fasting day 12. Don't settle for partial victory. Sometimes as Christians, we quit too early.   Honour is if you want to quit. There's nothing dishonoring about wanting to quit. Actually wanting to quit is a sign of success. How can you want to quit if you've never done anything? If somebody wants to quit, that means they're actually doing something. If somebody never wanted to quit, that means they're not doing anything. So there is no dishonor in wanting to quit. There's only dishonor in actually quitting. There's actually honor in wanting to quit. As long as you don't do it. You can enjoy the luxury of wanting to quit as long as you don't. You can enjoy the luxury of wanting to quit as long as you don't. But I want you to know something. Quitters don't win and winners don't quit. Now it's true. Winners quit some things. Bad things. Things they don't have fruit. Things that are not productive. But they don't quit on the things that are right. On the things that are important. On the things that are essential. You don't need persistence if you don't have resistance. If you don't have resistance to what you are doing, you will never develop persistence. You will never need persistence. In the Bible we see that Noah through his bulldog persistence build the ark. Jacob persisted in prayer. Moses persisted when Pharaoh did not want to let the Israel go. Joshua and Cable persisted on taking the land. 40 years they wandered in the wilderness but they still kept that vision and they still persisted. Persistent shepherd goes after the lost sheep until he finds it. Persistent woman searches for the lost peace until she finds it. Persistence means it's always too soon to quit. You are close to success. You're close to breakthrough. Persistence means it's too soon to quit. You're too close. Now maybe you stumbled during this fast or not only during this fast but just overall.  You can restart. You can resume. Now the verse I want to share with you today is from 2 Kings chapter 13 verse 17. And we see the prophet puts the hand on the king as he is pretty much putting a bow and arrow and then the prophet says and he said open the east window and he opened it and Elisha said shoot and he shot and he said the arrow of the Lord's deliverance and the arrow of deliverance from Syria for you must strike the Syrians at Apneck until you have destroyed them. Now go a few verses down verse 19 of the same chapter and the prophet says I want you to hit the ground with that arrow and the man of God it says was angry and said you have you must have struck five or six times then you have then you would have struck Syria until you have destroyed it but now you will strike Syria only three times. So the king hit the ground you know with his arrows only three times and the prophet got angry and he says you didn't go all the way. You will have partial victory but you will not have a complete victory over Syria. Now I wonder sometimes if this king's problem is also our problem. I wonder how many of us settle for partial victory how many of us don't strike until we receive complete victory. We strike until we get tired. We strike until we think well that's enough. You know a friend at midnight came asking for bread. He was persistent until he got bread. You know a widow kept asking the judge until she got the breakthrough. God is not pleased with partial victory. He wants to give you a complete victory. Don't settle for sea when God has an A for you. Don't settle for something that's partial when God wants you to receive a complete victory. Sometimes during the fast we get discouraged. We want to quit and as I mentioned there is an honor in wanting to quit because it means you're doing something right. There is no honor in quitting and for some people you know maybe during the water they fast they they they just can't do it anymore so they just switch to you know to a different type of fast and that's completely acceptable but what I want to speak against is this attitude that many of us have developed that has become part of our character and that is quitting too early quitting when it gets hard. There's an example of Abraham's father Tara. He stopped short of the land Canaan and settled in Huron Genesis 1131. See Tara settled for comfort. He was going to Canaan but he settled for Huron. He did not want to keep on going to the promised land. He got tired. He had a lot of animals. The family had to keep on moving. See God had great plans for Abraham's father but Tara settled for sea. He settled for the mediocrity. He settled for what's easy, what's comfortable because it was too hard to keep striving for God for what God had in store for him and so God took Abraham and continued that promise. What Tara failed in his son Abraham continued and Abraham continued to strive for that promised land. I want you to strive for a complete breakthrough this year. I want you to not give up. Sometimes one of the most common causes of our failure even in fasting is the habit of quitting we have developed for many many years. That's why for some of you this fast is hard and you want to quit every day but remember this is not just the fast you're doing. You're breaking a strong hold of quitting. When you're overtaken by temporary defeat, when you're overtaken by emotions that seem to take you down, maybe even physical discomfort, you have a choice. Yield to those cravings, yield to those pains, yield to those feelings or persevere. You don't need to be persistent if you don't have resistance. Resistance is natural. Anything worthwhile requires effort. If you want to climb a mountain, it's gonna take some effort. If you want to slide down and roll from the mountain, no effort is required and many of us want to climb higher. We're just not willing to pay the price because when things get hard, we settle. This is not to guilt-trip anybody. This is not to shame anybody or to make you feel bad for those of you who are, you know, maybe not wanting to persevere. I just want to provoke you and I want to tell you there is no luck. There's no easy thing. Easy way leads to destruction. Hard way leads to life. All of us are guilty of this mistake of quitting at one time or another. But I want to encourage you to develop a habit. Break down the stronghold of quitting. Don't settle for partial victory because God has a victory for you that's indeed. God has a victory for you that's a complete victory. So as we are more than halfway through this fast, this is when the quitting will become actually more persistent. The resistance will become more real. A lot of it will be in your head. A lot of it will be like, man, I already have done it. I did so good. I've never went this far. That's good. But the goal is not to go far. The goal is to go to the end. And so I want to challenge you to go to the end. I want to encourage you to whatever you do, however you are led by the Lord to fast, to do it till the end. Now if the Lord brings your end earlier, you know, that's between you and the Lord. He is our guide. But for those of you who join this fast and who are like, oh, it's 12th day already. I'm just not feeling it. I'm not, you know, I'm just gonna quit. I'm just gonna give up. You know, I've tried. I mentioned this yesterday and I want to repeat it again. You know, you will never know what God will do if you obey Him to the end. Not partial obedience, but complete obedience. And so when He leads you and you obey Him completely, you never know what's gonna be on the other side. But you already know what's going to happen if you don't. Nothing will change. Things just remain the same. We wish, we hope, but we lack faith and assurance that something is gonna change. And why we lack that? Because when there is no complete obedience, lack of complete obedience destroys our inner confidence in God's ability to do what He promised to do. You may say, no, I'm just gonna name it, claim it, blab it, grab it, confess it, possess it. That's not how that works. God is not a vending machine. If life is not backed up with obedience, inner world of faith, confidence, vision, expectation that comes from above.  I'm talking about faith that flows from life of obedience to the Lord. Trust. We must be obedient. So if you already stumbled, don't fall. Resume the fast. If you are wanting to quit, I want to encourage that and say, it's okay. It's normal. There's honor in that. There's honor in wanting to quit. Just don't do it. Just don't quit. And don't tell anybody you want to quit. Just let those feelings be there and and just persevere. Have honor in not quitting. There's honor in not quitting. And so honor in persevering. I understand sometimes we say, oh, but I just feel very weak. I just feel very tired. Yeah. But it's okay. You know, unless you're completely very, very, very difficult and you're throwing up blood and you just can't, you know, yeah, of course, you gotta take priority of your health and all of this stuff. But if it's just minor things where just you're just a little bit tired, you're fatigued, you're sleepy, you're not feeling great. All of those for most of us, I would say majority of us in this stream right now, that is our problem. It's this emotional, mental and fatigue. And while we feel great in our mind, yet this thing kind of settles in. I've done too far. I've went too far already. I'm going to quit. And so I just want to speak against that. And I want to encourage you to develop a resilient mind by doing what is right, even when you don't feel like doing it anymore, develop persistence, develop a habit of going to the end. The Lord dealt with me because about this, I'm going to tell you where that came from for me.  And, and I never promised this will be easy in stuff. So this will be challenging. And so then that's why, you know, we have each other. And you have me, you have the Lord, you have, you know, the fast forward book devotional to to strengthen you in this challenge. When 13 years ago, our church did a 21 day fast. You know, I was way skinnier at the time that I am right now was about 130 pounds. So very skinny. Same height. And we fasted, I just broke up with Lana like a week before the fast. So I've had some, you know, issues with my relationships. I was 24. I already had a house. I didn't live in that house. I rented it. I had a nice car. I had money saved for the wedding. And I attempted actually to get married. Before I met Lana, four years prior to that, I dated one person, things didn't work out. I dated the same person again, things didn't work out. And so it was my fault. And so I stopped completely the whole relationship thing I stopped and I said, man, I need to figure out what's happening with my own head. Something is not right. Why am I keep? Why do I keep doing these things?  I meet with her for the first time for a date. You know, she tells me everything about, you know, that date was more of an interrogation than actual date. And so I'm not sure that was the wisest thing to do. I probably won't recommend it for other people, but she lived three and a half hours away. So I didn't want to waste any time. And so I just went straight for the kill, you know, by asking her questions that usually people don't ask, not only on the first date, sometimes they don't even ask until they get married. And so I asked her everything. And so and afterwards I just felt this thing to quit, you know, and I, you know, I stopped that relationship to that night. We go into a fast. It was a 21 day fast. And on about 17th day, I stopped the fast, you know, actually quit. Things started to get harder for me. I definitely struggled with sleeping, which was sometimes normal when you're doing a 21 day fast. But I started to faint in the shower. So I made up this excuse that you know, I'm dying. When in reality, the reason why I was fainting in the shower is because I like to take hot showers.  Of course, you're gonna faint. And so like, I started to, you know, faint and I'm like, Oh, that's it. You know, and then I told my parents this sad story that I'm like, not feeling good. I'm like dying.  And so of course, I ate, you know, three days before I should have finished it. And now God still did some work. Don't get me wrong. That year, I got married to law and the things just changed. My mind changed everything. God just was a really great blessing. During that fast, the Lord led me to give a lot of my money away because I had money saved. You know, and for me, that was a lot of money for a lot of you. Maybe that's not a lot of money, but it was enough to get married, you know, honeymoon and buy simple furniture. And so during that fast, the Lord started to challenge really, that was the first time I experienced sacrifice, where above my tithe, I looked at my account and I said, man, I need to give this money away. And so I start praying during the fast, who to give the money away, man, I died million deaths. I think, you know, it was more painful actually giving the money than fasting. Because fasting, while it was hard, it's the giving part was way harder. For me, giving was always harder than fasting. And so now it's not as hard. So but something I've noticed, ever since that fast that I didn't finish, I was never able to go to more than seven day of fasting for 12 or 11 years, seven days for the max. I, I became scared of that. You know, and in reality, I didn't faint. I just took an extra hot shower for 20 minutes, which is not a wise thing to do. You know, so I mean, yeah, I was weak, but there was nothing really wrong with me. And, you know, and I should have finished it, I could have finished it, I just didn't. And so a few years ago, in December, as we were doing our typical three day fast, the Lord brought that memory and he said, you quit at that time. He says, you didn't go to the end. And I said, Lord, but you must understand, I was not feeling good. And he said, Lord, and I felt like the Lord said, he says, you sold one, you know, one thing to your parents, but in reality, you know, you should have not been doing that in the first place. That's not your body's fault. That's your decisions fault, your fault. And so, and the Lord really dealt with me harshly, you know, so the next month, which was a few years ago, we had a 21 day fast. And, and I was going into that fast to first to break that bad experience and say, and to say, I'm gonna go to the end. God will give me the grace. And I was convicted not to be a one who stops when it's hard. Now, when it's difficult and impossible to go, of course, that makes sense. Please hear me loud and clear, not telling anybody here to die. So, but what I'm saying is that a lot of us have laziness and procrastination. And we just would to we given too much into our fleshly desires. Okay. And while this may seem hard for some of you. Okay. Imagine if you would be persecuted right now. They'll be pulling nails out of you. Imagine what would happen if you would go through actually a time where they'll be beating you physically every single day for the cause of Christ to make you deny Jesus. Would you deny Jesus? For many of us, I'll be very honest with you. We would. Because we have a hard time obeying the Lord in simple things where nobody's beating us, where nobody's pulling our nails, where nobody's scorching us, where nobody is ripping our beard, where nobody's starving us. And so where nobody puts us in a frozen outside in the middle of snow for six hours naked, where we died of, you know, frostbites. And so so as I kind of was meditating, this was just my I'm just sharing my personal journey. As I was meditating and I said, man, if I quit, when this is hard, am I going to be ready if the Antichrist comes and the cost for preaching Jesus is my head cut off? You know, well, what would I do then? I'll definitely be preserving my head because I'll say, well, I can't. I can't go through this pain. I can't go through this suffering. God wouldn't want me to. Interesting. All 12 disciples with the exception of John got murdered, you know, Christians for hundreds of years.  If you read the Fox's book on martyrs, it's insane.  And I'm looking at my life and I said, Lord, the least I can do is prepare myself for doing what is right when I don't feel like doing it and to put your first above my desires, appetites, or a little funk that's going on in my own head sometimes. And so this is kind of my motivation is it's not only, hey, I want to be a finisher. Hey, I don't want to miss out on the breakthrough. But I also want to be a person that is prepared when the persecution comes that I can endure it. And one of the reasons I can endure it is because I've developed a muscle of doing things that not are always easy, but they're right for what the Lord wants me to do right now. I'm not saying self inflict yourself with some pain and purpose. I'm saying obey God and if it costs you pain, so what? Be like Moses. He chose to suffer with God's people. Did it cause him pain? The Bible says he had to embrace suffering. So what? As a good soldier, endure suffering. The fruit of this period is long suffering. And so a lot of us in our western culture, I'm just going to be very honest with you guys. Don't take this the wrong way. We are snowflakes. We melt too fast. We wither too fast. That's why the idea that Christians will not go through tribulation is only believed in the western world and in any other countries where American preachers or western preachers brought it to. Nobody outside of this believes in that stuff. Why? Because for everyone around the world suffering, enduring is part of living a Christian life. They really believe that Jesus said if you follow me, pick up the cross and deny yourself with us as westerns. We don't focus on suffering. All of our sermons are about how to be successful. This is how you become successful in your family. This is how you become successful in your marriage. This is how you become successful. This is how you avoid trouble. This is how you avoid stress. This is not that has its place. Don't get me wrong. I'm a pastor. Okay, so that has its place and I don't have a modern complex. But if I don't learn how to suffer, not just how to be successful, what kind of a Christian am I? And so, because I'm not being persecuted right now, I'm not being attacked right now, you know, but I can choose to deny myself of the things that the Lord wants me to deny for a season for His glory. So, don't quit. Don't give up. Persevere. Continue. God got you. Amen. Let's pray together. Let's ask the Holy Spirit right now to produce within us long suffering. Let's ask the Holy Spirit to help us go to the end, be a finisher. Drop it in the chat. I will be a finisher, not a quitter. I will not settle for partial victory. Lord, I stand in agreement with every person that is watching us right now. Every person that is going to be later on rewatching this. I stand in agreement with every person that will be listening this to this on the podcast. Dear Holy Spirit, many of us are too emotional. Lord, we quit too soon. We give up too early. We choose the easy instead of the right. We seek comfort instead of your calling. We settle for partial instead of going to the end. May this year be different. As we still have nine days to go, may you strengthen us. Lord, we want to quit. Our flesh wants to quit every single day. And there's an honor in that. But we refuse to let our flesh make the decisions. I pray for those that are physically maybe struggling right now and maybe they're feeling down emotionally. May you resurrect those the joy of the Lord. May you strengthen their resolve. I pray for those God that are genuinely right now. They just can't do it anymore because things in their health got so so much worse. Lord, as they change to a different fast, may you still meet them at the point of their need.  Lord, in Jesus' name, I bless them right now. Release your anointing. Release your favor. Release the strength and the peace of God. In Jesus' mighty name we pray. Amen. Amen. Amen. Drop that amen in the chat. Drop that drop that prayer emoji in the chat. Something that I wanted to ask you right now, if this was a word for you or this has been blessing to you, this stream, these these content, I want to ask you to to give today to partner with our ministry. There are different ways you can do that through cash app. You can give one time through Venmo. You can give through PayPal. We have other options on our website but I want to invite you to become a partner. Go to pastovlad.org forward slash partner. Partner with our ministry. We release content in video format, written content, reading plans, courses, books. We translate all of that as well to many languages and we also offer different video in different languages and so we support other ministries as well as we are able to and we have other plans to do great things for God. Our vision is to reach the world digitally and to globally and to disciple people digitally and so you are able to do that when you so you become a part of this ministry and so with your giving some of you cannot do what I do but God has blessed you with finances.  God has blessed you with your resources so I just want to invite you. You can give through YouTube chat. You can give through but I just want to invite you to become a partner and so many of us partner with a lot of other things and a lot of them are dumb and so but this will be a good good partnership to to do. Thank you Jessica, Brittany and Arlene who gave online as well as those of you Marcio who became a member. We appreciate you as well as Akausa gave on the chat. Thank you so much. God bless you. Let me just raise my music. Just a little bit more. Amen. Amen. So thank you for that. For those of you who gave Pamela thank you. God bless you. For those of you who gave on the website I'm gonna read just some donations who gave on the website today and so very much appreciate it for those of you that are today also giving on our website. So somebody's saying which app I should use for international payments or from where I can donate so the best way to do it is on our website.  You can give actually from any nation and so Leah thank you for your donation. Golden thank you for the nation James and Amber just became a partner on the website. We appreciate it. We're believing during this fast that the Lord will send us a hundred partners and so that we can give more content free of charge so that we can offer to more people. So really appreciate it. Pamela says I give monthly alpha k911. Very much appreciated for your extravagant extravagant gift on YouTube. Really appreciate it. Rachel God bless you as well for your gift. Lydia thank you as well. God bless you for that. Share the background music Irene so Everett just dropped the link and Nika is saying I became a partner this week. Thank you so much for doing that. Very much appreciated Nika. If the Lord puts a new heart to become a partner. You know people don't realize I actually londi thank you for that. I personally have been a partner with other ministries. Right now the Lord has put on my heart to partner with Hungry Jen. So I've been on a staff at Hungry Jen for 20 years and starting this month I actually give all of my salary that the Hungry Jen was giving me back to Hungry Jen as it's my partnership so and it's the most that I've ever you know given ever and it's every single month. So I am a firm believer something that I'm encouraging you guys to do. I do it myself so this is not something oh Lord just wants our money no that's not for me trust me if I wouldn't be doing a disc like releasing trying to bring more books and all of the last thing that I would be asking is for that but the Lord put on my heart he says he says give stuff for free make the best content possible build a strong team and he says and I will send partners and so you could be one of those people so thank you guys for doing that. Amen. Somebody is asking when am I speaking in San Antonio so this is going to be at the end of the month. Alicia thank you for becoming forgiving through the partnership link Roman Pavluk appreciate you bro Patricia God bless you as well amen. So my events in San Antonio are February 25 through 26 so that's where my events are in San Antonio. So one thing that yesterday we've mentioned and that I want to highlight right now that if you are a video editor but specifically we're looking for people who can make videos translate them in other languages maybe like Indian language or some other languages and you know how the photoshop works you're very savvy with that stuff you have your own equipment email Everett at pastovlad.org and another thing that I want to mention if you can format books if you have experience in formatting content for example we release books and we have a person that we're working with great guy and so but we're looking for more help in that if you know how to how the formatting works go ahead and send us an email and to send us some of your work that you've done we would love to maybe bring you along on our team as well in the near future. All right now let's if you have a question you can drop that in the chat we're gonna take some time right now to answer some of those questions will you be coming to the UK so I don't have any plans to go to the UK what if I am called to go to fast longer go longer as the Lord gives you that strength can a Christian be possessed by a demon and how can you prove this biblical the word possess does not exist actually in the original language when you say or I say possessed we always mean owned in the scripture every reference that deals with demonization has no reference to own being owned it has more reference of being inhabited controlled and influenced but it does not have the reference of ownership the bible does not separate possessed and oppressed it only really has one main word for all of the demonization it's demonized it's being under control of a demon it's not being influenced it's more controlled when a person has a demon now it doesn't mean that they're owned by a demon anymore than if you get a mouse in your house it doesn't mean that the mouse owns your house so that's what the that's what the difference is and so in the bible I don't have to prove that a Christian can have a demon you have to prove that a Christian cannot have a demon and so the burden of proof is on your end and you absolutely have now one shred of evidence in the scripture nor by church history where once a person gets saved that automatically all the demons they had before are gone for example if a person christian cannot have a demon because they have the holy spirit in them how come person has a flesh it's as evil as wicked and as nasty as any demon so a person can still have evil desires because the holy spirit lives in their spirit and the flesh is a war with the spirit you know and demons are not flesh demons are spiritual beings who have desires appetites who love to inhabit a body and when you become a Christian their legal right needs to be revoked and they need to be cast out Jesus only had one solution for demonization and that is expelling them it wasn't a sinner's prayer or even a water baptism or joining a church it was expelling those demons so demons need to be cast out and majority of deliverances that you see from any deliverance minister biblical christian deliverance minister they will tell you they do majority of deliverances on christians not on on christians so yeah i am on a fruit fast and there is a lot of disturbance from my kids my three-year-old daughter can't let me focus in my prayers what should i do should i stop the fast um no just fight those distractions not your daughter your daughter's problem not the problem but just fight those distractions and persevere you know you're also a mother so there's also a responsibility that you carry anastasia thank you so much for your donation dora thank you for your generous donation and through the vinmo or kasha one of those are you ever coming to new york yes i am coming to new york actually let me give you i've mentioned um earlier today that i'm coming to marriage conference on february 17th and so that's what i'm coming to new york now are you coming to california i do not have plans right now to come to california my 12th day of fasting going good but prayer times are less i don't know i'm getting busy with a lot of things i'm trying to keep myself with praying in tongues please advise me how i overcome this so when it comes to prayer during fasting there are there are times even when you're fasting where you're you're busy with other things and you might not be able to devote yourself to more prayer and so don't feel guilty about that do try to give as much time as you can to your prayer and if you're praying and your mind is filled with distractions on your duties and responsibilities my approach usually is this instead of trying to fight off those distractions i actually pray for those distractions for example i was praying today you know and i was thinking about this meeting that i have this opportunity that i have and so instead of saying i rebuke this i rebuke this i just simply start praying for it i said lord would you give me wisdom on this lord i i give you this problem right now lord i ask you for this meeting that i'm going to have lord would you guide me in this area so i pray for the things that my mind is concerned about and then as i turn them into prayer my worries into prayer they subside and god in return gives me his peace that passes understanding this is my day one can i proceed even after the challenge is over of course you can you can proceed if the if the challenge is over but honestly you can just you know go go for nine days if you've never done a fast and you're starting with a nine you know from now till the end of course go for it and you can go further and so and just rewatch the previous videos is it okay to give money to a sadhu and blessings from a sadhu sadhus are hindu spiritual leaders i wouldn't give money unless you're required by the law to pay taxes to the government but i would not give money to spiritual leaders who are not born again i wouldn't support ministries unless it's like you know maybe red cross or some kind of a charity organization but i would not put my money in that to advance those agendas that's why i always tell people you know support support god's kingdom support ministries like ours you know don't support garbage and then we complain sometimes oh why is this you know demonic agenda is advancing well it's advancing because somebody's paying for it and a lot of times guess who's paying for it we're paying for it so um metodi thank you for becoming a partner Tina god bless you for becoming a partner melissa god bless you for becoming a partner ramy and rebeca and jeffery god bless you for giving one time today and then the Rachel guy god bless you for becoming a partner um my arani lopez and marcia god bless you for becoming a partner today we appreciate so much for the Lord speaking to your hearts and you're responding to that. Can I trust the book of Enoch? I think you can read it for encouragement or inspiration, excuse me, not necessarily inspiration for education, but the book of Enoch has not been included in the canon and so I would discourage you from reading it as a Bible, but you can definitely read it. There's a lot of very interesting stuff in the book of Enoch and so read it as you would read, you know, fight back or fast forward, you know, it's not a Bible, but you can read it as a book and you compare everything with the Scripture.  In some actually sicknesses, it's helpful to fast, it helps you to recover faster, but again, it depends, it's depending on what you're sick. Can the devil give us dreams? 100%. The Bible says as they slept, the enemy planted sowed tears so he sowed certain things in the field. I believe the enemy can give us dreams, God can give us dreams and dreams can also come from pizza and so the cares of life and so if I would encourage you if you're getting demonic dreams, I would just double check on what you're watching, what you're listening, what you're putting yourself into. Also begin to examine is there other evidences that maybe you are under demonic oppression and you need deliverance and so if there's such, make sure that you, you know, seek deliverance. There's many ways you can, there's many places and many ways you can experience deliverance. You can come to Hungry Gen as I have mentioned already and I'm going to highlight it again that you can come to Hungry Gen's end of the fast. We have our deliverance service and as we're ending the fast, the last day of the fast, we're going to have our deliverance service, a sacrifice service.  So buy your ticket, fly to Hungry Gen. I'm going to be preaching. I would love to see you. Is it normal to feel spiritually dry? So I have videos on dealing with spiritual dryness. When you're fasting and you're feeling spiritual dry, watch my video yesterday what I dealt with how God rewards the fast. Is there a negative reaction to drinking cold water while fasting? Well typically during fasting you're already very cold so I would encourage to drink warmer water or room temperature or hot water. Are you coming to Wisconsin? No, I don't have any trips to Wisconsin. Are you coming to Germany? So I have an invitation to Germany right now that I am prayerfully considering but most likely not yet. Are you coming to Texas? Yes, I am coming to Texas. So I'm going to share this again. I'm coming to San Antonio on February 25th through 26th. Amy speaks. Thank you for your donation. Dora, God bless you for your giving on the cash up. Yes, on the cash up. And Hope Baker, thank you for becoming a partner on the website. We're very much appreciated. I am on day 12 of 21 water full fast. I really want to complete it but I woke up in the middle of the night to vomit and I feel sometimes sick. Is it normal or should I change my fast? So if you're already on the day 12 and you're vomiting and you're feeling sick, you know, I would just give maybe a day and then see if it doesn't improve then I might switch to just, you know, one meal a day or maybe a Daniel's fast and so but if within a day it improves, you know, add some salt fasting salt and complex be a vitamin, maybe take a complex be vitamin. Maybe that could help with that.   So I tossed and churned, tossed, I woke up like maybe like five times.   So maybe that's because of that. So but you know, and it's harder to walk. I feel like even though my body is very light. And so but the knees were kind of hurting. And so but I'm going to persevere just just drink those vitamins and drink water and stay hydrated.   Can God make anything possible? Do you think that he can make a real relationship happen where I live on the opposite side of the country as my potential future spouse? Yes, I believe that I believe God can make a relationship possible on the opposite side. My wife lived in three and a half hours away. Long distance relationships aren't more challenging. Sometimes you don't really get a chance to get to know the person better. But honestly, none of us really know the person that we're getting married to until we get married to them. So yeah, there's pros and cons to long distance relationships, but they're possible. Is there a negative effect of drinking cold water while fasting? I think I already mentioned that to try to drink warmer water. Marie, thank you for becoming a partner on the website. Very much appreciated. Can you differentiate overcoming flesh or a demon? So a flesh is crucified. A demon is cast out. The difference between the flesh and the demon is this is that the demon speaks. It has a voice. When it's inside of you, it starts to push you and it has a voice. And a demon does not get removed by crucifixion. It only gets removed by being expelled and a flesh has to be crucified. And so a flesh doesn't have a voice like a demon. And so those are the two main differences. So demons we cast out and the flesh we crucified. Can the demonic spirits bother me at night? I have terrible nightmares. Yes, demons can bother you at night and nightmares consistent, terrible nightmares are one of the signs that a person is demonized. And so just probably it means that you need to be seeking deliverance. You can watch my self-deliverance videos. You can take a deliverance prayer course on Vlad school and that could also begin that deliverance process. Join our monthly digital deliverance services online or you can make your way to Hungry Gen. We would love to pray for you. Can demons go away slowly through fasting? I've been fasting some days, not every day, but I feel like I'm getting better and I still feel that it's there. So fasting again does not remove demons. Fasting defeats the flesh. Deliverance removes demons. Fasting can weaken the demons and better prepare you for deliverance. But it's not a cure for demons. The only cure for demons is deliverance. It's driving them out by the power of the Holy Spirit in the name of Jesus. Therefore, I want to invite you to our end of the fast deliverance service where we are going to be doing exactly that. Because fasting, as I mentioned, defeats the flesh, but deliverance drives out demons. So at the end of the fast, not this Sunday, but next Sunday, we're having our deliverance Sunday, our sacrifice Sunday, and you are welcome to come for that deliverance. We would love to see you here. My bones hurt. Is there anything I can do to help? Add a little bit of sea salt into your water. Fasting salt or sea salt, hamaiamans, whatever the name for the salt, but fasting salt or also during complex B, compound B, or we have it in the comments below. I'm mixing my words now. And so that could help. Will we get delivered even online? 100%. I think that you can be delivered online as well. This is not a barrier. Pastor Vlad, can you kindly do a live with Pastor Lana sometime later about praying for the future spouse? Pastor Lana, who is in the chat? Would you be willing to do a live stream with me about praying for the future spouse? If Pastor Lana says yes, then yes. Yes. She said L-O-L. I don't know what that means. L-O-L. Does that mean yes or L-O-L means no? If you ask me very nicely, oh guys, this might be a challenge. So somebody is saying, hey, what is a sacrifice Sunday? So at the end of the fast, we usually bring God a sacrifice. We give something to God that hurts. And yeah, so that happens on the last day of the fast. And in our case, we're believing for $3 million for a building because we can fit all the people that God is bringing. And so that sacrifice will go for that. So and yeah, that's going to be the sacrifice Sunday, but it's also delivered on Sunday. It's going to be so many things happening on that Sunday. We're also anointing a pastor from Canada who was part of our internship. Pretty excited for that. So yeah, how is the flesh crucified when we deny ourselves? Fasting is one of the tools for that. Vera, thank you for your, I know this is a reoccurring donation. Okay, Miss Aguirre, thank you also for your donation and cash up. All right, guys. So this is, this has been an hour and 10 minutes. We are checking out for today. I will see you again tomorrow is Saturday. I know that a lot of you guys, well, if you're fasting, you are not going to be doing too many things. So I'm going to see you live and the wrecked tomorrow at the same time nine o'clock. Cannot wait to be with you guys tomorrow. And we're going to share tomorrow about day 13, how fasting unlocks closed doors. So it's going to be fire tomorrow. Come on. It's going to be fire. Pretty excited for that. So see you tomorrow. God bless you guys.


Have you ever felt like you're overly reliant on your partners or friends? Do your emotional needs go beyond what seems normal? And are you often called needy or clingy? If these questions ring true to you, it's possible that you're codependent. But what is codependency? It's defined as excessive emotional or psychological reliance on a partner or friend. One who typically needs a lot of support. You can put a lot of strain on both the relationship and the individuals involved in it. So, how can you tell if you're codependent? This video looks at 8 signs to watch out for. 1. Constant reassurance seeking Do you find yourself constantly asking for people's reassurances? Asking them to tell you they love you a lot or asking if they're mad at you constantly? This sort of constant reassurance seeking can be a sign of codependency. You may seek reassurance due to low self-worth and feeling like your partner is always just about to leave the relationship as you often don't feel you bring anything to the relationship. 2. Feeling responsible for problem solving Do you find yourself feeling it's your duty to solve all of your partner's problems? Not just that you want to help them but that you need to help them or they might leave? This is another common sign of codependency. It can be particularly damaging as it isn't possible for us to fix everything for someone all the time. And if your self-worth is tied up in this idea, it can set you up for a big fail. 3.   This is another classic sign of codependency. People with codependency issues can feel insecure in their relationships and feel like they're very fragile. Therefore, if you have codependency issues, you may feel something like simply saying no to your partner will be enough to ruin the relationship and that they'll leave you. This can be damaging as it's not good for us to ignore our own boundaries for others. 4. You need to avoid conflict Do you find yourself terrified of conflict arising in your relationship? As mentioned before, do you avoid asserting boundaries as you worry about conflict that might come if you do? As we've said, often people who are codependent worry their relationships aren't strong enough to withstand arguing or conflict and that if there is any, the other person will leave. This fear can often present itself as trying to strictly control relationships so that no conflict does appear. Are you enjoying this video? Finding it exciting and factual? If so, you'll love Psych2Go Magazine, the first psychology magazine by millennials for millennials. You can check out issue 14 on generalized anxiety disorder for free. Check the link in the description. The 100% discount will be applied at checkout. If you like it, consider pre-ordering at our Patreon. The link is in the description for as little as $1 per issue. Enjoy the rest of the video. 5. Would you do anything to hold onto the relationship? Do you find yourself doing and saying things you wouldn't usually do just to hold onto the relationship? Even if these things may be destructive or dangerous? This sign is very linked to the previous two, often arising due to a fear of conflict and a need to hold onto the relationship at all costs. It often means you'll ignore your own boundaries and do things you absolutely would not usually do, which is dangerous for many reasons. 6. You only feel valued when you're needed. Do you struggle to feel valued as a person if you're not fixing other people's problems? Do you believe you have very little to add to a relationship if you're not the problem solver? This is yet another sign of codependency. Due to low self-esteem, those with codependency often feel they're only useful to their partners as problem solvers. They feel if they don't fix every little problem their partner has, that they aren't doing enough to help, and that they have no value. This stems from their poor self-esteem and can also feed into their insecurity about their relationship. 7. Too concerned with others' feelings and needs. Do you find yourself always putting your partner first regardless of your own needs? Do you spend a long time preoccupied with your partner's feelings at the expense of your own? If so, it could be another sign of codependency. While concerning yourself with your partner is generally a normal feature of a relationship, with codependency, this concern goes beyond normality. You'll be obsessed with how the other person is feeling due to the need to be needed to feel valued that we just talked about. This often comes at the expense of yourself, a theme that's prominent in a lot of these signs of codependency. 8. Struggling to identify your own needs and emotions. The final sign of codependency links in with the previous one. Do you find that you can't work out what emotion you're feeling or what it is exactly you need? Due to the preoccupation with others and keeping the relationships alive, someone who's codependent may well struggle with actually identifying their own needs and working out their emotions. They become so fixated on their partner they lose sight on themselves and lose insight into their own needs and wants. This can lead to a whole host of issues, including burnout and not looking after yourself properly. We hope you enjoyed learning about codependency. Did you relate to any of these signs? Do you know someone you think might be codependent? Let us know what you think in the comments. And if you found this video useful, be sure to like, share and subscribe to our channel. As always, thanks so much for watching.