here, so that's all that's going to happen. All right, Dr. Warner's here so we can start. I just want you to know I have to leave early. That's okay. Very early, like right away. Well, then you leave right away. No, nothing against that. Hey, that's okay. All right, so I think we'll get started here. And I just want to say that I think I can speak on behalf of all the residents, fellows, and faculty that have worked with Dr. Chris Conraddy, that we're very glad he came here to the Moran to join our residency program. He came to us from the MD-PhD program at the Dean McGee Eye Center in Oklahoma, and he's been the VA intern for the last three to four months. In his role there, Dr. Conraddy has been incredibly efficient coming in after his morning runs at 6.30 to churn through the paperwork, and even being so ahead that he can see retina clinic patients in addition to his regular clinic load. He apparently was also a model of efficiency during his PhD years, turning out 10 first-author papers in three short years. And we really look forward to hearing more about that PhD work. But before we hear from Dr. Conraddy, I just want to share a little story about how I found out that his nickname was Kamikaze. As part of the resident retreat, Renee, Chris, and I went on a little mountain biking trip to the Flying Dog Jeremy Ranch. There's a particularly steep downhill switchback, and Dr. Conraddy cut it a little bit too close and hit his front brake a little too hard. He started a head-first dive over the handlebars. I'm behind him envisioning really bad consequences for him and for me who took him down this trail. However, mid-air, Dr. Conraddy managed to convert this head-first over the handlebars momentum into a flying hurdle of his handlebars and land on his feet in a run, five feet down the trail, and managed to avoid the aspen on the other side. I would say that if reaction times during mountain biking crashes have anything to do with surgical skill, Dr. Conraddy will turn out to be a very excellent cataract surgeon. With that, we look forward to hearing about his PhD work. Thanks, Dr. Wong. Like Dr. Wong said, I will be talking about some of my PhD work. I've entitled this talk, The Great Recognition of HSV-1, a view from within the cornea. We'll talk much deeper about this topic, probably to the point that you guys are sick of hearing about innate immunity, but just to kind of give you a good context of that, we'll go through it. Then I first got my interest in the scientific world from actually kind of a different background. I was actually first introduced to the sciences in the botanical world, and I was fascinated with science from actually the botanical side. For those of you that know flowers or don't know flowers, this is a lily of the Nile, and I've transposed some different colors on top of that. I haven't genetically modified the flowers, which is what Eileen was thinking I'd done to this flower, but no, I've not genetically modified it. First, let me tell you to every story, there's the good guy and the bad guy. I want to introduce you to a topic that we all interact with almost on a daily basis is herpes simplex virus type 1. It's a highly prevalent DNA virus. It's neurotropic. 60 to 90% of the population is seropositive for the actual virus, meaning that they've been infected at some point and likely harbor the virus, whether they actually show symptomatic signs of that is very, very broad spectrum, and we'll talk a little bit about that as we go. The reason that we were interested in this topic obviously from the Dean McGee, I Institute perspective was that it's the leading cause of corneal blindness to an infectious etiology in the developed world. So basically we see it quite a bit and that was the reason that we became interested in it. And just a little foreshadowing to the rest of the talk that I'll give is that herpes simplex is shown to activate tolite receptor 2, 3, 7 and 9 and several other innate sensors and we'll go in excruciating detail over this topic. But that's just a little foreshadowing. Then the interesting thing that you all know about herpes simplex is that it has a wide array of symptoms, clinical symptoms. And so we have patients that come in that don't have any symptoms at all. Then in the context of HSV2, a very close relative of HSV1, you can actually get females that asymptomatically shed HSV2 into the vaginal vault, basically spreading the virus without even having symptoms of the virus itself. Then that can progress or not necessarily progress. But in other cases, you can get kind of a nuisance cold sore. Then as we progress in increasing morbidity and mortality, we can get corneal infections where you get the kind of pathenumonic dendritic staining of the cornea and that's what we'll be focusing on today. And then a little bit of my work touched on herpes encephalitis where you actually can get mortality and actually in 70% of those that survive, you'll get long-term neurological deficits. And so it's a pretty broad spectrum when it comes to clinical symptomology. And then to introduce to you the good guys, and this will be something that we focus most of this talk on, is the immune system. So the immune system comes in two flavors to dust off all those cobwebs from immune classes in medical school or in graduate school. There's two flavors of immunity. The first, the innate nonspecific arm that's driven by NK cells, macrophages, and actual host cells themselves. And it's based on these conserved pathogen-associated molecular patterns where you have a pathogen that has a conserved sequence and then these innate sensors respond to those conserved sequences. Then that's in contrast to the adaptive or more specific arm of the immune system that's driven by T and B cells. And this is a very, very basic form of immunology, but just to kind of give you a basic grasp of it for those of you that haven't really thought about immune system in a while. But we're gonna focus on this first half, or the innate immune system that's driven by this, by toll-like receptors, DNA and RNA sensors, and we'll go in more detail as we go. So what exactly are innate sensors? So in the most basic form, think of a watchtower on a castle. So they're literally surveying the surrounding tissue inside the cell itself, depending on the sensor itself, and so they're trying to activate the earliest immune response to invading pathogens. And so that's driven by toll-like receptors, DNA sensors, RNA sensors, so there's a whole broad spectrum of innate sensors. And as over the last probably 20 years, toll-like receptors were first hypothesized in the late 1980s, then identified actually in the mid-1990s. However, in the last three years, three or four years we'll say, DNA and RNA sensors have become, I guess, a center part of the innate immune system. And then they, like I said, they've activated the innate immune system, and they do that by inducing the production of chemokines, so basically molecules that attract infiltrating leukocytes to the site of infection. They also drive a pro-inflammatory state with pro-inflammatory cytokines, and they up-regulate cell surface receptors, and probably the key that we'll talk about today is they drive interferon production. And from the context of toll-like receptors, I need you to kind of focus on this, and I'll boil down the innate immune system to three points I want you to hold on to, and this is one of them, but we'll rehash that here in a second. But all toll-like receptors signal through one of two adapter proteins, either a Mighty 88 or TRF. So all signaling through the 12 or 13 toll-like receptors, depending on who you're talking to, signal through one of these two adapter proteins. And then just to kind of overwhelm you with actual innate immunity, so this is showing just RNA and DNA sensors and the pathways that they drive doesn't show the 12 or 13 toll-like receptors that have been identified as well. But you can see that it's a very complex process, basically to lead to the production of the interferon and driving interferon products. And so to kind of boil that down, to kind of, I guess, dumb that down to just HSV-related sensors, and so in the literature, you can dig through the literature and see that there are three toll-like receptors that actually drive innate immunity in some context to HSV, and that's toll-like receptor two, three, and nine. And then there are three DNA sensors that have been identified, DAI, DDX-41, IFI-16, and then there's a controversial role of an RNA sensor, or maybe it's actually two sensors, RIGI, MDA-5. That still shows that this is a pretty complex process, and what exactly is happening is hard to identify in a lot of tissues due to the complexity of the innate immune system. But just to boil that down a little bit further, so if we want to look at basically the most basic innate immune response, all innate immunity basically drives an NF-Kappa-B signaling cascade to drive pro-inflammatory cytokine production to drive a pro-inflammatory state, and then from the antiviral perspective, you have IRF, so interferon regulatory factor, protein is phosphorylated, dimerizes, nuclear translocates to drive interferon production, the most downstream signaling portions of these innate sensors. And so we'll be looking at a lot of those signaling cascades, but we'll talk more about them, and I'll continue to rehash that as we go. Then, at least from an antiviral state, interferon is then released once the innate sensor is activated and activates interferon production. Interferon is released from the cell basically binds in an autocrine and paracrine manner, so to the cell itself and then to the surrounding cells to this heterodimeric interferon receptor, that then activates what you all are probably most familiar with, the jack-stat signaling cascade. We're not going to talk much about that today, but basically to say that the jack-stat signaling cascade is important in driving basically interferon regulatory proteins to drive antiviral immunity to viruses. Then interferons themselves, so what exactly are interferons? They're antiviral cytokines, they're produced following these innate sensor activation, and they activate a couple of proteins that are important in antiviral immunity, so PKR, RNA cell, they induce apoptosis, increase MHC Class 1 presentation, and just to dust off the cobwebs of immunology, that's important in cytotoxic CD8 T-cell surveillance of whether a cell needs to be killed because it's infected or whether it's actually just host tissue that's being presented and a cytotoxic T-cell goes on its way. All these proteins and pathways are basically, I guess, activated to reduce the amount of virus or the ability of the virus to replicate.  PKR and RNA cell both basically degrade all intracellular protein, so it's very difficult for a virus that hijacks intracellular proteins to replicate itself to do that when there are no proteins to replicate with. And then lastly, the sensitivity of MHC class I expression and this kind of shows the role of interferons bridging innate immunity with adaptive immunity. There's currently six subtypes of interferons known. Two of them we'll talk about today because they seem to be the predominant antiviral interferons. And like I said, I would blow this down to three key points that I need you to hold on to for the rest of the talks. I present some data. Something I've already basically hounded on and that's that all tolite receptors signal through either this TRF or Mighty 88 adapter protein. And don't worry if you forget some of these things, we'll hound on them a little bit more later. Then interferon production requires the nuclear translocation of these IRF subspecies, so these interferon regulatory factor proteins. And so if you don't get that nuclear translocation, you don't get interferon production. Then lastly, the activation of downstream interferon proteins results in increased cell death, decreased cell sensitivity to actual viral infection. So that's the key component of interferon. So then to take you through the literature up to date, what we knew going into this study, at least from tolite receptors in innate immunity in the cornea. And so it was very clear from literature that both bacterial and fungal infections require tolite receptor signals to clear the infection in the cornea. So if you knock out tolite receptors, you get an increased sensitivity to bacteria and fungus. Then it was widely assumed that interferon production was actually driven by tolite receptors in the cornea. And that was actually in part by a study that took human corneal epithelial cells and treated those with tolite receptor 3 agonists, and then they noted that interferon beta was actually produced. Then lastly, groups have gone through and taken corneal transplant, well, tissues that are of patients that are getting corneal transplants after HSV1 infection of the cornea, and noted that TLR mRNA is actually upregulated during HSV1 infections, suggesting that the virus, or at least the immune system, is upregulating its innate sensors to increase sensitivity to surveying the surrounding tissue. That would at least be the hypothesis from what they proposed. So then the two questions that kind of immediately popped up in our head was what drives the innate recognition of HSV1 in the cornea. Nobody had really nailed that down. And probably the more important question is why should we care? And so let me first target that second question. So for those of you that keep up with any sort of vaccine literature, there's no HSV1 or 2 vaccine on the market. Secondly, there have been multiple failed attempts most recently in 2012 where actually it showed an increased susceptibility to HSV infections. So not a great vaccine. Then there's emerging literature that would actually suggest, or actually not suggest, it's pretty firmly established at this point by multiple groups around the world that in order to get an efficacious vaccine, you have to activate the proper innate immune sensor. And so like I've already shown you, there's a dozen, and I didn't even show you all the innate immune sensors. There's a dozen of them. And so if you're making a vaccine with only a protein product but you actually need to activate the RNA sensor or the DNA sensor to create an efficacious vaccine, you're not going to get an efficacious vaccine. Then the other half of that, at least from the ophthalmology perspective, is that the therapeutic options and herpetic infections of the cornea are rather, let's say, I guess... You have two choices. You start antivirals, and then whether you start glucocorticoids or not later is kind of the question. So we'll talk about this as we go, but is there a more specific therapy possible? Because we all know the systemic... well, not necessarily systemic, but a lot of us have glucocorticoids, and then also I'll kind of elude on some of that as we go. And then let's return back to this question of what drives innate recognition of HSV1 and the cornea. And so to kind of introduce you to the first model, we basically used a mouse model in which we scarify corneas, topically inoculate HSV1 onto the cornea, and then we can look at specific time points compared to uninfected controls, and then we'll actually use global knockouts and proteins to look at the innate immune system in the cornea, at least in mice. Then we can do anything basically you could ever think of. We'll use plaque assays, basically where we grind up the corneas and then we can evaluate the amount of virus in the tissue by adding the tissue to Vero cells, and then HSV will actually form a plaque, and so we can actually count the amount of plaques in the tissue to give us a good logarithmic idea of how much virus is in the tissue. Then we can do all sorts of imaging, and we'll kind of walk through some of this data I actually won't show you. So then our first question became if interferon beta seems to be the predominant subtype produced during interferon infection, or at least when you treat cells with tolycperceptor-3 agonist, what is actually the predominant interferon subspecies when it comes to HSV1 infection? And so we looked at both interferon alpha and beta. They're predominant interferon or antiviral subtypes of the six interferons I suggested earlier. And so we looked at interferon beta. I don't show that data here due to time, but basically all interferon beta is restricted to the lumbus and infiltrating leukocytes. Then if we look at interferon alpha, you can see here in the middle panel, and this is kind of a busy panel, but I'll just kind of quickly highlight some of this, that if we look at HSV1 infection, so HSV infected cells are in red, blue is nuclei, so it's DAPI staining, and then interferon alpha is in green. We see that it seems to be interferon alpha that's actually produced by virally infected cells, and then also that it's the surrounding, as you see here with this yellow arrow, that cells that are uninfected will actually secrete the interferon alpha as well, and that's not a surprise since it binds in an autocrine and paracrine manner to basically reduce the ability of the virus to spread. So then at this point we're already scratching our heads thinking, okay, it's not interferon beta that seems to be driving the innate immune system in Acornia, but it seems to be interferon alpha. So then we took all the literature we had at the time and basically hypothesized what much of you would hypothesize at the time with the data that I've shown you, that the loss of toluic receptor signaling reduces HSV1 containment in Acornia due to loss of type 1 interferon production. And to first touch on that, we have, and I'll refer to it as our gold standard mouse, but it's a mouse that is deficient in one of the heterodimeric proteins of the interferon receptor, and so when you knock it out, you get no jack stat signaling and therefore you activate no antiviral pathways, at least when it comes to interferon. However, the mouse is still able to produce interferon, it just cannot respond to the signal. And so if we show you how susceptible these mice are to infection, this slide is very busy, but basically to boil it down to this. If you compare these knockout mice to wild type controls, you can clearly see that by three days post infection in almost any tissue you look at in a mouse's body, you have significantly more virus. So if you look in the brainstem, the trigeminal ganglia, the lymph node, the spleen, the thymus, then you can actually show that these mice, well, in these type 1 interferon receptor deficient mice that the virus is actually able to go by remake. These mice get extremely sick by day 5 or day 6 post infection. 90 to 100% of these mice are dead by day 5 post infection, likely from an encephalitis. So they're extremely susceptible to the virus and showing the extreme role of type 1 interferon in antiviral immunity. Then if we look at the complications of what happens when, at least from a pathological standpoint, of when we knockout type 1 interferon signaling an acornia, we can show you here on the left is a wild type control at 5 days post infection and then here start the image quality. It's not the best, but here on the right you can see that you get a ton of edema and actual stroma itself infiltrating neutrophils and I don't show the flow cytometry to confirm that, but we've confirmed that. Then you basically lose all epithelion of the actual CD118 mouse showing that these mice are extremely susceptible when it has pathological consequences on the cornea. Then we can basically conclude at this point that interferon is critical in host defense against the ocular HSC infection and we'll go down that road a little bit more. And then secondly that the loss of interferon signals results in rapid dissemination and death of a host. So these are absolutely critical signals. Then to get back to our hypothesis of whether toluic receptors are responsible for driving innate immunity we then took mice deficient in proteins and TRIF in Mighty 88, so the adapter proteins to all toluic receptors compared those to wild type controls and then our gold standard the type 100 interferon or CD118 deficient mouse. And we can see from this that the loss of those toluic receptors has no effect on viral containment. And then if we wanted to then that's at five days post infection and these are using those plaque assays to quantify the amount of virus in the cornea. If we then look at 24 hours post infection it appears that the role of type 100 interferons at least that we can appreciate from plaque assays is somewhere after 24 hours post infection. And so then at this point and this isn't actually a completely firm conclusion but the loss of toluic receptor signaling has no effect on HSV containment in the cornea when compared to CD118 mice five days post infection. And so your question probably or maybe you haven't really thought about this yet but quickly became apparent in my mind is what if a toluic receptor if you knock out that signal if another toluic receptor actually pick up the slack and actually recovers for missing toluic receptor. So if we're only knocking out one adapter protein there are other toluic receptors that can signal. And so I told you there's three toluic receptors that have all shown some role in HSV and aid immunity and so if we have others to cover for it if we're only knocking out one adapter protein then we may not be identifying the actual compensatory mechanism. So what we did is we actually back crossed toluic receptor adapter protein deficient mice into a double knockout and you see that when we knock out both toluic receptor adapter proteins it has no effect on viral containment in the cornea which would highly suggest that the loss of toluic receptors has no effect on the aid immunity in the cornea. And then just to kind of further solidify this I don't show the double knockout here but it has a basic finding that you see here you still see interferon production in all of these mice and like I said CD118 mice can still produce interferon they just can't respond to the signal because of the receptor. Then if we look down here and it's kind of small and I apologize for that but basically some of the interferon regulatory proteins we looked at a few of those by real-time PCR and they're activated in much the same way as wild type controls in our toluic receptor adapter protein deficient mice. And so basically we can conclude at that point that the loss of toluic receptors has no effect on downstream signaling at least when it comes to aid immunity. And I don't show this but the NF-Kappa B signaling cascade so this toluic receptor pathway or not toluic receptor pathway NF-Kappa B doesn't vary in that as well so it's NF-Kappa B and interferon production and it seemed to be completely fine even if you knock out toluic receptors. So then probably the biggest question that had us scratching our heads for quite some time is what is driving interferon production in NF-Kappa B signaling despite a loss in toluic receptors? So I've already told you that there are DNA sensors there's RNA sensors and probably any graduate student's worst nightmare is that you're going on a phishing experiment for an unknown protein. And so we decided to then look at what was known and then go with that. So I've already shown you that toluic receptor 2, 3, and 9 through the loss of toluic receptor adapter proteins seemed to have no effect in the cornea. So then that brought us to 4 sensors which seemed manageable unless it was an unknown sensor. And so we have, I'll first start with RIG-IMDA5 so that's an RNA sensor and in the literature if you dig back there's been a couple of reports in regards to HSV innate surveillance and they're not real strong reports in fact others have actually suggested that there's another sensor actually covering for the loss of RIG-IMDA5 and so it doesn't appear that they play a significant role in HSV surveillance. So then that brought us to the DNA sensors and so the first DNA sensor actually identified was DAI and I'm not going to tell you the actual full-length names of these proteins that are much easier just to give you the abbreviated because these are like four or five word-long proteins and it would just get very, very difficult to follow. So DAI had been shown that if you knock out DAI in macrophages you lose interferon beta production in regards to HSV infection. However if you take a mouse that's deficient in DAI has no effect on viral containment suggesting that DAI wasn't the innate sensor that we were looking for because we're looking for a sensor that makes the mouse exquisitely sensitive to the virus itself. Then DDX41 was actually a recently identified protein that had a similar role of DAI and been shown to play a role in macrophages but not much more than that. Then kind of the protein that we decided to hang our hats on at least from a research perspective was IFI-16. So I literally walked in so this paper was published in Nature Immunology as I'm trying to figure out the innate sensor responsible for HSV surveillance in Acornia. Paper was published in Nature Immunology the next morning I walk into my mentor's office and I say after six months of coming up and de-handed that I know what the innate sensor is and he was like Chris how can you be so confident in that? And it was due to the fact that this sensor had just been shown to drive an interferon response in regards to HSV infection in macrophages. So similar story that's how most DNA RNA sensors are identified in macrophages. However the other literature if you dig back to the late 1970s and early 1980s is that IFI-16 is actually expressed in prostate epithelium that when it is mutated prostate cancers now proliferate unabated and so that would actually suggest remember I told you interferons were critical in apoptosis and so it had been known for quite some time that if you lose interferon signaling and cancers that cancers proliferate and so we were looking at a protein that seemed to be regulating more than just interferon, beta production in macrophages in regards to HSV infection that it seemed to have some role in other tissues specifically in prostate epithelium and while the prostate is not the same as the cornea it at least was an epithelial tissue and so it was at least something that we could kind of grasp at that point. And so first we had to identify whether this protein was even expressed in corneal epithelium and so you can see here this is DAPI staining of the nuclei then P204 is the mouse homologue of IFI-16 and we have staining for that in red and you can clearly see that it's localized to both the nucleus and the cytoplasm and I didn't hound on this but this protein actually goes back and forth from the cytoplasm to the nucleus so it's no surprise that you see it in both places. Then to kind of further hang our hats on this we knew that the epithelial tissue was extremely sensitive to HSV infection and so this virus or this innate sensor was predominantly expressed in the corneal epithelium as you see here and let me use this instead as you see here in this 3D reconstruction of the corneal so this is the epithelial tissue here then the stroma here and then endothelium you can't really appreciate very well here but most of it's actually expressed in the epithelial tissue. Then we did something that we didn't actually think would work but that's a lot of science I guess so we took mice, we actually anesthetized them then transfected them with S-I-R-N-A to control and then also mice with P204 to knock down that protein to see if it had any effect on HSV surveillance because there was no global knockout of P204 IFI 16 and you can clearly see here that the loss of P204 results an increased susceptibility to HSV infection however you're probably thinking well that isn't near as drastic as what we saw earlier and so we first thought that maybe that was due to the fact that we were using a different model but probably the bigger thing was that when we look at the actual knockdown of how much knockdown we're actually getting we were getting anywhere from 40 to 50% knockdown of that protein of P204 so of IFI 16 in the corneal so that would probably mitigate a large portion of what we're seeing however we would then want it to look a little further and like I said IRF subspecies are translocated to the nucleus after activation of innate sensors and so if we look at the cytoplasmic portion of IRF3 which is the important IRF at least in regards to interferon alpha we can see that there's really no difference in the cytoplasmic portion of IRF3 however you can clearly see that when we knock out P204 with this 50% knockdown that you lose a good portion of the nuclear portion of IRF3 suggesting that we're losing the signal to drive interferon production with the loss of P204 then just to kind of further solidify that we then took mice, did the exact same thing transfected them, infected them and then did hole mounts and looked 48 hours post infection and you can see here that in our control you get interferon alpha production just like we saw previously however when we knock down P204 you lose that interferon alpha production in the cornea further solidifying the role of P204, IFI16 in interferon production in the cornea so then to take you back to this signaling cascade like I said we'd continue to return to this just to remind you, I have to remind myself as well IFI16 has an adapter protein STING STING then activates another protein that then activates another protein and there's probably five or six proteins that I don't show here needless to say it activates interferon regulatory factors so IRF nuclear translocation to drive interferon production so we did know that there was a STING deficient mouse out on the market so we actually contacted a group and wanted to look at what the loss of STING had in the cornea, so the downstream adapter protein of our IFI16 P204 so you can clearly see here that the loss of STING in CD118 there's no difference between their viral control at 48 hours post infection furthermore both of them are significantly elevated when it comes to the amount of virus you can recover from the cornea further solidifying the role of IFI16 P204 and viral surveillance of the cornea so then what probably any clinician then begins to ask is this just a mouse phenomenon so we wanted to kind of attest to that so we took an immortalized corneal epithelial cell line and wanted to first see if IFI16 was expressed in that cell line and you can see that there's a protein with a predicted size of IFI16 and then if we actually do confocal microscopy of these cells at least in this portion of the cell cycle most of the IFI16 protein is localized to the nucleus but it is expressed furthermore if we then look at IRF nuclear translocation so if we use control if we use SIRNA to knock down IFI16 and compare that to controls and we've quantified this but I'll show you the sexy images instead you get clear nuclear translocation as you can see here with purple staining where you have red and blue overlapping showing the nuclear portion of IRF however when you knock down IFI16 that protein is restricted to the cytoplasm showing that it's not activated and actually being nuclear translocated furthermore if you knock down IFI16 you have a significant rise the amount of recoverable virus from these cells then like any good Oklahoman we like to fish the picture up to this point in innate immunity had basically referred to sensors very specifically as sensors due to the fact that no one had actually shown that these innate sensors actually directly interact with the protein, DNA, or RNA that they're sensing or that they're just kind of prodding along, sensing the environment around and so what we did is we took these human corneal epithelial cells we transfected DNA very similar sequences to HSV into these human corneal epithelial cells within cross linked all proteins in the cells lice the cells then used IFI16 antibody to then pull down and then we could clearly show that when you have IFI16 you pull down DNA both single strand and DNA double strand DNA products that we transfected into the cell however when you don't have IFI16 or if you don't have DNA products you don't pull anything down suggesting that there is a direct actual sensing of IFI16 with DNA in the host cell whether that's actually cytoplasmic this would actually probably suggest that it's cytoplasmic because it's very difficult to get proteins into the nucleus or DNA into the actual nucleus this would suggest that it's a cytoplasmic interaction but it's still debatable at this point whether this interaction actually takes place in a cytoplasm where a virus would actually release its DNA products to then traffic into the nucleus or whether this is actually in the nucleus occurring so then the next question became does this happen with other pathogens are we just finding a nice phenomenon that just so happens to work that's not all that we've checked so far it didn't I'm just showing you the highlights there were a lot of negative data in this whole process and so if we look at an RNA virus vesicular stomatitis virus and I can clearly show you here with real-time PCR because it's not a plaque forming virus so this would be a way that we can quantify the amount of virus that when you lose our type 1 interferon signal you lose the ability to contain the virus so type 1 interferons are critical and the innate immune response to VSV however if you knock down P204 it does not have the same effect of losing P204 in regards to HSV infection suggesting that or I guess solidifying that this is a DNA sensor IFI 16 P204 and that at least at this point it's HSV specific but it's likely all DNA viruses then to kind of give you a model of what I've shown so far and some of this data I haven't shown you because just due to time constraints virus replicates epithelial tissues then activates P204 IFI 16 to then activate Sting and then probably a half dozen other proteins to then lead to the activation and dimerization and phosphorylation of IRF to then nuclear translocate to drive interferon alpha production and what I haven't shown is interferon alpha production while it drives PKR RNA cell apoptosis of cells and MHC class 1 expression we also show that it actually binds to the upstream promoter of a chemokine that's critical in driving inflammatory monocytes to the side of infection so while I'm showing you the major portion of innate immunity there is a bone marrow derived response while it is small it is significant it seems to be driven by inflammatory monocytes then I don't show the data on this but in the vaginal mucosa so in epithelial tissue it seems to be the same response so we actually knocked down IFI 16 P204 in mice and then infected with HSV2 and you get a significant rise in the amount of virus however we knew from literature that there's a group of children in France that are highly susceptible to recurrent herpetic encephalitis and that has been I guess remotely connected to a loss of tolite receptor 3 signaling and so we knew that when tolite receptors were first identified the person he actually didn't win the Nobel Prize and was kind of a controversial thing but a friend of his won the Nobel Prize for tolite receptors the guy that actually identified tolite receptors hypothesized that tolite receptors are omnipotent they actually drive all innate immunity and all cells and all tissues well it seems like a very strong statement and we were seeing something somewhat different and so we wanted to first test that and so if we look in the central nervous system and I'm only going to show briefly this just because I think it's interesting and the loss of type 1 interferon seems to be critical and you get HSV infection of appendable cells then you can further confirm that you lose the cilia of appendable cells you get some red blood cell hemorrhaging in those sites that you don't see in wild type controls and these mice get extremely swollen heads have neurological pathology that would be any neuropathologist dream but I'm not going to show any of that but to then go on to show that human appendable cells are actually extremely susceptible to the virus however it seems to be that if we take mouse brains and we actually take 200 micron sections of mouse brains and keep them alive on tissue culture and tissue culture we can show that if you lose the TRF adapter protein so the tolite receptor 3 adapter protein then when you lose that you lose innate immunity in the actual central nervous system then if you deplete macrophages with chloronate it's a very specific depletion that you actually lose that so it seems to be tolite receptor 3 in macrophages that's driving innate immunity in the central nervous system so to conclude the loss of p204-i5-16 results in enhanced viral susceptibility I've pretty clearly shown that then interferons play a crucial antiviral role in acornia as well as the entire host then the role of tolite receptor 3 in IFI-16 is tissue specific and that actually got us a lot of heat as we were trying to publish some of these papers as a Nobel Prize I guess non-winner but should have been winner was reviewing our papers and then lastly that p204-i5-16 drives recognition of HSV in acornia and it's I don't show well I didn't show as strong of data for the human side of that but I did show that in human corneal epithelium it seems to matter as well obviously you would have to do well you couldn't do those experiments then I'm going to leave you with a few questions just foods for thought and so first will inhibiting tolite receptors decrease complications of herpetic infections of acornia I don't show the data but groups have shown that tolite receptor 2 and even tolite receptor 9 seem to drive a very pro-inflammatory state so is it possible in acornia that when you have tolite receptor 2, 3, 9 IFI-16, p204 all being activated by HSV we clearly show that p204 is the antiviral innate sensor responsible for antiviral immunity but are these other sensors just driving a pro-inflammatory state so could you specifically target so instead of using glucocorticoids could you specifically target tolite receptors to decrease the pro-inflammatory state and hopefully the pathology that you see with herpetic infections of acornia that's anybody's guess at this point but that would be at least be you know something plausible with tolite receptor antagonists on the market at this point and then the second half of that question is the entire immune response a good thing and I've actually written on this subject before but in a central nervous system it's clear that the entire immune system if you add in the adaptive side of things that there's a pathological side of the immune system but then there's an important antiviral portion of the immune system patients they get long-term neurological pathology it's likely due not to the virus I mean the virus is activating the immune system but it's actually likely due to the immune system trying to clear the virus so you're actually getting that central nervous system pathology due to that and that's actually being tested in a huge clinical trial in Europe where they're using glucocorticoids and herpetic and cephalitis patients then the next question it's probably true but we haven't tested that so I can't say that it is but does p204-i5-16 drive an aid immunity to VSV so Zoster and so they're both alpha herpes viruses and so they're very very similar when it comes to structure of the viruses themselves and so it's likely that this is the same sensor for both viruses and so if you were to change therapy you would change it for both then lastly kind of the biggest question for thought is will HSV vaccination attempts to activate this innate sensor produce an efficacious vaccine and then furthermore could you actually design a vaccine that you could take into third world countries that's actually a topical eyedrop vaccine that would be much easier to take than a bunch of syringes only time would tell if you could actually do that and not induce pathology in the eye but it does seem plausible at this point my mentor is actually not necessarily testing well he is I guess testing a live attenuated virus very attenuated to see if he can actually activate this immune response then I'm only going to touch on one other thing because our lab has shown this quite clearly I've done a little bit of work on this but it was actually another graduate student in our lab that if you don't contain the virus early you get an increase in the amount of lymphatic vessels into the cornea and so from the pathological side early antiviral containment is crucial and interestingly this story actually goes back to HSV protein actually specifically drives VEGF-A production to drive lymphatic growth into the cornea and that's all been shown by this previous graduate student but it just hounds on the idea that you have to contain this virus early or you're going to have long-term complications and then here are my citations you can look those up if you'd like and then I have to give thanks to a lot of people and I boil it down to just a few people mainly from the people in my lab but let me just point out two people first my mentor Dr. Dan Carr at the D. McGee Institute and then a technician in the lab Min Zhang that was absolutely phenomenal let me do a lot of this work that I probably wouldn't have been able to do with my own hands and then with that said Dr. Wong asked me to put up some pictures of myself she actually wanted me as a kamikaze something I don't know when I was a kid I didn't have any of those and so this actually I guess my honeymoon with my wife were actually in Australia river rafting and here I am playing I guess a paddle guitar and my wife I guess right here is I guess about to bring the hammer down with somebody with her paddle so with that said any questions I can take at this point Dr. Harry so live attenuated so when you give live attenuated viruses if you actually look at almost every viral vaccine their live attenuated viruses the one example well there's two that come to mind that aren't so the flu vaccine you can actually get that intranasally or also polio and so I would actually argue that in the polio vaccine the live attenuated pill is actually better for you because it activates the proper innate system and actually now in the US for giving injections so it's not as efficacious and there's literature that actually shows that but it would probably be due to the fact that you're actually having that viral life cycle to activate the proper innate immune system and so if it is a YFI 16 P204 you would actually have to have DNA from the virus presented to this cytoplasmic nuclear sensor so the viral life cycle is absolutely critical and so in previous vaccine attempts with HSV they've done protein based vaccines and those like I said they make you actually more susceptible to the virus and so it's probably due to the fact that you're actually giving this actual viral life cycle to induce the immune system yes Dr. Wong they're coming around now we had to get to the point where we had to have someone pretty big in the field help us with a couple of experiments she was actually the one that identified YFI 16 P204 and so she's in a she works in a lab at UMass and so we had her do an experiment for us more is like hey can you check our work because reviewers are absolutely just slamming this idea and it was the same guy over and over again because you could tell from the writing in these reviews and so once she signed off on it and did this experiment for us it was like oh well if she signed off on it that must be true because this little Dean McGee lab in the middle of Oklahoma doesn't know what they're talking about but now that someone from UMass has used huge papers in the field now that she is actually signing off on it and it's done some of the work well I say she did some of the work she basically confirmed in her lab that what we were seeing was the same thing and so yeah from that they're finally catching on it's actually becoming more prominent in the field now so we'll see, time will tell yes well so P204 IFI16 hasn't been shown to directly activate those proteins but what it does is it activates interferon, interferon then binds to the heterodimeric interferon receptor then signals to the jack stat pathway to induce MHC class 1 expression keep in mind that MHC is always class 1 is always expressed on cells but it's actually upregulated in viral infections or anytime you have interferon production the virus actually has specific mechanisms to down regulate the MHC class 1 expression so it's a huge battle that I didn't describe here so it's a downstream effect of the sensor but it doesn't directly activate those two proteins or at least it hasn't been shown to this point which would be skipping a step in this long cascade so it's likely not directly driving those protein products or even apoptosis but it is having a downstream effect of that. Thanks, is that okay? So here is your goal down the road to some point to do cornea? I've thought about it but I'm kind of going back probably, I'll probably end up I'm pretty interested in uveitis from the innate side but honestly presentations I have a more basic science question of why I saved it so you mentioned that you believe that it was actually a DNA sensor and not an RNA sensor did I mishear it when it seemed like the IFI 16 was more with the DNA? P204 is the mouse homolog Oh that's right, that's right and that's a different name so same name and so all the names in innate literature there's a name like four names so then how did you then with RNA what was their respective thing oh rig I and that was the one there and then I noticed when you're showing the graphs


Good afternoon folks. Steve Rhodes coming to you live from the shores of sunny Delray Beach, Florida. This is your 2 p.m. Update and currently got the mixed bag out here with the Dow off 137 the S&P down to in the Nasdaq up 77. Russell's off 29 semis are up 31. Trendy's are down to 41. There's a lot to look at. Gold's off 2 bucks. Silver down 22 cents. Let's go ahead and begin looking at our industry charts out here. Give me a moment to just change over to that set of screens. We've got eight different charts that we can look at. The first one is going to be the Dow Jones industrial. So as we take a look at its chart, I'll just expand this out. And there really is no topping pattern or signal that I've got here in play. I don't have a completed a to be equal CD pattern. I got nothing doesn't mean that it didn't top or can't top. But what's important about this chart as long as price remains below this little green line, the oscillator and change line. This is saying over time price may be targeting its breakout level breakout levels 33 869 83. You can see that price has tagged that level once that was back in the July timeframe. So it may be targeting that level again 33 869 83. The S&P 500 closed below its breakout level yesterday of 44 886. Today has been a test and rejection of that. And that would then signal to me because price did not get back inside that range that the break from yesterday is real. Now price could rally into the close and close above 44 886. That would then offer the opposite message and say yesterday's move was a false breakdown. So watch 44 886 inside the NASDAQ 100. It has a rogement indicator top. But prices really just been trading sideways. Even as I pull this back here, just a sideways move. So now to the downside, its level of breakout support is 39 67. My saying that's where price is headed to. No, because we'd have to go take a look at the NQ and there'd be levels that would have to be broken broken there. The Russell just headed back to where we have prior lows. So that hasn't broken anything out. And this is the odd one. Not that it's odd. But if you were to ask me a question, and you were to say, Stevo, of these eight indices out here, which one would you take a long trade in? My answer right now would have to be the semiconductor index. Today is going to be bar number eight of ATD nine count and prices back inside as TD nine break out break a breakout level 32 16 98. Very interesting markets out here. Folks, stay tuned. Your favorite polar bears up next. I'll see you


The next item of business is an urgent question, and I call Brian Whittle. To ask the Scottish Government what its response is to the publication of the six-case study findings by the Scottish child abuse inquiry, in which the chair, Lady Smith, criticised the Scottish Government for a woeful and wholly avoidable delay in setting up an inquiry into accusations of historic abuse in Scotland. The Scottish Government apologises unreservedly that it did not respond more appropriately and sooner to the concerns of survivors of abuse and care who called for a public inquiry. Responding to survivors of abuse and care spanned different administrations between 2002 and 2014. Steps were taken by Government to respond in that period to the issues raised in the original petition, but this happened too slowly and did not go far enough. An inquiry was announced within weeks of the current First Minister assuming her office in 2014. We welcome the inquiry's on-going work, which is addressing the harrowing suffering experienced by survivors. We are grateful to survivors who have bravely come forward to participate and give their testimony. The Scottish Government will consider and address any future recommendations made by the inquiry to improve legislation, policy and practice. We are listening to all the issues and are determined to ensure that lessons are learned. I appreciate the Deputy First Minister's response to that question. I know that we have discussed this before, but Lady Smith said that the Scottish Government failed to grasp the fundamental importance that survivors appropriately and justifiably attach to their need for justice, accountability and redress. Furthermore, there was a failure of ministers to listen and engage with survivors. The redress bill focuses on those who have suffered abuse in a care home setting, with the Deputy First Minister stating that the bill was tackling abuse where institutions had illegal status and obligations of parents. Welcome, though the report and the redress bill are, they fail to recognise that there are victims with the same trauma from abuse within local authority buildings such as schools, where similar parental responsibilities are afforded to teachers. It says in the 1989 education bill the words, in local parentis, the definition being responsibility for a child where the child's parents are absent. In other words, while children are in school, teachers are legally in local parentis. I ask the Deputy First Minister why consideration was not given to those victims in the inquiry because their abuse and their trauma is no less acute. I understand entirely the sentiments that Mr Whittle puts to me, and in no way would I seek to differentiate in the suffering of individuals in any circumstances wherever a child was abused in whatever circumstance is wholly and utterly unacceptable and the perpetrator's risk is reprehensible for their conduct. The establishment of the abuse inquiry focused on essentially addressing the question of in-care settings where abuse had taken place. That was the substance of the original petition, and it was the basis of the extent of where the state essentially assumed the role of providing parental support to a child on an on-going basis. The example that Mr Whittle provides to me of schools is not on a permanent basis. It is on for a period of time during the day when parents remain in the role of parents in those circumstances. That explains the distinction, but I would not in any way want to seek to establish any lack of equivalence in the suffering of individuals in what they have endured in those circumstances. Brian Whittle, I appreciate very much that the Deputy First Minister's response to that. I have suggested to him in the past that the way in which the law has been drafted breaks the UNCRC law and creates inequality, and I have written to the UNCRC and await its response. I have witnessed the deterioration of one of my constituents' mental and physical health while trying to help her journey through the judicial system as she searches for justice, redress and closure. In dealing with sexual abuse cases, both currently and historically, for constituents and in both the Health and Sport Committee and the Petitions Committee, it is my conclusion that the system does little to support the victim in their search for justice. There are many agencies with a part to play who do not seem to be able to communicate with each other, so it is little wonder that the conviction rates of solo and alleged victims of such crimes are continually re-traumatised by the processes. Will the Deputy First Minister please take the time to look again at the journey of a victim through the justice system, consider the changes that are desperately needed and put the support in place to ensure that all victims' voices are heard? I am absolutely committed to ensuring that the voices of all victims are heard. That is precisely why the Government took the step. Despite there having been a number of different initiatives taken in the period during 2002-2014 to hear the voices of victims, I accept unreservedly that all of those steps were not good enough until we had established the inquiry. The inquiry, in my view, is fulfilling a difficult, painful but necessary role in Scottish society to oblige our country to face up to its past. The forensic nature of the interrogation of the evidence by Lady Smith, the power of her conclusions, I hope, provides some assurance to survivors that their voices are now being heard. Mr Whittle fairly raised the issues of the difficulties that individuals face, where they have already suffered in trying to pursue those issues through the judicial system. That is exactly why the Government established the redress system, because we want to try to minimise the suffering of individuals in trying to help them to achieve some form of redress. Indeed, the evidence that I now see from the advanced payment system that we have had in place now for around about two years is that individuals are appreciating a pathway that is enabling them to seek some form of redress for their suffering, to help them on the road to recovery. I assure Mr Whittle that the issues that he raises are taken very seriously by the Government, lies at the heart of our approach to establishing the inquiry and the thinking behind the redress bill that Parliament has already passed into statute. I would like to place on record my thanks to Lady Smith in her team for this very thorough report, which puts beyond any doubt the serious failure of Scotland's governing institutions and how badly survivors have been licked down. Can I ask the Scottish Government if it is satisfied that the catastrophic failures of process outlined in the report have been rectified and can ensure that they no longer exist as a barrier to justice in Scotland? When the first payments of the redress scheme will be made and what progress has been made in setting up the survivors forum, which will ensure that survivors are supported throughout the process? In relation to the process that we are going through, I assure Mr Marra that the Government is acting to rectify any of those past failures. I am not going to say in Parliament today that the journey is complete because I await further conclusions from Lady Smith's inquiry. Indeed, it would premature for me to say that the journey was complete because I do not know the scale of the challenge that Lady Smith will put to us, but I put on record my determination to make sure that that is properly addressed by the Government. In relation to the redress payments, all of the milestones for the establishment of redress Scotland are being met. The chair is in place, the chief executive officer is in place, and the steps to organising to open the scheme before the turn of the year will be met. I am confident of that. What then flows in relation to payments being made is slightly dependent on the applications that come forward and the nature of the process that has to be gone through to verify those applications. I assure Mr Marra that, based on the experience of the advanced payment scheme, I would be confident that payments could be made swiftly after the receipt of applications. By swiftly, I suspect that I should put some degree of time into that and that there is due time for consideration, but the advanced payment scheme has paid very quickly in relation to those payments. In relation to the survivor forum, various steps have been taken to interview individuals to be part of the survivor's forum, and, as I indicated in my earlier answer, the development work on that is on track. To ask the Deputy First Minister in what ways the redress for survivors historical childhood abuse and care Scotland act provides tangible support for those who have experienced abuse and care. As I have just explained to Mr Marra, the steps to establish the redress scheme are under way, as envisaged in statute. The advanced payment scheme will remain open for those eligible by either age or nature of their assessment of their health to enable them to participate in that scheme. I am confident that those arrangements are available on a timuous basis for individuals. I think that I have to say that, although the scheme can deliver payments to individuals, it is a question of whether that helps in the recovery of individuals from the trauma that they have suffered. However, I hope that the combination of the fact that the inquiry is able to provide findings of the power that Lady Smith has expressed, and this is the sixth report. The other case studies are focused on the individual settings in which abuse takes place, and they are, in my opinion, a very difficult reading indeed. I hope that the prominence that has been given to those issues, the reflection of the detail, the acknowledgement by Lady Smith of the veracity of the events that are accounted, helps, along with the redress payments, to provide a route to recovery for individuals who have been so badly let down by the state in those examples. I welcome the Deputy First Minister's apology and recognise his long-standing personal commitment to the issue that has been around for far too long. Lady Smith said that far too long survivors' voices were not listened to, not heard, they were treated as if their views did not matter and as if they were not worth listening to just as if they were abused in care. Lady Smith said that we must learn the lessons from this tragedy. I ask the Deputy First Minister to tell the Parliament what safeguarding issues are in place to ensure that the voices of survivors are never silenced by officials or ministers in this way ever again. Of all the comments that were made in the report by Lady Smith, the quote that Pauline McNeill has recounted to the Parliament is the one that I find the most difficult, because institutions of government handle issues in particular ways, but there is quite simply no defence for the treatment of the words, the circumstances that are led by Lady Smith to write those words. There is just no justification whatsoever. For example, I chair the national trauma training board with the Government personally, and it is designed to essentially address some of the behaviour that led to the dismissiveness of individuals. That is just one example of how we are trying to change the culture with the Government to ensure that the voices of individuals are listened to. I have to also say that at a time when our Parliament and its processes can often be attacked, one of the elements of this Parliament's processes, the Public Petitions Committee, is at the heart of providing a platform and a voice for the individuals that Pauline McNeill raised. If it had not been for Chris Daley's original petition to this Parliament, the seriousness with which it was taken by my Government man, when he was chair of the Petitions Committee, his tenacity in challenging Government about it, and the tenacity of other individuals such as David Whelan or Helen Holland to pursue those issues with ministers of successive administrations over many years, we would frankly not be in the position where we have to, as a Government and an institution, confront some of those hard and uncomfortable realities. I am hopeful that we will be the better for it in the way in which we deal with individuals and some of the practical steps that we are taking, such as the trauma training and work that I preside over, is designed to address exactly the circumstances that Pauline McNeill puts to me. That concludes the urgent question. The next item of business is a debate on motion 144 in the name of Angus Robertson on the impact of Brexit on Scotland's supply chain and labour market. I would ask members who wish to speak in the debate to press their request to speak buttons. I call on Angus Robertson to speak to and move the motion. Cabinet Secretary.


Item Number – SCP-334 Object Class – Euclid Special Containment Procedures SCP-334 is to be kept in a standard heat-treated containment cell, surrounded by magnetic field generators, positioned so that all six sides have an overlapping field, projecting at least 15 cm from the inner cell walls. Each generator is to have multiple redundant backups and power sources, including emergency batteries capable of maintaining full magnetic field output, for no less than 30 minutes. Maintenance of the anti-heat coating is to be carried on a weekly schedule, with subject to be transferred to a secondary cell, to allow for more extensive repairs if needed. Three live mice are to be introduced to its cell at a random time every day, to reduce chances of subject learning the pattern and attempting escape. Due to SCP-334's hunting and burrowing instincts, the floor of containment is to be reinforced with an anti-heat coating, or made of a substance with a melting point greater than 0°C, with the magnetic containment field underneath reinforced to approximately 1.5 times that of the other surfaces. Subject hunts in a fashion typical of foxes, with a strong downward pounce, which has been proven capable of penetrating a small distance into the containment fields, and has been the cause of numerous containment breaches. Description SCP-334 is a small cloud of superheated plasma, in the shape of a specimen, most closely resembling Volpe's Volpes, or the common red fox. Its body appears similar to waves of steam or fire, revealing gaps between the filaments of gas through which the opposite side is visible, its filaments fluoresce in the spectrum of red and orange, with eyes in the blue spectrum. Subject has a negligible measurable mass, and has no central body to speak of. SCP-334 apparently believes it is actually a red fox, and displays several normal Volpean behaviors such as hunting of prey, and in test 334F-03, whether it is an entity that imprinted on a Volpean, or is a Volpean which was transformed to its current state, is unknown and under investigation. Since it is composed of superheated ionized gas, containment of SCP-334 is effectively impossible without the use of magnetic containment fields, and is difficult even then, as the heat and energetic effects extend an average of 7 cm around its entire body, rapidly sublimating physical obstructions. Although its surface temperature is in excess of 0°C, the heat disperses rapidly, cooling to 0°C within 10 cm, far faster than the laws of thermodynamics would indicate. There is evidence that SCP-334 is capable of controlling its heat output. Subject is capable of bursts of speed measured at approximately 160 km per hour, for around 10 seconds. These bursts of speed disperse the subject's mass, tiring it out, making it need to rest while it reforms. Subject derives nourishment from converting matter into plasma and absorbing it, taking in a small amount of energy normally through conversion of air into ionized gas, but that alone is insufficient to feed it, making supplementary feeding necessary. Despite the fact that it does not touch the ground when it walks, and is immune to the pull of the Earth's gravity, it seems content to follow the floor's surface for walkable areas, and has yet to be seen actually flying. Whether it chooses not to, or simply does not know that it can, is unknown at this time. Due to its physical make-up, SCP-334 constantly emits a level of EM radiation, consistent with a clasp of solar flare, causing interference in unshielded electronics within 1 m, and making film or electronic surveillance problematic. Radiation is within tolerable limits for SCP personnel. Initial Containment SCP-334 was recovered several kilometers from a projected meteor impact site. Whether this was the subject's origin is still under investigation. The Foundation was alerted to the subject's existence when a routine sweep of emergency services reports found a pattern of strange burns, trails, and wood discovered in the woods, matching no publicly known source, but consistent with data expunged. A full Foundation combat team was sent in to contain the suspected wood, but found SCP-334 instead a far lesser threat. Utilizing a magnetic containment bottle and two live mice, subject was contained with an acceptable level of casualties. Addendum SCP-334 has a talent for escape, having escaped containment numerous times since initial retrieval, causing structural damage to site and the deaths of several personnel, mostly caused by the aforementioned structural damage. SCP-334 is wary of people, avoiding human contact if possible, yet prefers hiding in offices, barracks, and on-site quarters, where there is relatively little traffic and an abundance of small spaces. Despite its many escape events, SCP-334 has shown neither an obvious inclination towards leaving site nor towards unprovoked attack, and is not considered a direct threat. However, the structural damage caused had the potential to release SCP-334 and recapture should be considered a priority. Partial Testing Log Resulting matching expectations removed, available upon request. Experiment Log SCP-334-T1 through SCP-334-T48 Date SCP-210 Test Subject A selection of small prey animals, ranging from mice to medium-sized rabbits. Results Subjects stalked each one for varying lengths of time, apparently biding its time before pouncing. Each test subject sublimated into plasma, which was absorbed by SCP-334 through the mouth as it went through the motions of eating. Experiment Log SCP-334-F1 Date SCP-210 Test Subject A normal red fox, male. Results Test Subject displayed high levels of apprehension, with loud barking and growling. SCP-334 appeared disinterested. Test halted after 10 minutes of no appreciable change in either's reaction. Experiment Log SCP-334-F2 Date SCP-210 Test Subject A normal red fox, female. Results Identical to 334-F1 Experiment Log 334-F3 Date SCP-210 Test Subject A red fox, female. In heat. Results SCP-334 appeared to sniff the air for a moment, while test subject displayed reactions similar to previous two tests. Then, data expunged. Researcher note That was something I hope to never see again. Experiment Log 334-F1 Date SCP-210 Test Subject SCP-334 Note For this test, SCP-334's cell was flooded with ionized gas, identical to its own. Results SCP-334 appeared curious at first, then agitated as the pressure of ionized gas in the chamber started to rise. After five minutes, subject began glowing brightly, and data expunged. Containment was re-established, with SCP-334 apparently exhausted. Experiment Log 334-F2 Date SCP-310 Test Subject SCP-334 Note For this test, SCP-334 was subjected to a focused magnetic blade to attempt to sever a section of its mass. Results Data expunged Note Further experimentation with force-feeding or cutting SCP-334 is forbidden unless performed in a heavily shielded remote site. Experiment Log 334-D1 Date SCP-310 Test Subject D13487-334 Three live mice in standard cage Note D-Class was instructed to enter containment and feed SCP-334 after one day of withholding food from the subject. Results SCP-334 immediately leapt at the cage, incinerating it in the contents. As well as much of D13487-334's arm and torso. D13487-334 died instantly, while SCP-334 ignored the body, focusing on the converted mice in cage. Researcher Note Well, it certainly thinks it's a fox, and doesn't seem to notice or care that it can kill people by just getting too close. Experiment Log 334-E1-334-E Date D-2010 Test Subject SCP-334 Note This test was performed to determine SCP-334's environmental needs, if any. Results Containment made air tight, an internal atmosphere removed. Subjects showed no change from normal behavior patterns. Containment was then heated to an excess of 500 degrees Celsius. Subject showed no change from normal behavior patterns. Containment was then cooled to approximately negative 100 degrees Celsius. Once again, subject showed no change from normal behavior patterns. Containment was slowly pumped full of atmospheric gases in various combinations to a maximum pressure of three atmospheres. Subject became more energetic, consistent with behavior after being fed, and did not require supplemental feeding until the next day. Researcher note, it can survive in a vacuum or on the surface of Venus and doesn't need any supplemental food in high pressure. This and other data suggests data expunged. Lesson complete. If you missed the previous orientation, go watch SCP-333, City in a Symphony, right now, or for the complete course, watch this playlist.


Hi, I'm Lavi and this is Oli. We are attempting a new world record to become the youngest pair to circumnavigate the globe by motorcycle. Click the subscribe button to follow our adventure around the world. Good morning world, welcome back to the channel. It's day number seven on our world record attempt to become the youngest pair to circumnavigate the globe by motorcycle. Welcome! So we spent yesterday just trying to catch up with a bit of editing. We didn't get too much actually done, so we might need to take another day off editing pretty soon. But we managed to at least get the first episode up and on the channel, which is a big success for us. We are staying here in Dunkirk. I'm just going to take you outside. Look at that. There's a nice lake over there that we had a small walk around yesterday. As a sort of like congratulations to ourselves for getting the first video up. But yeah, we're staying at this premier class motel in Dunkirk. In this little room, our editing suite is just here. And let me show you where we are going to be heading today. So we are here, Dunkirk, after having crossed the channel two days ago. And we are going to be heading straight down towards Paris, in the small back roads all the way through the beautiful countryside of France. Just coming around the right side of Amiens. And then we are staying tonight here in Compiègne. And Compiègne is hopefully close enough to Paris that we can then shoot into Paris and through on our way south. But yeah, today we have about 143 miles to cover. If we manage to get all the way there today, then it will be our longest day yet on the road. Fingers crossed. So yeah, we are super excited and let's go. Let's go. Goodbye. I had a good night of rest. You know what? I was just ready to go in English mode. Yes. I mean I realised that we're on this side. That's all right. I've got it now. I've got it. Yeah, you've got it now. I don't know. We'll see. Okay. I was just about to cross over that I was like, there's some cars coming in the same direction that I want to go. That's weird. It's weird, eh? That's weird. They must be wrong. Oh, wait, it's me. Oh, there's the lake that we walked around yesterday. And if you can see it, but it's there. Goodbye, Dunkirk. Goodbye, Dunkirk.  Yeah.  Normally I don't have any issues like that. And then this morning we checked on Google Maps and we saw there's like a red mark over this town here. Yeah. Yeah. Like everywhere else around is like green or yellow. At least yellow, but just this area here, just this town had like a red mark over it. And we were like, okay. That's the reason that the room is so cheap. And I've got Garmin rooting us through hopefully all through nice little roads in the back villages through the countryside of France, avoiding all the big roads, avoiding all the payage.  And guys, before you forget, please hit the subscribe button right now to follow our journey here. Thank you guys. Thank you so much. You will help us a lot with this following. And yeah. Getting into the deep countryside of France now and there's not a cloud in the sky. And it's an absolutely beautiful day for riding through the deep countryside of France. I love it. And the thing about France is that there's just so much countryside. That's really nice because there's, it feels like there's a lot more open countryside. It feels like there's a lot more space here than in the UK. And it's really perfect for motorcycling. Yeah. And you can really see the spring is full on. The blossoms are out. Oh yeah. It's nice. It's starting to get nice and green. Hey guys, just a quick message. If you didn't know already, we are selling our very own t-shirt to help fund our round the world trip. It's called the Overlander and we designed it ourselves. It's got the awesome adventure motorcycle on it and the world of overlanding possibilities around. We absolutely love how it turned out and we hope you love it too. In buying one of these Overlander tees, you will be helping us to get a warm meal, a tank of fuel or a good night's sleep along the way. So all you have to do is click the link in the description to order your awesome adventure t-shirt. Thanks so much guys. Ride safe and we see you on the road. It's nice when the Garmin takes us on something a little bit smaller, a little bit more intimate than the sort of bigger roads that we've been on so far.  So good job Garmin for taking us down some smaller one track lanes. That's always appreciated. We have to adjust the suspension a little bit more even. Well make it a bit harder. Yeah maybe. Yeah yeah we can do it two clicks harder if you think it's still to bit too bouncy. I think so because it wasn't even like too bouncy the road no, but I think for this type of road I'm bouncing a little bit too much I would say. Yeah okay yeah. Just stopping for a break in the beautiful French countryside with the sun shining it's beautiful. And look what we just found on the road just in front of us just down there. It's a very it looks like a very weathered old coin. I can't really see anything on it to see what it is. But you know if you've ever heard the phrase find a penny pick it up all the day you'll have good luck. So we're going to keep it with us as a memento. We bought yesterday 500 grams of olives because we were so hungry and now today we have just to to eat them through. I put them in asparagus in the asparagus jar because the other jar was just huge. Did I have an olive? Yummy yummy. Look at this right road it's totally winding like a ribbon across the landscape and you can see all the rapeseed fields that's amazing.  So we've done about 83 miles already so far today and what's the time? It's just past one o'clock in the afternoon so we've done quite well this morning. Yes and we've routed to a Lidl which is in the town of Corbi which is coming up and it's 12 miles and that'll be our lunch break for the day. It's always good to have your lunch break at a Lidl because then you know whatever you have for lunch you know it's only going to be a couple of euros so it's good. Yeah and especially at Lidl here in France they have a nice bakery section with fresh croissants, fresh baguettes. Yeah yeah you always have to pick up a croissant, a baguette and a pan of chocolate. Low budget French patisserie travel tip. Lidl and my bum is sore so it's good time to stop. All right yes my bum is sore as well. Yeah we need to spend a little bit of time now. Off the bike we've had three solid hours. It's time for some land. Oh yeah. Look you've got a bag of salad. I like salad. I try to stay healthy on this trip. So I think pizza and salad counts eh? Salad on pizza. That's a good healthy meal. There you go it's half and half right there. That's lovely. My pizza doesn't have any salad on it. When you have a bike break it's like the opposite of a normal break.   We have to be getting out and about for them otherwise you get really sore bum. Okay yep we have to like step up and over. Yeah I'm not sure if that was easier for me or not. I think I can just about, oh no can you just about make it. I think I can just about get out by pushing us both. Very strong. I might have run out of space hold on let me just put that up. Oh no. I think there's a very slight bump. Do you think you can like wait one two three? One two three. No I think I can't. Yeah I think you'll have to. Ready? Yes. So easy when the engine's on and you get going. Right we're just gonna fill up our tank whilst we're here in town because once you hit the countryside we don't know how many petrol stations we're gonna pass. Wow look at this church. The church up there. Yes. Yeah this is one of those really nice historic towns in the middle of the countryside. Yeah in the middle of nowhere. Look the same like in the medieval times I'm sure. Look at that. Yeah that's beautiful I love it. Such a nice building. Yeah look at that it's massive for quite a small town. Incredible and their cobbled streets make it really feel like a rural French town. I just wait for quasi-moderate to look out. Look out from the windows. This is the Marie here. What is the Marie? Like the town hall. The Rathaus. So this is our petrol station. What's the price? 1 euro 68. 1 euro 68. Yep well that's really the maximum I can do. Last leg for the day. Wow yeah look at that. This whole area is full of these like red bricks with white in between. Oh there's the Marie. Hello Marie. This is a very pretty tree as well look at that. Very pretty. That's a charming little V. The V with a red road running through it. Magnifique. Magnifique. Oh my face. It's now four o'clock in the afternoon and after 140 miles everything still looks exactly the same as when we left this morning. It's the endless countryside of France. I can't believe we can be six hours on the bike and pretty much drive down the same road 500 times. I mean I'm not complaining then the roads are actually really really beautiful but anyway we're getting to the end of the day. We've probably got about 15-20 minutes left to go before we arrive in what's it called Camping where we've got a hotel called Hotel F1 booked. I can see it Hotel F1. Our home for the next days. Oh yeah yep it's this place right there. Nice. This is quite similar to where we just were. It's went from like one sort of motel to the next. Yeah we are not very good hotel vloggers. This is not the five-star hotel trip that's for sure. But at least you can see here very nice daisies along the roads so that's nice. Good evening guys we arrived in our beautiful new home for the next two days. Yeah the room is not too bad actually look at this apparently it's been recently renovated. Look at this a lot of inspiration. Yeah I've got all the travel posters there vintage travel posters and oh we've got our working desk there for the next few days so we're already getting started with editing the next episode so we're here in a town called Campien I think that's how you pronounce it Campien. It's not too far out of Paris. Total journey time was about six and a half hours so I think for us it was a good test but I think it was about our maximum that we could probably do at the moment in a day especially when we're not taking bigger highways because we wanted to just take all the little roads but there's only really so far you can get when you take the little roads. But it was a really nice experience nice to see the the french countryside but anyway guys we hope you enjoyed the episode if so please give us a thumbs up subscribe to the channel and comment and all the stuff below. Please do all of that. We will see you next time.


Over the last few weeks I've been trying to find an alternative to my to-do list application, and the to-do list application that I've been using for many, many years is Todoist. It is a very good application, but they've been pulling features out of the Linux version for a little while now, and I'm not all that happy about it, so I've been trying to find something different that has some of the same features. And I haven't had a lot of luck, despite there being a ton of to-do applications Most of them are either too simplistic or are too full-featured, they just have a ton of cruft in there that I don't really need note-taking and all this stuff. I don't need that in my to-do application. I just want something that gives me a list and gives me some ability to do recurring tasks and projects and stuff like that. So when I found an application called Sleek, I was a little skeptical because by this point I'd tried, like I said, about six of them or so, and I hadn't had much luck. So I didn't have high expectations for Sleek, but I found myself pretty impressed, so that's why I'm going to make a video about it today. So let me show you what Sleek looks like. This is the Sleek application, and I'm not sure if it's dark mode out of the box, so I think I might have changed that. So if you open this up in as light mode, just know that there is a dark mode. I'll talk about that in a minute, but other than that, I've done nothing to this application other than add a few tasks, and overall it's very simple. It uses the to-do.txt standard, so basically what it does is just creates a to-do.txt file somewhere on your system. You can choose where, and it saves all the information to that. That's all it does, and then it displays the information in that file here. And what's cool about that is that it means you can have different lists. So if you want to have a to-do list for work and a to-do list for grocery shopping, or around your house, or whatever, you can do that. All you'd have to do is open up that other application. So just hit the file button, find yourself another to-do.txt file here somewhere, or create a new one, and you're good to go. And it has tabs, so it'll actually show the list that you have open up at the top. Now at first, I thought that was just going to be way too simple, because how could you have all the features that I would personally need in a .txt file? It didn't really make all that much sense to me, but the application adds several things that to-do list has, but still keeps that simplicity of using a .txt file as the backend. So let me show you what I mean. So let's go ahead and open up the add task dialogue here just by hitting the plus button. There is a key binding, but I just used the button. And really what you can do here is several things. So first, you can set the priority, and you do that by choosing the letter of priority that you want. So the higher and the alphabet you have it, the higher the priority. So letter A would be the highest priority, letter Z would be the lowest priority. It's kind of pedestrian when it comes to priority, but it works, right? It's very simple just like you'd expect, and it's easy to choose. So you don't have to choose from the drop-down. You can just type in B, and it would be the B-level priority. You know what I mean? It's very easy to do it just that way. Like I said, it's a little pedestrian because if you have used some of the more full featured Todoist-like applications, they have flags and color coding and stuff like that. This one here, done by letters, and it does have color coding, but it's not anything special. But it works, right? That's really all you need, and it works just fine. So that's priority. So if you want to then do a task, we give the name of the task. So do VTRFS video, and then let's just say I wanted to have that thing a recurring video. I don't really want to do a recurring task on that particular task, but let's just say I wanted to do that. I could do REC and then colon, and then I can do a special code to tell it how often I wanted to recur. So if I wanted to do daily, I'd do D. If I wanted to do weekly, I could do W for weekly. If I wanted to do monthly, I could do M for monthly. If I wanted to do it every three weeks, I could do 3W, and it would recur every three weeks. Also, if I wanted to do it every three days, I could do every three days just like REC colon 3D. Now that's really cool because if you have ever used Todoist before, Todoist has a function where if you wanted to say schedule a video for a certain time. So let's just say I wanted to do, do VTRFS video on Thursday at noon. I could then save that and it would schedule that Todoist item for Thursday at noon. So it's obviously not as context aware as Todoist is, but you can do things like do and then the date. So 2022, 09, 01, whatever happens to be. The one thing that I haven't found is whether or not you can do a time. If you do a drop down here in the actual calendar, it doesn't show time anywhere. So I'm not sure if you can do that. I don't ever really need anything that specific. So if that's missing, that's not that big of a deal. Like I said, you can do recurrence with REC and then colon and then the shorthand for daily, weekly, monthly, and so on. Now, where do you find all that stuff? So you can find all that stuff in the help. So there is a very nice help page here that is always accessible. It will show you how to do things like recurrence. So you can do things like D for daily, B for each business day, W for weekly, M for monthly, year for annual annually. You can also specify a different interval. So like I said, 3M to repeat every third month or 3D for every third day. And that's really, really nice. Same thing for dates. You can do the date with your year, month, month, day, day. Now, the one thing that like I said, it's not here is the time. It doesn't seem to allow you to do time, which is okay for me, but it might be something that you miss out on. Now, another cool thing that it does do is it does do friendly date names. So like tomorrow next Tuesday or end of February will be converted into a perfect format of dates. So if we go back here and do a video do colon tomorrow and then hit save, it will have a video for tomorrow, which is the 30th, which is, you know, that's really cool. The fact that it has that context to where kind of like to do list has is very, very nice. Now I wish you didn't have to type in the do part. So if I do new video tomorrow and then save all it's going to do is say is do regular to do list apps with no data attached to them. So you have to do that do part first. I wish I didn't have to do that, but it's not that big of a deal. So outside of adding new tasks and things like that, you can also add things like contexts and projects. So if you click the plus button here, and let's just say you wanted to add a task tool project. So I would say rice, add new rice to collection. Now if I wanted to add that to a project, I would just do prices. And that would create a new project called Rises. And if I wanted to add some con context to that I do at and I could then do Rises for that as well. Then you could mess around with the due date weekly and stuff like that just like normal. And that shows the context and the project in color coded notations next to the application. Now you're probably wondering what context are. Contexts are things that have to do with the whole getting things done style of productivity. So if you're not familiar with that, which I'm not at all, it kind of gives you an idea of where those tasks are supposed to take place. So things like at home at work, things like that. So that's where really the context part of it for projects. That's more self explanatory when you have a gigantic piece of work that you need to get done. Each part of that project could be a to do list entry. And then you use the project tag to kind of put all those things together as part of one piece of work. So that is context and projects, which is again, something that I didn't know that it had when I first started and when I got into this to do this help page, that is something that to do list has. So again, this is a very good alternative to to do list. Now outside of that, that does have a few settings. So you has several languages that are available to you. It gives you notifications. So when you have something that is due, it will send you a notification. It'll minimize the application to the traybar. If that's something that you want by default, the error and event logging is turned off. I turned that on just to give them some information. So that is opt in. It has dark and light mode. So you can choose between those. It has a compact view, which means that makes it a little bit less big. So by default, this is what it looks like as all the bigger buttons and stuff. I turned that off made a little bit more compact, and it does have tabs. So if you have multiple to do files, you can have tabs. And then there's a default zoom here as well. So that is sleek. It is a very good little application that has a ton of features and way more features than I thought it would have given the fact that it uses uses a .txt file as the back end. So it gives you all these context and projects and giving you the ability to do recurrence and stuff like that. And it just is a very, very good application. I'm astonished really at how good it is. So that's going to be my new to do application. Goodbye to doists for now. So if you have thoughts on this, you can leave those in the comment section below. You can follow me on Twitter at Linuxcast. You can follow me on Maston or Odyssey. Those links will be in the video description. You can support me on Patreon at patreon.com slash Linuxcast, just like all these fine people. Thanks to everybody who does support me on Patreon and YouTube. You guys are all amazing. The channel would not be where it is without you. So thank you very, very much for your support. If you want to support me on Patreon, you can get 10% discount by supporting me a year in advance. Thanks everybody for watching. I'll see you next time.


We're here at CBS 2016 in Nairobi, Kenya, and I'm very pleased to be joined by Mr Pratik Marwar, who is the co-founder and COO of iOG Mentor Labs. Pratik, thank you very much for being with us in the studio today. Thank you, thank you so much. Now what, I know that you've been brought here as a group of young innovators, basically they're going to be discussing your particular innovations, technological innovations for socio-economic development, and I wanted to ask you about yours. Perhaps you could tell us a little bit about iOG Mentor Labs. Okay, so iOG Mentor is basically a personalized learning platform for the students. So we've created a mobile application. So because I've been into the industry of training and recruitment for a long time, I've seen employers talk about saying that students are able to crack the assessments, the examinations, but they are not able to speak to the clients. So the trouble with them is that they are not employable enough to be hired. But when we went to the universities and when we went to the colleges and talked to the students, one thing came into my mind that there has been only one medium of learning till now. People have been reading books or just trying to speak to the trainers or the faculty members. But we had identified as a youngster. I learned more through videos or maybe through cartoons, through animations, through stories. As a young kid, my grandfather made me learn mythology through stories, which made me understand more. So we've created a mobile application in which we've got all these mediums together at one place. So as I was talking about mediums that are cartoons, animations, stories, as well as videos, where you can learn and assess yourself and track and have progress reports, so which helps you to go and gain more. That's what we are trying to do. And how many people have you managed together to help you with this? So as of now we are a startup. We are just six months old. We have a 22 member team and we've recently got funded as well because of the new ideas. One of the most important features is that we have applied for a process patent on the technology that we are using. So that's about it. And in terms of where are you based, in terms of your basis? So I am from India and we are headquartered in Gurgaon, which is a satellite city of Delhi, which is the capital of India as of now. And so we have even expanded into Egypt, which is our first African country. We're going into Dubai. So we're making inroads. That's why we came into Kenya as well just to speak and interact more because we want to scale the product across the world. And just within six months we've reached more than 5,000 students and these guys are using it and they are enjoying it. And how are people getting to hear about it? So as of now the model is B2B that is business to business. We speak to the universities, their management, and we try to convince them to present our product in front of these students. And if they like it, they buy it. So that's about it. But later on we've decided that we'll open it for everybody. So anybody or anybody who's interested in using our platform can download the application and directly pay to us. So that's what we are trying to do. And you'll be updating it constantly? Yes, yes. So we have our own plans. Right now the application is totally digital. But later on we intend to introduce augmented reality as well as virtual reality with haptic feedback as well through wearable devices. So that's the next step. We have planned, we have technology plans for at least two years from now. And let's see how it goes. Right, we'll be looking out for you. And very good luck at the afternoon. Thank you, thank you so much. Thank you.