Hi everybody, it's Sam at Mixed Up Craft. Thank you for watching my video today. I'm going to be doing the unboxing for the latest Paper Craft Society kit. This is kit 4, box 4. Now I know, I was thinking about it, that I haven't shared any tutorials with the Bright Rosa, which is kit 3. Christmas has just completely kind of taken over. So the box came for me at a bit of a hard time because I was so into the Christmas tutorials and I had so many kind of just things that I had to share and I had to make and it's just kind of been sat there looking at me. So I promise you are going to have an influx of lovely tutorials with the Bright Rosa and also with this one here. I do feel that this one's come out sooner than the Bright Rosa one. I could be wrong, but I'm sure we've got this in the same month that Bright Rosa come and I thought it went slightly over. So anyway, it's not a bad thing. It's great to have all this lovely product. So this is the one, the latest one by Paper Discovery, which is created by Olga. So if you haven't already checked Olga out, go and head over to her YouTube channel and yeah, she's got amazing tutorials over there. So as always, beautifully packaged and this one says enjoy discovering the delights of the beautiful collection I've put together for you Olga. So the packaging, the color and everything is always a teaser to what the papers and everything's going to be like inside. So I did look at the the unboxing that they done on Craft Stash. It's got a real whimsical, magical, mystical kind of feel about it and it's something very different and I really like it. So you can see here you've got that silver foiling and you've got all these beautiful, these are the dandelions and then yeah, let's just get straight into it because I, it's like I said, I've seen that one so I haven't had a good look in this one yet. So we will do the, let's do the packaging first and we'll do the envelope afterwards. So this is Olga and again that lovely blue and you've got little bits, oh it's the sequins. Okay, right so we're looking at the moments in here, you've got your ink cube, so we have this blue and gray and there you go and these are always so juicy like there's usually ink, yeah there's like ink all around them so you're gonna, these will you know certainly last you. So they will match perfectly with what's in here in terms of your papers. So we've got some organza white ribbon there, which is really pretty, very delicate. So you can see there the organza ribbon. We've got our card, so again I hold punch mine and I have them all hanging together, I thought they were too stuck together. So this is a guide to paper sizes. That's handy, always good for when you want to get you know just, I don't know, sometimes I get like a brain fog and I can't think you know what size I need so it's good to have those little cards. So these have come out of the sequin box here, I can see the top where they've just kind of leaked out but it actually looks quite pretty as well. So these are your coordinating card, oh an acetate so let's just carefully take that one off and have a little look at the colors here. So we've got a gorgeous like sunshine yellow there, three of each, then we've got the gray and the blue. So you can see there how they're going to all match really nicely and it's lovely, nice thick cardstock there as well. So I'm just going to pop that one in there. I always keep mine all together and I put the papers, this cardstock I will end up putting it into the actual envelope so that'd be all right, I'll do that in a minute and then there's the sequins. So you can see there those lovely like holographic or iridescent ones, really really nice there. So I'm just going to keep all that neatly in there for the minute. Okay and then back to the envelopes, this is the main part, you get an envelope in every single box and on the side here on the spine it will say what it is. So this is the Papercraft Society box four and it's by Paper Discovery. You'll see now how lovely they start to look all stacked up. It would just look so nice on your shelf, you know all these lovely folders and you can just go to them and you know that everything's in there. So this is the first one card making magic, box two was simply made crafts, then you've got bright rows of a box three and then now the Paper Discovery. So let me just open this one up, I always do it the wrong way, yeah that's the right way. So we have the stamp set, so let me just grab, I'm just going to take the paper out and just use this here just so you can see them better. Straight away this jumps out to make such a beautiful stamp. You've got the beautiful dragonflies, the birds, you've got this bridge, you've got, I love these kind of border ones, love the toadstool there and the little frog. You've got deeper sympathies, you've got the only impossible journey is the one you never begin. Very very good, I like that one. Happy new home, good luck, get well soon. Hope you find strength with each new day and wishing you well. So it's got that, it's got a lot of motivational, supportive kind of quotes there which is nice, love the dandelions again and then this is that kind of whimsical magical with the silhouettes there, love this and I already have a really nice card idea so I can't wait to start that one. So I get so excited like this is you know genuine happiness with some paper and stamps, isn't it nice that you can just have that feeling from something like that. Then we have and this is a five by seven, just going to double check, pretty good, oh no this is bigger, so this is this is five and three quarters by eight. Olga does do big embossing folders, if I just show you, yeah so that's where it's come from so this is one of hers here, this is the elegant room embossing folder and you can see just how large those ones are. These are your different kind of scenes so you've got one there, one there and one there so you can cut them. So again we'll go through the inspirational book in a moment but it'd be nice to see how Olga's intended for those to be used in more detail. Then we've got some acetate, we've got another piece there so a two piece of acetate, look at that vellum, that is absolutely stunning, it's like it's almost looks like it's bamboo, I don't think it is, I think it's another kind of plant but it has got a bit of an oriental bamboo feel about it, absolutely stunning, really lovely quality, you could certainly use it as parchment because obviously vellum and parchment is very similar, parchment just tends to be thicker and this is on that thicker end, you could certainly do some really fun techniques with that. Then you've got some plain vellum here so again you could do some nice details with your embossing stylus and with the embossing folder so again can't wait to use that and then these are really fun, very very different but very much Olga's style so it's got that kind of, again I can see like an oriental feel coming through it, I think that's from my years living in Asia, I can kind of pick these bits out but that's what it does look like, this is beautiful, love that one. So you can imagine having these fairies and the butterflies kind of flying above, I think that would look really really sweet, so you've got those two and those two and then so it's a little bit different this time because in other ones you've had quite a lot of pattern papers but I think because you've got these beautiful vellums and you've got two sheets of the acetate there because I think this one here is used for like the dies and like aperture ones so to create windows and scenes so that's why you've got the acetate so that's those ones there and then onto the die set so I need to transfer this over onto my magnetic sheet but let me just again grab just a piece of this just you can see them a bit better but look for the wishing well, oh I haven't got a wishing well die, I have not of all the thousands of dies I have I do not have a wishing well that is so good absolutely love that, the bridge again the dandelion I don't have a dandelion like that love the fence think that's a stream not a path but then you could maybe depending on what color cardstock you use but I think that's water flowing that one and then here you'll see you've got this big die cut here it cuts this really lovely shape within your paper so that will give you your aperture your window for whatever it is you can do great shaker cards you can do shadow box cards you can do tunnel cards there's so many nice techniques to use this and then this one here will cut all this beautiful detail into your cards you're going to be left with all these lovely sprigs then you've got the moon and then you've got like a half moon there it's not quite half moon it's like a quarter moon the butterflies again the dragonfly love the bridge just think that's such a nice different kind of one and then all these sprigs and I can straight away I know it's got the fairies and everything but I see this as an underwater and I see this as like seaweed you know you take that all that middle bit out because none of that's attached but you do that in in you know your more underwater colors definitely definitely I can see that being used for underwater which as you all know is my favorite kind of theme when it comes to cards I just love all that so that is inside the envelope absolutely love it really really do and yeah I can't wait to start creating I've got two nice ideas for this so I will need to write them on a bit of paper and pop them inside because I forget my ideas they come to me and then they go again so let's just pop all this back in here though inside all of the booklets you always get I think it was 15 card ideas or project ideas so there will usually be a introduction by the designer then you have your contents and then there's always like a yeah five minutes a mini interview then there's the contents of the box and she will kind of tell you about the the embossing folder say she'll tell you about everything but here I've just seen the embossing folder so it's three different elements combined to make texture firstly use the straight lines to create your diorama concertina folds ah see already really really good and then then emboss using wavy lines either as a background or hillsides or gentle or gentle waterways to complete your scene so those lines are actually to do your concertina folds so that's really handy to note and here they show you how to use it so you're just running it through either ends of the paper to create that tunnel effect and you can see just look how beautiful is that what a lovely scene just so pretty really really love it and again there you can see there's less so you imagine you do that in green and then you're looking down like you're under the sea and you've got fish here so it's brilliant so you know these are any kind of sprigs and stuff as soon as you change the colors of them there's no reason why you can't be seaweed and plants that are under the water or even inside like a fish tank or something so I think that's going to be really cute then we've got what's some here so these are really pretty love that one there these are so these ones are by Linda so she's on the crash design team yeah I do like that that kind of tone on tone one with the wishing well I think the wishing was my favorite one I think it's really sweet and then these are your step-by-steps how to make the three cards there she got step-by-step there there and there so you you will know how to make all of them then this one here so there's the waves so that's really nice this is done by Claire and again she's just showing again all the like the step-by-steps are really really good then that's using oh so that piece there is Olga's used it behind the ferry and that's a shaker card and then that's that beautiful vellum with the sequins behind so again it's a shaker card that's been heat embossed in silver and then this is all of Olga's collection I do have most of this I think I've got everything yeah I've got everything apart from that one and I've got a really fun tutorial coming with this one and then we've got these are quick makes by Anne she's also on the design team so again if you want something a bit more simple then there's those of nice ideas there and again that wishing well which I just adore and then just a bit about other magazines that are available and we've got another one here this is by Carol and again she's used the vellum and kind of done a layered effect with the wishing well and it's got wishing you well oh did I miss that sentiment did I see wishing you well it must be on there because the cards are obviously there to be used with all the products so I love it and then Jeanette always does happy Jeanette she always does a really nice card at the end so this is using the vellum she's used the ribbon and she's done that silhouette effect using the ink and that's a lovely good luck card I like the way she's used that border with the grass actually all the way around so it's like that meadow again that real kind of whimsical feel about it and she shows you how to do it there as well and then yeah and then it kind of finishes so really really good there's always I will share all the links below the closing for this box is long gone now you won't be able to get this one unless they have any leftover they sometimes put them up on their website so the link that I share below you will be able to find if there are any more left of this but if you're lucky to get these then get them they're great value for money they ship worldwide and they're really really fun I do I really like them and I think if you're starting off these are a great way to start building your your stash and for you to get a feel about maybe the things you like because you get to try different things with each kit because every design is different so yeah thank you again craft stash for sending me this box and tune in for the tutorials now Christmas has done and dusted I can't wait I just love getting it like away from Christmas so nice to just have stuff that has no Santa Claus no snowmen no red or green so anyway I'm going to go now I'm just really excited for some reason I think might be in the chocolate bar I had before but anyway I'm going to go have a great day everybody and I'll be back again soon with another tutorial see you bye


Diffusion is one of the ways that things can pass in and out of the cell. Some things can diffuse directly through the cell membrane. Other things need help. And this is where facilitated diffusion comes into the mix. Facilitated diffusion is still diffusion. And diffusion, no energy required. You do not have to do anything to make diffusion happen. All you need is a concentration gradient. Really, concentration gradient. If you have a concentration gradient, you can make molecules move, assuming they can pass through the cell membrane. So if the molecule can't, like my friend Sodium, Sodium ion cannot pass through the cell membrane, then let's facilitate that, shall we? And you can facilitate the diffusion with a protein. This is awesome. You can put a channel in the cell membrane. And we actually have Sodium channels. And they literally look like this. And Sodium can come in and pass through. And the rest of this structure right here is, look at what I'm drawing you. This is cell membrane doggies, right? There's my phospholipid bilayer. I can do it. There we go. So accurate. It's a protein. It's almost like a little gap junction that doesn't connect two cells. That would be a fantastic compare and contrast question. Compare and contrast gap junctions and channels. It's facilitated diffusion because the movement will only happen through this channel if there's a concentration gradient present. So if there was a high concentration of Sodium on the outside in the extracellular fluid and there was a low concentration of Sodium in the intracellular fluid, then as long as I have a Sodium channel that's willing to do the job, meaning it's open, then Sodium will be able to diffuse down its concentration gradient and into the intracellular fluid. There are lots of different kinds of channels. There are some channels that are called gated channels.  I don't know why I can't see my colors here. I'm not cool with that. I'm having technical issues on this side. Oh, but they are mild technical issues, so I'm not complaining. Look, I just drew a lid on the channel. That's a gate. You can close channels. If I have a gated channel, then that channel is there, but the gate is closed. On gated channels? Dude, how awesome is this? If you feel like I will let the Sodium into the party in the intracellular fluid, come on in, fellas. Open the gate. And now the Sodium can pass through. Close the gate, and then we've made the cell impermeable again. The gates can be stimulated to open like, let's say, if there's an electric shock, open the gate. So that would be a voltage-gated Sodium channel. There are mechanical gated, different kinds of channels, so a mechanical stimulus can initiate the opening or closing of a gated channel. There are even light stimulated channels that will open with a stimulus of light or temperature or pH. There's all sorts of different kinds of stimuli that can cause the opening of gated channels. Not all channels are gated. Some of them just stay closed for good.  A significant point of the channel is that it's open to both sides. When it is open, it's open to both sides. So it's not like it has stages like here open up there and then close and then open up at the bottom. It's not like that. When it's open, it's open to both sides, so it's a little tunnel. There are other kinds of transporters that enable facilitated diffusion that don't operate that way. So channels are one type of transporter or protein in the cell membrane that enables facilitated diffusion. There's another one that we're going to talk about in the next section.


The primary phonological unit in many classical approaches is the phoneme. However, there is theoretical as well as empirical evidence that a unit smaller than the phoneme is more suitable. This unit is referred to as the distinctive feature. In order to understand today's system of distinctive features, this e-lecture introduces the history and the foundation of distinctive features. In a follow-up e-lecture about distinctive features, we will outline the advantages of a feature-based approach using today's feature system. So let's start with the history. There are three main approaches that have influenced the definition of distinctive features. The first is the theory of distinctive oppositions which goes back mainly to the Russian phonologist Nikolai Trubetskoy. The second is an acoustic approach which was first introduced by the Danish linguist Roman Jacobsen. And then there is the approach introduced by Noam Chomsky and Maurice Halle in their influential book The Sound Pattern of English. Let us look at these approaches in detail. The theory of distinctive oppositions goes back to Nikolai Trubetskoy in 1939. It was an attempt at classifying the properties of distinctive contrasts operative in natural languages. Trubetskoy defined a taxonomy of phonological contrasts that is of meaning-changing oppositions. There are oppositions within the system. There are relationships between opposite members. And there are types of oppositional extent. Thus Trubetskoy's approach was not a general theory of distinctive features. Nevertheless, he provided the framework for alternative views about phonological contrasts. So let us look at his taxonomy in detail. Oppositions within the system can be defined according to Trubetskoy in three ways. Segments stand in multilateral opposition if there is more than one phonetic property that keeps them apart. For example, in languages like Korean the system of plosives has always three members. Voiced unaspirated plosives, voiceless unaspirated plosives and voiceless aspirated plosives. There are two differentiating features, plus-minus voice and plus-minus aspiration. So a multilateral opposition. An opposition can be defined as proportional if the relation between its members is identical with the relation between the members of another opposition or several other oppositions within the system. The difference between T and D in English is identical with that of K and G in English. Thus we have a proportional relation. An opposition can be defined as isolated if no other segments stand in the same relation in the language. For example, the opposition between an alveolar approximate and an alveolar lateral consonant in English is isolated since no other segments stand in the same opposition. Opposite members can also be defined in terms of their relation or their opposition. In privateive oppositions one member carries a phonetic property which the other member lacks. In other words, privateive oppositions are relationships of the presence versus the absence of a feature, for example voicing. Oppositions in which the members are characterized by different degrees or gradations of the same properties are defined as gradual. This type of relationship holds for vowels where the property tongue height is a matter of degree rather than of binary. If the relationship between the members of an opposition can be considered logically equivalent it is defined as equipollant. A typical case is the opposition between English P versus T and T versus K. Unlike with vowels it is not possible to speak of a continuum from labial to vela and distinguish the consonants by means of degrees of backness. Rather, the different places of articulation constitute an equipollant relation. The extent of an opposition can also be defined in Troubet's Coise taxonomy. When two members of an opposition can occur in all positions the opposition is referred to as constant. This is almost the case as we will see in a second for English T and D. An opposition between segments is called neutralizable if there are contexts where only one of the opposition members can be found. A classic example of a neutralizable opposition comes from German where the opposition between voiced and voiceless consonants such as T and D disappears in final position. Or, and I will illustrate this right now, from present day English where the opposition between T and D is neutralized after S. Listen and see what's happening. Stick, stick, stick, stick, stick, stick, stick, stick, stick. And you see after S the opposition between T and D no longer exists. It is neutralized. Let's now turn our attention to the second approach towards distinctive features as defined by Roman Jacobson in the 1950s. Jacobson aimed at a description of the opposition's operative in languages. Together with his Swedish colleague Gunnar Funt and the American phonologist Maurice Halle he proposed a system of binary features now called distinctive that capture phonological contrasts rather than describe the phonetic segments themselves. But what exactly does binary mean? Well, a feature is defined as binary if it has two values. One of which is designated as plus the value and the other as minus the particular value. In many cases a binary approach is only phonologically significant in those oppositions which Troubet's Coy termed privative. For example, the feature nasal is binary phonologically. Phonetically, however, it is a matter of degree since some speech sounds can be more or less heavily nasalized. Probably due to the availability of the sonar graph, later referred to as spectrograph, which enabled phonologists to analyze speech sounds acoustically, Jacobson's features were acoustic in character. He defined these three feature groups, major class features, features for vowels, features for consonants and secondary articulation features. Let us look at Jacobson's acoustic features in detail. Now, the first group of features Jacobson defined are referred to as major class features. They have been introduced to distinguish the major classes of segments, vowels, consonants, and segments somewhat in between nasals, liquids and glides. However, there is disagreement about the number and type of major class features. Jacobson introduced the features consonantal and nasal. Later, as we will see, further features were added. So, in Jacobson's approach, all vowels were assigned the major class feature minus consonantal. To account for the parameters tongue height, tongue position, and lip rounding, additional features based on acoustic measurements came in. Here they are. They look complicated at first sight, but they aren't. The feature diffuse relates to all segments whose energy concentration can primarily be found in a narrow region central to the frequency spectrum. This defines vowels such as e and u, as well as most non-alveolar consonants as plus diffuse. The feature compact relates to all segments with low energy concentration in the narrow central regions of the spectrum. This defines the low vowels as compact. The feature grave relates to all segments whose energy concentration can primarily be found in a narrow region of the lower frequencies of the frequency spectrum. This defines all back vowels as plus grave. And the feature flat down here relates to all segments with weak or low energy concentration in the upper regions of the frequency spectrum. This defines most back vowels as plus flat. In Jacobson's feature system, all segments specified as plus consonantal qualify as a consonant. This feature distinguishes sounds with low energy and substantial obstruction from non-consonantal sounds. Among the features assigned to such segments are plus minus diffuse, plus minus grave, and plus minus flat. They were also used for vowels as we've just seen. Additional features were primarily articulatory in character. The feature voice correlates with vocal chord action. A speech sound is voiced and has the feature plus voice if the vocal chords vibrate. It has the feature minus voice if the glottis is open. Nasal sounds are produced by lowering the velum and allowing the air to pass outward through the nasal cavity. Non-nasal or oral sounds are produced with the velum raised to prevent the passage of air through the nose. Continuant sounds involve a primary constriction allowing the air to flow through the mid-sagittal region of the vocal tract. Sounds produced with a sustained occlusion are defined as minus-continued, hence vowels, glide trills and friccatives are plus-continued. Plosives and laterals are minus-continued. The feature plus-minus-continued thus by and large correlates with the distinction between plosives and non-plosives. The feature strident or sharpness of noise was introduced by Roman Jacobson to distinguish consonants involving high energy at high frequency from consonants involving low energy at low frequencies. Thus it distinguishes noisy consonants like ss from more mellow consonants like shh. So alveolar friccatives are classified as plus-strident whereas dental and labiodental ones and post-alveolar ones are defined as minus-strident. In order to capture aspects of secondary articulation such as labialization, palatalization, etc. The following acoustic features were introduced. Flat, a feature which refers to the weakening of upper frequencies, sharp, the strengthening of upper frequencies and checked the higher rate of energy discharge. The most influential approach of our time is the sound pattern of English approach which is essentially a book that was published in 1968 by Noam Chomsky and Morris Halle and they defined distinctive features in two ways as units that capture phonological contrast and patterns of languages and as means that describe the phonetic content of segments. The sound pattern of English system can be divided into three general groups of features. Major class features, primary placement features and additional features. So let's look at them. Like Jacobson, Chomsky and Halle used the feature Consonantal, here abbreviated as Cons, to distinguish consonants from vowels. They defined all those sounds as plus-consonantal that are produced with some sort of constriction in the vocal tract at least equal to that found in fricative consonants. Sounds that have the feature Syllabic are those which constitute peaks of syllables. Non-syllabic sounds are those which are in the margin of syllables. Assuming a non-linear format of phonological representation, especially the onset rhyme split of the syllable, this feature becomes redundant since it coincides with the peak of the syllable. So here are some examples. Let's take the word cattle. C, which is the onset, can be defined as plus-consonantal minus-syllabic. In cattle would be typically minus-consonantal plus-syllabic, it stands at the peak. And then we have T, which is another consonant, which is plus-consonantal minus-syllabic. And since the final L, the alveolar lateral constitutes a syllable in its own right, it can be defined as plus-consonantal plus-syllabic. Speech sounds, and that's the last major class feature, can be defined as sonorant if the pressure of air behind the constriction is approximately equal to the ambient air pressure. According to their amount of sonority, all speech sounds can be ranked, where one is the highest degree of sonority and five the lowest. The three highest, that is vowels, liquids and nasals, are defined as plus-sonorant. The two lowest, fricatives and plosives are minus-sonorant. A set of primary placement features supplements the major class features. Vowels can be defined using four such placement features. If they involve a high tongue position, they are referred to as plus-high. If the tongue is in a low position, the feature assigned to them is plus-low. And if the back of the tongue is involved, they are specified as plus-back. Finally, the distinctive feature round is used for vowels that involve lip rounding. It distinguishes the primary set of cardinal vowels from the secondary set. Here are some examples. E is defined as plus-high, minus-low, minus-back, minus-round. A cardinal four would be defined as minus-high, plus-low, minus-back, minus-round. And O would be defined as minus-high, minus-low, plus-back, and plus-round. We still need a fifth feature, but that will be shown in our second e-lecture about distinctive features. Consonants are defined by two features that define them on the basis of their place of constriction. That is, from labial to uvula. The feature, coronal, which you can see here, refers to the activity of the blade of the tongue. This involves all those sounds that are produced between the dental and the palatal alveolar region. The expression coronal is defined as pertaining to the blade of the tongue. A more transparent term would thus be bladele, yet this one doesn't exist in English. For this reason, the feature coronal relating to the crown of the tongue has been chosen instead. The feature anterior is associated with all those coronal consonants which are produced with the primary constriction located at or in front of the alveolar ridge. That is, with labial, dental, and alveolar consonants. Here are some examples. Per number one would be defined as minus anterior, minus coronal. It's outside the coronal region. Te is typically coronal and it is located in the anterior region. Ke, a palatal plosive, is minus anterior but still coronal. And ke, a vela plosive, would be minus anterior and minus coronal. Additional features in the sound pattern of English approach were taken over from Jacobson. For example, the features continent and strident. Whereas continent correlates with the distinction between plosives and nonplosives by and large, the feature strident distinguishes noisy fricatives from less noisy consonants. And then Chomsky and Halle added the feature delayed release, which was introduced to distinguish plain plosives from African consonants. The latter ones, the Africans, involve a delayed release of the oral closure due to the friction component preceding the plosives. Thus, Africans were defined as plus delayed release. Now, the three approaches defined in this e-lecture are by no means complete. However, they served as the input to the distinctive feature definitions that are used in modern phonology. Today, no serious phonologist defines phonological rules without reference to the theory of distinctive features. In a second e-lecture about distinctive features, we will thus first of all look at those features that are used in modern phonology and will then show how phonological rules can be defined by means of distinctive features. So, see you again in distinctive features too. Until then, thanks for your attention.


Cadets from the Hoosier Youth Challenge Academy recently traveled to Camp Atterbury Joint Maneuver Training Center in Edinburgh, Indiana to take the opportunity to utilize both the confidence course and the Repel Tower. The cadets, along with the cadre from the Hoosier Youth Challenge Academy, looked at this as an opportunity to not only face fears and overcome them, but to also increase their team building skills. You just got to motivate them, keep them going, keep them strived. Reporting from Camp Atterbury Joint Maneuver Training Center, I'm Tim Sprouls.


Well, welcome to this presentation then. So I'm going to talk about production of B-cell 2 proteins involved in apoptosis and in our lab we are interested in apoptosis in general, but in particular the B-cell 2 family of proteins that I'm going to talk about today. So I'm going to directly dive into the apoptotic pathways. I'm sure you're quite familiar with this map. I mean it's a quite complicated process, but it has to be complicated because this is the ultimate control and checkpoint for removal of cells. So we can differentiate between the extrinsic and the intrinsic pathway. So when the cell is doomed to die, that signal is going outside the membrane to form the disc complex and then follow up by activation of a lot of effector proteins such as pro-Caspace-8 is getting converted into Caspace-8. Bid is converted into t-bid, but the ultimate termination of the cell is taken care about with the Caspace-3 and 7 that will fragment DNA. But on the mitochondrial level we have a tight balance between B-cell 2, the protein I'm going to talk about today, which is a membrane protein, and also BACs. So you can say that these have opposite effects, as B-cell 2 will inhibit apoptosis, whereas BACs will drive apoptosis forward. So but eventually when the cell is doomed to die, BACs will create the pore in the membrane and you get the release of cytochrome C that will drive this apoptotic machinery forward. So you can guess that if this balance between these two proteins goes wrong, and this is of course a hot topic for cancer research because it's been observed that cancer patients that have an elevated and even balance of these proteins. So the protein B-cell 2 is actually a family of proteins, they are main alpha helical, and also they have conserved BH domains which are important for the interaction between targets such as BACs. So as you can see here, B-cell 2, the protein of today's talk has a transmembrane part which is hydrophobic, so it's a membrane protein. It has four BH domains. I would say BH3 is the most important since it binds to BACs or other protein of interest. And we also have example of BACs that only has three BH domains, and you also have some old examples that even lack this transmembrane part. So the question we want to ask in our lab is basically a few key points here. So first we are very interested in pinpoint the location of B-cell 2 when it interacts with BACs at the mitochondrial level. And one thing that's turned to be the holy grail of our research is that we are lacking the structure of B-cell 2, which is an important protein to study of course. So this is something we are investigating and trying to solve the full length B-cell 2 structure. And also this means that there are no complex structure between B-cell 2 and BACs present either. But you can see B-cell 2 is a rather small protein, but it's a membrane protein, so it still has its tricks to purify which I'm going to talk about. So to the left here we have an NMR structure of B-cell XL. And I want to point out that this is not B-cell 2, but this is a valuable mimic of B-cell 2 because it's soluble. So what people in general doing, they are shopping off the c-termal hydrophobic anchor of B-cell 2 in order to keep it soluble. And they also have modified and removed some loop regions. And thereby you can make actually this molecule soluble, but however it's not the full representation of the intact B-cell 2. So our goal of our research is to work with a full length B-cell 2. And of course when you work with biophysics in general you need a lot of protein. And especially when you turn into NMR you need milligram amounts. You need a steady supply of protein. And when I started in the lab a few years ago we were relying up on cell-free expression method. And honestly it worked pretty nice. But the drawback it's a quite fiddly process. And we got maximum I think we got like a half milligram, one milligram of each batch. And it's quite time-consuming. It's not very easy to scale up. So I thought maybe you can do this with a recombinant expression system. And now I would show you this slide, but keep in mind that this slide is, I mean it's based on several years of works. I know it has been optimized and went through a lot of iterations. But I hope this might serve as a generic protocol for, I mean if you're working something similar maybe you can use this as a as a guide at least to help you out with if you have some expression problems. So what we're doing we are overexpressed. I mean this is like a standard recombinant expression at least in the first stage. So we overexpressed the protein in the pet vector, pet 15b, that carries some entermal histag. And we grow the cells at 37 degrees. And I even grow them overnight at 37. And the reason I'm using M9 media here is simply because it seemed to help with the overall yield when the cell grows a little bit slower. And also you can mention in LB the yield is not very great. And then next day I follow up with the resuspension in a fairly neutral pH buffer. And this step is it's only to create the inclusion bodies. So in this step we are not recovering the protein. Instead this recovering step is done in the next stage which is in presence of urea. So we are unfolding the protein and we add 0.6 percent of sarcosyl or sodium sarcosynate. And this seems to do the trick. So this sarcosyl it helps to resolubilize the protein. And then we run dialysis overnight against the detergent. It in our case happened to be pre-35. The reason why we use pre-35 is simply because it's cheap and it's available. And you can imagine when you do dialysis you need quite a lot of buffer and it would be quite cost-effective if you were using something else. And then we just continue with fairly standard affinity purification. We run a nickel column just on the bench. And we do a detergent exchange here into TPC. The reason why we use TPC is also that it seems to be very compatible with NMR. So we screen a lot of different detergents and this dodecylphosphoridicoline seems to be the best candidate. And then we remove the histag basically standard procedure with trombine and then we run the infiltration. And I just want to show you some examples of gel cell from each step. So first we have the iMac step. I mean you can see it comes off more or less pure immediately. I mean keep in mind this has been optimized quite a lot. Next stage we separate the histag and the pool here is basically the merged fractions from the previous step. And then we run the infiltration. And the good thing about the M9 we can now adopt in this procedure to accommodate carbon-13 or 1915 label. We can even do deuteration of the protein but then of course the gel will drop. But on average we get around three milligrams per liter of cell culture which is great for membrane protein. So something we also did initially was to compare the 4uV seed spectrum with the B-cell tube we obtained from this cell-free expression method. And it matches also very well with the recommended expressed B-cell tube. And just keep in mind that the x-axis is slightly different here. But we have confirmed that the fault is unaffected. So I was already talking about the DPC. So here is just an example when the full-length B-cell tube is run with NMR. So I'm quite sure you're all familiar with NMR so I don't have to dig into details. But you get correlation between the imide and the nitriene. And each black dot here represents one amino acid. And this slide will just show you that we can obtain a fairly nice HSQC with the full-length B-cell tube in DPC. And we can also apply this T2 relaxation filter where we can pinpoint more flexible residues in the protein. So how this works is that the flexible residues that we have a longer relaxation rate and then you can set the cut off for the relaxation and you can fish these out. And these apparently must come from the membrane of the micellar core region. Something we also are working on quite heavily now is, I mean I already hinted that we want to solve the structure of the D-cell tube protein. But in order to do that we have to deal with the fact that this is a quite large protein when you consider the added molecular weight in the micelle. So we have a problem with overlap in the spectrum as you can see here. But we still managed to assign basically all the loop regions. So we need to find a way to overcome this crowd and a more spectrum. And to do this we have developed basically a strategy where we can cut off the protein in several parts which is probably not a very uncommon strategy. But the good thing here which I'm going to show is that we can cut these and they will still work as small puzzle pieces to get the full B-cell tube spectrum. So you can basically overlay these smaller fragments that will form the full length spectrum. So this means we have truncated the protein, we have deleted this transmembrane part and we also modified the entermal sections of the protein in a quite careful manner. We have taken account that we don't interfere with the secondary structure of course. So again we have checked this with 4uV. And to the top left here you have the full length B-cell tube spectrum. And this is alpha helical protein so you expect a dip at 222 and 208 nanometers which we have of course. And then you can see to the top middle here the B-cell tube delta TM where we have deleted the transmembrane region. This also yielded a partially soluble protein but we still retained the helical fold. And the same holds true for the rest of these mutations. You can for as an example bottom left here we have deleted the entermal first 82 residues. We still have a folded protein. The same holds true if we in addition delete the transmembrane part. And then we have chopped off quite a lot of the protein but we still have the helical shape. Maybe one example in the end here when we only have the BH4 domain and the flexible linker domain. Then we start to see a spectrum that represents both a random coil and a helical spectrum but that's not unexpected for this construct. So just to give you an example of how this looks when you put it in the NMR spectrometer. So you can take an example here either A or B but just to show you that if you have the B-cell tube therefore the full length in black here we can basically superimpose the red and the gray and reform pieces of the full length B-cell tube which is nice and that also proves that we can use this method as a tool to overcome this overlapping problem. And also we have done some efforts in the reconstitution of B-cell tube into DMPC vesicles which was the first attempt. So how this works you have your protein in detergent and then you add lipid in our case we use DMPC in the first try. You form mixed micelles and then you reconstitute and you remove this detergent with biobeats as a quite straightforward process at least when something works it's straightforward but it worked so after two days you get you start from a perfect transparent solution you end up with a cloudy solution and then you form your proton hyposomes and then you ultra centrifuge and this is just an example how it looks like this is the real sample in the centrifuge tube and you can see that the B-cell tube and DMPC proton hyposomes has been trapped between the 15 percent and the 30 percent layer here. And then STSGL further confirmed that we can do this for even for this delta TM when the transmembrane part has been deleted we can also do it in the PUPC and corylipid which better mimics the mitochondrion membrane. So just to summarize a little bit so maybe the most important thing here we have found an expression method that works with B-cell 2 that is also a membrane protein and now we can yield milligrams amounts of protein and we have also incorporated B-cell 2 into DMPC and other lipid systems as well. We also found that DPC is the candidate when we want to turn to NMR it gives a very nice NMR spectrum and also in addition we can use these shorter B-cell 2 constructs to together to aid us to form the full picture of how the B-cell 2 protein looks like in NMR. So to finalize this talk I want to thank the group leader which is Gary Grebner who is probably listening right now and the rest of the group Amik who is doing some mostly of the NMR work nowadays and also some previous members of the group and we have collaborators Gothenburg and Prague and also in Lund and together with ESS and Neutron experiments are going on in Harwell. And finally I want to thank you for your attention. Thank you so much Jörgen that was a very interesting talk and having the aspect of NMR as well that's interesting. Yeah this was actually a 30 minute talk that I had to squeeze into 15 but yeah but it's so we are again a little bit late but just one quick question. Jörgen you're here to unfold your membrane protein so we are commonly using Jörgen to wash of course a little bit lower concentration than you're using but commonly membrane proteins do not really always unfold so to speak so you have STS present. Yeah I can give some input on that the reason I mean when we started to establish this protocol I was checking several papers and most of them actually used urea and then also which I don't show here we have tried without urea it actually works without the urea so it doesn't necessarily you don't need to unfold the protein to recover it but I don't think it's hard so we just kept on going with urea but you're right actually you may not unfold it that was my question. Yeah I think you will unfold it but I mean honestly we haven't tested that to run a CD spectrum in presence of have a concentration of urea maybe you can form some residual I mean secondary structures in presence of high concentration of urea in even in that system but I wouldn't be too surprised if it's not entirely going to unfold that that is probably true.


That was the best Nintendo Direct we've ever had. You guys know how hyped I get after just a fantastic Nintendo Direct and that was just yet another one. I loved every second of it. So many cool announcements. I actually haven't seen it yet. I woke up this morning. I thought I would wake up this morning to a Nintendo Direct.  I thought it'd be better if I watch it with you. So subscribe if you're new. So I figured I'd at the very least, before even watching it, try and be positive about it. Be the one guy on the internet that thumbnail and title says he liked it, even though I'm guessing I'm probably not going to. How bad can it be? Really? Let's just watch it together. I'll hit play and I'll grab myself a bottle of... Oh, it's over? Oh, that was pretty quick. Blink and you miss it. All right, let's be honest. I wanted to do that gag of a look away for a second and I missed it because it was really short. I have seen it. Of course I've seen it. It's like 5 p.m. Of course I've seen it. It was like Ubisoft forward level of unimpressive. I'm not going to say disappointing. I'm going to say unimpressive, but here's the thing. It very clearly wasn't... It wasn't a Nintendo Direct mini. I know they called it a partner showcase. What it really was, in my opinion, was Nintendo's way of announcing Shin Megami Tensai 5 as well as Shin Megami, I know I pronounced it differently both times, a remake of the third game. I'm actually not totally up to date on my Shin Megami games. I like Persona a lot, but never played a SMT. Half of this freaking thing was Shin Megami Tensai News, which I think is really cool. I think it almost would have been cooler if Nintendo had said it's a Shin Megami Tensai Nintendo Direct mini with a little bit of other announcements from some of our other partners. I think that would have set expectations a lot more realistically, but this is almost, it's almost like a little clickbait just to kind of don't tell you anything so you kind of have to watch it and then whether you're a Shin Megami Tensai fan or not, you end up watching this direct. A lot of the fans of this franchise, and RPG fans were really excited, and everyone else, not so much. But there was some other kind of cool things in here we can take a look at. Cadence of Hyrule, indie game that I absolutely adored and speed sped run on the channel. I got it, I got under three hours, which I was pretty excited about. So I'm happy to dive back in with these three new DLC packs, a bunch of new characters like Shadow Link, Shadow Zelda, even Impa. I'm excited to dive back into that game. That was a cool announcement for sure. Also, it's getting a physical release, which I know a lot of us wanted and I'm definitely excited for that too. Oh, by the way, look what came in the mail. I forgot to mention. I haven't opened it yet. That's a video for another time. I just thought I'd show you. Oh, and speaking of milestones, I wanna thank everyone that went over and followed me on Twitch.   So if you don't wanna miss that and you wanna win some stuff, make sure to follow me. Links down below over on Twitch. And then the next night, Thursday night, I'm gonna stream the Xbox event only on Twitch. I know I usually do it on YouTube, but I'm gonna try doing it on Twitch this time. So if you wanna see my reaction to Xbox's event, make sure to watch me on Twitch Thursday night. So Wednesday and Thursday, I'll be live. Check it out. Oh, it's like a million degrees in here. Then they showed more of Rogue Company, which to be fair, this game doesn't look bad. And this trailer actually, it does seem like the games come a long way. I saw a big criticism this game received initially was just really stiff animations in the characters. And at least what I'm seeing here, a lot of that looks cleaned up and it looks a lot more smooth and animated. And I hope that game turns out okay. I actually like high res games quite a lot. They've done a lot of games I really enjoy on Switch like Smite and other things.  WWE Battlegrounds? I know offhand it looks bad and it does. But I wanna say that my favorite, me and my friends favorite wrestling game of all time, WWE All-Stars. I don't know who's played it. I don't know who remembers it. I am literally dripping with sweat. It was on Xbox 360, PlayStation 3. It is the best wrestling game. I and my friend, we all stand by it. It's so fun.   It's not, it doesn't try too hard to be super realistic both in the gameplay and the visuals. And what you end up with is just a really fun game. And this looks like that in a way. It doesn't have the flashy visuals that All-Stars has. It looks more grounded and honestly, it looks crap.  It just looks like a budget version of that game though. I don't know why they wouldn't, A, just port All-Stars over to the Switch because it's a fun game. Or B, make All-Stars too. But this looks pretty close to it. So who knows, I guess I'll check that out and see. Yeah, Blink if you miss it. That was it. We're caught up now. Cadence of Hyrule, some Rogue Company, some WWE, and then Shin Megami, Tensai News. Shin Megami is by far the best part out of this. Close runner up the Cadence of Hyrule stuff, but I know I've seen people online already today mocking YouTubers for their thumbnails and titles with worst direct ever. But if we're being technical, there's two ways to look at this. One, it falls into the category of Nintendo Direct Mini and in that category, yes, this is technically the worst Nintendo Direct we've ever had. The only new announcement we got was a remake of Shin Megami 3. Why do I keep trying? And then the fifth game, which I think that is pretty big news actually, I don't want to breeze over that. I am excited for that. As someone who has never played the series, I've been looking forward to this game ever since it was like one of the first Switch games announced to be coming to the system years ago and then disappeared. I honestly thought this game was scrapped or gone or I don't know what was happening. To see it's still happening and to see how excited the fans are and I assume the 71,000 likes, which is outweighing the dislikes must be the SMT fans. So I think that's really cool and really exciting for them. So technically anyway, from a Nintendo Direct Mini standpoint, yeah, it was the most, not the worst, just the most underwhelming one. But as far as a partner showcase, which it's apparently a new series in these Nintendo Directs, it's the first one. So it's also technically the best one we've ever had of a partner showcase. And really, it can only go up from here. You would hope. I think a lot of the frustration, in fact, I know a lot of the frustration coming from people that wanted more even though their expectations were probably low in the first place is just because we don't know much about this year. At all from anyone. Let's not single out Nintendo. We still don't have a PlayStation or an Xbox for a release date or price, let alone launch lineups. And with Nintendo, we know about a couple of games, like No More Heroes and Bravely Default, but that's about it for the whole year. And that can't be it. There has to be more. There's so many rumors and leaks about a Mario anniversary thing coming up with Sunshine and Galaxy, and I don't even know. But people are so hyped and ready and excited for something to break the boredom of 2020, this absolutely GOD awful year that we all can't wait to end. Everything's been postponed, everything's canceled, nothing's happening this year, it's all over, it's all terrible. We just want something. And we're not getting anything from any of these companies. And when you finally hear Nintendo Direct, even if it's a mini and a partner showcase, you think to yourself, oh, please, please, give me something to get me through the last five months of this stinky year. And when that something is really nothing, it's disappointing and it upsets a lot of people, but let's all chill. I mean, the year's not over, we're only about halfway through. There's time for more Directs and more announcements. We just need to be a little bit patient. Because I know we're all going a little cuckoo. That being said, Paper Mario and Ghost of Tsushima just came out, by the way. Have you finished both of them?  Do you have either one? If you like whatever this was, I'd appreciate it if you liked, if you are new, subscribe. Follow me on Twitch for those streams.  I'm just gonna get out of here. Bye, I'm melting. I feel like the wicked witch of the West right now. Love you all.


When I talk to Shaq, and I always miss William, I always say, I ain't got your kind of money yet, but what are you investing in?  Yeah, we on Boss Talk 101. I've been doing like entertainment business for a long time and they have so much knowledge to share. So why not? But what I love about what you said is the fact that you're able to call all these people, a lot of people meet these people, shake hand and then walk off, not getting that digit, not getting that number to keep that relationship going. No, when I talk to Shaq, and I always miss William, I always say, I ain't got your kind of money yet, but what are you investing in? Damn. Fuck's it's mine though. Damn. Fuck's it's mine though. That's real though. Like, what business, like, how do you get this, you know, how did you get this commercial? And he breaks it down to you? He breaks me, he breaks it down to me what I need to do and keep doing it. Damn. And cause his mind thing is keep being you and I got you. Man. You just shot you out, continue to put your face out. And the way he do it too. The way he do the same thing, I was just about to say. I found out a way to get people attention. Just like when I'm doing my sketch, like a lot of people are like, man, how can I get these big people attention? I'm in that inbox, where if you can meet them, start calling their name out. You gotta be funny though, and the way you do it.  In my own slick way. In my own slick way. In my own slick way, yeah. What did you say? I said, Dwayne Wade is rolling over in this fashion over. Think about it, think about it.  And he gonna know if that's a job or they just love. I'll never let the world know what it is. But I got his attention. He put it back on his store. That's dope, see, and the way you say in this catchy, we gotta get better at that. Maybe somebody will share out, but we can share it a lot. We ever did it. Bones Bones. Bones Bones, we ever did it. Y'all at Nick and Sam's steakhouse. No. Man, we was on a date, man. We were downtown. We'd be everywhere soon. I think we wanted all of us to do that. But man, and love doing it, love the travel, man. I got a question. Go ahead. Okay, when you coming out on stage, you used to come out to Nipsey, but now you come out to Young Doff. Why? Both motivation. Both of them the same to me. Both of them took care of people. Wow. Doff was hard. Taken away from us so young, so, so with Nipsey. See, we got to understand, man, when we go out here and kill people, we don't just kill them, we're killing 50 people lives when we do that. They take it, they were taking care of so many people. So, yeah, you're right. I come out to Nipsey because I listen to Nipsey before every show. That's motivation. They clear my thoughts. Nothing negative, all positive. Doff, he give me in that mode. Like sometimes I'm human. We all human. Sometimes I be feeling down. And where I was raised, I was raised to be humble. And I'm always gonna be humble.  You better act like it. What else do you do before a reason? What else do you do before a show to psych you up?  I'm laid-back. I heard something about a green tea. I'm dead, too. It gets from my vocals. It gets from my vocals. I know y'all who y'all got that from. I know who it is, I ask, too. He ain't got nothing to do with it.   But if I was a show, I'm probably talking to some people that got up under me. Some more comedians. I'm bringing on the road with me. And I wanna see them flirts.