We're back we're live, I'm Jay Fidel, it's a three o'clock block here on Think Tech. And we're talking about global connections, we're talking about Chinese nationals in American universities. Should they be barred from American universities? This is a serious problem. It's a problem for the United States because of our lack of foreign policy, and it's a problem for them of course. Chung Wang, the practicing lawyer in Minnesota, graduate of the University of Minnesota joins us with our old friend Russell Yu, a practicing lawyer in Hawaii, and who is very familiar with things in China because he spent something like 200 years over there, am I right? Something like that, Jay. Okay, we're going to conduct this entire show in Mandarin, if you don't mind, ready? Go. Okay. Okay. So the first thing I'd like to discuss is exactly what is the foreign policy of the United States toward China and toward Chinese students. Russell, why don't you talk about that? Sure. I think the policy of the United States since 2016 has been one of really no foreign policy except to really try to shut any kind of engagement or dialogue with China. It's not only interesting what the topic we're going to talk about, but many things, the U.S. trade war, now we have the technology war, and now we're having the whole concept of disengagement. So we've seen a gradual policy that says cut China out. And what's disturbing, Jay, is that now that it's pushed to a bipartisan political world in America where things are giving ominous sides, not only of the relation to China, but also our allies in Europe and around the world. So it's a little bit disconcerting that it is going this far, but there's no articulated policy of how we deal with this. Well, let's be optimistic for a minute. John, can you deal with this? Is this reversible, or is this already have sort of traction where this country and other countries are down on China for reasons that are really not valid? Well, before I answer your question, I just want to add one note to Russell's comments. I heard these comments from one diplomat from China and one diplomat from the United States from Foreign Service. When they talk about foreign policy, and they said the foreign policy is domestic policy. So the purpose of foreign policy is what's going to happen domestically. So we can say that we cannot figure out what exactly is the foreign policy, you know, the derogatory remark towards our allies, and then this very hostile attitude toward China. What exactly they want, their audience is inside the United States. So that is, you know, even it appears they have absolutely no strategy, no agenda, but they do have audience inside the United States. So here's my full note. Go back to your question, Jay. Is that reversible?  So even we change the leadership, you know, earlier next year. And this entire attitude, all these policy and executive order already in place, you can reverse the executive order, but you cannot reverse entire hostility in a culture inside a certain department, particularly department of state and department of justice and FBI. So that will go on for considerable while. On the other hand, the big, you know, talk about the larger picture, you know, there was a newly released pool in all the developing countries, developed countries, you know, Sweden, Australia, Japan, Korea, the United States, UK, and look at their attitude toward China and the Chinese. There is a dramatic increasing of the negative view of China in recent years and it's a very sharp decline of favorable views of China in the recent years. So in the foreseeable future, I would, I would put it about 10 years, 80 years mark, you know, this trend will, this overall trend and the overall environment will be pretty unfriendly to China. That's not healthy at all. It's not healthy at all, it could lead to fisticuffs. And so one question I would ask you, looking at all of that, let's suppose that we take a fresh look at American foreign policy to China, say in January, say January 20th, for example, what kind of changes would you make? Changes to deal with, you know, some of Xi Jinping's aggressive maneuvers, you know, in the South China Seas, in Hong Kong, human rights, all that. What could America do in its foreign policy to make things better between us and between China and the world? I would defer this to Russell first and I may add something later. Okay, Russell, your turn. Well, I think there is a de-escalation policy, which we should take. Again, national security is important as it articulated, but I think a de-escalation meaning that certain areas where we don't, like for this immigration topic we're talking about, we don't take a black view point, we take a very nuanced approach. I think having a de-escalation meaning that I think both sides will need to start to give up something, for example, again, this is one area where just even through the policies restricting immigration and all this, second is again, what we'd be seeing is an attack on Chinese businesses, more hostility towards WeChat, TikTok, again, there may be a better approach than what's happening now where we simply have a presidential ban and say, completely get out of here, you have to do it our way. So again, I think it's using rule of law or logic and more engagement. I think that word is engagement. It's a very important thing that we are really missing through this administration, a lack of engagement. Chang, you're nodding, yes. What would you add to that? Yes, I totally agree with Rosso that the Obama administration's policy was not perfect, but it worked. It worked very well, and the TPP should have worked, but TPP was killed by this administration. So the engagement largely has been criticized, but it's much, much better policy than decoupling and the current very hostile environment, hostile policy. But on the other hand, there's one thing I do want to point out, the current hospitality towards China and the Chinese, including Chinese Americans, are really part of the campaign strategy. GOP circulated a campaign memo in April 2020, 80 pages, something. The whole strategy is going down to one sentence, don't defend Trump, attack China. So that is part of the whole situation we are in right now, internationally, particularly with relation to China, it's part of the campaign. But a lot of people inside GOP, they keep attacking and attacking, and somehow they just forget it's part of the campaign. You know, if you want to lie, you're going to convince yourself first, right? And so they began to convince themselves that there's a real enemy, there's a real danger, and they kind of forget they mixed up the campaign rhetoric with their true belief because they do not believe in anything. They don't really have an ideology or principle to stick to. That's one thing. On the other hand, look at the human nature. We always pick up some disadvantaged group to blame and have a scapegoat. I'm on the board of Minnesota Zoo, so I went to the zoo and I talked to the horsekeepers. And the horsekeepers said among all the horses, 20 horses, they're almost always going to have one weakest horse to be the, you know, all these horses going to bully him or hurt little horse. And even these little horses, if the horsekeeper removed these little horses to another area, and they will quickly find another horse, little horse to bully too. So this is part of human nature. So now, you know, lucky us, now it's Chinese, you know, a couple of years ago was mostly, now it's just Chinese. And who, maybe next year, going to be somebody else. And that is a very, very sad situation. But, you know, we have to just accept that and the people just want to have somebody to bully too. I don't accept it. Right? Thinking people do not accept it. We are better than that. General Allen, what Chiang has said, again, Chiang and I had this discussion previously. And, you know, going back to the Obama administration and Bush administration, Robert Gates, the former Secretary of Defense, he coined the whole concept or strategy is basically United States should build a high fence around the small yard, meaning that we should selectively pick our little flights and, you know, look at, if we're going to do this scrutiny of the Chinese, but not do a blanket thing again. And this goes to part of the prior administration, which was really an engagement, constructive engagement, you know. And I think this is part of where we need to untrack and get on that track. What scares me more, Jay, is that, again, this is China's year in which they do a five-year plan. Okay. And the next five-year plan is going to be held this month with their Congress. And they're coming with the stool circulation policy. Okay. Two things that are going to concentrate. Well, one is the domestic economy consumption. But the other thing is to rapidly build their high tech. Okay. This is the accelerated brakes on their end, you know, which means that, again, it does no good for both sides. We have this thing that's going on. And so China goes into another gear. And the question is, then it's harder to de-accelerate later as we go down this road, this whole thing of disengagement will maybe impossible, you know. I think, to me, my personal opinion, as you know, Jay, just living in China, working at U.S. law firms, I think really what has been done is they put China back against the wall, where, as a lawyer, you know that when you litigate, you're going to have to leave some wiggle room for settlement. When you have no room, you're never going to settle. This is when I'm afraid we're at that crossroad, the most dangerous point of that intersection, where there's no room to negotiate anything. There's no settlement. There will never get something. And so China is the plan B. And this dovetails in our discussion today, because it means a lot of these intellectuals that would be helping our technical advantage or the technical world. China is going to keep them from going in. And they're going to say, we don't want to go to U.S. We're going to stay in China, help out this five-year plan. And you know what? There's going to be a big vacuum. As you know, most of Silicon Valley, all of our high tech is built upon a lot of foreign talent. Okay. And this is where we're going to bite ourselves in the foot. Okay.  Let's segue to that part of our discussion today. So we have a really non-functional foreign policy toward China. It's non-engagement. And it's a lot of xenophobia and isolationism and nationalism and all the wrong things. And of course, just as Chang Wang says, this is for the campaign. This is trying to appeal to Trump's base and tap into that racial hatred that he likes to tap into the base. So the question I put to you was, how does that affect the Chinese students that are here or potentially could come here and participate in the American academic world and the American business world, the American world in general? It's a put-off. You know, just looking at it from as far as we've gone in this discussion, it's not a friendly place for them. So query, you know, how offended are they? How affected are they? How ticked off are they? How disappointed are they? I mean, this is the Statue of Liberty doesn't work anymore. So Chang Wang, you know, are you in touch with the academic world? Could you speak to that? Yeah, I can't. I do can speak. I speak from my personal experience. I came to the United States 20 years ago as an international student on iPhone visa. Then for the first three, four years. And every time I go back to China, not actually the first six years. And I spent three years in graduate school and three years in middle school. At that time, before Obama administration, that was under George W. Bush administration. The visa policy is very strict. So every international student, every Chinese student, because of the visa policy, so how many, how you treat, how US government treated Chinese visa applicant will be exactly how the Chinese foreign ministry treated you American visa applicant. So at that time, the visa, even my graduate program was three years. My law school was three years. But my visa is only good for six months. So every time I travel, I go outside the United States. Either go to Europe or go back to China. I need to go to US embassy to reapply another visa stamp to enter the United States. So that was, I counted probably I have 15 visa stamps on my passport. Every time I leave, I need to, if out of the six months period, I need to reapply. I was determined. I was determined because I was a student of 1980s Chinese cultural renaissance. And his clothes is the same in China, compared to the US civil rights movement in the United States. So America is my, I did one post study in the United States. I was so determined. I don't mind waiting in line for three, four hours to get another visa stamp. And then Obama administration dramatically improved that.  You can travel back and forth without worrying about the visa.  And then we see the, you know, the huge increase of the Chinese student presence in the United States. And then now the Chinese students representing, I have the number from BBC right now, representing about 20%, there are nearly 400,000 Chinese students currently in the US colleges and universities in 2019, more than one-third of the country's international students. Think about it, one-third of all foreign students are Chinese students. And the Chinese students spent, listened to the number, $15 billion intuition payment to the US university and colleges. That not including rent and airline ticket and all the food and supply, everything they buy. I will not be surprised that even number easily get to a 20 billion. And the current visa policy toward Chinese students is basically to intimidate them to come to the United States to study. You know, not only they are going after STEM students, scientific, technology, engineering, mathematics, they are targeting also economic finance students and even political science students. So that, the Chinese student, I read a lot of reports from the BBC and from other news outlets in Chinese language and in English language. So the, the, the pervading feeling of the Chinese students in the United States is regret. And there are very few people like me, they are so determined and said, I just want to be here. And no matter what, you know, I trust the United States. And I think that this is just a detour of the United States. But a lot of students, you know, I hear this quote and I give you this quote. You know, I hear this quote from Chinese students, students. You know, Obama is what America pretends to be. And Trump is America really is. That's horrible. It is horrible. It is horrible. From the Chinese, not only Chinese students and not the, you actually, the Chinese student is not from him. He is quoting some European in the correspondent. So, and that is one is regret. You know, that's a few so sorry we made the wrong points to study in the country. Yeah, let me, let me talk to Russell about that.  So the question is, if I give you 400,000 students, Russell, and a good number of them have learned to have regret over the past few years, what does that mean on a macro basis? How do they conduct themselves differently now that they have understood regret? Well, Jay, I think what we're going to see is probably a really challenging fact, first of all, for students coming here. As Sean has mentioned, that's 15,000, 15 billion dollars. That was in 2018 now, okay. And, and that's going to really hurt us because 15 billion dollars, the only other export industry that outrances, outranks education is the aircraft and machinery, like the Boeing planes. So 15 billion is nothing to sneer at. It's interesting because this whole effort is a DOJ-led effort, not the FBI initiative. It's a DOJ effort. And where's the Department of Commerce? You know, again, that's just on the business side. But again, what I see getting back to your question is that we're going to see a lot of the students in China possibly going to the European countries. And again, it may never come back here for a while, you know, because of the concern. And as you know, Jay, just seeing the Chinese students in China, most of them have, their whole lives have studied English to get to this point. Their whole lives, the parents have been saving just to get that education. That's the culture, you know. And when it changes, it's going to really be a shakedown for many Chinese who realize that maybe this is not the dream and we're going to go elsewhere again. So I think it has a broader effect just purely on an economic effect for the American side, because it is bigger. And again, getting back to what I said earlier, the concern again is that after COVID-19, nations are scrambling to look at artificial intelligence, digital transformations. And Stanford had a study done in China this May of the top 135 executives that did over $1 trillion of global business. And their thing is prediction is the Chinese economy. They're going to go through digital transformation. And likewise, in the U.S., we're behind the rate, and we're going to get all of these kind of skilled talent to actually make our society kick in, to be more digitally connected, to transform our country. Because that's where the economic battlefield is headed on a macro level. For us to say, number one, if you look in that sense, we need the talent. We need the research. We need innovation. And that's so important by shutting this pipeline. They say once we shut it down or close it down, it's going to be another good eight to 10 years before it's going to restart. Eight to 10 years means many more years in the business world, from a macro point of view. We're just going to be way behind the curve at this point. Just being in China, seeing how they use digital payments in WeChat, and you've seen that, they're like 10, 15 years ahead of us using QR codes. For example, the success of controlling the pandemic in China, you had to scan a QR code before you could enter your subway. And that QR code made sure that if you sat next to somebody, maybe 10 feet away in your train, they had COVID-19, the government will find out who sat in this whole area and they will contact you and you will get tested. Real-time tracking, that's pretty creative. So, Chong, let me go to you for a minute and ask, what is the effect of this kind of restraint and intimidation of Chinese students? I keep thinking about Charles Dickens and the Christmas Carol, and the story of Christmas future, the nightmare, if you will, of Christmas future. So, I give you 10 years of this, maybe it can't be reversed, not right away. Chinese students, like everyone else, they see their lives as a valuable asset. They have to make a life, make a career, make a family, participate in commerce and the like. What happens to them? What happens to the country? If we intimidate them to the point where they have regret, and they turn around and they go somewhere else, what happens to them? What happens to us? What happens to our aspirations for American exceptionalism, although that's a term not in legitimate use right now? Well, part of your question actually was well answered by Russell's comments, and I just agree that every single word he said. On the other hand, let me clarify regret. There are a lot of Chinese students regret we came to the United States, instead of Canada, Australia, other English speaking countries, but these people are still believed, the vast majority of Chinese students and international students still believe in American exceptionalism. They believe, still believe America is exceptional. It's not, the reason they came to the US instead of Canada, Australia, or the UK, because they believe the United States is exceptional. So there, and on the one hand, there are still a lot of confidence in American system by the international students. On the other hand, they're stuck here. If they started their program, they're in the second year or in the third year, they can't believe, they can just simply leave. They're not a tourist because there are a lot of visitors. They're stuck here and they are baffled, perplexed, these are oriented, you're not scared, you name it.  If you're in the second year, third year, who cares? Go, they go home, go back to China. That is the intention. But just think, put yourself in their shoes. They can't simply leave. They pay two-year tuition, auto-state tuition. Remember, all the international students pay auto-state tuition. They don't pay new state tuition. So that is, U of M, it's about $40,000 a year. Most public universities are somewhere between $30,000 to $40,000 a year. They already paid. They cannot easily transfer their credits to another university in Canada. So they have to wait and see. So they just, that is a very, very sad situation. And I totally sympathize with them. But on the other hand, what about the foreign students? They are not here yet. They are in high school. They're preparing for study abroad. Because in Japan, in Korea, in China, in Hong Kong, in Taiwan, in the most, particularly Asian countries, is a no-brainer for middle-class families to send their kids to study abroad. It's just a part of the standard. Looking for a better life, yeah. Russell, in your notes on this, you mentioned an executive order that was particularly troublesome. Can you talk about that? And really, and I'll be asking Chang about this, too, what should the United States do to correct the errors here in attracting Chinese students? So but let's talk first about the executive order and how would you deal with that in a new administration? I think the order that was issued May 29th this year by President Trump, it bars the entry of these visas to Chinese students in the U.S. who, in the graduate program, who have been associated with or with any PRC type of military affiliation. And again, it also says that under this order that the State Department can use its powers to consider visa revocation, okay, and it can direct the State Department to come up and review immigration measures to deal with this issue. A lot of this is still right now unclear as to the procedure of the State Department, and a lot of it gives black discretion, to look at students entering in to apply for a visa. And it might be that somebody may have had a past affiliation years ago and no longer has any affiliation, and everybody's thrown into the same category, it's a blanket category. There's also question marks as to, essential part of the education also is for the students to get what is called OPT, optical trading after they graduate. And usually there's a time period that they hear, they do research with companies, which again, contributes to a better technology development research for us here. So that's what the order says, okay. The problem is it's too broad and second of all, the question is whether or not there's really justification for this, okay. From what I understand, they are already existing procedures that deals with this, that this order goes over, it overreaches them to it. And so again, so the question is whether... What would you do, Russell, to correct the era of this order? I mean, let's make you the president. I always wanted you to be the president. Let's make you the president on January 20th. What is your first action with regard to this executive order? Well, I think the problem is the executive order that grants these powers to the State Department to do a revocation of visa for one, the vetting process for the visa, especially what Chang has mentioned earlier, students cannot leave the country. Say it's a holiday of the fact that their visa can be rejected, okay, revoked. So this obviously certainly is something that we need to remove away. I think going back to what we had previously under the bottom of Bush administration would do a lot to get it back in track, okay. To provide clarity, to provide used existing procedures we have. And again, it's resetting the policy back again. I think that's important. Second, I think if it's national security interest, I think that the government has to work closely with the university to ensure best practices are followed. For example, there is a action brought against it. I believe it was a Harvard professor for being involved in some kind of program with the China side. Again, it's really having the universities to make sure that they follow best practices of even their own faculty. So it may not only just be a problem of the Chinese students that enter into the US. Again, it seems to me that really the problem is we're creating this chilling effect. We are also causing, I think, giving us economic problems. And Chang mentioned something a minute ago about many of the students come from middle class families. And this is their lifelong pursuit to get an education in America. And again, China middle class is between 500 to 70 million. And again, this class, usually people are generally coming to America for an education. And again, it's just a big market for the US from an economic broader point of view. And I think that that's important. So again, back to the question, I think we need to scale back that executive order. I think we need to go to an acceptable level. But we did before under Obama administration and working with the university to create better practices. The universities, they're in trouble financially, especially now. A lot of them are closed. They can't collect tuition. So they need help. And this is a source of funds for them. But we're almost out of time, Chang. And I'd like to ask you one last question. This is not an easy question. You know, we have learned here that American foreign policy translated into, as you mentioned, American political policy, American election campaign policy, really doesn't work very well. And it isn't working well now. But my question to you, it's in a larger sense, is we see here this administration taking off after the Chinese in so many ways, so many inappropriate ways. And we see other immigration policies that are profoundly affecting our image in the world as a liberal democracy. We have to deal with that. We have to recover from that. And my question is, what can we learn from the Chinese student phenomenon that we've been talking about in a larger sense to correct our image in the world, to have immigration policy that works for everybody, not only the Chinese, but every single group, every single individual who would like to find education or sanctuary in this country under the lamp of the Statue of Liberty? Can you talk about that for one minute? I'm sorry to limit you to that. You are there for one minute. The question, to be honest, I don't have a direct answer to that question. It's so profound. I still have this vivid memory of my first time where I stand on the land of the United States. And I just couldn't believe that I'm in America. And today, I'm part of the American community. And when I read the report from New York Times, I give you the title of the report. It's the world look at the United States with sympathy. And I think instead of regret, sympathy is a better world. The world look at the United States, don't see a leader, don't see a beacon, don't see freedom. They see a failed state. They see a dysfunctional state. And they sympathize with the United States. And so no matter how hard we treat foreign country, foreign students, international students, minorities, the most vast majority of these victims being bullied by us, they still believe in us. So that's something I don't take for granted and should be grateful. There was one problem for some of the people in the administration is they never feel grateful for anything. And they feel victimized. And don't you feel it's so ironic that people in power, they feel they are victim. And they're real victim. They don't feel, you know, hostility. Instead, they feel sorry for the abusers. Oh, wow.  Chan Wong, thank you so much for joining us. Russell, thank you for setting this up and coming on again. I hope we can do another show like this much more to discuss. And there's so many things happening worthy of discussion. Russell Liu, Chan Wong, Aloha. Thank you so much. Thank you.


5 facts about sleeping naked. 1. Increases sleep quality. Sleeping unclothed can lead to better sleep. This is because sleeping undressed can assist in regulating body temperature, making it simpler to fall asleep and stay asleep. Furthermore, pain and restricted movement from sleeping in uncomfortable or restricting clothing can interfere with your sleep. 2. Encourages skin health. Sleeping in a nude state might encourage skin health. This is because wearing clothing can create skin irritation such as acne and rashes by trapping sweat and bacteria against your skin. Your skin can breathe while you sleep naked, preventing these problems. 3. Increases self-confidence. Sleeping in your undies might increase your self-confidence. This is because it could make you feel more at ease with and accepting of your body.  4. May help you lose weight. Sleeping in your undies can aid in weight loss. This is so that you may burn more calories while you sleep since when it's cold outside, your body needs to work more to stay warm. In addition, regulating your cortisol levels can help you feel less stressed and avoid gaining weight. 5. Enhances fertility. Sleeping in your undies might enhance fertility. This is due to the fact that sleeping in constrictive clothing can make the testicles hotter, which can harm sperm and lower fertility. Sleeping unclothed can enhance sperm production and control testicular temperature. What are your views on this subject? Please share your thoughts and opinions in the comments.


Welcome to MOOC course on Introduction to Proteogenomics. So, we are proceeding from the protein quantification running on the SDS page gel, doing the sample cleanup peptide quantification. Now you are ready to inject the sample in the LC liquid chromatography followed by the MS analysis. So, now it comes the liquid chromatography. Here we are using reverse phase column C18 material, peptides are going to bind to the column and then you want to elude the peptide based on the hydrophobic hydrophilic properties. So, you will use a different gradient using estonitrile 5% to 80% or you can go even up to 90% with 0.1% formic acid and this concept I have already briefed in the beginning in the previous lecture. So, I am not repeating again, but you need to pay attention to the parameters for what should be the best gradient for doing the liquid chromatography. So, what is shown on the screen here is again a refresher we talked earlier as well that you will use different parameters depending on the kind of sample you have and you would like to achieve a good Gaussian distribution of the peptides which are eluted from the column. You would like to see that you know very soon maybe after 5 to 10 minutes time peptides start eluting out of the column, then after as you increase the gradient of estonitrile more and more peptides are coming out of the column and eventually once you have reached to the saturation level, then finally all the peptides are out of the column and you are then washing and reclubrating it being ready for the next injection. So, that ideal setup should give you a Gaussian distribution of good intensity of the all the peptides. One need to play with this parameters, but again shown here that you need to work on each of these and now let us we will talk more about these parameters in the lab session. Hello, so here I am going to explain about mass spectrometry. So basically there are main two components one is liquid chromatography another is mass spectrometry. So, I am going to explain first about liquid chromatography. So, here you can see there are main two solvent which is one solvent A which having a 0.1 percent formic acid, another is solvent B which is 80 percent AcN and 0.1 percent formic acid. Now here if you see this screen, now here you can see there are pump A and this is pump B basically which regulate the flow of solvent A and solvent B. This is another pump which is pump S it control the taking over sample that how much amount of the sample has to be injected that will be controlled by pump S. So, here I am ejecting the tray where you can keep your sample now here you can see this plate and this is while where you can keep your sample. So, generally here we are loading around 1 microgram accordingly you can calculate the amount of volume how much you have to inject in mass spectrometry. Now you are familiar with the liquid chromatography and how nanoLC can be used, how different parameters, different pumps are important keeping a watch on the pressure is very crucial. Now you are ready for injection with the electro spray ionization. This is a very crucial part because now all your peptides have converted to the gaseous ionized forms and they have to move it inside the mass analyzers for further analysis. So, various settings and again the voltage these criteria that important and you are going to learn more about them in the lab session, but briefly refreshing you here again that while all these ions are coming your major effort is most of them can you move them inside the mass analyzer and that is where the pressure and the charge these parameters are very crucial and you need to make sure that most of the ions are actually going inside the mass analyzer otherwise the proteome coverage will be affected. So, next let us assume that you know you are you have done a good electro spray ionization then you are ready to separate these ions inside the mass analyzer. There could be different type of mass analyzers and different type of mass spray configuration. For example, we can have you know ESI QTROF a popular configuration or orbitrap in this case we are going to talk about orbitrap fusion technology which is having a tri grid mass spectrometer and let us have a laboratory session about using orbitrap fusion technology and different parameters for MS and MS MS analysis. Once you have kept your sample in nano LC and then you will monitor through the software. So, your sample will go through this tube and this is first column which is three columns where your sample will start to clean up basically desalting and your peptide will start to bind in this column and then waste will go through this tube into a waste beaker. Now, once your sample will be cleaned in the pre column then second column which is analytical column then your sample will start pass through this analytical column and they will start to fractionate and slowly the first hydrophilic peptide will start to elute and then hydrophobic peptide will start to come. So, once your sample will start to elute from this tip of column few can see here and then it will start get ionized. Now, here we are applying voltage around 2.2 kilowatt and your sample like highly charge and they will start to elute. Now, here I am going to explain parameter for MS. So, this is MS 14. So, these are parameter for the MS where I kept orbitrap resolution around 60,000 scan range 375 to 1700 m by 0. So, generally for peptide it is you know that is that is optimized RF lens I have taken 16. AGC target is like automatic gain control like how many ions you want to accumulate so that you have to define which I am taking here around 4.0 into power 5 maximum injection time is 50 millisecond and quality is positive because our peptides ionized and they are positively charged. In intensity you have to define the intensity how much intensity threshold should be there which is 5.0 into power 3. Now, generally peptide is charged so I have to define how much charge should be there the range of charge basically. Now, here you can see 2 to 6 I am taking it will take a single charge and then more than 6 it will consider only 2 to 6 charge peptide and then I am defining dynamic exclusion which is 42nd here mass holdings should be KTM and this is the parameter for the MS MS. Now, where I am keeping isolation window is 1.2 now because the MS is happening on the peptide now those prepare have to be fragmented and for that I am using the SCD cell high collision dissociation and energy collision energy mode set that is fixed and this is the energy which I am applying for the fragmentation for our peptide and the detector is here orbit rate and resolution for the MS MS is 15 thousand and first mass which has to be detected 100. So, these are the like parameter which I am so new these are the like already optimized for the label 3 quantitation if you are using like different type of technique if you are using the label based in case of eye dragon and KMT accordingly those parameter will be changed. So, accordingly you can do it change. So, this was the parameter for the LC and MS parameter. So, now you are familiar that how you can use different parameters and settings for the MS and MS MS analysis after doing the a good run from these experiments from the same sample. Now, you will see these chromatograms which is shown here on the screen which shows that you know the time versus the intensity of these peptides as I mentioned you would like to see a good Gaussian distribution of the peptides coming out of you know from your sample. Now, from these sample how to make sense of this information that you know what these proteins are. So, if you remember that we talked briefly about you know looking at b and y ions. So, again if you keep looking at walking through the entire chromatograms you will see the pattern of the spectra which will give you a good idea for you know the b ion y ion various are generated and now you can use this information for the for the database search. So, let us have another lab session to discuss more about these chromatograms and looking at these data. So, till now we have seen the different liquid chromatography parameters and the mass spectrometry parameters required for the success of a LC-MS experiment. So, as you now know that the char species enter into the mass spectrometer and get fragmented. These fragments are then detected and by use of a suitable software the identification of the peptide is revealed. However, just by looking at the chromatogram one can easily reduce whether the sample run was good enough for it to be taken to further analysis. Let us now take a look at the example of a very good chromatogram. On the screen we see a very Gaussian distribution of the peptides. This is the MS-1 chromatogram that means all the peptides which have entered into the mass spectrometer as char species have got detected at the MS-1 level. Further based on the abundance of each of these peptides they are fragmented at the MS-2 level and detected. It is to be noted that most of the peptides which are less abundant are likely to be ignored and only the high abundant peptides get fragmented. So, the bottom panel shows the MS-2 fragmentation pattern for the selected peak. So, if you can see here this is the MS-2 pattern for the selected peak. So, you see different fragments which have been generated and also the signal is relatively less noisy. This helps in better interpretation of the data for the software. So, we now move to another chromatogram which is not that great. So, on the screen if you try to correlate if you try to compare this chromatogram to the chromatogram which is previously shown you can very clearly see that the distribution of the peptides is not a Gaussian distribution. Also you see that there are gaps in the chromatogram which are indicative of issues with the sample and with the electro spray ionization. So, there are certain segments of the chromatogram where probably nothing entered into the mass spectrometer or the peptides did not get ionized properly for them to be detected by the mass spectrometer. If you now look at the MS-2 pattern the MS-2 here has significantly less number of fragments which is a feature of a very bad chromatogram resulting from a very bad sample. This issue could have been due to improper handling of the sample or due to issues with the column but this is the basic information that one can reduce from merely looking at the chromatogram at the MS-1 level and the MS-2 level. The raw data that is generated is then further analyzed using specific software which can reduce the information in the chromatogram and reveal the identity of the peptides subsequently leading to the identification of the proteins. I hope you got a very good glimpse of doing the mass spec base proteomics workflow but how one could use the same set of information these you know workflow for a you know any case study for any biological problem. In this slide I have invited one of my PHA student Shulina Mukherjee to talk about how she has used this mass spectrometry based proteomics workflow in her own research. Very briefly she will walk you through the steps and the strategies for data analysis and in a nutshell that how it can give you some biologically meaningful insight from a clinical problem. So now that you have an idea about the chromatogram that comes out after your mass spectrometry experiment we will talk about how to interpret it and how to do the protein identification. So you can interpret the raw data coming out using several freely available software as well as commercial software. So one of the softwares is Maxquan and another software that I will be giving you a little glimpse of is proteome discoverer. So what you what the software does is it takes the raw data into account and then it does the spectral matching and counting and also it uses a database that is a background database. So if your sample is from human origin you give a Homo sapiens database and then it does the annotation of the peaks. Also in the process of sample annotation from raw data you can also do a grouped analysis for example in this case if you are looking at a cancer sample wherein you are comparing the normal or the non tumor samples with the tumor samples you can give different grades of the tumor for example this is grade 1 this is grade 2 and this is grade 3 of a tumor and these are the normals. So when you annotate the raw files in such a way that you already specify that which group it belongs to the software then takes into account these considerations and then we it gives you the details of how much of the protein abundance is present across these groups so that you can know whether you have some dysregulated proteins that can be subsequently used for identification of biomarkers. So for the setting up of the workflow first of all we do the database search as you can see here we have used sequence ht and also these are the steps that are followed that is you can annotate the parameters that is for example you have put a parameter of mass tolerance which is 10 ppm then you have used the charge states and then the retention time also here you can see the spectrum files will be taken and then it will go to the spectrum selector then it will go to the database and then there is another workflow which is called the percolator. Other than these things you can also use other search engines like you can use mascot in parallel with sequence ht and then if there are unmatched spectra it can go to the next search engine also for example in this case it is the spectrum confidence filter and then furthermore you can also use other softwares for knowing into the other modifications present in the peptide for example if you want to know whether your peptide is glycosylated or phosphorylated then you can use these kind of these kind of filtering to to annotate those changes in your peptide. So how does the data look? So as you can see after get going through a lot of filtering criteria the data that comes out is of high confidence because you have put on stringent filters so that whatever hits you get are the true data and not false positives. So here you can see that there are different tabs associated here for example this is the this is where you have annotated whether the protein FDR confidence how much is the level. So here you can see all of these proteins that have been identified have high FDR. So false discovery rate is the statistical value that estimates the number of false positive identifications among the peptide and it is also measure of certainty for the identification as in how much you are confident that the protein you have identified is a true match. Then we also have a contaminant database which we can plug in in the workflow the contaminant databases will actually indicate presence of keratin or serum albumin which are high abundant protein and often are responsible for giving false positives or masking your actual protein of interest. So as you can see here also in our data we have got serum albumin and the software is marked here as true. So while all the other proteins there is false in case of serum albumin it is marked as true so you can remove this kind of protein in case of your subsequent analysis. Other than that you will also have a plethora of information like the unique proteins. So the unique proteins again gives you an idea as to how confident you are of identifying the particular protein that has been ascribed to. For example, the first hit which is a neuroblaster differentiation protein has 370 unique peptides that means the mass spectrometer has encountered the peptide 370 times or it has actually annotated the protein the software has annotated the protein with very high confidence. Then you can also know about the score which is the score that is given to by the search engine and many other details. So thus you will have all the information available after you have run the protein through after you have run your raw file through a software and the software has different tabs that you can customize and you can set your parameters that you are looking for. So basically we have started from here that is we have taken a cancer sample and normal samples we have used a patient cohort then we have extracted the protein and then we have run the samples in the mass spectrometer. So after doing all the all these exercise you have got raw values you have got different peaks that you now you need to know that what are they. So you have used a software which I told you that you can either use a freely available software like Maxquant or a licensed software whichever resources available to you and then you can use the software and the various parameters to now do the data mining. So after the data mining what you are actually looking for is something like this wherein you can see a clear difference between condition A and condition B. So as you can see there are a signature list of proteins which are highly abundant in condition A and there are a signature list of proteins which are highly abundant in condition B. So these are indicative of the actual biological changes that are happening in the patient sample. Furthermore you can do the data curation and network analysis using again doing several bioinformatics software like string, DB, Metabo, Analyst etc and now you can see that after doing the whole exercise of using mass spectrometer and the software for annotating the data we get the we can map the proteins in various networks like this and then we can also see which are the ones which are classifying the different grades of tumor or the are different between the control and the cancer samples. For example these are these are a set of markers as you can see. So you can see in this one this is very high in the C sample which is the control sample and relatively low in the grade 2 samples. Similarly there is a reverse trend for this protein you can see QO 4637. So this is again showing a sequential increase that is it is low in the control samples and going high as the tumor progresses. So thus using these kind of tools you can answer a lot of biological questions. Alright so we started with a workflow of mass spectrometry based proteomics. We talked about how to do the protein quantification, protein digestion. Again we have talked about peptide quantification. Then you are ready for doing the LC-MS-MS based analysis. So liquid chromatography and MS parameters, what we generate the chromatograms, how to interpret chromatograms and how to review the whole data set make more meaningful insight from this data for the clinical case studies. I hope this gives you very basic of course it is not so detailed but you know a good glimpse of the workflow involved in doing MS-based proteomics. A lot can be done using mass spectrometry based proteomics. We have just talked about protein identification and label-free quantification workflows. You can also think about quantitative proteomics which I talked in the theory classes earlier about using iTrack or TMTs and those workflows can be very useful as well. But as long as you have done this workflow of sample preparation and their separation very well then you are ready for the quantitative proteomics based workflow as well. Thank you.


Why was ten minutes decided as the amount of time necessary for a steady and diminishing flow for each block? How is it guaranteed that a solution will be found within exactly this amount of time? That's a great question, Dimitri. Let's break that into two parts. First part is, why was ten minutes decided as the amount of time necessary for each block? It was really a matter of balancing two competing interests. When you have a heartbeat in a blockchain consensus algorithm as you do in Bitcoin, you've got to account for the fact that miners may be operating anywhere on the planet and may even discover blocks simultaneously in different parts of the world, which then are transmitted to the network at different speeds and may arrive at different times for different nodes. Let's think of an example. Let's say you have a miner in Australia and you have a miner in Canada, and they both simultaneously and by accident discover a new block, and then their blocks start propagating across the Bitcoin network and across the world. Now, as you can imagine, they're going to eventually be transmitted to the entire network. However, for some part of the Bitcoin network, the nodes will hear the block coming from Canada first, whereas for another part of the Bitcoin network, the nodes will hear the blocks coming from Australia first. In that case, some of the nodes will assume that the Australian block was found first, some of the nodes will assume that the Canada block was found first. What that causes is a temporary and a normal fork. That type of fork happens on average about once a week. It's a one-block divergence in the network, and it's fixed with the very next block, because the chance of two nodes simultaneously again finding a block is unlikely. So, in the next block, one miner is going to find a new solution to a block and transmit it. If they built on top of the Canadian block, that's part of the chain. If they built on top of the Australian block, because that's the one they heard first, that's part of the chain. The other block that wasn't made part of the longer chain will simply get recycled with all of its transactions, and all of those transactions will be re-included in subsequent blocks. This type of one-block fork happens, as I said, on average once a week. Imagine if you shortened the target time for a block. You were producing blocks every five minutes, or every one minute, or even every 30 seconds. Some blockchains do that. Ethereum is a good example. Ethereum has 15 to 30 second blocks and uses proof-of-work similarly. But in the case of Ethereum, the consensus algorithm anticipates that you're going to have these so-called orphaned blocks occurring much, much more often. They use a different consensus algorithm to anticipate that. The original Bitcoin consensus algorithm was fine-tuned to reduce the number of orphaned blocks that will occur naturally, so as to avoid retransmissions and re-convergence of the network. It only happens once a week. If you made it every five minutes, it might happen four or five times a week. If you made it every one minute, it may happen several times a day. This was a careful tuning. It was assumed at the time that ten minutes would be perfectly adequate for clearing and settlement of transactions on Bitcoin, and for most use cases, in fact, it is. That's really the trade-off. That's the first part of your question. The second part of your question was, how is it guaranteed that a resolution will be found within exactly this amount of time? That has to do with what is known as difficulty retargeting. Every two weeks, or every 2016 blocks more accurately, the difficulty of the Bitcoin consensus algorithm is adjusted so as to converge on to ten minutes, meaning ten minutes is the fixed heartbeat time of the Bitcoin network, and what is variable is the difficulty of hashing. If more miners are hashing, they'll find blocks a bit more often than every ten minutes on average, and then the difficulty will be increased in the next two-week period in order to adjust for that. What changes is the difficulty, and what remains fixed is the timing. The heartbeat is always targeted to ten minutes on average across many blocks, and what changes is the difficulty so that the heartbeat retargets to ten minutes. Then the ten-minute time period is hard-coded into the software. That is the one static heartbeat of Bitcoin. When more people become miners as they can earn Bitcoin, is there any effect to the Bitcoin ecosystem? If the miners are more than the transactions needed to be verified, will this cause competition between the miners? In fact, that's already the case. Miners do not split transactions between them. Miners all mine pretty much the same transactions, because all of the transactions that are available in the system and need to be mined are shared with everyone. Everyone has a copy of what's called the mempool, where they have the unconfirmed transactions. Think of it as a giant reservoir where unconfirmed transactions are waiting, and every miner has their own copy of this reservoir, and they use it to construct their candidate box that they're going to run the proof-of-work algorithm on. They try to cram as many of those transactions as they can fit into their candidate block. The competition isn't between miners as to what transactions they're going to mine. The competition between miners is how much hashing power they can put into their proof-of-work, so they can find a solution first. However, there is also competition in the transaction reservoir, or mempool, if you like, which is there's competition between transaction senders. If you send a transaction and you want it mined faster, you put a higher fee, and you're competing against everybody else who's trying to get space in the limited space of the block for their transaction, and you're competing through the pricing mechanism of fees. So all the users are competing for space in the block, and all miners are competing for speed in terms of how fast they solve the lock, so they can be first, because the Bitcoin mining algorithm is a winner takes all. So the first miner to win is the only one who gets rewarded.


to hear is true the names have been changed to protect the innocent you're a detective sergeant you're assigned a robbery detail a pair of bandits are staging a rapid-fire campaign of hold-ups in your city in 21 days 11 food markets have been robbed the hold-ups go on the suspects are still unidentified your job get them the documented drama of an actual crime for the next 30 minutes in cooperation with the Los Angeles Police Department you will travel step-by-step on the side of the law through an actual case transcribed from official police files from beginning to end from crime to punishment drag net is the story of your police force in action was Tuesday September 26 was hot in Los Angeles we're working the day watch out a robbery detail my partner's been Romero the boss is Captain diddy and my name is Friday was 9 18 a.m. when I got to the record Bureau Lieutenant Cunningham's office Joe what's doing grown up too much got a job you can do for us Frank like to have it as soon as you can get it out oh it's about only these market bandits we've already gotten out a dozen teletypes and bulletins on them like to get out some emigraphs too if we can general distribution man what do you want on them well I better lay out the whole thing for you a lot of the copies are going to the retail grosses association all their members no luck with those two thieves so far no I know closer than we were three weeks ago figure this out a plug another loophole how many copies you figure you want on this by a thousand yeah that's fine that ought to do it all right okay now what do you want to go in it Joe and you better lead off with the first job they pull that was on the third of this month makes a little better than three weeks now they hit a supermarket out on Pico ever since they've averaged about three jobs a week we know they're hitting nothing but supermarkets all of them on main thoroughfares no small neighborhood stuff so far they've confined their operations to the 77th Street division in the county territories generally the south end of town there are two men they got a pretty well-established M.O. they're hitting in the morning hours usually from the time the markets open up until around noon time I use a stolen car 95% of the time a car stolen from the industrial area cars they steal for the jobs are always either a Pontiac or an edge we also know they're dropping the stolen cars within a short radius of the holdup scene about a half a dozen blocks or so in some instances they use a second automobile or public transportation some kind of bus or streetcar you know to make their getaway after they drop the stolen I'm sorry a little faster yeah okay okay go ahead now they usually pull up and park directly in front of the market that they're gonna hold up one of the men always remains in the car behind the wheel apparently it's always the same man has a description meager we know he's good size MWA fairly large build here's about the best description we've been able to piece together on the second man picked up a little bit from each victim second man described as MWA also about 5 foot 8 inches medium build 155 to 160 pounds always wears a hat yeah round suit rough material of some kind maybe tweed what a small prank huh small point on when he enters the market for the holdup the second man always holds a white handkerchief holds a white handkerchief to his face as if he's wiping his eyes keeps most of his face covered with it during the robbery he's armed with a nickel plated revolver possibly 38 caliber we know the jobs are well cased because in almost every instance the suspect goes directly to the manager of the market directed manager hands the manager a brown paper sack tells him to put the money in it and then he takes off so far he's never attempted any rough stuff no gunplay on three occasions when the suspects got only small amounts of money at one market they hit again in 15 or 20 minutes at another store in the same general area they never make any attempt to disguise or conceal the license number on the getaway car as I say they always abandon the car a short distance from the holdup scene and see yeah okay I got it well that's about all of it Frank I guess you can boil that down into some kind of shape yeah okay I'll have it tapped up in the form I'll get your copy before we make the rod and see if it's okay with you fine thanks not going so good huh no it isn't Frank pretty slow I've been on top of the thing for a couple of weeks now we're still looking for some kind of a break so I understand Glenn Chandler from your office took off for some fishing down in Mexico yeah not doing bad but I hear I got a card from yesterday he's down by Kino Bay he put the bite on me to send him a dozen mountain plugs yeah fish must be hidden pretty good down that's what he says yeah it's a big brown sea bass especially I wouldn't mind getting in a couple of days down there myself excuse me yeah I give you a cunning am yes a minute you Joe thanks thanks yeah oh yeah man mm-hmm when they're right yeah okay right away yeah fine check that for me the later Frank might be a little change what's matter market bandits they hit again 938 a.m. then and I got on car and drove to the scene of the bandits latest holdup a new supermarket out on Swanson near the intersection of South Broadway after interviewing the manager of the store and all available witnesses to the robbery there wasn't much doubt about the identity of the suspects the description and MO fitted perfectly with that of the two men who'd been staging a whirlwind campaign of market robberies throughout the south end of the city for the past three weeks the approach and execution of the holdup was the same in every detail even to the stolen car they'd used which was found abandoned the short distance from the robbery scene there was only one deviation that was the first break we'd had in the case since it started a woman customer at the market a mrs. Thomas Swanson saw the holdup man as he left the store and ran to the getaway car she told us that the car door apparently stuck when he tried to pull it open and that he took away the handkerchief which he'd held to his face in order to pull the door open with both hands during those few seconds she got a good look at him after taking the crime report and making our preliminary investigation we brought mrs. Swanson downtown we had her check through mug books of all recent parolees from the state penitentiary all ex-convicts with robbery records after a couple of hours of checking mug shots she couldn't seem to make positive identification very sorry officer just gets a little confusing after a while looking all these pictures I did get a good look at the man's face I just can't settle on one of these pictures so well from what you did see of mrs. Swanson the lower part of the man's face especially you find a good likeness in any of the well yes I can find good likenesses for some of his features but none of them all together in any one of these pictures here like the mouth and chin on this man here yes ma'am is that pretty close most exactly I'd say and then the nose on this one here it's the very same I just haven't seen any picture that fits them all together you know what I mean don't you that's my my understand now these five pictures here that you've picked out you pretty much say that they incorporate all the features of the man that you saw getting that's right officer this man here and this one too I'd say the lower part of his face was exactly like that the small mouth and the stash just like that and the chin pointed like on this man and the upper part of his face would you say it resembled these others here yes these three here just the upper part you see around the eyes before it I'd say that was very close now just put the two sets of pictures together I think it has a man perfectly sorry I can't be more help to as I say looking at all these books of pictures it gets a little confusing well you've given us quite a bit of help ma'am thank you very much not at all is there anything else no thank you now right now we've got your address and telephone will probably be contacting you in a day or two if you'd like to wait right here we'll make arrangements to have one of the officers drive you back to your home all right thank you very much goodbye ma'am we'll be talking to you what do you think Joe it's more than we had yesterday her description is the best we've got him a guy so far how about getting a composite picture made up from the mug shots you picked that's what I was thinking you want to duck in and get somebody to drive miss Swanson home I want to go in here in the captain's office yeah okay thanks buddy how'd you make that could have been worse Captain woman find anything the mug books no positive identification she picked up five of them she thought were close I think maybe put off over the crime lab ought to be able to make up a pretty good composite from him she fairly positive about the man's description yeah it seems to have a clear idea good composite ought to help us quite a bit you get this new description that memory graph you haven't made up no not yet I'm gonna see Frank about it nice camper and got a hold of just Gonzalez Joey's gonna drive Mrs. Swanson home fine did you ask her if he checked on that last run we made through the stats office yeah he picked it up Ian Frank is strata gonna start checking the names this morning it's not much of a list and pretty well picked over already so you got some witnesses out there this morning description from this Mrs. Swanson yeah it's about it rest of it was their usual ammo Swanson woman was the only one who got a good look at the guy when he took the handkerchief away from his face how about the getaway car they found same as the others late in print checked it over clean two thieves had their share luck when we get eyes how about Metro division captain can we get any help out there what do you got in mind well we know the take that the two thieves got in that job this morning was pretty small for him three hundred and forty dollars yeah from their ammo in the past we know whenever they get a small take on a job they usually hit another place fast they work in a fairly concentrated area down the south end they only go after big markets on the main thoroughfare well I was thinking if we could get some help from Metro put maybe 15 or 20 crews down in the area for the next couple of days might stand a good chance of grabbing when we're talking to Dick Turen and over at the sheriff's office he says he could put some of his men down that area to cover the county on the on the fringes the territory around there what do you think I'm fine I can get Stevens at Metro right now set it up for you about 20 crews that right just about yeah with any kind of luck at all we ought to land them sure worth trying Lieutenant Stevens please yeah Joe this Harry Didion fine fine we've got a little stakeout problem here how about using some of your men about 30 all right I'll settle for 25 okay yeah a south end of town those market bandits fine would you have any report here at Ravi's seven o'clock tomorrow morning all right thanks a lot it's all set you got 25 men seven o'clock tomorrow morning okay skipper we'll give you a dozen teams to start with Gonzalez in the Stroud or one more how many figure working from the sheriff's office all about four or five teams I imagine all right it's your operation you told me in charge on our end you run the show you breathe the men if you need anything call me right how about the overall plan skipper you got any special ideas on it just one yeah get them 118 p.m. we got the revised description of one of the hold-up men until Lieutenant Frank Cunningham at the Record Bureau he told us a thousand copies of the special mimea graph form containing all available information on the two market bandits would be ready for us that night we met with Sergeant Putt off the artist at the crime lab and he went to work making up a composite picture of one of the suspects from the mugshot selected by the robbery witness Mrs. Swanson when the picture was completed we had Mrs. Swanson check it for similarity to the actual hold-up man and then we brought it back to the Record Bureau where a thousand copies were made of it the pictures along with a mimea graph forms were sent immediately to the retail grosses association for distribution to all market operators throughout the city additional copies of the picture and the information sheet were given to each man who was to work the stake out when we met in the squad room at seven o'clock the next morning besides the men from Metropolitan Division and the men from robbery who are going to work with us Lieutenant Dick Tannen and Sergeant Dave Terry from the Sheriff's Office who are also there for the briefing well briefly got some rundown on the two suspects and the M.O. they follow it's the same information and description on them you'll find those Mimeo forms we handed out Joe you want to take over give me a sign yeah well if you just take a look at this pin-up map here for a minute I'll try and brief you on how the stakeout's gonna go the way we got it figured anyway we've worked out a plan we think ought to be the easiest and the best way to work it if any of you have any suggestions as we go along what you're like to have them all right now as you can see from the map here the stakeout areas in the south end it runs from can you all see it from there yeah it runs from Santa Fe Avenue over here on the east the Vermont Avenue on the west running north and south now we'll have to cover from Santa Barbara Avenue on the north down to Manchester that'll be the southern boundary that's the area the thieves have been working and we're gonna have 14 of our own crews covering that area and Dick Tannen and Dave Terry from the Sheriff's Office will have their men covering and joining county territory now we've broken a map down into equal units as you can see here each unit will be covered by one car and each of you will be assigned a unit number Romero and myself will be an 80k we'll have a roving unit we'll cover the entire area you'll have three-way radios in your car so you'll be able to contact us directly if you have to now if you'll check the map again you can see these green pins we've placed here each one of them represents a market robbery now notice that every one of them is on a main thoroughfare they Manchester South Broadway Vernon South Hoover I don't have to tell you that that's where you'll have to keep a sharp watch on the main street each of the individual units here on the map will be covered by one car they said each one's roughly nine blocks square now you'll be responsible for the area assigned to you we haven't got too much time so before we go down the list to assign the areas if you got any questions any questions at all yeah there's supposed there's a 211 calling somebody else's area I mean other than the one you're assigned to do we leave our area and head over there no Jess I meant to bring that out if there's a robbery calling somebody else's area stay put did you get a call on the hand anything else yeah how about the managers of these markets they've been briefed on the stakeout I know we're gonna be covering the area yeah they've all been alerted now in case of a hold up they might try to contact you directly they'll be looking for you you'll be looking for them that it Bob's it's covered for you but yeah I'm just wondering about this number two suspect the one who drives the getaway car yeah it says here the dope sheet number one suspect carries a nickel plated revolver how about number two he carry a gun well I wish I could answer that we don't know all the information we got on Amazon a sheet right there I see well they never try any rough stuff anyway do they no gunplay they never hurt anyone no not yet don't let them make you the first 7 30 a.m. we finished handing out the assignments and the men left to take up their stakeout positions in the south end of the city two men to a team each team in a three-way radio car each car to patrol a designated nine square block unit there were 20 such units within the frame of the overall stakeout area at the same time cruiser cars from the sheriff's office operating on their own radio setup began patrolling the fringe positions which fell in county territory through control one at our communications division we kept in continual contact with them 8 a.m. all the cars were in position each of them patrolling their individual nine square block unit the first two hours passed no reports no sign of the market bandits between 10 a.m. and noon half a dozen possible suspects were stopped and questioned by different teams and then brought to the 77th Street Division for processing all of them were eliminated the afternoon of the first day went pretty much the same we had shakedowns on four-part cars in the area and more shakedowns on possible suspects nothing materialized the next morning Ben and I along the other 32 men in the stakeout picked up where we left off again no sign of the suspects no reports the morning of the third day the stakeout detail met in the squad room as usual and then left to take up their regular assigned positions 7.35 a.m. how about it Joey ready to go yeah just a minute okay let's go I get it okay robbery Friday yeah where when was that yeah right away oh come on we got a hurry what is it market bandits maybe we got them 7.50 a.m. Ben and I arrived at the intersection of 58th and San Pedro Street two of our stakeout cruiser cars were pulled up at the Southwest corner one of the teams was just Gonzalez and his partner Frank Estrada Gonzalez briefed us on what had happened victims the owner of the supermarket down the block in San Pedro is walking from his house to the store he passed the corner here about 7.35 a.m. at $340 on party yesterday's receipts from the store they got him right at the street they took every dollar of it you sure it was a market been an ammo description they check out all the way we had two teams on as soon as we got the word located the holdup car minute ago where is it down an Avalon Boulevard about the sleeves just car was empty no trace of Saturday 805 a.m. the latest victim of the market bandits the owner of the supermarket on San Pedro Street was taken to the 77th Street division where the crime report was made he told us that one of the men who robbed him bore a very close resemblance to the composite picture which we had drawn up of one of the suspects we figured we missed grabbing the holdup men by a matter of minutes even though some of our units were in the general area at the time of the robbery it did little good the victim had delayed reporting the holdup for a full five minutes because the bandits had threatened to find him and kill him if he did despite the fact that they changed their MO to the extent of robbing the store owner on the street instead of in the market proper we had no doubt that the same two men we were looking for it committed to hold up the abandoned car which they'd used in the robbery was gone over for fingerprints but it was clean mean time the stakeout went on Saturday past Sunday Monday nothing no leads on the suspects not a trace of them Tuesday the same thing all over again the operation settled down to a dull routine Tuesday night we checked the closing of the last market in our stakeout area 1045 p.m. ben and I drove back to 77th Street division every day seems to get a little longer these lousy stakeouts sure don't wear very well on me yeah I don't know what the setups gonna be if we don't get a break pretty soon we can't hang on to the gang from metro division forever join has been a bad month all I need now is a visit from the inlaw and do it up fine you talked to Jess at dinner didn't you know what do you say about those two men they picked up on South Maine this afternoon they brought them in for processing checked them out couldn't find anything wrong both of them came for it how about grabbing some lead after we check on okay we'll stop by Johnny Coke and what about a Frank's dip sandwich how's that sound haven't had one for a long time it suits me oh wait a minute he just came in it's for you Friday turn in from the shirts off oh yeah thanks Bob Friday talking yeah dick just now mm-hmm yeah we'll be over right away I got anything market bandits pulled a hold up a couple of minutes ago and store on Graham Avenue it's out in County territory yeah one of the sheriff's units took the call chased the hold up car two miles they get the things on your half right huh they got one of them 10 52 p.m. Ben and I drove to the sheriff's Firestone substation where the market bandit suspect was taken after his apprehension when we got there we talked to deputy sheriff sergeant Eddie Jones and Mel Viny who had made the arrest they said they arrived at the scene of the hold up the market on Graham Avenue a few moments after the bandits pulled away and they're stolen nash sedan Jones and Viny gave chase but when they finally forced the hold up car to a halt the driver was the only one in the car somewhere along the route of the two mile chase the second robbery suspect had succeeded in jumping out along with the loot taken in the robbery and making good his escape a broadcast and the bulletin was gotten out immediately the hold up suspect they'd apprehended had been fingerprinted and they were checking on his identification downtown Ben and I went in to talk to him Jones and Viny stood by while we questioned him I still don't know what it's all about property clerk will give you an itemizer seat for everything you have on take everything out of your wallet now spread it out into him making a pretty big thing out of nothing aren't I haven't done anything you want to check those things from his wallet been the cards it's some of those papers and yeah this your true name on this card here Grant B. Jameson all right that's right this your present address yeah you ever been arrested before why you ask that it's not going to take too much time to find out you might as well tell us we still don't know what this is all about I was speeding a little that's all I'm done anything now you're kidding yourself James only got everything on you that we need what do you think you're going to gain by stalling us must have me mixed up with somebody else I don't know what you're getting at this card here you took out of your wallet you recognize why these names and addresses you've got it down on the back of it mind checking them out for I don't know what you mean take a look these here whose addresses are they who are the people let's see no I don't think I even remember just something I scribble down that's all you don't even remember one of them no I don't think so I ask why don't you come off it mister what are you talking you know what we're talking about you know it as well as you know your own name 14 robberies down here in 28 days you're picked up on the hold up car after the last try tonight your partner gets away now you go right on kidding yourself James and play it real close you see how far it gets you I sure wish I knew what you were talking about I don't know the thing about any robberies all right then you stand by mister you're gonna know a lot more about him you're gonna know a lot in the hurry yeah just as soon as we pick up your partner 12 20 a.m. we continued to question the suspect Grant Jameson but he refused to admit anything even the fact that he'd stolen the car that he'd been apprehended in the only strong lead we could pick up from the articles found on his person was the list of four names and addresses jotted on the back of a card found in his wallet 12 30 a.m. Ben and I left the substation and drove to the address Jameson that told us was his present residence an apartment house on Vernon Avenue the manager told us he'd moved at least a year ago without leaving a forwarding address 1 a.m. we began checking out the list of four names and addresses which we'd found in the suspect's wallet the first was a jack williams his address a spring street rooming house the landlady told us he'd moved to Cleveland Ohio three months before the second was a John Gallagher his address a third-rate hotel in East Hollywood he hadn't been registered there in the past two months the third was a Matthew Sanford his address turned out to be an apartment house in the Crenshaw district want to place the light on the mailboxes over here Joe yeah here you go you see the name there yeah here it is ML Sanford that ought to be an apartment fire let's try it front door open sing for five should be done this way again mm-hmm okay let's give it a try police officers you Matthew Sanford get out of here get out of here you got it Joe all right get your hands on I'll cover him look around huh all right what is this anyway what's the idea you tell us you pulled a gun you got no business in here you got no right all right you just stand still we explain it downtown money sacks Joe found him in the desk same market they hit tonight all right Sanford you got nothing from me I don't know what you're talking about get his coat what you been yeah what are you trying to build anyway you haven't got anything on me 14 robberies mister that's what we got on you you think you got proof we got it yeah 14 victims come on let's go the names were changed to protect the innocent the two market banded suspects Grant Burton Jameson and Matthew Roy Sanford were tried and convicted on 14 counts of first degree robbery and received sentences as prescribed by law first degree robbery is punishable by imprisonment in the state penitentiary from five years to life you have just heard dragnet a series of authentic cases from official files technical advice comes from the office of chief of police W. H. Parker Los Angeles police department


My name is Amyak Sureshagar and I am representing my own company Aquarius Labs. Today I will be making an application which is going to help a most important problem faced by a newly let's say you go for an education to a new place and the first problem you face is like how to manage an expense and that too with a stranger. Let's say a group of three people they are managing their expenses like groceries too, let's say expensive alcohol and everything. So throughout the month they need to calculate their expenses, split up these four expenses. That's the most tedious job any bachelor can have during the study. So I'll be making an application like it's copyrighted with that service and you just upload this whole expense.


Boom, what's up fam? Anthony Johnston here today, founder of 21 studios got some big news to share that unfortunately is not so good So I'm gonna jump right into it now in December 29th 2021 about two weeks ago Our channel 21 studios was instantly kicked out of the YouTube partner program suspended What would seem to be indefinitely? No warning was given of any kind this happened about midday around noon poof The entire channel was demonetized and all 2,200 videos were demonetized over 2,000 Now for those of you don't know what the partner program is on YouTube It's what allows our channel 21 studios and all channels on YouTube to monetize What that means is like advertising super chat super. Thanks Memberships literally anything on YouTube that has to do with money has to go through the partner program and our channel found in 2007 by me 21 studios has been in that program since 2011 so well over 10 years at this point We never had a problem never had any warning for this either like I said and on top of that We didn't get a strike and we have never had a strike. So since 2007 never mind 2011 or 2021 We have never had a community strike. No kind of violations nothing like that not even a copyright strike Which is similar on YouTube. It's not good to have those So we had no warning and our entire channel was kicked out and we have no strikes and we continue to have no strikes and Even so this is a huge decision by YouTube that massively affected my company and our channel that you guys have come to know Love through our speeches and all the videos we post On top of that being kicked out of the program all the videos being demonetized all December money that we earned on YouTube instantly vanished from AdSense where YouTube pays out to you It's like a little process They have the Google to pay out to your bank account your company all that money disappeared poof It was over ten thousand dollars. It was like ten thousand nine hundred change instantly went to zero Google AdSense so that all disappeared overnight poof gone So again, we got no warning the zero warning the zero strikes and technically zero money from December all just went boom like that So what you're about to watch those is important is a five-minute appeal video that we were allowed to make for YouTube After this decision was made for our channel the appeal process is a new pilot program They started I think in 2021 so it's pretty new and there's very specific instructions to make a five-minute video with a couple Mechanics to it like aspects you need to cover and stuff. So where you're about to watch is that five-minute video? I'm extremely proud of it. I think it's the best video we've ever made. So I hope you enjoy it. I'll see you in a few minutes Hey, I'm Anthony Johnson founder of 21 studios. I appreciate your time and attention today I'll be narrating our appeal video. Make sure you hit like and subscribe and I'll see you soon 21 studios is a channel nearly as old as YouTube itself created by me fresh out of high school in July of 2007 at 18 years old as a student at the University of Central Florida in Orlando our first video is an event trailer from the under 21 convention that I created in 2007 a self-improvement event for young men from around the world reporters from my school newspaper would go on to interview me and report on these Early 21 conventions held each summer in 2010 I took a chance and dropped out of college to focus on my YouTube channel taking the big step of turning a hobby into a full-time business And expanding our events across the world as a college dropout and with only enough money for a one-way plane ticket I took a chance and expanded our convention to Stockholm, Sweden that year in the early years our channel videos are popular with young men Looking for mentorship guidance and how-to advice for relationships health and fitness I favor the TED Talks model of all speeches published free to the world to help millions of men improve their lives last year We celebrated the 15-year anniversary of these events which have been organized since 2006 around the world including our founding city of Orlando As well as Tampa Miami, Florida Austin, Texas Stockholm, Sweden London, England Warsaw, Poland and Melbourne, Australia The videos from our events have been the main purpose and content of our channel since day one over 95% of our 2000-plus channel videos are from these conventions and newer events for fathers and women in 2020 our videos and events hit the big time making national headlines including the New York Times New York Post Sky News and my own 20-minute TV interview with Piers Morgan a journalist who has interviewed world leaders like President Obama and President Trump among many others in front of over half a million people I got to represent my own company and YouTube channel that they broadcast on live TV Thanks to the news and social media. We reached over a hundred million people in 2020 our video production process is highly advanced for a channel This size we have a professional film team to film and edit from over 200 terabytes of video My admiration for Steve Jobs inspired me to pursue excellence in the quality of our videos and events I was also inspired by my tour of YouTube Space LA for designing our studio and editing space I believe our conference footage is some of the best and most beautiful of its kind on YouTube Even surpassing TED Talks and key aspects with only a fraction of the funding and subscriber base This includes filming three conventions Simultaneously plus an interview show all in 4k cinematic quality at multiple camera angles with professional audio Staging lighting and drones at 21 studios We film and edit everything ourselves for max quality and authenticity Ted Talks publishes their speeches free to the world and inspired me to do the same They have special events for women, but not men creating a content gap on YouTube We fill this gap with educational content Featuring over 170 professional speakers that includes psychologists like Dr. Sean Smith and Dr. Robert Glover Medical doctors like Dr. Doug McGuff FBI agents like Joan of Aro attorneys like Melissa Isak and Navy Seal veteran Rich Graham We've also featured YouTube stars like JP Sears and fitness youtuber Elliot Hulse Elliot is a friend and mentor who has inspired me to work relentlessly hard on my channel and never give up We've also worked well within the larger YouTube creator community Over 95% of our videos are professional original content I believe these videos are fully compliant with all of YouTube's policies to the point that they are exemplary in this regard All other content is always used with express written permission or on a fair use basis with a transformative nature to the video Our videos and events promote open debate free expression and traditional family values While not everyone will agree with our speakers We believe transparent civil discourse is the best way to navigate ideas and push the conversation forward in my mission to publish Our videos free to the world the partner program has been essential We have steadily relied on it for monetization for our videos since 2011 without the partner program our ability to film edit and publish Our videos free to the world like TED Talks is significantly jeopardized YouTube is always treated as fairly and we have always worked to abide by the terms you set to show for it We have a perfect 14-year track record for zero community strikes and zero copyright strikes This recent action to instantly remove our channel from the partner program with that warning came as a genuine shock And the generalized reason provided of reused content conflicts with YouTube's own human reviews of our content Including two fair use videos from December that fit this description They were reviewed and fully approved for monetization by YouTube These reviews informed my choices and led me to believe we were in full compliance with YouTube policy as always Sincerely I do not understand why the suspension has happened Especially given our long history of compliance with guidelines copyright and monetization policies Thank you for your time and attention today YouTube is a major part of my business And has been a daily part of my life since I was 18 years old I hope that you accept their appeal and reinstate 21 Studios to the partner program I believe this is the right decision due to the positive professional nature of our content and a long-term relationship Our channel has with YouTube. Thanks again, and I hope to hear from you soon All right, man, welcome back. I appreciate you watching that five minute video our official five minute appeal video to YouTube Per their instructions and guidelines for making a video like that and the comments below want to know a few things from you After you hit like and subscribe, of course So first of all in the first in the comments What do you think about the video we made from an artistic and creative standpoint and how effective it was a conveying information For an appeal was it effective? Was it artistic? Was it creative all that kind of stuff? Let me know in the comments below on top of that What do you think about YouTube even taking this action in the first place? Against any channel on YouTube 21 Studios or otherwise particularly with no warning And number three, do you think YouTube should accept the appeal? Yes or no because it's a binary decision at that point Right. They got to accept it or deny it So do you think it was creative artistic and effective first part second part? What do you think about them taking the action at all particularly with no warning and finally do you think they should accept our appeal? So let me know in the comments below. I appreciate it I will read them and then after this video and our next video. I'll see you next time I'm gonna let you know YouTube's official response to our appeal video. Thanks, and I'll see you soon


Everybody forced here ripping 2023 leaf autograph football jersey 10 box case number seven random team style Good luck. Here we go. There's a seven seven times on the random one two three four five six and seven Got Alan a down to William G and seven times on the teams one two three four five six and And seven Eagles down to Bengals Alan a Eagles Cameron C Cardinals Peter C Colts Dan C Dolphins Oliver H commanders Dan C Titans Shane C bucks Jeff G Cowboys Steve P Chargers Jenna with the Browns Alan Raiders Kyle our Lions Justin F Bears David M Texans Robert H Saints William G Ravens Peter C Panthers Michael T Jets Brandon J Seahawks William B Vikings William G Patriots Kyle our Niners Ryan W chiefs from Jeff G Falcons Dylan J The Broncos David M Giants Troy H Rams Brandon J Jags Kyle our bills Wayne P Steelers Jeff G Packers William G Bengals Flawless down to four down to four. All right, let's do this. I don't know why just that off.  And what are you watching now? Oh, that's sick Ray Lewis Ravens From we've got linebackers got better right there Ray Lewis back it authenticated for the Ravens. Oh No, get something. It's been okay. So we should stay here then. Oh Okay, okay, okay. I was gonna say Same thing. Yeah, I thought I thought the college game was on Which would make sense for a Friday night to be honest. Thank you Vaughan You got a Niners Joel Montana JSA authenticated Joel Montana Yes, that is a Niners Jersey in this Montaña, we've got a collegiate Jersey here. This is gonna be a Seahawks Jackson Smith and jigba. This is an Ohio State Jersey Seahawks Smith and jigba. All right, so Washington is the first team to officially make a playoff then I guess and they'll be If everyone if everybody that's supposed to be in right now if all stays the same, I guess they'll be number three a Justin Fields The Bears I can be science nice and full on the number there Beckett authenticated Justin Fields for the Bears two Bears jerseys now airy very nice Heck yeah, I'm glad to hear it man Save some money on Christmas joining Jersey breaks, huh? Let's go Raiders again Tim Brown Raiders double dipping now Tim Brown Beckett authenticated. Oh, we got Josh Allen for Buffalo Fire Beckett authenticated. What a case Holy cow, man Grats again Kyle you crushed it can't be many of those in this Even that Jersey Got them Another collegiate. We've got Falcons Bijan Robinson Falcons Congrats Texas has one of the better Burn orange and white Very good break here and a willy rope Saints willy rope Hull of Fame 2012 inscribed JSA authenticated this dude I beat him in the hot dog eating contest though I'd probably be Yeah, it's just extended Hundred he might beat me man. I got a lady probably, you know, I'm pro athlete four. I would assume a pro athlete All right y'all that'll do it fun case moving on now to one


On Friday, February 18th, tens of thousands of students hit the streets in over 40 cities across Italy. They were protesting the flawed policies on public education followed by the Mario Draghi government. The protests were organized mainly by student and youth organizations and leftist groups, including Pote Real Popolo, USB Italia, the Communist Youth Front and Italian Communist Youth Federation. In recent times, Italian students have been protesting across the country, demanding safer schools, higher investments in public education, and free public transport. The protests intensified last month following the death of Lorenzo Parelli, a student who died in a workplace accident while on an internship. The latest mobilization came after 16-year-old Giuseppe Linoce died on February 15th in yet another work-related accident. The protests blamed a 2015 mandatory government internship policy for the deaths of the students. We are here today to support the student struggle against Alternanza Scuola Lavoro, that is a sort of trainship during school hours, and during the last two months two kids died during Alternanza Scuola Lavoro. So we are here to say no to this, but there were already a lot of mobilization and demonstration during the past few months with the students, but the response of our politics is repression. And in a country that values democracy, this is not an acceptable response, because we want to talk, we want our politics to hear as, hear what are we saying, and also because this is not only a student struggle, it is also a worker struggle about the conditions that we are living. They also denounced this spike in unemployment and government insensitivity towards schools amid the COVID-19 crisis. The protests can name the policies on public education being implemented under the influence of big business led by the General Confederation of Italian Industry or Confindustria. In several cities, students march to the offices of the government, ruling political parties and Confindustria. The students have also called for the abolition of compulsory internships and resignations of Prime Minister Draghi along with the Education Minister and Interior Minister. High school students in Italy have to take up a mandatory internship where the student needs to spend a minimum number of education hours from 200 to 400 a year working without salary in private companies. Experts have noted that even after seven years of implementation of these mandatory internships, they have not brought about any improvements for students and young workers. At the end of 2021, the unemployment rate of those aged 125 increased to almost 30%, double compared to other EU 27 countries. Various forms of precarity such as irregular work, low wages, violence and harassment at the workplace affect one out of two workers under 35. Students across Italy were pushed into a breaking point by the devastating COVID-19 crisis. In 2020, it was estimated that 13.1% of young people did not finish school and left early. According to reports, a number of students who made it to graduation but acquired only a part of the expected skills in high school also increased. Various progressive student youth groups from Greece, Spain, Cyprus, Brazil, Pakistan, Turkey, Palestine, Portugal, Belgium and other countries also extended the solidarity to the protesting students of Italy.