00:01:00 This is the scene outside my hotel window. It's July 2nd, and I'm in Shanghai.

00:01:13 I arrived yesterday, spent the night. I'm waiting to catch a plane to Huangshan City where the Chinese Peptide Symposium is going to be held.

00:01:30 I'm amazed at how developed Shanghai is.

00:01:51 It's much more developed than Beijing, as I remember Beijing.

00:02:46 The total of 180 participants from various genres with this forum is impressive.

00:03:01 This symposium will provide an opportunity and a place for all participants to discuss the matters which they are interested in, and to make friends here.

00:03:17 We also provide a scientific program that covers natural development, eating, health, and health topics in peptide science.

00:03:31 The conference will also provide a rich social program.

00:03:40 The place of conference, Huangshan, is the most touristy country in China.

00:03:50 I invite you to take this opportunity to enjoy the inside of Huangshan.

00:03:57 Welcome to all of you, particularly those from abroad, because I know how tiring it is. It's a long trip, particularly for some of you.

00:04:07 I would also like to thank the Chinese participants for supporting this meeting.

00:04:16 For those of you who have not seen the movie, One Aspect, this meeting was started in mid-90s.

00:04:27 That's how the Peptide Symposium started.

00:04:32 The major theme of the meeting is not science, but culture.

00:04:38 An important cultural change so that we all understand each other in a more even ground.

00:04:45 With that, I wish every one of you enjoy both the scientific program as well as the social program.

00:04:55 Because Dr. Zhu, J.C. Zhu, our chair, and Dr. H.Y. Zhu, our vice chair, have put a great deal of care, time, and effort to make sure you enjoy.

00:05:09 So let's give both of them a big hand.

00:05:13 Thank you.

00:05:21 I'm sorry to say that the director of another department of the meeting is not able to be here.

00:05:32 Antimicrobial peptides can be classified or broadly classified into this family of active peptides,

00:05:43 which are active on membranes, and they are also antiparticulate.

00:05:48 Now, some of the properties of these kinds of peptides are the fact that they disrupt membrane permeability,

00:05:55 or they can self-assemble to form pores or channels.

00:05:59 The first property is pretty interesting by itself,

00:06:03 because it allows you to design peptides that could transport peptides, proteins, DNA, or genes,

00:06:14 from outside to inside of cells for intercellular transport.

00:06:20 The second property is that these peptides can be folded into antiparticulate structures

00:06:34 and with clusters of hydrophobic and charged residues.

00:06:38 And there are four major types.

00:06:41 Here they go.

00:06:43 Today I'm only going to concentrate on beta-beta-sheets.

00:06:48 And they contain end-to-end cyclic structures.

00:06:51 Now, thus far there are about 400 of these chaotic antimicrobial peptides identified,

00:06:58 from plants to animals to bacteria.

00:07:09 These types of peptides can also be part of a protein,

00:07:14 a protein from the transport membrane or from viral or bacterial proteins or toxins.

00:07:22 These are called pre-antimicrobial peptides.

00:07:38 The largest family of beta-stranded antimicrobial peptide is known as defensin.

00:07:46 It's first identified in glucoside by Robert Arum, and then it's called ALGO.

00:07:52 And then the second class is also found in bacterial tissues, which is called beta.

00:07:57 Beta is missing.

00:07:59 And it turns out that defensin, as a family, is very important for the mucosal defense system,

00:08:08 as part of the innate defense system.

00:08:11 And they're also found in insects and plants.

00:08:13 And so it's a big family.

00:08:15 Now, the ALGO defensin and beta-defensin contain three beta strands.

00:08:22 They're about 3 to 5 Kg.

00:08:24 They contain three disulfide bonds.

00:08:26 The insect and plant defensin contain additional alpha helix,

00:08:30 and they contain additional alpha bond.

00:08:33 These are cross-faction antibiotics.

00:08:36 They treat bacteria, fungi, virus.

00:08:39 But they have one disadvantage.

00:08:42 The disadvantage, they're so incentive,

00:08:45 because our body contains, the physiological condition contains approximately 100 millimolar sodium chloride.

00:08:54 And if it's outside the skin, it's even higher.

00:08:58 It's 180 millimolar.

00:09:00 So it's inactivated under physiological conditions, the defensin.

00:09:05 And it's also inactivated by serum proteins.

00:09:08 So what we would like to do is design defensins that are stable in physiological conditions

00:09:15 and stable in serum proteins.

00:09:17 Now, we started this problem approximately six years ago.

00:09:22 And our approach was pretty naive at that time.

00:09:25 What we did, we started with a defensin.

00:09:28 This is from rabbit.

00:09:30 It's MD1.

00:09:31 It contains about 8 or 9 charges.

00:09:34 And there are three disulfide bonds, as you can see here.

00:09:37 This one is right here, linking the end to C.

00:09:42 And so what we do is just simply replace these two systems.

00:09:46 We put in two glycine in there.

00:09:49 And just link it end-to-end to form an amide bond, like this.

00:09:52 So we call this phase 3.

00:09:55 It turns out this one works out very well.

00:09:59 We just published the result after six years.

00:10:02 It's that this becomes salt-insensitive.

00:10:07 Now, we don't know why.

00:10:10 We have no idea why it becomes salt-insensitive.

00:10:20 Once again, it's July 4th,

00:10:23 the second day of the 6th Chinese Peptide Symposium.

00:10:27 And today, we'll be interviewing some Chinese peptide chemists

00:10:31 that were involved with the insulin project.

00:10:34 This is a test of the microphone.

00:10:37 1, 2, 3, July 4th, 2000.

00:10:50 Just a free talk.

00:10:52 No, no, make no mistakes.

00:10:55 No problem?

00:10:56 No problem.

00:10:57 You can repeat it as many times as you want.

00:10:59 Okay.

00:11:02 Okay, do you understand?

00:11:03 Sure.

00:11:04 My name is Guishen Lu.

00:11:08 I worked in the Institute of Materia Medica

00:11:12 Chinese Academy of Medical Sciences

00:11:16 in the Department of Organic Synthetic Chemistry,

00:11:20 or we say Medicinal Chemistry.

00:11:23 I used to work in Dr. Merrifield's lab in 1979 to 1982.

00:11:30 I had a very nice time to stay over there.

00:11:34 Dr. Merrifield, under his guidance,

00:11:39 I have done some work around glucagon.

00:11:44 And he was my mentor of peptide chemistry

00:11:49 because before I was over there,

00:11:53 I worked on the contraceptives in our institute.

00:11:58 I never had a chance to work on peptides.

00:12:03 When I have this chance to go abroad

00:12:05 to work with him under his guidance,

00:12:08 I started to study on the peptide synthetic chemistry.

00:12:16 That's okay?

00:12:19 Probably, why did you want to join Bruce Merrifield?

00:12:26 I was introduced by the president of the academy,

00:12:32 Professor Huang Jiasi.

00:12:35 He introduced me to the director

00:12:38 of Publishing Council of Rockefeller Foundation,

00:12:44 and he introduced me to work with Dr. Merrifield.

00:12:48 At the first time I know that I will go to work with him,

00:12:52 he's a very famous peptide chemist.

00:12:56 I was, how to say,

00:13:00 at first I feel very lucky and very fortunate to work with him,

00:13:04 but also I was very worried, I worried a lot

00:13:09 because I'm a new man, never touched this field.

00:13:15 If I work with this famous chemist,

00:13:19 can I be well to work with him?

00:13:23 I have many worries about that.

00:13:27 Have you heard about solar phase before you joined Bruce?

00:13:32 I read some references of him,

00:13:37 and I asked my brother to call him,

00:13:40 what shall I prepare before I went to the United States.

00:13:44 And the first time I met him in the Rockefeller University,

00:13:48 he asked me, why you are so nervous and worried,

00:13:53 to ask your brother to ask me so many questions.

00:13:56 I said, I have never had this experience to work abroad,

00:14:03 so I worried.

00:14:05 Let me repeat the question again.

00:14:07 Have you heard of solar phase method before you joined Bruce Merrifield?

00:14:13 I know, I heard about it as PPS,

00:14:17 since Dr. Merrifield invented that method.

00:14:22 And so he was so famous since he invented this.

00:14:28 What did you think of solar phase then, not now, but then?

00:14:33 What was your feeling about solar phase synthesis at that time?

00:14:38 I almost have no idea,

00:14:41 because never tried these experiments in China.

00:14:45 So I don't know if it's easy for me,

00:14:48 or it's very difficult for me to study this new field.

00:14:54 Oh, it was new?

00:14:55 It's very new.

00:14:57 It's new for me.

00:14:58 So it's new in China too?

00:15:01 In China, not many people work in this field.

00:15:06 As I know, only some people in Shanghai,

00:15:09 and some people in Beijing, Peking University,

00:15:12 they have done...

00:15:14 Part-time work, but not solar phase?

00:15:17 They did solar phase, I think.

00:15:20 They published some papers, so I know that they have studied,

00:15:25 but not many people, only very few of them.

00:15:31 Well, is there anything else you want to say to Dr. Merrifield?

00:15:37 How was your experience in his lab?

00:15:41 I think the first talk to him, I feel very relaxed.

00:15:51 Before that, I was very nervous,

00:15:54 and anxious to work with this big boss.

00:15:59 But after the first talk with him,

00:16:03 I felt he is a very nice and kind person,

00:16:09 and very easy to talk with,

00:16:12 and very, very modest.

00:16:15 Something I have very impressed,

00:16:20 like once I asked a question about the lesson,

00:16:24 and he said, let's go to ask Jimmy.

00:16:28 His postgraduate thesis is work on resin.

00:16:34 That impressed me, because Jimmy is an assistant professor at that time,

00:16:40 and very young,

00:16:42 and the big boss went with me to his lab,

00:16:49 to Dr. Jimmy's lab, and to ask questions.

00:16:52 This is very unusual in China,

00:16:55 because big boss should know everything,

00:16:58 and never ask some people younger than him,

00:17:02 and even maybe his students.

00:17:04 So this is the first thing I was very impressed.

00:17:07 The second thing I was very impressed,

00:17:09 one student, his students worked with him for one year,

00:17:14 and then he changed mind.

00:17:16 He worked with another boss in the Rockefeller University,

00:17:22 and this student always came back to this lab

00:17:25 to try to find some materials he wanted to use,

00:17:32 and never thought he already did not belong to this lab.

00:17:38 So these two things made me think that Dr. Merrifield is a very kind person,

00:17:46 and in my opinion, in my experience,

00:17:51 if the student switched the supervisor from this professor to another professor,

00:18:03 they were very upset, not happy with these things happened.

00:18:16 Do you want to give a special greeting to Bruce Merrifield on his 80th birthday?

00:18:24 I joined the last celebration of his 70th birthday in Rockefeller University,

00:18:37 and the next year will be his 80th birthday.

00:18:44 So I would like to say happy birthday to him,

00:18:50 and also I would suggest him less work and more relax,

00:18:56 since he worked the whole life.

00:18:59 When I stayed over at his lab, only one year he had six times operation,

00:19:07 and every time after the operation,

00:19:09 he just went out of the hospital straightly to the lab,

00:19:16 and every day work in the lab,

00:19:18 and sometimes he pick up the medicine box and to take aspirin to decrease the pain,

00:19:31 and I said, are you very painful?

00:19:35 He laughed, I get used to take aspirin,

00:19:41 but I think he really is very painful and unpleasant for this sickness,

00:19:49 but he's very strong, and we say mind over mind, mind over matter,

00:20:02 he's a strong person.

00:20:05 So before I left the laboratory, returned to China,

00:20:10 I wrote a paper.

00:20:13 The title of the paper is A Great Scientist and a Common Person.

00:20:21 I wrote down what very impressed me,

00:20:26 what's his behavior, what's his good things I should learn from.

00:20:34 I wrote a paper before I left.

00:20:37 I give to Libby.

00:20:39 I said, I don't want to be flatter, means to say something good for the boss.

00:20:50 I said, I don't want to do that.

00:20:53 So before I left, I give you this paper.

00:20:58 If you can put on the paper of the Rockefeller University,

00:21:05 every week I think there is a published paper in black newspaper in Rockefeller University.

00:21:14 I said, after I leave, please you give the publisher of the Rockefeller University.

00:21:26 She said, she read this and almost tear up,

00:21:34 and she was very moved, emotion,

00:21:40 and she said, I think Dr. Merrifield won't let me do that.

00:21:47 And when I returned, she really didn't give to the publisher.

00:21:53 So I think they are very kind and very modest people as I know.

00:22:02 And so the whole life they contribute all energy and spirit for the science of the peptide chemistry.

00:22:15 So I hope they will get more relaxed and have a nice time and join their life over there.

00:22:27 It's okay?

00:22:32 I am Jiechen Xu from the Shanghai Institute of Organic Chemistry.

00:22:50 You can start over.

00:22:53 It doesn't matter.

00:22:55 Would you tell us your name?

00:22:59 I am Jiechen Xu from the Shanghai Institute of Organic Chemistry.

00:23:07 I am a professor.

00:23:11 I have been in charge of the department of biochemistry in this institute.

00:23:26 Can you tell us about your role in the insulin project?

00:23:33 In the 1960s, I was fortunate to have the chance to join the research group dealing with the synthesis of insulin.

00:23:54 I did some of the synthesis of peptide fragments of the etching of the insulin.

00:24:06 Who was the team leader?

00:24:08 At that time, the team leader was Dr. Low.

00:24:20 The group leader was not Wang Yu.

00:24:25 Wang Yu was in charge of the institute of leadership.

00:24:33 So your team is responsible for making fragments of etching?

00:24:41 Yes, etching.

00:24:43 At that time, I was a graduate student.

00:24:48 At that time, I was a graduate student under the supervision of Professor Wang Yu.

00:24:56 Professor Wang Yu was the overall leader of the project for the synthesis of etching.

00:25:07 How many people were in your team?

00:25:12 At the very beginning, almost half of the people of the Institute of Organic Chemistry were dealing with the synthesis of the insulin peptide synthesis.

00:25:30 But only for one or two months.

00:26:01 How many years? When did you start?

00:26:08 1960, May 1960.

00:26:16 The project was finished in 1965.

00:26:24 How many years did you work on the insulin project?

00:26:31 I worked on the insulin project from 1962 to 1965.

00:26:45 What fragments, how big are the peptides you are working on?

00:26:51 For me, I did the synthesis of the TEFAP pellet, the N-terminal of etching.

00:27:06 In large quantities?

00:27:09 Not so, maybe in grams.

00:27:16 And how many people, you said half the Institute was involved in the project.

00:27:25 How many people were there?

00:27:32 At the beginning, maybe more than 100 people.

00:27:44 All of them are working full-time?

00:27:48 Full-time.

00:27:54 One of the major things is the amino acid, because some of them need to be protected.

00:28:01 And for that, an amino acid factory was built.

00:28:07 When was the factory built, and what kind of material was it?

00:28:16 We built the amino acid factory in 1958.

00:28:26 Because at that time, we could not produce amino acids in China.

00:28:34 Most of the amino acids we got from abroad.

00:28:39 So we produced the amino acids ourselves.

00:28:48 This is amino acid from the unprotected amino acid?

00:28:56 First, we produced the amino acid, and we protected the amino acid in the laboratory.

00:29:05 So this is purely just producing like alanine?

00:29:12 Yeah, alanine.

00:29:14 This is from chemical synthesis or from fermentation?

00:29:19 For instance, for the isoleucine, it's the chemical synthesis, I think.

00:29:26 I think it's mostly by isoleucine.

00:29:30 I think it's only the isoleucine by the chemical synthesis.

00:29:37 Because isoleucine cannot be very difficult to isolate by the fraction.

00:29:45 What happened to the amino acid factory now?

00:29:50 Do they still maintain the amino acid factory?

00:29:53 Maybe you can ask this to Dr. Dui, because the amino acid factory

00:29:59 has been moved to the Institute of Bio-Organic Chemistry.

00:30:06 What was the major difficulty of your synthesis?

00:30:12 I think at that time, we were short of the experience in the peptide synthesis.

00:30:20 So I think for the peptide synthesis at that time,

00:30:25 how to choose the perfect protective group is very important.

00:30:34 Because at that time, we used the carboxylic acid for the protection.

00:30:50 And we said that we chose the master group for the side chain carboxyl protection.

00:31:04 And then we find that this is not a good protecting group, as good as the bulk.

00:31:17 During that time, are you aware of the competition from the United States and from Germany?

00:31:26 Do you know that the United States team was also working on insulin?

00:31:36 There's a team also working on insulin.

00:31:42 So do you know about the competition?

00:31:48 We know, yes. But maybe after...

00:31:52 After 1964.

00:31:55 After 1964?

00:31:58 I think before.

00:32:00 Before 1964.

00:32:02 We know the two groups, because we are missing.

00:32:06 We know the two groups, the teams in the Meinhof and Tsang group.

00:32:17 You think it's like this?

00:32:20 Not very interesting.

00:32:22 When a project was successfully completed, how did you celebrate?

00:32:32 Of course you graduated, but you got your Ph.D.

00:32:36 At the institute, how did you or the team celebrate?

00:32:47 I don't know.

00:32:49 No celebration at that time.

00:32:53 Did you get any award from the government?

00:32:56 No, no, no.

00:33:01 In the 80s.

00:33:04 Oh, yeah.

00:33:07 After the Cultural Revolution, maybe in 1982, the insulin census.

00:33:19 Right into the Cultural Revolution, isn't it?

00:33:23 Yeah, right after.

00:33:25 So after 1982, what did you get?

00:33:28 The insulin census.

00:33:30 I got a natural science award.

00:33:35 The first class.

00:33:42 That's very important.

00:33:44 Like the Chinese Nobel Prize.

00:33:46 Nice, yeah, like the Chinese Nobel Prize.

00:33:49 Academy Prize, yes.

00:33:53 Is there anything else you want to add?

00:33:57 Is there anything you want to say?

00:34:00 Say it in Chinese.

00:34:02 Yeah, you can say it in Chinese, or you can tell them about the meeting we have now.

00:34:08 The six CPMs.

00:34:11 You're in charge.

00:34:12 You're in charge.

00:34:15 Overwhelming building, whatever.

00:34:18 You can say whatever you want.

00:34:23 Chinese is easier.

00:34:26 You can say it in Chinese, or you can say it in Mandarin.

00:34:29 We'll help you translate it.

00:34:31 Translate, OK.

00:34:38 He's speaking half Mandarin, half English.

00:34:42 OK.

00:35:03 in the progress of the science of peptide science,

00:35:13 For instance, the synthesis of the...

00:35:22 Oxytocin.

00:35:23 Oxytocin.

00:35:25 Oxytocin, because it's the first time to synthesize a peptide

00:35:30 with a complicated structure.

00:35:33 And insulin synthesis,

00:35:38 because it's the first time to synthesize a protein.

00:35:44 And since then, for the people,

00:35:49 they are confident that peptide proteins

00:35:53 can be synthesized in the laboratory.

00:35:58 And another milestone is solid phase.

00:36:03 Solid phase peptide synthesis.

00:36:07 After that, there is a solid phase,

00:36:09 not only for peptide synthesis, organic synthesis.

00:36:13 And developed to the combinatorial synthesis

00:36:17 and the library.

00:36:20 So, see, the solid phase peptide senses

00:36:23 is very important in organic chemistry,

00:36:27 or in the biology.

00:36:39 progress of organic synthesis.

00:36:59 Would you like to say a special greeting to Bruce Merrifield on his eightieth birthday?

00:37:04 Chinese birthday.

00:38:23 of total synthesis insulin.

00:38:26 When I just graduated from Peking University in 1960.

00:38:34 And then, at that time,

00:38:38 I think I knew nothing about insulin.

00:38:42 And also knew nothing about peptide synthesis.

00:38:45 I learned from peptide synthesis

00:38:49 the project of total synthesis of insulin.

00:38:53 So I was so lucky.

00:38:55 And I was so happy to join this project.

00:38:59 Who was your team leader?

00:39:01 My team leader is Professor Qi Yixing.

00:39:04 You know him.

00:39:06 Qi Yixing is my advisor.

00:39:09 And he is leader of Peking University.

00:39:15 He is now 89 years old.

00:39:20 So he is still very active in his mind.

00:39:27 And what is the function of the team?

00:39:31 The function of the team from government.

00:39:35 What is the goal of the team?

00:39:37 Are you making A chain, B chain?

00:39:39 In the beginning, we made A chain.

00:39:43 And also from Shanghai Institute of Game Chemistry,

00:39:48 made A chain too.

00:39:50 Only for Shanghai Institute of Biochemistry,

00:39:53 they made B chain at the beginning.

00:39:56 But we put many, many students to work on the solvent

00:40:02 and also including the amino acid.

00:40:06 We isolated from serine, from silk, a lot of things.

00:40:15 But I think the level is lower at that time.

00:40:19 So after that, we know in Germany and the group

00:40:25 and in United States, the two groups

00:40:30 work on the insulin synthesis.

00:40:32 So after that, I think maybe in 1963,

00:40:41 all the leader, including the leader of the institute

00:40:47 and also from our university president,

00:40:52 they decided to collaboration to finish this work.

00:41:00 So basically, your department of chemistry at that time,

00:41:05 are you the only, is Professor Qi's lab the only lab

00:41:10 working on insulin?

00:41:12 Or there are many other labs?

00:41:14 Only organic division, but including

00:41:17 a lot of senior students at that time.

00:41:22 And how many people sit there?

00:41:24 I think more than 100.

00:41:29 The efforts are usually for starting material.

00:41:32 Yeah, yeah, yeah.

00:41:33 So after, I think we discussed the collaboration

00:41:40 at the end of 1963, I remember.

00:41:46 And so from 1964, Peking University

00:41:53 have a team to go to Shanghai Institute of Organic Chemistry.

00:41:59 So we have the same lab with Professor Xu.

00:42:04 I think our university send six people there,

00:42:11 except Professor Xin.

00:42:14 Except he was in Beijing, but for a period,

00:42:20 he came to Shanghai to discuss.

00:42:24 So what were the major difficulties

00:42:27 in accomplishing your insulin synthesis?

00:42:32 I think the biggest problem is every material we have known.

00:42:38 Every material we have made by ourselves.

00:42:43 For example, phosphate and hydrobromide in acetic acid.

00:42:50 Because at that time, only the amino acid

00:42:54 protected group for only CBC.

00:42:57 So we need to produce a lot of hydrobromide and acetic acid.

00:43:05 So I, Professor Chong Ximi and I, with a very big part

00:43:14 is to produce a lot of many kilogram to give everybody

00:43:21 to use.

00:43:22 And all the protected amino acid by ourself,

00:43:31 prepared by ourself.

00:43:33 And we couldn't find the selectivity protective group

00:43:41 at that time.

00:43:44 And every solvent we purified by ourself.

00:43:49 So a very big amount of work.

00:43:53 Because at that time, the solvents

00:43:56 are not in good quality.

00:43:58 So we must read distillation and dried.

00:44:04 So it's very, very big.

00:44:06 And also at that time, I think we knew a little

00:44:10 about peptide synthesis at that time.

00:44:13 So we need to learn.

00:44:15 We read a lot of literature at that time and discuss.

00:44:20 Were there any special problems since the teams

00:44:28 are in different locations?

00:44:31 And what were they?

00:44:33 And how were they being solved?

00:44:37 This is a big problem, especially for our team

00:44:42 from Peking University.

00:44:44 Because all of our family in Beijing,

00:44:48 but there are many.

00:44:50 I think at that time of the team, we are very young.

00:44:55 All of us less 30 years old.

00:44:58 I think I was 28 years old.

00:45:02 My child only one years old.

00:45:05 And my colleagues, their children

00:45:08 are also one or two years old.

00:45:12 So we left the children in Beijing or in other place.

00:45:19 And only ourselves in Shanghai.

00:45:22 We work till morning, until night.

00:45:26 And also in the weekend.

00:45:29 We never have relax in the weekend.

00:45:34 So every day we work very hard.

00:45:36 Why do you work so hard?

00:45:38 We hope to get the first total CSS for insulin in the world.

00:45:45 So there is the national pride involved?

00:45:50 Because you feel you are doing, contributing in competition,

00:45:54 the pride of being the first country or nation to produce?

00:45:59 Yes.

00:46:00 That is why.

00:46:03 When the project was successfully completed,

00:46:08 did your group in Beijing celebrate?

00:46:12 No.

00:46:13 At that time we were in Shanghai.

00:46:16 But I think we didn't celebrate.

00:46:20 I remember we didn't celebrate.

00:46:22 So how long were you in Shanghai?

00:46:25 I about one year and half year.

00:46:31 And then I went to the countryside.

00:46:37 What kind of countryside?

00:46:39 Beyond Beijing.

00:46:43 Why?

00:46:44 For some reason.

00:46:49 Is there anything else you want to add?

00:46:54 I hope to say that we know in the beginning,

00:46:58 when we know the STPS, we know this is new things.

00:47:04 But at that time it is difficult for us to use it.

00:47:10 Why?

00:47:11 Because we thought the product, the target compound,

00:47:18 is not in good purity.

00:47:22 We need to purify it.

00:47:25 It is very difficult to purify the mass, the compound.

00:47:31 This is question 16.

00:47:38 Is there anything else you want to add?

00:47:42 I want to say to Professor Merrifield,

00:47:49 80 years old, I wish a happy birthday to him.

00:47:56 And also I hope he is in good health, long life.

00:48:02 And I remember he gave a wonderful lecture in our university.

00:48:08 A lot of people stand there to listen to his lecture in 1994 in Beijing.

00:48:18 Before I met him, I thought Merrifield is a very tall, great person.

00:48:27 But after I met him, it's very, very easy to get along

00:48:35 and agree with Guixiang's opinion.

00:48:41 It's very modest.

00:48:43 He's a great man, but modest, very modest.

00:48:51 My name is Yuchang Du.

00:48:54 I'm a principal researcher, I think, or called a professor.

00:49:04 Actually, I'm not a professor in the university.

00:49:11 I work on the insulin project at the beginning from 1958.

00:49:23 Just a graduate student come from Peking University, biology department.

00:49:34 After I came to Shanghai, just at that time,

00:49:43 in Shanghai Institute of Biochemistry, there was a big argument.

00:49:51 Which topic should we take to do the so-called big job?

00:50:02 At that time, the government asked people to do a big job.

00:50:08 A great leap forward.

00:50:10 Yeah, a great leap forward.

00:50:13 Forget every small topic.

00:50:20 So the seed of the project started in 1958.

00:50:23 1958, yeah.

00:50:25 And that's because of the great leap forward.

00:50:29 At that time, the government encouraged the people to do the big work.

00:50:38 For example, should some satellite go to the moon,

00:50:47 or climb the mountain called Mount Everest.

00:51:00 Actually, at that time, the theme of climbing the mountain was the beginning time.

00:51:07 So we at that time argued with the people,

00:51:14 what should we do?

00:51:17 What topics?

00:51:19 For example, some high blood pressure, or cancer, or some disease, or something.

00:51:30 At last, they finished this argument.

00:51:36 Decided to do the synthesis of protein.

00:51:45 But the first protein known the structure, only one is insulin.

00:51:55 So we chose the insulin to synthesize.

00:51:58 Carbon nucleus was not known?

00:52:00 No, not known.

00:52:02 Yeah, because 1956, Sanger published this sequence.

00:52:14 So in my opinion, the other two groups in the world,

00:52:21 Americans and the Western Germans,

00:52:26 maybe they like to do this kind of thing at the same time.

00:52:32 Start about 1958?

00:52:34 I don't know the actual time, but I think they know that.

00:52:41 Because they want to do synthesis of protein too.

00:52:46 Does your institute or the team leaders know that the German group?

00:52:53 At that time, I don't know.

00:52:55 When did they know?

00:52:57 We know it should be after 1960.

00:53:02 After 1960.

00:53:05 Because at that time, they published a few papers,

00:53:11 synthesize some peptides, insulin peptide fragment.

00:53:19 Now we know that.

00:53:22 And in 1960, we know the combination of insulin by AHA and BHA.

00:53:30 One paper is from Canada.

00:53:35 It's called Dixon-Vedro.

00:53:39 They do that just before my paper came in.

00:53:48 How many teams were involved in this project?

00:53:52 That's hard to say.

00:53:55 Because if you vary the so-called teams,

00:54:02 at that time in 1960,

00:54:06 the big leap forward,

00:54:09 this time they collect lots of the people.

00:54:13 In Shanghai, I think including Physiology Institute,

00:54:17 Materia Medica, Institute of Materia Medica,

00:54:20 and Organic Chemistry Institute, and Physiology Institute.

00:54:27 And Plantar Physiology Institute, too.

00:54:32 Yeah.

00:54:33 So hard to measure the people for money.

00:54:37 But I think this is a story,

00:54:41 not the regular research work.

00:54:45 Actually, after 1961,

00:54:50 we have a so-called discussion.

00:54:55 Lots of the leaders in discussion.

00:54:59 Some people is very angry,

00:55:03 says this is not the research,

00:55:07 but just a factory.

00:55:09 Or some people just playing.

00:55:14 You know?

00:55:15 Playing car, maybe.

00:55:18 Use the...

00:55:24 Airflux.

00:55:25 Yeah, yeah.

00:55:26 And shaking it,

00:55:28 or fall down from the top of the building.

00:55:35 Everything is messy.

00:55:37 It's a big gamble.

00:55:39 Yeah, big gamble.

00:55:40 So, actually, the real teams

00:55:45 is from 1963.

00:55:51 1962 and 1963.

00:55:54 So who are the major teams

00:55:57 that do the total synthesis?

00:56:00 Can you tell us what are the major teams

00:56:02 in the synthesis part?

00:56:04 What's the character?

00:56:05 That's the three groups.

00:56:08 Should we say Shanghai Institute of Biochemistry,

00:56:12 there is two groups called

00:56:15 a combination of H&BJ,

00:56:18 from H&BJ,

00:56:20 and the BCH Synthesizing.

00:56:23 And the H&BJ Synthesizing

00:56:26 is collaboration by Peking University

00:56:30 and the Organic Chemistry Institute.

00:56:33 Who are the scientific leaders?

00:56:37 Tell us who lead, obviously.

00:56:39 I think at that time,

00:56:42 it's more democratic.

00:56:46 Real, they have the young men,

00:56:53 for example,

00:56:54 here, the people,

00:56:56 and Xu and me,

00:56:59 most of us are young people.

00:57:03 I think just 20 to 30 years old.

00:57:10 Very young.

00:57:12 But we can say what we want.

00:57:15 We can express our...

00:57:19 How about the more senior people,

00:57:20 like Professor Wang?

00:57:22 Yeah, that's mainly the four.

00:57:27 For example,

00:57:32 Jingyi Liu.

00:57:34 He is in the Institute of Biochemistry.

00:57:38 And Zhou Chenglu.

00:57:43 At that time,

00:57:45 he belongs to the Biochemistry Institute.

00:57:49 But afterwards,

00:57:50 he go to the Institute of Biophysics.

00:57:57 And the other is Wang Yu.

00:58:02 Professor Wang Yu.

00:58:03 Yeah, Professor Wang Yu.

00:58:04 And Professor Xin.

00:58:06 Jingyi Xin.

00:58:08 Yeah, Jingyi Xin.

00:58:09 The four people,

00:58:11 I think, concretely,

00:58:15 to discussion

00:58:18 and to decide what to do.

00:58:22 That's senior research work.

00:58:24 So the overall plan

00:58:26 has come from these four,

00:58:27 more or less, four senior...

00:58:30 I should say senior scientists.

00:58:31 Senior scientists, yes.

00:58:33 So they draw the scheme of what fragment to make.

00:58:37 Yeah, I should say

00:58:40 something is discussed

00:58:42 by the whole workers, researchers.

00:58:47 They make their decision.

00:58:52 I should say like that.

00:58:54 Because sometimes

00:58:58 some senior scientists

00:59:03 is not very understand

00:59:05 what's happen in the work process.

00:59:13 So sometimes they make some,

00:59:15 maybe some wrong decision or something.

00:59:20 Really happen such kind of things.

00:59:23 And then, actually,

00:59:28 modified and revised,

00:59:31 then become correct.

00:59:34 In your world,

00:59:36 how long did the entire project take?

00:59:41 So from, I'm not sure,

00:59:43 58, 60, 62?

00:59:46 Yeah, yeah.

00:59:47 From the end of 58

00:59:51 till the 65, 65.

00:59:56 Now, this is including recombination of two...

00:59:59 Yeah, yeah, total, total, yeah.

01:00:01 Total.

01:00:02 So seven years and then nine months.

01:00:07 But the early stages...

01:00:11 Yeah, early stages.

01:00:12 Not too many people are involved,

01:00:14 but you get more and more people involved

01:00:16 in the height of probably 63 and 64.

01:00:20 And more people involved in...

01:00:23 Yeah.

01:00:24 Now, that's like that.

01:00:27 Firstly, is a few people join such a group.

01:00:33 Then in the Big Leap Forward,

01:00:36 it's swollen,

01:00:42 became a bigger, maybe some army group.

01:00:47 62?

01:00:48 Yeah.

01:00:49 No, before 62.

01:00:52 Just, I think, 1960.

01:00:55 Just five months.

01:00:57 Just five months.

01:00:59 From May Day

01:01:02 till the day of October.

01:01:06 First day of October.

01:01:08 Just this time.

01:01:09 After that, stop.

01:01:11 Stop, then became a so-called very few people to do that.

01:01:17 Then to the 1963,

01:01:20 62 to 63,

01:01:23 then the people more and more.

01:01:26 At the last, I think it's 21.

01:01:30 29.

01:01:31 29.

01:01:32 29 people.

01:01:33 29 people.

01:01:34 Yeah.

01:01:35 But at the height of what you say,

01:01:36 between May of 1960 to...

01:01:41 It's October of 1960.

01:01:42 Yeah, yeah, October, yeah.

01:01:43 And then you say the army,

01:01:45 you're talking about 500 people?

01:01:47 1,000 people?

01:01:49 Not 1,000, but sure, hundreds.

01:01:53 Hundreds, in the hundreds.

01:01:54 Yeah, sure, there'll be hundreds.

01:01:56 Now, when did the agreement of collaboration

01:02:02 between Beijing University,

01:02:05 at that time, Peking University,

01:02:07 and the Shanghai Group,

01:02:10 which consists of

01:02:13 organic chemistry, biochemistry...

01:02:15 I think I already mentioned about that.

01:02:18 Yeah.

01:02:19 You can read...

01:02:20 Yeah, I think it's from 1963,

01:02:26 yeah, to 1965.

01:02:29 Yeah.

01:02:30 What were the major difficulties?

01:02:34 For what?

01:02:35 In accomplishing the insulin synthesis.

01:02:40 Now, talking about the major problems

01:02:45 for the synthesis of insulin,

01:02:48 in my opinion, I think,

01:02:50 it is the combination of peptide chains.

01:02:56 The A-chain and B-chain.

01:02:57 B-chain and the A-chain.

01:02:59 They built up the A-chain and the B-chain,

01:03:05 and then to ligation each other.

01:03:11 We call the constitution

01:03:15 of from A-chain and B-chain

01:03:18 to a whole molecule.

01:03:20 I see.

01:03:21 Yeah.

01:03:22 So, were there any surprises

01:03:24 or unexpected observations

01:03:26 during this research?

01:03:29 Sure.

01:03:30 What were they?

01:03:31 Lots of things.

01:03:34 Because at first stage,

01:03:38 for example, in 1959,

01:03:44 I joined this...

01:03:46 at the beginning,

01:03:47 joined this group

01:03:50 to do the recombination

01:03:52 from A-chain and B-chain.

01:03:54 But the A-chain and the B-chain,

01:03:56 you know,

01:03:58 already some people on the paper

01:04:03 said that's impossible

01:04:08 to build insulin

01:04:10 from the A-chain and the B-chain.

01:04:13 They already have this conclusion.

01:04:16 Why?

01:04:17 Because they do this experiment

01:04:19 using the native insulin,

01:04:22 put some, for example,

01:04:26 put some BAL

01:04:28 or put some glycol,

01:04:33 thioglycol,

01:04:34 and to reduce the A-chain,

01:04:37 the disulfide bond,

01:04:38 and then lose the activity.

01:04:42 Then to oxidize it

01:04:44 to see, yes,

01:04:46 if some activity recovery.

01:04:49 No.

01:04:50 No one success.

01:04:53 So they have this topics of the paper.

01:04:59 It's impossible.

01:05:00 Yeah, that's impossible.

01:05:02 So this is...

01:05:04 I met the first...

01:05:06 Actually, we follow this one.

01:05:08 We do this research,

01:05:11 the job.

01:05:13 Really, it's...

01:05:15 Because at that time,

01:05:17 I presume the ribonuclease

01:05:19 was done by other group

01:05:22 so that if you reduce a protein,

01:05:25 it will re-oxidize spontaneously

01:05:28 and give you back enzymatic activity.

01:05:31 But insulin is an exception.

01:05:32 No, no.

01:05:33 It was an exception at that time.

01:05:34 No.

01:05:35 At that time...

01:05:36 Done?

01:05:37 No, no.

01:05:38 This work, ribonuclease,

01:05:40 they don't have...

01:05:41 Not published yet.

01:05:43 I see, I see.

01:05:44 Yeah, yeah, before.

01:05:45 Because their work is published

01:05:47 in 1961 or 2.

01:05:50 I see.

01:05:51 So before that time,

01:05:52 when we do that work,

01:05:54 there's no reliable data

01:05:58 or some theory

01:06:00 because we worry about that.

01:06:03 Insulin is a protein.

01:06:05 They have their structure.

01:06:07 We know that the protein chemists,

01:06:10 they should have some stable structure.

01:06:15 But at that time,

01:06:18 how to build the structure

01:06:23 by chemical tools?

01:06:27 This is a big problem at the time.

01:06:30 No one knows.

01:06:33 How did you do it?

01:06:35 We just try.

01:06:37 Just try everything.

01:06:39 The 1960s for us is a very hard time.

01:06:45 More than 600 field experiments.

01:06:51 600 people doing experiments?

01:06:53 No, no, 600 times experiments.

01:06:57 Oh, 600.

01:06:58 No.

01:06:59 A lot of experiments doing the recombinant.

01:07:01 Recombinant.

01:07:02 I see.

01:07:03 You came up with the best method, right?

01:07:06 Yeah.

01:07:07 At that time, using the sulfonate

01:07:12 and in a ratio of two to one.

01:07:16 We found this after 1964.

01:07:22 In the 1964, we published the second paper

01:07:27 called the Conditions for Recombination.

01:07:32 Actually, we saw that the program,

01:07:34 firstly, is in 1960.

01:07:37 So we published the first paper in 1961.

01:07:42 I see.

01:07:43 So this is based on numerous experiments

01:07:49 that established this documentary ratio

01:07:51 of the A chain and B chain?

01:07:53 Yeah.

01:07:54 OK.

01:07:55 So what was, other than the hard work,

01:08:05 was there any particular reason why the ratio of two to one

01:08:09 is important?

01:08:10 Are we looking back now?

01:08:12 Yeah, looking back.

01:08:13 We can tell you.

01:08:16 No.

01:08:17 Actually, after lots of the experiment,

01:08:22 after that, in my mind, I know a little bit

01:08:27 is the experimental experience called nothing else

01:08:37 in the oxidation solution is good.

01:08:43 Because before that, some people say

01:08:47 we should catalyze the oxidation.

01:08:51 I use the copper ion, or I use some HO2O, like that.

01:09:02 Or a flash of the oxygen.

01:09:06 Actually, totally is wrong.

01:09:09 Because this pushed the build of the disulfide bond

01:09:17 very quick.

01:09:19 Actually, in the solution, the A chain and the B chain,

01:09:22 the conformation is fit each other.

01:09:30 Knead sometimes.

01:09:33 Not so quick as you think.

01:09:36 So if you push it like this, mostly

01:09:43 is the wrong conformation.

01:09:45 So basically, you want it to equilibrate.

01:09:49 But it's under a redox condition that favor more reducing.

01:09:55 That's why you have two equivalent of diol.

01:09:59 And then one equivalent of a sulfonated form.

01:10:03 Is that what the rationale is?

01:10:06 So looking back, it's more reducing condition.

01:10:10 Yeah, yeah.

01:10:13 I think the main is the oxidation time cost.

01:10:24 The time cost is neither 12 hours.

01:10:29 And the lower temperature, 4 degree.

01:10:35 4 degree.

01:10:36 Plus, not the minus.

01:10:39 Plus 4 degree is very important to the cost.

01:10:45 Why?

01:10:46 Because at that time, the conformation of peptide

01:10:52 is the most stable.

01:10:58 So they are complementary.

01:11:00 There's a conformation system.

01:11:02 Yeah, yeah.

01:11:03 And the disulfide formation, the change.

01:11:07 So after, I think used to be in the 1970s,

01:11:15 some people do the other proteins oxidation.

01:11:21 They are used usually by the low temperature.

01:11:29 And other, if you put some ion in the solution,

01:11:34 they will interact the right.

01:11:38 This is the correct information, yeah.

01:11:41 It's too fast.

01:11:42 Yeah.

01:11:45 What techniques that are available today

01:11:49 would have been the most valuable to you

01:11:55 and your team during the project?

01:11:58 If you wish something, look, I wish I have that method.

01:12:03 For what?

01:12:05 For do your insulin project.

01:12:08 For you or for the whole team.

01:12:10 Other than the starting material,

01:12:12 you can buy all the reagents now.

01:12:14 Yeah, yeah.

01:12:15 Then you make all the reagents.

01:12:17 But what techniques?

01:12:21 Today.

01:12:22 What techniques today would have been most valuable to you

01:12:29 and your team during the project?

01:12:31 I think if for the recombination,

01:12:35 I think that the conditions of recombination of insulin

01:12:41 still is the 1964 papers mentioned about.

01:12:47 I see.

01:12:48 So those information, but we eventually solve it.

01:12:51 That doesn't count.

01:12:52 But if you say, look, the technology,

01:12:56 techniques that you know today, right,

01:12:59 would be most useful to you

01:13:01 because the recombinant process worked out.

01:13:05 You conquered.

01:13:06 Yeah.

01:13:07 Okay, so that happened during.

01:13:10 Yeah.

01:13:11 How about today?

01:13:12 In 1981, I met the people in medicine.

01:13:22 Wisconsin.

01:13:23 Wisconsin medicine, you know.

01:13:25 At that time, the relay people.

01:13:31 One chance.

01:13:32 Chance.

01:13:33 They gave a talk about the recombination of insulin.

01:13:40 They proposed some conditions of recombination method.

01:13:44 I asked this question.

01:13:46 I said, what's the improvement you have for your recombination?

01:13:55 They said, yeah, we have some secret

01:13:58 because that's the commercial.

01:14:02 But afterwards, I know from the patent or something,

01:14:08 I think no much improvement.

01:14:11 I have to specify that the work that Ron Chan reported

01:14:18 at that time was the synthesis of insulin

01:14:23 by recombinant DNA method.

01:14:26 And of course, even you produce it by recombinant DNA,

01:14:30 you still need to do the combination.

01:14:34 Yeah, yeah.

01:14:35 Right?

01:14:38 How?

01:14:39 Well, I asked this question before already.

01:14:46 Yes?

01:14:47 I would like to answer your question.

01:14:52 Okay.

01:14:53 You want to sit over there?

01:15:01 Yeah, you can sit over there and then you can talk.

01:15:07 Maybe you can hand keep her mic?

01:15:09 Yeah, yeah, sure.

01:15:10 The microphone.

01:15:11 I would like to answer your question.

01:15:14 Now, I think...

01:15:15 What was my question?

01:15:17 The question is, what technique that are available today

01:15:21 would have been most valuable to you and your team

01:15:25 during the project?

01:15:27 I think the first is MS.

01:15:31 Mass spectrometry?

01:15:32 Yes.

01:15:33 At that time, we have no mass spectra.

01:15:37 This is very important for peptide synthesis.

01:15:40 So all the bioanalytical technique available today

01:15:44 would have been most useful?

01:15:46 Yes.

01:15:47 Correct?

01:15:48 Yes.

01:15:49 I would say so.

01:15:50 And the column, and the column,

01:15:54 and also...

01:15:55 Reverse phase.

01:15:56 Reverse phase.

01:15:57 All the bioanalytical techniques.

01:15:58 HPLC.

01:15:59 I remember that we...

01:16:01 At that time, we analysis our product by paper chromatography.

01:16:09 The paper...

01:16:11 Yes, paper chromatography.

01:16:12 Yeah.

01:16:13 It's very small.

01:16:15 It takes many hours, for example.

01:16:21 So now, it's very quickly.

01:16:23 And also, SPPS.

01:16:26 Solid-phase peptide synthesis.

01:16:27 Yes, solid-phase peptide synthesis is quickly.

01:16:30 And if we use the purification by HPLC, it's easy.

01:16:37 It make peptide synthesis easily.

01:16:41 I would like to say.

01:16:43 And also NMR.

01:16:45 2D NMR for the conformation for peptide

01:16:50 is very important.

01:16:52 And another...

01:16:54 I would like to follow Professor Du's talk just now.

01:17:02 In the beginning, there are some argue about the project.

01:17:07 Some professors know no way to synthesize the insulin

01:17:12 because we couldn't find the selective formation of disulfide bond.

01:17:22 So it's difficult to select the disulfide bridge.

01:17:30 So I think Professor Du's work is very important to tell us

01:17:38 we can separate the insulin to A chain and B chain

01:17:45 and then combine them.

01:17:47 We can get the insulin.

01:17:50 So the selective protection of 6-sulfhydryl was never attempted.

01:17:58 In other words, there's no scheme.

01:18:00 There's no plan to selectively protect the 6-sulfhydryl.

01:18:05 No, no.

01:18:08 At that time, we have some consideration.

01:18:13 But at that time, no such kind.

01:18:16 The three kinds of substrate can be debroke the protection group.

01:18:30 We have a lot of meeting, one or two weeks.

01:18:34 We have meeting in Institute of Biochemistry

01:18:41 and Institute of Organic Chemistry.

01:18:45 We discuss everything and decided by the meeting.

01:18:52 So at that time, I think we have very good collaboration.

01:18:58 We have hard time but very happy to work with this project.

01:19:04 In Shanghai Institute of Organic Chemistry,

01:19:08 we were responsible for synthesis of B chain.

01:19:14 For Peking University, we were responsible for the non-peptide N-terminal.

01:19:24 For Shanghai Institute of Organic Chemistry,

01:19:29 how to say short?

01:19:34 D-deca.

01:19:35 D-deca.

01:19:38 Peptide C-terminal.

01:19:40 And then combine to the H-chain.

01:19:43 So every day, we work together.

01:19:49 Every person was responsible for one reagent or some solvent,

01:19:57 purified the solvent.

01:19:59 So everybody has some big job there.

01:20:12 What do you think about Professor Merrifield and his work?

01:20:16 I met Merrifield in 1976 or 7,

01:20:25 very early before the opening policy.

01:20:31 At that time, he came to China and visited us

01:20:40 to see what's happening in China

01:20:43 and in our lab.

01:20:46 I met him.

01:20:50 So I know him from that time.

01:20:56 This is right after Nixon's,

01:20:58 soon after Richard Nixon's visit.

01:21:02 1972, you see.

01:21:04 Yes, right.

01:21:05 After that.

01:21:07 Because at that time,

01:21:10 firstly, he wants to see what's going on,

01:21:16 the project of insulin.

01:21:19 And secondly, we showed him about the synthesis of glucagon.

01:21:30 So then, we found each other.

01:21:36 I think he is very kind to the professors

01:21:42 and said,

01:21:45 I'm very kind and very friendly.

01:21:49 At that time, you know,

01:21:51 not many people from abroad to visit China.

01:21:59 Before the Cultural Revolution,

01:22:02 lots of Americans and Germans and Chinese,

01:22:07 so they came to visit us.

01:22:10 But cut it down.

01:22:14 The glucagon was synthesized by solutions in the solar phase.

01:22:19 Now, this is a story.

01:22:22 I synthesized it by the so-called solid phase,

01:22:27 but it's a...

01:22:28 Segment approach, right?

01:22:30 It's coated.

01:22:32 Coated beads.

01:22:35 Oh, okay, okay.

01:22:37 Use the fragment condensation on solid phase.

01:22:41 He was very impressed.

01:22:43 At the time.

01:22:44 Oh, that's good.

01:22:45 Is there anything else you want to add?

01:22:47 Yes.

01:22:49 From that time, I met him lots of times.

01:22:55 First, he was on medicine,

01:22:57 and then several times on the Peptide Symposium.

01:23:03 And afterwards, he came to China,

01:23:07 joined the Chinese Peptide Symposium,

01:23:11 and visited us several times.

01:23:14 I feel Dr. Merrifield is a real scientist.

01:23:23 He's very kind and very polite,

01:23:29 and encourages us to do and to try.

01:23:38 I think my peptide career is lots of encouragement from him.

01:23:45 Because I am actually not a peptide synthesizer.

01:23:51 I am just a catcher of peptides.

01:23:56 Afterwards, before, yes, do the enzymology.

01:24:04 Afterwards, we do some biochemistry of peptides.

01:24:11 But how to synthesize the peptides,

01:24:14 actually, I am not the expert.

01:24:19 Would you like to say a special greeting to Dr. Merrifield?

01:24:23 Yes, yes.

01:24:26 I think Professor Merrifield gave me

01:24:32 the most impressive image, yes, his spirit.

01:24:39 First time I met him, he was very active in medicine.

01:24:47 I found he had some skin cancer.

01:24:54 And in my mind, if people get some cancer,

01:25:00 the emotion will be down.

01:25:06 So when I met him again, I said,

01:25:10 oh, you are still very active.

01:25:13 He's very glad.

01:25:14 He said, yeah, why not?

01:25:16 And so one year by one year, two years, so long time now.

01:25:23 I think that's the two years before.

01:25:26 And so long time, still is very active.

01:25:32 Even now, already close to 80 years old.

01:25:42 More active than me, maybe 70 years old.

01:25:46 So that's very impressive to me.

01:25:50 So that's called dedication to the scientist.

01:25:56 Thank you.

01:25:57 Thank you very much.

01:25:58 Thank you.

01:25:59 OK.

01:26:00 Very good.

01:26:01 Thank you.

01:26:02 Thank you.